Structure-guided design and optimization of dipeptidyl inhibitors of norovirus 3CL protease. Structure-activity relationships and biochemical, X-ray crystallographic, cell-based, and in vivo studies

Article abstract of DOI:10.1021/jm5019934

Norovirus infection constitutes the primary cause of acute viral gastroenteritis. There are currently no vaccines or norovirus-specific antiviral therapeutics available for the management of norovirus infection. Norovirus 3C-like protease is essential for viral replication, consequently, inhibition of this enzyme is a fruitful avenue of investigation that may lead to the emergence of antinorovirus therapeutics. We describe herein the optimization of dipeptidyl inhibitors of norovirus 3C-like protease using iterative SAR, X-ray crystallographic, and enzyme and cell-based studies. We also demonstrate herein in vivo efficacy of an inhibitor using the murine model of norovirus infection.

Full text of DOI:10.1021/jm5019934

Article
pubs.acs.org/jmc
Structure-Guided Design and Optimization of Dipeptidyl Inhibitors
of Norovirus 3CL Protease. Structure−Activity Relationships and
Biochemical, X‑ray Crystallographic, Cell-Based, and In Vivo Studies
Anushka C. Galasiti Kankanamalage,† Yunjeong Kim,‡ Pathum M. Weerawarna,† Roxanne Adeline Z. Uy,†
Vishnu C. Damalanka,† Sivakoteswara Rao Mandadapu,† Kevin R. Alliston,† Nurjahan Mehzabeen,§
Kevin P. Battaile,∥ Scott Lovell,§ Kyeong-Ok Chang,*,‡ and William C. Groutas*,†
†Department of Chemistry, Wichita State University, 1845 North Fairmount Avenue, Wichita, Kansas 67260, United States
‡Department of Diagnostic Medicine & Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas
66506, United States
§Protein Structure Laboratory,The University of Kansas, Lawrence, Kansas 66047, United States
∥IMCA-CAT, Hauptman-Woodward Medical Research Institute, APS Argonne National Laboratory, Argonne, Illinois 60439, United
States
ABSTRACT: Norovirus infection constitutes the primary
cause of acute viral gastroenteritis. There are currently no
vaccines or norovirus-specific antiviral therapeutics available
for the management of norovirus infection. Norovirus 3C-like
protease is essential for viral replication, consequently,
inhibition of this enzyme is a fruitful avenue of investigation
that may lead to the emergence of antinorovirus therapeutics.
We describe herein the optimization of dipeptidyl inhibitors of
norovirus 3C-like protease using iterative SAR, X-ray crystallo-
graphic, and enzyme and cell-based studies. We also
demonstrate herein in vivo efficacy of an inhibitor using the
murine model of norovirus infection.
precursor is processed by a virus-encoded 3C-like protease
(3CLpro) to generate six mature nonstructural proteins,
including the viral protease (3CLpro or NS6Pro) and the
RNA dependent RNA polymerase (NS7Pol).12 Co- and post-
translational processing of the polyprotein by norovirus
3CLpro is essential for virus replication, consequently,
norovirus 3CLpro has emerged as a potential druggable target
for the discovery of antinorovirus small molecule therapeutics
and prophylactics.13,14
Norovirus 3CLpro is a chymotrypsin-like cysteine protease
with a Cys-His-Glu catalytic triad and an extended binding
site.11,15 The primary substrate specificity of the protease is for
a P1 glutamine residue and a strong preference for a −D/E-F-
X-L-Q-G-P− sequence (X is H, Q, or V), corresponding to the
subsites S5-S4-S3-S2-S1-S1′-S2′−, respectively.15,16 Cleavage is at
the P1−P1′ (Q-G) scissile bond. We have recently reported an
array of norovirus inhibitors, including acyclic and cyclic
sulfamide17−19 and piperazine20 derivatives. We have also
disclosed for the first time peptidyl transition state (TS)
inhibitors,13a−e TS mimics,13f as well as macrocyclic inhib-
itors13g effective in enzyme and cell-based assays. We have
furthermore described the first high throughput FRET assay of
INTRODUCTION
■
Human noroviruses are the primary cause of sporadic and
epidemic acute gastroenteritis in the U.S. and worldwide,1−3
consequently, they constitute an important public health
problem, as well as a potential bioterrorism threat. Although
the illness is generally considered to be mild and self-limiting, it
can incapacitate infected individuals, including military troops
on ships or war zones during the symptomatic phase.4
Noroviruses are very stable in the environment and refractory
to many common disinfectants, with only a few virions required
to initiate virus infection and shedding, which could be a source
for further contamination. Therefore, norovirus outbreaks are
hard to contain using routine sanitation, and even implementa-
tion of aggressive sanitary measures often fails to prevent
subsequent outbreaks.5,6 The problem is further compounded
by the current dearth of diagnostics, effective vaccines, and
norovirus-specific antiviral therapeutics and/or prophylac-
tics.7−9
Human noroviruses are single-stranded, positive sense RNA
viruses belonging to the Caliciviridae family.10 Genogroups I, II,
and IV of the six genogroups (GI-GVI) in the genus Norovirus
are known to infect humans. The norovirus genome (7−8 kb)
consists of three open reading frames that encode a 200 kDa
polyprotein (ORF1), a major capsid protein VP1 (ORF2), and
a small basic protein VP2 (ORF3).10,11 The mature polyprotein
Received: January 7, 2015
© XXXX American Chemical Society
A
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3CLpro from GI and GII noroviruses as a screening tool for
identifying potential protease inhibitors and have determined
high resolution X-ray crystal structures of Norwalk virus (NV, a
prototype strain of norovirus) 3CLpro in complex with peptidyl
transition state inhibitors,13c as well as the first solution
structure of the protease using high-field NMR.13h Finally, we
have demonstrated proof-of-concept using the mouse model of
murine norovirus (MNV) infection (vide infra).
complex showed that, under the crystallization conditions used,
the bisulfite reverted to the precursor aldehyde which
subsequently formed a tetrahedral adduct with the active site
cysteine (Cys139) (Figure 3). Inspection of the cocrystal
In continuing our foray in this area, we describe herein the
structure-based optimization of a series of dipeptidyl inhibitors
of NV 3CLpro represented by structure (I) (Figure 1) using an
array of X-ray crystallographic, structure−activity relationship,
biochemical, cell-based, and animal studies using the mouse
model of murine norovirus (MNV) infection.
Figure 3. Hydrogen bonding interactions between the inhibitor and
chain B for NV 3CLpro:ligand (precursor aldehyde of inhibitor 44)
Side chain residues are colored cyan, inhibitor is colored gray, and
water molecules are represented as red spheres. Hydrogen bonds are
represented as dashed lines and water mediated contacts as solid lines.
Figure 1. General structoure of dipeptidyl inhibitor (I).
RESULTS AND DISCUSSION
■
structure revealed opportunities for additional binding
interactions with a more efficient use of chemical structure.
Specifically, one such opportunity was recognized by observing
that there is a particular stretch of residues spanning Ala159,
Ala160, Thr161, and Lys162 that are within 4.0 Å from the
benzyl ring (Figure 4). Thus, it was envisaged that the
Inhibitor Design Rationale. We initially focused on the
design of peptidyl transition state inhibitors of NV 3CLpro that
incorporate in their structure a recognition element (a peptidyl
fragment) that is congruent with the known substrate specificity
of the enzyme (vide supra) and a warhead (aldehyde or α-
ketoamide), latent warhead (bisulfite adduct), or transition
state mimic (α-hydroxyphosphonate). In the case of inhibitors
incorporating an aldehyde or α-ketoamide functionality in their
structure, interaction with the active site cysteine (Cys139)
leads to the formation of a reversible adduct (Figure 2).17
Figure 4. Residues spanning Ala159-Lys162 located near the benzyl
ring of precursor aldehyde of inhibitor 44.
incorporation of an appropriate functional group into the
phenyl ring could serve as a locus for the formation of a
hydrogen bond with the Thr161 or Lys162 backbone and/or
side chains, stabilizing the flexible benzyl segment and
increasing binding affinity. The formation of a halogen bond
also appeared plausible.22 Thus, a small focused library of
compounds was synthesized to evaluate these hypotheses, the
ultimate goal being the identification of a dipeptidyl lead
candidate suitable for conducting preclinical studies.
Chemistry. The synthesis of compounds 13−44 is outlined
in Scheme 1. Refluxing cyclohexylalanine methyl ester hydro-
chloride (or leucine methyl ester hydrochloride) with
trichloromethyl chloroformate yielded the corresponding
isocyanate which was reacted with an appropriately substituted
benzyl alcohol to yield a carbamate adduct methyl ester that
was hydrolyzed to the corresponding acid with lithium
Figure 2. Interaction of a cysteine protease (E-Cys-SH) with a
peptidyl transition state inhibitor.
