antitumor activity of polycyano-substituted carbo

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M. D. Mashkovskii, Drugs [in Russian], RIA Novaya Volna,. Moscow (2007), pp. 981, 989, 990, 1013, 1014. 10. E. C. Taylor and R. W. Hendess, J. Am. Chem.
Pharmaceutical Chemistry Journal

Vol. 42, No. 12, 2008

ANTITUMOR ACTIVITY OF POLYCYANO-SUBSTITUTED CARBO- AND HETEROCYCLES PREPARED FROM 3-(2,2-DIALKYLHYDRAZINO)4-R-1,1,2,2-TETRACYANOCYCLOPENTANES V. P. Sheverdov,1 O. V. Ershov,1 and O. E. Nasakin1 Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 42, No. 12, pp. 13 – 15, December, 2008. Original article submitted May 6, 2008.

Anticancer activity tests have been carried out at the National Cancer Institute (USA) on a series of polycyano-substituted carbo- and heterocyclic compounds synthesized from 3-(2,2-dialkylhydrazino)-4-R1,1,2,2-tetracyanocyclopentanes. It is established that 1,1,2,2-tetracyano-substituted derivatives are the most active, showing a high activity comparable with that of reference drugs with respect to colon, ovarian, prostate, and renal cancer. The antitumor effect of the investigated compounds is explained by the presence of dialkylhydrazino- and 1,1,2,2-tetracyanoethyl moieties in their structures.

Cyano-containing compounds are attracting special attention at present in the search for new antitumor agents. Thus, the cyanogenetic glucosides linamarin, lotaustralin, heterodentrin, dhurrin, amygdalin, and tetraphyllin A, which were isolated from seeds and pits of certain plants of the family Rosaceae, subfamily plum, are used in experimental oncology [1]. Furthermore, several RTK-blockers [2], Ras oncogene inhibitors [3], transferase enzyme inhibitors [4], inhibitors of 1

signal transduction from oncogenic growth factor receptors and hormones from the cell surface to the nucleus [5], blockers of biosynthesis of hormones involved in the development of breast cancer [6, 7], and radio- and chemo-sensitizers for cancer therapy [8] have been found among cyano-containing compounds. Several drugs containing cyano groups are already used in medical practice to battle oncological diseases. These are the antiandrogens bicalutamide, anastrazole, and letrozole [9] and the antibiotic toyocamycin [10].

Ul’yanov Chuvash State University, Cheboksary, Chuvash Republic, Russia.

Scheme 1 R1

B(exc.) R 2=CH3 NC

NC

CN

N NC

II

NC NC

N(CH 3 )2

N CN H

NR 22

B(cat.) R2 =CH 3

NC N CN H

NC

N(CH3 )2

I KMnO4 (HCl, HBr) R1

CN

N NR 22 III

HNO2 HCl R 2=CH3 R1

CN CN

O

R1

R1

CN

KMnO4 (H2 SO4, CH3 COOH) R1 HNO2 HCl

H N

NC NC NC

N N CN V

CH 3

NC NC NC

N CN

IV

NR 22

I: a) R1 = CH3(a); b) R1 = C6H5; c) R1 = 2-Fu. II: a) R1 = CH3(a); b) R1 = C6H5; c) R1 = C3H7. III: a) R1 = R2 = CH3; b) R1 = CH3, R2 = C2H5; c) R1 = C3H7, R2 = CH3. IV: a) R1 = R2 = CH3; b) R1 = CH3, R2 = C2H5; c) R1 = C6H5, R2 = CH3. V: a) R1 = CH3; b) R2 = C6H5.

670 0091-150X/08/4212-0670 © 2008 Springer Science+Business Media, Inc.

