Design of novel HIV-1 protease inhibitors incorporating isophthalamide-derived P2-P3 ligands: Synthesis, biological evaluation and X-ray structural studies of inhibitor-HIV-1 protease complex

Article abstract of DOI:10.1016/j.bmc.2017.04.005

Based upon molecular insights from the X-ray structures of inhibitor-bound HIV-1 protease complexes, we have designed a series of isophthalamide-derived inhibitors incorporating substituted pyrrolidines, piperidines and thiazolidines as P2-P3 ligands for specific interactions in the S2-S3 extended site. Compound 4b has shown an enzyme K_i of 0.025 nM and antiviral IC₅₀ of 69 nM. An X-ray crystal structure of inhibitor 4b-HIV-1 protease complex was determined at 1.33 ? resolution. We have also determined X-ray structure of 3b-bound HIV-1 protease at 1.27 ? resolution. These structures revealed important molecular insight into the inhibitor–HIV-1 protease interactions in the active site.

Full text of DOI:10.1016/j.bmc.2017.04.005

Accepted Manuscript
Design of novel HIV-1 protease inhibitors incorporating isophthalamide-de-
rived P2-P3 ligands: synthesis, biological evaluation and X-ray structural studies
of inhibitor-HIV-1 protease complex
Arun K. Ghosh, Margherita Brindisi, Prasanth R. Nyalapatla, Jun Takayama,
Jean-Rene Ella-Menye, Sofiya Yashchuk, Johnson Agniswamy, Yuan-Fang
Wang, Manabu Aoki, Masayuki Amano, Irene T. Weber, Hiroaki Mitsuya
PII:
S0968-0896(17)30731-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.04.005
BMC 13674
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Accepted Date:
7 February 2017
4 April 2017
Please cite this article as: Ghosh, A.K., Brindisi, M., Nyalapatla, P.R., Takayama, J., Ella-Menye, J-R., Yashchuk,
S., Agniswamy, J., Wang, Y-F., Aoki, M., Amano, M., Weber, I.T., Mitsuya, H., Design of novel HIV-1 protease
inhibitors incorporating isophthalamide-derived P2-P3 ligands: synthesis, biological evaluation and X-ray structural
studies of inhibitor-HIV-1 protease complex, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/
10.1016/j.bmc.2017.04.005
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Design of novel HIV-1 protease inhibitors incorporating
Leave this area blank for abstract info.
isophthalamide-derived P2-P3 ligands: synthesis,
biological evaluation and X-ray structural studies of
inhibitor-HIV-1 protease complex
Arun K. Ghosh,*,a Margherita Brindisi,a Prasanth R. Nyalapatla,a Jun Takayama,a Jean-Rene Ella-Menye,a
Sofiya Yashchuk,a Johnson Agniswamy,b Yuan-Fang Wang,b Manabu Aoki,c Masayuki Amano,c Irene T.
Weber,b and Hiroaki Mitsuyac,d,e
aDepartment of Chemistry and Department of Medicinal Chemistry, Purdue University, West Lafayette, IN 47907 (USA); bDepartments of Biology and
Chemistry, Georgia State University, Atlanta, GA 30303 (USA); cDepartments of Hematology and Infectious Diseases, Kumamoto University of Medicine,
Kumamoto 860-8556 (Japan); dExperimental Retrovirology Section, HIV and AIDS Malignancy Branch National Cancer Institute, Bethesda, MD 20892
(USA); eCenter for Clinical Sciences, National Center for Global Health and Medicine, Shinjuku, Tokyo 162-8655 (Japan).

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Design of novel HIV-1 protease inhibitors incorporating isophthalamide-derived P2-
P3 ligands: synthesis, biological evaluation and X-ray structural studies of inhibitor-
HIV-1 protease complex
Arun K. Ghosh,*,a Margherita Brindisi,a Prasanth R. Nyalapatla,a Jun Takayama,a Jean-Rene Ella-Menye,a
Sofiya Yashchuk,a Johnson Agniswamy,b Yuan-Fang Wang,b Manabu Aoki,c Masayuki Amano,c Irene T.
Weber,b and Hiroaki Mitsuya c,d,e
aDepartment of Chemistry and Department of Medicinal Chemistry, Purdue University, West Lafayette, IN 47907 (USA); bDepartments of Biology and
Chemistry, Georgia State University, Atlanta, GA 30303 (USA); cDepartments of Hematology and Infectious Diseases, Kumamoto University of Medicine,
Kumamoto 860-8556 (Japan); dExperimental Retrovirology Section, HIV and AIDS Malignancy Branch National Cancer Institute, Bethesda, MD 20892 (USA);
eCenter for Clinical Sciences, National Center for Global Health and Medicine, Shinjuku, Tokyo 162-8655 (Japan).
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Based upon molecular insights from the X-ray structures of inhibitor-bound HIV-1 protease
complexes, we have designed a series of isophthalamide-derived inhibitors incorporating
substituted pyrrolidines, piperidines and thiazolidines as P2-P3 ligands for specific interactions
in the S2-S3 extended site. Compound 4b has shown an enzyme Ki of 0.025 nM and antiviral
IC50 of 69 nM. An X-ray crystal structure of inhibitor 4b-HIV-1 protease complex was
determined at 1.33 Å resolution. We have also determined X-ray structure of 3b-bound HIV-1
protease at 1.27 Å resolution. These structures revealed important molecular insight into the
inhibitor–HIV-1 protease interactions in the active site.
Available online
Keywords:
HIV-1 protease
Drug-resistance
X-ray structure
Synthesis
2009 Elsevier Ltd. All rights reserved
.
Isophthalamide
1. Introduction
Protein structure-based molecular design has had tremendous
impact in modern drug design and medicinal chemistry efforts.1,2
The design and development of HIV-1 protease inhibitors and
their combination with reverse transcriptase inhibitors, marked
the beginning of a new era of treatment of HIV infection and
AIDS.3,4 The combination antiretroviral therapies (ART)
significantly improved life expectancy and dramatically
improved the course of HIV management in developing
countries.5,6 Therapeutic protease inhibitors (PIs) block HIV-1
replication by inhibiting viral HIV-1 protease and generate
morphologically immature and noninfectious virions. However,
selective drug pressure blocks replication of viruses that are
susceptible to the drug and generates drug-resistant HIV-1
variants.7,8 A growing number of HIV/AIDS patients are
harboring multidrug-resistant HIV-1 variants that can be readily
transmitted. These drug-resistant viruses are difficult to treat and
raise a serious concern for the long-term treatment options of
HIV management.9,10 Therefore, one of the major current
therapeutic objectives is to design and develop novel PIs that
display broad-spectrum activity against drug-resistant HIV-1
variants and show improved drug-like properties.3,11
Figure 1. Structures of inhibitors 1,2, 3a and 4b.
In our continuing studies towards the design and
development of PIs to combat drug-resistance, we reported a
series of novel nonpeptide PIs that showed very potent enzyme

inhibitory and antiviral activity. Also, these PIs exhibited potent
antiviral activity3,12,13 against a panel of multidrug-resistant HIV-
1 variants.
R2
N
X
R
OH
H
R1
Y
N
N
O
One of our major design strategies is to incorporate cyclic
ether or polyether-like templates where ether oxygens are
positioned to form specific hydrogen bonds with the active site
backbone atoms.14,15 In essence, these ether oxygens mimic the
peptide carbonyl group in the active site. This design concept is
inspired by the occurrence of numerous bioactive natural
products with polyether subunits.16,17 Our laboratory reported a
range of exceptionally potent PIs with intriguing molecular
features such as TMC-114, TMC126, GRL-06579, GRL-02031,
and GRL-519.18,19,20 These inhibitors show broad-spectrum
antiviral activity against multidrug-resistant HIV-1 variants and
show high genetic barrier to resistance. Darunavir (DRV, 1) has
emerged as front-line treatment of HIV/AIDS patients.21,22 In
DRV, we have incorporated a stereochemically defined bis-
tetrahydrofuran (bis-THF) template to promote hydrogen
bonding interactions with the backbone atoms at S2-subsites.17,21
Our structure-based design efforts created numerous intriguing
ligands such as 3S-THF, bis-THF, Cp-THF, Tp-THF, and tris-
THF.12 All these ligands are involved in interactions in the
protease active site, especially extensive hydrogen bonding with
the protease backbone atoms.
Recently, we have explored a number of substituted benzoic
acid-based acyclic and macrocyclic inhibitors with good to
excellent potency.23,24 These inhibitors were designed based upon
the P2-ligand of FDA approved nelfinavir and P1′ and P2′
ligands inherent to DRV (1) and TMC-126 (2).17,25 In particular,
a series of PIs containing isophthalamide-based P2-P3 ligands
showed very potent enzyme inhibitory and antiviral activity.26
One of the important features of these PIs is that they contain
only two chiral centers and structural variants of these PIs can be
synthesized readily for structure-activity studies. Furthermore,
some of these ligand moieties have been utilized in the design
and synthesis of inhibitors of β-secretase (BACE-1), an aspartic
acid protease target for the treatment of Alzheimer’s disease.27-30
Our initial efforts led us to determine X-ray structures of HIV-1
protease complexes with a number of isophthalamide based
inhibitors.26 These X-ray structural studies provided molecular
insights into the ligand-binding site interactions in the HIV-1
protease active site. We have now further explored these
structural insights and herein we report the design, synthesis, and
S
O
O
3a-h and 4a-l
R
OH
X
R2
H2N
N
O
COOH
+
S
N
R1
X
O
5
6a
, R = OMe
, R = CH2OH
, R = NH2
6b
6c
O
OMe
X
O
O
HO
OMe
OH
OMe
O
O
7a
, X = CH
HO
O
O
7b
, X = N
8
9
7c
, X = C-N(Boc)Me
7d
, X = C-OH
Me
Me
N
Me
N
Me
O
S
O
N
Me
N
NH2
NH
NH2
NH2
NH2
10a
10b
10c
10d
S
NH
NH
NH
NH
N
N
N
O
S
N
N
S
S
S
Me
R
Me
Me
Me
11e
, R = Me
11f
11a,b
11c
11d
11g,h
, R = CH2OMe
Scheme 1. General synthetic approach for inhibitors 3a-h and 4a-l.
biological evaluation of
a series of PIs incorporating
isophthalamide-based P2-P3 ligands with substituted pirrolidines,
piperidines and thiazolidines for specific interactions in the S2-
S3 extended site.
2. Results and discussion
2.1. Synthesis
Our general strategy for the synthesis of HIV-1 protease
inhibitors 3a-h and 4a-l is illustrated in Scheme 1. We planned to
synthesize these inhibitors by amide coupling of the appropriate
isophthalic, phthalic or terephthalic acid derivatives 5 with the
amines belonging to hydroxyethylamine sulfonamide isosteres
(6a-c).19,28 The required acid derivatives were obtained by
coupling of readily available isophthalic (7a-d), phthalic (8) or
terephthalic (9) monoacids with amines 10a-d or cyclic amines
11a-h.28,31,32
The synthesis of oxazole- and thiazole-based P2-P3 ligands
12a-g is shown in Scheme 2. The known28 amine 10a was
coupled with isophthalic acid derivatives 7c,d using EDC, HOBt
in the presence of diisopropylethylamine to provide the
corresponding amide derivatives. The resulting amides were N-
Scheme 2. Reagents and conditions: (a) 7c or 7d, EDCI, HOBt, i-Pr2NEt,
CH2Cl2, 25 °C, 16 h, 80%; (b) MeI, NaH, dry THF, 25 °C, 1 h, 68%; (c)
LiOH.H2O, THF/MeOH/H2O (3:1:1), 25 °C, 16 h, 99%.

