Structure-Based Design of Highly Potent HIV-1 Protease Inhibitors Containing New Tricyclic Ring P2-Ligands: Design, Synthesis, Biological, and X-ray Structural Studies

Biological, and X ‑ray Structural Studies

Article

Structure-Based Design of Highly Potent HIV‑1 Protease Inhibitors

Containing New Tricyclic Ring P2-Ligands: Design, Synthesis,

Arun K. Ghosh,* Satish Kovela, Heather L. Osswald, Masayuki Amano, Manabu Aoki,

Johnson Agniswamy, Yuan-Fang Wang, Irene T. Weber, and Hiroaki Mitsuya

Cite This: https://dx.doi.org/10.1021/acs.jmedchem.0c00202

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sı Supporting Information

ABSTRACT: We describe here design, synthesis, and biological

evaluation of a series of highly potent HIV-1 protease inhibitors

containing stereochemically deﬁned and unprecedented tricyclic

furanofuran derivatives as P2 ligands in combination with a variety

of sulfonamide derivatives as P2′ ligands. These inhibitors were

designed to enhance the ligand-backbone binding and van der

Waals interactions in the protease active site. A number of

inhibitors containing the new P2 ligand, an aminobenzothiazole as the P2′ ligand and a diﬂuorophenylmethyl as the P1 ligand,

displayed very potent enzyme inhibitory potency and also showed excellent antiviral activity against a panel of highly multidrug-

resistant HIV-1 variants. The tricyclic P2 ligand has been synthesized eﬃciently in an optically active form using enzymatic

desymmetrization of meso-1,2-(dihydroxymethyl)cyclohex-4-ene as the key step. We determined high-resolution X-ray structures of

inhibitor-bound HIV-1 protease. These structures revealed extensive interactions with the backbone atoms of HIV-1 protease and

provided molecular insights into the binding properties of these new inhibitors.

INTRODUCTION

catalytic ﬁtness. Darunavir incorporated the stereochemically

deﬁned and conformationally constrained bicyclic polyether,

bis-tetrahydrofuran (bis-THF), which makes extensive inter-

■

HIV-1 protease inhibitors (PIs) are critical components of

combined antiretroviral therapy (cART) which dramatically

improved the HIV-related mortality and morbidity to patients

with HIV-1 infection and AIDS. PIs block the cleavage of

Gag-Pol polyproteins during viral maturation and result in

10,15,16

actions in the active site.

Numerous X-ray crystallo-

1,2

graphic studies of darunavir-bound HIV-1 protease revealed

that darunavir forms a network of hydrogen bonding

interactions with the backbone of HIV-1 protease in both S2

generation of noninfectious virions.

PI-based drugs in

7,18

and S2′ subsites.

The P2 bis-tetrahydrofuranyl urethane

general exhibit a relatively high genetic barrier to the

emergence of drug-resistant HIV-1 variants compared to

non-nucleoside reverse transcriptase inhibitors and integrase

(bis-THF) ligand is an intriguing pharmacophore where both

ring oxygens form strong hydrogen bonds with the Asp29 and

Asp30 backbone amide NHs. The bicyclic bis-THF ring also

−7

strand transfer inhibitors (INSTIs).

The last approved PI,

darunavir (1, DRV, Figure 1), is a ﬁrst-line therapy which is

often preferred for cART-naive HIV-1 infected patients

engages in van der Waals interaction with active site

19,20

residues.

In our continuing eﬀorts to optimize both P2

because of its tolerance and a high genetic barrier to the

and P2′ structural templates of darunavir, we have recently

−10

development of drug-resistant viruses.

However, there are

reported a number of very potent PIs with unprecedented

21−23

reports of failure of DRV-containing regimens and the

structural features.

In particular, we designed a crown-like

11,12

emergence of darunavir resistant HIV-1 variants.

Fur-

tetrahydropyrano-tetrahydrofuran in inhibitor 3 with a bridged

thermore, there are reports that a growing number of HIV/

AIDS patients are harboring highly multidrug-resistant HIV-1

methylene group as the P2 ligand to promote additional van

24,25

der Waals interactions in the active site.

Inhibitors

13,14

variants, and options for treating these patients are limited.

containing such crown-THF ligands are very potent inhibitors

Therefore, the development of potent and structurally novel

PIs with broad spectrum antiviral activity are necessary for the

future success of cART treatment regimens.

Received: February 4, 2020

Darunavir and its derivatives (2) were designed to promote

a network of hydrogen bonding interactions with the backbone

atoms at the active site of HIV-1 protease. The backbone

binding strategies are likely to slow development of drug

resistant HIV-1 variants because of possible reduction of

XXXX American Chemical Society

Journal of Medicinal Chemistry

J. Med. Chem. XXXX, XXX, XXX−XXX

Article

Table 1. HIV-1 Protease Inhibitory and Antiviral Activity of

PIs

Figure 1. Structures of HIV-1 PIs 1−4.

and maintained potent antiviral activity against multidrug-

25,26

resistant HIV-1 variants.

The X-ray structural analysis of

K values represents at least four data points. Standard error in all

crown-THF-derived inhibitors provided molecular insights to

cases was less than 7%. Darunavir exhibited K = 16 pM. Values are

24,25

further improve ligand-binding site interactions.

Herein,

means of at least three experiments. Standard error in all cases was

^l^e^s^s^t^h^aⁿ⁵^%^.^D^a^r^uⁿ^a^vⁱ^r^e^x^hⁱ^bⁱ^t^e^d^aⁿ^tⁱ^vⁱ^r^a^l^I^C50 = 3.2 nM, saquinavir

^I^C50 = 21 nM.

we report a new class of PIs containing cyclohexane fused bis-

tetrahydrofuran as the P2 ligands in combination with (R)-

hydroxyethylaminesulfonamide isosteres. A number of inhib-

itors exhibited exceptionally potent enzyme inhibitory and

antiviral activity. In particular, inhibitor 4d maintained

exceptional antiviral potency against selected multidrug-

resistant HIV-1 variants. Our high resolution X-ray structural

studies of inhibitor-bound HIV-1 protease revealed important

molecular insights into the ligand-binding site interactions

responsible for their potent activity. The design and synthesis

of new PIs have been an active area of our research. Several

potent inhibitors incorporating a variety of heterocyclic P2

ligands for improving interactions at the S2 subsite have been

new ligand with additional methylene groups around the bis-

THF may exhibit better van der Waals interactions in the

active site. Our preliminary model of the enantiomeric ligand

structure also showed good orientation for hydrogen bonding

interactions with backbone amide NHs of Asp29 and Asp30.

The conformationally constrained ligand also appeared to

engage in additional van der Waals contacts in the S2 subsite

compared to the bis-THF ligand to darunavir. While the

stereochemistry of the Chf-ligand in inhibitors 4a and 5a

appeared optimum, we planned to synthesize both enantio-

meric ligands as both enantiomers of bis-THF ligand and

crown-THF in inhibitor 3 showed comparable enzyme aﬃnity

and ligand binding site interactions. This is due to the fact that

the acetal oxygens in both enantiomers are in a similar

environment and both ligands can form strong hydrogen

bonds with the backbone amide residues in the S2 subsite.

Therefore, our plan is to synthesize both enantiomers and gain

molecular insights into their ligand-binding site interactions

using X-ray structural studies of the inhibitor-HIV-1 protease

complex. We also plan to examine various hydroxyethylsulfo-

namide isosteres containing varying P1 and P2′ ligands. In

particular, we plan to investigate an aminobenzothiazole(Abt)-

based P2′-ligand which has been showed to promote better

hydrogen bonding and van der Waals interactions compared to

7−30

reported.

A number of other design eﬀorts were focused

on optimizing both P1 and P2′ moieties to improve

7,28,31

potency.

Recently, bicyclic piperazine sulfonamide-

based inhibitors have been designed where the piperazine

NH forms the key interaction with the catalytic aspartic acids

32,33

of the HIV-1 protease.

RESULTS AND DISCUSSION

■

Based upon ligand-binding site interactions of the crown-THF

ligand in 3 in the HIV-1 protease active site, we have designed

a new tricyclic cyclohexane fused-tetrahydrofuranofuran (Chf-

THF) derivative as the P2 ligand shown in inhibitors 4a and 5a

(

Table 1). This new design may promote better hydrogen

bonding interactions with backbone atoms. Furthermore, the

J. Med. Chem. XXXX, XXX, XXX−XXX

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Article

the 4-aminobenzene-sulfonamide P2′-ligand of darunavir.

Structurally, the Abt P2′ ligand forms additional hydrogen

bonding and van der Waals interactions in the S2′ subsite. A

number of recent inhibitors containing these P2′-ligands

exhibited robust antiviral activity against highly multidrug-

Scheme 2. Synthesis of Substituted Tricyclic P2 Ligands 6

and ent-6

24,25

resistant HIV-1 variants.

For our studies, we plan to synthesize our newly designed

ligand in a stereodeﬁned manner. Our synthetic strategy for

optically active synthesis is shown in Scheme 1. Enantiomeric

Scheme 1. Synthetic Strategy for the Cyclohexane-Fused bis-

THF P2 Ligand Structure

ligand 6 can be obtained from the functionalized cyclohexane-

1,2-diol derivative 7. Structure 7 can be obtained from

cyclohexene derivative 8 by asymmetric dihydroxylation

4,35

reaction.

An optically active aldehyde derivative can be

derived conveniently from meso diol derivative 9 by enzymatic

desymmetrization as the key reaction. The meso-diol 9 can be

derived from commercially available and inexpensive 1,2,3,6-

tetrahydrophthalic anhydride 10.

Our synthesis of optically active ligand 6 is shown in Scheme

. meso-1,2,3,6-Tetrahydrophthalic anhydride 10 was reduced

by LiAlH in THF at 0 °C for 3 h to provide meso-diol

derivative 9 in a multigram scale. Diol 9 was subjected to

enzymatic desymmetrization reaction using porcine pancreatic

lipase (PPL) in ethyl acetate at 23 °C for 12 h to provide

monoacetate derivative 11 in gram scale in 82% yield and

Reagents and conditions. (a) LiAlH , THF, 0 °C, 3 h (85%); (b)

95% ee, as determined by high-performance liquid

PPL, EtOAc, 23 °C, 12 h (82%); (c) (COCl) , DMSO, TEA,

chromatography (HPLC) analysis (see Supporting Informa-

tion for further details). Swern oxidation of alcohol 11

provided the corresponding aldehyde which was reacted with

trimethylorthoformate in the presence of a catalytic amount of

, −78 to 0 °C, 1.5 h; (d) CH(OMe) , TBABr , MeOH, 23 °C,

3 3

h (76% over 2-steps for 12; 80% over 2-steps for ent-12); (e) AD

mix-β, CH SO NH , t-BuOH/H O (1:1), 0−23 °C; (f) 1 N NaOH,

MeOH, 0−23 °C, 3 h (90% over 2-steps for 13 & 14; 88% over 2-

steps for ent-13 & ent-14); (g) CSA, CH Cl , 0 °C, 1 h (82% for 6

tetrabutylammonium tribromide (TBABr ) at 23 °C for 8 h to

and 79% for ent-6); (h) Ac O, Py, DMAP, CH Cl , 0−23 °C (98%);

provide dimethylacetal derivative 12 in 76% yield over two

(i) PPL, 0.1 M phosphate buﬀer pH 7, 1 N NaHCO , 23 °C, 16 h

(84%).

steps. For diastereoselective dihydroxylation, we carried out

Sharpless asymmetric dihydroxylation using AD-mix-β.

Reaction of 12 with AD-mix-β in a mixture (1:1) of t-butanol

and water at 0−23 °C for 24 h resulted in a 1:1 mixture of

Our synthesis of enantiomeric ligand ent-6 from meso-diol 9

diastereomeric diol. The resulting diol was subjected to

saponiﬁcation using 1 N aqueous NaOH in MeOH at 0−23 °C

for 3 h to provide triol derivatives 13 and 14 in 90% yield over

two steps. These triol derivatives were separated by silica gel

chromatography using 5% MeOH in CH Cl as the eluent.

is shown in Scheme 2. Diol 9 was converted to diacetate

derivative 15 by reaction with acetic anhydride and pyridine in

the presence of a catalytic amount of DMAP at 23 °C for 16 h.

Exposure of diacetate to PPL in 0.1 M phosphate buﬀer at pH

7 in the presence of aqueous NaHCO at 23 °C for 16 h

37,39

Triol derivative 13 was reacted with a catalytic amount of

camphorsulfonic acid (CSA) in CH Cl at 0 °C for 1 h to

provided optically active alcohol ent-11 in 84% yield.

Alcohol ent-11 was converted to dimethylacetal ent-12 in 80%

yield as described above. Exposure of dimethylacetal ent-12 to

provide optically active tricyclic ligand alcohol 6 in 82% yield.

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Article

sharpless asymmetric dihydroxylation reaction with AD-mix-α

Scheme 4. Synthesis of PIs 4a−4f and 5a−f

aﬀorded a 1:1 mixture of diastereomeric diol. Deprotection

of the acetate derivative furnished triol derivatives ent-13 and

ent-14 which were separated by silica gel chromatography.

