Potent HIV-1 protease inhibitors incorporating squaramide-derived P2 ligands: Design, synthesis, and biological evaluation

Journal Pre-Proof Potent HIV-1 protease inhibitors incorporating squaramide-derived P2 ligands: Design, synthesis, and biological evaluation Arun K. Ghosh, Jacqueline N. Williams, Satish Kovela, Jun Takayama, Hannah M. Simpson, D. Eric Walters, Shin-ichiro Hattori, Manabu Aoki, Hiroaki Mitsuya PII: DOI: Reference:

S0960-894X(19)30532-3 https://doi.org/10.1016/j.bmcl.2019.08.006 BMCL 26602

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

21 June 2019 31 July 2019 4 August 2019

Please cite this article as: Ghosh, A.K., Williams, J.N., Kovela, S., Takayama, J., Simpson, H.M., Eric Walters, D., Hattori, S-i., Aoki, M., Mitsuya, H., Potent HIV-1 protease inhibitors incorporating squaramide-derived P2 ligands: Design, synthesis, and biological evaluation, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/ 10.1016/j.bmcl.2019.08.006

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier Ltd.

JOURNAL PRE-PROOF

Bioorganic & Medicinal Chemistry Letters

F

Potent HIV-1 protease inhibitors incorporating squaramide-derived P2 ligands: Design, synthesis, and biological evaluation

O

O

Arun K. Ghosh, *,a Jacqueline N. Williams,a Satish Kovela,a Jun Takayama,a Hannah M. Simpson,a D. Eric Walters,b Shin-ichiro Hattori,c Manabu Aoki,c,d and Hiroaki Mitsuya c,d,e

PR

a Department of Chemistry and Department of Medicinal Chemistry, Purdue University, West Lafayette, IN 47907 (USA); bDepartment of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, IL 60064; cDepartment of Refractory Viral Infections, National Center for Global Health and Medicine, Shinjuku, Tokyo 162-8655 (Japan); dExperimental Retrovirology Section, HIV and AIDS Malignancy Branch National Cancer Institute, Bethesda, MD 20892 (USA); eDivision of Clinical Sciences, Kumamoto University Hospital, Kumamoto 860-8556 (Japan).

A BS T RA C T

Article history: Received Revised Accepted Available online

We describe the design, synthesis, and biological evaluation of novel HIV-1 protease inhibitors containing a squaramide-derived scaffold as the P2 ligand in combination with a (R)hydroxyethylamine sulfonamide isostere. Inhibitor 3h with an N-methyl-3-(R)aminotetrahydrofuranyl squaramide P2-ligand displayed an HIV-1 protease inhibitory Ki value of 0.51 nM. An energy minimized model of 3h revealed the major molecular interactions between HIV-1 protease active site and the tetrahydrofuranyl squaramide scaffold that may be responsible for its potent activity.

AL

2013 Elsevier Ltd. All rights reserved .

JO

U

R N

Keywords: HIV-1 protease Inhibitor Squaramide Synthesis Bis-tetrahydrofuran

PR

E-

A RT I C L E I N F O

 Corresponding author. Tel.: +1-765-494-5323; fax: +1-765-496-1612; e-mail: [email protected] (AKG)

JOURNAL PRE-PROOF

AL

PR

E-

PR

O

O

F

Human immunodeficiency virus (HIV) protease inhibitors (PIs) are a class of antiviral drugs that are widely used in combination with reverse transcriptase inhibitors for the treatment of patients with HIV-1 infection and AIDS.1,2 The combination antiretroviral therapies significantly improve life spans and reduce mortality and morbidity in patients with HIV-1 infection.3,4 Our research efforts towards the design and synthesis of PIs with non-peptidic features, has led to a range of PIs with potent activity, particularly against a panel of highly multidrug-resistant HIV-1 variants.5-7 One of these PIs is darunavir (1, Figure 1), which was approved by the FDA as a first line therapy.8,9 Darunavir exhibits a high genetic barrier to the development of drug-resistant viruses relative to other PIs.10,11 However, the emergence of darunavir resistance is reported.12,13 While PIs are effective, structurally novel PIs with reduced drug-related toxicity and side effects are critical to long-term success of combined antiretroviral therapy.14,15 In darunavir, we incorporated a stereochemically defined 3(R),3a(S),6a(R)-bis-tetrahydrofuranyl urethane as the P2-ligand on an (R)-(hydroxyethylamino)-4-aminosulfonamide scaffold.16,17 The bicyclic polyether template was specifically designed with a urethane functionality to promote hydrogen bonding with the backbone atoms of HIV-1 protease in the S2 subsite.18,19 Indeed, the Xray structural studies of darunavir HIV-1 protease complex revealed that darunavir formed a network of hydrogen bonds throughout the HIV-1 protease active site.20,21 Of particular interest, the P2 bis-THF ligand formed two strong hydrogen bonds with the backbone amide NHs of Asp29 and Asp30 in the S2-subsite. Furthermore, the urethane NH formed a strong hydrogen bond with the Gly27 carbonyl and the urethane carbonyl formed a water-mediated tetracoordinated hydrogen bonding interaction with one of the sulfonamide oxygens and amide NHs of Ile50 and Ile50' in the flaps. On the basis of these binding site interactions, we have

