Identification of Highly Potent Human Immunodeficiency Virus Type-1 Protease Inhibitors against Lopinavir and Darunavir Resistant Viruses from Allophenylnorstatine-Based Peptidomimetics with P2 Tetrahydrofuranylglycine

Article abstract of DOI:10.1021/acs.jmedchem.7b01709

The emergence of drug-resistant HIV from a widespread antiviral 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 peptid

Full text of DOI:10.1021/acs.jmedchem.7b01709

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Article
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, 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
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Identification of Highly Potent Human
Immunodeficiency Virus Typeꢀ1 Protease Inhibitors
against Lopinavir and Darunavir Resistant Viruses from
AllophenylnorstatineꢀBased Peptidomimetics with P2
Tetrahydrofuranylglycine
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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,fꢀ JeffreyꢁTri Nguyen,b Yoshio Hayashi,g
and Yoshiaki Kisoh*
aLaboratory of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Kobe Gakuin University,
Kobe 650ꢀ8586, Japan; bDepartment of Medicinal Chemistry, Kyoto Pharmaceutical University, Kyoto
607ꢀ8412, 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 654ꢀ0142, Japan, eQuantum Beam Science Drectorate, National Institutes for Quantum
and Radiological Science and Technology, Tokai, Ibaraki, 319ꢀ1106, Japan, fQuantum Beam Science
Center, Japan Atomic Energy Agency, Tokai, Ibaraki, 319ꢀ1195, Japan gDepartment of Medicinal
Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192ꢀ0392, Japan; hLaboratory of
Peptide Sciences, Nagahama Institute of BioꢀScience and Technology, Nagahama 526ꢀ0829, Japan
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Abstract
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The emergence of drugꢀresistant HIV from a wideꢀspread antiꢀviral chemotherapy targeting HIV
protease in the past decades is unavoidable and provides a challenge to develop alternative inhibitors.
We synthesized a series of allophenylnorstatineꢀbased peptidomimetics with various P3, P2 and P2´
moieties. The derivatives with P2 tetrahydrofuranylglycine (Thfg) were found to be potent against wild
type HIVꢀ1 protease and the virus, leading to a highly potent compound 21f (KNIꢀ1657) against
lopinavir/ritonavirꢀ or darunavirꢀresistant strains. Coꢀcrystal structures of 21f and the wildꢀtype protease
revealed numerous key hydrogen bonding interactions with Thfg. These results suggest that the strategy
to design allophenylnorstatineꢀbased peptidomimetics combined with Thfg residue would be promising
for generating candidates to overcome multiꢀdrug resistance.
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KEYWORDS: allophenylnorstatine, antiꢀHIV agent, drug resistance, HIV protease inhibitor, HIV
protease variant.
Introduction
In the past decades, antiretroviral therapy for human immunodeficiency virus typeꢀ1 (HIVꢀ1) infection
using nucleoside and nonꢀnucleoside reverse transcriptase inhibitors, and protease inhibitors has
advanced to become convenient for patients because of the onceꢀdaily 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 longꢀterm 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
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of other HIVꢀ1 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 50ꢀfold 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 100ꢀfold.
Overcoming these resistances is the target of developing nextꢀgeneration inhibitors.
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Allophenylnorstatine [Apns: (2S,3S)ꢀ3ꢀaminoꢀ2ꢀhydroxyꢀ4ꢀphenylbutyric acid] is an unnatural αꢀ
hydroxyꢀβꢀamino acid residue that contains a hydroxymethylcarbonyl (HMC) group as a transitionꢀstate
isostere to act as a P1 residue in peptidic HIV protease inhibitors.13ꢀ15 The ideal binding interactions of
HMC isostere with two catalytic Asp residues of HIVꢀ1 protease have been disclosed by neutron
crystallography16 in addition to former Xꢀray crystallography17 and NMR analyses.18 Previously
developed Apnsꢀbased compounds 1 and 2, known as KNIꢀ272 and KNIꢀ764, 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 antiꢀviral activity against drug
resistant strains, waterꢀsolubility, and pharmacokinetic profile.21ꢀ24 Their effective potency against
resistances was unfortunately limited. To overcome the multidrugꢀresistant mutations, we planned a
series of Apnsꢀbased 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
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urethanes in amprenavir26 and darunavir,27 is newly applied to the Apnsꢀbased derivatives, leading to
compounds with highly potent antiꢀHIV activity, even against lopinavir/rꢀ or darunavirꢀresistant strains.
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[Please insert Figure 1.]
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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)ꢀ5,5ꢀdimethylthiazolidineꢀ4ꢀcarboxylic acid (Dmt) was also selected as the P1´
residue from the reported prolineꢀbased 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 nonꢀ
natural 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 structureꢀactivity relationship studies.24 Three types of
phenoxyacetyl moieties, including 5ꢀisoquinolinyloxyacetyl in compound 1 and the oxygenꢀmimicking
chromonylcarbonyl and bezofurancarbonyl groups, were chosen. The seven P2´ amide residues were
selected to include βꢀmethallylamide from a previous study on highly potent dipeptidic inhibitors such
as KNIꢀ1689.22
[Please insert Figure 2.]
Chemistry
The preparation of Thfg was performed as reported28 (Scheme 1). (R)ꢀ3ꢀHydroxytetrahydrofuran (3)
was converted to the substituted malonate 4, and then decarboxylated with cuprous oxide to generate
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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)ꢀΒοcꢀThfgꢀOH (9). In the case of (2S,3’S)ꢀisomer, (S)ꢀ3ꢀ
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)ꢀ2ꢀazidotetrahydrofuranylacetic acid 12.
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P3 phenoxyacetic acid analogues were synthesized from the phenol derivatives as described before.24
Scheme 2 illustrates the synthesis of Thfgꢀcontaining inhibitors. 15a and 15b were obtained from a
previously reported intermediate, Bocꢀprotected dipeptide carboxamides 13.20 The deprotection of the
Boc group using HClꢀdioxane 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 5ꢀ
isoquinolinyloxyacetic 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)ꢀΒοcꢀThfgꢀOH, 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 3ꢀ(dimethylamino)ꢀphenoxyacetic acid as shown in Scheme 4. Scheme 5 describes the
P2 derivative synthesis using BocꢀAsnꢀOH and BocꢀValꢀOH to couple with the amine generated from 19.
The subsequent deprotection and BOP coupling with benzofurancarboxylic acid derivatives gave the
compounds 27a-d.
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Results and Discussion
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To test the matching of Thfg residue with Apnsꢀbased peptidomimetics, we first combined a P3ꢀP2
structure based on Merck’s compounds,25 quinolinecarbonylꢀThfg, with ApnsꢀDmtꢀ(2ꢀ
methyl)benzylamide of 2. Compound 15a with P2 (2S,3’R)ꢀThfg exhibited highly potent HIVꢀ1 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 antiꢀHIV activity of 17a was weak (26 nM), in
agreement with its protease inhibition. The activities of these Thfgꢀcontaining 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 antiꢀHIV 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.
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[Please insert Table 1.]
Selection of the 5ꢀisoquinolinyloxyacetyl moiety was derived from inhibitor 1. The resultant
compound 18 exhibited sufficient HIVꢀ1 protease inhibition and antiꢀHIV 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 oꢀmethyl group, that is 2,6ꢀdimethylbenzylamide,
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 antiꢀHIV activity.
Interestingly, the addition of serum slightly affected the activity with a serum binding effect of 2.4.
Replacement with 3ꢀ(phenylamino)phenoxyacetyl (21b) increased the antiꢀHIV activity up to 5 nM. A
minor variant with the dimethylamino moiety in compound 21c improved antiꢀHIV inhibition (<3 nM).
The activity in the presence of human serum scored 15 nM, which is the most potent among the series.
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Other P3 modifications with chromonylcarbonyl, benzofurancarbonyl and 7ꢀmethoxybenzofurancarbonyl
were also successful in obtaining highly potent HIV protease inhibitors (21d-21f). The derivative 21d
with chromonylcarbonyl was equipotent to isoquinolinyloxyacetyl in inhibitory activity. Compounds
21e and 21f were slightly more effective than 21d for antiꢀHIV activity with or without human serum.
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[Please insert Table 2.]
The strong antiꢀHIV activity of compound 21c with low serum binding effect encouraged us to
perform combinatorial modification of the P2´ structure with a fixed P3 moiety, that is, 3ꢀ
(dimethylamino)phenoxyacetyl. Inhibitory activity of the derivatives with selected P2´ structures is
summarized in Table 3. HIV protease inhibitory activity ranged from 93% to 97% at 1 nM inhibitor
concentration. Antiꢀviral activity of compound 24a was potent with EC50 of 5 nM. The addition of
human serum had a moderate effect with a 6ꢀfold decrease. Modification with aminoindanol (24b),
which was reported to be effective for malarial plasmepsin inhibition,29 was also successful for antiꢀHIV
activity. In the presence of serum, the activity largely decreased approximately 20ꢀtimes. In our previous
paper, we presented relatively small P2´ structures that are effective in protease inhibition.
Cyclopentylamide derivative 24c exhibited 10 nM for viral inhibition and a moderate attenuation by
serum addition. P2´ allyl analogues 24d and 24e were slightly better than the cyclopentylamide
derivative for enzyme inhibition. Compound 24e with an additional methyl moiety was more potent than
24d for enzyme inhibition, which may be derived from interactions with hydrophobic side chains of
Ala28, Ile84 and Ile50’ in the S2´ pocket as previously reported.22 The antiꢀHIV activity had a positive
relationship with enzyme inhibitory potency. However, activity reductions in human serum were
different between the analogues, 24ꢀfold and 7ꢀfold, respectively. As a result, the
βꢀmethallylamide
derivative 24e was superior to the allyl derivative 24d. The extension of the hydroxymethyl group from
the allylamide structure was also effective for enzyme inhibition. As predicted from the previous results
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of dipeptideꢀtype inhibitors, the antiꢀHIV activity was relatively weak. The effect of the human serum
was small (3.6ꢀfold).
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We selected the topꢀfive inhibitors with high antiviral potency from the aboveꢀmentioned tests and
they were subjected to evaluation against the lopinavir/ritonavirꢀresistant HIV strain, A17 as shown in
Table 4. Lopinavir and 2 exhibited moderate potency with EC50 values of 60 nM and 30 nM,
respectively. Their potencies precipitously decreased against A17 up to the µM level. On the other hand,
the activity of Thfg analogues was still in the nM range, one order more potent compared to lopinavir
and compound 2. The most potent inhibitor against A17 was compound 21f with an EC50 value of 25
nM. The values of fold resistance were not much different from lopinavir and dipeptidic 2. Although the
strong potency of 21f against wildꢀtype (<3 nM) may also be largely attenuated, and the activity seemed
to be sustained against the resistant virus.