Furthermore, in previous studies, we demonstrated that
norovirus 3CLpro shows a strong preference for a P2
cyclohexylalanine and, consequently, a P2 cyclohexyl alanine
residue, as well as a glutamine surrogate,21 were incorporated in
the structures of the inhibitors. The key binding interactions
between norovirus 3CLpro and inhibitor were revealed by
determining the high resolution X-ray crystal structure of NV
3CLpro with bound inhibitor (I) (R1 = cyclohexylmethyl, R2 =
H, X = CH(OH)SO3Na). The cocrystal structure of the
B
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a
Scheme 1. Synthesis of inhibitors 13−44
a
(a) CH3OH, SOCl2, 45 °C, 3 h; (b) CCI3O(CO)Cl, dioxane, reflux, 12 h; (c) ArCH2OH, TEA, acetonitrile, reflux, 2 h; (d) 1 M LiOH(aq), THF,
RT, 3 h; (e) EDCI, HOBt, DIEA, DMF; (f) 2 M LiBH4, THF, CH3OH; (g) Dess−Martin periodinane, DCM; (h) diethylphosphite, DCM, DIEA;
(i) C2H5OH, EtOAc, NaHSO3; (j) EtOAc, HOAc, cyclopropyl isocyanide, K2CO3(aq), CH3OH, 18 h.
hydroxide in aqueous THF. Subsequent coupling with
glutamine surrogate methyl ester hydrochloride21 afforded the
desired dipeptidyl ester, which was then reduced to the
corresponding alcohol with lithium borohydride. Dess−Martin
oxidation followed by flash chromatography purification yielded
pure dipeptidyl aldehyde. The enantiomeric purity of the
aldehyde was consistently high, with the amount of epimerized
aldehyde ranging between 0 and 10%, depending on the
structure of the dipeptidyl aldehyde. Further reaction of the
aldehyde with diethyl phosphite in the presence of diisopropyl
ethyl amine yielded the corresponding α-hydroxyphosphonate
as a mixture of epimers.23 The corresponding bisulfite adducts
were readily obtained as white solids by stirring the aldehydes
with sodium bisulfite in an ethyl acetate/water mixture.24
Reaction of the aldehyde with cyclopropyl isonitrile followed by
Dess−Martin oxidation of the α-hydroxy cyclopropyl amide
yielded the desired α-ketoamides. The synthesized compounds
are listed in Table 1.
The synthesized compounds were evaluated for their
inhibitory activity against NV 3CLpro, as well as their
antinorovirus activity in a cell-based replicon system, as
previously described.13 The IC50 and ED50 values are the
average of at least two determinations and are listed in Table 1.
The enzyme selectivity of a select number of inhibitors was
evaluated against a panel of representative proteases, and the
results are summarized in Table 2. The methodologies
employed in conducting the enzyme assays and inhibition
studies were as described previously by us13 and others.25 The
in vivo efficacy of compound 16 was evaluated in the murine
model of norovirus infection.
X-ray crystallography was used to elucidate the nature of the
interaction of compound 17 with NV 3CLpro. The X-ray
crystal structure of NV 3CLpro revealed the presence of
prominent difference electron density with the substructure of
17 that is equivalent to precursor aldehyde inhibitor 16
covalently bound to Cys 139. However, no electron density was
observed for the hydroxyphosphonate group that should be
present for inhibitor 17 (Figure 5). Instead, the structure of the
NV 3CLpro−ligand complex was found to correspond to the
covalent adduct of precursor aldehyde inhibitor 16 and NV
3CLpro. In addition, the m-chlorobenzyl ring was partially
disordered so the electron density for this region of the
inhibitor was somewhat ambiguous. The interactions between
NV 3CLpro and inhibitor 16 are shown in Figure 6. The m-
chlorophenyl ring of inhibitor 16 occupies a hydrophobic
pocket near Ile 109 and Val 168. van der Waals and
electrostatic surface representations of the inhibitor and NV
3CLpro are shown in Figure 7.
Inspection of the results shown in Table 1 reveals that, in
general, dipeptidyl inhibitors that incorporate in their structure
an aldehyde or aldehyde bisulfite adduct (masked aldehyde)
display lower ED50 values than the corresponding compounds
bearing an α-hydroxyphosphonate or α-ketoamide moiety.
Furthermore, the aldehyde and aldehyde bisulfite adduct-
derived inhibitors had comparable ED50 values. These results
are consistent with previous studies which showed that the
activity of the aldehyde bisulfite adducts parallels the activity of
C
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Table 1. Activity of Compounds 13−44 against NV 3CL Protease and Norovirus in Cell-Based Replicon Cells
compd
R2
o-Cl
R1
X
IC50 (μM)
ED50 (μM)
13
14
15
Cha
CHO
0.8
>10
0.7
0.08
0.2
CH(OH)P(O)(OC2H5)2
CH(OH)(SO3−Na+)
0.07
16
17
18
19
20
21
22
23
m-Cl
Cha
CHO
0.1
>10
0.1
0.02
0.06
0.02
0.085
CH(OH)P(O)(OC2H5)2
CH(OH)(SO3−Na+)
C(O)C(O)NH(Cyc-Prop)
C(O)NHSO2CH3
CHO
0.25
>10
5.1
>2
Chg
Leu
0.15
0.1
CHO
0.9
C(O)C(O)NH(Cyc-Prop)
5.5
0.3
24
p-Cl
o-F
Cha
Cha
CHO
0.71
0.08
0.08
25
26
CHO
0.9
CH(OH)P(O)(OC2H5)2
>10
>2
27
28
m-F
Cha
Cha
CHO
1.2
3.5
0.09
0.3
C(O)C(O)NH(Cyc-Prop)
29
30
31
32
m-Br
CHO
0.3
0.03
0.15
0.03
0.2
CH(OH)P(O)(OC2H5)2
CH(OH)(SO3−Na+)
C(O)C(O)NH(Cyc-Prop)
6.5
0.15
1.5
33
34
m-I
Cha
CHO
CH(OH)(SO3−Na+)
0.35
0.15
0.05
0.04
35
36
37
38
39
o-OCH3
m-OCH3
m-CN
Cha
Cha
Cha
Cha
Cha
Leu
CHO
CHO
CHO
CHO
CHO
>10
1.4
0.1
0.15
0.2
>10
4.5
m-NHBoc
m-NO2
H
1.5
0.6
0.15
40
41
42
43
44
CHO
0.6
0.8
3.4
0.3
0.4
0.2
CH(OH)(SO3−Na+)
C(O)C(O)NH(Cyc-Prop)
CHO
0.3
1.1
Cha
0.06
0.06
CH(OH)(SO3−Na+)
Table 2. Selectivity of Selected Compounds against a Panel
of Proteases
potent than the corresponding cyclohexyl glycine or leucine
inhibitors (compare compounds 16, 21, and 22). As revealed
by the X-ray crystal structure (Figure 7), the cyclohexyl glycine
side chain optimally fills the hydrophobic S2 subsite, while the
leucine and t-butyl alanine side chains do not. A range of ring
substituents were explored to enhance pharmacological activity.
Compounds with a halogen at the meta position were
particularly effective (compounds 16, 27, 29, and 33). These
observations are suggestive of the involvement of a halogen
bond between the halogen and the CO of Ala160, however,
this could not be demonstrated with absolute certainty because
of the partially disordered m-chlorobenzyl moiety.
compound (% inhibition)
enzyme25
HNE
I/E
16
17
29
30
[I]f (μM)
50
250
250
250
250
250
250
15
0
86.5
2.4
8.8
0
26.5
0
63
0
15.4
2.5
chymotrypsin
trypsin
5
0
1
125
2.75
1.23
2.5
thrombin
0
0
0
factor Xa
1
0
0
6
plasmin
0
15
7
13
0
carboxypeptidase A
12
14.3
11.6
43
To evaluate the specific interactions between the enzyme and
the α-hydroxyphosphonate inhibitors, inhibitor 17 was
incubated with NV 3CLpro in Tris buffer, pH 8.0. Surprisingly,
the precursor aldehydes.13d,f Other things being equal,
inhibitors having a cyclohexyl alanine P2 residue are more
D
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and hydrophobic interactions in the enzyme−ligand complex
are near identical to those observed with aldehyde inhibitor (I),
where R1 = cyclohexylmethyl, R2 = H, and X = CHO (Figure
3).
An initial evaluation of selectivity was carried out using a
panel of proteases and four representative inhibitors (16, 17,
29, and 30). As shown in Table 2, these compounds displayed
minimal activity toward the proteases of the digestive and blood
coagulation cascade systems. Selectivity was uniformly lower
against the serine protease human neutrophil elastase (HNE).
Compound 16 was used to obtain a preliminary indication of
in vivo efficacy. All compounds that were effective against NV
in cell culture were also effective against MNV-1 in the range
0.08−5 μM. Among them, compound 16 was found to be the
most effective with an EC50 value of 80 nM. It is noted that
compound 16 was also most effective against NV (Table 1). In
mice, treatment with compound 16 significantly reduced the
virus titers in the small and large intestines at 3 days post virus
infection by 42.12- and 7.98-fold, respectively, compared to the
untreated control group (Figure 8). The overall virus titers in
the small intestine were higher than those in the large intestine,
and the reduction of viral titers was also greater in the small
intestine than the large intestine.
Figure 5. Fo − Fc map (green mesh) of precursor aldehyde derived
from compound 17 bound to NV 3CLpro contoured at 3σ.
CONCLUSION
■
In conclusion, these studies describe the optimization of the
pharmacological activity and selectivity of dipeptidyl inhibitors
of norovirus 3CL protease by employing iterative medicinal
chemistry/SAR studies, X-ray crystallography, in vitro and cell-
based screening, and in vivo efficacy studies. Importantly, these
studies have identified lead compounds that show efficacy in
the murine model of norovirus infection and, consequently, are
suitable for conducting further preclinical studies.
Figure 6. Hydrogen bond interactions between NV 3CLpro and
precursor aldehyde derived from compound 17. Hydrogen bonds are
represented as dashed lines, and water mediated contacts are shown as
solid lines.
