Structure-guided design, synthesis and evaluation of oxazolidinone-based inhibitors of norovirus 3CL protease

Article abstract of DOI:10.1016/j.ejmech.2017.12.014

Acute nonbacterial gastroenteritis caused by noroviruses constitutes a global public health concern and a significant economic burden. There are currently no small molecule therapeutics or vaccines for the treatment of norovirus infections. A structure-gu

Full text of DOI:10.1016/j.ejmech.2017.12.014

Accepted Manuscript
Structure-guided design, synthesis and evaluation of oxazolidinone-based inhibitors
of norovirus 3CL protease
Vishnu C. Damalanka, Yunjeong Kim, Anushka C. Galasiti Kankanamalage, Athri
D. Rathnayake, Nurjahan Mehzabeen, Kevin P. Battaile, Scott Lovell, Harry Nhat
Nguyen, Gerald H. Lushington, Kyeong-Ok Chang, William C. Groutas
PII:
S0223-5234(17)31020-6
10.1016/j.ejmech.2017.12.014
EJMECH 9992
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 July 2017
Revised Date: 18 November 2017
Accepted Date: 4 December 2017
Please cite this article as: V.C. Damalanka, Y. Kim, A.C. Galasiti Kankanamalage, A.D. Rathnayake,
N. Mehzabeen, K.P. Battaile, S. Lovell, H.N. Nguyen, G.H. Lushington, K.-O. Chang, W.C. Groutas,
Structure-guided design, synthesis and evaluation of oxazolidinone-based inhibitors of norovirus 3CL
protease, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.12.014.
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ACCEPTED MANUSCRIPT
R2
O
H
N
H
N
O
R3
O
R1
O
R2
O
H
N
peptidomimetic
N
R3
O
O
R1
O
S3-S4 subsites
EC50 - 6.7uM (compound 9)

ACCEPTED MANUSCRIPT
Structure-guided Design, Synthesis and Evaluation of
Oxazolidinone-based Inhibitors of Norovirus 3CL Protease
Vishnu C. Damalanka,a Yunjeong Kim,b Anushka C. Galasiti Kankanamalage,a Athri D.
Rathnayake,a Nurjahan Mehzabeen,c Kevin P. Battaile,d Scott Lovell,c Harry Nhat
Nguyen,a Gerald H. Lushington,e Kyeong-Ok Chang,b,** William C. Groutas,a,*
aDepartment of Chemistry, Wichita State University, Wichita, Kansas 67260, USA
bDepartment of Diagnostic Medicine & Pathobiology, College of Veterinary Medicine,
Kansas State University, Manhattan, Kansas 66506, USA
cProtein Structure Laboratory, The University of Kansas, Lawrence, Kansas 66047,
USA
dIMCA-CAT, Hauptman-Woodward Medical Research Institute, APS Argonne National
Laboratory, Argonne, IL 60439, USA
eLiS Consulting, Lawrence, KS 66046, USA
Authors to whom correspondence should be addressed.
*Department of Chemistry, Wichita State University, Wichita, KS 67260
Tel. (316) 978 7374; Fax: (316) 978 3431
e-mail: bill.groutas@wichita.edu
**Department of Diagnostic Medicine & Pathobiology, Manhattan, KS 66506
Tel. (785) 532 3849; Fax: (785) 532 4039
e-mail: kchang@vet.ksu.edu
1

