Discovery of a Conformationally Constrained Oxazolidinone with Improved Safety and Efficacy Profiles for the Treatment of Multidrug-Resistant Tuberculosis

Article abstract of DOI:10.1021/acs.jmedchem.0c00500

Tuberculosis (TB) remains a serious public health challenge, and the research and development of new anti-TB drugs is an essential component of the global strategy to eradicate TB. In this work, we discovered a conformationally constrained oxazolidinone 19c with improved anti-TB activity and safety profile through a focused lead optimization effort. Compound 19c displayed superior in vivo efficacy in a mouse TB infection model compared to linezolid and sutezolid. The druggability of compound 19c was demonstrated in a panel of assays including microsomal stability, cytotoxicity, cytochrome P450 enzyme inhibition, and pharmacokinetics in animals. Compound 19c demonstrated an excellent safety profile in a battery of safety assays, including mitochondrial protein synthesis, hERG K+, hCav1.2, and Nav1.5 channels, monoamine oxidase, and genotoxicity. In a 4 week repeated dose toxicology study in rats, 19c appeared to have less bone marrow suppression than linezolid, which has been a major liability of the oxazolidinone class.

Full text of DOI:10.1021/acs.jmedchem.0c00500

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Article
The Discovery of a Conformationally Constrained
Oxazolidinone with Improved Safety and Efficacy Profiles
for the Treatment of Multidrug-Resistant Tuberculosis
Hongyi Zhao, Bin Wang, Lei Fu, Gang Li, Haijia Lu, Yuke Liu, Li Sheng, Yan Li,
Baoxi Zhang, Yang Lu, Chen Ma, Haihong Huang, Dongfeng Zhang, and Yu Lu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00500 • Publication Date (Web): 15 Jul 2020
Downloaded from pubs.acs.org on July 15, 2020
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The Discovery of a Conformationally Constrained
Oxazolidinone with Improved Safety and Efficacy
Profiles for the Treatment of Multidrug-Resistant
Tuberculosis
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†
,‡
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⊥,‡
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⊥,‡
Hongyi Zhao, Bin Wang, Lei Fu, Gang Li, Haijia Lu, Yuke Liu, Li Sheng, Yan Li,
∥
∥
∥,‡
*,†,‡
Dongfeng Zhang*,†,‡ and Yu Lu *,§
Baoxi Zhang, Yang Lu, Chen Ma, Haihong Huang,
†Beijing Key Laboratory of Active Substance Discovery and Druggability Evaluation, Institute
of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, 1
Xian Nong Tan Street, Beijing 100050, P. R. China
‡Chinese Academy of Medical Sciences Key Laboratory of Anti-DR TB Innovative Drug
Research, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of
Medical Sciences, 1 Xian Nong Tan Street, Beijing 100050, P. R. China
§Beijing Key Laboratory of Drug Resistance Tuberculosis Research, Department of
Pharmacology, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing Chest
Hospital, Capital Medical University, 97 Ma Chang Street, Beijing 101149, P. R. China
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⊥
Beijing Key Laboratory of Non-Clinical Drug Metabolism and PK/PD Study, Institute of
Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, 1
Xian Nong Tan Street, Beijing 100050, P. R. China
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Beijing Key Laboratory of Polymorphic Drugs, Institute of Materia Medica, Peking Union
Medical College and Chinese Academy of Medical Sciences, 1 Xian Nong Tan Street, Beijing
1
00050, P. R. China
KEYWORDS. oxazolidinone, multidrug-resistant tuberculosis, mitochondrial protein synthesis,
myelosuppression, antitubercular agent
ABSTRACT. Tuberculosis (TB) remains a serious public health challenge, and the research and
development of new antituberculosis drugs is an essential component of the global strategy to
eradicate TB. In this work, we discovered a conformationally constrained oxazolidinone 19c with
improved antituberculosis activity and safety profile through a focused lead optimization effort.