Figure 1. Structures of inhibitors 1, 2, 3a, and 3h.

JO

U

R N

subsequently designed numerous structurally intriguing PIs that showed very potent antiviral activity against highly multidrugresistant HIV-1 variants. Based upon the X-ray structure of darunavir-bound HIV-1 protease, we now speculate that a squaramide template can mimic the binding properties of the urethane derived PIs. Particularly, squaramide carbonyls and NHs can form donor and acceptor hydrogen bonds similar to the urethane functionality.22 Overall, a squaramide template retains a very rigid structure that is mainly planar.23 It is also resilient to nucleophilic attack and maintains good stability. Thus, the squaramide functionality has now been utilized in drug design and medicinal chemistry.24,25 We describe here our preliminary work on the design and synthesis of a new class of HIV-1 protease inhibitors which incorporate a variety of squaramide derivatives to interact with residues in the S2 subsite of the HIV-1 protease active site.

JOURNAL PRE-PROOF

Our general strategy for the synthesis of scaffolds for HIV-1 protease inhibitors 3a-l with squaramides is shown in Scheme 1. We planned to synthesize these inhibitors by coupling the amine of the (R)-hydroxyethylamine sulfonamide isostere 426 with commercially available diethyl squarate 5. The resulting mixed squarate can be coupled with amines to provide squaramide derivatives. Alternatively, mixed squarate containing cyclic ethers can be prepared first by reacting respective cyclic ether-derived alcohol or amines with diethyl squarate to provide various mixed squarate derivatives 7. Reaction of these mixed squarate derivatives with amine 4 would provide squaramide containing inhibitors. Scheme 1. General synthetic approach for inhibitors 3a-l

R N

AL

PR

E-

PR

O

O

F

The synthesis of inhibitors 3a-d is shown in Scheme 2. Dipeptide isosteric amine 4 was reacted with diethyl squarate 5 in the presence of diisopropylethylamine (DIPEA) in ethanol at 23 °C for 1 h to provide mixed squarate derivative 3a in near quantitative yield. For the synthesis of methoxyethoxy squarate derivative 3b, diethyl squarate was stirred with excess of methoxyethanol in THF

JO

U

at at 23 °C for 12 h to provide squarate derivative 6. Reaction of amine 4 with squarate derivative 6 at 23 °C for 12 h afforded 3b in 90% yield. Enantiomeric squarate derivatives 7a and 7b were prepared by reaction of commercially available 3(S)- and 3(R)tetrahydrofuran-3-ol with diethyl squarate in ethanol at reflux for 48 h to provide mixed squarate derivatives in low yield (18-28%). Reactions of dipeptide isosteric amine 4 with mixed squarates 7a and 7b in the presence of DIPEA in ethanol at 23 °C for 1 h provided inhibitors 3c and 3d in 75% yield.

E-

PR

O

O

F

JOURNAL PRE-PROOF

Scheme 2. Reagents and conditions: (a) DIPEA, EtOH, 23 °C, 1 h, 99%; (b) EtOH, reflux, 48 h, 18 -28%; (c) 4, DIPEA, EtOH, 23 °C, 1 h, 75%.

