Design of Highly Potent, Dual-Acting and Central-Nervous-System-Penetrating HIV-1 Protease Inhibitors with Excellent Potency against Multidrug-Resistant HIV-1 Variants

Accepted Article

Title: Design of Highly Potent, Dual Acting and Central Nervous

System Penetrating HIV-1 Protease Inhibitors with Excellent

Potency against Multidrug-Resistant HIV-1 Variants

Authors: Arun K Ghosh, Kalapala Venkateswara Rao, Prasanth R.

Nyalapatla, Satish Kovela, Margherita Brindisi, Heather L.

Osswald, Bhavanam Sekhara Reddy, Johnson Agniswamy,

Yuan-Fang Wang, Manabu Aoki, Shin-ichiro Hattori, Irene T.

Weber, and Hiroaki Mitsuya

This manuscript has been accepted after peer review and appears as an

Accepted Article online prior to editing, proofing, and formal publication

of the final Version of Record (VoR). This work is currently citable by

using the Digital Object Identifier (DOI) given below. The VoR will be

published online in Early View as soon as possible and may be different

to this Accepted Article as a result of editing. Readers should obtain

the VoR from the journal website shown below when it is published

to ensure accuracy of information. The authors are responsible for the

content of this Accepted Article.

To be cited as: ChemMedChem 10.1002/cmdc.201700824

Link to VoR: http://dx.doi.org/10.1002/cmdc.201700824

A Journal of

www.chemmedchem.org

10.1002/cmdc.201700824

ChemMedChem

FULL PAPER

Design of Highly Potent, Dual Acting and Central Nervous System

Penetrating HIV-1 Protease Inhibitors with Excellent Potency

against Multidrug-Resistant HIV-1 Variants

Arun K. Ghosh*^[a], Kalapala Venkateswara Rao,^[a]Prasanth R. Nyalapatla_,^[a]Satish Kovela_,^[a]

Margherita Brindisi,^[a]Heather L. Osswald,^[a]Bhavanam Sekhara Reddy,^[a]Johnson Agniswamy,^[b]

Yuan-Fang Wang,^[b]Manabu Aoki,^[c,d,e]Shin-ichiro Hattori,^[e]Irene T. Weber,^[b]and Hiroaki Mitsuya ^[c,d,e]

[a]

Prof. Dr. A. K. Ghosh, Dr. K. V. Rao, Dr. P. R. Nyalapatla, Dr. S. Kovela, Dr. M. Brindisi, Dr. H. L. Osswald, Dr. B.S.Reddy

Department of Chemistry and Department of Medicinal Chemistry

Purdue University, West Lafayette, IN 47907

E-mail: akghosh@purdue.edu

[b]

[c]

Prof. Dr. I. T. Weber, Dr. J. Agniswamy, Y.-F. Wang

Departments of Biology and Chemistry, Molecular Basis of Disease

Georgia State University, Atlanta, GA 30303

Dr. H. Mitsuya, Dr. M. Aoki, Dr. M. Amano

Departments of Hematology and Infectious Diseases

Kumamoto University School of Medicine

Kumamoto 860-8556, Japan

[d]

[e]

Prof. Dr. H. Mitsuya

Experimental Retrovirology Section, HIV and AIDS Malignancy

Branch, National Cancer Institute, Bethesda, MD 20892

Dr. H. Mitsuya

Center for Clinical Sciences, National Center for Global Heath and Medicine

Shinjuku, Tokyo 162-8655, Japan

Supporting information for this article is given via a link at the end of the document

Abstract: We report here the design, synthesis, X-ray-structural,

and biological studies of an exceptionally potent HIV-1 protease

inhibitor 5. In this inhibitor, we incorporated an unprecedented

structure-based

designed

6-5-5-ring

the

fused

crown-like

tetrahydropyranofuran

as

P2-ligand,

a

cyclopropylaminobenzothiazole as the P2'-ligand and 3,5-

difluorophenylmethyl as the P1-ligand. The resulting inhibitor 5,

exhibited exceptional HIV-1 inhibitory and antiviral potency at

picomolar level. Furthermore, it displayed antiviral IC₅₀values in

picomolar range against a wide panel of highly multidrug-resistant

HIV-1 variants. The inhibitor shows an extremely high genetic barrier

against the emergence of drug-resistant variants. It also showed

extremely potent dimerization inhibitory activity and penetrated CNS

favourably. We have determined a high resolution X-ray structure of

inhibitor 5-bound HIV-1 complex which provided molecular insight

into the unprecedented activity profiles.

Introduction

The advent of combined antiretroviral therapy (cART) in the late

1990s is the most significant development in the treatment of

patients with HIV-1 infection and AIDS.^[1,2]The introduction of

these drug regimens for using

a combination of reverse

transcriptase and protease inhibitor drugs dramatically

suppressed viral replication, reduced plasma HIV-1 viral loads

and improved CD4 cell counts in HIV-1-infected patients.^[3,4]The

cART has significantly improved life expectancy for patients who

have access to these treatment regimens. There is no cure for

HIV-1 infection or AIDS at the moment.⁵However, mortality

rates of HIV/AIDS patients are approaching those of the general

population.^[6,7]Despite these advances, the growing emergence

Figure 1. Structures of HIV-1 protease inhibitors 1-5.

1

10.1002/cmdc.201700824

ChemMedChem

FULL PAPER

of multidrug-resistant HIV-1 variants has raised serious

concerns for the long-term viability of current treatment

options.^[8,9]Among cART, HIV-1 protease inhibitor (PI) drugs

generally show high genetic barrier against resistance. The

latest approved PI, darunavir (1, figure 1), is the only PI drug

recommended for first line therapy.^[10-12]It exhibits a high genetic

barrier and it is the most widely prescribed PI for treatment-

experienced and treatment-naïve patients with HIV-1 infection

and AIDS.^[13]This is possibly due to darunavir’s dual mechanism

of action as (i) an inhibitor of catalytic HIV-1 protease, and (ii) an

inhibitor of dimerization of protease monomers.^[14,15]The current

first-line cART with boosted PI-based and integrase inhibitor-

based regimens are effective against developing drug-resistant

HIV-1 variants over an extended period of time. However, there

are limitations, including side effects, toxicity, and rapid

development of drug-resistant variants for some HIV/AIDS

patient groups.^[16,17]Furthermore, current cART regimens are

ineffective for patients with HIV associated neurocognitive

disorders (HAND). A sub-therapeutic drug-concentration in the

central nervous system (CNS) may lead to development of

resistance and less virologic suppression in the CNS.^[18-20]

speculated that incorporation of fluorine atoms in the P1-ligand

would improve van der Waals contact in the S1-subsite. Our

hope is that these fluorine atoms in this PI would increase

lipophilicity which would improve drug penetration in the

CNS.^[28,29]Indeed, inhibitor 5 with a 3,5-difluorobenzyl moeity as

the P₁-ligand resulted in an exceptionally potent PI. This inhibitor

exhibited unprecedented drug properties.^[23]We have also

prepared mono-fluoro derivatives and compared their antiviral

activities against multidrug-resistant HIV-1 variants.

Synthesis of Fluoroisosteres and Inhibitors

Our

synthesis

of

3,5-difluorobenzene

containing

hydroxyethylamine sulfonamide isostere precursor in optically

active form (9) and its monofluoro counterparts (10, 11), is

shown in Scheme 1. Commercially available 1-bromo-3,5-

difluorobenzene 6a was converted to the corresponding

Grignard reagent. Reaction of the resulting 3,5-difluorophenyl

magnesium bromide with commercially available butadiene

monoxide in the presence of a catalytic amount of CuCN at -

78 °C provided allylic alcohol 7a in 96% yield.^[30]This

In an effort to address the above issues, our PI design

strategy is aimed at targeting the protein backbone to develop

PIs that are very potent with greater specificity to HIV-1, show

high genetic barrier to the emergence of multidrug-resistant HIV-

1

variants, and are capable of penetrating blood-brain

barrier.^[10,21-23]Herein, we report a novel HIV-1 PI containing a 6-

5-5-ring-fused crown-like-tetrahydropyranofuran as the P2-

ligand and an aminobenzothiazole as the P2' ligand and a two-

fluorine containing Phe-side chain as the P1-ligand. The

resulting PI exhibited an unprecedented antiviral activity against

a wide range of highly multidrug-resistant HIV-1 variants, and

displayed high genetic barrier against the emergence of

resistant variants. It also displayed very potent inhibition of

dimerization of protease monomers and favorable CNS-

penetration capability. We have carried out X-ray structural

studies of inhibitor-bound HIV-1 protease complex to gain

structural insight.

Since our design and discovery of DRV, our continuing

efforts focused on developing PIs that exhibit robust antiviral

potency against

a

variety of existing multi-PI-resistant

variants.^[10,24,25]Our objectives are that these PIs need to

significantly delay the emergence of HIV-1 variants resistant to

the same PI and show better CNS penetration capability and

exhibit potent dimerization inhibition activity.^[26]Towards these

goals, we have reported a variety of novel PIs that met many of

these critical objectives. Recently, we reported the design and

synthesis of a new class of PIs incorporating an unprecedented

6-5-5-ring-fused crown-like tetrahydropyranofuran (crn-THF) as

the P2-ligand and cyclopropylamino-benzothiazole (Cp-Abt) as

the P2'-ligand on a (R)-hydroxyethylsulfonamide isostere as the

transition-state mimic.^[23,27]Inhibitor 3, showed superior antiviral

activity and drug-resistance over darunavir (1, DRV). Our

detailed structural analysis also provided molecular insight into

its exceptional drug-resistance profiles. Based upon this

structural insight, we have further optimized ligand-binding site

interactions in the active site of HIV-1 protease to improve drug

properties of inhibitor 5. Our overall design objectives were to

preserve all critical hydrogen bonding interactions with the

backbone atoms of HIV-1 protease active site, while enhancing

van der Waals interaction in the active site. In particular, we

Scheme 1. Synthesis of azidoepoxide 9. Reagents and chemicals. (a) Mg, I₂,

THF, 23 °C; (b) butadienemonoxide, CuCN (catalytic), -78 °C (96% for 2-

steps); (c) ^tBuOOH, (-)-DET, Ti(O-iPr)₄, CH₂Cl₂, -20 °C; (d) Ti(O-iPr)₄, TMSN₃,

PhH, (71% for 2-steps); (e) AcOMe₂CCOCl, CHCl₃, 0 °C to 23 °C; (f) NaOMe,

THF, 23 °C, (63% for 2-steps).

was subjected to Sharpless asymmetric epoxidation^[31,32]using (-

)-diethyl-D-tartrate to provide the corresponding epoxide in very

good yield. Regioselective epoxide opening of the resulting

epoxide with TMSN₃and Ti(OiPr)₄afforded azido diol 8a in 71%

yield over two-steps.^[33]Reaction of this diol with 2-

acetoxyisobutyryl chloride in CHCl₃at 0 °C to 23 °C provided

chloroacetate derivative which was treated with NaOMe in THF

at 23 °C to afford azidooxirane 9 in 63% yield over two-steps.^[34]

Monofluoro oxirane derivatives 10 and 11 were prepared

following the above sequence of reactions utilizing commercially

available fluorobromides (please see Experimental Section).

2

10.1002/cmdc.201700824

ChemMedChem

FULL PAPER

Conversion of azidooxiranes to the corresponding

Scheme 3. As shown, reaction of oxiranes 10 and 11 with

isobutylamine furnished aminoalcohol derivatives 12b and 12c.

fluoroisosteric amines 13 and 17 is shown in Scheme 2.

Reaction of azidooxirane 9 with isobutylamine in isopropanol at

65 °C for 12 h afforded aminoalcohol 12a in excellent yield.

Treatment of aminoalcohol 12a with commercially available 4-

nitrobenzenesulfonyl chloride in the presence of Et₃N in CH₂Cl₂

at 23 °C for 12 h provided the corresponding sulfonamide

derivative in 67% yield from 9. Catalytic hydrogenation of the

resulting azidosulfonamide derivative with Pd(OH)₂in EtOAc at

23 °C for 12 h afforded diamine derivative 13 in 89% yield.^[35]

Aminoalcohol

12a

was

converted

to

cyclopropylaminobenzothiazole derivatives

These

Scheme 3. Synthesis of sulfonamide isosteres 18 and 19. Reagents and

chemicals. (a) i-BuNH₂, i-PrOH, 65 °C; (b) 14, CH₂Cl₂, Et₃N, 23 °C, (90% over

2-steps for 15a and 87% over 2-steps for 15b); (c) Ph₃P, THF-H₂O (3:1),

23 °C; (d) Boc₂O, THF-H₂O (1:1), NaHCO₃, 23 °C (83% over 2-steps for 16b

and 70% over 2-steps for 16c);

(e) m-CPBA, CH₂Cl₂, 23 °C; (f)

cyclopropylamine, THF, 65 °C, (97% over 2-steps 18 and 98% over 2-steps for

19); (g) TFA, CH₂Cl₂, 0 °C.

aminoalcohols were reacted with sulfonyl chloride 14 to provide

sulfonamide derivatives 15b and 15c in excellent yields.

Reduction of azide with Ph₃P followed by protection of the

resulting amines gave Boc-derivatives 16b and 16c. The

methylbenzothiazole derivatives were converted to mono-

fluorinated isosteric amines 18 and 19 by following the same

sequence of reactions as for the difluorinated isostere 17.