Treatment of the major triol derivative ent-13 with a catalytic

amount of CSA in CH Cl aﬀorded ligand alcohol ent-6 in 79%

yield.

The synthesis of the designed PIs was carried out in a two-

step sequence involving synthesis of activated carbonates

followed by reaction of these carbonates with appropriate

22,23

hydroxyethylaminesulfonamide isosteres.

The syntheses of

various activated carbonates are shown in Scheme 3. Optically

Scheme 3. Synthesis of Activated Carbonates 16 and ent-16

Reagents and Conditions. (a) DIPEA, CH CN, 23 °C, (59−87%)

yields (65−86%). Similarly, reactions of carbonate ent-16 with

amines 17−22 under similar conditions aﬀorded inhibitors

a−5f in very good yields (59−87%). The full structures of

these inhibitors are shown in Table 1.

Our preliminary model of the P2 Chf-THF ligand in

inhibitor 4a compared to the bis-THF ligand in inhibitor 2

indicated that both oxygens of the new Chf-THF ligand could

align optimally with Asp29 and Asp30 backbone amide NHs in

the S2 site. In addition, two methylene groups are expected to

provide van der Waals interactions surrounding Ile47, Val32,

Leu76, and Ile50′ residues in the S2 subsite. Because ligand

combinations are critical for overall aﬃnity, we investigated

enantiomeric Chf-THF with various P2′-ligands. The results of

compounds in HIV-1 protease inhibitory and antiviral assays

are shown in Table 1. The assay protocol for HIV-1 inhibition

Reagents and Conditions. (a) 4-NO PhOCOCl, Py, CH Cl , 0−23

°C, 8 h (87% for 16 and 88% for ent-16)

active ligand alcohols 6 and ent-6 synthesized above were

converted to their respective activated carbonates 16 and ent-

6. As shown, reaction of ligand alcohols 6 and ent-6 with 4-

nitrophenylchloroformate in the presence of pyridine in

CH Cl at 0−23 °C for 12 h provided activated carbonates

carbonates were then converted to urethane derivatives using

amines 17−22. The synthesis of various inhibitors containing

Chf-THF as the P2 ligands on the hydroxyethylamine

sulfonamide isostere is shown in Scheme 4. Reactions of

is similar to the reported procedure of Toth and Marshall.

The protocol for antiviral assay is described previously using

6 and ent-16 in 87 and 88% yields, respectively. These

MT-2 human T-lymphoid cells exposed to HIV-1_L_A_I

assess the stereochemical eﬀect, we ﬁrst examined the

enantiomeric ligands in combination with a sulfonamide

isostere with a 4-methoxy benzene sulfonamide as the P2′-

ligand in compounds 4a and 5a. Inhibitor 4a showed very

22,23

activated carbonate 16 with known

2 in the presence of diisopropylethylamine (DIPEA) in

CH CN at 23 °C for 72 h provided inhibitors 4a−f in good

amine derivatives 17−

potent HIV-1 protease inhibitory activity with a K value of 8

pM. The inhibitor 4a also exhibited a very good antiviral IC₅₀

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value of 38 nM (entry 1). Inhibitor 5a with enantiomeric Chf-

THF ligand is slightly less potent than 4a. It showed enzyme

ligand resulted in inhibitor 4d which displayed enzyme

inhibitory K of 54 pM and antiviral IC value of 86 pM.

Inhibitor 4d showed improved antiviral activity compared to

the corresponding inhibitor 4b containing a phenylmethyl

group as the P1-ligand. This improvement in antiviral activity

may be due to increased lipophilicity of inhibitor 4d (clogp

5.4) over inhibitor 4b (clogP 5.1). Inhibitor 5d containing the

inhibitory activity with a K of 34 pM and antiviral activity with

an IC₅₀value of 72 nM (entry 2). We then examined both

enantiomeric ligands in combination with other benzothiazoles

and benzoxazoles as the P2′-ligands. Inhibitor 4b showed very

potent enzyme K of 2 pM, which is nearly 10-fold better than

inhibitor 5b with the enantiomeric P2 ligand (entries 3 and 4).

However, both inhibitors exhibited very potent antiviral

activity with IC₅₀values of 3.3 and 1.8 nM. Inhibitors 4c

and 5c with enantiomeric Chf-THF P2 ligands and

benzoxazole P2′ ligands also showed very potent enzyme

inhibitors and antiviral activity (entries 5 and 6). Interestingly,

inhibitor 5c exhibited over 5-fold improvement of antiviral

activity (IC₅₀= 0.3 nM) compared to inhibitor 4c (IC₅₀= 1.7

nM).

We then examined inhibitors containing enantiomeric Chf-

THF ligands in combination with ﬂuorine-substituted phenyl-

methyl groups as the P1 ligands. As shown in Table 2,

incorporation of a 3,5-diﬂuoro phenylmethyl group as the P1-

enantiomeric P2 ligand also showed very potent enzyme K

value; however, it showed nearly 25-fold reduction of antiviral

activity compared to inhibitor 4d with an enantiomeric P2

ligand (entries 1 and 2). In comparison, darunavir and

^s^a^q^uⁱⁿ^a^vⁱ^r^e^x^hⁱ^bⁱ^t^e^d^aⁿ^tⁱ^vⁱ^r^a^l^I^C50 values of 3.2 and 21 nM,

respectively. We also examined the eﬀect of monoﬂuoro

substitution on the P1-ligand. As shown, incorporation of 3-

ﬂuorophenylmethyl as the P1 ligand resulted in inhibitors 4e

and 5e with only marginal improvement of antiviral activity

compared to unsubstituted P1 ligands in inhibitors 4b and 5b

(

Table 1). Incorporation of 4-ﬂuoro phenylmethyl as the P1

ligand resulted in inhibitors 4f and 5f with improvement of

antiviral activity, over 7-fold compared to inhibitor 4b and over

4-fold compared to inhibitor 5b (entry 3, Table 1). Overall,

inhibitor 4d with 3,5-bis-ﬂuorines on the P1 ligand showed the

best antiviral activity over methoxy sulfonamide and

monoﬂuoro derivatives.

Table 2. HIV-1 Protease Inhibitory and Antiviral Activity of

PIs

While the cART therapy and treatment guidelines are

updated regularly, PI-based drugs are important elements of

current cART regimens. In particular, PIs are widely used for

the treatment of naive and experienced HIV/AIDS patients. As

mentioned earlier, heavily-ART regimen-experienced HIV/

AIDS patients are reported to have drug-failure with the

11,12

currently available PIs including DRV.

Therefore, the

design of new classes of potent PIs with a high genetic barrier

to development of drug resistance is important for durable

treatment options. In this context, our long-standing objective

is to design PIs that can maintain robust potency against a

variety of existing multi-PI-resistant HIV-1 variants with better

selectivity index and safety proﬁles. Based upon preliminary

antiviral data, we selected two potent inhibitors (4d, antiviral

IC₅₀of 0.086 nM and 5c, antiviral IC₅₀of 0.3 nM) containing

enantiomeric Chf-THF ligands and evaluated their antiviral

activity against a panel of highly multidrug-resistant HIV-1

variants that had been selected in vitro with widely used FDA-

approved PIs, ATV and DRV. Each of these HIV-1 variants

were selected in vitro by propagating HIV-1_N_L₄_‑₃in the

presence of increasing concentrations of each PI (up to 5 μM)

41,42

in MT-4 cells, as described by us previously.

The results

are shown in Table 3. As can be seen, both widely used PI

drugs, ATV and DRV, lost activity against these resistant

variants. In particular, ATV lost signiﬁcant potency and is

unable to suppress the replication of highly PI-resistant HIV-1

variants examined. DRV showed relatively better results

compared to ATV; however, DRV lost 86-fold and 89-fold

activity against HIV-1_L_P_V_‑₅_μ_Mand HIV-1_D_R_V^R

variants.

P30

Inhibitor 5c containing the Chf-THF P2 ligand failed to

block replication of these highly drug-resistant HIV-1 variants.

Inhibitor 4d containing enantiomeric P2-ligand is signiﬁcantly

more potent than DRV and ATV against HIV-1_N_L₄_‑₃virus.

While this inhibitor maintained very good antiviral activity

(0.21−4.5 nM) against these highly drug-resistant HIV-1

variants, its fold-change of activity is comparable to DRV. The

reason for better overall antiviral activity is possibly due to

extensive hydrogen bonding and van der Waals interactions in

the S2 and S2′ subsites. Our X-ray crystallographic studies of

K values represents at least four data points. Standard error in all

cases was less than 7%. Darunavir exhibited K = 16 pM. Values are

means of at least three experiments. Standard error in all cases was

less than 5%. Darunavir exhibited antiviral IC₅₀= 3.2 nM, saquinavir

IC₅₀= 21 nM.

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with HIV-1 protease are highlighted in Figures 2 and 3,

Article

Table 3. Antiviral Activity of 5c and 4d and Other PIs against Highly PI-Resistant HIV-1 Variants^a^,^b^,^c

mean IC₅₀in nM ± SD (fold-change)

virus species

ATV

DRV

compound 5c

compound 4d

wild-type

in vitro HIV-1_P_R^R*

HIV-1_N_L₄_‑₃

4.0 ± 0.7

4.2 ± 0.9

38 ± 1

0.034 ± 0.026

HIV-1_A_T_V_‑₅_μ_M

HIV-1_L_P_V_‑₅_μ_M

>1000 (>250)

430 ± 10 (107)

>1000 (>250)

26 ± 6(6.1)

360 ± 50 (86)

370 ± 120 (89)

350 ± 30 (9.1)

>1000 (>26)

640 ± 510 (17)

0.21 ± 0.07 (6.3)

4.3 ± 0.2 (127)

4.5 ± 2.5 (133)

HIV-1_D_R_V^R

P30

The amino acid substitutions identiﬁed in protease of HIV-1_A_T_V_‑₅_μ_M, HIV-1_L_P_V_‑₅_μ_M, and HIV-1_D_R_V^Rcompared to the wild-type HIV-1_N_L₄_‑₃

P30

include L23I/E34Q/K43I/M46I/I50L/G51A/L63P/A71V/V82A/T91A, L10F/V32I/M46I/I47A/A71V/I84V, and L10I/I15V/K20R/L24I/

V32I/M36I/ M46L/L63P/K70Q/V82A/I84V/L89M, respectively. In vitro HIV-1_P_R, in vitro PI-selected HIV-1 variants. The EC (50%

eﬀective concentration) values were determined by using MT-4 cells as target cells. MT-4 cells (10 /mL) were exposed to 100 TCID s of each

HIV-1, and the inhibition of p24 Gag protein production by each drug was used as an endpoint. Numbers in parentheses represent fold changes in

IC s for each isolate compared to the IC s for wild-type HIV-1_N_L₄_‑₃. All assays were conducted in duplicate, and the data shown represent mean

values (±1 standard deviation) derived from the results of two independent experiments.

inhibitors 4a- and 5c-bound HIV-1 protease provided

molecular insights into the ligand-binding site interactions

responsible for their activity.

We determined the X-ray structures of inhibitors 4a and 5c

containing enantiomeric ligands in complex with wild-type

HIV-1 protease at a resolution of 1.25 and 1.3 Å,

respectively. Both complexes crystallized in the orthorhom-

bic space group P2 2 2 with one protease homodimer per

asymmetric unit. The inhibitors bind at the active site in two

alternate conformations related by 180° rotation with a relative

occupancy of 0.7/0.3 for inhibitor 4a and 0.55/0.45 for

inhibitor 5c. The overall dimer structure is very similar to the

17,18

HIV-1 protease−darunavir complex

with an rsmd for

superposition of 198 equivalent Cα atoms of 0.14 Å and 0.12

for protease complexes with inhibitors 4a and 5c, respectively.

The largest deviation of 0.45 Å in comparison to the protease−

darunavir complex occurs at residue Pro39 for 5c complex,

while ﬂap residue Phe53′ exhibits a maximum deviation of 0.51

Å in 4a complex. The key interactions of inhibitors 4a and 5c

Figure 3. Inhibitor 5c-bound HIV-1 protease X-ray structure is shown

(pdb code: 6VOE). The inhibitor carbon atoms are shown in cyan

and hydrogen bonds are shown by black dotted lines.

interactions with the main chain amide of Asp29 and Asp30

similar to the hydrogen bonds formed by the bis-THF oxygens

in darunavir. However, unlike the bis-THF of darunavir, the

bigger Chf-THF of inhibitors 4a and 5c form a number of

additional van der Waals interactions. We compared van der

Waals interactions of the enantiomeric Chf-THF ligands in

Figure 4. Interestingly, in one conformation, Chf-ligand in

compound 5c forms van der Waals contacts with Ile47. The

tricyclic core of the Chf-THF ligand forms van der Waals

interaction with the side chain atoms of Ile47, the main chain

amide of Gly48, and the Cδ1 atom of Ile50′. The two acetal

oxygens in the enantiomeric P2 Chf-THF of 4a are shifted by

0.8 Å in comparison to those of 5c. There is an additional

hydrogen bond interaction between one of the acetal oxygens

and the carboxylate side chain of Asp29. Further, the

enantiomeric change allows the 6-member ring of Chf-THF

in inhibitor 4a to shift toward Ile84. Because of this shift, the

P2 group of 4a form van der Waals interactions with Ile 84 in

addition to those with Ile47, Gly48, and Ile50′.