methylated with NaH and MeI in THF. Saponification of the
resulting methyl ester with aqueous lithium hydroxide afforded
isophthalic acid derivatives 12a,b. Acid derivatives 12c-f were
prepared using similar reaction sequences using suitable
commercially available amines 10b,c and oxazolylmethylamine
10d26 and carboxylic acids 7a, 8 or 9. The resulting amides were
N-methylated and subsequent saponification of the methyl ester
functionality afforded acids 12c-f.
The synthesis of various methylthiazolylpyrrolidine-
derived isophthalic acids is shown in Scheme 3. As shown,
amines 11a,b and isophthalic acid monomethyl esters 7a or 7b
were coupled together using standard EDC and HOBt coupling.
The resulting esters were saponified with aqueous lithium
hydroxide to provide carboxylic acids 13a-c. Compound 13a was
also employed for the synthesis of P2-P3 ligand 15 in which a
carbonyl group was converted to a methylene linker by reducing
the isophthalate carbonyl group. Thus, treatment of 13a with
LiAlH4 in dry THF at reflux provided aminoalcohol 14. The
hydroxymethyl group of 14 was oxidized with manganese
dioxide to aldehyde. Oxidation33 of this aldehyde with NaClO2
provided acid 15. Acid derivatives 16a-c were prepared by
following similar sequence of reactions as compound 13a.
Scheme 4. Reagents and conditions: (a) (R)-α-methylbenzylamine MeOH,
115 °C, 18 h, 75%; (b) BnOH, PPTS, toluene, reflux, 24 h, (S) 81%, (R) 73%;
(c) Lawesson’s reagent, toluene, 100 °C, 2 h, (S) 99%, (R) 67%; (d) MeI,
CH2Cl2, 25 °C, 24 h then NaBH4, EtOH, 0 °C, 1h, (S) 50%, (R) 38%; (e) H2,
10% Pd/C, 25 °C, 20 h, (S) 99%, (R) 99%; (f) CbzCl, 4 N NaOH, 0 °C, 2 h,
(S) 51%, (R) 39%; (g) Boc2O, pyridine, NH4HCO3, 1,4-dioxane, 25 °C, 16 h;
(h) Lawesson’s reagent, THF, 25 °C, 16 h; (i) chloroacetone, EtOH, 80 °C,
18 h, (S) 42%, (R) 56% (over 3 steps); (j) HBr in AcOH, 25 °C, 1 h, (S) 89%,
(R) 88%; (k) 7a, EDCI, HOBt, i-Pr2NEt, CH2Cl2, 25 °C, 16 h, (S) 55%, (R)
68%; (l) LiOH.H2O, THF/MeOH/H2O (3:1:1), 25 °C, 16 h, (S) 55%, (R) 54%.
derivatives 18a,b. These lactams were treated with Lawesson’s
reagent to provide thioamides 19a,b. Reduction of the thioamide
functionality by treatment with MeI followed by NaBH4 in EtOH
led to derivatives 20a,b. Simultaneous removal of benzyl and α-
methylbenzyl groups by catalytic hydrogenation was followed by
N-Cbz protection and conversion of the carboxylic functionality
to the corresponding amide to furnish carboxamide derivatives
21a,b. Treatment of these carboxamides with Lawesson’s reagent
afforded the corresponding thioamides, which underwent
cyclization upon treatment with chloroacetone in EtOH. Removal
of the Cbz group by exposure to HBr in AcOH, provided the
desired amine derivatives 11g,h. These ligands were coupled
with the isophthalic acid methyl ester 7a to furnish the
corresponding amide derivatives. Saponification of the methyl
ester with aqueous lithium hydroxide afforded acids 22a,b.
The synthesis of isophthalic acid derivative 25 is reported in
Scheme 5. Ester 23 was treated with LiBH4 in THF at 23 °C to
provide alcohol derivative 24. Alkylation of alcohol 24 with MeI
and NaH in THF provided the corresponding methyl ether.
Removal of the Cbz-group with HBr in acetic acid afforded
amine 11f. This amine was coupled with isophthalic acid
monomethyl ester 7a using EDC and HOBt in the presence of i-
Pr2NEt to provide the corresponding amide derivative.
Saponification of the methyl ester with aqueous LiOH afforded
acid 25.
Scheme 3. Reagents and conditions: (a) 7a or 7b, EDCI, HOBt, i-Pr2NEt,
CH2Cl2, 25 °C, 16 h, 80%; (b) LiOH.H2O, THF/MeOH/H2O (3:1:1), 25 °C,
16 h, 99%; (c) from 13a, LiAlH4, dry THF, reflux, 15 h, 88%; (d) MnO2,
CH2Cl2, 25 °C, 5 h; (e) NaClO2, NaH2PO4, t-BuOH/H2O, 2-methyl-2-butene,
25 °C, 4 h, 45% (over 2 steps).
The synthesis of pyrrolidin-3-yl-thiazole amine-derived P2-P3
ligands 11g,h is shown in Scheme 4. (3R)- and (3S)-pyrrolidines
were prepared by Michael addition of (R)-α-methylbenzylamine
with dimethyl itaconate (17) followed by cyclization of the
resulting amino-ester to give an equal mixture of lactams as
described previously.34 This mixture was separated by
chromatography.
Treatment of the resulting esters with
benzylalcohol in the presence of PPTS provided benzyl ester

cyclic and acyclic heterocycles could form hydrogen bonds with
backbone atoms as well as form stronger van der Waals
interactions in the active site. In particular, we first sought to
investigate the outcome of a focused series of derivatives bearing
oxazolylmethyl-
or
thiazolylmethyl-derived
P3-ligands
(compounds 3b-h, Table 1). HIV-1 protease inhibitory potency
of all inhibitors was first evaluated using the assay protocol
reported by Toth and Marshall.6 The antiviral activity of the new
inhibitors was determined based on a previously published assay
protocol using MT-2 cells exposed to HIV-1 LAI.37 These results
are shown in Tables 1 and 2. In compound 3b, we incorporated a
4-methyl oxazole as P3 to promote hydrogen bond through the
oxazole nitrogen. However, this compound lost significant
potency over compound 3a. Compounds 3c (entry 3) and 3d
(entry 4) contained a 2-methyloxazole and a 2-methylthiazole
ring, respectively. Derivative 3d displayed 5-fold enhanced
protease inhibitory activity over 3c. Nevertheless, the compounds
similarly performed in antiviral assay, displaying an over 20-fold
improvement with respect to their forerunner 3b (entry 2). The
dimethyloxazole moiety was also explored in combination with
polar substituents placed at the 3-position of the isophthalamide
moiety (entries 5 and 6). As it turned out, phenol derivative 3f
overtook its methylamino analogue 3e by 10-fold in terms of
protease inhibitory potency. However, the presence of a 3-
substituted isophthalamide portion was found detrimental for the
antiviral outcome of both compounds. Finally, the exploration of
the dimethyloxazole moiety linked to phthalamide- or
terephthalamide-based ligands (compounds 3g and 3h, entries 7
and 8, respectively) led to a strong drop in both the enzyme
inhibitory and the antiviral activities of the compounds, clearly
validating the key role of the isophthalamide moiety for
inhibitors’ efficacy. Taking into account the information gained
with the first set of compounds we sought to explore a novel
Scheme 5. Reagents and conditions: (a) LiBH4, dry THF, 25 °C, 4 h, 42%;
(b) MeI, NaH, dry THF, 25 °C, 1 h, 68%; (c) HBr in AcOH, 25 °C, 1 h, 67%;
(d) 7a, EDCI, HOBt, i-Pr2NEt, CH2Cl2, 25 °C, 16 h, 96%; (e) LiOH.H2O,
THF/MeOH/H2O (3:1:1), 25 °C, 16 h, 98%.
Our syntheses of the final inhibitors are shown in Scheme 6.
Synthesis of inhibitors 3a-h and 4a-l were carried out by
coupling of acids 12a-g, 13a-c, 15, 16a-c, 22a,b and 25 with
hydroxyethylamine sulfonamide isosteres (6a-c) using standard
EDC and HOBt coupling. Various isophthalamide-based
inhibitors were obtained in good to excellent yields (49-95%).
series
isophthalamide-based
extended
P2-P3
ligands
incorporating substituted pirrolidines, piperidines and
thiazolidines as the suitable support for thiazole or oxazole rings
(inhibitors 4a-l, Table 2). The incorporation of a 2-thiazolyl
pyrrolidine in both (S) or (R) configuration (compounds 4a and
4b, entries 1 and 2, respectively) clearly highlighted the key role
of stereochemistry for this series of compounds. Inhibitor 4b with
(R)-configuration displayed over 100-fold improved potency over
its (S) counterpart 4a. The same trend was also shown for the
antiviral outcome of the compounds. The combination of the (R)-
2-thiazolyl pyrrolidine moiety with different hydroxyethylamine
sulfonamide isosteres (compounds 4c and 4d, entries 3 and 4,
respectively) substantially led to a significant drop in both the
enzyme inhibitory and the antiviral activities. The increase of the
ring size in piperidine-based compound 4e (entry 5) significantly
reduced enzyme affinity and antiviral efficacy. Compound 4f
(entry 6) bearing a 2-thiazolyl thiazolidine moiety, although
characterized by a reduced enzyme inhibitory potency with
respect to 4a, displayed the best antiviral activity of this series of
compounds (IC50 = 32 nM). Compounds 4g and 4h (entries 7 and
8) containing a 2-oxazolyl pyrrolidine moiety were also explored.
In particular, methyloxazole derivative 4h resulted in a 10-fold
Scheme 6. Reagents and conditions: (a) EDCI, HOBt, i-Pr2NEt, CH2Cl2, 25
°C, 16 h (49-95%); (b) for 3e, TFA, CH2Cl2, 25 °C, 2 h, 98%.
2.2. Biological evaluation, structure-activity relationships, and
X-ray studies
On the basis of the X-ray studies reported for compounds 3a26
and darunavir-bound HIV-1 protease,35 we first sought to
investigate the outcome of a focused series of derivatives bearing
isophthalamide as the P2-ligand and various cyclic and acyclic
heterocycles as the P3-ligand. An overlay of the X-ray crystal
structures of inhibitors 1 (DRV) and 3a in the HIV-1 protease
active site is shown in Figure 2. As can be seen, the key
interactions in the S1’ and S2’ subsites are very similar. The
major differences are in the S2-S3 subsites. The bis-THF of
DRV forms two strong hydrogen bonds with Asp-29 and Asp-30
backbone amide NH’s in the S2-subsite. Inhibitor 3a forms a
strong hydrogen bond with Asp29 backbone amide NH and
forms strong van der Waals interactions with oxazolylmethyl
ligand. Based upon this molecular insight, we speculated that
improved
antiviral
profile
with
respect
to
its
methoxymethyloxazole analogue 4g. The exploration of
compounds 4i (entry 9) with a methylene replacing one of the
isophthalate carbonyl groups, and 4j (entry 10) bearing a pyridyl
ring in the isophthalic moiety led to a substantial loss of protease
inhibitory activity. The shift of the thiazole ring in 3-position of
the pyrrolidine was also investigated for both (R) and (S)
configurations (inhibitors 4k and 4l, entries 11 and 12,
respectively). Both compounds exhibited comparable protease
inhibitory profile and a moderate antiviral activity, indicating in

this case a negligible contribution of stereochemistry. To obtain
molecular insight into the inhibitor-HIV-1 protease
Table 1. Enzyme inhibitory and antiviral activity of inhibitors 3a-h.
a
Entry
Inhibitor structure
K
i
IC50
aDarunavir (1) exhibited Ki = 16 pM, antiviral IC50 = 3.2 nM

(nM)
Me
OMe
O
Me
N
OH
H
N
Me
N
1
2
0.21
0.63
31
N
S
O
O
O
O
Ph
3a
581
3b
Me
O
OMe
N
Me
N
OH
H
N
N
3
4
0.64
23
32
S
O
O
O
O
Ph
3c
Me
OMe
N
Me
N
OH
H
S
N
N
O
0.107
S
O
O
O
Ph
3d
5
6
5.08
0.51
600
290
3e
OH
Me
OMe
O
N
Me
N
OH
H
Me
N
N
O
S
O
O
O
Ph
3f
7
629
> 1000
3g
O
Me
OMe
N
Me
OH
O
H
N
N
8
N
O
70
> 1000
S
O
Me
O
Ph
3h
Table 2. Enzyme inhibitory and antiviral activity of inhibitors 4a-l.
a,b
K
i
IC50
Entry
Inhibitor structure
(nM)

1
2
3
4
30.8
0.025
1.61
> 1000
4a
69
4b
NH2
OH
H
N
N
N
S
189
O
O
O
O
N
S
Ph
4c
0.79
164
4d
5
0.53
221
4e
6
7
8
0.76
0.80
0.41
32
327
33
4f
4g
4h
a,b
K
i
IC50
Entry
Inhibitor structure
(nM)

9
23.6
nt
4i
10
2.50
nt
4j
11
12
0.49
0.40
280
109
4k
4l
aDarunavir (1) exhibited Ki = 16 pM, antiviral IC50 = 3.2 nM
interactions, we have determined the X-ray crystal structures of
the inhibitors 3b- and 4b-bound HIV-1 protease complexes.
bnt = not tested.