JO

U

R N

AL

PR

The synthesis of mixed squaramide inhibitors 3e and 3f containing 3(S)- and 3(R)- tetrahydrofuranamine is shown in Scheme 3. Reaction of mixed squarate derivative 3a with (S)-tetrahydrofuran-3-amine 927 in the presence of DIPEA in ethanol

Scheme 3. Reagents and conditions: (a) DIPEA, EtOH, 40 °C, 24 h, 60-62%; (a) Boc2O, Et3N, DMAP, CH 2Cl2, 24 h; (b) KHMDS, MeI, 23 °C, 16 h, 80% (2-steps); (c) TFA, CH2Cl2, 23 °C, 1 h; (d) 5, DIPEA, EtOH, 23 °C, 2 h, 97% (2-steps); (e) amine 4, DIPEA, 40 °C, 24 h, 72%.

at 40 °C for 24 h provided squaramide derivatives 3e in 62% yield. Similarly, reactions of (R)-tetrahydrofuran-3-amine furnished inhibitor 3f in 60% yield. For the synthesis of mixed squaramide derivatives 3g and 3h with N-methyl amide, optically active 3(S)amine 9 was protected as Boc-derivative by reaction with Boc2O and Et3N in the presence of a catalytic amount of DMAP in CH2Cl2 at 23 °C for 24 h. The resulting Boc-derivative was alkylated with MeI in the presence of KHMDS in THF at 23 °C for 16 h to provide methyl derivative 10 in 80% yield over 2-steps. Exposure of 10 to trifluoroacetic acid (TFA) in CH2Cl2 at 23 °C for 1 h provided the corresponding amine which was reacted with diethyl squarate to afford mixed squarate derivative 7c in 97% yield. Similarly, enantiomeric 3(R)-tetrahydrofuran-3-amine was converted to mixed squarate derivative 7d. Reactions of dipeptide isosteric amine 4

JOURNAL PRE-PROOF

R N

AL

PR

E-

PR

O

O

F

with mixed squarates 7c and 7d in the presence of DIPEA in ethanol at 40 °C for 24 h provided inhibitors 3g and 3h in 72% and 62 % yields, respectively. The synthesis of mixed squaramide derivatives containing various bis-tetrahydrofuranyl templates is shown in Scheme 4. Optically active (3R,3aS,6aR)-bis-tetrahydrofuran-3-ol 1117,28 was oxidized with a catalytic amount of TPAP in the presence of Nmethylmorpholine-N-oxide and 4Å molecular sieves in CH2Cl2 at 23 °C for 1 h to provide the corresponding ketone. Reaction of the resulting ketone with hydroxylamine hydrochloride in the presence of pyridine in ethanol at 23 °C for 30 min afforded oxime derivative 12 in 96% yield. Catalytic hydrogenation of oxime 12 over 10% Pd-C in MeOH at 23 °C under 60 psi hydrogen furnished amines 13 and 14 as a 2:1 mixture of diastereomers in 53% combined yield. Amine isomers were separated by silica gel chromatography and amine 13 was reacted with diethyl squarate in EtOH to furnish mixed squarate derivative 7e in 78% yield. Similarly, enantiomeric (3S,3aR,6aS)-bis-tetrahydrofuran-3-ol (ent-11) was converted to mixed squarate derivative 7f. For the synthesis of N-methyl derivative 7g, amine 13 was reacted with ethyl chloroformate in the presence of aqueous K2CO3 in CH2Cl2 at 23 °C to furnish the corresponding ethyl carbamate. Reduction of the resulting carbamate with lithium aluminum hydride (LAH) in THF at 45 °C furnished N-methylamine derivative 15 in 67% yield over 2-steps.29 Reaction of N-methyl amine 15 with diethyl squarate in ethanol furnished mixed squarate derivative 7g in 75% yield.

JO

U

Scheme 4: Reagents and conditions: (a) TPAP (cat), NMO, 4Å MS, CH2Cl2, 23 °C, 80%; (b) NH2OH•HCl, pyridine, EtOH, 23 °C, 96%; (c) H 2, 10% Pd-C, MeOH, 60 psi, 12 h, 53%; (d) 5, DIPEA, EtOH, 23 °C, (75-78%); (e) (EtO)COCl, aqueous K2CO3, CH2Cl2, 23 °C, 1 h, 97%; (f) LAH, THF, 45 °C, 2 h, 67%; (g) DIPEA, EtOH, 23 °C (50-75%).

JOURNAL PRE-PROOF

Similarly, enantiomeric amine ent-13 was converted to mixed squaramide 7h. Reactions of amine 4 with squaramide derivatives 7eTable 1. Enzyme inhibitory activity of inhibitors 3a-l. Entry

Inhibitor

463.3

2.

1957

O

F

1.

PR

O

3.

