Syntheses and biological evaluation of M-COPA analogs derived from pentadienoic Weinreb amide

Abstract

M-COPA (1), which contains diene and 3-picolylamine moieties in its side chain and seven stereogenic centers in a multisubstituted octalin skeleton, strongly inhibits the growth of several cancer cell lines. Expecting the improvement of conformational flexibility of basic and coordinating 3-pyridylmethylamino group on M-COPA and its physical properties, we efficiently synthesized its amine analogs by replacing its amide group with an amino group through the Weinreb amide-type Horner–Wadsworth–Emmons reaction. The cytotoxic properties of 1 and its analogs were evaluated against NCI-H226, a lung cancer cell line, HeLa, a cervical cancer cell line, and GIST-T1, a gastrointestinal stromal tumor cell line. The evaluation results indicated that the structural alteration from amide moiety to amine moiety lowered the pharmacological activity but remained strong cytotoxicity.

Broadening the Scope of Structure–Activity Relationships of M-COPA via an α,β,γ,δ-Unsaturated Weinreb Amide

1. Introduction

In 2005, Nippon Shinyaku Co., Ltd. semisynthesized AMF-26 (1) using AMF-14 (2), a naturally occurring norditerpenoid isolated from the Trichoderma strain NFS-9321 (Fig. 1).¹⁾ In 2012, we successfully conducted the enantioselective total syntheses of compounds 1 and 2 to unambiguously determine their absolute stereostructures and subsequently renamed the synthesized compound 1 as M-COPA.²⁾

Fig. 1.

Structures of M-COPA and trichodermic acid (TDA).

In 2005, Nippon Shinyaku Co., Ltd. reported that compound 2 inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) at a concentration of 59 µM in vitro.¹⁾ Notably, ICAM-1 is strongly expressed in inflamed vascular endothelium and is hence considered to be involved in inflammation. In 2012, the Li and Zhang groups also isolated AMF-14 and referred to it as trichodermic acid (TDA).³⁾ In addition to TDA, they also isolated its derivatives, TDA A and B, from the extracts of Trichoderma spirale and elucidated their structures using one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy and mass spectrometry data. In 2019, Zhao and coworkers isolated TDA along with three 3,4,6-trisubstituted α-pyrone derivatives from a solid-substrate fermentation culture of Penicillium ochrochloron.⁴⁾ They demonstrated that TDA exhibited antibacterial, antifungal, and cytotoxic activities against several human cancer cell lines. In 2021, Yang, Lin, and colleagues isolated TDA from the plant endophytic fungus Penicillium ochrochloron.⁵⁾ They demonstrated that TDA inhibited the proliferation of colon cancer cells in both in vitro and in vivo models.

Previous studies have also investigated the pharmacological activity of M-COPA (1). In 2005, Nippon Shinyaku Co., Ltd. used a collagen-induced arthritis mouse model to demonstrate that compound 1 was effective at treating rheumatoid arthritis in vivo. In a cell proliferation inhibition assay, compound 1 inhibited the growth of four cancer cell lines at lower concentrations than those required to inhibit three normal cell lines in vitro. In 2012, Watari and coworkers suggested that compound 1 inhibited vascular-endothelial-growth-factor (VEGF)-induced angiogenesis both in vitro and in vivo.⁶⁾ Moreover, upon stimulation by the VEGF or inflammatory cytokines, compound 1 was demonstrated to inhibit angiogenesis by suppressing both VEGF receptor 1/2 (VEGFR1/2) and nuclear factor-κB (NF-κB) signaling pathways. In 2012, Ohashi and coworkers reported that oral administration of compound 1 induced complete regression of human BSY-1 breast cancer xenografts in vivo.⁷⁾ Furthermore, compound 1 has been reported to prevent the activation of ADP-ribosylation factor 1 (ARF1) and to induce Golgi disruption, apoptosis, and cell growth inhibition. Moreover, in 2018, Abe, Obata, and colleagues reported that compound 1 demonstrated activity against imatinib-resistant gastrointestinal stromal tumors (GISTs), which evade imatinib-based treatment through secondary mutations in the KIT kinase domain.⁸⁾ Guanine nucleotide exchange factors and adenosine diphosphate ribose (ADP) ribosylation factors (ARFs) are known to play a critical role in the transport of receptor tyrosine kinases (RTKs) from the endoplasmic reticulum (ER) to the Golgi apparatus. Building on this understanding, Obata and coworkers reported in 2024 that compound 1 suppressed the growth of RTK-dependent cancer cells by inhibiting ARF1, ARF4, and ARF5.⁹⁾

The structure–activity relationship of imatinib, an FDA-approved drug for treating chronic myelogenous leukemia (CML) and GISTs, has been well developed. Mahboobi, Beckers, and coworkers reported the amine derivatives (4a, 4b) of imatinib (3) for the development of chimeric histone deacetylase- and tyrosine kinase-inhibitors and they revealed that the alteration from amide group to amino group did not lower the inhibitory activity against Abl (Fig. 2).¹⁰⁾ In addition, Go, Lam, and coworkers reported that modifying amide 5 to amine 6 improved its solubility in water and led to the emergence of new pharmacological activity.¹¹⁾

Fig. 2.