Figure 2. Inhibitor 4a-bound HIV-1 protease X-ray structure is shown

(

pdb code: 6VOD). The inhibitor carbon atoms are shown in green

and hydrogen bonds are shown by black dotted lines.

The aminobenzene group at the P2′ of darunavir is replaced

by the extended N-isopropylbenzo[d]oxazol-2-amine group in

inhibitor 5c. In the protease−darunavir complex, the P2′

amine of darunavir forms direct and water-mediated hydrogen

bonds with the two alternate conformations of Asp30′ side

chain. In the protease complex with 5c, the single

conformation of Asp30′ forms a direct hydrogen bond

interaction with the oxazole nitrogen of the inhibitor. In

addition, the extended P2′ amine group of inhibitor 5c forms a

new hydrogen bond with the carboxylate side chain of Asp30′.

respectively. Both inhibitors retain all the hydrogen bonds

observed between darunavir and the main chain atoms of the

HIV-1 protease. The new inhibitors also form a tetracoordi-

nated water-mediated hydrogen bond interaction involving one

of the sulfonamide oxygens and carbonyl oxygen with the

amide nitrogen of ﬂap residues Ile 50 and Ile 50′ similar to the

protease−darunavir complex.

Inhibitor 5c has a Chf-THF as P2 group and the two acetal

oxygens in the bulky Chf-THF form hydrogen bond

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Figure 4. Side by side comparison of the new Chf-THF moiety of inhibitor 4a (left, green carbon atoms) with the enantiomeric Chf-THF moiety of

inhibitor 5c (right, cyan carbon chain) inside the S2 subpocket. Both ligands form extensive van der Waals interactions (Val32, Ile47, Ile50′, and

Ile84 for 4a and Val32, Ile47, and Ile50′ for 5c) in the S2 subsite. Also, they are located close to the periphery of the protease active site and form

three strong hydrogen bonds in a similar fashion (black dotted lines).

Further, the terminal P2′ isopropyl group of 5c forms a C−H···

O interaction (2.8−3.2 Å) with the carboxylate side chain of

Asp29′ and van der Waals interaction with Ile47′. Asp29′ plays

a vital role in the structural integrity of the protease dimer by

forming a conserved inter-subunit ion pair with Arg8. The P2′

methoxy group of inhibitor 4a has a similar position as the P2′

aminobenzene of darunavir in the S2′ subsite of protease. The

oxygen atom of the P2′ methoxy group in inhibitor 4a forms a

hydrogen bond with the main chain amide of Asp30′. The Chf-

THF P2-ligand in inhibitor 4a appeared to form enhanced

ligand-binding site interactions over the enantiomeric ligand in

inhibitor 5c in the HIV-1 protease active site. The combination

of the Chf-THF P2-ligand, 3,5-diﬂuorinated P1 ligand and the

aminobenzothiazole P2′ ligand resulted in inhibitor 4d which

exhibited best antiviral activity and maintained very good

antiviral activity against a panel of highly drug-resistant HIV-1

variants.

better van der Waals interactions compared to the enantio-

meric ligands in the S2 site. Incorporation of the Chf-THF P2

ligand, the ﬂuorinated P1 ligand, and the aminobenzothiazole

P2′ ligand resulted in inhibitor 4d that showed best antiviral

activity. Further optimization of inhibitor properties and

ligand-binding site interactions are in progress in our

laboratory.

EXPERIMENTAL SECTION

■

General Methods. All chemicals and reagents were purchased

from commercial suppliers and used without further puriﬁcation

unless otherwise noted. The following reaction solvents were distilled

prior to use: dichloromethane from calcium hydride, diethyl ether and

tetrahydrofuran from Na/benzophenone, and methanol and ethanol

from activated magnesium under argon. All reactions were carried out

under an argon atmosphere in either ﬂame or oven-dried (120 °C)

glassware. 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

CONCLUSIONS

mesh, 60 Å pore diameter silica gel. H and C NMR spectra were

recorded at room temperature on a Bruker AV800, DRX-500 and AV-

■

In summary, based on X-ray structural data, we have designed

a new class of very potent HIV-1 PIs incorporating new

polycyclic cylcohexane-fused bis-tetrahydrofuran derivatives as

the P2 ligands. The new ligands have been speciﬁcally designed

to make favorable hydrogen bonding interactions and van der

Waals interactions with the residues at the S2-subsite. In

general, inhibitors containing enantiomeric ligands have shown

very potent activity, particularly inhibitors 4d and 5c.

However, inhibitor 4d with a diﬂuoro phenylmethyl P1 ligand

and an aminobenzothiazole as the P2′ ligand maintained very

potent activity against a panel of highly multidrug-resistant

HIV-1 variants. The results show that inhibitor 4d is superior

to darunavir and other approved PI drugs. The new polycyclic

ligand alcohols were synthesized eﬃciently in an enantiose-

lective manner using enzymatic desymmetrization of meso-diols

as the key steps. Because both enantiomeric ligands exhibited

very high enzyme aﬃnity, we determined high resolution X-ray

structures of inhibitor-bound HIV-1 protease containing

enantiomeric ligands. As it turned out, both oxygens of the

enantiomeric tricyclic ligands formed very strong hydrogen

bonds with the backbone amide NHs of Asp29 and Asp30 in

the S2 subsite. The P2 ligand in inhibitor 4a appears to make

00. Chemical shifts (δ values) are reported in parts per million and

are referenced to the deuterated residual solvent peak. NMR data are

reported as δ value (chemical shift, J-value (Hz), integration, where s

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

Optical rotations were recorded on a PerkinElmer 341 polarimeter.

HRMS and LRMS spectra were recorded at the Purdue University

Department of Chemistry Mass Spectrometry Center. HPLC analysis

and puriﬁcation were done on an Agilent 1100 series instrument using

a Chiralcel OZ-3 column of 4.6 mm ID for analysis. The purity of all

test compounds was determined by HPLC analysis to be ≥95% pure.

(

1R,3aS,7aR)-Octahydro-1,6-epoxyisobenzofuran-5-yl

(

(2S,3R)-3-Hydroxy-4-((N-isobutyl-4-methoxyphenyl)-

sulfonamido)-1-phenylbutan-2-yl)carbamate (4a). To a stirred

solution of activated alcohol 16 (15 mg, 0.046 mmol) and isostere 17

(21 mg, 0.051 mmol) in acetonitrile (2 mL) was added DIPEA (40

μL, 0.233 mmol) at 23 °C under an argon atmosphere. The reaction

mixture was stirred at 23 °C until completion. Upon completion,

solvents were removed under reduced pressure and the crude product

was puriﬁed by silica gel column chromatography (50% EtOAc in

hexane) to give 4a (22 mg, 81%) as an amorphous solid. R = 0.3

(

70% EtOAc/hexanes). H NMR (500 MHz, CDCl ): δ 7.72 (d, J =

.9 Hz, 2H), 7.30−7.23 (m, 4H), 7.22−7.17 (m, 1H), 6.97 (d, J = 8.9

Hz, 1H), 5.60 (d, J = 3.6 Hz, 1H), 5.02 (d, J = 9.3 Hz, 1H), 4.71 (t, J

= 6.2 Hz, 1H), 4.31 (t, J = 5.4 Hz, 1H), 4.10 (m, 1H), 3.87 (m, 1H),

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

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

.86 (s, 3H), 3.82−3.71 (m, 3H), 3.19−3.04 (m, 3H), 2.97 (dd, J =

3.4, 8.3 Hz, 1H), 2.83−2.75 (m, 2H), 2.72 (dt, J = 8.7, 3.9 Hz, 1H),

.50 (m, 1H), 1.96 (ddd, J = 12.5, 6.0, 4.2 Hz, 1H), 1.91−1.79 (m,

H), 1.70 (d, J = 12.5 Hz, 1H), 1.28 (m, 1H), 0.91 (d, J = 6.6 Hz,

C H F N O S , 707.2379; found, 707.2385.

7 2

(1R,3aS,7aR)-Octahydro-1,6-epoxyisobenzofuran-5-yl

((2S,3R)-4-((2-(Cyclopropylamino)-N-isobutylbenzo[d]-

thiazole)-6-sulfonamido)-1-(3-ﬂuorophenyl)-3-hydroxybutan-

H), 0.86 (d, J = 6.6 Hz, 3H); C NMR (125 MHz, CDCl ): δ

-yl)carbamate (4e). Activated alcohol 16 (12 mg, 0.037 mmol)

62.8, 155.8, 137.7, 129.9, 129.4, 129.4, 128.3, 126.3, 114.2, 108.7,

6.2, 75.3, 72.4, 58.6, 55.5, 55.1, 53.5, 42.4, 36.0, 34.1, 29.3, 29.0,

was treated with isostere amine 20 (21 mg, 0.041 mmol) by following

the procedure outlined for inhibitor 4a to give inhibitor 4e (18 mg,

70%) as an amorphous solid. R = 0.2 (80% EtOAc/hexanes). H

NMR (500 MHz, CDCl ): δ 8.13 (d, J = 1.9 Hz, 1H), 7.74 (dd, J =

7.1, 20.0, 19.8 LRMS-ESI (m/z): 589.2 [M + H] ; HRMS-ESI (m/

z): [M + H] calcd for C H N O S, 589.2578; found, 589.2572.

(

1R,3aS,7aR)-Octahydro-1,6-epoxyisobenzofuran-5-yl

.4, 2.0 Hz, 1H), 7.59 (d, J = 8.9 Hz, 1H), 7.26 (dd, J = 8.1, 6.2 Hz,

(

(2S,3R)-4-((2-(Cyclopropylamino)-N-isobutylbenzo[d]-

thiazole)-6-sulfonamido)-3-hydroxy-1-phenylbutan-2-yl)-

carbamate (4b). Activated alcohol 16 (8 mg, 0.024 mmol) was

treated with isostere amine 18 (14 mg, 0.027 mmol) by following the

1H), 7.18 (br s, 1H), 7.07 (d, J = 7.7 Hz, 1H), 7.00 (m, 1H), 6.92 (t, J

= 8.6 Hz, 1H), 5.63 (d, J = 3.7 Hz, 1H), 5.10 (d, J = 9.2 Hz, 1H), 4.75

(m, 1H), 4.35 (t, J = 5.6 Hz, 1H), 4.14 (t, J = 8.3 Hz, 1H), 3.94−3.77

(m, 4H), 3.23 (dd, J = 15.2, 8.4 Hz, 1H), 3.20−3.07 (m, 2H), 3.05

(dd, J = 13.4, 8.3 Hz, 1H), 2.91−2.71 (m, 4H), 2.54 (m, 1H), 2.07−

1.85 (m, 4H), 1.73 (d, J = 12.8 Hz, 1H), 1.34 (d, J = 10.0 Hz, 1H),

procedure outlined for inhibitor 4a to give inhibitor 4b (12.5 mg,

5%) as an amorphous solid. R = 0.15 (80% EtOAc/hexanes). H

NMR (500 MHz, CDCl ): δ 8.09 (s, 1H), 7.69 (d, J = 8.6 Hz, 1H),

.55 (d, J = 8.5 Hz, 1H), 7.39−7.15 (m, 5H), 5.59 (d, J = 3.6 Hz,

H), 5.09 (d, J = 9.7 Hz, 1H), 4.70 (m, 1H), 4.31 (t, J = 5.5 Hz, 1H),

.09 (t, J = 8.2 Hz, 1H), 3.94−3.71 (m, 4H), 3.21 (dd, J = 15.1, 8.9

1.00−0.88 (m, 8H), 0.84−0.80 (m, 2H); C NMR (200 MHz,

CDCl ): δ 172.9, 163.4 (d, J = 9.4 Hz), 162.2 (d, J = 10.1 Hz), 155.9,

155.5, 140.6 (d, J = 5.9 Hz), 131.2, 130.7, 129.8 (d, J = 8.3 Hz), 125.4

(d, J = 33.8 Hz), 121.0, 118.7, 116.4 (d, J = 21.0 Hz), 113.3 (d, J =

21.0 Hz), 108.9, 76.4, 75.4, 72.5, 72.5, 58.9, 55.1, 53.7, 42.5, 35.9,

34.2, 31.9, 29.7, 29.5, 29.4, 29.4, 29.1, 27.3, 26.7, 20.2, 19.9, 8.0.

Hz, 1H), 3.16−3.07 (m, 2H), 3.02 (dd, J = 13.4, 8.5 Hz, 1H), 2.86−

.68 (m, 4H), 2.50 (m, 1H), 1.99−1.80 (m, 4H), 1.69 (d, J = 12.6 Hz,

H), 1.26 (m, 1H), 0.97−0.90 (m, 5H), 0.87 (d, J = 6.5 Hz, 3H),

.81−0.77 (m, 2H); C NMR (125 MHz, CDCl ): δ 172.9, 155.8,

LRMS-ESI (m/z): 689.2 [M + H] ; HRMS-ESI (m/z): [M + H]

55.5, 137.7, 131.1, 130.4, 129.4, 128.3, 126.3, 125.3, 120.8, 118.4,

08.7, 76.2, 75.3, 72.4, 58.7, 55.1, 53.6, 42.4, 36.0, 34.0, 29.6, 29.3,

calcd for C H FN O S , 689.2474; found, 689.2466.