Compound 19c displayed superior in vivo efficacy in a mouse TB infection model compared to
linezolid and sutezolid. The druggability of compound 19c was demonstrated in a panel of assays
including microsomal stability, cytotoxicity, CYP450 enzyme inhibition and pharmacokinetics in
animals. Compound 19c demonstrated an excellent safety profile in a battery of safety assays,
+
including mitochondrial protein synthesis, hERG K , hCav1.2 and Nav1.5 channel, monoamine
oxidase, and genotoxicity. In a 4-week repeated dose toxicology study in rats, 19c appeared to
have less bone marrow suppression than linezolid, which has been a major liability of the
oxazolidinone class.
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INTRODUCTION
Tuberculosis (TB) is a major public health challenge, leading to about 1.2 million deaths in 2018.
Multidrug-resistant TB (MDR-TB) and extensively drug resistant TB (XDR-TB) have emerged
rapidly and make the treatment and prevention of tuberculosis extremely difficult. Three countries
with the largest share of the global MDR-TB burden were India (27%), China (14%) and the
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Russian Federation (9%). There is an urgent unmet medical need to develop new therapies with
a novel mechanism of action for the treatment of MDR- and XDR-TB.
Oxazolidinones is a relatively new class of antibacterial agents originally developed for the
treatment of Gram-positive bacterial infections. This class of compounds inhibits bacterial protein
synthesis by binding competitively to the 23S rRNA in the catalytic site of the bacterial 50S
ribosomes.2,3 Certain members of the oxazolidinone class have shown activity against M.
tuberculosis and are currently under clinical development for the treatment of MDR- and XDR-
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TB (Figure 1). Linezolid (1) is the first oxazolidinone approved for commercialization (2000) and
this compound has exhibited bacteriostatic activity against M. tuberculosis with a MIC of about 1
μg/mL.5,6 Linezolid is a key component of the Nix-TB regimen (bedaquiline, pretomanid and
linezolid) approved by the Food and Drug Administration (FDA) in 2019 for the treatment of
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XDR-TB or treatment-intolerant/non-responsive MDR-TB. Sutezolid (2), originally discovered
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by Upjohn company, is in Phase II studies for TB. Sutezolid differs from linezolid by having a
thiomorpholine substituent and has displayed a better antituberculosis activity and improved safety
profile compared to linezolid.9,10 In addition, delpazolid (3)11,12 and TBI-223 (4) are also under
clinical development for the treatment of TB. The development of AZD5847 (5) by AstraZeneca
for the treatment of Gram-positive infections and TB has been discontinued.14-16 The long duration
required for the treatment of TB with linezolid has been associated with severe side effects such
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as anemia, thrombocytopenia, optic and peripheral neuropathy. The toxicity of the oxazolidinone
class is thought to be mediated by the inhibition of mitochondrial protein synthesis (MPS) and
monoamine oxidase (MAO).17-19 Therefore discovery of new oxazolidinones with an improved
safety and efficacy profile is extremely important for the treatment of TB.20
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Figure 1. Chemical structure of oxazolidinones as anti-TB agents
A series of conformationally constrained tricyclic benzoxazinyl-oxazolidinones have been
reported to have excellent pharmacokinetics profile and potent antibacterial activity against Gram-
positive bacteria.21,22 These reports promoted us to take this novel series as lead to optimize for
antituberculosis activity. In our previous work, we have identified a novel series of
conformationally constrained oxazolidinones, as exemplified by compound 6, for the treatment of
MDR-TB.23 These compounds contain a common 3a,4-dihydro-1H,3H-benzo[b]oxazolo[3,4-
d][1,4]oxazin-1-one core and exhibited an improved pharmacokinetic profile compared to
linezolid. However, the in vitro anti-TB activity and MPS inhibitory activity of 6 were similar to
linezolid (Figure 2). In order to identify an oxazolidinone with improved anti-TB activity, reduced
toxicity and improved drug-like properties, we undertook a focused lead optimization effort based
on the 3a,4-dihydro-1H,3H-benzo[b]oxazolo[3,4-d][1,4]oxazin-1-one scaffold (Figure 2). The
stereochemistry of the core structure was first investigated, which indicated that the 3S,3aS
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configuration was optimal for antituberculosis activity. Further optimization was centered around
the (3S,3aS)-3a,4-dihydro-1H,3H-benzo[b]oxazolo[3,4-d][1,4]oxazin-1-one core and focused on
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the variation of the R group on the phenyl ring and the R group on the oxazolidinone ring.