Synthesis of inhibitors with fluoroisosteres is shown in

Scheme 4. Synthesis of optically active crown-THF alcohol and

its corresponding activated carbonate 20 has been described

recently.^[27]Reaction of 4-aminobenzene sulfonamide derivative

13 with 3,5-difluorophenylmethyl as the P1-ligand and activated

carbonate 20 in the presence of diisopropylethylamine (DIPEA)

in CH₃CN at 23 °C for 72 h furnished inhibitor 4 in 99% yield.

Similarly, reaction of activated carbonate 20 with fluoroisostere

17 in CH₃CN under similar conditions, afforded inhibitor 5 in 88%

yield. Monofluoro derivatives 18 and 19 were reacted with

activated carbonate 20 to provide inhibitors 21 and 22 in 95%

and 98% yields, respectively.

Scheme 2. Synthesis of sulfonamide isosteres 13 and 17. Reagents and

chemicals. (a) i-BuNH₂, i-PrOH, 65 °C; (b) 4-NO₂-PhSO₂Cl, Et₃N, CH₂Cl₂,

23 °C (67%, for 2-steps); (c) H₂, Pd(OH)₂-C, EtOAc, 23 °C (89%); (d) 14,

CH₂Cl₂, Et₃N, 23 °C, (90% over 2-steps); (e) Ph₃P, THF-H₂O (3:1), 23 °C; (f)

Boc₂O, THF-H₂O (1:1), NaHCO₃, 23 °C, (85% over 2-steps); (g) m-CPBA,

CH₂Cl₂, 23 °C; (h) cyclopropylamine, THF, 65 °C, (91% over 2-steps); (i) TFA,

CH₂Cl₂, 0 °C (99%).

as follows. Treatment of aminoalcohol 12a with sulfonyl chloride

14 in the presence of Et₃N at 23 °C for 12 h furnished

sulfonamide derivative 15a in excellent yield.^[27]This was

subjected to Staudinger reduction^[36]with Ph₃P in aqueous THF

at 23 °C for 24 h to provide the corresponding amine. The

resulting amine was reacted with (Boc)₂O in the presence of

NaHCO₃to provide Boc-derivative 16a in very good yield. This

was converted to aminobenzothiazole derivative 17 as follows.

Methyl sulfide was oxidized to its sulfone derivative with mCPBA

in CH₂Cl₂at 0 °C to 23 °C for 12 h. Treatment of the resulting

sulfone with cyclopropylamine in THF at 65 °C for 12 h afforded

the aminobenzothiazole derivative.^[27]Removal of the Boc group

by exposure to trifluoroacetic acid (TFA) in CH₂Cl₂at 0 °C to

23 °C for 1 h furnished difluoroisostere 17 in excellent yield. The

synthesis of monofluoro isosteres 18 and 19 is shown in

3

10.1002/cmdc.201700824

ChemMedChem

FULL PAPER

inhibitory potency with K_ivalue of 4 pM. Antiviral activity of this

inhibitor was determined in MT-2 human-T-lymphoid cells

[17]

exposed to HIV_LAI

.

Compound 4 displayed antiviral IC₅₀value

of 1.7 nM compared to IC₅₀values of 2.8 nM for DRV.

Incorporation of cyclopropylaminobenzothiazole as the P2'-

ligand resulted in inhibitor 5 with K_ivalue of 14 pM. It showed

antiviral IC₅₀value of 0.017 nM, a more than 150-fold potency

enhancement over DRV and a 100-fold improvement over

compound 4. Importance of bis-fluorines on the P1-ligand is

clearly evident as this inhibitor is over 15-fold more potent than

inhibitor

3

with no fluorines. Inhibitor 21 with 3-

fluorophenylmethyl derivative as the P1-ligand showed HIV-1

protease inhibitory K_iof 0.3 pM and antiviral IC₅₀value of 0.055

nM,

a

significant improvement over p-aminosulfonamide

derivative 4, but less potent than difluoro derivative 5. Antiviral

IC₅₀values for inhibitors 5 and 21 were 0.017±0.004 and

0.055±0.0024, respectively in the same assay. The

corresponding inhibitor 22 with a 4-fluorophenylmethyl as the P1

ligand showed reduction of enzyme K_iand antiviral IC₅₀values

compared to 3-fluoro derivative 21. Overall, inhibitor 5 with 3,5-

bis-fluorines on the P1-ligand displayed superior antiviral

potency over sulfonamide derivative

4

and monofluoro

derivatives 21 and 22.

Table 1. Structures and activity of inhibitors 3-5, 21-23.

Scheme 4. Synthesis of PIs. Reagents and chemicals. (a) amines 13, 17-19

and carbonate 20, DIPEA, CH₃CN, 23 °C.

Results and Discussion

We have recently reported a new class of crn-THF-derived

inhibitors as represented in PI 3.^[27]In this class of compounds

we were able to demonstrate how the replacement of a bis-THF

with a crown-THF P2 ligand led to enhanced van der Waals

interaction in the S2 site. These increased hydrophobic

interaction, in addition to the robust hydrogen bond interaction

pattern of a cyclopropylaminobenzothiazole-based P2' ligand,

led to inhibitor 3, which exhibited unprecedented potency

particularly against multidrug-resistant HIV-1 strains. Based

upon our examination of the X-ray crystal structure of DRV and

HIV-1 protease complex as well as our crown-THF-derived

inhibitor 3-bound HIV-1 protease, we planned further

modifications to improve ligand-binding site interactions.^[27,37]In

particular, we speculated that insertion of fluorine atom on the

P1-phenyl ring may lead to effective van der Waals interactions

with Pro81' and V82' residues in the active site. To test this

hypothesis, we designed and synthesized inhibitors 4 and 5 with

3,5-difluoro derivatives. We have also prepared inhibitors 21 and

22 with monofluorophenylmethyl derivatives as the P1-ligand.

The structure and activity of these inhibitors are shown in Table

1. For determination of HIV-1 protease inhibitory activity, we

utilized the assay protocol reported by Toth and Marshall.^[38]As

can be seen, inhibitor 4 with crown-THF as the P2-ligand and

two-fluoro-phenylmethyl as the P1-ligand showed enzyme

4

10.1002/cmdc.201700824

ChemMedChem

FULL PAPER

Table 2. Antiviral activity of three novel compounds against highly DRV-resistant HIV-1 variants.

Mean IC₅₀in nM ± SD (fold-change)

ATV

5.2 ± 0.3

>1,000 (>192)

DRV

3.7 ± 0.7

60 ± 6 (16)

134 ± 43 (36)

4,672 ± 1,263

4

3 (GRL-12113A)

0.26 ± 0.03

0.18 ± 0.1 (0.7)

1.4 ± 0.3 (5.3)

34 ± 10 (131)

5 (GRL-14213A)

0.016 ± 0.017

0.0016 ± 0.002 (0.1)

0.015 ± 0.015 (1)

2.8 ± 0.2 (175)

WT

^cHIV_nl4-3

HIV_DRV

5.9 ± 0.4

24 ± 9 (4)

11 ± 13 (1.9)

486 ± 52 (82)

R

P20

P30

P51

R

MT-4 cells (1 x 10⁴) were exposed to 50 TCID₅₀s of wild-type HIV-1_NL4-3or highly DRV resistant HIV-1 variants (HIV-1_{DRV P20}, HIV-

R

1_{DRV P30}, or HIV-1_{DRV P51}) and cultured in the presence of various concentrations of each compound, and the IC₅₀values were

determined by the p24 assay. All assays were conducted in triplicate, and the data shown represent mean values (±1 standard

deviation) derived from the results of two independent experiments.

R

The amino acid substitutions identified in protease of HIV-1_{DRV P20}, HIV-1_{DRV P30}, and HIV-1_{DRV P51}compared to HIV-1_NL4-3

include

L10I/I15V/K20R/L24I/V32I/M36I/M46L/L63P/A71T/V82A/I84V/L89M,

L10I/I15V/K20R/L24I/V32I/M36I/M46L/L63P/A71T/V82A/L89M,

and

L10I/I15V/K20R/L24I/V32I/L33F/M36I/M46L/I54M/L63P/A71T/V82I/I84V/L89M, respectively.

significantly improved (Table 2). Inhibior 3 containing Crn-THF

As mentioned earlier, PI drugs, particularly DRV are widely

used for the treatment of naïve and experienced patients.

Nonetheless, heavily-ART regimen experienced HIV/AIDS

patients are showing treatment-failure with currently approved

PIs, including DRV.^13,17As a result, our inhibitor design is

focused on developing PIs that can maintain robust antiviral

activity against the existing multi-PI-resistant HIV-1 variants.

Thus, we have evaluated inhibitor 5 against DRV-resistant HIV-1

variants and compared antiviral IC₅₀values against approved

PIs and inhibitors 3 and 4.

and Cp-Abt as the P2 and P2‘-ligands and no fluorines at the

P1-phenyl ring, blocked the replication of HIV-1_Nl4-314 to 23-fold

better than DRVand inhibitor 4. Also, this inhibitor more

effectively blocked the replication of all three highly DRV-

resistant HIV-1 variants than inhibitor 4.

Inhibitor 5, resulting from incorporation of two-fluorines on

the P1-ligand of inhibitor 4 showed remarkable antiviral activity

in low picomolar concentration against two DRV-resistant HIV-1

R

variants (HIV_DRV

and HIV_{DRV P30}) and IC₅₀values were 1.6

P20

In these studies, MT-4 cells (1x10⁴) were exposed to wild-

type HIV-1, HIV-1_NL4-3and three highly DRV-resistant HIV-1

pM and 15 pM, respectively. In contrast, DRV exhibited a 16-fold

increase in its IC₅₀values (IC₅₀= 60 nM) compared to its IC₅₀

R

variants, HIV_{DRV P20}, HIV_{DRV P30}, and HIV_DRV

.

^[17,39]These were

value against HIV-1_Nl4-3. Interestingly, inhibitor 5 displayed over

P5

R

10-fold improvement of its IC₅₀against HIV_DRV

compared to

P20

selected by propagating in the presence of increasing

concentration of DRV^.[17]These resistant variants are highly

resistant to all currently approved PIs, including DRV and

nucleoside-RT inhibitors such as tenofovir.^[17,23,39]Antiviral IC₅₀

values were determined using p24 assays and the results are

shown in Table 2. As observed in our investigation, ATV failed to

block the replication of these DRV-resistant HIV-1 variants.

Inhibitor 4 containing crn-THF and two-fluoro-phenylmethyl as

the P1 ligand and 4-aminobenzenesulfonamide as the P2‘ ligand

its IC₅₀against HIV-1_Nl4-3. Furthermore, inhibitor 5 exhibited

R

comparable IC₅₀values against HIV_DRV

(IC₅₀= 15 pM)

P30

compared to its IC₅₀value against HIV-1_Nl4-3. Against the most

R

highly DRV-resistant HIV variants, HIV_{DRV P51}, inhibitor 5 showed

175-fold loss of antiviral activity (IC₅₀= 2.8 nM) compared to its

IC₅₀value against wild type virus. In comparison, DRV displayed

R

>1000-fold increase in its IC₅₀values against HIV_DRV

(IC₅₀=

P51

4,672 nM). Inhibitor 5 also exerted very potent antiviral activity

against the HIV-2 variants.^[23]In addition, inhibitor 5 showed

much improved cytotoxicity profile with a selectivity index

showed comparable antiviral activity against wild-type HIV_Nl4-3

howeverits activity against DRV-resistant varuants was

,

Table 3. Antiviral activity of inhibitor 5 (GRL-14213A) against highly PI-resistant HIV-1 variants.

Mean IC₅₀in nM ± SD (fold-change)

Virus

ATV

DRV

3 (GRL-12113A)

5 (GRL-14313A)

WT

HIV_nl4-3

5.2 ± 0.3

548 ± 177(105) 22 ± 6 (5.9)

3.7 ± 0.7

0.39 ± 0.06^a

0.016 ± 0.017

HIV_SQV-5µM

HIV_IDV-5µM

HIV_NFV-5µM

HIV_APV-5µM

HIV_LPV-5µM

HIV_ATV-5µM

HIV_TPV-15µM

0.03 ± 0.01 (0.08)^a

0.012 ± 0.013 (0.03)^a

0.00020 ± 0.00002 (0.01)

0.00025 ± 0.00035 (0.02)

0.00068 ± 0.00045 (0.04)

0.00001 ± 0.00001 (0.0006)

0.0000024 ± 0.0000016 (0.0002)

0.02 ± 0.01 (1.3)

59 ± 13 (11)

19 ± 1 (3.7)

2.4 ± 0.7 (0.5)

36 ± 9 (7)

>1,000 (>192)

42 ± 1 (11)

6.1 ± 0.8 (1.6) 0.028 ± 0.02 (0.07)^a

43 ± 22 (12)

275 ± 64 (74)

26 ± 1 (7)

0.27 ± 0.1 (0.7)^a

0.0026 ± 0.0006 (0.007)^a

0.19 ± 0.06 (0.5)^a

41 ± 3 (11)

0.057 ± 0.03 (0.15)^a

0.00037 ± 0.00007 (0.02)

^aThe values are from [27].

