Bioorganic & Medicinal Chemistry Letters 25 (2015) 1880–1883
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel HIV-1 protease inhibitors using tertiary amine as P2-ligands Zhi-Heng Yang a, Xiao-Guang Bai a, Lei Zhou a, Ju-Xian Wang a, Hong-Tao Liu b, Yu-Cheng Wang a,⇑ a b
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China Department of Pharmacy, Hebei General Hospital, Hebei, Shijiazhuang 050051, PR China
a r t i c l e
i n f o
Article history: Received 6 January 2015 Revised 13 March 2015 Accepted 17 March 2015 Available online 24 March 2015
a b s t r a c t A series of tertiary amine derivatives exhibiting potent HIV-1 protease inhibiting properties were identified. These novel inhibitors were designed based on the structure of Darunavir with modification on the P2 and P20 position. This effort led to discovery of 35e and 38e, which exhibited excellent HIV-1 protease inhibition with IC50 values of 15 nM and 64 nM, respectively. Ó 2015 Elsevier Ltd. All rights reserved.
Keywords: HIV-1 protease Inhibitors HAART Darunavir
To date, the AIDS epidemic is still one of the most challenging problems.1,2 According to WHO reports, the number of people living with HIV has risen to 35.3 million, with 2.3 million new infections and 1.6 million AIDS-related deaths in 2012.3–6 Among many strategies to combat this disease, highly active antiretroviral therapy (HAART) containing at least one of HIV-1 protease inhibitors (PIs) was regarded as the most effective treatment for HIV infection.7–10 During the past 20 years, several HIV-1 PIs were discovered and widely used in the clinic, such as Saquinavir, Amprenavir, and Darunavir. However, drug resistance is still a major problem in the treatment of HIV/AIDS due to a high frequency mutation of the virus.11–13 Therefore, it is important to develop novel HIV-1 PIs with improved activity against multi-drug resistant variants. To develop new chemical entities against drug-resistant HIV, the strategy to design novel structures based on current approved drugs is an efficient way.14–16 Darunavir (Fig. 1, 1), approved by FDA in 2006, displayed an excellent antiviral activity against multidrug-resistant HIV-1 strains.17,18 A previous study showed that the binding potency was enhanced especially by the interaction between the oxygen in the fused ring of bis-THF and NH group in backbone of Asp29 and Asp30.19 While Amprenavir (2), contain-
⇑ Corresponding author. Tel./fax: +86 10 63165263. E-mail address: [email protected] (Y.-C. Wang). http://dx.doi.org/10.1016/j.bmcl.2015.03.047 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
ing single THF moiety, only exhibited weak O–H OH bonds with the main-chain amides of Asp29 and Asp30 in the S2 binding subsite.20 The tri-THF protease inhibitor, GRL-0519 (3), with excellent antiviral activity on drug resistant virus,21 showed the ability to make new interactions with the side chain of Asp30 in the wild type enzyme and Asn30 in the mutant variant. Thus, the P2 site substituent of the inhibitors has great relativity on the inhibitory activities. In this study, a series of PIs were designed based on the skeleton of 1, where the bis-THF moiety was replaced using scaffold-hopping strategy to identify new scaffolds. In this study, the scaffold was replaced based on the following strategies: (a) Split the bis-THF ring to two parts, tetrahydrofuran ring and alkane, which then were substituted by Ra and Rb, respectively (Fig. 2); (b) Ra imitates the P2 ligand of Lopinavir (4) and Nelfinavir (5), and Rb was substituted by acetamide structures. We envisioned that the new tertiary amine scaffold as the P2 ligand may interact strongly with enzyme backbone through the new flexible acetamide structure. Additionally, considering the modification in P2 site may alter the binding conformation of the whole designed compounds, both (RS)- and (SS)-enantiomers of novel inhibitors were synthesized and discussed. The synthesis of the tertiary amine intermediates 12a,b was outlined in Scheme 1. Protection of the 2-methyl-3-nitrophenol (6) with benzyl bromide in the presence of K2CO3 yielded benzyl derivative 7. Reduction of nitro group of 7, followed by coupling with ethyl chloroacetate gave compound 8. The compound 8 was further alkylated with bromoacetamides (9a,b) to obtain 10a, b.22
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Z.-H. Yang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1880–1883
O
H O
NH2
Ph
O O
O S N
N H
H
H
O
O
O
OH
NH 2
Ph
O N H
O S N
O O
O
N H
O
OH
O
Ph
O
O S N
O
OH
O 2 Amprenavir
1 Darunavir
O
H
Ph O
OH
H N
3 GRL-0519
N
N H
H N
NH HO O
Ph
OH
O S
4 Lopinavir
H
N H N H
O Ph
5 Nelfinavir
Figure 1. Structure of some marketed HIV PIs and GRL-0519.