U

R N

AL

PR

E-

4.

JO

Ki (nM)

31.1

226.4

5.

6099

6.

199.9

7.

396.6

8.

0.51

9.

54.8

10.

5.6

11.

4.5

12.

201

aDarunavir

(1) exhibited Ki = 16 pM, antiviral IC50 = 3.2 nM

JOURNAL PRE-PROOF

JO

U

R N

AL

PR

E-

PR

O

O

F

7h in the presence of DIPEA in ethanol at 23 °C afforded inhibitors 3i-3l in good to excellent yields (50-75%). Based upon the X-ray structure of darunavir and its methoxy derivative bound HIV-1 protease, we speculated that an appropriately functionalized squaramide derivative could mimic key ligand-binding site interactions in the S2 subsite of HIV-1 protease.20,30 One of the interesting features of the X-ray structure of darunavir-bound HIV-1 protease is the formation of watermediated tetracoordinated hydrogen bonding interactions involving the urethane carbonyl oxygen and one of the P2'-sulfonamide oxygens with the amide NHs of Ile50 and Ile50' in the flap region. We assumed that one of the two carbonyl functionalities of the planar squaramide mimics the urethane-carbonyl functionality of darunavir. We have created a model of methoxyethyl squaramide derivative 3b based upon the X-ray structure of darunavir’s methoxy derivative 2 and HIV-1 protease complex.30 A preliminary model is shown in Figure 2. As can be seen, the squaramide carbonyl adjacent to the P1-amine group is suitably positioned to form watermediated tetracoordinated hydrogen bonding interactions as seen in the X-ray structures of numerous inhibitor HIV-1 protease complexes.31,32 Furthermore, the ethyl group can form van der Waals interactions with Ile47, Arg8, and Ile84 in the S2-subsite. Based upon these possible ligand-binding site interactions, we sought to investigate a focused group of squaramide derivatives bearing stereochemically defined cyclic ether heterocycles, like tetrahydrofurans and bis-tetrahydrofurans, shown in Table 1. We examined HIV-1 protease inhibitory activity of all compounds using an assay developed by Toth and Marshall.33 The structure and inhibitory activity of inhibitors are shown in Table 1. As shown, the ethyl squaramide derivative 3a showed HIV-1 protease inhibitory Ki of 463 nM (entry 1). We incorporated a methoxy group to interact with backbone atoms in the S2 site, however, the resulting compound 3b showed over 4-fold loss of protease inhibitory activity. We then incorporated constrained ring cycles containing an oxygen and investigated their binding properties. Not surprisingly, the active site of S2 subsite clearly shows stereochemical preference. Inhibitor 3c with a 3(S)-tetrahydrofuran showed an enhanced Ki value of 31 nM compared to derivative 3d with a 3(R)-tetrahydrofuran as the P2 ligand (entries 3 and 4). Presumably, the tetrahydrofuran ring oxygen with the 3(S)-configuration is involved in hydrogen bonding interactions with Asp29 and Asp30 backbone NHs in the active site. Interestingly, 3(S)-aminotetrahydrofuran derivatives 3e showed significant reduction of enzyme Ki-value over the alkoxy derivative 3c (entries 5). The corresponding 3(R)-derivative 3f is slightly more potent than inhibitor 3d. The N-methylation of compound 3e furnished compound 3g which showed 15-fold potency enhancement over compound 3e (entry 7). However, the corresponding 3(R)-N-methyl derivative 3h displayed very potent HIV-1 protease inhibitory activity with a Ki value of 0.5 nM (entry 8). The 3(R)-derivative 3h is over 775-fold more potent than the 3(S) derivative 3g. We have also examined suitability of various bis-tetrahydrofuran derivatives which were specifically designed to form hydrogen bonds with the backbone NHs of Asp29 and Asp30 in the S2 subsite. Inhibitor 3i with (3R,3aS,6aR)-bis-THF ligand showed enzyme Ki of 55 nM compared to 3(S)-tetrahydrofuran derivative 3e (entries 5 and 9). Inhibitor 3j with (3S,3aR,6aS)-amino bis-THF ligand showed nearly 10-fold better inhibitory activity with a Ki of 5.6 nM (entry 10). Interestingly, N-methylation of amino-bis-THF ligand of inhibitor 3i resulted in

O

O

F

JOURNAL PRE-PROOF

PR

Figure 2. Stereoview of an overlay of energy-minimized squaramide-derived inhibitor 3b (carbon chain, turquoise) with the X-ray structure of inhibitor 2 (carbon chain, magenta)-bound HIV-1 protease (PDB : 3I7E). Possible hydrogen bonds between the inhibitor and active site of HIV-1 protease are shown in dotted lines.

