WO2003097635A1 - Processes for preparing 3-substituted 1-(chloromethyl)-1,2-dihydro-3h-[ring fused indol-5-yl(amine-derived)] compounds and analogues thereof, and to products obtained therefrom - Google Patents

Processes for preparing 3-substituted 1-(chloromethyl)-1,2-dihydro-3h-[ring fused indol-5-yl(amine-derived)] compounds and analogues thereof, and to products obtained therefrom Download PDF

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WO2003097635A1
WO2003097635A1 PCT/NZ2003/000094 NZ0300094W WO03097635A1 WO 2003097635 A1 WO2003097635 A1 WO 2003097635A1 NZ 0300094 W NZ0300094 W NZ 0300094W WO 03097635 A1 WO03097635 A1 WO 03097635A1
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dihydro
indole
carbonyl
benz
optionally substituted
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PCT/NZ2003/000094
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French (fr)
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William Alexander Denny
Shanjin Yang
Graham John Atwell
Scott Charles Jeffrey
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Auckland Uniservices Limited
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Priority to AU2003228173A priority Critical patent/AU2003228173A1/en
Priority to US10/514,893 priority patent/US7235578B2/en
Publication of WO2003097635A1 publication Critical patent/WO2003097635A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D401/00Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/06Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404Indoles, e.g. pindolol
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D209/00Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/56Ring systems containing three or more rings
    • C07D209/58[b]- or [c]-condensed
    • C07D209/60Naphtho [b] pyrroles; Hydrogenated naphtho [b] pyrroles
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D487/00Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04Ortho-condensed systems
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates generally to processes of preparing 3 -substituted 1- (chloromethyl)-l,2-dihydro-3H-[ring fused indol-5-yl(amine-derived)] compounds and analogues, particularly analogues substituted in the ring fused to the indole system, and the use of these compounds prepared from these processes as cytotoxins for cancer therapy and as prodrugs for gene-directed enzyme-prodrug therapy
  • GDEPT GDEPT
  • ADPT antibody-directed enzyme-prodrug therapy
  • 5-hydroxy and 5-arfiino-l,2-dihydro-3H-benzo[e]indoles are known to be very potent cytotoxins. Examples of these compounds are illustrated below as the 5-hydroxy compound A; (Boger & Johnson, Angew. Chem. Int. Ed. Engl , 1996, 35, 1438) and the 5-amino compound B; (A-twell et al., J Org. Chem., 1998, 63, 9414).
  • the present invention provides in a first aspect a method of preparing a compound of formula (I)
  • a and B together may represent a fused optionally substituted benzene, a fused optionally substituted naphthalene, a fused optionally substituted pyridine, a fused optionally substituted furan or a fused optionally substituted pyrrole ring, where the optional substituents are represented by Y; wherein Y is selected from one or more of H, a halogen, CH 3 , OR 1 , SR 1 , NR ⁇ 2 , SO2R 1 ,
  • R 1 or R 2 each independently represents H, lower Ci. 4 alkyl or a benzene ring, optionally substituted with one or more hydroxyl, NO 2 or amino groups, the amino groups being further optionally substituted with one or two C ⁇ -C 4 alkyl groups
  • X is halogen or OSO 2 R, where R represents H, lower C 1-4 alkyl or a benzene ring, optionally substituted with one or more hydroxyl, NO 2 or amino groups, the amino groups being further optionally substituted with one or two C ⁇ -C alkyl groups
  • W is selected from NO 2 , NHOH, N(R 3 ) 2 NHR 3 , NHCO 2 R 3 , N(phthaloyl) or NH 2 , where each R is selected from a C 1 alkyl group optionally substituted with one or more hydroxyl or amino groups, each amino group being optionally substituted with one or two C 1-4 alkyl groups; a C ⁇ alky
  • W is further selected from the group P , wherein J is selected from OH or R wherein R represents H, lower C 1- alkyl or a benzene ring, optionally substituted with one or more hydroxyl, NO 2 or amino groups, the amino groups being further optionally substituted with one or two C 1 -C alkyl groups, and
  • Pis a group which is a substrate suitable for a nitroreductase or carboxypeptidase enzyme such that one of said enzymes is able to bring about the removal of the group P which is selected from the following moieties:
  • each occurrence of Z' is independently H or CH 3 , n is 1 or 2
  • Ph is a phenyl moiety substituted in the moiety of (Ila) by a nitro group at the 2-position, and substituted in the moiety of (lib) by a nitro group in the 2- or 4-position
  • IMDZ is an imidazolyl moiety substituted in the moiety of (He) by a nitro group in the 2-, 4- or 5- position, and optionally further substituted by -CH 3 or Ci-C 4 alkyl at the 1 -position, wherein said alkyl is further substituted by OH
  • Phe is a phenylene (C 6 H 4 ) ring in which the group -L, which may represent O or NH, is para to the group -OCH 2
  • the Furyl, Thienyl, and Pyrrolyl groups in moieties, (Ilf), (Ilg) and (Ilh) respectively are optionally substituted by a nitro group, where
  • R represents H, lower C ⁇ . 4 alkyl or a benzene ring, optionally substituted with one or more hydroxyl or amino groups, each amino group being optionally substituted with one or two C ⁇ -C alkyl groups and where n represents 1, 2 or 3; or
  • HET may represent a 5- or 6-membered carbocycle or heterocycle containing one or two atoms independently selected fromN, O and S; or a physiologically functional derivative thereof, wherein the process for preparing a compound of formula I includes the steps of
  • R"' Halogen wherein the variables A, B, and Y are as defined above, and
  • variables A, B, Y, , . and Z are as. defined above.
  • A-B represent a fused benzo group. It is also preferred that X represents chloro.
  • Z represents one of following groups:
  • substituent -R may represent one or more substituents selected from -H, - OH, halogen, -NO 2, -NH 2, -N(C 1-4 alkyl) 2, -C 1-4 alkyl, -COOC 1 alkyl -OC 1-4 alkyl, and -NHCOC M alkyl.
  • NHCH 2 CH CH 2 , NH 2 , N(phthaloyl), NHCH 3 , N(CH 3 ) 2 , NO 2 and NHCO 2 CH 2 C 6 H 4 - v °x
  • the present invention provides compounds obtained by the processes defined above, preferably such compounds are selected from the following: N- Allyl- 1 -(chloromethyl)-3 - [(5,6,7-trimethoxy- lH-indol-2-yl)carbonyl]- 1 ,2-dihydro- IH-benzo [e]mdol-5-arnine; N-AUyl-l-(chloromethyl)-3-[(2E)-3-(4-methoxyphenyl)-2-propenoyl]-l,2-dihydro- lH-benzo [e]mdol-5-amine; l-(Cmoromethyl)-3-[(5,6,7-trimemoxy-lH-indol-2-yl)carbonyl]-l,2-dihydro-lH- benzo[e]mdol-5-arnine; l-(Chloromethyl)-3-[(2E)-3-(4-meth
  • the present invention relates to the use of the compounds obtained from one of the processes defined above as anticancer drugs.
  • the compounds so obtained are used for the selective killing of oxic and hypoxic tumor cells in methods of treatment of cancers, for example leukemias and particularly solid cancers including breast, bowel and lung tumors, including small cell lung carcinoma.
  • the present invention further relates to the use of some of the compounds obtained by the processes defined above that are suitable as substrates for nitroreductase or carboxypeptidase enzymes (for example, the aerobic NR2 nitroreductase isolated from E. coli) in methods of ADEPT and GDEPT therapy.
  • nitroreductase or carboxypeptidase enzymes for example, the aerobic NR2 nitroreductase isolated from E. coli
  • certain compounds produced by the methods of the present invention may exist in one of two different enantiomeric or diastereomeric forms. In such cases it is to be understood that the compounds prepared by the processes of the invention may represent either enantiomeric or diastereomeric form or a mixture of both.
  • halogen group or -Hal depiction used throughout the specification is to be taken as meaning a ffuoro, chloro, bromo or iodo group.
  • Physiologically functional derivatives of the compounds that are obtained by the processes defined above are to be understood as including salts, amides and esters.
  • Esters include carboxylic acid esters in which the non-carbonyl moiety of the ester grouping is selected from straight or branched chain C 1-6 alkyl, (methyl, n-propyl, n- butyl or t-butyl); or C 3-6 cyclic alkyl (e.g. cyclohexyl).
  • Salts include physiologically acceptable base salts, e.g. derived from an appropriate base, such as alkali metal (e.g. sodium), alkaline earth metal (e.g.
  • magnesium salts ammonium and NR 4 " (wherein R is Ci- 4 alkvp salts.
  • Other salts include acid addition salts, including the hydrochloride and acetate salts.
  • Amides include non-substituted and mono- and di- substituted derivatives. Such derivatives are prepared by techniques knownper se in the art of pharmacy.
  • the amino compounds of formula I can also be oxidised to the corresponding nitro compounds (such as those described in US 6,130,237) by a variety of methods, including peracetic acid (Sakaue et al, J Org. Chem., 1993, 58, 3633) or zirconium alkoxides (Kr ⁇ hn, Synthesis, 1997, 10, 1115; Krohh et al., J Frdkt. Chem. 1991, 339, 335).
  • peracetic acid Siokin, et al, J Org. Chem., 1993, 58, 3633
  • zirconium alkoxides Kr ⁇ hn, Synthesis, 1997, 10, 1115
  • Krohh et al. J Frdkt. Chem. 1991, 339, 335
  • GDEPT gene-directed enzyme prodrug therapy for cancer
  • the following GDEPT systems that are relevant to the com ⁇ nrr rlQ -nrnrl ⁇ ced ⁇ v +he ⁇ * r >ces ⁇ p ⁇ ⁇ 'f+he nresent invention include*
  • the vector for use in GDEPT therapies can be any suitable DNA or RNA vector.
  • Suitable viral vectors include those that are based upon a retrovirus. Such vectors are widely available in the art. ⁇ uber et al. (ibid) report the use of amphotropic retroviruses for the transformation of hepatoma, breast, colon or skin cells. Culver et al. (Science (1992) 256; 1550-1552) also describe the use of retroviral vectors in
  • retroviruses include rous sarcoma virus (RSN).
  • Englehardt et al. (Nature Genetics (1993) 4; 27-34) describe the use of adenovirus- based vectors in the delivery of the cystic fibrosis transmembrane conductance product (CFTR) into cells, and such adenovirus-based vectors may also be used.
  • Vectors utilising adeno virus promoter and other control sequences are of use in delivering a system according to the invention to cells in the lung, and hence useful in treating lung tumours.
  • vectors based on the Molony murine leukaemia virus are known (Ram, Z. et al., Cancer Research (1993) 53; 83-88; Dalton & Treisman, Cell (1992) 68; 597-612). These vectors contain the Murine Leukaemia virus (MLN) enhancer cloned upstream at a .beta.-globin minimal promoter. The .beta-globin 5' untranslated region up to the initiation ATG is supplied to direct efficient translation of the enzyme.
  • MSN Murine Leukaemia virus
  • Suitable promoters which can be used in vectors described above include MLN, CMN, RSN and adenovirus promoters.
  • Preferred adenovirus promoters are the adenovirus early gene promoters. Strong mammalian promoters may also be suitable.
  • An example of such a promoter is the EF-1.alpha, promoter which may be obtained by reference to Mi ⁇ xishima and ⁇ agata ((1990), Nucl. Acids Res. 18; 5322).
