CA2113156A1 - Lipid conjugates of therapeutic peptides and protease inhibitors - Google Patents
Lipid conjugates of therapeutic peptides and protease inhibitorsInfo
- Publication number
- CA2113156A1 CA2113156A1 CA002113156A CA2113156A CA2113156A1 CA 2113156 A1 CA2113156 A1 CA 2113156A1 CA 002113156 A CA002113156 A CA 002113156A CA 2113156 A CA2113156 A CA 2113156A CA 2113156 A1 CA2113156 A1 CA 2113156A1
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- Canada
- Prior art keywords
- glycero
- val
- amino
- phe
- peptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
- C07C69/96—Esters of carbonic or haloformic acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/10—Phosphatides, e.g. lecithin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/006—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length of peptides containing derivatised side chain amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1075—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of amino acids or peptide residues
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K11/00—Depsipeptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
- C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
- C07K5/0205—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)3-C(=0)-, e.g. statine or derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
- C07K5/06052—Val-amino acid
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/0606—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr
- C07K5/06069—Ser-amino acid
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06086—Dipeptides with the first amino acid being basic
- C07K5/06095—Arg-amino acid
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06104—Dipeptides with the first amino acid being acidic
- C07K5/06113—Asp- or Asn-amino acid
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/081—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
- C07K5/0823—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp and Pro-amino acid; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
- C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
-
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
- C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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Abstract
Compounds wherein therapeutic peptides, including HIV protease inhibitors, are covalently linked to phospholipids, glycerides or other membrane-targeting and membrane-anchoring species, and their pharmaceutically acceptable salts, together with processes for their preparation. The invention also provides novel HIV protease inhibitors. The compounds of the present invention possess useful pharmacological properties such as antiviral activity towards viral infection and inhibitory activity towards viral proteases.
Therefore, theses compounds can be used in the prophylaxis or treatment of viral infections, in particular infections caused by HIV
and other retroviruses. The targeting technology as described for the protease inhibitors is also applicable to a variety of inhibitors of other enzymes.
Therefore, theses compounds can be used in the prophylaxis or treatment of viral infections, in particular infections caused by HIV
and other retroviruses. The targeting technology as described for the protease inhibitors is also applicable to a variety of inhibitors of other enzymes.
Description
WO93/01828 2113 ~ 5 ~CT/US92/~153 LIPID CO~J~G~TE~ OF
T~ER~PE~TIC PEPTIDE~ ~ND~PROTEA8~ N~IBITOR8 This inv~ntion provides lipid ronjugates of therapeutic peptides. It also provides peptide inhibitors of HIY
protease.
Background of the Tnvention The uptake of therapeutic peptide~ into oells following in vivo administration is not efficient. A substantial amount of peptides are removed extremely rapidly from the plasma by a variety of mechanisms including cellular uptake and meta~olism, filtration by the kidney and~uri~ary excretion ~r destruction by the proximal tubule cells af :t.he kidney.
Furthermore the lipid bilayer of the cell me~brane presents a barrier to transport. ~et another disadvantage of these peptides is the need to a~minister them by injection because peptides in general po se55 very poor ~ral or nasaI
bioavailability.
The human immunodeficiency vi.~ s~(HIV) produces a long gag polyprotein which is cleav~d during Yiral ~uddi~g;into small~r~
proteins which ~have ~specific viral ~unctions. This proteolytic cleavage:is catalyzed by a ~peoifia~HIV aspartyl protease enzyme (~HIV P~). Small~;peptide fra ~ ents whi~h conformationally resem~le~the H~V pr~tease bind~i~g~si~e b~ :
have~the pl site~repia~ed by~non-hydro1yzable structures act ~:~ 25 ~-as~ inhi~it~rs of ~e~ HIV.~protease.~an~may be useful as~
therapeutic ayents in~AIDS,-~
. A.-::number~ of~ such prot~a~e,--i~hibitors ~ have~ een sy~the~ized. S~e ~or exampl~ erck European Pat~n~
:Application # EP 0 337 714 A,~publications by Upjohn, Sciençe ~47:454-456 (19~0), Roche European Patent Application EP 0 346 847 A2, Smith, Kline French International Pakent A~pli~a~ion WO 90/00399, Na~ure 343:90-92 ( 1 9 9 0 ~ t Proc.:Natl. Aca~d Sci.USA~ :
86:9752-9756 (1989),~Hoechst Europe~an patent Application~:EP 0 354 522 Al. All of:the~e pro~ease-inhibitors are peptides,~
and as peptides,~are subject to a g~ner~l problem of metabo}i ~ 3;1 5 6 PcT/us92lo6ls3 degradation and clearance which will prevent them from reaching the infected cell targets to ~ind HIV PR.
Protease inhibitory peptides, a~ well as other therapeutic peptides, la k targeting mechanisms in their na~ive foxm, and on administration to an animal they become distributed globally. For that reason they may not effectively treat infected tissues. For example, macrophage~
are believed to ~e a major s~te of HIV infection, and inhibitory peptides by themselves do not target to the macrophage reserYoir.
These problems can ~e overcome by preparing conjugates'of th~rapeutic peptid~s which resi~t clearance and degradation, and which can be targeted to the cell~
It is thereforP the object o~ the invention to overcome t~e problem of degradation and clearance of therapeutic peptides, including HIV protease inhibitory peptides, from the plasma. It is f~rther the object:of the invention to provide peptides comprising nonhydrolyzable groups capable o~
inhibiting HIV PR. ~nother object of ~his inv~ntion is to
T~ER~PE~TIC PEPTIDE~ ~ND~PROTEA8~ N~IBITOR8 This inv~ntion provides lipid ronjugates of therapeutic peptides. It also provides peptide inhibitors of HIY
protease.
Background of the Tnvention The uptake of therapeutic peptide~ into oells following in vivo administration is not efficient. A substantial amount of peptides are removed extremely rapidly from the plasma by a variety of mechanisms including cellular uptake and meta~olism, filtration by the kidney and~uri~ary excretion ~r destruction by the proximal tubule cells af :t.he kidney.
Furthermore the lipid bilayer of the cell me~brane presents a barrier to transport. ~et another disadvantage of these peptides is the need to a~minister them by injection because peptides in general po se55 very poor ~ral or nasaI
bioavailability.
The human immunodeficiency vi.~ s~(HIV) produces a long gag polyprotein which is cleav~d during Yiral ~uddi~g;into small~r~
proteins which ~have ~specific viral ~unctions. This proteolytic cleavage:is catalyzed by a ~peoifia~HIV aspartyl protease enzyme (~HIV P~). Small~;peptide fra ~ ents whi~h conformationally resem~le~the H~V pr~tease bind~i~g~si~e b~ :
have~the pl site~repia~ed by~non-hydro1yzable structures act ~:~ 25 ~-as~ inhi~it~rs of ~e~ HIV.~protease.~an~may be useful as~
therapeutic ayents in~AIDS,-~
. A.-::number~ of~ such prot~a~e,--i~hibitors ~ have~ een sy~the~ized. S~e ~or exampl~ erck European Pat~n~
:Application # EP 0 337 714 A,~publications by Upjohn, Sciençe ~47:454-456 (19~0), Roche European Patent Application EP 0 346 847 A2, Smith, Kline French International Pakent A~pli~a~ion WO 90/00399, Na~ure 343:90-92 ( 1 9 9 0 ~ t Proc.:Natl. Aca~d Sci.USA~ :
86:9752-9756 (1989),~Hoechst Europe~an patent Application~:EP 0 354 522 Al. All of:the~e pro~ease-inhibitors are peptides,~
and as peptides,~are subject to a g~ner~l problem of metabo}i ~ 3;1 5 6 PcT/us92lo6ls3 degradation and clearance which will prevent them from reaching the infected cell targets to ~ind HIV PR.
Protease inhibitory peptides, a~ well as other therapeutic peptides, la k targeting mechanisms in their na~ive foxm, and on administration to an animal they become distributed globally. For that reason they may not effectively treat infected tissues. For example, macrophage~
are believed to ~e a major s~te of HIV infection, and inhibitory peptides by themselves do not target to the macrophage reserYoir.
These problems can ~e overcome by preparing conjugates'of th~rapeutic peptid~s which resi~t clearance and degradation, and which can be targeted to the cell~
It is thereforP the object o~ the invention to overcome t~e problem of degradation and clearance of therapeutic peptides, including HIV protease inhibitory peptides, from the plasma. It is f~rther the object:of the invention to provide peptides comprising nonhydrolyzable groups capable o~
inhibiting HIV PR. ~nother object of ~his inv~ntion is to
2 0 provide ~or prodrugs o~ protease inhibitory peptides which treat the site of viral infection e~rec:tively~ Yet another o~ject of this inv~ntion is to improve the or3l and nasal bioa~ailability of therapeutic peptides.
SW~ARY OF THE I~ iTION: ~
The lnv~n~ion provides pepl:id~-lipid linking structurest protease ir~ibi~ors, and co~pou~ds ~h~r~iJ3 prot@as~ i~hibitor~
;: ` and o~her` ~h~rap~utic: peptides aré c:ovalently link~d ~o pho~pholipids, glyce~ides or other cell membrasle targeting or membrane anchorill~ moieties., Accordirlg to one aspect of the invention there are provided therapeutic peptides which are analogues of ~ubstrates for proteases. The~e peptides ia~clude the protea~e substrates of Group I d~scribed in detail in the specification. Pref~rred peptides of this group inhibit the
SW~ARY OF THE I~ iTION: ~
The lnv~n~ion provides pepl:id~-lipid linking structurest protease ir~ibi~ors, and co~pou~ds ~h~r~iJ3 prot@as~ i~hibitor~
;: ` and o~her` ~h~rap~utic: peptides aré c:ovalently link~d ~o pho~pholipids, glyce~ides or other cell membrasle targeting or membrane anchorill~ moieties., Accordirlg to one aspect of the invention there are provided therapeutic peptides which are analogues of ~ubstrates for proteases. The~e peptides ia~clude the protea~e substrates of Group I d~scribed in detail in the specification. Pref~rred peptides of this group inhibit the
3 5 HIV protease .
According ~o anoth r aspect of ~he invention there are proYided compounds capable of linking p ptides to lipid WO 93/01828 ~) 1131 5P&~/US92/06153 structures. These linking compounds include the amino ac:id phosphslipids of Group IIA D described in the specificatic~n, as well as other lipid-bearing linkers defined as X ~pe~::ies, suitable for attachment to an amino group of a peptide and C
spPcies, suita}: le fsr attachment to the carboxyl group of a peptide . .
According to yet another aspect of ~he invention there are provided peptide-lipid conjugates, comprising therapeutic peptides c:onjugated to any of the lipid-bearing linkers de~ined ~y the invention. Preferred species are lipid derivatives of protease inhibitory peptid~s. The peptide lipid conjugates can also com~prise spacers, compri~ing ~ifunctional alkyl groups def ined as Y, Z, and W spec:ies, which can modulate the distance between ~he peptide and the lS lipid linker. Preferreà peptide-lipid conjugates comprise peptide~ that are protease inhibitors. These conjugates of Group III- A-F are cle~cribed in detail in the specificationO
The invention also provides i.nte:~ediate ¢ompounds useful in the synthesis of t~e peptide~lipid c:ompounds oî the inven~ion, comprising deriva~ized linking c:ompounds suitable f or bind ing to the functional groups of lipids or peptideæ .
T~e invention al~o prcnride:s a m~tho~ for synthesizing peptideDlipid con~ugates, comprising the steps of selecting a therapeutic peptide having an available amino group or carbo~l group; c:hemic~lly bonding a }~ival2nt linker group to either the ami~o~;or th~ carboxyl groùp;i-and~
chemicaily bon~ding a : lipid species tc3 ~he ~ linker., In a preferred m~thod~ the p~p1:ide is a protease inhi~itor.~
The invention further provid~s a method of treating infectious disease caused by a virus, ~::omp:rising administ~ring to the infected person an effective virus~inhibiting amotll~t of a lipid derivative of a ~riral pro~ease inhibitory peptide.
The inhi~itory peptide-l ipid can b~ incorporated into a liposs:me prior to administration. In a preferred e~c:diment of the method, the disease treated is HIV infection, and the peptide is a HIV protease inhibitor.
WO93/01828 . . .. PCT/US92~061~3 '~ 113 1~ 6 DETAILED DESCRIPTION OF T~E INVENTION
The invention provides protease inhibitory peptides, lipid conjugates of therapeutic peptides, particularly peptides that are enzymP substrates, such as the HIV protease inhibitory peptides, linkers capable of ~ttaching lipid species to the peptides, intermediate compounds, and methods of synt'h~sis and use.
Group I: Protease i nhibitPrs:
Protea~e lnhibi~ors (PIs) are, in general, substrates of HIV 1 protease in which the amino acid residues at the pl site are replacPd by isosteric re~idues. Some of the HIV prote~se 5 substrates which ar~ the basis for the design of PIs are:
Sequence . _ __ Si~e _ ~ _ Ser-Gln-Asn-Tyr-Pro-Ile-Val pl7/p24 Ala-Arg-Val-Leu-Ala-Glu-Ala p24/p7 Ala-Thr-Ile-Met Met-Gln-~rg p24/p7 Ser-Phe-Asn-Phe-Pro-Gln-Ile HIV PR N-t~rm Detailed des~riptionæ of the design and~structures of individual :PIs are: discu~sed~ below.~ The present invention provides ~or the following:PIs~
Ser-Gln-Asn-Phe-Pro Ile-Val-NH2;
lS Ser-Gln-Asn-Tyr-Pro-IleoVal-N~2;~
Ser-Gln-~sn-Tyr-Achx-Ile-Va1~NH
Ser-Gln-Asn-Tyr-Acpr-Ile-Val-NH2;~
Ser-Gln-Asn-Tyr-Acpnt-Ile-Val-NH
Thr-I1e Leu ~(beta Ala~ Leu-Gln-Arg-NH2;~
2~ Se~-Gln A~n-Tyr-P~o~ -Val~Thr-Leu-Ava-Thr-Gln-Arg-NHz~;~
~Ac--~la-~la-~D-a Nal)-Pip-(a-~O~)-Leu)-Val-NH2;: : :
:Ac-Ala-Ala-Phe Pip-(a-(OH)-~eu)-Val-NH2; :~
Ac Ala-Ala-(DL-Phe(4-Cl))-Pip-(a-(OH)-Leu)-Val-NH2;
~la-Ala-Phe-(beta-~la)-Val-Val Gly-OH; : .
Ala-Ala-Phe-(beta-Ala)-Nva-Val-Gly-O~
Ala-Ala-Phe- (beta-~la ) ( a- ( ~H) ~iso-Valeryl ) -Val-Gly-OH;
~la-Ala-Phe-(beta-Ala)-Val-Val-Gly-O~e;
Ala-Ala-Phe~(beta-Ala)-Nva-Val-Gly-OMe;
Ala-~la~Phe-(beta-Ala)-~a-(OH)-iso-Valeryl)-Val-Gly:-OMe::
Boc-Ala-Ala-Phe-(beta-Ala)-Nva-Gly-OMe;
WO93/01828 2 1 1 3 1 ~FlUSg2/06153 .
-(a,e-di-~yristoyl-1ys)-D-Pro-Ava-Ala-Ala-Phe-~va-Val-Gly-O~e;
Ac-Ala-Ala-(D a-Nal)-Pip-OMe;
Ac-Ala-Ala-(D-a-Nal~-Pip-~HNH2;
~c-Ala-Ala-(L~a-Nal)-Pip-OMe;
Ac-Ala-Ala-(L-a-Nal)-Pip-NHNH2;
A~-Ala-Ala-(D-b~Nal~-Pip-OMe;
Ac-Ala Ala-(D-b-Nal)-Pip-NHNH2;
Chl-Ala~Ala-(L-aoNal) Pip OMe;
Chl-Ala-Ala~(L-a-Nal)-Pip-NHNH2;
~aa-Ala-Ala-(D-a-Nal) Pip-OMe;
Ac-Ala-Ala-(DL-Phe(4-Cl))-Pip-OH; .
Val-Ser-Gln-Asn-Tyr-Pro~ Val-~H2;
Val-Ser-Gln-Asn-(D-a-Mal)-Pip-(a-(OH)-Leu)-Val-NH2;
iBoc-(D-Phe)-(D-a-Nal)-Pip-(a-(OH)-~eu)-Val~NH2;
(iBoc-~D-Phe)~Leu )2-(3,5-di-Aba)-Pro-Leu-(~-Phe)-NH2;
iBoc-~D-Phe]-~D-a-Nal]-Pip-[L-a-OH)-Leu~ ValDNH2 i~oc-tD-Phe]-tD-a-Nal3-Pip~ a-OH~-Leu~-Val-OH
iBoc-Phe-tD-b ~al~-Pip-[L-a-(OH)~u]-~al-NH2 iBoc-Phe-~D-b-Nal]-Pip-~L-a-(OH)~eu~-Val-OH
iBoc-~Pbe(4-Br)3-~D-~-Nal] Pip-~L-a-(OH) L~u];Val OH
~Myristoyl-(D-Phe~ L~U-) 2 (3,5-di-Aba)-Pro-~eu-(D-Phe)-NH2;
iBoc-(3~5~di-Aba~-(D-a-Nal)-Pip-~a-~OH?-Leu)-Val-NH~;
Pro-Ile Val N~2; ~ .
Asn-Phe(CO-CH2N)Pip-Ile-NN2; ~
2~ Quinàldoyl-Asn-Phe(~O-CH2N3Pip r Ile-NH2;
~V l-S~r-Gln-A8n-Tyr-Pi~-Ile-Val-Gl~-NH2;
^ : Quinaldoyl-A~n-Ph~;(CHOH-C~2N~Diq-Ile-Val-Gln-NH2; : : :
Val-Ser-Gln-Asn-Tyr-Diq-Ile-Yal 1n-NH2;
Boc-Asn~Phe(CO-CH2N)~Diq-NHtBu;
Su~cinyl-S r Gln-Asn-Tyr-Pro-Ile-Val-NH2;
Succinyl Ser-Gln-Asn~Tyr;
Quinaldoyl-Asn~Phe(CHOH-CH2N)Pip-Ile NH2;
Phe(CHOH-CH~N)Phe-Ile-Phe-NH2:
Phe(CHOH-C~2N)Pro-Ile-Val-N~2;
His-Lys-Arg-Ala-Val Leu-Phe(4-NO2)-Glu~la-Nle-Ser-NH2;
[D-Phe]-~D-a-Nal~-Pip-~L-a-(OH)-Leu] Val-NH2;
iBoc-~D-Phe]-[D-b-Nal]-Pip-[L-a-(OH) Leu]-Val-NH2 WO93/01828 P~T/~S92/061S3 ~ 1 1 3 1 --6 ~ r~
iBoc-[D-Phe]-[D-b-Nal]-Pipo~-a (OH)-Leu]-Val-NH2 Boc-Phe(CHOH-CH2N)Phe-Ile-Phe-NH2;
Boc-Phe(CHOH-C~2N)Pro-Ile-Val-NH~;
Succinyl~-Phe(CHOH-CH2N)Phe-Ile-Phe-NH2;
[D-Phe]-[D-b-Nal]-Pip-[L~a-(OH~-Leu]-Val~-NH2;
iBoc-Tyr-Pro-Ile~&ly-OH; ..
(~oc-Phe-O-CH2-C~OH)2;
(Boc-Val-Phe-O-CH2oCHOH)2;
(Boc-Ser-Phe-O-CH2-CHOH) 2;
(Boc-Asn Phe-O-CH2-CHOH)2; and (Boc-Arg-Phe~O-CH2-CHOH) 2 .
LiE~id beari~ny Linkers The inYention also provides a means for improving the usefulness, e~ficacy, biolo~ical half life, transport across cellula~ membrane, oral and or nasal bioavailabilities of therapeu~ic peptides, including ~iral protea~e inhibitors by conjugating them to lipids via ester, amide, phosphate or phosphodiester bonds.
2 0 The protease i~hibitory peptides of the invention, as well as other therapeutic peptides, can ~e deri~atized with groups which help to increase their plasma half life a~d protect against tissue and renal clearance. By selecting . suitable anchoring groups, that is membrane seekin~, lipid associating groups, one can~achiev~ selectiYe ~argeting of:th~
peptide or protea~` inhibitor. A~ n ~ er of-n~tural and synthetic molecules such as glycolipids, phospholipids, fatty acids, diglycerides, and lipid associa~ing groups such as cholesterol~ lend thems~l~e~ as candidat~s ~or anchoring the protease inhibitors to the lipid bilayer of cell membranes.
Fur~hermore, the linkage between the anchoring group and the peptide or protease inhibitor can be modulated in -such a way as to obtain optimum rate of release of the drug or to position the dru~ in the most suitable conformationO These modified analogues of the viral protease inhibitory peptid~
may then be incorporated into liposomes, administered by injection (subcutaneously, intravenously, intraperitoneally or WO93/01828 PCT/~S92/06153 2~i315~
intramuscularly) to an animal. The derivatized p~ptides in liposomes will then have a much longer time of r~tention in the plasma; and will survive to be delivered to macrophages, a major site of HIV infection. In addition it can be expected that the lymphocyte reservoir of the HIV infectio~ will also receive a larger amount of the lipid-modlfi~d peptide by virtue of its ability to undergo exchange and tran fer to the surface membranes of cells in the circulation, such as the CD4+ lymphocytes. Finally, this approach will al~o be usefull~ applied to other protease inhibitory peptides, such as t~ose f or renin .
In order to prepare peptide-lipid conjugates, a number of linker groups were design~d. They offer a convenient way of conjugating lipids, phospholipid~, fat~y acids, or glycerides to small peptides or protea~e inhibitors. These linkers are designed to take advantage of natural biochemical proc~sses to release the active drug at or near the site of enzym~-action thus imparting improved biological functions to the parent drug. A~number of ~olecules containing multiple functional groups are suitable for linkers~ .
Particularly suitable for use as linker~ are the amino acids ha~ing hydro~yl functional group~. These hydroxyamino acids are attached to phosphat~ ~r~the phosphate group of phospholipids l:hrough: a phosphoester bond betwe~n the phosphat~ group and the~a~ino:acid hydroxyl,~hus l~ving both chara~t~ris~ic~amino acid groups, the~ami~o acid~N~2,~ and the - a~ino acid COOH, available ~o attach to th~peptide carboxyl and amino group~, respeetively, through amide bond~
HydrQxyamino acid phosphates can be attached to~ diacyl, dialkyl, monoacyl ur monoalkyl glycerols to provide peptide.
linked to lipids via a monopho~phate bond. Phosphatidyl-tyro ine, pho phatidyl-serine, phosphatidyl threonine, and phosphatidyl-~ydroxyproline are ~oms examples of prefgrred linkers. In a similar manner diacyl, dialkyl, monoaGyl or monoalkyl phospholipids could also be attachad to the phosphatidyl hydroxyamino acids to furnish peptides linked to lipids through a diphosphate bondO
WO~3/0182R PCT/US92/06153 3;1 5 6 ,~
The following linker moieties are prePerred for preparing the peptide-lipid conjugate~:
Group l I._Li~id-bearin Linker ~ompounds The inven~ion provides phospholipid amino acid compounds, 5 having the phosp~oester bond described above, wherein the phospholipid comprises one or two linear or.branched C4 - C24 alkyl chains, each chain having from 0 to 6 double bonds. C12 ~ C18 alkyl chains are pref~rred, and those ha~ing an e~en number of carbon atoms particularly preferred.
The alkyl chains ca~ be attached by means of the hydroxyl groups of the glyceryl portion of the phospholipid in 1,2-diradyl form, ~hat is through acyl/acyl, acyl/alkyl~
alkyl/acyl or alkyl~alkyl bonds. ~l~ernatively, the phospholipid may be a lyso species, having only one alkyl cha~n.
Accordingly, preferred species of lipid bearing linkers are~
Group II-A: 1,2diradyl-sn-~lycero-3 phospho-O-tyrosine, 1,2-diradyl-~n-glycero-3-phosphoo-se:ri~e, l,2-diradyl-sn-glyceroo 3-phospho-O~hydroxyproline and 1,2 diradyl-sn glycero-3-phospho-O-threonine:
Group II~B: 1,2-diradyl-sn-glycero-3-diphoæpho-o-tyrosin~, ~,2-diradyl-sn-glycero-3-diphospho-o-serine,:1,2diradyl-sn-glycero-3-diphospho O;hydxo~yproline and 1,2-diradyl-~n-~}ycero-3-diphospho-O-threonine~
Group II-C: l-O-acyl-~n-glycero-3 phospho-O tyrosi~e, l-~o acyl-~n-glycero-3-phospho-O-ser1ne~ O~acyl-sn ~lyceroo3-phospho-O-hydroxyproline and l-O acyl-~n-glycero-3-phospho O-threonine; and Group II-D: l-O-acyl-sn-glycero 3-dip~ospho-O-tyrosine, l-O-acyl-sn-glycero-3-diphospho-O se~ine, l-O-acyl-sn-glycero-3-diphospho-O-hydroxyproline and l-O-acyl-sn-glycero-3-diphospho-O-threonine.
Group II-E: l-O-alkyl-sn-glycero~3-phospho O-tyrosin~ O-3~ alkyl-sn-glycero-3-phospho-O-serine, 1-O-alkyl-sn-glycero-3-W~93/018~8 . PCT/U~92/06~3 9 2i~i3156 phospho-0-hydroxyproline, and 1-0-alkyl-sn-glycero-3-phospho-0-threonine; and Group II-F: 1 0-alkyl-sn-glycero-3-diphospho-0-tyrosine, 1-0-alkyl-sn-glycero 3 diphospho-0-serine,l-0 alkyl-sn-glycero-3-S diphospho-o-hydroxyproline, and 1 0-alkyl-sn-glycero-3-diphospho-0-threonine.
Particularly pr~ferred species of Group II-A are-phosphatidyl-tyrosine, phosphatidyl-serine, phosphatidyl-threonine, and phosphatidyl-hydroxyproline.
Particularly preferred species of Group IIB are:
tyrosine-O-diphosphate dipalmitoylglycerol, serine 0-diphosphate dipalmitoylglycerol, hydroxyproline-0-diphosphate dipalmitoylglycerol and threonine-O-diphosphate dipalmitoylglycerol:
Particularly preferred spec:ies of Group II- are: 1-0-h~xadecyl-sn-glycero-3phospho-O~tyrosine, l-0-hexadecyl-sn ylycero-3-phospho 0-serine, 1 0-hexadecyl-m-glycero-3-phospho-0-hydroxyproline and 1-0-hexad~cyl-sn-glycero-3-phospho-0-threonine; and Particularly preferred species of Group II F are:
l-O~hexadecyl sn -glycero-3-diphospho-0-tyrosine, 1-0-hexadecylosnoglycero-3-diphospho-0-serine, 1 0-hexadecyl-sn glycero-3-dipho~pho-O~hydroxyproline~: and l-0-hexadecyl-sn-~ :glycero-3-diphospho-0-threonine.
spa~ers .
The distanc~ between the lipid linker and the peptide can be adjusted by the insertion of pacer moieti~s. Spacers~
suitable for use in t~e peptide-lipids of the invention have two functional groups capable of binding to functional groups~
pr~sent on the lipid linkers and peptides and serving to conjugate them.
Preferred ~pacers are:
W: H2N-(CH23n-C00~, wherein n = 1 to 12, suitable for insertion at the carboxyl terminal of a peptide~ connecting it with a lipid linker and those moieties defined ~elow as C.
WO93/01828 PCT/US92/061S3 i Y: HOOC-(CH2)n-CQOH, wherein n - 1 to 1~, suitable ~or insertion at the amino ~erminal end o~ a pep~ide, to ~onnec~
it with a pho~pholipid having an amino functional group. See, for example, in Group III-A below~ wherein suc~înyl is a sp~cer conne~ting phosphatidyl-ethanolamine speci~s DPPE and DMPE with N-terminal amino acids of the peptide ~pecies.
Z: ~0- (CH2) n~COO~ wherein n = 1 to 12, suitable for ~:
insertion at the amino terminal end of a peptide, to con~e~t tha peptide with a phospholipid having a phosphate terminal ~roup, for example, phosphatidic acid species such as ~MPA and DPPA.
Group III: Pept de~l~pid_con~u~ates In order to improve the biological lives of the inhibitors in vivo and to achieve theix optimum targeting and rele`ase, we have designed the fol~owing series of deri~atives o protease inhibitory peptides.~ A number of linka~es betwe~n phospholipids and peptides are possible. For example, phosphatidic acid ca~ be linked to the~amino or the hydroxyl groups of peptide ~lternatively, carboxyl groups of the peptides can be attached to the amino ~roup of a phosphatidyl-ethanolamine.
Yet another strategy for obtaining the peptide ;~
phospholipid conjugates is t~ use suitable linkers:, su~h as - those:comprising hydroxy~mi~o acid~as d~scribed;previously.
25~ A variety of other moieties .lend~ themselve ~.as suitable~
linker~ for conjugating p~ptides to lipids. ~
The following peptide-lipi~conjugates are d~scri~ed as: ~:
examples, how~ver, other:therapeutic peptides: an~ enzyme inhi~itors similarly can; be convenientIy derivatized with~ :
appropriate lipid moieties by following the procedures pro~ided in the present invention~
Group III-A:
.
The following are examples of peptideolipid conjuga~es wherein the lipid,~ usually a pho-cpholipid, or species of the lipid linkers described aboYe (X), is attached at the terminal amino group of the peptide.
W0~3/01828 PCT/US92/06153 ` 21131~
iBoc-Tyr-Pro-Ile-Gly-DPPE
DPPA-Ser-Gln-Asn-Tyr-Pro-Ile Val-NH2 DPPA-Ser-~ln-Asn-Tyr-Acpnt-Ile Val-NH2 DPPE-Succinyl-AlaoAla-(D-b-Nal)-Pip-O~e DPPE-Succinyl-Val-Ser-Gln-Asn-Tyr-Pip-Ile-Val-Gln NH2 DMPE-Succinyl-VallSer-Gln-Asn-Tyr-Pip-Ile-Val-Gln-NH2 DMPE-Succinyl-V~ Ser-~:;ln-A~n-TyrDDiq-Ile-Vai-~;ln-NH2 DPP~-Succinyl-~D-Phe]-~D-a-Nal]~Pip-[~-a-(OH)-Leu~-Val-NH2 DPPE-Succinyl-Phe(C~OH-CH2N)Phe-Ile-Phe-NH2 DPP~:-Su~cinyl-Phe-tD-b-Nal3-Pip-[L-a (OH)-Leu]-Val-NH2 ~.
DPPE-Succinyl-Phe-O-CH2-CHOH
NH2-Phe-O~CH2-CHOH
Boc-Ser(DPP)-Phe-O-CH2-CHOH
Boc:-Ser-Phe-O-CH2-CHOH
X-Phe(CHOH-CH2N)Phe~-Ile-Phe-NH2 ~:
X-Phe(CHOH-CH2N)Pro-Ile-Val~N~2 abbrevi~lons: Ac = Acetyl; Boc = t-~utyIoxycarbonyl; Suc- ;
2 5 ~uccinic a~id 7 -O~e - ~ethyl ~ster, -NH2 = ~mide; -NHOH
.
= Hydroxylamide, -NHNH2 = Hydrazide; DPP~ = 1,2-di-Palmitoyl-phosphatidic acid; ~PPE ~ l~2-di-Palmitoyl-phosphatidyl ethAnolamine; Achx = 1-Amino, l-~yclohexane ~ carboxylic. ~acid~ ~Acpr = l-~mino,~ cycloprop~ne 30. .carboxylic acid;A~pnt~- l-Amino,: l-cyclopentane carboxylîc acid: Pip. =~ Pipe~lic acid ;(4 piperidine carboxylic acid): Ava = 5-~ ino valeric acid,:a-(OH)-Leu = L-Leucic acid ~2~0H-L-isocaproic acid); Nal ~
Naphthylalanine; Phe(4-Cl) = p-Chloro-phenylalani~e;
Phe(4-Br) = p-Bromo-phenylalanine; Nva = norvaline; Paa = Pho~phonoacetic acid; Chl = CAolic acid. 3,5-di-Aba~=
3,5-di-Aminobenzoic acid.
X represent phosphatidyl-tyr~sine, phosphati~yl~serine, phosphatidyl-threonine, phosphatidyl-hydroxyproli~e, l-0-hexadecyl-sn-glycero-3-phospho-0-tyrosine, 1-0~
hexadecyl~sn-glycero-3-phospho-0-serine, l-O-hexadecyl-~-W~93/01828 PCT/US92/061S3 2 11~3 f~ 6 -12-glycer~-3-phospho-O-hydroxyproline, 1-O-hexadecyl~sn-glycero-3-phospho-o-threonine, l-O-hexadecyl-sn-glycero 3-diphospho~O-tyrosine, 1-O-hexadecyl-sn-glycero 3-diphospho-serinej 1-O-hexadecyl-sn-glycero-3-diphospho-hydroxyproline, 1-O-hexadecyl-sn-glycero-3-diphospho~
threonine, or DPPE~Y or DPPA Z, wher~in Y is HOOC-(CH2~n-COOH;
Z is HO (C~2)n-COOH; and n = 1 to 12.
The linker moieties describ~d here are useful for conjugating lipid groups to a number of therapeutic peptides and protease inhibitor~ des~rib~d in, for example, the following publications or patent applicationæ: US Patent No.
According ~o anoth r aspect of ~he invention there are proYided compounds capable of linking p ptides to lipid WO 93/01828 ~) 1131 5P&~/US92/06153 structures. These linking compounds include the amino ac:id phosphslipids of Group IIA D described in the specificatic~n, as well as other lipid-bearing linkers defined as X ~pe~::ies, suitable for attachment to an amino group of a peptide and C
spPcies, suita}: le fsr attachment to the carboxyl group of a peptide . .
According to yet another aspect of ~he invention there are provided peptide-lipid conjugates, comprising therapeutic peptides c:onjugated to any of the lipid-bearing linkers de~ined ~y the invention. Preferred species are lipid derivatives of protease inhibitory peptid~s. The peptide lipid conjugates can also com~prise spacers, compri~ing ~ifunctional alkyl groups def ined as Y, Z, and W spec:ies, which can modulate the distance between ~he peptide and the lS lipid linker. Preferreà peptide-lipid conjugates comprise peptide~ that are protease inhibitors. These conjugates of Group III- A-F are cle~cribed in detail in the specificationO
The invention also provides i.nte:~ediate ¢ompounds useful in the synthesis of t~e peptide~lipid c:ompounds oî the inven~ion, comprising deriva~ized linking c:ompounds suitable f or bind ing to the functional groups of lipids or peptideæ .
T~e invention al~o prcnride:s a m~tho~ for synthesizing peptideDlipid con~ugates, comprising the steps of selecting a therapeutic peptide having an available amino group or carbo~l group; c:hemic~lly bonding a }~ival2nt linker group to either the ami~o~;or th~ carboxyl groùp;i-and~
chemicaily bon~ding a : lipid species tc3 ~he ~ linker., In a preferred m~thod~ the p~p1:ide is a protease inhi~itor.~
The invention further provid~s a method of treating infectious disease caused by a virus, ~::omp:rising administ~ring to the infected person an effective virus~inhibiting amotll~t of a lipid derivative of a ~riral pro~ease inhibitory peptide.
The inhi~itory peptide-l ipid can b~ incorporated into a liposs:me prior to administration. In a preferred e~c:diment of the method, the disease treated is HIV infection, and the peptide is a HIV protease inhibitor.
WO93/01828 . . .. PCT/US92~061~3 '~ 113 1~ 6 DETAILED DESCRIPTION OF T~E INVENTION
The invention provides protease inhibitory peptides, lipid conjugates of therapeutic peptides, particularly peptides that are enzymP substrates, such as the HIV protease inhibitory peptides, linkers capable of ~ttaching lipid species to the peptides, intermediate compounds, and methods of synt'h~sis and use.