H O O
O
O S N
N H
H
NH2
Ph
O
Removal of the benzyl group of 10a,b using 5% Pd-C and H2 in EtOH provided the corresponding phenol 11a,b, which were saponified with NaOH in MeOH/CH2Cl2 (1:3) afforded carboxylic acid derivatives 12a,b. The tertiary amine derivatives 12c–f were obtained using a similar strategy to the synthesis of 12a,b. As shown in Scheme 2, treatment of anilines 13 and 14 with ethyl chloroacetate followed by alkylation with bromoacetamides gave 11c–f. Saponification of 11c–f afforded the corresponding derivatives 12c–f. The synthetic route of designed PIs was illustrated in Scheme 3. Our synthesis was commenced with the commercial available epoxides 17 and 18. Treatment of epoxides 17 and 18 with isobutylamine followed by coupling with sulfonyl chloride affording sulfonamides 21–25 and 27.22 Subsequently, 25 and 27 were converted to hydroxymethylsulfonamide derivatives 26 and 28, respectively through deprotection of the acetates with K2CO3 and reduction of the corresponding aldehyde with NaBH4 in methanol.22 Finally, removal of the Boc protection of 21–24, 26, and 28 followed by the amidation reactions with 12a-f obtained tertiary amine derivatives of 35–37, 39, 41 and 42.23 The nitro group of compounds 37a–f and 39a–f was reduced using 5% Pd-C and H2 to provide 38a–f and 40a–f.22 The inhibition potency of the synthetic compounds against HIV1 protease was evaluated using a fluorescence resonance energy transfer (FRET) method.24,25 Indinavir and darunavir were used as positive control; and the results were presented in Tables 1 and 2, respectively. The data listed in Table 1 indicated that IC50 values of most of (RS)-enantiomeric compounds were in nanomolar. Whereas, the (RS)-enantiomeric compounds in Table 2
O
OH
Darunavir scaffold-hopping
Ra H O
N H
O
HO
NH2
Ph
O
O S N
O
OH
Rb
R
P2 R
a
b
N
Rc
Ph
O
P2'
O S
N H
N
O
OH (RS SS)
TM Ra =
HO O O
b
R =
N
N
O
O
Figure 2. Chemical structure of target molecular (TM).
HO
NO2
a
Bn
O
NO2
b,c
Bn
H N
O
O O
R3
Br
d
O 6
7
8
R3
R3
O Bn
O N
O
10a, 10b
9a, 9b
O
e O
R3 O N
HO
11a, 11b
O
f O
O N
HO
OH
12a, 12b a R 3 = morpholine b R3 = dimethylamine
Scheme 1. Reagents and conditions: (a) benzyl bromide, K2CO3, CH3CN, 80 °C, 3 h, 86%; (b) SnCl22H2O, EtOAc, 80 °C, 3.0 h, 62%; (c) AcONa, ethyl chloroacetate, EtOH, 80 °C, 6 h, 65%; (d) K2CO3, DMF, 90 °C, 1 h, 74% for 10a, 69% for 10b; (e) H2, Pd/C, EtOH, 30 psi, rt, 2.0 h, 89% for 11a, 76% for 11b; (f) MeOH/CH2Cl2 (1:3), NaOH, rt, 1 h, 68% for 12a, 63% for 12b.
1882
Z.-H. Yang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1880–1883 R3 NH 2 R'
O
H N
a R
13 R ' = 4-OMe 14 R ' = 2,6-di-Me
O
b
O
'
R3 O N
11c 11d 11e 11f
O N
O
R' 15 R ' = 4-OMe 16 R ' = 2,6-di-Me
O
c
R' = 4-OMe, R3 = morpholine R ' = 4-OMe, R 3 = dimethylamine R' = 2,6-di-Me, R 3 = morpholine R ' = 2,6-di-Me, R 3 = dimethylamine
R'
OH
12c 12d 12e 12f
R' = 4-OMe, R3 = morpholine R ' = 4-OMe, R 3 = dimethylamine R' = 2,6-di-Me, R 3 = morpholine R ' = 2,6-di-Me, R 3 = dimethylamine
Scheme 2. Reagents and conditions: (a) AcONa, ethyl chloroacetate, EtOH, 80 °C, 6 h, 62% for 15, 69% for 16; (b) 9a–b, K2CO3, DMF, 90 °C, 1 h, 56–72%; (c) MeOH/CH2Cl2 (1:3), NaOH, rt, 1 h, 54–71.0%.
Ph Ph Boc
O Boc
a N H
O
17 SS 18 RS
N H
NH
Cl
S
Boc
b
R1
OH 19 RS 20 SS
c
R1
R
N H
O S N
O
d 29 RS R1 = 4-OMe 30 SS R 1 = 4-OMe 31 RS R1 = 4-NO2 32 SS R 1 = 4-NO2 33 RS R1 = 4-OH 34 SS R 1 = 4-OH
R = Boc R=H
O
O N
OH
N H
O
OH
R3
R3
O
O S N
21 RS R1 = 4-OMe 22 SS R 1 = 4-OMe 23 RS R1 = 4-NO2 24 SS R 1 = 4-NO2 25 RS R1 = 4-di-CH(COCH 3) 2 26 RS R1 = 4-CH2 OH 27 SS R 1 = 4-di-CH(COCH 3 )2 28 SS R 1 = 4-CH 2OH
c
Ph
R1
Ph
O
R2
N
e R
OH
12a R 2 = 2-Me, 3-OH, R3 = morpholine 12b R2 = 2-Me, 3-OH, R 3 = dimethylamine 12c R 2 = 4-OMe, R3 = morpholine 12d R2 = 4-OMe, R 3 = dimethylamine 12e R 2 = 2,6-di-Me, R 3 = morpholine 12f R2 = 2,6-di-Me, R 3 = dimethylamine
2
f f
R1
Ph
O N H
O S
N
O
OH
35 RS a-f R 1 = 4-OMe 36 SS a-f R 1 = 4-OMe 37 RS a-f R 1 = 4-NO2 38 RS a-f R 1 = 4-NH2 39 SS a-f R 1 = 4-NO2 40 SS a-f R 1 = 4-NH 2 41 RS a-f R 1 = 4-CH2 OH 42 SS a-f R 1 = 4-CH 2OH
Scheme 3. Reagents and conditions: (a) i-BuNH2, CH3CN, 80 °C, 6 h, 83% for 19, 73% for 20; (b) THF, DIEA, DMAP, rt, 3 h, 74–82%; (c) (i) K2CO3, MeOH, rt, 1.5 h. (ii) NaBH4, MeOH, rt, 1 h, 86% for 26, 64% for 28; (d) CH2Cl2-CF3COOH (1:1), 3 h, 65.8–79.3%; (e) DMF, EDCHCl, HOBt, Et3N, rt, overnight, 45-73%; (f) H2, Pd/C, MeOH, 40 psi, rt, 1.5 h, 60– 78%.
