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Introduction

Presently, the urea moiety is a unique feature in the community of anticancer drugs. There are several groups of anticancer drugs that exploit the presence of urea functions as a major feature and, unexpectedly, each one of these groups has its own molecular mechanism of in vivo action. The simplest urea-based anticancer agent is hydroxyurea (HU), which is mainly used as a myelosuppressive agent in the treatment of chronic myelogenous leukemia (CML) [1]. HU exerts its cytotoxic effect through inhibition of ribonucleotide reductase, which is an important enzyme in DNA repair mechanism [2]. One of the major clinically used classes of drugs in cancer chemotherapy is the chloroethylnitrosourea (CENU) group. The parent CENU is carmustine, which can eradicate intracranially inoculated tumors because of its lipophilic character and ability to cross the blood – brain barrier [3]. Nitrosoureas show partial cross-resistance with other alkylating agents [4].

Nitrogen mustards and nitrosoureas are among the most powerful alkylating and carbamoylating agents for the treatment of leukemias and carcinomas [5 – 7]. Nitrosoureas are very potent, but unfortunately highly toxic, while aromatic nitrogen mustards are active and relatively well tolerated by the organism. There were trials for the develop-

ment of new antineoplastic agents designed as hybrids between these two classes of anticancer drugs.

1-Aryl-3-(2-chloroethyl)ureas (CEUs) represent a new class of compounds based on the conjugation of aromatic ring of chlorambucil as the prosthetic group and the cytotoxic 2-CENU moiety of carmustine as the pharmacophore. Only unnitrosated derivatives were found to be active [8]. 1-(4-tert-Butylphenyl)-3-(2-chloroethyl)urea exhibit higher cytotoxicity than the parent drugs (i.e., chlorambucil, carmustine) and showed significant antineoplastic activity in vivo [9]. 1-(4-Iodophenyl)-3-(2-chloroethyl)urea was found to efficiently block angiogenesis and tumor growth [10]. These CEU derivatives are nonmutagenic, nontoxic, and active even in cell lines that have developed resistance through P-glycoprotein overexpression, alteration in glutathione and3or glutathione-S-transferase activity, modification of topoisomerase I, or increase in DNA repair [11]. Investigations of the structure – activity relationships of CEU derivatives showed that the exocyclic urea function and 2-chloroethyl group were essential. The most favorable aryl groups were (i) a phenyl ring substituted at the 4 position with a branched alkyl chain or a halogen atom and (ii) an indanyl or fluorenyl group [12]. This new class was found to irreversibly alkylate -tubulin inside the cancer cell causing the cytotoxic effect [13].

From a synthetic point of view, compounds of low molecular weight are more attractive as leads [14]. The aim of the present investigation was to synthesize new simple 1-aroyl-4-(2-chloroethyl)semicarbazides (CESCs), which are structurally related to CEU derivatives, and to explore their in vitro cytotoxic activity against U251 and Hepg2 cell lines.

Results and discussion

Chemistry. In the present work, a series of new 1-( p-substituted benzoyl)-4-(2-chloroethyl)semicarbazide derivatives (8 – 14) were synthesized according to Scheme 1. The key hydrazide intermediates 1 – 7 were prepared by addition of the corresponding p-substituted ethyl benzoate ester to excess hydrazine hydrate in ethanol, followed by refluxing the reaction mixture for 2 h [15]. The acid hydrazides were then reacted with 2-chloroethyliso-

Another series of new 1-[(5-substituted-1H-in- dol-2-yl)carbonyl]-4-(2-chloroethyl)semicarbazide derivatives (20 – 24) were synthesized as illustrated in Scheme 2. The hydrazide intermediates (15 – 19) were prepared as described in [15]. These hydrazides were stirred with 2-chloroethylisocyanate in dioxane at room temperature for 12 h to afford the target compounds (20 – 24).

Cytotoxic evaluation. Compounds 8 – 14 and 20 – 24 were evaluated for in vitro cytotoxic activity against two cell lines, human brain carcinoma (U251) and human liver carcinoma (Hepg2). The results are displayed in Table 1. The drugs were tested at four concentrations (10, 25, 50, 100 g3ml) using the sulforhodamine B (SRB) assay [16]. A given compound is considered significantly active when its IC50 value is less than 100 g3ml.

