WO1993003729A1

WO1993003729A1 - N-substituted phenoxazines for treating multidrug resistant cancer cells

Info

Publication number: WO1993003729A1
Application number: PCT/US1992/006681
Authority: WO
Inventors: Peter J. Houghton; Julie K. Horton; Kuntebommanahalli N. Thimmaiah
Original assignee: Research Corporation Technologies, Inc.
Priority date: 1991-08-12
Filing date: 1992-08-10
Publication date: 1993-03-04
Also published as: US5371081A

Abstract

Phenoxazines, unsubstituted or N-substituted as defined herein, can potentiate the antitumor effectiveness of chemotherapeutic agents, particularly in multiple drug resistant (MDR) cells.

Description

N-SUBSTITUTED PHENOXAZINES FOR

TREATING MULTIDRUG RESISTANT CANCER CELLS

The present invention is directed to chemotherapy of cancer.

A major reason for failure of treatment of cancer patients is resistance to conventional chemo- therapeutic agents. One type of drug resistance, called multi-drug resistance (MDR) is characterized by cross- resistance to functionally and structurally unrelated chemotherapy drugs, such as doxorubicin, vincristine (VCR), vinblastine (VLB), colchicine, and actinomycin D. A number of drugs appear to be active in modifying MDR in model systems, including the calcium channel blocker, verapamil (VRP), the calmodulin inhibitor,

trifluoperazine, the anti-arrhythmic drug, guinidine, reserpine, cyclosporin A, Vinca alkaloid analogs, dihydropyridines, and pyridine analogs. Thus, it can be seen that agents that reverse MDR apparently do not seem to have common features. Although several of these MDR- reversing agents have been or are now being tested clinically in cancer patients, they have largely failed to enhance sensitivity to the chemotherapeutic agent. Instead, serious toxicities develop at or below plasma drug levels reguired for MDR reversal in vitro.

A tricyclic compound, phenoxazine, has been found to potentiate the uptake of VCR and VLB in MDR GC₃/Cl and KBCh^R-8-5 cells to a greater extent than verapamil. While this discovery has utility and holds promise, it would be desirable to identify derivatives of phenoxazine which would modulate MDR and which show even higher stability and lower toxicity. In one aspect, the present invention comprises compounds of formula (1)

and pharmacologically acceptable salts thereof,

wherein R is -[C(O)]_a-(CH₂)_b-A; wherein a is 0 or 1 and b is an integer from 0 to 6, provided that a and b are not both zero;

A is selected from the group consisting of -NR₁R₂ wherein R₁ and R₂ are independently alkyl having 1 to 4 carbon atoms, and either or both of R₁ and R₂ are optionally substituted with -OH; wherein X and Y are independently

alkylene having 1 to 4 carbon atoms, and Z is -O-,

-N(R₃)-or -CH(R₄)-, wherein R₃ is hydrogen or alkyl having 1 to 4 carbon atoms optionally substituted with a hydroxyl group, and wherein R₄ is hydrogen or alkyl having 1 to 4 carbon atoms optionally substituted with a hydroxyl groups;

halide; and trihalomethyl.

The present invention also relates to a method of potentiating the cytotoxicity of an agent cytotoxic to a tumor cell, comprising administering to said tumor cell, while it is exposed to said cytotoxic agent, a potentiating agent in an amount effective to potentiate the cytotoxicity of said cytotoxic agent to said cell, wherein said potentiating agent comprises a compound of formula (1):

or a pharmacologically acceptable salt thereof,

wherein R is -H or -[C(O)]_a-(CH₂)_b-A;

wherein a is 0 or 1 and b is an integer from 0 to 6, provided that a and b are not both zero; and

alkylene having 1 to 4 carbon atoms, and Z is -O-, - N(R₃)-or -CH(R₄)-, wherein R₃ is hydrogen or alkyl having 1 to 4 carbon atoms optionally substituted with a hydroxyl group, and wherein R₄ is hydrogen or alkyl having 1 to 4 carbon atoms optionally substituted with a hydroxyl group;

halide; and trihalomethyl.

The present invention further relates to a composition comprising cytotoxic agent toxic to tumor cells, and a potentiating agent which potentiates the cytotoxicity of said cytotoxic agent, wherein said potentiating agent comprises a compound of formula (1) and wherein said cytotoxic agent and potentiating agent are present in amounts effective to render the

composition cytotoxic to tumor cells.

The present invention still further relates to a method of killing a tumor cell which comprises administering to said cell a composition as described above in an amount effective to kill said cell.

As described in more detail below, the present invention provides novel and effective means for

potentiating the desired cytotoxic effect of anticancer drugs in tumor cells and especially in multidrug- resistant (MDR) cells.

One preferred group of compounds of the formula (1) is the N-alkyl derivatives, in which a is 0 in formula (1). Of those compounds wherein a is 0, the more preferred include those in which b is 3 or 4, denoting unbranched propylene and butylene moieties; R₁ and R₂ each are ethyl, n-propyl, ω-hydroxyethyl, or w- hydroxypropyl; X and Y are each -CH₂- or -CH₂CH₂- and, more preferably, both X and Y are -CH₂CH₂-; and R₃ and R₄ are each -H or ethyl, prowl, e.g. n-propyl, w- hydroxyethyl or ω-hydroxypropyl. Other more preferred embodiments when a is 0 are those derivatives wherein b is 3 or 4 and A is halogen, preferably chloro.

Another preferred group of compounds of formula (1) is the N-acyl derivatives, in which a is 1 in formula (1). Of those compounds wherexn a is 1, the more preferred include those in which b is 1 or 2, more preferably 1; R₁ and R₂ are each ethyl, n-propyl, ω- hydroxyethyl or ω-hydroxypropyl; X and Y are each -CH₂- or -CH₂CH₂-, and more preferably, both X and Y are -

CH₂CH₂-; each of R₃ and R₄ is -H or ethyl, n-propyl, ω- hydroxyethyl or ω-hydroxypropyl. Other more preferred embodiments are those in which b is 0 or 1 and A is trihalomethyl, preferably trichloromethyl or

trifluoromethyl; and in which b is 1 or 2 and A is halogen, preferably chloro.

As used herein, unless specified otherwise,

"alkyl" means saturated, branched or unbranched groups of the formula -(C_nH_2n+1); "halo" or "halogen" means fluoro, chloro, bromo, and/or iodo; and the optional hydroxyl and halo substituents disclosed herein can beon any carbon of an alkyl or alkylene group.

The compounds of this invention form salts, which are also within the scope of the invention, with various inorganic and organic acids. The

pharmacologically acceptable acid addition salts of the compounds of the present invention may be prepared by conventional means, such as by reacting with an

appropriate acid providing the desired anion, either in a solvent or medium in which the salt is insoluble, or in water. The salts of strong acids are preferred. As exemplary, but not limiting, of pharmacologically acceptable acid salts are the salts of hydrochloric, hydrobromic, sulfuric, nitric, acetic, fumaric, malic, maleic, tartaric and citric acids.

In general, the synthesis of the N-alkyl and

N-acyl derivatives is straightforward. N-alkylation can be achieved in the presence of basic condensing agents like sodium amide. The general procedure for preparing the N-alkyl derivatives of formula (1) consists of the condensation of phenoxazine with the appropriate α, ω- di-alkylhalide in such as Cl-(CH₂)_b-Br wherein b is 1 to

6, in the presence of sodium amide, either in liquid ammonia or in an anhydrous solvent such as toluene or benzene. For instance, the reaction of phenoxazine with mixed chlorobromoalkanes in the presence of sodium amide gives reactive N-chloroalkylphenoxazines, which can then be converted to the desired compound by reaction with an intermediate of the formula H-(CH₂)_b-A wherein b and

A have the meanings set forth above.