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ABSTRACT
Acute nonbacterial gastroenteritis caused by noroviruses constitutes a global public
health concern and a significant economic burden. There are currently no small
molecule therapeutics or vaccines for the treatment of norovirus infections. A structure-
guided approach was utilized in the design of a series of inhibitors of norovirus 3CL
protease that embody an oxazolidinone ring as a novel design element for attaining
optimal binding interactions. Low micromolar cell-permeable inhibitors that display anti-
norovirus activity have been identified. The mechanism of action, mode of binding, and
structural rearrangements associated with the interaction of the inhibitors and the
enzyme were elucidated using X-ray crystallography.
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1. Introduction
Human noroviruses are the primary causative agents of acute gastroenteritis and
have an increasing impact on public health worldwide [1-3]. While the illness is mild and
self-limiting in healthy individuals, it impacts disproportionately and most severely
immunocompromised individuals, and the young and elderly [4]. There are 19-21
million norovirus (NV) infections annually in the U.S., and these are associated with high
morbidity [5-7]. The toll exacted by NV infections among children <5 years old in
developing countries is more acute, resulting in an estimated 71000 deaths annually [8].
The problem is further exacerbated by the highly infectious nature of noroviruses, their
genetic diversity and copious virus shedding, as well as their high environmental
stability.
Despite the global burden of noroviruses, there are currently no therapeutics or
vaccines for the treatment of the disease [9-14], however, recent advances in norovirus
pathobiology [15-17], including the identification of proteinaceous receptors for murine
norovirus entry into cells [18-19], the nuanced interplay of norovirus pathogenesis and
the gut microbiome [20-21], and the development of new in vitro culture systems [22]
and animal models [23], have greatly illuminated our understanding of norovirus binding
and entry, as well as cell tropism, and have begun to lay a solid foundation for exploring
an array of conceptually-sound approaches toward the development of anti-norovirus
therapeutics.
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Human noroviruses are single-stranded, positive sense RNA viruses belonging to
the family Caliciviridae [24]. Of the seven genogroups (GI-GVII) in the genus Norovirus,
genogroups I, II and IV are known to infect humans. The norovirus genome (7-8 kb)
consists of three opening reading frames that encode a 200 kDa polyprotein (ORF1), a
major capsid protein VP1 (ORF2), and a small basic protein VP2 (ORF3) [25-26]. Co-
and post-translational processing of the mature polyprotein precursor by the virus-
encoded 3CL protease (NV 3CLpro) generates six mature non-structural proteins.
Norovirus 3CL protease (NV 3CLpro) plays a pivotal role in the life cycle of
norovirus through the cleavage of the viral polyprotein and is essential for viral
replication. NV 3CL pro is therefore an attractive target for the development of norovirus
therapeutics. NV 3CLpro is a chymotrypsin-like cysteine protease with an active site
comprised of a prototypical catalytic triad (Cys139-His30-Glu54). The protease
functions as an induced fit enzyme and has an extended binding cleft [27-29]. The
substrate specificity of the protease is for a P1 Gln [30] residue (or Gln surrogate) that
engages in critical H-bonding interactions with Thr134 and His157 located in close
proximity to the active site.
2. Results and Discussion
2.1 Inhibitor design rationale
NV 3CLpro has been the focus of exploratory investigations by us [31-36] and
others [29, 37] as a potential druggable target for the development of anti-norovirus
small molecule drugs. Inhibitors of NV 3CLpro reported by us include peptidyl and
macrocyclic transition state (TS) inhibitors and TS mimics shown to be effective in
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enzyme and cell-based assays, as well as efficacious in the mouse model of murine
norovirus infection [31]. Reduction of the peptidyl character of an inhibitor typically
enhances proteolytic stability, cellular permeability, and oral bioavailability.
Depeptidization of an inhibitor can be accomplished via the construction of a
macrocyclic inhibitor or a peptidomimetic capable of orienting recognition elements in a
specific vector relationship, thereby exploiting binding interactions with active site
residues. We hypothesized that the transformation of a peptidyl inhibitor to
peptidomimetic (I) (Figure 1) can be accomplished using a functionalized heterocyclic
ring (an oxazolidinone). The presence of a ring chiral center was furthermore
anticipated to provide directional control for optimal interactions between the S3-S4
subsites and recognition element R3. Since previous studies [31] showed that NV
3CLpro has a strong preference for a cyclohexylalanine (Cha) or Leu as the P2 residue
and a Gln or Gln surrogate [38] as the P1 residue, these recognition elements were
incorporated in the structure of inhibitor (I). The design, synthesis, and evaluation of a
series of oxazolidinone-derived inhibitors of NV 3CL protease are described herein.
2.2 Chemistry
Compounds 2a-c were synthesized via the epoxidation of the precursor alkenes with m-
chloroperbenzoic acid to yield the corresponding epoxide which was then treated with
(L) Leu-OCH3 or (L) Cha-OCH3 in trifluoroethanol (TFE) to yield compounds 3a-d
(Scheme 1). Reaction with carbonyl diimidazole (CDI) in dry THF yielded oxazolidinone
derivatives 4a-d which were hydrolyzed with LiOH in aqueous THF to yield the
corresponding acids 5a-d. Compounds 5a-d were coupled with a glutamine surrogate
[38] using EDCI/HOBt/DIEA/DMF to yield compounds 6a-d which, upon reduction with
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lithium borohydride, generated the corresponding alcohols 7a-d. Oxidation of the
alcohols using Dess-Martin periodinane yielded aldehydes 8-11. The corresponding
bisulfite adducts 12-15 were readily prepared by treating the precursor aldehydes 8-11
with NaHSO3/EtOAc/EtOH/H2O. The final compounds are listed in Table 1.
2.3 Biochemical studies
The inhibitory activity of the synthesized compounds against NV 3CLpro and
their anti-norovirus activity in a cell-based replicon system were evaluated as described
in the experimental section and the IC50, EC50, and CC50 values, are listed in Table 1.
These are the average of at least two determinations.
Inspection of the results shown in Table 1 reveals that oxazolidinone derivatives
are permeable and, furthermore, some of them display single digit potency (Table 1,
compounds 9, 13, 10, and 14). Considering that these compounds were screened as
mixtures of diastereomers, a further gain in potency may be realized by separating and
screening the individual diastereomers. Compounds with a P2 Cha were about 2-fold
more potent than those with a P2 Leu, confirming previous findings [31]. The position of
the phenyl ring, and by extension of the ring substituent, which are probably
accommodated in the S4 subsite of the enzyme, appear to impact potency moderately
(vide infra).
The mechanism of action and mode of binding of these compounds to the active
site of NV 3CLpro were elucidated by determining a high-resolution X-ray crystal
structure of inhibitor 8B bound to the enzyme. Examination of the active site revealed
the presence of prominent difference electron density consistent with inhibitor 8B
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covalently bound to Cys 139 (Figure 2A). However, the m-chlorobenzyl group of the
inhibitor (Figure 2B) was disordered and could not be modeled. The hydrogen bond
interactions between NV 3CLpro and 8B are shown in Figure 3A. The expected H-
bonds between the γ-lactam ring of the Gln surrogate with His157 and Thr134 are
clearly evident. Furthermore, the backbone hydrogen bonds with Ala158 and Ala160
which serve to correctly position the inhibitor in relation to the catalytic residues and
facilitate the reaction of Cys139 with the aldehyde warhead, are also evident. The
negatively-charged oxygen of the tetrahedral adduct is stabilized by a hydrogen bond
with His30. Dipeptidyl aldehyde inhibitors lacking an oxazolidinone ring form an
additional backbone H-bond with Gln110, which is not possible with the oxazolidinone
inhibitors (since the oxazolidinone ring N lacks a hydrogen) [31]. Comparison of a
previously determined inhibitor bound NV 3CLpro structure of the same crystal form
(5T6D) [39] revealed a high degree of similarity with an RMSD deviation of 0.71 Å (154
residues) between Cα atoms using GESAMT (Figure 3B) [40]. However, significant
differences are observed in β-strands bII and cII (Gly 92 to Leu 121) which contain Gln
110. The binding of 8B results in the displacement of Gln 110 by 6.89 Å relative to the
5T6D structure (Figure 4). As noted above, the m-chlorobenzyl group of 8B was
disordered and could not be modeled, however, modeling this portion of the inhibitor in
an idealized position would result in a steric clash with Gln 110 (Figure 5). It is surmised
that this results in the observed conformational change in the bII and cII strands. It is,
however, unclear how the conformational changes induced by the oxazolidinone ring of
inhibitor 8B impact potency since inhibitor 8B forms the same number of hydrogen
bonds as acyclic inhibitor 5T6D (Figure 3A-B). A likely explanation for this is that the
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active site conformational changes induced by inhibitor 8B result in sub-optimal
positioning of the inhibitor warhead for reaction with the active site Cys139.
3. Conclusion
There is currently a pressing need for the development of norovirus-specific
therapeutics and prophylactics for the management of norovirus infections. The studies
described herein disclose the structure-based design of the first series of oxazolidinone-
based peptidomimetic inhibitors of norovirus 3CL protease. Insights gained from these
studies have laid a solid foundation for conducting further optimization of potency and
PK characteristics.
4. Experimental section
4.1 General
Reagents and dry solvents were purchased from various chemical suppliers
(Aldrich, Oakwood chemicals, Acros Organics, Chem Impex, TCI America, Bachem,
and Fisher) and were used as obtained. Silica gel (230-450 mesh) used for flash
chromatography was purchased from Sorbent Technologies (Atlanta, GA). Thin layer
1
chromatography was performed using Analtech silica gel plates. The H spectra were
recorded in CDCl3 or DMSO-d6 on a Varian XL-400 NMR spectrometer. High resolution
mass spectra (HRMS) were 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 or a G6230B TOF MS
(Agilent Technologies, Santa Clara, CA). Visualization was accomplished using UV light
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and/ or iodine. All final compounds were purified to ≥95% purity as assessed by HPLC
and fully characterized by 1H NMR and HRMS.
4.1.1. Synthesis of compound 1 a–b. General procedure.
To a solution of alcohol (35 mmol) in anhydrous DMF (45 mL) was added sodium
hydride (1.09 g; 45.5 mmol) portionwise and the reaction mixture was stirred for 15 min
at room temperature, followed by the addition of allyl bromide (4.23 g; 35 mmol) and
stirring the reaction mixture overnight at room temperature. The solvent was removed in
vacuo and the residue was partitioned between ethyl acetate (150 mL) and 5% aqueous
HCl (2 x 50 mL). The layers were separated and the organic layer was washed with
saturated aqueous NaHCO3 (2 x 50 mL), followed by saturated NaCl (50 mL). The
organic layer was dried over anhydrous Na2SO4, filtered, and concentrated to yield an
oily product.
1
4.1.1.1. 1-(allyloxy)-3-chlorobenzene 1a. Oil (yield 83%); H NMR (400 MHz, CDCl3)
δ ppm 4.58-4.62 (d, J = 11.02, 2H), 5.46-5.31 (m, 2 H), 6.18-6.04 (m, 1 H), 7.35-7.03
(m, 4H). HRMS (ESI) calcd for C9H10ClO: [M+H] +: 169.0420 Found 169.0437.
1
4.1.1.2. 1-[(allyloxy)methyl]-3-chlorobenzene 1b. Oil (yield 84%); H NMR (400 MHz,
CDCl3) δ ppm 4.10-4.02 (d, J = 10.12, 2H), 4.78-4.73 (s, 2H), 5.38-5.28 (m, 2 H), 6.10-
6.05 (m, 1 H), 7.40-7.12 (m, 4H). HRMS (ESI) calcd for C10H12ClO: [M+H] +: 183.0577
Found 183.0595.
4.1.2. Synthesis of compounds 2a-c. General procedure.
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To a solution of the appropriate alkene (35 mmol) in dry DCM (100 mL) was
added m-CPBA (42 mmol) at 0oC. The ice bath was removed and the reaction mixture
was stirred at room temperature overnight (18 h). The consumption of starting material
was monitored by TLC. The reaction mixture was cooled to 0 – 5oC and quenched with
aqueous 1N NaOH solution (15 mL). The reaction mixture was partitioned between
water (100 mL) and DCM (100 mL) and the layers were separated. The aqueous layer
was extracted with DCM (100 mL) and the combined organic layers were washed with
brine (50 mL). The organic layer was dried over anhydrous Na2SO4, filtered and
concentrated to yield an oily product.
1
4.1.2.1. 2-(3-chlorobenzyl)oxirane 2a. Oil (yield (88%); H NMR (400 MHz, CDCl3)
δ ppm 2.54 (dd, J = 4.93, 2.62 Hz, 1H), 2.87-2.72 (m, 1H), 3.36 (d, J = 6.77 Hz, 1H),
+
3.16-3.07 (m, 2H), 7.67-7.03 (m, 4H). HRMS (ESI) calcd for C9H10ClO: [M+H] :
169.0420 Found 169.0427.
1
4.1.2.2. 2-[(3-chlorophenoxy) methyl] oxirane 2b. Oil (yield 85%); H NMR (400 MHz,
CDCl3) δ ppm 2.75 (dd, J = 4.90, 2.64 Hz, 1H), 3.00-2.89 (m, 1H), 3.35 (tdd, J = 5.70,
4.10, 2.86, 2.86 Hz, 1H), 3.93 (dd, J = 11.02, 5.73 Hz, 1H), 4.22 (dd, J = 11.02, 3.04 Hz,
+
1H), 7.35-7.03 (m, 4H). HRMS (ESI) calcd for C9H10ClO2: [M+H] : 185.0369 Found
185.0377.
1
4.1.2.3. 2-{[(3-chlorobenzyl) oxy] methyl} oxirane 2c. Oil (yield 84%); H NMR (400
MHz, CDCl3) δ ppm 2.61 (dd, J = 4.90, 2.64 Hz, 1H), 2.95-2.85 (m, 1H), 3.30 (tdd, J =
5.70, 4.10, 2.86, 2.86 Hz, 1H), 3.97 (dd, J = 11.02, 5.73 Hz, 1H), 4.25 (dd, J = 11.02,
3.04 Hz, 1H), 4.52 (s, 2H), 7.35-7.03 (m, 4H). HRMS (ESI) calcd for C10H12ClO2: [M+H]
+: 199.0526 Found 199.0534.
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4.1.3. Synthesis of compounds 3a–d. General procedure.
To a solution of compound 2 (29.65 mmol) in trifluoroethanol (50 mL) was added
the corresponding free amino acid ester (29.65 mmol) at room temperature. The
reaction mixture was refluxed for 6 h with stirring. The progress of the reaction was
monitored by TLC. The reaction mixture was cooled to room temperature and the
solvent was removed. The crude residue was purified by flash chromatography to yield
a colorless oil.
1
4.1.3.1. Methyl [3-(3-chlorophenyl)-2-hydroxypropyl]-L-leucinate 3a. Oil (yield 70%); H
NMR (400 MHz, CDCl3) δ ppm 1.06-0.89 (d, 6H), 1.53-1.49 (m, 1H), 1.77-1.70 (m, 2H),
3.30-2.44 (m, 4H), 3.71 (s, 3H), 4.12 (tt, J = 7.15, 7.15, 3.57, 3.57 Hz, 1H), 4.85-4.48
(m, 1H), 5.70—5-60 (br. s, 1H), 7.53-7.01 (m, 4H). HRMS (ESI) calcd for C16H25ClNO3:
[M+H] +: 314.1523 Found 314.1532.
4.1.3.2.
Methyl
(2S)-2-{[3-(3-chlorophenyl)-2-hydroxypropyl]
amino}-3-
1
cyclohexylpropanoate 3b. Oil (yield 75%); H NMR (400 MHz, CDCl3) δ ppm 1.93-0.76
(m, 13H), 2.71 (m, 4H), 3.52-3.47 (m, 1H), 3.70-3.60 (s, 3H), 3.95 (m, 1H), 5.18-5.04
+
(m, 1H), 7.49-7.03 (m, 4H). HRMS (ESI) calcd for C19H29ClNO3: [M+H] : 354.1836
Found 354.1845.
4.1.3.3. Methyl [3-(3-chlorophenoxy)-2-hydroxypropyl]-L-leucinate 3c. Oil (yield 72%);
1H NMR (400 MHz, CDCl3) δ ppm 0.96-0.89 (d, 6H), 1.52-1.49 (m, 1H), 1.78-1.72 (m,
2H), 2.84-2.61 (m, 3H), 3.68 (s, 3H), 4.24-3.98 (m, 3H), 4.85-4.78 (m, 1H), 5.70—5-60
(br. s, 1H), 7.53-7.01 (m, 4H). HRMS (ESI) calcd for C16H25ClNO4: [M+H] +: 330.1472
Found 330.1523.
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4.1.3.4. Methyl {3-[(3-chlorobenzyl) oxy]-2-hydroxypropyl}-L-leucinate 3d. Oil (yield
71%); 1H NMR (400 MHz, CDCl3) δ ppm 0.99-0.87 (d, 6H), 1.55-1.48 (m, 1H), 1.77-1.73
(m, 2H), 2.77-2.54 (m, 3H), 3.62-3.73 (m, 3 H), 3.71 (s, 3H), 4.64 (s, 2H), 5.25-5.18 (br.
s, 1H), 5.70—5-60 (br. s, 1H), 7.53-7.01 (m, 4H). HRMS (ESI) calcd for C19H27ClNO4:
[M+H] +: 344.1629 Found 344.1639.
4.1.4. Synthesis of compounds 4a–d. General procedure.
To a solution of compound 3 (21 mmol) in anhydrous THF (75 mL) kept at room
temperature was added carbonyl diimidazole (42 mmol) and the reaction mixture was
refluxed for 5 h. The disappearance of the starting material was monitored by TLC. The
reaction mixture was cooled to room temperature and the solvent was removed in
vacuo. The residue was partitioned between EtOAc (250 mL) and 5% aq HCl (100 mL).
The layers were separated and the organic layer was sequentially washed with 5% aq
HCl, saturated NaHCO3 (2x100 mL) and brine (100 mL). The organic layer was dried
over anhydrous Na2SO4, filtered and concentrated to yield a crude product which was
purified by flash chromatography.
4.1.4.1. Methyl (2S)-2-[5-(3-chlorobenzyl)-2-oxooxazolidin-3-yl]-4-methylpentanoate 4a.
Oil (yield 45%); 1H NMR (400 MHz, CDCl3) δ ppm 7.38-7.06 (m, 4H), 0.98-0.79 (d, 6H),
1.85-1.53 (m, 3H), 3.15-2.81 (m, 2H), 3.50-3.30 (m, 2H), 3.72 (s, 3H), 4.60-4.50 (m,
1H), 4.82-4.75 (m, 1H). HRMS (ESI) calcd for C17H23ClNO4: [M+H] +: 340.1316 Found
340.1321.
4.1.4.2. Methyl (2S)-2-[5-(3-chlorobenzyl)-2-oxooxazolidin-3-yl]-3-cyclohexylpropanoate
4b. Oil (yield 45%); 1H NMR (400 MHz, CDCl3) δ ppm 1.90-0.74 (m, 13H), 3.09-2.84 (m,
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2H), 3.82-3.63 (m, 2H), 4.85-4.55 (m, 2H), 7.37-7.05 (m, 4H). HRMS (ESI) calcd for
C20H27ClNO4: [M+H] +: 380.1629 Found 380.1642.
4.1.4.3.
Methyl(2S)-2-{5-[(3-chlorophenoxy)
methyl]-2-oxooxazolidin-3-yl}-4-
1
methylpentanoate 4c. Oil (yield 44%); H NMR (400 MHz, CDCl3) δ ppm 1.03-0.86 (d,
6H), 1.85-1.78 (m, 1H), 1.50-1.47 (m, 1H), 3.05-2.88 (m, 4H), 3.76 (s, 3H), 3.80-3.70
(m, 1H), 4.60-4.50 (m, 1H), 7.45-7.14 (m, 4H). HRMS (ESI) calcd for C17H23ClNO5:
[M+H] +: 356.1265 Found 356.1284.
4.1.4.4.
Methyl(2S)-2-(5-{[(3-chlorobenzyl)
oxy]
methyl}-2-oxooxazolidin-3-yl)-4-
1
methylpentanoate 4d. Oil (yield 40%); H NMR (400 MHz, CDCl3) δ ppm 1.05-0.76 (d,
6H), 1.87-1.77 (m, 1H), 1.50 (m, 1H), 2.95-2.85 (m, 4H), 3.73 (s, 3H), 3.77-3.60 (m, 1H),
4.59-4.50 (m, 1H), 4.76-4.68 (s, 3H), 7.42-7.16 (m, 4H). HRMS (ESI) calcd for
C18H25ClNO5: [M+H] +: 370.1421 Found 370.1437.
4.1.5. Synthesis of compounds 5a–d. General procedure.
A solution of ester 4 (9 mmol) in tetrahydrofuran (25 mL) was treated with 1M
aqueous LiOH (25 mL). The reaction mixture was stirred for 3 h at room temperature
while monitoring the disappearance of the ester by TLC. Most of the solvent was
evaporated off and the solution was acidified to pH ~3 using 5% hydrochloric acid (10
mL). The aqueous layer was extracted with ethyl acetate (2 x 100 mL) and the
combined organic layers were washed with brine (50 mL). The organic layer was dried
over anhydrous sodium sulfate, filtered, and concentrated to yield compound 5.
4.1.5.1. (2S)-2-[5-(3-chlorobenzyl)-2-oxooxazolidin-3-yl]-4-methylpentanoic acid 5a.
White solid (yield 93%); mp 129-1300C, 1H NMR (400 MHz, CDCl3) δ ppm 7.57-6.98 (m,
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4H), 5.06-4.67 (m, 1H), 4.77-4.42 (m, 1H), 3.34-2.72 (m, 3H), 3.48 (d, J = 7.94 Hz, 1H),
1.96-1.37 (m, 5H), 1.05-0.83 (d, 6H). HRMS (ESI) calcd for C16H20ClNNaO4: [M+Na] +:
348.0979 Found 348.0995.
4.1.5.2. (2S)-2-[5-(3-chlorobenzyl)-2-oxooxazolidin-3-yl]-3-cyclohexylpropanoic acid 5b.
Viscous oil (yield 94%); 1H NMR (400 MHz, CDCl3) δ ppm 1.03-0.74 (m, 6H), 1.26 (t, J =
7.16, 7.16 Hz, 2H), 1.85-1.39 (m, 5H), 2.50-2.25 (m, 2H), 3.00-2.75 (m, 2H), 3.80-3.65
(m, 1H), 4.54-4.39 (m, 1H), 4.87-4.76 (m, 1H), 7.38-7.01 (m, 4H). HRMS (ESI) calcd for
C19H25ClNNaO4: [M+Na] +: 388.1292 Found 388.1309.
4.1.5.3. 2-{5-[(3-chlorophenoxy) methyl]-2-oxooxazolidin-3-yl}-4-methylpentanoic acid
1
5c. Viscous oil (yield 95%); H NMR (400 MHz, CDCl3) δ ppm 1.03-0.86 (d, 6H), 1.85-
1.78 (m, 1H), 1.50-1.47 (m, 1H), 3.05-2.88 (m, 4H), 3.80-3.70 (m, 1H), 4.60-4.50 (m,
1H), 7.45-7.14 (m, 4H). HRMS (ESI) calcd for C16H21ClNO5: [M+H] +: 342.1108 Found
342.1125.
4.1.5.4. 2-(5-{[(3-chlorobenzyl) oxy] methyl}-2-oxooxazolidin-3-yl)-4-methylpentanoic
1
acid 5d. Viscous oil, yield (95%), H NMR (400 MHz, CDCl3) δ ppm 1.05-0.76 (d, 6H),
1.87-1.77 (m, 1H), 1.50 (m, 1H), 2.95-2.85 (m, 4H), 3.77-3.60 (m, 1H), 4.59-4.50 (m,
1H), 4.76-4.68 (s, 3H), 7.42-7.16 (m, 4H). HRMS (ESI) calcd for C17H23ClNO5: [M+H] +:
356.1265 Found 356.1278.
4.1.6. Synthesis of compounds 6a-d. General procedure.
To a solution of 5 (8.2 mmol) in dry DMF (20 mL) was added EDCI (10.6 mmol,
1.3 eq.) and HOBt (10.6 mmol, 1.3 eq) and the mixture was stirred for 30 minutes at
14