7 2

(1R,3aS,7aR)-Octahydro-1,6-epoxyisobenzofuran-5-yl

((2S,3R)-4-((2-(Cyclopropylamino)-N-isobutylbenzo[d]-

thiazole)-6-sulfonamido)-1-(4-ﬂuorophenyl)-3-hydroxybutan-

9.0, 27.2, 26.5, 20.1, 19.8, 7.9. LRMS-ESI (m/z): 671.2 [M + H] ;

HRMS-ESI (m/z): [M + H] calcd for C H N O S , 671.2568;

7 2

-yl)carbamate (4f). Activated alcohol 16 (12 mg, 0.037 mmol) was

found, 671.2574.

1R,3aS,7aR)-Octahydro-1,6-epoxyisobenzofuran-5-yl

treated with isostere amine 21 (21 mg, 0.041 mmol) by following the

(

[

(

(2S,3R)-3-Hydroxy-4-((N-isobutyl-2-(isopropylamino)benzo-

d]oxazole)-6-sulfonamido)-1-phenylbutan-2-yl)carbamate

4c). Activated alcohol 16 (10 mg, 0.031 mmol) was treated with

procedure outlined for inhibitor 4a to give inhibitor 4f (17.5 mg,

68%) as an amorphous solid. R = 0.2 (80% EtOAc/hexanes). H

NMR (500 MHz, CDCl ): δ 8.10 (d, J = 1.9 Hz, 1H), 7.70 (dd, J =

isostere amine 22 (16 mg, 0.034 mmol) by following the procedure

outlined for inhibitor 4a to give inhibitor 4c (17.5 mg, 86%) as an

8.5, 1.9 Hz, 1H), 7.57 (d, J = 8.5 Hz, 1H), 7.25−7.18 (m, 2H), 7.00−

6.94 (m, 2H), 6.89 (br s, 1H), 5.60 (d, J = 3.6 Hz, 1H), 5.02 (d, J =

9.0 Hz, 1H), 4.70 (t, J = 6.1 Hz, 1H), 4.33 (t, J = 5.4 Hz, 1H), 4.11 (t,

J = 8.2 Hz, 1H), 3.92−3.71 (m, 4H), 3.18 (dd, J = 15.1, 8.2 Hz, 1H),

amorphous solid. R = 0.4 (5% MeOH/CH Cl ). H NMR (800

MHz, CDCl ): δ 7.71 (d, J = 1.8 Hz, 1H), 7.64 (dd, J = 8.2, 1.8 Hz,

H), 7.41 (d, J = 8.3 Hz, 1H), 7.31−7.25 (m, 5H), 7.23−7.19 (m,

H), 5.61 (d, J = 3.6 Hz, 1H), 5.44 (br s, 1H), 5.09 (d, J = 9.3 Hz,

H), 4.72 (t, J = 6.3 Hz, 1H), 4.33 (t, J = 5.4 Hz, 1H), 4.16−4.09 (m,

H), 3.91−3.74 (m, 4H), 3.20 (dd, J = 15.1, 8.8 Hz, 1H), 3.15−3.07

.14−3.06 (m, 2H), 3.01 (dd, J = 13.4, 8.5 Hz, 1H), 2.83 (dd, J =

3.4, 6.7 Hz, 1H), 2.79−2.70 (m, 3H), 2.52 (m, 1H), 1.96 (dt, J =

2.5, 5.2 Hz, 1H), 1.92−1.81 (m, 2H), 1.70 (d, J = 12.6 Hz, 1H), 1.29

(

d, J = 10.0 Hz, 1H), 0.98−0.90 (m, 5H), 0.88 (d, J = 6.6 Hz, 3H),

(

m, 2H), 3.01 (dd, J = 13.4, 8.4 Hz, 1H), 2.85−2.79 (m, 2H), 2.74

.82−0.77 (m, 2H); C NMR (200 MHz, CDCl ): δ 172.9, 162.2,

(

dd, J = 8.8, 4.3 Hz, 1H), 2.52 (m, 1H), 1.97 (dt, J = 12.6, 4.7 Hz,

61.0, 156.0, 133.6, 131.0 (d, J = 8.0 Hz), 130.7, 125.4, 121.0, 118.7,

15.2 (d, J = 21.0 Hz), 108.9, 76.4, 75.4, 72.6, 72.5, 58.9, 55.3, 53.7,

2.6, 35.2, 34.2, 29.7, 29.4, 29.1, 27.3, 26.7, 20.2, 19.9, 8.0. LRMS-ESI

H), 1.90−1.85 (m, 2H), 1.81 (br s, 1H), 1.72 (d, J = 12.6 Hz, 1H),

.38 (d, J = 6.5 Hz, 6H), 1.31−1.27 (m, 1H), 0.94 (d, J = 6.6 Hz,

H), 0.89 (d, J = 6.6 Hz, 3H); C NMR (200 MHz, CDCl ): δ

(m/z): 689.2 [M + H] ; HRMS-ESI (m/z): [M + H] calcd for

63.3, 155.9, 147.9, 147.7, 137.8, 130.0, 129.5, 128.4, 126.4, 124.2,

16.0, 108.9, 108.5, 76.4, 75.4, 72.4, 58.8, 55.2, 53.7, 45.7, 42.6, 36.1,

4.2, 29.4, 29.1, 27.3, 23.0, 20.2, 19.9. LRMS-ESI (m/z): 657.2 [M +

C H FN O S , 689.2474; found, 689.2481.

7 2

(

1S,3aR,5S,7aS)-Octahydro-1,6-epoxyisobenzofuran-5-yl

((2S,3R)-3-Hydroxy-4-((N-isobutyl-4-methoxyphenyl)-

sulfonamido)-1-phenylbutan-2-yl)carbamate (5a). Activated

alcohol ent-16 (15 mg, 0.046 mmol) was treated with isostere

amine 17 (21 mg, 0.051 mmol) by following the procedure outlined

for inhibitor 4a to give inhibitor 5a (22 mg, 80%) as an amorphous

H] ; HRMS-ESI (m/z): [M + H] calcd for C H N O S, 657.2953;

found, 657.2947.

1R,3aS,7aR)-Octahydro-1,6-epoxyisobenzofuran-5-yl

(2S,3R)-4-((2-(Cyclopropylamino)-N-isobutylbenzo[d]-

(

thiazole)-6-sulfonamido)-1-(3,5-diﬂuorophenyl)-3-hydroxy-

butan-2-yl)carbamate (4d). Activated alcohol 16 (7 mg, 0.021

mmol) was treated with isostere amine 19 (13 mg, 0.023 mmol) by

following the procedure outlined for inhibitor 4a to give inhibitor 4d

solid. R = 0.3 (70% EtOAc/hexanes). H NMR (800 MHz, CDCl ):

δ 7.72 (d, J = 8.4 Hz, 2H), 7.33−7.25 (m, 4H), 7.22 (t, J = 7.2 Hz,

H), 6.98 (d, J = 8.4 Hz, 2H), 5.65 (s, 1H), 5.17 (d, J = 8.8 Hz, 1H),

.80−4.68 (m, 1H), 4.33 (t, J = 5.4 Hz, 1H), 4.13 (m, 1H), 3.88 (s,

H), 3.91−3.75 (m, 4H), 3.13−2.91 (m, 4H), 2.87−2.71 (m, 2H),

(

10 mg, 65%) as an amorphous solid. R = 0.1 (70% EtOAc/hexanes).

H NMR (500 MHz, CDCl ): δ 8.11 (s, 1H), 7.71 (d, J = 10.0 Hz,

2.56 (m, 1H), 2.06−1.68 (m, 5H), 1.52 (d, J = 15.8 Hz, 1H), 1.31 (m,

H), 7.57 (d, J = 8.5 Hz, 1H), 6.90 (br s, 1H), 6.80 (d, J = 7.6 Hz,

1H), 0.91 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.7 Hz, 3H); C NMR

2H), 6.64 (dd, J = 10.1, 7.7 Hz, 1H), 5.60 (d, J = 3.6 Hz, 1H), 5.13

(

200 MHz, CDCl ): δ 162.9, 156.2, 137.8, 130.1, 129.6, 129.5, 128.5,

26.4, 114.3, 108.9, 76.6, 75.7, 72.6, 72.1, 58.6, 55.6, 55.4, 53.5, 42.7,

(

d, J = 8.9 Hz, 1H), 4.73 (m, 1H), 4.32 (t, J = 5.2 Hz, 1H), 4.13 (t, J

8.1 Hz, 1H), 3.96−3.76 (m, 3H), 3.22−3.07 (m, 3H), 3.02 (dd, J =

5.2, 34.3, 29.7, 29.5, 29.5, 27.2, 20.1, 19.9. LRMS-ESI (m/z): 589.2

3.4, 8.4 Hz, 1H), 2.85 (dd, J = 13.4, 6.8 Hz, 1H), 2.81−2.70 (m,

H), 2.53 (m, 1H), 2.03−1.80 (m, 4H), 1.71 (d, J = 12.6 Hz, 1H),

.34 (d, J = 16.0 Hz, 1H), 0.97−0.91 (m, 5H), 0.89 (d, J = 6.5 Hz,

[

M + H] ; HRMS-ESI (m/z): [M + H] calcd for C H N O SNa,

30 40 2 8

H), 0.82−0.76 (m, 2H); C NMR (200 MHz, CDCl ): δ 172.8,

((2S,3R)-4-((2-(Cyclopropylamino)-N-isobutylbenzo[d]-

thiazole)-6-sulfonamido)-3-hydroxy-1-phenylbutan-2-yl)-

carbamate (5b). Activated alcohol ent-16 (15 mg, 0.046 mmol) was

treated with isostere amine 18 (25 mg, 0.051 mmol) by following the

procedure outlined for inhibitor 4a to give inhibitor 5b (22 mg, 70%)

63.5 (d, J = 12.7 Hz), 162.3 (d, J = 12.8 Hz), 155.8, 155.6, 142.2

(

m), 131.3, 130.6, 125.4, 121.0, 118.7, 112.4 (d, J = 20.6 Hz), 108.9,

01.9 (t, J = 25.1 Hz), 76.4, 75.3, 72.6, 72.4, 59.0, 55.0, 53.7, 42.5,

5.9, 34.2, 29.7, 29.4, 29.1, 27.4, 26.7, 20.2, 19.9, 8.0. LRMS-ESI (m/

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

as an amorphous solid. R = 0.2 (80% EtOAc/hexanes). H NMR

1.73 (d, J = 12.5 Hz, 1H), 1.50 (d, J = 15.8 Hz, 1H), 0.95−0.84 (m,

(

800 MHz, CDCl ): δ 8.08 (s, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.57 (d,

J = 8.5 Hz, 1H), 7.32−7.26 (m, 5H), 7.24−7.16 (m, 2H), 5.65 (d, J =

.6 Hz, 1H), 5.19 (d, J = 8.8 Hz, 1H), 4.78 (t, J = 6.1 Hz, 1H), 4.34

8H), 0.81−0.77 (m, 2H); C NMR (200 MHz, CDCl ): δ 173.0,

163.4, 162.2, 156.2, 155.6, 140.6, 131.2, 130.6, 130.5, 129.9−125.7

(m), 125.5−125.2 (m), 120.9 (d, J = 8.5 Hz), 118.6, 116.5 (d, J =

22.9 Hz), 113.4 (t, J = 19.0 Hz), 108.9, 76.5 (d, J = 36.1 Hz), 75.5 (d,

J = 35.0 Hz), 72.7, 72.3, 58.8, 55.4, 55.2, 53.6, 42.7, 42.5, 34.8, 34.3,

34.2, 29.7, 29.5, 29.4, 29.1, 27.3, 26.7, 20.2, 19.9, 8.0. LRMS-ESI (m/

(

t, J = 5.4 Hz, 1H), 4.13 (t, J = 8.3 Hz, 1H), 3.98−3.81 (m, 4H), 3.15

dd, J = 15.0, 8.7 Hz, 1H), 3.10−2.95 (m, 3H), 2.85 (dd, J = 13.5, 6.9

Hz, 1H), 2.80−2.70 (m, 2H), 2.55 (m, 1H), 2.05−1.67 (m, 5H), 1.52

(

d, J = 15.8 Hz, 1H), 0.97 (d, J = 6.5 Hz, 2H), 0.92 (d, J = 6.6 Hz,

z): 689.2 [M + H] ; HRMS-ESI (m/z): [M + H] calcd for

H), 0.88 (d, J = 6.6 Hz, 3H), 0.81 (m, 2H); C NMR (200 MHz,

CDCl ): δ 172.98, 156.19, 155.60, 137.81, 131.20, 130.60, 129.62,

C H FN O S , 689.2474; found, 689.2481.