  • the enzyme is a non-mammalian nitroreductase enzyme, such as a bacterial nitroreductase.
  • AnE. coli nitroreductase as disclosed in WO93/08288 is particularly preferred.
  • the enzyme may be modified by standard recombinant DNA techniques, e.g. by cloning the enzyme, determining its gene sequence and altering the gene sequence by methods such as truncation, substitution, deletion or insertion of sequences for example by site-directed mutagenesis. Reference may be made to "Molecular Cloning' ' by Sambrook et al. (1989, Cold Spring Harbor) for discussion of standard recombinant DNA techniques.
  • the modification made may be any which still leaves the enzyme with the ability to reduce the nitro group of the protecting group P in the compound of formula (I) or the nitro or arnine N-oxide groups when these are represented by Y in formula (I) but alters other properties of the enzyme, for example its rate of reaction or selectivity.
  • small truncations in the N- and/or C-terminal sequence may occur as a result of the manipulations required to produce a vector in which a nucleic acid sequence encoding the enzyme is linked to the various other vector sequences.
  • the enzyme is preferably a bacterial carboxypeptidase, especially carboxypeptidase CPG2 or Pseudomonas ⁇ -glutamylhydrolas ⁇ EC3.4.22.12 (Levy, C. C. & Goldman, P., J. Biol. Chem. 242; p 2933 (1967).
  • Carboxypeptidase G2 (CPG2) is disclosed in O88/07378.
  • native CPG2 is preferred, alterations to its sequence which are amino acid substitutions, deletions or insertions (e.g. of about 1, 2, 3, 4, 5, 10 or 20 residues in each case) are also possible.
  • the alteration will be such that the enzyme retains its ability to convert a prodrug to an active drug at substantially the same rate as the native enzyme.
  • substantially the same rate will desirably be within 1 order of magnitude, and preferably from about 50-fold e.g. about 2-fold less to 2, 5 or 10 f ⁇ ld ' more.
  • the enzyme may otherwise be altered by truncation, substitution, deletion or insertion as long as the activity of the enzyme is substantially unchanged as defined above.
  • small truncations in the N- and/or C- ter inal sequence may occur as a result of the manipulations required to produce a vector in which a nucleic acid sequence encoding the enzyme is linked to a suitable promoter.
  • ADEPT antibody-directed enzyme prodrug therapy for cancer
  • an antibody directed against a tumour specific marker is linked to the nitroreductase or carboxypeptidase enzyme, which may be modified as described above.
  • the antibody may be monoclonal or polyclonal.
  • the term "antibody”, unless specified to the contrary, includes fragments of whole antibodies which retain their binding activity for a tumour target antigen. Such fragments include Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies.
  • the antibodies and fragments thereof may be humanised antibodies, e.g. as described in EP-A-239400.
  • the antibodies may be produced by conventional hybridoma techniques or, in the case of modified antibodies or fragments, by recombinant DNA technology, e.g. by the expression in a suitable host vector of a DNA construct encoding the modified antibody or fragment operably linked to a promoter.
  • suitable host cells include bacterial (e.g. E. coli), yeast, insect and mammalian.
  • the enzyme may be produced by linking a nucleic acid sequence encoding the enzyme (optionally modified as described above) to the 3' or 5' end of the sequence of the construct encoding the antibody or fragment thereof.
  • the compounds of formula (I) obtained from the processes of the present invention can be used in a method of treatment of the human or animal body.
  • Such treatment includes a method of treating the growth of neoplastic cells in a patient with neoplastic disease which comprises administering to a patient in need of treatment compounds of formula (I) of the invention, or where the compounds of formula (I) obtained are suitable as substrates as part of an ADEPT or GDEPT therapy system.
  • Neoplastic diseases include leukaemia and solid tumours such as breast, bowel and lung tumours including small cell lung carcinoma.
  • treatment includes any measure taken by the physician to alleviate the effect of the tumour on a patient.
  • effective treatment will also include any measures capable of achieving partial remission of the tumour as well as a slowing down in the rate of growth of a tumour including metastases. Such measures can be effective in prolonging and/or enhancing the quality of life and relieving the symptoms of the disease.
  • dose of the compound will be at the discretion of the physician, taking account of the condition and needs of the patient, typical doses will be in the range of from about 0.1 to 200 mg/Kg, preferably about from 10 to 100 mg/Kg per patient per day.
  • the antibody/enzyme conjugate for ADEPT can be administered simultaneously but it is often found preferable, in clinical practice, to administer the enzyme/agent conjugate before the prodrug, e.g. up to 72 hours or even 1 week before, in order to give the enzyme/agent conjugate an opportunity to localise in the region of the tumour target.
  • administer the enzyme/agent conjugate before the prodrug e.g. up to 72 hours or even 1 week before
  • conversion of the prodrug to the cytotoxic agent tends to be confined to the regions where the enzyme/agent conjugate is localised, i.e. the region of the target tumour the premature release of the toxic fragment of formula (I) is minimised.
  • the degree of localisation of the enzyme/agent conjugate (in terms of the ratio of localized to freely circulating active conjugate) can be further enhanced using the clearance and/or inactivation systems described in WO89/10140.
  • a component may include an antibody to the enzyme component of the system which is capable of inactivating the enzyme.
  • the second component may be linked to a macromolecule such as dextran, a liposome, albumin, macroglobulin or a blood group O erythrocyte so that the second component is restrained from leaving the vascular compartment.
  • the second component may include a sufficient number of covalently bound galactose residues, or residues of other sugars such as lactose or mannose, so that it can bind the conjugate in plasma but be removed together with the conjugate from plasma by receptors for galactose or other sugars in the liver.
  • the second component should be administered and designed for use such that it will not, to any appreciable extent, enter the extravascular space of the tumour where it could inactivate localised conjugate prior to and during administration of the prodrug.
  • the dose of the prodrug and conjugate will ultimately be at the discretion of the physician, who will take into account such factors as the age, weight and condition of the patient. Suitable doses of prodrug and conjugate are given in Bagshawe et al. Antibody, Immunoconjugates, and Radiopharmaceuticals (1991), 4, 915-922.
  • a suitable dose of conjugate may be from 500 to 200,000 enzyme units/m 2 (e.g. 20,000 enzyme units/m 2 ) and a suitable dose of prodrug may be from about 0.1 to 200 mg/Kg, preferably about from 10 to 100 mg/Kg per patient per day.
  • the ADEPT system when used with nitroreductase also preferably comprises a suitable cofactor for the enzyme.
  • suitable cofactors include a riboside or ribotide of nicotinic acid or nicotinamide.
  • the antibody/enzyme conjugate may be administered by any suitable route usually used in ADEPT therapy.
  • the vectors will usually be packaged into viral particles and the particles delivered to the site of the tumour, as described in for example Ram et al. (ibid).
  • the viral particles may be modified to include an antibody, fragment thereof (including a single chain) or tumour-directed ligand to enhance targeting of the tumour.
  • the vectors may be packaged into liposomes.
  • the liposomes may be targeted to a particular tumour. This can be achieved by attaching a tumour-directed antibody to the liposome.
  • Viral particles may also be incorporated into liposomes.
  • the particles may be delivered to the tumour by any suitable means at the disposal of the physician.
  • the viral particles will be capable of selectively infecting the tumour cells.
  • selective infecting it is meant that the viral particles will primarily infect tumour cells and that the-proportion of non-tumour cells infected is such that the ' damage to non-tumour " cells by administration of a prodrug will be acceptably low, given the nature of the disease being treated. Ultimately, this will be determined by the physician.
  • Viruses for example isolated from packaging cell lines may also be adrninistered by regional perfusion or direct intratumoral direction, or direct injection into a body cavity (intracaviterial administration), for example by intraperitoneum injection.
  • the amount of virus or other vector delivered will be such as to provide a similar cellular concentration of enzyme as in the ADEPT system mentioned above.
  • the vector will be administered to the patient and then the uptake of the vector by transfected or infected (in the case of viral vectors) cells monitored, for example by recovery and analysis of a biopsy sample of targeted tissue. This may be determined b v clinical trials which involve administerin ⁇ a rano'e of trial doses to a patient and measuring the degree of infection or transfection of a target cell or tumour.
  • the amount of prodrug required will be similar to or greater than that for ADEPT systems.
  • prodrug In using a GDEPT system the prodrug will usually be administered following acimir ⁇ stration of the vector encoding an enzyme. Suitable doses of prodrug are from about 0.1 to 200 mg/Kg, preferably about from 10 to 100 mg/Kg per patient per day.

Abstract

The invention provides processes of preparing 3-substituted 1-(chloromethyl)-1,2-dihydro-3H-[ring fused indol-5-yl(amine-derived)] compounds of formula (I) and its analogues, or a physiologically functional derivative thereof, (I), wherein A and B together may represent a fused optionally substituted benzene, naphthalene, pyridine, furan or a pyrrole ring, where the optional substituents are represented by Y; X is halogen or OSO2R , and W is selected from NO2, NHOH, N(R3)2NHR3, NHCO2R3, N(phthaloyl) or NH2, or W is further selected from the group (a) , wherein J is selected from OH or R, and P is a group which is a substrate suitable for a nitroreductase or carboxypeptidase enzyme. The invention is also directed to the use of compounds of formula (I) prepared by the processes of the invention as cytotoxins for cancer therapy and as prodrugs for gene-directed enzyme-prodrug therapy (GDEPT) and antibody-directed enzyme-prodrug therapy (ADEPT).

Description

PROCESSES OF PREPARING 3-SUBSTITUTED l-(CHLOROMETHYL)-l,2- DfflYDRO-3H-[RING FUSED INDOL-5-YL(AMINE-DERιVED)] COMPOUNDS AND ANALOGUES THEREOF, AND TO PRODUCTS OBTAINED THEREFROM
The present invention relates generally to processes of preparing 3 -substituted 1- (chloromethyl)-l,2-dihydro-3H-[ring fused indol-5-yl(amine-derived)] compounds and analogues, particularly analogues substituted in the ring fused to the indole system, and the use of these compounds prepared from these processes as cytotoxins for cancer therapy and as prodrugs for gene-directed enzyme-prodrug therapy
(GDEPT) and antibody-directed enzyme-prodrug therapy (ADEPT).
Background to the invention
5-hydroxy and 5-arfiino-l,2-dihydro-3H-benzo[e]indoles are known to be very potent cytotoxins. Examples of these compounds are illustrated below as the 5-hydroxy compound A; (Boger & Johnson, Angew. Chem. Int. Ed. Engl , 1996, 35, 1438) and the 5-amino compound B; (A-twell et al., J Org. Chem., 1998, 63, 9414).
Figure imgf000002_0001
These compounds have IC50S in cell culture in the low nM range. It has also been shown that both the 5-hydroxy- (Boger et al., Tetrahedron, 1991, 47, 2661) and the 5- amino (Gieseg et al.,AntiCancer Drug Design, 1999, 14, 77) compounds bind in the minor groove of DNA and alkylate at the N3 of adenine in a highly regio- and sequence-selective manner. A number of analogues of the 5-hydroxy compounds have been reported in the literature, where the 5-hydroxy group has been protected as a carbamate. For potency to be expressed the carbamate is cleaved by rapid and non- specific enzymatic hydrolysis releasing the corresponding phenol. These compounds include carzelesin (C; Li et al., Cancer Res., 1992, 52, 4904)
Figure imgf000003_0001
and KW-2189 (D; Kobayashi et al., Cancer Res., 1994, 54, 2404).