Antitumor Activity of Polycyano-Substituted Carbo- and Heterocycles

671

TABLE 1. Antitumor Activity of I – VII Compound/Criterion Panel/Cel l Line

Ia

IIa

GI50

TGI

LG50

GI50

IIIa TGI

LG50

GI50

IVa TGI

LG50

GI50

Va TGI

LG50

VI

VII

GI50

TGI

LG50

GI50

TGI

LG50

GI50

TGI

LG50

5.21b

3.87b >1.00a >1.00a >1.00a >1.00a >1.00a

Colon cancer COLO 205 HCC2998 HCT-116 HCT-15 HT 29 KM 12 SW-620

3.83c

1.61b

4.01b >3.87b >1.00a >1.00a 2.03c

4.79d

1.73b

1.45c

3.06c

6.46c

4.34c

1.82b

1.72b

3.21b

6.00b >1.00a >1.00a >1.00a 1.66b

3.29b

6.50b

1.60b

3.07b

5.90b

2.15b

5.06b >1.00a >1.00a >1.00a >1.00a >1.00a >1.00a >1.00a

7.78b 2.15b 2.15b 6.08b >1.00a

1.37b 1.78b 1.65b >1.00a 7.37c

3.17b 3.48b 3.55b >1.00a 2.42b

7.33b 6.82b 7.63b >1.00a 6.79b

2.21b 2.19b 1.75b >1.00a 2.21b

5.13b 4.17b 3.79b >1.00a 4.89b

9.40b >1.00a >1.00a >1.00a 1.92b 2.05b

3.63b 4.46b

6.83b 9.73b

8.87c 2.49b 6.58b 7.14c 2.18b 5.44b 3.42c 1.14b 6.07b 7.28b >1.00a >1.00a 9.13c >1.00a >1.00a Ovarian cancer 1.99b 3.69b 6.83b 1.31b 3.39b 8.73b

1.95b 2.63b

3.97b 8.08b >1.00a >1.00a >1.00a >1.00a >1.00a >1.00a 9.12b >1.00a

> 1.00a > 1.00a 3.32b > 1.00a > 1.00a 1.83b

3.34b

6.08b

1.26b

2.00b

3.48b

1.10b 2.92b 7.38b 2.13b 1.54b 2.13b 1.66b 3.18b 1.78b >1.00a

IGROV1 1.78b OVCAR3 OVCAR- 3.07b 4 OVCAR- 2.33b 5 OVCAR- 2.63b 8 SK-OV-3 > 1.00a

4.09b

4.74b

9.63b

>1.00a >1.00a >1.00a >1.00a 3.54b

2.56b

5.16b

>1.00a >1.00a >1.00a >1.00a 3.54b

>1.00a >1.00a >1.00a >1.00a >1.00a

>1.00a >1.00a >1.00a >1.00a >1.00a

>1.00a >1.00a >1.00a >1.00a >1.00a

>1.00a >1.00a >1.00a >1.00a >1.00a

>1.00a >1.00a >1.00a >1.00a >1.00a

6.07b >1.00a >1.00a >1.00a

5.07b > 1.00a 3.79b > 1.00a 9.63b > 1.00a > 1.00a > 1.00a > 1.00a > 1.00a > 1.00a

4.19b

1.81b

3.38b

6.33b

1.55b

3.30b

> 1.00a > 1.00a 5.01b > 1.00a > 1.00a 2.06b

4.21b

8.59b

2.20b 4.53b 9.33b > 1.00a > 1.00a > 1.00a > 1.00a > 1.00a > 1.00a > 1.00a > 1.00a > 1.00a Renal cancer 3.34c 1.16b 4.52b 1.96b 3.69b 6.92b > 1.00a > 1.00a > 1.00a > 1.00a > 1.00a > 1.00a

> 1.00a > 1.00a 2.11b

2.56b 4.30b 3.46b 7.00c 3.13b 2.73b 3.40b 2.93b

PC-3 DU-145

2.04b 1.57b

4.66b > 1.00a 1.90b 3.83b 7.27b 2.10b

b

2.56b

>1.00a 7.96b 8.20b >1.00a >1.00a

5.28b > 1.00a 2.29b

9.00c 2.09b 1.33b 1.71c 1.30b 1.41b 1.70b 1.35b

´ 10 – 4,

>1.00a >1.00a >1.00a >1.00a >1.00a

5.91b > 1.00a 2.54b

786-Î A-498 ACHN CAKI-1 RXF 393 SN12C TK-10 UO-31

a

>1.00a >1.00a >1.00a >1.00a >1.00a

´ 10 – 5,

6.83b 8.87b 9.04b 2.68b 7.50b 5.28b 6.82b 6.35b

c

1.77b 1.82b 1.89b 8.62c 2.65b 2.08b 1.75b 1.86b

8.33b

4.23b > 1.00a 3.38b 6.27b 3.48b 6.39b 2.19b 5.15b 5.45b > 1.00a 3.69b 6.57b 3.24b 5.96b 3.44b 6.36b

1.00b

2.26b

5.11b

1.37b 1.91c 6.92c 2.09b 1.37b 1.66b

2.66b 4.04c 2.14b 3.78b 2.70b 3.03b

5.19b 8.54c 5.35b 6.81b 5.30b 5.52b

3.56b 3.62b

1.83b 1.75b

3.62b 3.16b

7.17b 5.73b

6.66b 6.24b

7.06b

4.34c 1.59b 3.99b 1.80c 3.31c 6.07c 8.60c 2.31b 5.64b 1.66b 3.16b 6.00b 1.14b 2.41b 5.06b 1.72b 3.10b 5.58b Prostate cancer 2.33b 5.68b 1.00a 1.80b 3.26b 5.93b