More specifically, compounds such as those described in Examples 1-14 below can be prepared by first alkylating phenoxazine with 1-bromo-3- chloropropane or 1-bromo-4-chloropropane to produce 10-

(3'-chloropropyl) phenoxazine or 10-(4'- chlorobutyl)phenoxazine, alkylation being accomplished by first converting phenoxazine to the anionic species using the strong base, sodium amide. Iodide-catalyzed nucleophilic substitution of the propyl or butyl

chloride with various secondary amines (e.g. N,N- diethylamine, N,N-diethanolamine, morpholine,

piperidine, pyrrolidine and β-hydroxyethyl-piperazine) by refluxing for about 20 hours with potassium carbonate in anhydrous acetonitrile affords the free bases of formula (1).

The acyl derivatives of formula ( 1) can be synthesized by acylating phenoxazine with a compound of the formula Cl-C(O)-C(CH₂)_0-6-Cl and then reacting the product with an amine of the formula H-A, wherein A has the meaning given above in anhydrous acetonitrile containing potassium iodide. The haloacetylphenoxazine can be prepared by reacting phenoxazine with the

anhydride (C(halo)₃CO)₂O.

All the compounds described in Examples 1-14 were separated and purified by column chromatography or recrystallization and dried under high vacuum. The structures were established by UV-, IR, ¹H- and ¹³C-NMR and EIMS spectral data, and by elemental analyses. The physical properties of the compounds are given in Table

I. The UV-spectral data of N-substituted phenoxazines are in close agreement with the spectral characteristics of analogous heterocycles. The IR bands also indicate the presence of characteristic functional groups, and peaks at 1670-1695 cm^-1 indicated the presence of >C=O group in the acyl derivatives. The ¹H-NMR in CDCl₃, typical of phenoxazine compound, showed eight aromatic protons and the data are in accordance with the

structures assigned. The assignment of protons is fully supported by the integration curves. The ¹³C-NMR spectrum of each N-substituted phenoxazine exhibitedsize signals representing 12 aromatic carbons. The GC-

Mass spectrum showed an intense molecular ion peak (M⁺) for each of the compounds characteristic of the

phenoxazine type of structure. The spectral data are consistent with the assigned structures.

SYNTHESIS AND ANALYSIS

In the syntheses and experiments described below, melting points were recorded on a Perkin-Elmer

Model 1320 spectrophotometer, as KBr pellets; UV-spectra were recorded in MeOH on a Perkin-Elmer Lambda 3B spectrophotometer. Elemental analyses were performed and found values within 0.4% of theoretical, unless otherwise noted. Reactions were monitored by tic. For tlc, Analtech silica gel GF plates (20 x 20 cm, 250 microns, glass-backed), with petroleum ether- ethylacetate (9.7:0.3 by volume, system A), and

ethylacetate-methanol (9.9:0.1 by volume, system B) as solvents were used. Column chromatography utilized silica gel Merc grade 60 (230-400 mesh, 60Å). ¹H- and

¹³C-NMR spectra were recorded in CDCl₃ solution in a 5- mm tube on an IBM NR 200 AF Fourier transform

spectrometer with tetramethylsilane as internal

standard. Chemical shifts are expressed as "δ" (ppm) values. The spectrometer was internally locked to the deuterium frequency of the solvent. Electron-impact mass spectra (EIMS) were recorded on a Ribermag R10-10C

GC-mass spectrometer with an upper mass limit of 1500

AMU. All chemicals and supplies were obtained from standard commercial sources unless otherwise indicated.

Phenoxazine, secondary amines indicated in the text, and anhydrous organic solvents were purchased from Aldrich

Chemical Co. (Milwaukee, WI). Vincristine sulfate

(oncovin) was purchased from Eli Lilly and Co.

(Indianapolis, IN), and vinblastme sulfate was from

Cetus Corporation (Emeryville, CA). [G-³H]vincristine

(sp. act. (specific activity) 7.1 Ci/mmol), and [G-

³H]vinblastine (sp. act. 10.1 Ci/mmol) were obtained from Amersham Corporation (Arlington Heights, IL).

Verapamil hydrochloride, colchicine, RPMI-1640 medium, powder with glutamine and without sodium bicarbonate were purchased from the Sigma Chemical Co. (St. Louis,

MO).

The synthesis of representative compounds of formula (1) is described below. Each of the indicated compounds in these Examples is considered a preferred embodiment of the present invention.

EXAMPLE 1

10-(3'-chloropropyl)-phenoxazine. To a suspension of sodium amide (1.72 g) in 100 ml of liquid ammonia, 7g (0.038 mol) of phenoxazine was added. After stirring for 30 minutes, 6.3 g (0.04 mol., 3.96 mL) of 1-bromo-3-chloropropane was added slowly with constant stirring. After one more hour, ammonia was allowed to evaporate and solid ice pieces were added carefully followed by cold water. When the reaction ceased, the mixture was extracted three times with ether. The ether solution was washed three times with water, dried over anhydrous sodium sulfate and evaporated. The residue was chromatographed on silica gel. Petroleum ether- ethylacetate (9 mL + 3 mL) eluted the pure title

compound (7.94 g) as white crystals. VU-λ_{ma x} (MeOH)

218, 238 and 321 nm; IR (KBr) 3070, 2860, 1630, 1490,

1380, 1275, 920, 815 and 740 cm^-1-; ¹H-NMR (δ) 6.47-6.82

(m, 8H, ArH, H₁-H₄ and H₆-H₉), 2.11 (m, 2H, H₁), 3.63

(m, 2H, H_k), and 3.69 (m, 2H, H_m) ; ¹³C-NMR (¹H

decoupled) 111.23 (C₁ and C₉), 115.50 (C₄ and C₆),

121.07 (C₃ and C₇ ) , 123.70 (C₂ and C₈), 133.03 (C₉, and

C₉, ), 144.92 (C₄, and C₆,), 27.82 (C₁), 41.09 (C_k) and

42.63 (C_m); EIMS (m/z) 259 (M⁺).

EXAMPLE 2

10- (3'-diethylaminopropyl)phenoxazine. lg

(4.31 mmol) of the product of Example 1 was dissolved in

150 mL of anhydrous acetonitrile, and 1.5 g KI, 2.13 gK₂CO₃ and 1.6 mL (15.4 mmol) of N,N-diethylamine were added. The mixture was refluxed overnight until a substantial amount of product was formed (TLC, System B,

R_f = 0.40). The reaction mixture was diluted with water and extracted with ether three times. The ether layer was washed with water and dried over anhydrous Na₂SO₄ and evaporated. The crude oil was subjected to column chromatography for purification. Ethylacetate-petroleum ether (50 mL + 50 mL) eluted the title compound as the free base as a colorless oil, which was dried and used for NMR studies. An ethereal solution of the free base was treated with an excess of tartaric acid to separate the hygroscopic tartrate salt (1.2 g). UV-λ_{ma x} (MeOH)

215, 238 and 320 nm; IR (CHCl₃) 3378, 2974, 2838, 1453,

1375, 1155, 973 and 722 cm^-1; ¹H-NMR ('δ') 6.51-6.80 (m,

8H, ArH, H₁-H₄ and H₆-H₉), 1.16 (t, 6H, H_c and H_d), 1.70

(m, 2H, H₁), 2.50 (q, 4H, H_a and H_b, J=7 Hz), 3.42-3.63

(m, 4H, H_k and H_m); ¹³C-NMR 111.54 (C₁ and C₉ ) , 115.49

(C₄ and C₆), 121.21 (C₃ and C₇ ) , 123.85 (C₂ and C₈),

132.72 ( C₁ , and C₉, ), 144.95 (C₄, and C₆,), 8.21 (C_c and

C_d) , 19.90 (C₁), 40.72 (C_a and C_b), 45.87 (C_m), and

48.50 (C_k); EIMS (m/z) 296 (M⁺).

