Identification of Highly Potent Human Immunodeficiency Virus Type-1

Subscriber access provided by Kaohsiung Medical University

Article

Identification of Highly Potent Human Immunodeficiency Virus Type-1 Protease Inhibitors against Lopinavir and Darunavir Resistant Viruses from AllophenylnorstatineBased Peptidomimetics with P2 Tetrahydrofuranylglycine Koushi Hidaka, Tooru Kimura, Rajesh Sankaranarayanan, Jun Wang, Keith F. McDaniel, Dale J. Kempf, Masanori Kameoka, Motoyasu Adachi, Ryota Kuroki, Jeffrey-Tri Nguyen, Yoshio Hayashi, and Yoshiaki Kiso J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01709 • Publication Date (Web): 31 May 2018 Downloaded from http://pubs.acs.org on May 31, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

Identification of Highly Potent Human Immunodeficiency Virus Type-1 Protease Inhibitors against Lopinavir and Darunavir Resistant Viruses from Allophenylnorstatine-Based Peptidomimetics with P2 Tetrahydrofuranylglycine Koushi Hidaka,a Tooru Kimura,b Rajesh Sankaranarayanan,b Jun Wang,b Keith F. McDaniel,c Dale J. Kempf,c Masanori Kameoka,d Motoyasu Adachi,e Ryota Kuroki,f∆ Jeffrey-Tri Nguyen,b Yoshio Hayashi,g and Yoshiaki Kisoh* a

Laboratory of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Kobe Gakuin University,

Kobe 650-8586, Japan; bDepartment of Medicinal Chemistry, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan; cGlobal Pharmaceutical Research and Development, AbbVie, North Chicago, Illinois 60064, U.S.A.; dDepartment of International Health, Kobe University Graduate School of Health Sciences, Kobe 654-0142, Japan, eQuantum Beam Science Drectorate, National Institutes for Quantum and Radiological Science and Technology, Tokai, Ibaraki, 319-1106, Japan, fQuantum Beam Science Center, Japan Atomic Energy Agency, Tokai, Ibaraki, 319-1195, Japan gDepartment of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan; hLaboratory of Peptide Sciences, Nagahama Institute of Bio-Science and Technology, Nagahama 526-0829, Japan

ACS Paragon Plus Environment

1

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 49

Abstract

The emergence of drug-resistant HIV from a wide-spread anti-viral chemotherapy targeting HIV protease in the past decades is unavoidable and provides a challenge to develop alternative inhibitors. We synthesized a series of allophenylnorstatine-based peptidomimetics with various P3, P2 and P2´ moieties. The derivatives with P2 tetrahydrofuranylglycine (Thfg) were found to be potent against wild type HIV-1 protease and the virus, leading to a highly potent compound 21f (KNI-1657) against lopinavir/ritonavir- or darunavir-resistant strains. Co-crystal structures of 21f and the wild-type protease revealed numerous key hydrogen bonding interactions with Thfg. These results suggest that the strategy to design allophenylnorstatine-based peptidomimetics combined with Thfg residue would be promising for generating candidates to overcome multi-drug resistance.

KEYWORDS: allophenylnorstatine, anti-HIV agent, drug resistance, HIV protease inhibitor, HIV protease variant.

Introduction In the past decades, antiretroviral therapy for human immunodeficiency virus type-1 (HIV-1) infection using nucleoside and non-nucleoside reverse transcriptase inhibitors, and protease inhibitors has advanced to become convenient for patients because of the once-daily dosing regimen and reduced numbers of orally administered tablets and capsules.1 Recent approvals of entry and integrase inhibitors provide new options for patients struggling with drug resistance.2,3 HIV/AIDS has been, accordingly, recognized as a controllable chronic illness.4 Despite the therapeutic success, drug resistance and side effects still remain a great concern in long-term treatment. Resistance to HIV protease inhibitors is one of the problems because of the market for generic drugs.5 The situation is complicated by the existence