exhibited dissatisfactory IC50 values that were in the micromolar levels. These results clearly showed that (RS)-enantiomer of hydroxyethylamine isostere exhibits more remarkable inhibition to HIV-1 protease, which is consistent with the enantiomer of darunavir. Thus, only the Structural–Activity Relationship (SAR) study of (RS)-enantiomer of novel compounds in Table 1 were discussed in the next. As a novel scaffold, we synthesized and tested series of analogues with variations at both the P2 and P20 positions. The first series of inhibitors, bearing a 4-methoxyphenyl at P20 site (35a– f), showed generally higher potency than the corresponding substituted with that of 4-aminophenyl (38a–f), 4-nitrophenyl (37a–f) and 4-hydroxymethylphenyl (41a–f). Compounds bearing 2,6dimethyl phenyl group at P2 position showed impressive potency to inhibit HIV-1 protease, especially with a morpholine (R3) substituent (35e, 37e, 38e, 41e). Among them, compounds 35e and 38e showed excellent activity to inhibit HIV-1 protease with IC50 values of 15 nM and 64 nM, respectively. Whereas, the presence of 4-methoxy or 2-methyl-3-hydroxy of the R2 substituent is
unsatisfactory with relatively low inhibitory activity. Besides, the compounds bearing a dimethylamine (R3) group showed higher potency than the corresponding substituted morpholine analogues (35b vs 35a, 35d vs 35c, 37b vs 37a, 37d vs 37c, 38b vs 38a, 38d vs 38c, 41b vs 41a, 41d vs 41c). However, it is interestingly to note an opposite result when R2 was the 2,6-dimethyl group (35e vs 35f, 37e vs 37f, 38e vs 38f, 41e vs 41f). It indicated that the substituents of R2 on the phenyl group as P2 ligand are crucial to the activity against the HIV-1 protease. In summary, a series of forty-eight novel HIV-1 PIs containing different P2 and P20 ligands were designed and synthesized. The enzyme activity assay showed the inhibitors with (R)-(hydroxyethylamino)-sulfonamide skeleton more effectively bind to the HIV-1 protease, and the substituents of R2 on the phenyl group are crucial to the activity. Compounds 35e and 38e showed excellent activities against HIV-1 protease with IC50 values of 15 nM and 64 nM, respectively. As a novel HIV-1 PI scaffold, further structural modifications of the tertiary amine moiety at P2 site are undergoing in our lab.
Z.-H. Yang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1880–1883 Table 1 Inhibitory of compounds against wild-type HIV-1 protease R3 O
O N
We are grateful to the financial support from the National Natural Science Foundation of China (Nos. 81302644, 81473099, and 81473098).
O N H
R2
Acknowledgment
R1
Ph N
S O
OH
Supplementary data
(RS)
a
Entry
R1
R2
R3
IC50a (lM)
35a 35b 35c 35d 35e 35f 37a 37b 37c 37d 37e 37f 38a 38b 38c 38d 38e 38f 41a 41b 41c 41d 41e 41f Indinavir Darunavir
4-OMe 4-OMe 4-OMe 4-OMe 4-OMe 4-OMe 4-NO2 4-NO2 4-NO2 4-NO2 4-NO2 4-NO2 4-NH2 4-NH2 4-NH2 4-NH2 4-NH2 4-NH2 4-CH2OH 4-CH2OH 4-CH2OH 4-CH2OH 4-CH2OH 4-CH2OH — —
2-Me, 3-OH 2-Me, 3-OH 4-OMe 4-OMe 2,6-Di-Me 2,6-Di-Me 2-Me, 3-OH 2-Me, 3-OH 4-OMe 4-OMe 2,6-Di-Me 2,6-Di-Me 2-Me, 3-OH 2-Me, 3-OH 4-OMe 4-OMe 2,6-Di-Me 2,6-Di-Me 2-Me, 3-OH 2-Me, 3-OH 4-OMe 4-OMe 2,6-Di-Me 2,6-Di-Me — —
Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine — —
1.38 ± 0.03 0.54 ± 0.006 0.29 ± 0.01 0.22 ± 0.01 0.015 ± 0.002 0.18 ± 0.006 6.67 ± 0.26 0.61 ± 0.02 1.02 ± 0.07 0.21 ± 0.01 0.073 ± 0.001 0.29 ± 0.02 2.73 ± 0.19 0.59 ± 0.01 1.36 ± 0.09 0.14 ± 0.006 0.064 ± 0.002 1.36 ± 0.09 1.21 ± 0.16 0.15 ± 0.00 0.89 ± 0.07 0.37 ± 0.01 0.073 ± 0.001 0.29 ± 0.02 0.88 ± 0.04 nM 0.061 ± 0.02 nM
Values are means of three independent experiments.
