Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 64 No. 1 pp. 27ñ33, 2007

ISSN 0001-6837 Polish Pharmaceutical Society

SYNTHESIS AND EVALUATION OF ETHYLNITROSOUREAS OF SUBSTITUTED NAPHTHALIMIDES AS ANTICANCER COMPOUNDS ANINDITA PAIN1, SUVA SAMANTA1, SUSHANTA DUTTA1, AJIT K. SAXENA2, MUTIAH SHANMUGAVEL2, MADHUNIKA SHARMA2, GULAM N. QAZI2 and UTPAL SANYAL1* Department of Anticancer Drug Development, Chittaranjan National Cancer Institute, Kolkata ñ 700026, India. 2 Pharmacology Division, Regional Research Laboratory, Canal Road, Jammu-Tawi ñ 180001, India. 1

Abstract: Four new ethylnitrosourea derivatives of substituted naphthalimides 3a-d have been synthesized from the respective N-(2-ethylamino)naphthalimides. Their chemical alkylating activity compared with the clinical drug CCNU and an experimental compound Mitonafide indicated that they possess lower alkylating activity than CCNU and comparable activity with the latter. Their anti-tumor efficacies were assessed in vivo in two murine ascites tumors namely Sarcoma-180 (S-180) and Ehrlich ascites carcinoma (EAC) by measuring the increase in median survival times (MST) of drug treated (T) over untreated control (C) mice. CCNU and Mitonafide were used as positive controls for comparison. The representative compound 3a has displayed marginal anti-tumoral activity in these tumors. Three compounds were further screened in vitro in 4 different human tumor cell lines but no significant activity was observed in those lines. These compounds moderately inhibit the synthesis of DNA and RNA in S-180 tumor cells. Keywords: New anticancer agent, synthesis, screening, anticancer activity.

It is hypothesized that upon in vivo enzymatic degradation these compounds may exert synergistic activity since the naphthalimide ring moiety may bind to DNA, while the ethyl-NU group will exert cytotoxicity through carbamoylation and alkylation of biological nucleophiles. The amino compounds 1a-d obtained by treating substituted 1,8-naphthalic anhydride with 1,2-diaminoethane were converted to the ureido compounds 2a-d by treating with ethyl isocyanate at room temperature, which in turn upon nitrosation with sodium nitrite in the presence of formic acid at 0OC furnished nitrosoureido compounds 3a-d in good yield (Scheme 1).

An important family of alkylating agents are the nitrosourea (NU) compounds (1) that contain the functionality (R-NH-CO-N(NO)CH2R1). Among these chemotherapeutic agents 2-chloroethylnitrosourea (2-ClEtNU) compounds [R1 = CH2Cl] are best represented by Chlorozotocin, BCNU (Carmustine), CCNU (Lomustine), Me-CCNU (Semustine) etc., whereas methylnitrosourea compounds [R1 = H] are best exemplified by Streptozotocin (Figure 1). Streptozotocin has been in clinical use since 1967 (2) and finds application in the carcinoid tumor and endocrine tumors of pancreas. Evaluation of its activity in different cancers is an area of research interest till date (3). It is also used in medical research for its diabetogenic effect in animals (4). According to the literature ethylnitrosourea (EtNU) compounds [R1 = CH3] are less active and less toxic than 2-chloroethylnitrosoureas. The anticancer activities of two 2-chloroethylnitrosoureas of substituted naphthalimide were earlier reported from this laboratory (5). The rationale of choosing naphthalimide moiety is because some of them as Mitonafide and Amonafide (Figure 2) are DNA binder (6) and have undergone clinical trials (7). In the present report we describe the preparation of four new naphthalimido ethylnitrosoureas 3a-d (Scheme 1) and their anti-tumoral activity.

Streptozotocin Figure 1.

* Corresponding author: Fax: +91-33-24757606. E-mail: HYPERLINK mailto:[email protected]

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ANINDITTA PAIN et al. stirred further for 3 h. The solid was filtered and washed with chloroform to furnish the desired product, 2a-d, as white or light yellow powder, which was further purified by crystallization from methanol. Step 2. General procedure for preparation of 2{2-[3-(2-ethyl)-3-nitrosoureido]ethyl}-1Hbenzo[d,e]isoquinoline-1,3-diones (compounds 3ad)]. To a cooled (0OC) solution of compounds 2a-d (1 mmol) in formic acid (6 cm3), sodium nitrite solution (245 mg, 3.6 mmol) in water (0.7 cm3) was added dropwise. A solid precipitate began to appear

