Semi-synthesis and cytotoxicity evaluation of pyrimidine, thiazole, and indole analogues of argentatins A–C from guayule (Parthenium argentatum) resin

Argentatins A–C (1–3), the major cycloartane-type triterpenoids of guayule resin, a byproduct of commercial rubber production, were converted into their pyrimidine (7–12), thiazole (13–15), and indole (16–18) analogues by a molecular hybridization approach. The cytotoxic activities of these fused heterocyclic analogues 7–18 were compared with those of argentatins A–C (1–3) against a panel of three sentinel human cancer cell lines [NCI-H460 (non-small cell lung), MCF-7 (breast adenocarcinoma), and SF-268 (central nervous system glioma)], and normal human fibroblast (WI-38) cells. The cytotoxicity data suggest that the pyrimidine analogues 7 and 8 (derived from 1), 9 and 10 (derived from 2), and 12 (derived from 3) had significantly enhanced activity compared to the parent compounds or their thiazole (13–15) and indole (16–18) analogues. These findings indicate that triterpenoid constituents of guayule resin may be exploited to obtain value-added products with potential applications in anticancer drug discovery.


Introduction
Natural products (NPs) continue to be valuable sources of structurally diverse and biologically active compounds for drug discovery [1][2][3]. Many NPs have also served as promising lead compounds for drug development, especially as anticancer agents, after chemical modifications [4,5]. Triterpenoids, one of the major classes of NPs, have been reported to exhibit a variety of biological activities [6,7]. Among these, cycloartane-type triterpenoids are found widely in plants and algae, some of which are known to display cytotoxic activity [8,9]. Cycloartane-type triterpenoids, such as argentatins A-C, have been found to be the major chemical components in Parthenium argentatum Gray (guayule) resin [10,11], which is one of the byproducts in the manufacture of natural rubber from guayule, a widely-investigated arid land crop of potential industrial importance [12][13][14]. Among argentatins, argentatin A has been found to have antimicrobial activity [15], whereas argentatin B is known to inhibit proliferation of colon (HCT-15) and prostate (PC-3) cancer cells [16]. Detailed studies on argentatin B has revealed that it is a noncompetitive inhibitor of 3H-estradiol-binding receptors of hormone-dependent breast cancer [17]. In addition, ring A modification of argentatins A and B have provided several analogues with enhanced cytotoxic activity against some selected cancer cell lines [18,19]. Therefore, it was of interest to investigate the effect of structural modifications of argentatins A-C (1-3) on their potential anticancer activity for the purpose of obtaining value-added products from this byproduct of guayule rubber production.
Heteroaromatic moieties such as pyrimidines, thiazoles, and indoles are important fragments representing useful pharmacophores for the enhancement of therapeutic activity. Compounds containing these moieties have been found to exhibit a wide variety of biological activities, including anticancer, anti-inflammatory, antimicrobial, anti-HIV, antitubercular, antioxidant, and anti-hypertensive activities [20][21][22][23][24][25]. Molecular hybridization involving the linking of two structural moieties as a single hybrid is an important strategy in medicinal chemistry to enhance biological activity and pharmacokinetic properties [26,27]. We envisaged that argentatins A-C (1-3) with a carbonyl group at C-3 in ring A and some differences in ring D and the side chain could serve as suitable starting materials for conversion into the corresponding heteroaromatic hybrids which may then display enhanced biological activities. Herein we report the semi-synthesis of novel pyrimidine, thiazole, and indole analogues 7-18 of argentatins A-C (1-3) and the investigation of their cytotoxic activity against three sentinel human cancer cell lines and normal human fibroblast cells.

