Synthesis of Acetogenin Analogs Comprising Pyrimidine Moieties Linked by Amine Bonds and Their Inhibitory Activity against Human Cancer Cell Lines

Abstract

Here, we synthesized three acetogenin analogs containing pyrimidine moieties linked by amine bonds, which represent the skeleton structure of pyrimidifen, a mitochondrial complex I-inhibiting insecticide. Replacing the pyrimidine moiety linked by the amine bond remarkably enhanced growth-inhibitory activity of the analogs against several human cancer cell lines. Moreover, these analogs selectively and potently inhibited the growth of these human cancer cell lines regardless of the pyrimidine substituents. Furthermore, COMPARE analyses suggested that these analogs inhibited cancer growth by inhibiting mitochondrial complex I. Our study provides insights into the design of acetogenin analogs as novel antitumor agents.

Introduction

Mitochondrial complex I (reduced nicotinamide-adenine dinucleotide-ubiquinone oxidoreductase) inhibitors have recently garnered the interest of medicinal chemists and biologists engaged in developing novel anticancer agents.¹⁾ The fact that mitochondrial complex I inhibitors selectively suppress tumor growth even though complex I is essential to the survival of normal tissues is a very intriguing research topic. Kawada and colleagues reported that complex I inhibitors exhibit selective antiproliferation effects through concomitant acidification of the intra- and extra-cellular environment since tumor tissues comprise more stromal cells in which energy production depends on oxidative phosphorylation in the mitochondria.²⁾

Our research group is interested in characterizing the antitumor activity of molecules that are hybrids^3–5) of annonaceous acetogenins and mitochondrial complex I-targeting insecticides. Annonaceous acetogenins are polyketides isolated from Annona plants growing in tropical and subtropical regions; the molecules exhibit antitumor activity through mitochondrial complex I inhibition.^6,7) Most annonaceous acetogenins contain one to three 2,5-disubstituted tetrahydrofurans (THFs) in the center of the long hydrocarbon chain and an α,β-unsaturated-γ-lactone at the end of the molecule (Fig. 1). The structure of solamin is shown in Fig. 1, as an example acetogenin structure.^8–10) In contrast, mitochondrial complex I-targeting insecticides are composed of N-containing heterocycles with lipophilic side chains.¹¹⁾ Inspired by the structural and biological similarities between acetogenins and mitochondrial complex I-targeting insecticides, we synthesized acetogenin analogs with N-methylpyrazole originating from tebufenpyrad (a mitochondrial complex I-inhibiting insecticide) in place of γ-lactone, and revealed that these analogs potentially inhibited the growth of human cancer cell lines.¹²⁾ Interestingly, the antitumor activity of these analogs depends on the connecting groups between N-methylpyrazole and the alkyl linker bearing the THF moiety.^13,14) In contrast, mitochondrial complex I-inhibiting insecticides, such as pyrimidifen, contain pyrimidines with a lipophilic side chain linked by an amine bond. However, the activity of analogs with heterocycles linked by amine bonds has not been evaluated. Therefore, we aimed to investigate the activity of hybrid acetogenins linked by the amine bonds, including an analog 1a. This study reports (1) the synthesis of analogs containing pyrimidines linked by amine bonds in place of γ-lactone, (2) their growth inhibitory activity, and (3) the mechanisms that are predicted to underlie their effects.

Fig. 1. Design of a Hybrid Molecule 1a of Solamin and Pyrimidifen

Results and Discussion

The analog 1a was synthesized from α-tetrahydrofuranyl aldehyde (2) prepared using our previously developed methodology^15–20) (Chart 1). The asymmetric alkynylation of 2 with 11-azido-1-undecyne (3) using a Zn(OTf)₂-Et₃N-N-methylephedrine system reported by Carreira and colleagues²¹⁾ resulted in a high yield of propargyl alcohol (4), which shows high diastereoselectivity. We reduced the alkyne and azide moieties in 4, resulting in a high yield of primary amine 5. Then, primary amine 5 was condensed to 4,5-dichloro-6-ethylpyrimidine in the presence of K₂CO₃ to give rise to secondary amine 6a. Finally, deprotection of t-butyldimethylsilyl ether in 6a under acidic conditions resulted in a good yield of the desired pyrimidine analog 1a. The isomers 1b and 1c were designed to examine the effects of replacing γ-lactone in acetogenins by pyrimidine moieties linked by amine bonds, as these isomers can easily be prepared since their corresponding dichloropyrimidines are commercially available. They were synthesized using the same reaction that was used for synthesizing 1a.