ACCEPTED MANUSCRIPT
room temperature. In a separate flask, a solution of boc-deprotected glutamine
surrogate (8.2 mmol) in dry DMF (20 mL) cooled to 0-5ºC was treated with
diisopropylethylamine (32.8 mmol, 4.0 eq) and the solution was stirred for 30 minutes. It
was then added to the solution above and the reaction mixture was stirred for 16 h while
monitoring the reaction by TLC. The solvent was removed in vacuo and the residue was
partitioned between ethyl acetate (200 mL) and 10% citric acid (75 mL). The layers
were separated and the organic layer was washed with saturated aqueous NaHCO3 (2 x
75 mL), followed by saturated NaCl (75 mL). The organic layer was dried over
anhydrous Na2SO4, filtered, and concentrated. The crude residue was purified by flash
chromatography to yield the product as a white solid.
4.1.6.1.
Methyl
(2S)-2-{(2S)-2-[5-(3-chlorobenzyl)-2-oxooxazolidin-3-yl]-4-
methylpentanamido}-3-[(S)-2-oxopyrrolidin-3-yl] propanoate 6a. diastereomers A and B
were separated using flash chromatography. (A) White solid (yield 32%); mp 47-480C,
1H NMR (400 MHz, CDCl3) δ ppm 0.93 (ddd, J = 25.59, 17.70, 6.33 Hz, 6H), 1.63 (td, J
= 14.14, 7.53, 7.53 Hz, 1H), 1.85 (ddd, J = 13.52, 7.86, 2.99 Hz, 2H), 2.37 (m, 4H), 2.89
(dd, J = 14.02, 6.52Hz, 1H), 3.12 (dd, J = 14.09, 7.06 Hz, 1H), 3.50-3.31 (m, 2H), 3.69-
3.50 (m, 4H), 3.77-3.67 (s, 3H), 4.33 (d, J = 6.05 Hz, 1H), 4.88-4.54 (m, 1H), 6.63 (d,
1H), 7.49-7.12 (m, 4H), 8.34 (d, J = 4.87Hz, 1H). HRMS (ESI) calcd for
C24H32ClN3NaO6: [M+Na] +: 516.1877 Found 516.1981.
(B) White solid (yield 25%); mp 51-520C, 1H NMR (400 MHz, CDCl3) δ ppm 0.95 (d, J =
20.12 Hz, 6H), 1.72-1.66 (td, J = 14.14, 7.53, 7.53 Hz, 1H), 1.90-1.83 (dd, J = 11.14,
7.12 Hz, 2H), 2.50-2.37 (m, 4H), 2.90-2.79 (dd, J = 14.02, 6.52Hz, 1H), 3.26-3.11 (d,
15