EXPERIMENTAL SECTION
■
General. Reagents and dry solvents were purchased from various
chemical suppliers (Aldrich, Acros Organics, Chem-Impex, TCI
America, and Bachem) and were used as obtained. Silica gel (230−
450 mesh) used for flash chromatography was purchased from Sorbent
Technologies (Atlanta, GA). Thin layer chromatography was
performed using Analtech silica gel plates. Visualization was
accomplished using UV light and/or iodine. NMR spectra were
recorded in CDCl3 or DMSO-d6 using a Varian XL-400 spectrometer.
Melting points were recorded on a Mel-Temp apparatus and are
uncorrected. High resolution mass spectrometry (HRMS) was
performed at the University of Kansas Mass Spectrometry Lab using
an LCT Premier mass spectrometer (Waters, Milford, MA) equipped
with a time-of-flight mass analyzer and an electrospray ion source. The
purity of the compounds was established using HPLC and was >95%.
Synthesis of Amino Acid Methyl Esters 1. General Procedure.
To a 250 mL RB flask (oven-dried and purged with nitrogen) was
added absolute methanol (30 mL), and the solution was cooled to 0
°C in an ice bath while kept under a nitrogen atmosphere. Thionyl
chloride (8 mL) was added to the cooled methanol with stirring,
followed by the addition of the amino acid (100 mmol). The ice bath
was replaced by a water bath, and the reaction mixture was heated to
∼50 °C for 3 h with stirring. Removal of the solvent left a white
residue which was washed with diethyl ether (250 mL) and collected
by vacuum filtration to yield the amino acid methyl ester hydro-
chloride 1 as a white solid.
Figure 7. Surface representation of precursor aldehyde of compound
17 bound to NV 3CLpro. Neighboring residues of NV 3CLpro are
colored yellow (nonpolar), white (weakly polar), and cyan (polar).
the enzyme−inhibitor complex formed corresponded to the
complex formed between aldehyde 16 and 3CL protease. The
formation of this complex indicates initial nonenzymatic
(buffer)- or enzyme-catalyzed conversion of α-hydroxyphosph-
onate 17 to the corresponding aldehyde 16, followed by the
formation of a tetrahedral adduct with the active site cysteine
(Cys139). Studies aimed at ascertaining the validity of this
hypothesis are in progress. The ensemble of hydrogen bonding
Synthesis of Amino Acid Methyl Ester Isocyanates 2. General
Procedure. Amino acid methyl ester hydrochloride (100 mmol) was
placed in a dry 500 mL RB flask and then dried overnight on the
vacuum pump. The flask was flushed with nitrogen, and dry dioxane
(200 mL) was added followed by trichloromethyl chloroformate
(29.67 g, 150 mmol), and the reaction mixture was refluxed for 10 h.
E
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Figure 8. Protease inhibitor suppresses norovirus replication in the intestinal tract in mice (effects of a 3CLpro inhibitor treatment on murine
norovirus titers in the intestinal tracts of mice). Balb/c mice were orally infected with MNV-1 at 2 × 106 TCID50 and intraperitoneally given
compound 16 at 10 mg/kg twice daily with the first dose starting 4 h prior to virus infection. At 72 h post virus infection, the intestinal tract tissues
were harvested and processed for determination for virus titers by the TCID50 assay. Mean and the standard error of the mean of virus titers in the
small intestine (A) or large intestine (B) at 3 day post MNV-1 infection are shown. Each filled circle indicates a control mouse that was given drug
vehicle, and each empty box indicates a mouse given compound 16. Asterisk indicates P < 0.01.
The solvent was removed on the rotary evaporator, and the residue
was vacuum distilled to yield pure isocyanate 2 as a colorless oil.
Synthesis of Substituted Benzyl Carbamates 3. General
Procedure. A solution of substituted benzyl alcohol (20 mmol) in dry
acetonitrile (15 mL) was treated with triethylamine (4.05 g, 40 mmol),
followed by the amino acid methyl ester isocyanate (20 mmol). The
resulting solution was refluxed for 2 h and then allowed to cool to
room temperature. The solution was concentrated and the residue was
taken up in ethyl acetate (75 mL). The organic layer was washed with
5% HCl (2 × 20 mL) and brine (20 mL). The organic layer was dried
over anhydrous sodium sulfate, filtered, and concentrated, leaving a
colorless oil (compound 3).
Synthesis of Acids 4. General Procedure. A solution of ester 3
(20 mmol) in tetrahydrofuran (30 mL) was treated with 1 M LiOH
(40 mL). The reaction mixture was stirred for 3 h at room
temperature, and the disappearance of the ester was monitored by
TLC. Most of the solvent was evaporated off, and the residue was
diluted with water (25 mL). The solution was acidified to pH ∼3 using
5% hydrochloride acid (20 mL), and the aqueous layer was extracted
with ethyl acetate (3 × 100 mL). The combined organic layers were
dried over anhydrous sodium sulfate, filtered, and concentrated to
yield compound 4 as a colorless oil.
Synthesis of Compounds 5. General Procedure. To a solution
of compound 4 (10 mmol) in dry DMF (20 mL) was added EDCI
(2.40 g, 12.5 mmol, 1.25 equiv), HOBt (1.92 g, 12.5 mmol, 1.25
equiv), and the mixture was stirred for 30 min at room temperature. In
a separate flask, a solution of deprotected glutamine surrogate 12 (2.23
g, 10 mmol) in DMF (15 mL) cooled to 0−5 °C was treated with
diisopropylethylamine (DIEA) (9.5 g, 40 mmol, 4 equiv), stirred for
30 min, and then added to the reaction mixture containing acid. The
reaction mixture was stirred for 12 h while monitoring the reaction by
TLC. The solvent was removed, and the residue was partitioned
between ethyl acetate (200 mL) and 10% citric acid (2 × 40 mL). The
ethyl acetate layer was further washed with saturated aqueous
NaHCO3 (40 mL), followed by saturated NaCl (50 mL). The organic
layer was dried over anhydrous sodium sulfate, filtered, and
concentrated to yield a yellow-colored oily product. Purification by
flash chromatography yielded ester 5 as a white solid.
Synthesis of Alcohols 6. General Procedure. To a solution of
ester 5 (5 mmol) in anhydrous THF (30 mL) was added lithium
borohydride (2 M in THF, 7.5 mL, 15 mmol) dropwise, followed by
absolute ethyl alcohol (15 mL), and the reaction mixture was stirred at
room temperature overnight. The reaction mixture was then acidified
by adding 5% HCl and the pH adjusted to ∼2. Removal of the solvent
left a residue which was taken up in ethyl acetate (100 mL). The
organic layer was washed with brine (25 mL), dried over anhydrous
sodium sulfate, filtered, and concentrated to yield compound 6 as a
white solid.
Synthesis of Aldehydes 7. General Procedure. Compound 6 (5
mmol) was dissolved in anhydrous dichloromethane (50 mL) under a
nitrogen atmosphere and cooled to 0 °C. Dess−Martin periodinane
reagent (3.18 g, 7.5 mmol, 1.5 equiv) was added to the reaction
mixture with stirring. The ice bath was removed and the reaction
mixture was stirred at room temperature for 3 h (monitoring by TLC
indicated complete disappearance of the starting material). A solution
of 10% aqueous sodium thiosulfate (20 mL) was added, and the
solution was stirred for another 15 min. The aqueous layer was
removed, and the organic layer was washed with 10% aqueous sodium
thiosulfate (20 mL), followed by saturated aqueous sodium
bicarbonate (2 × 20 mL), water (2 × 20 mL), and brine (20 mL).
The organic layer was dried over anhydrous sodium sulfate, filtered,
and concentrated. The yellow residue was purified by flash
chromatography (silica gel/methylene chloride/ethyl acetate/meth-
anol) to yield a white solid 7.
2-Chlorobenzyl ((S)-3-Cyclohexyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-ox-
opyrrolidin-3-yl)propan-2-yl)amino)propan-2-yl)carbamate (13).
Yield (78%), mp 58−60 °C. 1H NMR (400 MHz, DMSO-d6) δ
9.41 (s, 1H), 8.47−8.52 (m, 1H), 7.57−7.69 (m, 1H), 7.44−7.51 (m,
2H), 7.32−7.40 (m, 2H), 5.74−5.77 (m, 1H), 5.02−5.19 (m, 2H),
4.06−4.22 (m, 2H), 3.00−3.17 (m, 2H), 2.19−2.31 (m, 1H), 2.08−
2.17 (m, 1H), 1.84−1.94 (m, 1H), 1.54−1.76 (m, 8H), 1.39−1.52 (m,
3H), 1.08−1.19 (m, 2H), 0.81−0.93 (m, 2H). HRMS (ESI) calcd for
C24H32ClN3O5Na [M + Na]+, 500.1928; found, 500.1914.
3-Chlorobenzyl ((S)-3-Cyclohexyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-ox-
opyrrolidin-3-yl)propan-2-yl)amino)propan-2-yl)carbamate (16).
1
Yield (86%), mp 54−56 °C. H NMR (400 MHz, CDCl3) δ 9.46
(s, 1H), 8.35 (d, J = 5.47 Hz, 1H), 7.33 (br s, 1H), 7.27−7.31 (m,
1H), 7.22−7.27 (m, 1H), 7.16−7.21 (m, 1H), 6.36−6.49 (m, 1H),
5.60 (d, J = 8.20 Hz, 1H), 5.05 (s, 2H), 4.17−4.41 (m, 2H), 3.15−3.38
(m, 2H), 2.24−2.50 (m, 2H), 1.84−2.06 (m, 2H), 1.74−1.83 (m, 2H),
1.45−1.73 (m, 6H), 1.30−1.43 (m, 1H), 1.02−1.27 (m, 3H), 0.76−
1.01 (m, 2H). HRMS (ESI) calcd for C24H32ClN3O5Na [M + Na]+,
500.1928; found, 500.1917.
