Synthesis and antiproliferative activity of novel limonene derivatives with a substituted thiourea moiety

ARTICLE

Synthesis and antiproliferative activity of novel limonene derivatives with a substituted thiourea moiety

Isis M. Figueiredo^I; Luciane V. dos Santos^I; Willian F. da Costa^I; João E. de Carvalho^II; Cleuza C. da Silva^I; Juliana L. Sacoman^II; Luciana K. Kohn^II; Maria H. Sarragiotto^{*, I}

^IDepartamento de Química, Universidade Estadual de Maringá, Avenida Colombo 5790, 87020-900 Maringá-PR, Brazil

^IICentro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas, Universidade Estadual de Campinas, CP 6171, 13083-970 Campinas-SP, Brazil

ABSTRACT

A series of R-(+)-limonene derivatives bearing a substituted thiourea moiety (3-13) and five S-methyl analogs (14-18) were synthesized and evaluated for their in vitro antiproliferative activity against human cancer cell lines. Compounds bearing aromatic substituents (3-6) exhibit cytotastic activity in the full panel of cell lines tested, with GI₅₀ values in the range of 2.5 to 24 µmol L^-1. Compounds 3, 10, 12 and 16 were the most active with GI₅₀ values in the range of 0.41 to 3.0 µmol L^-1, against different cell lines.

Keywords: limonene derivatives, thioureas, antiproliferative activity

RESUMO

No presente trabalho descrevemos a síntese e a avaliação da atividade antiproliferativa, frente a linhagens de células tumorais humanas, de derivados do R-(+)-limoneno (3-18) contendo uma unidade tiouréia substituída. Os derivados com substituintes arílicos (3-6) exibiram atividade citostática frente a todas linhagens testadas, com inibição de 50% do crescimento celular (GI₅₀) em concentrações na faixa de 2,5 a 24 µmol L^-1. Os compostos 3, 10, 12 e 16 foram os mais ativos, com GI₅₀ na faixa de 0,41 a 3,0 mmol L^-1, frente a diferentes linhagens celulares.

Introduction

In the last years several approaches have been employed for cancer therapy, and to discover and develop novel therapeutic agents for the treatment of malignancy. In this context, the use of natural products as prototypes has been pointed out as one of the successful approaches to discover novel anticancer drugs.

Monoterpenes are a class of compounds, which occurs naturally in plant, and possess a range of pharmacological properties. Several studies have demonstrated the efficacy of this class of compounds as potential anticancer agents.^1-3 D-Limonene, a monoterpene found in a variety of foods and essential oils, have been shown to exert chemopreventive and chemotherapeutic activities in a variety of carcinogen-induced animal tumor models.^4-7 Dietary administration of D-limonene causes complete regression of N-nitrosourea (NMU)-induced and 7,12-dimethylbenzyl[a]anthracene (DMBA)-induced mammary carcinomas with minimal toxicity.^8,9

As a result of its chemopreventive and chemotherapeutic potential, limonene is under clinical trials. Phase I and pharmacokinetic study in patients with advanced cancer confirm its low toxicity and support D-limonene as prototype of a novel class of chemotherapeutic drugs.¹⁰

The potential use of limonene as an anticancer agent led us to focus our attention on the synthesis and antiproliferative activity evaluation of new limonene derivatives, as part of our research program in this area. We present herein the synthesis and the results of the antiproliferative activity evaluation of a series of limonene derivatives (3-13) bearing a substituted thiourea moiety, and of five their S-methyl analogues (14-18).