1.76b

3.88b

6.49b > 1.00a > 1.00a > 1.00a > 1.00a > 1.00a > 1.00a

1.66b 6.25b 1.86b 1.53b 1.49b 1.60b

3.02b 1.87b 4.29b 3.01b 2.81b 3.15b

5.50b 4.32b 9.88b 5.91b 5.30b 6.23b

> 1.00a 2.34b > 1.00a > 1.00a > 1.00a > 1.00a

> 1.00a 4.83b > 1.00a > 1.00a > 1.00a > 1.00a

> 1.00a 9.96b > 1.00a > 1.00a > 1.00a > 1.00a

> 1.00a > 1.00a > 1.00a > 1.00a > 1.00a > 1.00a

> 1.00a > 1.00a > 1.00a > 1.00a > 1.00a > 1.00a

> 1.00a > 1.00a > 1.00a > 1.00a > 1.00a > 1.00a

2.88b 2.41b

8.98b 5.62b

1.00a > 1.00a > 1.00a > 1.00a > 1.00a > 1.00a > 1.00a 1.00a > 1.00a > 1.00a > 1.00a > 1.00a > 1.00a > 1.00a

´ 10 – 6 M.

The promise of synthesizing new cyano-containing compounds and studying their antitumor activity has been demonstrated [11]. The synthesis of Diels-Alder adducts of vinylporphyrins with dimethylacetylenedicarboxylate and with tetracyanoethylene was reported. The products were effective in photodynamic therapy of rat bladder tumors induced by N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide. We have previously synthesized pyrrolidin-3,3,4,4tetracarbonitriles, 2-amino-2-pyrrolin-3,3,4-tricarbonitriles, and pyrrol-3,4-dicarbonitriles. In addition, the 4-oxoalkan1,1,2,2-tetracarbonitriles 6-imino-2,7-dioxabicyclo[3, 2, 1]octan-4,4,5-tricarbonitriles, 9-oxo-1,2,3,4b,5,6,8a,9-octahydropyrido[31,41:3, 4]pyrrolo[1, 2-a]-[1, 3, 5]triazin-4b,8a-dicarbonitriles, 1-oxo-3a,4,5,7a-tetrahydro-1H-pyrrolo[3, 4-c]pyridin-3a,7a-dicarbonitriles, and 1,2,3,4-tetrahydropyridin3,3,4,4-tetracarbonitriles were tested for antitumor activity [12]. The most active among them was 2-(4-bromophenyl)-5,6-tetramethylen-1,2,3,4-tetrahydropyridin-3,3,4,4-tetr acarbonitrile.

In searching for new biologically active compounds of this type, we synthesized new polycyano-containing carboand heterocyclic compounds I – V (Scheme 1) based on 3-(2,2-dialkylhydrazino)-4-R-1,1,2,2-tetracyanocyclopentan es, taking into account their unique ability to cleave the C(2)-C(3) single bond of the ring, even upon dissolution at room temperature in organic solvents. The reactions occurred quickly, in several minutes, and in one step [13 – 15]. Screening results showed that all studied compounds I – V exhibited antitumor properties (Table 1). The antitumor activity was on average greater than that of carbo- and heterocycles [12] prepared from pyrrolidin-3,3,4,4-tetracarbonitriles and 4-oxoalkan-1,1,2,2-tetracarbonitriles. The antitumor activity of I – V can be classified by three distinct signatures. 1) Tetracyano-substituted III – V, which contain the most nitriles, have the highest activity (with LC50 values approaching 10 – 6 M). 2) The activity of I – V depends little on the nature of the substituents. 3) All com-

672

V. P. Sheverdov et al.

pounds are characteristically more effective for renal and prostate cancer. The therapeutic index, i.e., the gap between possible therapeutic and lethal doses, was greatest for 1-dialkylamino-5,5,6,6-tetracyano-4-R-piperidin-2-ones III. The LC50 for III was the lowest whereas the LD50 was highest (1600 – 1700 mg/kg). The antitumor effect of piperidin-2ones III showed a clear trend of growth suppression and complete killing of prostate, renal, colon, and ovarian tumor cells on changing their concentration from 10 – 6 to 10 – 4 M (Table 1). We synthesized derivatives of piperidin-2-ones III, bicycles VI and VIII (Scheme 2), in order to determine the factors responsible for their high antitumor activity. Scheme 2 CH 3 CN