[Please insert Table 4.]
P2 preference was explored on the P3 benzofurancarbonyl derivatives, which exhibited promising antiꢀ
HIV activity. Table 5 summarizes a comparison of Asn, Val and Thfg at the P2 position. The Asn
analogues 27a and 27c were equipotent with Thfg analogues in HIV protease inhibitory activity with 96
and 97% inhibition at a 1 nM concentration (27a and 21e, 27c and 21f). The Val derivatives 27b and
27d were slightly less potent than that of the Thfg and Asn derivatives. As expected, the potency of
analogues was reversed in the antiviral assay, the Asn derivatives were less potent and the Val analogues
were equipotent compared with the Thfg analogues. The hydrophilic and hydrophobic balance of the
Asn analogue, which is caused by an increase of one amide group, may not be suitable for membrane
permeability. The results against A17 disclosed another discrepancy between the P2 structures. The
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potency of the Val analogues against A17 was drastically weakened with 60 to 100ꢀfold resistance
values. In contrast, the potency of Asn derivatives was sustained with 1.4 and 1.6ꢀfold values. A
dramatic decrease in potency was observed in compound 21e similar to that of the Val analogues 27b
and 27d (Table 4). The result suggests that 21f, with the structural similarity of the fiveꢀmembered ring
of Thfg compared to the bulkiness of branched isopropyl group, would also exhibit diminished potency.
These results suggest that the flexible hydrogen bonding ability of the Asn carbamoylmethyl side chain
could maintain the interactions in the mutated S2 pocket. Other than the sustained activity of the Asn
derivatives, the Thfg analogue 21f was superior against A17 because of the intrinsic high potency
against wildꢀtype HIV.
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[Please insert Table 5.]
To further examine the potency of 21f, we prepared twoꢀtypes of darunavirꢀresistant viral assays based
on a lentivirus vector infection with 293T cells.30 The mutation sites in the protease region were derived
from an original report by Koh et al.12 First, we tested darunavir, which showed a strong inhibition
against wildꢀtype with an EC50 value of 0.9 nM (Table 6). The potency was attenuated to the virus
containing V32I/L33F/I54M/V82I mutations approximately 15ꢀfold. Another mutation of
V32I/L33F/I54M/I84V precipitously dropped the susceptibility of darunavir by 137ꢀfold. These
observed activity reductions were similar to the reported results. Compound 21f also exhibited strong
potency against wildꢀtype virus (1.71 nM) with three times less potency than that of darunavir.
Interestingly, the activity of 21f was enhanced approximately five times against the virus containing
V32I/L33F/I54M/V82I with an EC50 of 0.30 nM. On the other hand, the mutations, including I84V,
presented a slight influence on the activity, with an EC50 of 1.21 nM and a 0.7ꢀfold decrease compared
with the wildꢀtype virus. These results indicate that compound 21f did not show crossꢀresistance to
darunavir.
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[Please insert Table 6.]
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To determine the prominent potency of compound 21f, Xꢀray crystallographic analysis of the complex
with the wildꢀtype protease (WTm5) was performed (Figure 4). The result with a resolution of 0.85 Å
showed an interaction between the HMC and the catalytic two Asp residues similar to the previous
analysis using Xꢀray and neutron crystallography.16,17 Direct hydrogen bonding interactions were formed
between the oxygen of the THF ring and amide NH group of Asp30’, as well as the NH of the Thfg
residue and carbonyl group of Gly48’ (Figure 4a). We identified the three bridging water molecules.
One existed between the two carbonyl groups of the inhibitor and the NH groups of the Ile50 and Ile50’
residues in the flap region, which was known as water 301. The second water molecule interacted with
the NH of the benzylamide moiety, the carbonyl of Gly27 and the amide NH of Asp29 in the entrance of
the S2’ and S3’ pockets. Another water molecule was placed between the two carbonyl groups of the P3
cap and Gly27’, and the carboxylate side chain of Asp29. There were ten hydrogen bonding interactions
overall, including indirect bridging water molecules. The hydrogen bonding interactions were also
observed in the active sites of the A17m5 protease, which has six mutations of V32I, I47V, and I84V to
form S2 and S2’ pockets, retaining the binding orientation of 21f (Figure 4b). The strong hydrogen
bonding interactions would explain the sustained activities against drugꢀresistant viruses. On the other
hand, the I47V and I84V mutations in these pockets resulted in a removal of the four methyl groups
(green surfaces in Figure 4b) in each pocket, generating unoccupied space. The loss of hydrophobic
contacts may cause the decrease in the EC50 value against A17 strain. Other mutations and the protein
backbone structure in A17m5 protease seemed to not influence the binding mode of 21f (see
Supplementary Figure S1). The affinity experiments using A17m5 protease are ongoing to discuss the
contribution of the unchanged conformation.
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[Please insert Figure 4.]
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Conclusion
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Highly potent Apnsꢀcontaining inhibitors with P2 tetrahydrofuranylglycine (Thfg) were synthesized
and evaluated against lopinavir/ritonavirꢀ or darunavirꢀresistant HIV strains to identify compound 21f
(KNIꢀ1657) with a strong inhibitory activity among the derivatives. Interestingly, compound 21f did not
show crossꢀresistance to darunavir. Xꢀray coꢀcrystal analysis revealed ten hydrogen bonding interactions,
suggesting the stability of the pocket to changes from multiple mutations. These results provide useful
knowledge for designing next generation HIV protease inhibitors that are highly potent against drug
resistant HIV strains.
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Experimental Section
Reagents and solvents used were purchased from Wako Pure Chemical Ind., Ltd. (Osaka, Japan),
Nacalai Tesque (Kyoto, Japan), Aldrich Chemical Co. Inc. (Milwaukee, WI), and Tokyo Chemical
Industry Co., Ltd. (Tokyo, Japan) used without further purification. Column chromatography was
performed on silica gel 60 (70–230 mesh, Merck 107734). The purities of the desired compounds were
confirmed by HPLC with simple integration >95%. The purity of compound 27a was determined to be
90.2%. Analytical HPLC was performed using C18 reversedꢀphase column (4.6 × 150 mm; YMC Pack
ODS AM302) with binary solvent systems: (A) linear gradient of CH3CN 40–100 % in 0.1 % aqueous
TFA in 15 min, (B) linear gradient of CH3CN 20–80 % in 0.1 % aqueous TFA in 30 min at a flow rate
of 0.9 mL/min, detected at 230 nm. Preparative HPLC was carried out on a C18 reversedꢀphase column
(20 × 250 mm; YMC Pack ODS SH343ꢀ5) with a binary solvent system: a linear gradient of CH3CN in
1
0.1% aqueous TFA with a flow rate of 5.0 mL/min and detection at 230 nm. H NMR spectra were
recorded on JNMꢀAL 300 (JEOL, Ltd.), UNITY INOVA 400NB (Varian, Inc.) or DPX400 (Brucker,
Ltd.) spectrometers with TMS as an internal standard. Mass spectra with electrospray ionization using
50% aqueous methanol as mobile phase were recorded on a micrOTOFꢀQ II spectrometer (Brucker,
Ltd.).
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(S)-2-tert-butoxycarbonylamino-2-((R)-tetrahydrofuran-3-yl)acetic acid (9)
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1H NMR (400 MHz, DMSOꢀd6):
δ (ppm): 12.56 (br s, 1H), 7.23 (d, J = 7.9 Hz, 1H), 3.78 (t, J = 8.8
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7
8
Hz, 1H), 3.74ꢀ3.69 (m, 2H), 3.61 (dd, J = 15.8, 7.3 Hz, 1H), 3.39 (dd, J = 8.4, 7.0 Hz, 1H), 2.45ꢀ2.44
(m, 1H), 1.91ꢀ1.85 (m, 1H), 1.70ꢀ1.63 (m, 1H), 1.38 (s, 9H); TOFꢀMS (ESI) m/z: calcd for
C11H19NO5Na [M + Na]+ 268.1155; found 268.1140.
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(S)-2-azido-2-((S)-tetrahydrofuran-3-yl)acetic acid (12)
1H NMR (300 MHz, CDCl3)
δ (ppm): 3.99ꢀ3.92 (m, 2H), 3.91 (d, J = 7.5 Hz, 1H), 3.80 (dd, J = 16.0,
7.5 Hz, 1H), 3.72 (dd, J = 9.2, 6.6 Hz, 1H), 2.74 (dt, J = 14.9, 7.5 Hz, 1H), 2.18ꢀ2.07 (m, 1H), 1.98ꢀ1.86
(m, 1H); TOFꢀMS (ESI) m/z: calcd for C6H8N3O3 [M − H]− 170.0571; found 170.0567.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((S)-2-(tert-butoxycarbonyl)amino-2-((R)-tetrahydrofuran-
3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (14)
Compound 14 was prepared from BocꢀApnsꢀDmtꢀNH(2ꢀmethylbenzyl) 1320 in a similar manner to
that described for compound 20; yield 65%; 1H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.38 (br s, 1H),
7.91 (d, J = 8.6 Hz, 1H), 7.32ꢀ7.30 (m, 1H), 7.28 (d, J = 7.3 Hz, 3H), 7.20ꢀ7.07 (m, 6H), 6.93 (d, J = 9.2
Hz, 1H), 5.26 (d, J = 7.0 Hz, 1H), 5.01 (d, J = 9.0 Hz, 1H), 4.93 (d, J = 9.2 Hz, 1H), 4.50 (s, 1H), 4.43
(d, J = 6.2 Hz, 1H), 4.40ꢀ4.34 (m, 2H), 4.27ꢀ4.2 (br s, 1H), 4.17 (dd, J = 14.8, 4.6 Hz, 1H), 3.76 (t, J =
9.2 Hz, 1H), 3.65ꢀ3.59 (m, 1H), 3.56 (t, J = 8.1 Hz, 1H), 3.46 (dd, J =15.4, 7.5 Hz, 1H), 3.27 (t, J = 7.0
Hz, 1H), 2.73 (br d, J = 11.7 Hz, 1H), 2.63 (br d, J = 13.6 Hz, 1H), 2.27 (s, 3H), 2.28ꢀ2.25 (m, 1H),
1.50 (s, 3H), 1.44 (m, 2H), 1.37 (s, 9H), 1.36 (s, 3H); TOFꢀMS (ESI) m/z: calcd for C35H49N4O7S [M +
H]+ 669.3316; found 669.3288.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((S)-2-(quinoline-2-carbonyl)amino-2-((R)-
tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (15a, KNI-1640)
A mixture of intermediate 14 (84.9 mg, 0.13 mmol), anisole (27.6 ꢀL, 0.25 mmol), and 4M HCl in
dioxane (1.27 mL) was stirred for 60 min at rt. After removal of the solvent in vacuo, the residue was
precipitated from ether to give the hydrochloride salt. To a solution of the salt in DMF (2 mL) were
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added triethylamine (35.4 ꢀL, 0.25 mmol), quinolineꢀ2ꢀcarboxylic acid (24.2 mg, 0.14 mmol) and BOP
(67.2 mg, 0.15 mmol) in an ice bath and the mixture was stirred overnight at room temperature. After
removal of the solvent in vacuo, the residue was added into EtOAc, washed sequentially with 5%
NaHCO3 and brine, dried over MgSO4, and concentrated in vacuo to give a product as a solid, 78 mg.