Figure 3. Stereoview of the X-ray structure of inhibitor 3b (torquois)-bound HIV-1 protease (PDB code: 5UPZ). All strong
active site hydrogen bonding interactions of inhibitor 3b with HIV-1 protease are shown as dotted lines.
Figure 2. Stereoview of the overlay X-ray structures of inhibitor 3a (green) and darunavir (1, torquois)-bound HIV-1 protease.
All strong active site hydrogen bonding interactions of darunavir with HIV-1 protease are shown as dotted lines.
.
Inhibitor 3b contains a 4-methyloxazolyl-2-methylamide as
the P3-ligand and a stereoview of the active site interactions is
shown in Figure 3. The P2-isophthalamide carbonyl group of the
inhibitor maintains a strong hydrogen bonding with backbone
amide and side chain of Asp29. However, the oxazole ring
flipped toward the S2-extended site and was unable to form any
hydrogen bond in the active site. The isophthalamide group in
P2 is sandwiched in a cavity formed by the hydrophobic side
chains of Ile50’, Ala28, Val32, Ile47 and Ile84. The extended P3
group (4-methyl-2-oxazole) in inhibitor 3b forms hydrophobic
interactions with the side chains of Val82’. In comparison, the
2,5-dimethyl-4-oxazole in in inhibitor 3a fits in two
conformations with occupancy ratio of 50:50.26 The ligand-
binding site interactions of inhibitor 3b in the S1, S1’ and S2’
subsites are similar to daruanvir. The hydroxymethyl group on
the P2’-ligand form a strong hydrogen bond with the backbone
amide NH of Asp30’ in the S2’-site.
The crystal structures of wild type HIV-1 protease in complex
with the inhibitors 3b (GRL-0518A) and 4b (GRL-1118A) were
determined and refined at 1.27 Å and 1.33 Å resolution,
respectively. Both crystal structures contain the protease dimer
and inhibitor bound in a single orientation. The overall structures
are comparable to the structure of HIV-1 protease and darunavir35
with low root mean square differences (RMSD) of 0.18 Å and
0.21 Å, respectively, for Cα atoms. Most differences between
corresponding Cα atoms are less than 0.4 Å. Only residues 47’-
51’ exhibit root mean square differences of about 0.5-0.6 Å,
likely due to displacement by the P2 group. For residues 78’-81’,
the larger RMSD of about 0.6-0.9 Å occurs due to the opposite
effect since the 80’s loop moves in to generate more contacts
with the single smaller isobutyl group in P1’ position of these
two complexes. The third larger RMSD of about 0.7-0.8 Å in
residue 4 may be due to the rotation of its side chain. Both
inhibitors form hydrogen bonding interactions of their urethane
NH with the carbonyl oxygen of Gly27. In addition, water-
mediated interactions connect the inhibitor carbonyl oxygen and
sulfonamide oxygen with the amides of Ile50 and 50’ in the flaps.
Similar interactions are shared by the protease-darunavir complex
and are considered responsible for the high affinity of darunavir
for HIV protease and its potency against drug resistant HIV-1
variants.35
Inhibitor 4b contains a thiazolylpyrrolidine moiety as the
P3-liagand and a stereoview of the active site interactions is
shown in Figure 4. Like compound 3b, the carbonyl group of
inhibitor 4b in the P2 position maintains polar hydrogen bonding
interactions with the amide and side chain of Asp29’ with
interatomic distances of 2.9 Å. The pyrrolidine group and the
larger sulfur atom in thiazoline of inhibitor 4b may constrain the
conformation of the extended P3 group (methyl-thiazole) to form

π-π stacking interactions with the guanidine group of Arg8’.
These extended interactions are expected to stabilize the inhibitor
in the binding site. Furthermore, the P2 isophthalamide phenyl
ring forms van der Waals contacts with Ile50’, Val32, Ile47 and
Ile84.38
hydrate (HOBt) (50 mg, 0.322 mmol) were added followed by
amine 10a (27 mg, 0.215 mmol) and diisopropylethylamine (225
µL, 1.290 mmol). The reaction mixture was stirred at 25 °C for
16 h then CH2Cl2 was added and the resulting solution was
washed with saturated aqueous NH4Cl and water. The organic
layers were dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (EtOAc/n-hexane 1:4) to furnish methyl 3-((tert-
butoxycarbonyl)(methyl)amino)-5-(((2,5-dimethyloxazol-4-yl)
3. Conclusion
In summary, we have designed and synthesized potent HIV-1
protease inhibitors incorporating isophthalamide derivatives as
the P2-P3 ligands for the HIV-1 protease active site. These
inhibitors were specifically designed to interact with the extended
S2 subsite, particularly to form hydrogen bonds with the
backbone atoms as well as to promote van der Waals interactions
with residues in the S3 subsite. In this context, we have
synthesized and evaluated a range of heterocyclic derivatives of
isophthalamides to occupy the extended S2-subsite. In particular,
we have investigated the suitability of oxazolyl and thiazolyl
isophthalamides as well as pyrrolidinyl thiazolyl and pyrrolidinyl
oxazole amide derivatives of isophthalamides with donor and
acceptor groups to serve as P2-P3 ligands. A number of
compounds showed very potent enzyme inhibitory and antiviral
activity. Inhibitor 4b with a pyrrolidinyl thiazole isophthalamide
derivative showed very potent enzyme inhibitory activity (Ki =
25 pM) and antiviral IC50 value of 69 nM in MT-2 cells exposed
to HIV-1 LAI. Further optimization efforts led to inhibitor 4h
with a pyrrolidinyl oxazole isophthalamide derivative which
showed an improved antiviral IC50 value of 33 nM. We have
determined X-ray structures of inhibitor 3b-bound HIV-1
protease and inhibitor 4b-bound HIV-1 protease. These structures
provided molecular insights into the key ligand-binding site
interactions responsible for inhibitors potency. The
isophthalamide P2-P3 ligand amide carbonyl formed very strong
hydrogen bonds with Asp29 backbone amide NH and the
pyrrolidinylthiazole heterocycles are involved in van der Waals
interactions with Leu23’ and Val82’ in the S2 extended site.
These ligand-binding site interactions can be further exploited to
design more potent and less complex protease inhibitors.
1
methyl)carbamoyl)benzoate (80%). H NMR (400 MHz, CDCl3)
δ 8.13 (t, 1H, J = 1.5 Hz), 8.03-8.00 (m, 1H), 7.93 (t, 1H, J = 1.8
Hz), 7.32 (t, 1H, J = 5.2 Hz), 4.38 (d, 2H, J = 5.3 Hz), 3.87 (s,
3H), 3.24 (s, 3H), 2.32 (s, 3H), 2.30 (s, 3H), 1.42 (s, 9H); 13C
NMR (100 MHz, CDCl3) δ 166.0, 165.9, 159.8, 154.3, 145.2,
144.5, 135.2, 130.8, 129.2, 128.5, 124.1, 81.2, 52.5, 37.0, 35.4,
28.3, 13.8, 10.1.
To a solution of this latter (35 mg, 0.084 mmol) in dry THF (2
mL), NaH (60% suspension in mineral oil, 7 mg, 0.168 mmol)
was added and the reaction mixture was stirred at 25 °C for 1 h.
The reaction was quenched by adding water at 0 °C, diluted with
CH2Cl2 and washed twice with water. The organic layers were
dried over Na2SO4, filtered and concentrated under reduced
pressure. The residue was purified by silica gel column
chromatography (EtOAc/n-hexane 1:5) providing methyl methyl
3-((tert-butoxycarbonyl)(methyl)amino)-5-(((2,5-dimethyloxazol-
1
4-yl) methyl)(methyl)carbamoyl)benzoate (68%). H NMR (400
MHz, CDCl3) δ 8.14-7.90 (m, 1H), 7.88-7.72 (m, 1H), 7.53 (s,
1H), 4.50 (s, 1H), 4.22 (s, 1H), 3.90 (s, 3H), 3.27 (s, 3H), 3.04 (s,
3H), 2.46-2.22 (m, 6H), 1.43 (s, 9H); 13C NMR (100 MHz,
CDCl3) δ 166.1, 159.4, 154.3, 146.3, 144.3, 137.0, 130.8, 130.4,
128.6, 124.7, 81.1, 52.5, 42.4, 37.8, 37.1, 28.4, 14.0, 10.2.
To a solution of the methyl ester (17 mg, 0.039 mmol) in a in
THF/MeOH/H2O mixture (1.5 mL: 0.5 mL: 0.5 mL) at 0 °C was
added LiOH·H2O (3 mg, 0.059 mmol). The mixture was stirred at
25 °C for 16 h. THF and MeOH were evaporated under reduced
pressure and the residue was diluted with water, acidified with
1N HCl to pH = 2 and extracted with EtOAc. The organic layers
were dried over Na2SO4, filtered and concentrated under reduced
pressure providing pure 12a (99%). 1H NMR (400 MHz, CDCl3)
δ 8.07-7.92 (m, 1H), 7.80 (m, 1H), 7.53 (s, 1H), 4.50 (s, 1H),
4.22 (s, 1H), 3.27 (s, 3H), 3.04 (s, 3H), 2.44-2.25 (m, 6H), 1.43
(s, 9H).
4. Experimental
4.1 General
All moisture-sensitive reactions were carried out in oven-dried
glassware under an argon atmosphere unless otherwise stated.
Anhydrous solvents were obtained as follows: Diethyl ether and
tetrahydrofuran were distilled from sodium metal/benzophenone
under argon. Toluene and dichloromethane were distilled from
calcium hydride under argon. All other solvents were reagent
grade. Column chromatography was performed using Silicycle
4.2.2. 3-(((2,5-Dimethyloxazol-4-yl)methyl)(methyl)
carbamoyl)-5-hydroxybenzoic acid (12b).
Compound 12b was prepared from 10a and 7d following the
procedure described for 12a. 1H NMR (400 MHz, CDCl3) δ 7.61-
7.34 (m, 2H), 7.06-6.80 (m, 1H), 4.42 (s, 1H), 4.19 (s, 1H), 2.95-
2.84 (m, 3H), 2.41-2.16 (m, 6H).
−400 mesh silica gel. Thin
SiliaFlash F60 230
-layer
chromatography was carried out using EMD Millipore TLC silica
gel 60 F254 plates. 1H NMR and 13C NMR spectra were recorded
on a Varian INOVA300 or a Bruker ARX400. Low-resolution
mass spectra were collected on a Waters 600 LCMS or by the
Purdue University Campus-Wide Mass Spectrometry Center.
High-resolution mass spectra were collected by the Purdue
University Campus-Wide Mass Spectrometry Center.
4.2.3. 3-(Methyl((2-methyloxazol-4-yl)methyl)carbamoyl)
benzoic acid (12c).
Compound 12c was prepared from 10b and 7a following the
procedure described for 12a.1H NMR (400 MHz, CDCl3) δ 8.54
(t, 1H, J = 1.5 Hz), 8.07 (dt, 1H, J = 7.7 and 1.4 Hz), 8.02-7.97
(m, 1H), 7.52 (s, 1H), 7.40 (q, J = 7.9 Hz, 1H), 4.46 (s, 1H), 4.37
(s, 1H), 2.95 (m, 3H), 2.34 (s, 3H).
4.2 Experimental procedures
4.2.1. 3-((tert-Butoxycarbonyl)(methyl)amino)-5-(((2,5-
dimethyloxazol-4-yl)methyl)(methyl)carbamoyl)benzoic acid
(12a).
To a solution of 7c (45 mg, 0.215 mmol) in dry CH2Cl2 (2 mL),
N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride
(EDCI) (62 mg, 0.322 mmol), and 1-hydroxybenzotriazole
4.2.4. 3-(Methyl((2-methylthiazol-4-yl)methyl)carbamoyl)
benzoic acid (12d).
Compound 12d was prepared from 10c and 7a following the
procedure described for 12a.1H NMR (300 MHz, CDCl3) δ 11.83
(br, 1H), 8.20-8.06 (m, 2H), 7.64 (d, 1H, J = 6.8 Hz), 7.47-7.42