Table 2 Inhibitory of compounds against wild-type HIV-1 protease R3 O N R2
R1
Ph
O
O N H
N
S O
OH (SS)
a
1883
Entry
R1
R2
R3
IC50a (lM)
36a 36b 36c 36d 36e 36f 39a 39b 39c 39d 39e 39f 40a 40b 40c 40d 40e 40f 42a 42b 42c 42d 42e 42f Indinavir Darunavir
4-OMe 4-OMe 4-OMe 4-OMe 4-OMe 4-OMe 4-NO2 4-NO2 4-NO2 4-NO2 4-NO2 4-NO2 4-NH2 4-NH2 4-NH2 4-NH2 4-NH2 4-NH2 4-CH2OH 4-CH2OH 4-CH2OH 4-CH2OH 4-CH2OH 4-CH2OH — —
2-Me, 3-OH 2-Me, 3-OH 4-OMe 4-OMe 2,6-Di-Me 2,6-Di-Me 2-Me, 3-OH 2-Me, 3-OH 4-OMe 4-OMe 2,6-Di-Me 2,6-Di-Me 2-Me, 3-OH 2-Me, 3-OH 4-OMe 4-OMe 2,6-Di-Me 2,6-Di-Me 2-Me, 3-OH 2-Me, 3-OH 4-OMe 4-OMe 2,6-Di-Me 2,6-Di-Me — —
Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine — —
>20 lM 5.78 ± 0.16 >20 lM >20 lM 8.65 ± 0.25 >20 lM >20 lM 7.41 ± 0.07 19.21 ± 0.08 >20 lM 5.87 ± 0.05 5.88 ± 0.12 16.07 ± 0.24 5.92 ± 0.12 6.81 ± 0.20 6.96 ± 0.34 2.59 ± 0.06 0.49 ± 0.04 6.12 ± 0.16 >20 lM >20 lM 5.34 ± 0.59 8.94 ± 0.58 17.3 ± 1.11 0.88 ± 0.04 nM 0.061 ± 0.02 nM
Values are means of three independent experiments.
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.03. 047. These data include MOL files and InChiKeys of the most important compounds described in this article. References and notes 1. Shafer, R. W.; Rhee, S. Y.; Pillay, D.; Miller, V.; Sandstrom, P.; Schapiro, J. M.; Kuritzkes, D. R.; Bennett, D. AIDS 2007, 21, 215. 2. Menendez-Arias, L. Antiviral Res. 2010, 85, 210. 3. Marx, J. L. Science 1982, 217, 618. 4. Joint United Nations Programme on HIV/AIDS. Global Report: UNAIDS Report on the Global AIDS Epidemic 2013; UNAIDS/WHO: Geneva. 5. Yu, Z.; Kabashima, T.; Tang, C.; Shibata, T.; Kitazato, K.; Kobayashi, N.; Lee, M. K.; Kai, M. Anal. Biochem. 2010, 397, 197. 6. Lines, J. A.; Yu, Z.; Dedkova, L. M.; Chen, S. Biochem. Biophys. Res. Commun. 2014, 443, 308. 7. Sepkowitz, K. A. N. Engl. J. Med. 2001, 344, 1764. 8. Pomerantz, R. J.; Horn, D. L. Nat. Med. 2003, 9, 867. 9. Bartlett, J. A.; Fath, M. J.; Demasi, R.; Hermes, A.; Quinn, J.; Mondou, E.; Rousseau, F. AIDS 2006, 20, 2051. 10. MacArthur, R. D.; Novak, R. M.; Peng, G.; Chen, L.; Xiang, Y.; Hullsiek, K. H.; Kozal, M. J.; van den Berg-Wolf, M.; Henely, C.; Schmetter, B.; Dehlinger, M. Lancet 2006, 368, 2125. 11. Condra, J. H.; Schleif, W. A.; Blahy, O. M.; Gabryelski, L. J.; Graham, D. J.; Quintero, J.; Rhodes, A.; Robbins, H. L.; Roth, E.; Shivaprakash, M.; Titus, D.; Yang, T.; Tepplert, H.; Squires, K. E.; Deutsch, P. J.; Emini, E. A. Nature 1995, 374, 569. 12. Mehellou, Y.; De Clercq, E. J. Med. Chem. 2010, 53, 521. 13. Johnson, V. A.; Brun-Vezinet, F.; Clotet, B.; Gunthard, H. F.; Kuritzkes, D. R.; Pillay, D.; Schapiro, J. M.; Richman, D. D. Top. HIV Med. 2007, 15, 119. 14. Kempf, D. J.; Marsh, K. C.; Kumar, G.; Rodrigues, A. D.; Denissen, J. F.; McDonald, E.; Kukulka, M. J.; Hsu, A.; Granneman, G. R.; Baroldi, P. A.; Sun, E.; Pizzuti, D.; Plattner, J. J.; Norbeck, D. W.; Leonard, J. M. Antimicrob. Agents Chemother. 