ACCEPTED MANUSCRIPT
1H), 3.45-3.32 (m, 2H), 3.68-3.52 (m, 4H), 3.72-3.68 (s, 3H), 4.51 (d, J = 6.25 Hz, 1H),
4.90-4.85 (m, 1H), 6.83 (d, 1H), 7.51-7.22 (m, 4H), 8.34 (d, J = 4.82 Hz, 1H). HRMS
(ESI) calcd for C24H32ClN3NaO6: [M+Na] +: 516.1877 Found 516.1862.
4.1.6.2.
Methyl
(2S)-2-{(2S)-2-[5-(3-chlorobenzyl)-2-oxooxazolidin-3-yl]-3-
cyclohexylpropanamido}-3-[(S)-2-oxopyrrolidin-3-yl] propanoate 6b. White solid (yield
58%); mp 88-890C, 1H NMR (400 MHz, CDCl3) δ ppm 1.34-0.77 (m, 11H), 1.67 (m, 2H),
3.11-2.66 (m, 4H), 2.68-2.27 (m, 4H), 3.54-3.25 (m, 4H), 3.72 (s, 3H), 5.01-4.36 (m,
2H), 7.43-7.08 (m, 4H), 9.85-9.45 (br. s, 1H). HRMS (ESI) calcd for C27H36ClN3NaO6:
[M+Na] +: 556.2190 Found 556.2204.
4.1.6.3. Methyl (2S)-2-[(2S)-2-{5-[(3-chlorophenoxy) methyl]-2-oxooxazolidin-3-yl}-4-
methylpentanamido]-3-{(S)-2-oxopyrrolidin-3-yl} propanoate 6c. White solid (yield 65%);
mp 45-460C,1H NMR (400 MHz, CDCl3) δ ppm 1.08-0.89 (d, 6H), 1.53-1.47 (m, 1H)
1.93-1.71 (m, 2H), 2.51-2.25 (m, 2H), 3.37 (dd, J = 6.18, 3.43 Hz, 2H), 3.57 (dd, J =
8.95, 6.07 Hz, 2H), 3.79-3.71 (s, 3H), 4.12 (dtd, J = 14.73, 10.42, 10.40, 5.24 Hz, 2H),
4.68 (t, J = 7.77, 7.77 Hz, 1H), 4.89 (m, 1H), 7.01-6.65 (m, 2H), 7.31-7.12 (m, 2H), 8.44
(d, J = 6.05 Hz, 1H). HRMS (ESI) calcd for C24H32ClN3NaO7: [M+Na] +: 532.1826 Found
532.1730.
4.1.6.4. Methyl (2S)-2-[(2S)-2-(5-{[(3-chlorobenzyl) oxy] methyl}-2-oxooxazolidin-3-yl)-4-
methylpentanamido]-3-{(S)-2-oxopyrrolidin-3-yl} propanoate 6d. White solid (yield 68%);
1
mp 47-480C, H NMR (400 MHz, CDCl3) δ ppm 1.04-0.79 (d, 6H), 1.91-1.53 (m, 3H),
2.48-2.13 (m, 4H), 3.42-3.18 (m, 4H), 3.81-3.60 (m, 2H), 3.79-3.71 (s, 3H), 4.12 (d, J =
7.14 Hz, 1H), 4.42-4.31 (m, 2H), 4.65-4.49 (s, 2H), 6.36 (br.s, 1H), 7.36-7.13 (m, 4H),
16