Figure imgf000003_0002
The lability of these carbamates in plasma would be desirable for systemic release of a drug. However, in contrast to the rapid and non-specific cleavage of these carbamates, the corresponding carbamates of the 5-amino chloromethyl benzoindoles (CBIs) such as for example compound E; (Hay et al., Bioorg. Med. Chem. Lett., l-999,-l-5-,-2237)
Figure imgf000003_0003
have been found to be stable and relatively non-toxic until the nitro group is reduced by a localized enzymatic-activated step that causes the f agmentation of the cytotoxic 5-aminoCBI. The minor aerobic nitroreductase (NTR) from E. coli (Parkinson et al., J Med. Chem., 2000, 43, 3624) might for example be used for the enzymatic- activated step. The 5-amino compounds are thus of interest as potent cytotoxins
(Atwell et al., J Med. Chem., 1999, 42, 3400), and also for the formation of prodrugs for GDΕPT (Hay et al., Bioorg. Med. Chem. Lett., 1999, 15, 2237).
The reported (Atwell et al., JOrg Chem., 1998, 63, 9414) 15-step synthesis of the 5- amino compounds is from l-hydroxynaphthalene-2-carboxylic acid, employing a nitro group as a protecting group for the eventual 5-amino group and gives the 5- amino compound B in only a 3% overall yield. The synthetic scheme is not straightforward and includes some difficult synthetic steps.
It is therefore an object of the invention to provide a synthetic method that provides better yields for these 5-aminoCBI compounds, or to at least provide the public with a useful alternative.
Summary of the Invention The present invention provides in a first aspect a method of preparing a compound of formula (I)
Figure imgf000004_0001
wherein A and B together may represent a fused optionally substituted benzene, a fused optionally substituted naphthalene, a fused optionally substituted pyridine, a fused optionally substituted furan or a fused optionally substituted pyrrole ring, where the optional substituents are represented by Y; wherein Y is selected from one or more of H, a halogen, CH3, OR1, SR1, NR^2, SO2R1,
CONHR1, CN or CO2R1 where R1 or R2 each independently represents H, lower Ci. 4alkyl or a benzene ring, optionally substituted with one or more hydroxyl, NO2 or amino groups, the amino groups being further optionally substituted with one or two Cι-C4 alkyl groups, X is halogen or OSO2R, where R represents H, lower C1-4alkyl or a benzene ring, optionally substituted with one or more hydroxyl, NO2 or amino groups, the amino groups being further optionally substituted with one or two Cι-C alkyl groups, W is selected from NO2, NHOH, N(R3)2 NHR3, NHCO2R3, N(phthaloyl) or NH2, where each R is selected from a C1 alkyl group optionally substituted with one or more hydroxyl or amino groups, each amino group being optionally substituted with one or two C1-4 alkyl groups; a Cμ alkylene group optionally substituted with hydroxyl or amino groups, each a ino group being optionally substituted with one or two C1- alkyl groups; or a benzene ring, optionally substituted with one or more hydroxyl, NO2 or amino groups, the amino groups being further optionally substituted with one or two Cι-C4 alkyl groups, or
— N— J
1
W is further selected from the group P , wherein J is selected from OH or R wherein R represents H, lower C1- alkyl or a benzene ring, optionally substituted with one or more hydroxyl, NO2 or amino groups, the amino groups being further optionally substituted with one or two C1-C alkyl groups, and
Pis a group which is a substrate suitable for a nitroreductase or carboxypeptidase enzyme such that one of said enzymes is able to bring about the removal of the group P which is selected from the following moieties:
-C(O)-(CZ'2)n-Ph (Ha) .
-C(O)-O-CH2 -Ph (lib) -C(O)-NH-C(COOH)-(CH2)2-COOH (He)
-C(O)-O-CH2 -Phe-L-C(O)-NH-C(COOH)-CH2-COOH (lid)
-C(O)-O-CH2 -IMDZ (He)
-C(O)-O-CH2 -Furyl (Ilf)
-C(O)-O-CH2 -Thienyl (Hg)
-C(O)-O-CH2 -Pyrrolyl (Hh)
wherein each occurrence of Z' is independently H or CH3, n is 1 or 2, Ph is a phenyl moiety substituted in the moiety of (Ila) by a nitro group at the 2-position, and substituted in the moiety of (lib) by a nitro group in the 2- or 4-position, IMDZ is an imidazolyl moiety substituted in the moiety of (He) by a nitro group in the 2-, 4- or 5- position, and optionally further substituted by -CH3 or Ci-C4alkyl at the 1 -position, wherein said alkyl is further substituted by OH, Phe is a phenylene (C6 H4) ring in which the group -L, which may represent O or NH, is para to the group -OCH2, wherein the Furyl, Thienyl, and Pyrrolyl groups in moieties, (Ilf), (Ilg) and (Ilh) respectively are optionally substituted by a nitro group, wherein the Pyrrolyl group of (Ilh) is further optionally substituted at the 1 -position by CH3 and/or CO2CH2CH3 at the 2-position, the groups Ph and Phe, being further optionally substituted by a group R1 which is a group selected from R, CONHR, NHCOR, NHR, OR or SO2R where R represents H, lower C1-4alkyl optionally substituted by morpholino, or a benzene ring, optionally substituted with one or more hydroxyl, NO or amino groups, the amino groups being further optionally substituted with one or two Ci-C4 alkyl groups Z is selected from the following structures (la, lb, Ic or Id)
Figure imgf000006_0001
Ia lb Ic Id wherein E is selected from — N=, or -CH= , G is O, S, or H, Q is either (a) absent or independently selected from one to three of R, -OR, halogen, -NRR, -NO2, -COOR, -CONRR, -NRCOR, OP(O)(OH)2, O(CH2)nP(O)(OH)2,
(CH2)„OP(O)(OH)2, -NRCO
Figure imgf000007_0001
, or a fused > — ' group; where R represents H, lower Cι.4alkyl or a benzene ring, optionally substituted with one or more hydroxyl or amino groups, each amino group being optionally substituted with one or two Cι-C alkyl groups and where n represents 1, 2 or 3; or
(b) is an additional group of formulae (la, lb. Ic or Id);and
HET may represent a 5- or 6-membered carbocycle or heterocycle containing one or two atoms independently selected fromN, O and S; or a physiologically functional derivative thereof, wherein the process for preparing a compound of formula I includes the steps of
(a) converting the NO2 groups of a compound of formula (F) into NHBOC groups of a compound of formula (IF)
Figure imgf000007_0002
R'=OH R"=H
R'=OTf R"=Halogen
R'=Halogen
wherein the variables A, B, and Y are as defined above,
(b) converting a compound of formula (IF) by allylation of the NHBOC groups into a compound of formula .(JJF)
Figure imgf000008_0001
4: R"=H
5: R"=Halogen
wherein the variables A, B, and Y are as defined above,
(c) converting a compound of formula (IIP) into an indole derivative of formula (TV')
Figure imgf000008_0002
wherein the variables A, B, and Y are as defined above,
(d) reacting the compound of Formula (rv ') in the presence of Zn powder to give a compound of Formula (V')
Figure imgf000008_0003
R"'=Halogen wherein the variables A, B, and Y are as defined above, and
(e) reacting the NBOC and allyl groups of Formula (V') as desired to give a compound of Formula (I)
Figure imgf000009_0001
R'"=OH R'"=Halogeπ
wherein the variables A, B, Y, , .and Z are as. defined above.
In the process defined above, preferably A-B represent a fused benzo group. It is also preferred that X represents chloro.
More preferably, Z represents one of following groups:
Figure imgf000010_0001
wherein the substituent -R may represent one or more substituents selected from -H, - OH, halogen, -NO2, -NH2, -N(C1-4alkyl)2, -C1-4alkyl, -COOC1 alkyl -OC1-4alkyl, and -NHCOCMalkyl.
It is also preferred that is selected from one of the following
NHCH2CH=CH2, NH2, N(phthaloyl), NHCH3, N(CH3)2, NO2 and NHCO2CH2C6H4- v °x
In another aspect, the present invention provides compounds obtained by the processes defined above, preferably such compounds are selected from the following: N- Allyl- 1 -(chloromethyl)-3 - [(5,6,7-trimethoxy- lH-indol-2-yl)carbonyl]- 1 ,2-dihydro- IH-benzo [e]mdol-5-arnine; N-AUyl-l-(chloromethyl)-3-[(2E)-3-(4-methoxyphenyl)-2-propenoyl]-l,2-dihydro- lH-benzo [e]mdol-5-amine; l-(Cmoromethyl)-3-[(5,6,7-trimemoxy-lH-indol-2-yl)carbonyl]-l,2-dihydro-lH- benzo[e]mdol-5-arnine; l-(Chloromethyl)-3-[(2E)-3-(4-methoxyphenyl)-2-ρropenoyl]-l,2-dihydro-lH- benzo[e]mdol-5-arnine;
1 -(Chloromethyl)-5-phthalirnido-3 - [(5 ,6,7-trimethoxyindol-2-yl)carbonyl]- 1 ,2- dihydro-3H-benz[e]indole l-CMoromemyl-5-mfro-3-[5,6,7-trime oxyindol-2-yl)-carbonyl]-l,2-dihydro-3Η- benz[e]indole; l-(CMorome yl)-3-[[7-i2-(dime ylamino)ethoxy]-5-methoxyindol-2-yl]carbonyl]-
5-nitro- 1 ,2-dihydro-3H-benz[e]indole; l-(CMorome yl)-3-[[6-[2-(dime ylammo)emoxy]-5-me oxyindol-2-yl]carbonyl]-
5-nitro- 1 ,2-dihydro-3h-benz[e]indole; l-(CMorome yl)-3-[(Ε)-3-me oxycinnamoyl]-5-mfro-l,2-dihydro-3H- benz[e]indole;
(R)-l-(ChJoromemyl)-5-mtxo-3-[(556,7-frimemoxyindol-2-yl)carbonyl]-l,2-dihydro-
3H-benz[e]indole;
(S)-l-(CMorome yl)-5-m1xo-3-[(5,6,7-tιime oxyindol-2-yl)carbonyl]-l,2-dihydro- 3H-benz[e]indole;
Methyl l-(cUoromemyl)-5-mfro-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-l,2- dihydro-3H-pyrrolo [3 ,2-e]indole-7-carboxylate;
5-Ammo-l-(cMoromemyl)-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-l,2-dihyd ro-
3H-benz[e]indole; l-(C orome yl)-5-methylamino-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-l,2 - dihydro-3H-benz[e]indole;
5-A rώιo- 1 -(cUoromemyl)-3-[[7 2-dime ylammo)emoxy]-5-memoxyindol-2- yl] carbonyl] - 1 ,2-dihydro-3H-benz[e]indole;
5-Am o-l-(cMoromethyl)-3-[[6-[2-dime ylammo)emoxy]-5-me oxyindol-2- yl]carbonyl]-l,2-dihydro-3H-benz[e]indole;
5-A-tnino-l-(cMoromethyl)-3-[[7-[2-dimemylammo)emoxy]-5-me oxyindol-2- yl]carbonyl]-l,2-dihydro-3H-benz[e]indole; 5-Arnino- 1 -[(E)-4~butvrylamino- 1 -methyl-2-pyrroleacryloyl] - 1 -(chloromethyl)- 1 ,2- dihydro-3H-benz[e]indole;
5-An mo-l-(cMorome yl)-3-[(E)-3-memoxycinnamoyl]-l,2-dihydro-3H- benz[e]indole; (R)-5-Ammo-l-(cMbromemyl)-3-[(5,6,7-1xime1hoxyindol-2-yl)carbonyl]-l,2- dihydro-3 H-benz [ejindole;
(S)-5-A nmo-l-(cWoromemyl)-3-[(5,6,7-frimemoxyindol-2-yl)carbonyl]-l,2-dihydro-
3H-benz[e]indole;
Me yl 5-ammo-l-(c orome yl)-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-l,2- dihydro-3H-pyrrolo[3 ,2-e]indole-7-carboxylate;
Methyl l-(cMorome yl)-5-me ylammo-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]- l,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate;
Methyl l-(cMoromemyl)-5-dimemylammo-3-[(5,6,7-frimemoxyindol-2-yl)carbonyl]-
1 ,2-dihydro-3H-pyrrolo [3 ,2-e]indole-7-carboxylate; Methyl l-(cMoromemyl)-5-[(4-rnfroben2yloxycarbonyl)arnino]-3-[(5,6,7- trimemoxyindol-2-yl)carbonyl]-l,2-dmydro-3H-pyrrolo[3,2-e]indole-7-carboxylate; and
1 -(Chloromethyl)-5- [(4- frobenzyloxycarbonyl)amino] -3 - [(5 , 6,7-trimethoxyindol-2- yl)carbonyl] - 1 ,2-dihydro-3H-benz[e]indole; or a physiologically functional derivative thereof.