Group I: Protease i nhibitPrs:
Protea~e lnhibi~ors (PIs) are, in general, substrates of HIV 1 protease in which the amino acid residues at the pl site are replacPd by isosteric re~idues. Some of the HIV prote~se 5 substrates which ar~ the basis for the design of PIs are:
Sequence . _ __ Si~e _ ~ _ Ser-Gln-Asn-Tyr-Pro-Ile-Val pl7/p24 Ala-Arg-Val-Leu-Ala-Glu-Ala p24/p7 Ala-Thr-Ile-Met Met-Gln-~rg p24/p7 Ser-Phe-Asn-Phe-Pro-Gln-Ile HIV PR N-t~rm Detailed des~riptionæ of the design and~structures of individual :PIs are: discu~sed~ below.~ The present invention provides ~or the following:PIs~
Ser-Gln-Asn-Phe-Pro Ile-Val-NH2;
lS Ser-Gln-Asn-Tyr-Pro-IleoVal-N~2;~
Ser-Gln-~sn-Tyr-Achx-Ile-Va1~NH
Ser-Gln-Asn-Tyr-Acpr-Ile-Val-NH2;~
Ser-Gln-Asn-Tyr-Acpnt-Ile-Val-NH
Thr-I1e Leu ~(beta Ala~ Leu-Gln-Arg-NH2;~
2~ Se~-Gln A~n-Tyr-P~o~ -Val~Thr-Leu-Ava-Thr-Gln-Arg-NHz~;~
~Ac--~la-~la-~D-a Nal)-Pip-(a-~O~)-Leu)-Val-NH2;: : :
:Ac-Ala-Ala-Phe Pip-(a-(OH)-~eu)-Val-NH2; :~
Ac Ala-Ala-(DL-Phe(4-Cl))-Pip-(a-(OH)-Leu)-Val-NH2;
~la-Ala-Phe-(beta-~la)-Val-Val Gly-OH; : .
Ala-Ala-Phe-(beta-Ala)-Nva-Val-Gly-O~
Ala-Ala-Phe- (beta-~la ) ( a- ( ~H) ~iso-Valeryl ) -Val-Gly-OH;
~la-Ala-Phe-(beta-Ala)-Val-Val-Gly-O~e;
Ala-Ala-Phe~(beta-Ala)-Nva-Val-Gly-OMe;
Ala-~la~Phe-(beta-Ala)-~a-(OH)-iso-Valeryl)-Val-Gly:-OMe::
Boc-Ala-Ala-Phe-(beta-Ala)-Nva-Gly-OMe;
WO93/01828 2 1 1 3 1 ~FlUSg2/06153 .
-(a,e-di-~yristoyl-1ys)-D-Pro-Ava-Ala-Ala-Phe-~va-Val-Gly-O~e;
Ac-Ala-Ala-(D a-Nal)-Pip-OMe;
Ac-Ala-Ala-(D-a-Nal~-Pip-~HNH2;
~c-Ala-Ala-(L~a-Nal)-Pip-OMe;
Ac-Ala-Ala-(L-a-Nal)-Pip-NHNH2;
A~-Ala-Ala-(D-b~Nal~-Pip-OMe;
Ac-Ala Ala-(D-b-Nal)-Pip-NHNH2;
Chl-Ala~Ala-(L-aoNal) Pip OMe;
Chl-Ala-Ala~(L-a-Nal)-Pip-NHNH2;
~aa-Ala-Ala-(D-a-Nal) Pip-OMe;
Ac-Ala-Ala-(DL-Phe(4-Cl))-Pip-OH; .
Val-Ser-Gln-Asn-Tyr-Pro~ Val-~H2;
Val-Ser-Gln-Asn-(D-a-Mal)-Pip-(a-(OH)-Leu)-Val-NH2;
iBoc-(D-Phe)-(D-a-Nal)-Pip-(a-(OH)-~eu)-Val~NH2;
(iBoc-~D-Phe)~Leu )2-(3,5-di-Aba)-Pro-Leu-(~-Phe)-NH2;
iBoc-~D-Phe]-~D-a-Nal]-Pip-[L-a-OH)-Leu~ ValDNH2 i~oc-tD-Phe]-tD-a-Nal3-Pip~ a-OH~-Leu~-Val-OH
iBoc-Phe-tD-b ~al~-Pip-[L-a-(OH)~u]-~al-NH2 iBoc-Phe-~D-b-Nal]-Pip-~L-a-(OH)~eu~-Val-OH
iBoc-~Pbe(4-Br)3-~D-~-Nal] Pip-~L-a-(OH) L~u];Val OH
~Myristoyl-(D-Phe~ L~U-) 2 (3,5-di-Aba)-Pro-~eu-(D-Phe)-NH2;
iBoc-(3~5~di-Aba~-(D-a-Nal)-Pip-~a-~OH?-Leu)-Val-NH~;
Pro-Ile Val N~2; ~ .
Asn-Phe(CO-CH2N)Pip-Ile-NN2; ~
2~ Quinàldoyl-Asn-Phe(~O-CH2N3Pip r Ile-NH2;
~V l-S~r-Gln-A8n-Tyr-Pi~-Ile-Val-Gl~-NH2;
^ : Quinaldoyl-A~n-Ph~;(CHOH-C~2N~Diq-Ile-Val-Gln-NH2; : : :
Val-Ser-Gln-Asn-Tyr-Diq-Ile-Yal 1n-NH2;
Boc-Asn~Phe(CO-CH2N)~Diq-NHtBu;
Su~cinyl-S r Gln-Asn-Tyr-Pro-Ile-Val-NH2;
Succinyl Ser-Gln-Asn~Tyr;
Quinaldoyl-Asn~Phe(CHOH-CH2N)Pip-Ile NH2;
Phe(CHOH-CH~N)Phe-Ile-Phe-NH2:
Phe(CHOH-C~2N)Pro-Ile-Val-N~2;
His-Lys-Arg-Ala-Val Leu-Phe(4-NO2)-Glu~la-Nle-Ser-NH2;
[D-Phe]-~D-a-Nal~-Pip-~L-a-(OH)-Leu] Val-NH2;
iBoc-~D-Phe]-[D-b-Nal]-Pip-[L-a-(OH) Leu]-Val-NH2 WO93/01828 P~T/~S92/061S3 ~ 1 1 3 1 --6 ~ r~
iBoc-[D-Phe]-[D-b-Nal]-Pipo~-a (OH)-Leu]-Val-NH2 Boc-Phe(CHOH-CH2N)Phe-Ile-Phe-NH2;
Boc-Phe(CHOH-C~2N)Pro-Ile-Val-NH~;
Succinyl~-Phe(CHOH-CH2N)Phe-Ile-Phe-NH2;
[D-Phe]-[D-b-Nal]-Pip-[L~a-(OH~-Leu]-Val~-NH2;
iBoc-Tyr-Pro-Ile~&ly-OH; ..
(~oc-Phe-O-CH2-C~OH)2;
(Boc-Val-Phe-O-CH2oCHOH)2;
(Boc-Ser-Phe-O-CH2-CHOH) 2;
(Boc-Asn Phe-O-CH2-CHOH)2; and (Boc-Arg-Phe~O-CH2-CHOH) 2 .
LiE~id beari~ny Linkers The inYention also provides a means for improving the usefulness, e~ficacy, biolo~ical half life, transport across cellula~ membrane, oral and or nasal bioavailabilities of therapeu~ic peptides, including ~iral protea~e inhibitors by conjugating them to lipids via ester, amide, phosphate or phosphodiester bonds.
2 0 The protease i~hibitory peptides of the invention, as well as other therapeutic peptides, can ~e deri~atized with groups which help to increase their plasma half life a~d protect against tissue and renal clearance. By selecting . suitable anchoring groups, that is membrane seekin~, lipid associating groups, one can~achiev~ selectiYe ~argeting of:th~
peptide or protea~` inhibitor. A~ n ~ er of-n~tural and synthetic molecules such as glycolipids, phospholipids, fatty acids, diglycerides, and lipid associa~ing groups such as cholesterol~ lend thems~l~e~ as candidat~s ~or anchoring the protease inhibitors to the lipid bilayer of cell membranes.
Fur~hermore, the linkage between the anchoring group and the peptide or protease inhibitor can be modulated in -such a way as to obtain optimum rate of release of the drug or to position the dru~ in the most suitable conformationO These modified analogues of the viral protease inhibitory peptid~
may then be incorporated into liposomes, administered by injection (subcutaneously, intravenously, intraperitoneally or WO93/01828 PCT/~S92/06153 2~i315~
intramuscularly) to an animal. The derivatized p~ptides in liposomes will then have a much longer time of r~tention in the plasma; and will survive to be delivered to macrophages, a major site of HIV infection. In addition it can be expected that the lymphocyte reservoir of the HIV infectio~ will also receive a larger amount of the lipid-modlfi~d peptide by virtue of its ability to undergo exchange and tran fer to the surface membranes of cells in the circulation, such as the CD4+ lymphocytes. Finally, this approach will al~o be usefull~ applied to other protease inhibitory peptides, such as t~ose f or renin .
In order to prepare peptide-lipid conjugates, a number of linker groups were design~d. They offer a convenient way of conjugating lipids, phospholipid~, fat~y acids, or glycerides to small peptides or protea~e inhibitors. These linkers are designed to take advantage of natural biochemical proc~sses to release the active drug at or near the site of enzym~-action thus imparting improved biological functions to the parent drug. A~number of ~olecules containing multiple functional groups are suitable for linkers~ .
Particularly suitable for use as linker~ are the amino acids ha~ing hydro~yl functional group~. These hydroxyamino acids are attached to phosphat~ ~r~the phosphate group of phospholipids l:hrough: a phosphoester bond betwe~n the phosphat~ group and the~a~ino:acid hydroxyl,~hus l~ving both chara~t~ris~ic~amino acid groups, the~ami~o acid~N~2,~ and the - a~ino acid COOH, available ~o attach to th~peptide carboxyl and amino group~, respeetively, through amide bond~
HydrQxyamino acid phosphates can be attached to~ diacyl, dialkyl, monoacyl ur monoalkyl glycerols to provide peptide.
linked to lipids via a monopho~phate bond. Phosphatidyl-tyro ine, pho phatidyl-serine, phosphatidyl threonine, and phosphatidyl-~ydroxyproline are ~oms examples of prefgrred linkers. In a similar manner diacyl, dialkyl, monoaGyl or monoalkyl phospholipids could also be attachad to the phosphatidyl hydroxyamino acids to furnish peptides linked to lipids through a diphosphate bondO
WO~3/0182R PCT/US92/06153 3;1 5 6 ,~
The following linker moieties are prePerred for preparing the peptide-lipid conjugate~:
Group l I._Li~id-bearin Linker ~ompounds The inven~ion provides phospholipid amino acid compounds, 5 having the phosp~oester bond described above, wherein the phospholipid comprises one or two linear or.branched C4 - C24 alkyl chains, each chain having from 0 to 6 double bonds. C12 ~ C18 alkyl chains are pref~rred, and those ha~ing an e~en number of carbon atoms particularly preferred.
The alkyl chains ca~ be attached by means of the hydroxyl groups of the glyceryl portion of the phospholipid in 1,2-diradyl form, ~hat is through acyl/acyl, acyl/alkyl~
alkyl/acyl or alkyl~alkyl bonds. ~l~ernatively, the phospholipid may be a lyso species, having only one alkyl cha~n.
Accordingly, preferred species of lipid bearing linkers are~
Group II-A: 1,2diradyl-sn-~lycero-3 phospho-O-tyrosine, 1,2-diradyl-~n-glycero-3-phosphoo-se:ri~e, l,2-diradyl-sn-glyceroo 3-phospho-O~hydroxyproline and 1,2 diradyl-sn glycero-3-phospho-O-threonine:
Group II~B: 1,2-diradyl-sn-glycero-3-diphoæpho-o-tyrosin~, ~,2-diradyl-sn-glycero-3-diphospho-o-serine,:1,2diradyl-sn-glycero-3-diphospho O;hydxo~yproline and 1,2-diradyl-~n-~}ycero-3-diphospho-O-threonine~
Group II-C: l-O-acyl-~n-glycero-3 phospho-O tyrosi~e, l-~o acyl-~n-glycero-3-phospho-O-ser1ne~ O~acyl-sn ~lyceroo3-phospho-O-hydroxyproline and l-O acyl-~n-glycero-3-phospho O-threonine; and Group II-D: l-O-acyl-sn-glycero 3-dip~ospho-O-tyrosine, l-O-acyl-sn-glycero-3-diphospho-O se~ine, l-O-acyl-sn-glycero-3-diphospho-O-hydroxyproline and l-O-acyl-sn-glycero-3-diphospho-O-threonine.
Group II-E: l-O-alkyl-sn-glycero~3-phospho O-tyrosin~ O-3~ alkyl-sn-glycero-3-phospho-O-serine, 1-O-alkyl-sn-glycero-3-W~93/018~8 . PCT/U~92/06~3 9 2i~i3156 phospho-0-hydroxyproline, and 1-0-alkyl-sn-glycero-3-phospho-0-threonine; and Group II-F: 1 0-alkyl-sn-glycero-3-diphospho-0-tyrosine, 1-0-alkyl-sn-glycero 3 diphospho-0-serine,l-0 alkyl-sn-glycero-3-S diphospho-o-hydroxyproline, and 1 0-alkyl-sn-glycero-3-diphospho-0-threonine.
Particularly pr~ferred species of Group II-A are-phosphatidyl-tyrosine, phosphatidyl-serine, phosphatidyl-threonine, and phosphatidyl-hydroxyproline.
Particularly preferred species of Group IIB are:
tyrosine-O-diphosphate dipalmitoylglycerol, serine 0-diphosphate dipalmitoylglycerol, hydroxyproline-0-diphosphate dipalmitoylglycerol and threonine-O-diphosphate dipalmitoylglycerol:
Particularly preferred spec:ies of Group II- are: 1-0-h~xadecyl-sn-glycero-3phospho-O~tyrosine, l-0-hexadecyl-sn ylycero-3-phospho 0-serine, 1 0-hexadecyl-m-glycero-3-phospho-0-hydroxyproline and 1-0-hexad~cyl-sn-glycero-3-phospho-0-threonine; and Particularly preferred species of Group II F are:
l-O~hexadecyl sn -glycero-3-diphospho-0-tyrosine, 1-0-hexadecylosnoglycero-3-diphospho-0-serine, 1 0-hexadecyl-sn glycero-3-dipho~pho-O~hydroxyproline~: and l-0-hexadecyl-sn-~ :glycero-3-diphospho-0-threonine.
spa~ers .
The distanc~ between the lipid linker and the peptide can be adjusted by the insertion of pacer moieti~s. Spacers~
suitable for use in t~e peptide-lipids of the invention have two functional groups capable of binding to functional groups~
pr~sent on the lipid linkers and peptides and serving to conjugate them.
Preferred ~pacers are:
W: H2N-(CH23n-C00~, wherein n = 1 to 12, suitable for insertion at the carboxyl terminal of a peptide~ connecting it with a lipid linker and those moieties defined ~elow as C.
WO93/01828 PCT/US92/061S3 i Y: HOOC-(CH2)n-CQOH, wherein n - 1 to 1~, suitable ~or insertion at the amino ~erminal end o~ a pep~ide, to ~onnec~
it with a pho~pholipid having an amino functional group. See, for example, in Group III-A below~ wherein suc~înyl is a sp~cer conne~ting phosphatidyl-ethanolamine speci~s DPPE and DMPE with N-terminal amino acids of the peptide ~pecies.
Z: ~0- (CH2) n~COO~ wherein n = 1 to 12, suitable for ~:
insertion at the amino terminal end of a peptide, to con~e~t tha peptide with a phospholipid having a phosphate terminal ~roup, for example, phosphatidic acid species such as ~MPA and DPPA.
Group III: Pept de~l~pid_con~u~ates In order to improve the biological lives of the inhibitors in vivo and to achieve theix optimum targeting and rele`ase, we have designed the fol~owing series of deri~atives o protease inhibitory peptides.~ A number of linka~es betwe~n phospholipids and peptides are possible. For example, phosphatidic acid ca~ be linked to the~amino or the hydroxyl groups of peptide ~lternatively, carboxyl groups of the peptides can be attached to the amino ~roup of a phosphatidyl-ethanolamine.
Yet another strategy for obtaining the peptide ;~
phospholipid conjugates is t~ use suitable linkers:, su~h as - those:comprising hydroxy~mi~o acid~as d~scribed;previously.
25~ A variety of other moieties .lend~ themselve ~.as suitable~
linker~ for conjugating p~ptides to lipids. ~
The following peptide-lipi~conjugates are d~scri~ed as: ~:
examples, how~ver, other:therapeutic peptides: an~ enzyme inhi~itors similarly can; be convenientIy derivatized with~ :
appropriate lipid moieties by following the procedures pro~ided in the present invention~
Group III-A:
.
The following are examples of peptideolipid conjuga~es wherein the lipid,~ usually a pho-cpholipid, or species of the lipid linkers described aboYe (X), is attached at the terminal amino group of the peptide.
W0~3/01828 PCT/US92/06153 ` 21131~
iBoc-Tyr-Pro-Ile-Gly-DPPE
DPPA-Ser-Gln-Asn-Tyr-Pro-Ile Val-NH2 DPPA-Ser-~ln-Asn-Tyr-Acpnt-Ile Val-NH2 DPPE-Succinyl-AlaoAla-(D-b-Nal)-Pip-O~e DPPE-Succinyl-Val-Ser-Gln-Asn-Tyr-Pip-Ile-Val-Gln NH2 DMPE-Succinyl-VallSer-Gln-Asn-Tyr-Pip-Ile-Val-Gln-NH2 DMPE-Succinyl-V~ Ser-~:;ln-A~n-TyrDDiq-Ile-Vai-~;ln-NH2 DPP~-Succinyl-~D-Phe]-~D-a-Nal]~Pip-[~-a-(OH)-Leu~-Val-NH2 DPPE-Succinyl-Phe(C~OH-CH2N)Phe-Ile-Phe-NH2 DPP~:-Su~cinyl-Phe-tD-b-Nal3-Pip-[L-a (OH)-Leu]-Val-NH2 ~.
DPPE-Succinyl-Phe-O-CH2-CHOH
NH2-Phe-O~CH2-CHOH
Boc-Ser(DPP)-Phe-O-CH2-CHOH
Boc:-Ser-Phe-O-CH2-CHOH
X-Phe(CHOH-CH2N)Phe~-Ile-Phe-NH2 ~:
X-Phe(CHOH-CH2N)Pro-Ile-Val~N~2 abbrevi~lons: Ac = Acetyl; Boc = t-~utyIoxycarbonyl; Suc- ;
2 5 ~uccinic a~id 7 -O~e - ~ethyl ~ster, -NH2 = ~mide; -NHOH
.
= Hydroxylamide, -NHNH2 = Hydrazide; DPP~ = 1,2-di-Palmitoyl-phosphatidic acid; ~PPE ~ l~2-di-Palmitoyl-phosphatidyl ethAnolamine; Achx = 1-Amino, l-~yclohexane ~ carboxylic. ~acid~ ~Acpr = l-~mino,~ cycloprop~ne 30. .carboxylic acid;A~pnt~- l-Amino,: l-cyclopentane carboxylîc acid: Pip. =~ Pipe~lic acid ;(4 piperidine carboxylic acid): Ava = 5-~ ino valeric acid,:a-(OH)-Leu = L-Leucic acid ~2~0H-L-isocaproic acid); Nal ~
Naphthylalanine; Phe(4-Cl) = p-Chloro-phenylalani~e;
Phe(4-Br) = p-Bromo-phenylalanine; Nva = norvaline; Paa = Pho~phonoacetic acid; Chl = CAolic acid. 3,5-di-Aba~=
3,5-di-Aminobenzoic acid.
X represent phosphatidyl-tyr~sine, phosphati~yl~serine, phosphatidyl-threonine, phosphatidyl-hydroxyproli~e, l-0-hexadecyl-sn-glycero-3-phospho-0-tyrosine, 1-0~
hexadecyl~sn-glycero-3-phospho-0-serine, l-O-hexadecyl-~-W~93/01828 PCT/US92/061S3 2 11~3 f~ 6 -12-glycer~-3-phospho-O-hydroxyproline, 1-O-hexadecyl~sn-glycero-3-phospho-o-threonine, l-O-hexadecyl-sn-glycero 3-diphospho~O-tyrosine, 1-O-hexadecyl-sn-glycero 3-diphospho-serinej 1-O-hexadecyl-sn-glycero-3-diphospho-hydroxyproline, 1-O-hexadecyl-sn-glycero-3-diphospho~
threonine, or DPPE~Y or DPPA Z, wher~in Y is HOOC-(CH2~n-COOH;
Z is HO (C~2)n-COOH; and n = 1 to 12.
The linker moieties describ~d here are useful for conjugating lipid groups to a number of therapeutic peptides and protease inhibitor~ des~rib~d in, for example, the following publications or patent applicationæ: US Patent No.
4,743,585: DE 3913272; DE 3840452: DE 3819846; DE 3800233:
EP Q 387 231; EP 0 337 714; EP 0 354 522; EP 0 356 223: EP
0 373 576; EP 0 373 549; EP 0 372 537; EP 0 365:992; EP 0 361 41; EP 0 386 611; EP 0 357 332; EP 0 342 541; EP 0 337 714; FR 264~353; WO 9101327; WO 9012804; WO 9000399:
WO 8910920, WO 8809815~ The ~pep~ide and ~he procedur~s described for the~preparation o~ th~ said: compounds and rela~ed interm~dia~es are inco ~ orat~d~h~rein by ref~renc~.
In the examples that f~ollow, X and th~ abbreviations are as de:fined above for Gro~p III-A,~:and ~ ~
C~ represents H, OH:, O~e~ NH2,:~H-~ where~Rl~repre~ents ~C~ 12 a1kyl,`~benzy1, substitùted b~nzyl, ~2)n pheny~ wh~r~:n 25:~ 2 methyl: pyri~y~ pho ~ tidyl-~y~o~ine~~
-~ phosphatidyl- ~erine~ ph~ospha~i~dyl-~hreonime, phospha~idyl-hy~roxyproline, 1-OQhexadecyl-sn-glyc~ro 3~
phospho-O~tyro~ine, l-O-hexadecyl-m~g1ycero-3-phospho-serine, 1-3 hexadecyl-sn-glycero-3-phospho-0-~ hydroxyproline and l-O-hexadecyl-sn lycero-3-phospho-O-threonine, 1-O-hexadecyl-sn-g1ycero-3-diphospho-O-tyrosine, l-O-hexadecyl-sn-gly ero-3-diphospho-o-serir~e, l-O-hexadecyl -sn -glycero-3-diphospho-0-hydroxyp~oline, W093/01828 PCT/~92/06153 1 5 ~
1O0-hexadecy1-sn-g1ycero-3-diphospho-0-threonine, or DPPE-W, wherein W is H2N-~CH2)n-COOH, a~d n = 1 to 12.
Group III B: Pepti~a-lip~ ~o~iug~tes_ba~ on tb~ ~ptid~ of ~
The fo110wing are examp1es o~ peptide~1ipid conjugates wherein the lipid, a species of the lipid linkers describ~d abQve (X~, is attached at the te~inal a~ino group of the peptide or aspecies (C) i5 attached at the carbo~l end of thepeptide. Inpreferredspecieso~thisclass~,thepeptide islinkedtoonelipidspe~ies,thusXispresentonlywhenC
is absent or is not a lipid. ~en C is present, it can:be separated from the peptide by the in~ertion of a W t~e spacer, ~escribed above. ~en X i5 present, it can be separated from the peptide by a ~ or Z t~e sp er, also describedabove.
EP Q 387 231; EP 0 337 714; EP 0 354 522; EP 0 356 223: EP
0 373 576; EP 0 373 549; EP 0 372 537; EP 0 365:992; EP 0 361 41; EP 0 386 611; EP 0 357 332; EP 0 342 541; EP 0 337 714; FR 264~353; WO 9101327; WO 9012804; WO 9000399:
WO 8910920, WO 8809815~ The ~pep~ide and ~he procedur~s described for the~preparation o~ th~ said: compounds and rela~ed interm~dia~es are inco ~ orat~d~h~rein by ref~renc~.
In the examples that f~ollow, X and th~ abbreviations are as de:fined above for Gro~p III-A,~:and ~ ~
C~ represents H, OH:, O~e~ NH2,:~H-~ where~Rl~repre~ents ~C~ 12 a1kyl,`~benzy1, substitùted b~nzyl, ~2)n pheny~ wh~r~:n 25:~ 2 methyl: pyri~y~ pho ~ tidyl-~y~o~ine~~
-~ phosphatidyl- ~erine~ ph~ospha~i~dyl-~hreonime, phospha~idyl-hy~roxyproline, 1-OQhexadecyl-sn-glyc~ro 3~
phospho-O~tyro~ine, l-O-hexadecyl-m~g1ycero-3-phospho-serine, 1-3 hexadecyl-sn-glycero-3-phospho-0-~ hydroxyproline and l-O-hexadecyl-sn lycero-3-phospho-O-threonine, 1-O-hexadecyl-sn-g1ycero-3-diphospho-O-tyrosine, l-O-hexadecyl-sn-gly ero-3-diphospho-o-serir~e, l-O-hexadecyl -sn -glycero-3-diphospho-0-hydroxyp~oline, W093/01828 PCT/~92/06153 1 5 ~
1O0-hexadecy1-sn-g1ycero-3-diphospho-0-threonine, or DPPE-W, wherein W is H2N-~CH2)n-COOH, a~d n = 1 to 12.
Group III B: Pepti~a-lip~ ~o~iug~tes_ba~ on tb~ ~ptid~ of ~
The fo110wing are examp1es o~ peptide~1ipid conjugates wherein the lipid, a species of the lipid linkers describ~d abQve (X~, is attached at the te~inal a~ino group of the peptide or aspecies (C) i5 attached at the carbo~l end of thepeptide. Inpreferredspecieso~thisclass~,thepeptide islinkedtoonelipidspe~ies,thusXispresentonlywhenC
is absent or is not a lipid. ~en C is present, it can:be separated from the peptide by the in~ertion of a W t~e spacer, ~escribed above. ~en X i5 present, it can be separated from the peptide by a ~ or Z t~e sp er, also describedabove.
5(S)-X-Amino-4(S)-hy~droxy~6phenyl 2(R)-(phenylmethyl)hexanoyl-~u-Phe-C;~
5 ( S ) - X - A m i~n o - 4 ~S)-hy~droxy-6-p~he~ny1~-:2(R)-(phenylmethyl) heYanoyl-~u-c;
S(S)-X-amin~-4`(~:S)-hy:droxy-6-~yelohex~yl-2(R):~
(phenylmethyl)hexanoyl-C~
5 (sj - X - A m i:n o -~4 ( R~) h y d r:ox:y - 6 - p h e n y 1 -:2 (:R) ~pheny1m~hy13h~xanoy1- ~ u-Phe-C;~
~ (S) - X - ~ m i n o -4:( R) -h y d:r o x y - 6 - p h e n y l -2 (R):~ :
30~ `` (ph~ny1methy1th~xan~y1-Leu~ and:~
5(S) -X-ami~o-:4 (~ hydroxy-6:-cyc1~ohex~y~1-2 (R)~
: (pheny1methy1)hexanoy1-C.
wherein X and~C~are as defined aboYe.
roup III-C: Pept~ a co~juqate~ ba3e~ on the pept~e~
of ~iohn publicatiQ~: 8~IE~aE~9o~ 247 P~ ~5~-456~ Ig90: an~
: : ~ :
ThePollowingareex~amples~fpeptidelipidconjugatesof thesametypeas:those~presentedinGroup IIIB.~
: 5(S)-X-Amino-4;(S)-hydroxy:-6-cyc1cohexy1-2(~)-isopropy1- : .
hexanoyl~Ile-C:
. , , WO93/018~8 , PCT/US92/~153 "'`' ,'2`~13156 ,,~ .., ~ (S) -X-Amino-4 (R) -hydroxy-6-cycl ohexyl-2(S)-isopropyl-hexanoyl-Ile-C;
5(S)-X-Amino-4(5)-hydroxy-6-phenyl-2(R~-isopropyl-hexanoyl-Ile-C:
5(S)-X-Amino;4(R)-hydroxy 6-phenyl 2(S) is~propyl-hexanoyl-Ile-C;
wherein X and C are as defined above. ~
t~e ROC~ Europe~ P~tent Applica~ 0 3~6 847 A2 The following are examples of peptide-lipid conjugates of the same type as those presented in ~roup IIIB.
(3(S)-X-Asparaginyl)-amino-2tR)-hydroxy-4-phenylbutyl-Pro-C;
(3(S)-X-Asparaginyl)-amino-2(S~-hydroxy-4-phenylbutyl-Pro-C;
(3(S)-X-Asparaginyl)-amino-2 (R,S)-hydroxy-~-phenylbutyl-Pro-C;
(3tS) X-Asparaginyl)-amino~2(~s)-hydroxy-4-phenylbutyl-pr Ile-C:
~3~S)-X-Leucyl-Aæparaginyl)-amino-2(R,S)-hydroxy-4-phenylbutyl-Pro-Ile-C;
(3tS~-X-Asparaginyl~-amino-2(R) hydroxy-4 phenylbutyl-N-1,2,3,4 tetrahydro(R,S)iso~uinoline car~oxyl-C:
3~
~3(S~-X-Asparaginyl)-amino-2(S)-hydroxy-4-phenylbutyl-N-1,2,3,4-tetrahydro(R,S)isoquinol ine carboxyl-C;~
(3~S)-XwAsparaginyl3-amino-2(R,5j-hydroxy-4-phenylbutyl-N-3~ 1,2,3,4-tetrahydro(R,S)iso~uinoline car~oxyl-C;
(3~S)-X~A~paraginyl)-amino-2(~:,S)~-hydroxy-4-phPnylbutyl-N-decahydro-3~S)-isoquino1ine carboxyl-C;
(3(S~-X-Asparaginyl)-amino-2(R3-hydroxy-4-phe~nyl~utyl-N-decahydro-3tS)-isoquinoline carboxyl-C; and (3(S)~X-Asparaginyl)-amino-2(S)-hydroxy-4-phenylbuty~-N-decahydro-3(S)-isoquinoline carboxyl-C;
wherein X and C are as defined above. ~ :
' 5~
Grou~ E: _ Pep~i~e-lipid coniU~ate~ ~3ed on t ~D~L:--of th.e 8K~F ~nternation l P~tent Applicatio~ ~ WO_ so~off3ss;
and Pu~licatîon~: ~AT~RE 343:90-92_~1990~: PNAS. VOL 86:9752-9756 Llg89):_ WO93/01828 ~ . PCT/US92/0~i53 1'3 1 S 6 The following are examples of peptide-lipid conjugates of the s~me type as tho~e presented in Group III-B.
4(S) (X-Alanyl)amino-3(5)-hydroxy-5-phenyl-penta~oyl-Val-Val-C
4(S~-(X-Alanyl-alanyl)amino-3(S)-hydroxy-5-phenyl-pentanoyl- !
Yal-Val~C
4(S)-(X-Seryl-alanyl-alanyl)amino-3(S)-hydroxy-5-phenyl- ;
pentanoyl-Val-Yal-C
4(S)-(X-Alanyl)amino 3~R)-hydroxy-5-phenyl-pentanoyl-Val-Va1~C
4(S) (X-Alanyl-alanyl)amino-3(R)~hydroxyS-phenyl-pentanoyl-Val Val-C
- ~ .
4(S)-(X-Seryl-alanyl-alanyl)amino-3(~-hydroxy- 5-phenyl~
pentanoyl Val-Val C;
wherein X and C are as defined above.
Group III-F:_P~pti~e-liDi~ ~o~tu~at~ on th~ ~e~t~e~ o~
the ~OE~ uroDe~ Patent ~_~P 0354 522~
The following are examples of pepti~e-lipid conjugates of the same type as those presented in Group III-Bo X~Val-Phe-Nva-(cycloh~xyl~ethyl.(4,4,5,5-te~ram~thyl-1,3,2-dioxo~orlan-2-yl)methylamide: :~
X-Val-Phe-Nva-(ry~loh~xylmethyl,dihydroxyboronyl~methylamide;
~ :
X-Valo(L a-Nal)-Nvaotcycl~hexylmethyl(4,4,5,5 tetramethyl-1,3,2-dioxoborlan-2-yl)methylamide;
X-Val-(L a-~al~-Nva~
6cyclohexylmethy1,dihydrox ~oronyl)methylamide;~
X-Nva-(~yclohexylmethyl,dihydroxyboronyl)~me~hyl;~mide~ :
XDVal- ( cyc} ohexylm~thyl,dihydroxyboronyl)methylamide;
:4~ :
wherein X is as defined abo~e.
As described in the experim~ntal section, many of he lipid linker species, particularly a phosphatidyI-hydroxyamino acid, are bifunGtional, and can be positioned~either at the C-terminal, N-terminal or in the midd~e at internal sites of~the protease inhibitory peptide as desired. That species of lipid linker can also be attached to two sites of the same peptide, using appropriate spacers, if necessary. It is also within W093/01828 PCT/US92/~6tS3 31~i6 ,~
-16- .
the contemplation of the inven~ion to conjugate a monoglyceride or diglyceride dir~ctly to a fxee carboxyl, preferably the terminal carboxyl, of a peptide through the hydroxy group of the lipid.
PeDtide S~nthesis Peptides of the inven~ion can be produced by any of the peptide synthesis proc~dures known to those in th~ art~ for example, solution phase syn~hesis, fra~ment cond~nsation, enzyme synthesi~, or any of the me~ho~s o~ solid phase ln synthesis. These peptides can also be produced by recombinant DNA technology. The solution phase method and the solid pha~e methods are preferred. The solid phase me~hods are particularly preferred. These m~thods are well known to those skilled in the ar~ and described in detail in the li~erature;
eg. Barany, G. and R.~. Merrifield, in 7~e Pe~es, Vol. 2; Eo Gross & J. Meienhoff~r, eds.; ~cad~mic Press, New York, pp 3 284 (1979). Commercially available deri~atized amino acid~
used for the ~ynthesis of peptid~; describ~d in this invention are: Boc Ala-OH, Boc-Arg(To~-OH~ Boc sn-O~, Boc-Asp(O
~yclohexyl~-OH, Boc Asp(OBzl)-O~, Boc-Cys(S-4-MeBzI)-OH,:Boc-Gln-OH, Boc~Glu(O-~yelohexyl)-OH r Boc-Glu(OBzl)-OH, Boc-Glyo OH, 80c-Hi~(Tos)-OH, Boc-HistBom)~;OH, Boc-Ile OH~ Boc-L~u-OH, Boc-Lys~Cl-2) ~H, Boc-Norleucine, Boc-Norvaline, Noc-Mek-OH, ~oc~Phe-O~, Boc;Pro-OH~ Boc- ~r(Bzl)-O~, Boc-~hr(Bzl~-O~, Boe~
Thr (Bzl j -OHOi80c-~rp-OH,Boc-Trp(Fon~yl)-0~,30c-Tyr(Br-Z)-O~, and B~c-Val~
P~ptid~s of ~he inven~ion containing basic amino a~ids such a~ lysine, arg~nine, and histidine may exist in the~form of salts ~uch as chloride, acetate, phosphate, citrate, succinate, oxalate, etc. Acetate and hydrochloride salt ~orm5 are particularIy pr~ferred. Peptides of the invention containing aspartic acid, glutamic acids or phosphate linker moieties may exist in the form of salts such as sodium, ~potassium, calcium, bari~m, ammonium or other acceptable cataion. For the purposes of this invention, peptides of the WO 93/01828 r 2 1 1 3 1 s~/US92/06153 . ~ , :; .
invention and their acid addition salts are con~;idered to be one and the same.
The peptides and peptide-lipid conjuga e5 of the present invention are useful as inhibitors of viral pr~teases and therefore could be used as therapeutic agents ~or exaraple for the prevention or treatment of infeçtion by ,human immunodef iciency virus (HIV), and ~;ubs~quent disease conditions sllch as acquired immunodef ici2nc::y syndrome tAIDS ) and AIDS related c:omplex (a~RC~.
lûThe peptide lipid c:on~ugates are also us;eful in improving the effica~y OI other protease înhibitors, such as is~hibitors of renin. Renin is a protease of approximately 40, 000 MW
secretsd in the kidney by juxtaglomerular cells surrounding the afferent arterioles of the cortical glomeruli. Renin 15itself has no activity, ~ut ac:ts on a protein s~abstrate, angiotensinogen/ to split off the inactive decapeptide, ~ngiotensin I, which is l:hen ~::c?nve~ed hrs:~ugh ~he splitting of f of two C-terminal peptide~, to the active pr~ssor agerlt, - ~:he octapeptide angiote~sin II. l~ngio~ensin; II is the mo t ~O potent pressor made in the body and it exerts ~his p~s~;or action ~y a direct effect on arteriolar smooth muscle~.