3-Chlorobenzyl ((S)-1-Cyclohexyl-2-oxo-2-(((S)-1-oxo-3-((S)-2-ox-
opyrrolidin-3-yl)propan-2-yl)amino)ethyl)carbamate (21). Yield
1
(81%), mp 51−53 °C. H NMR (400 MHz, CDCl3) δ 9.49 (s, 1H),
8.49−8.56 (m, 1H), 7.38 (br s, 1H), 7.27−7.30 (m, 2H), 7.23 (d, J =
4.30 Hz, 1H), 5.71−5.78 (m, 1H), 5.44−5.51 (m, 1H), 5.08 (d, J =
13.28 Hz, 2H), 4.25−4.33 (m, 1H), 4.14−4.21 (m, 1H), 3.37 (d, J =
9.37 Hz, 2H), 2.36−2.52 (m, 2H), 1.95 (d, J = 6.64 Hz, 1H), 1.88 (br
s, 1H), 1.76 (d, J = 10.94 Hz, 2H), 1.68 (br s, 2H), 1.58 (s, 4H), 1.21−
F
DOI: 10.1021/jm5019934
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1.34 (m, 2H), 1.13 (d, J = 12.89 Hz, 2H). HRMS (ESI) calcd for
C23H30ClN3O5Na [M + Na]+, 486.1772; found, 486.1758.
1H), 1.08−1.30 (m, 3H), 0.85−1.04 (m, 2H). HRMS (ESI) calcd for
C25H35N3O6Na [M + Na]+, 496.2424; found, 496.2432.
3-Methoxybenzyl ((S)-3-Cyclohexyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-
3-Chlorobenzyl ((S)-4-Methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxo-
oxopyrrolidin-3-yl)propan-2-yl)amino)propan-2-yl)carbamate (36).
pyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (22).
1
1
Yield (76%), mp 60−62 °C. H NMR (400 MHz, CDCl3) δ 9.48 (s,
Yield (82%), mp 53−55 °C. H NMR (400 MHz, CDCl3) δ 9.45
1H), 8.32 (d, J = 5.95 Hz, 1H), 7.22−7.26 (m, 1H), 6.88−6.95 (m,
2H), 6.84 (d, J = 8.24 Hz, 1H), 6.48 (br s, 1H), 5.56 (d, J = 8.39 Hz,
1H), 5.08 (s, 2H), 4.22−4.44 (m, 2H), 3.80 (s, 3H), 3.24−3.36 (m,
2H), 2.29−2.51 (m, 2H), 1.87−2.08 (m, 2H), 1.76−1.86 (m, 1H),
1.59−1.75 (m, 6H), 1.46−1.58 (m, 1H), 1.38 (br s, 1H), 1.06−1.28
(m, 3H), 0.82−1.04 (m, 2H). HRMS (ESI) calcd for C25H35N3O6Na
[M + Na]+, 496.2424; found, 496.2418.
(s, 1H), 8.51−8.54 (m, 1H), 7.90−7.94 (m, 1H), 7.36−7.43 (m, 1H),
7.13−7.33 (m, 3H), 5.26−5.32 (m, 1H), 5.02−5.15 (m, 2H), 4.25−
4.37 (m, 2H), 3.29−3.41 (m, 2H), 2.39−2.47 (m, 1H), 1.83−1.99 (m,
2H), 1.66−1.78 (m, 3H), 1.57 (m, 2H), 0.97 (d, J = 6.16 Hz, 6H).
HRMS (ESI) calcd for C21H28ClN3O5Na [M + Na]+, 460.1615; found,
460.1611.
4-Chlorobenzyl ((S)-3-Cyclohexyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-ox-
3-Cyanobenzyl ((S)-3-Cyclohexyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-ox-
opyrrolidin-3-yl)propan-2-yl)amino)propan-2-yl)carbamate (24).
1
opyrrolidin-3-yl)propan-2-yl)amino)propan-2-yl)carbamate (37).
Yield (67%), mp 56−58 °C. H NMR (400 MHz, CDCl3) δ 9.48
1
Yield (73%), mp 58−60 °C. H NMR (400 MHz, CDCl3) δ 9.45
(s, 1H), 8.31 (d, J = 5.47 Hz, 1H), 7.27−7.35 (m, 4H), 6.01 (br s,
1H), 5.34 (d, J = 8.20 Hz, 1H), 5.03−5.11 (m, 2H), 4.28−4.39 (m,
2H), 3.35 (t, J = 8.20 Hz, 2H), 2.35−2.51 (m, 2H), 1.90−1.98 (m,
1H), 1.79−1.90 (m, 2H), 1.60−1.76 (m, 5H), 1.53 (ddd, J = 5.47,
8.98, 14.06 Hz, 1H), 1.23−1.30 (m, 2H), 1.08−1.22 (m, 2H), 0.81−
1.03 (m, 3H). HRMS (ESI) calcd for C24H32ClN3O5Na [M + Na]+,
500.1928; found, 500.1917.
(s, 1H), 8.53 (d, J = 5.47 Hz, 1H), 7.65−7.76 (m, 2H), 7.54−7.62 (m,
1H), 7.43−7.50 (m, 1H), 6.24−6.50 (m, 1H), 5.60−5.75 (m, 1H),
5.02−5.35 (m, 2H), 4.45−4.56 (m, 1H), 4.24−4.41 (m, 1H), 3.18−
3.43 (m, 2H), 2.29−2.59 (m, 2H), 2.07−2.25 (m, 2H), 1.49−1.99 (m,
6H), 1.30−1.46 (m, 1H), 1.08−1.23 (m, 4H), 0.83−1.01 (m, 3H).
HRMS (ESI) calcd for C25H32N4O5Na [M + Na]+, 491.2270; found,
491.2258.
2-Fluorobenzyl ((S)-3-Cyclohexyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-ox-
3-((tert-Butoxycarbonyl)amino)benzyl ((S)-3-Cyclohexyl-1-oxo-1-
opyrrolidin-3-yl)propan-2-yl)amino)propan-2-yl)carbamate (25).
1
(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)propan-
Yield (81%), mp 61−63 °C. H NMR (400 MHz, CDCl3) δ 9.49
1
2-yl)carbamate (38). Yield (65%), mp 106−110 °C. H NMR (400
(s, 1H), 8.35−8.53 (m, 1H), 8.24−8.30 (m, 1H), 7.40 (t, J = 7.23 Hz,
1H), 7.27−7.34 (m, 1H), 7.10−7.17 (m, 1H), 7.05 (t, J = 9.18 Hz,
1H), 5.82 (br s, 1H), 5.27−5.34 (m, 1H), 5.14−5.25 (m, 2H), 4.25−
4.43 (m, 2H), 3.26−3.44 (m, 2H), 2.31−2.57 (m, 2H), 1.93−2.00 (m,
2H), 1.79−1.91 (m, 2H), 1.47−1.76 (m, 5H), 1.38 (d, J = 2.73 Hz,
1H), 1.07−1.29 (m, 3H), 0.84−1.03 (m, 2H). HRMS (ESI) calcd for
C24H32FN3O5Na [M + Na]+, 484.2224; found, 484.2214.
MHz, CDCl3) δ 9.52 (s, 1H), 9.41−9.46 (m, 1H), 8.40−8.46 (m, 1H),
7.32−7.38 (m, 1H), 7.24 (br s, 1H), 7.22 (s, 1H), 7.20 (s, 1H), 6.92−
6.98 (m, 1H), 5.42−5.51 (m, 1H), 4.98 (s, 2H), 4.22−4.41 (m, 2H),
3.30 (d, J = 7.20 Hz, 2H), 2.22−2.50 (m, 2H), 2.03 (s, 1H), 1.86−1.94
(m, 1H), 1.76 (d, J = 8.09 Hz, 3H), 1.56−1.72 (m, 4H), 1.48 (s, 9H),
1.30−1.40 (m, 1H), 1.04−1.25 (m, 4H), 0.91 (br s, 2H). HRMS (ESI)
calcd for C29H42N4O7Na [M + Na]+, 581.2951; found, 581.2957.
3-Nitrobenzyl ((S)-3-Cyclohexyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxo-
pyrrolidin-3-yl)propan-2-yl)amino)propan-2-yl)carbamate (39).
3-Fluorobenzyl ((S)-3-Cyclohexyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-ox-
opyrrolidin-3-yl)propan-2-yl)amino)propan-2-yl)carbamate (27).
1
Yield (66%), mp 53−55 °C. H NMR (400 MHz, CDCl3) δ 9.48
1
Yield (70%), mp 52−54 °C. H NMR (400 MHz, CDCl3) δ 9.45
(s, 1H), 8.40 (d, J = 5.08 Hz, 1H), 7.27−7.35 (m, 1H), 7.06−7.14 (m,
1H), 6.99 (dt, J = 2.34, 8.59 Hz, 2H), 5.97 (br s, 1H), 5.41 (d, J = 8.20
Hz, 1H), 5.06−5.14 (m, 2H), 4.25−4.41 (m, 2H), 3.29−3.41 (m, 2H),
2.27−2.54 (m, 2H), 1.90−2.00 (m, 1H), 1.79−1.89 (m, 2H), 1.60−
1.78 (m, 6H), 1.48−1.58 (m, 1H), 1.32−1.46 (m, 1H), 1.08−1.30 (m,
3H), 0.85−1.04 (m, 2H). HRMS (ESI) calcd for C24H32FN3O5Na [M
+ Na]+, 484.2224; found, 484.2207.