EXPERIMENTAL Chemistry Melting points: Thomas-Hoover Unimelt capillary apparatus. IR spectra (KBr pellets): Perkin Elmer RX-1 FTIR. UV spectra (MeOH): Shimadzu UV-160A. 1H-NMR spectra: Bruker 300-DPX (δ values in ppm, TMS as internal standard, solvent ñ CDCl3). HPLC: Waters (m-Bondapak C18 steel column; 30 cm ( 3.9 mm; isocratic mobile phase: acetonitrile ñ water in varying proportions (up to 50 : 50, v/v), at a flow rate of 1.0 mL/min, at ambient temperature, UV detection at 250 nm). TLC: Silica gel G (Merck), solvent system CHCl3 ñ MeOH (90:10, v/v). Physical, analytical and IR spectroscopic data of compounds are described in Table 1 whereas 1H-NMR spectral data in Table 2. Compounds 1a (8); 1b (5); 1c (5); and 1d (6) were prepared according to the reported literature procedures. Step 1. General procedure for preparation of 2{2-[3-(2-ethyl)ureido]ethyl}-1H-benzo[d,e]isoquinoline-1,3-diones (compounds 2a-d)]. To a clear solution of the respective compound, 1a-d, (3 mmol) in chloroform, ethylisocyanate (0.27 cm3, 3.3 mmol) was added at room temperature. A yellow precipitate appeared immediately. It was

R = NO2, Mitonafide NH2, Amonafide Figure 2.

Table 1. Physical, analytical and IR spectroscopic data of compounds.

Comp. M. p. OC No. Yield %

Formula Mol. weight

Analysis % (Calc. / Found)

TLC Rf

IR (KBr) cm-1

C

H

N

2a

230-231 80

C17H17N3O3 311.18

65.5 65.0

5.5 5.7

13.5 13.1

0.70

3373 (NH); 2924 (CH stretching); 1706 (amide); 1662 (urea); 1532, 1479 (arom. ring).

2b

233-235 60

C17H16BrN3O3 390.15

52.3 51.9

4.1 3.8

10.8 11.0

0.71

3313 (NH); 2975, 2863 (CH stretching); 1696 (amide); 1660 (urea); 1619, 1585 (arom. ring).

2c

228-230 57

C17H16ClN3O3 345.70

59.0 59.5

4.6 4.8

12.1 11.8

0.61

3340 (NH); 2973 (CH stretching); 1696 (amide); 1659 (urea); 1585 (arom. ring).

2d

235-237 48

C17H16N4O5 356.17

57.3 56.9

4.5 4.4

15.7 16.1

0.69

3332 (NH); 3070, 2981 (CH stretching); 1711 (amide); 1674 (urea); 1580, 1536 (arom. ring).

3a

140-142 75

C17H16N4O4 340.18

60.0 59.6

4.7 4.3

16.5 16.2

0.90

3366 (NH); 2952, 2918 (CH stretching); 1700, 1663 (amide); 1588 (arom. ring).

3b

154-155 52

C17H15BrN4O4

48.7 48.4

3.6 3.2

13.4 13.0

0.92

419.15

3356 (NH); 3070, 2985, 2864 (CH stretching); 1660 (amide); 1590 (arom. ring).

3c

143-145 50

C17H15ClN4O4 374.70

54.4 54.0

4.0 3.7

14.9 14.6

0.79

3379 (NH); 3072, 2982 (CH stretching); 1704, 1656 (amide); 1591 (arom. ring).

3d

140-142 32

C17H15N5O6 385.17

52.9 52.1

3.9 3.6

18.1 18.4

0.82

3386 (NH); 3080, 2924 (CH stretching); 1709, 1666 (amide); 1599 (arom. ring).

29

Synthesis and evaluation of ethylnitrosoureas...

a:R=H b : R = 6-Br c : R = 6-Cl d : R = 5-NO2 Scheme 1.

Table 2. 1H NMR spectral data of compounds.

Comp. No. 2a

0.84 (t, 3H, CH3), 2.89 (m, 2H, CH2N), 3.31 (m, 2H, CH2N), 4.12 [t, 2H, CH2N(CO)2], 5.71 and 5.91 (t, 2H, 2 × NH), 7.88 (t, 2H, arom. H), 8.47 (m, 4H, arom H).

