Discovery of (E)-1-amino-4-phenylbut-3-en-2-ol derivatives as novel neuraminidase inhibitors

Bioorganic & Medicinal Chemistry Letters 28 (2018) 2003–2007

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl

Discovery of (E)-1-amino-4-phenylbut-3-en-2-ol derivatives as novel neuraminidase inhibitors Cheng Lu, Yan Yin ⇑, Fanli Meng, Yongbin Dun, Keke Pei, Chenlu Wang, Xu Xu, Fanhong Wu School of Chemical and Environmental Engineering, Shanghai Institute of Technology, 100 Hai Quan Rd., Shanghai, China

a r t i c l e

i n f o

Article history: Received 30 January 2018 Revised 25 April 2018 Accepted 2 May 2018 Available online 4 May 2018 Keywords: Neuraminidase Neuraminidase inhibitor 1-Amino-4-phenylbut-3-en-2-ol derivatives Influenza Molecular docking

a b s t r a c t Neuraminidase has been considered as an important target for designing agents against influenza viruses. In a discovery of anti-influenza agents with epigoitrin as the initial lead compound, a series of 1-amino-2alkanols were synthesized and biologically evaluated. The in vitro evaluation indicated that (E)-1-amino4-phenylbut-3-en-2-ol (C1) had better inhibitory activities than 2-amino-1-arylethan-1-ol derivatives. To our surprise, sulfonation of C1 with 4-methoxybenzenesulfonyl chloride afforded more active inhibitor II with up to 6.4 lM IC50 value against neuraminidase. Furthermore, docking of inhibitor II into the active site of NA found that the H atoms in both NH2 and OH groups of inhibitor II were the key factors for potency. Molecular docking research did not explained very well the observed structure-activity relationship (SAR) from amino acid residue level, but also aided the discovery of (E)-1-amino-4-phenylbut-3en-2-ol derivatives as novel and potent NA inhibitors. Ó 2018 Published by Elsevier Ltd.

Influenza is an acute infectious respiratory disease caused by the influenza virus, leading to serious economic and social problems worldwide.1–3 There are two different types of influenza viral integral glycoproteins, namely, hemagglutinin and neuraminidase (NA).4,5 As the influenza viral surface protein, NA promotes cleavage of glycosidic bonds of sialic acids from viral and cell surfaces to release mature virions from infected cells,6–8 which plays a key role during virus infection and replication. Therefore, as a potential target for influenza virus infection,9 NA has attracted global attention.10,11 Currently, neuraminidase inhibitors (NAIs) as commercially available anti-influenza drugs including oseltamivir12, zanamivir13 and peramivir,14 comprise the main class of antiviral drugs for clinical prevention and treatment of influenza,15 but there is a urgent need to explore NAIs for the rapid emergence of drug-resistant mutants.16–21 Epigoitrin, which mainly exists in the radix of Isatis indigotica Fort, has been reported to exhibit various biological capabilities including antithyroid and antiviral activities.22,23 It showed moderate anti-influenza virus activity (IC50 = 269 lΜ against neuraminidase, Fig. 1).24 In a champion of discovery of bioactive organic small compounds, some enzymatic inhibitors were designed, synthesized, and biological evaluated in our research group recently.24–28 Inhibitor I was designed on the basis of a combined computational study with epigoitrin as the lead compound,

⇑ Corresponding author. E-mail address: [email protected] (Y. Yin). https://doi.org/10.1016/j.bmcl.2018.05.002 0960-894X/Ó 2018 Published by Elsevier Ltd.