1997, 41, 654. 15. Youle, M. J. Antimicrob. Chemother. 2007, 60, 1195. 16. Zeldin, R. K.; Petruschke, R. A. J. Antimicrob. Chemother. 2004, 53, 4. 17. King, N. M.; Prabu-Jeyabalan, M.; Nalivaika, E. A.; Wigerinck, P.; de Bethune, M. P.; Schiffer, C. A. J. Virol. 2004, 78, 12012. 18. Kovalevsky, A. Y.; Tie, Y.; Liu, F.; Boross, P. I.; Wang, Y. F.; Leshchenko, S.; Ghosh, A. K.; Harrison, R. W.; Weber, I. T. J. Med. Chem. 2006, 49, 1379. 19. Ghosh, A. K.; Kincaid, J. F.; Walters, D. E.; Chen, Y.; Chaudhuri, N. C.; Thompson, W. J.; Culberson, C.; Fitzgerald, P. M.; Lee, H. Y.; McKee, S. P.; Munson, P. M.; Duong, T. T.; Darke, P. L.; Zugay, J. A.; Schleif, W. A.; Axel, M. G.; Lin, J.; Huff, J. R. J. Med. Chem. 1996, 39, 3278. 20. Weber, I. T.; Waltman, M. J.; Mustyakimov, M.; Blakeley, M. P.; Keen, D. A.; Ghosh, A. K.; Langan, P.; Kovalevsky, A. Y. J. Med. Chem. 2013, 56, 5631. 21. Ghosh, A. K.; Xu, C. X.; Rao, K. V.; Baldridge, A.; Agniswamy, J.; Wang, Y. F.; Weber, I. T.; Aoki, M.; Miguel, S. G.; Amano, M.; Mitsuya, H. ChemMedChem 1850, 2010, 5. 22. Ghosh, A. K.; Sridhar, P. R.; Leshchenko, S.; Hussain, A. K.; Li, J.; Kovalevsky, A. Y.; Walters, D. E.; Wedekind, J. E.; Grum-Tokars, V.; Das, D.; Koh, Y.; Maeda, K.; Gatanaga, H.; Weber, I. T.; Mitsuya, H. J. Med. Chem. 2006, 49, 5252. 23. Maillard, M. C.; Hom, R. K.; Benson, T. E.; Moon, J. B.; Mamo, S.; Bienkowski, M.; Tomasselli, A. G.; Woods, D. D.; Prince, D. B.; Paddock, D. J.; Emmons, T. L.; Tucker, J. A.; Dappen, M. S.; Brogley, L.; Thorsett, E. D.; Jewett, N.; Sinha, S.; John, V. J. Med. Chem. 2007, 50, 776. 24. Matayoshi, E. D.; Wang, G. T.; Krafft, G. A.; Erickson, J. Science 1990, 247, 954. 25. Jorissen, R. N.; Reddy, G. S.; Ali, A.; Altman, M. D.; Chellappan, S.; Anjum, S. G.; Tidor, B.; Schiffer, C. A.; Rana, T. M.; Gilson, M. K. J. Med. Chem. 2009, 52, 737.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel HIV-1 protease inhibitors using tertiary amine as P2-ligands Zhi-Heng Yang a, Xiao-Guang Bai a, Lei Zhou a, Ju-Xian Wang a, Hong-Tao Liu b, Yu-Cheng Wang a,⇑ a b
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China Department of Pharmacy, Hebei General Hospital, Hebei, Shijiazhuang 050051, PR China
a r t i c l e
i n f o
Article history: Received 6 January 2015 Revised 13 March 2015 Accepted 17 March 2015 Available online 24 March 2015
a b s t r a c t A series of tertiary amine derivatives exhibiting potent HIV-1 protease inhibiting properties were identified. These novel inhibitors were designed based on the structure of Darunavir with modification on the P2 and P20 position. This effort led to discovery of 35e and 38e, which exhibited excellent HIV-1 protease inhibition with IC50 values of 15 nM and 64 nM, respectively. Ó 2015 Elsevier Ltd. All rights reserved.