Secretion of renin is stimula~ed in various disorders and causes hypertension~ An~ approach to ~the therapy of:
- hypertension comprises the administration of renin inhibitory 25 ' ~:peptides which are- renin su~strate~:analogues,~ a:strategy t"~comparable to the~a~ini~r~tion o~H~V pro~eas~:inhib~ito~ in the~therapy:~o~ HIV in ection.~ Lipid con3ugates~`o~ r~nin ~ ;
inhibito~y peptides, ha~ing the~composi~ion of the~eptide-~
lipids ~f the invention, can similarly contribut~ to ~he effecti~e ther py of hypertension by providing renin peptides in a form that resist~ clearance and de~radation xnd promotes e~ficient uptake by the cells. ~
The present in~ention also relat~s to the use of the therapeutic peptide5 and peptide conjugates of the invention and their physiologically acceptable salts for the preparation of pharmaceutical formulations which can be employed as medicaments in human and veterinary medicine. For this WO 9~ 18~X . PCr/US92/06153 ~il3ls6 i~ ~
purpose, it is possible to convert them into a suitable form for administration tog~ther with at least one vehicle or auxiliary. Suit~ble vehi ::les are organic and inorgarlic substances which are suitable for enteral (for example, oral), paren~eral, topical, transdermal or nasal adminis ration and which do not react with the ~active dnlg supstances. The indicated formulations can be sterilized and/or contain auxiliaries, such as ~ lubricants, preservative~ s~abilizers, wetting agents, emulsiiers,buffer~, colorin~s and flavorins3.
The active peptides may be administered parenterally, that is by subcutanec~us, intramuscular, intraperitoneal ~ or intravenous injection. The ph~rmacPutical formulations suitable for injectable use include aqueous solutions o~
dispersions and powders for the extemporaneous preparatiorl of inj ectable ~;~lution~ or disp~rsions . It is possible also to freeze-dry the peptides and to use the lyophilizates o3~tained, for ~xamplel for the preparation of products for i~ection~
In all cases ~ the form must be st~rile and the solution must be fluid to the ~xt~nt that easy s;yrInga3:)ility ~exists. It ~u t be stabl~ und~r the conditions of ~nanufacture and ~3torage and must be pr ~erved again t cont~Dlinations of miar~organisms, such as for example, bacteria and fuIlgi. The carrier can be a solvent or a dispersion medium containing; for example, water, or a polyol such as glycerol, and suita~le mi~tures --thereo~O~ Compositions~for i~tramu ¢ular use ~ay-:~l o::contain minor amounts o~-salt~"- acids,- ~nd ba~s to.adjust tonicity ? and buffer the solution. Suit~ble buffering ~nd isotonicity agents are readily d~terminable by persons skill~d in the art.
Oral or nasal administration is~al~o po.~ible especially with peptide lipid conjugat~. Formulations for oral ingestion are in the form of ta~lets, capsules, pills~
ampoules of powdered active agent, or oily ~or aqueous suspensions o~ solutions. Tablet~ or other non-liquid oral compositions may contain acceptable excipients, Xnown to the art for the manufacture of pharmaceutical compo~itions, comprising diluents, such as lactose or calcium oarbonate;
binding agents such as qelatin or starch; and one or more W~ ~3/018~ , ; . PCI~US92/06~53 `211315~
agents selected from the grcup consisting of sweetening agents, flavoring agents, coloring or preserving agents to provide a palatable preparatic: n. ~oreover, such oral preparations may be coated by known techrliques tc further delay disin~egration and absorp~ion in the intestinal tract.
Such oral compositions and preparations should ~ontain at least 0.1% of active peptide, although the percentagas o~ the compositions may vary widely . The amc: unt c~f therapeutic:ally active compound in suc:h ~ompositions i~ such that a suitable dosage will be obtained in a convenient volume for inge~tion.
Formulation~ for nasal admini~3tration may be in th~ form of liquids and optionally may ~:ontain absorption promoting suhstances, for example, a lactone of a water-soluble organic acid, and other compounds of similar function well known to those trained in the art. ~rhe nasal formulatio~s may also be in the form of an aerosol comprisiJlg the peptide together with an ex ende~ which may be an amino acid, for exampl~, methionine.
~ransdermal application fol~ulations can comprise the peptides and their lipid conjugates, optiona;lly inco~orated into a suitable topical carrie!r ~ or a dermal patch. q~he compounds can be combined with ~ penetration-enhancing agen~, for ~xample, dimethylsulfoxide ~DMSO), dime~hyl~onmamide, dimethylacetamide, or Azone~, azacycloheptane~2-o~e. Both 25: i~ topical carriers and~the u~ o-~ pen~ration ~enhanc~rs are isclosed~iby~Blaug~ 5A~ Chap. a7 ~
~, l5th~d.~ Ma~k ;Pu~ h~ng :Co., Eas~on~ ~A 180~2 (1975~
In the therapy of HIV~i~fected patient~, ~he lipid conjugated protease inhibiting p~ptide will be a~minister~d parent~rally, orally, or na~ally. Parenteral do~es are usually from 0.01 to 1 gm ev~ ~ 3O8 hours. Na~al or oral doses may be ~rom 2 to lO times the parenteral dose, depending on bioavailabilit~ of the peptide, and it~ retention in the - plasma and tissues. The most e~icacious dose ean be readily determinPd by standard pharmacokinetic and toxicological studies in animals and humans.
W0~3/01B28 æ ¦l3 15 6 PCT/US92/Q6153 The amino acid abbreviations used are those commonly employed in ths pep~ide art and described in the literature, eg. IUPAC-IUB Commission on ~iochemical Nomenclature, J.
Biol. Chem. 247, 97~-982 (1972). Additional abbreviations used are: Ac = Acetyl; ~oc = t-Butyloxycarbonyl; Suc succinic acid; -0~ = Methyl ester, -NH2 = Amide7 -NHOH
Hydroxylamide; -NHNH2 = Hydrazide; DPPA - 1,2-dip~l~itoyl-phosphatidic acid; DPPE = 1,2-dipalmitoyl phosphatidyl ethanolamine; Achx = l Amino, l-cyclohexane carboxylic acid;
Acpr = 1-Amino, l-cyclopropane car~oxylic acid; Acpnt ='1-Amino, l-cyclopentane carboxylic acid; Pip = Pipecolic acid (4-piperidine car~oxylic acid); Ava = 5-Amino valeri¢ acid, a-(OH)-Leu = L-Leucic acid (2-OH-L isocaproic acid); Nal =
Naphthylalanine; Phe(4-Cl) ~ p-Chloro phenylalanine; Nva -norvaline; Paa = Phosphonoacetic acid; Chl = Cholic acid.
3 9 5-di-Aba = 3,5-diaminobenzoic acid. Amino acids discussed herein are of the L-form unle~s otherwise me~tioned.
All the t~mperatures d~scrib~d are in degree Celsius and are unco~rected. Evaporations were carried out und~r~va~uu~
below 35C. TLC was carried out using E.M~rck precoated plates and the spot~ were visuali~ed by exposure to W lightf iodin~, : ninhyd:rin spray, phosphorous spray~ or sul~uric acid spray ~;
followed by charring as appr~priate. Analytical HPL~ was carried out us~ing:~a Be kman ~system and Vydac re~erce phase:
- column~ (C-4-o~ C-18 as ~ppropriat~). .Prepar~ati~e~i~HPLC~wa~
carried ou~ usin~g.-,a~:Wa~ers Deltapr~p ~ystem ~usl~ eith~r reverse phase or silica columns. Purity and~a~thenticity of:
~he ompounds were established by TLC, analytical ~PLC, amino acid analysis, elemental analysis, UV spectra, NMR, F~-MS as required and appropriate.
The chemical reactions de~cribed below are generally disclosed in terms of their ~eneral application to the preparation o:peptides of the invention. Occasionally~ the reaction may not be applicable as described to each pep~ide included within the disclo~ed scope. The peptides ~cr which this occurs will be readily recognized by those skilled in the WO ~3/01828 ' ~. PCr/US92/061~3 21131~S ~
art. In all such cases, either the reaction~ c~n be successfully performed by c:onventional modifications known to those skilled in the art, e. g. by appropriate protertion of interfering groups, by changing to alternative conventiorlal reagents, or by routine ms:ldi~ication of reac:tion conditions.
Alternatively, other reactions disclosed herein or otherwise conventional will be applicable to the prep~ration o~ the corresponding peptide~ of the inv~ntion. In all preparative methods, all ~tarting ma~erials 2Ire known or readi~y preparable from known starting m terials.
~ithout further elaboration, it is believed that one skilled in the ar~ can, using the preceding description ~
utilize the invention to i~s full~st extentO The following preferred embodiments are, therefore, to be construed a~
merely illustrative and not limit:a~iYe or the remainder of th~
disclosure in any way whatsoever~ In ~he following examples, all temperatureæ are ~et forth uncorrec~ed in degree~ Cel~iu;
unless otherwise indicated, all parts and perc:entages are by weight .
The present invention is de~ribed below in d~ail u~ing the following examples, but the methods d~scribed below are applicable for the preparation o~ all peptides covered by th~
scope of the int~ention and are not limited to the Examples si~ren below, ..:
DeDe¦d~ ~ :
1,2-Dipalmitoyl-sn ~lycero-3 phosphatidic acid.2Na 3Q (DPPA.2Na: Avanti Polar Lipids, Birmingham AL; MW: 697.84; 698 mg, 1 mmol) was partitioned betw~en chlorofo~m:metha~ol (2:1 ~v/v); 200 ml) and cold 1 N HCl (50 ml). The aqueous layer was re-extracted with chloroform methanol (2:1 (~/v): 100 ~1).
The combined organic phase was evaporated and dried under vacuum over P20s. The resulting free phosphatidic a id was dissolved in a mi~ture of DMF (2 ml) and pyridine ~2 ml) and to the solution were added the appropriate peptide having a W093/01828 P~T/USg2/~153 71131~6 `
free amino group (1 mmol) followed by NN'~
dicyclohexylcarbodiimide (DCC; Aldrich Chemical Co. Milwaukee WI, MW: 206, 620 mg, 3 mmol). The reaction mixture was stirred for 24 hours at room temperatur~. The solvents were 5 evaporated and the product was purified by fl~sh chromato~raphy over silica gel column (2.5 x 50 cm) using a linear gradient of 0 ~o 50% methanol in chloro~orm. Fractions containing the desired product a~ indicated by TLC and HPLC
were pooled and evaporat~d. The product wa~ further purified, if necessary, by pr~parative HPLC or by crystalli~ation.
EXAMPL~ lA: DPPA-Ser~Gln-Asn-TyrePxo-Ile-Val-NH2 and E~MP~
lB: DPPA-Ser-Gln-Asn-Tyr-Acpnt Ile-Val-NH2 were prepared by the above procedure.
~ _ ~ 2 General procedure for couplinq phosphatidic acid to the hydroxy~ro~p of~a peptide 1,2-~ipalmitoyl-sn-glycero-3-phosphatidic acid (l mmol) prepared as above was dissolved in a mix~ur~ ~f DMF (2 ml) and pyridine (2 ml) and to the solution were added the appropriate peptide having a free hydroxyl group (1 mmol) followed by DCC
~620 mg, 3 mmol). The reaction was carried out and the product was isolated as~described in Example 1 o 2~
The condensation of~t~e:~phosphatidic acid and ~he hydroxyl group o~ a peptide was also conveniently carried out by u~ing 2,4,6-triisopropylbenzenesulfonyl chloride (TPS-Cl;~Aldrich Chemical CoO, Milwaukee WI; MW: 302.86: 758 mg, 205 mmol) as a coupling agent in place of DCC.
WO93/01828 PCT/US92/~1S3 211315~
~ PLE 3 General proc~dure_for couplinq a pepti.de conta _ na a fr~ee carboxyl group_ to the amino arou _o~ _a phosphatidyl-ethanolamine A mixture of the appropriate peptide (1 mmol), phosphatidyl-ethanolamine (l mmol~ were dissol~ed in pyridine (5 ml~ and DCC ~3 mmol) ~ollowed by 1-hydroxybenzotriazole (HOBt: Aldrich Chemical Co., HOBt, MW: 153; 450 mg, 3 mmol) were added. The reaction mixture was stirred for 24 hours at r~om temperature and the praduct was purified by sil~ica gel chromatography as descrîbed in Exampl 14 ~XANP~E 3A:
iBoc-Tyr-Pro-Ile-Gly~DPPE was prepared by the above procedure.
~ANP~E 4 General_ ~rocedure for coupli~ peptide containina ~a free carboxyl_~rouP to the hydFox~
~l~cerol ~A mix~ure of the appropriate peptide (1 mmol), diacyl or dialkyl glycerol (1 mmolj were dissolved in pyridi~e (~ ml) and DCC (3 mmol) followed by~4-dimethylaminopyridine (Aldrich Chemical Co., D~AP, MW: 122.17; 122 mg, 1 mmol) were added.
The~ reaction mixture was stirred ~for 24 hours at ~room ~. :temperature~ and~ -the ~ product ~was~ purified- ~y-silica gel:
chromatography as de~cribed.in~iExampl~e~
: : EX$~PL~ 5:
Pre;~aration. ~of ~ N-ter~Jlp~ oxyc~Lrbonyl-Serin~
Hydroxy uccinimide Ester [Boc~-S~r-OSu) MW: 302.26 ~ : :
Boc-Ser-OH ( 20.52 g, 100 mmol) and N-hydroxysuccinimide (Aldrich Chemical Co.~Milwauk~e WI; MW: 115:.09; 23.3 g~ 200 mmol) were dissolved in dry ~HF (400 ml). The solution was:
cooled to ~-10C, DCC (MW: 206.33; 30010 g; 150 mmol) was added. The mixture was stirred for 2 hours at -10C and overni~ght at room temperature. The solvents were evaporated~
under vacuum, and the residue was stirred in ethyl acetate (l l~ and filtered to remove insoluble material. The resulting W093~01~28 PCT~US92/06153 2l1'3''l1~'6 ,i',~''?~ ;
filtrate was washed successively with water (3 x 250 ml~, cold lN HCl (3 x 250 ml), water (3 x 250 ml), cold 10% NaHC03 (3 x 250 ml~ and water (3 x 250 ml) and dried over anhyd~ous sodium sulfate. After evaporation the residue was crystallized from ~:
isopropyl alcohol.
Boc-Tyr~OS~, Boc-Hyp-OSu and Boc-Thr-OSu were prepared similarly.
:
Ser-OSu: ~oG ~ a~ ~W: 850.17 1,2-Dipalmitvyl-~n-glycero-3-phosphatidic acid (1 ~mol) ~-was dissolved in anhydrous pyridine (5 ml) and 2,4,6- ~
1~ triisopropylbenzenesulfonyl chloride (TPS Cl, 758 mg, 2.5 :
mmol) followed by Boc-Ser-OSu (~W: 302026; 302 mg, l mm~l~ was added. The reaction mix~ure.was ~tirred under dry nitrog~n atmosphere for 16 hours at room tlsmperature. The reaction wa~
then quenched by adding water ~1 ml) and the solYents wer~
evaporated under. vacuum~ The re idue wa~:dissolv~d :in chloro~orm ~5 ml) and lo~d~d onto a silica gel 60 column (2.5 cm x 4:5 cm~ eg~ilibrat~d with chloroform~ The colu~n was~
eluted with a gradient of chloroform~(500 ml) to 15% MeOH in ~ :
chloroform (: 500 ~ml).~ Frac~ions~ eontain~ng the desired .. product (as..indicated.by TLC):.were pooled and-evaporate~to furnish ~oc~SertOD~ OSu.o ~
Boc-Tyr~ODPP)-OSu Boc-Hyp(ODPP)-OSu and Boc Thr(ODPPj-OSu were prepared ~imilarly. :~
~en~ral Procedure for__couplina phos~hatidyl-serine,_ to a pePtide contain~in a free aminP unction.
Boc-Ser(ODPPj-OSu:(~O.l mmol) and the peptide in ~ues~ion 35 containi~g a f~ee~a~ine function (0.1 mmol) were dissolved in DMF (2 ml~ and~pyridine (2 ml) and stirred for 24 hours at room tempera~ure~ The solYents were evaporated and the product was purified by flash chromatography over silica gel column (2.5 x 50 cm) using a liner gradient of 0 to 50%
methanol in chloroform. Fractions containing the desired product as indicated by TLC and HPLC were pooled and evaporated. The product was further purified, if necessary, by preparative HPLC or by crystallization and deprotected by treatment with trifluoroacetic acid (2ml) for 12 hours at room temperature. The acid was removed by evaporation under vacuum and the product was isolated by lyophilization from water.
Peptides linked to phosphatidyl-tyrosine, phosphatidyl-hydroxyproline and phosphatidyl-threonine were prepared similarly.
EXAMPLE 7A:
was prepared according to the above procedure.
Preparation of 1.2-Dipalmitoyl-sn-glycero-3-phospho-O-(N-Fmoc)-Ser-OBzl. Fmoc-Ser(ODPP)-OBz}
Starting from Fmoc-Ser-OBzl the title compound was prepared following the procedure described for Boc-Ser(ODPP)-OSu. Fmoc-Tyr(ODPP)-OBzl, Fmoc-Hyp(ODPP)-OBzl and Fmoc-Thr(ODPP)-OBzl were prepared similarly.
General procedure for coupling a peptide containing a free caboxyl group to phosphatidyl-sering Fmoc-Ser(ODPP)-OBzl (0.1 mmol) was dissolved in DMF (2 ml) and piperidine (0.2 ml) and the mixture was stirred under dry nitrogen atmosphere for 4 hours and the solvents were evaporated under vacuum. The residue was dissolved in a dry DMF (5 ml) and the peptide in question containing a free WO93/01828 PCT/US92/~61~3 `2i`i'3U'~'6 ,<..................................... ~ -26-carboxyl group (0.1 mmol) was added to it followed by DCC ~0.1 mmol) and HOBt (O.~ mmol). The rea~tion mixture was stirred for 2~ hvurs at room temperature. The solvents were evaporated and the product was purified by flash chromatography over silica gel column (2.5 x 50 cm~ using a linear gradient of O to 50% methanol in chloro~rm. Fractions co~taining the desired produc~ as indicated by ~C and HP~C
were pooled and evaporat~d~ The produc~ was furthex`purified, if necessary, ~y preparative HPLC or by crystallizatlon. The product was then dissolved in DMF ( 5 ml~ a~d hydrogenated in the pre~ence of palladium-carbon (5~) catalyst (200 mg). The catalyst was removed by fil~ration and th~ product was purified by silica gel chromatography as described above.
Peptides containi~g phosphatidyl-tyrosine, phosphatidyl hydroxyproline and phosphatidyl-threonine were prepar~d similarly.
N-hyd~oxysuccini~ide ester Boc-Ser(O-DP~DPG)-ONSu ':
Boc-Ser-OSu (~W: 302.26: 302 mg, 1 mmol) was dissolv~d in 2S triethyl phosphate (5 ml) and cooled to 5 C. POC13 (~W
153.33; 460 mg, 3 mmol) was a~ded to the above ~olu~ion ~d `:
th~ reaction mixture was stirred under dry nitrogen a~mo~p~re for 16 hours at -10 C. Eth@r (20 ml) was added and the resulting precipitate was s~parated from the supar~atant by decantation and the residue was wa~hed with ether (20 ml each). The residue was resuspended in ice ~old water (lO ml3 and the pH was immediately adjus~ed to 7.5 by adding lN ~aOH.
The solution was stirred for 1 hour at 0 C while maintaining the pH at 7.S and lyophiliæed. The resulting product was dissolved in water (5 ml) and load~d onto a DEAE Sephadex A~2 column (1 cm x 10 cm) equilibrated with O.01 M ammonium bicarbonate (pH 7). The column was eluted with a gradient of W093/01X2~ .q., PCT/US92~061~3 2~L~31$6 0.01 M ammonium bicar~onate to 0.3 ~ ammonium bicarbonate (pH
7). Frac~ions containing the desired product (as indi~ated by TLC) were pooled and lyophilized to furnish Bo~-Ser(0-phosphate)-Os~
B. 1. 2-Dipalmitoyl-sn-~lycero-3-hs~bon~
MoFphol~ate) 1,2Dipalmitoyl-sn-glycero-3 phosphatidicacid(DPPA,650 mg, 1 mmol) and morpholine (350 ~g, 4 ~ol) were dissol~ed in chlorofoxm (lO ml), ~ butanol glO ml ) and w~ter ( 1 ~1 ) . The solution wa~ stirred under gentle reflux for 2 hours while adding a solution of DCC (~5 mg, 4 mmol) in t-butanol (lO
ml). The mixture was stirred ~or an addi~ional 4 hours and ev~porated to dryness and suspended in water (lO0 ml) and was ~.xtracted three ~imes with eth~r (100 ml), evaporate~ to dryness and lyophilized.
. .
C. Çouplin~ of_DPPA-Morpho.l~d~
Osu DPPA-~orpholidate (O.5 mmol) and Bo~-Ser~0-phosphate)-Osu (0.3 mmol) w~re dissolv~d in anhydrous pyridine (25 ml). The solution was e~aporated to dryness in vacuo five times fr~
anhydrous pyridine, followed by the addition of anhydrous pyridine (5 ml). The reaction ~ixture ~as~stirr~d at 40 C
and evaporated to dryness~.~ The product was dissolve~ in --~ chloro~om:. ~ethanol:~;water (2:3~ and-.~:loaded onto a DEAE
S~phadex A-25 col~mn ~1 cm x:~lO ~cmj~e~uilibr~ed with chlorofom::methanol: water t2:3:13.~ The column was eluted with a linear gradiant of O to 0.3 M ammonium bi~arbonate in the sam~ solvenk. Fractions con~aining ~h~ desired product (as indi~at~d by T~C) wera pooled, concentrated to a 60 ml volume and extract~d 5 times with chlorsfoxm (50 ml each).
Th cloroform solution was evaporated to ~urnish Boc-Serine(O-diphosphate dipalmitoylglycerol)-N-hydroxysuc~inimide ester.
Boc-Tyr(O-DP-DPG)-ONSu, Boc-H~p(O-DP DPG) ONSu an~ Boc-Thr(O-DP-DPG~-ONSu were prepared similarly.
WO93~01828 ' i; ~ 113 15 6 PCT/US92/06153 E~MPLE 11 Gener.al procedure for_ coupli~s Serine-O-d~hosp~e dipalmitoylglycerol to a p~.eptide c~ontaininq~ free amine function.
Boc-Ser(O-DP-DPG)-ONSu ( 0.1 mmol) and the peptide in guestion containing a free amine function (0.1 mmol) were d~ssolved in D~F (2 ml) and pyridine (2 ml~ and stirred for 24 hours at room temperature. The solvents were evaporated and the product was purified by ~lash chromatsgraphy over silica gel column (2.5 x 50 cm) using a linear gradient of O to 50~
methanol in chloroform. Fractions containing the desired product as indicated by TLC and ~PLC were pooled and evaporated~ The product was further purified, if necessary, by preparative HPLC or by crystallization and deprotected by treatment with trifluoroacetic acid- (2 ml) for 12 hours at room temperature. The acid was removed by evaporation under ~acuum a~d the product was isolated by lyophilization from water.
Tyrosine-O-dip~osphate dipalmitoy}~lycerol, hydroxyproline-O-diphosphate dipalmitoy1~1ycerol and threonine-O-d~iphosphate : dipalmitoylglycerol containing~ peptides were prepared similarly. . :;
~ANPLE:12 : Starting from Fmoc-Ser~OBæl and DPPA-Morpholidate ~he title compound was prepared following the pro~edure described~
in Example 11.
Fmoc-Tyr(O-DP DPG)-OBzl, Fmoc-Hyp(O-DP-DPG~-OBzl and Fmoc-Thr(O-DP~DPG)-OBzl were prepared similarly. : ~
:
~MP~E 13 Starting from Fmoc-Ser(V-DP-DP&~-OBzl, deprotection of 5 th2 Fmoc- group, the coupling reaction, catalytic hydrogenation followed by puri~ication as described abo~e.
Corresponding tyrosine-O-diphosphate dipalmitoylglycerol, hydroxyproline-O-diphospha~e dipalmitoylglycerol and threonine-O~diphosphate dipalmitoylglycerol containing peptides were prepared similarly. g ~A~PLE 14 ser-oSu Boc-Ser(O-phosphate)-OSu (l mm~l~ was dissolv~d in anhydrous pyridine ~5 ml~ and 2,:4, 6Q
~riisopropylbenzenesulfonyl chloride (TPS-Cl~;Aldrich Chemi~al Col, Milwaukee WI; MW: 302.86;: 758 ~g, 205: ~mol) was added fcllowed by l-OoHexadecyl~sn-glyceroln The:~reactlon mixture was stirred under dry ni~rogen atmosphere for 16 hours at room temper~ture. The reaction was then qu~nched by addin~ water (l ~i ml) and the solvents were evaporated under~acuum. The residue was dissolved:i~ chloroform (S ml) an~loaded onto a silica gel 60 column~(Z.5 cm x -45 cm) -equilibrated with chloroform. The ~column was ~luted~ with~ ~a gradi~nt of chlor~fonm (5~0 ml) ~to:~15~ MeOH in chloroform ~ 500 ml).
Fractions containing the dç~ired product ~as indicated by TLC)~
were pooled and evaporated to f~rni~h ~h~ title compound.
Preparation of l-O-h~xadecyl-~n glycero-3-phospho-0-(N-~oc~
Tyr~Osu,l O-hexadecy1-sn-glycero-:3-phospho~O-(N-Boc)-Hyp-Osu and l-O-hexadecyl-sn~ cero-3 phospho-O-(N-Boc)-Thr-O~u were carried out in a similar manner.
:
WO93/01B28 PCT/US92/061~3 ~1131S6 _30~
~ P~E i5 General_~rocedure for couplin~ o-hexadecyl sn-~lycero 3-phospho-serine to a_~eptide co~taininq free ~minÇ fun~tion ~his coupling reaction, subsequent deprotec~ion and purification of the product was carried out following the procedure described in Example g except for using 1 O-hexadecyl-sn-glycero-3-phospho-O-(N-Boc)-Ser-OSu in place of Boc S~r(ODPP)-OSu.
Peptides containing l-O-hexad~cyl-sn~glycero-3-ph~spho O
tyrosine,l-O-h~xadecyl-sn-glycero-3-phospho-O-hydroxyprol~ne and 1-O-hexade~yl-sn-glycero-3-phospho~O-thrPonine were prepared in a similar manner.
~AM~E ~6 Preparation of 1o~O-hexadecyl-sn-~lycero-3-phos~ho-O-(Fmoc~-Ser-OBzl Starting f rom Fmoc-Ser-OBzl the title compound was prepared~following the procedure~d~scribed ~or the preparation of 1 O hexadecyl-sn-91ycero-3-phospho-O-(N~Boc)-Ser-OSu.
Preparation of l-O hexadecyl-sn-glycero-3-phospho-O-(Fmoc)-Tyr OBzl~ l-O-hexadecyl-~n-glycero-3-phospho-O-(Fmoc)-Hyp-OBzl and 1-O-hexadecyl-s~-glycero-3-ph~spho-O~(Fmoc~-Thr-OBzl~were c rri~d out similarly.
BSaMP~ 17~
Starting from l-O-hexadecyl-sn-glycero-3-phospho-O-(Fmoc)-S~r OBzl, d~protection of the~Fmoc- group, the coupling reaction, catalytic hydrogena~ion followed by purification as~
described in Example l0. : :
:~
Pep~ides containing 1 O-hexadecyl-sn-glycero-3-phosph~-~
tyrosine, 1-O-hexadecyl-sn-glycero-3:-phospho-hydroxyproline W093/0182~ PCT/US92/06153 31- :~ 7 ~
andl O-hexadecyl sn-glycero-3-pho~pho-threo~inewereprepared in a similar mznner.
~XA~ 18 preparation~of_l-O~hexadecyl-sn glycero~-diphospho-O-(N~Bocl-Ser~OSu Boc Ser(O-phosphate)-OSu (1 mmol) was converted to the corresponding morpholidate by reacting with DCC and morpholine. The product was then reac~e~ with 1-0-hexadecylglycerol-3-phosphate as descri~ed for the preparation of Bo~Ser(O-DP-DPG)-OSu. : ' Preparation of l-O-hexadecyl-sn-glycero-3-diphospho-0-(N-Boc)-Tyr-Osu, 1-0-hexadecyl-sn-glycero-3-diph~spho-0-(N-Boc~-Hyp-Osu and l-O-hexadecyl~sn-glycero~3~diphospho-0-(N-Boc)-Thr-Osu w~re carried out in a similar manner.
, ~X~PL~
~ .. :
~
This coup~:ing~ reaction,~subse ~ ent deprotect~ion ~`and purif ication of:the product was~ carried out o:110wing the : procedure ~described~in~ Example ~9:~except ~:for using 1-0-hexadecyl-sn-glycero-3-d:iphospho-0-~N-Boc)-S~er~OSuinplaceof :~ :
Boc-Ser(O-DP-DP~)-OSu. :
Peptides: containing l-O-hexadecyl-sn-glycero-3-diphospho-0 tyrosine, l-O-hexa~ecyi-sn-glycero-3-diphos~pho-0:~
hydroxyprolIne a~d -~-hexadecyl-snt~glyc~ro-3-diphQspho~O~
threonine were prepar~d in a similar manner.
~ ~ANP~X 2 PreParation of~ hexadecyl-s~-lycero-3-diphospho-o-~Fmoc~- ~
Ser-08zl : ~-:~Starting ;from Fmoc-Ser-OBzl the title compoun~d was ~ :
prepared following the procedure described for the preparation of 1-0-hexadecyl-sn-glycero-3-diphospho-0-(N-Boc)-Ser-OSu.
W093/01828 PCTJUS92/~6153 2 113i5 6 -32;
Preparation of l-O-hex~decyl~sn-glycero 3 dipho~pho-O-(F~oe)-Tyr~OBzl, 1 O~hexadecyl-~n-glycero-3-diphospho-0 (Fmoc~-Hyp-OBzl and l-O-hexadecyl-sn-glycero-3-diphospho-O-(Fmor~Thr-OBzl were carried out in a similar manner.
~X~L~ 2l Starting from l-O-hexade~yl-sn~glycero-3-diphospho 0-(Fmoc3-SerOBzl, deprotection of the Fmo¢- group, the coupling reaction, catalytic hydrogen2tion followed by purification'as described in Example 9.
Peptides containing l-O-hexadecyl-sn-glyc2ro 3~diphosp~o 0 lS tyrosine, l O-hexadecyl sn-glycero-3-diphospho-0-hydroxyproline and l O-hexad~cyl-sn-glycero-3-diphospho-0 threonine were prepared in a simi.1ar manner~
~8A~P~æ 22 Succinyl!-ethanolamine: DPPE SUCCIN~C ~ID NM: 79l.03 To a solution o~ l,2-Dipalmitoyl- n-glycerc-3-phosphatidyl ethanolamine (D~PE; Avan~i Polar: Lipids, Bixmingh~m AL; MW: 690.96; 346 mg, 0.~5 ~mol) in chloroform (lO
ml), succinic anhydride (Sig~a Chemical ~o.,~St. ~Louis MOr lOO.7, lOO ~g, 1 mmo1~ dis o1ved:in ch10ro~orm (lO ml) and~
- -~tri~thylamine (O.l ml)~were~added. The reaction mixture waæ
stirred under dry nitrogen atmosphere for 16 hours at~ room temper~ture. The reaction was monitored by running TLC at various intarvals. After ~he reaction i~ completed, the solvents were evaporated under vacuum and the residue was di solved in chloroform:(5 ml) and loaded onto a silica gel 60 column (2.5 cm x 45 cm) eguilibrated with chlorofonm~ The column was eluted~with a gradient of chloro~o~m (500 ml) to I5% MeOH in chloroform ( 500 ml). Fractions containing the desired product (as indicated by TLC) were pooled and evaporated to furnish DPPE-SUCCINIC ACIDu ~ 2113~6 Preparation of 1,2-Dimyri~toyl-sn-glycero-3-phospho-O (N-Succinyl) ethanolami~e, DMPE-SUCCINIC ACID, was carried out similarly.
~A~PL~ 23 Preparation of ~oc-Phe O-CH2~CHOH
Boc-Phe-O-CH2-CHOH MW: 616 A mixture o~ Boc-Phe-OH (5~3 g, 20 mmol), triethylamine (TEA; MW: 101.19; 3.1 g, 31 mmol), znhydrous KF (1.2 g, 20 mmol) and 1,3-dibromobutane-2,3-diol (Br-CH2-CHOH-CHOH~CH2-Br, 2.5 g, 10 mmol) was dissolved in DMF (25 ml) and the solution was stirr2d for 24 hr at 45 C. The solvent was evaporated under vacuum and residue was di~solved in EtAc (330 ml!, extracted with 10% sodium bicarbonate (3 x 50 ml), water (3 x 50 ml), 10% ci ric acid (3 x 50 ml~ and water (3 x 50 ml3.
The organic phase was dried over a~hydrous sodium sulphate and evaporat~d under ~acuum and the :residue was crystalIized from acetonitrile to furnish th~ prod~ct as wh~te amo~phous powder.
~ P~ 2 : Pre~aration of : ~ .
Bo~-Val Phe-O-CHz-CHOH
~: Boc-Val-Phe-O~ [2-C~HOH
The com~ound. r3m !~examp}e.~23 (616~mg,..~1 ~ ol) was~
dissolv~d in.a~hydrou~FA ~5~ and:stirred~or 15;.m~inutes.
The solvent wa$~ evaporat~d~:a~nd the ~residue~;~riturated~with :30 ether and filtered. The product was dissolved in DMF;(20~ml) ~ :
and cooled to -10C. Boc-~al-OH (MW: 217~.14, 696~mg, :3 mmol~, HOBt (456 =g, 3~mol) and DCC (620 mg,~ 3 mmol) were added to th above solution. The reaction mixture was stirred~
for 2 hours at ~10C;and for 16~hours at~ room tempera~ure.
The solvents were~then~ evaporated under vacuum,~ and ~the ~ :
residue was dissolved in ~thyl ace~ate (250 ml)~ and filt ed .
to rem~ve ins~oluble material. The resulting ~iItrate was washed successively wi~h water (3~x 50 ml), cold lN ~Cl~ (3 x WO93/0182~ PCT/US92/06153 ~ i ~ 3 f S 6 ,~'?~ .
50 ml~, water (3 x 50 ml), cold 10~ NaHCO3 (3 x 50 ml~ and water (3 x 50 ml) and dried over anhydrous sodium lfateO
Evaporation of the solvent provided the crude produ t which was purified by crystallization from acetonitrile.
~A~PLæ 2s Preparation of Boc-Ser-Phe-O-CH2-CHOH
~oc-Ser-Phe O-CH2-CHOH
The title compound was prepared f~llowing tho procedure descri~ed for Example 24 except u~ing ~oc--Ser-OH instead of Boc-Val-OH.
~a~PL~ 26 Pre~aration of Boc-Asn-Phe 0 CH2-CHOH
Boc sn-Phe-0-CH2-CHOH
The title compound was prepared followin~ the procedure described for Examlple 24~ exGept: using Boc-A5n 0H insgead o~
.~
~oo-Val-OH.
~ANP~;27 : :
A. ;~ ~Ç i~ de_Ey~h i~ Boc~amino ~acyl-ben:zyl ~ester~
~: resin or Boc~-amino a~yl-~4:-me~yl:)benzhydrylàmin~;r~sin:~(Boc~
esini~2 g~ 1~mmol) placed~:in a reaGtion~essel~of~Béckman~
~ -990 B Pép~ide Synthesizer~(Beo~man~Instruments,~Palo Alto, C~
:~ and subjected to the gollowing operations.~ Each tep~is ~ca~ried out one time unless~specified othe~wise:,~ and reagents~
and solvents after each step are~æeparated from ~he peptide ~:
resin by filtration under nitrogen Ste~ Rea~ent/sl vent~No~æ~ y~mes Mix Time (minute-s~
1 : DCM ~(30 ml, 3 times) : : 1.5~
2 : TF~-DCM (1~ (3Q ml) : 1.5 ~ : :
3 TF~-~CM (1~:1) (30 ml) ~30.0 4 DC~:~(30 ml, 3:~imes~ ~ 1.5 : ~
S ~ Methanol (30 ml, 3 times) 1.5 ~::
5 ( S ) - X - A m i~n o - 4 ~S)-hy~droxy-6-p~he~ny1~-:2(R)-(phenylmethyl) heYanoyl-~u-c;
S(S)-X-amin~-4`(~:S)-hy:droxy-6-~yelohex~yl-2(R):~
(phenylmethyl)hexanoyl-C~
5 (sj - X - A m i:n o -~4 ( R~) h y d r:ox:y - 6 - p h e n y 1 -:2 (:R) ~pheny1m~hy13h~xanoy1- ~ u-Phe-C;~
~ (S) - X - ~ m i n o -4:( R) -h y d:r o x y - 6 - p h e n y l -2 (R):~ :
30~ `` (ph~ny1methy1th~xan~y1-Leu~ and:~
5(S) -X-ami~o-:4 (~ hydroxy-6:-cyc1~ohex~y~1-2 (R)~
: (pheny1methy1)hexanoy1-C.
wherein X and~C~are as defined aboYe.
roup III-C: Pept~ a co~juqate~ ba3e~ on the pept~e~
of ~iohn publicatiQ~: 8~IE~aE~9o~ 247 P~ ~5~-456~ Ig90: an~
: : ~ :
ThePollowingareex~amples~fpeptidelipidconjugatesof thesametypeas:those~presentedinGroup IIIB.~
: 5(S)-X-Amino-4;(S)-hydroxy:-6-cyc1cohexy1-2(~)-isopropy1- : .
hexanoyl~Ile-C:
. , , WO93/018~8 , PCT/US92/~153 "'`' ,'2`~13156 ,,~ .., ~ (S) -X-Amino-4 (R) -hydroxy-6-cycl ohexyl-2(S)-isopropyl-hexanoyl-Ile-C;
5(S)-X-Amino-4(5)-hydroxy-6-phenyl-2(R~-isopropyl-hexanoyl-Ile-C:
5(S)-X-Amino;4(R)-hydroxy 6-phenyl 2(S) is~propyl-hexanoyl-Ile-C;
wherein X and C are as defined above. ~
t~e ROC~ Europe~ P~tent Applica~ 0 3~6 847 A2 The following are examples of peptide-lipid conjugates of the same type as those presented in ~roup IIIB.