In another aspect, the present invention relates to the use of the compounds obtained from one of the processes defined above as anticancer drugs. The compounds so obtained are used for the selective killing of oxic and hypoxic tumor cells in methods of treatment of cancers, for example leukemias and particularly solid cancers including breast, bowel and lung tumors, including small cell lung carcinoma.
In a further aspect, the present invention further relates to the use of some of the compounds obtained by the processes defined above that are suitable as substrates for nitroreductase or carboxypeptidase enzymes (for example, the aerobic NR2 nitroreductase isolated from E. coli) in methods of ADEPT and GDEPT therapy. It is recognised that certain compounds produced by the methods of the present invention may exist in one of two different enantiomeric or diastereomeric forms. In such cases it is to be understood that the compounds prepared by the processes of the invention may represent either enantiomeric or diastereomeric form or a mixture of both.
A halogen group or -Hal depiction used throughout the specification is to be taken as meaning a ffuoro, chloro, bromo or iodo group.
Physiologically functional derivatives of the compounds that are obtained by the processes defined above are to be understood as including salts, amides and esters. Esters include carboxylic acid esters in which the non-carbonyl moiety of the ester grouping is selected from straight or branched chain C1-6 alkyl, (methyl, n-propyl, n- butyl or t-butyl); or C3-6 cyclic alkyl (e.g. cyclohexyl). Salts include physiologically acceptable base salts, e.g. derived from an appropriate base, such as alkali metal (e.g. sodium), alkaline earth metal (e.g. magnesium) salts, ammonium and NR4" (wherein R is Ci-4 alkvp salts. Other salts include acid addition salts, including the hydrochloride and acetate salts. Amides include non-substituted and mono- and di- substituted derivatives. Such derivatives are prepared by techniques knownper se in the art of pharmacy.
Further aspects of the present invention will become apparent from the following description given by way of example only and with reference to the accompanying synthetic schemes.
Detailed Description of the Invention
With reference to the following Scheme 1 a synthetic route to the compounds of Formula I is outlined as defined in the first aspect of the invention above.
In Scheme 1, conversion of 2,4-dinitro-l-hydroxynaphthalene (Martius Yellow; 1) to the iodide (3) is carried out by conversion to the trifluorosulfonate 2, followed by reaction with Nal. Concomitant reduction of the two nitro groups and removal of the iodine from 3 with SnCl2 then gave the 1,3-diarmnonaphthalene 4. Protection of the amines with BOC anhydride, followed by electrophilic iodination with NIS (general method of Boger et al., J Org. Chem., 2001 66, 5163), gave iodide 5. Allylation of the carbamate (NaH/allyl bromide) to give 6, followed by a Bu3SnH promoted 5-exo- trig free-radical cyclization of this with in situ TEMPO (general method of Boger et al; J Org. Chem., 2001, 66, 5163) gave 7, which was subsequently reduced (Zn/AcOH) to the alcohol 8 in quantitative yield. The alcohol was converted to the chloride 9 with PI13P/CC 1.. Acid-catalyzed deprotection (TFA) of 9 followed by regioselective coupling with 4-methoxysuccinic acid using EDCI gave the required
10a. This was deblocked by a suitable reagent, (for example, Grubb's carbene [Alcaide et al., Org. Lett., 2001, 3623] or preferably, sodium phenylsulfonate/camphorsulfonic acid/Pd(Pb.3P)4 (modification of the method of Honda et al., J. Org. Chem. 1997, 62 8932) to the required 11a. This provides a 10- step synthesis of 11a from 1, with an overall yield of 32%. A similar synthesis gave the analogue lib in 19% overall yield.
Scheme 1
Figure imgf000015_0001
(i) TFSA/E13N; (iv) NIS then TsOH; (vii) Zn powder;
(ii) Nal; (v) NaH, then CH2=CHCH2CI; (viii) Ph3P then CCI4;
(iii) SπCI2, then BOC20; (vi) TEMPO/Bu3SnH; (ix) TFA, then appropriate acid/EDCI; (x) Grubb's carbene.
Example 1: Synthesis of 10a and 10b by the process of Scheme 1.
A solution of Martius Yellow (1) (5.0 g, 21.4 mmol) in CH2C12 (100 mL) was treated with Et3N (8 mL, 57 mmol), and the resulting solution was cooled with ice-salt bath and treated dropwise with trifluoromethanesulfonic anhydride (5 mL, 28 mmol). After stirring at room temperature for 2 h, 0.5 N HC1 (100 mL) was added in one portion and the mixture was stirred for a further 30 min. The aqueous phase was separated and extracted with CH2C12 (2x50 mL). The combined organic phase was washed with sat. aq. NaHCO3, brine, and was dried and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel. Elution with CH2C12, gave 2,4-dinitro-l-naphthyl trifluoromethanesulfonate (2) (7.0 g, 89%) as yellow needles: mp (EtOAc/CH2Cl2) 105-107 °C; 1H NMR [(CD3)2SO] δ 8.90 (s, 1 H), 8.59 (m, 1 H), 8.55 (m, 1 H), 7.98 (m, 1 H), 7.78 (m, 1 H); 13C NMR δ 158.24,
135.07, 133.15, 127.92, 127.85, 127.73, 127.26, 125.54, 123.32, 122.42. Anal. (CιιH5F3N2O7S) C, H, N.
A solution of (2) (5.0 g, 13.7 mmol) and Nal (7.0 g) in EtOAc (200 mL) was heated under reflux for 2 h. The mixture was cooled to room temperature and washed with sat. aq. Na2S2O3. The organic phase was dried, concentrated under reduced pressure, and the residue was purified by column chromatography on silica gel. Elution with CH2C12, gave l-iodo-2,4-dinitronaphthalene (3) (4.3 g. 91%) as yellow needle: mp (EtOAc/CH2Cl2) 194-195 °C; 1H NMR [(CD3) SO] δ 8.76 (s, 1 H, H-3), 8.54 (m, 1 H), 8.33 (m, 1 H), 8.00 (m, 2 H); 13C NMR δ 151.81, 147.05, 134.97, 132.18, 131.24,
123.88, 123.26, 117.50, 102.66. Anal. (C10H5IN2O4) C, H, N.
A suspension of (3) (1.0 g, 2.9 mmol) and SnCl22H2O (9.8 g, 436 mmol) in EtOAc (100 mL) was heated at reflux for 30 min. The white suspension was poured onto ice (ca. 100 g), and sat. aq. NaHC03 was added until the aqueous layer was basic as shown by litmus paper inspection. The mixture was extracted with ethyl acetate, the organic layer was washed with water, dried and the solvent was removed under reduced pressure to give crude 1,3-naphmalenech'arrιine. This material was dissolved in THF (30 mL) and treated with BOC2O (3.0 g). The mixture was heated under reflux for 5 hrs. The solvent was removed and the residue was purified by column chromatography on silica gel. Elution with CH C12 gave naphthalene- 1,3 -(bis- tertbutoxycarbamate) (4) (0.916 g, 88%) as a brown solid: mp (petroleum ether) 129- 131 °C; 1H NMR (CDC13) δ 7.94 (s, 1 H), 7.78 (s, 1 H), 7.74 (m, 2 H), 7.41 (m, 2 H), 6.94 (s, 1 H), 6.69 (s, 1 H), 1.55 (s, 9 H), 1.54 (s, 9 H); 13C NMR δ 153.11, 152.75, 135.75, 134.75, 133.69, 128.47, 126.46, 124.36, 122.28, 119.67, 110.57, 110.47,
80.88, 80.57, 28.33. Anal (C2oH26N2O4) C, H, N. A solution of 4 (8.63 g, 24.1 mmol) in THF-CH3OH (200 mL, 1:1) at -78 °C was treated wimN-iodosuccinimide (NIS, 8.5 g, 33.7 mmol) in THF (10 mL) followed by TsOH'H2O (9.5 g, 50.2 mmol) in CH3OH (10 mL). The reaction mixture was allowed to slowly warm to room temperature over 4 h and then diluted with 5% aqueous
Νa2S2U3 and stirred at room temperature for 15 min. The reaction mixture was extracted with EtOAc (3x100 mL) and the combined extracts were dried and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel. Elution with CH2C12 and crystallization from EtO Ac/petroleum ether afforded l-iodonaphthalene-2,4-(bis-tertbutoxycarbamate) (5)
(9.46 g, 81%) as a brown solid: mp 154-156 °C (dec); 1H NMR (CDCI3) δ 8.65 (s, 1 H), 8.12 (m, 1 H), 7.76 (m, 1 H), 7.52 (m, 1 H), 7.45 (m, 1 H), 7.19 (s, 1 H), 6.83 (s, 1 H), 1.57 (s, 9 H), 1.55 (s, 9 H); 13C NMR δ 153.14, 152.63, 138.20, 134.80, 134.59, 132.58, 128.16, 125.31, 124.78, 121.33, 113.52, 81.27, 81.04, 28.34. Anal. (Cι0H5lN2O4) C, H, N.
A solution of 5 (4.86 g, 10.0 mmol) in anhydrous DMF (150 mL) under nitrogen was treated withNaH (1.2 g, 30 mmol, 60% oil dispersion), and the reaction mixture was stirred for 30 min at 0°C. Allyl bromide (12.1 g, 100 mmol) was added dropwise over 5 min, and the solution was allowed to warm to room temperature and stirred for 2 h.