Results and Discussion

Synthesis

The synthetic route for the preparation of limonene derivatives is presented in Scheme 1. The limonene isothiocyanate (2) was obtained from the reaction of the limonene (1) with HSCN, in chloroform, according to a previously reported procedure.^11,12 Treatment of 2 with different amines (primary, heterocyclic and aromatic) afforded compounds 3-13. For primary and heterocyclic amines the reactions were carried out at 25 ºC for 15 h, by using 2 equivalents of the amine in chloroform as solvent, and the title thioureas were obtained in 67 – 97% yield. However, limited yields (20-30%) were observed for the reaction of 2 with aromatic amines such as aniline, o-toluidine, o-anisidine and p-bromoaniline. In these cases, reactions were performed with a large excess of amine, at 100 ºC for 24 h. The products (3-13) were characterized by analysis of their spectroscopic data. The presence of the C=S group was evidenced by the IR absorption bands at 1523-1566 and 1250-1410 cm^-1, together with the signal at d 177.3 – 181.8 in the ¹³C NMR spectra. The monoterpene moiety was characterized by the signals at d_H/d_C 5.30-5.40 (1H, brs, H-2)/119.7-120.5 (C-2), 1.30-1.49 (3H, s, H-8)/24.1-24.9 (C-8), 1.34-1.50 (3H, s, H-9)/24.2-25.4 (C-9) and 1.61-1.73 (3H, s, H-10)/23.0-23.5 (C-10) in the ¹H/¹³C NMR spectra. The NMR data were also consistent for the substituents attached to the nitrogen atom N-1, as showed in the Experimental Section. The EI-mass spectra showed peaks at m/z 58 and at m/z (M^+• - 135) as main fragments. The proposed mechanisms for the fragmentation are shown in Figure 1.

The S-methylated derivatives (14-18) were prepared from the reaction of the corresponding thioureas with methyl iodide at 0 ºC, in chloroform, for 24 h in quantitative yields. The ¹H NMR spectra of the S-methylthioureas showed signal at d 2.78 – 2.85 corresponding to the SCH₃ group. The formation of S-methythiourea was also evidenced by the presence of the signals at d 16.9 – 18.5 (S-CH₃) and d 143.7 – 169.2 (C=N) in the ¹³C NMR spectra, besides of the absorption at 1600-1690 cm^-1 (C=N), in the IR spectra.

Antiproliferative activity

The results of the antiproliferative assays are showed in Tables 1 and 2. The response parameter GI₅₀ (Table 1) refers to the drug concentration that produce a 50% reduction of cellular growth when compared to untreated control cells. Table 2 includes data for the compounds that reached TGI and LC₅₀ values. The TGI and LC₅₀ parameters refer, respectively, to the drug concentration for total growth inhibition, and that for killing 50% of the cells.

As shown in Table 1, the compounds bearing aromatic substituents (3-6) exhibit cytotastic activity against all cancer cell lines tested, with GI₅₀ values in the range of 2.5 to 24 mmol L^-1. Analysis of TGI and LC₅₀ data presented in Table 2 show that compounds 3-5 had higher potency for total growth inhibition, and for killing 50% of the cells. From this series, compound 3 was the most active, with GI₅₀ and TGI values of 2.5 mmol L^-1 and 22.5 mmol L^-1, respectively, against breast resistant NCI/ADR cancer cell line. On the other hand, compounds 7-18 of the aliphatic series exhibit different profiles and dependence on the nature of the substituent on the nitrogen atom. Compared to aromatic series, the specificity was increased with the most of the compounds of the aliphatic series exhibiting smaller GI₅₀ values for leukemic K-562 cell line. From the thiourea aliphatic series, compounds 9 and 12 showed potent activity and particular selectivity against leukemic K-562 cell line with GI₅₀ values of 6.6 and 3.0 mmol L^-1, respectively. The replacement of the 1-N-methyl group of the compound 12 for an oxygen results in the inactive compound 13. The thiourea 10 exhibits potent antiproliferative activity against ovarian OVCAR and breast resistant NCI/ADR with GI₅₀ 3.0 and 6.1 mmol L^-1, respectively. S-methylation of thioureas bearing aliphatic substituents results in changes in the antiproliferative activity profiles. Conversion of the thiourea 10 to the S-methylthiourea analogue 16, led to the most potent compound, with GI₅₀ value of 0.41 mmol L^-1 and high selectivity against leukemia K-562 cell lines.

Conclusions

In this paper we report the synthesis and cytotoxic evaluation of a series of new limonene derivatives containing a substituted thiourea moiety. The results show the potentiality of some compounds, particularly 3, 10, 12 and 16, as inhibitors of tumor cells proliferation. Some compounds bearing aromatic substituents were also able to kill 50% of the breast resistant NIC/ADR (compounds 3 and 4), melanoma UACC-62 (compound 4), and renal 786-0 (compounds 4 and 5) cancer cell lines.