EXAMPLE 3

10-(3'-bishydroxyethylaminopropyl)phenoxazine.

The procedure used for Example 2 was repeated with lg,

(4.31 mmol) of the product of Example 1, 1.5 g KI, and1.62 g (15.4 mmol, 1.5 mL) of diethanolamine.

Recrystallization of the solid in ethylacetate and petroleum ether gave (1.14 g) of the title compound in the pure form. UV-λ_max (MeOH) 218, 239, and 322 nm; IR

(KBr) 3300, 2960, 2880, 1590, 1490, 1440, 1375, 1270,

1190, 1125, 1075, 1040, 890, 840, and 740 cm^-3; ¹H-NMR

('δ') 6.44-6.78 (m, 8H, ArH, H₁-H₄ and H₆-H₉), 1.71-1.82

(m, 2H, H₁), 2.54-2.61 (t, 4H, H_a and H_b, J = 6 Hz),

3.39 - 3.68 (m, 8H, H_k, H_c; and H_d and H_m), and 2.95

(s,H_e and H_f, disappearing on D₂O exchange); ¹³C-NMR111.37 ( C₁ and C₉), 115.33 (C₄ and C₆), 120.80 (C₃ and

C₇), 123.66 (C₂ and C₈), 133.25 (C₁, and C₉ , ) , 144.99

(C₄, and C₆,), 22.42 ( C₁ ) , 41.83 (C_a and C_b), 52.38

(C_m), 55.91 (C_k) and 59.64 (C_c and C_d); EIMS (m/z) 328

(M⁺).

EXAMPLE 4

10-(3'-N-morpholinopropyl)phenoxazine. The procedure used for Example 2 was repeated with 1g of the product of Example 1, 1.5 g KI, 2.0 g K₂CO₃ and 1.4 g (15.40 mmol, 1.34 mL) of morpholine. The oily residue was purified by column chromatography to give the title compound as a brown oil. An ethereal solution of the free base was treated with ethereal hydrochloride to give the hydro-chloride salt (1.07 g). UV-λ_max (MeOH) 216, 239, and 320 nm; IR (KBr) 3200, 1495, 1380, 1280, 1230, 1135, 1100, 1050, 1020, 980, 870, 830, 760 and 735 cm^-1; ¹H-NMR ('δ') 6.63-6.81 (m, 8H, ArH, H₁-H₄ and H₆- H₉), 1.78 (m, 2H, H₂), 2.40 (t, 4H, H_a and H_b, J = 12 Hz), 3.45-3.80 (m, 8H, K_k, H_m, H_c and H_d); ¹³C-NMR

111.64 (C₁ and C₉), 115.80 (C₄ and C₆), 121.59 (C₃ and C₇), 123.91 (C₂ and C₈), 133.50 ( C₁ , and C₉.), 145.11 (C₄, and C₆,), 20.06 (C₁), 40.93 (C_a and C_b), 51.91 (C_m), 55.20 (C_k), and 63.50 (C_c and C_d); EIMS (m/z) 310 (M⁺).

EXAMPLE 5

10-(3'-N-piperidinopropyl)phenoxazine. The procedure used for Example 2 was used with 1.12 g (4.31 mmol) of the product of Example 1, 1.5 g IH, 2.4 g K₂CO₃and 1.5 g (17.62 mmol, 1.74 mL) of piperidine. The product was chromatographed on silica gel with petroleum ether-ethylacetate (1:1 by volume) to obtain the pure title compound in the form of an oil. By adding

ethereal hydrochloride to the ether solution of the free base, the hydrochloride salt (1.15 g) was obtained. UV-

*_max (MeOH) 218, 238 and 320 nm; IR (KBr) 3300, 2940,

2680, 1595, 1495, 1385, 1275, 1160, 1050, 825 and 745 cm^-1; ¹H-NMR ('δ') 6.56-6.86 (m, 8H, ArH, H₁-H₄ and H₆-

H₉), 1.53 (m, 6H, H_c, H_d and H_e), 2.30 (m, 2H, H₁),

2.56-2.67 (m, 4H, H_a and H_b), and 3.45-3.70 (m, 4H, H_k and H_m); ¹³C-NMR 111.65 (C₁ and C₉), 115.62 (C₄ and C₆),

121.38 (C₃ and C₇ ) , 123.88 (C₂ and C₈), 132.73 (C₁, and

C₉,), 144.98 (C₄, and C₆,), 20.21 (C_e), 21.93 (C_c and

C_d) , 22.50 (C₁), 41.05 (C_a and C_b), 53.18 (C_m), and

54.62 (C_k); EIMS (m/z) 308 (M⁺).

EXAMPLE 6

10-(3'-β-hydroxyethylpiperazinopropyl) phenoxazine. The procedure used for Example 2 was repeated with 1 g (4.31 mmol) of the product of Example

1, 1.5 g KI, 2.12 g K₂CO₃ and 2 g (15.4 mmol, 1.9 mL) of β-hydroxyethylpxperazxne. The free base was

recrystallized in petroleum ether-ether mixture (7:3 by volume) to give 1.16 g of the title compound. W-λ_max

(MeOH) 217, 239 and 322 nm; IR (KBr) 3060, 2820, 1630,

1595, 1495, 1385, 1270, 1160, 1070, 980, 850, 810 and

735 cm^-1; ¹H-NMR ('δ') 6.46-6.76 (m, 8H, ArH, H₁-H₄ and

H₆-H₉), 1.74 (m, 2H, H₁), 2.33-2.80 (M, 12H, H_a and H_b,

H_c and H_d, H_e and H_m), 2.79 (s, 1H, Hg, disappearing on

D₂O exchange), 3.47-3.65 (m, 4H, H_k and H_f); ¹³C-NMR

111.34 (C₁ and C₉), 115.24 (C₄ and C₆), 120.66 (C₃ and

C₇), 123.50 (C₂ and C₈), 133.30 (C₁, and C₉ ,), 144.83

(C₄, and C₆,), 22.58 (C₁), 41.72 (C_m), 52.96 (C_a and

C_b), 53.28 (C_c and C_d) , 55.19 (C_k); 57.77 (C_e), and

59.34 (C_f); MS (m/z) 353 (M⁺).

EXAMPLE 7

10-(3'-N-pyrrolidinopropyl)phenoxazine. The procedure used for Example 2 was repeated with lg of the title product of Example 1, 1.5 g KI, 2g K₂CO₃ and 1.1g (15.5 mmol, 1.3 mL) of pyrrolidine. The product was purified by column chromatography and the oil was converted into the hydrochloride salt (1.02g). W-λ_max

(MeOH) 217, 239, and 319 nm; IR (KBr) 3300, 2660, 1590,

1490 , 1375 , 1270 , 1130 , 920 , 820 and 745 cm^-1 ; ¹H-NMR

('δ') 6.46-6.77 (m, 8H, ArH, H₁-H₄ and H₆-H₉), 2.01-2.17

(t, 4H, H_c and H_d, J = 13 Hz), 2.21 (m, 2H, H₁), 3.06-

3.14 (t, 4H, H_a and H_b), and 3.60-3.67 (m, 4H, H_k and

H_m); ¹³C-NMR 111.60 (C₁ and C₉), 115.66 (C₄ and C₆),

121.40 (C₃ and C₇), 123.85 (C₂ and C₈), 132.73 (C₁, and

C₉,), 144.98 (C₄, and C₆,), 22.25 (C_c and C_d) , 23.30

(C₁), 40.90 (C_a and C_b), 52.80 (C_m), and 53.63 (C_k); MS

(m/z) 294 (M⁺).