Saturated aqueous NaHCO3 (100 mL) was added, and the aqueous phase was extracted with EtOAc (3 x 100 mL). The combined extracts were dried, concentrated under reduced pressure, and the residue was purified by column chromatography on silica gel. Elution with EtO Ac/petroleum ether (from 1:10 to 1:5), crystallization from EtO Ac/petroleum ether afforded l-iodonaphthalene-2,4-(bis~tert-butyl allylcarbamate) (6) (5.64 g, 100%) as a white solid: mp 150-152 °C; 1H NMR (CDCI3) δ 8.23 (m, 1 H), 7.77 (m, 1 H), 7.58 (m, 2 H), 7.10 (m, 1 H), 5.93 (m, 2 H), 5.07 (m, 4 H), 4.62 (m, 2 H), 3.87 (m, 2 H), 1.33 (s, 9 H), 1.25 (s, 9 H); 13C NMR δ 154.66, 153.74, 133.59, 133.45, 131.63, 128.06, 127.33, 127.25, 123.39, 123.32, 120.53, 118.26, 80.39, 52.76, 28.11. Anal. (C26H33IN2O4) C, H, N. A solution of (6) (2.82 g, 5.0 mmol) in benzene (200 mL) was treated sequentially with TEMPO (2.34 g, 3.0 equiv) and BusSnH (1.0 equiv). The reaction mixture was warmed to 60 °C. After 30 min, an additional 1 equiv of BuSnH was added. After 30 min, additional TEMPO (2 equiv) and BusSnH (1.0 equiv) were added. After 30 min, 2 equiv of TEMPO and 1 equiv of BU3S11H in two separate portions at 20 min intervals were added. After 45 min at 60 °C, the solvent was removed by evaporation, and the residue was purified by column chromatography on silica gel. Elution with EtOAc/petroleum ether (from 1:20 to 1:5), afforded N-allyl-(l-{ [(2,2,6,6-tetramethyl- 1 -piperidinyl)oxy]methyl } - 1 ,2-dihydro-3H-benzo [e]indole)-3 ,5 -bis(carbamate) (7) (2.97 g, 100%) as a yellow gum: 1H NMR (CDC13) δ 7.76 (m, 2 H), 7.43 (m, 1 H),
7.32 (m, 1 H), 7.06 (s, 1 H), 5.95 (m, 1 H), 5.07 (m, 2 H), 4.23 (m, 2 H), 4.09 (m, 2 H), 3.86 (m, 2 H), 1.59 (s, 18 H), 1.45-1.0 (m, 19 H); 13C NMR δ 152.46, 134.04, 133.81, 129.89, 128.90, 126.35, 126.02, 123.82, 123.35, 117.51, 79.98, 59.87, 52.43, 39.66, 33.11, 28.51, 28.17, 20.11, 17.05.
A solution of (7) (2.97 g, 5.0 mmol) in THF-HOAc-H2O (3:1:1, 200 mL) was treated with Zn powder (30 g, 80 equiv), and the mixture was warmed at 70 °C for 10 h. The Zn powder was removed by filtration through Celite, and the mixture was concentrated under reduced pressure The residue was extracted with CH2C12 (3x100 mL) and the combined extracts were dried, concentrated under reduced pressure, and the crude product was purified by column chromatography on silica gel. Elution with EtO Ac/petroleum ether (from 1 :4 to 1 :1), and crystallization from EtO Ac/petroleum ether afforded N-allyl-(l -hydroxymethyl- 1 ,2-dihydro-3H-benzo [e]indole)-3 ,5- bis(carbamate) (8) (2.13 g, 94%) as a white solid: mp 125-128 °C; 1H NMR (CDC13) δ 7.75 (m, 2 H), 7.43 (m, 1 H), 7.34 (m, 1 H), 7.06 (s, 1H), 5.98 (m, 1 H), 5.11 (m, 2
H), 4.20 (m, 2 H), 4.11 (m, 2 H), 3.90 (m, 2 H), 1.59 (s, 18 H), 1.26 (m, 1 H); 13C NMR δ 152.99, 152.46, 133.94, 128.89, 126.79, 126.00, 124.37, 123.91, 117.59, 64.69, 60.38, 52.11, 28.49, 28.15, 21.03. Anal. (Cι0H5IN2O4) C, H, N.
A solution of (8) (4.55 g, 10.0 mmol) in CH2C12 (100 mL) under nitrogen was treated sequentially with Ph3P (7.9 g, 30.0 mmol, 3 equiv) and CC (14.0 g, 90 mmol, 9 equiv). The reaction mixture was stirred at room temperature for 2 h. The solvent was evaporated under reduced pressure, and the crude product was purified by column chromatography on silica gel. Elution with EtO Ac/petroleum ether (from 1:4 to 1:1), and crystallization from EtO Ac/petroleum ether afforded N-allyl-(l-chloromethyl- l,2-dihydro-3H-benzo[e]indole)-3,5-bis(carbamate) (9) (4.46 g, 90%) as a yellow oil:
1HNMR (CDC13) δ 7.78 (m, 1 H), 7.70 (m, 1 H), 7.50 (m, 2 H), 7.38 (m, 1H), 5.98 (m, 1 H), 5.10 (m, 2 H), 4.20-3.40 (m, 6 H), 1.60 (s, 18 H), 1.20 (m, 1 H); 13C NMR δ 154.90, 152.36, 133.88, 132.04, 128.43, 127.17, 124.42, 123.86, 122.46, 117.66, 60.38, 52.73, 46.30, 28.47, 28.15, 21.03. HRMS m/z required for C26H33ClN2O4: 472.2129. Found: 472.2129.
A solution of 9 (406 mg, 0.858 mmol) in 50 mL of trifluoroacetic acid (TFA) was stirred under nitrogen at 0 °C for 2 h . The reaction mixture was then concentrated under reduced pressure, benzene (30 mL) was added and the mixture was concentrated once more. The resulting residue was treated with sat. aq. NaHCO3, extracted with CH2C1 (3 x 50 mL) and the combined extracts were dried and concentrated unuer reduced pressure. To the resulting residue was added 5,6,7- trimethoxyindole-2-carboxylic acid (220 mg) and EDCIHC1 (411 mg), followed by DMA (10 mL). The reaction mixture was stirred at room temperature overnight and was diluted with sat. aq. NaHCO3. The resulting mixture was extracted with EtOAc
.(3 x 50 mL) and the combined extracts were dried and concentrated under reduced pressure. The product was purified by column chromatography on silica gel. Elution with EtOAc/CH Cl (1:5), and crystallization from CH2Cl /diisopropyl ether afforded N-allyl- 1 -(chloromethyl)-3 -[(5,6,7-trimethoxy- lH-indol-2-yl)carbonyl] - 1 ,2-dihydro- 3H-benzo[e]indol-5-amine (10a) (355 mg, 80%) as a yellow solid: mp 107-112 °C;
1H ΝMR (CDCI3) δ 9.45 (s, 1 Η), 7.82 (d, J= 8.7 Ηz, 1 Η), 7.81 (s, 1 Η), 7.70 (d, J= 8.3 Ηz, 1 Η), 7.51 (m, 1 Η), 7.37 (m, 1 Η), 7.00 (d, J= 2.3 Ηz, 1 Η), 6.88 (s, 1 Η), 6.05 (m, 1 Η), 5.40 (m, 1 Η), 5.26 (m, 1 Η), 4.76 (m, 2 Η), 4.61 (m, 1 Η), 4.07 (s, 3 Η), 4.02 (m, 3 Η), 3.95 (s, 3 Η), 3.91 (s, 3 Η), 3.42 (m, 1 Η), 1.64 (br, 1 Η) J= 2.7 Ηz; 13C ΝMR δ 160.40, 150.13, 144.91, 142.71, 140.41, 138.87, 134.49, 130.12,
130.04, 127.11, 125,48, 123.64, 123.41, 123.14, 121.25, 121.18, 116.99, 113.18, 106.39, 97.72, 96.88, 61.46, 61.09, 56.28, 55.14, 46.72, 46.14, 43.28. Anal. (CιoH5IN2O4) C, H, N.
Benzenesulfinic acid, sodium salt (25 mg, 0.15 mmol) and camphorsulfonic acid (40 mg, 0.17 mmol) were added to a solution of 10a (30 mg, 0.059 mmol) in dichloromethane (5 mL). Pd(Pli3P)4 (5 mg, 0.004 mmol) was added and the reaction mixture was stirred at an ambient temperature for 30 min. The mixture was purified directly via a silica gel column. Elution with EtO Ac/petroleum ether (1:1) gave 1- (chloromethyl)-3 - [(5 , 6,7-trimethoxy- lH-indol-2-yl)carbonyl] - 1 ,2-dihydro-3H- benzo[e]indol-5-amine (11a) (22 mg, 80%), spectroscopically identical to an authentic sample (Atwell et al., J Org. Chem., 1998, 63, 9414).
Alternatively, a solution of 10a (70 mg, 0.138 mmol) in anhydrous toluene (5 mL) (under N2 and protected from light), was heated at reflux and treated every 20 min with small portions of Cl2(Cy3p)2Ru=CΗPh (Alcaide et al., Org. Lett., 2001, 3623)
(overall total 0.02 mmol). After 8 h, the resulting mixture was concentrated under reduced pressure. Chromatography of the residue eluting with EtOAc/CH2Cl2 gave 17 mg of recovered starting material and 11a (27 mg, 55% based on consumption of starting material). The product was crystallized from CH2Cl2/diisopropyl ether and was spectroscopically identical to an authentic sample (Atwell et al., J Org. Chem. ,
1998, 63, 9414).
Similarly, 9 (2.74 g, 5.81 mmol) was treated with TFA (50 mL) 2 h and worked up as above. The crude product was stirred at an ambient temperature overnight with 4- methoxycinnamic acid (1.14 g, 6.4 mmol) and EDC1.HC1 (2.77 g) in DMA (10 mL).
Workup, and column chromatography of the product on silica gel, eluting with EtOAc/CH2Cl2 (1:5), followed by crystallization from CH2Cl2/diisopropyl ether, gave N-allyl-l-(chloromethyl)-3-[(2E)-3-(4-methoxyphenyl)-2-propenoyl]-l,2-dihydro- lH-benzo[e]indol-5-amine (10b) (980 mg, 39%) as a yellow solid: mp 152-154 °C; 1H ΝMR (CDC13) δ 7.95 (br, 1Η), 7.80 (s, 1 Η), 7.79 (d, J= 8.5 Ηz, 1 Η), 7.68 (d, J=
8.2 Ηz, 1 Η), 7, 57 (s, 1 Η), 7.55 (s, 1 Η), 7.48 (m, 1 Η), 7.33 (m, 1 Η), 6.93 (s, 1 Η),
6.91 (s, 1 Η), 6.80 (br, 1 Η), 6.09 (m, 1 Η), 5.40 (m, 1 Η), 5.26 (m, 1 Η), 4.49 (m, 1 H), 4.36 (m, 1 H), 4.00 (m, 5 H), 3.85 (s, 3 H), 3.41 (m, 1 H); 13C NMR δ 164.95, 161.23, 144.95, 143.49, 142.64, 134.56, 130.11, 129,77, 127.77, 127.02, 123,16, 123.02, 121.15, 121.00, 116.99, 116,30, 114.32, 96.91, 55.38, 53.39, 46.73, 46.33, 42.55.
A solution of 10b (691 mg, 1.6 mmol) in dichloromethane (20 mL) was treated with benzenesulfinic acid, sodium salt (524 mg, 3.2 mmol), camphorsulfonic acid (742 mg, 3.2 mmol) and Pd(Pli3P)4 (92 mg, 0.08 mmol) as described above. The mixture was stirred under an inert atmosphere at an ambient temperature until all starting material was consumed (typically 15 min, however the reaction is dependent on the quality of the Pd(Pli3P)4 used and it is sometimes necessary to add an extra portion of the catalyst). The mixture was directly aspirated onto a 4 mm Chromatotron radial chromatography plate and the product was eluted as the major yellow band with 25% EtOAc/hexanes. The combined fractions were concentrated under reduced pressure to give 1 -(chloromethyl)-3 - [(2E)-3 -(4-methoxyphenyl)-2-propenoyl] - 1 ,2-dihydro-3H- benzo[e]indol-5-amine (lib) (547 mg, 87%), identical with an authentic sample 1 τ.tweιt et ai., >. wj.