(s, 1H), 8.67 (d, J = 5.08 Hz, 1H), 8.30 (s, 1H), 8.16 (d, J = 8.20 Hz,
1H), 7.66 (d, J = 7.81 Hz, 1H), 7.50−7.55 (m, 1H), 5.85 (br s, 1H),
5.38 (d, J = 8.20 Hz, 1H), 5.11−5.30 (m, 2H), 4.31−4.40 (m, 1H),
4.24 (d, J = 6.25 Hz, 1H), 3.37 (dd, J = 6.05, 8.79 Hz, 2H), 2.33−2.52
(m, 2H), 1.86−1.97 (m, 2H), 1.47−1.84 (m, 6H), 1.31−1.44 (m, 2H),
1.06−1.28 (m, 3H), 0.82−1.04 (m, 3H). HRMS (ESI) calcd for
C24H32N4O7Na [M + Na]+, 511.2169; found, 511.2153.
3-Bromobenzyl ((S)-3-Cyclohexyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-ox-
Benzyl ((S)-4-Methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-
yl)propan-2-yl)amino)pentan-2-yl)carbamate (40). Yield (83%), mp
57−59 °C. 1H NMR (400 MHz, CDCl3) δ 9.53 (br s, 1H), 8.32−8.45
(m, 1H), 7.32−7.46 (m, 5H), 5.78−5.85 (m, 1H), 5.34 (br s, 1H),
5.09−5.18 (m, 2H), 4.35 (br s, 2H), 3.30−3.45 (m, 2H), 2.30−2.59
(m, 2H), 1.99 (d, J = 6.64 Hz, 1H), 1.83−1.93 (m, 1H), 1.69−1.81
(m, 1H), 1.58−1.69 (m, 2H), 1.23−1.30 (m, 1H), 0.94 (d, J = 6.15
Hz, 6H). HRMS (ESI) calcd for C21H29N3O5Na [M + Na]+,
426.2005; found, 426.1997.
opyrrolidin-3-yl)propan-2-yl)amino)propan-2-yl)carbamate (29).
1
Yield (83%), mp 52−55 °C. H NMR (400 MHz, CDCl3) δ 9.48
(s, 1H), 8.24−8.67 (m, 1H), 7.53 (br s, 1H), 7.35−7.47 (m, 1H),
7.25−7.29 (m, 2H), 7.22 (d, J = 7.42 Hz, 1H), 5.66−5.74 (m, 1H),
5.08 (br s, 2H), 4.23−4.40 (m, 2H), 3.36 (d, J = 8.59 Hz, 2H), 2.32−
2.53 (m, 2H), 1.95 (br s, 1H), 1.84 (br s, 2H), 1.69 (d, J = 9.37 Hz,
5H), 1.57 (br s, 3H), 1.32−1.46 (m, 1H), 1.08−1.28 (m, 2H), 0.84−
1.05 (m, 2H). HRMS (ESI) calcd for C24H32BrN3O5Na [M + Na]+,
544.1423; found, 544.1410.
Benzyl ((S)-3-Cyclohexyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrroli-
3-Iodobenzyl ((S)-3-Cyclohexyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxo-
din-3-yl)propan-2-yl)amino)propan-2-yl)carbamate (43). Yield
1
pyrrolidin-3-yl)propan-2-yl)amino)propan-2-yl)carbamate (33).
(74%), mp 53−56 °C. H NMR (400 MHz, CDCl3) δ 9.48 (s, 1H),
1
Yield (80%), mp 56−58 °C. H NMR (400 MHz, CDCl3) δ 9.48
8.31 (d, J = 5.86 Hz, 1H), 7.73 (br s, 1H), 7.34 (br s, 5H), 5.50−5.59
(m, 1H), 5.04−5.15 (m, 2H), 4.20−4.53 (m, 2H), 3.20−3.41 (m, 2H),
2.23−2.51 (m, 2H), 1.88−2.03 (m, 1H), 1.74−1.86 (m, 2H), 1.66 (d, J
= 5.86 Hz, 6H), 1.45−1.58 (m, 1H), 1.30−1.43 (m, 1H), 1.18 (d, J =
16.12 Hz, 3H), 0.79−1.03 (m, 2H). HRMS (ESI) calcd for
C24H33N3O5Na [M + Na]+, 466.2318; found, 466.2299.
(s, 1H), 8.38−8.44 (m, 1H), 7.73 (br s, 1H), 7.64 (d, J = 7.81 Hz,
1H), 7.31 (d, J = 7.81 Hz, 1H), 7.05−7.11 (m, 1H), 5.86 (br s, 1H),
5.34 (d, J = 7.81 Hz, 1H), 5.05 (s, 2H), 4.27−4.39 (m, 2H), 3.31−3.40
(m, 2H), 2.32−2.53 (m, 2H), 1.91−1.99 (m, 2H), 1.79−1.90 (m, 2H),
1.69 (d, J = 7.81 Hz, 6H), 1.47−1.58 (m, 1H), 1.38 (br s, 1H), 1.09−
1.29 (m, 2H), 0.84−1.04 (m, 2H). HRMS (ESI) calcd for
C24H32IN3O5Na [M + Na]+, 592.1284; found, 592.1295.
Synthesis of α-Hydroxyphosphonates 8. General Procedure.
To a solution of diethylphosphite (135 mg, 1 mmol) in dry
dichloromethane (3.5 mL) was added DIEA (129 mg, 1 mmol),
followed by a solution of aldehyde 7 (1 mmol) in dichloromethane (5
mL). The reaction mixture was stirred at room temperature for 20 h.
The reaction mixture was diluted with dichloromethane (100 mL) and
washed with 5% HCl (2 × 20 mL) and brine (30 mL). The organic
layer was dried over anhydrous sodium sulfate, filtered, and
concentrated, leaving a light-yellow solid which was purified by flash
chromatography to yield compound 8 as a white solid.
2-Methoxybenzyl ((S)-3-Cyclohexyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-
oxopyrrolidin-3-yl)propan-2-yl)amino)propan-2-yl)carbamate (35).
Yield (64%), mp 56−58 °C. 1H NMR (400 MHz, CDCl3) δ 9.49 (br s,
1H), 8.16−8.24 (m, 1H), 7.30−7.37 (m, 2H), 7.29 (s, 1H), 6.93 (t, J =
7.42 Hz, 1H), 6.88 (d, J = 8.20 Hz, 1H), 5.80 (br s, 1H), 5.11−5.24
(m, 2H), 4.28−4.40 (m, 2H), 3.83 (s, 3H), 3.27−3.39 (m, 2H), 2.40
(d, J = 12.89 Hz, 2H), 1.96 (br s, 2H), 1.78−1.90 (m, 2H), 1.64−1.76
(m, 4H), 1.60 (s, 1H), 1.53 (dd, J = 4.10, 9.57 Hz, 1H), 1.39 (br s,
G
DOI: 10.1021/jm5019934
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2-Chlorobenzyl ((2S)-3-Cyclohexyl-1-(((2S)-1-(diethoxyphosphor-
yl)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-1-ox-
opropan-2-yl)carbamate (14). Yield (79%), mp 103−106 °C. 1H
NMR (400 MHz, CDCl3) δ 7.66−7.71 (m, 1H), 7.41 (d, J = 3.12 Hz,
1H), 7.33−7.37 (m, 1H), 7.28−7.32 (m, 1H), 7.21−7.26 (m, 3H),
5.49−5.57 (m, 1H), 5.15−5.24 (m, 2H), 4.06−4.21 (m, 4H), 3.97−
4.04 (m, 1H), 3.86−3.95 (m, 1H), 3.41−3.52 (m, 1H), 1.77−1.86 (m,
2H), 1.56−1.71 (m, 6H), 1.50−1.56 (m, 1H), 1.25−1.37 (m, 7H),
1.23 (s, 2H), 1.17 (dt, J = 2.15, 7.13 Hz, 4H), 0.86 (br s, 4H). HRMS
(ESI) calcd for C28H43ClN3O8PNa [M + Na]+, 638.2374; found,
638.2379.
3-Chlorobenzyl ((2S)-3-Cyclohexyl-1-(((2S)-1-(diethoxyphosphor-
yl)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-1-ox-
opropan-2-yl)carbamate (17). Yield (81%), mp 58−61 °C. 1H NMR
(400 MHz, CDCl3) δ 7.77−7.83 (m, 1H), 7.30−7.38 (m, 1H), 7.24−
7.29 (m, 2H), 7.16−7.23 (m, 1H), 6.39−6.48 (m, 1H), 5.86−5.94 (m,
1H), 5.33−5.42 (m, 1H), 4.98−5.12 (m, 2H), 4.22−4.42 (m, 2H),
4.07−4.20 (m, 4H), 3.91−4.01 (m, 1H), 3.17−3.35 (m, 2H), 2.30−
2.47 (m, 2H), 2.10−2.23 (m, 2H), 1.66 (d, J = 11.33 Hz, 6H), 1.41−
1.54 (m, 2H), 1.28−1.36 (m, 6H), 1.05−1.22 (m, 3H), 0.79−1.00 (m,
3H). HRMS (ESI) calcd for C28H43ClN3O8PNa [M + Na]+, 638.2374;
found, 638.2367.