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Purification of the product by preparative HPLC gave compound 15a as a white powder; yield 85%; H
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NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.78 (d, J = 9.3 Hz, 1H), 8.59 (d, J = 8.2 Hz, 1H), 8.44 (d, J = 8.4
Hz, 1H), 8.40 (d, J = 6.0 Hz, 1H), 8.18ꢀ8.15 (m, 2H), 8.10 (dd, J = 8.4, 1.1 Hz, 1H), 7.88 (dt, J = 7.6,
1.5 Hz, 1H), 7.74 (dt, J = 7.4, 1.1 Hz, 1H), 7.31ꢀ7.29 (m, 1H), 7.27 (d, J = 7.1 Hz, 2H), 7.17ꢀ7.06 (m,
6H), 6.96 (t, J = 7.5 Hz, 1H), 5.34 (d, J = 7.1 Hz, 1H), 5.06 (d, J = 9.2 Hz, 1H), 4.98 (d, J = 9.0 Hz, 1H),
4.53 (t, J = 8.8 Hz, 1H), 4.51 (s, 1H), 4.46ꢀ4.38 (m, 2H), 4.3ꢀ4.23 (m, 1H), 4.18 (dd, J = 15.0, 4.8 Hz,
1H), 3.71 (dt, J = 7.9, 5.1 Hz, 1H), 3.64 (dd, J = 8.8, 7.3 Hz, 1H), 3.54 (dd, J = 15.8, 7.7, Hz, 1H), 3.45
(dd, J = 8.8, 6.4 Hz, 1H), 2.75 (dd, J = 13.6, 2.2 Hz, 1H), 2.67ꢀ2.61 (m, 1H), 2.26 (s, 3H), 1.66ꢀ1.59 (m,
2H), 1.51 (s, 3H), 1.36 (s, 3H); TOFꢀMS (ESI) m/z: calcd for C40H46N5O6S [M + H]+ 724.3163; found
724.3161.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((S)-2-(quinoxaline-2-carbonyl)amino-2-((R)-
tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (15b, KNI-1639)
Compound 15b was prepared from compound 14 using quinoxalineꢀ2ꢀcarboxylic acid in a manner
1
similar to that described for compound 15a; yield 61%; H NMR (300 MHz, DMSOꢀd6) δ (ppm): 9.48
(s, 1H), 8.77 (d, J = 9.1 Hz, 1H), 8.45ꢀ8.35 (m, 2H), 8.27ꢀ8.20 (m, 2H), 8.04ꢀ7.97 (m, 2H), 7.33ꢀ7.25
(m, 3H), 7.13ꢀ7.05 (m, 6H), 6.98ꢀ6.92 (m, 1H), 5.34 (d, J = 7.2 Hz, 1H), 5.06 (d, J = 8.9 Hz, 1H), 4.98
(d, J = 9.2 Hz, 1H), 4.56ꢀ4.49 (m, 2H), 4.47ꢀ4.38 (m, 2H), 4.31ꢀ4.25 (m, 1H), 4.18 (dd, J = 15.0, 4.77
Hz, 1H), 3.75ꢀ3.69 (m, 1H), 3.65 (dd, J = 8.7, 7.2 Hz, 1H), 3.54 (q, J = 7.6 Hz, 1H), 3.46ꢀ3.42 (m, 1H),
2.79ꢀ2.73 (m, 1H), 2.69ꢀ2.61 (m, 2H), 2.26 (s, 3H), 1.65ꢀ1.58 (m, 2H), 1.51 (s, 3H), 1.36 (s, 3H); TOFꢀ
MS (ESI) m/z: calcd for C39H45N6O6S [M + H]+ 725.3116; found 725.3091. Anal. calcd for
C39H44N6O6S·1CF3COOH·0.4H2O: C, 58.2; H, 5.46; N, 9.93. Found: C, 58.26; H, 5.56; N, 9.81.
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(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((S)-2-azido-2-((S)-tetrahydrofuran-3-yl)acetyl)amino-2-
hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (16)
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Compound 16 was prepared from 13 in a similar manner to that described for compound 20; yield
quantitative; 1H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.50 (d, J = 8.6 Hz, 1H), 8.41 (t, J = 5.4 Hz, 1H),
7.33ꢀ7.24 (m, 3H), 7.23ꢀ7.17 (m, 2H), 7.17ꢀ7.10 (m, 4H), 5.41 (d, J = 7.1 Hz, 1H), 5.05ꢀ4.93 (m, 2H),
4.51 (s, 1H), 4.45ꢀ4.37 (m, 2H), 4.27ꢀ4.16 (m, 2H), 3.73ꢀ3.70 (m, 1 H), 3.69ꢀ3.65 (m, 1H), 3.62ꢀ3.57
(m, 2H), 3.44ꢀ3.40 (m, 1H), 2.77ꢀ2.71 (m, 1H), 2.66ꢀ2.59 (m, 1H), 2.27 (s, 3H), 1.95ꢀ1.87 (m, 1H), 1.73
(s, 9H), 1.69ꢀ1.55 (m, 2H), 1.50 (s, 3H), 1.36 (s, 3H); TOFꢀMS (ESI) m/z: calcd for C30H39N6O5S [M +
H]+ 595.2697; found 595.2672.
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(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((S)-2-(quinoline-2-carbonyl)amino-2-((S)-
tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (17a, KNI-1603)
A mixture of compound 16 (680 mg, 1.14 mmol), 5% Pd(OH)2 (50 mg) in ethanolꢀTHF (1: 1, 40 mL)
was stirred under hydrogen gas for 10 h at rt. After removal of the solvent in vacuo, the residue was
purified by silica gel column chromatography to give the amino intermediate. To a solution of the
intermediate in DMF were added triethylamine, quinolineꢀ2ꢀcarboxylic acid and BOP in an ice bath and
the mixture was stirred overnight at room temperature. After removal of the solvent in vacuo, the
residue was added into EtOAc, washed sequentially with 5% NaHCO3 and brine, dried over MgSO4,
and concentrated in vacuo to give a product as a solid. Purification of the product by preparative HPLC
gave compound 17a as a white powder; yield 83%; 1H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.80 (d, J
= 9.2 Hz, 1H), 8.60 (d, J = 8.6 Hz, 1H), 8.43ꢀ8.36 (m, 2H), 8.18ꢀ8.15 (m, 2H), 8.10 (dd, J = 8.1, 0.7 Hz,
1H), 7.88 (dt, J = 7.9, 1.1 Hz, 1H), 7.74 (t, J = 7.1 Hz, 1H), 7.31ꢀ7.29 (m, 1H), 7.25 (d, J = 7.3 Hz, 2H),
7.15ꢀ7.08 (m, 4H), 7.06 (d, J = 7.5 Hz, 2H), 6.94 (t, J = 7.3 Hz, 1H), 5.39 (d, J = 7.6 Hz, 1H), 5.05 (d, J
= 9.0 Hz, 1H), 4.98 (d, J = 9.2 Hz, 1H), 4.54 (d, J = 8.8 Hz, 1H), 4.51 (s, 1H), 4.46ꢀ4.38 (m, 2H), 4.29ꢀ
4.23 (m, 1H), 4.17 (dd, J = 15.0, 4.8 Hz, 1H), 3.70 (dt, J = 8.1, 4.9 Hz, 1H), 3.57 (dd, J = 15.4, 7.5 Hz,
1H), 3.50 (t, J = 7.5 Hz, 1H), 3.43ꢀ3.42 (m, 1H), 2.76ꢀ2.61 (m, 3H), 2.26 (s, 3H), 1.90ꢀ1.83 (m, 1H),
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1.67ꢀ1.59 (m, 1H), 1.51 (s, 3H), 1.36 (s, 3H); TOFꢀMS (ESI) m/z: calcd for C40H46N5O6S [M + H]+
724.3163; found 724.3147; Anal. calcd for C40H45N5O6S·0.1H2O: C, 66.20; H, 6.28; N, 9.65. Found: C,
66.22; H, 6.19; N, 9.47.