Structure–activity relationship (SAR) of imatinib (3) and indole derivative (5).

Inspired by these findings, we designed analogs of compound 1, which is a promising target for the development of new CML- and GISTs-drugs, by replacing their amide groups with amino groups expecting the improvement of conformational flexibility of the basic and coordinating 3-pyridylmethylamino group on 1 and its physical properties.

The resulting amine analogs 7–9 are presented in Fig. 3. Among these, secondary amine 7 and tertiary amine 8 share the core structure of compound 1 and contain 3-picolylamine and 3-(methylaminomethyl)pyridine moieties, respectively, instead of the original amide group. Secondary amine 9 also contains a 3-picolylamine moiety in place of the amide group and is a regioisomer of amine 8. In this study, we investigate the in vitro anti-proliferative activities of these three analogs of compound 1 against cancer cells to explore the relationship between the biological activities of amide- and amino-containing compounds.

Fig. 3.

Amine analogs 7–9 of M-COPA (1).

2. Results and discussion

Amines 7–9 share a substructure with 1, which contains a diene and a 3-picolylamine moiety in the side chain, along with seven stereogenic centers in a multisubstituted octalin skeleton. Given this structural similarity, we reasoned that an intermediate of a previously synthesized derivative of 12 could serve as an intermediate for amines 7–9 (Fig. 4).

Fig. 4.

Retrosynthetic analysis of amines 7–9.

Amines 7 and 8 were retrosynthetically traced back to aldehyde 10, while amine 9 was traced to ketone 11 via Borch reductive amination.¹²⁾ Aldehyde 10 and ketone 11 can be prepared from Weinreb amide (E)-12.¹³⁾ This Weinreb amide (E)-12 can be obtained from key intermediate 13 via the Horner–Wadsworth–Emmons (HWE) reaction.^14),15)

As described above, key intermediate 13 was previously prepared during the synthesis of compound 1. Using a previously reported intermediate, we leveraged the (E)-selective HWE reaction between aldehyde 13 and Weinreb amide-type HWE reagent 14¹⁶⁾ (Table 1). In a previous study, we investigated a mild and highly (E)-selective HWE reaction using reagent 14.¹⁷⁾ In that study, we established standard reaction conditions using isopropylmagnesium chloride (ⁱPrMgCl) as the base, tetrahydrofuran (THF) as the solvent, and room temperature as the reaction temperature. Under these conditions, we examined the conversion of aldehyde 13 to Weinreb amide (E)-12. As an initial attempt, we selected lithium bis(trimethylsilyl)amide, previously used in the HWE reaction for the synthesis of 1, as the base for the HWE reaction (Table 1, entry 1). The desired (E)-12 was obtained in 90% yield with a (2E/2Z) geometric isomer ratio of 96:4. Subsequently, we used a Grignard reagent as the base, which played a crucial role in achieving both high yield and selectivity in the (E)-selective Weinreb amide-type HWE reaction (entries 2, 3). Although the desired compound (E)-12 was obtained in moderate yield, the (2E/2Z) geometric isomer ratio improved compared to that in entry 1 (Table 1, entry 2). When the equivalents of Weinreb amide-type HWE reagent 14 and ⁱPrMgCl increased, the yield of (E)-12 rose substantially to 94%. These results confirmed that the previously reported standard conditions—using ⁱPrMgCl as the base and THF as the solvent—were optimal for this reaction.

Table 1. (E)-selective Weinreb amide-type HWE reaction of an α,β-unsaturated aldehyde

Given that (E)-12 was obtained in high yield with excellent (E)-selectivity, we proceeded to synthesize amines 7–9 from (E)-12 by leveraging the Weinreb amide, which enables a one-step transformation to the corresponding aldehyde or ketone. First, we synthesized amines 7 and 8, as indicated in Scheme 1. The Weinreb amide (E)-12 was then reduced to the corresponding aldehyde 10. Borch reductive amination of aldehyde 10 with 3-picolylamine afforded secondary amine 15. Subsequent reductive amination of aldehyde 10 with amine 15 yielded tertiary amine 16. Deprotection of secondary amine 15 afforded amine 7. Similarly, removal of the TBS group from tertiary amine 16 afforded amine 17. In parallel, Borch reductive amination of aldehyde 10 with 3-(methylaminomethyl)pyridine provided tertiary amine 18 in high yield, and its subsequent deprotection afforded amine 8.