ACCEPTED MANUSCRIPT
8.15 (d, J = 6.48 Hz, 1H). HRMS (ESI) calcd for C25H34ClN3NaO7: [M+Na] +: 546.1983
Found 546.1894.
4.1.7. Synthesis of compounds 7a–d. General procedure.
To a solution of ester (1.87 mmol) in anhydrous THF (10 mL) was added
dropwise lithium borohydride (2M in THF, 2.8 mL, 5.61 mmol) followed by anhydrous
methanol (10 mL), and the reaction mixture was stirred at room temperature overnight.
The reaction mixture was then acidified by adding 5% aqueous HCl until the pH of the
solution was ~3. 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 the desired alcohol.
4.1.7.1.
(2S)-2-[5-(3-chlorobenzyl)-2-oxooxazolidin-3-yl]-N-{(S)-1-hydroxy-3-[(S)-2-
oxopyrrolidin-3-yl] propan-2-yl}-4-methylpentanamide 7a. (A) White solid (yield 93%);
1
mp 59-600C, H NMR (400 MHz, CDCl3 δ 1.02-0.71 (d, 6H), 2.06-1.38 (m, 4H), 2.67-
2.17 (m, 4H), 2.98-2.75 (m, 4H), 3.55-3.05 (m, 4H), 3.74-3.45 (m, 1H), 3.87 (t, J = 8.75,
8.75 Hz, 1H), 4.36 (m, 1H), 5.06-4.63 (m, 1H), 6.41-5.91 (d, 1H), 7.51-6.93 (m, 4H),
8.20-7.78 (d, 1H). HRMS (ESI) calcd for C23H32ClN3NaO5: [M+Na] +: 488.1928 Found
488.1835.
1
(B) White solid (yield 95%); mp 65-660C, H NMR (400 MHz, CDCl3) δ 0.94-0.85 (d,
6H), 2.36-1.72 (m, 4H), 2.57-2.25 (m, 4H), 3.02-2.85 (m, 4H), 3.60-3.15 (m, 4H), 3.75-
3.45 (m, 1H), 3.92-3.88 (t, J = 8.68, 8.68 Hz, 1H), 4.52 (m, 1H), 5.12-4.78 (m, 1H), 6.45-
6.12 (d, 1H), 7.55-7.03 (m, 4H), 8.01-7.94 (d, 1H). HRMS (ESI) calcd for
C23H32ClN3NaO5: [M+Na] +: 488.1928 Found 488.1835.
17

