Design and synthesis of novel 7-[(N-substituted-thioureidopiperazinyl)-methyl]-camptothecin derivatives as potential cytotoxic agents

Abstract As part of continuing our research on diverse C-7 derivatives of camptothecin (CPT), 16 CPT derivatives bearing piperazinyl-thiourea chemical scaffold and different substituent groups have been designed, synthesized and evaluated in vitro for cytotoxicity against five tumor cell lines (A-549, MDA-MB-231, MCF-7, KB and KBvin). As a result, all the synthesized compounds showed promising in vitro cytotoxic activity against the five tumor cell lines tested, and were more potent than irinotecan. Importantly, compounds 13 g (IC50 = 0.514 μM) and 13o (IC50 = 0.275 μM) possessed similar or better antiproliferative activity against the multidrug-resistant (MDR) KBvin subline than that of topotecan (IC50 = 0.511 μM) and merit further development as anticancer candidates for clinical trail. With these results in hand, we have a reason to conclude that incorporating piperazinyl-thiourea motifs into position-7 of camptothecin confers well cytotoxic activity against cancer cell lines, probably resulting in new anticancer drugs. Graphical Abstract

Camptothecin (CPT) is one of the natural quinoline-class alkaloids extracted from the wood and bark of Camptotheca acuminata (happy tree in Chinese). Its powerful antitumor activity is induced by its directly binding to topoisomerase I (Topo I), which results in interference with the catalytic cycle of DNA-Topo I and stabilization of the DNA-Topo I binary complex, the molecular basis for the cytotoxic activity of CPT (Burke 1996;Li et al. 2006;Liu et al. 2015). Extensive chemical modifications of camptothecin on its C-7-, 9-or 10-position led to the clinical introduction of three semisynthetic analogues, that is, irinotecan (2), topotecan (3) and belotecan (4), and they, exhibiting better solubility and pharmacokinetic profiles, are now widely used in clinical cancer chemotherapy, alone or in combination, for the treatment of ovarian, colon, breast, lung and leukemia cancers (Zunino et al. 2002;Liew and Yang 2008;Liu et al. 2018;Song et al. 2018). Particularly, during early chemical modification studies, numerous studies found that the introduction of lipophilic substituents at the 7-position provides favorable molecular interactions and improved pharmacological features that could have potential therapeutic advantages (Wang et al. 1994;Jew et al. 1996;Dallavalle et al. 2000Dallavalle et al. , 2001Dallavalle et al. , 2006Wang et al. 2014a;Pisano et al. 2008;Niizuma et al. 2009;Liu et al. 2012). Accordingly, various substitutions, such as ethyl (Sawada et al. 1991), aryliminomethyl (Dallavalle et al. 2004), alkylsilyl (Josien et al. 1997;Du et al. 2003), and cycloalkyl (Li et al. 2009), were introduced at the 7-position of CPT leading to either enhanced or comparable activity. Through C-7 modification, some analogues were found to exhibit superior pharmacological properties to 1, and several clinical trial drug candidates, including gimatecan (5), BNP-1350 (6), lurtotecan (7) and sinotecan (8) emerged as alternatives to overcome the drawbacks of 1 . Their clinical success, along with extensive structure-activity and pharmacology studies, reenergized interest in further modification studies on the C-7 substituted CPT to achieve better antitumor activity. A binding model of CPT with biological macromolecules also further demonstrated that the C-7 molecular area could accommodate considerable structural diversities (Fan et al. 1998;Staker et al. 2002).
In our recent search for camptothecin-derived antitumor agents, we have been involved in chemical transformations and isolation of camptothecin analogues and have prepared a large number of camptothecin derivatives by modifications of different positions in the camptothecin skeleton, some of which displayed significant antitumor activities (Skehan et al. 1990;Wang et al. 2014b;Song et al. 2016;Zhu et al. 2017). As highlights, most of 7-substituted camptothecin analogs displayed potent cytotoxic activities with distinctively different drug-resistance profiles from those of 1, which suggested an important role of various C-7 substitutions in the activity profiles of 1 analogs and the feasibility of optimizing this compound class through rational C-7 modifications. This critical structure-activity relationship (SAR) basis prompted us urgently to further extend our investigation by synthesizing a novel series of 7-[(Nsubstituted-thioureidopiperazinyl)-methyl]-camptothecin derivatives. The two functional fragments, thiourea and piperazine, were chosen based on the facts that these groups are commonly abundant not only in all kinds of drugs but also in substantial bioactive natural products and usually introduction of an intrinsic antineoplastic thioureidopiperazinyl group can usually potentiate the biochemical or pharmacological properties of the original molecule (Chen et al. 2018;Seelam et al. 2018). With the intention of advancing our previous work and exploring more potent camptothecinderived antitumor agents, in this paper, we used camptothecin as lead compound and introduced the two functional parts, thiourea and piperazine, into 1 at the C-7 position via a coupling reaction according to Figure 1  .