Keywords: HIV-1 protease Inhibitors HAART Darunavir
To date, the AIDS epidemic is still one of the most challenging problems.1,2 According to WHO reports, the number of people living with HIV has risen to 35.3 million, with 2.3 million new infections and 1.6 million AIDS-related deaths in 2012.3–6 Among many strategies to combat this disease, highly active antiretroviral therapy (HAART) containing at least one of HIV-1 protease inhibitors (PIs) was regarded as the most effective treatment for HIV infection.7–10 During the past 20 years, several HIV-1 PIs were discovered and widely used in the clinic, such as Saquinavir, Amprenavir, and Darunavir. However, drug resistance is still a major problem in the treatment of HIV/AIDS due to a high frequency mutation of the virus.11–13 Therefore, it is important to develop novel HIV-1 PIs with improved activity against multi-drug resistant variants. To develop new chemical entities against drug-resistant HIV, the strategy to design novel structures based on current approved drugs is an efficient way.14–16 Darunavir (Fig. 1, 1), approved by FDA in 2006, displayed an excellent antiviral activity against multidrug-resistant HIV-1 strains.17,18 A previous study showed that the binding potency was enhanced especially by the interaction between the oxygen in the fused ring of bis-THF and NH group in backbone of Asp29 and Asp30.19 While Amprenavir (2), contain-
⇑ Corresponding author. Tel./fax: +86 10 63165263. E-mail address: [email protected] (Y.-C. Wang). http://dx.doi.org/10.1016/j.bmcl.2015.03.047 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
ing single THF moiety, only exhibited weak O–H OH bonds with the main-chain amides of Asp29 and Asp30 in the S2 binding subsite.20 The tri-THF protease inhibitor, GRL-0519 (3), with excellent antiviral activity on drug resistant virus,21 showed the ability to make new interactions with the side chain of Asp30 in the wild type enzyme and Asn30 in the mutant variant. Thus, the P2 site substituent of the inhibitors has great relativity on the inhibitory activities. In this study, a series of PIs were designed based on the skeleton of 1, where the bis-THF moiety was replaced using scaffold-hopping strategy to identify new scaffolds. In this study, the scaffold was replaced based on the following strategies: (a) Split the bis-THF ring to two parts, tetrahydrofuran ring and alkane, which then were substituted by Ra and Rb, respectively (Fig. 2); (b) Ra imitates the P2 ligand of Lopinavir (4) and Nelfinavir (5), and Rb was substituted by acetamide structures. We envisioned that the new tertiary amine scaffold as the P2 ligand may interact strongly with enzyme backbone through the new flexible acetamide structure. Additionally, considering the modification in P2 site may alter the binding conformation of the whole designed compounds, both (RS)- and (SS)-enantiomers of novel inhibitors were synthesized and discussed. The synthesis of the tertiary amine intermediates 12a,b was outlined in Scheme 1. Protection of the 2-methyl-3-nitrophenol (6) with benzyl bromide in the presence of K2CO3 yielded benzyl derivative 7. Reduction of nitro group of 7, followed by coupling with ethyl chloroacetate gave compound 8. The compound 8 was further alkylated with bromoacetamides (9a,b) to obtain 10a, b.22
1881
Z.-H. Yang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1880–1883
O
H O
NH2
Ph
O O
O S N
N H
H
H
O
O
O
OH
NH 2
Ph
O N H
O S N
O O
O
N H
O
OH
O
Ph
O
O S N
O
OH
O 2 Amprenavir
1 Darunavir
O
H
Ph O
OH
H N
3 GRL-0519
N
N H
H N
NH HO O
Ph
OH
O S
4 Lopinavir
H
N H N H
O Ph
5 Nelfinavir
Figure 1. Structure of some marketed HIV PIs and GRL-0519.
H O O
O
O S N
N H
H
NH2
Ph
O
Removal of the benzyl group of 10a,b using 5% Pd-C and H2 in EtOH provided the corresponding phenol 11a,b, which were saponified with NaOH in MeOH/CH2Cl2 (1:3) afforded carboxylic acid derivatives 12a,b. The tertiary amine derivatives 12c–f were obtained using a similar strategy to the synthesis of 12a,b. As shown in Scheme 2, treatment of anilines 13 and 14 with ethyl chloroacetate followed by alkylation with bromoacetamides gave 11c–f. Saponification of 11c–f afforded the corresponding derivatives 12c–f. The synthetic route of designed PIs was illustrated in Scheme 3. Our synthesis was commenced with the commercial available epoxides 17 and 18. Treatment of epoxides 17 and 18 with isobutylamine followed by coupling with sulfonyl chloride affording sulfonamides 21–25 and 27.22 Subsequently, 25 and 27 were converted to hydroxymethylsulfonamide derivatives 26 and 28, respectively through deprotection of the acetates with K2CO3 and reduction of the corresponding aldehyde with NaBH4 in methanol.22 Finally, removal of the Boc protection of 21–24, 26, and 28 followed by the amidation reactions with 12a-f obtained tertiary amine derivatives of 35–37, 39, 41 and 42.23 The nitro group of compounds 37a–f and 39a–f was reduced using 5% Pd-C and H2 to provide 38a–f and 40a–f.22 The inhibition potency of the synthetic compounds against HIV1 protease was evaluated using a fluorescence resonance energy transfer (FRET) method.24,25 Indinavir and darunavir were used as positive control; and the results were presented in Tables 1 and 2, respectively. The data listed in Table 1 indicated that IC50 values of most of (RS)-enantiomeric compounds were in nanomolar. Whereas, the (RS)-enantiomeric compounds in Table 2
O
OH
Darunavir scaffold-hopping
Ra H O
N H
O
HO
NH2
Ph
O
O S N
O
OH
Rb
R
P2 R
a
b
N
Rc
Ph
O
P2'
O S
N H
N
O
OH (RS SS)
TM Ra =
HO O O
b
R =
N
N
O
O
Figure 2. Chemical structure of target molecular (TM).
HO
NO2
a
Bn
O
NO2
b,c
Bn
H N
O
O O
R3
Br
d
O 6
7
8
R3
R3
O Bn
O N
O
10a, 10b
9a, 9b
O
e O
R3 O N
HO
11a, 11b
O
f O
O N
HO
OH
12a, 12b a R 3 = morpholine b R3 = dimethylamine
Scheme 1. Reagents and conditions: (a) benzyl bromide, K2CO3, CH3CN, 80 °C, 3 h, 86%; (b) SnCl22H2O, EtOAc, 80 °C, 3.0 h, 62%; (c) AcONa, ethyl chloroacetate, EtOH, 80 °C, 6 h, 65%; (d) K2CO3, DMF, 90 °C, 1 h, 74% for 10a, 69% for 10b; (e) H2, Pd/C, EtOH, 30 psi, rt, 2.0 h, 89% for 11a, 76% for 11b; (f) MeOH/CH2Cl2 (1:3), NaOH, rt, 1 h, 68% for 12a, 63% for 12b.