EXAMPLE 8

10-(4'-chlorobutyl)phenoxazine, (8.4 g) in the pure form was prepared following the procedure used for

Example 1 with 7g phenoxazine, 1.63 g sodium amide and4.36 mL of 1-bromo-4-chlorobutane (0.038 mol) to produce the title compound. W-λ_max (MeOH) 200, 212, 238, and

320 nm; IR (KBr) 3060, 2980, 1630, 1590, 1495, 1380,

1280, 1130, 915, 840 and 730 cm^-1; ¹H-NMR ('δ') 6.36-

6.74 (m, 8H, ArH, H₁-H₄ and H₆-H₉), 1.75 (broad, 4H, H₁ and H_m), and 3.38-3.50 (m, 4H, H_k and H_n), ¹³C-NMR

111.43 (C₁ and C₉), 115.53 (C₄ and C₆), 121.01 (C₃ and

C₇ ) , 123.83 (C₂ and C₈), 133.27 (C₁, and C₉,), 145.10

(C₄, and C₆,), 22.60 (C_m), 29.87 ( C₁ ), 43.27 (C_k), and

44.61 ( C_n ) ; EIMS (m/z) 273 (M⁺).

EXAMPLE 9

10-(4'-diethylaminobutyl)phenoxazine. The procedure used for Example 2 was followed with lg (3.65 mmol) of the product of Example 8, 1.5g KI, 2g K₂CO₃ and1.07 g (14.63 mmol, 1.5 mL) of N,N-diethylamine to obtain the indicated product. The oily product was chromato-graphed on the silica gel with CH₃OH-CHCl₃

(3:1) and the hydrochloride salt (.076g) was obtained in the pure form. W-λ_max (MeOH) 201, 213, 239 and 320 nm;

IR (KBr) 3300, 2940, 1590, 1495, 1380, 1270, 1130, 1040,

925 and 750 cm^-1; ¹H-NMR ('δ') 6.47-6.80 (m, 8H, ArH,

H₁-H₄ and H₆-H₉), 1.33 (broad, 6H, H_c and H_d), 1.66-1.91

(m, 4H, H₁ and H_m), 3.05 (very broad, 6H, H_a, H_b and

H_n), and 3.50 (m, 2H, H_k); ¹³C-NMR 111.51 (C₁ and C₉),

115.31 (C₄ and C₆), 120.99 (C₃ and C₇), 123.75 (C₂ and

C₈), 132.78 ( C₁ , and C₉,), 144.78 (C₄, and C₆,), 8.54

(C_c and C_d ) , 21.02 (C_m), 22.46 (C₁), 43.05 (C_a and C_b),

46.50 (C_n), and 51.26 (C_k); MS (m/z) 310 (M⁺).

EX AMPLE 10

10-(4'-bishydroxyethylaminobutyl) phenoxazine, as its hydrochloride salt (l.llg) was obtained by following the procedure of Example 3 with lg of the product of Example 8, 1.5g KI and 1.54 g (14.65 mmol, 1.4 mL) of N,N-diethanolamine followed by column

chromato-graphy. W-λ_{ma x} (MeOH) 204, 210, 238 and 321 nm; IR (KBr) 3280, 2850, 1630, 1590, 1490, 1375, 1270,

1135, 1095, 1065, 1045, 1020, 925, 890, 845, and 740 cm-

¹; ¹H-NMR ('δ') 6.52-6.84 (m, 8H, ArH, H₁-H₄ and H₆-H₉),

1.70-1.98 (m, 4H, H₁, and H_m), 3.35-3.57 (broad, 10H,

H_a, H_b, H_n, H_k, H_e and H_f), 3.95 (t, 4H, H_c and H_d; J =

7 Hz), and 10.3 (H⁺); ¹³C-NMR 110.53 (C₁ and C₉), 114.17

(C₄ and C₆), 119.83 (C₃ and C₇), 122.76 (C₂ and C₈),

131.85 (C₁, and C₉,), 143.60 (C₄, and C₆,), 19.98 (C_m),

21.10 (C₁), 42.06 (C_n), 52.92 (C_a and C_b), 54.78 (C_k), and 54.96 (C_c and C_d); EIMS (m/z) 342 (M⁺).

EXAMPLE 11

10-(4'-N-morpholinobutyl)phenoxazine. The procedure used for Example 4 was repeated with 1 g of the product of Example 8, 1.5g KI, 2g of K₂CO₃ and 1.273 g (14.61 mmol, 1.3 mL) of morpholine. The product was recrystallized in ether-petroleum ether mixture (3:1) to give the title compound (0.95g). W-λ-_max 202, 213,

239, and 321 nm; IR (KBr) 2960, 2810, 1630, 1595, 1495,

1380, 1295, 1220, 1130, 1070, 1010, 970, 920, 870, 855,

825, 765 and 745 cm^-1; ¹H-NMR ('δ') 6.53-7.29 (m, 8H,

ArH, H₁-H₄ and H₆-H₉), 1.61-1.74 (m, 4H, H₁ and H_m),

2.40-2.50 (m, 6H, H_a, H_b, and H_n), 3.49 (m, 2H, H_k), and

3.49-3.78 (t, 4H, H_c and H_d, J = 12 Hz); ¹³C-NMR 111.28

(C₁ and C₉), 115.28 (C₄ and C₆), 120.67 (C₃ and C₇),

123.52 (C₂ and C₈), 133.30 (C₁, and C₉,), 144.99 (C₄, and C₆,), 22.34 (C_m), 23.50 (C₁), 43.63 (C_n), 53.67 (C_a and C_b), 57.91 (C_k), and 66.97 (C_c and C_d); EIMS (m/z)

324 (M⁺).

EXAMPLE 12

10-(4'-N-piperidinobutyl)phenoxazine. 1g of the product of Example 8, 1.5g of KI, 2g K₂CO₃ and 1.45g

(17.03 mmol, 1.5 mL) of piperidine were refluxed and processed according to the procedure used for Example 10. Purification by column chromatography afforded the free amine as a brown oil which was converted into the hydrochloride salt (1.18 g) . W-λ_mΛX 203, 210, 238, and

320 nm; IR (KBr) 3320, 2940, 1625, 1590, 1490, 1380,

1270, 1130, 1060, 955, 840, 820, and 730 cm^-1; ¹H-NMR

('δ') 6.42-6.81 (m, 8H, ArH, H₁-H₄ and H₆-H₉), 1.44-1.82

(m, 6H, H_c, H_d and H_e), 1.98-21.8 (m, H₁ and H_m), 2.70-

2.97 (m, 4H, H_a and H_b), 3.39-3.45 (m, 4H, H_k and H_n) and 11.54 (H⁺); ¹³C-NMR 111.42 (C₁ and C₉), 115.32 (C₄ and C₆), 120.98 (C₃ and C₇), 123.71 (C₂ and C₈), 132.78

(C₁, and C₉,), 144.73 (C₄, and C₆,), 20.96 (C_e), 21.79

(C_c and C_d), 22.48 (C_l and C_m), 43.08 (C_a and C_b), 52.91

(C_n), and 56.70 (C_k); EIMS (m/z) 322 (M⁺).