and I exhibited about three folds higher inhibitory activity against neuraminidase compared with epigoitrin in the following synthesis and biological evaluation (IC50 = 85.5 lM for I vs. 269 lM for epigoitrin, Fig. 1). Furthermore, docking of both epigoitrin and I into the active site of NA found that the H atom in NH group was good for inhibitory activity, which was substituted in compound I.24 Therefore, oxazolidine-2-thione five-member ring in compound I was opened and led to N-Mbs sulfonated (E)-1-amino-4phenylbut-3-en-2-ol (II) as a new NA inhibitor. To our exciting, inhibitor II showed 13 times increased NA inhibitory activity compared with inhibitor I (IC50 = 6.4 lM for II vs. 85.5 lM for I, Fig. 1). Herein, the discovery process including synthesis, in vitro NA inhibitory activity evaluation, and molecular docking of (E)-1-amino4-phenylbut-3-en-2-ol derivatives as novel NAIs were reported in detail. The synthesis of targeted compounds II, C1–C5, D1–D13, E1–E3, and F1–F4 was presented in Scheme 1. With commercial available aromatic aldehyde A as the starting materials, 1-amino-2-alkanols C were obtained through cyanosilylation, hydrolysis reaction, and cyanide reduction. Then protection of amino alcohols C with different acyl chlorides (or sulfonyl chlorides) gave the target compounds II, D1–D11 and E1–E3 in high yields. Under alkaline condition, the treatment of (E)-1-amino-4-phenylbut-3-en-2-ols C1 with benzyl bromides produced tertiary amines F1–F4. With Et3N as the base, reaction of inhibitor C1, di-Boc-thiourea, and NIS gave compound D12, which was subsequently treated with HCl to remove the Boc protecting group to afford compound D13 as a HCl salt.

2004

C. Lu et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 2003–2007

Fig. 1. Transition of the lead compounds.

Scheme 1. Reagents and condition: (a) TMSCN, LiClO4
3H2O, rt; (b) NaBH4, CF3COOH, THF, 0 °C; (c) Sulfonyl chlorides (or Acyl chlorides), Et3N, CH2Cl2, rt; (d) NIS, di-Bocthiourea, Et3N, CH2Cl2, rt; (e) HCl, CH2Cl2, 80 °C; (f) Benzyl bromide derivatives, K2CO3, MeCN, reflux.

All synthesized compounds (II, C1–C5, D1–D13, E1–E3, and F1– F4) were dissolved with DMSO at 5  10 3 mol/L concentration and stored in a 20 °C fridge, then diluted with the buffer to the concentration of 5  10 4 mol/L, 5  10 5 mol/L, 5  10 6 mol/L, and 5  10 7 mol/L. Inhibitory effects on influenza virus neuraminidase were determined by Neuraminidase Inhibitors Screen Kit, which is a fluorescent enzyme immunoassay purchased from Beyotime Institute of Biotechnology.29,30 The 50% inhibitory concentrations (IC50s) were summarized in Table 1, and used as indicators to research the structure-activity relationship (SAR). SAR information of substitutions on N atom was firstly investigated with (E)-1-amino-4-phenylbut-3-en-2-ol C1 with free NH2 as the reference compound (Table 1). N benzene sulfonation was benefit for inhibitory activities compared inhibitors D1 with C1 probably due to the increased hydrogen bonds or hydrophobic interaction sites (IC50 = 7.6 lM for D1 vs. 207 lM for C1, entries 1 and 2). Addition of small groups including F, Cl, Br, CH3, and OCH3 to the benzenesulfonyl group cause no obvious effect on the IC50 values and all inhibitors (D2–D5, and II) showed single digital lM inhibition (IC50 = 7.9 lM for D2 with F, 9.2 lM for D3 with Cl, 9.4 lM for D4 with Br, 7.6 lM for D5 with CH3, 6.4 lM for II with OCH3 vs. 7.6 lM for D1, entries 2–7). Adding one more big group such as benzyloxy to the benzenesulfonyl group seen to be no good for activity (IC50 = 11.1 lM for D6 vs. 7.6 lM for D1, entries 2 and 8). Benzoylation of inhibitor C1 had the similar improved inhibitions as benzene sulfonation (IC50 = 7.2 lM for D7 with 3-OCH3, 8.0 lM for D8 with 2-OCH3, 11.4 lM for D9 with 4-CN, 13.3 lM for D10 with 4-COOCH3 vs. 7.6 lM for D1, entries 2, and 9–12). It was obvious that substitution on N atom with neither Boc nor guanidyl group could increase the inhibition (IC50 = 106 lM for D11 with Boc, 103 lM for D12 with Boc protected guanidine, and 214 lM for D13 with guanidyl group vs. 207 lM for C1, entries 13–15) probable due to the similar binding interactions formed in D11-D13 as in C1. The following results could be indicated from Table 1: (A) More than one hydrogen atom in NH2 group was not necessary; (B) Benzene sulfonation and benzoylation on N atom cause the improved inhibition might by the incensement of hydrophobic interaction sites. Then the importance of carbon-carbon double bond was investigated and the results showed that C@C unit was necessary for potency in this series of inhibitors otherwise worse inhibition