MT-4 cells (1 x 10⁴) were exposed to 50 TCID₅₀s of wild-type HIV-1_NL4-3or highly PI resistant HIV-1 variants (HIV-1_SQV-5mM, HIV-1_IDV-5mM, HIV-1_NFV-

_5mM, HIV-1_APV-5mM, HIV-1_LPV-5mM, HIV-1_ATV-5mM, or HIV-1_TPV-5mM) and cultured in the presence of various concentrations of each compound, and the IC₅₀

values were determined by the p24 assay. All assays were conducted in triplicate, and the data shown represent mean values (±1 standard deviation)

derived from the results of two independent experiments.

The amino acid substitutions identified in protease of HIV-1_SQV-5mM, HIV-1_IDV-5mM, HIV-1_NFV-5mM, HIV-1_APV-5mM, HIV-1_LPV-5mM, HIV-1_ATV-5mM, and HIV-

1_TPV-15mMcompared

to

HIV-1_NL4-3

include

L10I/N37D/G48V/I54V/L63P/G73C/I84V/L90M,

L10F/L24I/M46I/I54V/L63P/A71V/G73S/V82T,

L10F/K20T/D30N/M46I/A71V/T74S, L10F/M46I/I50V/I84V, L10F/V32I/M46I/I47A/A71V/I84V, L23I/E34Q/K43I/M46I/L50L/G51A/L63P/A71V/V82T, and

L10I/L33I/M36I/M46I/I54VK55R/I62V/L63P/A71V/G73S/V82T/L90M/I93L, respectively.

5

10.1002/cmdc.201700824

ChemMedChem

FULL PAPER

(CC₅₀/IC₅₀) as high as 2,473,684 compared to DRV (41,562).^[23]

We further investigated the ability of both inhibitors 3 and 5

against seven resistant variants selected in vitro against each of

seven FDA-approved PIs. These drug-resistant HIV-1 variants

were selected by propagating HIV-1_NL4-3or a mixture of 11 multi-

drug resistant HIV-1 clinical isilates in the presence of increasing

concentrations (up to 5 or 15 µM) of each FDA-approved PI in

MT-4 cells.^[17,40]The results are shown in Table 3. ATV failed to

block the replication of these variants effectively. DRV

contrasted favorably compared to ATV and DRV variants with

IC₅₀values ranging from 6.1 nM to 275 nM with fold changes 1.6

to 74-fold. Inhibitor 3 without fluorines, exhibited very potent

antiviral activity against all seven resistant HIV-1 variants,

showing IC₅₀values ranging from 2.6 pM to 0.27 nM.^[27]

Inihibitor 5 exhibited unprecedented antiviral activity against all

seven highly P1-resistant HIV-1 variants. This inhibitor showed

antiviral IC₅₀values, ranging from 2.4 fM (0.0000024 nM) to 0.02

nM against these drug-resistant HIV-1 variants. While inhibitor 3

very effectively blocked the replication of highly PI-resistant HIV-

1 variants, the corresponding fluorinated inhibitor 5 has shown

superior antiviral activity compared to inhibitor 3 without fluorine.

Of particular note, inhibitor 5 has shown an extremely high

genetic barrier to the emergence of HIV-1 variants resistant to

this inhibitor.^[23]

For the acquisition of proteolytic activity of HIV-1 protease,

dimerization of HIV-1 protease subunits is an essential process.

Thus, disruption of the dimerization process can inhibit HIV-1

maturation. We previously reported that DRV potently inhibited

dimerization of HIV-1 protease monomer subunits^.[39,40]This was

demonstrated by the fluorescence resonance energy transfer

(FRET)-based HIV-1 expression assay.^[15]The majority of other

PIs do not show dimerization inhibition activity. We examined

the dimerization inhibition properties of inhibitor 5. As shown in

Figure 2, we observed that in the absence of inhibitor, the mean

CFP^A/Bratio was 1.07, which indicates that protease dimerization

clearly occurred. In the presence of 100 nM concentration of

DRV, the ratio decreased to 0.89, demonstrating that DRV

blocked the dimerization of the protease subunits. However,

inhibitor 5 blocked dimerization of protease subunits much more

effectively at 0.1 nM concentration, significantly lower than in the

case of DRV. It appears that inhibitor 5 very potently inhibited

dimerization by at least a factor of 1000-fold compared to DRV.

This data demonstrate that inhibitor 5, like DRV, exhibits dual

mechanism of action, (i) inhibition of HIV-1 protease dimerization

and (ii) inhibition of proteolytic activity of HIV-1 protease.

We have examined CNS penetration of inhibitor 5 in rats

and compared that with DRV.^[23]Both inhibitor 5 and DRV were

administered orally in two rats at a dose of 5 mg/kg with RTV

(8.33 mg/kg). The C_maxwere achieved at 360 min for inhibitor 5

and 90 min for DRV. The concentrations of inhibitor 5 in brain

were 7.24 ± 9.65 and 32.6 ± 1.4 nM in 60 and 360 min,

respectively. A brain concentration of 32.6 nM indicates an

approximately 1880-fold greater drug level with respect to its

IC₅₀value and an approximately 114-fold greater than its IC₉₅

value for inhibitor 5.^[23]This data strongly suggest that inhibitor 5

would potently block HIV-1 replication in the brain.

X-ray description

To gain molecular insight into the ligand-binding site

interactions of inhibitor 5 (GRL14213), we co-crystallized this

inhibitor with wild-type HIV-1 protease and the X-ray structure

was refined at a resolution of 1.67 Å. The structure was

determined at a higher resolution than our recently reported

structure with 2.0 Å resolution.^[23]The complex crystallized in

orthorhombic space group P2₁2₁2 with one protease homodimer

per asymmetric unit. The inhibitor binds in the active site in two

alternate conformations related by 180° rotation with relative

occupancy of 0.55/0.45. The overall dimer structure can be

compared to HIV-1 protease and DRV complex^[37]with a low

RMSD of 0.2 Å for 198 equivalent Ca atoms. The largest

deviation of 0.7 Å on comparison of the two structures occurs at

residue Phe53¢ in the flap region. Inhibitor 5 retains all the

hydrogen bonds observed between DRV and the main chain

atoms of protease. Our previously reported inhibitor 5-HIV-1

protease complex was determined in P6₁space group at 2 Å

resolution and the structure shows good similarity with a RMSD

of 0.6 Å for 198 equivalent Ca atoms.^[23]Large deviations of 1.8

Å and 1.9 Å occur at Gly40 and Pro44¢, respectively, likely due

to differences in the crystal packing contacts of the two

structures. The key inhibitor-binding site interactions are shown

in Figure 3. An overlay of X-ray structures of DRV-bound HIV-1

protease and inhibitor 5-bound HIV-1 protease is shown in

Figure 4.

Figure 2. Inhibition of HIV-1 protease dimerization inhibition activity by GRL-14213A. COS7 cells were exposed to various concentrations (0.01 to

100 nM) of GRL-142 or DRV and were subsequently co-transfected with two plasmids, pHIV-PRWT-CFP and pHIV- PRWT-YFP, respectively. After 72

hr, cultured cells were examined in the FRET-based HIV-1 expression assay and the CFPA/B ratios (Y axis) were determined. The arithmetic mean

values of the ratios obtained are shown as horizontal bars. A CFPA/B ratio that is greater than one signifies that protease dimerization occurred,

whereas a ratio that is less than one signifies that the disruption of protease dimerization occurred. All the experiments were conducted in a blind

6

fashion. The P values were determined using the Wilcoxon rank-sum test (JMP software, SAS, Cary, NC) and were 0.3816 for the CFPA/B ratio in the

absence of drug (CFPA/BNo-Drug) versus the CFPA/B ratio in the presence of 10 nM DRV (CFPA/B10-DRV), 0.004 for CFPA/BNo-Drug versus

CFPA/B100-DRV, 0.8192 for CFPA/BNo-Drug versus CFPA/B0.01- GRL-142, 0.0459 for CFPA/BNo-Drug versus CFPA/B0.1-GRL-142, 0.0197 for

CFPA/BNo-Drug versus CFPA/B1-GRL-142, 0.0248 for CFPA/BNo-Drug versus CFPA/B10-GRL-142, and 0.0003 for CFPA/BNo-Drug versus CFPA/

B100-GRL-142.

10.1002/cmdc.201700824

ChemMedChem

FULL PAPER

In the present high resolution structure, two fluorine atoms

forms hydrophobic interactions with Gly48, Ile50 and Pro81¢.

The second fluorine atom forms multipolar interactions with the

guanidium group of Arg8¢, which is involved in a critical

intersubunit ion pair with Asp29. The second fluorine atom also

has van der Waals contact with Val82¢. The P2 group contains

the crn-THF instead of bis-THF in DRV. Both oxygen atoms in

the crn THF form hydrogen bond interactions with the main

chain amide of Asp29 and Asp30 similar to those observed for

on the P1 ligand of inhibitor 5 form important halogen bond

interactions with the protease. As shown in Figure 5, one of the

fluorine atoms interacts with the tips of both flaps by forming a

strong polar interaction (2.8 Å distance) with the main chain NH

group of Ile50 (C-F…H-N) and an orthogonal multipolar

interaction (C-F…C-O) with the main chain carbonyl of Gly49

with an F…C distance of 2.8-2.9 Å.^[41,42]In addition, this fluorine

Figure 3. Inhibitor 5-bound X-ray structure of HIV-1 protease. The major orientation of the inhibitor is shown. The inhibitor carbon

atoms are shown in green, water molecules are red spheres, and the hydrogen bonds are indicated by dotted lines (PDB ID: 6BZ2).

Halogen interactions of the fluorines are omitted for clarity.

Figure 4. An overlay of X-ray crystal structure of DRV-bound HIV-1 protease (green) with the X-ray structure of inhibitor 5 (orange)-

bound HIV-1 protease.

7

10.1002/cmdc.201700824

ChemMedChem

FULL PAPER

Figure 5. Side view of the S1 subsite. The protein surface is shown in transparent gray. The van der Waals surface for the fluorinated

P1-ligand for inhibitor 5 is shown in orange wire mesh and van der Waals contacts are shown by dotted lines.

DRV. However, unlike DRV, the crn-THF ligand has van der

Waals interactions with all the side chain atoms of Ile47.

opening as the key steps. This synthetic route enabled us to

synthesize both bis-fluoro- and mono-fluoro derivatives very

efficiently. We have recently reported that crn-THF containing

inhibitor 3 is a potent HIV-1 protease inhibitor with robust drug-

resistance properties. We have specifically incorporated 3,5-

difluorophenylmethyl group on this inhibitor to further modulate

van der Waals interactions in the S1 subpocket of HIV-1

protease. Inhibitor 5 displayed enhanced enzyme inhibitory and

antiviral potency compared to inhibitor 3 and DRV. Particularly,

inhibitor 5 exhibited unprecedented antiviral activity against a

variety of highly multidrug-resistant HIV-1 variants with antiviral

activity ranging from picomolar to femtomolar levels. Inhibitor 5

displayed an extremely high genetic barrier to resistance. Also, it

showed dual mechanism of action with potent dimerization

inhibition of HIV-1 protease monomers as well as potent

inhibition of catalytic proteolytic enzyme HIV-1 protease. In fact,

inhibitor 5 showed nearly 1000-fold more potent dimerization

inhibition activity than DRV. Furthermore, this compound

showed much improved CNS penetration over DRV and its brain

level in rats strongly suggest that this inhibitor would block

replication of HIV-1 effectively in the brain. Furthermore, it

showed significantly higher selectivity (S:2,473,684) than DRV

(41,562). We have obtained high resolution X-ray structure of

inhibitor 5-HIV-1 protease complex. Inhibitor 5 forms extensive

interactions in the HIV-1 protease active site. In particular, it

forms a network of hydrogen bonding with the backbone atoms

of HIV-1 protease. Our structural analysis provided the

molecular basis for enhanced potency of inhibitor 5. Our present

studies suggest that inhibitor 5 would be a favorable agent for

development. Further studies are ongoing.

The P2¢ position of inhibitor 5 has Cp-Abt moiety instead of

aminobenzene in DRV. The extended P2¢ moiety results in a

deviation of ~1Å in position relative to P2’ of DRV as measured

by the distance between the thiazole nitrogen of inhibitor 5 and

the 4-aminobenzene nitrogen of DRV in both major and minor

conformations (Figure 4). The thiazole nitrogen of 5 forms a

hydrogen bond interaction with the main chain amide of Asp30¢

similar to the interaction of 4-aminobenzene of DRV. The P2¢

amino group of inhibitor 5 has strong hydrogen bond interaction

(2.5 Å length) with the side chain of Asp30¢. Further, the

cyclopropyl group juts out and forms C-H…O interactions with

the side chain of Asp29¢. Thus, P1 and P2¢ modifications in

inhibitor 5 significantly contribute additional interactions with Arg

8¢/8 and Asp29/29¢, which stabilize the dimer of HIV-1 protease

by a critical inter-subunit ion pair. In addition, the halogen bond

interactions between the flap residues of protease and P1

fluorine of inhibitor 5 together with the van der Waals contacts

between crn-THF and Ile47 increase the favorable inhibitor-HIV-

1 protease interactions in comparison to the DRV complex.

These new interactions are likely to account for the improved

antiviral activity of inhibitor 5.