2

Page 3 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of other HIV-1 subtypes and circular recombinant forms because of the altered susceptibility to drugs.6,7 Therefore, the development of alternative HIV protease inhibitors is still a challenge for researchers.8 Progress on recently approved HIV protease inhibitors led to drugs that possess a high genetic barrier against the virus. Lopinavir, as an example, requires more than 6 mutations to lose ten times its antiviral activity in vitro.9 Another example, darunavir, is now the first option for therapeutic regimens, and would take more than one hundred days to induce drug resistance.10 Nevertheless, a research group of Abbott Laboratories has generated a lopinavir/ritonavir resistant HIV strain, A17.11 The mutant clone displayed approximately 50-fold resistance to lopinavir. Although the mutation sites were identified in the protease coding region, detailed study on the decreasing activity was not reported. Koh et al. reported high resistance to darunavir using mixed clones from four patients with therapeutic failure.12 A combination of the four mutations surprisingly attenuated the activity approximately 100-fold. Overcoming these resistances is the target of developing next-generation inhibitors. Allophenylnorstatine [Apns: (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid] is an unnatural αhydroxy-β-amino acid residue that contains a hydroxymethylcarbonyl (HMC) group as a transition-state isostere to act as a P1 residue in peptidic HIV protease inhibitors.13-15 The ideal binding interactions of HMC isostere with two catalytic Asp residues of HIV-1 protease have been disclosed by neutron crystallography16 in addition to former X-ray crystallography17 and NMR analyses.18 Previously developed Apns-based compounds 1 and 2, known as KNI-272 and KNI-764, respectively, are highly potent against the protease and the virus, and both were tested orally in clinical studies (Figure 1).19,20 Modifications of these inhibitors were extensively studied for improving anti-viral activity against drug resistant strains, water-solubility, and pharmacokinetic profile.21-24 Their effective potency against resistances was unfortunately limited. To overcome the multidrug-resistant mutations, we planned a series of Apns-based tripeptidic compounds with a combination of residues and terminal caps that were selected from previous experiments exhibiting high protease inhibitory potency. Especially, unnatural P2 residue tetrahydrofuranylglycine (Thfg), originally designed by Ghosh et al25 and further exploited in


3


Page 4 of 49

urethanes in amprenavir26 and darunavir,27 is newly applied to the Apns-based derivatives, leading to compounds with highly potent anti-HIV activity, even against lopinavir/r- or darunavir-resistant strains.

[Please insert Figure 1.]

Ligand selections for combinational modifications The general structure of the HIV protease inhibitor synthesized in this study is depicted in Figure 2. Our combinational modifications were intended to be structurally focused on yielding high potency against the protease. First, Apns was chosen as the P1 residue from a previous comparison with phenylnorstatine.15 (R)-5,5-dimethylthiazolidine-4-carboxylic acid (Dmt) was also selected as the P1´ residue from the reported proline-based modifications.19 Three residues were selected to optimize the P2 residue. Asn was known as the most effective for protease inhibition among many natural and nonnatural amino acid residues. Val was also selected with a little less potency than Asn. In addition to the above branched residues, Thfg, which is a hybrid structure of Asn and Val, was applied. The P3 cap was searched among our results from previous structure-activity relationship studies.24 Three types of phenoxyacetyl moieties, including 5-isoquinolinyloxyacetyl in compound 1 and the oxygen-mimicking chromonylcarbonyl and bezofurancarbonyl groups, were chosen. The seven P2´ amide residues were selected to include β-methallylamide from a previous study on highly potent dipeptidic inhibitors such as KNI-1689.22

[Please insert Figure 2.]

Chemistry The preparation of Thfg was performed as reported28 (Scheme 1). (R)-3-Hydroxytetrahydrofuran (3) was converted to the substituted malonate 4, and then decarboxylated with cuprous oxide to generate ACS Paragon Plus Environment

4

Page 5 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


tetrahydrofuranylacetic acid 5. The acid was coupled to (S)-Evans auxiliary, and then subjected to stereoselective

and

electrophilic

azidation

using

trisyl

azide

to

afford

(2S,3’R)-

azidotetrahydrofuranylacetate 7. The intermediate was subjected to reduction using SnCl2 in ethanol and in situ Boc protection to give a mixture of 8a and 8b in 45 and 30% yields, respectively. Both were hydrolyzed

to

give

(2S,3’R)-Βοc-Thfg-OH

(9).