JO

U

R N

AL

PR

E-

N-methyl derivative 3k which showed over 10-fold improvement of enzyme affinity compared to 3i (entry 11). The corresponding enantiomeric N-methyl inhibitor 3l is nearly 35-fold less potent than inhibitor 3j. As can be seen in Table 1, a number of inhibitors, particularly compounds 3h, 3j, and 3k, show potent enzyme inhibitory activity and they are expected to exhibit potent antiviral activity. We have then determined antiviral activity of all squaramide derivatives listed in Table 1 using our previously published assay protocol using MT-4 cells exposed to HIV-1NL4-3.34 However, none of these squaramide derivatives show any appreciable antiviral activity (IC50 > 1 μM). The lack of antiviral activity of inhibitors, particularly for compounds 3h, 3j, and 3k, is not clear. It may be due to low solubility and high protein binding properties of squaramide derivatives.35 We are further investigating the reason for the poor antiviral activity. To obtain molecular insight into the specific ligand-binding site interactions, we created an energy-minimized active model of inhibitor 3h based upon the X-ray structure of darunavair derivate 2-bound HIV-1 protease.20,30 A stereoview of the model is shown in Figure 3. Modeling was carried out using Molecular Operating Environment, version 2019.0101 (Chemical Computing Group, Montreal) and the MMFF94 forcefield.36 Inhibitor 3h was modeled using PDB structure 3I7E as the starting point.30 The structure was minimized, solvated in a box of explicit water molecules, re-minimized, and then subjected to several picoseconds of molecular dynamics simulation under NPA conditions at 300K, with rigid water molecules and 2 femtosecond timesteps. Representative configuration was selected and minimized to produce the final structures. As it appears in Figure 3, one of the squaramide carbonyl oxygens can form the water-mediated tetracoordinated hydrogen bonding interactions similar to the reported X-ray structures of previous inhibitor-bound HIV-1 protease.30,31 Furthermore, it seems that the stereochemistry of the tetrahydrofuran ring is important for the ring oxygen to form hydrogen bonds with the backbone amide NHs of Asp29 and Asp30. The corresponding inhibitor 3g with

Figure 3. Stereoview of the model of inhibitor 3h (green carbon chain) with HIV-1 protease. Possible hydrogen bonds between the inhibitor and active site of HIV-1 protease are shown in black dotted lines.

JOURNAL PRE-PROOF

O

O

F

3(S)-tetrahydrofuran ring does not make these key interactions with Asp29 and Asp30. This may explain the reason for potent HIV1 inhibitory activity for inhibitor 3h. In summary, we have designed a series of novel HIV-1 protease inhibitors incorporating mixed squaramide functionalities to link various cyclic ether-derived P2-ligands and a hydroxyethylamine isostere. We specifically designed squaramide derivatives to replace the urethane functionality with a planar squaramide template to form hydrogen bonds and promote van der Waals interactions with residues in the S2 subsite. We have particularly incorporated cyclic ether derived heterocycles such as tetrahydrofuran and bistetrahydrofurans as the P2 ligands. A number of compounds showed very potent enzyme inhibitory activity, however, these compounds are not potent in antiviral assay. Inhibitor 3h with a 3(R)-N-methyltetrahydrofuran as the P2 ligand displayed the most potent enzyme inhibitory activity with a Ki value of 0.5 nM. Inhibitors 3j and 3k containing amino-bis-THF ligands also showed single digit nanomolar enzyme inhibitory activity. To obtain molecular insights into the ligand-binding site interactions, we have created an energy-minimized active model of inhibitor 3h. These models suggested that one of the squaramide carbonyl oxygens forms water-mediated tetracoordinated hydrogen bonds similar to the urethane carbonyl of darunavir. Furthermore, both amino-THF and amino-bis-THF ligands form hydrogen bonds with the backbone residues in the S2 subsite. These preliminary results are being utilized for further optimization of potency and inhibitor properties.

PR

Acknowledgement: This research was supported by the National Institutes of Health (Grant GM53386). The authors would like to thank the Purdue University Center for Cancer Research, which supports the shared NMR and mass spectrometry facilities. Supplementary data Supplementary data associated with this article can be found in the online version.