Figure imgf000021_0001
iWo, U , -riτ7.
Alternatively, deprotection of 10b (90 mg) with Cl2(Cy3P)2Ru=CΗPh as described above followed by chromatography of the residue, eluting with ΕtOAc/CH2Cl2, gave lib (34 mg, 42%). Crystallization from CH2Cl2/diisopropyl ether gave a product spectroscopically identical to an authentic sample.
The amino compounds of formula I can also be oxidised to the corresponding nitro compounds (such as those described in US 6,130,237) by a variety of methods, including peracetic acid (Sakaue et al, J Org. Chem., 1993, 58, 3633) or zirconium alkoxides (Krόhn, Synthesis, 1997, 10, 1115; Krohh et al., J Frdkt. Chem. 1991, 339, 335). Thus oxidation of 1 la with sodium perborate/acetic acid in THF gives the corresponding nitro compound.
It will be apparent to those skilled in the art that the processes of the present invention could be readily applied without undue experimentation and with appropriate selection of starting material so that other examples of Formula (I) could be expeditiously prepared. For example, many of the carbamate protecting groups envisaged that could be prepared for the arnine of group W are described in O00/64864. The disclosure of O00/64864 is incorporated herein in its entirety. Similarly, it is to be appreciated that all the compounds described in US 6,130,237 could be prepared by the methods of the present invention. The disclosure of US 6,130,237 is also incorporated herein in its entirety.
It is an advantage of the processes of the present invention that the processes described above of Schemes 1 and 2 provide significantly higher-yielding synthetic routes to the general class of 3 -substituted 1 -(chloromethyl)- 1,2-dihydro-lH- benzo [e] indol-5 -ylamines .
GDEPT (gene-directed enzyme prodrug therapy for cancer) is a tool suitable for use with some of the compounds that are capable of being produced by the processes of the present invention. The following GDEPT systems that are relevant to the comηnrr rlQ -nrnrlπced ^v +he ιτ*r>cesαpα ^'f+he nresent invention include*
(i) Vector Systems In general, the vector for use in GDEPT therapies can be any suitable DNA or RNA vector.
Suitable viral vectors include those that are based upon a retrovirus. Such vectors are widely available in the art. Ηuber et al. (ibid) report the use of amphotropic retroviruses for the transformation of hepatoma, breast, colon or skin cells. Culver et al. (Science (1992) 256; 1550-1552) also describe the use of retroviral vectors in
GDEPT. Such vectors or vectors derived from them may also be used. Other retroviruses may also be used to make vectors suitable for use in the present invention. Such retroviruses include rous sarcoma virus (RSN).
Englehardt et al. (Nature Genetics (1993) 4; 27-34) describe the use of adenovirus- based vectors in the delivery of the cystic fibrosis transmembrane conductance product (CFTR) into cells, and such adenovirus-based vectors may also be used. . Vectors utilising adeno virus promoter and other control sequences are of use in delivering a system according to the invention to cells in the lung, and hence useful in treating lung tumours.
Other vector systems including vectors based on the Molony murine leukaemia virus are known (Ram, Z. et al., Cancer Research (1993) 53; 83-88; Dalton & Treisman, Cell (1992) 68; 597-612). These vectors contain the Murine Leukaemia virus (MLN) enhancer cloned upstream at a .beta.-globin minimal promoter. The .beta-globin 5' untranslated region up to the initiation ATG is supplied to direct efficient translation of the enzyme.
Suitable promoters which can be used in vectors described above, include MLN, CMN, RSN and adenovirus promoters. Preferred adenovirus promoters are the adenovirus early gene promoters. Strong mammalian promoters may also be suitable. An example of such a promoter is the EF-1.alpha, promoter which may be obtained by reference to Mi∑xishima and Νagata ((1990), Nucl. Acids Res. 18; 5322). Variants nf σπch ηmmntpro rf-tαininσ cπfoe+cmtiial similar tτaηocτ, tirynal
Figure imgf000023_0001
mnv nlon Tnp used.
(ii) Nitroreductase
Compounds obtained by the present invention of the formula (I) which is a substrate suitable for a nitroreductase or carboxypeptidase enzyme in which P is group (Ila) or (lib) or (IΙe)and compounds of the formula (I) in which Y is a group NO2 or N(O)RR can be activated by reduction of the group P or Y (as defined above) by nitroreductase.
Preferably, the enzyme is a non-mammalian nitroreductase enzyme, such as a bacterial nitroreductase. AnE. coli nitroreductase as disclosed in WO93/08288 is particularly preferred. The enzyme may be modified by standard recombinant DNA techniques, e.g. by cloning the enzyme, determining its gene sequence and altering the gene sequence by methods such as truncation, substitution, deletion or insertion of sequences for example by site-directed mutagenesis. Reference may be made to "Molecular Cloning' ' by Sambrook et al. (1989, Cold Spring Harbor) for discussion of standard recombinant DNA techniques. The modification made may be any which still leaves the enzyme with the ability to reduce the nitro group of the protecting group P in the compound of formula (I) or the nitro or arnine N-oxide groups when these are represented by Y in formula (I) but alters other properties of the enzyme, for example its rate of reaction or selectivity.
In addition, small truncations in the N- and/or C-terminal sequence may occur as a result of the manipulations required to produce a vector in which a nucleic acid sequence encoding the enzyme is linked to the various other vector sequences.
(iii) Carboxypeptidase
Compounds obtained by the present invention of the formula (I) in which P is group (lie) can be activated by removal of the group P by a carboxypeptidase. The enzyme is preferably a bacterial carboxypeptidase, especially carboxypeptidase CPG2 or Pseudomonas γ-glutamylhydrolasε EC3.4.22.12 (Levy, C. C. & Goldman, P., J. Biol. Chem. 242; p 2933 (1967).
Carboxypeptidase G2 (CPG2) is disclosed in O88/07378. Although native CPG2 is preferred, alterations to its sequence which are amino acid substitutions, deletions or insertions (e.g. of about 1, 2, 3, 4, 5, 10 or 20 residues in each case) are also possible. In any event, the alteration will be such that the enzyme retains its ability to convert a prodrug to an active drug at substantially the same rate as the native enzyme. In this context, "substantially the same rate" will desirably be within 1 order of magnitude, and preferably from about 50-fold e.g. about 2-fold less to 2, 5 or 10 fόld'more.
In addition to specific changes the enzyme may otherwise be altered by truncation, substitution, deletion or insertion as long as the activity of the enzyme is substantially unchanged as defined above. For example, small truncations in the N- and/or C- ter inal sequence may occur as a result of the manipulations required to produce a vector in which a nucleic acid sequence encoding the enzyme is linked to a suitable promoter.
ADEPT (antibody-directed enzyme prodrug therapy for cancer) is a tool suitable for use with some of the compounds that are capable of being produced by the processes of the present invention.
For applications in ADEPT systems, an antibody directed against a tumour specific marker is linked to the nitroreductase or carboxypeptidase enzyme, which may be modified as described above. The antibody may be monoclonal or polyclonal. For the purposes of the present invention, the term "antibody", unless specified to the contrary, includes fragments of whole antibodies which retain their binding activity for a tumour target antigen. Such fragments include Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies. Furthermore, the antibodies and fragments thereof may be humanised antibodies, e.g. as described in EP-A-239400.
The antibodies may be produced by conventional hybridoma techniques or, in the case of modified antibodies or fragments, by recombinant DNA technology, e.g. by the expression in a suitable host vector of a DNA construct encoding the modified antibody or fragment operably linked to a promoter. Suitable host cells include bacterial (e.g. E. coli), yeast, insect and mammalian. When the antibody is produced by such recombinant techniques the enzyme may be produced by linking a nucleic acid sequence encoding the enzyme (optionally modified as described above) to the 3' or 5' end of the sequence of the construct encoding the antibody or fragment thereof.
Applications of the compounds obtained by the processes of the present invention
The compounds of formula (I) obtained from the processes of the present invention can be used in a method of treatment of the human or animal body. Such treatment includes a method of treating the growth of neoplastic cells in a patient with neoplastic disease which comprises administering to a patient in need of treatment compounds of formula (I) of the invention, or where the compounds of formula (I) obtained are suitable as substrates as part of an ADEPT or GDEPT therapy system. Neoplastic diseases include leukaemia and solid tumours such as breast, bowel and lung tumours including small cell lung carcinoma.
It will be understood that where treatment of tumours is concerned, treatment includes any measure taken by the physician to alleviate the effect of the tumour on a patient. Thus, although complete remission of the tumour is a desirable goal, effective treatment will also include any measures capable of achieving partial remission of the tumour as well as a slowing down in the rate of growth of a tumour including metastases. Such measures can be effective in prolonging and/or enhancing the quality of life and relieving the symptoms of the disease.
(i) Compounds of the Formula (I) obtained by a process of the present invention
Compounds of the formula (I) obtained by the processes of the present invention can be used in a method of treatment of neoplastic disease in a patient, which method
Crymrvri sec nrlmiriiote inσ rvf fre*:»+ττιeτ"'t SH effective nmn nt nf n
Figure imgf000026_0001
compound of formula (i). The compounds obtained may be administered in the form of a pharmaceutical composition.
While the exact dose of the compound will be at the discretion of the physician, taking account of the condition and needs of the patient, typical doses will be in the range of from about 0.1 to 200 mg/Kg, preferably about from 10 to 100 mg/Kg per patient per day.
(ii) ADEPT Therapy
The antibody/enzyme conjugate for ADEPT can be administered simultaneously but it is often found preferable, in clinical practice, to administer the enzyme/agent conjugate before the prodrug, e.g. up to 72 hours or even 1 week before, in order to give the enzyme/agent conjugate an opportunity to localise in the region of the tumour target. By operating in this way, when the prodrug is administered, conversion of the prodrug to the cytotoxic agent tends to be confined to the regions where the enzyme/agent conjugate is localised, i.e. the region of the target tumour the premature release of the toxic fragment of formula (I) is minimised.
In ADEPT the degree of localisation of the enzyme/agent conjugate (in terms of the ratio of localized to freely circulating active conjugate) can be further enhanced using the clearance and/or inactivation systems described in WO89/10140. This involves, usually following administration of the conjugate and before administration of the prodrug, the administration of a component (a "second component") which is able to bind to the part of the conjugate so as to inactivate the enzyme and/or accelerate the clearance of the conjugate from the blood. Such a component may include an antibody to the enzyme component of the system which is capable of inactivating the enzyme.
The second component may be linked to a macromolecule such as dextran, a liposome, albumin, macroglobulin or a blood group O erythrocyte so that the second component is restrained from leaving the vascular compartment. In addition or as an alternative the second component may include a sufficient number of covalently bound galactose residues, or residues of other sugars such as lactose or mannose, so that it can bind the conjugate in plasma but be removed together with the conjugate from plasma by receptors for galactose or other sugars in the liver. The second component should be administered and designed for use such that it will not, to any appreciable extent, enter the extravascular space of the tumour where it could inactivate localised conjugate prior to and during administration of the prodrug.