(m, 1H), 6.87 (s, 1H), 5.04 (s, 1H), 4.74 (s, 1H), 3.11-2.94 (m,
3H), 2.40-2.31 (m, 3 H); 13C NMR (75 MHz, CDCl3) δ 170.7,
168.6, 166.1, 151.6, 135.3, 131.7, 131.4, 130.6, 128.7, 128.5,
115.2, 48.3, 37.7, 16.4. LRMS-ESI (m/z): 313.0 [M+Na].
4.2.8. 3-[(R)-2-(4-Methylthiazol-2-yl)pyrrolidine-1-
carbonyl]benzoic acid (13a).
To a solution of isophthalic acid mono methyl ester 7a (133 mg,
0.74 mmol), EDCI (283 mg, 1.48 mmol) and HOBt (200 mg,
1.48 mmol) in anhydrous CH2Cl2 (8 mL) was added a solution of
(R)-2-(4-methylthiazol-2-yl)pyrrolidine 11a (124 mg, 0.74
mmol) and diisopropylethylamine (0.77 mL, 4.44 mmol) in
anhydrous CH2Cl2 (2 mL) at 0 °C under argon atmosphere and it
was allowed to stir for 16 h at 23 °C. The reaction mixture was
quenched with water and extracted with CH2Cl2. The organic
layers were dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (CH2Cl2/MeOH 9:1) to furnish 3-[(R)-2-(4-
methylthiazol-2-yl)pyrrolidine-1-carbonyl]benzoic acid methyl
ester (80%) as a colorless oil. 1H NMR (300 MHz, CDCl3) δ 8.26
(s, 1H), 8.13 (d, 1H, J = 7.5 Hz), 7.80 (d, 1H, J = 7.5 Hz), 7.53 (t,
1H, J = 7.5 Hz), 6.80 (s, 1H), 5.68 (dd, 1H, J = 7.2 and 5.4 Hz),
3.94 (s, 3H), 3.82-3.74 (m, 1H), 3.57-3.49 (m, 1H), 2.51-2.35 (m,
2H), 2.45 (s, 3H), 2.17-2.03 (m, 2H); 13C NMR (75 MHz, CDCl3)
δ 171.5, 169.0, 166.1, 152.4, 136.6, 131.6, 131.0, 130.0, 128.5,
128.1, 112.7, 58.6, 52.1, 50.0, 32.1, 24.9, 17.0.
4.2.5. 4-(((2,5-Dimethyloxazol-4-yl)methyl)(methyl)
carbamoyl)benzoic acid (12e).
Compound 12e was prepared from 10a and 9 following the
1
procedure described for 12a. H NMR (400 MHz, CDCl3) δ 8.09
(d, 2H, J = 8.2 Hz), 7.69-7.63 (m, 1H), 7.49 (d, 1H, J = 7.2 Hz),
4.56 (s, 1H), 4.27 (s, 1H), 3.01-2.97 (m, 3H), 2.51-2.27 (m, 6H).
4.2.6. 2-(((2,5-Dimethyloxazol-4-yl)methyl)(methyl)
carbamoyl)benzoic acid (12f).
Compound 12f was prepared from 10a and 8 following the
1
procedure described for 12a. H NMR (400 MHz, CDCl3) δ 8.08-
8.01 (m, 1H), 7.54 (q, 1H, J = 7.9 Hz), 7.46-7.36 (m, 1H), 7.28-
7.22 (m, 1H), 4.61 (s, 1H), 4.06 (s, 1H), 2.79-2.72 (m, 3H), 2.45-
2.34 (m, 6H).
4.2.7. 3-(Methyl((4-methyloxazol-2-yl)methyl)carbamoyl)
benzoic acid (12g).
To
a
stirring solution of 3-[(R)-2-(4-methylthiazol-2-
yl)pyrrolidine-1-carbonyl]benzoic acid methyl ester (190 mg,
0.58 mmol) in a THF/MeOH/H2O mixture (12 mL: 4 mL: 4 mL)
at 0 °C was added solid LiOH·H2O (36 mg, 0.86 mmol), and the
resulting solution was stirred at 23 °C for 16 h. After this period,
the reaction mixture was evaporated until 1 mL and the mixture
was extracted with toluene to remove organic impurities. The
aqueous layer was cooled to 0 °C, acidified with 1N HCl to pH =
Compound 12g was prepared from 10d and 7a following the
procedure described for 12a.1H NMR (400 MHz, CDCl3) δ 9.82
(br, 1H), 8.23 (s, 1H), 8.14-8.09 (m, 1H), 7.72-7.67 (m, 1H), 7.49
(t, 1H, J = 7.7 Hz), 7.40 (s, 1H), 4.89 (s, 1H), 4.55 (s, 1H), 3.18-
3.03 (m, 3H), 2.19 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 170.9,
159.6, 136.2, 135.4, 135.2, 131.9, 131.4, 130.3 (2), 128.8, 128.6,
60.4, 37.9, 14.1.
3, extracted with EtOAc, and dried over anhydrous Na2SO4. The
solvent was evaporated to furnish title compound 13a (99%). H
1
NMR (400 MHz, CDCl3) δ 8.30 (s, 1H), 8.12 (d, 1H, J = 7.6 Hz),
7.78 (d, 1H, J = 7.6 Hz), 7.48 (t, 1H, J = 7.6 Hz), 6.77 (s, 1H),
5.65 (t, 1H, J = 6.8 Hz), 3.82-3.76 (m, 1H), 3.53-3.47 (m, 1H),
2.49-2.39 (m, 2H), 2.40 (s, 3H), 2.14-2.02 (m, 2H); 13C NMR
(100 MHz, CDCl3) δ 172.4, 169.6, 168.7, 152.3, 136.2, 132.0,
131.8, 130.9, 130.5, 128.6, 113.1, 58.7, 50.5, 32.8, 25.2, 16.6.
LRMS-ESI (m/z): 338.9 [M+Na].
4.2.9. 3-[(S)-2-(4-Methylthiazol-2-yl)pyrrolidine-1-carbonyl]
benzoic acid (13b).
Compound 13b was prepared from 11b and 7a following the
procedure described for 13a. 1H NMR and 13C NMR data are
identical to those reported for compound 13a.
4.2.10. (R)-5-(2-(4-methylthiazol-2-yl)pyrrolidine-1-carbonyl)
nicotinic acid (13c).
Compound 13c was prepared from 11a and 7b following the
1
procedure described for 13a. H NMR (400 MHz, CD3OD) δ
9.22 (s, 1H), 8.90 (s, 1H), 8.54 (s, 1H), 6.88 (s, 1H), 5.58-5.54
(m, 1H), 3.90-3.82 (m, 1H), 3.60-3.54 (m, 1H), 2.52-2.40 (m,
4H), 2.25-2.08 (m, 2H), 2,02-1.96 (m, 1H); 13C NMR (100 MHz,
CD3OD) δ 173.5, 172.9, 168.6, 153.8, 153.3, 152.3, 137.8, 137.1,
115.5, 114.6, 60.6, 51.7, 34.2, 26.3, 17.7. LRMS-ESI (m/z):
340.0 [M+Na].
4.2.11. (R)-(3-((2-(4-methylthiazol-2-yl)pyrrolidin-1-yl)
methyl)phenyl)methanol (14).
To a solution of 13a (146 mg, 0.442 mmol) in dry THF (8 mL)
LiAlH4 (42 mg, 1.106 mmol) was added portionwise at 0 °C and
the reaction mixture was stirred at reflux temperature for 15 h.
The reaction was quenched with MeOH and filtered through a
Celite® pad. The filtrate was concentrated and the residue was
purified by silica gel column chromatography (EtOAc/n-hexane
1:1) affording compound 14 (88%). 1H NMR (300 MHz, CDCl3)
δ 7.38-7.27 (m, 4H), 6.83 (s, 1H), 4.72 (s, 2H), 4.08-3.97 (m,
2H), 3.41-3.36 (m, 1H), 3.10-3.05 (m, 1H), 2.44 (s, 3H), 2.38-
2.28 (m, 2H), 1.95-1.79 (m, 4H); 13C NMR (75 MHz, CDCl3) δ

177.5, 152.1, 141.2, 139.0, 128.2, 127.6, 127.1, 125.5, 113.2,
65.6, 64.7, 58.6, 53.2, 34.2, 22.9, 16.9.
4.2.16. Benzyl (R)-5-oxo-1-((R)-1-phenylethyl)pyrrolidine-3-
carboxylate (18a) and benzyl (S)-5-oxo-1-((R)-1-
phenylethyl)pyrrolidine-3-carboxylate (18b).
To a solution of dimethyl itaconate 17 (3 g, 18.97 mmol) in
MeOH (4 mL) was added (R)-α-methylbenzylamine (3.14 mL,
26.66 mmol). The solution was stirred for 1 h at 80 °C and for 18
h at 115 °C. The reaction mixture was then concentrated under
reduced pressure and water was added. The aqueous solution was
adjusted to pH = 2 with 10% HCl and extracted with CH2Cl2. The
organic layers were dried over Na2SO4, filtered and concentrated
under reduced pressure. The residue was purified by silica gel
column chromatography (EtOAc/pentane 3:7 to 1:1) providing
4.2.12. (R)-3-((2-(4-methylthiazol-2-yl)pyrrolidin-1-yl)methyl)
benzoic acid (15).
To a solution of alcohol 14 (112 mg, 0.388 mmol) in dry CH2Cl2
(4 mL) MnO2 (1.18 g, 12.86 mmol) was added at 0 °C and the
rection mixture was stirred for 5 h at 25 °C. The reaction mixture
was then filtered through a Celite® pad. The filtrate was
concentrated and the residue was purified by silica gel column
chromatography (EtOAc/n-hexane 1:3) affording (R)-3-((2-(4-
methylthiazol-2-yl)pyrrolidin-1-yl)methyl)benzaldehyde. 1H NMR
(300 MHz, CDCl3) δ 9.97 (s, 1H), 7.84 (s, 1H), 7.71 (d, 1H, J =
7.6 Hz), 7.62 (d, 1H, J = 7.6 Hz), 7.43 (t, 1H, J = 7.6 Hz), 6.76
(s, 1H), 4.09-3.97 (m, 2H), 3.43-3.39 (m, 1H), 3.03-2.98 (m, 1H),
2.37 (s, 3H), 2.36-2.25 (m, 2H), 1.92-1.74 (m, 3H); 13C NMR (75
MHz, CDCl3) δ 192.4, 176.8, 152.4, 140.4, 136.4, 134.6, 129.6,
128.9, 128.4, 113.2, 66.1, 58.3, 53.4, 34.2, 23.1, 17.1.
To a solution of the aldehyde (72 mg, 0.251 mmol) in 3-methyl-
2-butene (4 mL) and t-BuOH (2 mL), a solution of NaClO2 (284
mg, 3.140 mmol) and NaH2PO4 (208 mg, 1.5 mmol) in water (1
mL) was added. The reaction mixture was stirred for 4 h at 25 °C
then acidified to pH = 3 with 10% HCl and extracted with
EtOAc. The organic layers were dried over Na2SO4, filtered and
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (EtOAc/ n-hexane 1:1)
providing compound 15 (45% over 2 steps). 1H NMR (300 MHz,
CDCl3) δ 8.13 (s, 1H), 8.03 (d, 1H, J = 7.5 Hz), 7.67 (d, 1H, J =
7.5 Hz), 7.66-7.42 (m, 2H), 6.86 (s, 1H), 4.19-4.10 (m, 2H), 3.51
(d, 1H, J = 12.9 Hz), 3.16-3.09 (m, 1H), 2.49-2.32 (m, 5H), 1.96-
1.87 (m, 3H); 13C NMR (75 MHz, CDCl3) δ 177.8, 170.7, 152.3,
139.4, 133.5, 130.6, 130.2, 128.9, 128.4, 113.6, 65.8, 58.6, 53.5,
34.4, 23.2, 17.0.
isolated
diastereoisomers
methyl
(R)-5-oxo-1-((R)-1-
phenylethyl)pyrrolidine-3-carboxylate (32%) and methyl (S)-5-
oxo-1-((R)-1-phenylethyl)pyrrolidine-3-carboxylate (43%).
The isolated methyl esters (219 mg, 0.886 mmol) were dissolved
in dry toluene (1.8 mL). Benzyl alcohol (140 µL, 1.328 mmol)
and PPTS (45 mg, 0.179 mmol) were sequentially added and the
reaction mixture was stirred under reflux for 24 h under argon
atmosphere. The reaction mixture was quenched with saturated
aqueous NaHCO3 and extracted with EtOAc. The organic layers
were dried over Na2SO4, filtered and concentrated under reduced
pressure. The residue was purified by silica gel column
chromatography (EtOAc/n-hexane 1:2).
1
18a (73% yield): H NMR (300 MHz, CDCl3) δ 7.40-7.27 (m,
10H), 5.55 (q, 1H, J = 6.9 Hz), 5.12 (s, 2H), 3.64-3.55 (m, 1H),
3.33-3-22 (m, 2H), 2.87-2.69 (m, 2H), 1.58 (d, 3H, J = 7.2 Hz);
13C NMR (75 MHz, CDCl3) δ 172.5 172.0, 139.6, 135.3, 128.7
(2), 128.5 (2), 128.3 (2), 127.7 (2), 127.1 (2), 67.1, 49.2, 44.6,
36.2, 34.4, 16.3.
1
18b (81% yield): H NMR (300 MHz, CDCl3) δ 7.48-7.32 (m,
10H), 5.56 (q, 1H, J = 6.9 Hz), 5.22 (s, 2H), 3.65-3.57 (m, 1H),
3.26-3-15 (m, 2H), 2.89-2.70 (m, 2H), 1.58 (d, 3H, J = 7.2 Hz);
13C NMR (75 MHz, CDCl3) δ 172.7, 171.8, 139.8, 135.4, 128.7
(3), 128.4 (2), 127.7 (2), 127.2 (2), 67.2, 49.2, 44.4, 36.2, 34.6,
16.0.
4.2.13. (R)-3-(4-(4-methylthiazol-2-yl)thiazolidine-3-carbonyl)
benzoic acid (16a).
Compound 16a was prepared from 11d and 7a following the
1
4.2.17. Benzyl (R)-1-((R)-1-phenylethyl)-5-thioxopyrrolidine-
3-carboxylate (19a).
procedure described for 13a H NMR (400 MHz, CDCl3) δ 8.79
(br, 1H), 8.28-8.05 (m, 2H), 7.80-7.32 (m, 2H), 6.86 (s, 1H), 6.10
(br, 1H), 4.84 (d, 2H, J = 8.9 Hz), 3.66-3.56 (m, 2H), 2.59-2.30
(m, 3H); 13C NMR (100 MHz, CDCl3) δ 168.9, 166.0, 153.0,
135.8, 131.7, 130.5, 128.8, 128.3, 113.8, 52.3, 39.6, 17.0.
To a solution of lactam 18a (2 g, 6.184 mmol) in dry toluene (39
mL) Lawesson’s reagent (2.0 g, 4.945 mmol) was added and the
reaction mixture was stirred for 3 h at 100 °C and then
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (EtOAc/n-hexane 1:6)
4.2.14. (R)-3-(2-(4-methyloxazol-2-yl)pyrrolidine-1-carbonyl)
benzoic acid (16b).
1
providing 19a (67%). H NMR (400 MHz, CDCl3) δ 7.39-7.22
Compound 16b was prepared from 11e and 7a following the
(m, 8H), 7.07-6.99 (m, 2H), 6.34 (q, 1H, J = 7.0 Hz), 5.05 (m,
2H), 3.88-3.84 (m, 1H), 3.79-3.74 (m, 1H), 3.55-3.51 (m, 1H),
3.39-3.34 (m, 1H), 3.29-2.20 (m, 1H), 1.59 (d, 3H, J = 7.1 Hz);
13C NMR (100 MHz, CDCl3) δ 198.1, 171.7, 138.0 135.0, 128.8
(2) , 128.4 (2), 128.3, 128.0, 127.2, 126.9, 67.1, 54.1, 53.9, 51.2,
47.5, 37.6, 37.4, 15.2.
1
procedure described for 13a. H NMR (400 MHz, CDCl3) δ 8.38
(s, 1H), 8.13-8.00 (m, 1H), 7.77 (d, 1H, J = 7.5 Hz), 7.48 (t, 1H,
J = 7.7 Hz), 7.34-7.20 (m, 2H), 5.36 (t, 1H, J = 6.5 Hz), 3.89-
3.83 (m, 1H), 3.56-3.50 (m, 1H), 2.46-2.39 (m, 1H), 2.34-2.04
(m, 5H), 1.99-1.91 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 169.3,
168.4, 164.8, 136.2, 135.9, 131.7, 130.8, 130.0, 128.7, 128.4,
127.4, 54.8, 50.1, 31.2, 25.3, 10.9.
4.2.18. Benzyl (S)-1-((R)-1-phenylethyl)-5-thioxopyrrolidine-
3-carboxylate (19b).
4.2.15. (R)-3-(2-(4-methylthiazol-2-yl)piperidine-1-carbonyl)
benzoic acid (16c).
Compound 19b was prepared following the procedure described
1
for 19a (99%). H NMR (400 MHz, CDCl3) δ 7.41-7.29 (m,
Compound 16c was prepared from 11c and 7a following the
procedure described for 13a. 1H NMR (300 MHz, CDCl3) δ
11.45 (br, 1H), 8.24 (s, 1H), 8.12 (d, 1H, J = 7.5 Hz), 7.57-7.48
(m 1H), 6.90 (s 1H), 6.27 (br, 1H), 5.16 (br, 0.5H), 4.69 (br,
0.5H), 3.73-3.61 (m, 1H), 3.39-3.25 (m, 1H), 2.66-2.60 (m, 1H),
2.44 (s, 3H), 2.06-2.00 (m, 1H), 1.78-1.57 (m, 4H); 13C NMR (75
MHz, CDCl3) δ 170.2, 169.6, 168.6, 152.3, 135.5, 131.0 (2),
130.2, 128.5, 127.0 113.9, 60.2, 51.3, 44.9, 28.0, 25.4, 19.6, 16.6.
LRMS-ESI (m/z): 353.0 [M+Na].
10H), 6.34 (q, 1H, J = 7.1 Hz), 5.14 (s, 2H), 3.90-3.85 (m, 1H),
3.43-3.32 (m, 3H), 3.19-3.10 (m, 1H), 1.57 (d, 3H, J = 7.0 Hz);
13C NMR (100 MHz, CDCl3) δ 197.7, 171.9, 138.3, 135.1, 128.7
(2), 128.6 (2), 128.5 (2), 128.3 (2), 128.1, 127.1, 67.2, 54.0, 50.9,
47.6, 37.6, 14.7.
4.2.19.
Benzyl
(R)-1-((R)-1-phenylethyl)pyrrolidine-3-
carboxylate (20a).
To a solution of thioamide 19a (1.44 g, 4.242 mmol), in dry
CH2Cl2 (21 mL) MeI (2.6 mL, 42.40 mmol) was added and the
solution was stirred for 24 h at 25 °C. The reaction mixture was