First of all, an analysis of the data presented in Table 1 shows that variable results concerning the cytotoxic activity of compounds 8 – 14, and 20 – 24 are obtained. The unsubstituted compound 8 and its p-fluorosubstituted analog 10 were the only derivatives that showed weak cytotoxic activity against both U251 and Hepg2 cell lines. Compound 9 exhibited no activity against U251 cell line. Compounds 12 – 14 showed no cytotoxic activity against Hepg2. The chloro derivative 11 produced no cytotoxic effect upon both U251 and Hepg2. We can also suggest that (i) substituting the p-position of compound 8 by different groups does not markedly affect the cytotoxic activity against the assigned cell lines and (ii) the p-fluoro group (compound 10) is a better halogen substituent than the

p-chloro group (compound 11). On the other hand, the replacement of the unsubstituted phenyl group in compound 8 by the unsubstituted 2-indolyl group (compound 20) slightly increases the cytotoxic activity, which is confirmed by comparing the IC50 values of compound 8 (IC50 = 96 g3ml against U251 and 100 g3ml against Hepg2) and compound 20 (IC50 = 89 g3ml against U251 and 90 g3ml against Hepg2). The last fact also applies if we compare the activity patterns of the fluoro derivatives 10 and 21, but with the additional observation of a dramatic increase in the activity against Hepg2 cells: from

IC50 = 100 g3ml for compound 10 to 32 g3ml for compound 21. Such a dramatic increase in the activity against

Hepg2 cell line becomes much clearer when we compare the corresponding values of IC50 obtained for derivatives with electron-donor substituents like methoxy and benzyloxy groups (cf. 12, 13 against 23, 24). Moreover, an increase in the cytotoxic activity against U251 cell line resulted from replacing the phenyl group in compounds 12 and 13 by 2-indolyl moiety in compounds 23 and 24, which can be seen from Table 1.

Conclusions

The main aim of this study was to generate new simple anticancer derivatives through exploration of the cytotoxic activity of new 1-aroyl-4-(2-chloroethyl)semicarbazides. The conclusions that can be most likely drawn from the obtained results are as follows: (i) CESCs are constituting a new class of compounds exhibiting a promising cytotoxic activity profile; (ii) the (2-indolyl) carbonyl substituent usually leads to more cytotoxic derivatives than does the benzoyl substituent; (iii) electron-donor substituents like methoxy and benzyloxy groups at position 5 of the indole ring greatly enhance the cytotoxic activity against U251 and Hepg2 cell lines; (iv) the most active derivatives 23 and 24 (Fig. 1) can be recommended for further optimiza-

tion and for the investigation of molecular mechanisms of the drug action.

Experimental chemical part

Melting points were determined in open capillaries with a Gallenkamp digital apparatus and are uncorrected. The IR spectra were recorded in KBr pellets using a Bruker spectrophotometer. The 1H NMR spectra were determined on a Varian Gemini 200 MHz instrument using DMSO-d6 as the solvent and TMS as the internal standard (with chemical shifts expressed in ppm). The mass spectra were measured on a Shimadzu QP1000EX GC3MS instrument operating at an electron-impact ionization energy of 70 eV. Elemental analyses were performed at the Microanalytical Centre (National Research Centre, Cairo, Egypt). Their results corresponded to the calculated values within experimental error. TLC was performed on silica gel G (Fluka), and spots were visualized under UV irradiation (254 nm). Starting materials were purchased from Lancaster Synthesis Corporation (United Kingdom). Hydrazide intermediates (1 – 7, 15 – 19) were synthesized using a method described in [15].

General procedure for the synthesis of compounds 8 – 14. To a solution of the initial acid hydrazide 1 – 7 (1 mmole) in EtOH (10 ml) was added an equimolar amount of 2-chloroethylisocyanate. The reaction mixture was stirred for 12 h and diluted with water (30 ml). The precipitated product was filtered under vacuum, washed with water, and crystallized from EtOH.

1-Benzoyl-4-(2-chloroethyl)semicarbazide (8): yield, 27%; m.p., 176 – 178°C; IR spectrum (max, cm – 1): 3393, 3329, 3210 (NH), 1704, 1675 (C=O); mass spectrum [m3z

(Irel., %)]: [M]+ 242 (1.2), 205 (2.3), 162 (1.0), 136 (16.5), 105 (100), 77 (52.2); anal. calcd. for C10H12ClN3O2 (%):

C, 49.70; H, 5.00; N, 17.39; found (%): C, 49.67; H, 4.79; N, 17.48.

4-(2-Chloroethyl)-1-(4-methylbenzoyl)semicarbazi- de (9): yield,37%; m.p., 192 – 193°C; IR spectrum (max,

T a b l e 1

Cytotoxicity of the Synthesized Compounds against U251 and Hepg2 Carcinomas

Notes: a IC50 is a dose required to inhibit the cell growth by 50%; b NA = no activity (i.e., IC50 > 100 g3ml); c ND = not determined.

cm – 1): 3379, 3343, 3307 (NH), 1710, 1652 (C=O); 1H NMR spectrum ( , ppm): 2.35 (s, 3H, CH3), 3.37 (m, 2H, CH2), 3.58 (t, 2H, CH2), 6.71 (bs, 1H, NHCH2, exch.), 7.27 (d, 2H, J 7.8 Hz, ArH), 7.78 (d, 2H, J 8.1 Hz, ArH), 8.00 (s, 1H, NHNHCO, exch.), 10.05 (s, 1H, ArCONH, exch.); anal. calcd. for C11H14ClN3O2 (%): C, 51.67; H, 5.52; N, 16.43; found (%): C, 51.25; H, 5.90; N, 16.03.

