Synthesis and Antiproliferative Activity of Novel Limonene Derivatives with a Substituted Thiourea Moiety

Isis M. Figueiredo, Luciane V. dos Santos, Willian F. da Costa, João E. de Carvalho, Cleuza C. da Silva, Juliana L. Sacoman, Luciana K. Kohn and Maria H. Sarragiotto* Departamento de Química, Universidade Estadual de Maringá, Avenida Colombo 5790, 87020-900 Maringá-PR, Brazil Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas, Universidade Estadual de Campinas, CP 6171, 13083-970 Campinas-SP, Brazil


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. 1-3D-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. 4-7Dietary 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)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13) bearing a substituted thiourea moiety, and of five their S-methyl analogues (14-18).
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 o C, in chloroform, for 24 h in quantitative yields.The 1 H NMR spectra of the Smethylthioureas showed signal at δ 2.78 -2.85 corresponding to the SCH 3 group.The formation of Smethythiourea was also evidenced by the presence of the signals at δ 16.9 -18.5 (S-CH 3 ) and δ 143.7 -169.2 (C=N) in the 13 C 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 GI 50 (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 LC 50 values.The TGI and LC 50 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)(4)(5)(6) exhibit cytotastic activity against all cancer cell lines tested, with GI 50 values in the range of 2.5 to 24 μmol L -1 .Analysis of TGI and LC 50 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 GI 50 and TGI values of 2.5 μmol L -1 and 22.5 μmol 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 GI 50 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 GI 50 values of 6.6 and 3.0 μmol 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 GI 50 3.0 and 6.1 μmol 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 GI 50 value of 0.41 μmol 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

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. 1 H and 13 C NMR spectra were recorded on a Varian Mercury Plus 300 MHz in CDCl 3 and TMS as internal reference.Column chromatography was performed on silica gel Merck 230-400 mesh ASTM.

General procedure for S-methylthioureas 14-18
To a solution of thiourea (1 mmol) in CHCl 3 (10 mL) was added methyl iodide (5 mmol) at 0 o C. The mixture was kept at 0 o C 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.

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. 17Doxorubicin 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 doseresponse curves and three dose response parameters (GI 50 , TGI and LC 50 ) were calculated.