The synthetic route to target compounds 13a-p is depicted in Figure 2. Briefly, the starting material CPT was converted into 7-chloromethyl-CPT (10) first in excellent yields by reaction with ClCH 2 CHO in 75% sulfuric acid in the presence of hydrogen peroxide and ferrous sulfate heptahydrate (Sawada et al. 1991). Then treatment of 10 with N-Boc protected piperazine in dry DMF solution furnished precursor 11 in 80% yields. The N-Boc group of 11 was removed with trifluoroacetic acid (TFA) in CH 2 Cl 2 (1:1) to form the key intermediate TFA salts 12 ). Subsequently, intermediate 12 was coupled with the appropriate isothiocyanates in dry CH 2 Cl 2 to provide the target compounds 13a-p in 61-77% yields.
The sixteen new camptothecin derivatives were evaluated for in vitro cytotoxic activity against five tumor cell lines, that is, KB (nasopharyngeal Carcinoma), KBvin (MDR KB subline), A-549 (lung Carcinoma), MDA-MB-231 (breast Carcinoma) and MCF-7 (breast adenocarcinoma) by using a sulforhodamine B colorimetric assay with triplicate experiments (Zhu et al. 2017). Irinotecan and topotecan were used as positive controls. The screening results are shown in Table 1.
As displayed in Table 1, all the synthesized compounds exhibited noticeable in vitro activity against the tested five tumor cell lines, and the overwhelming majority of them were more potent than 2, which is a commercial available and clinical applied CPT-derived chemotherapeutic drug. Apparently, all the new derivatives of CPT showed good to moderate cytotoxic activity in vitro against A-549, which was generally sensitive to various CPT derivatives. Among these new compounds, 13g and 13o were of the best cytotoxicity against all five tested tumor cell lines, with R group ¼ omethoxyphenyl/1-naphthyl, respectively. For compounds with aliphatic substitution, benzyl substituted compound 13n is less cytotoxic than the other four aliphatic derivatives (13a~13d) against A-549, MDA-MB-231 and KB, except for almost similar cytotoxicity of 13b with 13n when against MDA-Mb-231. With respect to aromatic substituted derivatives, there seemed somewhat elusory in terms of their cytotoxicity, which was affected obviously by various substituent groups on C-7 of CPT, in that substituted groups on phenyl would either decrease or increase the cytotoxic activity of the derivatives (13f$13m, 13o and 13p) compared to the compound without substituted group on the benzene ring (13e). Unintelligibly, 13g, with o-methoxyphenyl ring on C-7, exhibited remarkably better cytotoxicity than that of p-methoxyphenyl ring substituted compound (13f), whereas through replacing electron-donating methoxy with less electron-donating methyl, a reverse result is obtained (13h and 13i). Moreover, when investigating the activity of halogen substituted compounds against these three cell lines (MDA-MB-231, KB and MCF-7), the rank order of cytotoxic potency, 13j (p-fluorobenzene) > 13k (p-chlorobenzene) > 13m (p-bromobenzene), was achieved, apart from a little better activity of 13m than 13k against MDA-MB-231. On the contrary, the opposite result (13j < 13k < 13m) was obtained when they were evaluated for cytotoxicity against A-549. However, 13l (m-chlorobenzene) was the most potent one distinguishing from the other three halogen substituted derivatives (13j, 13k and 13m) when combating with A-549 and KB. These results suggested that the size and position of substituents were crucial for activity. Importantly, 13o, a fused ring substituted CPT compound, was one of the most potent compounds among these 16 CPT derivatives, besides 13g, against the tested five tumor cell lines, especially compared to topotecan when fighting with multidrug-resistant (MDR) KBvin subline. This encouraging result indicated that the new derivative could kill the MDR phenotype by suppressing its overexpressing P-glycoprotein and that a fused ring substitution on the C-7 might be favoured which could be responsible for its excellent activity. Consequently, we plan to synthesize additional fused ring substituted thioureidopiperazinyl derivatives of 1 based on our preliminary results so as to produce compounds with greater potency. Taken together, these results implied that both the identity and substitution patterns in the R group on C-7 could influence the cytotoxicity of the new CPT derivatives to a great extent.
In summary, as an extension of our work on C-7 modified CPT compounds, 16 new C-7 thioureidopiperazinyl CPT derivatives (13a$13p) were designed, synthesized, and evaluated for antiproliferative activity against five human tumor cell lines (A-549, MDA-MB-231, MCF-7, KB and KBvin) by using a sulforhodamine B colorimetric assay. All the synthesized compounds showed superior cytotoxicity activity compared with 2.
In particular, compounds 13o exhibited superior activity against MDR KB subline KBvin, with two folds greater potency than 3. With an efficient synthesis and potent cytotoxic profiles, the new CPT-derivative, 13o, of the greatest cytotoxicity against the MDR KB-VIN cell line, merits further development as preclinical drug candidate for treating cancer, especially for MDR phenotype. Furthermore, SAR analysis indicated that the size, electron density, and distribution of the substituents within the thioureidopiperazinyl side chain are critical to the derivatives' activity. Accordingly, these findings will facilitate our further optimization of 1 to develop potential cancer chemotherapeutic candidates.

Disclosure statement
No potential conflict of interest was reported by the authors.