4-(2-Chloroethyl)-1-(4-fluorobenzoyl)semicarbazide (10): yield: 43%; m.p., 216 – 218°C; IR spectrum (max, cm – 1): 3308, 3240, 3125 (NH), 1664, 1644 (C=O); 1H NMR spectrum ( , ppm): 3.35 (m, 2H, CH2), 3.55 (t, 2H, CH2), 6.70 (bs, 1H, NHCH2), 7.30 – 7.80 (m, 4H, ArH), 8.10 (s, 1H, NHNHCO), 10.20 (s, 1H, ArCONH); mass

spectrum [m3z (Irel., %)]: [M–36]+ 223 (2.6), 181 (1.4), 154 (22), 123 (100), 95 (34.6); anal. calcd. for

C10H11ClFN3O2 (%): C, 46.25; H, 4.27; N, 16.18; found (%): C, 45.97; H, 3.94; N, 16.18.

1-(4-Chlorobenzoyl)-4-(2-chloroethyl)semicarbazide (11): yield, 77%; m.p., 219 – 220°C; IR spectrum (max, cm – 1): 3389, 3324, 3218 (NH), 1704, 1676 (C=O); mass

spectrum [m3z (Irel., %)]: [M]+ 275 (0.9), 239 (2.6), 196 (1.7), 139 (100), 75 (20.5); anal. calcd. for C10H11Cl2N3O2

(%): C, 43.50; H, 4.02; N, 15.22; found (%): C, 43.48; H, 4.08; N, 15.28.

4-(2-Chloroethyl)-1-(4-methoxybenzoyl)semicarba- zide (12): yield, 58%; m.p., 192 – 194°C; IR spectrum (max, cm – 1): 3300, 3240, 3110 (NH), 1644 (C=O); 1H NMR spectrum ( , ppm): 3.35 (m, 2H, CH2), 3.55 (t, 2H, CH2), 3.8 (s, 3H, OCH3), 6.75 (bs, 1H, NHCH2), 7.00 (d, 2H, J 7.8 Hz, ArH), 7.75 (d, 2H, J 7.9 Hz, ArH), 8.00 (s, 1H, NHNHCO), 10.05 (s, 1H, ArCONH); anal. calcd. for C11H14ClN3O3 (%): C, 48.63; H, 5.19; N, 15.47; found (%): C, 48.51; H, 5.36; N, 15.42.

1-(4-Benzyloxybenzoyl)-4-(2-chloroethyl)semicar- bazide (13): yield, 76%; m.p., 191 – 193°C; IR spectrum (max, cm – 1): 3395, 3329, 3209 (NH), 1702, 1675 (C=O); anal. calcd. for C17H18ClN3O3: C, 58.71; H, 5.22; N, 12.08; found (%): C, 58.62; H, 5.27; N, 11.98.

4-(2-Chloroethyl)-1-(4-nitrobenzoyl)semicarbazide (14): yield, 45%; m.p., 207 – 208°C; IR spectrum (max, cm – 1): 3316, 3264, 3109 (NH), 1649 (C=O), 1490, 1314

(NO2); mass spectrum [m3z (Irel., %)]: [M–36]+ 250 (13.7), 207 (9.4), 181 (29.8), 150 (87.6), 76 (100); anal. calcd. for

C10H11ClN4O4: C, 41.90; H, 3.87; N, 19.54; found (%): C, 41.77; H, 3.99; N, 19.10.

General procedure for the preparation of compounds 20 – 24. To a solution of the appropriate acid hydrazide 15 – 19 (1 mmole) in dioxane (10 ml) was added an equimolar amount of 2-chloroethylisocyanate. The reaction mixture was stirred for 12 h at room temperature and then poured into water (30 ml). The precipitated product was filtered under vacuum, washed with water, and finally crystallized from EtOH.

4-(2-Chloroethyl)-1-[(1H-indol-2-yl)carbonyl]semi- carbazide (20): yield, 43%; m.p., 230 – 231°C; IR spectrum (max, cm – 1): 3365, 3304 (NH), 1702, 1659 (C=O);

mass spectrum [m3z (Irel., %)]: [M]+ 280 (7.2), 244 (10.9), 202 (8.7), 175 (17.1), 144 (100), 116 (18.4), 63 (25.5);

anal. calcd. for C12H13ClN4O2 (%): C, 51.34; H, 4.67; N, 19.96; found (%): C, 51.25; H, 4.94; N, 19.66.