Experimental

IR spectra were recorded on KBr pellets in a Bomem model MB-100 spectrophotometer. Mass spectra were measured on Shimadzu GC/MS, QP 2000A, at 70 eV. ¹H and ¹³C NMR spectra were recorded on a Varian Mercury Plus 300 MHz in CDCl₃ and TMS as internal reference. Column chromatography was performed on silica gel Merck 230-400 mesh ASTM.

General procedure for thioureas 3 – 13

To a solution of limonene isothiocyanate 2 (1 mmol) in CHCl₃ (10 mL) was added drop-wise the amine (2 mmol).^11,12 The solution was kept at room temperature for 15 hours and then the solvent was removed under reduced pressure. For aromatic amines, reactions were performed with a large excess of amine, without solvent, at 100 ºC for 24 h. The residue was purified by column chromatography using hexane and a mixture of hexane-ethyl acetate in increasing polarity as solvent.

N-[1-(4R)-(4-isopropyl-1-methylcyclohexenyl)]-N'-[2 -(phenyl)]thiourea (3). Yield: 30%; IR n_max/cm^-1: 3387 (N-H), 3156 (C-H arom), 1590 (C=C arom), 1540 and 1298 (C=S); EI-MS m/z (rel. int.): 288 (M^+•, 5), 153 (100, M^+•-135, [H₂NCSNHC₆H₅]⁺); ¹H NMR (300 MHz, CDCl₃): d 1.18 and 1.62 (1H each, m, H-5), 1.40 (3H, s, H-8), 1.45 (3H, s, H-9), 1.61 (3H, s, H-10), 1.84 and 1.90 (4H, m, H-3 and H-6), 2.59 (1H, m, H-4), 5.36 (1H, brs, H-2), 7.20 (2H, d, J 7.5 Hz, H-2' and H-6'), 7.29 (1H, t, J 7.5 Hz, H-4'), 7.43 (2H, t, J 7.5 Hz, H-3' and H-5'); ¹³C NMR (75.5 MHz, CDCl₃): d 23.5 (C-10), 24.2 (C-9), 24.3 (C-5), 24.5 (C-8), 26.7 (C-3), 31.2 (C-6), 41.1 (C-4), 59.5 (C-7), 120.5 (C-2), 125.2 (C-2' and C-6'), 127.1 (C-4'), 130.2 (C-3' and C-5'), 134.3 (C-1), 136.8 (C-1'), 179.5 (C=S).

N-[1-(4R)-(4-isopropyl-1-methylcyclohexenyl)]-N'-[2-(2 -methylphenyl)]thiourea (4). Yield: 30%. IR n_max/cm^-1: 3367 (N-H), 3173 (C-H arom), 1643 (C=C arom), 1621 (C=C), 1533 and 1251 (C=S); EI-MS m/z (rel. int.): 302 (M^+•, 10), 167 (100, M^+• - 135, [H₂NCSNH-2-methylphenyl]⁺), 91 (45), 58 (70), 41 (46); ¹H NMR (300 MHz, CDCl₃): d 1.39 (3H, s, H-8), 1.45 (3H, s, H-9), 1.61 (3H, s, H-10), 1.75 (2H, m, H-5), 1.93 (4H, m, H-3 and H-6), 2.29 (3H, s, CH₃-Ar), 2.48 (1H, m, H-4), 5.30 (1H, brs, H-2), 7.20 (1H, m, H-6'), 7.25 (1H, m, H-5'), 7.28 (1H, m, H-4'), 7.31 (1H, m, H-3'); ¹³C NMR (75.5 MHz, CDCl₃): d 18.1 (CH₃-Ar), 23.5 (C-10), 24.1 (C-8), 24.2 (C-5), 24.4 (C-9), 26.6 (C-3), 31.2 (C-6), 41.4 (C-4), 59.2 (C-7), 120.5 (C-2), 127.7 (C-6'), 127.8 (C-5'), 127.9 (C-2'), 128.7 (C-4'), 131.9 (C-3'), 134.3 (C-1), 136.0 (C-1'), 179.9 (C=S).