7 2

(1S,3aR,5S,7aS)-Octahydro-1,6-epoxyisobenzofuran-5-yl

28.49, 126.49, 125.40, 120.93, 118.55, 108.88, 76.59, 75.67, 72.67,

2.20, 58.71, 55.48, 53.63, 42.72, 35.24, 34.32, 29.71, 29.50, 27.31,

((2S,3R)-4-((2-(Cyclopropylamino)-N-isobutylbenzo[d]-

thiazole)-6-sulfonamido)-1-(4-ﬂuorophenyl)-3-hydroxybutan-

-yl)carbamate (5f). Activated alcohol ent-16 (4 mg, 0.012 mmol)

6.68, 20.18, 19.91, 7.97. LRMS-ESI (m/z): 671.2 [M + H] ; HRMS-

was treated with isostere amine 21 (7 mg, 0.013 mmol) by following

ESI (m/z): [M + H] calcd for C H N O S , 671.2568; found,

7 2

the procedure outlined for inhibitor 4a to give inhibitor 5f (6.6 mg,

71.2563.

(

7%) as an amorphous solid. R = 0.2 (80% EtOAc/hexanes). H

1S,3aR,5S,7aS)-Octahydro-1,6-epoxyisobenzofuran-5-yl

(

[

(

(2S,3R)-3-Hydroxy-4-((N-isobutyl-2-(isopropylamino)benzo-

d]oxazole)-6-sulfonamido)-1-phenylbutan-2-yl)carbamate

5c). Activated alcohol ent-16 (8 mg, 0.024 mmol) was treated with

NMR (800 MHz, CDCl ): δ 8.07 (s, 1H), 7.67 (d, J = 8.6 Hz, 1H),

7.56 (d, J = 8.5 Hz, 1H), 7.25−7.20 (m, 2H), 6.98−6.95 (m, 2H),

.83 (br s, 1H), 5.63 (d, J = 3.5 Hz, 1H), 5.10 (d, J = 9.1 Hz, 1H),

.74 (m, 1H), 4.32 (t, J = 5.5 Hz, 1H), 4.11 (t, J = 7.9 Hz, 1H), 3.90−

.72 (m, 4H), 3.12 (dd, J = 15.0, 8.5 Hz, 1H), 3.07−3.03 (m, 1H),

isostere amine 22 (13 mg, 0.027 mmol) by following the procedure

outlined for inhibitor 4a to give inhibitor 5c (14 mg, 86%) as an

amorphous solid. R = 0.4 (5% MeOH/CH Cl ). H NMR (800

3.01−2.96 (m, 2H), 2.91 (dd, J = 14.2, 8.2 Hz, 1H), 2.83 (dd, J =

MHz, CDCl ): δ 7.66 (s, 1H), 7.60 (d, J = 8.2 Hz, 1H), 7.39 (d, J =

13.5, 6.8 Hz, 1H), 2.77−2.69 (m, 2H), 2.53 (m, 1H), 2.03−1.95 (m,

.2 Hz, 1H), 7.30−7.23 (m, 5H), 7.20 (t, J = 7.1 Hz, 1H), 5.63 (d, J =

.5 Hz, 1H), 5.23 (br s, 1H), 5.07 (d, J = 8.9 Hz, 1H), 4.76 (m, 1H),

.32 (m, 1H), 4.15−4.05 (m, 2H), 3.87−3.72 (m, 4H), 3.13−2.92

H), 1.89−1.80 (m, 2H), 1.73 (d, J = 12.5 Hz, 1H), 1.48 (d, J = 15.8

Hz, 1H), 0.96−0.93 (m, 2H), 0.91 (d, J = 6.6 Hz, 3H), 0.86 (d, J =

6.7 Hz, 3H), 0.81−0.78 (m, 2H); C NMR (200 MHz, CDCl3): δ

172.9, 162.3, 161.1, 156.1, 155.5, 133.4, 131.2, 131.1−131.0 (m),

130.6, 125.4, 120.9, 118.7, 115.2 (d, J = 20.9 Hz), 108.9, 76.6, 75.5,

72.7, 72.1, 58.8, 55.4, 53.7, 42.7, 34.5, 34.3, 29.7, 29.5, 29.4, 27.3,

(

m, 4H), 2.85−2.67 (m, 2H), 2.53 (m, 1H), 2.03−1.95 (m, 2H),

.92−1.77 (m, 2H), 1.73 (d, J = 12.5 Hz, 1H), 1.50 (d, J = 15.8 Hz,

H), 1.36 (d, J = 6.5 Hz, 6H), 0.90 (d, J = 6.6 Hz, 3H), 0.86 (d, J =

.6 Hz, 3H); ¹³C NMR (200 MHz, CDCl ): δ 163.3, 156.2, 147.9,

26.7, 20.2, 19.9, 8.0. LRMS-ESI (m/z): 689.2 [M + H]+; HRMS-ESI

37.7, 130.1, 129.6, 128.5, 126.5, 124.2, 116.0, 108.9, 108.4, 76.6,

5.7, 72.7, 72.1, 58.7, 56.0, 55.4, 53.7, 45.8, 42.7, 35.3, 34.3, 29.7,

9.5, 27.3, 23.0, 20.2, 19.9. LRMS-ESI (m/z): 657.2 [M + H] ;

(

m/z): [M + H] calcd for C H FN O S , 689.2474; found,

33 42 4 7 2

89.2478.

(

1R,3aS,5S,7aR)-Octahydro-1,6-epoxyisobenzofuran-5-ol

HRMS-ESI (m/z): [M + H] calcd for C H N O S, 657.2953;

(6). To a stirred solution of triol 13 (340 mg, 1.54 mmol) in

dichloromethane (16 mL) was added 10-camphorsulfonic acid (36

mg, 0.15 mmol) at 0 °C for 1 h. The crude residue was puriﬁed by

silica gel column chromatography (40% EtOAc/hexanes) to aﬀord

found, 657.2948.

1S,3aR,5S,7aS)-Octahydro-1,6-epoxyisobenzofuran-5-yl

(2S,3R)-4-((2-(Cyclopropylamino)-N-isobutylbenzo[d]-

thiazole)-6-sulfonamido)-1-(3,5-diﬂuorophenyl)-3-hydroxy-

butan-2-yl)carbamate (5d). Activated alcohol ent-16 (12 mg, 0.037

mmol) was treated with isostere amine 19 (21 mg, 0.041 mmol) by

following the procedure outlined for inhibitor 4a to give inhibitor 5d

(

alcohol 6 (198 mg, 82%) as a white amorphous solid. R = 0.3 (70%

EtOAc/hexanes). [α] + 45.5 (c 0.77, CHCl ). H NMR (400 MHz,

CDCl ): δ 5.64 (d, J = 4.2 Hz, 1H), 4.30 (t, J = 5.7 Hz, 1H), 4.03−

3.87 (m, 3H), 3.21 (d, J = 10.3 Hz, 1H), 2.82 (dt, J = 8.8, 4.4 Hz,

(

15.5 mg, 59%) as an amorphous solid. R = 0.3 (5% MeOH/

CH Cl ). H NMR (800 MHz, CDCl ): δ 8.11 (s, 1H), 7.72 (d, J =

1H), 2.56−2.48 (m, 1H), 1.93 (dt, J = 12.4, 5.0 Hz, 1H), 1.83−1.73

(

m, 2H), 1.56 (dt, J = 15.4, 1.1 Hz, 1H); C NMR (100 MHz,

.6 Hz, 1H), 7.59 (d, J = 8.5 Hz, 1H), 6.98 (br s, 1H), 6.87−6.80 (m,

H), 6.67 (m, 1H), 5.65 (d, J = 3.6 Hz, 1H), 5.25 (d, J = 8.8 Hz, 1H),

.83−4.73 (m, 1H), 4.36 (t, J = 5.4 Hz, 1H), 4.16−4.05 (m, 2H),

.95−3.73 (m, 3H), 3.18−2.93 (m, 4H), 2.89 (dd, J = 13.3, 6.9 Hz,

H), 2.84−2.72 (m, 3H), 2.56 (q, J = 8.2 Hz, 1H), 2.05−1.95 (m,

H), 1.94−1.83 (m, 2H), 1.76 (d, J = 12.6 Hz, 1H), 1.72 (m, 1H),

.52 (d, J = 15.9 Hz, 1H), 0.97 (d, J = 6.6 Hz, 2H), 0.94 (d, J = 6.5

CDCl ): δ 108.6, 76.8, 76.6, 67.5, 42.1, 34.7, 31.8, 29.6. LRMS-ESI

(

m/z): 157 [M + H] .

1S,3aR,7aS)-Octahydro-1,6-epoxyisobenzofuran-5-ol (ent-

). The title compound ent-6 (62 mg, 79%) was obtained from ent-

(

3 (110 mg, 0.5 mmol) by following the procedure outlined for

compound 6. R = 0.3 (70% EtOAc/hexanes); [α] = −47.3 (c 0.76,

CHCl ). H and C NMR data is identical with 6.

Hz, 3H), 0.90 (d, J = 6.9 Hz, 3H), 0.83−0.79 (m, 2H). C NMR

cis-Cyclohex-4-ene-1,2-dimethanol (9). To a slurry of lithium

aluminum hydride in THF was added cis-4-cyclohexene-1,2-

dicarboxylic anhydride 10 (7 g, 46.00 mmol) in THF (300 mL) at

(

200 MHz, CDCl ): δ 173.0, 163.6 (d, J = 11.8 Hz), 162.3 (d, J =

2.1 Hz), 156.2, 155.6, 142.2 (m), 131.3, 130.4, 125.4, 120.9, 118.7,

12.4 (d, J = 20.2 Hz), 108.9, 102.0 (t, J = 24.2 Hz), 76.5, 75.5, 72.7,

2.3, 59.0, 55.3, 53.7, 42.6, 34.8, 34.3, 29.7, 29.5, 27.4, 26.7, 20.2,

9.9, 8.0. LRMS-ESI (m/z): 707.2 [M + H] ; HRMS-ESI (m/z): [M

H] calcd for C H F N O S , 707.2379; found, 707.2384.

1S,3aR,5S,7aS)-Octahydro-1,6-epoxyisobenzofuran-5-yl

(2S,3R)-4-((2-(Cyclopropylamino)-N-isobutylbenzo[d]-

thiazole)-6-sulfonamido)-1-(3-ﬂuorophenyl)-3-hydroxybutan-

-yl)carbamate (5e). Activated alcohol ent-16 (4 mg, 0.012 mmol)

was treated with isostere amine 20 (7 mg, 0.013 mmol) by following

the procedure outlined for inhibitor 4a to give inhibitor 5e (7.5 mg,

°C over 20 min. The reaction mixture was stirred at 0 °C for 3 h

and quenched by dropwise addition of methanol over a period of 30

min at 0 °C. The reaction mixture was allowed to warm to room

temperature, and aq solution of sodium sulfate was added and stirred

at 23 °C overnight. The resulting slurry was ﬁltered, and the solid was

rinsed with ethyl acetate. The layers were separated, and the aqueous

layer was extracted with ethyl acetate. The combined organic layers

7 2

(

were dried over Na

reduced pressure to give cis-diol 9 (g, 85%) as liquid. H NMR (400

MHz, CDCl ): δ 5.62−5.59 (m, 2H), 3.71 (dd, J = 11.0, 6.5 Hz, 2H),

3.58 (dd, J = 11.1, 3.3 Hz, 2H), 3.27 (br s, 2H), 2.18−1.96 (m, 6H);

SO , ﬁltered, and the solvent was removed under

2 4

7%) as an amorphous solid. R = 0.2 (80% EtOAc/hexanes). H

NMR (800 MHz, CDCl ): δ 8.14−8.07 (m, 1H), 7.73−7.65 (m, 1H),

(

.56 (dd, J = 8.3, 2.8 Hz, 1H), 7.23 (m, 1H), 7.08−7.00 (m, 2H), 6.98

d, J = 10.2 Hz, 1H), 6.89 (td, J = 8.8, 3.3 Hz, 1H), 5.63 (d, J = 3.6

Hz, 1H), 5.21 (d, J = 8.8 Hz, 1H), 4.77 (t, J = 6.3 Hz, 1H), 4.32 (t, J =

C NMR (101 MHz, CDCl ): δ 125.4, 64.0, 37.7, 26.8. LRMS-ESI

(m/z): 143.0 [M + H] .