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4.1.7.2. (2S)-2-[5-(3-chlorobenzyl)-2-oxooxazolidin-3-yl]-3-cyclohexyl-N-{(S)-1-hydroxy-
3-[(S)-2-oxopyrrolidin-3-yl] propan-2-yl} propanamide 7b. White solid (yield 94%); mp
133-1340C, 1H NMR (400 MHz, DMSO-d6) δ ppm 1.20-0.76 (m, 9H), 1.81-1.53 (m, 6H),
2.78-2.68 (m, 4H), 3.42-3.13 (m, 4H), 3.93-3.78 (m, 4H), 4.46-4.26 (m, 1H), 4.95-4.75
(m, 1H), 7.17-7.09 (s, 1H), 7.27-7.14 (m, 3H), 7.97-7.88 (br. s, 1H), 9.80-9.65 (br. s,
1H). HRMS (ESI) calcd for C26H36ClN3NaO5: [M+Na] +: 528.2241 Found 528.22321.
4.1.7.3. (2S)-2-{5-[(3-chlorophenoxy) methyl]-2-oxooxazolidin-3-yl}-N-[(S)-1-hydroxy-3-
{(S)-2-oxopyrrolidin-3-yl} propan-2-yl]-4-methylpentanamide 7c. White solid (yield 93%);
1
mp 58-590C, H NMR (400 MHz, CDCl3 δ 1.03-0.82 (d, 6H), 1.55-1.50 (m, 2H), 2.65-
2.22 (m, 6H), 3.30-3.21 (m, 2H), 3.80-3.50 (m, 4H), 4.39-4.05 (m, 2H), 5.02-4.76 (m,
1H), 7.07-6.90 (m, 1H), 7.46-7.13 (m, 3H), 8.21-8.01 (br. s, 1H). HRMS (ESI) calcd for
C23H32ClN3NaO6: [M+Na] +: 504.1877 Found 504.1804.
4.1.7.4.
(2S)-2-(5-{[(3-chlorobenzyl)
oxy]
methyl}-2-oxooxazolidin-3-yl)-N-[(S)-1-
hydroxy-3-{(S)-2-oxopyrrolidin-3-yl} propan-2-yl]-4-methylpentanamide 7d. White solid
1
(yield 93%); H NMR (400 MHz, CDCl3 δ 1.14-0.79 (d, 6H), 1.50 (dd, J = 28.65, 20.93
Hz, 2H), 2.71-2.20 (m, 6H), 3.34 (m, 2H), 3.82-3.45 (m, 4H), 4.36-3.85 (m, 2H), 4.52 (s,
2H), 5.13-4.78 (m, 1H), 7.07-6.70 (m, 1H), 7.40-7.10 (m, 3H), 8.20-7.93 (br. s, 1H).
HRMS (ESI) calcd for C24H33ClN3O6: [M-H] +: 494.2058 Found 494.2402.
Synthesis of aldehydes 8-11. General procedure.
A
representative alcohol (2.07 mmol) was dissolved in anhydrous
dichloromethane (30 mL) under a nitrogen atmosphere and cooled to 0oC. Dess-Martin
periodinane (1.75 g, 4.14 mmol, 2.0 eq.) was added to the reaction mixture with stirring.
18

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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 40 mM sodium thiosulfate in saturated aqueous NaHCO3 (50 mL) was added
and the solution was stirred for another 15 minutes. The aqueous layer was removed
and the organic layer was washed with sodium bicarbonate (25 mL), water (2 x 25 mL)
and brine (25 mL). The organic layer was dried over anhydrous sodium sulfate, filtered
and concentrated. The crude aldehyde was purified by flash chromatography (silica
gel/methylene chloride/ethyl acetate/methanol).
(2S)-2-[5-(3-chlorobenzyl)-2-oxooxazolidin-3-yl]-4-methyl-N-[(S)-1-oxo-3-{(S)-2-
oxopyrrolidin-3-yl} propan-2-yl] pentanamide (8): (A) White solid, mp 60-610C, yield
1
(75%), H NMR (400 MHz, CDCl3) δ ppm 1.05-0.81 (d, 6H), 1.32-1.27 (m, 1H), 1.75-
1.49 (m, 2H), 2.38-2.12 (m, 4H), 2.74-2.54 (m, 4H), 3.21-3.13 (m, 2H), 3.40 (d, J = 4.42
Hz, 1H), 3.61 (d, J = 39.11 Hz, 1H), 4.29-4.20 (m, 1H), 6.16-5.99 (m, 1H), 7.34-7.09 (m,
4H), 8.55-8.45 (d, 1H), 9.48 (s, 1H), 9.78-9.76 (br. s, 1H). HRMS (ESI) calcd for
C23H29ClN3O5: [M-H] +: 462.1796 Found 462.2080.
1
(B) White solid, mp 54-550C ,yield (70%), H NMR (400 MHz, CDCl3) δ ppm 0.98-0.77
(d, 1H), 1.60 (ddd, J = 15.25, 9.74, 5.00 Hz, 1H), 1.84 (ddd, J = 6.61, 5.82, 3.56 Hz,
2H), 2.60-2.40 (m, 5H), 2.99 (dd, J = 7.47, 5.78 Hz, 2H), 3.20 (dd, J = 8.77, 6.54 Hz,
2H), 3.40-3.27 (m, 2H), 3.96-3.66 (m, 1H), 4.29-3.97 (m, 1H), 4.52 (dd, J = 10.39, 5.38
Hz, 1H), 6.34 (s, 1H), 7.19-7.09 (m, 1H), 7.39-7.16 (m, 3H), 8.64 (s, 1H), 9.44 (s, 1H).
HRMS (ESI) calcd for C23H29ClN3O5: [M-H] +: 462.1796 Found 462.1825.
19