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(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((S)-2-(quinoxaline-2-carbonyl)amino-2-((S)-
tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (17b, KNI-1609)
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Compound 17b was prepared from compound 16 in a manner similar to that described for compound
1
17a; yield 86%; H NMR (400 MHz, DMSOꢀd6) δ (ppm): 9.48 (s, 1H), 8.80 (d, J = 9.0 Hz, 1H), 8.40ꢀ
8.38 (m, 2H), 8.26ꢀ8.2 (m, 2H), 7.31ꢀ7.29 (m, 1H), 7.25 (d, J = 7.0 Hz, 2H), 7.12ꢀ7.07 (m, 3H), 7.05 (d,
J = 7.7 Hz, 2H), 6.94 (t, J = 7.3 Hz, 1H), 5.4 (d, J = 7.3 Hz, 1H), 5.05 (d, J = 9.0 Hz, 1H), 4.98 (d, J =
9.3 Hz, 1H), 4.52 (t, J = 9.0 Hz, 1H), 4.51 (s, 1H), 4.45ꢀ4.38 (m, 2H), 4.28ꢀ4.23 (m, 1H), 4.18 (dd, J =
14.8, 5.0 Hz, 1H), 3.70 (dt, J = 8.1, 5.0 Hz, 1H), 3.57 (dd, J = 15.6, 7.5 Hz, 1H), 3.52ꢀ3.32 (m, 2H,
overlapped with water), 2.77ꢀ2.63 (m, 3H), 2.25 (s, 3H), 1.91ꢀ1.86 (m, 1H), 1.67ꢀ1.58 (m, 1H), 1.50 (s,
3H), 1.35 (s, 3H); TOFꢀMS (ESI) m/z: calcd for C39H45N6O6S [M + H]+ 725.3116; found 725.3099;
Anal. calcd for C39H44N6O6S: C, 64.62; H, 6.12; N, 11.59. Found: C, 64.33; H, 6.14; N, 11.30.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((S)-2-(isoquinolin-5-yloxyacetyl)amino-2-((R)-
tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (18, KNI-1641)
Compound 18 was prepared from compound 14 in a manner similar to that described for compound
15a; yield 66%; 1H NMR (400 MHz, DMSOꢀd6) δ (ppm): 9.29 (s, 1H), 8.54 (d, J = 5.9 Hz, 1H), 8.38 (t,
J = 6.0 Hz, 1H), 8.20 (d, J = 8.8 Hz, 2H), 8.02 (dt, J = 5.9, 0.9 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.57 (t,
J = 8.1 Hz, 1H), 7.31ꢀ7.29 (m, 1H), 7.26 (d, J = 7.7 Hz, 2H), 7.17 (t, J = 7.5 Hz, 3H), 7.15ꢀ7.09 (m, 4H),
5.4 (d, J = 7.1 Hz, 1H), 5.03 (d, J = 9.0 Hz, 1H), 4.96 (d, J = 9.2 Hz, 1H), 4.80 (d, J = 14.7 Hz, 1H),
4.72 (d, J = 14.7 Hz, 1H), 4.49 (s, 1H), 4.43ꢀ4.37 (m, 2H), 4.32 (t, J = 8.4 Hz, 1H), 4.26ꢀ4.21 (m, 1H),
4.17 (dd, J = 14.8, 4.6 Hz, 1H), 3.66 (dt, J = 8.1, 4.8 Hz, 1H), 3.57ꢀ3.48 (m, 2H), 3.40ꢀ3.30 (m, 1H,
overlapped with water), 2.77 (dd, J = 14.1, 2.8 Hz, 1H), 2.69ꢀ2.63 (m, 2H), 2.26 (s, 3H), 1.68ꢀ1.61 (m,
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1H), 1.59ꢀ1.52 (m, 1H), 1.50 (s, 3H), 1.34 (s, 3H); TOFꢀMS (ESI) m/z: calcd for C41H48N5O7S [M + H]+
754.3269; found 754.3244; Anal. calcd for C41H47N5O7S·1.5H2O: C, 63.06; H, 6.45; N, 8.97. Found: C,
63.02; H, 6.21; N, 8.85.
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(R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(tert-butoxycarbonyl)amino-2-((R)-
tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (20)
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A mixture of BocꢀApnsꢀDmtꢀNH(2,6ꢀdimethylbenzyl) 1922 (789 mg, 1.42 mmol), anisole (307 ꢀL,
2.84 mmol), and 4M HCl in dioxane (4.6 mL) was stirred for 30 min at rt. After removal of the solvent
in vacuo, the residue was precipitated from ether to give a white solid. To a solution of the
hydrochloride salt in DMF (15 mL) were triethylamine (198 ꢀL, 1.29 mmol), HOBt·H2O (198 mg, 1.29
mmol), Bocꢀ(2S,3R)ꢀTfgꢀOH 9 (316 mg, 1.29 mmol) and EDC·HCl (247 mg, 1.29 mmol) in an ice bath
and the mixture was stirred overnight at room temperature. After removal of the solvent in vacuo, the
residue was added into EtOAc, washed sequentially with 10% citric acid, 5% NaHCO3 and brine, dried
over MgSO4, and concentrated in vacuo to give the titled compound as an amorphous solid; quantitative
yield; 1H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.15ꢀ8.07 (m, 1H), 7.97 (d, J = 8.6 Hz, 1H), 7.35 (d, J =
7.2 Hz, 2H), 7.25ꢀ7.19 (m, 2H), 7.17ꢀ7.13 (m, 1H), 7.10ꢀ7.05 (m, 1H), 7.03ꢀ6.99 (m, 2H), 6.92 (d, J =
9.2 Hz, 1H), 5.15 (d, J = 7.5 Hz, 1H), 5.03 (d, J = 9.1 Hz, 1H), 4.90 (d, J = 8.9 Hz, 1H), 4.54 (s, 1H),
4.47 (dd, J = 13.9, 6.1 Hz, 1H), 4.37 (dd, J = 7.3, 2.0 Hz, 1H), 3.78 (t, J = 9.2 Hz, 1H), 4.24ꢀ4.13 (m,
2H), 3.62 (td, J = 8.0, 4.8 Hz, 1H), 3.55 (t, J = 7.9 Hz, 1H), 3.46 (q, J = 7.7 Hz, 1H), 3.28ꢀ3.24 (m, 1H),
2.73ꢀ2.61 (m, 2H), 2.31 (s, 6H), 2.24 (dd, J = 16.3, 7.9 Hz, 1H), 1.46 (s, 3H), 1.41ꢀ1.24 (m, 14H); TOFꢀ
MS (ESI) m/z: calcd for C36H51N4O7S [M + H]+ 683.3473; found 683.3440.
(R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(isoquinolin-5-yloxyacetyl)amino-2-((R)-
tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (21a, KNI-1648)
Compound 21a was prepared from compound 20 in a manner similar to that described for compound
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15a; yield 76%; H NMR (400 MHz, DMSOꢀd6) δ (ppm): 9.56 (s, 1H), 8.62 (d, J = 6.0 Hz, 1H), 8.29
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(br s, 1H), 8.28 (d, J = 6.8 Hz, 1H), 8.24 (d, J = 8.8 Hz, 1H), 8.12 (dd, J = 5.9, 3.7 Hz, 1H), 7.88 (d, J =
8.2 Hz, 1H), 7.73 (t, J = 8.0 Hz, 1H), 7.34 (d, J = 7.1 Hz, 2H), 7.29 (d, J = 7.7 Hz, 1H), 7.21 (t, J = 7.3
Hz, 2H), 7.15ꢀ7.05 (m, 2H), 7.01 (d, J = 7.3 Hz, 2H), 5.07 (d, J = 8.8 Hz, 1H), 4.92 (d, J = 8.8 Hz, 1H),
4.85 (d, J = 14.7 Hz, 1H), 4.78 (d, J = 14.7 Hz, 1H), 4.53 (s, 1H), 4.47ꢀ4.42 (m, 2H), 4.35 (t, J = 8.4 Hz,
1H), 4.22ꢀ4.17 (m, 1H), 4.17 (dd, J = 13.7, 4.2 Hz, 1H), 3.67 (dt, J = 8.4, 5.0 Hz, 1H), 3.58ꢀ3.32 (m, 3H,
overlapped with water), 2.75ꢀ2.60 (m, 3H), 2.30 (s, 6H), 1.70ꢀ1.64 (m, 1H), 1.62ꢀ1.55 (m, 1H), 1.45 (s,
3H), 1.35 (s, 3H); TOFꢀMS (ESI) m/z: calcd for C42H50N5O7S [M + H]+ 768.3425; found 768.3383;
Anal. calcd for C42H49N5O7S·0.75H2O: C, 64.55; H, 6.51; N, 8.96. Found: C, 64.57; H, 6.56; N, 8.94.
(R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(3-(phenylamino)phenoxyacetyl)amino-2-((R)-
tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (21b, KNI-1650)
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Compound 21b was prepared from compound 20 in a manner similar to that described for compound
15a; yield 57%;1H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.24 (d, J = 8.6 Hz, 1H), 8.20 (br s, 1H), 8.11
(dd, J = 5.6, 3.7 Hz, 1H), 7.94 (d, J = 9.0 Hz, 1H), 7.33 (d, J = 7.3 Hz, 1H), 7.24 (d, J = 8.1 Hz, 1H),
7.23ꢀ7.22 (m, 3H), 7.20 (d, J = 7.7 Hz, 1H), 7.01ꢀ7.05 (m, 3H), 7.01 (d, J = 7.5 Hz, 2H), 6.84 (t, J = 7.0
Hz, 1H), 6.67 (dd, J = 8.1, 1.8 Hz, 1H), 6.63 (t, J = 2.2 Hz, 1H), 6.36 (dd, J = 8.2, 2.4 Hz, 1H), 5.05 (d,
J = 9.0 Hz, 1H), 4.91 (d, J = 9.0 Hz, 1H), 4.53 (s, 1H), 4.50 (d, J = 14.8 Hz, 1H), 4.46ꢀ4.40 (m, 1H),
4.41 (d, J = 15.0 Hz, 1H), 4.40 (d, J = 2.6 Hz, 1H), 4.27 (t, J = 8.6 Hz, 1H), 4.21ꢀ4.15 (m, 1H), 4.17 (dd,
J = 13.9, 3.3 Hz, 1H), 3.60 (dd, J = 13.4, 8.4 Hz, 1H), 3.50ꢀ3.42 (m, 2H, overlapped with water), 3.25ꢀ
3.21 (m, 1H), 2.71ꢀ2.61 (m, 2H), 2.41ꢀ2.36 (m, 1H), 2.30 (s, 6H), 1.51ꢀ1.45 (m, 1H), 1.45 (s, 3H), 1.40ꢀ
1.29 (m, 1H), 1.35 (s, 3H); TOFꢀMS (ESI) m/z: calcd for C45H54N5O7S [M + H]+ 808.3738; found
808.3704; Anal. calcd for C45H53N5O7S·0.1CF3COOH: C, 66.25; H, 6.53; N, 8.55. Found: C, 66.21; H,
6.66; N, 8.41.
(R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(3-(dimethylamino)phenoxyacetyl)amino-2-((R)-
tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (21c, KNI-1652)
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Compound 21c was prepared from compound 20 in a manner similar to that described for compound
15a; yield 45%; 1H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.26 (d, J = 8.0 Hz, 1H), 8.13ꢀ8.09 (m, 1H),
7.92 (d, J = 9.2 Hz, 1H), 7.33 (d, J = 7.0 Hz, 2H), 7.21 (t, J = 7.3 Hz, 2H), 7.12ꢀ7.06 (m, 3H), 7.01 (d, J
= 7.5 Hz, 2H), 6.37 (d, J = 7.6 Hz, 1H), 6.27 (s, 1H), 6.22 (d, J = 8.0 Hz, 1H), 5.06 (d, J = 8.7 Hz, 1H),
4.92 (d, J = 9.0 Hz, 1H), 4.54 (s, 1H), 4.50 (d, J = 15.2 Hz, 1H), 4.43 (m, 1H), 4.41 (d, J = 15.2 Hz, 1H),
4.408 (d, J = 2.0 Hz, 1H), 4.03 (t, J = 8.8 Hz, 1H), 4.21ꢀ4.17 (m, 2H), 3.64ꢀ3.59 (m, 1H), 3.52ꢀ3.46 (m,
2H), 3.28ꢀ3.22 (m, 1H), 2.87 (s, 6H), 2.74ꢀ2.58 (m, 2H), 2.30 (s, 6H), 2.02ꢀ1.95 (m, 1H), 1.55ꢀ1.49 (m,
2H), 1.46 (s, 3H), 1.35 (s, 3H); TOFꢀMS (ESI) m/z: calcd for C41H54N5O7S [M + H]+ 760.3738; found
760.3723; Anal. calcd for C41H53N5O7S·0.2CF3COOH: C, 63.52; H, 6.85; N, 8.95. Found: C, 63.46; H,
6.94; N, 9.01.