N-[1-(4R)-(4-isopropyl-1-methylcyclohexenyl)]-N'-[2 -(2-methoxyphenyl)]thiourea (5). Yield: 31%; IR n_max/cm^-1: 3370 (N-H), 3048 (C-H arom), 1599 and 1535 (C=C), 1460 and 1250 (C=S); EI-MS m/z (rel. int.): 318 (M^+•, 7), 183 (27, M^+• - 135, [H₂NCSNH-2-methoxyphenyl]⁺ ); 123 (93, [H₂N-2-methoxyphenyl]^+·), 93 (27), 58 (100), 41 (43); ¹H NMR (300 MHz, CDCl₃): d 1.42 (3H, s, H-8), 1.48 (3H, s, H-9), 1.63 (3H, s, H-10), 1.77 (2H, m, H-5), 1.90 (4H, m, H-3 and H-6), 2.00 (1H, m, H-4), 3.84 (3H, s, OCH₃), 5.36 (1H, brs, H-2), 6.92 (1H, m, H-3'), 6.94 (1H, d, J 7.8 Hz, H-6'), 6.98 (1H, t, J 7.8 Hz, H-5'), 7.20 (1H, t, J 7.8 Hz, H4'); ¹³C NMR (75.5 MHz, CDCl₃): d 23.4 (C-10), 24.3 (C-5), 24.6 (C-8), 24.6 (C-9), 26.7 (C-3), 31.2 (C-6), 41.7 (C-4), 55.8 (OCH₃), 59.3 (C-7), 110.3 (C-4'),120.5 (C-2), 121.0 (C- 5' and C-6'), 124.5 (C-3'), 134.1 (C-1'), 134.2 (C-1), 154.6 (C-2'), 179.3 (C=S).

N-[1-(4R)-(4-isopropyl-1-methylcyclohexenyl)]-N'-[2-(4 -bromophenyl)]thiourea (6). Yield: 20%; IR n_max/cm^-1: 3281 (N-H), 3103 (C-H), 1578 and 1530 (C=C, arom.), 1488 and 1364 (C=S); EI-MS m/z (rel. int.): 366 (M^+•, 7), 215 (23), 58 (100); ¹H NMR (300 MHz, CDCl₃): d 1.24 and 1.68 (1H each, m, H-5'), 1.42 (3H, s, H-8), 1.48 (3H, s, H-9), 1.63 (3H, s, H-10), 1.68 and 1.97 (1H each, m, H-3), 1.96 (2H, m, H-6), 2.59 (1H, m, H-4), 5.36 (1H, brs, H-2), 7.09 (2H, d, J 8.7 Hz, H-2' and H-6'), 7.54 (2H, d, J 8.7 Hz, H-3' and H-5'); ¹³C NMR (75.5 MHz, CDCl₃): d 23.5 (C-10), 24.4 (C-5), 24.6 (C-8), 24.6 (C-9), 26.7 (C-3), 31.2 (C-6), 40.9 (C-4), 59.7 (C-7), 120.4 (C-2), 120.6 (C-4'), 126.8 (C2' and C-6'), 133.4 (C3' and C-5'), 134.4 (C-1), 136.0 (C-1'), 179.4 (C=S).

N-[1-(4R)-(4-isopropyl-1-methylcyclohexenyl)]-N'-[2 -(butyl)]thiourea (7). Yield: 97%; IR n_max/cm^-1: 3269 (N-H), 1543 and 1342 (C=S); EI-MS m/z (rel. int.): 268 (M^+•, 10), 133 (100, M^+• - 135, [H₂NCSNH-butyl]⁺), 58 (68), 41 (60); ¹H NMR (300 MHz, CDCl₃): d 0.95 (3H, t, J 7.2 Hz, H-4'); 1.35 (3H, s, H-8), 1.39 (3H, s, H-9), 1.41 (2H, m, H-3'), 1.60 (2H, m, H-6), 1.64 (3H, s, H-10), 1.82 (2H, m, H-5), 1,99 (3H, m, H-3 and H-4), 2.01 (2H, m, H-2'), 3.54 (2H, q, J 6.0 Hz, H-1'), 5.40 (1H, brs, H-2); ¹³C NMR (75.5 MHz, CDCl₃): d 13.7 (C-4'), 20.1 (C-3'), 23.2 (C-10), 24.2 (C-5), 24.8 (C-8), 25.0 (C-9), 26.5 (C-3), 30.9 (C-2'), 31.2 (C-6), 58.3 (C-7), 42.2 (C-4), 45.1 (C-1'), 120.0 (C-2), 133.9 (C-1), 180.8 (C=S).