In

the

case

of

(2S,3’S)-isomer,

(S)-3-

hydroxytetrahydrofuran was used as the starting material and transformed in a manner similar to (R)form, and the intermediate azide 11 with Evans auxiliary attached was hydrolyzed using LiOH to yield (2S,3’S)-2-azidotetrahydrofuranylacetic acid 12. P3 phenoxyacetic acid analogues were synthesized from the phenol derivatives as described before.24 Scheme 2 illustrates the synthesis of Thfg-containing inhibitors. 15a and 15b were obtained from a previously reported intermediate, Boc-protected dipeptide carboxamides 13.20 The deprotection of the Boc group using HCl-dioxane gave the amine component to react with 9 using EDC–HOBt coupling reagents, and the subsequent deprotection and BOP coupling with quinolinecarboxylic acid derivatives afforded the desired compounds. The (2S,3’S)-Thfg isomer was incorporated as an azido form to give the intermediate 16. The reduction to amine and BOP coupling with the P3 carboxylic acids gave compounds 17a and 17b in good yields. The intermediate 14 was deprotected and used to combine 5isoquinolinyloxyacetic acid to give compound 18. Preparation of a series of P3 modifications was started from P2´-dimethylbenzyl intermediate 19 (Scheme 3). The Boc group was removed to couple with (2S,3’R)-Βοc-Thfg-OH, and then removal of the Boc group and condensations with six types of P3 carboxylic acids afforded compounds 21a-f. Similarly, compounds 24a-f were synthesized from P2´-intermediates except for the last BOP coupling procedure with P3 3-(dimethylamino)-phenoxyacetic acid as shown in Scheme 4. Scheme 5 describes the P2 derivative synthesis using Boc-Asn-OH and Boc-Val-OH to couple with the amine generated from 19. The subsequent deprotection and BOP coupling with benzofurancarboxylic acid derivatives gave the compounds 27a-d.


5


Page 6 of 49

Results and Discussion To test the matching of Thfg residue with Apns-based peptidomimetics, we first combined a P3-P2 structure

based

on

Merck’s

compounds,25

quinolinecarbonyl-Thfg,

with

Apns-Dmt-(2-

methyl)benzylamide of 2. Compound 15a with P2 (2S,3’R)-Thfg exhibited highly potent HIV-1 protease inhibitory activity, 96% inhibition at 1 nM inhibitor concentration (Table 1). As expected from the results in Merck’s report, compound 17a with P2 (2S,3’S)-Thfg was less potent than compound 15a. The EC50 value of compound 15a was less than 3 nM. The anti-HIV activity of 17a was weak (26 nM), in agreement with its protease inhibition. The activities of these Thfg-containing derivatives were relatively potent compared with the parent compounds 1 and 2 (176 and 60 nM, respectively). Replacement of P3 quinolinecarbonyl with quinoxalinecarbonyl resulted in a slight increase in protease inhibition. An improvement was also observed in the anti-HIV activity under 50% human serum. Additional aromatic nitrogen may increase the basicity affecting the cellular membrane permeability. These successful results on the combination with Thfg urged us to optimize the P3 position.

[Please insert Table 1.]

Selection of the 5-isoquinolinyloxyacetyl moiety was derived from inhibitor 1. The resultant compound 18 exhibited sufficient HIV-1 protease inhibition and anti-HIV activity with EC50 of 21 nM (Table 2). However, the activity under 50% human serum was dramatically attenuated to 4.35 µM. We shifted to use the P2´ structure with an additional o-methyl group, that is 2,6-dimethylbenzylamide, which is the most favorable P2´ for enzyme inhibition among the previously reported dipeptidic inhibitors.22 Compound 21a exhibited 98% enzyme inhibition and 60 nM of anti-HIV activity. Interestingly, the addition of serum slightly affected the activity with a serum binding effect of 2.4. Replacement with 3-(phenylamino)phenoxyacetyl (21b) increased the anti-HIV activity up to 5 nM. A minor variant with the dimethylamino moiety in compound 21c improved anti-HIV inhibition (

Identification of Highly Potent Human Immunodeficiency Virus Type-1

Recommend Documents