Conclusions

We have designed, synthesized, and evaluated fluorine-

containing inhibitors with novel 6-5-5-ring-fused crown-like

tetrahydropyranofuran

aminobenzothiazole

difluorophenylmethyl

as

moiety

fluorine-containing

the

P2-ligand,

P2'-ligand

cyclopropyl

and

3,5-

The

as

the

P1-ligand.

corresponding

hydroxyethylamine

sulfonamide isostere was synthesized utilizing Sharpless

asymmetric epoxidation followed by regioselective epoxide

8

10.1002/cmdc.201700824

ChemMedChem

FULL PAPER

Experimental Section

After cooling to room temperature, 5% H₂SO₄(8 mL) was added

and the solution was stirred at room temperature for 1 hour. The

layers were separated and the aqueous portion was extracted

with ethyl acetate (3×). The organic solution was dried with

Na₂SO₄, filtered, and concentrated under reduced pressure. The

residue was purified by silica gel column chromatography (50%

General

All reactions were carried out under an argon atmosphere in

either flame or oven-dried (120 C) glassware. All reagents and

o

1

chemicals were purchased from commercial suppliers and used

without further purification unless otherwise noted. Anhydrous

solvents were obtained as follows: Dichloromethane from

calcium hydride, diethyl ether and tetrahydrofuran from

Na/Benzophenone, methanol and ethanol from activated

magnesium under argon. All purification procedures were

carried out with reagent grade solvents (purchased form VWR)

in air. TLC analysis was conducted using glass-backed Thin-

Layer Silica Gel Chromatography Plates (60 Å, 250 µm

thickness, F-254 indicator). Column chromatography was

performed using 230-400 mesh, 60 Å pore diameter silica gel.

¹H, ¹³C NMR spectra were recorded at room temperature on a

Bruker ARX-400 and DRX-500. Chemical shifts (δ values) are

reported in parts per million, and are referenced to the

deuterated residual solvent peak. NMR data is reported as: δ

value (chemical shift, J-value (Hz), integration, where s = singlet,

d = doublet, t = triplet, q = quartet, brs = broad singlet). LRMS

and HRMS spectra were recorded at the Purdue University

Department of Chemistry Mass Spectrometry Center.

EtOAc in hexanes) to afford 8a (558 mg, 68%). H NMR (400

MHz, CDCl₃+CD₃OD) δ 6.78-6.72 (m, 2H), 6.63 (tt, J = 2.0, 11.0

Hz, 1H), 3.69-3.57 (m, 2H), 3.56-3.48 (m, 2H), 3.02 (dd, J = 3.0,

14.0 Hz, 1H), 2.66 (dd, J = 9.1, 14.0 Hz, 1H); ¹³C NMR (100

MHz, CDCl₃+CD₃OD) δ 164 (d, J = 13.0 Hz), 161.6 (d, J = 13.0

Hz), 141.6, 112.1 (d, J = 6.0 Hz), 112.0 (d, J = 7.0 Hz), 102.0 (t,

J = 25 Hz), 72.9, 64.5, 62.9, 36.4; LRMS-APCI (m/z): 266 [M +

Na] ⁺, 216 [M - N₂] ⁺.

(S)-2-((S)-1-Azido-2-(3,5-difluorophenyl)ethyl)oxirane (9):

To a stirred solution of 8a (243 mg, 1.0 mmol) in

chloroform (5 mL) was added 1-chlorocarbonyl-1-methylethyl

acetate (215 μL, 1.50 mmol) at 0 °C and the solution was stirred

at 0 °C for 30 min. The reaction mixture was warmed to room

temperature and stirred for 1h. A saturated solution of NaHCO₃

(aq) was added and the mixture stirred for 15 minutes. The

crude chloroacetate was extracted with chloroform, dried with

Na₂SO₄, filtered, and concentrated in vacuo. The residue was

dissolved in dry THF (8 mL) and cooled to 0 °C. Solid NaOMe

(65 mg, 1.20 mmol) was added and the reaction mixture was

stirred at room temperature for 2 h. The reaction was quenched

with saturated NH₄Cl(aq) and extracted with ethyl acetate.

Drying over Na₂SO₄, filtered, and concentrating under reduced

pressure afforded a residue which was purified by flash column

chromatography (12% EtOAc in hexanes) to afford pure azido

epoxide 9 as a liquid (174 mg, 77% yield over two steps). ¹H

NMR (400 MHz, CDCl₃) δ 6.82-6.76 (m, 2H), 6.71 (tt, J = 2.3,

11.3 Hz, 1H), 3.54 (quintet, J = 4.3, 5.5 Hz, 1H), 3.05 (m, 1H),

2.96 (dd, J = 4.0, 14.0 Hz, 1H), 2.85 (m, 1H), 2.82-2.73 (m, 2H);

¹³C NMR (100 MHz, CDCl₃) δ 164.1 (d, J = 13.0 Hz), 161.6 (d, J

= 13.0 Hz), 140.3 (t, J = 9.0 Hz), 112.2 (d, J = 6.0 Hz), 112.1 (d,

J = 7.0 Hz), 102.5 (t, J = 25.0 Hz), 63.1, 52.7, 45.2, 37.8; LRMS-

APCI (m/z): 198 [M - N₂] ⁺.

(E)-4-(3,5-Difluorophenyl)but-2-en-1-ol (7a):

To a suspension of magnesium turnings (450 mg, 18.54

mmol) in THF (15 mL) was added a solution of 1-bromo-3,5-

difluorobenzene 6a (3.3 g, 17.11 mmol) in THF (3 mL). The

mixture was heated at 65 °C for 1 h and the solution was cooled

to 23 °C. The resulting Grignard solution was added dropwise to

a mixture of CuCN (128 mg, 1.426 mmol) and butadiene

monoxide (1.0 g, 14.26 mmol) in 80 mL of anhydrous THF at

−78 °C. The reaction mixture was stirred for 1 h at −78 °C, after

which it was quenched with 15 mL of saturated NH₄Cl solution

followed by 15 mL of NH₄OH. The aqueous layer was extracted

twice with ethyl acetate. The combined organic phases were

dried over sodium sulfate and concentrated in vacuo. The

residue was purified by silica gel column chromatography (20%

EtOAc in hexane) to give 7a (1.9 g, 60% yield over two steps).

¹H-NMR (400 MHz, CDCl₃) δ 6.72-6.67 (m, 2H), 6.63 (tt, J = 4.0,

8.0 Hz, 1H), 5.79 (dtt, J = 1.2, 6.5, 15.5 Hz, 1H), 5.71 (dtt, J =

1.2, 6.5, 16.0 Hz, 1H), 4.13 (dd, J = 1.0, 5.5 Hz, 2H), 3.35 (d, J =

6.5 Hz, 2H), 1.70 (s, 1H); ¹³C-NMR (100 MHz, CDCl₃) δ 164.1 (d,

J =13.0 Hz), 161.7 (d, J = 13.0 Hz), 143.8 (t, J = 9.0 Hz), 131.5,

129.3, 111.2 (d, J = 7.0 Hz), 111.1 (d, J = 6.0 Hz), 101.5 (t, J =

25 Hz), 63.0, 38.1; LRMS-APCI (m/z): 167 [M - OH]⁺.

(E)-4-(3-Fluorophenyl)but-2-en-1-ol (7b):

The title compound 7b (942 mg, 67% yield over two steps)

was obtained from 6b (1.47 g, 8.4 mmol) by following the

procedure outlined for compound 7a. ¹H NMR (500 MHz, CDCl₃)

δ 7.24 (m, 1H), 6.96 (d, J = 7.6 Hz), 6.92-6.86 (m, 2H), 5.83 (m,

1H), 5.71 (m, 1H), 4.13 (dd, J = 1.1, 5.6 Hz, 2H), 3.38 (d, J = 6.5

Hz, 2H), 1.56 (s, 1H).

(2S,3S)-3-Azido-4-(3-fluorophenyl)butane-1,2-diol (8b):

The title compound 8b (1.0 g, 78% yield over two steps)

was obtained from 7b (940 mg, 5.66 mmol) by following the

procedure outlined for compound 8a. ¹H NMR (400 MHz, CDCl₃)

δ 7.29 (m, 1H), 7.05 (d, J = 7.6 Hz, 1H), 7.02-6.93 (m, 2H), 3.86-

3.73 (m, 2H), 3.72-3.62 (m, 2H), 3.07 (dd, J = 3.4, 14.0 Hz, 1H),

2.79 (dd, J = 8.8, 14.0 Hz, 1H), 2.49 (brs, 2H).

(2S,3S)-3-Azido-4-(3,5-difluorophenyl)butane-1,2-diol (8a):

Molecular sieves (500 mg) were flame dried in a flask and

then allowed to cool to 23 °C. Under argon, dry dichloromethane

(30 mL) and D-DET (439 mg, 2.13 mmol) were added and the

suspension was cooled to −25 °C. To this, Ti(OⁱPr)₄(318 μL,

1.06 mmol) and TBHP (4.3 mL, 5.5 M solution in decane, 23.43

mmol) were added and the mixture was stirred at −25 °C for 30

minutes. A solution of 7a (1.96 g, 10.65 mmol) in dry CH₂Cl₂(20

mL) was added to the above mixture and run for 12 h at −25 °C.

To the reaction mixture H₂O (10 mL) was added and stirred at

0 °C for 30 minutes. A 10% aqueous NaOH solution was then

added and the mixture was warmed to room temperature for 1 h.

The product was extracted with DCM (3×), dried with Na₂SO₄,

filtered, and concentrated under reduced pressure. Purification

by silica gel column chromatography (30–40% EtOAc in

hexanes) afforded epoxy alcohol (1.77 g, 83%) as a liquid.

Dry benzene (20 mL), Ti(OⁱPr)₄(1.52 mL, 5.10 mmol), and

TMSN₃(1.35 mL, 10.20 mmol) were refluxed for 5 h. A solution

of above epoxy alcohol (680 mg, 3.40 mmol) in dry benzene (10

mL) was added and the solution was refluxed for 30 minutes.

(S)-2-((S)-1-Azido-2-(3-fluorophenyl)ethyl)oxirane (10):

The title compound 10 (265 mg, 69% yield over two steps)

was obtained from 8b (417 mg, 1.85 mmol) by following the

procedure outlined for compound 9. ¹H NMR (400 MHz, CDCl₃)

δ 7.29 (m, 1H), 7.03 (d, J = 7.7 Hz, 1H), 7.00-6.93 (m, 2H), 3.57

(quintet, J = 4.2, 5.6 Hz, 1H), 3.06 (ddd, J = 2.6, 3.7, 6.2 Hz, 1H),

2.99 (dd, J = 4.2, 14.0 Hz, 1H), 2.87-2.76 (m, 3H); ¹³C NMR (100

MHz, CDCl₃) δ 164.1, 161.7, 139.2 (d, J = 7.2 Hz), 130.1 (d, J =

8.2 Hz), 125.1, 116.4 (d, J = 21.0 Hz), 114.0 (d, J = 20.8 Hz),

108.8, 63.5, 53.0, 45.3, 38.0.

(E)-4-(4-Fluorophenyl)but-2-en-1-ol (7c):

The title compound 7c (1.6 g, 61%) was obtained from 6c

(2.74 g, 15.68 mmol) by following the procedure outlined for

1

compound 7a. H NMR (400 MHz, CDCl₃) δ 7.15-7.10 (m, 2H),

9

10.1002/cmdc.201700824

ChemMedChem

FULL PAPER

7.00-6.94 (m, 2H), 5.87-5.78 (m, 1H), 5.73-5.64 (m, 1H), 4.15-

4.08 (m, 2H), 3.35 (d, J = 6.4 Hz, 2H), 1.51 (brs, 1H); ¹³C NMR

(100 MHz, CDCl₃) δ 162.6, 160.1, 135.4 (d, J = 2.6 Hz), 131.2,

130.4, 129.8 (d, J = 7.7 Hz), 115.1(d, J = 21.0 Hz), 63.3, 37.6;

LRMS-APCI (m/z): 149 [M - OH]⁺.

¹H NMR (400 MHz, CDCl₃) δ 7.53 (d, J = 8.7 Hz, 2H), 6.76 –

6.71 (m, 2H), 6.68 – 6.62 (m, 3H), 4.26 (brs, 2H), 3.68 (dt, J =

7.5, 4.8 Hz, 1H), 3.19 – 3.12 (m, 2H), 3.03 (dt, J = 9.2, 4.3 Hz,

1H), 2.99 – 2.91 (m, 2H), 2.80 (dd, J = 13.4, 6.8 Hz, 1H), 2.47

(dd, J = 13.5, 9.7 Hz, 1H), 1.85 (dt, J = 13.6, 6.8 Hz, 1H), 0.88

(dd, J = 14.4, 6.6 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 164.2

(d, J = 12.8 Hz), 161.7 (d, J = 12.9 Hz), 151.2, 143.1 (t, J = 9.0

Hz), 129.3, 125.6, 114.0, 112.5 – 110.9 (m), 101.8 (t, J = 25.3

Hz), 77.4, 73.0, 58.6, 55.3, 52.8, 38.8, 27.1, 20.1, 19.9; LRMS-

ESI (m/z): 428.1 [M+H]⁺.