((1S,6R)-6-(Hydroxymethyl)cyclohex-3-en-1-yl)methyl Ace-

tate (11). A mixture of diol 9 (0.5 g, 3.52 mmol) and PPL (porcine

pancreatic lipase Sigma type 2, 2.27 g) in ethyl acetate (50 mL) was

stirred at 23 °C for 12 h. After completion of diol by TLC, the

.2 Hz, 1H), 4.11 (td, J = 8.9, 8.5, 3.2 Hz, 1H), 4.02−3.73 (m, 4H),

.15−2.92 (m, 4H), 2.85 (dd, J = 13.3, 6.7 Hz, 1H), 2.79−2.70 (m,

H), 2.57−2.48 (m, 1H), 2.01−1.93 (m, 2H), 1.89−1.81 (m, 2H),

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

reaction mixture was ﬁltered and the solvent was removed in vacuo to

give a crude residue which was puriﬁed by column chromatography

over silica gel (30% EtOAc/hexanes) to aﬀord the mono acetate 11

methyl)-5-(hydroxymethyl)cyclohexane-1,2-diol (14). AD-mix-

β (3.0 g) was dissolved in 1:1 tert-butyl alcohol/water (22 mL), and

the mixture was stirred for 10 min. MeSO NH (208 mg, 2.19 mmol)

(

0.53 g, 82%) along with di acetate (40 mg, 5%) as the minor

was then added, and stirring was continued for a further 10 min. After

the mixture was cooled to 0 °C, 12 (500 mg, 2.19 mmol) in t-BuOH

(2 mL) was added. The reaction was slowly warmed to ambient

temperature and stirred for 24 h. At this time, solid Na SO was

product. R = 0.5 (50% EtOAc/hexanes). [α] + 17.5 (c 2.2, CHCl ),

{

literature data: [α]_D+ 19.0 (c 5.85, CHCl )}; H NMR (400 MHz,

CDCl ): δ 5.67−5.57 (m, 2H), 4.17 (dd, J = 11.0, 6.0 Hz, 1H), 3.94

(

dd, J = 11.0, 8.0 Hz, 1H), 3.68−3.53 (m, 2H), 2.27−2.20 (m, 1H),

added, and the reaction was stirred for an additional 30 min. The

reaction was then partitioned between EtOAc/water and the aqueous

layer extracted with EtOAc. The combined organic layers were

washed brine solution, dried over Na SO , ﬁltered, and concentrated

.17−2.05 (m, 3H), 2.04 (s, 3H), 2.03−1.82 (m, 3H). C NMR (101

MHz, CDCl ): δ 171.3, 125.5, 125.0, 64.9, 63.5, 37.1, 33.1, 26.9, 25.9,

0.9. LRMS-ESI (m/z): 185.1 [M + H] . Spectral data was identical

with the reported data.

under reduced pressure to yield inseparable mixture of diols which

(

(1R,6S)-6-(Hydroxymethyl)cyclohex-3-en-1-yl)methyl Ace-

were used for the next step with further puriﬁcation.

tate (ent-11). To a stirred solution of diacetate 15 (18.6 g, 82.30

mmol) in 0.1 M phosphate buﬀer (242 mL, pH 7) was added PPL

(

The stirred solution of above diol was dissolved in methanol (6

mL), and 1 N NaOH (0.6 mL) at 0 °C was added. The reaction

mixture was slowly warmed to ambient temperature and stirred for 3

h. After completion of the starting material, methanol was evaporated

and extracted with dichloromethane (3 × 30 mL). The combined

organic layers were dried over Na SO , ﬁltered, and concentrated

1.86 g, Sigma type II, crude) at 23 °C. NaHCO solution (93 mL) (1

N) was added dropwise, and the heterogeneous mixture was stirred

for 16 h. The mixture was then ﬁltered through a pad of Celite. The

ﬁltrate was extracted with dichloromethane (×3). The combined

organic layers were washed with brine, dried over Na SO , ﬁltered,

under reduced pressure. The crude residue was puriﬁed by column

chromatography (5% MeOH/CH Cl ) over silica gel to aﬀord triol

and the solvent was evaporated in vacuo. The residue was puriﬁed by

chromatography over silica gel (30% EtOAc/hexanes) to obtain ent-

1 (12.75 g, 84%) as colorless oil. R = 0.5 (50% EtOAc/hexanes).

13 (207 mg, 43%) and 14 (227 mg, 47%) as an oily liquids.

Compound 13. R = 0.4 (10% MeOH/CH Cl ). [α] − 3.2 (c

[

α] = −17.5 (c = 1.43, CHCl ); LRMS-ESI (m/z): 185.1 [M + H] .

0.58, CHCl ). H NMR (400 MHz, methanol-d ): δ 4.46 (d, J = 8.3

H and C NMR spectral data were identical with 11.

(1S,6R)-6-(Dimethoxymethyl)cyclohex-3-en-1-yl)methyl

Acetate (12). Oxalyl chloride (1.34 mL, 15.21 mmol) in dry CH Cl

Hz, 1H), 3.82−3.72 (m, 2H), 3.60 (dd, J = 11.4, 4.3 Hz, 1H), 3.55

(m, 1H), 3.36 (s, 3H), 3.35 (s, 3H), 2.00−1.82 (m, 3H), 1.81−1.69

(

(m, 2H), 1.62 (dt, J = 12.9, 4.3 Hz, 1H); C NMR (100 MHz,

(

60 mL) was cooled to −78 °C under a nitrogen atmosphere.

Dimethyl sulfoxide (2.2 mL, 30.43 mmol) was added dropwise. After

5 min, alcohol 11 (1.4 g, 7.60 mmol) in dry CH Cl (20 mL) was

added to the reaction mixture via cannula and stirred for 30 min at

78 °C. Then, Et N (5.3 mL, 38.04 mmol) was added and the

mixture stirred for 15 min. Further reaction was carried out at 0 °C.

The solvent was concentrated, extracted with EtOAc (2 × 100 mL),

and washed with H O and brine, and the organic layer was dried over

methanol-d ): δ 104.9, 70.7, 68.4, 62.9, 52.4, 52.1, 39.0, 34.6, 33.2,

27.4. LRMS-ESI (m/z): 243.1 [M + Na] .

Compound 14. R = 0.2 (10% MeOH/CH Cl ). [α] − 0.77 (c

3.49, CHCl ). H NMR (400 MHz, methanol-d ): δ 4.30 (d, J = 7.8

−

Hz, 1H), 3.89 (dt, J = 6.0, 3.0 Hz, 1H), 3.77 (dt, J = 10.2, 3.7 Hz,

1H), 3.63 (dd, J = 10.9, 6.0 Hz, 1H), 3.50 (dd, J = 11.0, 8.8 Hz, 1H),

3.33 (s, 3H), 3.31 (s, 3H), 2.26 (ddt, J = 12.0, 8.3, 4.2 Hz, 1H), 2.15−

2.07 (m, 1H), 1.87−1.79 (m, 1H), 1.78−1.65 (m, 2H), 1.63−1.51

Na SO , ﬁltered, and concentrated under reduced pressure. The

(m, 1H). C NMR (100 MHz, MeOD): δ 105.2, 68.4, 67.3, 60.3,

residue was puriﬁed by ﬂash column chromatography (15% EtOAc/

hexanes) to aﬀord aldehyde S1 (1.25 g, 90%) as colorless oil. R = 0.5

52.7, 51.7, 35.8, 34.3, 28.8, 28.5. LRMS-ESI (m/z): 243.1 [M + Na] .

(1S,2R,4S,5R)-4-(Dimethoxymethyl)-5-(hydroxymethyl)-

cyclohexane-1,2-diol (ent-13) and (1R,2S,4S,5R)-4-(Dimethox-

ymethyl)-5-(hydroxymethyl)cyclohexane-1,2-diol (ent-14).

Triol ent-13 (405 mg, 42%) and ent-14 (445 mg, 46%) were

synthesized from ent-12 (1 g, 4.38 mmol) by following the procedure

outlined for compound 13 and 14.

(

30% EtOAc/hexanes). [α]_D− 47.9 (c 0.73, CHCl ); H NMR (400

MHz, CDCl ): δ 9.71 (s, 1H), 5.73−5.61 (m, 2H), 4.12−4.03 (m,

H), 2.65−2.55 (m, 2H), 2.33−2.24 (m, 3H), 2.07−1.96 (m, 4H);

C NMR (100 MHz, CDCl ): δ 203.2, 170.6, 125.3, 124.7, 64.4,

7.2, 32.7, 26.8, 22.6, 20.6. LRMS-ESI (m/z): 183.0 [M + H] .

To a stirred solution of above aldehyde (1.25 g, 6.86 mmol) in

cis-Cyclohex-4-ene-1,2-diylbis(methylene) Diacetate (15).

To a stirred solution of diol 9 (11.5 g, 80.98 mmol) were added

pyridine (26.1 mL, 323.94 mmol), acetic anhydride (15.3 mL, 161.97

mmol), and followed by DMAP (495 mg, 4.05 mmol) at 0 °C. The

resulting mixture was stirred at 23 °C overnight. Upon completion,

the reaction mixture was quenched with water and extracted with

EtOAc (2 × 100 mL). The combined organic layers were dried over

NaSO4, ﬁltered, and concentrated under reduced pressure. The crude

residue was puriﬁed by silica gel column chromatography (20%

methanol (20 mL) was added trimethyl orthoformate (7.5 mL, 68.60

mmol) followed by tetrabutylammonium tribromide (66 mg, 0.137

mmol) at 23 °C. The reaction mixture was stirred for 8 h at 23 °C.

After this period, the reaction mixture was quenched by the addition

of saturated aqueous NH Cl solution. Methanol was removed under

reduced pressure and the reaction mixture was diluted with ethyl

acetate. The layers were separated, the aqueous layer was extracted

with EtOAc, and combined organic extracts were dried over Na SO₄

and concentrated under reduced pressure. The crude product was

puriﬁed by silica gel column chromatography (10% EtOAc/hexanes)

EtOAc/hexanes) to aﬀord alcohol 15 (18.1 g, 98%). R = 0.8 (50%

EtOAc/hexanes). H NMR (400 MHz, chloroform-d): δ 5.63−5.60

(m, 2H), 4.12−4.07 (m, 2H), 4.04−3.98 (m, 2H), 2.28−2.11 (m,

to aﬀord 12 (1.32 g, 84%). R = 0.5 (10% EtOAc/hexanes, 3 times).

[

α] + 5.9 (c 1.2, CHCl ). H NMR (400 MHz, CDCl ): δ 5.70−

4H), 2.05 (s, 6H), 1.97−1.89 (m, 2H). C NMR (101 MHz,

.54 (m, 2H), 4.33 (dd, J = 8.3, 1.9 Hz, 1H), 4.12 (ddd, J = 10.8, 5.4,

.8 Hz, 1H), 3.99 (ddd, J = 10.8, 9.0, 1.9 Hz, 1H), 3.36−3.30 (m,

H), 2.29 (m, 1H), 2.21−1.99 (m, 7H), 1.88 (dddd, J = 16.1, 7.9, 4.3,

CDCl ): δ 170.9, 125.0, 65.0, 33.6, 26.5, 20.9. LRMS-ESI (m/z):

227.1 [M + H] .

4-Nitrophenyl ((1R,3aS,7aR)-Octahydro-1,6-epoxyisobenzo-

furan-5-yl) Carbonate (16). To a stirred solution of 6 (22 mg, 0.14

mmol) in dichloromethane (1.0 mL) were added pyridine (30 μL,

0.32 mmol) and 4-nitrophenylchloroformate (63 mg, 0.31 mmol) at 0

_.₂_H_z_,₁_H₎_;13C NMR (100 MHz, CDCl ): δ 171.1, 125.7, 124.7,

05.0, 64.3, 53.5, 52.5, 37.4, 31.9, 27.5, 24.5, 21.0. LRMS-ESI (m/z):

51.1 [M + Na] .

C under an argon atmosphere. The reaction mixture was warmed to

(

(1R,6S)-6-(Dimethoxymethyl)cyclohex-3-en-1-yl)methyl

Acetate (ent-12). The title compound ent-12 (9 g, 80% over two

steps) was obtained from ent-11 (9 g, 48.91 mmol) by following the

23 °C and stirred for 12 h. Upon completion, solvent was removed

under reduced pressure. The crude product was puriﬁed by silica gel

column chromatography (35% EtOAc in hexane) to aﬀord 16 (39.5

procedure outlined for compound 12. R = 0.5 (10% EtOAc/hexanes,

times). [α]²− 5.5 (c 1.0, CHCl ). H and C NMR spectral data

mg, 87%) as an amorphous solid. R

= 0.5 (70% EtOAc/hexanes).

were identical with 12.

1R,2S,4R,5S)-4-(Dimethoxymethyl)-5-(hydroxymethyl)-

cyclohexane-1,2-diol (13) and (1S,2R,4R,5S)-4-(Dimethoxy-

[α]

+ 77.4 (c 0.7, CHCl

). H NMR (500 MHz, CDCl ): δ 8.27−

(

8.23 (m, 2H), 7.43−7.38 (m, 2H), 5.69 (d, J = 3.6 Hz, 1H), 4.91

(ddt, J = 7.4, 5.0, 1.0 Hz, 1H), 4.62 (t, J = 5.4 Hz, 1H), 4.18 (t, J = 8.3

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

orcid.org/0000-0003-4876-7393

Article

Hz, 1H), 3.93 (dd, J = 8.8, 2.9 Hz, 1H), 2.82 (dt, J = 8.6, 3.9 Hz, 1H),

Masayuki Amano − Departments of Infectious Diseases and

Hematology, Kumamoto University Graduate School of

Biomedical Sciences, Kumamoto 860-8556, Japan

Manabu Aoki − Department of Medical Technology, Kumamoto

Health Science University, Kumamoto 861-5598, Japan;

Experimental Retrovirology Section, HIV and AIDS Malignancy

Branch, National Cancer Institute, National Institutes of

Health, Bethesda, Maryland 20892, United States; Department

of Clinical Sciences, Kumamoto University Hospital, Kumamoto

.68−2.59 (m, 1H), 2.11−2.01 (m, 2H), 1.80 (d, J = 12.7 Hz, 1H),

.71 (d, J = 14.7 Hz, 1H); C NMR (125 MHz, CDCl ): δ 155.7,

51.7, 145.3, 125.2, 121.9, 109.1, 77.2, 76.2, 73.9, 42.3, 34.0, 29.2,

8.5. LRMS-ESI (m/z): 344 [M + Na] .

4-Nitrophenyl ((1S,3aR,7aS)-Octahydro-1,6-epoxyisobenzo-

furan-5-yl) Carbonate (ent-16). The title compound ent-16 (100

mg, 88%) was obtained from ent-6 (55 mg, 0.352 mmol) by following

the procedure outlined for compound 16. R = 0.5 (70% EtOAc/

hexanes). [α]²− 79.1 (c 0.8, CHCl ). H and C NMR data are

identical with 16.