(3(S)-X-Asparaginyl)-amino-2tR)-hydroxy-4-phenylbutyl-Pro-C;
(3(S)-X-Asparaginyl)-amino-2(S~-hydroxy-4-phenylbutyl-Pro-C;
(3(S)-X-Asparaginyl)-amino-2 (R,S)-hydroxy-~-phenylbutyl-Pro-C;
(3tS) X-Asparaginyl)-amino~2(~s)-hydroxy-4-phenylbutyl-pr Ile-C:
~3~S)-X-Leucyl-Aæparaginyl)-amino-2(R,S)-hydroxy-4-phenylbutyl-Pro-Ile-C;
(3tS~-X-Asparaginyl~-amino-2(R) hydroxy-4 phenylbutyl-N-1,2,3,4 tetrahydro(R,S)iso~uinoline car~oxyl-C:
3~
~3(S~-X-Asparaginyl)-amino-2(S)-hydroxy-4-phenylbutyl-N-1,2,3,4-tetrahydro(R,S)isoquinol ine carboxyl-C;~
(3~S)-XwAsparaginyl3-amino-2(R,5j-hydroxy-4-phenylbutyl-N-3~ 1,2,3,4-tetrahydro(R,S)iso~uinoline car~oxyl-C;
(3~S)-X~A~paraginyl)-amino-2(~:,S)~-hydroxy-4-phPnylbutyl-N-decahydro-3~S)-isoquino1ine carboxyl-C;
(3(S~-X-Asparaginyl)-amino-2(R3-hydroxy-4-phe~nyl~utyl-N-decahydro-3tS)-isoquinoline carboxyl-C; and (3(S)~X-Asparaginyl)-amino-2(S)-hydroxy-4-phenylbuty~-N-decahydro-3(S)-isoquinoline carboxyl-C;
wherein X and C are as defined above. ~ :
' 5~
Grou~ E: _ Pep~i~e-lipid coniU~ate~ ~3ed on t ~D~L:--of th.e 8K~F ~nternation l P~tent Applicatio~ ~ WO_ so~off3ss;
and Pu~licatîon~: ~AT~RE 343:90-92_~1990~: PNAS. VOL 86:9752-9756 Llg89):_ WO93/01828 ~ . PCT/US92/0~i53 1'3 1 S 6 The following are examples of peptide-lipid conjugates of the s~me type as tho~e presented in Group III-B.
4(S) (X-Alanyl)amino-3(5)-hydroxy-5-phenyl-penta~oyl-Val-Val-C
4(S~-(X-Alanyl-alanyl)amino-3(S)-hydroxy-5-phenyl-pentanoyl- !
Yal-Val~C
4(S)-(X-Seryl-alanyl-alanyl)amino-3(S)-hydroxy-5-phenyl- ;
pentanoyl-Val-Yal-C
4(S)-(X-Alanyl)amino 3~R)-hydroxy-5-phenyl-pentanoyl-Val-Va1~C
4(S) (X-Alanyl-alanyl)amino-3(R)~hydroxyS-phenyl-pentanoyl-Val Val-C
- ~ .
4(S)-(X-Seryl-alanyl-alanyl)amino-3(~-hydroxy- 5-phenyl~
pentanoyl Val-Val C;
wherein X and C are as defined above.
Group III-F:_P~pti~e-liDi~ ~o~tu~at~ on th~ ~e~t~e~ o~
the ~OE~ uroDe~ Patent ~_~P 0354 522~
The following are examples of pepti~e-lipid conjugates of the same type as those presented in Group III-Bo X~Val-Phe-Nva-(cycloh~xyl~ethyl.(4,4,5,5-te~ram~thyl-1,3,2-dioxo~orlan-2-yl)methylamide: :~
X-Val-Phe-Nva-(ry~loh~xylmethyl,dihydroxyboronyl~methylamide;
~ :
X-Valo(L a-Nal)-Nvaotcycl~hexylmethyl(4,4,5,5 tetramethyl-1,3,2-dioxoborlan-2-yl)methylamide;
X-Val-(L a-~al~-Nva~
6cyclohexylmethy1,dihydrox ~oronyl)methylamide;~
X-Nva-(~yclohexylmethyl,dihydroxyboronyl)~me~hyl;~mide~ :
XDVal- ( cyc} ohexylm~thyl,dihydroxyboronyl)methylamide;
:4~ :
wherein X is as defined abo~e.
As described in the experim~ntal section, many of he lipid linker species, particularly a phosphatidyI-hydroxyamino acid, are bifunGtional, and can be positioned~either at the C-terminal, N-terminal or in the midd~e at internal sites of~the protease inhibitory peptide as desired. That species of lipid linker can also be attached to two sites of the same peptide, using appropriate spacers, if necessary. It is also within W093/01828 PCT/US92/~6tS3 31~i6 ,~
-16- .
the contemplation of the inven~ion to conjugate a monoglyceride or diglyceride dir~ctly to a fxee carboxyl, preferably the terminal carboxyl, of a peptide through the hydroxy group of the lipid.
PeDtide S~nthesis Peptides of the inven~ion can be produced by any of the peptide synthesis proc~dures known to those in th~ art~ for example, solution phase syn~hesis, fra~ment cond~nsation, enzyme synthesi~, or any of the me~ho~s o~ solid phase ln synthesis. These peptides can also be produced by recombinant DNA technology. The solution phase method and the solid pha~e methods are preferred. The solid phase me~hods are particularly preferred. These m~thods are well known to those skilled in the ar~ and described in detail in the li~erature;
eg. Barany, G. and R.~. Merrifield, in 7~e Pe~es, Vol. 2; Eo Gross & J. Meienhoff~r, eds.; ~cad~mic Press, New York, pp 3 284 (1979). Commercially available deri~atized amino acid~
used for the ~ynthesis of peptid~; describ~d in this invention are: Boc Ala-OH, Boc-Arg(To~-OH~ Boc sn-O~, Boc-Asp(O
~yclohexyl~-OH, Boc Asp(OBzl)-O~, Boc-Cys(S-4-MeBzI)-OH,:Boc-Gln-OH, Boc~Glu(O-~yelohexyl)-OH r Boc-Glu(OBzl)-OH, Boc-Glyo OH, 80c-Hi~(Tos)-OH, Boc-HistBom)~;OH, Boc-Ile OH~ Boc-L~u-OH, Boc-Lys~Cl-2) ~H, Boc-Norleucine, Boc-Norvaline, Noc-Mek-OH, ~oc~Phe-O~, Boc;Pro-OH~ Boc- ~r(Bzl)-O~, Boc-~hr(Bzl~-O~, Boe~
Thr (Bzl j -OHOi80c-~rp-OH,Boc-Trp(Fon~yl)-0~,30c-Tyr(Br-Z)-O~, and B~c-Val~
P~ptid~s of ~he inven~ion containing basic amino a~ids such a~ lysine, arg~nine, and histidine may exist in the~form of salts ~uch as chloride, acetate, phosphate, citrate, succinate, oxalate, etc. Acetate and hydrochloride salt ~orm5 are particularIy pr~ferred. Peptides of the invention containing aspartic acid, glutamic acids or phosphate linker moieties may exist in the form of salts such as sodium, ~potassium, calcium, bari~m, ammonium or other acceptable cataion. For the purposes of this invention, peptides of the WO 93/01828 r 2 1 1 3 1 s~/US92/06153 . ~ , :; .
invention and their acid addition salts are con~;idered to be one and the same.
The peptides and peptide-lipid conjuga e5 of the present invention are useful as inhibitors of viral pr~teases and therefore could be used as therapeutic agents ~or exaraple for the prevention or treatment of infeçtion by ,human immunodef iciency virus (HIV), and ~;ubs~quent disease conditions sllch as acquired immunodef ici2nc::y syndrome tAIDS ) and AIDS related c:omplex (a~RC~.
lûThe peptide lipid c:on~ugates are also us;eful in improving the effica~y OI other protease înhibitors, such as is~hibitors of renin. Renin is a protease of approximately 40, 000 MW
secretsd in the kidney by juxtaglomerular cells surrounding the afferent arterioles of the cortical glomeruli. Renin 15itself has no activity, ~ut ac:ts on a protein s~abstrate, angiotensinogen/ to split off the inactive decapeptide, ~ngiotensin I, which is l:hen ~::c?nve~ed hrs:~ugh ~he splitting of f of two C-terminal peptide~, to the active pr~ssor agerlt, - ~:he octapeptide angiote~sin II. l~ngio~ensin; II is the mo t ~O potent pressor made in the body and it exerts ~his p~s~;or action ~y a direct effect on arteriolar smooth muscle~.
Secretion of renin is stimula~ed in various disorders and causes hypertension~ An~ approach to ~the therapy of:
- hypertension comprises the administration of renin inhibitory 25 ' ~:peptides which are- renin su~strate~:analogues,~ a:strategy t"~comparable to the~a~ini~r~tion o~H~V pro~eas~:inhib~ito~ in the~therapy:~o~ HIV in ection.~ Lipid con3ugates~`o~ r~nin ~ ;
inhibito~y peptides, ha~ing the~composi~ion of the~eptide-~
lipids ~f the invention, can similarly contribut~ to ~he effecti~e ther py of hypertension by providing renin peptides in a form that resist~ clearance and de~radation xnd promotes e~ficient uptake by the cells. ~
The present in~ention also relat~s to the use of the therapeutic peptide5 and peptide conjugates of the invention and their physiologically acceptable salts for the preparation of pharmaceutical formulations which can be employed as medicaments in human and veterinary medicine. For this WO 9~ 18~X . PCr/US92/06153 ~il3ls6 i~ ~
purpose, it is possible to convert them into a suitable form for administration tog~ther with at least one vehicle or auxiliary. Suit~ble vehi ::les are organic and inorgarlic substances which are suitable for enteral (for example, oral), paren~eral, topical, transdermal or nasal adminis ration and which do not react with the ~active dnlg supstances. The indicated formulations can be sterilized and/or contain auxiliaries, such as ~ lubricants, preservative~ s~abilizers, wetting agents, emulsiiers,buffer~, colorin~s and flavorins3.
The active peptides may be administered parenterally, that is by subcutanec~us, intramuscular, intraperitoneal ~ or intravenous injection. The ph~rmacPutical formulations suitable for injectable use include aqueous solutions o~
dispersions and powders for the extemporaneous preparatiorl of inj ectable ~;~lution~ or disp~rsions . It is possible also to freeze-dry the peptides and to use the lyophilizates o3~tained, for ~xamplel for the preparation of products for i~ection~
In all cases ~ the form must be st~rile and the solution must be fluid to the ~xt~nt that easy s;yrInga3:)ility ~exists. It ~u t be stabl~ und~r the conditions of ~nanufacture and ~3torage and must be pr ~erved again t cont~Dlinations of miar~organisms, such as for example, bacteria and fuIlgi. The carrier can be a solvent or a dispersion medium containing; for example, water, or a polyol such as glycerol, and suita~le mi~tures --thereo~O~ Compositions~for i~tramu ¢ular use ~ay-:~l o::contain minor amounts o~-salt~"- acids,- ~nd ba~s to.adjust tonicity ? and buffer the solution. Suit~ble buffering ~nd isotonicity agents are readily d~terminable by persons skill~d in the art.
Oral or nasal administration is~al~o po.~ible especially with peptide lipid conjugat~. Formulations for oral ingestion are in the form of ta~lets, capsules, pills~
ampoules of powdered active agent, or oily ~or aqueous suspensions o~ solutions. Tablet~ or other non-liquid oral compositions may contain acceptable excipients, Xnown to the art for the manufacture of pharmaceutical compo~itions, comprising diluents, such as lactose or calcium oarbonate;
binding agents such as qelatin or starch; and one or more W~ ~3/018~ , ; . PCI~US92/06~53 `211315~
agents selected from the grcup consisting of sweetening agents, flavoring agents, coloring or preserving agents to provide a palatable preparatic: n. ~oreover, such oral preparations may be coated by known techrliques tc further delay disin~egration and absorp~ion in the intestinal tract.
Such oral compositions and preparations should ~ontain at least 0.1% of active peptide, although the percentagas o~ the compositions may vary widely . The amc: unt c~f therapeutic:ally active compound in suc:h ~ompositions i~ such that a suitable dosage will be obtained in a convenient volume for inge~tion.
Formulation~ for nasal admini~3tration may be in th~ form of liquids and optionally may ~:ontain absorption promoting suhstances, for example, a lactone of a water-soluble organic acid, and other compounds of similar function well known to those trained in the art. ~rhe nasal formulatio~s may also be in the form of an aerosol comprisiJlg the peptide together with an ex ende~ which may be an amino acid, for exampl~, methionine.
~ransdermal application fol~ulations can comprise the peptides and their lipid conjugates, optiona;lly inco~orated into a suitable topical carrie!r ~ or a dermal patch. q~he compounds can be combined with ~ penetration-enhancing agen~, for ~xample, dimethylsulfoxide ~DMSO), dime~hyl~onmamide, dimethylacetamide, or Azone~, azacycloheptane~2-o~e. Both 25: i~ topical carriers and~the u~ o-~ pen~ration ~enhanc~rs are isclosed~iby~Blaug~ 5A~ Chap. a7 ~
~, l5th~d.~ Ma~k ;Pu~ h~ng :Co., Eas~on~ ~A 180~2 (1975~
In the therapy of HIV~i~fected patient~, ~he lipid conjugated protease inhibiting p~ptide will be a~minister~d parent~rally, orally, or na~ally. Parenteral do~es are usually from 0.01 to 1 gm ev~ ~ 3O8 hours. Na~al or oral doses may be ~rom 2 to lO times the parenteral dose, depending on bioavailabilit~ of the peptide, and it~ retention in the - plasma and tissues. The most e~icacious dose ean be readily determinPd by standard pharmacokinetic and toxicological studies in animals and humans.
W0~3/01B28 æ ¦l3 15 6 PCT/US92/Q6153 The amino acid abbreviations used are those commonly employed in ths pep~ide art and described in the literature, eg. IUPAC-IUB Commission on ~iochemical Nomenclature, J.
Biol. Chem. 247, 97~-982 (1972). Additional abbreviations used are: Ac = Acetyl; ~oc = t-Butyloxycarbonyl; Suc succinic acid; -0~ = Methyl ester, -NH2 = Amide7 -NHOH
Hydroxylamide; -NHNH2 = Hydrazide; DPPA - 1,2-dip~l~itoyl-phosphatidic acid; DPPE = 1,2-dipalmitoyl phosphatidyl ethanolamine; Achx = l Amino, l-cyclohexane carboxylic acid;
Acpr = 1-Amino, l-cyclopropane car~oxylic acid; Acpnt ='1-Amino, l-cyclopentane carboxylic acid; Pip = Pipecolic acid (4-piperidine car~oxylic acid); Ava = 5-Amino valeri¢ acid, a-(OH)-Leu = L-Leucic acid (2-OH-L isocaproic acid); Nal =
Naphthylalanine; Phe(4-Cl) ~ p-Chloro phenylalanine; Nva -norvaline; Paa = Phosphonoacetic acid; Chl = Cholic acid.
3 9 5-di-Aba = 3,5-diaminobenzoic acid. Amino acids discussed herein are of the L-form unle~s otherwise me~tioned.
All the t~mperatures d~scrib~d are in degree Celsius and are unco~rected. Evaporations were carried out und~r~va~uu~
below 35C. TLC was carried out using E.M~rck precoated plates and the spot~ were visuali~ed by exposure to W lightf iodin~, : ninhyd:rin spray, phosphorous spray~ or sul~uric acid spray ~;
followed by charring as appr~priate. Analytical HPL~ was carried out us~ing:~a Be kman ~system and Vydac re~erce phase:
- column~ (C-4-o~ C-18 as ~ppropriat~). .Prepar~ati~e~i~HPLC~wa~
carried ou~ usin~g.-,a~:Wa~ers Deltapr~p ~ystem ~usl~ eith~r reverse phase or silica columns. Purity and~a~thenticity of:
~he ompounds were established by TLC, analytical ~PLC, amino acid analysis, elemental analysis, UV spectra, NMR, F~-MS as required and appropriate.
The chemical reactions de~cribed below are generally disclosed in terms of their ~eneral application to the preparation o:peptides of the invention. Occasionally~ the reaction may not be applicable as described to each pep~ide included within the disclo~ed scope. The peptides ~cr which this occurs will be readily recognized by those skilled in the WO ~3/01828 ' ~. PCr/US92/061~3 21131~S ~
art. In all such cases, either the reaction~ c~n be successfully performed by c:onventional modifications known to those skilled in the art, e. g. by appropriate protertion of interfering groups, by changing to alternative conventiorlal reagents, or by routine ms:ldi~ication of reac:tion conditions.
Alternatively, other reactions disclosed herein or otherwise conventional will be applicable to the prep~ration o~ the corresponding peptide~ of the inv~ntion. In all preparative methods, all ~tarting ma~erials 2Ire known or readi~y preparable from known starting m terials.
~ithout further elaboration, it is believed that one skilled in the ar~ can, using the preceding description ~
utilize the invention to i~s full~st extentO The following preferred embodiments are, therefore, to be construed a~
merely illustrative and not limit:a~iYe or the remainder of th~
disclosure in any way whatsoever~ In ~he following examples, all temperatureæ are ~et forth uncorrec~ed in degree~ Cel~iu;
unless otherwise indicated, all parts and perc:entages are by weight .
The present invention is de~ribed below in d~ail u~ing the following examples, but the methods d~scribed below are applicable for the preparation o~ all peptides covered by th~
scope of the int~ention and are not limited to the Examples si~ren below, ..:
DeDe¦d~ ~ :
1,2-Dipalmitoyl-sn ~lycero-3 phosphatidic acid.2Na 3Q (DPPA.2Na: Avanti Polar Lipids, Birmingham AL; MW: 697.84; 698 mg, 1 mmol) was partitioned betw~en chlorofo~m:metha~ol (2:1 ~v/v); 200 ml) and cold 1 N HCl (50 ml). The aqueous layer was re-extracted with chloroform methanol (2:1 (~/v): 100 ~1).
The combined organic phase was evaporated and dried under vacuum over P20s. The resulting free phosphatidic a id was dissolved in a mi~ture of DMF (2 ml) and pyridine ~2 ml) and to the solution were added the appropriate peptide having a W093/01828 P~T/USg2/~153 71131~6 `
free amino group (1 mmol) followed by NN'~
dicyclohexylcarbodiimide (DCC; Aldrich Chemical Co. Milwaukee WI, MW: 206, 620 mg, 3 mmol). The reaction mixture was stirred for 24 hours at room temperatur~. The solvents were 5 evaporated and the product was purified by fl~sh chromato~raphy over silica gel column (2.5 x 50 cm) using a linear gradient of 0 ~o 50% methanol in chloro~orm. Fractions containing the desired product a~ indicated by TLC and HPLC
were pooled and evaporat~d. The product wa~ further purified, if necessary, by pr~parative HPLC or by crystalli~ation.
EXAMPL~ lA: DPPA-Ser~Gln-Asn-TyrePxo-Ile-Val-NH2 and E~MP~
lB: DPPA-Ser-Gln-Asn-Tyr-Acpnt Ile-Val-NH2 were prepared by the above procedure.
~ _ ~ 2 General procedure for couplinq phosphatidic acid to the hydroxy~ro~p of~a peptide 1,2-~ipalmitoyl-sn-glycero-3-phosphatidic acid (l mmol) prepared as above was dissolved in a mix~ur~ ~f DMF (2 ml) and pyridine (2 ml) and to the solution were added the appropriate peptide having a free hydroxyl group (1 mmol) followed by DCC
~620 mg, 3 mmol). The reaction was carried out and the product was isolated as~described in Example 1 o 2~
The condensation of~t~e:~phosphatidic acid and ~he hydroxyl group o~ a peptide was also conveniently carried out by u~ing 2,4,6-triisopropylbenzenesulfonyl chloride (TPS-Cl;~Aldrich Chemical CoO, Milwaukee WI; MW: 302.86: 758 mg, 205 mmol) as a coupling agent in place of DCC.
WO93/01828 PCT/US92/~1S3 211315~
~ PLE 3 General proc~dure_for couplinq a pepti.de conta _ na a fr~ee carboxyl group_ to the amino arou _o~ _a phosphatidyl-ethanolamine A mixture of the appropriate peptide (1 mmol), phosphatidyl-ethanolamine (l mmol~ were dissol~ed in pyridine (5 ml~ and DCC ~3 mmol) ~ollowed by 1-hydroxybenzotriazole (HOBt: Aldrich Chemical Co., HOBt, MW: 153; 450 mg, 3 mmol) were added. The reaction mixture was stirred for 24 hours at r~om temperature and the praduct was purified by sil~ica gel chromatography as descrîbed in Exampl 14 ~XANP~E 3A:
iBoc-Tyr-Pro-Ile-Gly~DPPE was prepared by the above procedure.
~ANP~E 4 General_ ~rocedure for coupli~ peptide containina ~a free carboxyl_~rouP to the hydFox~
~l~cerol ~A mix~ure of the appropriate peptide (1 mmol), diacyl or dialkyl glycerol (1 mmolj were dissolved in pyridi~e (~ ml) and DCC (3 mmol) followed by~4-dimethylaminopyridine (Aldrich Chemical Co., D~AP, MW: 122.17; 122 mg, 1 mmol) were added.
The~ reaction mixture was stirred ~for 24 hours at ~room ~. :temperature~ and~ -the ~ product ~was~ purified- ~y-silica gel:
chromatography as de~cribed.in~iExampl~e~
: : EX$~PL~ 5:
Pre;~aration. ~of ~ N-ter~Jlp~ oxyc~Lrbonyl-Serin~
Hydroxy uccinimide Ester [Boc~-S~r-OSu) MW: 302.26 ~ : :
Boc-Ser-OH ( 20.52 g, 100 mmol) and N-hydroxysuccinimide (Aldrich Chemical Co.~Milwauk~e WI; MW: 115:.09; 23.3 g~ 200 mmol) were dissolved in dry ~HF (400 ml). The solution was:
cooled to ~-10C, DCC (MW: 206.33; 30010 g; 150 mmol) was added. The mixture was stirred for 2 hours at -10C and overni~ght at room temperature. The solvents were evaporated~
under vacuum, and the residue was stirred in ethyl acetate (l l~ and filtered to remove insoluble material. The resulting W093~01~28 PCT~US92/06153 2l1'3''l1~'6 ,i',~''?~ ;
filtrate was washed successively with water (3 x 250 ml~, cold lN HCl (3 x 250 ml), water (3 x 250 ml), cold 10% NaHC03 (3 x 250 ml~ and water (3 x 250 ml) and dried over anhyd~ous sodium sulfate. After evaporation the residue was crystallized from ~:
isopropyl alcohol.
Boc-Tyr~OS~, Boc-Hyp-OSu and Boc-Thr-OSu were prepared similarly.
:
Ser-OSu: ~oG ~ a~ ~W: 850.17 1,2-Dipalmitvyl-~n-glycero-3-phosphatidic acid (1 ~mol) ~-was dissolved in anhydrous pyridine (5 ml) and 2,4,6- ~
1~ triisopropylbenzenesulfonyl chloride (TPS Cl, 758 mg, 2.5 :
mmol) followed by Boc-Ser-OSu (~W: 302026; 302 mg, l mm~l~ was added. The reaction mix~ure.was ~tirred under dry nitrog~n atmosphere for 16 hours at room tlsmperature. The reaction wa~
then quenched by adding water ~1 ml) and the solYents wer~
evaporated under. vacuum~ The re idue wa~:dissolv~d :in chloro~orm ~5 ml) and lo~d~d onto a silica gel 60 column (2.5 cm x 4:5 cm~ eg~ilibrat~d with chloroform~ The colu~n was~
eluted with a gradient of chloroform~(500 ml) to 15% MeOH in ~ :
chloroform (: 500 ~ml).~ Frac~ions~ eontain~ng the desired .. product (as..indicated.by TLC):.were pooled and-evaporate~to furnish ~oc~SertOD~ OSu.o ~
Boc-Tyr~ODPP)-OSu Boc-Hyp(ODPP)-OSu and Boc Thr(ODPPj-OSu were prepared ~imilarly. :~
~en~ral Procedure for__couplina phos~hatidyl-serine,_ to a pePtide contain~in a free aminP unction.
Boc-Ser(ODPPj-OSu:(~O.l mmol) and the peptide in ~ues~ion 35 containi~g a f~ee~a~ine function (0.1 mmol) were dissolved in DMF (2 ml~ and~pyridine (2 ml) and stirred for 24 hours at room tempera~ure~ The solYents were evaporated and the product was purified by flash chromatography over silica gel column (2.5 x 50 cm) using a liner gradient of 0 to 50%
methanol in chloroform. Fractions containing the desired product as indicated by TLC and HPLC were pooled and evaporated. The product was further purified, if necessary, by preparative HPLC or by crystallization and deprotected by treatment with trifluoroacetic acid (2ml) for 12 hours at room temperature. The acid was removed by evaporation under vacuum and the product was isolated by lyophilization from water.
Peptides linked to phosphatidyl-tyrosine, phosphatidyl-hydroxyproline and phosphatidyl-threonine were prepared similarly.
EXAMPLE 7A:
was prepared according to the above procedure.
Preparation of 1.2-Dipalmitoyl-sn-glycero-3-phospho-O-(N-Fmoc)-Ser-OBzl. Fmoc-Ser(ODPP)-OBz}
Starting from Fmoc-Ser-OBzl the title compound was prepared following the procedure described for Boc-Ser(ODPP)-OSu. Fmoc-Tyr(ODPP)-OBzl, Fmoc-Hyp(ODPP)-OBzl and Fmoc-Thr(ODPP)-OBzl were prepared similarly.
General procedure for coupling a peptide containing a free caboxyl group to phosphatidyl-sering Fmoc-Ser(ODPP)-OBzl (0.1 mmol) was dissolved in DMF (2 ml) and piperidine (0.2 ml) and the mixture was stirred under dry nitrogen atmosphere for 4 hours and the solvents were evaporated under vacuum. The residue was dissolved in a dry DMF (5 ml) and the peptide in question containing a free WO93/01828 PCT/US92/~61~3 `2i`i'3U'~'6 ,<..................................... ~ -26-carboxyl group (0.1 mmol) was added to it followed by DCC ~0.1 mmol) and HOBt (O.~ mmol). The rea~tion mixture was stirred for 2~ hvurs at room temperature. The solvents were evaporated and the product was purified by flash chromatography over silica gel column (2.5 x 50 cm~ using a linear gradient of O to 50% methanol in chloro~rm. Fractions co~taining the desired produc~ as indicated by ~C and HP~C
were pooled and evaporat~d~ The produc~ was furthex`purified, if necessary, ~y preparative HPLC or by crystallizatlon. The product was then dissolved in DMF ( 5 ml~ a~d hydrogenated in the pre~ence of palladium-carbon (5~) catalyst (200 mg). The catalyst was removed by fil~ration and th~ product was purified by silica gel chromatography as described above.
Peptides containi~g phosphatidyl-tyrosine, phosphatidyl hydroxyproline and phosphatidyl-threonine were prepar~d similarly.
N-hyd~oxysuccini~ide ester Boc-Ser(O-DP~DPG)-ONSu ':
Boc-Ser-OSu (~W: 302.26: 302 mg, 1 mmol) was dissolv~d in 2S triethyl phosphate (5 ml) and cooled to 5 C. POC13 (~W
153.33; 460 mg, 3 mmol) was a~ded to the above ~olu~ion ~d `:
th~ reaction mixture was stirred under dry nitrogen a~mo~p~re for 16 hours at -10 C. Eth@r (20 ml) was added and the resulting precipitate was s~parated from the supar~atant by decantation and the residue was wa~hed with ether (20 ml each). The residue was resuspended in ice ~old water (lO ml3 and the pH was immediately adjus~ed to 7.5 by adding lN ~aOH.
The solution was stirred for 1 hour at 0 C while maintaining the pH at 7.S and lyophiliæed. The resulting product was dissolved in water (5 ml) and load~d onto a DEAE Sephadex A~2 column (1 cm x 10 cm) equilibrated with O.01 M ammonium bicarbonate (pH 7). The column was eluted with a gradient of W093/01X2~ .q., PCT/US92~061~3 2~L~31$6 0.01 M ammonium bicar~onate to 0.3 ~ ammonium bicarbonate (pH
7). Frac~ions containing the desired product (as indi~ated by TLC) were pooled and lyophilized to furnish Bo~-Ser(0-phosphate)-Os~
B. 1. 2-Dipalmitoyl-sn-~lycero-3-hs~bon~
MoFphol~ate) 1,2Dipalmitoyl-sn-glycero-3 phosphatidicacid(DPPA,650 mg, 1 mmol) and morpholine (350 ~g, 4 ~ol) were dissol~ed in chlorofoxm (lO ml), ~ butanol glO ml ) and w~ter ( 1 ~1 ) . The solution wa~ stirred under gentle reflux for 2 hours while adding a solution of DCC (~5 mg, 4 mmol) in t-butanol (lO
ml). The mixture was stirred ~or an addi~ional 4 hours and ev~porated to dryness and suspended in water (lO0 ml) and was ~.xtracted three ~imes with eth~r (100 ml), evaporate~ to dryness and lyophilized.
. .
C. Çouplin~ of_DPPA-Morpho.l~d~
Osu DPPA-~orpholidate (O.5 mmol) and Bo~-Ser~0-phosphate)-Osu (0.3 mmol) w~re dissolv~d in anhydrous pyridine (25 ml). The solution was e~aporated to dryness in vacuo five times fr~
anhydrous pyridine, followed by the addition of anhydrous pyridine (5 ml). The reaction ~ixture ~as~stirr~d at 40 C
and evaporated to dryness~.~ The product was dissolve~ in --~ chloro~om:. ~ethanol:~;water (2:3~ and-.~:loaded onto a DEAE
S~phadex A-25 col~mn ~1 cm x:~lO ~cmj~e~uilibr~ed with chlorofom::methanol: water t2:3:13.~ The column was eluted with a linear gradiant of O to 0.3 M ammonium bi~arbonate in the sam~ solvenk. Fractions con~aining ~h~ desired product (as indi~at~d by T~C) wera pooled, concentrated to a 60 ml volume and extract~d 5 times with chlorsfoxm (50 ml each).
Th cloroform solution was evaporated to ~urnish Boc-Serine(O-diphosphate dipalmitoylglycerol)-N-hydroxysuc~inimide ester.
Boc-Tyr(O-DP-DPG)-ONSu, Boc-H~p(O-DP DPG) ONSu an~ Boc-Thr(O-DP-DPG~-ONSu were prepared similarly.
WO93~01828 ' i; ~ 113 15 6 PCT/US92/06153 E~MPLE 11 Gener.al procedure for_ coupli~s Serine-O-d~hosp~e dipalmitoylglycerol to a p~.eptide c~ontaininq~ free amine function.
Boc-Ser(O-DP-DPG)-ONSu ( 0.1 mmol) and the peptide in guestion containing a free amine function (0.1 mmol) were d~ssolved in D~F (2 ml) and pyridine (2 ml~ and stirred for 24 hours at room temperature. The solvents were evaporated and the product was purified by ~lash chromatsgraphy over silica gel column (2.5 x 50 cm) using a linear gradient of O to 50~
methanol in chloroform. Fractions containing the desired product as indicated by TLC and ~PLC were pooled and evaporated~ The product was further purified, if necessary, by preparative HPLC or by crystallization and deprotected by treatment with trifluoroacetic acid- (2 ml) for 12 hours at room temperature. The acid was removed by evaporation under ~acuum a~d the product was isolated by lyophilization from water.
Tyrosine-O-dip~osphate dipalmitoy}~lycerol, hydroxyproline-O-diphosphate dipalmitoy1~1ycerol and threonine-O-d~iphosphate : dipalmitoylglycerol containing~ peptides were prepared similarly. . :;
~ANPLE:12 : Starting from Fmoc-Ser~OBæl and DPPA-Morpholidate ~he title compound was prepared following the pro~edure described~
in Example 11.
Fmoc-Tyr(O-DP DPG)-OBzl, Fmoc-Hyp(O-DP-DPG~-OBzl and Fmoc-Thr(O-DP~DPG)-OBzl were prepared similarly. : ~
:
~MP~E 13 Starting from Fmoc-Ser(V-DP-DP&~-OBzl, deprotection of 5 th2 Fmoc- group, the coupling reaction, catalytic hydrogenation followed by puri~ication as described abo~e.
Corresponding tyrosine-O-diphosphate dipalmitoylglycerol, hydroxyproline-O-diphospha~e dipalmitoylglycerol and threonine-O~diphosphate dipalmitoylglycerol containing peptides were prepared similarly. g ~A~PLE 14 ser-oSu Boc-Ser(O-phosphate)-OSu (l mm~l~ was dissolv~d in anhydrous pyridine ~5 ml~ and 2,:4, 6Q
~riisopropylbenzenesulfonyl chloride (TPS-Cl~;Aldrich Chemi~al Col, Milwaukee WI; MW: 302.86;: 758 ~g, 205: ~mol) was added fcllowed by l-OoHexadecyl~sn-glyceroln The:~reactlon mixture was stirred under dry ni~rogen atmosphere for 16 hours at room temper~ture. The reaction was then qu~nched by addin~ water (l ~i ml) and the solvents were evaporated under~acuum. The residue was dissolved:i~ chloroform (S ml) an~loaded onto a silica gel 60 column~(Z.5 cm x -45 cm) -equilibrated with chloroform. The ~column was ~luted~ with~ ~a gradi~nt of chlor~fonm (5~0 ml) ~to:~15~ MeOH in chloroform ~ 500 ml).
Fractions containing the dç~ired product ~as indicated by TLC)~
were pooled and evaporated to f~rni~h ~h~ title compound.
Preparation of l-O-h~xadecyl-~n glycero-3-phospho-0-(N-~oc~
Tyr~Osu,l O-hexadecy1-sn-glycero-:3-phospho~O-(N-Boc)-Hyp-Osu and l-O-hexadecyl-sn~ cero-3 phospho-O-(N-Boc)-Thr-O~u were carried out in a similar manner.
:
WO93/01B28 PCT/US92/061~3 ~1131S6 _30~
~ P~E i5 General_~rocedure for couplin~ o-hexadecyl sn-~lycero 3-phospho-serine to a_~eptide co~taininq free ~minÇ fun~tion ~his coupling reaction, subsequent deprotec~ion and purification of the product was carried out following the procedure described in Example g except for using 1 O-hexadecyl-sn-glycero-3-phospho-O-(N-Boc)-Ser-OSu in place of Boc S~r(ODPP)-OSu.
Peptides containing l-O-hexad~cyl-sn~glycero-3-ph~spho O
tyrosine,l-O-h~xadecyl-sn-glycero-3-phospho-O-hydroxyprol~ne and 1-O-hexade~yl-sn-glycero-3-phospho~O-thrPonine were prepared in a similar manner.
~AM~E ~6 Preparation of 1o~O-hexadecyl-sn-~lycero-3-phos~ho-O-(Fmoc~-Ser-OBzl Starting f rom Fmoc-Ser-OBzl the title compound was prepared~following the procedure~d~scribed ~or the preparation of 1 O hexadecyl-sn-91ycero-3-phospho-O-(N~Boc)-Ser-OSu.