EXAMPLE 13

10-(4'-β-hydroxyethylpiperazinobutyl) phenoxazine. The procedure used for Example 6 was repeated with 1 g of the product of Example 8, 1.5g KI, and 1.9g (14.6 mmol, 1.8 mL) of β- hydroxyethylpxperazx.ne. The ox.ly resx.due was treated with 500 μl of ethylacetate first and then with

petroleum ether (20 mL), when a white crystalline solid separated out. The solid was recrystallized to give the pure title compound (1.21g). W-λ_max (MeOH) 202, 239, and 320 nm; IR (KBr) 3060, 2940, 2860, 1590, 1495, 1380,

1225, 1135, 1020, 1005, 935, 880, 830, 780, and 740 cm-

¹; ¹H-NMR ('δ') 6.46-6.75 (m, 8H, ArH, H₁-H₄ and H₆ and

H₉), 1.58 (broad, 4H, H₁ and H_m), 2.36-2.51 (m, 12H, H_a,

H_b, H_c, H_d, H_e and H_n), 3.42 (broad, 3H, H_k, and H_g), and 3.58-3.63 (t, 2H, H_f, J = 7 Hz); ¹³C-NMR 111.39 (C₁ and C₉), 115.26 (C₄ and C₆), 120.64 (C₃ and C₇), 123.61

(C₂ and C₈), 133.30 (C₁, and C₉,), 144.95 (C₄, and C₆,),

22.28 (C_l and C_m), 23.72 (C_n), 43.60 (C_a and C_b), 53.11

(C_c and C_d) , 57.38 (C_k), 57.96 (C_e) and 59.76 (C_f); EIMS

(m/z) 367 (M⁺).

EXAMPLE 14

10-(4'-N-pyrrolidinobutyl)phenoxazine. The experimental steps used for Example 2 were repeated using 1g of the product of Example 8, 1.5g KI, 2g K₂CO₃ and 1.04g (14.6 mmol, 1.22 mL) of pyrrolidine as reactants. The product was chromatographed on silica gel with CHCl₃-MeOH (1:1) to give the free amine as a brown oil. An ether solution of this oil was treated with ethereal hydrogen chloride to secure the pure

(0.9g) hydrochloride salt. W-λ_max (MeOH) 205, 211, 238 and 320 nm; IR (KBr) 3060, 2840, 1590, 1495, 1380, 1295,

1270, 1160, 1090, 1045, 915, 840, 830, 795, and 740 cm-

¹; ¹H-NMR ('δ') 6.43-6.79 (m, 8H, ArH, H₁-H₄ and H₆-H₉),

1.64-2.10 (m, 8H, H₁, H_m, H_c and H_d), 2.97-3.17 (m, 6H,

H_a, H_b and H_n), 3.45-3.54 (m, 2H, H_k) and 10.10 (H⁺);

¹³C-NMR 111.43 ( C₁ and C₉), 115.41 (C₄ and C₆), 121.01

(C₃ and C₇), 123.73 (C₂ and C₈), 132.89 (C₁, and C₉,),

144.87 (C₄, and C₆,), 22.47 (C_c and C_d) , 23.27 (C₁ and

C_m), 43.14 (C_a and C_b), 53.50 (C_n), and 54.91 ( C_k ); EIMS

(m/z) 308 (M⁺).

EXAMPLE 15

10-(chloroacetyl)phenoxazine. To a solution of 5g (0.03 mol) of phenoxazine dissolved in 100 mL anhydrous acetonitrile containing 10 mL of anhydrous ether, was added dropwise 7 mL (9.926 g, 0.088 mol) of chloroacetyl-chloride with constant stirring. The reaction mixture was stirred at room temperature for 5H when white crystalline solid separated out (TLC, system

A, R_f=0.030). The crystals were filtered, washed several times with petroleum ether-ether mixture (9:1) and dried under high vacuum to get 6.03g of the product.

UV-λ_max (MeOH) 218, 249, and 287 nm; IR (KBr) 3070,

1675, 1580, 1480, 1410, 1350, 1260, 1210, 1115, 1040,

860, 815, 750 and 660 cm^-1; ¹H-NMR ('δ') 7.55-7.61 (m,

2H, ArH, H₁ and H₉), 7.12-7.25 (m, 6H, ArH, H₂-H₄ and

H₆-H₈), 4.32 (s, 2H, H₁); ¹³C-NMR 110.04 (C₁ and C₉),

117.11 (C₄ and C₆), 123.75 (C₃ and C₇), 124.32 (C₂ and

C₈), 127.60 (C₁, and C₉,), 150.95 (C₄, and C₆,), 41.51

(C₁), and 170 (C_k); EIMS (m/z) 259 (M⁺).

EXAMPLE 16

10-(diethylaminoacetyl)phenoxazine. 1g (3.9 mmol) of the product of Example 15 was dissolved in 150 mL of anhydrous acetonitrile and 1.5g of KI and 1.13 g (15.45 mmol, 1.6 mL) of N,N-diethylamine were added to it. The reaction mixture was refluxed for In when substantial amount of the product was formed (TLC, system B, R_f=0.40). The mixture was processed as in

Example 2 to get a white crystalline solid which was further recrystallized in ethylacetate and petroleum ether mixture to get the pure compound (0.86g). UV-λ_max

(MeOH) 220, 246, and 287 nm; IR (KBr) 2800, 1685, 1580,

1480, 1320, 1210, 1150, 1060, 1035, 940, 860, 810, 755 and 670 cm^-1; ¹H-NMR ('δ') 7.53-7.59 (m, 2H, ArH, H₁ and

H₉), 7.05-7.20 (m, 6H, ArH, H₂-H₄ and H₆-H₈), 0.95 (t,

6H, H_c and H_d, J=7 Hz), 2.60 (q, 4H, H_a and H_b), and

3.55 (s, 2H, H₁); ¹³C-NMR 116.79 (C₁ and C₉), 123.31 (C₄ and C₆), 125.02 (C₃ and C₇), 126.82 (C₂ and C₈), 129.62

(C₁, and C₉,), 151.07 (C₄, and C₆,), 12.08 (C_c and C_d) ,

47.04 (C_a and C_b), 54.99 (C₁), and 169.84 (C_k); MS (m/z)

296 (M⁺).

EXAMPLE 17

10-(N-morpholinoacetyl)phenoxazine. The same procedure used for Example 16 was employed with 1g of the product of Example 15, 1.5g KI and 1.347g (16 mmol,1.4 mL) of morpholine. The solid product was

recrystallized in a mixture of ethylacetate, petroleum ether and ether and the free base was converted into hydrochloride salt (1.07g) using ethereal hydrochloride.

W-λ_max 213, 246, and 287 nm; IR (KBr) 2980, 2860, 1690,

1485, 1440, 1355, 1270, 1180, 1120, 1070, 1005, 900,

870, 855, 760 and 640 cm^-1; ¹H-NMR ('δ') 7.60 (broad,

2H, ArH, H₁ and H₉), 7.12-7.34 (m, 6H, ArH, H₂-H₄ and

H₆-H₈), 2.40-2.60 (t, 64, H_a and H_b, J=12 Hz), 3.35 (s,

2H, H₁) and 3.50-3.70 (t, 4H, H_c and H_d); ¹³C-NMR 117.03 (C₁ and C₉), 123.90 (C₄ and C₆), 124.98 (C₃ and C₇),

126.95 (C₂ and C₈), 127.91 (C₁, and C₉,), 150.54 (C₄, and C₆,), 52.41 (C_a and C_b), 57.01 (C₁), 63.23 (C_c and

C_d), and 163.40 (C_k); MS (m/z) 310 (M⁺).