CH3 CN NH2 H 2O N O

(H 3 C) 2N

CN O

VI

CH 3OH

N

CN O

N

CH3 CN NH 2 CN

N

CN N(CH 3) 2 III

O

N

(H3 C) 2N VII

(50 mL) and propan-2-ol (50 mL). Yield of VI, 90%, mp 185 – 186°C (dec.). C12H14N6O2. IR spectrum (cm – 1): 3380, 3320, 3270, 3200, 2270, 1715, 1670, 1680, 1600, 1560. 13 C NMR spectrum (d, ppm): 32.79, 36.18, 54.96, 71.52, 114.08, 114.43, 165.46, 176.54, 176.89. 5-Amino-1-(dimethylamino)-7,7-dimethoxy-4-methyl -2-oxo-3,4-dihydro-1H-pyrrolo[3, 4-b]pyridin-4a,7a-(2H, 7H)-dicarbonitrile (VII). Piperidin-2-one IIIa (2.56 g, 0.01 mol) was mixed with methanol (10 mL) and treated dropwise with diethylamine or piperidine (1 – 2 drops). Compound IIIa dissolved after 5 – 10 min. The solution was cooled to 0 – 5°C. The resulting precipitate was filtered off and washed with methanol:water (1:1, 20 mL). Yield of VII, 77%, mp 164 – 165°C (dec.). C14H20N6O3. IR spectrum (cm – 1): 3410, 2258, 1710, 1670, 1610. 13C NMR spectrum (d, ppm): 33.78, 37.28, 54.93, 71.42, 114.07, 114.38, 120.37, 166.46, 170.79.

O CN O

Bicycle VI formed after heating IIIa in acetic acid for 10 – 20 sec in the presence of a catalyst. Piperidone IIIa reacted with methanol in 1 – 3 min in the presence of base and produced VII. Tests of VI and VII for antitumor activity showed that it was low (Table 1). Thus, the study of the antitumor activity of polycyano-containing carbo- and heterocycles prepared from tetracyanoethylene suggests that they all exhibit antitumor properties to a certain extent. The 1,1,2,2-tetracyano-substituted compounds are the most active. The facility of the conversions VI ¬ IIIa ® VII indicates that 1,1,2,2-tetracyano-substituted carbo- and heterocycles react with nucleophilic centers of cellular targets, primarily with DNA. This leads to incorrect information transcription from DNA. The tumor cells perish because of interference with DNA replication and mitosis. EXPERIMENTAL CHEMICAL PART The course of reactions and purity of products was monitored by TLC on Silufol UV-254 plates with detection in UV light, iodine vapor, and heating. IR spectra in mineral oil were recorded on a UR-20 instrument. 13C NMR spectra in DMSO-d6 were recorded on a Gemini-300 (Varian) spectrometer at 75 MHz. Elemental analyses (C, H, N) agreed with those calculated. 5-Amino-1-(dimethylamino)-4-methyl-2,7-dioxo-3,4dihydro-1H-pyrrolo[3, 4-b]pyridin-4a,7a-(2H,7H)-dicarb onitrile (VI). Piperidin-2-one IIIa (2.56 g, 0.01 mol), N,N-dimethylhydrazine (0.06 g, 0.01 mol), and ZnCl2 (1.35 g, 0.01 mol) were mixed in acetic acid (50 mL), heated to 80°C, and immediately diluted with water (100 mL). The resulting precipitate was filtered off and washed with water

EXPERIMENTAL BIOLOGICAL PART The synthesized compounds were tested for antitumor activity at the National Cancer Institute in Maryland (USA). An in vitro model that enabled the experimental conditions to be standardized for the repetitive series was used. Tests were conducted on 60 cell lines obtained from solid tumors of lung, colon, brain, ovary, kidney, prostate, and breast in addition to human leukemia and melanoma. The percent tumor growth and criteria GI50, TGI, and LC50 were determined (GI50, concentration at which tumor cell growth was inhibited by 50%; TGI, concentration totally inhibiting growth of tumor cells; LC50, lethal concentration for 50% of tumor cells). Tumor cells were stored on plates with microwells in the presence of the studied compounds and without them. The antitumor activity was manifested as growth inhibition (death) of tumor cells and was determined spectrophotometrically. The anionic dye sulforhodamine B, which binds only to living cells and was fixed by trichloroacetic acid, was used to determine the end point of cell growth. The bound dye was determined spectrophotometrically after rinsing. The analysis was based on the cytotoxic activity of the test compounds for the tumor cells. The number of living cells in the culture was determined from the fluorescence intensity of rhodamine B in the rinsings. The greater the fluorescence intensity was, the fewer living cells there were to absorb the dye from the solution. The acute toxicity of I – VII for white mongrel rats (250 g) showed that LD50 for them was 350 – 1700 mg/kg for i.p. administration. Thus, they have low toxicity or are practically nontoxic according to the Sidorov classification. REFERENCES 1. V. V. Plemenkov, Introduction to the Chemistry of Natural Compounds [in Russian], Kazan (2001), pp. 59 – 60.

Antitumor Activity of Polycyano-Substituted Carbo- and Heterocycles

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