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(R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(3-(chromon-2-carbonyl)amino-2-((R)-
tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (21d, KNI-1654)
Compound 21d was prepared from compound 20 in a manner similar to that described for compound
15a; yield 42%; 1H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.88 (d, J = 8.6 Hz, 1H), 8.33 (d, J = 8.2 Hz,
1H), 8.10 (br. s, 1H), 8.06 (dd, J = 7.9, 1.7 Hz, 1H), 7.90 (dt, J = 7.9, 1.8 Hz, 1H), 7.78 (d, J = 7.9 Hz,
1H), 7.55 (dt, J = 7.6, 0.9 Hz, 1H), 7.33 (d, J = 7.1 Hz, 2H), 7.15 (d, J = 7.5 Hz, 2H), 7.07ꢀ7.03 (m, 2H),
7.00 (d, J = 7.3 Hz, 2H), 6.84 (s, 1H), 5.3 (d, J = 7.1 Hz, 1H), 5.06 (d, J = 9.0 Hz, 1H), 4.94 (d, J = 9.0
Hz, 1H), 4.53 (s, 1H), 4.47ꢀ4.43 (m, 2H), 4.40 (t, J = 9.0 Hz, 1H), 4.23ꢀ4.2 (m, 1H), 4.17 (dd, J = 13.9,
3.3 Hz, 1H), 3.75 (dt, J = 8.1, 5.1 Hz, 1H), 3.69 (dd, J = 8.4, 7.0 Hz, 1H), 3.59 (dt, J = 15.1, 7.5 Hz,
1H), 3.40 (dd, J = 8.8, 6.4 Hz, 1H), 2.75 (dt, J = 13.7, 2.4 Hz, 1H), 2.69ꢀ2.59 (m, 2H), 2.30 (s, 6H),
1.72ꢀ1.69 (m, 1H), 1.68ꢀ1.62 (m, 1H), 1.45 (s, 3H), 1.34 (s, 3H); TOFꢀMS (ESI) m/z: calcd for
C41H47N4O8S [M + H]+ 755.3109; found 755.3094; Anal. calcd for C41H46N4O8S·0.75H2O: C, 64.09; H,
6.23; N, 7.29. Found: C, 64.06; H, 6.13; N, 7.27.
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(R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(3-(benzofuran-2-carbonyl)amino-2-((R)-
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tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (21e, KNI-1666)
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Compound 21e was prepared from compound 20 in a manner similar to that described for compound
15a; yield 39%; 1H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.55 (d, J = 8.8 Hz, 1H), 8.26 (d, J = 8.4 Hz,
1H), 8.15ꢀ8.08 (m, 1H), 7.79 (d, J = 7.0 Hz, 1H), 7.70 (d, J = 8.1 Hz, 1H), 7.62 (s, 1H), 7.49 (t, J = 8.2
Hz, 1H), 7.38ꢀ7.34 (m, 3H), 7.15 (d, J = 7.3 Hz, 2H), 7.06 (dd, J = 14.8, 6.8 Hz, 2H), 7 (d, J = 7.5 Hz,
2H), 5.05 (d, J = 9.0 Hz, 1H), 4.93 (d, J = 9.3 Hz, 1H), 4.53 (s, 1H), 4.47ꢀ4.36 (m, 3H), 4.25ꢀ4.14 (m,
2H), 3.72ꢀ3.63 (m, 2H), 3.52 (dd, J = 15.2, 10.4 Hz, 1H), 3.46ꢀ3.31 (m, 1H, overlapped with water),
2.74ꢀ2.59 (m, 2H), 2.49ꢀ2.45 (m, 1H), 2.30 (s, 6H), 1.54ꢀ1.47 (m, 2H), 1.45 (s, 3H), 1.35 (s, 3H), 1.31ꢀ
1.20 (m, 1H); TOFꢀMS (ESI) m/z: calcd for C40H47N4O7S [M + H]+ 727.3160; found 727.3143; Anal.
calcd for C40H46N4O7S·0.6H2O: C, 65.13; H, 6.45; N, 7.59. Found: C, 65.03; H, 6.43; N, 7.75.
(R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(3-(7-methoxybenzofuran-2-carbonyl)amino-2-
((R)-tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-
thiazolidine-4-carboxamide (21f, KNI-1657)
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Compound 21f was prepared from compound 20 in a manner similar to that described for compound
15a; yield 57%; 1H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.39 (d, J = 9.5 Hz, 1H), 8.22 (d, J = 8.4 Hz,
1H), 8.06 (br s, 1H), 7.54 (d, J = 2.0 Hz, 1H), 7.28 (t, J = 7.9 Hz, 3H), 7.22 (t, J = 7.9 Hz, 1H), 7.1 (t, J
= 7.5 Hz, 2H), 7.05ꢀ6.95 (m, 5H), 5.00 (d, J = 9.0 Hz, 1H), 4.89 (d, J = 8.6 Hz, 1H), 4.49 (s, 1H), 4.43ꢀ
4.32 (m, 3H), 4.18ꢀ4.1 (m, 2H), 3.93 (s, 3H), 3.67ꢀ3.64 (m, 2H), 3.51ꢀ3.48 (m, 2H, overlapped with
water), 2.70ꢀ2.56 (m, 3H), 2.25 (s, 6H), 1.48ꢀ1.44 (m, 2H), 1.40 (s, 3H), 1.30 (s, 3H); 13C NMR (101
MHz, DMSOꢀd6) δ (ppm): 170.12, 169.92, 167.72, 157.57, 148.49, 145.26, 143.62, 139.21, 137.29,
134.45, 129.44, 128.62, 127.89, 127.79, 127.26, 125.77, 124.59, 114.45, 110.64, 108.76, 72.51, 72.28,
71.64, 70.88, 69.82, 67.19, 63.08, 60.25, 55.72, 55.26, 53.96, 53.12, 51.14, 47.68, 41.34, 37.23, 33.11,
30.32, 28.38, 24.57,19.41; TOFꢀMS (ESI) m/z: calcd for C41H49N4O8S [M + H]+ 757.3266; found
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757.3248; Anal. calcd for C40H51N5O6S·0.6H2O: C, 64.14; H, 6.46; N, 7.30. Found: C, 64.07; H, 6.43;
N, 7.38.
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(R)-N-[(S)-Indan-1-yl]-3-[(2S,3S)-3-((S)-2-(tert-butoxycarbonyl)amino-2-((R)-tetrahydrofuran-3-
yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (23a)
Compound 23a was prepared from 22a22 in a manner similar to that described for compound 20; yield
82%; 1H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.36 (d, J = 8.1 Hz, 1H), 7.93 (d, J = 8.6 Hz, 1H), 7.28ꢀ
7.25 (m, 3H), 7.23ꢀ7.21 (m, 2H), 7.17 (d, J = 7.3 Hz, 2H), 7.14ꢀ7.12 (m, 2H), 6.92 (d, J = 8.8 Hz, 1H),
5.31 (dd, J = 15.4, 7.7 Hz, 1H), 5.19 (d, J = 7.3 Hz, 1H), 5.01 (d, J = 9.2 Hz, 1H), 4.95 (d, J = 9.2 Hz,
1H), 4.46 (s, 1H), 4.39 (dd, J = 7.3, 2.2 Hz, 1H), 4.25 (br. s., 1H), 3.76 (t, J = 9.2 Hz, 1H), 3.62 (dt, J =
5.8 Hz, 1H), 3.55 (t, J = 7.7 Hz, 1H), 3.46 (dd, J = 15.4, 7.7 Hz, 1H), 3.31ꢀ3.24 (m, 1H), 2.94 (ddd, J =
15.9, 8.6, 3.2 Hz, 1H), 2.84ꢀ2.78 (m, 2H), 2.72ꢀ2.68 (m, 2H), 2.42ꢀ2.34 (m, 1H), 2.27ꢀ2.22 (m, 1H),
1.91ꢀ1.80 (m, 1H), 1.51 (s, 3H), 1.46 (s, 3H), 1.37 (s, 9H), 1.36ꢀ1.27 (m, 2H); TOFꢀMS (ESI) m/z: calcd
for C36H49N4O7S [M + H]+ 681.3316; found 681.3275.
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(R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-((S)-2-(tert-butoxycarbonyl)amino-2-((R)-
tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (23b)
Compound 23b was prepared from 22b22 in a manner similar to that described for compound 20;
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quantitative yield; H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.10 (d, J = 8.80 Hz, 1H), 7.97 (d, J = 8.6
Hz, 1H), 7.35 (d, J = 7.2 Hz, 2H), 7.27 (d, J = 7.3 Hz, 1H), 7.25ꢀ7.05 (m, 7H), 6.92 (d, J = 9.1 Hz, 1H),
5.28 (dd, J = 8.8, 5.1 Hz, 1H), 5.22ꢀ5.14 (m, 1H), 5.10ꢀ4.99 (m, 2H), 4.95 (d, J = 9.1 Hz, 1H), 4.75 (s,
1H), 4.48ꢀ4.38 (m, 2H), 4.27ꢀ4.19 (m, 1H), 3.77 (t, J = 9.2 Hz, 1H), 3.64ꢀ3.58 (m, 1H), 3.54 (t, J = 7.8
Hz, 1H), 3.47ꢀ3.42 (m, 1H), 3.28ꢀ3.22 (m, 1H), 3.08ꢀ3.02 (m, 1H), 2.86ꢀ2.75 (m, 2H), 2.68ꢀ2.62 (m,
2H), 2.22 (dt, J = 16.1, 8.0 Hz, 1H), 1.56 (s, 3H), 1.48 (s, 3H), 1.37 (s, 9H), 1.31ꢀ1.21 (m, 2H); TOFꢀMS
(ESI) m/z: calcd for C36H49N4O8S [M + H]+ 697.3266; found 697.3209.