1882
Z.-H. Yang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1880–1883 R3 NH 2 R'
O
H N
a R
13 R ' = 4-OMe 14 R ' = 2,6-di-Me
O
b
O
'
R3 O N
11c 11d 11e 11f
O N
O
R' 15 R ' = 4-OMe 16 R ' = 2,6-di-Me
O
c
R' = 4-OMe, R3 = morpholine R ' = 4-OMe, R 3 = dimethylamine R' = 2,6-di-Me, R 3 = morpholine R ' = 2,6-di-Me, R 3 = dimethylamine
R'
OH
12c 12d 12e 12f
R' = 4-OMe, R3 = morpholine R ' = 4-OMe, R 3 = dimethylamine R' = 2,6-di-Me, R 3 = morpholine R ' = 2,6-di-Me, R 3 = dimethylamine
Scheme 2. Reagents and conditions: (a) AcONa, ethyl chloroacetate, EtOH, 80 °C, 6 h, 62% for 15, 69% for 16; (b) 9a–b, K2CO3, DMF, 90 °C, 1 h, 56–72%; (c) MeOH/CH2Cl2 (1:3), NaOH, rt, 1 h, 54–71.0%.
Ph Ph Boc
O Boc
a N H
O
17 SS 18 RS
N H
NH
Cl
S
Boc
b
R1
OH 19 RS 20 SS
c
R1
R
N H
O S N
O
d 29 RS R1 = 4-OMe 30 SS R 1 = 4-OMe 31 RS R1 = 4-NO2 32 SS R 1 = 4-NO2 33 RS R1 = 4-OH 34 SS R 1 = 4-OH
R = Boc R=H
O
O N
OH
N H
O
OH
R3
R3
O
O S N
21 RS R1 = 4-OMe 22 SS R 1 = 4-OMe 23 RS R1 = 4-NO2 24 SS R 1 = 4-NO2 25 RS R1 = 4-di-CH(COCH 3) 2 26 RS R1 = 4-CH2 OH 27 SS R 1 = 4-di-CH(COCH 3 )2 28 SS R 1 = 4-CH 2OH
c
Ph
R1
Ph
O
R2
N
e R
OH
12a R 2 = 2-Me, 3-OH, R3 = morpholine 12b R2 = 2-Me, 3-OH, R 3 = dimethylamine 12c R 2 = 4-OMe, R3 = morpholine 12d R2 = 4-OMe, R 3 = dimethylamine 12e R 2 = 2,6-di-Me, R 3 = morpholine 12f R2 = 2,6-di-Me, R 3 = dimethylamine
2
f f
R1
Ph
O N H
O S
N
O
OH
35 RS a-f R 1 = 4-OMe 36 SS a-f R 1 = 4-OMe 37 RS a-f R 1 = 4-NO2 38 RS a-f R 1 = 4-NH2 39 SS a-f R 1 = 4-NO2 40 SS a-f R 1 = 4-NH 2 41 RS a-f R 1 = 4-CH2 OH 42 SS a-f R 1 = 4-CH 2OH
Scheme 3. Reagents and conditions: (a) i-BuNH2, CH3CN, 80 °C, 6 h, 83% for 19, 73% for 20; (b) THF, DIEA, DMAP, rt, 3 h, 74–82%; (c) (i) K2CO3, MeOH, rt, 1.5 h. (ii) NaBH4, MeOH, rt, 1 h, 86% for 26, 64% for 28; (d) CH2Cl2-CF3COOH (1:1), 3 h, 65.8–79.3%; (e) DMF, EDCHCl, HOBt, Et3N, rt, overnight, 45-73%; (f) H2, Pd/C, MeOH, 40 psi, rt, 1.5 h, 60– 78%.
exhibited dissatisfactory IC50 values that were in the micromolar levels. These results clearly showed that (RS)-enantiomer of hydroxyethylamine isostere exhibits more remarkable inhibition to HIV-1 protease, which is consistent with the enantiomer of darunavir. Thus, only the Structural–Activity Relationship (SAR) study of (RS)-enantiomer of novel compounds in Table 1 were discussed in the next. As a novel scaffold, we synthesized and tested series of analogues with variations at both the P2 and P20 positions. The first series of inhibitors, bearing a 4-methoxyphenyl at P20 site (35a– f), showed generally higher potency than the corresponding substituted with that of 4-aminophenyl (38a–f), 4-nitrophenyl (37a–f) and 4-hydroxymethylphenyl (41a–f). Compounds bearing 2,6dimethyl phenyl group at P2 position showed impressive potency to inhibit HIV-1 protease, especially with a morpholine (R3) substituent (35e, 37e, 38e, 41e). Among them, compounds 35e and 38e showed excellent activity to inhibit HIV-1 protease with IC50 values of 15 nM and 64 nM, respectively. Whereas, the presence of 4-methoxy or 2-methyl-3-hydroxy of the R2 substituent is
unsatisfactory with relatively low inhibitory activity. Besides, the compounds bearing a dimethylamine (R3) group showed higher potency than the corresponding substituted morpholine analogues (35b vs 35a, 35d vs 35c, 37b vs 37a, 37d vs 37c, 38b vs 38a, 38d vs 38c, 41b vs 41a, 41d vs 41c). However, it is interestingly to note an opposite result when R2 was the 2,6-dimethyl group (35e vs 35f, 37e vs 37f, 38e vs 38f, 41e vs 41f). It indicated that the substituents of R2 on the phenyl group as P2 ligand are crucial to the activity against the HIV-1 protease. In summary, a series of forty-eight novel HIV-1 PIs containing different P2 and P20 ligands were designed and synthesized. The enzyme activity assay showed the inhibitors with (R)-(hydroxyethylamino)-sulfonamide skeleton more effectively bind to the HIV-1 protease, and the substituents of R2 on the phenyl group are crucial to the activity. Compounds 35e and 38e showed excellent activities against HIV-1 protease with IC50 values of 15 nM and 64 nM, respectively. As a novel HIV-1 PI scaffold, further structural modifications of the tertiary amine moiety at P2 site are undergoing in our lab.