then concentrated and the residue dissolved in EtOH (7 mL).
NaBH4 (1.4 g, 37.00 mmol) was added portionwise at 0 °C and
the reaction mixture was stirred at 0 °C for 1 h. The reaction was
quenched with water and extracted with CH2Cl2. The organic
layers were dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue was purified by silica gel column
To a solution of amide 21a (254 mg, 1.023 mmol) in THF (5 mL)
Lawesson’s reagent (228 mg, 0.564 mmol) was added
portionwise at 0 °C and the reaction mixture was allowed to stir
at 25 °C for 16 h. The reaction was concentrated under reduced
pressure, the residue was diluted with saturated aqueous NaHCO3
and extracted with diethyl ether. The organic layers were dried
over Na2SO4, filtered and concentrated under reduced pressure.
The residue containing (R)-benzyl 3-carbamothioylpyrrolidine-1-
carboxylate was used for the next step without further
purification. Accordingly, this latter (109 mg, 0.412 mmol) was
dissolved in EtOH (2 mL), chloroacetone (49 µL, 0.618 mmol)
was added at 25 °C and the resulting solution was allowed to stir
at 80 °C for 18 h. The reaction mixture was the cooled to 25 °C
and saturated aqueous NaHCO3 was added. EtOH was
evaporated under reduced pressure and the aqueous layer was
extracted with EtOAc. The organic layers were dried over
Na2SO4, filtered and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography
(EtOAc/ n-hexane 1:3) affording benzyl (R)-3-(4-methylthiazol-2-
yl)pyrrolidine-1-carboxylate. 1H NMR (400 MHz, CDCl3) δ
7.35-7.26 (m, 5H), 6.74 (s, 1H), 5.16-5.09 (m, 2H), 3.95-3.89 (m,
1H), 3.76-3.57 (m, 3H), 3.51-3.44 (m, 1H), 2.39 (s, 3H), 2.37-
2.33 (m, 1H), 2.26-2.15 (m, 1H); 13C NMR (100 MHz, CDCl3) δ
169.6, 154.5, 152.5, 136.7, 136.6, 128.4, 127.9, 127.8 (2), 112.6,
66.8, 51.9/51.4, 45.7/45.3, 42.5/41.7, 32.8/32.0, 17.0. This latter
was submitted to Cbz deprotection as described for amine 11f
1
chromatography (EtOAc/pentane 1:6) providing 20a (38%). H
NMR (300 MHz, CDCl3) δ 7.58-7.32 (m, 10H), 5.27-5.18 (m,
2H), 3.38-3.31 (m, 1H), 3.20-3.09 (m, 1H), 2.97-2.86 (m, 2H),
2.77-2.72 (m, 1H), 2.29-2.17 (m, 2H), 1.50 (d, 3H, J = 7.2 Hz);
13C NMR (75 MHz, CDCl3) δ 174.9, 145.5, 136.2, 128.7 (2),
128.5 (2), 128.3, 128.2 (2), 127.2 (2), 127.1, 66.4, 65.4, 55.9,
52.4, 42.2, 27.7, 23.3.
4.2.20. Benzyl
carboxylate (20b).
(S)-1-((R)-1-phenylethyl)pyrrolidine-3-
Compound 20b was prepared following the procedure described
1
for 20a (50%). H NMR (300 MHz, CDCl3) δ 7.51-7.31 (m,
10H), 5.25 (s, 1H), 3.35 (q, 1H, J = 6.3 Hz), 3.25-3.15 (m, 1H),
3.11-3.05 (m, 1H), 2.81-2.76 (m, 1 H), 2.72-2.54 (m, 2H), 2.27-
2.12 (m, 2H), 1.50 (d, 3H, 6.3 Hz); 13C NMR (75 MHz, CDCl3) δ
175.0, 145.4, 136.2, 128.7 (2), 128.5 (2), 128.3, 128.2 (2), 127.2
(2), 127.1, 66.5, 65.5, 55.6, 52.7, 42.2, 27.7, 23.3.
4.2.21. Benzyl (R)-3-carbamoylpyrrolidine-1-carboxylate
(21a).
To a solution of 20a (500 mg, 1.616 mmol) in MeOH (13 mL)
10% Pd/C (179 mg) was added and the mixture was allowed to
stir for 20 h at 25 °C under H2 atmosphere. The reaction was
filtered on Celite® and the filtrate was concentrated under
reduced pressure quantitatively providing (R)-pyrrolidine-3-
carboxylic acid submitted to the next step without further
purification. To a solution of this latter (460 mg, 3.995 mmol) in
2N NaOH (2 mL) benzylchloroformate (740 µL, 5.194 mmol)
and 4N NaOH (1.5 mL) were added and the mixture was stirred
at 0 °C for 2 h. The reaction was extracted with diethyl ether. The
aqueous layer was adjusted to pH = 2 with 10% HCl at 0 °C and
extracted with EtOAc. The organic layers were dried over
Na2SO4, filtered and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography
1
providing compound 11g. H NMR (400 MHz, CD3OD) δ 6.96
(s, 1H), 3.82-373 (m, 1H), 3.49-3.44 (m, 1H), 3.32-3.26 (m, 1H),
3.19-3.11 (m, 2H), 2.53-2.44 (m, 4H), 2.19-2.06 (m, 1H); 13C
NMR (100 MHz, CD3OD) δ 173.3, 152.2, 112.7, 53.6, 46.5,
43.2, 33.9, 16.4. LRMS-ESI (m/z): 169.0 [M+H].
4.2.24. (S)-4-methyl-2-(pyrrolidin-3-yl)thiazole (11h).
1H NMR and 13C NMR data are identical to those reported for
compound 11g.
4.2.25. (R)-3-(3-(4-methylthiazol-2-yl)pyrrolidine-1-carbonyl)
benzoic acid (22a).
Compound 21a was prepared from 11g and 7a following the
1
procedure described for 13a. H NMR (400 MHz, CDCl3) δ 12.0
(CH2Cl2/MeOH
9:1)
affording
(R)-1-
(br, 1H), 8.20-8.03 (m, 2H), 7.72 (s, 1H), 7.46-7.42 (m, 1H),
6.90-6.83 (m, 1H), 4.20-3.63 (m, 5H), 2.46-2.22 (m, 5H); 13C
NMR (100 MHz, CDCl3) δ 169.4, 169.2, 168.9, 152.4, 136.5,
132.1, 131.9, 131.6, 130.1, 128.7, 113.0, 54.6, 45.7, 42.2, 31.3,
16.2. LRMS-ESI (m/z): 339.0 [M+Na].
((benzyloxy)carbonyl)pyrrolidine-3-carboxylic acid (39%). 1H
NMR (300 MHz, CDCl3) δ 9.17 (br, 1H), 7.44-7.32 (m, 5H), 5.18
(s, 2H), 4.73 (s, 1H), 3.73-3.47 (m, 3H), 3.19-3.09 (m, 1H), 2.24-
2.13 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 177.0, 155.0, 136.6,
129.6, 128.6, 128.1, 127.6, 127.1, 67.2, 48.3/47.9, 45.7/45.3,
43.1/42.3, 28.8/28.2.
4.2.26. (S)-3-(3-(4-methylthiazol-2-yl)pyrrolidine-1-carbonyl)
benzoic acid (22b).
To a solution of this latter (307 mg, 1.231 mmol) in 1,4-
dioxane (4 mL) pyridine (62 µL, 0.763 mmol), di-tert-
butyldicarbonate (370 mg, 1.695 mmol) and NH4HCO3 (127 mg,
1.606 mmol) were sequentially added. The reaction mixture was
stirred at 25 °C for 16 h and then concentrated under reduced
pressure. The residue was diluted with EtOAc. The organic layer
was washed with water, 5% H2SO4 and brine, dried over Na2SO4,
filtered and concentrated under reduced pressure. The residue
containing 21a was used for the next step without further
Compound 22b was prepared from 11h and 7a following the
1
procedure described for 13a H NMR and 13C NMR data are
identical to those reported for compound 22a.
4.2.27. Benzyl (R)-2-(4-(hydroxymethyl)oxazol-2-yl)
pyrrolidine-1-carboxylate (24).
To a solution of ester 23 (1.4 g, 4.238 mmol) in dry THF (10 mL)
LiBH4 (277 mg, 12.72 mmol) was added portionwise at 0 °C and
the reaction mixture was allowed to stir at 25 °C for 4 h. EtOAc
(5 mL) was added and the resulting solution was cooled to °C.
1N HCl (10 mL) and water (20 mL) were then added and the
reaction mixture was extracted with EtOAc. The organic layers
were dried over Na2SO4, filtered and concentrated under reduced
pressure. The residue was purified by silica gel column
chromatography (CH2Cl2/MeOH 97:3) providing compound 24
1
purification. H NMR (300 MHz, DMSO-d6) δ 7.43 (m, 1H),
7.34 (m, 4H), 7.30 (br, 1H), 6.94 (br, 1H), 5.04 (s, 1 H), 3.51-
3.21 (m, 4 H), 2.93-2.87 (m, 1 H), 2.00-1.88 (m, 2 H).
4.2.22. Benzyl (S)-3-carbamoylpyrrolidine-1-carboxylate
(21b)
1
Compound 21b was prepared following the procedure described
for 21a 1H NMR data are identical to those reported for
compound 21a.
(42%). H NMR (300 MHz, CDCl3) δ 7.56-7.21 (m, 5H), 5.25-
5.05 (m, 3H), 4-85-4.80 (m, 1H), 4.61-4.57 (m, 1H), 3.79-3.72
(m, 1H), 3.67-3.55 (m, 1H), 2.32-1.85 (m, 5H); 13C NMR (75
MHz, CDCl3) δ 164.6, 154.5, 140.5, 136.5, 128.5, 128.4, 128.0,
127.9, 127.7, 127.2, 66.9, 55.1, 54.7, 46.9, 32.4, 24.3.
4.2.23. (R)-4-methyl-2-(pyrrolidin-3-yl)thiazole (11g).