(2S,3S)-3-Azido-4-(4-fluorophenyl)butane-1,2-diol (8c):

The title compound 8c (760 mg, 56% yield over two steps)

was obtained from 7c (1.0 g, 6.02 mmol) by following the

procedure outlined for compound 8a. ¹H NMR (400 MHz,

CDCl₃+CD₃OD) δ 7.21-7.15 (m, 2H), 6.98-6.92 (m, 2H), 3.73-

3.58 (m, 2H), 3.57-3.52 (m, 2H), 3.05-2.99 (m, 1H), 2.68 (dd, J =

9.1, 14.2 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃+CD₃OD) δ 162.9,

160.5, 133.2 (d, J = 2.8 Hz), 130.7 (d, J = 7.0 Hz), 115.2 (d, J =

21.0 Hz), 72.9, 65.2, 63.0, 35.9; LRMS-APCI (m/z): 248 [M +

Na], 198 [M - N₂] ⁺.

N-((2R,3S)-3-Azido-4-(3,5-difluorophenyl)-2-hydroxybutyl)-N-

isobutyl-2-(methylthio)benzo[d]thiazole-6-sulfonamide

(15a):

To a stirred solution of amine 12a in dichloromethane was

added sulfonyl chloride 14 (248 mg, 0.89 mmol) and triethyl

o

amine (0.37 mL, 2.66 mmol) at 23 C under argon atmosphere.

(S)-2-((S)-1-Azido-2-(4-fluorophenyl)ethyl)oxirane (11):

The title compound 11 (700 mg, 76% over two steps) was

obtained from 8c (1.0 g, 4.44 mmol) by following the procedure

outlined for compound 9. ¹H NMR (400 MHz, CDCl₃) δ 7.23-7.18

(m, 2H), 7.01 (tt, J = 2.1, 11.7 Hz, 1H), 3.53 (ddd, J = 4.5, 5.5,

8.7 Hz, 1H), 3.04 (ddd, J = 2.5, 3.8, 5.5 Hz, 1H), 2.97 (dd, J =

4.5, 14.1 Hz, 1H), 2.85-2.75 (m, 3H); ¹³C NMR (100 MHz,

CDCl₃) δ 163.1, 160.6, 132.1, 130.7 (d, J = 8.0 Hz), 115.3 (d, J =

21.0 Hz), 63.6, 52.7, 45.1, 37.3.; LRMS-APCI (m/z): 180 [M - N₂]

The reaction mixture was stirred at 23 ^oC for 12 h. Upon

completion, solvent was removed under reduced pressure and

the crude product was purified by silica gel column

chromatography (15% EtOAc in hexane) to give 15a (433 mg,

1

90% over two steps). H NMR (500 MHz, CDCl₃) δ 8.23 – 8.21

(m, 1H), 7.89 (d, J = 8.6 Hz, 1H), 7.80 – 7.76 (m, 1H), 6.79 (d, J

= 6.2 Hz, 2H), 6.65 (t, J = 8.9 Hz, 1H), 3.76 (d, J = 17.6 Hz, 2H),

3.56 (ddd, J = 9.5, 6.1, 3.7 Hz, 1H), 3.27 – 3.17 (m, 2H), 3.05

(ddd, J = 21.8, 13.7, 5.8 Hz, 2H), 2.89 (dd, J = 13.4, 6.8 Hz, 1H),

2.76 (s, 3H), 2.74 – 2.70 (m, 1H), 1.88-1.78 (m, 1H), 0.87 (d, J =

6.6 Hz, 3H), 0.84 (d, J = 6.6 Hz, 3H); ¹³C NMR (100 MHz,

CDCl₃) δ 173.6, 164.4 (d, J = 12.7 Hz), 161.9 (d, J = 12.9 Hz),

156.1, 141.3 (t, J = 9.1 Hz), 136.0, 133.7, 125.1, 121.9, 121.1,

112.6, 112.3, 102.6 (t, J = 25.3 Hz), 71.9, 66.0, 59.1, 53.2, 36.6,

27.3, 20.2, 19.9, 16.1; LRMS-ESI (m/z): 542.1 [M+H]⁺.

+

.

(2R,3S)-3-Amino-4-(3,5-difluorophenyl)-1-(isobutylamino)-

butan-2-ol (12): To a stirred solution of 9 (200 mg, 2.66 mmol)

in isopropanol was added isobutylamine (0.27 mL, 2.66 mmol) at

23 ^oC under argon atmosphere. The reaction mixture was

o

stirred at 65 C for 12 h. After this period, isopropanol was

N-((2R,3S)-3-Azido-4-(3-fluorophenyl)-2-hydroxybutyl)-N-

isobutyl-2-(methylthio)benzo[d]thiazole-6-sulfonamide

(15a): The title compound (15a) was obtained by following the

procedure outlined for compound 15a, (129 mg, 87% yield over

two steps). ¹H NMR (500 MHz, CDCl₃) δ 8.24 (d, J = 1.7 Hz, 1H),

7.94 (d, J = 8.6 Hz, 1H), 7.81 (dd, J = 8.6, 1.8 Hz, 1H), 7.30 –

7.24 (m, 1H), 7.05 (d, J = 7.6 Hz, 1H), 7.01 – 6.92 (m, 2H), 3.78

(td, J = 6.2, 3.2 Hz, 1H), 3.63 – 3.55 (m, 2H), 3.28 (dd, J = 15.2,

9.1 Hz, 1H), 3.17 (dd, J = 15.2, 2.5 Hz, 1H), 3.11 (dd, J = 14.3,

3.6 Hz, 1H), 3.08 – 3.04 (m, 1H), 2.88 (dd, J = 13.4, 6.6 Hz, 1H),

2.81 (s, 3H), 2.79 – 2.75 (m, 1H), 1.84 (tt, J = 13.3, 6.6 Hz, 1H),

0.92 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.6 Hz, 3H); ¹³C NMR (125

MHz, CDCl₃) δ 173.5, 163.9, 161.9, 155.9, 139.8 (d, J = 7.2 Hz),

135.8, 133.6, 130.2 (d, J = 8.2 Hz), 125.3 – 125.0 (m), 121.8,

121.0, 116.3 (d, J = 21.2 Hz), 113.9 (d, J = 20.9 Hz), 71.8, 66.2,

58.9, 52.9, 36.6, 27.2, 20.2, 19.8, 16.1; LRMS-ESI (m/z): 546.2

[M+Na]⁺.

removed under reduced pressure. ¹H NMR (400 MHz, CDCl₃)

d 6.83-6.77 (m, 2H), 6.70 (tt, J = 2.3, 11.3 Hz, 1H), 3.59-3.51 (m,

2H), 3.01 (dd, J = 3.2, 14.3 Hz, 1H), 2.87 (dd, J = 3.0, 12.0 Hz,

1H), 2.86 (brs, 1H), 2.76-2.64 (m, 2H), 2.45 (dd, J = 2.0, 6.9 Hz,

2H), 1.74 (m, 1H), 0.93 (d, J = 1.4 Hz, 3H), 0.92 (d, J = 1.4 Hz,

3H); ¹³C NMR (100 MHz, CDCl₃) d 164.1 (d, J = 13.0 Hz), 161.6

(d, J = 13.0 Hz), 141.5 (t, J = 9.0 Hz), 112.2 (d, J = 6.0 Hz),

112.0 (d, J = 6.0 Hz), 102.2 (t, J = 25.0 Hz), 70.0, 66.2, 57.5,

50.8, 36.7, 28.2, 20.3.; LRMS-APCI (m/z): 299 [M+H]+.

4-Amino-N-((2R,3S)-3-amino-4-(3,5-difluorophenyl)-2-

hydroxybutyl)-N-isobutylbenzenesulfonamide (13): To

a

stirred solution of crude product 12a (331 mg) in

dichloromethane was added 4-nitrobenzene sulfonyl chloride

(246 mg, 1.11 mmol) and triethyl amine (0.5 mL, 3.33 mmol) at

23 ^oC under argon atmosphere. The reaction mixture was stirred

at 23 ^oC for 12 h. Solvent was removed under reduced pressure

and the crude product was purified by silica gel column

chromatography (55% EtOAc in hexane) to give azide (348 mg,

67% over two steps). ¹H NMR (400 MHz, CDCl₃) δ 8.39 – 8.35

(m, 2H), 8.02 – 7.98 (m, 2H), 6.81 (d, J = 7.2 Hz, 2H), 6.70 (t, J

= 8.9 Hz, 1H), 3.76 (t, J = 6.8 Hz, 1H), 3.57 (ddd, J = 9.4, 6.2,

4.1 Hz, 1H), 3.32 (brs, 1H), 3.29 – 3.20 (m, 2H), 3.12 – 3.03 (m,

2H), 2.94 (dd, J = 13.5, 6.9 Hz, 1H), 2.77 (dd, J = 14.3, 9.2 Hz,

1H), 1.86 (tt, J = 13.8, 6.9 Hz, 1H), 0.90 (d, J = 6.6 Hz, 3H), 0.87

(d, J = 6.5 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 164.4 (d, J =

13.0 Hz), 161.9 (d, J = 13.0 Hz), 150.3, 144.4, 141.0 (t, J = 9.1

Hz), 128.6, 124.6, 114.3 – 110.5 (m), 102.6 (t, J = 25.2 Hz), 71.6,

66.0, 58.5, 52.6, 36.6, 27.1, 20.1, 19.8.

To a stirred solution of above azide (338 mg, 0.7 mmol) in

EtOAc (5 mL) was added 20% Pd(OH)₂/C (80 mg). The resulting

solution was stirred at 23 ^oC under 1 atm H₂gas over 20 h.

Upon completion, the mixture was filtered through a plug of

Celite and solvents were removed under reduced pressure. The

crude product was purified by silica gel column chromatography

(5% NH₃in MeOH in CH₂Cl₂) to afford 13 (265 mg, 89% yield).

N-((2R,3S)-3-Azido-4-(4-fluorophenyl)-2-hydroxybutyl)-N-

isobutyl-2-(methylthio)benzo[d]thiazole-6-sulfonamide

(15b): The title compound (15b) was obtained by following the

procedure outlined for compound 15a, (171 mg, 93% yield over

two steps). ¹H NMR (500 MHz, CDCl₃) δ 8.24 (d, J = 1.5 Hz, 1H),

7.94 (d, J = 8.6 Hz, 1H), 7.81 (dd, J = 8.6, 1.7 Hz, 1H), 7.23 (dd,

J = 8.4, 5.5 Hz, 2H), 7.00 (t, J = 8.6 Hz, 2H), 3.79 – 3.72 (m, 1H),

3.59 – 3.51 (m, 2H), 3.27 (dd, J = 15.2, 9.1 Hz, 1H), 3.16 (dd, J

= 15.2, 2.3 Hz, 1H), 3.12 – 3.04 (m, 2H), 2.87 (dd, J = 13.4, 6.6

Hz, 1H), 2.81 (s, 3H), 2.77 (dd, J = 14.2, 9.2 Hz, 1H), 1.83 (dp, J

= 13.3, 6.6 Hz, 1H), 0.92 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.6 Hz,

3H); ¹³C NMR (125 MHz, CDCl₃) δ 173.6, 162.9, 161.0, 156.0,

135.9, 133.6, 132.9 (d, J = 2.5 Hz), 131.0 (d, J = 7.8 Hz), 125.1,

121.8, 121.1, 115.5 (d, J = 21.2 Hz), 71.8, 66.6, 58.9, 53.0, 36.1,

27.2, 20.2, 19.8, 16.1; LRMS-ESI (m/z): 524.0 [M+H]⁺.

tert-Butyl((2S,3R)-1-(3,5-difluorophenyl)-3-hydroxy-4-((N-

isobutyl-2-(methylthio)benzo[d]thiazole)-6-

sulfonamido)butan-2-yl)carbamate (16a): To a stirred solution

of 15a (275 mg, 0.5 mmol) in THF/H₂O (3:1 ratio, 4 mL) was

10

10.1002/cmdc.201700824

ChemMedChem

FULL PAPER

added triphenylphosphine (160 mg, 0.61 mmol) at 23 ^oC. The

reaction mixture was stirred at 23 C for 24 h. After this period,

EtOAc in hexane) to give cyclopropylamine (880 mg, 91% over

two steps). H NMR (400 MHz, CDCl₃+CD₃OD) δ 7.93 (s, 1H),

o

1

to the reaction mixture Boc anhydride (133 mg, 0.61 mmol) and

7.57 – 7.52 (m, 1H), 7.38 (d, J = 8.5 Hz, 1H), 6.63 (d, J = 6.6 Hz,

2H), 6.49 (t, J = 9.0 Hz, 1H), 5.47 (d, J = 9.4 Hz, 1H), 3.68 –

3.60 (m, 1H), 3.58 – 3.47 (m, 1H), 3.21 – 3.10 (m, 1H), 2.83 (dtd,

J = 39.5, 15.1, 13.3, 5.5 Hz, 4H), 2.61 – 2.50 (m, 2H), 1.84-1.71

(m, 1H), 1.18 (s, 9H), 0.75 (dd, J = 9.7, 5.8 Hz, 8H), 0.62 – 0.57

(m, 2H); ¹³C NMR (100 MHz, CDCl₃+CD₃OD) δ 173.3, 164.1 (d,

J = 13.3 Hz), 161.7 (d, J = 12.5 Hz), 156.7, 155.5, 131.0, 130.4,

125.4, 120.9, 118.3, 112.4 (d, J = 24.1 Hz), 101.9 (d, J = 26.8

Hz), 79.9, 72.8, 60.6, 58.6, 54.5, 53.5, 35.4, 28.2, 27.1, 26.6,

20.0, 7.7; LRMS-ESI (m/z): 625.2 [M+H]⁺.