N-[1-(4R)-(4-isopropyl-1-methylcyclohexenyl)]-N'-[2 -(isopropyl)]thiourea (8). Yield: 84%; IR n_max/cm^-1: 3272 (N-H), 1538 and 1325 (C=S); EI-MS m/z (rel. int.): 254 (M^+•, 7), 119 (100, M^+• - 135, [H₂NCSNH-isopropyl]⁺), 58 (65), 41 (60); ¹H NMR (300 MHz, CDCl₃): d 1.23 (6H, d, J 6.0 Hz, NHCH(CH₃)₂), 1.35 (3H, s, H-8), 1.40 (3H, s, H-9), 1.65 (3H, s, H-10), 1.78 (2H, m, H-5), 1.99 (5H, m, H-3, H-4 and H-6), 4.40 (1H, m, H-1'), 5.40 (1H, brs, H-2); ¹³C NMR (75.5 MHz, CDCl₃): d 22.6 (NHCH(CH₃)₂), 23.2 (C-10), 24.2 (C-5), 24.9 (C-8), 25.2 (C-9), 26.6 (C-3), 30.9 (C-6), 42.5 (C-4), 47.2 (C-1'), 58.3 (C-7), 120.0 (C-2), 134.0 (C-1), 179.8 (C=S).

N-[1-(4R)-(4-isopropyl-1-methylcyclohexenyl)]-N'-[2 -(isopentyl)]thiourea (9). Yield: 80%; IR n_max/cm^-1: 3258 (N-H), 1643 (C=C), 1537 and 1322 (C=S); (KBr); EI-MS m/z (rel. int.): 282 (M^+·•, 10), 147 (100, M^+• - 135, [H₂NCSNH-isopentyl]⁺), 58 (70); 41 (65); ¹H NMR (300 MHz, CDCl₃): d 0.93 (6H, t, J 7.5 Hz, NHCH(CH₂CH₃)₂ ), 1.30 and 1.83 (1H each, m, H-5), 1.34 (3H, s, H-8), 1.38 (3H, s, H-9), 1.51 (4H, m, NHCH(CH₂CH₃)₂ ), 1.61(2H, m, H-3), 1.65 (3H, s, H-10), 1.99 (3H, m, H-4 and H-6), 4.23 (1H, m, H-1'), 5.36 (1H, brs, H-2); ¹³C NMR (75.5 MHz, CDCl₃): d 10.0 (NHCH(CH₂CH₃)₂ ), 23.3 (C-10), 24.3 (C-5), 25.1 (C-8), 25.4 (C-9), 26.6 (C-3), 27.0 (NHCH(CH₂CH₃)₂ ), 31.0 (C-6), 42.8 (C-4), 58.2 (C-7), 58.3 (C-1'), 119.7 (C-2), 134.1 (C-1),180.7 (C=S).

N-[1-(4R)-(4-isopropyl-1-methylcyclohexenyl)]-N'-[2 -(cyclohexyl)]thiourea (10). Yield: 80%; IR n_max/cm^-1: 3258 (N-H); 1540 and 1338 (C=S); EI-MS m/z (rel. int.): 294 (M^+•, 10), 58 (100), 41 (60); ¹H NMR (300 MHz, CDCl₃): d 1.37 (3H, s, H-9), 1.34 (3H, s, H-8), 1.60 (m, H-4'), 1.65 (3H, s, H-10), 1.67 (4H, m, H-3'and H-5'),1.80 (2H, m, H-6), 1.81 (4H, m, H-3 and H-5), 1,93 (1H, m, H-4), 2.02 (4H, m, H-2'and H-6'), 4.12 (1H, m, H-1'), 5.36 (1H, brs, H-2); ¹³C NMR (75.5 MHz, CDCl₃): d 23.3 (C-10), 24.2 (C-5), 24.7 (C-3'and C-5'), 24.9 (C-8), 25.2 (C-9), 25.4 (C-4'), 26.6 (C-3), 31.0 (C-6), 32.9 (C-2'and C-6'), 42.7 (C-4), 53.9 (C-1'), 58.3 (C-7), 120.0 (C-2), 134.0 (C-1), 179.7 (C=S).