2-Fluorobenzyl ((2S)-3-Cyclohexyl-1-(((2S)-1-(diethoxyphosphor-
yl)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-1-ox-
opropan-2-yl)carbamate (26). Yield (73%), mp 98−101 °C. 1H
NMR (400 MHz, DMSO-d6) δ 7.80 (br s, 1H), 7.65 (d, J = 8.98 Hz,
1H), 7.49−7.57 (m, 1H), 7.33−7.48 (m, 2H), 7.14−7.25 (m, 2H),
5.90−5.97 (m, 1H), 5.71−5.84 (m, 1H), 5.07 (d, J = 6.35 Hz, 2H),
3.95−4.09 (m, 5H), 3.75−3.93 (m, 1H), 2.96−3.24 (m, 3H), 2.00−
2.26 (m, 2H), 1.50−1.75 (m, 7H), 1.36−1.47 (m, 2H), 1.19−1.33 (m,
6H), 1.01−1.15 (m, 3H), 0.84 (d, J = 7.91 Hz, 3H). HRMS (ESI)
calcd for C28H43FN3O8PNa [M + Na]+, 622.2670; found, 622.2658.
3-Bromobenzyl ((2S)-3-Cyclohexyl-1-(((2S)-1-(diethoxyphosphor-
yl)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-1-ox-
opropan-2-yl)carbamate (30). Yield (78%), mp 48−52 °C. 1H NMR
(400 MHz, CDCl3) δ 8.35−8.40 (m, 1H), 7.46 (d, J = 3.32 Hz, 1H),
7.33−7.39 (m, 1H), 7.21 (br s, 1H), 7.11−7.18 (m, 2H), 5.88 (s, 1H),
5.26−5.31 (m, 1H), 5.01 (d, J = 3.32 Hz, 2H), 4.02−4.15 (m, 6H),
3.27 (d, J = 6.44 Hz, 3H), 1.88 (br s, 1H), 1.76 (d, J = 9.37 Hz, 2H),
1.60 (br s, 7H), 1.46 (d, J = 1.95 Hz, 1H), 1.23−1.34 (m, 6H), 1.19 (s,
1H), 0.98−1.16 (m, 3H), 0.75−0.96 (m, 3H). HRMS (ESI) calcd for
C28H43BrN3O8PNa [M + Na]+, 682.1869; found, 682.1896.
Synthesis of Bisulfite Adducts 9. General Procedure. To a
solution of aldehyde 7 (5 mmol) in dry ethyl acetate (20 mL) was
added absolute ethanol (12 mL) with stirring, followed by a solution
of sodium bisulfite (540 mg; 5 mmol) in water (5 mL). The reaction
mixture was stirred for 3 h at 50 °C. The reaction mixture was allowed
to cool to room temperature and then vacuum filtered. The solid was
thoroughly washed with absolute ethanol, and the filtrate was dried
over anhydrous sodium sulfate, filtered, and concentrated to yield
yellowish oil. The oily product was treated with ethyl ether (2 × 50
mL) to form white solid. The white solid was stirred with ethyl ether
(30 mL) and ethyl acetate (15 mL) for 5 min. Careful removal of the
solvent using a pipet left compound 9 as a white solid.
2.95−3.05 (m, 1H), 2.01−2.25 (m, 3H), 1.51−1.78 (m, 6H), 1.37−
1.48 (m, 1H), 1.28 (br s, 1H), 1.15−1.21 (m, 1H), 1.08−1.14 (m,
2H), 0.74−0.94 (m, 2H). HRMS (ESI) calcd for C24H33ClN3O8S,
[M−], 558.1677; found, 558.1656.
Sodium (2S)-2-((S)-2-((((3-Bromobenzyl)oxy)carbonyl)amino)-3-
cyclohexylpropana mido)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)-
1
propane-1-sulfonate (31). Yield (86%), mp 132−135 °C. H NMR
(400 MHz, DMSO-d6) δ 7.68 (d, J = 8.98 Hz, 1H), 7.60 (d, J = 9.37
Hz, 1H), 7.53−7.58 (m, 1H), 7.50 (d, J = 7.81 Hz, 1H), 7.45 (d, J =
4.30 Hz, 1H), 7.29−7.41 (m, 2H), 5.54 (d, J = 5.47 Hz, 1H), 5.36 (d, J
= 5.08 Hz, 1H), 5.03 (s, 2H), 4.18−4.27 (m, 1H), 3.91−4.10 (m, 1H),
2.96−3.15 (m, 2H), 2.05−2.23 (m, 2H), 1.89−2.05 (m, 1H), 1.49−
1.75 (m, 6H), 1.38−1.47 (m, 1H), 1.23−1.35 (m, 1H), 1.02−1.21 (m,
5H), 0.75−0.95 (m, 2H). HRMS (ESI) calcd for C24H33BrN3O8S
[M−], 602.1172; found, 602.1168.
Sodium (2S)-2-((S)-3-Cyclohexyl-2-((((3-iodobenzyl)oxy)-
carbonyl)amino)propane mido)-1-hydroxy-3-((S)-2-oxopyrrolidin-
3-yl)propane-1-sulfonate (34). Yield (78%), mp 135−137 °C. 1H
NMR (400 MHz, DMSO-d6) δ 8.48−8.52 (m, 1H), 7.73 (br s, 1H),
7.66 (d, J = 7.62 Hz, 1H), 7.51−7.59 (m, 1H), 7.45 (d, J = 3.52 Hz,
1H), 7.36 (br s, 1H), 7.13−7.21 (m, 1H), 5.45−5.52 (m, 1H), 5.28−
5.35 (m, 1H), 4.92−5.14 (m, 2H), 4.13−4.29 (m, 1H), 3.88−4.08 (m,
1H), 2.97−3.19 (m, 2H), 1.91 (s, 3H), 1.37−1.78 (m, 7H), 1.02−1.24
(m, 5H), 0.73−0.97 (m, 3H). HRMS (ESI) calcd for C24H33IN3O8S
[M−], 650.1033; found, 650.1019.
Sodium (2S)-2-((S)-2-(((Benzyloxy)carbonyl)amino)-4-methylpen-
tanamido)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propane-1-sulfo-
nate (41). Yield (73%), mp 109−113 °C. 1H NMR (400 MHz,
DMSO-d6) δ 7.57−7.62 (m, 1H), 7.47−7.52 (m, 1H), 7.26−7.40 (m,
5H), 5.47−5.51 (m, 1H), 5.31−5.36 (m, 1H), 4.96−5.05 (m, 3H),
4.19−4.28 (m, 1H), 3.83−4.06 (m, 2H), 3.07−3.16 (m, 1H), 2.97−
3.06 (m, 1H), 2.04−2.23 (m, 2H), 1.71−1.81 (m, 1H), 1.52−1.68 (m,
2H), 1.39−1.50 (m, 2H), 0.85 (ddd, J = 3.03, 6.52, 9.40 Hz, 6H).
HRMS (ESI) calcd for C21H30N3O8S [M−], 484.1754; found,
484.1754.
Sodium (2S)-2-((S)-2-(((Benzyloxy)carbonyl)amino)-3-cyclohexyl-
propanamido)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propane-1-
1
sulfonate (44). Yield (78%), mp 131−133 °C. H NMR (400 MHz,
DMSO-d6) δ 7.62−7.69 (m, 1H), 7.43−7.51 (m, 1H), 7.27−7.40 (m,
5H), 5.82−5.86 (m, 1H), 5.52 (d, J = 6.25 Hz, 1H), 5.36 (d, J = 5.96
Hz, 1H), 5.02 (s, 2H), 3.83−4.08 (m, 2H), 2.97−3.20 (m, 3H), 1.86−
2.25 (m, 3H), 1.51−1.82 (m, 6H), 1.37−1.48 (m, 2H), 1.29 (br s,
1H), 1.02−1.22 (m, 3H), 0.75−0.94 (m, 2H). HRMS (ESI) calcd for
C24H34N3O8S [M−], 524.2067; found, 524.2045.
Synthesis of Compound 10. General Procedure. A solution of
aldehyde 7 (5 mmol) in ethyl acetate (30 mL) kept at 0 °C was treated
with acetic acid (0.34 g; 5.75 mmol), followed by cyclopropyl
isocyanide (0.37 g; 5.5 mmol), and the reaction mixture was stirred at
room temperature for 18 h. The solution was concentrated in vacuo,
and the residue was dissolved in methanol (30 mL) and treated with a
solution of K2CO3 (1.72 g; 12.5 mmol) in water (25 mL). The
reaction mixture was stirred at room temperature for 2 h. Methanol
was evaporated off, and the aqueous layer was extracted with ethyl
acetate (3 × 100 mL). The combined organic layers were washed with
5% HCl (2 × 50 mL) and brine (75 mL). The organic layer was dried
over anhydrous sodium sulfate, and the solvent was removed to yield
compound 10 as a white solid. This was used in the next step without
further purification.