Scheme 1.

Syntheses of secondary amine 7 and tertiary amines 17 and 8.

Next, amine 9 was synthesized from Weinreb amide (E)-12, as indicated in Scheme 2. Specifically, Weinreb amide (E)-12 was first converted to ketone 11 through the Weinreb ketone synthesis reaction with methylmagnesium bromide. Borch reductive amination of ketone 11 with 3-picolylamine then afforded secondary amine 19 as a diastereomeric mixture. Finally, removal of the TBS group from secondary amine 19 afforded amine 9.

Scheme 2.

Synthesis of secondary amine 9.

Finally, amide analogs were synthesized from the Weinreb amide (E)-12 (Scheme 3). Hydrolysis of (E)-12 followed by MNBA-mediated amidation furnished compound 21. These results indicated that Weinreb amide can be used not only for reduction and Borch reductive amination sequence but also for the precursor of carboxylic acid. Subsequent removal of the TBS group from 21 afforded 2-demethyl-M-COPA (22), in which the methyl group at the C2 position is absent. In addition, deprotection of the TBS group in (E)-12 provided the corresponding Weinreb amide analogue (23).

Scheme 3.

Syntheses of amide derivatives 22 and 23.

As described above, we synthesized M-COPA (1) analogs 7–9, along with tertiary amine 17, which was derived from byproduct 16 of the Borch reductive amination, and amide analogs 22 and 23. We evaluated the in vitro biological activities of these compounds alongside several commercially available anticancer drugs (5-FU; Cisplatin; Gefitinib; and Osimertinib). The cytotoxic properties of 1, its analogs, and the anticancer drugs were evaluated against NCI-H226, a lung cancer cell line, HeLa, a cervical cancer cell line, and GIST-T1, a gastrointestinal stromal tumor cell line.¹⁸⁾ The results of this activity evaluation are summarized in Table 2 and highlight nine key findings. (1) The methyl group at the C2 position of compound 1 was not essential for demonstrating biological activity (1 vs. 22, 23). (2) The biological activities of amines 7–9 were approximately 100-fold weaker than that of 1. (3) The biological activity of compound 8 was approximately 10-fold greater than that of tertiary amine analog 17, indicating that the steric bulk of the amino group influences biological activity. (4) Notably, the biological activity of Weinreb amide 23 was comparable to that of tertiary amine 8. This finding indicates that the amide structure of compound 1 contributes to its biological activity. (5) Our results also demonstrated that the amide moiety of 1 is not essential for biological activity. (6) The biological activities of 1 and its analogs 7–9, 17, 22, and 23 were stronger than those of the commercially available anticancer drugs. (7) The biological activities of 1 and 17 against NCI-H226 were more potent than those against HeLa and GIST-T1. (8) The biological activities of 7–9 and 23 against NCI-H226 and HeLa are more potent than GIST-T1. Additionally, the biological activities of 7–9 and 23 against HeLa are twice as potent as those against NCI-H226. (9) Finally, the amide 22 has high cytotoxicity against NCI-H226, HeLa, and GIST-T1 in an almost uniform manner.

Table 2. IC₅₀ values of M-COPA (1); its analogs 7–9, 17, 22, and 23; and commercially available anticancer drugs against NCI-H226, HeLa, and GIST-T1

In conclusion, our previously reported (E)-selective HWE reaction involving Weinreb amide-type reagent 14 efficiently produced Weinreb amide (E)-12 in high yield and with excellent (E)-selectivity. Because Weinreb amide (E)-12 could be readily converted into the corresponding aldehyde, ketone, and carboxylic acid, it enabled further investigation of the SAR of 1. In fact, the current SAR study revealed that the amide moiety of 1 is not essential for retaining biological activity. Further synthesis of 1 analogs and evaluations of their biological activities are currently underway.

Acknowledgments

This paper is dedicated to the memory of Professor Teruaki Mukaiyama, who led us into the field of synthetic organic chemistry. This work was partly supported by the Research Institute for Science and Technology (RIST) of Tokyo University of Science.

Conflict of interest

The authors declare no conflicts of interest.

Supplementary materials

Supplementary materials are available at https://doi.org/10.2183/pjab.101.029.

Notes

Edited by Takao SEKIYA, M.J.A.

Correspondence should be addressed to: T. Murata, Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan (e-mail: [e-mail]); I. Shiina, Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan (e-mail: [e-mail]).

Footnotes

H.T. and D.U. contributed equally.

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