Synthesis and biological evaluation of water-soluble derivatives of chiral gossypol as HIV fusion inhibitors targeting gp41

Accepted Manuscript Synthesis and biological evaluation of water-soluble derivatives of chiral gossypol as HIV fusion inhibitors targeting gp41 Jian Yang, Long-Long Li, Ju-Rong Li, Jing-Xiang Yang, Fang Zhang, Gang Chen, Rui Yu, Wen-Jie Ouyang, Shu-Wen Wu PII: DOI: Reference:

S0960-894X(17)30851-X http://dx.doi.org/10.1016/j.bmcl.2017.08.049 BMCL 25243

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

18 April 2017 18 August 2017 22 August 2017

Please cite this article as: Yang, J., Li, L-L., Li, J-R., Yang, J-X., Zhang, F., Chen, G., Yu, R., Ouyang, W-J., Wu, S-W., Synthesis and biological evaluation of water-soluble derivatives of chiral gossypol as HIV fusion inhibitors targeting gp41, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl. 2017.08.049

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Synthesis and biological evaluation of water-soluble derivatives of chiral gossypol as HIV fusion inhibitors targeting gp41 Jian Yanga, *, Long-Long Lia, c, Ju-Rong Lia, c, Jing-Xiang Yanga, c, Fang Zhanga, c, Gang Chena, c, Rui Yua, c, Wen-Jie Ouyangb, Shu-Wen Wub a

Department of Pharmacy, Renmin Hospital of Wuhan University, Wuhan 430060,

China b

The College of Life Sciences, State Key Laboratory of Virology, Modern Virology

Research Center, Wuhan University, Wuhan 430072, China c

Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China

*Corresponding authors. Jian Yang is to be contacted at Department of Pharmacy, Renmin Hospital of Wuhan University, China E-mail address: [email protected] (J. Yang). Phone: 0086-27-88041911-88381. Abstract: A series of novel or known water-soluble derivatives of chiral gossypol were synthesized and screened in vitro for their anti-HIV-1 activity. (-)-gossypol derivative was more active against HIV-1 than the corresponding (+)-gossypol derivative, respectively. Among these derivatives, D-glucosamine derivative of (-)-gossypol, oligopeptide derivative of (-)-gossypol and taurine derivative of (-)-gossypol, such as compounds 1a, 3a and 14a, showed significant inhibitory activities against HIV-1 replication, HIV-1 mediated cell-cell fusion and HIV gp41 6-helix bundle formation as some amino acid derivatives of (-)-gossypol. Keywords: chiral gossypol derivative; amino acid derivative; D-glucosamine derivative; oligopeptide derivative; taurine derivative; anti-HIV-1; gp41

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein transmembrane subunit gp41 plays a crucial role in the early steps of viral entry into target cells and may serve as important target for the development of HIV-1 entry inhibitors. T20 (brand name, Fuzeon) was the first and only clinically approved HIV-1 fusion inhibitor that being used for the treatment of patients with HIV-1 infection and AIDS, especially those infected by virus resistant to the current antiretroviral drugs. However, the clinical application of T20 was constrained due to its lack of oral bioavailability and high production costs. Therefore, it is critical to develop novel non-peptide small molecule HIV-1 fusion inhibitors of gp41.1, 2 Gossypol is a polyphenolic aldehyde extracted from cottonseed that has been used as a male contraceptive.3Gossypol is also marginally toxic, and this toxicity has been associated with the aldehyde groups, thus stimulating interest in exploration of derivatives or analogs of gossypol in which the aldehyde groups are altered. As a result, a large number of gossypol derivatives have been prepared and characterized. Many of these derivatives and analogs possess a diverse array of unusual disease-inhibiting activities as antimalarial, antiparasitic, anticancer and antiviral activities.4, 5 Gossypol has been shown to inactivate HIV in an in vitro system, albeit with poor efficiency.6 Then, a few of gossypol derivatives were synthesized and tested for anti-HIV-1 activity, Nevertheless, these gossypol derivatives were less active against HIV-1 than some analogs of gossypol.4, 7 Thereafter, there were not almost new reports on the anti-HIV-1 activity of gossypol derivatives. We postulated that the higher cytotoxicity of gossypol might limit its further study on antiviral effect, so we tried to modify the aldehyde groups of gossypol by using hydrophilic groups in order to reduce its cytotoxicity. The study results indicated that gossypol and its enantiomers did not exhibit potent anti-HIV-1 activity, but replacement of the