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Morpholine, 2-oxa-6-azaspiro[3.3]heptane, 4,4-difluoro-piperidine and thiomorpholine were
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introduced to R position. In addition, the known sulfoxide and sulfone metabolites of the
thiomorpholine were also prepared. To increase the metabolic stability of the thiomorpholine
group, a bridge-thiomorpholine was also prepared with the goal to increase steric hindrance. At
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the R position, various substituents, including amides, heterocycles, carbamates and sulfonamides
were introduced. Compound 19c was identified through this effort as a promising development
candidate for the treatment of MDR-/XDR-TB.
Figure 2. Lead optimization strategy of the 3a,4-dihydro-1H,3H-benzo[b]oxazolo[3,4-
d][1,4]oxazin-1-one
CHEMISTRY
As shown in Scheme 1, the key epoxides 9a−d were synthesized from but-2-ene-1,4-diol (7a
and 7b). 7a was reacted with trityl chloride (TrCl) in the presence of triethylamine and 4-
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dimethylaminopyridine (DMAP) to afford compound 8a. (E)-but-2-ene-1,4-diol (7b) reacted with
TrCl in the presence of 2,4,6-collidine and tetrabutylammonium iodide (TBAI) to yield compound
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8b. Compounds 8a and 8b were converted to the racemic epoxides 9a and 9b via oxidation
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reaction using 3-chloroperoxybenzoic acid (m-CPBA). A Sharpless asymmetric epoxidation
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reaction was carried out to convert 8a and 8b into epoxides 9c and 9d respectively. Nucleophilic
substitution of 10a and 10b with different amines afforded compounds 11a−f, followed by
reduction of the nitro group using catalytic hydrogenation or Zinc/acetic acid, and then the aniline
product was protected with benzyl chloroformate (CbzCl) to afford compounds 12a−f (Scheme 2).
Scheme 1. Synthesis of Compounds 9a−da
a
o
Reagents and conditions: (i) TrCl, Et
3
N, DMAP, DCM, 0 C-rt for 8a; TrCl, 2,4,6-Collidine,
o
o
TBAI, DCM, 0 C-rt, for 8b; (ii) m-CPBA, DCM, 30 C; (iii) TBHP, Ti(OiPr)
4
, 4Å MS, DCM, -
o
o
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0 C to -20 C, D-(-)-DET for 9c, L-(+)-DET for 9d.
Scheme 2. Synthesis of Compounds 12a−fa
a
o
Reagents and conditions: (i) amine, N-methylmorpholine (NMM), MeCN, 80 C; (ii) H
2
, Raney
o
Ni, THF, 20-50 psi, rt for 12a, 12c−e; Zn, AcOH, THF, 30-40 C for 12b and 12f; (iii) CbzCl,
NaHCO
o
3
, THF/H O, 0 C.
2
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The synthesis of intermediates 15a−i was depicted in Scheme 3. Compound 12a reacted with
epoxides 9a−d via Mitsunobu reaction to furnish compounds 13a−d respectively. Subsequently,
treatment of compounds 13a−d with t-BuOLi or n-BuLi afforded the key tricyclic intermediates
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4a−d. Cleavage of trityl protective group yielded products 15a−d. Compounds 12b−f reacted
with epoxide 9c via Mitsunobu reaction to furnish compounds 13e−i, subsequently, compounds
5e−i were obtained via cyclization and deprotection.
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Scheme 3. Synthesis of Compounds 15a−ia
a
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3
o
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4b and 14d; n-BuLi, THF, -78 C-rt for 14c and 14e−i; (iii) TFA, DCM, rt for 15a−f and 15h;
TsOH·H O, DCM, MeOH, rt for 15g and 15i.
