Design, semisynthesis and potent cytotoxic activity of novel 10-fluorocamptothecin derivatives

Abstract

Fluorination is a well-known strategy for improving the bioavailability of bioactive molecules in the lead optimization phase of drug discovery projects. In an attempt to improve the antitumor activity of camptothecins (CPTs), novel 10-fluoro-CPT derivatives were designed, synthesized and evaluated for cytotoxicity against five human cancer cell lines (A-549, MDA-MB-231, KB, KB-VIN and MCF-7). All of the derivatives showed more potent in vitro cytotoxic activity than the clinical CPT-derived drug irinotecan against the tumor cell lines tested, and most of them showed comparable or superior potency to topotecan. Remarkably, compounds 16b (IC₅₀, 67.0 nM) and 19b (IC₅₀, 99.2 nM) displayed the highest cytotoxicity against the multidrug-resistant (MDR) KB-VIN cell line and merit further development as preclinical drug candidates for treating cancer, including MDR phenotype. Our study suggested that incorporation of a fluorine atom into position 10 of CPT is an effective method for discovering new potent CPT derivatives.

Graphical abstract

A novel type of 10-fluorocamptothecin derivatives with excellent cytotoxicity activity were designed and synthesized.

Fluorine, a highly active element, is widely used in drugs with high electronegativity and small size.^1,2 Fluorine incorporation often improves drug-like properties by blocking undesired metabolism at a specific site, increasing lipophilicity or binding affinity, or altering drug absorption, distribution, or excretion. The unique and complex physicochemical and biological properties provided by incorporation of fluorine atoms or fluorinated moieties into bioactive molecules have resulted in a wide application of fluorinated compounds for construction of new drugs.^3,4 As a result, more than 20% of the currently marketed drugs contain at least one fluorine atom. Particularly, an increasing number of fluorinated antitumor agents such as fluorouracil derivatives, enzalutamide, teriflunomide, ponatinib, sorefenib, gefitinib and vandetanib are becoming available for cancer treatment.⁵ Therefore, derivatization of current pharmaceuticals to fluorinated analogues has become an increasingly attractive strategy to obtain more potent drugs for drug discovery.

Camptothecin (CPT, 1), a naturally occurring quinoline alkaloid, exhibits significant antitumor activity against a broad spectrum of cancers via inhibition of DNA enzyme topoisomerase I (topo I).^6,7 Extensive structural modifications on the A-, B-, E-ring or acylation of the 20-hydroxy group of CPT led to the successful identification and development of the antitumor drugs irinotecan (2), topotecan (3) and belotecan (4), as well as several candidates such as lurtotecan (5), gimatecan (6), rubitecan (7), and prothecan (8), which are in various stages of preclinical development.^8–16 Significantly, newly emerging fluorinated derivatives, including exatecan (9) and BN-80915 (10), where a fluorine atom has been introduced on the A ring of CPT, have shown excellent antitumor activities both in vitro and in vivo and are currently undergoing clinical trials.^17,18 Recently, Zhang et al reported the synthesis of 11 via a new strategy to improve the metabolic stability of CPT’s lactone by replacing it with an α-fluoro ether as a lactone bioisostere.¹⁹ The clinical successes and accumulated SAR studies have stimulated great interest in further exploration of CPT-derivatives with better antitumor activity.

In our continuous efforts to produce CPT-derived antitumor agents, we recently found that a series of novel 20-sulfonylamidine-CPT derivatives displayed potent antitumor activity with significantly different drug-resistance profiles from those of CPT. Among them, YQL-9a (12) showed excellent antitumor activities both in vitro and in vivo, indicating it to be a promising antitumor lead.^20,21 In addition, in prior SAR studies on CPT, addition of an electronegative substituent at C-10 has always resulted in improved cytotoxicity. Based on these facts, we postulated that the addition of fluorine at the C-10 positions of CPT and 12 would be an efficient way to increase the cytotoxicity of both compounds (Fig. 2). Also, as mentioned earlier, fluorination has the potential to improve the pharmacological profile of drugs. Therefore, herein, we report our synthesis of C-10 fluorinated CPT and 12 derivatives, as well as evaluation of their cytotoxic activity.

Fig. 2.

Design strategy for 10-fluorocamptothecin derivatives.

The synthetic routes to target compounds 16a–b and 19a–b are outlined in Scheme 1. Briefly, 10-(trifluoromethanesulfonyloxy)camptothecins 14a and 14b were prepared from 13a and 13b by a classical synthetic method using N-phenylbis(trifluoromethanesulfonimide) and triethylamine in DMF. Next, both 14a and 14b were converted to 10-(tributylstannyl)camptothecins (15a and 15b) in excellent yields by reaction with bis(tri-n-butyltin) in the presence of tetrakis(triphenylphosphine)palladium/lithium chloride.^22,23 Treatment of 15a and 15b with1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octanebis(hexafluorophosphate) (F-TEDA-PF6) and silver triflate (AgOTf) in dry acetone solution at 23 °C afforded the desired compounds 16a–b in moderate yields.²⁴ Furthermore, the 20-hydroxy groups of 16a–b were converted to N-Boc protected glycine esters (17a–b) using a combination of DIPC (N,N'-diisopropylcarbodiimide) and DMAP (4-dimethylaminopyridine). Deprotection of 17a and 17b with TFA/CH₂Cl₂ afforded the TFA salts (18a–b). Subsequently, the key intermediates (18a–b) were successfully reacted with 4-ethynylanisole and p-toluenesulfonyl azide in a Cu-catalyzed three-component reaction to produce the corresponding target compounds (19a–b) in good yields.²⁵

Scheme 1.