Preparation of l-O hexadecyl-sn-glycero-3-phospho-O-(Fmoc)-Tyr OBzl~ l-O-hexadecyl-~n-glycero-3-phospho-O-(Fmoc)-Hyp-OBzl and 1-O-hexadecyl-s~-glycero-3-ph~spho-O~(Fmoc~-Thr-OBzl~were c rri~d out similarly.
BSaMP~ 17~
Starting from l-O-hexadecyl-sn-glycero-3-phospho-O-(Fmoc)-S~r OBzl, d~protection of the~Fmoc- group, the coupling reaction, catalytic hydrogena~ion followed by purification as~
described in Example l0. : :
:~
Pep~ides containing 1 O-hexadecyl-sn-glycero-3-phosph~-~
tyrosine, 1-O-hexadecyl-sn-glycero-3:-phospho-hydroxyproline W093/0182~ PCT/US92/06153 31- :~ 7 ~
andl O-hexadecyl sn-glycero-3-pho~pho-threo~inewereprepared in a similar mznner.
~XA~ 18 preparation~of_l-O~hexadecyl-sn glycero~-diphospho-O-(N~Bocl-Ser~OSu Boc Ser(O-phosphate)-OSu (1 mmol) was converted to the corresponding morpholidate by reacting with DCC and morpholine. The product was then reac~e~ with 1-0-hexadecylglycerol-3-phosphate as descri~ed for the preparation of Bo~Ser(O-DP-DPG)-OSu. : ' Preparation of l-O-hexadecyl-sn-glycero-3-diphospho-0-(N-Boc)-Tyr-Osu, 1-0-hexadecyl-sn-glycero-3-diph~spho-0-(N-Boc~-Hyp-Osu and l-O-hexadecyl~sn-glycero~3~diphospho-0-(N-Boc)-Thr-Osu w~re carried out in a similar manner.
, ~X~PL~
~ .. :
~
This coup~:ing~ reaction,~subse ~ ent deprotect~ion ~`and purif ication of:the product was~ carried out o:110wing the : procedure ~described~in~ Example ~9:~except ~:for using 1-0-hexadecyl-sn-glycero-3-d:iphospho-0-~N-Boc)-S~er~OSuinplaceof :~ :
Boc-Ser(O-DP-DP~)-OSu. :
Peptides: containing l-O-hexadecyl-sn-glycero-3-diphospho-0 tyrosine, l-O-hexa~ecyi-sn-glycero-3-diphos~pho-0:~
hydroxyprolIne a~d -~-hexadecyl-snt~glyc~ro-3-diphQspho~O~
threonine were prepar~d in a similar manner.
~ ~ANP~X 2 PreParation of~ hexadecyl-s~-lycero-3-diphospho-o-~Fmoc~- ~
Ser-08zl : ~-:~Starting ;from Fmoc-Ser-OBzl the title compoun~d was ~ :
prepared following the procedure described for the preparation of 1-0-hexadecyl-sn-glycero-3-diphospho-0-(N-Boc)-Ser-OSu.
W093/01828 PCTJUS92/~6153 2 113i5 6 -32;
Preparation of l-O-hex~decyl~sn-glycero 3 dipho~pho-O-(F~oe)-Tyr~OBzl, 1 O~hexadecyl-~n-glycero-3-diphospho-0 (Fmoc~-Hyp-OBzl and l-O-hexadecyl-sn-glycero-3-diphospho-O-(Fmor~Thr-OBzl were carried out in a similar manner.
~X~L~ 2l Starting from l-O-hexade~yl-sn~glycero-3-diphospho 0-(Fmoc3-SerOBzl, deprotection of the Fmo¢- group, the coupling reaction, catalytic hydrogen2tion followed by purification'as described in Example 9.
Peptides containing l-O-hexadecyl-sn-glyc2ro 3~diphosp~o 0 lS tyrosine, l O-hexadecyl sn-glycero-3-diphospho-0-hydroxyproline and l O-hexad~cyl-sn-glycero-3-diphospho-0 threonine were prepared in a simi.1ar manner~
~8A~P~æ 22 Succinyl!-ethanolamine: DPPE SUCCIN~C ~ID NM: 79l.03 To a solution o~ l,2-Dipalmitoyl- n-glycerc-3-phosphatidyl ethanolamine (D~PE; Avan~i Polar: Lipids, Bixmingh~m AL; MW: 690.96; 346 mg, 0.~5 ~mol) in chloroform (lO
ml), succinic anhydride (Sig~a Chemical ~o.,~St. ~Louis MOr lOO.7, lOO ~g, 1 mmo1~ dis o1ved:in ch10ro~orm (lO ml) and~
- -~tri~thylamine (O.l ml)~were~added. The reaction mixture waæ
stirred under dry nitrogen atmosphere for 16 hours at~ room temper~ture. The reaction was monitored by running TLC at various intarvals. After ~he reaction i~ completed, the solvents were evaporated under vacuum and the residue was di solved in chloroform:(5 ml) and loaded onto a silica gel 60 column (2.5 cm x 45 cm) eguilibrated with chlorofonm~ The column was eluted~with a gradient of chloro~o~m (500 ml) to I5% MeOH in chloroform ( 500 ml). Fractions containing the desired product (as indicated by TLC) were pooled and evaporated to furnish DPPE-SUCCINIC ACIDu ~ 2113~6 Preparation of 1,2-Dimyri~toyl-sn-glycero-3-phospho-O (N-Succinyl) ethanolami~e, DMPE-SUCCINIC ACID, was carried out similarly.
~A~PL~ 23 Preparation of ~oc-Phe O-CH2~CHOH
Boc-Phe-O-CH2-CHOH MW: 616 A mixture o~ Boc-Phe-OH (5~3 g, 20 mmol), triethylamine (TEA; MW: 101.19; 3.1 g, 31 mmol), znhydrous KF (1.2 g, 20 mmol) and 1,3-dibromobutane-2,3-diol (Br-CH2-CHOH-CHOH~CH2-Br, 2.5 g, 10 mmol) was dissolved in DMF (25 ml) and the solution was stirr2d for 24 hr at 45 C. The solvent was evaporated under vacuum and residue was di~solved in EtAc (330 ml!, extracted with 10% sodium bicarbonate (3 x 50 ml), water (3 x 50 ml), 10% ci ric acid (3 x 50 ml~ and water (3 x 50 ml3.
The organic phase was dried over a~hydrous sodium sulphate and evaporat~d under ~acuum and the :residue was crystalIized from acetonitrile to furnish th~ prod~ct as wh~te amo~phous powder.
~ P~ 2 : Pre~aration of : ~ .
Bo~-Val Phe-O-CHz-CHOH
~: Boc-Val-Phe-O~ [2-C~HOH
The com~ound. r3m !~examp}e.~23 (616~mg,..~1 ~ ol) was~
dissolv~d in.a~hydrou~FA ~5~ and:stirred~or 15;.m~inutes.
The solvent wa$~ evaporat~d~:a~nd the ~residue~;~riturated~with :30 ether and filtered. The product was dissolved in DMF;(20~ml) ~ :
and cooled to -10C. Boc-~al-OH (MW: 217~.14, 696~mg, :3 mmol~, HOBt (456 =g, 3~mol) and DCC (620 mg,~ 3 mmol) were added to th above solution. The reaction mixture was stirred~
for 2 hours at ~10C;and for 16~hours at~ room tempera~ure.
The solvents were~then~ evaporated under vacuum,~ and ~the ~ :
residue was dissolved in ~thyl ace~ate (250 ml)~ and filt ed .
to rem~ve ins~oluble material. The resulting ~iItrate was washed successively wi~h water (3~x 50 ml), cold lN ~Cl~ (3 x WO93/0182~ PCT/US92/06153 ~ i ~ 3 f S 6 ,~'?~ .
50 ml~, water (3 x 50 ml), cold 10~ NaHCO3 (3 x 50 ml~ and water (3 x 50 ml) and dried over anhydrous sodium lfateO
Evaporation of the solvent provided the crude produ t which was purified by crystallization from acetonitrile.
~A~PLæ 2s Preparation of Boc-Ser-Phe-O-CH2-CHOH
~oc-Ser-Phe O-CH2-CHOH
The title compound was prepared f~llowing tho procedure descri~ed for Example 24 except u~ing ~oc--Ser-OH instead of Boc-Val-OH.
~a~PL~ 26 Pre~aration of Boc-Asn-Phe 0 CH2-CHOH
Boc sn-Phe-0-CH2-CHOH
The title compound was prepared followin~ the procedure described for Examlple 24~ exGept: using Boc-A5n 0H insgead o~
.~
~oo-Val-OH.
~ANP~;27 : :
A. ;~ ~Ç i~ de_Ey~h i~ Boc~amino ~acyl-ben:zyl ~ester~
~: resin or Boc~-amino a~yl-~4:-me~yl:)benzhydrylàmin~;r~sin:~(Boc~
esini~2 g~ 1~mmol) placed~:in a reaGtion~essel~of~Béckman~
~ -990 B Pép~ide Synthesizer~(Beo~man~Instruments,~Palo Alto, C~
:~ and subjected to the gollowing operations.~ Each tep~is ~ca~ried out one time unless~specified othe~wise:,~ and reagents~
and solvents after each step are~æeparated from ~he peptide ~:
resin by filtration under nitrogen Ste~ Rea~ent/sl vent~No~æ~ y~mes Mix Time (minute-s~
1 : DCM ~(30 ml, 3 times) : : 1.5~
2 : TF~-DCM (1~ (3Q ml) : 1.5 ~ : :
3 TF~-~CM (1~:1) (30 ml) ~30.0 4 DC~:~(30 ml, 3:~imes~ ~ 1.5 : ~
S ~ Methanol (30 ml, 3 times) 1.5 ~::
6 DCM :(30 ml, 3 times) 1.5
7 TFA-DCM (1:1) (30 ml) 1.5
8 TFA-DCM (1:1) (30 ml) 5.0 WO93/01828 PCT/U~2/061~3 _35 2113~5~ i g DCM (30 ml~ 3 times) 1.5 10 DMF (30 ~1, 3 times) 1.5 11 Boc-Thr~B l)-OH~HOBt/DCC (~ mmol ea~h) in DMF (20 ml) 240.0*
12 DCM (30 ml, 3 times) 1.5 13 Methanol (30 ml, 3 times~ 1.5 14 DCM ~30 ml, 3 times~ 1.5 Alternativ~ly, the resin was subjected to the following oper~tions: :
1. DCM (30 ml; 1 time) 1.0 2. Neat TFA [30 ml; 1 time) lo O
3 . Neat TFA (30 ml; 1 time~ 5.0 4. DCM (30 ml; 2 times) 1.0 5. DMF (3U ml; 2 times:) 1.0 6. DIEA-DMF (30 ml; I time) 1.0 7. DIEA-DMF ~30 ml; 1 time) S.0:
8. DMF (30 ml; 3 times) : 1.0 20 9. Boc-~mino acid-OH/HOBt/D~C (4 mmol each~
in DMF (20 ml; 1 time) 120.0 10. D~F (30 ml; 3 times) ~ 1.0 * Coupling reaction was carried out for~an ~ average ~of 4 hoursl as in this case, or until a~ninhydrin test:(~Kaiser:E.T.
et al., Anal.;~Bio~h~. 34:595-8~ 196~ showed a negativ@
result i~dicating the~absence of free~amino groups.:~ The s2me seq~en~e of r~aotions was ~repeated using appropriate~amino acid~ derivatives~ u~til :the~:requir~d peptide :~chain:~was~
:: 30 ~ assembled on the~resin. ~After completion~of:~:the~;synthesiæ, :
. the~resin was removed~ rom the.:vessel~and~dried under vacuum.
(HF): Peptides containin~free~carboxyl groups~or carboxamid:: 35 ~unction at the~ C-terminal are prepared~by: trea~ing~:~:the corresponding benzyl ester;~inked~or~4-methylbenzhyd~yl;~ine~
linked peptide resins according to the fo1lowing pro~edure.
The~dried peptide~rssin (l g)~, anisole (1 ml)and p~cresol.(O.l~
g) were;placed~in~a Bel:~F:reaction v~ssel.~ The~:vessel was ~:
placed in a~bath of:liquid ni*rogen ànd anhydrous~HF (15~ml)~
was condensed into~: the~ ~e:s~el. The: reaction :mixture~ was : - ~ :stirred at -10~C~for l:hour:and~HF was removed by evaporation - under vacuum.: The residue was triturated with dry ether~(50 W093/01~28 PCT/U~92/~61~3 ~1131S6 3~
ml), filtered and washed with additional ~uantity of ether (3 x 50 ml~. Peptide product in the mixture was isolated by extractin~ with glacial acetic acid (3 x 50 ml) followed 3:~y lyophilization of the solvent.
C. Preparation of peptide _ methyL__ esters bY
transesterifacation: Boc-peptidyl-b . nzyl ester resin (1 g) was s~irred with me~hanol~10 ml) and triethylamine (1 ml~ for 18 hours at room temperature . The resin was f iltered and washed three times with methanol (20 ml each time) and the combined ~iltrate was evaporated to provide peptide methyl esters. Some of the peptides prepared by this method contain protecting groups for other functional groups whlch are very conveniently depro~ected by trea~ment with liquid HF as described above. The resulting products w~re purified as described below.
D. razides:: ~oc-peptidyl-benzyl ester resin (1 g) was stirred wit~ methanol~10 ml~ and anhydrou~ hydrazine (1 ml~ for 18 hours at room te~p~rature.
The resin was filtsred and washed three times with methanol (20 ml each time) and t~e co~ ined filtrat~ was evaporated to pro~ide peptîde hydrazides esters. Some of the peptide prepared by this method contain protecting groups :for other functional groups which are very conveniently deprot~cted by - ~::- tr@atment~.with-.liguid HF as described:aboYe~ The resulting ~roducts wer~ purified :8~ described belowu E~ ~ Peptide powder obtained above (200 mg) was dissolved in lN acetic acid (3 mL), loaded to a Sephadex G 25 (superfine) column tl.5 cm x lO0 cm) and eluted with lN acetic acid. The eluent fractions; containing the peptide were pooled and freeze dried. The resulting pepti~e ~50 mg) was further purified by preparative reverse phase high performance chromatography (RP HPLC) usin~ a Waters C-4 column and a buffer gradient of 0.1% TFA in water to 70% acetonitrile in o.1~ TFA in water. The fractions containing pure peptide WO93/018~8 PCT/US92/06153 ~, 211;33L'5;6i (determined by analytical ~LC) were combined and the product isolated by lyophilization. Purity of the p~ptide was better than 95% by HPLC: and amino acid analysis followed by acid hydrolysis (6N H~l, 110 C, 24 hr) gave expected amino acid ratios.
PeptidPs of the foregoing examples listed below were prepar~d by the solid phase method.
E2aNP~g 27-01 Ser-Gln-Asn-Phe-Pro-Ile-Val-NH2 ;~
~PL~ 27-02 Ser-Gln-Asn-~yr-Pro-Ile-Val-NH2 E~AMPLE 27-03 Ser-Gln-Asn-Tyr-Achx-Ile-Val-NHz EX~P~ 27-04 Ser-Gln-Asn-Tyr-Acpr-Ile-Val-NH2 ~AN~E 27-05 Ser-~ln-Asn~Tyr-~cpnt-Ile--Val-NH2 EXA~P~E 27-06 Thr-Ile-Leu-(bet2=Ala) Leu-Gln-Arg-N~2 ~MP~E 27-07 Ser-Gln-Asn-Tyr-Pxo-Ile Valal Thr Leu-A~a Thr-Q~Ar~-NH2 : :.
E~MP~E 27-15 Ac-A~a ~la-(D-a-Nal)-Pip-~a-~O~)-~eu)-Val-NH2 E~MPLE 27-16 Ac-A}a-Ala-Phe-Pi]p ~a-(OH)-Leu3-Val-NH2 E2~PLE 27-17 Ac-Ala Ala-(DL-Phe(4-Cl))-Pip-(z-(OH)-LeU) Val-NH2~ . ~
E~A~P~E 27-18 Ala-Ala-Phe-(b~a-Ala)-Val-~al-Gly-OH ~ :
EXA~P~E 27019 Ala Ala-Phe-(~eta--~la)-Nva-Val-Gly-OH
. EX~P~E 27-20 Ala-Ala-Phe-(beta-Ala)-(a-(OH)-iso-Valeryl)-~ Val-G~y-OH~
EXA~PL~ 27-21 ~Ala-AlaoPhe ~bata~Ala)~-~al-Val-Gly O~e~
XAMPL~ 27~`~2 Ala-Ala-Phe-(b~ta-Ala~Nv~-Val-Gly-OMe L~ 27-23 Al~-Ala-Phe-~beta-Ala)-~a-~OH)-i~o-Va:leryl) VaI-Gly-O~e :~
E~AMP~ 27-24 Boc-Ala Ala-P~e-(beta Ala)~N~a-Gly-O~e EXA~PLE 27-25 (a,e-dimyris~oyl-K) dP-Ava-Ala-Ala-Phe-Ava- `~
Val-Gly-OMe EXAN~E 27-26 ~c-Ala-~la-~D-a-Nal~-Pip-o~e ~XA~LE 27-27 Ac-Ala-Ala(D a-Nal)-Pip-NHNH2 :
Æ~MP~E 27-28 Ac-Ala-Ala-~L-a-Nal)-Pip OMe EX~M~E 27 29 Ac-Ala-Ala-(L a-Nal)-Pip-NHNH2 EXAMP~E 27-30 Ac Ala-Ala-(D-b Nal)-Pip-OMe .
W093/0182X ~ 1 13 1~ ~ PCT/US92/06153 . . , , , .~ .
! -38 E~AMPLE 27-31 Ac-Ala-Ala~(D-b-Nal)-Pip-NHNH2 E~M~LE 27-32 Chl-Ala-Ala-(L-a-Nal)-Pip-OMe EXA~PLE 27-33 Chl-Ala-Ala-(L-a-Nal)-Pip-NHNH2 EX~MP~E 2.7-34 Paa-Ala ~la(D-a-Nal)-Pip-OMe EX~MPLE 27-36 Ac-Ala-Ala-(DL-Phe(4-Cl~)-Pip-OH
EX~MPLE 27-38 Val-Ser-Gln-Asn-Tyr-Pro~ Val-NH2 E~AMPL~ 27-39 Val-Ser-Gln-Asn-(D a-Nal)-Pip-(a-(OH)-Leu3-Va,l -NH2 E~AMPLE 27-40 iBoc-(D-Ph )-(D-a~Nal)~Pip-(a-(OH)-Leu) Val-EX~NPLE 27-41 (iBoc-(D-Phe)-Leu-) 2- (3,5-di-Aba)-Pro-Leu ~D-Ph~)-NH2 EXAMPLE 27~42 (Myristoyl-(D-Phe)-Leu-) 2-( 3,5-di-Aba~-Pro~
Leu-(D Phe)-NH2 EXANPLE 27-43 iBoc-(3,5-di~Aba)-(D-a-Nal)-Pip-~a-(OH) Leu)-~al-NH2 EX~PL~ 27-46 Pro-Ile~Val-NH2 EXANP~E 27-47 Ser-Gln-A~n-Tyr ~XAMP~E 27~48 Asn-Phe(CO-~HzN)P.ip-Ile NH2 EX~MP~ 27-49 Quinaldoyl-A~n-Phe;(CO~CH2N)Pip-Ile;NH2 SX~X~ 27-S0 Val-Ser-Gln-Asn-Tyr-Pip~ Val-Gln-NH2 EX~E 27-51 Quinaldoyl-Asn-Phe(CHOH-CH~)Diq-Ile-Val-Gln-NH2 EXANPLE 27-52 Val-Ser-Gln-Asn-Tyr-Diq-Ile-Val-Gln-NH2 EX~P~E 27-53 Boc-Asn-Phe(CO 2N)Diq-NHtBu~
EX~M~E 27-54 Su~cinyl-5er-Gln~As~-Tyr-Pro-Ile-Val NH2~ :
` EX~P~E 2~-55~Succinyl-Ser-&ln-Asn-Tyr~
EX~P~E-27-56~Quinaldoyl-~sn-Phe:(C~OH H2N~Pip-T~e-NH2 ; EXa~L~ 27-60 Phe(CH~H-CH2N)Phe-Ile-Phe-NH2: -~ :
EX~MP~E 27-61 Phe(CHOH-CH2N)Pro-Ile-Val-NH2: : :
EXA~P~E 27-62 His-Lys-Arg-Ala-Val-Leu-Phe(4-NO2)-Glu~Ala-Nle~Ser-NH2 EXAMP~E 27-69 [D~Phej~-[D-a Nalj-Pip-[L-a-(OH)-Leu]-Val-NH2 EXAMP~E 27-70 Boc-Phe(CHOH-CHzN)Phe Ile-Phe-NH2 EXAMPLE 27-71 B~c-Phe(CHOH-CH2N)Pro-Ile Val-NH2 EXAMPLE 27-74 :Succlny}-Phe(CHOH-CH2N~Phe-Ile~Phe-NH2 EX~MPLE 27-77 [D-Phe]-tD-b-Nal] Pip-~L-a-(OH)-Leu]-Val-NH2 EXA~PLE 27-79 iBoc-Tyr-Pro-Ile-~ly-OH
~39~ ~113i5~
EXAMP~E 27-80 iBoc-[D-Phe]-[D-a-Mal~-Pip~L-a (OH) Leu]-Val-OH
E~ANPL~ 27-8l iBoc-Phe~D-b-Nal~-Pip-[L-a-(OH)-Leu]-Val-NH2 E~ANPL~ 27-~2 iBoc-Phe-~D-b-Nal]-Pip-[L-a (OH)-LRu]-Val-OH
S ~NPLE 27-83 IBoc;~Phe(4-Br)]-~D-b-Nal]-Pip-~Lraw(OH)-Leu]
Val-OH
General procedure for the coup~_nq of DMPE-SUCCINIC ACI~ or DPPE-SUCCINIC ACID or D~PE~;SUCCINIC ACID tl mmol) and the required peptide having a ~ree amine function t1 mmol) we~e dissol~ed in DMF (5 ml) and DCC (3 mmol) followed by 1-hydroxybenzotriazole ~HOBt; Aldrich Chemical Co., HOBt, MW:
153; 450 mg, 3 mmol~ were added. The r~action mixture was stirred for 24 hours at room temperature and the product was purifisd by silica gel chromatography as described in Example 1 . .
This proc~dure was u~ed to prepare the following peptides:
E ~ P~E ~8A: DPPE-Suc-~la-Ala-(D-~-Nal) Pip-O~e0 ~a~ 288: DPPE-Succinyl-Val~Ser~Gln-Asn-Tyr-Pip-Ile-Val-Gln-NH2 E~AMPL~ 28C: D~PE-Succinyl-Val-Ser-Gln-Asn-Tyr-Pip-Ile-Val-Gln-N~2 E~MP~R 28D: DMPE-Su~cinyl-Val-Ser-Gln-Asn-Tyr-Diq-Ile-Val- ~ ;
~ Gln-NH2 X~ E<~28E:.DPPE-Succiny1-CD-Ph~]-~D-a-Nal~ Pip- E L-a-(OH)- :
Leu]-Va1-NH2 k~aV~ 28F: DPPE-Succinyl-Phe(CHOH~CH2N)Phe-I1e Phe-NH2 ~ ;
XA~P~ 28-: DPPE-Succinyl Phe-O-CH2-CHOH
NNz-Phe-O-CH2-CHOH
~MP~ 2g :
AssaY For The Reduction o:f_HIV Sy~ytia Formed In HT4-6C CQlls The syncytia~reduction assay as described~by La~der~ B.
et al~, Science 243:1731-1734(1989) was used to measure the antiviral effect of protease inhibitor. A HeLa cell line, HT4-6C, expressing the human OD4 receptor on its surface were WO93/01~28 PCT/US92/061S3 ~ 113 15 6 .~;'?~, .
trypsinized with 0.25% trypsin for 5 min~ Cells were centrifug~d to remove the residual tr~psin, and the cell pellet was resuspended in DMEM with 10% FCS. HeL~ c~lls were plated in 96 well plate (lx105 cells/well) overnight. C~
cultures ware infected with ~IV (approx. 100 PFU/well) for one hour. The infected cells were then prepared in stock solution~, there wer~ then two fold dilut~d in 2% DMEM wi~h 0.5% methylcellulose. 100 ~1 of ea~h dilu~ed antiviral agent is added into each well o~ HIV in~ec~ed cells. The treated cell cultures were incuba~ed in 37C C2 incubator for 24 hours. Plate of HIV-infected cell culture~ were fixed with methanol and stained wi~h 1% crystal violet for 10 min., the dye is rinsed off with tap water. Plate is dried and syncytia were counted. The antiviral effect of pro~ease inhibitor is c~lculated by 50% syncytia reduction.
Anti~iral activities for the compounds of the pre~ent invention are summarized in the following table. :
,:
~o EXAMP~ 27 30: ~ >100 ~M~
Ac-Ala-Ala-(D-b-Nal)-pip-oMe PLE 28A: 10 ~M
DPPE-Suc-Ala-Ala (D-b-Nal)-Pip-OMe ~ ~
E~M~LE 27-40~ 0 ~M ~i Boc-~D-Phe~j-(D-a-Nal~-Pip-:(a7(OH)-Leu)-Val~-NH
~XA~LB 28E~ 2 ~
DPPE-Succinyl - [ D-Phe ] - ~ D-a Nal ~ Pip- [ L-a ~ i ( OH j -~eu ] -Va1 -NHz PI.E: 24 ~
3~ (8c-Phe-o-~2-cHo~)2 ~XA~P~E 25: :3 ~M
(Boc-Val-Phe-~O-CH2-CHOH)2 ~ -~.
...
WO93/01828 . PCT/~S92/0~1~3 SEQU~NCE LISTING
(1) GENE~AL INFORMATION:
(i) APPLICANT: Basava, ~han~a Hostel r, Karl Y.
(ii) TITLE OF INVENTION: LIPID CONJUGATES..OF THERAPEUTIC
PEPTIDES AND PROTEASE INHIBITORS
NUMBER OF SEQUENCES: 35 (iv) COR~ESPONDEN~E ADDRESS: :
(A) ADDRESSEEs VI~AL, I~CD
(B) STREET: 9373:Towne Centre Drive (C) CITY: San Diego, (D) STATE: Cali~ornia ~ :
(E) COUNTRY: USA
(F) ZIP: 92121 ~ :
(v) COMPUTER ~EADABhE FOR~
(A~ ~EDIUM T~PE: Floppy disk : :
< (B) COMPUTER: IB~ PC co~patible (C) OPERATING SYSTE~: PC~DOS/~S-DOS:
~ (D) SOFTWARE:~PatentIn Release #1.0,:Version #1.25 ~:
(~i) CUR~ENT APPLICATION ~TA~
(A) ~PPLICAT~ON NU~B : US 07~734,434 tB) FI~ING~;D~TE~ 3-JUL~l:991 : ~ :
(C~CLASS~FI:CATION~
(viii) ATTO~NEY~AGENT IN~ORMATION~
(A) NAME: Kir~pa~rick~ Anita M. :~
(B~ REjGISTR~TION NUMBER:~:32,617 (C) REFEREN~E/~GCKET~N~MBER::~ICAL:.019 (2) INFORMATION FOR~SEQ~ID NO~
: (~i3 SEQUENCE CHARACTERISTTCS~
(A) LENG~H: 7 amino acid ~B) TYPE: amino ac:id~
(C) STR~NDEDNESS: single ~D) TOPOLOGY: linear~
(ii)~ MOLECULE TYPE: p~ptide~
(iii) HYPOTHETICAL: NO
, (iv) ~NTI-SENSE: NO ~ : : : :
(v) FRAGMENT TYPE: internal ::
~, WO93/01828 PCT/U~92/06153 &
--42-- ~
(ix~ FEATURE:
(A) NAME/K2Y: misc-difference ~:
~B) LOCATION: 7 (D~ OTHER INFORMATION: carboxyl grQup of terminal Val-7 re idue is in amide form. : -(xi) SEQUENCE DESCRIP~ION: SEQ ID NO~
5er Gly Asn Phe Pro Ile Val l 5 ~,.
( 2 ) INFORMATION Fl:)R SEQ ID NO: 2: ~ :
( i ) SEQUENCE C~iARACTERISTICS
~A~ LENGTH: 7 amino acids (B~ TYPE: amino acid (C) 5TRANDEDNESS: single tD) TOPOL~GY: linea~r ;
(ii) MOLECULE T~PE: ~A) peptide (iii) HYPO~ETICAL: NO
(iv) ANTI-SENSE: NO
~(v)~FRA~ME~T TYPE:~internal;~
(1X~FEATURE~
(A~:NAME/XEY. misc-di~ference (~) LO~ATI~N~ 7~
(D? ~THER~INFOR~ATION;~ arboxyI group~of terminal : Val-7 residue is in:ami~e~form~
: (xij~SEQUENCE DES:CRIPTION::~SEQ~:ID M~:2 Ser Gln A n:::~yr~Pro~le;Val~
: (2)~INFO~ATION~F~R~SEQ ID NO:3 ~i) SEQUENCE:CHARA TERISTICS~
(~) LENGTH: 7 ami~o acids `
:: (8~: TYPE:~ ~mino a~:id :) STRANDEDNESS:~single~
D)~TOPOLOGY~ inear ::
OLEULE~;TYPE: peptide~
~(iii~3;HYPOTHETICAL-; NO
:~(iv) ANTI-SE~SE: NO ` :
.:
:
WO93/01828 PCT~USg2/~6153-`" ~113~L51~
~v) FRAGMENT TYPE: internal (ix) FEATURE:
(A~ NA~E/XEY: Unusual amino acids; mis~. differences (B) LOCATION: S, 7 (D) OTHER INFORMATION: Xaa=l-amino, l-cyclohexahe carboxylic acid (Achx); carbo ~1 group of terminal Val-7 residue is in amide form.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Ser Gln ~sn Tyr Xaa Ile Val (2) INFORMATION FOR SEQ ID NO:4: ' (i) SEQUENCE CH~RACTERISTXCS: `~
(A) LENGTH: 7 amino acids : :
(B:'~ TYPE: amino acid ~ :
t~) STRANDEDNEss:~sins~le (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide~
(iii) HYPOTBETlCAL: NO
~(iv) A~TI-SENSE: NO~
(v) FR~GMENT~TYPE: internal (:ix) FEATURE~
: ~: (A) N~ME~KEY: Unusual a~ino:acid~ misc. differences~
(B3~L0C~ATION~ 5,~7~
: (D~ OTHER:INFORN~TION: Xaa=~l-;amin~ cycloprop~ne càrboxyli~ aeid~N~ pr):;: :carboxyl :~r~up ..te ~ ~al V~1 7 ~es~idue~is in~amide form~
(xi)~:SEQUEN OE~DXSC~PTION~ SEQ~:~ID NO~4 Ser Gln Asn Tyr Xaa~Ile Val~
(2) INFORMATION FOR SEQ I~ NO:5:~
~(i)`SEQUENCE:~CHARACTERISTI~S~
: (A) LENG~H::~7 amin~o~ac:ids :: ;:~
(~) TYPE::amino:~a~id~
~C~ TRANDEDNESS:: singl:e~
~:- : (D) ~OPO~0GY: line r~
(il) MOLECULE~TYPE:::peptide ;~
HYPOTHETICA1: NO
,~
WO 93/01B28 PCI/U$92/06153 .., q ~ ., ~ii31SC _44 ( iv ) ANTI -SENSE: No (v) FRAGNENT TYPE: internal (ix) FEATURE: `
(A) NAME/~EY: Unusual amino acids; misc. dif~erences (B) LOCATION 5, 7 (D3 OT~ER INFORMATION: Xaa=1-amino, l~cyclopentane carboxylic: acid (Acpnt3; c:arboxyl group of terminal Val 7 r2sidue is in amide foxm.
( xi ) SEQU~:NCE DE5CRIPTION: SE52 II) NO: 5:
Ser Gln A,sn Tyr Xaa Ile: Val ( 2 ) INFORMATION FOR SEQ ID NO: 6: :
( i ) SEQUENCE CHARACTERISTICS: :
tA) LENGTH: 7 amino ac:ids tB3 TYPE: amino acid (C) ST~A21DEDNESS: sing~le ( D) TOPOLOGY: linear (ii) MOLECUI.E ~YPE: peptidç ~ :
(iii3 HYPOTHETICAL: NO
( iy) ANTI-SENSE :~ NO
: (v j ~ ~FRA~MENT: ~TYPE ~ ternal (A) N~5EfKEY~ Unusual amino~acids~;~ mi c~ d:ifferences 8~ CATION ~ 4 D):; O~IE~;;INFl)~ION~ Xa~=bAla;~ carbo~ roup ~o~
:t~r~inal P~rg~ residue :i5 in~ am de~ ~Eo~. :
~xi) SEQUEN~E~ DES~P~IPTION:~ SEQ ~ID NO.6 mr Ile ~ Leu :Xaa I,eu Gln Arg (2) IN~O~IATION: FOR SEQ ID NO:7 SEQUENCE C~ARAcTERISTICS ~
(Pa3 LEN6TH: 13 :amino acids ::
(B~ ~TY~E::~ amino acid: ~ : :
~C~ :STRANDEDNESS: single ~D) ~OPOLOGY: linear ( ii ) MOLECULE TYPE: peptide : : , ::
WO 93/018~8 PCr/VS92/061~3 :: 2`~ 6 iii ) HYPOTHETICAL: NO
( iv ) ANTI-SENSE: NO
(v) FR~GMENT TYPE: internal ( ix ) FEATURE ~
(A) NAME~KEY: Unusual amino acids; misc. differenc s ~ B) LOCATION: 10, 13 (D) OTHER INFORMATION: Xaa=S-aminoisovaleric acid(~va); carboxyl group of t~rminal Arg-13 residue is in amide ~orm.
(xi~ SEQUENCE DESCRIPTION: SEQ ~ID NO:7~
Ser Gln ~sn Tyr Pro: I1e Va1 Thr X,eu Xaa Thr G1rl Arg ~ :
5 ~ 1 0 ( 2 ) INFORMATION FOR ~ SEQ ID NO: 8: :
i ) SEQUENCE CHARACTERISTIC:S:
(A) LEN~;TH: 6 amino: acids tB~ TYPE: amino :acid (C) ST~NDEDNF,S~;: sin~le (D) TOPOLOGY: 1inear ~ : :
: ~ (ii) MOLECULE :~YPE:~ peptide ( iii:) HYPO~HETICAL: NO
(~ iv ) ~TI-SENSE: NO
( v ) F~AGNENT~ ~ TYPE: :: interna l ~
(A) NAME~KEY: ~Unusuàl~ a~in~o ;acid ;;~ misc.~ d:i~feren6:es `D) OT~ER INF~RM~TI~N~: Xaa3~ p~enylalani~e~(Phe)~
Xaa4~4-p ~ridine~`iaar~oxyl~ic~a~id~:~`ÇPip)~ Xa~5:~
hydroxyl~ucine(~`~H-Leu~ amino ~up~:of~
ini~îal ~1~ l residue:~is:acetylat~d~ ar~oxy~
: `group of terminal~ al-S;~resi~ue~ i~s ln~:~amide (xi~ SEQUENCE~DESCRIPTION: SEQ ID~NO:8 Ala Ala~:Xaa Xaa:Xaa Val (2)~INFORMATION~FOR~SEQ ID NO:9 : ~(i)~ SEQUENCE:~HAR~CTERISTICS~
, , :
, W~93/n1~2~ PCT/US92/06153 ~ 1 13 1 i 61 ~ 5~
(A) LENGTH: 7 amino acids ~B) TYPE: amino acid (C) STRANDEDNESS: ingle (D) TOPO,~OGY~ ear (ii) MOLECULE TYPE: peptide (iii) HYPOT~ET:lCAL: NO .