4-(2-Chloroethyl)-1-[(5-fluoro-1H-indol-2-yl)carbo- nyl]semicarbazide (21): yield, 38%; m.p., 225 – 227°C; IR spectrum ( max, cm – 1): 3391, 3349, 3295, 3200 (NH),

1704, 1668 (C=O); mass spectrum [m3z (Irel., %)]: [M–36]+ 262 (9.5), 219 (11.5), 193 (15.7), 162 (100), 116

(18.4), 134 (21.5), 63 (15.1); anal. calcd. for C12H12ClFN4O2 (%): C, 48.25; H, 4.05; N, 18.76; found (%): C, 48.07; H, 4.27; N, 18.76.

4-(2-Chloroethyl)-1-[(5-chloro-1H-indol-2-yl)carbo- nyl]semicarbazide (22): yield, 37%; m.p., 230 – 232°C;

mass spectrum [m3z (Irel., %)]: [M–36]+ 278 (14.5), 209 (10.1), 178 (100), 150 (15.8), 123 (49.4), 63 (7.8); anal.

calcd. for C12H12Cl2N4O2 (%): C, 45.73; H, 3.84; N, 17.78; found (%): C, 45.44; H, 3.63; N, 17.49.

4-(2-Chloroethyl)-1-[(5-methoxy-1H-indol-2-yl)car- bonyl]semicarbazide (23): yield, 79%; m.p., 218 – 220°C; IR spectrum ( max, cm – 1): 3364, 3299 (NH), 1704, 1652 (C=O); 1H NMR spectrum ( , ppm): 3.30 (m, 2H, CH2), 3.58 (m, 2H, CH2), 3.76 (s, 3H, OCH3), 6.83 (bs, 1H, NHCH2, exch.), 6.86 – 7.31 (m, 4H, ArH), 8.10 (s, 1H, NHNHCO, exch.), 10.10 (s, 1H, ArCONH, exch.), 11.5 (s, 1H, NH of indole, exch.); mass spectrum [m3z

(Irel., %)]: [M]+ 310 (4.3), 274 (5.2), 231 (16.3), 205 (20.3), 174 (100), 146 (35.6), 119 (44.4), 63 (10.5); anal.

calcd. for C13H15ClN4O3b (%): C, 50.25; H, 4.87; N, 18.03; found (%): C, 50.21; H, 4.82; N, 17.91.

1-[(5-Benzyloxy-1H-indol-2-yl)carbonyl]-4-(2-chlo- roethyl)semicarbazide (24): yield, 79%; m.p., 205 – 207°C; IR spectrum ( max, cm – 1): 3340, 3216 (NH), 1704, 1655 (C=O); 1H NMR spectrum ( , ppm): 3.35 (m, 2H, CH2), 3.55 (m, 2H, CH2), 5.10 (s, 2H, OCH2), 6.75 (bs, 1H, NHCH2), 6.92 – 7.85 (m, 9H, ArH), 8.10 (s, 1H, NHNHCO), 10.10 (s, 1H, ArCONH), 11.50 (s, 1H, NH of

indole); MS: mass spectrum [m3z (Irel., %)]: [M – 35.5] + 351 (0.4), 308 (3.3), 281 (12.9), 250 (8.4), 91 (100), 63

(6.1); anal. calcd. for C19H19ClN4O3 (%): C, 58.99; H, 4.95; N, 14.48; found (%): C, 58.70; H, 5.00; N, 14.14.

Experimental biological part

Cytotoxic activity evaluation. The SRB assay of Skehan [16] was used to evaluate the cytotoxic activity of compounds 8 – 14 and 20 – 24 against two cell lines, human brain carcinoma (U251) and human liver carcinoma (Hepg2). These cytotoxic activity assays were performed at the Cancer Biology Department (Pharmacology Unit, National Cancer Institute, Cairo, Egypt). Cells were plated in 96-multiwell plates (104 cells3well) and kept for 24 h

before treatment with test compounds to allow the attachment of cells to the wall of the plate. Using DMSO and saline solution as solvent system, various concentrations of the test compounds (10, 25, 50, and 100 g3ml) were added to the cell monolayer. Triplicate wells were used for each dose. Monolayer cells were incubated with the test compounds for 48 h at 37°C in atmosphere containing 5% CO2. After incubation, the cells were fixed, washed, and stained with Sulforhodamine B stain. Excess stain was washed with acetic acid and the attached stain was recovered with Tris EDTA buffer. Coloration intensity was measured in an ELISA reader. A relation between the percentage survival fraction and the drug concentration was plotted for each tumor cell line treated with particular compounds (Fig. 1).

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Sabmitted 26.12.05