EXAMPLE 18

10-(N-piperidinoacetyl)phenoxazine. The method employed for Example 17 was used with 1g of the product of Example 15, 1.5g KI and 1.31g (15.4 mmol,1.52 mL) of piperidine to get 0.95g of the title compound. W-λ_max (MeOH) 218, 246 and 287 nm; IR (KBr)

2960, 1670, 1610, 1580, 1480, 1370, 1330, 1260, 1190,

1120, 1040, 940, 890, 855, 810, 765, and 655 cm^-1; ¹H-

NMR ('δ') 7.57-7.61 (m, 2H, ArH, H₁ and H₉), 7.12-7.16

(m, 6H, ArH, H₂-H₄ and H₆-H₈), 1.51 (very broad, 6H, H_c,

H_d and H_e), 2.44 (m, 4H, H_a and H_b) and 3.34 (s, 2H,

H₁); ¹³C-NMR 116.72 (C₁ and C₉), 123.28 (C₄ and C₆),

124.97 (C₃ and C₇), 126.79 (C₂ and C₈), 129.48 (C₁, and

C₉,), 151.01 (C₄, and C₆ '), 23.92 (C_e), 25.93 (C_c and

C_d) , 54.15 (C_a and C_b), 60.80 (C₁), and 168.92 (C_k);

EIMS (m/z) 308 (M⁺).

EXAMPLE 19

10-(β-hydroxyethylpiperazinoacetyl) phenoxazine. The procedure used for Example 17 was repeated with lg of the product of Example 15, 1.5g KIand 2g (15.4 mmol, 1.9 mL) of β-hydroxyethylpiperazine. Recrystallization of the whxte solxd yielded 1.17 g of the title compound. UV_max (MeOH) 213, 246 and 287 nm;

IR (KBr) 3200, 2940, 1685, 1665, 1480, 1265, 1190, 1160,

945, 855, 765 and 640 cm^-1; ¹H-NMR ('δ') 7.53-7.58 (m,

2H, ArH, H₁ and H₉), 7.08-7.25 (m, 6H, ArH, H₂-H₄ and

H₆-H₈), 2.48 (m, 10H, H_a, H_b, H_c, H_d and H_e), 2.70 (s,

1H, H_g, disappearing on D₂O exchange), 3.39 (s, 2H, H₁) and 3.60 (t, 2H, H_f, J=7 Hz); ¹³C-NMR 116.85 (C₁ and

C₉), 123.34 (C₄ and C₆), 124.86 (C₃ and C₇), 126.99 (C₂ and C₈), 129.25 (C₁, and C₉,), 151.04 (C₄, and C₆,),

52.70 (C_a and C_b), 52.90 (C_c and C_d) , 57.70 (C_g), 59.23

(C₁), 59.80 (C_f), and 168.43 (C_k); EIMS (m/z) 353 (M⁺).

EXAMPLE 20

10-(N-pyrrolidinoacetyl)phenoxazine. The experimental procedure used for Example 17 was employed with 1g of the product of Example 15, 1.5g KI and 1.095g (15.4 mmol, 1.3 mL) of pyrrolidine. Purification by recrystallization afforded 1.02 g of the title compound.

UV-λ_max (MeOH) 214, 240, and 286 nm; IR (KBr) 2980,

2820, 1695, 1670, 1480, 1455, 1340, 1270, 1180, 1100,

1040, 985, 905, 855, 755 and 640 cm^-1; ¹H-NMR ('δ')

7.58-7.63 (m, 2H, ArH, H₁ and H₉), 7.07-7.18 (m, 6H,

ArH, H₂-H₄ and H₆-H₈), 1.77 (t, 4H, H_c and H_d, J=7 Hz),

2.64 (t, 4H, H_a and H_b) and 3.51 (s, 2H, H₁); ¹³C-NMR

116.80 (C₁ and C₉), 123.33 (C₄ and C₆), 125.06 (C₃ and

C₇), 126.85 (C₂ and C₈), 129.28 (C₁, and C₉,), 151.00

(C₄, and C₆,), 23.73 (C_c and C_d), 53.83 (C_a and C_b),

57.24 (C₁), and 168.92 (C_k); EIMS (m/z) 294 (M⁺).

EXAMPLE 21

10-(trifluoroacetyl)phenoxazine. To a solution of 200 mg of phenoxazine in 10 mL anhydrous chloroform and 4 mL anhydrous ether, was added 50 μl of (0.7435g, 3.54 mmol) trifluoroacetic anhydride. The resulting mixture was stirred at room temperature for 8 hours. The formation of the product was monitored by

TLC (system A). The product solution was then extracted with chloroform and evaporated. The residue was

subjected to column chromatography which afforded the pure title compound. W-λ_max (MeOH) 212, 238, and 252 nm: IR (KBr) 3375, 1695, 1580, 1480, 1455, 1390, 1290,

1170, 1110, 1030, 965, 890, 850, 800, 760, 730, and 670 cm^-1; ¹H-NMR ('δ') 7.57-7.61 (m, 2H, ArH, H₁ and H₉), 7.14-7.32 (m, 6H, ArH, H₂-H₄ and H₆-H₈); ¹³-C-NMR 117.20

(C₁ and C₉), 123.83 (C₄ and C₆), 124.34 (C₃ and C₇),

128.34 (C₂ and C₈), 151.04 (C₁. and C₉., and C₄. and

C₆.), and >200 ppm (C_k and C₁); EIMS (m/z) 279 (M⁺).

The potentiating agent is preferably

administered by infusion in solution in sterile water.

The potentiating agents as hydrochloride salts can be dissolved in sterile water. The agents as bases can be solubilized in IN hydrochloric acid, following which the solution is back titrated with sodium hydroxide to provide a final pH between 7 and 8.

Cytotoxic agents whose cytotoxicity would be potentiated by agents within the scope of this invention include VCR, VLB, doxorubicin, colchicine, actinomycin D, daunomycin, M-AMSA, and other anthracyclic compounds.

The potentiating agent is administered to tumor cells which are exposed to one or more cytotoxic agents. By "exposed" is meant that the cytotoxic agent has been administered simultaneously with the

potentiating agent, and/or is administered subsequently to the administration of the potentiating agent, so long as at least some of the cytotoxic agent(s) is present in the tumor cell when the potentiating agent is present in the tumor cell. The cytotoxic agent should not be administered before the potentiating agent. Preferably, the cytotoxic agent is administered when the

potentiating agent concentration reaches steady state during administration by infusion.

It will be recognized that the amount of potentiating agent to be administered will vary between hosts, between cytotoxic agents and between potentiating agents, but the effective amounts can readily be

ascertained by those of ordinary skill in this field.

As guidance one can refer to the data in Examples 22-24 as well as the following Table. In general, though, effective amounts to potentiate cytotoxic agents are about 2000-3000 moles of potentiating agent per mole of

VCR; about 1,000-2,000 moles of potentiating agent per mole of VLB; and about 25-35 moles of potentiating agent per mole of VP-16 (Etoposide). These values, and thecorresponding values for any other cytotoxic agents, can readily be converted if desired into dosages per host body weight by calculation based on the dosages for the cytotoxic agent of interest. The in vitro techniques described herein can be employed to determine the effectiveness of any particular potentiating agent with any given cytotoxic agent or agents.