4.2.28. (R)-4-(methoxymethyl)-2-(pyrrolidin-2-yl)oxazole
(11f).
ESI (m/z): 701.4 [M+Na]. HRMS-ESI (m/z) [M+Na]+ calculated
for C35H42N4O6S2Na 701.2444, found 701.2438.
To a solution of alcohol 24 (138 mg, 0.456 mmol) in dry THF (4
mL) NaH (60% suspension in mineral oil, 33 mg, 0.825 mmol)
and MeI (34 µL, 0.547 mmol) were sequentially added at 0 °C
and the reaction mixture was allowed to stir at 25 °C for 1 h. The
reaction was quenched with saturated aqueous NH4Cl at 0 °C and
extracted with EtOAc. The organic layers were dried over
Na2SO4, filtered and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography
4.2.32.
N1-((2,5-dimethyloxazol-4-yl)methyl)-N3-((2S,3R)-3-
hydroxy-4-((N-isobutyl-4-methoxyphenyl)sulfonamido)-1-
phenylbutan-2-yl)-N1-methyl-5-(methylamino)isophthalamide
(3e).
1H NMR (400 MHz, CDCl3) δ 7.81-7.60 (m, 3H), 7.50 (s, 1H),
7.38-7.03 (m, 5H), 6.96 (d, 2H, J = 8.7 Hz,), 6.85-6.61 (m, 1H),
6.38 (br, 1H), 4.93 (s, 1H), 4.49 (s, 1H), 4.42-4.15 (m, 2H), 4.16-
3.90 (m, 3H), 3.86 (s, 3H), 3.41-2.66 (m, 8H), 2.44-2.24 (m, 6H),
2.16 (s, 3H), 1.99-1.80 (m, 1H), 0.86 (dd, 6H, J = 12.9 and 6.8
Hz). LRMS-ESI (m/z): 728.5 [M+Na]. HRMS-ESI (m/z)
[M+Na]+ calculated for C37H47N5O7SNa 728.3096, found
728.3088.
(EtOAc/n-hexane
3:2)
affording
benzyl
(R)-2-(4-
1
(methoxymethyl)oxazol-2-yl)pyrrolidine-1-carboxylate (68%). H
NMR (400 MHz, CDCl3) δ 7.50 (s, 1H), 7.41 (s, 1H), 7.40-7.29
(m, 3H), 7.23-7.13 (m, 1H), 5.16-4.94 (m, 3H), 4.31 (d, 2H, J =
17.4 Hz), 3.70-3.65 (m, 1H), 3.58-3.43 (m, 1H), 3.37 (s, 3H),
2.27-2.00 (m, 3H), 1.93-1.88 (m, 1H); 13C NMR (100 MHz,
CDCl3) δ 164.7, 154.7/154.3, 137.7, 136.6/136.4, 136.0/135.7,
128.3, 128.2, 127.8, 127.6, 127.5, 66.9/66.7, 66.3/66.2, 58.4,
54.9/54.6, 46.8/46.4, 32.3/31.2, 24.1/23.3.
4.2.33.
N1-((2,5-dimethyloxazol-4-yl)methyl)-5-hydroxy-N3-
((2S,3R)-3-hydroxy-4-((N-isobutyl-4-methoxyphenyl)
sulfonamido)-1-phenylbutan-2-yl)-N1-methylisophthalamide
(3f).
1H NMR (400 MHz, CDCl3) δ 8.01 (s, 1H), 7.77-7.61 (m, 3H),
7.36-6.85 (m, 8H), 6.72 (br, 1H), 4.64-3.92 (m, 2H), 3.84 (s, 3H),
3.36-2.71 (m, 12H), 2.52-2.24 (m, 3H), 2.20-1.68 (m, 2H), 0.97-
0.72 (m, 6H). LRMS-ESI (m/z): 715.5 [M+Na]. HRMS-ESI (m/z)
[M+Na]+ calculated for C36H44N4O8SNa 715.2778, found
715.2773.
This latter (98 mg, 0.310 mmol) was dissolved in a solution of
HBr in acetic acid (1.0 mL) and the resulting mixture was stirred
at 25 °C for 1 h. The addition of ether (2 mL) caused the amine
salt to precipitate. The solvent was drawn off and the resulting
residue retained. To the etheral solution was added petroleum
ether (4 mL) to give a further precipitate. The solvent was
evaporated under reduced pressure and the two batches of solid
1
were combined and dried to provide amine 11f (67%). H NMR
4.2.34.
N1-((2,5-dimethyloxazol-4-yl)methyl)-N2-((2S,3R)-3-
(400 MHz, CDCl3) δ 7.48 (s, 1H), 4.28 (m, 3H), 3.35 (s, 3H),
3.09-3.03 (m, 1H), 2.96-2.90 (m, 1H), 2.54 (br, 1H), 2.16-2.07
(m, 1H), 2.02-1.94 (m, 1H), 1.89-1.71 (m, 2H); 13C NMR (100
MHz, CDCl3) δ 166.7, 137.2, 136.0, 66.1, 58.3, 55.4, 46.7, 30.7,
25.3.
hydroxy-4-((N-isobutyl-4-methoxyphenyl)sulfonamido)-1-
phenylbutan-2-yl)-N1-methylphthalamide (3g).
1H NMR (400 MHz, CDCl3) δ 7.72 (d, 2H, J = 8.8 Hz,), 7.61-
7.23 (m, 7H), 7.23-7.12 (m, 2H), 7.03 (d, 1H, J = 8.3 Hz,), 6.94
(d, 2H, J = 8.7 Hz), 4.55 (br, 1H), 4.49-4.28 (m, 2H), 4.22 (br,
1H), 4.06 (m, 2H), 3.88-3.79 (m, 3H), 3.24-3.01 (m, 3H), 3.01-
2.82 (m, 4H), 2.72 (s, 3H), 2.38 (s, 3H), 2.12-1.80 (m, 2H), 0.91-
0.79 (m, 6H). LRMS-ESI (m/z): 699.6 [M+Na]. HRMS-ESI (m/z)
[M+H]+ calculated for C36H45N4O7S 677.3009, found 677.2994.
4.2.29. (R)-3-(2-(4-(methoxymethyl)oxazol-2-yl)pyrrolidine-1-
carbonyl)benzoic acid (25).
Compound 25 was prepared from 11f and 7a following the
1
procedure described for 13a. H NMR (400 MHz, CDCl3) δ 9.39
(br, 1H), 8.13-8-08 (m, 1H), 8.00 (s, 1H), 7.79-7.75 (m, 1H), 7.57
(s, 1H), 7.49-7.45 (m, 1H), 5.40 (t, 1H, J = 6.3 Hz), 4.44 (s, 2H),
3.93-3.78 (m, 2H), 3.56-3.51 (m, 1H), 3.37 (s, 3H), 2.44-2.36 (m,
1H), 2.28-2.12 (m, 2H), 1.98-1.90 (m, 1H); 13C NMR (100 MHz,
CDCl3) δ 169.2, 168.6, 164.8, 137.5, 136.5, 136.1, 132.0, 131.7,
131.0, 130.3, 128.7, 66.0, 58.4, 54.8, 50.1, 31.0, 25.2.
4.2.35.
N1-((2,5-dimethyloxazol-4-yl)methyl)-N4-((2S,3R)-3-
hydroxy-4-((N-isobutyl-4-methoxyphenyl)sulfonamido)-1-
phenylbutan-2-yl)-N1-methylterephthalamide (3h).
1H NMR (400 MHz, CDCl3) δ 7.65 (d, 2H, J = 8.9 Hz), 7.62-7.48
(m, 2H), 7.35 (d, 1H, J = 7.8 Hz), 7.31-7.21 (m, 4H), 7.21-7.12
(m, 1H), 6.91 (d, 2H, J = 8.9 Hz), 6.75-6.57 (m, 1H), 4.49 (s,
1H), 4.42-4.25 (m, 2H), 4.13 (s, 1H), 3.98 (dd, 1H, J = 7.5 and
3.6 Hz,), 3.83 (s, 3H), 3.23-2.89 (m, 6H), 2.84 (d, 2H, J = 7.5
Hz), 2.35 (m, 5H), 2.02 (s, 1H), 1.90-1.80 (m, 1H), 0.83 (dd, 6H,
J = 6.5 and 3.3 Hz). LRMS-ESI (m/z): 699.6 [M+Na]. HRMS-
ESI (m/z) [M+Na]+ calculated for C36H44N4O7SNa 699.2829,
found 699.2820.
4.2.30. N1-((2S,3R)-3-hydroxy-4-((N-isobutyl-4-
methoxyphenyl) sulfonamido)-1-phenylbutan-2-yl)-N3-
methyl-N3-((2-methyloxazol-4-yl)methyl)isophthalamide (3c).
1H NMR (400 MHz, CDCl3) δ 8.08 (m, 1H), 8.17-8.04 (m, 1H),
8.01-7.91 (m, 1H), 7.77 (d, 1H, J = 7.8 Hz,), 7.73-7.61 (m, 3H),
7.43 (t, J = 7.7 Hz, 2H), 7.47-7.04 (m, 4H), 7.34-7.02 (m, 2H),
7.10-6.75 (m, 2H), 4.56 (s, 1H), 4.46 (d, J = 5.4 Hz, 1H), 4.42-
4.21 (m, 2H), 4.07-3.94 (m, 1H), 3.89-3.78 (m, 3H), 3.21-2.70
(m, 5H), 2.49 2.35 (m, 2H), 2.10 (br, 1H), 1.92-1.78 (m, 2H),
1.66-1.50 (m, 1H), 0.84 (dd, J = 6.4, 1.9 Hz, 6H). LRMS-ESI
(m/z): 685.5 [M+Na]. HRMS-ESI (m/z) [M+Na]+ calculated for
C35H42N4O7SNa 685.2672, found 685.2650.
4.2.36. N-((2S,3R)-3-hydroxy-4-((N-isobutyl-4-
methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)-3-((S)-2-
(4-methylthiazol-2-yl)pyrrolidine-1-carbonyl)benzamide (4a).
To a solution of 3-[(S)-2-(4-methylthiazol-2-yl)pyrrolidine-1-
carbonyl]benzoic acid 13b (40 mg, 0.13 mmol), EDCI (37.4 mg,
0.20 mmol) HOBt (26.4 mg, 0.20 mmol) in anhydrous CH2Cl2 (3
mL) a solution of amine 6a (52 mg, 0.13 mmol) and
diisopropylethylamine (0.14 mL, 0.78 mmol) in anhydrous
CH2Cl2 (1 mL) were added at 0 °C under argon atmosphere and it
was allowed to stir for 16 h at 23 °C. The reaction mixture was
quenched with water and extracted with CH2Cl2. The organic
layers were dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (CH2Cl2/MeOH 9:1) to furnish inhibitor 4a
4.2.31. N1-((2S,3R)-3-hydroxy-4-((N-isobutyl-4-
methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)-N3-methyl-
N3-((2-methylthiazol-4-yl)methyl)isophthalamide (3d).
1H NMR (300 MHz, CDCl3) δ 8.07 (s, 1H), 7.99 (s, 1H), 7.96 (s,
1H), 7.77-7.59 (m, 4H), 7.49-7.40 (m, 1H), 7.27-7.16 (m, 3H),
6.97-6.82 (m 3H), 6.54 (d, 1H, J = 7.8 Hz), 4.96 (s, 1H), 4.88 (d,
1H, J = 5.4 Hz), 4,38-4.18 (m, 2H), 4.03-3.97 (m, 1H), 3.86 (s,
3H), 3.17-2.98 (m, 4H), 2.87 (d, 2H, J = 6.6 Hz), 2.47-2.39 (m,
2H), 1.91-1.83 (m, 1H), 0.86 (dd, J = 6.4, 1.9 Hz, 6H). LRMS-
1
(95%) as a white solid. H NMR (400 MHz, CDCl3) δ 7.81 (s,