60-8556, Japan

Determination of X-ray structure of HIV-1 Protease−

Johnson Agniswamy − Department of Biology, Georgia State

University, Atlanta, Georgia 30303, United States

Yuan-Fang Wang − Department of Biology, Georgia State

University, Atlanta, Georgia 30303, United States

Irene T. Weber − Department of Biology, Georgia State

University, Atlanta, Georgia 30303, United States;

Inhibitor Complex. The HIV-1 protease was expressed and puriﬁed,

as described previously. The PR/5c complex was crystallized by the

hanging drop vapor diﬀusion method with a well solution of 1.25 M

NaCl and 0.1 M sodium acetate, pH 4.8, while PR/4a crystals were

grown with a reservoir solution of 0.65 M NaCl and 0.1 M sodium

acetate, pH 6.0. Diﬀraction data were collected on a single crystal

cooled to 90 K at SER-CAT (22-BM beamline), Advanced Photon

Source, Argonne National Lab (Chicago, USA) with an X-ray

wavelength of 1.0 Å. The two data sets were processed by HKL-

Hiroaki Mitsuya − Department of Refractory Viral Infection,

National Center for Global Health and Medicine Research

Institute, Tokyo 162-8655, Japan; Experimental Retrovirology

Section, HIV and AIDS Malignancy Branch, National Cancer

Institute, National Institutes of Health, Bethesda, Maryland

20892, United States; Department of Clinical Sciences,

Kumamoto University Hospital, Kumamoto 860-8556, Japan

000 to a Rmerge of 5.6 and 7.9%. The complex structures were

48−50

solved by PHASER in CCP4i Suite

determined isomorphous structure with PDB code 3NU3 as the

using the previously

start model. The PR/5c and PR/4a complexes were reﬁned by

2,53

SHELX-2014

up to 1.3 Å resolution and by REFMAC5 to 1.25

55 56

Å resolution. PRODRG-2 and Jligand were used to construct

inhibitors and the restraints for reﬁnement. COOT

57,58

was used for

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c00202

model building. Anisotropic 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,

Notes

two acetate ion, and 189 water molecules for PR/5c and one Na ion,

two Cl ions, one glycerol, one formic acid, and 222 water molecules

−

The authors declare no competing ﬁnancial interest.

for PR/4a . The crystallographic statistics are provided in the

Supporting Information section. The coordinates and structure

factors of PR/5c and PR/4a were deposited in the Protein Data

ACKNOWLEDGMENTS

■

This research was supported by the National Institutes of

Health (Grant AI150466, A.K.G. and Grant AI150461,

I.T.W.). X-ray data were collected at the Southeast Regional

Collaborative Access Team (SER-CAT) beamline 22BM at the

Advanced Photon Source, Argonne National Laboratory. Use

of the Advanced Photon Source was supported by the US

Department of Energy, Basic Energy Sciences, Oﬃce of

Science, under contract no. W-31-109-Eng-38. This work was

also supported by the Intramural Research Program of the

Center for Cancer Research, National Cancer Institute,

National Institutes of Health (H.M.), and in part by grants

for the promotion of AIDS research from the Ministry of

Health; grants from Welfare and Labor of Japan (H.M.); grants

for the Research Program on HIV/AIDS from the Japan

Agency for Medical Research and Development (AMED)

under grant numbers JP15fk0410001 and JP16fk0410101

Bank with code 6VOE and 6VOD, respectively.

ASSOCIATED CONTENT

sı Supporting Information

■

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00202.

Molecular formula strings and some data (CSV)

Full NMR spectroscopic data for all ﬁnal compounds X-

ray structural data for inhibitors 4a and 5c-bound HIV-1

Protease (PDF)

Accession Codes

The PDB accession codes for X-ray structures of inhibitor 4a

and 5c-bound HIV-1 protease are: 6VOD and 6VOE.

(

H.M.); a grant from the National Center for Global Health

AUTHOR INFORMATION

■

and Medicine (NCGM) Research Institute (H.M.); and a

Grant-in-Aid for Scientiﬁc Research (Priority Areas) from the

Ministry of Education, Culture, Sports, Science, and

Technology of Japan (Monbu Kagakusho) (H.M.). The

authors would like to thank the Purdue University Center

for Cancer Research, which supports the shared NMR and

mass spectrometry facilities. The authors also thank M.A. for

discussion.

Corresponding Author

Arun K. Ghosh − Department of Chemistry, Department of

Medicinal Chemistry and Molecular Pharmacology, Purdue

University, West Lafayette, Indiana 47907, United States;

orcid.org/0000-0003-2472-1841 ; Phone: (765)-494-

323; Email: akghosh@purdue.edu; Fax: (765)-496-1612

Authors

Satish Kovela − Department of Chemistry, Department of

ABBREVIATIONS

Medicinal Chemistry and Molecular Pharmacology, Purdue

University, West Lafayette, Indiana 47907, United States

Heather L. Osswald − Department of Chemistry, Department of

Medicinal Chemistry and Molecular Pharmacology, Purdue

University, West Lafayette, Indiana 47907, United States

■

ART, antiretroviral therapies; ATV, atazanavir; Abt, amino-

benzothiazole; bis-THF, bis-tetrahydrofuran; Chf-THF, cyclo-

hexane fused bis-tetrahydrofuran; DIPEA, N,N-diisopropyletyl-

amine; DRV, darunavir; PI, protease inhibitor

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

1) Lohse, N.; Hansen, A.-B. E.; Gerstoft, J.; Obel, N. Improved

Article

Combating Drug-Resistant HIV. Angew. Chem., Int. Ed. 2012, 51,

778−1802.

17) Tie, Y.; Boross, P. I.; Wang, Y.-F.; Gaddis, L.; Hussain, A. K.;

REFERENCES

■

(

Survival in HIV-Infected Persons: Consequences and Perspectives. J.

Antimicrob. Chemother. 2007, 60, 461−463.

2) Montaner, J. S.; Lima, V. D.; Barrios, R.; Yip, B.; Wood, E.; Kerr,

T.; Shannon, K.; Harrigan, P. R.; Hogg, R. S.; Daly, P.; Kendall, P.

Association of Highly Active Antiretroviral Therapy Coverage,

Population Viral Load, and Yearly New HIV Diagnoses in British

Columbia, Canada: A Population-Based Study. Lancet 2010, 376,

Leshchenko, S.; Ghosh, A. K.; Louis, J. M.; Harrison, R. W.; Weber, I.

T. High Resolution Crystal Structures of HIV-1 Protease with a

Potent Non-peptide Inhibitor (UIC-94017) Active Against Multi-

Drug-resistant Clinical Strains. J. Mol. Biol. 2004, 338, 341−352.

(

(18) Kovalevsky, A. Y.; Liu, F.; Leshchenko, S.; Ghosh, A. K.; Louis,

J. M.; Harrison, R. W.; Weber, I. T. Ultra-High Resolution Crystal

Structure of HIV-1 Protease Mutant Reveals Two Binding Sites for

Clinical Inhibitor TMC114. J. Mol. Biol. 2006, 363, 161−173.

(

32−539.

3) Kohl, N. E.; Emini, E. A.; Schleif, W. A.; Davis, L. J.; Heimbach,

(19) Ghosh, A. K.; Chapsal, B. D. Aspartic Acid Proteases as

J. C.; Dixon, R. A.; Scolnick, E. M.; Sigal, I. S. Active Human

Immunodeficiency Virus Protease is Required for Viral Infectivity.

Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 4686−4690.

Therapeutic Targets; Ghosh, A. K., Ed.; Wiley-VCH: Weinheim,

Germany, 2010; pp 169−204.

(20) Ghosh, A. K.; Chapsal, B. D.; Weber, I. T.; Mitsuya, H. Design

4) Smith, R.; Brereton, I. M.; Chai, R. Y.; Kent, S. B. H. Ionization

of HIV Protease Inhibitors Targeting Protein Backbone: An Effective

Strategy for Combating Drug Resistance. Acc. Chem. Res. 2008, 41,

States of the Catalytic Residues in HIV-1 Protease. Nat. Struct. Mol.

Biol. 1996, 3, 946−950.

5) Blanco, J.-L.; Varghese, V.; Rhee, S.-Y.; Gatell, J. M.; Shafer, R.

W. HIV-1 Integrase Inhibitor Resistance and Its Clinical Implications.

J. Infect. Dis. 2011, 203, 1204 −1214.

6) Naggie, S.; Hicks, C. Protease Inhibitor-Based Antiretroviral

Therapy in Treatment-Naive HIV-1-Infected Patients: The Evidence

Behind the Options. J. Antimicrob. Chemother. 2010, 65, 1094−1099.

7) Koh, Y.; Nakata, H.; Maeda, K.; Ogata, H.; Bilcer, G.;

8−86.

21) Ghosh, A. K.; Osswald, H. L.; Prato, G. Recent Progress in the

Development of HIV-1 Protease Inhibitors for the Treatment of

HIV/AIDS. J. Med. Chem. 2016, 59, 5172−5208.

F.; Weber, I. T.; Mitsuya, H. Design and Synthesis of Highly Potent

(22) Ghosh, A. K.; R. Nyalapatla, P.; Kovela, S.; Rao, K. V.; Brindisi,

M.; Osswald, H. L.; Amano, M.; Aoki, M.; Agniswamy, J.; Wang, Y.-

HIV-1 Protease Inhibitors Containing Tricyclic Fused Ring Systems

as Novel P2 Ligands: Structure −Activity Studies, Biological and X-ray

Structural Analysis. J. Med. Chem. 2018, 61, 4561−4577.

(

Devasamudram, T.; Kincaid, J. F.; Boross, P.; Wang, Y.-F.; Tie, Y.;

Volarath, P.; Gaddis, L.; Harrison, R. W.; Weber, I. T.; Ghosh, A. K.;

Mitsuya, H. Novel bis-Tetrahydrofuranylurethane-Containing Non-

peptidic Protease Inhibitor (PI) UIC-94017 (TMC114) with Potent

Activity Against Multi-PI-Resistant Human Immunodeficiency Virus

In Vitro. Antimicrob. Agents Chemother. 2003, 47, 3123−3129.

(23) Ghosh, A. K.; Martyr, C. D.; Osswald, H. L.; Sheri, V. R.;

Kassekert, L. A.; Chen, S.; Agniswamy, J.; Wang, Y.-F.; Hayashi, H.;

Aoki, M.; Weber, I. T.; Mitsuya, H. Design of HIV-1 Protease

Inhibitors with Amino-bis-Tetrahydrofuran Derivatives as P2-Ligands

to Enhance Backbone-Binding Interactions: Synthesis, Biological

Evaluation and Protein-Ligand X-ray Studies. J. Med. Chem. 2015, 58,

(

8) De Meyer, S.; Azijn, H.; Surleraux, D.; Jochmans, D.; Tahri, A.;

Pauwels, R.; Wigerinck, P.; de Bethune, M.-P. TMC114, a Novel

Human Immunodeficiency Virus Type 1 Protease Inhibitor Active

Against Protease Inhibitor-Resistant Viruses, Including a Broad Range

of Clinical Isolates. Antimicrob. Agents Chemother. 2005, 49, 2314−

(

994−7006.

24) Ghosh, A. K.; Rao, K. V.; Nyalapatla, P. R.; Osswald, H. L.;

Martyr, C. D.; Aoki, M.; Hayashi, H.; Agniswamy, J.; Wang, Y.-F.;

Bulut, H.; Das, D.; Weber, I. T.; Mitsuya, H. Design and

Development of Highly Potent HIV-1 Protease Inhibitors with a

Crown-like Oxotricyclic Core as the P2-Ligand to Combat Multidrug-

Resistant HIV Variants. J. Med. Chem. 2017, 60, 4267−4278.

(

321.

9) de Bethune, M. P.; Sekar, V.; Spinosa-Guzman, S.; Vanstockem,

M.; De Meyer, S.; Wigerinck, P.; Lefebvre, E. Darunavir (Prezista,

TMC114): From Bench to Clinic. Improving Treatment Options for

HIV-Infected Patients in Antiviral Drugs: From Basic Discovery through

Clinical Trials; John Wiley & Sons, Inc.: New Jersey, 2011; pp 31−45.

(25) Ghosh, A. K.; Rao, K. V.; Nyalapatla, P. R.; Kovela, S.; Brindisi,

M.; Osswald, H. L.; Sekhara Reddy, B.; Agniswamy, J.; Wang, Y.-F.;

Aoki, M.; Hattori, S.-i.; Weber, I. T.; Mitsuya, H. Design of Highly

Potent, Dual Acting and Central Nervous System Penetrating HIV-1

Protease Inhibitors with Excellent Potency against Multidrug-

Resistant HIV-1 Variants. ChemMedChem 2018, 13, 803−815.