( iv ) ,~TI-SENSE: NO
tv~) FRA~MENT TYPE: in~ernal ( ix ) FEATURE ~
(A) NAME/XEY: Unusual amino acids; mis dif~ferences (B~ LOCAT ON: 4, 7 (D) OTHER I:NFORM~TION: ~aa:~alanine(bAla~;
terminal Gly-7 residue is~ hydroxylated (xi) SEQUEN~E DESCRIPTION: SEQ ID NO:9:
Ala Ala Phe Xaa Val Val Gly (2) INFORN~TION FOR SEQ~ N0~10~
(i) ~EQUENCE~CHAR~CTER~STI~S- -~(A) :LENGTH~ 7:amino::~a:cids ~ B3 TYP~ amino~aci~
:~C) STRA~DED~ESS:~single ~(~) TOPOL~GY~ lin~ar~
: ~ (ii) MO~ECULE~TYPE: p~ptide~
: ; (i:ii)~HYPOTHETICAL:~NO~
)' ~ G~ENT T~PE~ :in~r~
(Aj NAME/KEY~ nusual ami~o acids;:misc~ differen~s ) LOC~TI9N::4,~:.:5:,~7 ~D~ OT~ER INFO ~ TION: Xaa4=bAla; Xaa5=Nva~
carbo~y ~ terminal Gly-7 residue ~ is hydroxylate~
(xi) SEQUENCE:~DESCRIPTION::SEQ~ID~:NO:10 Ala Ala ~h~Xaa Xaa~Yal Gly ~
1 ~ 5: : :: ; ::
~: ` `:: ::: ~ : : :
~ ~ :
: :
WO93/01828 , PCT/U~92/~1~3 ~7~ ~113i~
(2) INFORMATION FOR SE4 ID NO~
(i) SEQUENCE CH~RACTERISTICS:
(A) LE~GTH: 7 amino acids (B) TYPE: amino acid (C~ STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) NYPOTHETICAL: N~
(iv~ ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (ix) FEATURE~
(A) NAME/KEY: Unusual amino acid~; misc. differences (B) L~CATION: 4, ~, 7 : ~ ~
(D) OTHER INFORM~TION: Xaa~=bAla: ~-Xaa5~ H-isov~leryl;~ carboxy terminal Gly-7 ~:
residue is hydroxylated.
(xi) S QUENCE~DESCRIPTION: SEQ ID NO~
Ala Ala Phe Xaa Xaa Val Giy :(2) INFO~ATION~FOR~SEQ~ID~NO~:12 (i3 5EQUENCE:CHARACTERISTICS:~
: (A) LENGTH:::7 amino a~:ids :
(B:) T~PE:~amino aci~
(C)~STRANDEDNESS~ s~i~gle D)~OPO~oGY:~lin MO~ ~TYPE~ pép~Ide~
(iii) HYP4THETIC~L~ NO~
iv) ~ TI-sENSE~: NO~
(Y) FRAGMENT TYPE::internal~
(ix~ FEA~ RE~
N~E/K~Y: Unusual ami~o::~acids:~misc. dif~ere~es (D) OTHER:INFORM~TI~N:~ Xaa:~alanine~(b~la~
arboxy te~rminal~Gly-?~ re~idue~ is a methyl~
(xi)~SEQUENCE DESCRIPTION: SEQ~ID~NO:12 : : :
:~
:~
3 135i~6 `, Ala Ala Phe Xaa Val Val Gly (2) INFORMATION FOR SEQ ID NO:13: .
(i) SEQUENCE CH~RACTERIS~ICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid ...
(C) STR~NDED~ESS: single (D~ TOPO~OGY: linear ( ii ) MOLECULE TYPE: peptide ~ :
r (iii) HYPOTHETICAL: NO
(iv) ANTI SENSE: NO :
(v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME/KEY: Unusual amino acids; misc. difference~
(B) LOCATION: 4, 5, 7 (D) OTHER INFO~M~TI~N: Xaa4-bAla; Xaa5=N~a c~rboxy~terminal Gly-7 ::residue is a m~thyl ~ster.
(xi) SEQUEN~E~DESCRIPTION: SEQ ID NO:13 Ala Aia Ph~ Xaa~Xaa: Yal Gly :: :~
(2) INFO~MATION FQR SEQ~ D~NO:~14 i) SEQUENCE~C~AR~TER~5TI~S~
: (A) L~NGTH~ 7~ ino acids : :~
D~D~ESS::~sin~le (D) TOPO~OGY:~linear MOLE ~ LE~TYPE:~peptide:
(iii~ HYPOTHETIC~L: NO~
(iv) ANTI-SENSE: NO
) FR~GMENT TYPE:~ internal~
(ix~ FEATU~
(A)~N~E/KEY::Unusual amino acids; misc. differences (B) LOCATION:~4,~`~5, 7 : : ,`
::
WO93/0l82g : PCTJUS92~061~3 ~113156 -4g-(D) OTHER IN~ORMATION: Xaa4=bAla;
Xaa5=~ OH~isovaleryl; carboxy terminal Gly~7 residue is a methyl ester.
( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Ala Ala Phe Xaa Xaa Val Gly . ..
(2) INFORMATI0N FOR SEQ ID NO:15:
. (i) SEQUENCE CHARACTERISTICS~
(~) LE~GTH: 6 amino acids : ,~
(B) TYPE: amino acid (C) STRANDEDNESS: single (D) T0PO~GY: linear (ii) MOLECUIE TYPE: peptide (iii) HYPOTHETICAL: NO
(i~) AN~I-SENSE: NO
(v) F~ GMENT:TYPE: i~ternal :
:
(ix) FEATU~E~
: (A~ NAME/KEY~::Unus~al aimino a~ids; misc. differences :
: (B) L~C~TIO~:: 4~: 5, 6:~
(~) o~HER~INFaRM~TIoN: Xaa4=bAla~
Xaa5=norvaline(N~a)~;~amino ~ terminal ~a~
residue is ~ modi~ied~ by :t-bu~yloxy~ar~onyl ~ :
: t~ached~ to::ami~o~:~:group; :térmi~al Gly-6 residue~is~:a~methyl~ ester.
xi)~SEQUEN~E~DEgCRIPTION:~S~Q I~NO:~15~
- ~ :.Al-a~ la Phe Xaa:~àa~Gly~ ~Y;:~ 7' (~2) INFORMATION FOR SEQ ID NQ:16 (i) SEQUENCE CH~RA~ERISTICS~
(A) LENGTH: 4:amino acids (B3 TYPE: amino acid : :~ :. :
~C) STRAN~EDNESS:~sin~le ::
:~D~:~TOP~L0GY~::linear: :~
~ ) MOLECULE~TYPE: peptide~
(iii):~HYPOTHETICAL: ~O
(iv3 ~NTI-~ENSE: NO
~::
: ~ '.
::
~.
~.
WO~3/0182~ P~T/US92/~61~3 3~S6 "~"''`': ' (v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME/KEY: Unusual amino acids; misc. differences (B) LOCATION: 3, 4 ( D ~ O T H E R I N F O R M A T I O N : ~
Xaa3=L-~naphthylalanine(L ~-Nal); `.
~aa4=4-pip~ridine carboxylic acid (Pip~; `;
initial amino terminal Ala-~ residue is an ac:etamide; c:arboxy terminal pipecolic acid, Pip-7, is a methyl e~;ter.
~xi) SEQUENCE DESCRIPTION: SEQ ~ID No: 16 Ala Ala Xaa Xaa '`~.`? `.
(2) INFORMATIOM FOR SEQ ID NO:17 (i) SEQUENCE CHARACTERISTXCS~
~A~ L~ENGTH: 4 aminb acids ( B) TYPE:: amino acid (C~ Sl~Nl~:~D~ESS : single : ::
(D) TOPOLOGY:~ :lin~ar ~ii) M0LECULE ~YPE: peptide ~(iiL) HYPOTHETIC~L:~ NO
: :~i~3 ~NTI-SENSE:: NO~
~(~) ER~GMENT~TYP~: internal~
: : (ix):~:FEATU
N~ME/~EY:~Unusual amino acids~; misc.:~diferenc~s~
~Xaa3=I,~ naph~hyla~anin~ Nal~
~ Xa~4 :- 4,-pi~d~ine~ arboxyl~c~ :ac:id~ (Pip):~
: i~itIa~ ino:~te~mina~ a~ 'res:idue`~ an:~
acetamide~ : ,earbo~ ens:inal ~ ipecol ic ~ ~ ac~
Pip-4 / is :-~a~: hydra~ide.
xi ) SEQUENCE DE5CR3:PTION - SEQ ID No: 17 ~: ~ Ala Ala Xaa:~:~Xaa:
(2) INFORMATION FOR SEQ ID NO:18 (i) SEQUENCE:CH~RA~TERISTICS~
~ (A) LENGTH::~:4 amino:acids ~ ; : : :
:
.~:
;
,, W~g3/01%2~ P~TJUS92/~6153 ;~` 2I~3156 ~-s 1-- .
(B) TYPE: amino acid (C) STRAND~DNESS: single ~D) TOPOL~GY: linear -(ii) MOLECUiE TYP~: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI~SENSE: ~O . - -(v) FRAGMENT TYPE: internal (ix) FEATURE: :
(A~ NAN~/~EY: Unusual amino acids; misc. differences (B) LOCATION: 3, 4 ( D ) O T H E R I~N F O R M A T I O N
Xaa3~L ~-naphthylalanine(L~-Nal~
Xaa4- 4-piperidine :car~oxylic acid (Pip);
amino terminal: Ala-l ~esi~ue is a: cholic amide, carboxy terminal pipecolic acid, Pip 4 ~ :~
is a methyl ester.
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:}8 , ~.
Ala Ala Xaa Xaa ~ i.
(2) INFORMATION FOR SEQ ID NO~:l9 SEQUENCE:C~ARA~TERISTICS:~
: : ::(A) LENGTH: 4 ~mino:a~ids : :(B): TY~E:~:a~i~o acid~
: :~(C) STR~ND~DNES~ single : : ~: (D) TOPOLOGY.~linear : : (ii) MOLE~ULE~TYPE::~pept~de ~YPOTHETICA~ NO
: ~ (i~): ANTI-5ENSE::NO ~
: ~(v~ FRAGMENT TYPE: internal (ix~ FEATURE:
(A) NAME/KEY: Unusual amino acids: misc. diferences (~ LOC~TION:~3, 4 Q~T H~E~ N F O R:~M~A T:I O:~N :
~aa3=L-~ naphthylalanine(L-~-Nal); : ~ ~:
: Xaa4~4-pi~eridi~e carboxyllc a~id~: ~Pip);
ini~ial amino terminal :Ala-l Yesidue is:: a ; cholic ~amide; carboxy~ ~ter~inal pipecolic acid,Pip-4, is a hydrazide.
:~
:,, ;~
: : ~, WO93/01828 , , PCT/V$92/06153 .21'1;~1~6. ``
(xi3 SEQUENCE DESCRIPTION: SEQ ID NO;l9: ;~
Ala Ala Xaa Xaa '.' (2) INFORMATION FOR SEQ ID NO:20: ~;
(i) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: ~ amino acids /.
(B) TYPE: amino acid .
~C) STRANDEDNESS: single (D) TOPOL0GY: linear ;~
(ii) MOLE~ULE TYPE: peptide : ~ .
, (iii) HYPOTH~TICAL. N~ ~
(iv) ~NTI-SENSE: NO : ;.
(v) F~A~E~T TYPE: internal tiX) FEATU~E: ~
(A) NANE/KEY: Unu ual amino aci~s; misc. differences `:
(B) L0CATION: 8 (D) OTHER I~FORMATION: c:arboxy terminal Val-8 ~:
residue is an amide.
(Xl~ SEQUENCE~DE8CRIPTION:~SEQ ID NO:20 Val Ser Gln Asn Tyr Pro~Ile~V~
l 5 (2) INFORNATION FOR SEQ ID:NO~21 SEQUENCE~C~ RACTERI5TICS~
LE~G~ 4 a~ino aci~
TYPX:~:ami~ cid;~
(~) STRANDE~NE8S~ si~g ) TOPOL~GY: lin~ar::
(ii) MOLECULE TYPE: peptide .~
) H~POTHETIC~L: NO
: (iv):ANTI-SENSE: NO
(v)~ FRAGMENT TYPE:;~ineernal :
: (ix)~ F~ATURE~
: ~ (A) NA~E/KEY: U~usual amino acids; misc. differenc~s~
~ :(8) LOCA~ION: 3,: 4 ~ 3 : (~D~ OTHER INFORMAT~ON: Xaa=4-piperidine carboxylic ;
.:
W~93~0182X P~T/US92/~61~3 -53- ~.
acid(Pip); Phe-2 is joined to Xaa by an isosteric bond ha~ing the structure -C(O)-CH2-N ; carboxy te~minal Ile-4 is an amide. .
[xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: .
Asn Phe Xaa Ile ~.
''' ~ ,.
.~ .
( 2 ~ INFOR~TION FOR SEQ ID NO: 2 2: :
( i ) SEQUENCE CHARACTERISTICS: -(A) LENGTH:: 4 amino aGids ( B) TYPE: amino acid (C) ~ STRANDEDNESS: single - (D) TOPOLOGY: linear : ~ -( i i ) MOLECULE TYPE: peptide : ~ -( i i i ) HYPOTHETI CAL: NO
ANTIoSENSE: NO
(v) FRAGMENT~TYPE: internal (ix): FEATURE~
: (~) N~ME/ ~ Y: Unu~u 1 am:ino a~ids, mi~c~dif~erences :-~
)~Loc~TIoN:;2-3:~ 3,:~
D) OTHER~INFO~ArION: Xaa-4piperidine:;carboxylic : : i--;.
~acid~:(Pip~ Phe-2 :is~join~d:to~ Xaa~ by~-a~
ost~ c ~ lin~: ha~ing :~:~he~ : s~ruc~u~é~
~-CH2-N~ ;am~ino ~exminal~;A~n~ rasidue~is ~ a~qul~oloyl derivatiYe~ ar;box~y termina~ 4 : ~ is ~n a~ide (xi) SEQUEN~E DESCRIPTI~N~`5EQ~ID N~:22 Asn~P e~Xaa:~I~le (2) INFORMATION FOR SEQ ID NO:23 : (i) SEQUENC~C ~ CTERISTICS~
(A:)~:LENG~H:~9~amino:acids~
(B~ TYPE:~i:amino acid (~): STRANDEDNESS: single : (D) TOPOLOGY: linear (il) ~OLECULE~::TYPE::peptide:
, ~ . "
(~iii):~:HYPOTHETICAL: NO
i ;~
~''' WO93~018~8 P~T/USg2/061~3 ~li3i~6 _54_ (iv) ~NTI-SENSE: NO ~-(v) F~AG~ENT TYPE: internal (ix) FEATVRE:
(A) NAME/KEY: Untlsual amino acids misc:. differerlces (B) LOC~T3:ON: 6, 9 (D) OTHER INFORM~TION: Xaa-4-piperidine carboxylic acid lPip); carboxy t~r~inal ~ln-9 resi~ue is an amid~
~xi3 SEQUENCE DESCRIPTION: SEQ ID NO:23:
Val Ser Gln Aæn T~r Xaa Ile Val Gln l 5: -(2) INFORMATION FOR SEQ ID NO:24:~
(i) SEQUEN OE CHARACTERISTICS~
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPO~OGY: linear~
(ii) MOLE~ULE TYPE: peptide : :
(iii~ HYPOTHETICAL: NO
iv) ANTI-SENSE:::NO
(;v:) ~RAGMENT~:TYPE: internal~
(ix) FEAT~RE~
(A) N ~E/KEY: Vnusual:amino acI~ds; ~is~.~diff~rences ~: :
(D)~ OTHER I~FORM~TION~ Xaa~deca~droisoquinolIne~
ca~boxyli~:acid (:Di~ Ph~-2: i~ joined;~o Xaa~
: ~ : by~ an ~isosteri~;~lin~ ha~i~g~th~ ruc~ure~
HOH~C~2-N-;~amino~ rminal ~sn~ 2sidu~ a : ~ : quinoloyl deriYatiYe~ :carbo~y~:terminal Gln~6 (xi) SEQUENCE~DESC~IPTION:~SEQ ID NO:24 Asn Phe Xaa Ile Val;Gln :~
(2) INFORMATION FOR SEQ ID NO:25 :(i)~SEQUENCE C~ARACTERISTICS~
~ A3 LENGTH: 9:amino aci~s : ~ : : (B) TYPE: amin~ acid :: (C) STRANDEDNESS: single: : : ,~
: .
: ~ ~ : ',:,' ~::
WO 93/01828 PCr/US92/061~3 ~li315~ `
(D) TOPOLOGY: linear ~.
( ii ) ~OLECULE TYPE: peptide (iii) ~YPOTHETICAL: NO
iv) ANTI-SE~5E: NO .
(v) FRAG~:NT TYPE: internal ~.
ix ~ Fl :ATllBE ~
~A) N~E/~EY: Unusual amino acids; ~m~sc. differences ..
(B) L9CATION: 6, 9 : : ~
(D) OT~ER IN~O~TION: Xaacd~cahydroisoquinoline '-:.
c:ar~oxylic :acid: (Diq); Garboxy terminal Gln-9 reGidue i8 an amide.
(xi) ~EQUENCE DESCRIPTION: SEQ :ID NO:25:
Val Ser Gln Asn Tyr Xaa Ile Val Gln :
( 2 ) INFORMATION FOR SEQ ID NO: 2 6 (i) SEQUENCE :C~hRACTERISTICS~
(A3: ~ENGTH :~ ~7 amirlo ac:ids :(B:) T Y~ : amiYIo acid ~
(C) :: STRANDl:r~NE~S: single ~: ; ( D) TOPOL~GY: ~l inear ~ ( ii ) MO~E~UT E ~ ~YPE~ peptide (iii) HYP~TH~:TICAI.~ ;NO
(iv): ~NTI-sENSE~:; NO~
(v~ F~G~N~ ~T~PE~ internal~
~: ~ (A) N~ME/KEY- misc:~ldi:fference ::
(D) Ol~IER ~:INFOl~ATION: amia~o ter~o:inal SeE~ is a~
succinyl:: deriYative;: carboxyl ~group: ~of termina1~ ~Val-7 :~residue is in ~ amide~ form.
(xi~ SE~2UENCE~-DES~RIPTION~ SEQ: ~ID~ NO:26 ~ :Ser Gly ~Asn Tyr~ Pro ~Ile Va:l (~2 ) ~INFORMATIt:)N ~ FOR ~ SEQ ID NO: 27 . ,:
,:
:
WO 93/01,'B2# PC~/US92/061S3 ~1131~6 -56-i ) SEQUENCE C~RACTERISTICS
(A) T!~NGTH ~a amino aci.ds :`~
(B) TYPE: amino ac:id ~:
(C) STRANDEDNESS: single (D) TO~OLOGY: linear .
( ii ) MOLECULE TYPE: peptide ( iii ) HYPOTHETICAL: NO
. .. ,~ ~, ( i~) ANTI-SENSE : NO ~ ~:
(v) FRA~MENT TYPE: internal ( ix) FEATURE:
(A) N~MEJKE,Y:~ misc-difference ' ( B) LOCATION
(D) O~HER INFQRMATION: amino t~rmirlal Ser-l is a ~:
succinyl deriva~ive.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27~
Ser Gly Asn Tyr ~ ~ -(2) INFOIU5ATION FOR SEQ ID NO:28 ( i ) SEQUEN~E ~ C~9CTERISTICS
(A) LENGTEI: 4 a~ino aeids (B) TYPE:~:a~:ino acid ,~C) STRANDEDNESS: single ~ ~-~D) TOPOL~GY: linear~
(ii) MOIE'~LE';TYPE:~ pep~ide ( i i i ) ~HYPOT~ET I C~L: ~ NO~
( iv ); ;~TI -s ENSE: NO ~
(v) FRAGME'NT TYPE:~ ~:internal (A~ NAME,/~OEY: Unusual amino ac:ids; misc. dif~erent:es ( B) LO~ATION: l, 3, 4 (D) OT~E,R INFORM~TIO~ Caa=4-piperidine carboxylic :
: ~ acld (Pip), Phe is~ oined~to ~Xaa ~by~ an: ;~
lsosteric link :~ havi~g : the : structure ;~
-CH~H CH2-N-; amino:terminal:Asn 1 residue is :
a:quinoloylderiYa~ive; carboxy terminal Ile 4 ~ l5 an amide.
: (~xi)~SEQUENCE~DESCRIPTION~: SEQ ID NO:28 , , ~, ::
:- ~
WO 93/01828 . P~/US92/06153 21131~6 Asn Phe Xaa Ile .
(2) INFORNATION FOR SEQ ID U0:29:
(i) SEQUENCE CHARACTERISTICS: ~ ..
(A) LENGTH: ~ amino acids (B) ~YPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~-;
(ii) MOLECULE ~YPE: peptide ~-(iii) ~YPOTHETICAL: NO ~ -(iv) ANTI-SENSE: NO
(v) FR~GMXNT TYPE: internal :
(ixj FEATURE~
(A) ~A~E/KEY Mi~c. di~ferences (B~ ~OCATION: 1-2:,:4 ~ ~-(D) OTHER IN~O~MATION~: Phe-l i 5 ~joined to~Phe~2:by .. :`~
an isos:t~ric linkagP having ~ the~ ~ s~ ture -CHOH-CH2-N- :; carboxy terminal Ile-~ ~is an : :-amid~O~
(xi) SEQ~ENCE~DESC~IPTION: SEQ~ID NO.~29 Ph~ ~he Il2~Phe (2) INFORMATION FOR~SEQ I:D:~NO:~30 SE~UENCÉ~ ~ E~ISTICS~
( B)~ TYP~ i'no ~cid C~ S'rR;ANDEI3P~ESS~ single (D~ ~ TOPOI,OGY~ 1iDear ~oLE~ pE: p~aptide~
(iii) HYPOTHETICAL: NO
(iv~) ANTI-SEN81~ NO ;~ a`~
(v3 FRAGMENT :TYPE: inter~al : ~ ~(A) NA~EfKEY: Misc. dif:ferences: :~
(B)~ ~OCATI~N~ 2, 4 :~
(D) OTH:ER`~INFORMATION: ~Phe-1 is :joined to Phe-? ~by~
:-. . ~.
",. :, , '~
W093/01828 PCT/USg2/06153 : , 2113 1S6 _ an isosteric linkage having the structure ~~
-CHOH-C~2-~-; carboxy terminal Val-4 is an amide.
(xi~ SEQUENCE DESCRIPTION: SEQ ID NO:30:
Phe Pro Ile Val ( 2 3 INFORMP.TIO~ FOR SEQ ID NO: 31:
( i ) SEQUENCE: CHARACTERISTIC5: - `
(A~ LENGTH: 11 amino ac:~ids : ~ :
(B~ TYPE: a~ino acid (C) STRANDEI:~ESS: single (D~ TOPOLOGl~: 1inear ( ii ) MOLECWLE TYPE :; peptide (iii) HYPOTHETICAL: NO
(iY) ANTI-SENSE: NO :
(v) F~A~;~3NT TYPE~ te~nal tix) FEATURE~
;(A) NAME/XEY: Unusual:ami.no:~aci~s,~mlsc.~dif~2rènces~
: (~) ~OC~TI~N~ 7,~10,:11 ~D) OTHER INFO~MATION:~:Xaa7~ ~-N~2 p:~h~ n y~ :a~l a:~n~i n:~e t 4 ~ N ~ 2:~-~P:h e~
Xaa~lOanorleu~ine(Nle):~carboxy termiinal:Ser~
(~i); SEQUEN~E DESCRIPT~ION:~;SE~ID~NO:31.
His Lys~A~g~Ala~Val~Ru~`Xaà~Glu Ala~Xaa~Ser~
: t2~ INFO ~ TION FOR~SEQ~ID~0 32 (i) SEQUENCE~:~HARACTERISTICS:~
(A); LENGTH:~4 amino acids (B) TYPE~. :ami~o acid ::~STRANDEDNESS: single~
~Dj TOPQ ~ GY: linear (ii) MOLECULE TYPE: peptlde (ii~i)~ HYPOTHETI~AL:~NO
(iv) ~NTI-SENSE:~No ;
: ::
' :
W~9~/01828 ~T/US92/061~3 21 1315b -59~
~v) FRAGMENT TYPE: internal (ix) FEATURE:
(A~ N~ME~KEYo Misc. differences (B) LOCATION: l, 1 2, 4 (D) OTH~R INFOR~TION: amino terminal Phe-l residue is a t-butyloxycarbonyl derivative; Phe-l and Phe 2 are joined by an isosteric lin~ having ~`
the structure -C~OH-CH2N-;.carboxy ~e~minal Phe-4 is an ~mide. : -:
(xi~ SEQUE~CE DESCRIPTION: SEQ ID NO:32:: ~ ~
Phe Phe Ile Phe ~ -: ~ : ."
(2) INFORMATION FOR SE~ ID NO:33:
(i) SEQUEN~E C~ARACTERISTI~S~
(~): LENGT~I: 4 amino aci~s (B) ~YP~ amino~acid (C) STR~NDEDNESS~: single :
(D) TOPOL~GY;:~line~ar (ii) MO~ECULE TYPE:~pep~ld~
~iii)~HYPOTHE~ICAL:~NO~
: :~(iv) ANTI-SENSE:~NO~
(v):~FR~G~ENT~TYPE~:: internal ix) FE~TURE:~
NA~E/~EY~ nusua}~am~nQ aoids~ misc.~di~ferenl:es~
OT~R ~ :~amino te~mina1 A1a~ residue~
a t ~ ~:oxyà~rbo~yl derivat~ Phe~ and~
Pro-~;ar~oirOe~ an:iso~t~eri¢~ nk~ àY`in~
he~ st~ucture~: 6HO~ 2-N~ carboxy~:terminàl (xi~ SE~UENCE~DES~CRIPTIO~ SEQ~ID NO:33 Phe Pro Ile Val (2) INFORM~TI~N~:FOR~SEQ~ID;NO:34 SEQUEN~E~:~HARA~TERISTICS~
: (A)~LENGTH~ amino~a~ids ~ }-~
:~
(C~):STRAN~DNESS::~single (D) TOPOLOGY: linear : ~ : : :: : :;
:. , : ' i, ~ .
:: 1,~,' ~,~'`.
WO 93/01828 . , ~ ~ P~/US92/06153 i~1i5 6 -60- '`'""'~``
(ii) MOLECULE TYPE: peptide ( iii ) HYPO~HE~ICAL: NO :
( i~,r ) ANTI -SENSE: NO
~v) FRAG~ENT TYPE: internal (A) NA~E/KEY: Unusual ~mino acids; mis~. diff r~nc:es t B) LOCATION: l, 4 (D) I:)THER INFOR~A~ION: amino tenninal:Pheol residue is ~odi~Eied by ~uccinyl attached to a~ino group ; carboxy terminal Phe-4 is an amide . ~ ~:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34 Phe Phe Ile Phe ~ :~
( 2 ) INFORMATIO~ FOR SEQ ID NO: 3 5 ( i ) SEQUENCE CHARACTERISTICS
(A) LE~3GTH: 4 amino acid~
(8) TYPE: amino a ::id: :
~C) STRANDi:DNESS: singl~
(D) TOPOLOGY:~ linear MO~CU~ TYPE: ~ peptide ( iii~ HYPOT}IETI~L: NO
( iv ) ~ ANTI--SENS~: NO
~: (tJ) :~FR~GME~T q~YPE: int2rnal "~:..(A)~MI~E/XEY:~Misc..dif~erences ::~
:: : ~ (D~:OT~E~ INFOR~ATION~ ami~o~t~rminal Ty~-l r~sidue :: ~ is~modified:~by:~but~loxycarbonyl:attached ~o ::~ :~
: ~amino g~oup; :~ carboxy:: texminal ~ ~ly-~ i is hydroxylated~
~. ~
(~xi) SEQUENCE DESCRIPTION::SEQ ID NO:35 Tyr Pro Ile:Gly~
::
' ' ; ,;'~sj:
:
12 DCM (30 ml, 3 times) 1.5 13 Methanol (30 ml, 3 times~ 1.5 14 DCM ~30 ml, 3 times~ 1.5 Alternativ~ly, the resin was subjected to the following oper~tions: :
1. DCM (30 ml; 1 time) 1.0 2. Neat TFA [30 ml; 1 time) lo O
3 . Neat TFA (30 ml; 1 time~ 5.0 4. DCM (30 ml; 2 times) 1.0 5. DMF (3U ml; 2 times:) 1.0 6. DIEA-DMF (30 ml; I time) 1.0 7. DIEA-DMF ~30 ml; 1 time) S.0:
8. DMF (30 ml; 3 times) : 1.0 20 9. Boc-~mino acid-OH/HOBt/D~C (4 mmol each~
in DMF (20 ml; 1 time) 120.0 10. D~F (30 ml; 3 times) ~ 1.0 * Coupling reaction was carried out for~an ~ average ~of 4 hoursl as in this case, or until a~ninhydrin test:(~Kaiser:E.T.
et al., Anal.;~Bio~h~. 34:595-8~ 196~ showed a negativ@
result i~dicating the~absence of free~amino groups.:~ The s2me seq~en~e of r~aotions was ~repeated using appropriate~amino acid~ derivatives~ u~til :the~:requir~d peptide :~chain:~was~
:: 30 ~ assembled on the~resin. ~After completion~of:~:the~;synthesiæ, :
. the~resin was removed~ rom the.:vessel~and~dried under vacuum.
(HF): Peptides containin~free~carboxyl groups~or carboxamid:: 35 ~unction at the~ C-terminal are prepared~by: trea~ing~:~:the corresponding benzyl ester;~inked~or~4-methylbenzhyd~yl;~ine~
linked peptide resins according to the fo1lowing pro~edure.
The~dried peptide~rssin (l g)~, anisole (1 ml)and p~cresol.(O.l~
g) were;placed~in~a Bel:~F:reaction v~ssel.~ The~:vessel was ~:
placed in a~bath of:liquid ni*rogen ànd anhydrous~HF (15~ml)~
was condensed into~: the~ ~e:s~el. The: reaction :mixture~ was : - ~ :stirred at -10~C~for l:hour:and~HF was removed by evaporation - under vacuum.: The residue was triturated with dry ether~(50 W093/01~28 PCT/U~92/~61~3 ~1131S6 3~
ml), filtered and washed with additional ~uantity of ether (3 x 50 ml~. Peptide product in the mixture was isolated by extractin~ with glacial acetic acid (3 x 50 ml) followed 3:~y lyophilization of the solvent.
C. Preparation of peptide _ methyL__ esters bY
transesterifacation: Boc-peptidyl-b . nzyl ester resin (1 g) was s~irred with me~hanol~10 ml) and triethylamine (1 ml~ for 18 hours at room temperature . The resin was f iltered and washed three times with methanol (20 ml each time) and the combined ~iltrate was evaporated to provide peptide methyl esters. Some of the peptides prepared by this method contain protecting groups for other functional groups whlch are very conveniently depro~ected by trea~ment with liquid HF as described above. The resulting products w~re purified as described below.
D. razides:: ~oc-peptidyl-benzyl ester resin (1 g) was stirred wit~ methanol~10 ml~ and anhydrou~ hydrazine (1 ml~ for 18 hours at room te~p~rature.
The resin was filtsred and washed three times with methanol (20 ml each time) and t~e co~ ined filtrat~ was evaporated to pro~ide peptîde hydrazides esters. Some of the peptide prepared by this method contain protecting groups :for other functional groups which are very conveniently deprot~cted by - ~::- tr@atment~.with-.liguid HF as described:aboYe~ The resulting ~roducts wer~ purified :8~ described belowu E~ ~ Peptide powder obtained above (200 mg) was dissolved in lN acetic acid (3 mL), loaded to a Sephadex G 25 (superfine) column tl.5 cm x lO0 cm) and eluted with lN acetic acid. The eluent fractions; containing the peptide were pooled and freeze dried. The resulting pepti~e ~50 mg) was further purified by preparative reverse phase high performance chromatography (RP HPLC) usin~ a Waters C-4 column and a buffer gradient of 0.1% TFA in water to 70% acetonitrile in o.1~ TFA in water. The fractions containing pure peptide WO93/018~8 PCT/US92/06153 ~, 211;33L'5;6i (determined by analytical ~LC) were combined and the product isolated by lyophilization. Purity of the p~ptide was better than 95% by HPLC: and amino acid analysis followed by acid hydrolysis (6N H~l, 110 C, 24 hr) gave expected amino acid ratios.
PeptidPs of the foregoing examples listed below were prepar~d by the solid phase method.
E2aNP~g 27-01 Ser-Gln-Asn-Phe-Pro-Ile-Val-NH2 ;~
~PL~ 27-02 Ser-Gln-Asn-~yr-Pro-Ile-Val-NH2 E~AMPLE 27-03 Ser-Gln-Asn-Tyr-Achx-Ile-Val-NHz EX~P~ 27-04 Ser-Gln-Asn-Tyr-Acpr-Ile-Val-NH2 ~AN~E 27-05 Ser-~ln-Asn~Tyr-~cpnt-Ile--Val-NH2 EXA~P~E 27-06 Thr-Ile-Leu-(bet2=Ala) Leu-Gln-Arg-N~2 ~MP~E 27-07 Ser-Gln-Asn-Tyr-Pxo-Ile Valal Thr Leu-A~a Thr-Q~Ar~-NH2 : :.
E~MP~E 27-15 Ac-A~a ~la-(D-a-Nal)-Pip-~a-~O~)-~eu)-Val-NH2 E~MPLE 27-16 Ac-A}a-Ala-Phe-Pi]p ~a-(OH)-Leu3-Val-NH2 E2~PLE 27-17 Ac-Ala Ala-(DL-Phe(4-Cl))-Pip-(z-(OH)-LeU) Val-NH2~ . ~
E~A~P~E 27-18 Ala-Ala-Phe-(b~a-Ala)-Val-~al-Gly-OH ~ :
EXA~P~E 27019 Ala Ala-Phe-(~eta--~la)-Nva-Val-Gly-OH
. EX~P~E 27-20 Ala-Ala-Phe-(beta-Ala)-(a-(OH)-iso-Valeryl)-~ Val-G~y-OH~
EXA~PL~ 27-21 ~Ala-AlaoPhe ~bata~Ala)~-~al-Val-Gly O~e~
XAMPL~ 27~`~2 Ala-Ala-Phe-(b~ta-Ala~Nv~-Val-Gly-OMe L~ 27-23 Al~-Ala-Phe-~beta-Ala)-~a-~OH)-i~o-Va:leryl) VaI-Gly-O~e :~
E~AMP~ 27-24 Boc-Ala Ala-P~e-(beta Ala)~N~a-Gly-O~e EXA~PLE 27-25 (a,e-dimyris~oyl-K) dP-Ava-Ala-Ala-Phe-Ava- `~
Val-Gly-OMe EXAN~E 27-26 ~c-Ala-~la-~D-a-Nal~-Pip-o~e ~XA~LE 27-27 Ac-Ala-Ala(D a-Nal)-Pip-NHNH2 :
Æ~MP~E 27-28 Ac-Ala-Ala-~L-a-Nal)-Pip OMe EX~M~E 27 29 Ac-Ala-Ala-(L a-Nal)-Pip-NHNH2 EXAMP~E 27-30 Ac Ala-Ala-(D-b Nal)-Pip-OMe .
W093/0182X ~ 1 13 1~ ~ PCT/US92/06153 . . , , , .~ .