10. 11. 12. 13. 14. 15. 16.

JO

U

17. 18. 19. 20.

PR

8. 9.

AL

6. 7.

Ghosh, A. K.; Osswald, H. L.; Prato, G. J. Med. Chem. 2016, 59, 5172-5208. Esté, J. A.; Cihlar, T. C. Antivir. Res. 2010, 85, 25-33. Cohen, M. S.; Chen, Y.Q.; McCauley, M. N. Engl. J. Med. 2011, 365, 493-505. Diffenbach, C. W.; Fauci, A. S. Ann Intern Med. 2011, 154, 766-771. Ghosh, A. K.; Rao, K. V.; Nyalapatla, P. R.; Kovela, S.; Brindisi, M.; Osswald, H. L.; Reddy, B. S.; Agniswamy, J.; Wang, Y.-F.; Aoki, M.; Hattori, S.-i.; Weber, I. T.; Mitsuya, H. ChemMedChem 2018, 13, 803-815. Ghosh, A. K. J. Med. Chem. 2009, 52, 2163-2176. Ghosh, A. K.; Chapsal, B. D. Design of the anti-HIV protease inhibitor darunavir. In ‘From Introduction to Biological and Small Molecule Drug Research and Development’ Ed. Ganellin, C. R.; Roberts, S. M.; Jefferis, R. 2013, 355-384. Ghosh, A. K.; Dawson, Z. L.; Mitsuya, H. Bioorg. Med. Chem. 2007, 15, 756-7580. De Meyer, S.; Lathouwers, E.; Dierynck, I.; De Paepe, E.; Van Baelen, B.; Vangeneugden, T.; Spinosa-Guzman, S.; Lefebvre, E.; Picchio, G.; de Béthune, M. P. AIDS 2009, 23, 1829–1840. Koh, Y.; Matsumi, S.; Das, D.; Amano, M.; Davis, D. A.; Li, J.; Leschenko, S.; Baldridge, A.; Shioda, T.; Yarchoan, R.; Ghosh , A. K.; Mitsuya, H. J. Biol. Chem. 2007. 282, 28709–28720. Hayashi, H.; Takamune, N.; Nirasawa, T.; Aoki, M.; Morishita, Y.; Das, D.; Koh, Y.; Ghosh, A. K.; Misumi, S.; Mitsuya, H. Proc. Natl. Acad. Sci. U. S. A. 2007 111, 12234–12239. Mitsuya, Y.; Liu, T. F.; Rhee, S. Y.; Fessel, W. J.; Shafer, R. W. J. Infect. Dis. 2007, 196, 1177–1179. Tang, M. W.; Shafer, R. W. Drugs 2012, 72, e1– e25. Pennigs, P. S. Infect. Dis. Rep. 2013, 5, 21-25. Wilhelmsson, P.; Lindgreen, P. E. The Lancet. 2016, 16, 512-513. Ghosh, A. K.; Chapsal, B.; Mitsuya, H. Darunavir, a New PI with Dual Mechanism: From a Novel Drug Design Concept to New Hope Against Drug-Resistant HIV. In ‘Aspartic Acid Proteases as Therapeutic Targets’ Ed. Ghosh, A. K. Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, 2010, 205-243. Ghosh, A. K.; Sridhar, P. R.; Kumaragurubaran, N.; Koh, Y.; Weber, I. T.; Mitsuya, H. ChemMedChem 2006, 1, 939-950. Ghosh, A. K.; Anderson, D. D.; Weber, I. T.; Mitsuya, H Angew. Chem. Int. Ed. 2012, 51, 1778-1802. Ghosh, A. K.; Chapsal, B.; Weber, I. T.; Mitsuya, H. Acc. Chem. Res. 2008, 41, 78-86. Y. Tie, P. I. Boross, Y.-F. Wang, L. Gaddis, A. K. Hussian, S. Leshchenko, A. K. Ghosh, J. M. Louis, R. W. Harrison, I. T. Weber, J. Mol. Biol. 2004, 338, 341–352. Kovalevsky, A. Y.; Liu, F.; Leshchenko, S.; Ghosh, A. K.; Louis, J. M.; Harrison, R. W.; Weber, I. T., J. Mol. Biol. 2006, 363, 161-173. Ian Storer, R.; Aciro, C.; Jones, L. H., Chem. Soc. Rev. 2011, 40, 2330-2346. Kijima, H.; Isobe, Y.; Kiuchi, Y.; Kajita, S.; Saito, T.; Fukushima, K.; Sato, M.; Kimura, M.; Kaneda, Y.; Akashi, T., Effect of a histamine H2receptor antagonist pibutidine hydrochloride on the healing of acetic acid-induced gastric ulcer. 1999; 27, p 61-68. (a) Storer, R. I.; Aciro, C.; Jones, L. H.; Chem. Soc. Rev. 2011, 40, 2330-2346; (b) Amendola, V.; Bergamaschi, G.; Boiocchi, M.; Fabbrizzi, L.; Milani, M. Chem. Eur. J. 2010, 16, 4368-4380. Salchow, K.; Bond, M.; Evans, S.; Press, N.; Charlton, S.; Hunt, P.; Bradley, M., Brit. J. Pharmacol. 2010, 159, 1429-1439. Ghosh, A. K., Leshchenko, S., Noetzel, M. J. Org. Chem. 2004, 69, 7822-7829 Ramanujam, R.; Ganjihal, S.; Kalyanam, N.; Majeed, M. Tetrahedron: Asymmetry 2013, 24, 663-668. Ghosh, A. K.; Chen, Y. Tetrahedron Lett. 1995, 36, 505-508. Webb, R. R.; Venuti, M. C.; Eigenbrot, C. J. Org. Chem. 1991, 56, 4706-4713. Ghosh, A. K; Kulkarni, S.; Anderson, D. D.; Hong, L.; Baldridge, A.; Wang, Y. F.; Chumanevich, A. A.; Kovalevsky, A. Y.; Tojo, Y.; Koh, Y.; Tang, J.; Weber, I. T.; Mitsuya, H. J. Med. Chem. 2009, 52, 7689-7705. Wlodawer, A. Annu. Rev. Med. 2002, 53, 595–614. Pokorná, J.; Machala, L.; Řezáčová, P.; Konvalinka, J. Viruses 2009, 1, 1209-1239;