2b

0.83 (m, 3H, CH3), 2.85 (m, 2H, CH2N), 3.3 (m, 2H, CH2N), 4.1 [t, 2H, CH2N(CO)2], 5.72 and 5.91 (t, 2H, 2 × NH), 8.02 (m, 1H, arom. H), 8.24 (m, 1H, arom H), 8.33 (m, 1H, arom. H), 8.56 (m, 2H, arom H).

2c

0.83 (t, 3H, CH3), 2.88 (m, 2H, CH2N), 3.3 (d, 2H, CH2N), 4.1 [t, 2H, CH2N(CO) 2], 5.72 and 5.91 (t, 2H, 2 × NH), 8.04 (m, 2H, arom.H), 8.43 (d, 1H, arom H), 8.59 (t, 2H, arom H).

2d

0.80 (t, 3H, CH3), 2.83 (m, 2H, CH2N), 3.32 (d, 2H, CH2N), 4.13 [t, 2H, CH2N(CO) 2], 5.74 and 5.93 (t, 2H, 2 × NH), 8.1 (d, 1H, arom.H), 8.67 (d, 1H, arom H), 8.76 (d, 1H, arom H), 8.95 (d, 1H, arom H), 9.47 (d, 1H, arom H).

3a

0.91 (t, 3H, CH3), 3.8 (m, 2H, CH2N), 3.88 (m, 2H, CH2N), 4.54 [t, 2H, CH2N(CO)2], 7.45 (s, 1H, NH), 7.76 (t, 2H, arom.H), 8.23 (d, 2H, arom H), 8.6 (d, 2H, arom H).

3b

0.91 (t, 3H, CH3), 3.82 (m, 2H, CH2N), 3.9 (m, 2H, CH2N), 4.51 [t, 2H, CH2N(CO)2], 7.46 (s, 1H, NH), 7.84 (t, 2H, arom.H), 8.46 (m, 1H, arom H), 8.61 (m, 2H, arom H).

3c

0.91 (t, 3H, CH3), 3.8 (m, 2H, CH2N), 3.85 (m, 2H, CH2N), 4.5 [t, 2H, CH2N(CO)2], 7.42 (m, 1H, NH), 7.82 (m, 2H, arom.H), 8.47 (t, 1H, arom H), 8.61 (m, 2H, arom H).

3d

0.89 (t, 3H, CH3), 3.77 (m, 2H, CH2N), 3.9 (m, 2H, CH2N), 4.55 [t, 2H, CH2N(CO)2], 7.33 (s, 1H, NH), 7.94 (m, 1H, arom.H), 8.43 (m, 1H, arom H), 8.77 (m, 1H, arom H), 9.15 (d, 1H, arom H), 9.29 (m, 1H, arom H).

in CDCl3

a

H NMR δ (ppm)a

1

30

ANINDITTA PAIN et al.

Table 3. Determination of chemical alkylating activity*.

Conc. (µmol/mL) Compound

0.25

0.50

3a

0.17 ± 0.08

0.29 ± 0.11

3b

0.12 ± 0.10

0.23 ± 0.12

3c

0.16 ± 0.05

0.26 ± 0.10

3d

0.09 ± 0.08

0.16 ± 0.11

CCNU

0.37 ± 0.04

0.61 ± 0.07

Mitonafide

0.15 ± 0.03

0.30 ± 0.06

Blank

0.03 ± 0.01

0.03 ± 0.01

For details see Experimental section. *Expressed in O.D. values

after 1 h. It was stirred further for 3 h. The yellow solid was filtered, washed with water and dried. This was purified by column chromatography on silica gel (8 g). Elution with chloroform followed by repeated crystallization from chloroform furnished light yellow colored solids, 3a-d, homogeneous in TLC and HPLC. Determination of chemical alkylating activity The procedure described earlier (5) was essentially followed. Thus, a solution of the samples 3ad, CCNU and Mitonafide in different concentrations in acetone (1 cm3), distilled water (2 cm3) and acetate buffer (1 cm3, 0.25 M, pH 6.0) were incubated at 100OC for 20 min with a solution of 4-(4nitrobenzyl)pyridine (5% w/v) in acetone (0.4 cm3) and cooled to 25OC. After the addition of acetone (2 cm3), EtOAc (5 cm3) and sodium hydroxide solution (0.25 M, 1.5 cm3), the reaction mixtures were vortexed and allowed to stand to separate the organic layers. The absorbance in the organic layers were immediately determined (within 2 min of NaOH addition) at 540 nm. The experiments were carried out in triplicate. The results were expressed in optical density values (mean ± S.E.M., n = 3 in all cases) (Table 3). In vivo screening S-180 and EAC cells freshly obtained from the National Center for Cell Sciences, Pune, India, were used and maintained in Swiss mice by serial transplantation. Tumor cell suspension was aspirated under aseptic conditions with a hypodermic syringe from a mouse bearing tumor for 10-11 days. The tumor cells were diluted in physiological saline and counted under light microscope employing a hemocytometer plate after trypan blue dye exclusion test as usual. Cell suspensions were prepared to final concentrations of 5 × 106 viable cells/cm3 as described (5). Swiss male mice of about 6 weeks age