In ADEPT systems, the dose of the prodrug and conjugate will ultimately be at the discretion of the physician, who will take into account such factors as the age, weight and condition of the patient. Suitable doses of prodrug and conjugate are given in Bagshawe et al. Antibody, Immunoconjugates, and Radiopharmaceuticals (1991), 4, 915-922. A suitable dose of conjugate may be from 500 to 200,000 enzyme units/m2 (e.g. 20,000 enzyme units/m2) and a suitable dose of prodrug may be from about 0.1 to 200 mg/Kg, preferably about from 10 to 100 mg/Kg per patient per day. In order to secure maximum concentration of the conjugate at the site of desired treatment, it is normally desirable to space apart administration of the two components by at least 4 hours. The exact regime will be influenced by various factors including the nature of the tumour to be targeted and the nature of the prodrug, but usually there will be an adequate concentration of the conjugate at the site of desired treatment within 48 hours.
The ADEPT system when used with nitroreductase also preferably comprises a suitable cofactor for the enzyme. Suitable cofactors include a riboside or ribotide of nicotinic acid or nicotinamide.
The antibody/enzyme conjugate may be administered by any suitable route usually used in ADEPT therapy.
(iii) GDEPT Therapy
For use of the vectors in therapy, the vectors will usually be packaged into viral particles and the particles delivered to the site of the tumour, as described in for example Ram et al. (ibid). The viral particles may be modified to include an antibody, fragment thereof (including a single chain) or tumour-directed ligand to enhance targeting of the tumour. Alternatively the vectors may be packaged into liposomes. The liposomes may be targeted to a particular tumour. This can be achieved by attaching a tumour-directed antibody to the liposome. Viral particles may also be incorporated into liposomes. The particles may be delivered to the tumour by any suitable means at the disposal of the physician. Preferably, the viral particles will be capable of selectively infecting the tumour cells. By "selectively infecting" it is meant that the viral particles will primarily infect tumour cells and that the-proportion of non-tumour cells infected is such that the' damage to non-tumour" cells by administration of a prodrug will be acceptably low, given the nature of the disease being treated. Ultimately, this will be determined by the physician.
One suitable route of administration is by injection of the particles in a sterile solution. Viruses, for example isolated from packaging cell lines may also be adrninistered by regional perfusion or direct intratumoral direction, or direct injection into a body cavity (intracaviterial administration), for example by intraperitoneum injection.
The exact dosage regime for ADEPT will, of course, need to be determined by individual clinicians for individual patients and this, in turn, will be controlled by the exact nature of the prodrug and the cytotoxic agent to be released from the prodrug but some general guidance can be given. Chemotherapy of this type will normally involve parenteral administration of modified virus and administration by the intravenous route is frequently found to be the most practical.
In GDEPT systems the amount of virus or other vector delivered will be such as to provide a similar cellular concentration of enzyme as in the ADEPT system mentioned above. Typically, the vector will be administered to the patient and then the uptake of the vector by transfected or infected (in the case of viral vectors) cells monitored, for example by recovery and analysis of a biopsy sample of targeted tissue. This may be determined bv clinical trials which involve administerinσ a rano'e of trial doses to a patient and measuring the degree of infection or transfection of a target cell or tumour. The amount of prodrug required will be similar to or greater than that for ADEPT systems.
In using a GDEPT system the prodrug will usually be administered following acimirύstration of the vector encoding an enzyme. Suitable doses of prodrug are from about 0.1 to 200 mg/Kg, preferably about from 10 to 100 mg/Kg per patient per day.
Wherein the foregoing description reference has been made to reagents, or integers having" known equivalents thereof, then" those" equivalents are herein incorporated as" if individually set forth.
While this invention has been described with reference to certain embodiments and examples, it is to be appreciated that further modifications and variations may be made, to embodiments and examples without departing from the spirit or scope of the invention.

Claims

Claims
1. A process of preparing a compound of formula (I)
Figure imgf000031_0001
wherein
A and B together may represent a fused optionally substituted benzene, a fused optionally substituted naphthalene, a fused optionally substituted pyridine, a fused optionally substituted furan or a fused optionally substituted pyrrole ring, where the optional substituents are represented by Y; wherein
Y is selected from one or more of H, a halogen, CH3, OR1, SR1, M^R2, SO2R1, CONHR1, CN or CO2R: where R1 or R2 each independently represents H, lower Ci. 4alkyl or a benzene ring, optionally substituted with one or more hydroxyl, NO2 or amino groups, the amino groups being further optionally substituted with one or two -C4 alkyl groups,
X is halogen or OSO2R, where R represents H, lower C1-4alkyl or a benzene ring, optionally substituted with one or more hydroxyl, NO2 or amino groups, the amino groups being further optionally substituted with one or two Ci-C4 alkyl groups, W is selected from NO2, NHOH, N(R3)2 NHR3, NHCO2R3, N(phthaloyl). or NH2, where each R3 is selected from a Cμ alkyl group optionally substituted with one or more hydroxyl or amino groups, each amino group being optionally substituted with one or two Cμ alkyl groups; a Cμ alkylene group optionally substituted with hydroxyl or amino groups, each amino group being optionally substituted with one or two Cμ alkyl groups; or a benzene ring, optionally substituted with one or more hydroxyl, NO2 or ar ino groups, the amino groups being further optionally substituted with one or two -C4 alkyl groups, or
-N-J W is further selected from the group , wherein J is selected from OH or R wherein R represents H, lower Cμalkyl or a benzene ring, optionally substituted with one or more hydroxyl, NO or amino groups, the amino groups being further optionally substituted with one or two C1-C4 alkyl groups, and
P is a group which is a substrate suitable for a nitroreductase or carboxypeptidase enzyme such that one of said enzymes is able to bring about the removal of the group P which is selected from the following moieties:
-C(O)-(CZ'2)n-Ph (Ila)
-C(O)-O-CH2 -Ph (lib)
-C(O)-NH-C(COOH)-(CH2)2-COOH (lie) -C(O)-O-CH2 -Phe-L-C(O)-NH-C(COOH)-CH2-COOH (lid)
-C(O)-O-CH2 -TMDZ (He)
-C(O)-O-CH2 -Furyl (Ilf)
-C(O)-O-CH2 -Thienyl (Ilg)
-C(O)-O-CH2 -Pyrrolyl (Ilh)
wherein each occurrence of Z' is independently H or CH3, n is 1 or 2, Ph is a phenyl moiety substituted in the moiety of (Ila) by a nitro group at the 2-position, and substituted in the moiety of (lib) by a nitro group in the 2- or 4-position, IMDZ is an imidazolyl moiety substituted in the moiety of (He) by a nitro group in the 2-, 4- or 5- position, and optionally further substituted by -CH3 or Cι-C4alkyl at the 1 -position, wherein said alkyl is further substituted by OH, Phe. is a phenylene (C6 B ). ring in which the group -L, which may represent O or NH, is para to the group -OCH2, wherein the Furyl, Thienyl, and Pyrrolyl groups in moieties, (Ilf), (Ilg) and (Ilh) respectively are optionally substituted by a nitro group, wherein the Pyrrolyl group of (Ilh) is further optionally substituted at the 1 -position by CH3 and/or CO2CH2CH3 at the 2-position, the groups Ph and Phe, being further optionally substituted by a group R1 which is a group selected from R, CONHR, NHCOR, NHR, OR or SO2R where R represents H, lower Cμalkyl optionally substituted by morpholino, or a benzene ring, optionally substituted with one or more hydroxyl, NO2 or amino groups, the amino groups being further optionally substituted with one or two C1-C4 alkyl groups Z is selected from the following structures (la, lb, Ic or Id)
Figure imgf000033_0001
la lb Ic Id wherein E is selected from -N=, or -CH=, G is O, S, orNH, Q is either
(a) absent or independently selected from one to three of R, -OR, halogen, -NRR, -NO2, -COOR, -CONRR, -NRCOR, OP(O)(OH)2, O(CH2)nP(O)(OH)2,
(CH2)nOP(O)(OH)2, -NRCO
Figure imgf000033_0002
group; where R represents H, lower Cμalkyl or a benzene ring, optionally substituted with one or more hydroxyl or amino groups, each amino group being optionally substituted with one or two C1-C4 alkyl groups and where n represents 1, 2 or 3; or
(b) is an additional group of formulae (la, lb. Ic or Id);and
HET may represent a 5- or 6-membered carbocycle or heterocycle containing one or two atoms independently selected from N, O and S; or a physiologically functional derivative thereof, wherein the process for preparing a compound of formula I includes the steps of
(a) converting the NO groups of a compound of formula (F) into NHBOC groups of a compound of formula (IF)
Figure imgf000034_0001
R'=OH R"=H
R'=OTf R"=Halogen
R -Halogen
wherein the variables A, B, and Y are as defined above in this claim,
(b) converting a compound of formula (IF) by allylation of the NHBOC groups into a compound of formula (IIF)
Figure imgf000034_0002
4: R"=H
5: R"=Halogen
wherein the variables A, B, and Y are as defined above in this claim,
(c) converting a compound of formula (IIF) into an indole derivative of formula (IV)
Figure imgf000034_0003
wherein the variables A, B, and Y are as defined above, (d) reacting the compound of Formula (IV') in the presence of Zn powder to give a compound of Formula (V')
Figure imgf000035_0001
R"'=Halogen
wherein the variables A, B, and Y are as defined above in this claim, and
(ε) reacting the NBOC and allyl groups of Formula (V') as desired to give a compound of Formula (I)
Figure imgf000035_0002
R«"=OH R'"=Halogen
wherein the variables A, B, Y, W, and Z are as defined above in this claim.
2. The process according to claim 1 wherein A-B represents a fused benzo group.
3. The process according to claim 1 or claim 2 wherein X represents chloro.
4. The process according to any one of claims 1 to 3 wherein Z represents one of following groups:
Figure imgf000036_0001
wherein the substituent -R may represent one or more substituents selected from -H, -OH, halogen, -NO2, -NH , -N(Cμalkyl)2, -Cμalkyl, -COOCμalkyl, -OCμalkyl, and -NHCOCμalkyl.
5. The process according to any one of claims 1 to 4 wherein W is selected from one of the following NHCH2CH=CH2, NH2, N hthaloyl), NHCH3, N(CH3)2, NO , andNHCO2CH2C6H4-p-NO2.