Synthesis of α-Ketoamides 11. General Procedure. To a
solution of compound 10 (5 mmol) in anhydrous dichloromethane
(50 mL) cooled to 0 °C and kept under a nitrogen atmosphere was
added Dess−Martin periodinane reagent (3.18 g, 7.5 mmol, 1.5 equiv)
with stirring. The ice bath was removed, and the reaction mixture was
stirred at room temperature for 3 h. The reaction was monitored by
TLC until the starting material disappeared. A solution of 10%
aqueous sodium thiosulfate (20 mL) was added, and the solution was
stirred for 15 min. The solution was poured into a separatory funnel,
and the aqueous layer was removed. The organic layer was washed
with 10% aqueous sodium thiosulfate (20 mL), followed by saturated
aqueous sodium bicarbonate (2 × 20 mL), water (2 × 20 mL), and
brine (20 mL). The organic layer was dried over anhydrous sodium
Sodium (2S)-2-((S)-2-((((2-Chlorobenzyl)oxy)carbonyl)amino)-3-
cyclohexylpropana mido)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)-
1
propane-1-sulfonate (15). Yield (60%), mp 128−132 °C. H NMR
(400 MHz, CDCl3) δ 7.51−7.53 (m, 1H), 7.40−7.45 (m, 1H), 7.35−
7.39 (m, 1H), 7.26 (s, 3H), 6.99−7.00 (m, 1H), 5.18−5.29 (m, 2H),
3.68−3.78 (m, 2H), 3.48 (d, J = 7.03 Hz, 3H), 3.29−3.40 (m, 1H),
1.92−1.98 (m, 1H), 1.80−1.91 (m, 2H), 1.71 (br s, 7H), 1.24−1.28
(m, 5H), 0.79−0.96 (m, 2H), 0.68−0.76 (m, 1H). HRMS (ESI) calcd
for C24H33ClN3O8S [M−], 558.1677; found, 558.1648.
Sodium (2S)-2-((S)-2-((((3-Chlorobenzyl)oxy)carbonyl)amino)-3-
cyclohexylpropana mido)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)-
1
propane-1-sulfonate (18). Yield (67%), mp 124−126 °C. H NMR
(400 MHz, DMSO-d6) δ 7.66 (d, J = 8.98 Hz, 1H), 7.58 (dd, J = 9.18,
11.52 Hz, 1H), 7.49 (d, J = 8.20 Hz, 1H), 7.42 (d, J = 4.30 Hz, 1H),
7.40 (d, J = 1.95 Hz, 1H), 7.30−7.39 (m, 2H), 5.41 (d, J = 5.86 Hz,
1H), 5.25 (d, J = 5.86 Hz, 1H), 5.03 (s, 2H), 4.17−4.26 (m, 1H),
3.89−3.99 (m, 2H), 3.81−3.87 (m, 1H), 3.10 (t, J = 9.96 Hz, 1H),
H
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Table 3. Crystallographic Data for NV 3CLpro:Inhibitor Structures
NV 3CLpro:17
NV 3CLpro:44-h
NV 3CLpro:44-o
Data Collection
unit-cell parameters (Å, deg)
space group
a = 124.23, c = 49.97
P6522
a = 121.85, c = 51.50
P6522
a = 37.47 b = 66.91 c = 126.4
P212121
a
resolution (Å)
45.32−1.85 (1.89−1.85)
1.0000
46.28−1.60 (1.63−1.60)
1.0000
45.94−1.45 (1.48−1.45)
1.0000
wavelength (Å)
temperature (K)
obsd reflns
100
100
100
384657
579016
371208
unique reflns
19899
30219
57354
a
⟨I/σ(I)⟩
14.9 (2.4)
16.3 (2.0)
100 (100)
19.2 (16.9)
16.6 (169.2)
17.0 (174.5)
3.9 (42.3)
99.9 (71.1)
18.9 (1.8)
a
completeness (%)
100 (100)
19.3 (18.5)
19.7 (150.5)
20.3 (154.7)
4.6 (35.8)
100 (100)
a
multiplicity
6.5 (6.5)
a,b
Rmerge (%)
4.7 (105.4)
5.1 (114.5)
2.0 (44.4)
a,d
Rmeas (%)
a,d
Rpim (%)
a,e
CC1/2
0.998 (0.763)
100 (63.5)
Refinement
a
resolution (Å)
45.32−1.85
18907/968
16.3/19.7
36.85−1.60
28660/1530
16.3/17.5
35.65−1.45
54369/2905
17.3/19.9
a
reflns (working/test)
a,c
R
factor/Rfree (%)
no. of atoms (protein/ligand/water)
Model Quality
1236/33/129
1319/32/160
2444/58/218
rmsd
bond lengths (Å)
bond angles (deg)
average B-factor (Å2)
all atoms
0.009
1.229
0.009
1.234
0.008
1.020
21.1
19.9
25.7
29.9
0.16
17.2
15.6
18.5
19.0
0.18
24.3
23.6
25.4
32.1
0.15
protein
ligand
water
coordinate error (maximum likelihood) (Å)
Ramachandran plot
most favored (%)
additionally allowed (%)
98.8
98.3
1.7
98.5
1.5
1.2
a
b
Values in parentheses are for the highest resolution shell. Rmerge = ∑hkl ∑i |Ii(hkl) − ⟨I(hkl)⟩|/∑hkl ∑i Ii(hkl), where Ii(hkl) is the intensity
c
measured for the ith reflection and ⟨I(hkl)⟩ is the average intensity of all reflections with indices hkl. Rfactor = ∑hkl||Fobs (hkl) | − |Fcalcd (hkl)||/∑hkl|
d
Fobs (hkl)|; Rfree is calculated in an identical manner using 5% of randomly selected reflections that were not included in the refinement. Rmeas
=
28,37
38,39 e
redundancy-independent (multiplicity-weighted) Rmerge
.
R
= precision-indicating (multiplicity-weighted) Rmerge
.
CC1/2 is the correlation
pim
coefficient of the mean intensities between two random half-sets of data.40,41
sulfate, filtered, and concentrated, leaving a yellow solid which was
purified by flash chromatography (silica gel/methylene chloride/ethyl
acetate/methanol) to yield 11 as a white solid.
HRMS (ESI) calcd for C25H33ClN4O6Na [M + Na]+, 543.1986; found,
543.1989.
3-Fluorobenzyl ((S)-3-Cyclohexyl-1-(((S)-4-(cyclopropylamino)-
3,4-dioxo-1-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)amino)-1-oxopro-
pan-2-yl)carbamate (28). Yield (74%), mp 95−98 °C. 1H NMR (400
MHz, CDCl3) δ 8.41−8.49 (m, 1H), 7.28−7.36 (m, 2H), 7.25 (d, J =
2.34 Hz, 1H), 7.10 (t, J = 7.42 Hz, 2H), 6.90−7.02 (m, 1H), 5.83 (br s,
1H), 5.04−5.13 (m, 2H), 4.25−4.39 (m, 2H), 3.36 (dd, J = 4.49, 8.79
Hz, 2H), 2.76 (dd, J = 3.52, 7.42 Hz, 1H), 2.51−2.61 (m, 1H), 2.39−
2.50 (m, 1H), 2.06−2.14 (m, 1H), 1.94 (d, J = 10.94 Hz, 2H), 1.80 (br
s, 1H), 1.58−1.74 (m, 6H), 1.48 (br s, 1H), 1.31−1.42 (m, 1H), 1.07−
1.29 (m, 3H), 0.79−1.03 (m, 3H), 0.61 (ddd, J = 1.56, 4.00, 5.76 Hz,
2H). HRMS (ESI) calcd for C28H37FN4O6Na [M + Na]+, 567.2595;
found, 567.2592.
3-Chlorobenzyl ((S)-3-Cyclohexyl-1-(((S)-4-(cyclopropylamino)-
3,4-dioxo-1-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)amino)-1-oxopro-
1
pan-2-yl)carbamate (19). Yield (88%), mp 135−138 °C. H NMR
(400 MHz, CDCl3) δ 8.42 (br s, 1H), 7.36 (br s, 1H), 7.27−7.29 (m,
2H), 7.19−7.24 (m, 2H), 6.97 (br s, 1H), 5.97 (br s, 1H), 5.08 (d, J =
4.30 Hz, 2H), 4.24−4.42 (m, 2H), 3.27−3.40 (m, 2H), 2.76 (dd, J =
3.52, 7.42 Hz, 1H), 2.35−2.60 (m, 2H), 2.07 (dd, J = 3.32, 5.27 Hz,
1H), 1.89−1.99 (m, 2H), 1.77−1.87 (m, 2H), 1.59−1.75 (m, 4H),
1.43−1.56 (m, 1H), 1.38 (br s, 1H), 1.09−1.28 (m, 3H), 0.86−1.04
(m, 2H), 0.81−0.85 (m, 2H), 0.56−0.66 (m, 2H). HRMS (ESI) calcd
for C28H37ClN4O6Na [M + Na]+, 583.2299; found, 583.2293.
3-Chlorobenzyl ((S)-1-(((S)-4-(Cyclopropylamino)-3,4-dioxo-1-
((S)-2-oxopyrrolidin-3-yl)butan-2-yl)amino)-4-methyl-1-oxopentan-
2-yl)carbamate (23). Yield (72%), mp 66−68 °C. H NMR (400
3-Bromobenzyl ((S)-3-Cyclohexyl-1-(((S)-4-(cyclopropylamino)-
3,4-dioxo-1-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)amino)-1-oxopro-
pan-2-yl)carbamate (32). Yield (70%), mp 124−127 °C. H NMR
1
1
MHz, CDCl3) δ 8.40 (d, J = 5.47 Hz, 1H), 7.24 (br s, 1H), 7.16−7.22
(m, 1H), 7.11 (br s, 2H), 6.75 (br s, 1H), 5.63−5.75 (m, 1H), 5.10−
5.21 (m, 1H), 4.93−5.05 (m, 2H), 4.22−4.36 (m, 2H), 3.15−3.30 (m,
2H), 2.69 (d, J = 3.12 Hz, 1H), 2.39−2.51 (m, 1H), 2.31 (d, J = 3.91
Hz, 1H), 1.80−2.01 (m, 5H), 1.50−1.72 (m, 3H), 1.38−1.49 (m, 2H),
0.86 (d, J = 4.69 Hz, 2H), 0.74 (d, J = 7.42 Hz, 2H), 0.54 (br s, 2H).