(R)-N-Cyclopentyl-3-[(2S,3S)-3-((S)-2-(tert-butoxycarbonyl)amino-2-((R)-tetrahydrofuran-3-
yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (23c)
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Compound 23c was prepared from 22c22 in a manner similar to that described for compound 20; yield
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92%; 1H NMR (400 MHz, DMSOꢀd6) δ (ppm): 7.92 (d, J = 7.1 Hz, 2H), 7.31 (d, J = 7.3 Hz, 2H), 7.20
(d, J = 7.6 Hz, 2H), 7.15ꢀ7.13 (m, 1H), 6.91 (d, J = 9.2 Hz, 1H), 5.14 (d, J = 7.3 Hz, 1H), 4.98 (d, J =
9.0 Hz, 1H), 4.92 (d, J = 9.0 Hz, 1H), 4.42 (s, 1H), 4.38 (dd, J = 7.5, 2.3 Hz, 1H), 4.26ꢀ4.20 (br. s., 1H),
4.05ꢀ3.97 (m, 1H), 3.76 (t, J = 9.2 Hz, 1H), 3.61 (dt, J = 7.9, 4.8 Hz, 1H), 3.54 (t, J = 7.7 Hz, 1H), 3.42
(dd, J = 15.4, 7.5 Hz, 1H), 3.29ꢀ3.23 (m, 1H), 2.69ꢀ2.65 (m, 1H), 2.62ꢀ2.58 (m, 1H), 2.26ꢀ2.18 (m, 1H),
1.80ꢀ1.72 (m, 2H), 1.65ꢀ1.60 (s, 2H), 1.49ꢀ1.37 (s, 19H), 1.34ꢀ1.25 (m, 2H); TOFꢀMS (ESI) m/z: calcd
for C32H49N4O7S [M + H]+ 633.3316; found 633.3289.
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(R)-N-Allyl-3-[(2S,3S)-3-((S)-2-(tert-butoxycarbonyl)amino-2-((R)-tetrahydrofuran-3-
yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (23d)
Compound 23d was prepared from 22d22 in a manner similar to that described for compound 20; yield
94%; 1H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.16 (t, J = 5.8 Hz, 1H), 7.89 (d, J = 8.6 Hz, 1 ), 7.29 (d,
J = 7.2 Hz, 2H), 7.15ꢀ7.11 (m, 1H), 7.22ꢀ7.17 (m, 2 H), 6.94 (d, J = 8.8 Hz, 1H), 5.28ꢀ5.17 (m, 2H),
5.83ꢀ5.73 (m, 1H), 5.03 (dq, J = 10.3, 1.67 Hz, 1H), 4.42 (s, 1H) 5.04ꢀ4.92 (m, 3H), 4.36 (dd, J = 7.2,
3.42 Hz, 1H), 4.24 (t, J = 9.4 Hz, 1H), 3.65ꢀ3.53 (m, 2H) 3.78ꢀ3.69 (m, 3H), 3.28ꢀ3.23 (m, 1H) 3.49ꢀ
3.42 (m, 1H), 2.73ꢀ2.63 (m, 2H), 2.24 (dd, J = 15.9, 7.6 Hz, 1H), 1.51 (s, 3H), 1.41ꢀ1.33 (m, 12H), 1.30ꢀ
1.24 (m, 2H); TOFꢀMS (ESI) m/z: calcd for C30H45N4O7S [M + H]+ 605.3003; found 605.2969.
(R)-N-(2-Methylallyl)-3-[(2S,3S)-3-((S)-2-(tert-butoxycarbonyl)amino-2-((R)-tetrahydrofuran-3-
yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (23e)
Compound 23e was prepared from 22e22 in a manner similar to that described for compound 20; yield
99%; 1H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.17 (t, J = 5.8 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.29
(d, J = 7.2 Hz, 2H), 7.19 (t, J = 7.3 Hz, 2H), 7.16ꢀ7.12 (m, 1H), 6.94 (d, J = 9.1 Hz, 1H), 5.25 (d, J = 7.5
Hz, 1H), 5.02ꢀ4.96 (m, 1H), 4.96ꢀ4.88 (m, 2H), 4.74 (s, 1H), 4.45 (s, 1H), 4.36 (dd, J = 7.3, 3.2 Hz, 1H),
4.23 (t, J = 9.4 Hz, 1H), 3.77ꢀ3.72 (m, 2H), 3.64ꢀ3.53 (m, 3H), 3.46 (q, J = 7.7 Hz, 1H), 3.28ꢀ3.23 (m,
1H), 2.73ꢀ2.62 (m, 2H), 2.24 (dd, J = 16.3, 7.7 Hz, 1H), 1.51 (s, 3H) 1.66 (s, 3H), 1.41ꢀ1.23 (m, 14H);
TOFꢀMS (ESI) m/z: calcd for C31H47N4O7S [M + H]+ 619.3160; found 619.3148.
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(R)-N-(cis-4-Hydroxy-2-buten-1-yl)-3-[(2S,3S)-3-((S)-2-(tert-butoxycarbonyl)amino-2-((R)-
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tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (23f)
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Compound 23f was prepared from 22f22 in a manner similar to that described for compound 20; yield
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85%; H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.14 (br. s., 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.30 (d, J =
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7.3 Hz, 2H), 7.24ꢀ7.08 (m, 5H), 6.93 (d, J = 9.1 Hz, 1H), 5.57ꢀ5.50 (m, 1H), 5.37ꢀ5.29 (m, 1H), 5.23 (d,
J = 7.3 Hz, 1H), 4.99ꢀ4.89 (m, 2H), 4.66 (t, J = 5.3 Hz, 1H), 4.40ꢀ4.32 (m, 2H), 4.23 (br. s., 1H), 4.03 (t,
J = 5.8 Hz, 2H), 3.80ꢀ3.70 (m, 3H), 3.65ꢀ3.52 (m, 2H), 3.49ꢀ3.43 (m, 1H), 3.29ꢀ3.23 (m, 1H), 2.73 ꢀ2.65
(m, 2H), 2.28ꢀ2.19 (m, 1H), 1.53ꢀ1.47 (m, 3H), 1.42ꢀ1.26 (m, 14H); TOFꢀMS (ESI) m/z: calcd for
C31H47N4O8S [M + H]+ 635.3109; found 635.3078.
(R)-N-[(S)-Indan-1-yl]-3-[(2S,3S)-3-((S)-2-(3-(dimethylamino)phenoxyacetyl)amino-2-((R)-
tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (24a, KNI-1665)
Compound 24a was prepared from compound 23a in a manner similar to that described for compound
15a; yield 40%; 1H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.36 (d, J = 8.1 Hz, 1H), 8.22 (d, J = 8.6 Hz,
1H), 7.92 (d, J = 8.8 Hz, 1H), 7.26 (d, J = 7.1 Hz, 2H), 7.23ꢀ7.15 (m, 6H), 7.13ꢀ7.08 (m, 2H), 6.43 (d, J
= 7.0 Hz, 1H), 6.35 (br s, 1H), 6.29 (d, J = 7.5 Hz, 1H), 5.30 (dd, J = 15.6, 7.9 Hz, 1H), 5.03 (d, J = 9.2
Hz, 1H), 4.97 (d, J = 9.2 Hz, 1H), 4.51 (d, J = 14.8 Hz, 1H), 4.46 (s, 1H), 4.43 (d, J = 14.8 Hz, 1H),
4.42 (d, J = 3.1 Hz, 1H), 4.28 (t, J = 8.6 Hz, 1H), 4.25ꢀ4.19 (m, 1H), 3.73ꢀ3.45 (m, 4H, overlapped with
water), 3.25 (dd, J = 8.6, 7.0 Hz, 1H), 2.96ꢀ2.91 (m, 1H), 2.89 (s, 6H), 2.83ꢀ2.58 (m, 3H), 2.42ꢀ2.35 (m,
2H), 1.86ꢀ1.81 (m, 1H), 1.56ꢀ1.47 (m, 2H), 1.50 (s, 3H), 1.45 (s, 3H); TOFꢀMS (ESI) m/z: calcd for
C41H52N5O7S [M + H]+ 758.3582; found 758.3556; Anal. calcd for C41H51N5O7S·1CF3COOH: C, 59.23;
H, 6.01; N, 8.03. Found: C, 59.38; H, 6.16; N, 8.33.
(R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-((S)-2-(3-
(dimethylamino)phenoxyacetyl)amino-2-((R)-tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-
phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (24b, KNI-1749)
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Journal of Medicinal Chemistry
Compound 24b was prepared from compound 23b in a manner similar to that described for compound
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15a; yield 31%; 1H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.26 (d, J = 7.9 Hz, 1H), 8.11 (d, J = 9.0 Hz,
1H), 7.92 (d, J = 9.0 Hz, 1H), 7.33 (d, J = 7.3 Hz, 2H), 7.26 (d, J = 7.0 Hz, 1H), 7.22ꢀ7.14 (m, 4H),
7.11ꢀ7.05 (m, 3H), 6.37 (d, J = 8.8 Hz, 1H), 6.28 (br s, 1H), 6.23 (d, J = 7.9 Hz, 1H), 5.27 (dd, J = 9.0,
5.5 Hz, 1H), 5.04 (d, J = 9.2 Hz, 1H), 4.95 (d, J = 9.0 Hz, 1H), 4.74 (s, 1H), 4.49 (d, J = 15.0 Hz, 1H),
4.46 (d, J = 2.8 Hz, 1H), 4.42 (d, J = 14.7 Hz, 1H), 4.41 (m, 1H), 4.27 (t, J = 8.8 Hz, 1H), 4.20ꢀ4.19 (m,
1H), 3.61 (dd, J = 13.0, 7.7 Hz, 1H), 3.50ꢀ3.29 (m, 2H, overlapped with water), 3.24ꢀ3.20 (m, 1H), 3.04
(dd, J = 15.8, 4.6 Hz, 1H), 2.87 (s, 6H), 2.83ꢀ2.79 (m, 3H), 2.70ꢀ2.63 (m, 1H), 2.43ꢀ2.33 (m,1H), 1.55
(s, 3H), 1.54ꢀ1.49 (m, 2H), 1.48 (s, 3H); TOFꢀMS (ESI) m/z: calcd for C41H52N5O8S [M + H]+
774.3531; found 774.3512.