6. The process according to claim 1 wherein the compound of Formula 1 is selected from the following: N-AJlyl-l-(cMoromethyl)-3-[(5,6,7-trimethoxy-lH-indol-2-yl)carbonyl]-l,2- d ydro-3H-benzo[e]indol-5-amine;
N-Allyl-l-(chloromethyl)-3-[(2E)-3-(4-methoxyρhenyl)-2-propenoyl]-l,2- d ydro-3H-benzo[e]indol-5-amine; l-(Cωorome l)-3-[(5,6,7-trimethoxy-lH-indol-2-yl)carbonyl]-l,2-dihydro-3H- benzo [e]indol-5-amine; l-(CMoromethyl)-3-[(2E)-3-(4-methoxyρhenyl)-2-proρenoyl]-l,2-dihydro-3H- benzo [e]mdol-5-amine; l-(CUoromemyl)-5-phthdimido-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-l,2- dihydro-3JJ-benz[e]indole
1 -CMoromethyl-5-mfro-3-[5,6,7-trimemoxyindol-2-yl)-carbonyl]- 1 ,2-dihydro-
3Η-benz[e]indole; l-(CHoromemyl)-3-[[7-[2-(dimemylamino)ethoxy]-5-methoxyindol-2- yl]carbonyl]-5-nitro- 1 ,2-dihydro-3H-benz[e]indole; 1 -(CWoromemyl)-3-[[6-[2-(dήnemylammo)emoxy]-5-methoxyindol-2- yl]carbonyl]-5-nitro-l,2-dihydro-3H-benz[e]indole;
1 -(CMoromemyl)-3-[(Ε)-3-memoxycinnamoyl]-5-nitro- 1 ,2-dihydro-3H- benz[e]indole;
(R)-l-(CMoromethyl)-5-mtro-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-l,2- dihydro-3JJ-benz[e]indole;
(S)-l-(CMoromethyl)-5-mfro-3-[(5,6,7-frimethoxyindol-2-yl)carbonyl]-l,2- dihydro-3H-benz[e]indole;
Methyl l-(c oromemyl)-5-mtio-3-[(5,6,7-frimethoxyindol-2-yl)carbonyl]-l,2- dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate; 5-Amino-l -(cMoromethyl)-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-l ,2-dihyd ro-3JJ-benz[e]indole; l-(GMoromethyl)-5-methylammo-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-l,2 - dihydro-3H-benz[e]indole;
5 -Amino- 1 -(chloromethyl)-3 - [ [7-[2-dimethylamino)ethoxy] -5-methoxyindol-2- ' yl]carbonyl]-l,2-dihydro-3H-benz[e]indole;
5-Amino-l-(cMoromemyl)-3-[[6-[2-dime ylammo)emoxy]-5-methoxyindol-2- yljcarbonyl]- 1 ,2-dihydro-3JJ-benz[e]indole; 5-Amino- 1 -(cMoromethyl)-3-[[7-[2-dimeτh.ylanιmo)ethoxy]-5-methoxyindol-2- yl] carbonyl] - 1 ,2-dihydro-3H-benz[e]indole;
5-Arnino- 1 -[(E)-4-butyrylamino-l -methyl-2-pyrroleacryloyl]- 1 -(chloromethyl)- 1 ,2-dihydro-3H-benz[e]indole; 5-Amino-l -(cMoromethyl)-3-[(E)-3-methoxycinnamoyl]- 1 ,2-dihydro-3H- benz[e]indole;
(R)-5-Ammo-l-(cMorome yl)-3-[(5,6,7-frimethoxyindol-2-yl)carbonyl]-l,2-
. dihydro-3H-benz[e]indole;
(S)-5-Ammo-l-(cMoromemyl)-3-[(5,6,7-trimeτ ιoxyindol-2-yl)carbonyl]-l,2- dihydro-3H-benz[e]indole;
Methyl 5-ammo-l-(chloromethyl)-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-l,2- dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate; ■
Methyl l-(cMoromethyl)-5-me ylammo-3-[(5,6,7-trimethoxyindol-2- yl)carbonyl]- 1 ,2-dihydro-3H-pyrrolo [3 ,2-e]indole-7-carboxylate; Methyl 1 -(cMorome yl)-5-dimemylamino-3-[(5,6,7-trimethoxyindol-2- yl)carbonyl] - 1 ,2-dihydro-3H-pyrrolo [3 ,2-e]indole-7-carboxylate;
Methyl l-(chloromethyl)-5-[(4-mfrobenzyloxycajbonyl)3jrιino]-3-[(5,6,7- trimemoxymdol-2-yl)carbonyl]-l,2-dihydro-3H-pyrrolo[3,2-e]indole-7- carboxylate; and 1 -(Chloromethyl)-5-[(4-nitrobenzyloxycarbonyl)amino]-3-[(5,6,7- trimethoxyindol-2-yl)carbonyl]- 1 ,2-dihydro-3H-benz[e]indole; or a physiologically functional derivative thereof.
7. The process according to claim 6 wherein the compound of Formula 1 is selected from the following:
N-Allyl-l-(chlorometh-yl)-3-[(5,6,7-trimethoxy-lH-indol-2-yl)carbonyl]-l,2- d ydro-3H-benzo [e]mdol-5-amine;
N-Allyl-l-(chloromethyl)-3-[(2E)-3-(4-methoxyphenyl)-2-propenoyl]-l,2- dihydro-3H-benzo [e]indol-5 -amine; l-(C oromethyl)-3-[(5,6,7-trimemoxy-lH- dol-2-yl)carbonyl]-l,2-dihydro-3JJ- benzo[e]mdol-5-amine; and l-(CMoromethyl)-3-[(2E)-3-(4-methoxyphenyl)-2-propenoyl]-l,2-dihydro-3H- benzo[e]indol-5-amine.
8. A compound obtained by the process according to any one of claims 1 to 5 selected from the following:
N- yl-l-(chloromethyl)-3-[(5,6,7-trimethoxy-lH-indol-2-yl)carbonyl]-l,2- dihydro-3H-benzo[e]indol-5-amine;
N- Allyl- 1 -(chloromethyl)-3 - [(2E)-3 -(4-methoxyphenyl)-2-propenoyl]- 1 ,2- dihydro-3H-benzo[e]indol-5-amine; l-(Cmoromethyl)-3-[(5,6,7-trimethoxy-lH-indol-2-yl)carbonyl]-l,2-dihydro-3H- benzo[e]indol-5-amine; l-(Cmoromethyl)-3-[(2E)-3-(4-methoxyphenyl)-2-propenoyl]-l,2-dihydro-3H- benzo[e]indol-5-amine; l-(CMoromemyl)-5-phmalimido-3-[(5,6,7-frimeτ ιoxymdol-2-yI)carbonyI]- dihydro-3H-benz[e]indole
1 -Chloromethvl-5-nitro-3 - r5,6.7-trimethoxvindol-2- vlVcarbonvll - 1.2-dihvdro-
3H-benz[e]indole; l-(CMoromethyl)-3-[[7-[2-(dimethiylammo)ethoxy]-5-methoxyindol-2- yl]carbonyl]-5-nitro-l ,2-dihydro-3Η-benz[e]indole; l-(Chlorome yl)-3-[[6-[2-(dimethylamino)ethoxy]-5-methoxyindol-2- yl]carbonyl]-5-nitro- 1 ,2-dihydro-3H-benz[e]indole;
1 -(Chloromethyl)-3-[(Ε)-3-methoxycinnamoyl]-5-nitro-l ,2-dihydro-3H- benz[e]indole; (R)-l-(CMoromemyl)-5-mfro-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-l,2- dihydro-3H-benz[e]indole;
(S)-l-(eMoromethyl)-5-mfro-3-[(5,6,7^imethoxyindol-2-yl)carbonyl]-l,2- dihydro-3H-benz[e]indole;
Methyl 1 -(cMoromethyl)-5-mtro-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-l ,2- dihydro-3H-pyrrolo [3 ,2-e]indole-7-carboxylate;
5-Ammo-l-(c oromethyl)-3-[(5,6,7-trimemoxyindol-2-yl)carbonyl]-l,2-dihyd ro-3H-benz[e]indole; 1 -(CHoromethyl)-5-methylammo-3-[(5,6,7-1rimethoxyιndol-2-yl)carbonyl]- 1 ,2 • dihydro-3H-benz[e]indole;
5-Amino- 1 -(cMoromemyl)-3-[[7-[2-dimethylaιrrmo)emoxy]-5-memoxyindol-2- yl]carbonyl]-l,2-dihydro-3H-benz[e]indole; 5-Amino-l-(cHoromemyl)-3-[[6-[2-dimethylammo)emoxy]-5-methoxyindol-2- yl]carbonyl]-l,2-dihydro-3H-benz[e]indole;
5-Ammo-l-(cMoromemyl)-3-[[7-[2-dimethylammo)ethoxy]-5-methoxyindol-2- yl]carbonyl]-l,2-dihydro-3H-benz[e]indole;
5-Amino- 1 - [(E)-4-butyrylamino- 1 -methyl-2-pyrroleacryloyl]- 1 -(chloromethyl)- l,2-dihydro-3H-benz[e]indole;
5-Am o-l-(cMoromemyl)-3-[(E)-3-methoxycirmamoyl]-l,2-dihydro-3H- benz[e]indole;
(R)-5-Ajnmo-l-(cMoromethyl)-3-[(5,6,7-frin emoxyindol-2-yl)carbonyl]-l,2- dihydro-3H-benz[e]indole; (S)-5-Ammo-l-(cMoromet ιyl)-3-[(5,6,7-frimethoxyindoi-2-yl)carbonyI]-l,2- dihydro-3H-benz[e]indole;
Methyl 5 -amino- 1 -(chloromethyl)-3 - [(5 , 6,7-trimethoxyindol-2-yl)carbonyl] - 1 ,2- dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate;
Methyl 1 -(cΗoromet ιyl)-5-me ylamino-3-[(5,6,7-trimethoxyindol-2- yl)carbonyl]-l,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate; and
Methyl 1 -(chlorome yl)-5-dimethylamino-3-[(5,6,7-trimethoxyindol-2- yl)carbonyl]- 1 ,2-dihydro-3H-pyrrolo[3 ,2-e]indole-7-carboxylate;
Methyl 1 -(cMoromethyl)-5-[(4-mfrobenzyloxycarbonyl)amino]-3-[(5,6,7-
1rimemoxymdol-2-yl)carbonyl]-l,2-d ydro-3H-pyrrolo[3,2-e]indole-7- carboxylate; and l-(CMoromethyl)-5-[(4-mfrobenzyloxycarbonyl)arnino]-3-[(5,6,7- trimethoxyindol-2-yl)carbonyl] - 1 ,2-dihydro-3H-benz[e]indo"le; or a physiologically functional derivative thereof.
9. A compound obtained by the process according to any one of claims 1 to 5 selected from the following: N- Allyl- 1 -(chloromethyl)-3 -[(5,6,7-trimethoxy- lH-indol-2-yl)carbonyι]- 1 ,2- d ydro-3H-beήzo[e] dol-5-amine;
N-Allyl-l-(chloromethyl)-3-[(2E)-3-(4-methoxyρhenyl)-2-ρropenoyl]-l,2- d&ydro-3H-benzo[e] dol-5-amine; l-(CMoromethyl)-3-[(5,6,7-trimemoxy-lH-mdol-2-yl)carbonyl]-l,2-dihydro-3H- benzo[e] dol-5-amine; and l-(Chloromethyl)-3-[(2E)-3-(4-methoxyphenyl)-2-propenoyl]-l,2-dihydro-3H- berιzo[e]indol-5-aιrιine.
10. The use of one or more compounds obtained from the process according to any one of claims 1 to 7 as one or more anticancer drugs.
11. The use of the compounds according to claim 10 as anticancer drugs for a selective killing of oxic and hypoxic tumor cells in the treatment of one or more cancers.
12. The use according to claim 11 wherein the cancer is a leukemia.
13. The use according to claim 11 wherein the cancer is a solid cancer tumor selected from breast, bowel and lung tumors, including small cell lung carcinoma.
14. The use of the compounds obtained by the process according to any one of claims 1 to 6 that are suitable as substrates for nitroreductase or carboxypeptidase enzymes in methods of ADEPT and GDEPT therapy.
15. The use of the compounds according to claim 14 wherein the nitroreductase enzyme is an aerobic ΝR2 nitroreductase- isolated from E. coli.
PCT/NZ2003/000094 2002-05-17 2003-05-19 Processes for preparing 3-substituted 1-(chloromethyl)-1,2-dihydro-3h-[ring fused indol-5-yl(amine-derived)] compounds and analogues thereof, and to products obtained therefrom WO2003097635A1 (en)

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