Z.-H. Yang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1880–1883 Table 1 Inhibitory of compounds against wild-type HIV-1 protease R3 O
O N
We are grateful to the financial support from the National Natural Science Foundation of China (Nos. 81302644, 81473099, and 81473098).
O N H
R2
Acknowledgment
R1
Ph N
S O
OH
Supplementary data
(RS)
a
Entry
R1
R2
R3
IC50a (lM)
35a 35b 35c 35d 35e 35f 37a 37b 37c 37d 37e 37f 38a 38b 38c 38d 38e 38f 41a 41b 41c 41d 41e 41f Indinavir Darunavir
4-OMe 4-OMe 4-OMe 4-OMe 4-OMe 4-OMe 4-NO2 4-NO2 4-NO2 4-NO2 4-NO2 4-NO2 4-NH2 4-NH2 4-NH2 4-NH2 4-NH2 4-NH2 4-CH2OH 4-CH2OH 4-CH2OH 4-CH2OH 4-CH2OH 4-CH2OH — —
2-Me, 3-OH 2-Me, 3-OH 4-OMe 4-OMe 2,6-Di-Me 2,6-Di-Me 2-Me, 3-OH 2-Me, 3-OH 4-OMe 4-OMe 2,6-Di-Me 2,6-Di-Me 2-Me, 3-OH 2-Me, 3-OH 4-OMe 4-OMe 2,6-Di-Me 2,6-Di-Me 2-Me, 3-OH 2-Me, 3-OH 4-OMe 4-OMe 2,6-Di-Me 2,6-Di-Me — —
Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine — —
1.38 ± 0.03 0.54 ± 0.006 0.29 ± 0.01 0.22 ± 0.01 0.015 ± 0.002 0.18 ± 0.006 6.67 ± 0.26 0.61 ± 0.02 1.02 ± 0.07 0.21 ± 0.01 0.073 ± 0.001 0.29 ± 0.02 2.73 ± 0.19 0.59 ± 0.01 1.36 ± 0.09 0.14 ± 0.006 0.064 ± 0.002 1.36 ± 0.09 1.21 ± 0.16 0.15 ± 0.00 0.89 ± 0.07 0.37 ± 0.01 0.073 ± 0.001 0.29 ± 0.02 0.88 ± 0.04 nM 0.061 ± 0.02 nM
Values are means of three independent experiments.
Table 2 Inhibitory of compounds against wild-type HIV-1 protease R3 O N R2
R1
Ph
O
O N H
N
S O
OH (SS)
a
1883
Entry
R1
R2
R3
IC50a (lM)
36a 36b 36c 36d 36e 36f 39a 39b 39c 39d 39e 39f 40a 40b 40c 40d 40e 40f 42a 42b 42c 42d 42e 42f Indinavir Darunavir
4-OMe 4-OMe 4-OMe 4-OMe 4-OMe 4-OMe 4-NO2 4-NO2 4-NO2 4-NO2 4-NO2 4-NO2 4-NH2 4-NH2 4-NH2 4-NH2 4-NH2 4-NH2 4-CH2OH 4-CH2OH 4-CH2OH 4-CH2OH 4-CH2OH 4-CH2OH — —
2-Me, 3-OH 2-Me, 3-OH 4-OMe 4-OMe 2,6-Di-Me 2,6-Di-Me 2-Me, 3-OH 2-Me, 3-OH 4-OMe 4-OMe 2,6-Di-Me 2,6-Di-Me 2-Me, 3-OH 2-Me, 3-OH 4-OMe 4-OMe 2,6-Di-Me 2,6-Di-Me 2-Me, 3-OH 2-Me, 3-OH 4-OMe 4-OMe 2,6-Di-Me 2,6-Di-Me — —
Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine Morpholine Dimethylamine — —
>20 lM 5.78 ± 0.16 >20 lM >20 lM 8.65 ± 0.25 >20 lM >20 lM 7.41 ± 0.07 19.21 ± 0.08 >20 lM 5.87 ± 0.05 5.88 ± 0.12 16.07 ± 0.24 5.92 ± 0.12 6.81 ± 0.20 6.96 ± 0.34 2.59 ± 0.06 0.49 ± 0.04 6.12 ± 0.16 >20 lM >20 lM 5.34 ± 0.59 8.94 ± 0.58 17.3 ± 1.11 0.88 ± 0.04 nM 0.061 ± 0.02 nM
Values are means of three independent experiments.
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