(400 MHz, CDCl3) δ 8.39−8.45 (m, 1H), 7.52 (br s, 1H), 7.43 (d, J =
7.62 Hz, 1H), 7.27 (br s, 1H), 7.22 (d, J = 7.62 Hz, 2H), 6.90−6.95
(m, 1H), 5.71−5.75 (m, 1H), 5.18−5.27 (m, 1H), 5.07 (s, 2H), 4.28−
4.35 (m, 1H), 3.35 (dd, J = 4.10, 8.98 Hz, 3H), 2.72−2.81 (m, 2H),
2.06−2.15 (m, 2H), 1.86−2.01 (m, 2H), 1.69 (d, J = 10.94 Hz, 5H),
1.58 (s, 3H), 1.25 (s, 2H), 0.87−1.02 (m, 2H), 0.84 (d, J = 7.42 Hz,
I
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2H), 0.55−0.67 (m, 2H). HRMS (ESI) calcd for C28H37BrN4O6Na
[M + Na]+, 627.1794; found, 627.1791.
against MNV-1 in cell culture was determined with RAW264.7 cells.
Each compound was dissolved in DMSO to make a 10 mM stock
solution. The amount of DMSO in cell culture did not exceed 0.5%.
The stock solution was serially diluted with cell culture media prior to
addition to confluent monolayer of RAW264.7 cells in 24-well plates.
Cells were immediately infected with virus at a multiplicity of infection
(MOI) of 0.05−0.1. After further incubation of the cells at 37 °C for
48 h, cells were frozen and thawed, and virus titers were determined by
the 50% tissue culture infectious dose (TCID50) method. The 50%
effective concentration (EC50) values were determined by GraphPad
Prism (GraphPad Software, San Diego, CA) by nonlinear regression
analyses of dose−response curves of the inhibition of virus replication
against log inhibitor concentration (variable slope).
Benzyl ((S)-1-(((S)-4-(Cyclopropylamino)-3,4-dioxo-1-((S)-2-oxo-
pyrrolidin-3-yl)butan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)-
1
carbamate (42). Yield (78%), mp 75−78 °C. H NMR (400 MHz,
CDCl3) δ 8.43 (d, J = 5.68 Hz, 1H), 7.33 (s, 5H), 7.08 (d, J = 3.48 Hz,
1H), 6.57 (s, 1H), 5.53 (d, J = 8.79 Hz, 1H), 5.19−5.26 (m, 1H), 5.08
(s, 2H), 4.40 (dt, J = 5.49, 8.88 Hz, 1H), 3.22−3.36 (m, 2H), 2.71−
2.80 (m, 1H), 2.48−2.58 (m, 1H), 2.35−2.46 (m, 1H), 1.88−2.10 (m,
3H), 1.59−1.79 (m, 2H), 1.45−1.55 (m, 1H), 0.95 (d, J = 6.41 Hz,
6H), 0.79−0.86 (m, 2H), 0.57−0.66 (m, 2H). HRMS (ESI) calcd for
C25H34N4O6Na [M + Na]+, 509.2376; found, 509.2364.
Enzyme Assays and Inhibition Studies against NV with
3CLpro and Cell-Based Replicon System. These studies were
carried out as described previously.13c
Animal Experiments.36 The animal study was performed in
accordance with a protocol approved by the Institutional Animal Care
and Use Committee (IACUC) at Kansas State University. BALB/c
mice were purchased from Charles River Lab (Wilmington, MA).
Seven-to-eight-week-old female BALB/c mice were inoculated intra-
peritoneally with MNV-1 at 2 × 106 TCID50/mouse. Mice were
peritoneally given 50 μL of drug vehicle (10% EtOH and 90%
PEG400) or compound 16 (20 mg/kg/day) divided into two doses
per day. Compound administration started 4 h prior to virus infection
and continued daily until mice were euthanized. At 3 days post
infection (dpi), mice were sacrificed and the small and large intestinal
tracts were collected. The intestines were placed in PBS, and the
intestinal contents were gently removed by squeezing. The tissues
were homogenized in PBS (5 or 3 mL for small and large intestines,
respectively) and frozen at −70C until ready to use. Virus titers of the
homogenized supernatant were determined by the TCID50 method,
and statistical analysis was performed on log-transformed TCID50
values using a two-tailed Student t test (p < 0.05). Fold changes in the
geometric mean liver virus titers in each group were calculated by
dividing virus titers in control group by the treated group.
X-ray Crystallographic Studies. Crystallization and Data
Collection. Purified NV 3CLpro in 100 mM NaCl, 50 mM PBS, pH
7.2, and 1 mM DTT at a concentration of 10 mg/mL was used for
preparation of the norovirus 3CL protease−compound 17 complex. A
stock solution of 100 mM compound 17 or 44 was prepared in
DMSO, and each NV 3CLpro:inhibitor complex was prepared by
mixing 12 μL of 17 or 44 (3 mM) with 388 μL (0.49 mM) of NV
3CLpro and incubating on ice for 1 h. The mixture was loaded onto a
Superdex 75 10/300 GL column equilibrated with 100 mM NaCl and
20 mM Tris, pH 8.0, and the sample was concentrated to 8.0 mg/mL
in a Vivaspin-20 (Vivaproducts, Inc.) concentrator for crystallization
screening. All crystallization experiments were conducted Compact Jr.
(Rigaku Reagents) sitting drop vapor diffusion plates at 20 °C using
equal volumes of protein and the crystallization solution were
equilibrated against 75 μL of the latter. Crystals of NV 3CLpro−
ligand 17 that displayed a needle morphology were obtained overnight
from the Proplex HT screen (Molecular Dimensions) condition D4
(20% (w/v) PEG 5000 MME, 100 mM Tris, pH 7.5, and 200 mM
ammonium sulfate) (Table 3). Samples were transferred to a fresh
drop composed of 80% crystallization solution and 20% PEG glycerol
and stored in liquid nitrogen. Two crystal forms of the NV 3CLpro:44
complex that displayed a prismatic morphology were observed in 2
days from the Wizard 3 and 4 screen (Emerald Biosystems). The
primitive hexagonal (NV 3CLpro:44-h) form was obtained from
condition A1 (20% w/v PEG3350, 200 mM ammonium citrate) and a
primitive orthorhombic (NV 3CLpro:44-o) from A10 (20% w/v
PEG3350, 200 mM sodium thiocyanate). Samples were transferred to
a fresh drop composed of 80% crystallization solution and 20% PEG
400 and stored in liquid nitrogen prior to data collection. X-ray
diffraction data were collected at the Advanced Photon Source
beamline 17-ID using a Dectris Pilatus 6 M pixel array detector.
Structure Solution and Refinement. Intensities were integrated
using XDS,26,27 and the Laue class analysis and data scaling were
performed with Aimless,28 which suggested that the highest probability
Laue class was 6/mmm and possible space groups were P6122 or P6522
for NV 3CLpro, NV 3CLpro:17, and NV 3CLpro:44-h. The
Matthew’s coefficient29 suggested that there was a single molecule in
the asymmetric unit (Vm = 2.8 Å3/Da, % solvent = 56%). Structure
solution was conducted by molecular replacement with Phaser30 using
a previously determined structure of inhibitor bound NV 3CLpro
(PDB: 3UR9)13c as the search model, and all space groups with 622
point symmetry were tested. The top solution was obtained in the
space group P6522, which was used from this point forward. NV
3CLpro:44-o was isomorphous with PDB 3UR9. Structure refinement
using and manual model building were conducted with Phenix31 and
Coot,32 respectively. Disordered side chains were truncated to the
point for which electron density could be observed. Structure
validation was conducted with Molprobity,33 and figures were
prepared using the CCP4MG package.34
ASSOCIATED CONTENT
Accession Codes
■
The coordinates and structure factors for norovirus 3CL
protease in complex with inhibitors have been deposited to the
Protein Data Bank with the accession codes NVpro:17 (4XBB),
NVPro:44-h (4XBC) and NVPro:44-o (4XBD).
AUTHOR INFORMATION
Corresponding Authors
■
*For W.C.G.: Phone, (316) 978-7374; fax, (316) 978-3431; E-
mail, bill.groutas@wichita.edu; address, Department of Chem-
istry, Wichita State University, Wichita, Kansas 67260, United
States.
*For K.-O.C.: phone, (785) 532-3849; fax, (785) 532-4039; E-
mail, kchang@vet.ksu.edu; address, Department of Diagnostic
Medicine & Pathobiology, Manhattan, Kansas 66506, United
States.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The generous financial support of this work by the National
Institutes of Health (AI109039) is gratefully acknowledged.
Use of the University of Kansas Protein Structure Laboratory
was supported by grants from the National Center for Research
Resources (5P20RR017708-10) and the National Institute of
General Medical Sciences (8P20GM103420-10). Use of the
IMCA-CAT beamline 17-ID at the Advanced Photon Source
was supported by the companies of the Industrial Macro-
molecular Crystallography Association through a contract with
Hauptman−Woodward Medical Research Institute. Use of the
Advanced Photon Source was supported by the U.S. Depart-
Study with MNV.35 RAW264.7 cells, mouse macrophage cell line,
were maintained in Dulbecco’s Minimum Essential Medium (DMEM)
containing antibiotics of chlortetracycline (25 μg/mL), penicillin (250
U/ml), and streptomycin (250 μg/mL) in the presence of 2−5% fetal
bovine serum. MNV-1 was obtained from H. Virgin at Washington
University (St. Louis, MO). Antiviral activity of each compound
J
DOI: 10.1021/jm5019934
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Article
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