Chart 1. Synthesis of Pyrimidine Analogs 1a–1c

Reagents and conditions: (i) Zn(OTf)₂, (1R,2S)-(−)-N-methylephedrine, Et₃N, toluene, r.t., 86%, dr=>97 : 3; (ii) H₂ (3 atm), Pd(OH)₂–C, THF, r.t., 99%; (iii) Ar-Cl, K₂CO₃, DMF/H₂O, r.t., 56% from 4; (iv) Ar-Cl, K₂CO₃, THF/H₂O, r.t., 70% (6b), 59% (6c); (v) 48% HF aq., THF/MeCN, r.t., 73% (1a), 99% (1b), 58% (1c). DMF = N,N-dimethylformamide, dr = diastereomeric ratio, r.t. = room temperature, TBS = t-butyldimethylsilyl, Tf = trifluoromethanesulfonyl, THF = tetrahydrofuran.

We then evaluated the growth inhibitory activities of pyrimidine analogs 1a–1c against 39 human cancer cell lines (JFCR39).^22–24) The results of experiments involving 1a–1c and solamin²⁵⁾ as control are presented in Fig. 2. Pyrimidine 1a, with substituents identical to those of pyrimidifen, selectively inhibited growth of several cancer cell lines. For example, solamin moderately inhibited the growth of DMS114, LOX-IMVI, MKN-B, and MKN-A cells, whereas this inhibition was enhanced by pyrimidine 1a by approximately 430-, 100-, 1300-, and 1200-fold, respectively (<0.01, 0.29, <0.01, and <0.01 µM, respectively). In addition, growth inhibition of HCT-116, NCI-H23, and MKN74 cell lines was not observed in response to solamin but in response to pyrimidine 1a (<0.01, 0.42, and 0.17 µM, respectively). Similarly, the monochloropyrimidine analogs 1b and 1c elicited cytotoxic effects against the DMS114, MKN-B, and MKN-A cell lines, indicating that the connecting groups might exert a higher impact than the substituents on the pyrimidine moiety in our analogs.¹³⁾ However, this is difficult to conclusively infer because the positions of chlorine in these analogs are not the same as in 1a. The reason why our analogs 1a–1c showed more potent activities against specific cell lines, such as HCT-116, DMS114, MKN-B, and MKN-A than others remains unknown. Since cellular sensitivity to mitochondrial perturbation may depend on the level of intracellular reactive oxygen species (ROS) and the expression of apoptosis-related proteins,²⁶⁾ these factors might also affect the sensitivity towards our analogs. Salgia and colleagues reported that DMS114 cells depend on aerobic metabolism, unlike other small cell lung cancer cell lines,²⁷⁾ indicating that the high sensitivity of DMS114 cells to mitochondrial complex I inhibitors can be explained by metabolic dependence of the mitochondrial respiratory chain.

Fig. 2. Cell Growth Inhibition Assessed Using the Sulforhodamine B Colorimetric Assay by Measuring Changes in Total Cellular Protein Levels Following 48 h of Treatment with a Given Test Compound

Fingerprints were obtained by calculating 50% growth inhibitory concentration (GI₅₀) values for JFCR39 cell lines. The x-axis represents the logarithm of the 1/GI₅₀ values for 39 human cancer cell lines. Absence of a bar signifies that the GI₅₀ value of the corresponding cell line is higher than 10⁻⁴ M. CNS = central nervous system.

We predicted the mechanisms underlying the antitumor effects of 1a–1c using COMPARE analysis.^22–24,28) The Pearson correlation coefficients (r) between various combinations of compounds, including known mitochondrial complex I inhibitors (deguelin, buformin, and phenformin), are shown in Table 1. In a COMPARE analysis, the r value >0.75 strongly suggested that both compounds utilize the same mechanisms to exert their effects. We previously revealed that the thiophene carboxamide analog of solamin (JCI-20679) exhibits potent antitumor activity by inhibiting mitochondrial complex I.^29–36) The pyrimidine analogs 1a–1c were also significantly correlated with mitochondrial complex I inhibitors, suggesting that analogs 1a–1c inhibit cancer cell growth through mitochondrial complex I inhibition. It is unclear why analogs 1a–1c showed higher inhibitory activity than solamin, but the results discussed above suggest that the pyrimidine moiety linked by the amine bond strongly binds to the target molecule in mitochondrial complex I. On the other hand, differences in cell membrane permeability of 1a–1c and solamin are unlikely to affect their activities because their overall molecular structures have very slight structural differences. Although the cytotoxicity of 1a–1c against normal cells was not examined, present analogs are expected not to show acute toxicity against vital organs in vivo because JCI-20679, a solamin analog, did not show body weight loss in mice at the dose with significant antitumor activity.³⁶⁾ A few studies apart from ours have reported the synthesis of heterocyclic acetogenin analogs by replacing γ-lactone.^37–42) The insights provided by the present study will be valuable to the design of acetogenin analogs as novel antitumor agents.