would be obtained (IC50 = 870 lM for C2, 5200 lM for C3, 6100 lM for C4, and 8900 lM for C5 vs. 207 lM for D1, entries 16–19, Table 1). For the increased hydrophobic interaction sites on N atom were helpful for inhibitory activity, inhibitors with disulfonyl or diacyl groups on N atom were planned to synthesize with C1 as the starting material for the further improvement of activities. Unfortunately, the synthesis did not succeed. Then we turned to the synthesis of N,N-dialkylated tertiary amines. Luckily, F1–F4 were obtained smoothly through the dibenzylation reaction between C1 and benzyl bromides in the presence of K2CO3 as the inorganic base (Table 2). F1 with N,N-dibenzyl groups exhibited around 5 times higher inhibitory activities compared with C1, about 5 times lower activities compared with D1, and the similar activities as I (IC50 = 41.5 lM for F1 vs. 207 lM for C1, 7.6 lM for D1, and 85.5 lM for I, entry 1). Neither electron-donating groups nor electronwithdrawing groups on the benzyl group had significant effect on the inhibitions (IC50 = 50.7 lM for F2 with CH3, 49.8 lM for F3 with CN, and 32.8 lM for F4 with CF3, entries 2–4). The results according to Table 2 indicated that the bulk groups in the amino group might be involved in the favorable interactions in the active site. The findings also revealed that the hydrogen atom on NH2 group influenced the activity of the compound and should be kept in new designs of NAIs. Since N,O-dibenzoylated products (E1–E3) were also obtained in the benzoylation of C1, E1–E3 were submitted the in vitro investigation. As shown in Table 3, E1–E3 had a little bit better activities than C1 with free NH2 and OH groups (IC50 = 61.5 lM for E1, 33.0 lM for E2, and 40.5 lM for E3 vs. 207 lM for C1, Table 3). Compounds E2 with CN and E3 with COOCH3 exhibited about a threefold decrease in inhibitory activity compared with D9 and D10, respectively (IC50 = 33.0 lM for E2 and 40.5 lM for E3 vs. 11.4 lM for D9 and 13.3 lM for D10, entries 2 and 3). So, substitution on the hydroxyl group was disadvantageous for antiviral activity. To explain the activity difference of lead compounds listed in Fig. 1, epigoitrin, I, and II were docked into the active site of NA (PDB code: 2HU0) and the obtained docking results were shown in Fig. 2. The main interactions between epigoitrin and NA activity sites were two hydrogen bonds (NAH


Glu277 and HCAH


Glu227)31,32 and two hydrophobic interactions (C@CHAH


Arg152, and C@C


Trp178) (Fig. 2a). In I-NA complex

2005

C. Lu et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 2003–2007 Table 2 IC50 values of inhibitors F1–F4.

Table 1 IC50 values of compounds II, C1–C5, and D1–D13.

R2

IC50(lM)a

IC50(lM)a

Entry

Compd.

C1

207 ± 43.3

1

F1

41.5 ± 8.8

2

D1

7.6 ± 1.2

2

F2

50.7 ± 9.2

3

D2

7.9 ± 1.4

3

F3

49.8 ± 9.1

4

D3

9.2 ± 2.0 4

F4

32.8 ± 6.2

5

D4

9.4 ± 1.9

6

D5

7.6 ± 1.4

7

II

6.4 ± 0.8

8

D6

11.1 ± 1.6

9

D7

7.2 ± 1.2

10

D8

8.0 ± 1.9

Entry

Compd.

1

Ar

R1

11

D9

11.4 ± 2.2

12

D10

13.3 ± 2.1

13

D11

106 ± 21.3

14

D12

103 ± 20.2

15

D13

214 ± 45.4

16

C2

870 ± 240

17

C3

5200 ± 1200

18

C4

6100 ± 1400

19

C5

8900 ± 2400

a Values were means of three or more experiments. The error was within +30% of the mean.