EX AMPLE 22

Table II below gives representative in vivo values of the molar ratios (shown below as "compound:

(cytotoxic agent)") of potentiating agent to cytotoxic agent for compounds within the scope of this invention. Vincristine (VCR) was administered to mice at 3 mg/kg

(3.25 μmol/kg); vinblastine (VLB) was at 5 mg/kg (5.5 μmol/kg); VP-16 (Etoposide) was at 50 mg/kg/day for 3 days (0.255 mmol/kg total). The compound number is the number of the example in which the potentiating agent was prepared.

EX AMPLE 23

Evaluation of N-substituted

Phenoxazines For Anti-MDR activity

A cloned line of human colon adenocarcinoma,GC₃/Cl³¹, which is intrinsically resistant to VCR (≈ 4- fold relative to KB-3-1), was routinely grown at 37°C in antibiotic-free RPMI-1640 medium supplemented with 2 mM glutamine and 10% FBS (Hyclone Laboratories, Inc.,

Logan, UT) in a humidified atmosphere of 5% CO₂ and 95% air. Human epidermoid carcinoma KB-3-1 cells and a colchicine selected MDR variant, KBCh^R-8-5, were

obtained which was cross-resistant to VCR (45-fold) and

VLB (6.3-fold); it was grown in monolayer culture at

37°C in DMEM with 10% FBS and L-glutamine in a

humidified atmosphere of 10% CO₂ in air. The resistance of the KBCh^R-8-5 cells was maintained by culturing them with colchicine (10 ng/ml).

Then, 2 mL of cell suspensions (2 x 10⁶) were plated in 35 x 10 mm style "easy grip" culture dishes

(Beσton Dickinson Co., Lincoln Park, NJ). Cells were allowed to attach to plastic overnight at 37°C. Medium was aspirated and cells were washed with (2 x 2 mL) physiologic tris (PT) buffer. Monolayers were incubated at room temperature for 10 minutes in PT buffer prior to aspiration and adding 1 mL of serum-free RPMI-1640 Hepes buffer (10.4g RPMI-1640 medium in IL of 25 mM Hepes, pH

7.4) containing 70.4 nm [³H] VCR (sp.act. 7.1 ci/mmol) or 49.5 nm [³H] VLB (sp. act. 10.1 Ci/mmol) with or without a compound of Examples 1-21 (100 uM) or VRP dissolved in H₂O dissolved in DMSO (final culture concentration <0.1% DMSO). After 2h of incubation at room temperature, medium was rapidly aspirated to terminate drug accumulation, and monolayers were washed four times with ice-cold PBS (g/L: NaCL 8.0;

Na₂HPO₄.12H₂O, 2.9; KCl 0.2; KH₂PO₄, 0.2) and drained.

To each dish, 1 ml of trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA) was added. After 1 minute, monolayers were triturated to give a uniform suspension of cells, and radioactivity in 0.75 ml was determined by scintillation counting. Cell number per dish was determined on 200 μl of suspension using the method of Butler, and amounts of intracellular VCR or VLB were determined. The results are set forth in Table III, in which the compound number is the number of the Example in which the compound (or

"modulator" or "potentiating agent") was prepared.

EXAMPLE 24

Evaluation of N-substituted Phenoxazines

Cytotoxicity To Tumor Cells

The KBCh^R-8-5 cells were plated in triplicate at a density of 1000 cells per well and GC₃ at 3000 cells per well in Falcon 6-well flat-bottom tissue culture plates (Becton Dickinson Co., Lincoln Park, NJ).

After 24h, incubation medium was replaced with 3 mL of fresh medium containing compounds 1-4 or 10-14 or 18 at concentrations ranging from 1-100 μm (final culture concentration, 0.1% DMSO), and cells were incubated at

37ºC for a further 7 days. The medium was aspirated and cells were washed once with 2 mL of 0.9% saline and dried overnight. Colonies were stained with 1 mL of

0.1% crystal violet followed by washing twice with distilled water and were counted using an automated

ARTEK Model 880 colony counter. The IC₅₀ values were determined from concentration-percent-cell-survival curves and were defined as the concentrations of

phenoxazines required for 50% reduction in colonies compared to controls. The results of these measurements are set forth in Table IV.

EXAMPLE 25

Effect Of N-substituted Phenoxazines On In Vitro Cytotoxicity of VLB And VCR

Tumor cells were treated with graded concentrations of VCR and VLB in the absence or presence of nontoxic concentrations of the products of Examples 1, 3, 4 and 18. The plates were then transferred to a CO₂ incubator and, after further incubation for 7 days at 37°C, colonies were enumerated as described in

Example 23. The results are set forth in Table V.

Claims

WHAT IS CLAIMED IS:

1. A method of potentiating the cytotoxicity of an agent cytotoxic to a tumor cell, comprising administering to said tumor cell, while it is exposed to said cytotoxic agent, a potentiating agent in an amount effective to potentiate the cytotoxicity of said

cytotoxic agent to said cell, wherein said potentiating agent comprises a compound of the formula (1):

or a pharmacologically acceptable salt thereof,

wherein R is -H or -[C(O)]_a-(CH₂)_b-A; wherein a is 0 or 1 and b is an integer from 0 to 6, provided that a and b are not both zero; and

alkylene having 1 to 4 carbon atoms, and Z is -O-,

-N(R₃)-or -CH(R₄)-, wherein R₃ is hydrogen or alkyl having 1 to 4 carbon atoms optionally substituted with a hydroxyl group, and wherein R₄ is hydrogen or alkyl having 1 to 4 carbon atoms optionally substituted with a hydroxyl group;

halide; and trihalomethyl.

2. The method of Claim 1 wherein said tumor cell is present in a living host.

3. The method of Claim 1 wherein said

cytotoxic agent is selected from the group consisting of vincristine, vinblastine, etoposide, doxorubicin, colchicxne, actinomycin D, daunomycin, m-AMSA, and mixtures thereof.

4. The method of Claim 1 wherein said tumor cell exhibits multiple drug resistance.

5. The method of Claim 1 wherein a is zero; b is 3 or 4; R₁ and R₂ are independently selected from the group consisting of ethyl, propyl, ω-hydroxyethyl, and ω-hydroxypropyl; X and Y are each independently selected from the group consisting of -CH₂- and -CH₂CH₂-; and R₃ and R₄ are independently selected from the group

consisting of -H, ethyl, propyl, ω-hydroxyethyl, and ω- hydroxypropyl.

6. The method of Claim 5 wherein said

potentiating agent is 10-(3'-chloropropyl)-phenoxazine,

10-(3'-diethylaminopropyl)-phenoxazine, 10-(3'- bishydroxyethylaminopropyl)-phenoxazine, 10-(3'-N- morpholinopropyl)-phenoxazine, 10-(3'-N- piperidinopropyl)-phenoxazine, 10-(3'-β- hydroxyethylpiperazinopropyl)-phenoxazine, (10-(3'-N- pyrrolidinopropyl)-phenoxazine, 10-(4'-chlorobutyl)- phenoxazine, 10-(4'-diethylaminobutyl)-phenoxazine, 10-

(4'-bishydroxyethylaminobuty1)-phenoxazine, 10-(4'-N- morpholinobutyl)-phenoxazine,10-(4'-piperidinobutyl)- phenoxazine, 10-(4'-β-hydroxyethylpiperazinobutyl)- phenoxazine, 10-(4'-N-pyrrolidinobutyl)-phenoxazine or pharmacologically acceptable salts thereof.