(

10) Ghosh, A. K.; Chapsal, B. D. 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.

(

11) Lambert-Niclot, S.; Flandre, P.; Canestri, A.; Peytavin, G.;

(26) Aoki, M.; Hayashi, H.; Rao, K. V.; Das, D.; Higashi-Kuwata, N.;

Blanc, C.; Agher, R.; Soulie, C.; Wirden, M.; Katlama, C.; Calvez, V.;

Bulut, H.; Aoki-Ogata, H.; Takamatsu, Y.; Yedidi, R. S.; Davis, D. A.;

Hattori, S.-i.; Nishida, N.; Hasegawa, K.; Takamune, N.; Nyalapatla,

P. R.; Osswald, H. L.; Jono, H.; Saito, H.; Yarchoan, R.; Misumi, S.;

Ghosh, A. K.; Mitsuya, H. A Novel Central Nervous System-

Penetrating Protease Inhibitor Overcomes Human Immunodeficiency

Virus 1 Resistance with Unprecedented aM to pM potency. eLife

2017, 6, No. e28020.

Marcelin, A.-G. Factors Associated with the Selection of Mutations

Conferring Resistance to Protease Inhibitors (PIs) in PI-experienced

Patients Displaying Treatment Failure on Darunavir. Antimicrob.

Agents Chemother. 2008, 52, 491−496.

(

12) de Meyer, S.; Vangeneugden, T.; van Baelen, B.; de Paepe, E.;

van Marck, H.; Picchio, G.; Lefebvre, E.; de Bethune, M.-P. Resistance

Profile of Darunavir: Combined 24-Week Results from the POWER

Trials. AIDS Res. Hum. Retroviruses 2008 , 24 , 379 −388.

(27) Hohlfeld, K.; Tomassi, C.; Wegner, J. K.; Kesteleyn, B.; Linclau,

B. Disubstituted Bis-THF Moieties as New P2 Ligands in Non-

peptidal HIV-1 Protease Inhibitors. ACS Med. Chem. Lett. 2011, 2,

461−465.

13) Este, J. A.; Cihlar, T. Current Status and Challenges of

Antiretroviral Research and Therapy. Antiviral Res. 2010, 85, 25−33.

(14) Saylor, D.; Dickens, A. M.; Sacktor, N.; Haughey, N.; Slusher,

B.; Pletnikov, M.; Mankowski, J. L.; Brown, A.; Volsky, D. J.;

McArthur, J. C. HIV-Associated Neurocognitive Disorder 

Pathogenesis and Prospects for Treatment. Nat. Rev. Neurol. 2016,

(28) Hohlfeld, K.; Wegner, J. K.; Kesteleyn, B.; Linclau, B.; Unge, J.

Disubstituted Bis-THF Moieties as New P2 Ligands in Nonpeptidal

HIV-1 Protease Inhibitors (II). J. Med. Chem. 2015, 58, 4029−4038.

(29) Bai, X.; Yang, Z.; Zhu, M.; Dong, B.; Zhou, L.; Zhang, G.;

Wang, J.; Wang, Y. Design and Synthesis of Potent HIV-1 Protease

Inhibitors with (S)-Tetrahydrofuran-Tertiary Amine-Acetamide as

P2 −Ligand: Structure −Activity Studies and Biological Evaluation.

Eur. J. Med. Chem. 2017, 137, 30−44.

(

2, 234−248.

15) Ghosh, A. K.; Ramu Sridhar, P.; Kumaragurubaran, N.; Koh, Y.;

Weber, I. T.; Mitsuya, H. Bis-Tetrahydrofuran: A Privileged Ligand

for Darunavir and a New Generation of HIV Protease Inhibitors That

Combat Drug Resistance. ChemMedChem 2006, 1, 939−950.

(16) Ghosh, A. K.; Anderson, D. D.; Weber, I. T.; Mitsuya, H.

(30) Yang, Z.-H.; Bai, X.-G.; Zhou, L.; Wang, J.-X.; Liu, H.-T.;

Wang, Y.-C. Synthesis and Biological Evaluation of Novel HIV-1

Enhancing Protein Backbone Binding A Fruitful Concept for

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Mutant PRS17 Shows Enhanced Binding to Substrate Analogues. ACS

Article

Protease Inhibitors Using Tertiary Amine as P2-Ligands. Bioorg. Med.

Chem. Lett. 2015, 25, 1880−1883.

Omega 2019, 4, 8707−8719.

(31) Yan, J.; Huang, N.; Li, S.; Yang, L.-M.; Xing, W.; Zheng, Y.-T.;

(46) Otwinowski, Z.; Minor, W. Processing of X-ray Diﬀraction Data

Collected in Oscillation Mode. In Methods in Enzymology, 276:

Macromolecular Crystallography, Part A.; Carter, C. W., Jr., Sweet, R.

M., Eds.; Academic Press: New York, 1997; pp 307−326.

(47) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M.

D.; Storoni, L. C.; Read, R. J.; Phaser Crystallographic Software.

Phasercrystallographic software. J. Appl. Crystallogr. 2007, 40, 658−

Hu, Y. Synthesis and Biological Evaluation of Novel Amprenavir-

Based P1-Substituted Bi-Aryl Derivatives as Ultra-Potent HIV-1

Protease Inhibitors. Bioorg. Med. Chem. Lett. 2012, 22, 1976−1979.

(32) Bungard, C. J.; Williams, P. D.; Ballard, J. E.; Bennett, D. J.;

Beaulieu, C.; Bahnck-Teets, C.; Carroll, S. S.; Chang, R. K.; Dubost,

D. C.; Fay, J. F.; Diamond, T. L.; Greshock, T. J.; Hao, L.; Holloway,

M. K.; Felock, P. J.; Gesell, J. J.; Su, H.-P.; Manikowski, J. J.; McKay,

D. J.; Miller, M.; Min, X.; Molinaro, C.; Moradei, O. M.; Nantermet,

P. G.; Nadeau, C.; Sanchez, R. I.; Satyanarayana, T.; Shipe, W. D.;

Singh, S. K.; Truong, V. L.; Vijayasaradhi, S.; Wiscount, C. M.; Vacca,

J. P.; Crane, S. N.; McCauley, J. A. Discovery of MK-8718, an HIV

Protease Inhibitor Containing a Novel Morpholine Aspartate Binding

Group. ACS Med. Chem. Lett. 2016, 7, 702−707.

(

74.

48) Winn, M. D.; Ballard, C. C.; Cowtan, K. D.; Dodson, E. J.;

Emsley, P.; Evans, P. R.; Keegan, R. M.; Krissinel, E. B.; Leslie, A. G.

W.; McCoy, A.; McNicholas, S. J.; Murshudov, G. N.; Pannu, N. S.;

Potterton, E. A.; Powell, H. R.; Read, R. J.; Vagin, A.; Wilson, K. S.

Overview of the CCP4 Suite and Current Developments. Acta

Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 235−242.

(49) Collaborative Computational Project, Number 4. The CCP4

(33) Bungard, C. J.; Williams, P. D.; Schulz, J.; Wiscount, C. M.;

Suite: Programs for Protein Crystallography. Acta Crystallogr., Sect. D:

Biol. Crystallogr. 1994, 50, 760−763.

Holloway, M. K.; Loughran, H. M.; Manikowski, J. J.; Su, H.-P.;

Bennett, D. J.; Chang, L.; Chu, X.-J.; Crespo, A.; Dwyer, M. P.;

Keertikar, K.; Morriello, G. J.; Stamford, A. W.; Waddell, S. T.;

Zhong, B.; Hu, B.; Ji, T.; Diamond, T. L.; Bahnck-Teets, C.; Carroll,

S. S.; Fay, J. F.; Min, X.; Morris, W.; Ballard, J. E.; Miller, M. D.;

McCauley, J. A. Design and Synthesis of Piperazine Sulfonamide

Cores Leading to Highly Potent HIV-1 Protease Inhibitors. ACS Med.

Chem. Lett. 2017, 8, 1292−1297.

(50) Potterton, E.; Briggs, P.; Turkenburg, M.; Dodson, E. A

Graphical User Interface to the CCP4 Program Suite. Acta

Crystallogr., Sect. D: Biol. Crystallogr. 2003, 59, 1131−1137.

(51) Shen, C.-H.; Wang, Y.-F.; Kovalevsky, A. Y.; Harrison, R. W.;

Weber, I. T. Amprenavir Complexes with HIV-1 Protease and its

Drug-Resistant Mutants Altering Hydrophobic Clusters. FEBS J.

010, 277, 3699−3714.

52) Sheldrick, G. M. Short History of SHELX. Acta Crystallogr.,

Sect. A: Found. Crystallogr. 2008 , 64 , 112 −122.

53) Sheldrick, G. M. Crystal Structure Refinement with SHELXL.

Acta Crystallogr. 2015, 71, 3−8.

54) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Refinement of

35) Jacobsen, E. N.; Marko, I.; Mungall, W. S.; Schroeder, G.;

34) Kolb, H. C.; VanNieuwenhze, M. S.; Sharpless, K. B. Catalytic

Asymmetric Dihydroxylation. Chem. Rev. 1994, 94, 2483−2547.

(

Sharpless, K. B. Asymmetric Dihydroxylation via Ligand-accelerated

Catalysis. J. Am. Chem. Soc. 1988, 110, 1968−1970.

Palmisano, G. Indole Alkaloids. Enantioselective Synthesis of

(36) Riva, R.; Banfi, L.; Danieli, B.; Guanti, G.; Lesma, G.;

Macromolecular Structures by the Maximum-likelihood Method. Acta

−)-Alloyohimbane by a Chemoenzymatic Approach. J. Chem. Soc.,

Chem. Commun. 1987, 1987, 299−300.

37) (a) Danieli, B.; Lesma, G.; Mauro, M.; Palmisano, G.;

Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53, 240 −255.

(55) Schuettelkopf, A. W.; van Aalten, D. M. F. PRODRG: a Tool

for High-Throughput Crystallography of Protein −Ligand Complexes.

Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 1355−1363.

(56) Lebedev, A. A.; Young, P.; Isupov, M. N.; Moroz, O. V.; Vagin,

A. A.; Murshudov, G. N. Ligand: A Graphical Tool for the CCP4

Template-restraint Library. Acta Crystallogr., Sect. D: Biol. Crystallogr.

2012, 68, 431−440.

(

Passarella, D. First Enantioselective Synthesis of (-)-Akagerine by a

Chemoenzymatic Approach. J. Org. Chem. 1995 , 60 , 2506 −2513.

(b) Adams, G. L.; Carroll, P. J.; Smith, A. B., III Total Synthesis of

(+)-Scholarisine A. J. Am. Chem. Soc. 2012 , 134 , 4037 −4040.

(-)-Alloyohimbane and (-)-Yohimbane by an Efficient Enzymatic

(57) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features

and Development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr.

2010, 66, 486−501.

Desymmetrization Process. Eur. J. Org. Chem. 2016, 2016, 6001−

009.

38) Dihydroxylation of 12 with OsO and NMO resulted in diols

(58) Emsley, P.; Cowtan, K. Coot: Model-Building Tools for

Molecular Graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004,

60, 2126−2132.

(

3 and 14 in 1:3 ratio.

39) Von Langen, D. J.; Tolman, R. L. Resolution and Stereo-

selective Synthesis of the Herpes Thymidine Kinase Inhibitor L-

53180. Tetrahedron: Asymmetry 1997, 8, 677 −681.

40) Toth, M. V.; Marshall, G. R. A Simple, Continuous

Fluorometric Assay for HIV Protease. Int. J. Pept. Protein Res. 1990,

6, 544−550.

41) Koh, Y.; Amano, M.; Towata, T.; Danish, M.; Leshchenko-

(59) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T.

N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E. The Protein Data

Bank. Nucleic Acids Res. 2000, 28, 235−242.

Mitsuya, H. In Vitro Selection of Highly Darunavir-Resistant and

Yashchuk, S.; Das, D.; Nakayama, M.; Tojo, Y.; Ghosh, A. K.;

Replication-Competent HIV-1 Variants by Using a Mixture of Clinical

HIV-1 Isolates Resistant to Multiple Conventional Protease

Inhibitors. J. Virol. 2010, 84, 11961−11969.

(42) Aoki, M.; Danish, M. L.; Aoki-Ogata, H.; Amano, M.; Ide, K.;

Das, D.; Koh, Y.; Mitsuya, H. Loss of the Protease Dimerization

Inhibition Activity of Tipranavir (TPV) and Its Association with the

Acquisition of Resistance to TPV by HIV-1. J. Virol. 2012, 86,

(

3384−13396.

43) For details of X-ray studies, please see Supporting Information .

(44) Wlodawer, A.; Vondrasek, J. Inhibitors of HIV-1 Protease: A

Major Success of Structure-assisted Drug Design. Annu. Rev. Biophys.

Biomol. Struct. 1998, 27, 249−284.

(45) Agniswamy, J.; Kneller, D. W.; Brothers, R.; Wang, Y.-F.;

Harrison, R. W.; Weber, I. T. Highly Drug-Resistant HIV-1 Protease