(Fig. 2b), there were six hydrogen bonds (C@S


Arg156, S@CAO


Arg152, C@CAPh


Ser246, and H3CAO


Arg118), one hydrophobic interactions (O2SAPh


Tyr406), and one electrostatic interaction (O2SAPh


Glu277). There were six hydrogen bonds (OCH2AH


Glu276, O@S@O


Ser179, NAH


Glu227, HAO


Arg156, and OAH


Arg119), three hydrophobic interactions (C@CAPh


Tyr406, SO2APh


Ile222, and SO2APh


Arg224), and two electrostatic interactions (C@CAPh


Arg118, and SO2APh


Arg152) were observed in II-NA complex (Fig. 2c). More

a Values were means of three or more experiments. The error was within +30% of the mean.

Table 3 IC50 values of compounds E1–E3.

R1

IC50(lM)a

Entry

Compd.

1

E1

61.5 ± 12.2

2

E2

33.0 ± 6.5

3

E3

40.5 ± 8.5

a Values were means of three or more experiments. The error was within +30% of the mean.

binding interactions including hydrogen bonds, hydrophobic interactions and electrostatic interactions should be the reason for the improved inhibition among epigoitrin, I, and II, and the hydrogen bond between H atom of NH group and NA in II-NA complex was the key contribution for 13-fold higher inhibition of II over I. To understand the SAR information obtained from Tables 2 and 3, N-benzoylated inhibitor D10, N,O-dibenzoylated inhibitor E3, and N, N-dibenzoylated inhibitor F2 were submitted the docking studies (Fig. 3). Six hydrogen bonds (CH3AOAH


Glu119, HAO


Arg156, NAH


Asp151, NHC@O


Tyr406, and PhC(OCH3)@O


Arg152) and one hydrophobic interaction (C@CHAPh


Arg224) were formed in D10-NA complex (Fig. 3a). In E3-NA complex (Fig. 3b), there were three H bonds (PhCH@CHCHAO


Arg118, CH@CHCHC@O


Tyr347, and NAH


Asp151), two hydrophobic interactions (CH@CHAPh


Arg118, and O@CAPhACOOCH2AH


Trp403), and two electrostatic interactions (CH3OOCAPh


Arg371, and CH3OOCAPh


Asp151). In F2-NA complex (Fig. 3c), there were three H bonds (OAH


Glu119, PhC@CC(OH)A H


Glu119, and NCHAH


Tyr406), three hydrophobic interactions (C@CAPh


Arg224, NCH2APh


Ile222, and NCH2PhACH2H


Tyr347), and one electrostatic interaction (C@CAPh


Glu277). Although benzoylation of OH cause increased binding sites which

2006

C. Lu et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 2003–2007

Fig. 2. Docking results. (a) Epigoitrin-NA complex; (b) I-NA complex; (c) II-NA complex. Hydrogen bonds, hydrophobic interactions and electrostatic interactions were shown as green, pink and orange dotted lines, respectively.

Fig. 3. Docking results. (a) D10-NA complex; (b) E3-NA complex; (c) F2-NA complex. Hydrogen bonds, hydrophobic interactions and electrostatic interactions were shown as green, pink and orange dotted lines, respectively.

C. Lu et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 2003–2007

were good for potency, the elimination of hydrogen bond between H atom of OAH and protein led to the decreased potency compared E3 with D10 (Fig. 3a and b). More hydrogen bonds especial between H atom of NAH and protein contribute the better potency of D10 compared with F2 although N,N disubstitution brought more interactions (Fig. 3a and c). So molecular docking also explained very well the disadvantage of substitution on the hydroxyl group and disubstitution on NH2 for antiviral activity, at the same time docking results also pointed out that H atom in OH group and one of the H atom in NH2 group should be kept. In summary, a small library of 1-amino-2-propanols was synthesized and in vitro biological evaluated as anti-influenza agents. In Neuraminidase Inhibitors Screen Kit test, Mbs-sulfonated (E)-1amino-4-phenylbut-3-en-2-ol II exhibited up to 6.4 lM IC50 value. Molecular docking indicated that the hydrogen atoms on the amino group and hydroxyl group significantly influenced the inhibitory activities of inhibitors and should be kept. The discovery of more active neuraminidase inhibitors from 1-amino-2-propanol scaffold is underway in our lab and will be reported in due course. Acknowledgments This work was supported by National Natural Science Foundation of China (Grant No.21502117), the collaboration Innovation Foundation of Shanghai Institute of Technology (No. XTCX2016-3), and Shanghai Municipal Education Commission (Plateau Discipline Construction Program). We are also grateful to Prof. Gang Zhao and Prof. Guanjun Wang for helpful discussion. A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.bmcl.2018.05.002.