aldehyde groups of gossypol with some water soluble substituents not only reduced the cytotoxicity of gossypol but also enhanced their anti-HIV-1 activity, such as some amino acids.8 The further study indicated that amino acid derivatives of gossypol were novel small molecule HIV-1 fusion inhibitors of gp41.9 Recently, we found that some oligopeptides and D-glucosamine were also important fragments to prepare gossypol derivatives as HIV-1 entry inhibitors besides some amino acids. 2 Because of restricted rotation around the C2-C20 internaphthyl bond, gossypol had two optically active forms: the (-) and the (+)-enantiomers, respectively. Previous studies suggested that (-)-gossypol was usually more potent in most biological evaluations in comparison with (+)-gossypol or racemic gossypol.10-12 However, our study results showed that some (+)-gossypol derivatives and its analogs were more active against H5N1 than the corresponding (-)-gossypol derivatives and its analogs, respectively. 13 On the basis of the results, in order to carry out comprehensive comparison of the anti-HIV-1 activity of chiral gossypol derivatives and avoid the male antifertility activity of gossypol, some novel or known water-soluble chiral gossypol derivatives were synthesized and tested in vitro anti-HIV-1 activity as well as in vitro spermicidal assays. The procedure for synthesis of the title compounds is outlined in Scheme 1. (-)-Gossypol and (+)-gossypol were simply prepared by acid hydrolysis of the Schiff bases (+)-gossypol bis-(L-tryptophan methyl ester) and (-)-gossypol bis-(L-tryptophan methyl ester) in 90% and 95% yield with ee>95%, respectively.13 These novel or known chiral gossypol derivatives were synthesized by treating chiral gossypol with the corresponding amino acids, oligopeptides, D-glucosamine and taurine in a suitable solvent to form adducts in high yields (more than 85%). The analytical and spectral

data of all synthesized compounds agreed with the desired chemical structures. RHN

OH

CHO OH

RNH2

HO

OH

OH

O

O

OH OH

HO

CHO

OH

HO

(-)/(+)-Gossypol

NHR

(-)/(+)-Gossypol Schiff base

RNH2= CH2OH O HO HO

N H

NH2 (-)-1a and (+)-1b

O N H

COONa

(-)-5a and (+)-5b

COONa

H2N

(-)-6a and (+)-6b

COONa NH2

(-)-9a and (+)-9b

COONa NH2 (-)-13a and (+)-13b

N H

(-)-10a and (+)-10b

H2N

O

COONa

H N

COONa NaOOC

NH2 O

(-)-3a and (+)-3b

COONa NH N H O

COONa NH2

H 2N

(-)-2a and (+)-2b

O H 2N

O

O OH H2N

(-)-4a and (+)-4b

COONa

COONa

NH N H O

(-)-7a and (+)-7b

COONa NH2 (-)-11a and (+)-11b

NH2 (-)-8a and (+)-8b

H N

COONa NH2

(-)-12a and (+)-12b

SO3Na NH2 (-)-14a and (+)-14b

Scheme 1. Reagents and conditions: (i) for 1: methanol, r.t., 3h, nitrogen; (ii) for 2-7: methanol, NaOH, PH=7.4, r.t., 3h, nitrogen; (iii) for 8-14: methanol, NaOH, PH=7.4, 80℃, 3h, nitrogen.

As shown in Table 1, chiral gossypol might have no evident anti-HIV activity, but (-)-gossypol derivatives (compounds 1a-14a, IC50=0.35-6.99μM, SI[5.39-183.29]) showed more active against HIV-1 than (-)-gossypol. Likewise, (+)-gossypol derivatives (compounds 1b-14b, IC50=1.97- 9.06μM, SI [4.30-27.81]) also exhibited more active against HIV-1 than (+)-gossypol. We could observe that in each pair, the (-)-gossypol derivative was obviously more active against HIV-1 than the corresponding (+)-gossypol derivative, which was different from some chiral gossypol derivatives against H5N1.13 Notably, oligopeptide derivatives of (-)-gossypol(compounds 2a-7a) and taurine derivative of (-)-gossypol (compound 14a) showed potent anti-HIV-1 activity besides amino acids derivative of (-)-gossypol (compounds 8a-13a), while D-glucosamine derivative of (-)-gossypol (compound 1a, IC50=0.72μM, SI=32.12) with the absence of the COONa group also had much better