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(2S)-2-[5-(3-chlorobenzyl)-2-oxooxazolidin-3-yl]-3-cyclohexyl-N-[(S)-1-oxo-3-{(S)-2-
oxopyrrolidin-3-yl} propan-2-yl] propanamide (9): White solid, mp 76-770C, yield
(70%),1H NMR (400 MHz, CDCl3) δ ppm 1.15-0.75 (m, 9H), 2.17-1.37 (m, 6H), 2.51-
2.31 (m, 2H), 3.09-2.89 (m, 4H), 3.47-3.24 (m, 2H), 3.94-3.67 (m, 2H), 4.27-4.09 (m,
1H), 4.71-4.38 (m, 1H), 5.02-4.71 (m, 1H), 7.37-7.05 (m, 4H), 8.75-8.57 (d, 1H), 9.50-
9.46 (s, 1H). HRMS (ESI) calcd for C26H33ClN3O5: [M-H] +: 502.2109 Found 502.2212.
(2S)-2-{5-[(3-chlorophenoxy) methyl]-2-oxooxazolidin-3-yl}-4-methyl-N-[(S)-1-oxo-3-{(S)-
2-oxopyrrolidin-3-yl} propan-2-yl] pentanamide (10): White solid, mp 53-540C, yield
(65%),1H NMR (400 MHz, CDCl3) δ ppm 1.08-0.89 (d, 6H), 1.53-1.47 (m, 1H) 1.93-1.71
(m, 2H), 2.51-2.25 (m, 2H), 3.37 (dd, J = 6.18, 3.43 Hz, 2H), 3.57 (dd, J = 8.95, 6.07 Hz,
2H), 3.79-3.71 (s, 3H), 4.12 (dtd, J = 14.73, 10.42, 10.40, 5.24 Hz, 2H), 4.68 (t, J = 7.77,
7.77 Hz, 1H), 4.89 (m, 1H), 7.01-6.65 (m, 2H), 7.31-7.12 (m, 2H), 8.44 (d, J = 6.05 Hz,
1H). HRMS (ESI) calcd for C23H29ClN3O6: [M-H] +: 478.1745 Found 478.2065.
(2S)-2-(5-{[(3-chlorobenzyl) oxy] methyl}-2-oxooxazolidin-3-yl)-4-methyl-N-[(S)-1-oxo-3-
{(S)-2-oxopyrrolidin-3-yl} propan-2-yl] pentanamide (11): White solid, mp 45-460C, yield
1
(55%), H NMR (400 MHz, CDCl3) δ ppm 1.14-0.81 (d, 6H), 1.26 (t, J = 7.15, 7.15 Hz,
1H), 1.80 (ddd, J = 25.33, 24.66, 17.19 Hz, 2H), 2.41-2.10 (m, 5H), 3.43-3.20 (m, 6H),
4.62-4.43 (m, 2H), 4.70 (s, 2H), 5.70-5.58 (m, 1H), 5.94-5.86 (m, 1H), 7.44-7.11 (m,
+
4H), 8.35-8.25 (d, 1H), 9.42 (s, 1H). HRMS (ESI) calcd for C24H31ClN3O6: [M-H] :
492.1901 Found 492.2206.
Synthesis of aldehyde bisulfite salts 12-15. General procedure.
20

ACCEPTED MANUSCRIPT
To a solution of aldehyde 8-11 (0.20 mmol) in dry ethyl acetate (1.0 mL) was
added absolute ethanol (0.5 mL) with stirring, followed by a solution of sodium bisulfite
0
(0.20 mmol) in water (0.20 mL). The reaction mixture was stirred for 3 h at 50 C,
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 a yellowish oil. The oily product was
treated with ethyl ether (2 x 5 mL) to form a white solid. The white solid was sequentially
stirred with ethyl ether (3 mL) and ethyl acetate (1.5 mL) for 5 minutes. Careful removal
of the solvent using a pipette left behind the product as a white solid.
Sodium (2S)-2-{(2S)-2-[5-(3-chlorobenzyl)-2-oxooxazolidin-3-yl]-4-methylpentan amido}-
1-hydroxy-3-[(S)-2-oxopyrrolidin-3-yl] propane-1-sulfonate (12): (A) White solid, mp 99-
1000C, yield (67%),1H NMR (400 MHz, DMSO-d6) δ ppm 0.90-0.83 (d, 6H), 1.75-1.32
(m, 4H), 2.28-2.06 (m, 2H), 2.94-2.80 (m, 3H), 3.83-3.67 (m, 4H), 3.99-3.91 (m, 2H),
4.35-3.96 (m, 2H), 4.99-4.73 (m, 2H), 7.35 (d, J = 11.88 Hz, 1H), 7.75-7.62 (m, 3H),
+
8.50-8.47 (br. s, 1H). HRMS (ESI) calcd for C23H31ClN3O8S: [M-] : 544.1526 Found
544.1537.
(B) White solid, mp 103-1040C, yield (70%),1H NMR (400 MHz, DMSO-d6) δ ppm 0.92-
0.85 (d, 6H), 1.74-1.43 (m, 4H), 2.28-2.02 (m, 2H), 2.98-2.81 (m, 3H), 3.85-3.72 (m,
4H), 3.99-3.95 (m, 2H), 4.32-4.06 (m, 2H), 5.01-4.79 (m, 2H), 7.36 (d, 1H), 7.75-7.63
(m, 3H), 8.52-8.49 (br. s, 1H). HRMS (ESI) calcd for C23H31ClN3O8S: [M-] +: 544.1526
Found 544.1529.
21

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Sodium
(2S)-2-{(2S)-2-[5-(3-chlorobenzyl)-2-oxooxazolidin-3-yl]-3-cyclohexylprop
anamido}-1-hydroxy-3-[(S)-2-oxopyrrolidin-3-yl] propane-1-sulfonate (13): White solid,
1
yield (67%), mp 123-1240C, H NMR (400 MHz, DMSO-d6) δ ppm 1.29-0.96 (m, 4H),
1.79-1.46 (m, 5H), 2.29-2.05 (m, 5H), 3.17-2.90 (m, 2H), 3.78-3.66 (m, 3H), 4.35-4.27
(m, 1H), 4.96-4.82 (br. s, 1H), 5.67-5.56 (m, 1H), 7.35 (d, J = 10.46 Hz, 1H), 7.66-7.63
(m, 3H), 8.47-8.40 (br. s, 1H). HRMS (ESI) calcd for C26H35ClN3O8S: [M-] +: 584.1839
Found 584.1858.
Sodium
(2S)-2-[(2S)-2-{5-[(3-chlorophenoxy)methyl]-2-oxooxazolidin-3-yl}-4-methyl
pentanamido]-1-hydroxy-3-[(S)-2-oxopyrrolidin-3-yl] propane-1-sulfonate (14): White
solid, yield (68%), mp 69-700C, 1H NMR (400 MHz, DMSO-d6) δ ppm 0.92-0.82 (d, 6H),
1.21-1.02 (m, 1H), 2.36-1.88 (m, 7H), 3.21-2.94 (m, 5H), 4.06-3.64 (m, 2H), 4.55-4.10
(m, 2H), 5.06-4.86 (m, 2H), 7.03 (d, J = 9.48 Hz, 1H), 7.34-7.22 (m, 1H), 7.73-7.60 (m,
2H), 9.52-9.47 (br. s, 1H). HRMS (ESI) calcd for C23H31ClN3O9S: [M-] +: 560.1475 Found
560.1502.
Sodium
(2S)-2-[(2S)-2-(5-{[(3-chlorobenzyl)oxy]methyl}-2-oxooxazolidin-3-yl)-4-
(15):
methylpentanamido]-1-hydroxy-3-[(S)-2-oxopyrrolidin-3-yl]propane-1-sulfonate
White solid, mp 40-410C, yield (67%),1H NMR (400 MHz, DMSO-d6 δ 0.90-0.81 (dd, J =
18.13, 7.78 Hz, 6H), 1.71-1.40 (m, 1H), 2.21-2.00 (m, 6H), 3.20-2.90 (m, 2H), 3.64-3.54
(m, 3H), 3.78-3.64 (m, 2H), 4.55 (m, 1H), 4.85-4.75 (s, 2H), 7.39 (m, 2H), 7.71-7.63 (m,
2H), 8.51-8.46 (m, 1H). HRMS (ESI) calcd for C24H33ClN3O9S: [M-] +: 574.1632 Found
574.1658.
4.2 X-ray crystallographic studies. Crystallization and data collection.
22