2007

References 1. Short KR, Reading PC, Brown LE, et al. Infect Immun. 2013;81:645–652. 2. Enkhtaivan G, John KMM, Ayyanar M, Sekar T, Jin KJ, Kim DH. Saudi J Biol Sci. 2015;22:532–538. 3. Gruta NLL, Kedzierska K, Stambas J, Doherty PC. Immunol Cell Biol. 2007;85:85–92. 4. Plemper RK, Lakdawala AS, Gernert KM, Snyder JP, Compans RW. Biochem. 2003;42:6645–6655. 5. Wang Y, Curtis-Long MJ, Yuk HJ, Kim DW, Tan XF, Park KH. Bioorg Med Chem. 2013;21:6398–6404. 6. Skehel JJ, Wiley DC. Annu Rev Biochem. 2000;69:531–569. 7. Wagner R, Matrosovich M, Klenk HD. Rev Med Virol. 2002;12:159–166. 8. Yu M, Wang Y, Tian L, et al. RSC Adv. 2015;5:94053–94066. 9. Anne Moscona MD. N Engl J Med. 2005;353:1363–1373. 10. Zhang L, Cheng YX, Liu AL, Wang HD, Wang YL, Du GH. Molecules. 2010;15:8507. 11. Recker M, Pybus OG, Nee S, Gupta S. Proc Natl Acad Sci. 2007;104:7711–7716. 12. De CE. Nat Rev Drug Discovery. 2006;5:1015–1025. 13. McClellan K, Perry CM. Drugs. 2001;61:263. 14. Dunn CJ, Goa KL. Drugs. 1999;58:761–784. 15. Jain S, Fry AM. Clin Infect Dis. 2011;52:707–709. 16. Hwang BS, Lee IK, Choi HJ, Yun BS. Bioorg Med Chem Lett. 2015;25:3256–3260. 17. Wang PC, Fang JM, Tsai KC, et al. J Med Chem. 2016;59:5297–5310. 18. Hay AJ, Hayden FG. Lancet. 2013;381:2230–2232. 19. Xie YC, Huang B, Yu KX, Shi FY, Liu TQ, Xu WF. Bioorg Med Chem Lett. 2013;23:3556–3560. 20. Thorlund K, Awad T, Boivin G, Thabane L. BMC Infect Dis. 2011;11:134. 21. Das A, Adak AK, Ponnapalli K, et al. Eur J Med Chem. 2016;123:397–406. 22. Zhou W, Zhang XY. Am J Chin Med. 2013;4:743. 23. Xu LH, Huang F, Cheng T, Wu J. Chin J Nat Med. 2005;6:359–360. 24. Meng FL, Xu JY, Yin Y, et al. Bioorg Med Chem. 2018;70. 25. Ding M, Yin Y, Wu FH, et al. Bioorg Med Chem Lett. 2015;23:2505–2517. 26. Cui JX, Ding M, Deng W, et al. Bioorg Med Chem Lett. 2015;23:7464–7477. 27. Zhao Z, Cui JX, Yin Y, et al. Chin J Chem. 2016;1–8. 28. Duan YB, Yin Y, Meng FL, et al. Chem J Chinese Univ. 2017;9:1568–1577. 29. Li HB, Yan D, Wang JB, et al. Acta Pharmacol Sin. 2009;2:162–166. 30. Lou J, Yang XY, Rao ZG, et al. Eur J Med Chem. 2014;83:466–473. 31. Abdelsamie AS, Bey E, Gargano EM, van Koppen CJ, Empting M, Frotscher M. Eur J Med Chem. 2015;103:56–68. 32. Javid MT, Rahim F, Taha M, et al. Bioorg Chem. 2018;78:201–209.