selectivity index and comparable potency, next to those of amino acid derivatives of (-)-gossypol(compounds 8a-11a). On the other hand, aliphatic amino acid derivatives of (-)-gossypol (compounds 8a-11a) were more active than aromatic amino acids derivatives of (-)-gossypol (compounds12a and 13a), while dipeptide derivatives of (-)-gossypol (compounds 2a-5a) had similar antiviral activities to tripeptide derivatives of (-)-gossypol (compounds 6a and 7a). Additionally, taurine derivative of (+)-gossypol (compound14b, IC50=1.97μM, SI=27.81) exhibited the strongest activity against HIV-1 among (+)-gossypol derivatives (compounds1b-14b), close to some (-)-gossypol derivatives (compounds 2a-7a). Table 1 Effects of chiral gossypol derivatives on the replication of HIV-1IIIB in TZM-bl cells. Compound AZT T20 (-)-Gossypol (+)-Gossypol (-)-1a (+)-1b (-)-2a (+)-2b (-)-3a (+)-3b (-)-4a (+)-4b (-)-5a (+)-5b (-)-6a (+)-6b (-)-7a (+)-7b (-)-8a (+)-8b (-)-9a (+)-9b (-)-10a (+)-10b (-)-11a (+)-11b (-)-12a (+)-12b (-)-13a (+)-13b (-)-14a (+)-14b a

ICa50 (μmol/L) 0.0028 0.0905 — — 0.72 7.64 1.56 4.28 1.43 5.82 1.25 5.35 1.62 6.32 1.38 6.62 1.45 6.39 0.35 4.13 0.49 5.92 0.68 7.16 0.47 4.22 1.08 9.06 6.99 8.21 1.17 1.97

CCb50 (μmol/L) ND ND 5.22 18.12 23.13 32.87 40.31 61.12 35.74 59.51 42.63 51.16 33.33 50.34 43.76 62.39 39.42 58.72 64.15 72.63 49.89 63.82 53.32 60.15 62.18 73.86 48.23 57.04 37.69 53.81 37.84 54.78

SIc — — — — 32.12 4.30 25.84 14.28 24.99 10.22 34.10 9.56 20.57 7.97 31.71 9.42 27.19 9.19 183.29 17.59 101.82 10.78 78.41 8.40 132.29 17.50 44.66 6.29 5.39 6.55 32.34 27.81

IC50: concentration of compound required to produce 50% of the relative luciferase activity of the control wells with HIV-1IIIB infected TZM-bl cells in the absence of compound. b CC50: compound concentration required to cause 50% death of uninfected TZM-bl cells, as determined by the MTT method. c SI: selectivity index as CC50/ IC50.ND: no determination.—: no calculation. All data represent average values for three separate experiments. The variation of these results under the standard procedures is below ±10%.

Considering that some amino acid derivatives of (-)-gossypol were novel small molecule HIV-1 fusion inhibitors of gp41,9 we also wanted to know whether D-glucosamine derivative, oligopeptide derivatives and taurine derivative could target to gp41 protein. Therefore, compounds 1a, 3a and 14a were chosen for further study. Then, time-of-addition assay was carried out to investigate the possible target phase of these compounds in the HIV-1 life cycle. From Figure 1, time-of-addition assay suggested that the test compounds and T20 inhibited HIV-1 replication when they were added to the cells with virus together, but they showed no inhibitory activity if they were added 2h or longer after virus was added to the cells. Nevertheless, AZT(3’-azido-3’-deoxythymidine, zidovudine) was still effective in inhibiting HIV-1 replication even when it was added 6 h after post-infection. The results showed that compounds 1a, 3a and 14a could block the entry of HIV-1IIIB into target cell, which was similar to that of T20, likely targeting to HIV-1gp41 as some amino acid derivatives of (-)-gossypol.9

Figure1. Compounds 1a, 3a and 14a inhibited HIV-1IIIB entry. Inhibition of HIV-1 IIIB entry was determined by a time-of-addition assay. For the time-of-addition assay, TZM-bl cells, seeded in 48-well dishes, were incubated with HIV-1 IIIB at 37℃ for 0, 2, 4 and 6 h, respectively, before the addition of a test compound (10μM). AZT and T20 with the same molarity were used as controls.

Thus, compounds1a, 3a and 14a were screened for their inhibitory activities against 1) HIV-1 replication(p24), 2) HIV-1-mediated cell-cell fusion and 3) HIV gp41 six-helix bundle (6-HB) formation.9 In this preliminary study, compounds 1a, 3a and 14a showed significant potency against HIV-1 replication with IC50 values of 1.22, 1.72 and 1.56μM, respectively, and HIV-1-mediated cell-cell fusion with IC50 values of 6.19, 7.14 and 6.67μM, respectively, and gp41 6-HB formation with IC50 values of 6.36, 7.87 and 7.34μM, respectively. In contrast, (-)-gossypol had no evident effects in above assays. The results indicated that these chiral gossypol derivatives might target to gp41. To compare the spermicidal activity of compounds 1a, 3a and 14a with (-)-gossypol, an in vitro spermicidal assay was conducted.9 The results showed that exposure of sperm to either compounds 1a, 3a and 14a or (-)-gossypol at a concentration of 100μM for up to 1h resulted in less spermicidal activity when compounds 1a, 3a and 14a were used than when (-)-gossypol was used. Furthermore, we used HIV-1 gp41 N-peptide bundle (PDB ID: 2r5d) 14 as a reference for our modeling work. Using Molegro Virtual Docker (version 4.3.0), the key interactions between these compounds and gp41 protein residues were summarized in Table 2. The binding modes of compounds 1a, 3a and 14a were shown in Figure 2, respectively. As in shown in Table 2, the MolDockScores of (-)-gossypol derivatives (compounds 1a, 3a and 14a) were super to those of the corresponding (+)-gossypol derivatives (compounds 1b, 3b and 14b), respectively, so (-)-gossypol derivative might have stronger binding affinities to gp41 protein when compared with