o

sodium bicarbonate (85 mg, 1 mmol) was added at 23 C. The

o

reaction mixture was stirred at 23 C for 20 h. Upon completion,

the mixture was concentrated under reduced pressure and

diluted with EtOAc. The reaction mixture was washed with brine,

dried (Na₂SO₄) and concentrated under reduced pressure. The

crude product was purified by silica gel column chromatography

(30% EtOAc in hexane) to give 16a (270 mg, 87% over two

1

steps). H NMR (400 MHz, CDCl₃) δ 8.22 (d, J = 1.4 Hz, 1H),

7.92 (d, J = 8.6 Hz, 1H), 7.79 (dd, J = 8.6, 1.6 Hz, 1H), 6.77 (d, J

= 6.2 Hz, 2H), 6.64 (tt, J = 9.2, 2.2 Hz, 1H), 4.74 (d, J = 8.7 Hz,

1H), 4.05 (s, 1H), 3.84 (s, 1H), 3.71 (tt, J = 9.4, 4.9 Hz, 1H), 3.14

(qd, J = 15.1, 5.9 Hz, 2H), 3.01 – 2.84 (m, 4H), 2.81 (s, 3H),

1.94-1.79 (m, 1H), 1.34 (s, 9H), 0.87 (dd, J = 10.4, 6.6 Hz, 6H);

¹³C NMR (100 MHz, CDCl₃) δ 173.5, 164.3 (d, J = 12.7 Hz),

To a stirred solution of above cyclopropylamine (870 mg,

1.39 mmol) in dichloromethane (15 mL) was added TFA (5mL)

o

at 0 C under argon atmosphere and the mixture was stirred at

23 ^oC for 1 h. Upon completion, solvent was removed under

reduced pressure to give 17 (730 mg) used for next coupling

161.8 (d, J = 13.2 Hz), 156.0 (d, J = 7.8 Hz), 142.3 (t, J = 8.9 Hz), reaction without further purification. LRMS-ESI (m/z): 525.2

135.9, 133.9, 125.1, 121.8, 121.1, 112.6, 112.3, 108.8, 102.0 (t,

J = 25.4 Hz), 80.2, 73.0, 58.9, 54.7, 53.8, 35.2, 28.3, 27.3, 20.2,

20.0, 16.1; LRMS-ESI (m/z): 638.1 [M+Na]⁺.

[M+H]⁺.

N-((2R,3S)-3-Amino-4-(3-fluorophenyl)-2-hydroxybutyl)-2-

(cyclopropylamino)-N-isobutylbenzo[d]thiazole-6-

tert-Butyl

((2S,3R)-1-(3-fluorophenyl)-3-hydroxy-4-((N-

sulfonamide (18): The cyclopropylamine was obtained from

16b by following the procedure outlined for compound 17, (116

mg, 97% yield over two steps). ¹H NMR (500 MHz, CDCl₃) δ

8.08 (s, 1H), 7.68 (d, J = 8.2 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H),

7.25 – 7.18 (m, 1H), 7.03 (d, J = 6.3 Hz, 1H), 6.95 (d, J = 9.1 Hz,

isobutyl-2-(methylthio)benzo[d]thiazole)-6-

sulfonamido)butan-2-yl)carbamate (16b): The title compound

(16b) was obtained by following the procedure outlined for

compound 16a, (123 mg, 83% yield over two steps). H NMR

1

(500 MHz, CDCl₃) δ 8.24 – 8.18 (m, 1H), 7.90 (d, J = 8.6 Hz, 1H), 1H), 6.89 (t, J = 7.9 Hz, 1H), 4.78 (d, J = 7.4 Hz, 1H), 3.85 (s,

7.77 (dd, J = 8.6, 1.7 Hz, 1H), 7.21 (q, J = 7.8 Hz, 1H), 7.00 (d, J

1H), 3.76 (brs, 1H), 3.20 – 3.08 (m, 2H), 3.00 (dt, J = 22.3, 10.4

= 7.6 Hz, 1H), 6.94 (d, J = 9.8 Hz, 1H), 6.88 (td, J = 8.4, 2.1 Hz,

Hz, 2H), 2.88 (dt, J = 18.6, 12.0 Hz, 2H), 2.75 (s, 1H), 1.93 –

1H), 4.75 (d, J = 8.3 Hz, 1H), 3.99 (s, 1H), 3.83 (brs, 1H), 3.73 (tt, 1.80 (m, 1H), 1.34 (s, 9H), 0.91 (td, J = 17.8, 6.1 Hz, 8H), 0.79 (s,

J = 9.3, 5.1 Hz, 1H), 3.13 (td, J = 16.3, 15.1, 9.7 Hz, 2H), 3.04 –

2.93 (m, 2H), 2.89 (dd, J = 13.4, 7.1 Hz, 2H), 2.79 (s, 3H), 1.86

(dp, J = 13.7, 6.7 Hz, 1H), 1.32 (s, 9H), 0.86 (dd, J = 13.5, 6.6

Hz, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 173.3, 163.8, 161.9,

156.1, 155.9, 140.9 – 140.6 (m), 135.7, 133.9, 129.9 (d, J = 8.2

Hz), 125.2 (d, J = 18.0 Hz), 121.7, 121.0, 116.4 (d, J = 21.0 Hz),

113.3 (d, J = 20.9 Hz), 79.9, 72.9, 58.6, 54.7, 53.6, 35.2, 28.3,

27.2, 20.16, 20.0, 16.1; LRMS-ESI (m/z): 620.4 [M+Na]⁺.

2H); ¹³C NMR (125 MHz, CDCl₃) δ 173.4, 163.9, 161.9, 156.1,

155.8, 140.8 (d, J = 6.8 Hz), 131.3, 130.4, 129.9 (d, J = 7.6 Hz),

125.4 (d, J = 20.3 Hz), 121.0, 118.5, 116.5 (d, J = 20.8 Hz),

113.4 (d, J = 20.9 Hz), 80.0, 72.9, 59.0, 54.7, 53.9, 35.2, 28.4,

27.4, 26.6, 20.3, 20.0, 8.0; LRMS-ESI (m/z): 629.5 [M+Na]⁺.

The title compound (18) was obtained by following the de-

Boc procedure outlined for compound 17, (97 mg) used for next

coupling reaction without further purification. LRMS-ESI (m/z):

507.3 [M+H]⁺.

tert-Butyl

((2S,3R)-1-(4-fluorophenyl)-3-hydroxy-4-((N-

isobutyl-2-(methylthio)benzo[d]thiazole)-6-

sulfonamido)butan-2-yl)carbamate (16c): The title compound

N-((2R,3S)-3-Amino-4-(4-fluorophenyl)-2-hydroxybutyl)-2-

(cyclopropylamino)-N-isobutylbenzo[d]thiazole-6-

(16c) was obtained by following the procedure outlined for

compound 16a, (127 mg, 70% yield over two steps). H NMR

sulfonamide (19): The cyclopropylamine was obtained form

16c by following the procedure outlined for compound 17, (125

mg, 98% yield over two steps). ¹H NMR (500 MHz, CDCl₃) δ

8.08 (d, J = 1.5 Hz, 1H), 7.68 (dd, J = 8.5, 1.8 Hz, 1H), 7.56 (d, J

= 8.5 Hz, 1H), 7.24 – 7.18 (m, 2H), 6.97 (t, J = 8.6 Hz, 2H), 4.68

(d, J = 8.7 Hz, 1H), 4.05 (s, 1H), 3.82 (brs, 1H), 3.77 – 3.69 (m,

1H), 3.11 (d, J = 6.5 Hz, 2H), 3.03 – 2.94 (m, 2H), 2.92 – 2.81

(m, 2H), 2.76 (tt, J = 6.7, 3.5 Hz, 1H), 1.85 (dp, J = 12.9, 6.4 Hz,

1H), 1.34 (s, 9H), 0.97 – 0.93 (m, 2H), 0.91 (d, J = 6.6 Hz, 3H),

0.87 (d, J = 6.6 Hz, 3H), 0.79 (p, J = 5.0 Hz, 2H); ¹³C NMR (125

MHz, CDCl₃) δ 173.2, 162.7, 160.8, 156.1, 155.8, 134.0 – 133.6

(m), 131.4, 131.1 (d, J = 7.7 Hz), 130.5, 125.5, 121.0, 118.7,

115.3 (d, J = 21.1 Hz), 79.9, 73.0, 59.0, 54.8, 54.0, 34.6, 28.4,

27.4, 26.8, 20.3, 20.0, 8.1; LRMS-ESI (m/z): 607.2 [M+H]⁺.

The title compound (19) was obtained by following the de-

Boc procedure outlined for compound 17, (166 mg) used for

next coupling reaction without further purification. LRMS-ESI

(m/z): 507.4 [M+H]⁺.

1

(500 MHz, CDCl₃) δ 8.21 (s, 1H), 7.91 (d, J = 8.6 Hz, 1H), 7.77

(d, J = 8.6 Hz, 1H), 7.19 (dd, J = 8.0, 5.6 Hz, 2H), 6.96 (t, J = 8.6

Hz, 2H), 4.68 (d, J = 8.6 Hz, 1H), 3.98 (s, 1H), 3.81 (brs, 1H),

3.76 – 3.67 (m, 1H), 3.18 – 3.09 (m, 2H), 2.97 (t, J = 10.6 Hz,

2H), 2.92 – 2.84 (m, 2H), 2.81 (s, 3H), 1.86 (dp, J = 13.4, 6.5 Hz,

1H), 1.32 (s, 9H), 0.87 (dd, J = 13.9, 6.6 Hz, 6H); ¹³C NMR (125

MHz, CDCl₃) δ 173.4, 162.7, 160.7, 156.1, 156.0, 135.8, 133.7

(d, J = 31.9 Hz), 131.0 (d, J = 7.6 Hz), 125.1, 121.7, 121.0,

115.3 (d, J = 21.1 Hz), 79.9, 72.8, 58.7, 54.8, 53.7, 34.6, 28.3,

27.2, 20.2, 20.0, 16.1; LRMS-ESI (m/z): 598.2 [M+H]⁺.

N-((2R,3S)-3-Amino-4-(3,5-difluorophenyl)-2-hydroxybutyl)-

2-(cyclopropylamino)-N-isobutylbenzo[d]thiazole-6-

sulfonamide (17): To a stirred solution of 16a (960 mg, 1.56

mmol) in dichloromethane (10 mL) was added mCPBA (807 mg,

4.68 mmol) at 0 ^oC under argon atmosphere and the mixture

was stirred at 23 ^oC for 12 h. After this period, the reaction

mixture was quenched by the addition of saturated aqueous

Na₂S₂O₃(2 mL) and extracted with dichloromethane. The

extracts were washed with saturated aqueous NaHCO₃, dried

(Na₂SO₄) and concentrated under reduced pressure. To the

crude product in dry THF (10 mL) at 23 ^oC under argon

atmosphere was added cyclopropylamine (0.35 mL, 4.68 mmol)

(3S,7aS,8S)-Hexahydro-4H-3,5-methanofuro[2,3-b]pyran-8-yl

((2S,3R)-4-((4-amino-N-isobutylphenyl)sulfonamido)-1-(3,5-

difluorophenyl)-3-hydroxybutan-2-yl)carbamate (4): To

a

stirred solution of activated alcohol 20 (12 mg, 0.037 mmol) and

isoster 13 (19 mg, 0.045 mmol) in acetonitrile (2 mL) was added

DIPEA (33 µL, 0.19 mmol) at 23 ^oC under argon atmosphere.

The reaction mixture was stirred at 23 ^oC until completion. Upon

completion, solvents were removed under reduced pressure and

o

and the mixture was stirred at 65 C for 12 h. Upon completion,

solvent was removed under reduced pressure and the crude

product was purified by silica gel column chromatography (50%

11

10.1002/cmdc.201700824

ChemMedChem

FULL PAPER

crude product was purified by silica gel column chromatography

(55% EtOAc in hexane) to give inhibitor 4 (20 mg, 88% yield). ¹H

NMR (400 MHz, CDCl₃) δ 7.54 (d, J = 8.5 Hz, 2H), 6.78 (d, J =

6.4 Hz, 2H), 6.68 (d, J = 8.5 Hz, 2H), 6.64 (dt, J = 9.2, 2.4 Hz,

1H), 5.44 (d, J = 6.6 Hz, 1H), 5.26 (d, J = 9.0 Hz, 1H), 4.83 (dd,

J = 8.9, 5.8 Hz, 1H), 4.19 (brs, 2H), 3.92 – 3.79 (m, 4H), 3.62

(ddd, J = 19.0, 10.2, 7.2 Hz, 2H), 3.05 (dt, J = 14.4, 4.8 Hz, 2H),

2.97 (d, J = 16.3 Hz, 1H), 2.94 – 2.89 (m, 1H), 2.86 – 2.64 (m,

5H), 2.38 – 2.32 (m, 1H), 1.82 (d, J = 12.0 Hz, 2H), 1.45 (dd, J =

12.0, 3.7 Hz, 1H), 0.92 (d, J = 6.6 Hz, 3H), 0.88 (d, J = 6.5 Hz,

3H); ¹³C NMR (100 MHz, CDCl₃) δ 164.3 (d, J = 12.9 Hz), 161.9

(3S,7aS,8S)-Hexahydro-4H-3,5-methanofuro[2,3-b]pyran-8-yl

((2S,3R)-4-((2-(cyclopropylamino)-N-

isobutylbenzo[d]thiazole)-6-sulfonamido)-1-(4-

fluorophenyl)-3-hydroxybutan-2-yl)carbamate

(22):

Compound 20 (15 mg, 0.047 mmol) was treated with fluoro

isosters 19 (28 mg, 0.056 mmol) by following the procedure

outlined for inhibitor 4 to give inhibitor 22 (30 mg, 94% yield). ¹H

NMR (500 MHz, CDCl₃) δ 8.10 – 8.06 (m, 1H), 7.68 (dd, J = 8.5,

1.7 Hz, 1H), 7.55 (d, J = 8.5 Hz, 1H), 7.45 (brs, 1H), 7.20 (dd, J

= 8.1, 5.5 Hz, 2H), 6.97 (t, J = 8.6 Hz, 2H), 5.42 (d, J = 6.7 Hz,

1H), 5.32 – 5.27 (m, 1H), 4.79 (dd, J = 8.9, 5.8 Hz, 1H), 3.93 –

(d, J = 13.1 Hz), 155.7, 151.0, 142.2 (t, J = 9.0 Hz), 129.6, 126.1, 3.83 (m, 4H), 3.76 (d, J = 9.2 Hz, 1H), 3.57 (dt, J = 9.1, 7.1 Hz,

114.3, 113.1 – 111.8 (m), 104.5, 102.2 (t, J = 25.1 Hz), 75.2,

72.9, 68.5, 60.1, 59.1, 55.0, 53.8, 45.0, 42.1, 37.5, 35.3, 27.5,

23.7, 20.3, 20.1; LRMS-ESI (m/z): 610.2 [M+H]⁺; HRMS-ESI

(m/z): [M+H]⁺calcd for C₂₉H₃₈F₂N₃O₇S, 610.2399; found

610.2394.