Figure 3. Effects of compounds on DNA and RNA synthesis in S180 tumor cells (for details refer to the experimental section).

weighing 20 ± 2 g were inoculated at an injection volume of 0.2 cm3 on day 0 thus having 106 cells/mouse for in vivo study. Physiological saline containing 2% Tween ñ 80 (Sigma Inc., USA) was used as vehicle for drug administration by injection through intraperitoneal route. The drug solutions were prepared daily just prior to the experiment. The compounds in respective doses were administered to each mouse in treated groups (Table 4). The control groups received equal volumes of vehicle (0.2 cm3) on those days. The testing was evaluated by calculating the median survival times (M.S.T.) (9) of drug-treated (T) and control (C) tumor bearing ani-

31

Synthesis and evaluation of ethylnitrosoureas...

Table 4. In vivo screening data.

Comp. No.

Tumor System

Expt. No. 1

Dose (mg/kg)

No. of injns.

Injection days

MST (days)

Survivors > 60 days

T/C %

60

7

1ñ7

30.0

ñ

133

40

7

1ñ7

31.5

ñ

140

22.5

ñ

100

Control 3a 2

60

5

1ñ5

28.0

ñ

130

40

5

1ñ5

29.0

ñ

134

21.5

ñ

100

5

1,3,5,7,9

32.5

ñ

135

24.0

ñ

100

Control 3 S-180

40 Control

CCNU 4

50

5

1ñ5

49.5

2

230

25

5

1-5

>60.0

4

>279

21.5

-

100

Control Mitonafide

1

7

1-7

25.0

-

128

5

0.5

7

1-7

36.5

1

187

19.5

-

100

60

7

1ñ7

29.5

ñ

128

40

7

1ñ7

31.0

ñ

134

23.0

ñ

100

Control 6

Control 7 3a

40

5

1ñ5

Control

EAC 8

40

5

1,3,5,7,9

Control CCNU

9

25 Control

5

1-5

32.0

ñ

125

25.5

ñ

100

27.0

ñ

129

21.0

ñ

100

>60.0

4

>240

23.5

-

100

% T/C value > 125 is considered as significant

mals and expressed as percent T/C value. A T/C percentage value greater than 125 is considered as significant. CCNU [1-(2-chloroethyl)-3-cyclohexyl-1nitrosourea] and Mitonafide were used as positive controls for comparison. In vitro screening in human tumor lines Compounds 3a, 3b and 3d were screened in the following human tumor cell lines as SiHa cervix, T47D Breast, SNB-78 CNS and HOP-62 Lung by sulforhodamine-B (SRB) semi-automated assay (10). A stock solution of 2 × 10-2 M of each compound was prepared in DMSO (HYBRIMAX grade, Sigma, USA). Each compound was tested in 3 sets of experiment in triplicate at 10-4 to 10-7 molar concentrations. CCNU and Mitonafide were included as positive controls to ascertain authenticity of the experiment. Growth inhibition value = 50% at 1×10-5 M was considered as significant. 3

H-thymidine and 3H-uridine incorporation in S180 cells in vitro

The assay was conducted similarly as described earlier (11). Briefly, the tumor cells aspirated from a mouse bearing S-180 at the log phase of growth (7th day tumor) after transplantation were washed twice in Hankís balanced salt solutions and resuspended in RPMI-1640 medium supplemented with 10% heat inactivated fetal calf serum, streptomycin (100 µg/ cm3) and penicillin (100 units/ cm3), following a viable cell count. Cell suspensions taken in glass tubes were adjusted in such a fashion so that the final cell count became 1.0 × 106/0.1 cm3 after the addition of 3H-thymidine or 3H-uridine (activity 10 µCi each) dissolved in 0.01 cm3 of sterile saline and addition of compounds 3a-d at 8 µM concentration (data presented for compound 3a). For comparison, the same concentration of Mitonafide and CCNU were used as the standard. The tubes were incubated at 37OC for 30 min and 60 min. Cell viability assessed by trypan blue dye exclusion test was of the order of 95 %. The cells were harvested at 0, 30 and 60 min of incubation and absorbed onto 25-mm discs of Whatman 3MM filter papers. The discs