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The synthetic route of the target compounds 19a−l was illustrated in Scheme 4. Compounds
5a−d reacted with 4-methylbenzenesulfonyl chloride to obtain compounds 16a−d. Nucleophilic
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substitution of 16a−d with potassium phthalimide afforded compounds 17a−d, which reacted with
methylamine in MeOH to yield 18a−d. Finally, the target compounds 19a−d were prepared from
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8a−d by acetylation. Compound 19e was prepared by reaction of intermediate 15c with acetyl
chloride and Et N in DCM. The target compounds 19f−i were synthesized by amidation of 18c
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with different acyl chlorides. Compound 18c was reacted with isonicotinic acid, pyrazine-2-
carboxylic acid and MeOH in the presence of EDCI/HOBt and CDI to afford the final products
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Scheme 4. Synthesis of Compounds 19a−la
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Reagents and conditions: (i) TsCl, Et N, DMAP, DCM, 0 C-rt; (ii) potassium phthalimide,
3
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DMF, 80 C; (iii) MeNH , MeOH, reflux; (iv) acetic anhydride, pyridine, DCM, 0 C-rt; (v) acetyl
2
o
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chloride, Et
Et N, DCM, 0 C for 19g−i; isonicotinic acid, EDCI, HOBt, Et
carboxylic acid, EDCI, HOBt, Et N, DMF, rt for 19k; CDI, THF, rt, then MeOH, THF, rt for 19l.
3
N, DCM, 0 C-rt; (vi) methylsulfonyl chloride, Et
3
N, DCM, 0 C for 19f; acyl chloride,
o
3
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N, DMF, rt for 19j; pyrazine-2-
3
Scheme 5 displayed the synthesis of the target compounds 19m−o. Intermediate 16c was reacted
with MeNH in a sealed tube to furnish compound 19m. Compound 19n was obtained from the
2
2
6
reaction of 16c with tert-butyl isoxazol-3-yl-carbamate in the presence of NaH, followed by
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removing the amino-protective group using HCl in EtOAc. Nucleophilic substitution of 16c with
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sodium azide at 80 C in DMF, followed by cycloaddition reaction with bicyclo[2.2.1]hepta-2,5-
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diene, gave compound 19o.
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The synthesis of compounds 19p−s was outlined in Scheme 6. 19c could be readily acetylated
and methylated to afford compounds 19p and 19q, employing acetic anhydride/DMAP and
MeI/NaH, respectively. The corresponding sulfoxide 19r was prepared by treating 19c with NaIO ,
4
and the sulfone 19s was prepared by treating 19r with NaIO
4
at higher temperature.
Scheme 5. Synthesis of Compounds 19m−oa
a
o
Reagents and conditions: (i) MeNH
2
, MeOH, THF, sealed tube, 100 C; (ii) N-Boc-3-
o
aminoisoxazole, NaH, DMF, 0-70 C; (iii) HCl, EtOAc rt then H
2
O, NaHCO
3
, rt; (iv) NaN , DMF,
3
o
8
0 C; (v) bicyclo[2.2.1]hepta-2,5-diene, dioxane, reflux.
Scheme 6. Synthesis of Compounds 19p−sa
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a
o
Reagents and conditions: (i) acetic anhydride, DMAP, 100 C; (ii) MeI, NaH, THF, reflux; (iii)
o
NaIO
4
, MeOH, H
2
O, rt; (iv) NaIO
4
, MeOH, H O, 60 C.
2
28
The synthetic procedure of compounds 19t, 20a−g (containing bridge-thiomorpholine ), 21, 22
and 23a−d were shown in Scheme 7. Compounds 15e−h were reacted with 4-
methylbenzenesulfonyl chloride to obtain compounds 16e−h. Nucleophilic substitution of 16e−h
with ammonium hydroxide afforded compounds 18e−h. Compounds 19t, 20a, 21 and 22 were
prepared from 18e−h by the acetylation, respectively. The desired products 20b−g were
synthesized by the same sequence of steps as compounds 19g−l. The hydroxyl of 15i was reacted
with methylsulfonyl chloride to obtain compound 16i. Next, substitution of the sulphonate group
in the presence of sodium azide afforded compound 18i, followed by hydrogenation to afford
amine intermediate, and then compounds 23a−c were synthesized via condensation. Compound
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
8i underwent cycloaddition to give compound 23d. The target compounds 20h and 20i were
synthesized utilizing the synthetic route shown in Scheme 8.