General synthetic procedure for 10-fluorocamptothecin derivatives 16a–b and 19a–b.

The 10-fluorinated derivatives of CPT and 12 were evaluated for in vitro cytotoxic activity against five tumor cell lines, A-549 (lung carcinoma), MDA-MB-231 (triple-negative breast carcinoma), KB (originally isolated from epidermoid carcinoma of the nasopharynx), KB-VIN (MDR KB subline) and MCF-7 (breast adenocarcinoma) by using a sulforhodamine B colorimetric assay.^26,27 Irinotecan (2) and topotecan (3) were used as the positive controls. The screening results are shown in Table 1.

Table 1.

In vitro cytotoxicity data for compounds 16a–b and 19a–b against five tumor cell lines.

Entry	IC50 (nM)
	A-549	MDA-MB-231	KB	KB-VIN	MCF-7
16a	58.6 ± 0.23	579 ± 2.41	185 ± 0.02	2954 ± 20.9	431 ± 67.7
16b	9.91 ± 0.36	319 ± 107	69.6 ± 2.37	67.0 ± 0.77	76.9 ± 5.11
19a	62.2 ± 1.36	601 ± 57.0	280 ± 50.6	497 ± 37.6	649 ± 71.8
19b	8.72 ± 0.03	238 ± 28.3	47.0 ± 6.20	99.2 ± 17.7	80.8 ± 3.48
1	8.63 ± 0.66	307 ± 118	50.0 ± 9.7	15.7 ± 1.1	87.5 ± 6.4
2	8,300 ± 280	15,600 ± 302	7,990 ± 312	>20,000	11,300 ± 249
3	112 ± 28.4	773 ± 36.0	102 ± 8.1	287 ± 25.3	666 ± 26.7

As shown in Table 1, all target compounds exhibited significant in vitro cytotoxic activity against the five tested tumor cell lines, with IC₅₀ values ranging from 8.72 to 649 nM, and they exhibited more potent in vitro cytotoxic activity than 2, while most of the new derivatives also showed comparable or superior potency to 3. The data also revealed that the A-549 cell line was more sensitive than the other four cell lines to these compounds, which is consistent with the clinical behavior of other derivatives of CPT.²⁸ Remarkably, all of the compounds were more potent than 2 (IC₅₀ >20,000 nM) against the MDR KB-VIN cell line, with 16b (IC₅₀ 67.0 nM) and 19b (IC₅₀ 99.2 nM) showing the greatest cytotoxicity against this cell line. They also showed increased cytotoxic potency against the triple-negative breast cancer (MDA-MB-231) cell line compared with 2 or 3. This result implied that the introduction of a fluorine atom at C-10 position or a sulfonylamidine group at C-20 might combat the tumor MDR phenotype caused by P-glycoprotein overexpression. Compared with 16a and 19a, the corresponding 7-ethyl derivatives 16b and 19b exhibited greater in vitro cytotoxic activity against the five tested tumor cell lines. Thus, the introduction of a C-7 ethyl group contributed to improved cytotoxicity.

It is well known that therapeutic use of unmodified CPT is hindered by its poor solubility, high toxicity, and rapid inactivation through lactone ring hydrolysis in vivo. In addition, the carboxylate form readily binds to human serum albumin, making it less accessible for cellular uptake. This behavior gives rise to a drop in therapeutic efficacy, along with formulation difficulties. Therefore, approaches that stabilize the lactone ring prior to entry into the site of action are a critical feature in order to maximize therapeutic activity and minimize toxicity. It is widely known that fluorine incorporation is an efficient strategy for improving the bioavailability of bioactive molecules in the lead optimization phase. Additionally, early efforts by clinicians and research scientists to reduce the side effects of CPT and improve the metabolic stability of CPT’s lactone were directed at synthesis of modified esters at position 20. It is interesting to note from Table 1 that, in general, the introduction of an ester moiety at the 20 position of CPT (compounds 19a, 19b) caused a loss of cytotoxic activity, which is a common feature of prodrugs. Also, these target compounds may improve the bioavailability of CPT by such modified approaches.

In summary, CPT and our previous lead compound 12 are effective cytotoxic agents that merit considerable attention. In response to the need to design novel CPT-derived antitumor agents with improved activity, we incorporated fluorine, an atom with unique properties, into CPT and 12. In a preliminary in vitro cytotoxicity assay, the four new fluorinated compounds showed potent cytotoxicity (IC₅₀ 8.72–649 nM) and were more effective than 2. Notably, 7-ethyl 10-fluoro compounds 16b (IC₅₀ 67.0 nM) and 19b (IC₅₀ 99.2 nM) showed the greatest cytotoxicity against the MDR KB-VIN cell line cell line (cf, 2, IC₅₀ >20,000 nM) and were selected as lead molecules for further development. The results in this study provide the first convincing evidence that the introduction of fluorine at the C-10 position of CPT is favorable for enhanced cytotoxic activity. Meanwhile, the mechanism of action of these new derivatives is unknown, and further investigation will be required to understand the effect of fluorine. A better understanding of its role could provide meaningful insights for optimization with fluorine in further drug design. With a concise synthesis and potent cytotoxic profiles, we hope that 10-fluorinated CPT-derivatives will be promising templates for further preparation of improved anticancer agents in the future. Continuing studies to substantiate and improve activity profiles are underway in our laboratories and will be reported in due course.

Fig. 1.

Structures of CPT-derivatives