32

ANINDITTA PAIN et al.

were dried, washed twice with ice-cold 10% trichloroacetic acid followed by absolute alcohol. The discs were dried in air, placed in scintillation vials containing scintillation fluid and the radioactivity was counted. For background counts, filter papers were soaked with 0.01 cm3 of 10 µCi of 3H-thymidine or 3 H-uridine and washed as described before and the radioactivity was counted. Actual incorporation of the isotopes in the drug-treated groups was calculated by subtracting the background count from the observed counts. The results are expressed as a percentage of the incorporation in the appropriate control without drug (Figure 3). RESULTS AND DISCUSSION It is hypothesized that there is a correlation between the chemical alkylating activity and antitumor activity (12). Hence the alkylating activity of 3a-d was determined and compared with CCNU and Mitonafide (Table 3). The results indicate that the compounds possess lower chemical alkylating activity than CCNU and comparable activity with Mitonafide. The in vivo anti-tumoral activity of the compounds in S-180 and EAC were determined following treatment at different doses and schedules. The result obtained for the representative compound 3a is presented (Table 4). The optimum dose confirmed by duplicate screening experiments was found to be 40.0 mg/kg for the schedule of 1-7 days having the maximum T/C values of 140 and 134 in S-180 and EAC respectively (Table 4). Thus, the compound was marginally active. Similar results were obtained for other compounds (data not presented). None of the compounds has exhibited curative effects in these tumors. The animals were autopsied after their death. It was found that the internal organs like heart, spleen, liver and kidney all retained their usual distinctive colors and appearances in the treated groups on different days as of normal control mouse. CCNU administered at the optimum dose of 25 mg/kg in the treatment schedule (QD1-5, i.p.) exhibited very high T/C values of > 279 and > 240 in S180 and EAC, respectively, having long-term survivors. Mitonafide has also demonstrated significant anticancer activity in S-180 (T/C value 187). At the optimum dose of 3a, no significant depression in body weights of treated host animals was noted. Toxic symptoms were not externally

observed in animals in general appearance, with respect to skin and hair texture and normal behavioral pattern with respect to food and water intake and the activity. Three compounds: 3a, 3b and 3d were subjected to in vitro cytotoxicity assay in the four cell lines mentioned in the experimental section. Since the growth inhibition value = 50% at 1x10-5M was not reached, the compounds have not displayed significant activity (data not presented). Studies were conducted to ascertain their inhibitory effect on nucleic acid synthesis. Hence, 3 H-thymidine and 3H-uridine uptake by S-180 cells harvested from untreated mouse were evaluated after treating the cells in vitro. The untreated S-180 cells demonstrated an almost linear pattern of 3Hthymidine and 3H-uridine uptake over a period of 60 min incubation. Simultaneous exposure of tumor cells to compounds 3a-d at concentration of 8 (µM resulted in moderate inhibition of 3H-thymidine and 3 H-uridine uptake, less than that of Mitonafide and CCNU at the same concentration (8 µM). Thus at the end of 1 h incubation time, with respect to control, Mitonafide and CCNU exhibited 99% and 94% of DNA synthesis inhibitions, while the maximum inhibition (65%) was shown by compound 3a among the EtNU compounds (Figure 3). Again, after 1 h Mitonafide and CCNU exhibited 94 and 90% of RNA synthesis inhibition, while the maximum inhibition (60%) was shown by compound 3a (Figure 3). An exposure to lower drug concentration (4 µM) of 3a, CCNU and Mitonafide caused less inhibition as expected (data not presented). Thus our presumption regarding the DNA and RNA inhibitory properties of these EtNU compounds were supported from the obtained data, since the compounds had actually exhibited such properties to a moderate extent (Figure 3). They have exhibited greater inhibitory effect on DNA synthesis than RNA synthesis. From the above results, it can be concluded that compared to the corresponding 2-chloroethylnitrosourea derivatives (5), ethylnitrosourea derivatives 3a-d have exhibited marginal anticancer activity as a new class of anticancer agents. Acknowledgments We sincerely thank the Director of CNCI, for her encouragement, and the Council of Scientific & Industrial Research (CSIR), New Delhi, India, for financial assistance.

Synthesis and evaluation of ethylnitrosoureas...

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