N-[1-(4R)-(4-isopropyl-1-methylcyclohexenyl)]-N'-[2 -(pyrrolidyl)]thiourea (11). Yield: 67%; IR n_max/cm^-1: 3395 (N-H), 1643 (C=C), 1531 and 1347 (C=S); EI-MS m/z (rel. int.): 266 (M^+•); 233 (28); 131 (100, M^+• - 135; [H₂NCSNH-pyrrolidyl]⁺), 114 (92); 93 (30); 58 (38); 41 (58); ¹H NMR (300 MHz, CDCl₃): d 1.25 and 1.78 (1H each, m, H-5), 1.49 (3H, s, H-8), 1.55 (3H, s, H-9), 1.64 (3H, s, H-10), 1.77 and 2.04 (1H each, m, H-3), 1.98 (2H, m, H-6), 1.98 (4H, m, H-2'and H-3'), 2.70 (1H, tdd, J 12.0, 2.4 and 2.1, H-4), 3.55 (4H, m, H-1' and H-4'), 5.37 (1H, brs, H-2); ¹³C NMR (75.5 MHz, CDCl₃): d 23.3 (C-10), 24.1 (C-5), 24.6 (C-8), 24.9 (C-9), 25.5 (C-2'and C-3'), 26.6 (C-3), 31.1 (C-6), 40.8 (C-4), 49.2 (4H, m, C-1' and C-4'), 58.9 (C-7), 120.6 (C-2), 134.0 (C-1), 177.3 (C=S).

N-[1-(4R)-(4-isopropyl-1-methylcyclohexenyl)]-N'-[2-(N -methylpiperazyl)]thiourea (12). Yield: 90%; IR n_max/cm^-1: 3438 (N-H), 1629 (C=C), 1566 and 1410 (C=S); EI-MS m/z (rel. int.): 295 (M^+•); 195 (20), 121 (34), 93 (64), 58 (100), 41 (97); ¹H NMR (300 MHz, CDCl₃): d 1.46 (3H, s, H-8), 1.53 (3H, s, H-9), 1.63 (3H, s, H-10), 1.73 (2H, m, H-5), 1.81 and 1.96 (1H each, m, H-3), 1.96 (2H , m, H-6), 2.30 (3H, s, NCH₃), 2.43 (4H, t, J 5.1 Hz, H-2'and H-3'), 2.81 (1H, m, H-4), 3.78 (4H, t, J 5.1 Hz, H-1'and H-4'), 5.36 (1H, brs, H-2); ¹³C NMR (75.5 MHz, CDCl₃): d 23.0 (C-10), 23.8 (C-5), 24.2 (C-9), 24.3 (C-8), 26.2 (C-3), 30.7 (C-6), 39.9 (C-4), 45.4 (NCH₃), 46.8 (C-1' and C-4'), 54.2 (C-2' and C-3'), 58.9 (C-7), 120.3 (C-2), 133.5 (C-1), 180.8 (C=S).

N-[1-(4R)-(4-isopropyl-1-methylcyclohexenyl)]-N'-[2 -(morpholyl)]thiourea (13). Yield: 88%; IR n_max/cm^-1: 3408 (N-H), 1523 and 1347 (C=S); ¹H NMR (300 MHz, CDCl₃): d 1.48 (3H, s, H-8), 1.54 (3H, s, H-9), 1.65 (2H, m, H-5), 1.73 (3H, s, H-10), 2.02 (4H, m, H-3 and H-6), 2.75 (1H, m, H-4), 3.73 (8H, m, H-1', H-2', H-3'and H-4'), 5.36 (1H, brs, H-2); ¹³C NMR (75.5 MHz, CDCl₃): d 23.3 (C-10), 24.3 (C-5), 24.6 (C-8), 24.7 (C-9), 26.5 (C-3), 30.7 (C-6), 44.5 (C-4), 47.4 (C-1'and C-4'), 59.5 (C-7), 66.3 (C-2'and C-3'), 119.7 (C-2), 134.1 (C-1), 181.8 (C=S).

General procedure for S-methylthioureas 14-18

To a solution of thiourea (1 mmol) in CHCl₃ (10 mL) was added methyl iodide (5 mmol) at 0 ºC. The mixture was kept at 0 ºC for 24 h and then the solvent and excess of methyl iodide were removed under reduced pressure to give the salt of the corresponding S-methylthiourea in quantitative yield for all compounds.