Table 1. Pearson Correlation Coefficients (r) between the Fingerprints of Solamin, Analogs 1a–1c, and Complex I Inhibitors

	Solamin	1a	1b	1c	Deguelin	Buformin	Phenformin
Solamin	1
1a	0.756	1
1b	0.849	0.922	1
1c	0.825	0.878	0.935	1
Deguelin	0.764	0.848	0.848	0.845	1
Buformin	0.811	0.758	0.898	0.896	0.748	1
Phenformin	0.789	0.780	0.893	0.881	0.756	0.952	1

The chemical structures of known inhibitors are shown in Supplementary Fig. S1.

Conclusion

Here, we report the synthesis of three acetogenin analogs with pyrimidine moieties linked by amine bonds. These analogs selectively and potentially inhibited the growth of several human cancer cell lines regardless of the pyrimidine substituents. COMPARE analyses suggested that these analogs inhibit tumor growth by inhibiting mitochondrial complex I. Currently, we are performing further studies examining this structure–activity relationship.

Acknowledgments

The in vitro antiproliferative activities of acetogenin analogs against human cancer cell lines were examined by the Screening Committee of Anticancer Drugs, supported by a Grant-in-Aid for Scientific Research on Innovative Areas, Scientific Support Programs for Cancer Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT). We thank Dr. Tetsuya Fushimi for his technical assistance in the preliminary part of this study and Prof. Tetsuaki Tanaka and Prof. Takehiko Yoshimitsu for their helpful discussions. This study was supported in part by JSPS KAKENHI (Grant Nos. 25460159, 16K08330 [N.K.], 16H05105 [S.D. and A.A.]), the MEXT-Supported Program for the Strategic Research Foundation at Private Universities, 2015–2019 (Grant No. S1511024L [N.K.]), a Kyoto Pharmaceutical University Fund for the Promotion of Scientific Research (N.K.), and a research grant from the Nippon Foundation (S.D.).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