1H NMR (400 MHz, CDCl3) δ 7.79 (s, 1H), 7.63 (d, 2H, J = 8.7
Hz), 7.60 (d, 1H, J = 7.7 Hz), 7.35 (t, 1H, J = 7.7 Hz), 7.30-7.11
(m, 6H), 6.90 (d, 2H, J = 8.7 Hz), 6.73 (s, 1H), 5.60 (dd, 1H, J =
7.3 and 4.9 Hz), 4.38-4.31 (m, 1H), 4.01-3.96 (m, 1H), 3.80 (s,
3H), 3.63-3.58 (m, 1H), 3.41-3.35 (m, 1H), 3.20 (dd, 1H, J = 3.0
and 15.0 Hz), 3.12-2.96 (m, 3H), 2.91-2.79 (m, 2H), 2.38 (s, 3H),
2.36-2.25 (m, 3H), 2.07-1.99 (m, 1H), 1.91-1.81 (m, 4H), 0.83
(dd, 6H, J = 6.3 and 2.8 Hz); 13C NMR (100 MHz, CDCl3) δ
171.7, 169.3, 167.1, 162.8, 152.6, 137.9, 136.6, 134.4, 130.1,
129.7, 129.3 (2), 128.6 (2), 128.4 (2), 126.3, 125.7, 114.2 (2),
112.8, 72.7, 58.7, 58.4, 55.5, 54.5, 53.3, 50.1, 35.0, 32.3, 27.0,
25.7, 24.9, 20.0, 19.9, 17.0. LRMS-ESI (m/z): 757.6 [M+K].
HRMS-ESI (m/z) [M+Na]+ calculated for C38H46N4O6S2Na
741.2757, found 741.2749.
1H), 7.64-7.59 (m, 3H), 7.36 (t, 1H, J = 7.7 Hz), 7.29-7.09 (m,
7H), 6.91 (d, 2H, J = 8.7 Hz), 6.72 (s, 1H), 5.59 (dd, 1H, J = 7.4
and 4.8 Hz), 4.38-4.32 (m, 2H), 4.02-3.97 (m, 1H), 3.81 (m, 4H),
3.67-3.62 (m, 1H), 3.43-3.37 (m, 1H), 3.24-3.19 (m, 1H), 3.15-
2.98 (m, 3H), 2.92-2.80 (m, 2H), 2.38 (s, 3H), 2.35-2.27 (m, 2H),
2.08-1.98 (m, 1H), 1.90-1.81 (m, 2H), 0.83 (dd, 6H, J = 6.5 and
3.2 Hz); 13C NMR (100 MHz, CDCl3) δ 171.7, 171.1, 169.2,
167.1, 162.8, 152.5, 138.0, 136.7, 134.3, 130.2, 129.8, 129.3 (2),
129.2, 128.6, 128.4 (2), 126.4, 125.8, 114.3 (2), 112.9, 72.6, 58.8,
58.5, 55.5, 54.7, 53.2, 50.1, 34.8, 32.4, 27.0, 24.9, 20.0, 19.9,
17.1. LRMS-ESI (m/z): 727.6 [M+Na]. HRMS-ESI (m/z) [M+H]+
calculated for C37H45N4O6S2 705.2781, found 705.2784.
4.2.37. N-((2S,3R)-3-hydroxy-4-((N-isobutyl-4-
methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)-3-((R)-2-
(4-methylthiazol-2-yl)pyrrolidine-1-carbonyl)benzamide (4b).
1H NMR (400 MHz, CDCl3) δ 7.79 (s, 1H), 7.63 (d, 2H, J = 8.7
Hz), 7.60 (d, 1H, J = 7.7 Hz), 7.35 (t, 1H, J = 7.7 Hz), 7.30-7.11
(m, 6H), 6.90 (d, 2H, J = 8.7 Hz), 6.73 (s, 1H), 5.60 (dd, 1H, J =
7.3 and 4.9 Hz), 4.38-4.31 (m, 1H), 4.01-3.96 (m, 1H), 3.80 (s,
3H), 3.63-3.58 (m, 1H), 3.41-3.35 (m, 1H), 3.20 (dd, 1H, J =
15.0 and 3.0 Hz), 3.12-2.96 (m, 3H), 2.91-2.79 (m, 2H), 2.38 (s,
3H), 2.36-2.25 (m, 2H), 2.07-1.99 (m, 1H), 1.91-1.81 (m, 2H),
0.83 (dd, 6H, J = 6.4 and 2.8 Hz); 13C NMR (100 MHz, CDCl3) δ
171.7, 169.3, 167.1, 162.8, 152.6, 137.9, 136.6, 134.4, 130.1,
129.7, 129.3, 128.6, 128.4, 126.3, 125.7, 114.2, 112.8, 72.7, 58.7,
58.4, 55.5, 54.5, 53.3, 50.1, 35.0, 32.3, 27.0, 24.9, 20.0, 19.9,
17.0. LRMS-ESI (m/z): 705.7 [M+H]. HRMS-ESI (m/z) [M+Na]+
calculated for C37H44N4O6S2Na 727.2600, found 727.2592.
4.2.41. N-((2S,3R)-3-hydroxy-4-((N-isobutyl-4-
methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)-3-((R)-4-
(4-methylthiazol-2-yl)thiazolidine-3-carbonyl)benzamide (4f).
1H NMR (400 MHz, CDCl3) δ 7.80 (s, 1H), 7.75-7.58 (m, 3H),
7.41 (br, 1H), 7.26-7.21 (m, 4H), 7.19-7.15 (m, 1H), 6.94 (d, 2H,
J = 8.6 Hz), 6.82-6.76 (m, 2H), 6.13 (br, 1H), 4.63-4.58 (m, 1H),
4.38-4.24 (m, 2H), 4.05-3.98 (m, 1H), 3.83 (s, 3H), 3.49 (m, 2H),
3.19-3.03 (m, 5H), 2.86 (d, 2H, J = 7.4 Hz), 2.40 (s, 3H), 1.90-
1.82 (m, 1H), 0.85 (m, 6H); 13C NMR (100 MHz, CDCl3) δ
168.8, 166.8, 162.9, 153.0, 137.8, 135.8, 134.5, 129.7, 129.3 (3),
129.2 (3), 128.9, 128.5 (2), 126.5, 125.8, 114.3 (2), 113.0, 72.7,
60.3, 58.6, 55.6, 54.5, 53.4, 34.9, 27.1, 21.0, 20.0, 19.9, 17.0.
LRMS-ESI (m/z): 745.5 [M+Na]. HRMS-ESI (m/z) [M+Na]+
calculated for C36H42N4O6S3Na 745.2165, found 745.2161.
4.2.42. N-((2S,3R)-3-hydroxy-4-((N-isobutyl-4-
4.2.38. N-((2S,3R)-4-((4-amino-N-isobutylphenyl)
methoxyphenyl) sulfonamido)-1-phenylbutan-2-yl)-3-((R)-2-
(4-(methoxymethyl)oxazol-2-yl)pyrrolidine-1-carbonyl)
benzamide (4g).
sulfonamido)-3-hydroxy-1-phenylbutan-2-yl)-3-((R)-2-(4-
methylthiazol-2-yl)pyrrolidine-1-carbonyl)benzamide (4c).
1H NMR (400 MHz, CDCl3) δ 7.79 (s, 1H), 7.62-7.53 (m, 2H), 7-
39-7.32 (m, 2H), 7.27-7.12 (m, 6H), 6.88 (d, 1H, J = 8.6 Hz),
6.74 (s, 1H), 6.49 (d, 2H, J = 8.6 Hz), 5.61 (dd, 1H, J = 7.5 and
4.8 Hz), 4.35-4.30 (m, 3H), 4.03-3.97 (m, 1H), 3.63-3.57 (m,
1H), 3.42-3.36 (m, 1H), 3.17-3.08 (m, 2H), 3.00-2.84 (m, 3H),
2.76-2.71 (m, 1H), 2.40-2.24 (m, 5H), 2.06-1.99 (m, 1H), 1.92-
1.82 (m, 1H), 0.85 (m, 6H); 13C NMR (100 MHz, CDCl3) δ
171.7, 169.4, 167.0, 152.6, 151.1, 137.9, 136.7, 134.5, 130.0,
129.3 (3), 128.7 (3), 128.4 (2), 126.4, 125.9, 125.0, 113.9 (2),
112.9, 72.8, 58.8, 54.3, 53.6, 50.1, 35.4, 32.4, 29.6, 27.0, 24.9,
20.0 (2), 17.1. LRMS-ESI (m/z): 690.7 [M+H]. HRMS-ESI (m/z)
[M+Na]+ calculated for C36H43N5O5S2Na 712.2604, found
712.2601.
1H NMR (400 MHz, CDCl3) δ 7.82 (s, 1H), 7.74-7.69 (m, 3H),
7.52 (s, 1H), 7.39-7.30 (m, 1H), 7.26-7.13 (m, 6H), 6.96-6.85 (m,
3H), 5.37 (dd, 1H, J = 7.3 and 4.9 Hz), 4.36-4.30 (m, 3H), 4.11
(m, 1H), 4.02-3.97 (m, 1H), 3.82 (s, 3H), 3.68-2.62 (m, 1H),
3.36-3.40 (m, 1H), 3.38 (s, 3H), 3.19-2.97 (m, 4H), 2.92-2.80 (m,
2H), 2.38-2.28 (m, 1H), 2.22-2.04 (m, 2H), 1.95-1.83 (m, 2H),
0.84 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 168.9, 167.0,
164.3, 162.9, 137.9, 137.7, 136.5, 136.0, 134.4, 134.2, 130.3,
129.7, 129.3 (3), 128.7, 128.6, 128.4 (2), 126.4, 125.8, 114.2 (2),
72.7, 66.2, 58.6, 58.4, 55.5, 54.6, 54.4, 53.4, 49.9, 35.0, 30.8,
27.0, 25.1, 20.0, 19.9. LRMS-ESI (m/z): 741.7 [M+Na]. HRMS-
ESI (m/z) [M+Na]+ calculated for C38H46N4O8SNa 741.2935,
found 741.2932.
4.2.39.
N-((2S,3R)-3-hydroxy-4-((4-(hydroxymethyl)-N-
4.2.43. N-((2S,3R)-3-hydroxy-4-((N-isobutyl-4-
isobutylphenyl)sulfonamido)-1-phenylbutan-2-yl)-3-((R)-2-(4-
methylthiazol-2-yl)pyrrolidine-1-carbonyl)benzamide (4d).
1H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.72-7.56 (m, 3H),
7.37-7.14 (m, 9H), 6.95 (d, 1H, J = 8.6 Hz), 6.75 (s, 1H), 5.57
(dd, 1H, J = 7.5 and 4.9 Hz), 4.68-4.58 (m, 2H), 4.31-4.23 (m,
1H), 4.02-3.95 (m, 1H), 3.64-3.57 (m, 1H), 3.42-3.36 (m, 1H),
3.21-2.72 (7H), 2.38-2.24 (m, 5H), 2.06-1.99 (m, 1H), 1.92-1.86
(m, 2H), 0.86 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 171.7,
169.6 167.1, 152.6, 146.9, 137.9, 136.5, 136.4, 134.4, 129.9,
129.3 (2), 128.7 (2), 128.5 (2), 127.3 (2), 126.9 (2), 126.4, 125.9,
112.9, 72.8, 63.6, 58.8, 58.5, 54.4, 53.3, 50.2, 35.3, 32.5, 26.9,
24.9, 20.0 (2), 17.0. LRMS-ESI (m/z): 705.6 [M+H]. HRMS-ESI
(m/z) [M+Na]+ calculated for C37H44N4O6S2Na 727.2600, found
727.2595.
methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)-3-((R)-2-
(4-methyloxazol-2-yl)pyrrolidine-1-carbonyl)benzamide (4h).
1H NMR (400 MHz, CDCl3) δ 7.85 (s, 1H), 7.75-7.64 (m, 3H),
7.40-7.18 (m, 8H), 6.97-6.90 (m, 2H), 6.82 (d, 1H, J = 7.0 Hz),
5.36-5.33 (m, 1H), 4.39-4.32 (m, 1H), 4.02-3.97 (m, 1H), 3.84-
3.82 (m, 3H), 3.68-3.61 (m, 1H), 3.49-3.44 (m, 1H), 3.24-2.95
(m, 4H), 2.86 (d, 2H, J = 7.1 Hz), 2.36-2.31 (m, 1H), 2.18-2.09
(m, 4H), 1.97 (br, 1H), 1.93-1.82 (m, 2H), 0.85 (m, 6H); 13C
NMR (100 MHz, CDCl3) δ 168.9, 167.0, 163.7, 162.9, 137.9
136.6, 136.3, 134.1, 130.3, 129.7 (2), 129.3 (3), 128. 7 (2), 128.5
(3), 126.4, 125.8, 114.2 (2), 72.7, 58.6, 55.5, 54.6, 54.4, 53.4,
49.9, 35.0, 30.9, 27.1, 25.1, 20.0, 19.9, 14.1. LRMS-ESI (m/z):
711.6 [M+Na]. HRMS-ESI (m/z) [M+Na]+ calculated for
C37H44N4O7SNa 711.2829, found 711.2833.
4.2.44. N-((2S,3R)-3-hydroxy-4-((N-isobutyl-4-
methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)-3-(((R)-2-
(4-methylthiazol-2-yl)pyrrolidin-1-yl)methyl)benzamide (4i).
4.2.40. N-((2S,3R)-3-hydroxy-4-((N-isobutyl-4-
methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)-3-((R)-2-
(4-methylthiazol-2-yl)piperidine-1-carbonyl)benzamide (4e).