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Purified norovirus 3CL protease (NV 3CLpro) (10 mg/mL) in 100 mM NaCl, 50
mM PBS buffer, pH 7.2, and 1 mM dithiothreitol (DTT) was used for the preparation of
the enzyme:8B complex. A stock solution of 100 mM inhibitor 8B was prepared in
DMSO and the NV 3CLpro:inhibitor complex was prepared by mixing 7 µL of inhibitor
8B (3mM) with 243 µL (0.49 mM) of NV 3CLpro and incubating on ice for 1 h. The buffer
was exchanged with 100 mM NaCl, 20 mM Tris pH 8.0 using a Zeba spin desalting
column (MWCO=7 kDa, Life Technologies) and the sample was concentrated to 11.0
mg/mL for crystallization screening. All crystallization experiments were conducted with
o
Compact Jr. (Rigaku Reagents) sitting drop vapor diffusion plates at 20 C using equal
volumes of protein and crystallization solution equilibrated against 75 µL of the latter.
Crystals that displayed a prismatic morphology were obtained in 2-3 days from the
Index HT screen (Hampton Research) condition G2 (25% (w/v) polyethylene glycol
3350, 100 bis-Tris pH 5.5, 200 mM Li2SO4). Samples were transferred to a fresh drop
composed of 80% crystallization solution and 20% (v/v) PEG 200 and stored in liquid
nitrogen. X-ray diffraction data were collected at the Advanced Photon Source
beamline 17-ID using a Dectris Pilatus 6M pixel array detector.
4.2.1 Structure Solution and Refinement
Intensities were integrated using XDS [41-k42] via Autoproc [43] and the Laue
class analysis and data scaling were performed with Aimless [44]. Structure solution
was conducted by molecular replacement with Phaser [45] using a previously
determined isomorphous structure of inhibitor bound NV 3CLpro (PDB: 5T6D) [39] as
the search model. Structure refinement and manual model building were conducted
with Phenix [46] and Coot [47], respectively. Disordered side chains were truncated to
23

ACCEPTED MANUSCRIPT
the point for which electron density could be observed. Structure validation was
conducted with Molprobity [48] and figures were prepared using the CCP4MG package
[49].
4.3 FRET protease assays
The FRET protease assay was performed by preparing stock solutions of the
substrate (Edans-DFHLQ/GP-Dabcyl) and inhibitor in DMSO and diluting into assay
buffer which was comprised of 20 mM HEPES buffer, pH 8, containing NaCl (200 mM),
0.4 mM EDTA, glycerol (60%), and 6 mM dithiothreitol (DTT). The protease was mixed
with serial dilutions of each compound up to 100 µM or with DMSO in 25 µL of assay
o
buffer and incubated at 37 C for 30 min, followed by the addition of 25 µL of assay
buffer containing substrate. Fluorescence readings were obtained using an excitation
wavelength of 360 nm and an emission wavelength of 460 nm on a fluorescence
microplate reader (FLx800; Biotec, Winoosk, VT) for 1 h following the addition of
substrate. Relative fluorescence units (RFU) were determined by subtracting
background values (substrate-containing well without protease) from the raw
fluorescence values, as described previously [31-36]. The dose-dependent FRET
inhibition curves were fitted with a variable slope using GraphPad Prism software
(GraphPad, La Jolla, CA) in order to determine the IC50 values of the inhibitors.
4.4 Cell-based inhibition assays
The effects of each inhibitor on virus replication were examined against NV in the
NV replicon harboring cells (HG23 cells) [31]. Briefly, confluent and semi-confluent cells
were incubated with medium containing DMSO (<0.1%) or each compound (up to 100
µM) for 48 h. After the incubation, total RNA was extracted and viral genome was
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quantitated with real-time quantitative RT-PCR (qRT-PCR). The EC50 values were
determined by GraphPadPrism software [31].
4.5 Nonspecific cytotoxic effects
The cytotoxic dose for 50% cell death (CC50) for each compound was determined
for HG23 cells. Confluent cells grown in 96-well plates were treated with various
concentrations (1-100 µM) of each compound for 72 h. Cell cytotoxicity was measured
using a CytoTox 96 nonradioactive cytotoxicity assay kit (Promega, Madison, WI) and
crystal violet staining. The in vitro therapeutic index was calculated by dividing the CC50
by the EC50.
Accession codes
Coordinates and structure factors were deposited to the Worldwide Protein Data Bank
(wwPDB) with the accession code: 5WEJ
Acknowledgements
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 a grant from the National Institute of General
Medical Sciences (P30GM110761) of the National Institutes of Health. Use of the IMCA-
CAT beamline 17-ID at the Advanced Photon Source was supported by the companies
of the Industrial Macromolecular Crystallography Association through a contract with
Hauptman-Woodward Medical Research Institute. Use of the Advanced Photon Source
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was supported by the U.S. Department of Energy, Office of Science, Office of Basic
Energy Sciences under contract no. DE-AC02-06CH11357.
Appendix A. Supplementary data
Supplementary data related to this article can be found at …….
ABBREVIATIONS USED
PK, pharmacokinetics; DMF, N,N-dimethyl formamide; DCM, dichloromethane; CDI,
carbonyl diimidazole; TLC, thin layer chromatography; EDCI, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide;
HOBt,
N-hydroxybenzotriazole;
DIEA,
diisopropylethylamine; DTT, dithiothreitol; DMSO, dimethyl sulfoxide; IC50, 50%
inhibitory concentration in the enzyme assay; EC50, 50% effective concentration in cell
culture; CC50, 50% cytotoxic concentration in cell-based assays; GESAMT, general
efficient structural alignment of macromolecular targets; rmsd, root mean-square
deviation; XDS, X-ray detector software; ESI, electron spray ionization. RFU, relative
fluorescence units;
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