2H), 3.17 (dd, J = 15.0, 8.6 Hz, 1H), 3.07 – 2.97 (m, 3H), 2.88 –

2.79 (m, 2H), 2.75 (dq, J = 6.8, 3.4 Hz, 1H), 2.73 – 2.67 (m, 1H),

2.67 – 2.61 (m, 1H), 2.35 – 2.28 (m, 1H), 1.86 (dt, J = 14.3, 7.2

Hz, 1H), 1.81 (d, J = 12.3 Hz, 1H), 1.46 – 1.40 (m, 1H), 0.97 –

0.94 (m, 2H), 0.92 (d, J = 6.6 Hz, 3H), 0.88 (d, J = 4.2 Hz, 3H),

0.81 – 0.76 (m, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 173.3, 162.8,

160.8, 155.8 (d, J = 9.2 Hz), 133.5, 131.4, 131.0 (d, J = 7.6 Hz),

130.4, 125.5, 121.0, 118.6, 115.5 (d, J = 21.1 Hz), 104.4, 75.1,

72.8, 68.4, 60.0, 59.0, 55.3, 53.9, 45.0, 42.1, 37.5, 31.7, 27.5,

26.8, 23.6, 22.8, 20.3, 20.0, 8.0; LRMS-ESI (m/z): 689.2 [M+H]⁺;

HRMS-ESI (m/z): [M+H]⁺calcd for C₃₃H₄₂FN₄O₇S₂, 689.2479;

found 689.2474.

(3S,7aS,8S)-Hexahydro-4H-3,5-methanofuro[2,3-b]pyran-8-yl

((2S,3R)-4-((2-(cyclopropylamino)-N-isobutylbenzo

[d]thiazole)-6-sulfonamido)-1-(3,5-difluorophenyl)-3-

hydroxybutan-2-yl)carbamate (5): Compound 20 (200 mg,

0.62 mmol) was treated with fluoro isoster 17 (392 mg, 0.75

mmol) by following the procedure outlined for inhibitor 4 to give

inhibitor 5 (434 mg, 99% yield). ¹H NMR (500 MHz, CDCl₃) δ

8.27 (s, 1H), 8.10 – 8.07 (m, 1H), 7.73 – 7.67 (m, 1H), 7.52 (d, J

= 8.5 Hz, 1H), 6.76 (d, J = 6.3 Hz, 2H), 6.61 (t, J = 8.9 Hz, 1H),

5.84 (d, J = 9.2 Hz, 1H), 5.41 (d, J = 6.7 Hz, 1H), 4.82 (dd, J =

8.9, 5.8 Hz, 1H), 4.16 (brs, 1H), 3.98 – 3.92 (m, 1H), 3.85 (dt, J

= 12.6, 6.1 Hz, 3H), 3.60 (dd, J = 9.0, 6.5 Hz, 1H), 3.54 (dd, J =

11.1, 7.9 Hz, 1H), 3.17 – 3.11 (m, 2H), 3.06 (dd, J = 14.0, 3.4 Hz,

1H), 2.96 (dd, J = 13.3, 8.0 Hz, 1H), 2.89 (dd, J = 13.4, 7.1 Hz,

1H), 2.79 (dd, J = 13.9, 10.5 Hz, 1H), 2.75 – 2.68 (m, 2H), 2.67 –

2.61 (m, 1H), 2.34 – 2.27 (m, 1H), 1.86 (dp, J = 14.5, 6.9 Hz,

1H), 1.78 (d, J = 11.9 Hz, 1H), 1.43 (dd, J = 8.0, 4.0 Hz, 1H),

0.93 (t, J = 6.7 Hz, 2H), 0.87 (dd, J = 12.6, 7.1 Hz, 6H), 0.79 –

0.75 (m, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 173.7, 163.9 (d, J =

12.7 Hz), 161.9 (d, J = 12.8 Hz), 155.7 (d, J = 18.0 Hz), 142.4 (t,

J = 8.8 Hz), 131.1, 130.2, 125.5, 121.0, 118.2, 112.5 – 112.0 (m),

104.3, 102.0 (t, J = 25.1 Hz), 74.8, 72.9, 68.3, 59.9, 58.8, 55.1,

53.5, 44.9, 42.2, 37.4, 31.6, 27.3, 26.7, 23.6, 22.7, 20.2, 20.0,

7.8; LRMS-ESI (m/z): 707.2 [M+H]⁺; HRMS-ESI (m/z): [M+H]⁺

calcd for C₃₃H₄₁F₂N₄O₇S₂, 707.2385; found 707.2379.

Determination of X-ray structure of HIV-1 protease and

inhibitor 5 complex:

The HIV-1 protease was expressed and purified as described.^[43]

The protease-inhibitor complex was crystallized by the hanging

drop vapor diffusion method with well solutions of 1.65 M NaCl,

0.1 M Na Acetate, pH 5.5. Diffraction data were collected on a

single crystal cooled to 90 K at SER-CAT (22-ID beamline),

Advanced Photon Source, Argonne National Lab (Chicago,

USA) with X-ray wavelength of 0.8 Å, and processed by HKL-

2000^[44]to give an Rmerge of 7.1%. The crystal structure was

solved by PHASER^[45]in CCP4i Suite^[46-48]using one of the

previously reported isomorphous structures^[49]as the initial

model, and refined by SHELX-97^[50,51]with 1.67 Å resolution

diffraction data. PRODRG-2^[52]was used to construct the

inhibitor and the restraints for refinement. COOT^[53,54]was used

for modification of the model. Alternative conformations were

modeled, isotropic atomic displacement parameters (B factors)

were applied for all atoms including solvent molecules. The final

refined solvent structure comprised one Na⁺ion, two Cl^-ions,

one acetate ion and 169 water molecules. The crystallographic

statistics are listed in Table 1 (supplemental material). The

coordinates and structure factors of the protease complex with

inhibitor 5 have been deposited in the Protein Data Bank^[55]with

code 6BZ2.

(3S,7aS,8S)-Hexahydro-4H-3,5-methanofuro[2,3-b]pyran-8-yl

((2S,3R)-4-((2-(cyclopropylamino)-N-isobutylbenzo

[d]thiazole)-6-sulfonamido)-1-(3-fluorophenyl)-3-

hydroxybutan-2-yl)carbamate (21): Compound 20 (7 mg,

0.022 mmol) was treated with fluoro isostere 18 (13 mg, 0.026

mmol) by following the procedure outlined for inhibitor 4 to give

1

inhibitor 21 (13.5 mg, 90% yield). H NMR (500 MHz, CDCl₃) δ

8.09 – 8.07 (m, 1H), 7.70 – 7.67 (m, 1H), 7.56 (d, J = 8.5 Hz,

1H), 7.24 (d, J = 7.8 Hz, 1H), 7.06 (brs, 1H), 7.04 – 7.00 (m, 1H),

6.96 (d, J = 9.4 Hz, 1H), 6.94 – 6.88 (m, 1H), 5.42 (d, J = 6.6 Hz,

1H), 5.23 (d, J = 8.7 Hz, 1H), 4.81 (dd, J = 9.0, 5.8 Hz, 1H), 3.94

– 3.85 (m, 4H), 3.82 – 3.76 (m, 1H), 3.59 (dd, J = 7.9, 4.8 Hz,

2H), 3.17 (dd, J = 15.0, 8.5 Hz, 1H), 3.10 – 2.98 (m, 3H), 2.89 –

2.81 (m, 2H), 2.78 – 2.69 (m, 2H), 2.68 – 2.62 (m, 1H), 2.33 (q, J

= 4.6, 4.1 Hz, 1H), 1.86 (dd, J = 13.3, 6.6 Hz, 1H), 1.81 (d, J =

12.2 Hz, 1H), 1.44 (dt, J = 12.0, 3.8 Hz, 1H), 0.97– 0.93 (m, 5H),

0.88 (d, J = 6.5 Hz, 3H), 0.82 – 0.78 (m, 2H); ¹³C NMR (125

MHz, CDCl₃) δ 173.1, 164.0, 162.0, 155.8 (d, J = 11.0 Hz),

140.5 (d, J = 7.1 Hz), 131.5, 130.4, 130.1 (d, J = 8.3 Hz), 125.4

(d, J = 33.7 Hz), 121.0, 118.8, 116.4 (dd, J = 20.9, 8.3 Hz),

113.7 (dd, J = 20.7, 8.2 Hz), 104.5, 75.2, 72.9, 68.5, 60.0, 59.1,

55.1, 53.9, 45.0, 42.1, 37.5, 35.2, 29.8, 27.5, 26.8, 23.7, 20.3,

20.0, 8.1; LRMS-ESI (m/z): 689.3 [M+H]⁺; HRMS-ESI (m/z):

[M+H]⁺calcd for C₃₃H₄₂FN₄O₇S₂, 689.2479; found 689.2474.

Acknowledgements

This research was supported by the National Institutes of Health

(Grant GM53386, AKG and Grant GM62920, ITW). This work

was also supported by the Intramural Research Program of the

Center for Cancer Research, National Cancer Institute, National

Institutes of Health, and in part by a Grant-in-Aid for Scientific

Research (Priority Areas) from the Ministry of Education, Culture,

Sports, Science, and Technology of Japan (Monbu Kagakusho),

a Grant for Promotion of AIDS Research from the Ministry of

Health, Welfare, and Labor of Japan, and the Grant to the

Cooperative Research Project on Clinical and Epidemiological

Studies of Emerging and Reemerging Infectious Diseases

(Renkei Jigyo) of Monbu-Kagakusho. The authors would like to

12

10.1002/cmdc.201700824

ChemMedChem

FULL PAPER

thank the Purdue University Center for Cancer Research, which

supports the shared NMR and mass spectrometry facilities.

[15] Y. Koh, S. Matsumi, D. Das, M. Amano, D. A. Davis, J. Li, S.

Leschenko, A. Baldridge, T. Shioda, R. Yarchoan, A. K. Ghosh, H.

Mitsuya, J. Biol. Chem. 2007, 282, 28709–28720.

[16] B. Clotet, J. Feinberg, J. van Lunzen, M. A. Khuong-Josses, A. Antinori,

I. Dumitru, V. Pokrovskiy, J. Fehr, R. Ortiz, M. Saag, J. Harris, C.

Brennan, T. Fujiwara, S. Min, Lancet 2014, 383, 2222–2231.

[17] Y. Koh, M. Amano, T. Towata, M. Danish, S. Leshchenko-Yashchuk, D.

Das, M. Nakayama, Y. Tojo, A. K. Ghosh, H. Mitsuya, J. Virol. 2010, 84,

11961–11969.

Keywords: HIV-1 protease inhibitors • brain penetration • Drug

resistance • dimerization inhibitor • Structure-based design •

synthesis • genetic barrier.

[18] D. Saylor, A. M. Dickens, N. Sacktor, N. Haughey, B. Slusher, M.

Pletnikov, J. L. Mankowski, A. Brown, D. J. Volsky, J. C. McArthur, Nat.

Rev. Neurol. 2016, 12, 234-248.

[19] R. K. Heaton, D. B. Clifford, D. R. Franklin, S. P. Woods, C. Ake, F.

Vaida, R. J. Ellis, S. L. Letendre, T. D. Marcotte, J. H. Atkinson, M.

Rivera-Mindt, O. R. Vigil, M. J. Taylor, A. C. Collier, C. M. Marra, B. B.