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(R)-N-Cyclopentyl-3-[(2S,3S)-3-((S)-2-(3-(dimethylamino)phenoxyacetyl)amino-2-((R)-
tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (24c, KNI-1729)
Compound 24c was prepared from compound 23c in a manner similar to that described for compound
1
15a; Yield 66%; H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.22 (d, J = 8.4 Hz, 1H), 7.94ꢀ7.91 (m, 2H),
7.30 (d, J = 7.0 Hz, 2H), 7.20 (t, J = 7.5 Hz, 2H), 7.15ꢀ7.08 (m, 2H), 6.43 (br. d, J = 7.3 Hz, 1H), 6.34
(br s, 1H), 6.28 (d, J = 7.9 Hz, 1H), 5.00 (d, J = 9.0 Hz, 1H), 4.94 (d, J = 9.2 Hz, 1H), 4.31 (d, J = 15
Hz, 1H), 4.43 (d, J = 13.2 Hz, 1H), 4.428 (s, 1H), 4.41 (m, 1H), 4.29 (t, J = 8.8 Hz, 1H), 4.23ꢀ4.19 (br.
s, 1H), 4.00 (dt, J = 12.1, 5.1 Hz, 1H), 3.62 (dt, J = 8.1, 5.3 Hz, 1H), 3.52ꢀ3.46 (m, 2H, overlapped with
water), 3.24 (dd J = 8.8, 7.0 Hz, 1H), 2.89 (s, 6H), 2.70 (dd, J = 13.7, 2.4 Hz, 1H), 2.63 (dd, J = 13.9,
2.9 Hz, 1H), 2.44ꢀ2.37 (m,1H), 1.78ꢀ1.74 (m, 2H), 1.64ꢀ1.59 (m, 2H), 1.56ꢀ1.38 (m, 6H), 1.49 (s, 3H),
1.36 (s, 3H); TOFꢀMS (ESI) m/z: calcd for C37H52N5O7S [M + H]+ 710.3582; found 710.3553.
(R)-N-Allyl-3-[(2S,3S)-3-((S)-2-(3-(dimethylamino)phenoxyacetyl)amino-2-((R)-tetrahydrofuran-
3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (24d,
KNI-1742)
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Journal of Medicinal Chemistry
Page 24 of 49
Compound 24d was prepared from compound 23d in a manner similar to that described for compound
15a; Yield 56%; 1H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.22ꢀ8.09 (m, 2H), 7.93 (d, J = 8.9 Hz, 1H),
7.32ꢀ7.23 (m, 2H), 7.22ꢀ7.16 (m, 2H), 7.15ꢀ7.06 (m, 2H), 6.39 (d, J = 8.3 Hz, 1H), 6.30 (br. s., 1H), 6.25
(d, J = 8.1 Hz, 1H), 5.82ꢀ5.72 (m, 1H), 5.25ꢀ5.19 (m, 1H), 5.06ꢀ4.92 (m, 3H), 4.53ꢀ4.48 (m, 1H), 4.46ꢀ
4.35 (m, 3H), 4.29ꢀ4.19 (m, 2H), 3.78ꢀ3.58 (m, 4H), 3.52ꢀ3.47 (m, 2H), 3.27ꢀ3.23 (m, 1H), 2.88 (s, 6
H), 2.76ꢀ2.70 (m, 1H), 2.68ꢀ2.63 (m, 1H), 2.43ꢀ2.37 (m, 1H), 1.58ꢀ1.44 (m, 5H), 1.36 (s, 3H); TOFꢀMS
(ESI) m/z: calcd for C35H48N5O7S [M + H]+ 682.3269; found 682.3234.
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(R)-N-(2-Methylallyl)-3-[(2S,3S)-3-((S)-2-(3-(dimethylamino)phenoxyacetyl)amino-2-((R)-
tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (24e, KNI-1728)
Compound 24e was prepared from compound 23e in a manner similar to that described for compound
15a; Yield 37%; 1H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.18ꢀ8.16 (m, 2H), 7.95 (d, J = 8.8 Hz, 1H),
7.27 (d, J = 7.1 Hz, 2H), 7.19 (t, J = 7.3 Hz, 2H), 7.15ꢀ7.08 (m, 2H), 6.42 (d, J = 7.9 Hz, 1H), 6.3 (s,
1H), 6.27 (d, J = 8.1 Hz, 1H), 5.01 (d, J = 9.2 Hz, 1H), 4.94 (d, J = 9.3 Hz, 1H), 4.90 (s, 1H), 4.74 (s,
1H), 4.51 (d, J = 14.8 Hz, 1H), 4.44 (s, 1H), 4.43 (d, J = 14.7 Hz, 1H), 4.38 (d, J = 3.7 Hz, 1H), 4.27 (t,
J = 8.8 Hz, 1H), 4.23ꢀ4.18 (m, 1H), 3.74 (dd, J = 15.4, 6.2 Hz, 1H), 3.64ꢀ3.57 (m, 2H), 3.24 (dd, J = 7,
8.6 Hz, 1H), 2.88 (s, 6H), 2.74ꢀ2.60 (m, 2H), 2.43ꢀ2.37 (m, 1H), 1.65 (s, 3H), 1.56ꢀ1.45 (m, 2H), 1.51
(s, 3H), 1.36 (s, 3H); TOFꢀMS (ESI) m/z: calcd for C36H50N5O7S [M + H]+ 696.3425; found 696.3409.
(R)-N-(cis-4-Hydroxy-2-buten-1-yl)-3-[(2S,3S)-3-((S)-2-(3-(dimethylamino)phenoxyacetyl)amino-
2-((R)-tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-
thiazolidine-4-carboxamide (24f, KNI-1653)
Compound 24f was prepared from compound 23f in a manner similar to that described for compound
15a; Yield 24%; 1H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.22ꢀ8.08 (m, 2H), 7.93 (d, J = 8.93 Hz, 1H),
7.32ꢀ7.24 (m, 2H), 7.24ꢀ7.17 (m, 2H), 7.14ꢀ7.07 (m, 2H), 6.40 (d, J = 8.2 Hz, 1H), 6.30 (br. s., 1H), 6.25
(d, J = 7.7 Hz, 1H), 5.58ꢀ5.50 (m, 1H), 5.36ꢀ5.29 (m, 1H), 4.96 (q, J = 9.2 Hz, 2H), 4.50 (d, J = 14.9 Hz,
1H), 4.43 (d, J = 14.8 Hz, 1H), 4.40ꢀ4.34 (m, 2H), 4.29ꢀ4.18 (m, 2H), 4.05ꢀ4.00 (m, 2H), 3.81ꢀ3.69 (m,
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Journal of Medicinal Chemistry
4H), 3.62 (d, J = 4.9 Hz, 2H), 3.27ꢀ3.23 (m, 1H), 2.88 (s, 6 H), 2.76ꢀ2.71 (m, 1H), 2.68ꢀ2.60 (m, 2H),
2.40 (dd, J = 15.5, 7.7 Hz, 1H), 1.58ꢀ1.42 (m, 5H), 1.35 (s, 3H); TOFꢀMS (ESI) m/z: calcd for
C36H50N5O8S [M + H]+ 712.3375; found 712.3343.
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(R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(tert-butoxycarbonyl)amino-3-
carbamoylpropionyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (25)
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Compound 25 was prepared from 1922 using BocꢀAsnꢀOH in a manner similar to that described for
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compound 20; yield 94%; H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.09 (br. s., 1H), 7.89 (d, J = 8.4
Hz, 1H), 7.32 (d, J = 7.7 Hz, 2H), 7.26ꢀ7.06 (m, 4H), 7.03ꢀ7.00 (m, 2H), 6.87 (br. s., 2H), 6.80 (d, J =
8.3 Hz, 1H), 5.22 (d, J = 7.2 Hz, 1H), 4.95 (d, J = 8.9 Hz, 1H), 4.87 (d, J = 9.1 Hz, 1H), 4.50 (s, 1 H),
4.45 (dd, J = 13.9, 5.9 Hz, 1H), 4.36 (dd, J = 7.2, 2.8 Hz, 1H), 4.22ꢀ4.11 (m, 3H), 2.72ꢀ2.61 (m, 2H),
2.31 (s, 6H), 2.29ꢀ2.20 (m, 2H), 1.45 (s, 3H), 1.41ꢀ1.32 (m, 12H); TOFꢀMS (ESI) m/z: calcd for
C34H48N5O7S [M + H]+ 670.3269; found 670.3244.
(R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(tert-butoxycarbonyl)amino-3-
methylbutanoyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (26)
Compound 26 was prepared from 1922 using BocꢀValꢀOH in a manner similar to that described for
compound 20; yield 77%; 1H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.12 (dd, J = 5.7, 3.2 Hz, 1H), 7.89
(d, J = 8.4 Hz, 1H), 7.36 (d, J = 7.2 Hz, 2H), 7.23ꢀ7.18 (m, 2H), 7.16ꢀ7.05 (m, 2H), 7.04ꢀ6.99 (m, 2H),
6.65 (d, J = 9.7 Hz, 1H), 5.14ꢀ5.03 (m, 2H), 4.89 (d, J = 9.1 Hz, 1H), 4.54 (s, 1H), 4.48 (dd, J = 13.9,
6.2 Hz, 1H), 4.38 (dd, J = 7.4, 1.9 Hz, 1H), 4.23ꢀ4.13 (m, 2H), 3.70ꢀ3.65 (m, 1H), 2.71ꢀ2.60 (m, 2H),
2.31 (s, 6H), 1.78ꢀ1.68 (m, 1H), 1.46 (s, 3H), 1.41ꢀ1.32 (m, 12H), 0.67 (d, J = 6.7 Hz, 3H), 0.56 (d, J =
6.6 Hz, 3H); TOFꢀMS (ESI) m/z: calcd for C35H51N4O6S [M + H]+ 655.3524; found 655.3492.
(R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(3-(benzofuran-2-carbonyl)amino-3-
carbamoylpropionyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (27a, KNI-1539)
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Compound 27a was prepared from compound 25 in a manner similar to that described for compound
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2
3
1
15a; yield 33%; purity 90.2%; H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.60 (d, J = 8.2 Hz, 1H), 8.11
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(dd, J = 5.8, 3.7 Hz, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.83ꢀ7.78 (m, 1H), 7.69 (dd, J = 8.4, 0.8 Hz, 1H),
7.58 (s, 1H), 7.51ꢀ7.46 (m, 1 H), 7.38ꢀ7.28 (m, 4H), 7.09ꢀ7.03 (m, 3H), 7.02ꢀ6.96 (m, 2H), 6.90 (br. s.,
2H), 5.74 (br. s., 1H), 5.01 (d, J = 8.9 Hz, 1H), 4.89 (d, J = 9.1 Hz, 1H), 4.80ꢀ4.73 (m, 1H), 4.52 (s, 1H),
4.50ꢀ4.40 (m, 2H), 4.17 (dd, J = 13.9, 3.4 Hz, 2H), 2.70ꢀ2.62 (m, 2H), 2.56ꢀ2.53 (m, 2H), 2.30 (s, 6H),
1.45 (s, 3H), 1.34 (s, 3H); TOFꢀMS (ESI) m/z: calcd for C38H44N5O7S [M + H]+ 714.2956; found
714.2951.