Results and discussion
Chemistry Argentatins A-C (1-3) obtained from guayule resin [28], were used for the synthesis of their target pyrimidine, thiazole, and indole analogues as outlined in Schemes 1 and 2. Depicted in Scheme 1 is the synthesis of pyrimidine analogues (7)(8)(9)(10)(11)(12). Claisen-Schmidt condensation [22] of 1-3 with benzaldehyde afforded their corresponding benzylidene intermediates 4-6 in 74-77% yield, which were the key precursors for the synthesis of their pyrimidine analogues. Presence of the benzylidene moiety in 4-6 were inferred from their characteristic absorption bands of the α, β-unsaturated carbonyl group at 1678 cm −1 in their IR spectra and the presence of a signal at δ C~2 08 in their 13 C NMR spectra. The stereochemistry of the newly formed double bond was determined to be of E-configuration from the 1 H NMR coupling constant (ca. 3.0 Hz) observed for one of the protons at C-1 due to allylic coupling ( 4 J trans ) [29] and by comparison with the 1 H NMR data of H 2 -1 and the olefinic proton of the benzylidene group of 2-arylidenedihydrotestosterones for which E-configuration of the benzylidene double bond has been confirmed by X-ray crystallography [30]. Intermediates 4-6 were separately treated with guanidine hydrochloride, thiourea, and urea in the presence of ethanolic KOH under reflux to give crude products which on treatment with DDQ (2,3dichloro-5,6-dicyano-p-benzoquinone) in dry 1,4-dioxane at room temperature afforded 2′-amino-pyrimidine analogues (7, 9, and 11) in 49-79% yield and the 2′-oxo-3′Hpyrimidine analogues (8, 10, and 12) in 31-58% yield. The structures of pyrimidine analogues were confirmed by the characteristic peaks in their 13 C NMR spectra. Signals around δ C 175, 166, 161, and 116 ppm in 2′-amino-pyrimidine analogues 7, 9, and 11 were assigned to C-3, C-4′, C-2′ and C-2, respectively by the HMBC correlations of H 3 -29/C-3, H 3 -30/C-3, H 2 -1/C-3, H 2 -1/C-4′, and H 2 -1/C-2. The presence of the 2′-amino-pyrimidine moiety in these was further confirmed by the 1 H NMR signal (δ H 4.80-4.90, brs, 2H) due to the NH 2 group. It is noteworthy that the pyrimidine analogues 8, 10, and 12 exhibited only two 13 C NMR signals for the pyrimidine moiety (at δ C~1 59 and 110-112 ppm), assigned for C-2′ and C-2 respectively, and the chemical shift of C-2 indicated the presence of 2′-oxo-substituted pyrimidine moiety in these [31]. The actual tautomer of pyrimidine moiety was suggested to be 3′H-pyrimidine because C-3 of 10 was detected at δ C 164.4 ppm by the HMBC experiment although the signals due to C-3 and C-4′ of the pyrimidine ring were not detected or appeared as very weak peaks [32].
Synthesis of thiazole and indole analogues (13-18) of argentatins A-C (1-3) is outlined in Scheme 2. The target thiazole analogues were obtained by a two-step protocol. Compounds 1-3 were brominated at C-2 by the reaction with liquid bromine in acetic acid at 0°C-25°C [18]. The resulting mixture of bromoketones (α and β epimers) were used without further purification for the next step involving cyclization with thiourea [31] in ethanol at reflux temperature to afford fused amino-thiazoles (13)(14)(15) in 46-58% yield. The identities of these products were confirmed by their NMR data. The characteristic broad singlet proton signals at δ H~4 .78 ppm of these in their 1 H NMR spectra were assigned to the NH 2 group, whereas the carbon signals in their 13 C NMR spectra at δ C~1 64-168 (C-2′),~154 (C-3), and~116 (C-2) corresponding to thiazole rings were assigned with the help of HMBC correlations of H 2 -1/C-3, H 2 -1/C-2 and H 3 -29/C-3, H 3 -30/C-3.

Conclusion
In summary, we designed and synthesized twelve new fused heterocyclic analogues 7-18 of argentatins A-C (1-3) by utilizing the C-3 keto functionality of ring A of the parent compounds. All analogues were evaluated for their cell proliferation inhibitory activity against the three sentinel cancer cell lines NCI-H460, MCF-7, SF-268, and towards normal fibroblast cells, WI-38. The bioassay results demonstrated that the analogues 7, 8, 9, 10, and 12 exhibited enhanced cytotoxic activity against NCI-H460 and  3.8 ± 0.8 >10.0 5.7 ± 0.6 8.1 ± 1.1 10 3.9 ± 0.3 7.6 ± 0.7 3.6 ± 0.1 5.9 ± 0.5 12 3.4 ± 0.7 Among all compounds tested, the pyrimidine analogue 10 was unique in displaying single-digit micro-molar activity against SF-268 cells. The preliminary structure-activity relationship (SAR) study suggested that the 2′-amino-pyrimidine and 2′-oxo-3′H-pyrimidine derivatives were more active than the thiazole and the indole analogues against the cell lines tested. These findings support the possibility of development of potential anticancer agents from some triterpenoid constituents of the guayule resin. The following abbreviations were used to indicate the NMR signals: s = singlet, brs = broad singlet, d = doublet, t = triplet, m = multiplet, J = coupling constant. All compounds used for cytotoxicity assays were purified by HPLC and the purity of each compound was determined to be ≥95% by HPLC analysis.