N-[1-(4R)-(4-isopropyl-1-methylcyclohexenyl)]-N'-[2 -(isopropyl)]-S-methylthiourea (14). IR n_max/cm^-1: 3193 (N-H), 1600 (C=N); EI-MS m/z (rel. int.): 268 (7, M^+•), 220 (68, M^+• – CH₃SH), 205 (57), 128 (100), 127 (60), 93 (77), 58 (91), 43 (96), 41 (85); ¹H NMR (300 MHz, CDCl₃): d 1.49 (6H, d, J 6.3 Hz, NCH(CH₃)₂), 1.50 (3H, s, H-8), 1.58 (3H, s, H-9), 1.65 (3H, s, H-10), 1.76 (2H, m, H-5), 1.93 (4H, m, H-3 and H-6), 2.27 (1H, m, H-4), 2.85 (3H, s, SCH₃), 4.30 (1H, m, H-1'), 5.35 (1H, brs, H-2); ¹³C NMR (75.5 MHz, CDCl₃): d 18.4 (SCH₃), 22.7 (NCH(CH₃)₂), 23.3 (C-10), 24.4 (C-5), 25.1 (C-8), 25.7 (C-9), 26.7 (C-3), 30.8 (C-6), 42.7 (C-4), 51.2 (C-1'), 57.4 (C-7), 120.1 (C-2), 134.1 (C-1), 143.7 (C=N).

N-[1-(4R)-(4-isoproyl-1-methylcyclohexenyl)]-N'-[2 -(isopentyl)]-S-methylthiourea (15). IR n_max/cm^-1: 3347 (N-H), 1690 (C=N); EI-MS m/z (rel. int.): 248 (40, M^+• – CH₃SH), 233 (50), 219 (55), 128 (81), 127 (49), 93 (61), 58 (74); 43 (100), 41 (86); ¹H NMR (300 MHz, CDCl₃): d 0.99 (6H, t, J 7.3 Hz, NCH(CH₂CH₃)₂ ), 1.33 (1H, m, H-5), 1.55 (3H, s, H-8), 1.58 (3H, s, H-9), 1.65 (3H, s, H-10), 1.77 (1H, m, H-5'), 1.79 (4H, m, NCH(CH₂CH₃)₂ ), 1.94 (2H, m, H-3), 2.01 (2H, m, H-6), 2.26 (1H, m, H-4), 2.78 (3H, s, SCH₃), 3.78 (1H, m, H-1'), 5.37 (1H, brs, H-2); ¹³C NMR (75.5 MHz, CDCl₃): d 10.9 (NCH(CH₂CH₃)₂ ), 18.4 (SCH₃), 23.3 (C-10), 24.4 (C-5), 25.1 (C-8), 25.6 (C-9), 26.7 (C-3), 27.5 (NCH(CH₂CH₃)₂ ), 30.8 (C-6), 42.8 (C-4), 61.5 (C-7), 63.4 (C-1'), 119.4 (C-2),134.4 (C-1), 169.2 (C=N).

N-[1-(4R)-(4-isopropyl-1-methylcyclohexenyl)]-N'-[2 -(cyclohexyl)]-S-methyl-thiourea (16). IR n_max/cm^-1: 3186 (N-H), 1685 (C=N), 1591 (C=C); EI-MS m/z (rel. int.): 308 (M^+•), 261 (31, M^+• – CH₃S^·), 128 (63), 127 (39), 93 (53), 58 (55), 55 (54), 43 (57), 41 (100); ¹H NMR (300 MHz, CDCl₃): d 1.34 and 1.81 (1H each, m, H-6), 1.55 (3H, s, H-8), 1.57 (3H, s, H-9), 1.62 (2H, m, H-4'), 1.65 (3H, s, H-10), 1.81 (2H, m, H-5), 1.81 (4H, m, H-3'and H-5'), 1.93 (4H, m, H-2'and H-6'), 1.93 (2H, m, H-3), 2.25 (1H, m, H-4), 2.83 (3H, s, SCH₃), 3.88 (1H, m, H-1'), 5.37 (1H, brs, H-2); ¹³C NMR (75.5 MHz, CDCl₃): d 18.5 (SCH₃), 23.3 (C-10), 24.4 (C-4'), 24.6 (C-5), 24.8 (C-3' and C-5'), 25.6 (C-8 and C-9), 26.7 (C-3), 30.7 (C-6), 32.6 (C-2'and C-6'),42.7 (C-4), 56.8 (C-1'), 63.4 (C-7), 119.4 (C-2), 134.3 (C-1), 168.1 (C=N).