the corresponding (+)-gossypol derivative. As expected, the docking results also verified the ideas that chiral gossypol derivative with some water soluble substituents replacing the aldehyde groups, such as oligopeptides, D-glucosamine and taurine, could increase Van der Waals interaction, hydrophobic contacts, hydrogen bonding network and electrostatic interactions, and could have higher binding affinities to gp41 in comparison with the corresponding parent compound. From Figure2, compound 14a had the strong electrostatic interaction with the active residues Lys38, while compound 3a formed the strong electrostatic interaction with the other active residues Arg43. Unlike the binding modes of compounds 3a, 14a and other gp41 fusion inhibitors,15,16 two D-glucosamine moieties of compound 1a were closed to the important active residues Lys38 and Gln41, respectively, which could generate more favorable hydrogen bonding networks and increase affinities with the gp41 protein. These interactions, identified by docking simulations, rationalized the anti-HIV-1 activity of chiral gossypol derivatives. Table 2 The key interactions between some compounds and gp41 protein Interactions with gp41

(-)-Gossypol

(+)-Gossypol

MolDockScore

-101.985

-96.0613

Van der Waals interaction

√

Hydrophobic contacts

√

(-)-1a

(+)-1b

(-)-3a

(+)-3b

(-)-14a

(+)-14b

-114.737

-110.153

-159.185

-148.207

-158.789

-146.224

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

electrostatic interaction with Lys38 √

electrostatic interaction withArg43 √

Hydrogen bond with Val34 √

Hydrogen bond with Lys38 Hydrogen bond with Gln39

√

√

√(2)

√

√

√

√

√

√

√

Hydrogen bond with Leu40 √(2) Hydrogen bond with Gln41

√

√(2) √

√(4)

√

√(3)

√(3) (3) √

Hydrogen bond with Ala42 Hydrogen bond with Arg43

√

√

MolDockScore: The energy score using docking (arbitrary units).

1a

3a

14a Figure 2. The calculated most favorable binding modes of compounds 1a, 3a and 14a inside the hydrophobic pocket of HIV-1 gp41 N-peptide bundle respectively, and residues of the pocket surrounding ligands. Compounds 1a, 3a and 14a are shown as a stick model respectively and hydrogen bonds with HIV-1 gp41 N-peptide bundle are in yellow dashed lines. The strong electrostatic interactions between these compounds and HIV-1 gp41 N-peptide bundle are shown as red hemispheres respectively.

In conclusion, a series of novel or known water-soluble derivatives of chiral gossypol were synthesized and screened in vitro for their anti-HIV-1 activity. Interestingly, (-)-derivative was obviously more active against HIV-1 than the corresponding (+)-derivative, which was different from some chiral gossypol derivatives against H5N1. Among (-)-gossypol derivatives, compound 1a, 3a and 14a promised good anti-HIV-1 activity and appeared to be significant inhibitory activities against HIV gp41 6-helix bundle formation and less spermicidal activity. The results further supported our ideas that some water soluble substituents were important fragments to prepare gossypol derivatives as HIV-1 entry inhibitors targeting to gp41 protein besides some amino acids. Acknowledgment We gratefully acknowledge the National Natural Science Foundation of China (No. 30770228), and Collaborative Innovation Project, Department of Medicine, Wuhan University for financial support.

A. Supplementary data Supplementary data associated with this article can be found in the online version.

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Graphical Abstract RHN

OH

OH

O

O OH

HO

NHR

(-)-Gossypol Schiff base

CH2OH O RNH2=

HO HO NH2 (-)-1a

O OH

H2 N

SO3Na N H

(-)-3a

COONa NH2 (-)-14a

Compounds 1a, 3a and 14a showed significant inhibitory activities against HIV-1 replication, HIV-1 mediated cell-cell fusion and HIV gp41 6-helix bundle formation.