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(R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(3-(benzofuran-2-carbonyl)amino-3-
methylbutanoyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (27b, KNI-1540)
Compound 27b was prepared from compound 25 in a manner similar to that described for compound
15a; yield 46%; 1H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.26 (d, J = 8.2 Hz, 1H), 8.19 (d, J = 9.3 Hz,
1H), 8.12 (dd, J = 3.5, 6.0 Hz, 1H), 7.78 (td, J = 7.9, 1.3 Hz, 1H), 7.70 (dd, J = 8.4, 0.7 Hz, 1H), 7.62 (d,
J = 0.9 Hz, 1H), 7.48 (dt, J = 7.3, 1.3 Hz, 1H), 7.37ꢀ7.32 (m, 3H), 7.16 (t, J = 7.5 Hz, 2H),7.09ꢀ7.05 (m,
2H), 7.01 (d, J = 7.7 Hz, 2H), 5.20 (br s, 1H), 5.09 (d, J = 8.8 Hz, 1H), 4.93 (d, J = 9.0 Hz, 1H), 4.54 (s,
1H), 4.47 (dd, J = 14.1, 6.2 Hz, 1H), 4.42 (br s, 1H), 4.28ꢀ4.14 (m, 3H), 2.74ꢀ2.60 (m, 2H), 2.3 (s, 6H),
2.02ꢀ1.97 (m, 1H), 1.45 (s, 3H), 1.36 (s, 3H), 0.82 (d, J = 6.6 Hz, 3H), 0.68 (d, J = 6.8 Hz, 3H); TOFꢀ
MS (ESI) m/z: calcd for C39H47N4O6S [M + H]+ 699.3211; found 699.3201.
(R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(7-methoxybenzofuran-2-carbonyl)amino-3-
carbamoylpropionyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (27c, KNI-1565)
Compound 27c was prepared from compound 26 in a manner similar to that described for compound
15a; yield 29%; 1H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.54 (d, J = 8.2 Hz, 1H), 8.10 (dd, J = 5.8, 3.6
Hz, 1H), 8.06 (d, J = 8.6 Hz, 1H), 7.57ꢀ7.53 (m, 1H), 7.36ꢀ7.24 (m, 5H), 7.12ꢀ7.04 (m, 4H), 7.01ꢀ6.97
(m, 2H), 6.90 (br. s., 1H), 5.75 (br. s., 1H), 5.00 (d, J = 8.9 Hz, 1H), 4.89 (d, J = 8.9 Hz, 1H), 4.75 (td, J
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Journal of Medicinal Chemistry
= 7.8, 5.8 Hz, 1H), 4.52 (s, 1 H), 4.48ꢀ4.41 (m, 2H), 4.20ꢀ4.12 (m, 2H), 3.98 (s, 3H), 2.72ꢀ2.62 (m, 2H),
2.57ꢀ2.53 (m, 2H), 2.30 (s, 6H), 1.45 (s, 3H), 1.34 (s, 3H); TOFꢀMS (ESI) m/z: calcd for C39H46N5O8S
[M + H]+ 744.3062; found 744.3040.
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(R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(7-methoxybenzofuran-2-carbonyl)amino-3-
methylbutanoyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (27d, KNI-1566)
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Compound 27d was prepared from compound 26 in a manner similar to that described for compound
15a; yield 40%;1H NMR (400 MHz, DMSOꢀd6) δ (ppm): 8.28 (d, J = 8.4 Hz, 1H), 8.12 (dd, J = 6, 3.5
Hz, 1H), 8.07 (d, J = 9.3 Hz, 1H), 7.60 (s, 1H), 7.35 (d, J = 7.1 Hz, 2H), 7.31 (dd, J = 7.9, 1.1 Hz, 1H),
7.26 (t, J = 7.9 Hz, 1H), 7.16 (t, J = 7.7 Hz, 2H), 7.1ꢀ7.03 (m, 3H), 7.01 (d, J = 7.5 Hz, 2H), 5.19 (d, J =
7.1 Hz, 1H), 5.08 (d, J = 8.8 Hz, 1H), 4.93 (d, J = 9.2 Hz, 1H), 4.54 (s, 1H), 4.47 (dd, J = 14.1, 6.4 Hz,
1H), 4.42 (dd, J = 7.1, 2.7 Hz, 1H), 4.26 (t, J = 8.1 Hz, 1H), 4.24ꢀ4.20 (m, 1H), 4.17 (dd, J = 13.9, 3.3
Hz, 1H), 3.97 (s, 3H), 2.72 (dd, J = 13.9, 2.6 Hz, 1H), 2.64 (dd, J = 13.6, 2.6 Hz, 1H), 2.30 (s, 6H),
2.04ꢀ1.97 (m, 1H), 1.45 (s, 3H), 1.36 (s, 3H), 0.82 (d, J = 6.8 Hz, 3H), 0.69 (d, J = 6.8 Hz, 3H); TOFꢀ
MS (ESI) m/z: calcd for C40H49N4O7S [M + H]+ 729.3316; found 729.3297; Anal. calcd for
C40H48N4O7S·0.1H2O: C, 65.75; H, 6.65; N, 7.67. Found: C, 65.72; H, 6.71; N, 7.76.
HIV protease inhibition
Recombinant HIVꢀ1 protease was purchased from Bachem AG, Bubendorf, Switzerland. In the
inhibition assay, 25 ꢀL of 200 mM 2ꢀ(Nꢀmorpholino)ethanesulfonic acid (MES)–NaOH buffer (pH 5.5),
containing 2 mM dithiothreitol, 2 mM EDTAꢀ2Na and 1M NaCl was mixed with 5 ꢀL of the inhibitor
(10 nM) dissolved in DMSO and 10 ꢀL of HIVꢀ1 protease (40 ng/mL) in 50 mM AcOH (pH 5.0)
containing 1 mM EDTAꢀ2Na, 25 mM NaCl, 0.2% 2ꢀmercaptoethanol, 0.2% Nonidet Pꢀ40, and 10%
glycerol. The mixture was preincubated for 5 min at 37 °C, and the reaction was initiated by adding of
10 ꢀL of a 1.0 mM substrate solution (HꢀLysꢀAlaꢀArgꢀValꢀTyrꢀPhe(pꢀNO2)ꢀGluꢀAlaꢀNleꢀNH2). After
incubation for 3 hours at 37 °C, the reaction was terminated by the addition of 1N HCl, and the Nꢀ
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Page 28 of 49
terminal cleavage fragment (HꢀLysꢀAlaꢀArgꢀValꢀTyrꢀOH) was separated by reversedꢀphase HPLC on a
C18 column (3.0 × 75 mm; YMC Pack ODS ASꢀ3E7) with a linear gradient of CH3CN in 0.1% aqueous
TFA at a flow rate of 1.0 mL/min and its quantity was determined by monitoring fluorescence intensity
(Ex, 275nm; Em, 305 nm).
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Anti-HIV Activity
AntiꢀHIV activity of test compounds was determined based on inhibition of HIVꢀinduced cytopathic
effect in MTꢀ4 cells.31 Wildꢀtype HIVꢀ1 (pNL4ꢀ3) or LPVꢀresistant strain A1711 was inoculated to MTꢀ4
cells in a 96ꢀwell plate. The resulting culture was treated with an equal volume of a 1% DMSO solution
of each test compound with several concentrations and 10% fetal bovine serum, and was incubated for 5
days in a CO2 incubator at 37 °C, in triplicate. After treatment with 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazolium bromide (MTT), the optical density of the plate was measured, and percent
cytopathic effect reduction was calculated, then EC50 values were estimated by fitting the data to a
medianꢀeffect equation. Cytotoxicity (TD50) was determined by incubation in the absence of the virus.
In addition, pNL4ꢀ3ꢀbased, wildꢀtype or DRVꢀresistant luciferase reporter lentiviral vector30 was
generated by transfecting 293T cells with pCMVꢀꢁR8.91 or the derivatives containing
V32I/L33F/I54M/V82I or V32I/L33F/I54M/I84V mutations together with pLenti CMV Puro LUC
(w168ꢀ1) (17477, Addgene, Cambridge, MA) and pHit/G32 using FuGENE HD Transfection Regent
(Promega, Madison, WI) in the presence or absence of HIVꢀ1 protease inhibitor. Fortyꢀeight hours later,
293T cells were infected with lentiviral vector. Twentyꢀfour hours after infection, luciferase activity in
infected cells was measured using a SteadyꢀGlo Luciferase Assay System (Promega, Madison, WI) with
an LB962 microplate luminometer (Berthold, Bad Wildbad, Germany). Drug susceptibility was
evaluated as a reduction in luciferase activity in infected cells. The 50% inhibitory concentration (IC50)
of protease inhibitors for suppressing the transduction of luciferase gene was calculated from the dose–
response curve using a standard function of GraphPad Prism 5 software (GraphPad Software, San
Diego, CA).
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Journal of Medicinal Chemistry
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Preparation and X-Ray Crystallographic Analysis of WTm5 and A17m5
The chemically synthesized DNA encoded the gene for the initial methionine and the 99 amino acid
including five mutations of Q7K, L33I, L63I C67A and C95A (WTm5) and the gene derived from A17
type HIVꢀ1 protease11 with additional lopinavir/ritonavirꢀresistant mutations of L10F, V32I, M46I,
I47V, Q58E, I84V (A17m5) were used for expression in E. coli. Preparation of WTm5 and A17m5 for
crystallization was performed as reported.16 WTm5 and A17m5/21f complexes were prepared with a 2ꢀ
fold molar excess of inhibitors to protease at 5.0 and 2.0 mg/mL protein concentrations, respectively.
The both complexes were crystallized using hanging drop vapor diffusion method by mixing in equal
volumes (2 ꢁl each) of protease solution and crystallization buffer containing 126 mM phosphate buffer
at pH 5.0, 63 mM sodium citrate. For crystallization of A17m5/21f complex, PEG4000 was used as
precipitant for crystallization at the final concentration of 20% (w/v). The obtained crystals were flashꢀ
frozen under a N2 gas cryoꢀstream (100 K), and subjected to Xꢀray at BL41XU beamline in SPringꢀ8.
Data for WTm5/21f and A17m5/21f crystals belongs to the same space group of P21212 with similar cell
constants, and were processed to the resolution of 0.85 and 1.50 Å resolution, respectively, by using
HKL2000.33 The structures of WTm5/21f and A17m5/21f complexes were refined to a crystallographic
Rꢀfactor of 10.3% (free Rꢀfactor = 12.5%) using program SHELXꢀ9734, and Rꢀfactor of 17.1% (free Rꢀ
factor = 18.8%) using program Refmac535 and PHENIX 1.936, respectively (see Supporting Information
for Xꢀray diffraction data).
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Associated Content
Supporting Information: The supporting information is available free of charge on the ACS
publication website. HPLC analysis of target compounds; Xꢀray diffraction data collection and
refinement statistics; Supplementary figure (PDF) Molecular formula strings (CSV)
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