! -38 E~AMPLE 27-31 Ac-Ala-Ala~(D-b-Nal)-Pip-NHNH2 E~M~LE 27-32 Chl-Ala-Ala-(L-a-Nal)-Pip-OMe EXA~PLE 27-33 Chl-Ala-Ala-(L-a-Nal)-Pip-NHNH2 EX~MP~E 2.7-34 Paa-Ala ~la(D-a-Nal)-Pip-OMe EX~MPLE 27-36 Ac-Ala-Ala-(DL-Phe(4-Cl~)-Pip-OH
EX~MPLE 27-38 Val-Ser-Gln-Asn-Tyr-Pro~ Val-NH2 E~AMPL~ 27-39 Val-Ser-Gln-Asn-(D a-Nal)-Pip-(a-(OH)-Leu3-Va,l -NH2 E~AMPLE 27-40 iBoc-(D-Ph )-(D-a~Nal)~Pip-(a-(OH)-Leu) Val-EX~NPLE 27-41 (iBoc-(D-Phe)-Leu-) 2- (3,5-di-Aba)-Pro-Leu ~D-Ph~)-NH2 EXAMPLE 27~42 (Myristoyl-(D-Phe)-Leu-) 2-( 3,5-di-Aba~-Pro~
Leu-(D Phe)-NH2 EXANPLE 27-43 iBoc-(3,5-di~Aba)-(D-a-Nal)-Pip-~a-(OH) Leu)-~al-NH2 EX~PL~ 27-46 Pro-Ile~Val-NH2 EXANP~E 27-47 Ser-Gln-A~n-Tyr ~XAMP~E 27~48 Asn-Phe(CO-~HzN)P.ip-Ile NH2 EX~MP~ 27-49 Quinaldoyl-A~n-Phe;(CO~CH2N)Pip-Ile;NH2 SX~X~ 27-S0 Val-Ser-Gln-Asn-Tyr-Pip~ Val-Gln-NH2 EX~E 27-51 Quinaldoyl-Asn-Phe(CHOH-CH~)Diq-Ile-Val-Gln-NH2 EXANPLE 27-52 Val-Ser-Gln-Asn-Tyr-Diq-Ile-Val-Gln-NH2 EX~P~E 27-53 Boc-Asn-Phe(CO 2N)Diq-NHtBu~
EX~M~E 27-54 Su~cinyl-5er-Gln~As~-Tyr-Pro-Ile-Val NH2~ :
` EX~P~E 2~-55~Succinyl-Ser-&ln-Asn-Tyr~
EX~P~E-27-56~Quinaldoyl-~sn-Phe:(C~OH H2N~Pip-T~e-NH2 ; EXa~L~ 27-60 Phe(CH~H-CH2N)Phe-Ile-Phe-NH2: -~ :
EX~MP~E 27-61 Phe(CHOH-CH2N)Pro-Ile-Val-NH2: : :
EXA~P~E 27-62 His-Lys-Arg-Ala-Val-Leu-Phe(4-NO2)-Glu~Ala-Nle~Ser-NH2 EXAMP~E 27-69 [D~Phej~-[D-a Nalj-Pip-[L-a-(OH)-Leu]-Val-NH2 EXAMP~E 27-70 Boc-Phe(CHOH-CHzN)Phe Ile-Phe-NH2 EXAMPLE 27-71 B~c-Phe(CHOH-CH2N)Pro-Ile Val-NH2 EXAMPLE 27-74 :Succlny}-Phe(CHOH-CH2N~Phe-Ile~Phe-NH2 EX~MPLE 27-77 [D-Phe]-tD-b-Nal] Pip-~L-a-(OH)-Leu]-Val-NH2 EXA~PLE 27-79 iBoc-Tyr-Pro-Ile-~ly-OH
~39~ ~113i5~
EXAMP~E 27-80 iBoc-[D-Phe]-[D-a-Mal~-Pip~L-a (OH) Leu]-Val-OH
E~ANPL~ 27-8l iBoc-Phe~D-b-Nal~-Pip-[L-a-(OH)-Leu]-Val-NH2 E~ANPL~ 27-~2 iBoc-Phe-~D-b-Nal]-Pip-[L-a (OH)-LRu]-Val-OH
S ~NPLE 27-83 IBoc;~Phe(4-Br)]-~D-b-Nal]-Pip-~Lraw(OH)-Leu]
Val-OH
General procedure for the coup~_nq of DMPE-SUCCINIC ACI~ or DPPE-SUCCINIC ACID or D~PE~;SUCCINIC ACID tl mmol) and the required peptide having a ~ree amine function t1 mmol) we~e dissol~ed in DMF (5 ml) and DCC (3 mmol) followed by 1-hydroxybenzotriazole ~HOBt; Aldrich Chemical Co., HOBt, MW:
153; 450 mg, 3 mmol~ were added. The r~action mixture was stirred for 24 hours at room temperature and the product was purifisd by silica gel chromatography as described in Example 1 . .
This proc~dure was u~ed to prepare the following peptides:
E ~ P~E ~8A: DPPE-Suc-~la-Ala-(D-~-Nal) Pip-O~e0 ~a~ 288: DPPE-Succinyl-Val~Ser~Gln-Asn-Tyr-Pip-Ile-Val-Gln-NH2 E~AMPL~ 28C: D~PE-Succinyl-Val-Ser-Gln-Asn-Tyr-Pip-Ile-Val-Gln-N~2 E~MP~R 28D: DMPE-Su~cinyl-Val-Ser-Gln-Asn-Tyr-Diq-Ile-Val- ~ ;
~ Gln-NH2 X~ E<~28E:.DPPE-Succiny1-CD-Ph~]-~D-a-Nal~ Pip- E L-a-(OH)- :
Leu]-Va1-NH2 k~aV~ 28F: DPPE-Succinyl-Phe(CHOH~CH2N)Phe-I1e Phe-NH2 ~ ;
XA~P~ 28-: DPPE-Succinyl Phe-O-CH2-CHOH
NNz-Phe-O-CH2-CHOH
~MP~ 2g :
AssaY For The Reduction o:f_HIV Sy~ytia Formed In HT4-6C CQlls The syncytia~reduction assay as described~by La~der~ B.
et al~, Science 243:1731-1734(1989) was used to measure the antiviral effect of protease inhibitor. A HeLa cell line, HT4-6C, expressing the human OD4 receptor on its surface were WO93/01~28 PCT/US92/061S3 ~ 113 15 6 .~;'?~, .
trypsinized with 0.25% trypsin for 5 min~ Cells were centrifug~d to remove the residual tr~psin, and the cell pellet was resuspended in DMEM with 10% FCS. HeL~ c~lls were plated in 96 well plate (lx105 cells/well) overnight. C~
cultures ware infected with ~IV (approx. 100 PFU/well) for one hour. The infected cells were then prepared in stock solution~, there wer~ then two fold dilut~d in 2% DMEM wi~h 0.5% methylcellulose. 100 ~1 of ea~h dilu~ed antiviral agent is added into each well o~ HIV in~ec~ed cells. The treated cell cultures were incuba~ed in 37C C2 incubator for 24 hours. Plate of HIV-infected cell culture~ were fixed with methanol and stained wi~h 1% crystal violet for 10 min., the dye is rinsed off with tap water. Plate is dried and syncytia were counted. The antiviral effect of pro~ease inhibitor is c~lculated by 50% syncytia reduction.
Anti~iral activities for the compounds of the pre~ent invention are summarized in the following table. :
,:
~o EXAMP~ 27 30: ~ >100 ~M~
Ac-Ala-Ala-(D-b-Nal)-pip-oMe PLE 28A: 10 ~M
DPPE-Suc-Ala-Ala (D-b-Nal)-Pip-OMe ~ ~
E~M~LE 27-40~ 0 ~M ~i Boc-~D-Phe~j-(D-a-Nal~-Pip-:(a7(OH)-Leu)-Val~-NH
~XA~LB 28E~ 2 ~
DPPE-Succinyl - [ D-Phe ] - ~ D-a Nal ~ Pip- [ L-a ~ i ( OH j -~eu ] -Va1 -NHz PI.E: 24 ~
3~ (8c-Phe-o-~2-cHo~)2 ~XA~P~E 25: :3 ~M
(Boc-Val-Phe-~O-CH2-CHOH)2 ~ -~.
...
WO93/01828 . PCT/~S92/0~1~3 SEQU~NCE LISTING
(1) GENE~AL INFORMATION:
(i) APPLICANT: Basava, ~han~a Hostel r, Karl Y.
(ii) TITLE OF INVENTION: LIPID CONJUGATES..OF THERAPEUTIC
PEPTIDES AND PROTEASE INHIBITORS
NUMBER OF SEQUENCES: 35 (iv) COR~ESPONDEN~E ADDRESS: :
(A) ADDRESSEEs VI~AL, I~CD
(B) STREET: 9373:Towne Centre Drive (C) CITY: San Diego, (D) STATE: Cali~ornia ~ :
(E) COUNTRY: USA
(F) ZIP: 92121 ~ :
(v) COMPUTER ~EADABhE FOR~
(A~ ~EDIUM T~PE: Floppy disk : :
< (B) COMPUTER: IB~ PC co~patible (C) OPERATING SYSTE~: PC~DOS/~S-DOS:
~ (D) SOFTWARE:~PatentIn Release #1.0,:Version #1.25 ~:
(~i) CUR~ENT APPLICATION ~TA~
(A) ~PPLICAT~ON NU~B : US 07~734,434 tB) FI~ING~;D~TE~ 3-JUL~l:991 : ~ :
(C~CLASS~FI:CATION~
(viii) ATTO~NEY~AGENT IN~ORMATION~
(A) NAME: Kir~pa~rick~ Anita M. :~
(B~ REjGISTR~TION NUMBER:~:32,617 (C) REFEREN~E/~GCKET~N~MBER::~ICAL:.019 (2) INFORMATION FOR~SEQ~ID NO~
: (~i3 SEQUENCE CHARACTERISTTCS~
(A) LENG~H: 7 amino acid ~B) TYPE: amino ac:id~
(C) STR~NDEDNESS: single ~D) TOPOLOGY: linear~
(ii)~ MOLECULE TYPE: p~ptide~
(iii) HYPOTHETICAL: NO
, (iv) ~NTI-SENSE: NO ~ : : : :
(v) FRAGMENT TYPE: internal ::
~, WO93/01828 PCT/U~92/06153 &
--42-- ~
(ix~ FEATURE:
(A) NAME/K2Y: misc-difference ~:
~B) LOCATION: 7 (D~ OTHER INFORMATION: carboxyl grQup of terminal Val-7 re idue is in amide form. : -(xi) SEQUENCE DESCRIP~ION: SEQ ID NO~
5er Gly Asn Phe Pro Ile Val l 5 ~,.
( 2 ) INFORMATION Fl:)R SEQ ID NO: 2: ~ :
( i ) SEQUENCE C~iARACTERISTICS
~A~ LENGTH: 7 amino acids (B~ TYPE: amino acid (C) 5TRANDEDNESS: single tD) TOPOL~GY: linea~r ;
(ii) MOLECULE T~PE: ~A) peptide (iii) HYPO~ETICAL: NO
(iv) ANTI-SENSE: NO
~(v)~FRA~ME~T TYPE:~internal;~
(1X~FEATURE~
(A~:NAME/XEY. misc-di~ference (~) LO~ATI~N~ 7~
(D? ~THER~INFOR~ATION;~ arboxyI group~of terminal : Val-7 residue is in:ami~e~form~
: (xij~SEQUENCE DES:CRIPTION::~SEQ~:ID M~:2 Ser Gln A n:::~yr~Pro~le;Val~
: (2)~INFO~ATION~F~R~SEQ ID NO:3 ~i) SEQUENCE:CHARA TERISTICS~
(~) LENGTH: 7 ami~o acids `
:: (8~: TYPE:~ ~mino a~:id :) STRANDEDNESS:~single~
D)~TOPOLOGY~ inear ::
OLEULE~;TYPE: peptide~
~(iii~3;HYPOTHETICAL-; NO
:~(iv) ANTI-SE~SE: NO ` :
.:
:
WO93/01828 PCT~USg2/~6153-`" ~113~L51~
~v) FRAGMENT TYPE: internal (ix) FEATURE:
(A~ NA~E/XEY: Unusual amino acids; mis~. differences (B) LOCATION: S, 7 (D) OTHER INFORMATION: Xaa=l-amino, l-cyclohexahe carboxylic acid (Achx); carbo ~1 group of terminal Val-7 residue is in amide form.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Ser Gln ~sn Tyr Xaa Ile Val (2) INFORMATION FOR SEQ ID NO:4: ' (i) SEQUENCE CH~RACTERISTXCS: `~
(A) LENGTH: 7 amino acids : :
(B:'~ TYPE: amino acid ~ :
t~) STRANDEDNEss:~sins~le (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide~
(iii) HYPOTBETlCAL: NO
~(iv) A~TI-SENSE: NO~
(v) FR~GMENT~TYPE: internal (:ix) FEATURE~
: ~: (A) N~ME~KEY: Unusual a~ino:acid~ misc. differences~
(B3~L0C~ATION~ 5,~7~
: (D~ OTHER:INFORN~TION: Xaa=~l-;amin~ cycloprop~ne càrboxyli~ aeid~N~ pr):;: :carboxyl :~r~up ..te ~ ~al V~1 7 ~es~idue~is in~amide form~
(xi)~:SEQUEN OE~DXSC~PTION~ SEQ~:~ID NO~4 Ser Gln Asn Tyr Xaa~Ile Val~
(2) INFORMATION FOR SEQ I~ NO:5:~
~(i)`SEQUENCE:~CHARACTERISTI~S~
: (A) LENG~H::~7 amin~o~ac:ids :: ;:~
(~) TYPE::amino:~a~id~
~C~ TRANDEDNESS:: singl:e~
~:- : (D) ~OPO~0GY: line r~
(il) MOLECULE~TYPE:::peptide ;~
HYPOTHETICA1: NO
,~
WO 93/01B28 PCI/U$92/06153 .., q ~ ., ~ii31SC _44 ( iv ) ANTI -SENSE: No (v) FRAGNENT TYPE: internal (ix) FEATURE: `
(A) NAME/~EY: Unusual amino acids; misc. dif~erences (B) LOCATION 5, 7 (D3 OT~ER INFORMATION: Xaa=1-amino, l~cyclopentane carboxylic: acid (Acpnt3; c:arboxyl group of terminal Val 7 r2sidue is in amide foxm.
( xi ) SEQU~:NCE DE5CRIPTION: SE52 II) NO: 5:
Ser Gln A,sn Tyr Xaa Ile: Val ( 2 ) INFORMATION FOR SEQ ID NO: 6: :
( i ) SEQUENCE CHARACTERISTICS: :
tA) LENGTH: 7 amino ac:ids tB3 TYPE: amino acid (C) ST~A21DEDNESS: sing~le ( D) TOPOLOGY: linear (ii) MOLECUI.E ~YPE: peptidç ~ :
(iii3 HYPOTHETICAL: NO
( iy) ANTI-SENSE :~ NO
: (v j ~ ~FRA~MENT: ~TYPE ~ ternal (A) N~5EfKEY~ Unusual amino~acids~;~ mi c~ d:ifferences 8~ CATION ~ 4 D):; O~IE~;;INFl)~ION~ Xa~=bAla;~ carbo~ roup ~o~
:t~r~inal P~rg~ residue :i5 in~ am de~ ~Eo~. :
~xi) SEQUEN~E~ DES~P~IPTION:~ SEQ ~ID NO.6 mr Ile ~ Leu :Xaa I,eu Gln Arg (2) IN~O~IATION: FOR SEQ ID NO:7 SEQUENCE C~ARAcTERISTICS ~
(Pa3 LEN6TH: 13 :amino acids ::
(B~ ~TY~E::~ amino acid: ~ : :
~C~ :STRANDEDNESS: single ~D) ~OPOLOGY: linear ( ii ) MOLECULE TYPE: peptide : : , ::
WO 93/018~8 PCr/VS92/061~3 :: 2`~ 6 iii ) HYPOTHETICAL: NO
( iv ) ANTI-SENSE: NO
(v) FR~GMENT TYPE: internal ( ix ) FEATURE ~
(A) NAME~KEY: Unusual amino acids; misc. differenc s ~ B) LOCATION: 10, 13 (D) OTHER INFORMATION: Xaa=S-aminoisovaleric acid(~va); carboxyl group of t~rminal Arg-13 residue is in amide ~orm.
(xi~ SEQUENCE DESCRIPTION: SEQ ~ID NO:7~
Ser Gln ~sn Tyr Pro: I1e Va1 Thr X,eu Xaa Thr G1rl Arg ~ :
5 ~ 1 0 ( 2 ) INFORMATION FOR ~ SEQ ID NO: 8: :
i ) SEQUENCE CHARACTERISTIC:S:
(A) LEN~;TH: 6 amino: acids tB~ TYPE: amino :acid (C) ST~NDEDNF,S~;: sin~le (D) TOPOLOGY: 1inear ~ : :
: ~ (ii) MOLECULE :~YPE:~ peptide ( iii:) HYPO~HETICAL: NO
(~ iv ) ~TI-SENSE: NO
( v ) F~AGNENT~ ~ TYPE: :: interna l ~
(A) NAME~KEY: ~Unusuàl~ a~in~o ;acid ;;~ misc.~ d:i~feren6:es `D) OT~ER INF~RM~TI~N~: Xaa3~ p~enylalani~e~(Phe)~
Xaa4~4-p ~ridine~`iaar~oxyl~ic~a~id~:~`ÇPip)~ Xa~5:~
hydroxyl~ucine(~`~H-Leu~ amino ~up~:of~
ini~îal ~1~ l residue:~is:acetylat~d~ ar~oxy~
: `group of terminal~ al-S;~resi~ue~ i~s ln~:~amide (xi~ SEQUENCE~DESCRIPTION: SEQ ID~NO:8 Ala Ala~:Xaa Xaa:Xaa Val (2)~INFORMATION~FOR~SEQ ID NO:9 : ~(i)~ SEQUENCE:~HAR~CTERISTICS~
, , :
, W~93/n1~2~ PCT/US92/06153 ~ 1 13 1 i 61 ~ 5~
(A) LENGTH: 7 amino acids ~B) TYPE: amino acid (C) STRANDEDNESS: ingle (D) TOPO,~OGY~ ear (ii) MOLECULE TYPE: peptide (iii) HYPOT~ET:lCAL: NO .
( iv ) ,~TI-SENSE: NO
tv~) FRA~MENT TYPE: in~ernal ( ix ) FEATURE ~
(A) NAME/XEY: Unusual amino acids; mis dif~ferences (B~ LOCAT ON: 4, 7 (D) OTHER I:NFORM~TION: ~aa:~alanine(bAla~;
terminal Gly-7 residue is~ hydroxylated (xi) SEQUEN~E DESCRIPTION: SEQ ID NO:9:
Ala Ala Phe Xaa Val Val Gly (2) INFORN~TION FOR SEQ~ N0~10~
(i) ~EQUENCE~CHAR~CTER~STI~S- -~(A) :LENGTH~ 7:amino::~a:cids ~ B3 TYP~ amino~aci~
:~C) STRA~DED~ESS:~single ~(~) TOPOL~GY~ lin~ar~
: ~ (ii) MO~ECULE~TYPE: p~ptide~
: ; (i:ii)~HYPOTHETICAL:~NO~
)' ~ G~ENT T~PE~ :in~r~
(Aj NAME/KEY~ nusual ami~o acids;:misc~ differen~s ) LOC~TI9N::4,~:.:5:,~7 ~D~ OT~ER INFO ~ TION: Xaa4=bAla; Xaa5=Nva~
carbo~y ~ terminal Gly-7 residue ~ is hydroxylate~
(xi) SEQUENCE:~DESCRIPTION::SEQ~ID~:NO:10 Ala Ala ~h~Xaa Xaa~Yal Gly ~
1 ~ 5: : :: ; ::
~: ` `:: ::: ~ : : :
~ ~ :
: :
WO93/01828 , PCT/U~92/~1~3 ~7~ ~113i~
(2) INFORMATION FOR SE4 ID NO~
(i) SEQUENCE CH~RACTERISTICS:
(A) LE~GTH: 7 amino acids (B) TYPE: amino acid (C~ STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) NYPOTHETICAL: N~
(iv~ ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (ix) FEATURE~
(A) NAME/KEY: Unusual amino acid~; misc. differences (B) L~CATION: 4, ~, 7 : ~ ~
(D) OTHER INFORM~TION: Xaa~=bAla: ~-Xaa5~ H-isov~leryl;~ carboxy terminal Gly-7 ~:
residue is hydroxylated.
(xi) S QUENCE~DESCRIPTION: SEQ ID NO~
Ala Ala Phe Xaa Xaa Val Giy :(2) INFO~ATION~FOR~SEQ~ID~NO~:12 (i3 5EQUENCE:CHARACTERISTICS:~
: (A) LENGTH:::7 amino a~:ids :
(B:) T~PE:~amino aci~
(C)~STRANDEDNESS~ s~i~gle D)~OPO~oGY:~lin MO~ ~TYPE~ pép~Ide~
(iii) HYP4THETIC~L~ NO~
iv) ~ TI-sENSE~: NO~
(Y) FRAGMENT TYPE::internal~
(ix~ FEA~ RE~
N~E/K~Y: Unusual ami~o::~acids:~misc. dif~ere~es (D) OTHER:INFORM~TI~N:~ Xaa:~alanine~(b~la~
arboxy te~rminal~Gly-?~ re~idue~ is a methyl~
(xi)~SEQUENCE DESCRIPTION: SEQ~ID~NO:12 : : :
:~
:~
3 135i~6 `, Ala Ala Phe Xaa Val Val Gly (2) INFORMATION FOR SEQ ID NO:13: .
(i) SEQUENCE CH~RACTERIS~ICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid ...
(C) STR~NDED~ESS: single (D~ TOPO~OGY: linear ( ii ) MOLECULE TYPE: peptide ~ :
r (iii) HYPOTHETICAL: NO
(iv) ANTI SENSE: NO :
(v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME/KEY: Unusual amino acids; misc. difference~
(B) LOCATION: 4, 5, 7 (D) OTHER INFO~M~TI~N: Xaa4-bAla; Xaa5=N~a c~rboxy~terminal Gly-7 ::residue is a m~thyl ~ster.
(xi) SEQUEN~E~DESCRIPTION: SEQ ID NO:13 Ala Aia Ph~ Xaa~Xaa: Yal Gly :: :~
(2) INFO~MATION FQR SEQ~ D~NO:~14 i) SEQUENCE~C~AR~TER~5TI~S~
: (A) L~NGTH~ 7~ ino acids : :~
D~D~ESS::~sin~le (D) TOPO~OGY:~linear MOLE ~ LE~TYPE:~peptide:
(iii~ HYPOTHETIC~L: NO~
(iv) ANTI-SENSE: NO
) FR~GMENT TYPE:~ internal~
(ix~ FEATU~
(A)~N~E/KEY::Unusual amino acids; misc. differences (B) LOCATION:~4,~`~5, 7 : : ,`
::
WO93/0l82g : PCTJUS92~061~3 ~113156 -4g-(D) OTHER IN~ORMATION: Xaa4=bAla;
Xaa5=~ OH~isovaleryl; carboxy terminal Gly~7 residue is a methyl ester.
( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Ala Ala Phe Xaa Xaa Val Gly . ..
(2) INFORMATI0N FOR SEQ ID NO:15:
. (i) SEQUENCE CHARACTERISTICS~
(~) LE~GTH: 6 amino acids : ,~
(B) TYPE: amino acid (C) STRANDEDNESS: single (D) T0PO~GY: linear (ii) MOLECUIE TYPE: peptide (iii) HYPOTHETICAL: NO
(i~) AN~I-SENSE: NO
(v) F~ GMENT:TYPE: i~ternal :
:
(ix) FEATU~E~
: (A~ NAME/KEY~::Unus~al aimino a~ids; misc. differences :
: (B) L~C~TIO~:: 4~: 5, 6:~
(~) o~HER~INFaRM~TIoN: Xaa4=bAla~
Xaa5=norvaline(N~a)~;~amino ~ terminal ~a~
residue is ~ modi~ied~ by :t-bu~yloxy~ar~onyl ~ :
: t~ached~ to::ami~o~:~:group; :térmi~al Gly-6 residue~is~:a~methyl~ ester.
xi)~SEQUEN~E~DEgCRIPTION:~S~Q I~NO:~15~
- ~ :.Al-a~ la Phe Xaa:~àa~Gly~ ~Y;:~ 7' (~2) INFORMATION FOR SEQ ID NQ:16 (i) SEQUENCE CH~RA~ERISTICS~
(A) LENGTH: 4:amino acids (B3 TYPE: amino acid : :~ :. :
~C) STRAN~EDNESS:~sin~le ::
:~D~:~TOP~L0GY~::linear: :~
~ ) MOLECULE~TYPE: peptide~
(iii):~HYPOTHETICAL: ~O
(iv3 ~NTI-~ENSE: NO
~::
: ~ '.
::
~.
~.
WO~3/0182~ P~T/US92/~61~3 3~S6 "~"''`': ' (v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME/KEY: Unusual amino acids; misc. differences (B) LOCATION: 3, 4 ( D ~ O T H E R I N F O R M A T I O N : ~
Xaa3=L-~naphthylalanine(L ~-Nal); `.
~aa4=4-pip~ridine carboxylic acid (Pip~; `;
initial amino terminal Ala-~ residue is an ac:etamide; c:arboxy terminal pipecolic acid, Pip-7, is a methyl e~;ter.
~xi) SEQUENCE DESCRIPTION: SEQ ~ID No: 16 Ala Ala Xaa Xaa '`~.`? `.
(2) INFORMATIOM FOR SEQ ID NO:17 (i) SEQUENCE CHARACTERISTXCS~
~A~ L~ENGTH: 4 aminb acids ( B) TYPE:: amino acid (C~ Sl~Nl~:~D~ESS : single : ::
(D) TOPOLOGY:~ :lin~ar ~ii) M0LECULE ~YPE: peptide ~(iiL) HYPOTHETIC~L:~ NO
: :~i~3 ~NTI-SENSE:: NO~
~(~) ER~GMENT~TYP~: internal~
: : (ix):~:FEATU
N~ME/~EY:~Unusual amino acids~; misc.:~diferenc~s~
~Xaa3=I,~ naph~hyla~anin~ Nal~
~ Xa~4 :- 4,-pi~d~ine~ arboxyl~c~ :ac:id~ (Pip):~
: i~itIa~ ino:~te~mina~ a~ 'res:idue`~ an:~
acetamide~ : ,earbo~ ens:inal ~ ipecol ic ~ ~ ac~
Pip-4 / is :-~a~: hydra~ide.
xi ) SEQUENCE DE5CR3:PTION - SEQ ID No: 17 ~: ~ Ala Ala Xaa:~:~Xaa:
(2) INFORMATION FOR SEQ ID NO:18 (i) SEQUENCE:CH~RA~TERISTICS~
~ (A) LENGTH::~:4 amino:acids ~ ; : : :
:
.~:
;
,, W~g3/01%2~ P~TJUS92/~6153 ;~` 2I~3156 ~-s 1-- .
(B) TYPE: amino acid (C) STRAND~DNESS: single ~D) TOPOL~GY: linear -(ii) MOLECUiE TYP~: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI~SENSE: ~O . - -(v) FRAGMENT TYPE: internal (ix) FEATURE: :
(A~ NAN~/~EY: Unusual amino acids; misc. differences (B) LOCATION: 3, 4 ( D ) O T H E R I~N F O R M A T I O N
Xaa3~L ~-naphthylalanine(L~-Nal~
Xaa4- 4-piperidine :car~oxylic acid (Pip);
amino terminal: Ala-l ~esi~ue is a: cholic amide, carboxy terminal pipecolic acid, Pip 4 ~ :~
is a methyl ester.
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:}8 , ~.
Ala Ala Xaa Xaa ~ i.
(2) INFORMATION FOR SEQ ID NO~:l9 SEQUENCE:C~ARA~TERISTICS:~
: : ::(A) LENGTH: 4 ~mino:a~ids : :(B): TY~E:~:a~i~o acid~
: :~(C) STR~ND~DNES~ single : : ~: (D) TOPOLOGY.~linear : : (ii) MOLE~ULE~TYPE::~pept~de ~YPOTHETICA~ NO
: ~ (i~): ANTI-5ENSE::NO ~
: ~(v~ FRAGMENT TYPE: internal (ix~ FEATURE:
(A) NAME/KEY: Unusual amino acids: misc. diferences (~ LOC~TION:~3, 4 Q~T H~E~ N F O R:~M~A T:I O:~N :
~aa3=L-~ naphthylalanine(L-~-Nal); : ~ ~:
: Xaa4~4-pi~eridi~e carboxyllc a~id~: ~Pip);
ini~ial amino terminal :Ala-l Yesidue is:: a ; cholic ~amide; carboxy~ ~ter~inal pipecolic acid,Pip-4, is a hydrazide.
:~
:,, ;~
: : ~, WO93/01828 , , PCT/V$92/06153 .21'1;~1~6. ``
(xi3 SEQUENCE DESCRIPTION: SEQ ID NO;l9: ;~
Ala Ala Xaa Xaa '.' (2) INFORMATION FOR SEQ ID NO:20: ~;
(i) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: ~ amino acids /.
(B) TYPE: amino acid .
~C) STRANDEDNESS: single (D) TOPOL0GY: linear ;~
(ii) MOLE~ULE TYPE: peptide : ~ .
, (iii) HYPOTH~TICAL. N~ ~
(iv) ~NTI-SENSE: NO : ;.
(v) F~A~E~T TYPE: internal tiX) FEATU~E: ~
(A) NANE/KEY: Unu ual amino aci~s; misc. differences `:
(B) L0CATION: 8 (D) OTHER I~FORMATION: c:arboxy terminal Val-8 ~:
residue is an amide.
(Xl~ SEQUENCE~DE8CRIPTION:~SEQ ID NO:20 Val Ser Gln Asn Tyr Pro~Ile~V~
l 5 (2) INFORNATION FOR SEQ ID:NO~21 SEQUENCE~C~ RACTERI5TICS~
LE~G~ 4 a~ino aci~
TYPX:~:ami~ cid;~
(~) STRANDE~NE8S~ si~g ) TOPOL~GY: lin~ar::
(ii) MOLECULE TYPE: peptide .~
) H~POTHETIC~L: NO
: (iv):ANTI-SENSE: NO
(v)~ FRAGMENT TYPE:;~ineernal :
: (ix)~ F~ATURE~
: ~ (A) NA~E/KEY: U~usual amino acids; misc. differenc~s~
~ :(8) LOCA~ION: 3,: 4 ~ 3 : (~D~ OTHER INFORMAT~ON: Xaa=4-piperidine carboxylic ;
.:
W~93~0182X P~T/US92/~61~3 -53- ~.
acid(Pip); Phe-2 is joined to Xaa by an isosteric bond ha~ing the structure -C(O)-CH2-N ; carboxy te~minal Ile-4 is an amide. .
[xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: .
Asn Phe Xaa Ile ~.
''' ~ ,.
.~ .
( 2 ~ INFOR~TION FOR SEQ ID NO: 2 2: :
( i ) SEQUENCE CHARACTERISTICS: -(A) LENGTH:: 4 amino aGids ( B) TYPE: amino acid (C) ~ STRANDEDNESS: single - (D) TOPOLOGY: linear : ~ -( i i ) MOLECULE TYPE: peptide : ~ -( i i i ) HYPOTHETI CAL: NO
ANTIoSENSE: NO
(v) FRAGMENT~TYPE: internal (ix): FEATURE~
: (~) N~ME/ ~ Y: Unu~u 1 am:ino a~ids, mi~c~dif~erences :-~
)~Loc~TIoN:;2-3:~ 3,:~
D) OTHER~INFO~ArION: Xaa-4piperidine:;carboxylic : : i--;.
~acid~:(Pip~ Phe-2 :is~join~d:to~ Xaa~ by~-a~
ost~ c ~ lin~: ha~ing :~:~he~ : s~ruc~u~é~
~-CH2-N~ ;am~ino ~exminal~;A~n~ rasidue~is ~ a~qul~oloyl derivatiYe~ ar;box~y termina~ 4 : ~ is ~n a~ide (xi) SEQUEN~E DESCRIPTI~N~`5EQ~ID N~:22 Asn~P e~Xaa:~I~le (2) INFORMATION FOR SEQ ID NO:23 : (i) SEQUENC~C ~ CTERISTICS~
(A:)~:LENG~H:~9~amino:acids~
(B~ TYPE:~i:amino acid (~): STRANDEDNESS: single : (D) TOPOLOGY: linear (il) ~OLECULE~::TYPE::peptide:
, ~ . "
(~iii):~:HYPOTHETICAL: NO
i ;~
~''' WO93~018~8 P~T/USg2/061~3 ~li3i~6 _54_ (iv) ~NTI-SENSE: NO ~-(v) F~AG~ENT TYPE: internal (ix) FEATVRE:
(A) NAME/KEY: Untlsual amino acids misc:. differerlces (B) LOC~T3:ON: 6, 9 (D) OTHER INFORM~TION: Xaa-4-piperidine carboxylic acid lPip); carboxy t~r~inal ~ln-9 resi~ue is an amid~
~xi3 SEQUENCE DESCRIPTION: SEQ ID NO:23:
Val Ser Gln Aæn T~r Xaa Ile Val Gln l 5: -(2) INFORMATION FOR SEQ ID NO:24:~
(i) SEQUEN OE CHARACTERISTICS~
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPO~OGY: linear~
(ii) MOLE~ULE TYPE: peptide : :
(iii~ HYPOTHETICAL: NO
iv) ANTI-SENSE:::NO
(;v:) ~RAGMENT~:TYPE: internal~
(ix) FEAT~RE~
(A) N ~E/KEY: Vnusual:amino acI~ds; ~is~.~diff~rences ~: :
(D)~ OTHER I~FORM~TION~ Xaa~deca~droisoquinolIne~
ca~boxyli~:acid (:Di~ Ph~-2: i~ joined;~o Xaa~
: ~ : by~ an ~isosteri~;~lin~ ha~i~g~th~ ruc~ure~
HOH~C~2-N-;~amino~ rminal ~sn~ 2sidu~ a : ~ : quinoloyl deriYatiYe~ :carbo~y~:terminal Gln~6 (xi) SEQUENCE~DESC~IPTION:~SEQ ID NO:24 Asn Phe Xaa Ile Val;Gln :~
(2) INFORMATION FOR SEQ ID NO:25 :(i)~SEQUENCE C~ARACTERISTICS~
~ A3 LENGTH: 9:amino aci~s : ~ : : (B) TYPE: amin~ acid :: (C) STRANDEDNESS: single: : : ,~
: .
: ~ ~ : ',:,' ~::
WO 93/01828 PCr/US92/061~3 ~li315~ `
(D) TOPOLOGY: linear ~.
( ii ) ~OLECULE TYPE: peptide (iii) ~YPOTHETICAL: NO
iv) ANTI-SE~5E: NO .
(v) FRAG~:NT TYPE: internal ~.
ix ~ Fl :ATllBE ~
~A) N~E/~EY: Unusual amino acids; ~m~sc. differences ..
(B) L9CATION: 6, 9 : : ~
(D) OT~ER IN~O~TION: Xaacd~cahydroisoquinoline '-:.
c:ar~oxylic :acid: (Diq); Garboxy terminal Gln-9 reGidue i8 an amide.
(xi) ~EQUENCE DESCRIPTION: SEQ :ID NO:25:
Val Ser Gln Asn Tyr Xaa Ile Val Gln :
( 2 ) INFORMATION FOR SEQ ID NO: 2 6 (i) SEQUENCE :C~hRACTERISTICS~
(A3: ~ENGTH :~ ~7 amirlo ac:ids :(B:) T Y~ : amiYIo acid ~
(C) :: STRANDl:r~NE~S: single ~: ; ( D) TOPOL~GY: ~l inear ~ ( ii ) MO~E~UT E ~ ~YPE~ peptide (iii) HYP~TH~:TICAI.~ ;NO
(iv): ~NTI-sENSE~:; NO~
(v~ F~G~N~ ~T~PE~ internal~
~: ~ (A) N~ME/KEY- misc:~ldi:fference ::
(D) Ol~IER ~:INFOl~ATION: amia~o ter~o:inal SeE~ is a~
succinyl:: deriYative;: carboxyl ~group: ~of termina1~ ~Val-7 :~residue is in ~ amide~ form.
(xi~ SE~2UENCE~-DES~RIPTION~ SEQ: ~ID~ NO:26 ~ :Ser Gly ~Asn Tyr~ Pro ~Ile Va:l (~2 ) ~INFORMATIt:)N ~ FOR ~ SEQ ID NO: 27 . ,:
,:
:
WO 93/01,'B2# PC~/US92/061S3 ~1131~6 -56-i ) SEQUENCE C~RACTERISTICS
(A) T!~NGTH ~a amino aci.ds :`~
(B) TYPE: amino ac:id ~:
(C) STRANDEDNESS: single (D) TO~OLOGY: linear .
( ii ) MOLECULE TYPE: peptide ( iii ) HYPOTHETICAL: NO
. .. ,~ ~, ( i~) ANTI-SENSE : NO ~ ~:
(v) FRA~MENT TYPE: internal ( ix) FEATURE:
(A) N~MEJKE,Y:~ misc-difference ' ( B) LOCATION
(D) O~HER INFQRMATION: amino t~rmirlal Ser-l is a ~:
succinyl deriva~ive.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27~
Ser Gly Asn Tyr ~ ~ -(2) INFOIU5ATION FOR SEQ ID NO:28 ( i ) SEQUEN~E ~ C~9CTERISTICS
(A) LENGTEI: 4 a~ino aeids (B) TYPE:~:a~:ino acid ,~C) STRANDEDNESS: single ~ ~-~D) TOPOL~GY: linear~
(ii) MOIE'~LE';TYPE:~ pep~ide ( i i i ) ~HYPOT~ET I C~L: ~ NO~
( iv ); ;~TI -s ENSE: NO ~
(v) FRAGME'NT TYPE:~ ~:internal (A~ NAME,/~OEY: Unusual amino ac:ids; misc. dif~erent:es ( B) LO~ATION: l, 3, 4 (D) OT~E,R INFORM~TIO~ Caa=4-piperidine carboxylic :
: ~ acld (Pip), Phe is~ oined~to ~Xaa ~by~ an: ;~
lsosteric link :~ havi~g : the : structure ;~
-CH~H CH2-N-; amino:terminal:Asn 1 residue is :
a:quinoloylderiYa~ive; carboxy terminal Ile 4 ~ l5 an amide.