References

• 1) Bedi M., Ray M., Ghosh A., Mol. Cell. Biochem., 477, 345–361 (2022).
• 2) Yoshida J., Ohishi T., Abe H., et al., iScience, 24, 103497 (2021).
• 3) Kumar H. M. S., Herrmann L., Tsogoeva S. B., Bioorg. Med. Chem. Lett., 30, 127514 (2020).
• 4) Bérubé G., Expert Opin. Drug Discov., 11, 281–305 (2016).
• 5) Nepali K., Sharma S., Sharma M., Bedi P. M. S., Dhar K. L., Eur. J. Med. Chem., 77, 422–487 (2014).
• 6) Neske A., Hidalgo J. R., Cabedo N., Cortes D., Phytochemistry, 174, 112332 (2020).
• 7) Kojima N., Tanaka T., Molecules, 14, 3621–3661 (2009).
• 8) Nakanishi Y., Chang F.-R., Liaw C.-C., Wu Y.-C., Bastow K. F., Lee K.-H., J. Med. Chem., 46, 3185–3188 (2003).
• 9) Sinha S. C., Keinan E., J. Am. Chem. Soc., 115, 4891–4892 (1993).
• 10) Myint S. H., Cortes D., Laurens A., Hocquemiller R., Lebȩuf M., Cavé A., Cotte J., Quéro A.-M., Phytochemistry, 30, 3335–3338 (1991).
• 11) Murai M., Miyoshi H., Biochim. Biophys. Acta, Bioenerg., 1857, 884–891 (2016).
• 12) Kojima N., Fushimi T., Maezaki N., Tanaka T., Yamori T., Bioorg. Med. Chem. Lett., 18, 1637–1641 (2008).
• 13) Ohta K., Fushimi T., Okamura M., Akatsuka A., Dan S., Iwasaki H., Yamashita M., Kojima N., RSC Adv., 12, 15728–15739 (2022).
• 14) Kojima N., Fushimi T., Tatsukawa T., Yoshimitsu T., Tanaka T., Yamori T., Dan S., Iwasaki H., Yamashita M., Eur. J. Med. Chem., 63, 833–839 (2013).
• 15) Maezaki N., Kojima N., Tanaka T., Synlett, 2006, 993–1003 (2006).
• 16) Kojima N., Yakugaku Zasshi, 124, 673–681 (2004).
• 17) Kojima N., Maezaki N., Tominaga H., Yanai M., Urabe D., Tanaka T., Chem. Eur. J., 10, 672–680 (2004).
• 18) Kojima N., Maezaki N., Tominaga H., Asai M., Yanai M., Tanaka T., Chem. Eur. J., 9, 4980–4990 (2003).
• 19) Maezaki N., Kojima N., Tominaga H., Yanai M., Tanaka T., Org. Lett., 5, 1411–1414 (2003).
• 20) Maezaki N., Kojima N., Asai M., Tominaga H., Tanaka T., Org. Lett., 4, 2977–2980 (2002).
• 21) Frantz D. E., Fässler R., Carreira E. M., J. Am. Chem. Soc., 122, 1806–1807 (2000).
• 22) Dan S., Okamura M., Seki M., Yamazaki K., Sugita H., Okui M., Mukai Y., Nishimura H., Asaka R., Nomura K., Ishikawa Y., Yamori T., Cancer Res., 70, 4982–4994 (2010).
• 23) Yaguchi S., Fukui Y., Koshimizu I., Yoshimi H., Matsuno T., Gouda H., Hirono S., Yamazaki K., Yamori T., J. Natl. Cancer Inst., 98, 545–556 (2006).
• 24) Yamori T., Matsunaga A., Sato S., Yamazaki K., Komi A., Ishizu K., Mita I., Edatsugi H., Matsuba Y., Takezawa K., Nakanishi O., Kohno H., Nakajima Y., Komatsu H., Andoh T., Tsuruo T., Cancer Res., 59, 4042–4049 (1999).
• 25) Kojima N., Morioka T., Urabe D., Yano M., Suga Y., Maezaki N., Ohashi-Kobayashi A., Fujimoto Y., Maeda M., Yamori T., Yoshimitsu T., Tanaka T., Bioorg. Med. Chem., 18, 8630–8641 (2010).
• 26) Fulda S., Galluzzi L., Kroemer G., Nat. Rev. Drug Discov., 9, 447–464 (2010).
• 27) Mambetsariev I., Mirzapoiazova T., Lennon F., Jolly M. K., Li H., Nasser M. W., Vora L., Kulkarni P., Batra S. K., Salgia R., J. Clin. Med., 8, 1038 (2019).
• 28) Paull K. D., Shoemaker R. H., Hodes L., Monks A., Scudiero D. A., Rubinstein L., Plowman J., Boyd M. R., J. Natl. Cancer Inst., 81, 1088–1092 (1989).
• 29) Ando S., Kojima N., Moyama C., Fujita M., Ohta K., Ii H., Nakata S., Mol. Med. Rep., 26, 238 (2022).
• 30) Ohta K., Akatsuka A., Dan S., Iwasaki H., Yamashita M., Kojima N., Chem. Pharm. Bull., 69, 1029–1033 (2021).
• 31) Ando S., Moyama C., Kojima N., Fujita M., Ohta K., Kohno Y., Ii H., Nakata S., Biochem. Biophys. Res. Commun., 591, 62–67 (2022).
• 32) Matsumoto T., Akatsuka A., Dan S., Iwasaki H., Yamashita M., Kojima N., Tetrahedron, 76, 131058 (2020).
• 33) Matsumoto T., Kojima N., Akatsuka A., Yamori T., Dan S., Iwasaki H., Yamashita M., Tetrahedron, 73, 2359–2366 (2017).
• 34) Akatsuka A., Kojima N., Okamura M., Dan S., Yamori T., Pharmacol. Res. Perspect, 4, e00246 (2016).
• 35) Kojima N., Suga Y., Matsumoto T., Tanaka T., Akatsuka A., Yamori T., Dan S., Iwasaki H., Yamashita M., Bioorg. Med. Chem., 23, 1276–1283 (2015).
• 36) Kojima N., Fushimi T., Tatsukawa T., Tanaka T., Okamura M., Akatsuka A., Yamori T., Dan S., Iwasaki H., Yamashita M., Eur. J. Med. Chem., 86, 684–689 (2014).
• 37) Gould E. R., King E. F. B., Menzies S. K., Fraser A. L., Tulloch L. B., Zacharova M. K., Smith T. K., Florence G. J., Bioorg. Med. Chem., 25, 6126–6136 (2017).
• 38) Liu Y., Xiao Q., Liu Y., Li Z., Qiu Y., Zhou G.-B., Yao Z.-J., Jiang S., Eur. J. Med. Chem., 78, 248–258 (2014).
• 39) Chen Y.-J., Jin S., Xi J., Yao Z.-J., Tetrahedron, 70, 4921–4928 (2014).
• 40) Duval R. A., Lewin G., Peris E., Chahboune N., Garofano A., Dröse S., Cortes D., Brandt U., Hocquemiller R., Biochemistry, 45, 2721–2728 (2006).
• 41) Duval R. A., Poupon E., Romero V., Peris E., Lewin G., Cortes D., Brandt U., Hocquemiller R., Tetrahedron, 62, 6248–6257 (2006).
• 42) Duval R., Lewin G., Hocquemiller R., Bioorg. Med. Chem., 11, 3439–3446 (2003).