R N

1. 2. 3. 4. 5.

E-

References and notes

21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

JOURNAL PRE-PROOF

33. Toth, M. V.; Marshall, G. R. Protease. Int. J. Pept. Protein Res. 1990, 36, 544-550. 34. Koh, Y.; Amano, M.; Towata, T.; Danish, M.; Leshchenko-Yashchuk, S.; Das, D.; Nakayama, M.; Tojo, Y.; Ghosh, A. K.; Mitsuya, H. J. Virol. 2010, 84, 11961-11969.

35. McCleland, B. W.; Davis, R. S.; Palovich, M. R.; Widdowson, K. L.; Werner, M. L.; Burman, M.; Foley, J. J.; Schmidt, D. B.; Sarau, H. M.;

JO

U

R N

AL

PR

E-

PR

O

O

F

36.

Rogers, M.; Salyers, K. L.; Gorycki, P. D.; Roethke, T. J.; Stelman, G. J.; Azzarano, L. M.; Ward, K. W.; Busch-Petersen, J. Bioorg. & Med. Chem. Lett. 2007, 17, 1713-1717. Halgren, T. A. J. Comp. Chem. 1996, 17, 490-641.

JOURNAL PRE-PROOF

Graphical Abstract To create your abstract, type over the instructions in the template box below. Fonts or abstract dimensions should not be changed or altered.

Leave this area blank for abstract info.

F

Potent HIV-1 protease inhibitors incorporating squaramide-derived P2 ligands: Design, synthesis, and biological evaluation

O

Arun K. Ghosh,*,a Jacqueline N. Williams,a Satish Kovela,a Jun Takayama,a Hannah M. Simpson,a D. Eric Walters,b Shinichiro Hattori,c Manabu Aoki,c,d and Hiroaki Mitsuya c,d,e

JO

U

R N

AL

PR

E-

PR

O

a Department of Chemistry and Department of Medicinal Chemistry, Purdue University, West Lafayette, IN 47907 (USA); bDepartment of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, IL 60064; cDepartment of Refractory Viral Infections, National Center for Global Health and Medicine, Shinjuku, Tokyo 162-8655 (Japan); dExperimental Retrovirology Section, HIV and AIDS Malignancy Branch National Cancer Institute, Bethesda, MD 20892 (USA); eDivision of Clinical Sciences, Kumamoto University Hospital, Kumamoto 860-8556 (Japan).