: (~xi)~SEQUENCE~DESCRIPTION~: SEQ ID NO:28 , , ~, ::
:- ~
WO 93/01828 . P~/US92/06153 21131~6 Asn Phe Xaa Ile .
(2) INFORNATION FOR SEQ ID U0:29:
(i) SEQUENCE CHARACTERISTICS: ~ ..
(A) LENGTH: ~ amino acids (B) ~YPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~-;
(ii) MOLECULE ~YPE: peptide ~-(iii) ~YPOTHETICAL: NO ~ -(iv) ANTI-SENSE: NO
(v) FR~GMXNT TYPE: internal :
(ixj FEATURE~
(A) ~A~E/KEY Mi~c. di~ferences (B~ ~OCATION: 1-2:,:4 ~ ~-(D) OTHER IN~O~MATION~: Phe-l i 5 ~joined to~Phe~2:by .. :`~
an isos:t~ric linkagP having ~ the~ ~ s~ ture -CHOH-CH2-N- :; carboxy terminal Ile-~ ~is an : :-amid~O~
(xi) SEQ~ENCE~DESC~IPTION: SEQ~ID NO.~29 Ph~ ~he Il2~Phe (2) INFORMATION FOR~SEQ I:D:~NO:~30 SE~UENCÉ~ ~ E~ISTICS~
( B)~ TYP~ i'no ~cid C~ S'rR;ANDEI3P~ESS~ single (D~ ~ TOPOI,OGY~ 1iDear ~oLE~ pE: p~aptide~
(iii) HYPOTHETICAL: NO
(iv~) ANTI-SEN81~ NO ;~ a`~
(v3 FRAGMENT :TYPE: inter~al : ~ ~(A) NA~EfKEY: Misc. dif:ferences: :~
(B)~ ~OCATI~N~ 2, 4 :~
(D) OTH:ER`~INFORMATION: ~Phe-1 is :joined to Phe-? ~by~
:-. . ~.
",. :, , '~
W093/01828 PCT/USg2/06153 : , 2113 1S6 _ an isosteric linkage having the structure ~~
-CHOH-C~2-~-; carboxy terminal Val-4 is an amide.
(xi~ SEQUENCE DESCRIPTION: SEQ ID NO:30:
Phe Pro Ile Val ( 2 3 INFORMP.TIO~ FOR SEQ ID NO: 31:
( i ) SEQUENCE: CHARACTERISTIC5: - `
(A~ LENGTH: 11 amino ac:~ids : ~ :
(B~ TYPE: a~ino acid (C) STRANDEI:~ESS: single (D~ TOPOLOGl~: 1inear ( ii ) MOLECWLE TYPE :; peptide (iii) HYPOTHETICAL: NO
(iY) ANTI-SENSE: NO :
(v) F~A~;~3NT TYPE~ te~nal tix) FEATURE~
;(A) NAME/XEY: Unusual:ami.no:~aci~s,~mlsc.~dif~2rènces~
: (~) ~OC~TI~N~ 7,~10,:11 ~D) OTHER INFO~MATION:~:Xaa7~ ~-N~2 p:~h~ n y~ :a~l a:~n~i n:~e t 4 ~ N ~ 2:~-~P:h e~
Xaa~lOanorleu~ine(Nle):~carboxy termiinal:Ser~
(~i); SEQUEN~E DESCRIPT~ION:~;SE~ID~NO:31.
His Lys~A~g~Ala~Val~Ru~`Xaà~Glu Ala~Xaa~Ser~
: t2~ INFO ~ TION FOR~SEQ~ID~0 32 (i) SEQUENCE~:~HARACTERISTICS:~
(A); LENGTH:~4 amino acids (B) TYPE~. :ami~o acid ::~STRANDEDNESS: single~
~Dj TOPQ ~ GY: linear (ii) MOLECULE TYPE: peptlde (ii~i)~ HYPOTHETI~AL:~NO
(iv) ~NTI-SENSE:~No ;
: ::
' :
W~9~/01828 ~T/US92/061~3 21 1315b -59~
~v) FRAGMENT TYPE: internal (ix) FEATURE:
(A~ N~ME~KEYo Misc. differences (B) LOCATION: l, 1 2, 4 (D) OTH~R INFOR~TION: amino terminal Phe-l residue is a t-butyloxycarbonyl derivative; Phe-l and Phe 2 are joined by an isosteric lin~ having ~`
the structure -C~OH-CH2N-;.carboxy ~e~minal Phe-4 is an ~mide. : -:
(xi~ SEQUE~CE DESCRIPTION: SEQ ID NO:32:: ~ ~
Phe Phe Ile Phe ~ -: ~ : ."
(2) INFORMATION FOR SE~ ID NO:33:
(i) SEQUEN~E C~ARACTERISTI~S~
(~): LENGT~I: 4 amino aci~s (B) ~YP~ amino~acid (C) STR~NDEDNESS~: single :
(D) TOPOL~GY;:~line~ar (ii) MO~ECULE TYPE:~pep~ld~
~iii)~HYPOTHE~ICAL:~NO~
: :~(iv) ANTI-SENSE:~NO~
(v):~FR~G~ENT~TYPE~:: internal ix) FE~TURE:~
NA~E/~EY~ nusua}~am~nQ aoids~ misc.~di~ferenl:es~
OT~R ~ :~amino te~mina1 A1a~ residue~
a t ~ ~:oxyà~rbo~yl derivat~ Phe~ and~
Pro-~;ar~oirOe~ an:iso~t~eri¢~ nk~ àY`in~
he~ st~ucture~: 6HO~ 2-N~ carboxy~:terminàl (xi~ SE~UENCE~DES~CRIPTIO~ SEQ~ID NO:33 Phe Pro Ile Val (2) INFORM~TI~N~:FOR~SEQ~ID;NO:34 SEQUEN~E~:~HARA~TERISTICS~
: (A)~LENGTH~ amino~a~ids ~ }-~
:~
(C~):STRAN~DNESS::~single (D) TOPOLOGY: linear : ~ : : :: : :;
:. , : ' i, ~ .
:: 1,~,' ~,~'`.
WO 93/01828 . , ~ ~ P~/US92/06153 i~1i5 6 -60- '`'""'~``
(ii) MOLECULE TYPE: peptide ( iii ) HYPO~HE~ICAL: NO :
( i~,r ) ANTI -SENSE: NO
~v) FRAG~ENT TYPE: internal (A) NA~E/KEY: Unusual ~mino acids; mis~. diff r~nc:es t B) LOCATION: l, 4 (D) I:)THER INFOR~A~ION: amino tenninal:Pheol residue is ~odi~Eied by ~uccinyl attached to a~ino group ; carboxy terminal Phe-4 is an amide . ~ ~:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34 Phe Phe Ile Phe ~ :~
( 2 ) INFORMATIO~ FOR SEQ ID NO: 3 5 ( i ) SEQUENCE CHARACTERISTICS
(A) LE~3GTH: 4 amino acid~
(8) TYPE: amino a ::id: :
~C) STRANDi:DNESS: singl~
(D) TOPOLOGY:~ linear MO~CU~ TYPE: ~ peptide ( iii~ HYPOT}IETI~L: NO
( iv ) ~ ANTI--SENS~: NO
~: (tJ) :~FR~GME~T q~YPE: int2rnal "~:..(A)~MI~E/XEY:~Misc..dif~erences ::~
:: : ~ (D~:OT~E~ INFOR~ATION~ ami~o~t~rminal Ty~-l r~sidue :: ~ is~modified:~by:~but~loxycarbonyl:attached ~o ::~ :~
: ~amino g~oup; :~ carboxy:: texminal ~ ~ly-~ i is hydroxylated~
~. ~
(~xi) SEQUENCE DESCRIPTION::SEQ ID NO:35 Tyr Pro Ile:Gly~
::
' ' ; ,;'~sj:
:
Claims (29)
1. A peptide selected from the group consisting of:
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; and
2. A peptide-lipid conjugate selected from the group consisting of:
;
;
;
;
;
;
;
X-Succinyl-(D-Phe)-(D-a-Nal)-Pip-(L-a-(OH)-Leu)-Val-C;
X-Succinyl-Phe(CHOH-CH2N)Phe-Ile-Phe-C:
;
;
;
;
;
; and wherein X represents phosphatidyl-tyrosine; phosphatidyl-serine;
phosphatidyl-threonine; phosphatidyl-hydroxypoline;
1-0-alkyl-sn-glycero-3-phospho-0-tyrosine; 1-0-acyl-sn-glycero-3-phospho-0-tyrosine; 1-0-alkyl-sn-glycero-3-phospho-0-serine; 1-0-acyl-sn-glycero-3-phospho-0-serine; 1-0-alkyl-sn-glycero-3-phospho-0-hydroxyproline;
1-0-acyl-sn-glycero-3-phospho-0-hydroxyproline; 1-0-alkyl-sn-glycero-3-phospho-0-threonine;
1-0-acyl-sn-glycero-3-phospho-0-threonine;1-0-alkyl-sn-glycero-3-diphospho-0-tyrosine; 1-0-acyl-sn-glycero-3-diphospho-0-tyrosine; 1-0-alkyl-sn-glycero-3-diphospho-0-serine; 1-0-acyl-sn-glycero-3-diphospho-0-serine; 1-0-alkyl-sn-glycero-3-diphospho-0-hydroxyproline; 1-0-acyl-sn-glycero-3-diphospho-0-hydroxyproline; 1-0-alkyl-sn-glycero-3-diphospho-0-threonine; 1-0-acyl-sn-glycero-3-diphospho-0-threonine; phosphatidyl ethanolamine-Y;
phosphatidic acid, or phosphatidic acid-Z, (DMPE, DPPA, DPPE, DMPE-Y, DPPE-Y or DPPA-Z), wherein Y is HOOC-(CH2)n-COOH;
Z is NO-(CH2)n-COOH: and n = 1 to 12.
C represents H, OH, OMe, NH2, NH-R where R1 represents C1-12 alkyl, benzyl, substituted benzyl, (CH2)n-phenyl where n = 1 to 12, 2-methyl pyridyl, phosphatidyl-tyrosine, phosphatidyl-serine, phosphatidyl-threonine, phosphatidyl-hydroxyproline, 1-0-alkyl-sn-glycero-3-phospho-0-tyrosine, 1-0-acyl-sn-glycero-3-phospho-0-tyrosine, 1-0-alkyl-sn-glycero-3-phospho-0-serine, 1-0-acyl-sn-glycero-3-phospho-0-serine, 1-0-alkyl-sn-glycero-3-phospho-0-hydroxyproline; 1-0-acyl-sn-glycero-3-phospho-0-hydroxyproline; 1-0-alkyl-sn-glycero-3-phospho-0-threonine, 1-0-acyl-sn-glycero-3-phospho-0-threonine,1-0-alkyl-sn-glycero-3-diphospho-0-tyrosine, -0-acyl-sn-glycero-3-diphospho-0-tyrosine, 1-0-alkyl-sn-glycero-3-diphospho-0-serine, 1-0-acyl-sn-glycero-3-diphospho-0-serine, 1-0-alkyl-sn-glycero-3-diphospho-0-hydroxyproline, 1-0-acyl-sn-glycero-3-diphospho-0-hydroxyproline, 1-0-alkyl-sn-glycero-3-diphospho-0-threonine, 1-0-acyl-sn-glycero-3-diphospho-0-threonine, phosphatidyl ethanolamine, or phosphatidyl ethanolamine-W (DPPE-W), wherein W is H2n-(CH2)n-COOH, and n = 1 to 12.
;
;
;
;
;
;
;
X-Succinyl-(D-Phe)-(D-a-Nal)-Pip-(L-a-(OH)-Leu)-Val-C;
X-Succinyl-Phe(CHOH-CH2N)Phe-Ile-Phe-C:
;
;
;
;
;
; and wherein X represents phosphatidyl-tyrosine; phosphatidyl-serine;
phosphatidyl-threonine; phosphatidyl-hydroxypoline;
1-0-alkyl-sn-glycero-3-phospho-0-tyrosine; 1-0-acyl-sn-glycero-3-phospho-0-tyrosine; 1-0-alkyl-sn-glycero-3-phospho-0-serine; 1-0-acyl-sn-glycero-3-phospho-0-serine; 1-0-alkyl-sn-glycero-3-phospho-0-hydroxyproline;
1-0-acyl-sn-glycero-3-phospho-0-hydroxyproline; 1-0-alkyl-sn-glycero-3-phospho-0-threonine;
1-0-acyl-sn-glycero-3-phospho-0-threonine;1-0-alkyl-sn-glycero-3-diphospho-0-tyrosine; 1-0-acyl-sn-glycero-3-diphospho-0-tyrosine; 1-0-alkyl-sn-glycero-3-diphospho-0-serine; 1-0-acyl-sn-glycero-3-diphospho-0-serine; 1-0-alkyl-sn-glycero-3-diphospho-0-hydroxyproline; 1-0-acyl-sn-glycero-3-diphospho-0-hydroxyproline; 1-0-alkyl-sn-glycero-3-diphospho-0-threonine; 1-0-acyl-sn-glycero-3-diphospho-0-threonine; phosphatidyl ethanolamine-Y;
phosphatidic acid, or phosphatidic acid-Z, (DMPE, DPPA, DPPE, DMPE-Y, DPPE-Y or DPPA-Z), wherein Y is HOOC-(CH2)n-COOH;
Z is NO-(CH2)n-COOH: and n = 1 to 12.
C represents H, OH, OMe, NH2, NH-R where R1 represents C1-12 alkyl, benzyl, substituted benzyl, (CH2)n-phenyl where n = 1 to 12, 2-methyl pyridyl, phosphatidyl-tyrosine, phosphatidyl-serine, phosphatidyl-threonine, phosphatidyl-hydroxyproline, 1-0-alkyl-sn-glycero-3-phospho-0-tyrosine, 1-0-acyl-sn-glycero-3-phospho-0-tyrosine, 1-0-alkyl-sn-glycero-3-phospho-0-serine, 1-0-acyl-sn-glycero-3-phospho-0-serine, 1-0-alkyl-sn-glycero-3-phospho-0-hydroxyproline; 1-0-acyl-sn-glycero-3-phospho-0-hydroxyproline; 1-0-alkyl-sn-glycero-3-phospho-0-threonine, 1-0-acyl-sn-glycero-3-phospho-0-threonine,1-0-alkyl-sn-glycero-3-diphospho-0-tyrosine, -0-acyl-sn-glycero-3-diphospho-0-tyrosine, 1-0-alkyl-sn-glycero-3-diphospho-0-serine, 1-0-acyl-sn-glycero-3-diphospho-0-serine, 1-0-alkyl-sn-glycero-3-diphospho-0-hydroxyproline, 1-0-acyl-sn-glycero-3-diphospho-0-hydroxyproline, 1-0-alkyl-sn-glycero-3-diphospho-0-threonine, 1-0-acyl-sn-glycero-3-diphospho-0-threonine, phosphatidyl ethanolamine, or phosphatidyl ethanolamine-W (DPPE-W), wherein W is H2n-(CH2)n-COOH, and n = 1 to 12.
3. A peptide-lipid conjugate selected from the group consisting of:
5 ( S ) - X - A m i n o - 4 ( S ) - h y d r o x y - 6 - p h e n y l - 2 ( R ) -(phenylmethyl)hexanoyl-Leu-Phe-C;
5 ( S ) - X - A m i n o - 4 ( S ) - h y d r o x y - 6 - p h e n y l - 2 ( R ) -(phenylmethyl)hexanoyl-Leu-C;
5 ( S ) - X - a m i n o - 4 ( S ) - h y d r o x y - 6 - c y c l o h e x y l - 2 ( R ) -(phenylmethyl)hexanoyl-C;
5 ( S ) - X - A m i n o - 4 ( R ) - h y d r o x y - 6 - p h e n y l - 2 ( R ) -(phenylmethyl)hexanoyl-Leu-Phe-C;
5 ( S ) - X - A m i n o - 4 ( R ) - h y d r o x y - 6 - p h e n y l - 2 ( R ) -(phenylmethyl)hexanoyl-Leu-C; and 5(S)-X-amino-4(R)-hydroxy 6-cyclohexyl-2(R)-(phenylmethyl)hexanoyl-C;
5 ( S ) - X - A m i n o - 4 ( S ) - h y d r o x y - 6 - p h e n y l - 2 ( R ) -(phenylmethyl)hexanoyl-Leu-Phe-C;
5 ( S ) - X - A m i n o - 4 ( S ) - h y d r o x y - 6 - p h e n y l - 2 ( R ) -(phenylmethyl)hexanoyl-Leu-C;
5 ( S ) - X - a m i n o - 4 ( S ) - h y d r o x y - 6 - c y c l o h e x y l - 2 ( R ) -(phenylmethyl)hexanoyl-C;
5 ( S ) - X - A m i n o - 4 ( R ) - h y d r o x y - 6 - p h e n y l - 2 ( R ) -(phenylmethyl)hexanoyl-Leu-Phe-C;
5 ( S ) - X - A m i n o - 4 ( R ) - h y d r o x y - 6 - p h e n y l - 2 ( R ) -(phenylmethyl)hexanoyl-Leu-C; and 5(S)-X-amino-4(R)-hydroxy 6-cyclohexyl-2(R)-(phenylmethyl)hexanoyl-C;
4. A peptide-lipid conjugate selected from the group consisting of:
5(S)-X-Amino-4(S)-hydroxy-6-cyclcohexyl-2(R)-isopropyl;
hexanoyl-Ile-C:
5(S)-X-Amino-4(R)-hydroxy-6-cyclcohexyl-2(S)-isopropyl-hexanoyl-Ile-C;
5(S)-X-Amino-4(S)-hydroxy-6-phenyl-2(R)-isopropyl-hexanoyl-Ile-C; and 5(S)-X-Amino-4(R)-hydroxy-6-phenyl-2(S)-isopropyl-hexanoyl-Ile-C;
wherein X and C are as defined above.
5(S)-X-Amino-4(S)-hydroxy-6-cyclcohexyl-2(R)-isopropyl;
hexanoyl-Ile-C:
5(S)-X-Amino-4(R)-hydroxy-6-cyclcohexyl-2(S)-isopropyl-hexanoyl-Ile-C;
5(S)-X-Amino-4(S)-hydroxy-6-phenyl-2(R)-isopropyl-hexanoyl-Ile-C; and 5(S)-X-Amino-4(R)-hydroxy-6-phenyl-2(S)-isopropyl-hexanoyl-Ile-C;
wherein X and C are as defined above.
5. A peptide-lipid conjugate selected from the group consisting of:
(3(S)-X-Asparaginyl)-amino-2(R)-hydroxy-4-phenylbutyl-Pro-C;
(3(S)-X-Asparaginyl)-amino-2(S)-hydroxy-4-phenylbutyl-Pro-C;
(3(S)-X-Asparaginyl)-amino-2(R,S)-hydroxy-4-phenylbutyl-Pro-C;
(3(S)-X-Asparaginyl)-amino-2(R,S)-hydroxy-4-phenylbutyl-Pro-Ile-C;
(3(S)-X-Leucyl-Asparaginyl)-amino-2(R,S)-hydroxy-4-phenylbutyl-Pro-Ile-C;
(3(S)-X-Asparaginyl)-amino-2(R)-hydroxy-4-phenylbutyl-N-1,2,3,4-tetrahydro(R,S)isoquinoline carboxyl-C;
(3(S)-X-Asparaginyl)-amino-2(S)-hydroxy-4-phenylbutyl-N-1,2,3,4-tetrahydro(R,S)isoquinoline carboxyl-C;
(3(S)-X-Asparaginyl)-amino-2(R,S)-hydroxy-4-phenylbutyl-N-1,2,3,4-tetrahydro(R,S)isoquinoline carboxyl-C;
(3(S)-X-Asparaginyl)-amino-2(R,S)-hydroxy-4-phenylbutyl-N
decahydro-3(S)-isoquinoline carboxyl-C;
(3(S)-X-Asparaginyl)-amino-2(R)-hydroxy-4-phenylbutyl-N-decahydro-3(S)-isoquinoline carboxyl-C; and (3(S)-X-Asparaginyl)-amino-2(S)-hydroxy-4-phenylbutyl-N-decahydro-3(S)-isoquinoline carboxyl-C;
wherein X and C are as defined above.
(3(S)-X-Asparaginyl)-amino-2(R)-hydroxy-4-phenylbutyl-Pro-C;
(3(S)-X-Asparaginyl)-amino-2(S)-hydroxy-4-phenylbutyl-Pro-C;
(3(S)-X-Asparaginyl)-amino-2(R,S)-hydroxy-4-phenylbutyl-Pro-C;
(3(S)-X-Asparaginyl)-amino-2(R,S)-hydroxy-4-phenylbutyl-Pro-Ile-C;
(3(S)-X-Leucyl-Asparaginyl)-amino-2(R,S)-hydroxy-4-phenylbutyl-Pro-Ile-C;
(3(S)-X-Asparaginyl)-amino-2(R)-hydroxy-4-phenylbutyl-N-1,2,3,4-tetrahydro(R,S)isoquinoline carboxyl-C;
(3(S)-X-Asparaginyl)-amino-2(S)-hydroxy-4-phenylbutyl-N-1,2,3,4-tetrahydro(R,S)isoquinoline carboxyl-C;
(3(S)-X-Asparaginyl)-amino-2(R,S)-hydroxy-4-phenylbutyl-N-1,2,3,4-tetrahydro(R,S)isoquinoline carboxyl-C;
(3(S)-X-Asparaginyl)-amino-2(R,S)-hydroxy-4-phenylbutyl-N
decahydro-3(S)-isoquinoline carboxyl-C;
(3(S)-X-Asparaginyl)-amino-2(R)-hydroxy-4-phenylbutyl-N-decahydro-3(S)-isoquinoline carboxyl-C; and (3(S)-X-Asparaginyl)-amino-2(S)-hydroxy-4-phenylbutyl-N-decahydro-3(S)-isoquinoline carboxyl-C;
wherein X and C are as defined above.
6. A peptide-lipid conjugate selected from the group consisting of:
4(S)-(X-Alanyl)amino-3(S)-hydroxy-5-phenyl-pentanoyl-Val Val-C;
4(S)-(X-Alanyl-alanyl)amino-3(S)-hydroxy-5-phenyl-pentanoyl-Val-Val-C;
4(S)-(X-Seryl-alanyl-alanyl)amino-3(S)-hydroxy-5-phenyl-pentanoyl-Val-Val-C;
4(S)-(X-Alanyl)amino-3(R)-hydroxy-5-phenyl-pentanoyl-Val-Val-C;
4(S)-(X-Alanyl-alanyl)-amino-3-(R)-hydroxy-5-phenylopentanoyl-Val-Val-C; and 4(S)-(X-Seryl-alanyl-alanyl)amino-3(R)-hydroxy-5-phenyl-pentanoyl-Val-Val-C;
wherein X and C are as defined above.
4(S)-(X-Alanyl)amino-3(S)-hydroxy-5-phenyl-pentanoyl-Val Val-C;
4(S)-(X-Alanyl-alanyl)amino-3(S)-hydroxy-5-phenyl-pentanoyl-Val-Val-C;
4(S)-(X-Seryl-alanyl-alanyl)amino-3(S)-hydroxy-5-phenyl-pentanoyl-Val-Val-C;
4(S)-(X-Alanyl)amino-3(R)-hydroxy-5-phenyl-pentanoyl-Val-Val-C;
4(S)-(X-Alanyl-alanyl)-amino-3-(R)-hydroxy-5-phenylopentanoyl-Val-Val-C; and 4(S)-(X-Seryl-alanyl-alanyl)amino-3(R)-hydroxy-5-phenyl-pentanoyl-Val-Val-C;
wherein X and C are as defined above.
7. A peptide-lipid conjugate selected from the group consisting of:
X-Val-Phe-Nva-(cyclohexylmethyl(4,4,5,5-tetramethyl-1,3,2 dioxoborlan-2-yl)methylamide;
X-Val-Phe-Nva-(cyclohexylmethyl,dihydroxyboronyl)methylamide;
X-Val-(L-a-Nal)-Nva-(cyclohexylmethyl(4,4,5,5-tetramethy}-1,3,2-dioxoborlan-2-yl)methylamide;
X-Val-(L-a-Nal)-Nva-(cyclohexylmethyl,dihydroxyboronyl)methylamide;
X-Nva-(cyclohexylmethyl,dihydroxyboronyl)methylamide; and X-Val-(cyclohexylmethyl,dihydroxyboronyl)methylamide;
wherein X and C are as defined above.
X-Val-Phe-Nva-(cyclohexylmethyl(4,4,5,5-tetramethyl-1,3,2 dioxoborlan-2-yl)methylamide;
X-Val-Phe-Nva-(cyclohexylmethyl,dihydroxyboronyl)methylamide;
X-Val-(L-a-Nal)-Nva-(cyclohexylmethyl(4,4,5,5-tetramethy}-1,3,2-dioxoborlan-2-yl)methylamide;
X-Val-(L-a-Nal)-Nva-(cyclohexylmethyl,dihydroxyboronyl)methylamide;
X-Nva-(cyclohexylmethyl,dihydroxyboronyl)methylamide; and X-Val-(cyclohexylmethyl,dihydroxyboronyl)methylamide;
wherein X and C are as defined above.
8. An amino acid-phospholipid composition selected from the group consisting of phosphatidyl-tyrosine, phosphatidyl-threonine, and phosphatidyl-hydroxyproline.
9. An amino acid phospholipid composition selected from the group consisting of tyrosine diphosphate diglyceride; threonine diphosphate diglyceride; and hydroxyproline diphosphate diglyceride.
10. An intermediate compound selected from the group consisting of:
Boc-Tyr(O-Phosphate)-OSu, Boc-Ser(O-Phosphate)-OSu, Boc-Thr(O-Phosphate)-OSu, Boc-Hyp(O-Phosphate)-OSu, Fmoc-Tyr(O-Phosphate)-OBzl, Fmoc-Ser(O-Phosphate)-OBzl, Fmoc-Thr(O-Phosphate)-OBzl, and Fmoc-Hyp(O-Phosphate)-OBzl.
Boc-Tyr(O-Phosphate)-OSu, Boc-Ser(O-Phosphate)-OSu, Boc-Thr(O-Phosphate)-OSu, Boc-Hyp(O-Phosphate)-OSu, Fmoc-Tyr(O-Phosphate)-OBzl, Fmoc-Ser(O-Phosphate)-OBzl, Fmoc-Thr(O-Phosphate)-OBzl, and Fmoc-Hyp(O-Phosphate)-OBzl.
11. An intermediate compound selected from the group consisting of:
Fmoc-Tyr(O-DP-DPG)-OBzl, Fmoc-Ser(O-DP-DPG)-OBzl, Fmoc-Hyp(O-DP-DPG)-OBzl, Fmoc-Thr(O-DP-DPG)-OBzl, Boc-Tyr(O-DP-DPG)-OSu, Boc-Hyp(O-DP-DPG)-OSu and Boc-Thr(O-DP-DPG)-OSu.
Fmoc-Tyr(O-DP-DPG)-OBzl, Fmoc-Ser(O-DP-DPG)-OBzl, Fmoc-Hyp(O-DP-DPG)-OBzl, Fmoc-Thr(O-DP-DPG)-OBzl, Boc-Tyr(O-DP-DPG)-OSu, Boc-Hyp(O-DP-DPG)-OSu and Boc-Thr(O-DP-DPG)-OSu.
12. A compound selected from the group consisting of:
1-O-alkyl-sn-glycero-3-phospho-O-tyrosine, 1-O-acyl-sn-glycero-3-phospho-O-tyrosine, 1-O-alkyl-sn-glycero-3-phospho-O-serine, 1-O-acyl-sn-glycero-3-phospho-O-serine, 1-O-alkyl-sn-glycero-3-phospho-O-hydroxyproline, 1-O-acyl-sn-glycero-3-phospho-O-hydroxyproline, 1-O-alkyl-sn-glycero-3-phospho-O-threonine and 1-O-acyl-sn-glycero-3-phospho-O-threonine.
1-O-alkyl-sn-glycero-3-phospho-O-tyrosine, 1-O-acyl-sn-glycero-3-phospho-O-tyrosine, 1-O-alkyl-sn-glycero-3-phospho-O-serine, 1-O-acyl-sn-glycero-3-phospho-O-serine, 1-O-alkyl-sn-glycero-3-phospho-O-hydroxyproline, 1-O-acyl-sn-glycero-3-phospho-O-hydroxyproline, 1-O-alkyl-sn-glycero-3-phospho-O-threonine and 1-O-acyl-sn-glycero-3-phospho-O-threonine.
13. A compound selected from the group consisting of:
1-O-alkyl-sn-glycero-3-diphospho-O-tyrosine, 1-O-acyl-sn-glycero-3-diphospho-O-tyrosine, 1-O-alkyl-sn-glycero-3-diphospho-O-serine, 1-O-acyl-sn-glycero-3-diphospho-O-serine, 1-O-alkyl-sn-glycero-3-diphospho-O-hydroxyproline, 1-O-acyl-sn-glycero-3-diphospho-O-hydroxyproline1-O-alkyl-sn-glycero-3-diphospho-O-threonine, and 1-O-acyl-sn-glycero-3-diphospho-O-threonine.
1-O-alkyl-sn-glycero-3-diphospho-O-tyrosine, 1-O-acyl-sn-glycero-3-diphospho-O-tyrosine, 1-O-alkyl-sn-glycero-3-diphospho-O-serine, 1-O-acyl-sn-glycero-3-diphospho-O-serine, 1-O-alkyl-sn-glycero-3-diphospho-O-hydroxyproline, 1-O-acyl-sn-glycero-3-diphospho-O-hydroxyproline1-O-alkyl-sn-glycero-3-diphospho-O-threonine, and 1-O-acyl-sn-glycero-3-diphospho-O-threonine.
14. A compound comprising a therapeutic peptide conjugated to the amino group of linkers in Claims 8, 9, 12, or 13.
15. A compound comprising a therapeutic peptide conjugated to the carboxyl group of linkers in Claims 8, 9, 12, or 13.
16. A compound comprising a viral protease inhibitor conjugated to the amino group of linkers in Claims 8, 9, 12, or 13.
17. A compound comprising a viral protease inhibitor conjugated to the carboxyl group of linkers in Claims 8, 9, 12, or 13.
18. A method for the preparation of a lipid derivative of a peptide, comprising the steps of:
selecting a peptide having an available amino group or carboxyl group;
chemically bonding a bivalent linker group to either said amino or said carboxyl group; and chemically bonding a lipid species to said linker.
selecting a peptide having an available amino group or carboxyl group;
chemically bonding a bivalent linker group to either said amino or said carboxyl group; and chemically bonding a lipid species to said linker.
19. The method of Claim 18 wherein said peptide is a viral protease inhibitor.
20. A method for the treatment of viral disease comprising administering to a virally infected person an effective viral inhibiting amount of a viral protease inhibitory peptide linked to a lipid.
21. A method for the treatment of viral disease, comprising incorporating into a liposome a viral protease inhibiting peptide linked to a lipid, and administering an effective viral inhibitory amount of said liposomally incorporated peptide to a virally infected person.
22. The method of Claim 20 or 21 wherein said lipid-linked viral protease inhibitory peptide is administered orally.
23. The method of Claim 20 or 21 wherein said viral disease is an HIV infection.
24. A compound which is a peptide covalently linked to a lipid.
25. The compound of Claim 24, wherein said lipid is a phospholipid.
26. The compound of Claim 24, wherein said protease inhibitor and said lipid are joined by an enzymatically cleavable bond.
27. A peptide-lipid conjugate according to Claim 2 selected from the group consisting of:
iBoc-Tyr-Pro-Ile-Gly-DPPE;
DPPA-Ser-Gln-Asn-Tyr-Pro-Ile-Val-NH2;
DPPA-Ser-Gln-Asn-Tyr-Acpnt-Ile-Val-NH2;
DPPE-Succinyl-Ala-Ala-(D-b-Nal)-Pip-OMe;
DPPE-Succinyl-Val-Ser-Gln-Asn-Tyr-Pip-Ile-Val-Gln-NH2;
DMPE-Succinyl-Val-Ser-Gln-Asn-Tyr-Pip-Ile-Val-Gln-NH2;
DMPE-Succinyl-Val-Ser-Gln-Asn-Tyr-Diq-Ile-Val-Gln-NH2;
DPPE-Succinyl-(D-Phe)-(D-a-Nal)-Pip-(L-a-(OH)-Leu)-Val-NH2;
DPPE-Succinyl-Phe(CHOH-CH2N)Phe-Ile-Phe-NH2;
;
;
, X-Phe(CHOH-CH2N)Phe-Ile-Phe-NH2; and X-Phe(CHOH-CH2N)Pro-Ile-Val-NH2, wherein DMPE is dimyristoyl phophatidyl ethanolamine;
DPPE is dipalmitoyl ehtanolamine; and DPPA is dipalmitoyl phosphatidic acid; and Ser(DPP) is dipalmitoyl phosphatidyl serine;
iBoc-Tyr-Pro-Ile-Gly-DPPE;
DPPA-Ser-Gln-Asn-Tyr-Pro-Ile-Val-NH2;
DPPA-Ser-Gln-Asn-Tyr-Acpnt-Ile-Val-NH2;
DPPE-Succinyl-Ala-Ala-(D-b-Nal)-Pip-OMe;
DPPE-Succinyl-Val-Ser-Gln-Asn-Tyr-Pip-Ile-Val-Gln-NH2;
DMPE-Succinyl-Val-Ser-Gln-Asn-Tyr-Pip-Ile-Val-Gln-NH2;
DMPE-Succinyl-Val-Ser-Gln-Asn-Tyr-Diq-Ile-Val-Gln-NH2;
DPPE-Succinyl-(D-Phe)-(D-a-Nal)-Pip-(L-a-(OH)-Leu)-Val-NH2;
DPPE-Succinyl-Phe(CHOH-CH2N)Phe-Ile-Phe-NH2;
;
;
, X-Phe(CHOH-CH2N)Phe-Ile-Phe-NH2; and X-Phe(CHOH-CH2N)Pro-Ile-Val-NH2, wherein DMPE is dimyristoyl phophatidyl ethanolamine;
DPPE is dipalmitoyl ehtanolamine; and DPPA is dipalmitoyl phosphatidic acid; and Ser(DPP) is dipalmitoyl phosphatidyl serine;
28. The compound of Claim 26 wherein said peptide is a protease inhibitor.
29. The compound of Claim 24 wherein said peptide and said lipid are linked through an amino acid.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US07/734,434 US5554728A (en) | 1991-07-23 | 1991-07-23 | Lipid conjugates of therapeutic peptides and protease inhibitors |
| US734,434 | 1991-07-23 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2113156A1 true CA2113156A1 (en) | 1993-02-04 |
Family
ID=24951684
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002113156A Abandoned CA2113156A1 (en) | 1991-07-23 | 1992-07-22 | Lipid conjugates of therapeutic peptides and protease inhibitors |
Country Status (6)
| Country | Link |
|---|---|
| US (2) | US5554728A (en) |
| EP (1) | EP0596024A4 (en) |
| JP (1) | JPH07501316A (en) |
| AU (1) | AU671078B2 (en) |
| CA (1) | CA2113156A1 (en) |
| WO (1) | WO1993001828A1 (en) |
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-
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- 1991-07-23 US US07/734,434 patent/US5554728A/en not_active Expired - Lifetime
-
1992
- 1992-07-22 WO PCT/US1992/006153 patent/WO1993001828A1/en not_active Application Discontinuation
- 1992-07-22 CA CA002113156A patent/CA2113156A1/en not_active Abandoned
- 1992-07-22 AU AU24251/92A patent/AU671078B2/en not_active Ceased
- 1992-07-22 JP JP5503064A patent/JPH07501316A/en active Pending
- 1992-07-22 EP EP92917096A patent/EP0596024A4/en not_active Withdrawn
-
1995
- 1995-06-02 US US08/458,401 patent/US5804552A/en not_active Expired - Lifetime
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|---|---|
| EP0596024A1 (en) | 1994-05-11 |
| AU671078B2 (en) | 1996-08-15 |
| US5804552A (en) | 1998-09-08 |
| AU2425192A (en) | 1993-02-23 |
| US5554728A (en) | 1996-09-10 |
| EP0596024A4 (en) | 1996-07-17 |
| WO1993001828A1 (en) | 1993-02-04 |
| JPH07501316A (en) | 1995-02-09 |
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Legal Events
| Date | Code | Title | Description |
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| FZDE | Discontinued |