1H NMR (400 MHz, CDCl3) δ 7.66 (d, 2H, J = 8.8 Hz), 7.60 (s,
1H), 7.51-7.45 (m, 2H), 7.34-7.25 (m, 5H), 7.21-7.17 (m, 1H),
6.92 (d, 2H, J = 8.8 Hz), 6.78 (s, 1H), 6.55 (d, 1H, J = 8.2 Hz),
4.41-4.34 (m, 1H), 4.04-3.96 (m, 3H), 3.84 (s, 3H), 3.36 (d, 1H, J
= 13.0 Hz), 3.23-2.98 (m, 5H), 2.89-2.83 (m, 2H), 2.40 (s, 3H),
2.40-2.24 (m, 3H), 1.92-1.76 (m, 4H), 0.86 (dd, 6H, J = 6.5 and
1.8 Hz); 13C NMR (100 MHz, CDCl3) δ 176.9, 168.1 162.9,
152.4, 139.8, 137.8, 133.9, 131.9, 129.7, 129.4 (4), 128.5 (3),
127.0 126.5, 125.5, 114.2 (2), 113.3, 72.8, 66.0, 58.7, 58.4, 55.5,
54.5, 53.4, 34.9, 34.2, 29.6, 27.1, 23.1, 20.0, 19.9, 17.1. LRMS-
ESI (m/z): 691.7 [M+H]. HRMS-ESI (m/z) [M+H]+ calculated for
C37H47N4O5S2 691.2988, found 691.2983.
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, 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. We thank Mr.
Emilio Cardenas (Purdue University) for helpful discussions.
The authors would like to thank the Purdue University Center for
Cancer Research, which supports the shared NMR and mass
spectrometry facilities.
4.2.45. N-((2S,3R)-3-hydroxy-4-((N-isobutyl-4-
methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)-5-((R)-2-
(4-methylthiazol-2-yl)pyrrolidine-1-carbonyl)nicotinamide
(4j).
1H NMR (400 MHz, CDCl3) δ 8.82 (s, 1H), 8.78 (s, 1H), 8.09 (s,
1H), 7.65 (d, 2H, J = 8.7 Hz), 7.23-7.09 (m, 6H), 6.92 (d, 2H, J =
8.7 Hz), 6.76 (s, 1H), 5.64-5.58 (m, 1H), 4.39-4.33 (m, 1H),
4.10-4.06 (1H), 3.82 (s, 3H), 3.71-3.62 (m, 1H), 3.45-3.38 (m,
1H), 3.24-3.05 (m, 3H), 2.96-2.81 (m, 3H), 2.43-2.28 (m, 4H),
2.19-2.01 (m, 2H), 1.97-1.85 (m, 2H), 0.89-0.78 (m, 6H); 13C
NMR (100 MHz, CDCl3) δ 171.1, 166.9, 165.2, 162.9, 152.7,
150.1, 149.3, 138.0, 133.9, 131.9, 129.9 129.7, 129.3 (2), 129.2,
128.6, 128.4 (2), 126.4, 114.3 (2), 113.0, 72.7, 59.0, 58.5, 55.6,
54.6, 53.3, 50.1, 32.4, 29.6, 27.0, 24.9, 20.0, 19.9, 17.1. LRMS-
ESI (m/z): 728.6 [M+Na]. HRMS-ESI (m/z) [M+H]+ calculated
for C36H44N5O6S2 706.2733, found 706.2723.
Supplementary data
Supplementary data associated with this article can be found in
the online version.
References and notes
1. Inhibitor, Dabigatran Etexilate, as an Anticoagulant Drug"
Chapter 15 in: Structure-based Design of Drugs and Other
Bioactive Molecules: Tools and Strategies; Wiley-VCH;
2014:337-354.
2. Hubbard, R. E. Structure-based Drug Discovery: An Overview
RSC Publishing 2006.
4.2.46. N-((2S,3R)-3-hydroxy-4-((N-isobutyl-4-
3. Ghosh, A. K.; Osswald, H. L.; Prato, G. J. Med. Chem. 2016, 59,
5172-5208.
4. Hue, S.; Gifford, R. J.; Dunn, D.; Fernhill, E.; Pillay, D. J. Virol.
2009, 83, 2645-2654.
5. Cohen, M. S.; Chen, Y.Q.; McCauley, M. N. Engl. J. Med. 2011,
365, 493-505.
6. Diffenbach, C. W.; Fauci, A. S. Ann Intern Med. 2011, 154, 766-
771.
7. Gupta, R.; Hill, A.; Sawyer, A. W.; Pillay, D., Clin. Infect. Dis.
methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)-3-((R)-3-
(4-methylthiazol-2-yl)pyrrolidine-1-carbonyl)benzamide (4k).
1H NMR (400 MHz, CDCl3) δ 7.76 (s, 1H), 7.67-7.58 (m, 3H),
7.43-7.38 (m, 1H), 7.26-7.14 (m, 5H), 6.8-6.88 (m, 3H), 6.79-
6.70 (m, 2H), 4.40-4.33 (m, 1H), 4.13-4.09 (m, 1H), 4.03-3.97
(m, 1H), 3.85-3.64 (m, 6H), 3.58-3.48 (m, 2H), 3.19-3.03 (m,
4H), 2.87-2.79 (m, 2H), 2.42-2.23 (m, 3H), 1.92-1.84 (m, 2H),
0.93-0.85 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 168.7, 168.5,
167.1, 162.9, 152.7, 137.8, 136.9, 134.3, 130.1, 129.7, 129.3 (2),
129.2 (2), 128.7 (2), 128.5 (2), 126.5, 125.6, 114.3 (2), 112.9,
72.7, 58.7, 55.6, 54.6, 53.4, 45.7, 42.8, 41.2, 34.9, 29.6, 27.1,
20.0, 19.9, 16.9. LRMS-ESI (m/z): 743.6 [M+K]. HRMS-ESI
(m/z) [M+Na]+ calculated for C37H44N4O6S2Na 727.2600, found
727.2604.
2008, 47, 712-722.
8. Patel, K.; Hernán, M. A.; Williams, P. L.; Seeger, J. D.; McIntosh,
K.; Van Dyke, R. B.; Seage III, G. R. Clin. Infect. Dis. 2008, 46,
507-515.
9. Pennigs, P. S. Infect. Dis. Rep. 2013, 5, 21-25.
10. Wilhelmsson, P.; Lindgreen, P. E. The Lancet. 2016, 16, 512-513.
11. Cihlar, T.; Fordyce, M. Curr. Opin. Virol. 2016, 18, 50-56.
12. Ghosh, A. K.; Anderson, D. D.; Weber, I. T.; Mitsuya, H Angew.
Chem. Int. Ed. 2012, 51, 1778-1802.
13. Ghosh, A. K. J. Med. Chem. 2009, 52, 2163-2176.
14. Ghosh, A. K.; Chapsal, B. D. Design of the anti-HIV protease
inhibitor darunavir. In ‘From Introduction to Biological and Small
Molecule Drug Research and Development’ Ed. Ganellin, C. R.;
Roberts, S. M.; Jefferis, R. 2013, 355-384.
15. Ghosh, A. K.; Sridhar, P. R.; Kumaragurubaran, N.; Koh, Y.;
Weber, I. T.; Mitsuya, H. ChemMedChem 2006, 1, 939-950.
16. Ghosh, A. K. J. Org. Chem. 2010, 75, 7967-7989.
17. Ghosh, A. K.; Chapsal, B.; Mitsuya, H. Darunavir, a New PI with
Dual Mechanism: From a Novel Drug Design Concept to New
Hope Against Drug-Resistant HIV. In ‘Aspartic Acid Proteases as
Therapeutic Targets’ Ed. Ghosh, A. K. Wiley-VCH Verlag GmbH
& Co. KGaA: Weinheim, 2010, 205-243.
4.2.47. N-((2S,3R)-3-hydroxy-4-((N-isobutyl-4-
methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)-3-((S)-3-
(4-methylthiazol-2-yl)pyrrolidine-1-carbonyl)benzamide (4l).
1H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.67-7.59 (m, 4H),
7.42-7.37 (m, 1H), 7.27-7.14 (m, 5H), 6.93 (d, 2H, J = 8.8 Hz),
6.80-6.75 (m, 2H), 4.38-4.31 (m, 2H), 4.03-3.98 (m, 1H), 3.90-
3.77 (m, 5H), 3.75-3.66 (m, 2H), 3.58-3.49 (m, 1H), 3.19-3.04
(m, 4H), 2.86 (d, 2H, J = 7.4 Hz), 2.47-2.20 (m, 4H), 1.90-1.84
(m, 2H), 0.91-0.84 (m, 6H); 13C NMR (100 MHz, CDCl3) δ
168.7, 168.6, 167.1, 162.9, 152.6, 137.8, 136.9, 134.3, 130.1,
129.7, 129.3 (2), 129.2 (2), 128.7 (2), 128.5 (2), 126.5, 125.6,
114.2 (2), 112.8, 72.7, 58.7, 55.5, 54.6, 53.4, 45.7, 42.8, 41.2,
34.9, 29.6, 27.1, 20.0, 19.9, 16.9. LRMS-ESI (m/z): 727.6
[M+Na]. HRMS-ESI (m/z) [M+Na]+ calculated for
C37H44N4O6S2Na 727.2600, found 727.2596.
18. Ghosh, A. K.; Chapsal, B.; Weber, I. T.; Mitsuya, H. Acc. Chem.
Res. 2008, 41, 78-86.
19. Ghosh, A. K.; Sridhar, P. R.; Leshchenko, S.; Hussain, A. K.; Li,
J.; Kovalevsky, A. Y.; Walters, D. E.; Wedekind, J. E.; Grum-
Tokars, V.; Das, D.; Koh, Y.; Maeda, K.; Gatanaga, H.; Weber, I.
T.; Mitsuya, H. J. Med. Chem. 2006, 49, 5252-5261.
20. Ghosh, A. K.; Xu, C.-X.; Rao, K. V.; Baldridge, A.; Agniswamy,
J.; Wang, Y.-F.; Weber, I. T.; Aoki, M.; Miguel, S. G. P.; Amano,
M.; Mitsuya, H. ChemMedChem 2010, 5, 1850-1854.
21. Ghosh, A. K.; Dawson, Z. L.; Mitsuya, H. Bioorg. Med. Chem.
2007, 15, 756-7580.
Acknowledgement: This research was supported by the National
Institutes of Health (Grant GM53386, AKG and Grant
GM62920, ITW). X-ray data were collected at the Southeast
Regional Collaborative Access Team (SER-CAT) beamline
22BM at the Advanced Photon Source, Argonne National

22. Koh, Y.; Nakata, H.; Maeda, K.; Ogata, H.; Bilcer, G.;
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. Antimicrob. Agent Chemother 2003, 47, 3123-
3129.
23. Ghosh. A. K.; Schiltz, G. E.; Rusere, L. N.; Osswald, H. L.
Walters, D. E.; Amano, M.; Mitsuya, H. Org. Biomol. Chem.
2014, 12, 6842-6854.
24. Ghosh, A. K.; Swanson, L. M.; Cho, H.; Hussain, K. A.;
Leschenko, S.; Kay, S.; Walters, D. E.; Mitsuya, H. J. Med. Chem.
2005, 48, 3576.
25. Kaldor, S. W; Kalish, V. J.; Davies, J. F.; Shetty, B. V.; Fritz, J.
E.; Appelt, K.; Burgess, J. A.; Campanale, K. M.; Chirgadze, N.
Y.; Clawson, D. K.; Dressman, B. A.; Hatch, S. D.; Khalil, D. A.;
Kosa, M. B.; Lubbehusen, P. P.; Muesing, M. A.; Patick, A. K.;
Reich, S. H.; Su, K. S.; Tatlock, J. H. J. Med. Chem. 1997, 40,
3979-3985.
26. Ghosh, A. K.; Takayama, J.; Kassekert, L. A.; Ella-Menye, J. R.;
Yashchuk, S.; Agniswamy, J.; Wang, Y. F.; Aoki, M.; Amano,
M.; Weber, I. T.; Mitsuya, H. Bioorg. Med. Chem. Lett. 2015, 25,
4903-4909.
27. Ghosh, A.K.; Kumaragurubaran, N.; Hong, L.; Kulkarni, S.; Xu,
X.; Miller, H. B.; Reddy, D. S.; Weerasena, V.; Turner, R.; Chang,
W.; Koelsch, G.; Tang, J. Bioorg. Med. Chem. Lett. 2008, 18,
1031-1036.
28. Ghosh, A. K.; Kumaragurubaran, N.; Hong, L.; Kulkarni, S. S.;
Xu, X.; Chang, W.; Weerasena, V.; Turner, R.; Koelsch, G.;
Bilcer, G.; Tang, J. J. Med. Chem. 2007, 50, 2399-2407.
29. Horn, R. K.; Gailunas, M.; Fang, L. Y.; Tung, J. S.; Walker, D.
D.; Thorsett, E. D.; Jewette, N. E.; Moon, J. B.; Varghese, J. J.
Med. Chem. 2004, 47, 158-164.
30. Beck, J. P.; Mergott, D. J. Peptidic, peptidomimetic, and HTS-
derived BACE Inhibitors. In BACE Lead Target for Orchestrated
Therapy of Alzheimer’s Disease, Ed. John, V., Chapter 4, 59-105.
Wiley, NJ, 2010.
31. Ghosh, A. K.; Liu, C.; Devasamudram, T.; Lei, H.; Swanson, L.
M.; Ankala, S. V.; Lilly, J. C.; Bilcer, G. M. Preparation of (3-
hydroxy-4-amino-butan-2-yl)-3-[2-(thiazol-2-yl)pyrrolidine-1-
carbonyl]benzamide derivatives and related compounds as
selective beta-secretase inhibitors. PCT Int Appl WO2009042694,
2009.
32. Ghosh, A. K. Aryldicarboxamide derivatives as non-peptide HIV-
1
protease inhibitors and their preparation, pharmaceutical
compositions and use in the treatment of HIV, AIDS, and AIDS-
related diseases. PCT Int Appl WO2010002994, 2010.
33. Lindgren, Bengt O.; Nilsson, Torsten; Husebye, Steinar; Mikalsen,
ØYvind; Leander, Kurt; Swahn, Carl-Gunnar (1973). "Preparation
of Carboxylic Acids from Aldehydes (Including Hydroxylated
Benzaldehydes) by Oxidation with Chlorite". Acta Chem. Scan.
1973, 27: 888-890.
34. Culbertson, T. P.; Domagala, J. M.; Nichols, J. B.; Priebe, S.;
Skeean, R. W., J. Med. Chem. 1987, 30, 1711-1715.
35. Tie, Y.; Boross, P. I.; Wang, Y. F.; Gaddis, L.; Hussain, A. K.;
Leshchenko, S.; Ghosh, A. K.; Louis, J. M.; Harrison, R.W.;
Weber, I.T. J.Mol.Biol. 2004, 338, 341-352.
36. Toth, M. V.; Marshall, G. R. Protease. Int. J. Pept. Protein Res.
1990, 36, 544-550.
37. Koh, Y.; Amano, M.; Towata, T.; Danish, M.; Leshchenko-
Yashchuk, S.; Das, D.; Nakayama, M.; Tojo, Y.; Ghosh, A. K.;
Mitsuya, H. J. Virol. 2010, 84, 11961-11969.
38. The coordinates and structure factors for the structure of HIV-1
protease with inhibitors 3b and 4b have been deposited in Protein
Data Bank with codes: 5UPZ and 5UOV.
39. For details of X-ray studies, please see Supplementary
information.