N-[1-(4R)-(4-isopropyl-1-methylcyclohexenyl)]-N'-[2 -(pyrrolidyl)]-S-methyl-thiourea (17). IR n_max/cm^-1: 1677 (C=N), 1584 (C=C); EI-MS m/z (rel. int.): 280 (M^+•),142 (52), 128 (93), 127 (69), 84 (44), 70 (42), 57 (42), 43 (100), 42 (87), 41 (40); ¹H NMR (300 MHz, CDCl₃): d 1.36 and 1.82 (1H each, m, H-5), 1.60 (3H, , H-9), 1.62 (3H, s, H-8), 1.65 (3H, s, H-10), 1.82 (m, H-5), 1.96 (2H, m, H-3), 2.01 (2H, m, H-6), 2.18 (4H, m, H-2'and H-3'), 2.35 (1H, m, H-4), 2.70 (3H, s, SCH₃), 4.04 (4H, m, H-1'and H-4'), 5.37 (1H, brs, H-2); ¹³C NMR (75.5 MHz, CDCl₃): d 17.6 (SCH₃), 23.5 (C-10), 24.6 (C-5), 25.4 (C-2'and C-3'), 26.2 (C-8), 26.6 (C-9), 27.1 (C-3), 31.1 (C-6), 43.3 (C-4), 53.5 (C-1'and C-4'), 64.1 (C-7),119.9 (C-2), 134.4 (C-1), 164.2 (C=N).

N-[1-(4R)-(4-isoproyl-1-methylcyclohexenyl)]-N'-[2-(N -methypiperazyl)]-S-methyl-thiourea (18). IR n_max/cm^-1: 1675 (C=N), 1610 (C=C); EI-MS m/z (rel. int.): 304 (M^+•), 195 (43), 136 (55), 121 (28), 93 (100), 81 (53), 58(16), 44 (56), 41 (75); ¹H NMR (300 MHz, CDCl₃): d 1.27 (3H, s, H-8), 1.28 (3H, s, H-9), 1.65 (3H, s, H-10), 1.76 (2H, m, H-5), 1.80 and 1.97 (1H each, m, H-3), 1.97 (3H, m, H-4 and H-6), 2.34 (3H, s, NCH₃), 2.73 (3H, s, SCH₃), 3.59 (4H, m, H-1'and H-4'), 3.70 (4H, m, H-2'and H-3'), 5.37 (1H, brs, H-2); ¹³C NMR (75.5 MHz, CDCl₃): d 16.9 (SCH₃), 23.4 (C-10), 24.3 (C-5), 24.6 (C-8), 24.8 (C-9), 26.9 (C-3), 31.3 (C-6), 42.9 (C-1'and C-4'), 46.2 (C-4), 58.8 (C-7), 61.5 (C-2'and C-3'), 52.2 (NCH₃), 121.1 (C-2), 133.9 (C-1), 151.1 (C=N).

Antiproliferative assays

Synthesized compounds were evaluated in vitro against a nine-cell panel lines consisting of melanoma UACC-62, breast MCF7, lung NCI-460, leukemia K-562, ovarian OVCAR, prostate PCO-3, colon HT29, renal 786-0 and breast resistant NCI/ADR according NCI standard protocol.¹⁷ Doxorubicin was used as positive control. Assays were performed in a 96-well plate using four concentrations at 10-fold dilutions (0.25 mg mL^-1 to 250 mg mL^-1) for each test compound. The anticancer activity was deduced from dose-response curves and three dose response parameters (GI₅₀, TGI and LC₅₀) were calculated.

Acknowledgments

We are grateful to CAPES (Brazil) for the fellowship to I. M. Figueiredo.

Received: January 9, 2006

Published on the web: July 6, 2006

FAPESP helped in meeting the publication costs of this article.

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Publication Dates

• Publication in this collection
  01 Dec 2006
• Date of issue
  Oct 2006

History

• Accepted
  06 June 2006
• Received
  09 Jan 2006