Gelman, J. C. McArthur, S. Morgello, D. M. Simpson, J. A. McCutchan,

I. Abramson, A. Gamst, C. Fennema-Notestine, T. L. Jernigan, J. Wong,

I. Grant, Neurology 2010, 75, 2087–2096.

References:

[1]

J. L. Marcus, C. R. Chao, W. A. Leyden, L. Xu, C. P. Quesenberry, D. B.

Klein, W. J. Towner, M. A. Horberg, M. J. Silverberg, J. Acquir. Immune

Defic. Syndr. 2016, 73, 39–46.

[2]

H. Samji, A. Cescon, R. S. Hogg, S. P. Modur, K. N. Althoff, K. Buchacz,

A. N. Burchell, M. Cohen, K. A. Gebo, M. J. Gill, A. Justice, G. Kirk, M.

B. Klein, P. T. Korthuis, J. Martin, S. Napravnik, S. B. Rourke, T. R.

Sterling, M. J. Silverberg, S. Deeks, L. P. Jacobson, R. J. Bosch, M. M.

Kitahata, J. J. Goedert, R. Moore, S. J. Gange, PLoS One 2013, 8,

e81355.

[20] V. Tozzi, P. Balestra, D. Serraino, R. Bellagamba, A. Corpolongo, P.

Piselli, P. Lorenzini, U. Visco-Comandini, C. Vlassi, M. E. Quartuccio, M.

Giulianelli, P. Noto, S. Galgani, G. Ippolito, A. Antinori, P. Narciso,

AIDS Res. Hum. Retroviruses 2005, 21, 706–713.

[21] A. K. Ghosh, B. D. Chapsal, I. T. Weber, H. Mitsuya, Acc. Chem. Res.

2008, 41, 78−86.

[3]

[4]

A. K. Ghosh, H. L. Osswald, G. Prato, J. Med. Chem. 2016, 59,

5172−5208.

[22] A. K. Ghosh, B. D. Chapsal, Design of the Anti-HIV-1 Protease Inhibitor

Darunavir. In Introduction to Biological and Small Molecule Drug

Research and Development: Theory and Case Studies; Ganellin, C. R.,

Jefferis, R., Roberts, S., Eds.; Elsevier: London, 2013; pp 355−384.

[23] M. Aoki, H. Hayashi, K. V. Rao, D. Das, N. Higashi-Kuwata, H. Bulut, H.

Aoki-Ogata, Y. Takamatsu, R. S. Yedidi, D. A. Davis, S.-i. Hattori, N.

Nishida, K. Hasegawa, N. Takamune, P. R. Nyalapatla, H. L. Osswald,

H. Jono, H. Saito, R. Yarchoan, S. Misumi, A. K. Ghosh, H. Mitsuya,

eLife 2017, 6, e28020.

J. S. G. Montaner, V. D. Lima, R. Barrios, B. Yip, E. Wood, T. Kerr, K.

Shannon, P. R. Harrigan, R. S. Hogg, P. Daly, P. Kendall, Lancet 2010,

376, 532−539.

[5]

S. G. Deeks, S. R. Lewin, A. L. Ross, J. Ananworanich, M. Benkirane,

P. Cannon, N. Chomont, D. Douek, J. D. Lifson, Y. R. Lo, D. Kuritzkes,

D. Margolis, J. Mellors, D. Persaud, J. D. Tucker, F. Barre-Sinoussi, G.

Alter, J. Auerbach, B. Autran, D. H. Barouch, G. Behrens, M.

Cavazzana, Z. Chen, É. A. Cohen, G. M. Corbelli, S. Eholié, N. Eyal, S.

Fidler, L. Garcia, C. Grossman, G. Henderson, T. J. Henrich, R.

Jefferys, H. P. Kiem, J. McCune, K. Moodley, P. A. Newman, M. Nijhuis,

M. S. Nsubuga, M. Ott, S. Palmer, D. Richman, A. Saez-Cirion, M.

Sharp, J. Siliciano, G. Silvestri, J. Singh, B. Spire, J. Taylor, M. Tolstrup,

S. Valente, J. van Lunzen, R. Walensky, I. Wilson, J. Zack, Nat. Med.

2016, 22, 839–850.

[24] A. K. Ghosh, B. D. Chapsal, A. Baldridge, M. P. Steffey, D. E. Walters,

Y. Koh, M. Amano, H. Mitsuya, J. Med. Chem. 2011, 54, 622−634.

[25] A. K. Ghosh, P. R. Sridhar, S. Leshchenko, A. K. Hussain, J. Li, A. Y.

Kovalevsky, D. E. Walters, J. Wedekind, V. Grum-Tokars, D. Das, Y.

Koh, K. Maeda, H. Gatanaga, I. T. Weber, H. Mitsuya, J. Med. Chem.

2006, 49, 5252−5261.

[26] A. K. Ghosh, S. Yashchuk, A. Mizuno, N. Chakraborty, J. Agniswamy,

Y.-F. Wang, M. Aoki, P. M. Salcedo Gomez, M. Amano, I. T. Weber, H.

Mitsuya, ChemMedChem 2015, 10, 107-115.

[6]

[7]

N. Lohse, A. B. Hansen, J. Gerstoft, N. Obel, J. Antimicrob. Chemother.

2007, 60, 461−463.

A. Edmonds, M. Yotebieng, J. Lusiama, Y. Matumona, F. Kitetele, S.

Napravnik, S. R. Cole, A. Van Rie, F. Behets, PLoS Med. 2011, 8,

e1001044.

[27] A. K. Ghosh, K. V. Rao, P. R. Nyalapatla, H. L. Osswald, C. D. Martyr,

M. Aoki, H. Hayashi, J. Agniswamy, Y. F. Wang, H. Bulut, D. Das, I. T.

Weber, H. Mitsuya, J. Med. Chem. 2017, 60, 4267–4278.

[28] B. K. Park, N. R. Kitteringham, P. M. O’Neill, Annu. Rev. Pharmacol.

Toxicol. 2001, 41, 443– 470.

[8]

Guidelines for the Use of Antiretroviral Agents in HIV-1-Infected Adults

and

Adolescents,

July

14,

2016.

https://aidsinfo.nih.gov/contentfiles/lvguidelines/adultandadolescentgl.p

[29] B. E. Smart, J. Fluor. Chem. 2001, 109, 3–11.

df.

[30] A. K. Ghosh, W. J. Thompson, M. K. Holloway, S. P. McKee, T. T.

Duong, H. Y. Lee, P. M. Munson, A. M. Smith, J. M. Wai, P. L. Darke, J.

Med. Chem. 1993, 36, 2300–2310.

[9]

S. Hue, R. J. Gifford, D. Dunn, E. Fernhill, D. Pillay, J. Virol. 2009, 83,

2645−2654.

[10] A. K. Ghosh, D. D. Anderson, I. T. Weber, H. Mitsuya, Angew. Chem.

Int. Ed. 2012, 51, 1778−1802.

[31] T. Katsuki, K. B. Sharpless, J. Am. Chem. Soc. 1980, 102, 5974−5976.

[32] Y. Gao, R. M. Hanson, J. M. Klunder, S. Y. Ko, H. Masamue, K. B.

Sharpless, J. Am. Chem. Soc. 1987, 109, 5765–5780.

[11] Y. Koh, M. Aoki, M. L. Danish, H. Aoki-Ogata, M. Amano, D. Das, R. W.

Shafer, A. K. Ghosh, H. Mitsuya, J. Virol. 2011, 85, 10079–10089.

[11] Y. Koh, H. Nakata, K. Maeda, H. Ogata, G. Bilcer, T. Devasamudram, J.

F. Kincaid, P. Boross, Y. F. Wang, Y. Tie, P. Volarath, L. Gaddis, R. W.

Harrison, I. T. Weber, A. K. Ghosh, H. Mitsuya, Antimicrob. Agents

Chemother. 2003, 47, 3123–3129.

[33] M. Caron, K. Carlier, K. B. Sharpless, J. Org. Chem. 1988, 53, 5185–

5187.

[34] A. K. Ghosh, S. P. McKee, H. Y. Lee, W. T. Thompson, J. Chem. Soc.

Chem. Commun. 1992, 1992, 273–274.

[35] A. K. Ghosh, S. Leshchenko, M. Noetzel, J. Org. Chem. 2004, 69,

7822–7829.

[12] A. K. Ghosh, B. D. Chapsal, H. Mitsuya, 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;

Ghosh, A. K., Ed.; Methods and Principles in Medicinal Chemistry, Vol.

[36] Y. G. Gololobov, I. N. Zhmurova, L. F. Kasukhin, Tetrahedron 1981, 37,

437−472.

[37] 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.

45; Wiley-VCH: Weinheim, Germany, 2010; pp 205−243.

[13] S. De Meyer, E. Lathouwers, I. Dierynck, E. De Paepe, B. Van Baelen,

T. Vangeneugden, S. Spinosa-Guzman, E. Lefebvre, G. Picchio, M. P.

de Béthune, AIDS 2009, 23, 1829-1840.

[38] M. V. Toth, G. R. Marshall, Int. J. Pept. Protein Res. 1990, 36, 544−550.

[39] M. Aoki, H. Hayashi, R. S. Yedidi, C. D. Martyr, Y. Takamatsu, H. Aoki-

Ogata, T. Nakamura, H. Nakata, D. Das, Y. Yamagata, A. K. Ghosh, H.

Mitsuya, J. Virol. 2016, 90, 2180-2194.

[14] H. Hayashi, N. Takamune, T. Nirasawa, M. Aoki, Y. Morishita, D. Das,

Y. Koh, A. K. Ghosh, S. Misumi, H. Mitsuya, Proc. Natl. Acad. Sci. 2014,

111, 12234–12239.

13

10.1002/cmdc.201700824

ChemMedChem

FULL PAPER

[40] M. Aoki, M. L. Danish, H. Aoki-Ogata, M. Amano, K. Ide, D. Das, Y.

Koh, H. Mitsuya, J. Virol. 2012, 86, 13384-13396.

[41] M. Zürcher, F. Diederich, J. Org. Chem. 2008, 73, 4345-4361.

[42]

K. Müller, C. Faeh, F. Diederich, Science 2007, 317, 1881-1886.

[43] B. Mahalingam, J. M. Louis, J. Hung, R. W. Harrison, I. T. Weber,

Proteins 2001, 43, 455–464.

[44] Z. Otwinowski, W. Minor, Processing of X-ray Diffraction Data Collected

in Oscillation Mode. Methods in Enzymology, 276: Macromolecular

Crystallography, Part A; Carter, C.W., Jr., Sweet, R. M., Eds.;

Academic Press: New York, 1997; pp 307-326.

[45] A. J. McCoy, R. W. Grosse-Kunstleve, P. D. Adams, M. D. Winn, L. C.

Storoni, R. J. Read, J. Appl. Crystallogr. 2007, 40, 658–674.

[46] M. D. Winn, C. C. Ballard, K. D. Cowtan, E. J. Dodson, P. Emsley, P. R.

Evans, R. M. Keegan, E. B. Krissinel, A. G. W. Leslie, A. McCoy, S. J.

McNicholas, G. N. Murshudov, N. S. Pannu, E. A. Potterton, H. R.

Powell, R. J. Read, A. Vagin, K. S. Wilson, Acta Crystallogr., Sect. D:

Biol. Crystallogr. 2011, 67, 235-242.

[47] Collaborative Computational Project, Number 4 Acta Crystallogr., Sect.

D: Biol. Crystallogr., 1994, 50, 760-763.

[48] E. Potterton, P. Briggs, M. Turkenburg, E. Dodson, Acta Crystallogr.,

Sect. D: Biol. Crystallogr. 2003, 59, 1131–1137.

[49] C.-H. Shen, Y.-F. Wang, A. Y. Kovalevsky, R. W. Harrison, I. T. Weber,

FEBS J. 2010, 277, 3699-3714.

[50] G. M. Sheldrick Acta Crystallogr., Sect. A: Found. Crystallogr. 2008, 64,

112–122.

[51] G. M. Sheldrick, T. R. Schneider, Meth. Enzymol. 1997, 277, 319–343.

[52] A. W. Schuettelkopf, D. M. F. van Aalten, Acta Crystallogr., Sect. D:

Biol. Crystallogr. 2004, 60, 1355–1363.

[53] P. Emsley, B. Lohkamp, W. G. Scott, K. Cowtan, Acta Crystallogr., Sect.

D: Biol. Crystallogr. 2010, 66, 486-501.

[54]

P. Emsley, K. Cowtan, Acta Crystallogr., Sect. D: Biol. Crystallogr.

2004, 60, 2126–2132.

[55] H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H.

Weissig, I. N. Shindyalov, P. E. Bourne, Nucleic Acids Res. 2000, 28,

235–242.

14

10.1002/cmdc.201700824

ChemMedChem

FULL PAPER

Entry for the Table of Contents

Insert graphic for Table of Contents here.

We report structure-based design, synthesis, biological evaluation and X-ray structural studies of a series of exceptionally potent HIV-

1 protease inhibitors containing novel P1, P2 and P2' –ligands to interact with active site residues.

15

Article Doi

Design of Highly Potent, Dual-Acting and Central-Nervous-System-Penetrating HIV-1 Protease Inhibitors with Excellent Potency against Multidrug-Resistant HIV-1 Variants

DOI: 10.1002/cmdc.201700824

Source and publish data:

Authors:

Article abstract of DOI:10.1002/cmdc.201700824

Full text of DOI:10.1002/cmdc.201700824

Products guided by the article

R&D Labs maybe for 1452139-09-1

Relevant to this article

Hot Product