7. The method of Claim 1 wherein a is 1.

8. The method of Claim 7 wherein b is 1 or 2; R₁ and R₂ are independently selected from the group consisting of ethyl, propyl, ω-hydroxyethyl, and ω- hydroxypropyl; X and Y are each independently selected from the group consisting of -CH₂- and -CH₂CH₂-; and R₃ and R₄ are independently selected from the group

consisting of -H, ethyl, propyl, ω-hydroxyethyl, and ω- hydroxypropyl.

9. The method of Claim 8 wherein said

potentiating agent is 10-(chloroacetyl)-phenoxazine, 10- (diethylaminoacetyl)-phenoxazine, 10-(N- morpholinoacetyl)-phenoxazine, 10-(N-piperidinoacetyl)- phenoxazine, 10-(β-hydroxyethylpiperazinoacetyl)- phenoxazine, 10-(N-pyrrolidinoacetyl)-phenoxazine, 10-

(trifluoroacetyl)-phenoxazine or pharmacologically acceptable salts thereof.

10. A composition comprising a cytotoxic agent toxic to tumor cells, and a potentiating agent which potentiates the cytotoxicity of said cytotoxic agent, wherein said potentiating agent comprises a compound of the formula (1)

or a pharmacologically acceptable salt thereof,

wherein R is -H or -[C(O)]_a-(CH₂)_b-A;

alkylene having 1 to 4 carbon atoms, and Z is -O-, -

N(R₃)-or -CH(R₄)-, wherein R₃ is hydrogen or alkyl having 1 to 4 carbon atoms optionally substituted with a hydroxyl group, and wherein R₄ is hydrogen or alkyl having 1 to 4 carbon atoms optionally substituted with a hydroxyl group;

halide; and trihalomethyl;

wherein said cytotoxic agent and potentiating agent are present in amounts effective to render the composition cytotoxic to tumor cells.

11. The composition of Claim 10 wherein said cytotoxic agent is selected from the group consisting of vincristine, vinblastine, etoposide, doxorubicin, colchicine, actinomycin D, daunomycin, m-AMSA, and mixtures thereof.

12. The composition of Claim 10 wherein a is zero; b is 3 or 4; R₁ and R₂ are independently selected from the group consisting of ethyl, propyl, ω- hydroxyethyl, and ω-hydroxypropyl; X and Y are each independently selected from the group consisting of -

CH₂- and -CH₂CH₂-; and R₃ and R₄ are independently selected from the group consisting of -H, ethyl, propyl, ω-hydroxyethyl, and ω-hydroxypropyl.

13. The composition of Claim 12 wherein said potentiating agent is 10-(3--chloropropyl)-phenoxazine. 10-(3'-diethylaminopropyl)-phenoxazine, 10-(3'- bishydroxyethylaminopropyl)-phenoxazine, 10-(3'-N- morpholinopropyl)-phenoxazine, 10-(3'-N- piperidinopropyl)-phenoxazine, 10-(3'-β- hydroxyethylpiperazinopropyl)-phenoxazine, (10-(3'-N- pyrrolidinopropyl)-phenoxazine, 10-(4'-chlorobutyl)- phenoxazine, 10-(4'-diethylaminobutyl)-phenoxazine, 10-

(4'-bishydroxyethylaminobutyl)-phenoxazine, 10-(4'-N- morpholinobutyl)-phenoxazine,10-(4'-piperidinobutyl)- phenoxazine, 10-(4'-β-hydroxyethylpiperazinobutyl)- phenoxazine, 10-(4'-N-pyrrolidinobutyl)-phenoxazine or pharmacologically acceptable salts thereof.

14. The composition of Claim 10 wherein a is

1; b is 1 or 2; R₁ and R₂ are independently selected from the group consisting of ethyl, propyl, w- hydroxyethyl, and ω-hydroxypropyl; wherein X and Y are each independently selected from the group consisting of

-CH₂- and -CH₂CH₂-; and R₃ and R₄ are independently selected from the group consisting of -H, ethyl, propyl, ω-hydroxyethyl, and w-hydroxypropyl.

15. The composxtxon of Claxm 14 wherexn saxd potentiating agent is 10-(chloroacetyl))phenoxazine, 10- (diethylaminoacetyl)-phenoxazine, 10-(N- morpholinoacetyl)-phenoxazine, 10-(N-piperidinoacetyl)- phenoxazine, 10-(β-hydroxyethylpiperazinoacetyl)- phenoxazine, 10-(N-pyrrolxdxnoacetyl)-phenoxazxne, 10-

(trifluoroacetyl)-phenoxazine or pharmacologically acceptable salts thereof.

16. A method of killing a tumor cell which comprises administering to said cell a composition according to Claim 10 in an amount effectxve to kill said cell.

17. The method of Claim 16 wherein said tumor cell is present in a living host.

18. The method of Claim 16 wherein said tumor cell exhibits multiple drug resistance.

19. A compound of the formula (1)

and pharmacologically acceptable salts thereof,

wherein R is -[C(O)]_a-(CH₂)_b-A; wherein a is 0 or 1 and b is an integer from 0 to 6, provided that a and b are not both zero; and

halide; and trihalomethyl.

20. A compound or salt according to Claim 19 wherein a is zero; b is 3 or 4; R₁ and R₂ are independently selected from the group consisting of ethyl, propyl, w-hydroxyethyl, and ω-hydroxypropyl; X and Y are each independently selected from the group consisting of -CH₂- and -CH₂CH₂-; and R₃ and R₄ are independently selected from the group consisting of -H, ethyl, propyl, w-hydroxyethyl, and ω-hydroxypropyl.

21. The compound according to Claim 20 which is 10-(3'-chloropropyl)-phenoxazine, 10-(3'- diethylaminopropyl)-phenoxazine, 10-(3'- bishydroxyethylaminopropyl)-phenoxazine, 10-(3'-N- morpholinopropyl)-phenoxazine, 10-(3'-N- piperidinopropyl)-phenoxazine, 10-(3'-β- hydroxyethylpiperazinopropyl)-phenoxazine, (10-(3'-N- pyrrolidinopropyl)-phenoxazine, 10-(4'-chlorobutyl)- phenoxazine, 10-(4'-diethylaminobutyl)-phenoxazine, 10-

(4'-bishydroxyethylaminobutyl)-phenoxazine, 10-(4'-N- morpholinobutyl)-phenoxazine, 10-(4'-piperidinobutyl)- phenoxazine 10-(4'-β-hydroxyethγlpiperazinobutyl)- phenoxazine, 10-(4'-N-pyrrolidinobutyl)-phenoxazine or pharmacologically acceptable salts thereof.

22. A compound or salt according to Claim 19 wherein a is 1; b is 1 or 2; R₁ and R₂ are independently selected from the group consisting of ethyl, propyl, ω- hydroxyethyl, and w-hydroxypropyl; X and Y are each independently selected from the group consisting of -

CH₂- and -CH₂CH₂-; and R₃ and R₄ are xndependently selected from the group consisting of -H, ethyl, propyl, w-hydroxyethyl, and δ)-hydroxypropyl.

23. The compound according to Claim 22 which is 10-(chloroacetyl)-phenoxazine, 10-

(diethylaminoacetyl)-phenoxazine, 10-(N- morpholinoacetyl)-phenoxazine, 10-(N-piperidinoacetyl)- phenoxazine, 10-(β-hydroxyethylpiperazinoacetγl)- phenoxazine, 10-(N-pyrrolidinoacetyl)-phenoxazine, 10- (trifluoroacetyl)-phenoxazine or pharmacologically acceptable salts thereof.