Scheme 7. Synthesis of Compounds 19t, 20a−g, 21, 22 and 23a−d a
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2
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2
2
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0
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8
9
0
a
Reagents and conditions: (i) TsCl, Et N, DMAP, DCM, rt; (ii) ammonium hydroxide, THF,
3
o
o
sealed tube, 100 C; (iii) acetic anhydride, pyridine, DCM, 0 C-rt for 19t, 20a, 21 and 22; CDI,
o
THF, rt, then MeOH, THF, rt for 20b; acyl chloride, Et
EDCI, HOBt, Et N, DMF, rt for 20f; pyrazine-2-carboxylic acid, EDCI, HOBt, Et
0g; (iv) MsCl, NMM, DCM, rt; (v) NaN
pyridine, THF, 0 C-rt for 23a; acyl chloride, Et
,5-diene, dioxane, reflux.
3
N, DCM, 0 C for 20c−e; isonicotinic acid,
N, DMF, rt for
, Pd/C, THF; (vii) acetic anhydride,
N, THF, 0 C for 23b−c; (viii) bicyclo[2.2.1]hepta-
3
3
o
2
3
, DMF, 70 C; (vi) H
2
o
o
3
2
Scheme 8. Synthesis of Compounds 20h and 20ia
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a
o
Reagents and conditions: (i) N-Boc-3-aminoisoxazole, NaH, DMF, 0-70 C then HCl, EtOAc rt
o
then H
2
O, NaHCO
3
, rt; (ii) NaN , DMF, 80 C then bicyclo[2.2.1]hepta-2,5-diene, dioxane, reflux.
3
The absolute configuration of 19c was confirmed by X-ray crystallography (Figure 3). The
lattice parameters were a = 12.150, b = 26.738, c = 23.805 Å and the lattice volume was 7713.78
1
1
1
1
1
1
1
1
1
1
2
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2
2
2
2
2
2
2
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0
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1
2
3
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5
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8
9
0
1
2
3
4
5
6
7
8
9
0
3
Å . There were 8 molecules in one lattice. The configurations of chiral centers are (3S,3aS).
Figure 3. X-ray crystallographic structure of compound 19c
RESULTS AND DISCUSSION
All final compounds were evaluated for their inhibitory activity against M. tuberculosis H37Rv
in microplate alamar blue assay (MABA) and cytotoxicity against Vero cell line. Selected
compounds were also tested for their MPS inhibitory activity.
Firstly, the impact of the stereochemistry of the 3a,4-dihydro-1H,3H-benzo[b]oxazolo[3,4-
d][1,4]oxazin-1-one core on the activity was investigated. Linezolid and other oxazolidinone
currently under clinical development possess only one chiral center and the 5-S configuration is
5
known to be important for antibacterial activity. Due to two chiral centers existed in this novel
benzoxazinyl-oxazolidinone scaffold, it is essential to explore the relationship between the
configurations and anti-TB activity. As shown in Table 1, compounds containing the racemate
trans configuration (15a, 18a and 19a) appeared significantly more potent than those with the
racemate cis configuration (15b, 18b and 19b) against M. tuberculosis H37Rv. It is anticipated
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
that the (3S,3aS) isomer 19c with trans configuration appeared much more potent than its isomer
1
9d with (3R,3aR) configuration. This result is consistent with our previous finding that the
(3S,3aS) configuration is essential for activity.23
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. In Vitro Antitubercular Activity of 3a,4-Dihydro-1H,3H-benzo[b]oxazolo[3,4-
d][1,4]oxazin-1-ones Containing Thiomorpholine
H37Rv
MIC (μg/mL)
Compds
Structure
15a
3.79
1
5b
8a
>32
0.10
>32
1
18b
19a
0.16
1
9b
>32
0.04
>32
1
9c
19d
Linezolid
Sutezolid
0.88
0.12
Subsequently, further optimization was centered around the (3S,3aS)-3a,4-dihydro-1H,3H-
benzo[b]oxazolo[3,4-d][1,4]oxazin-1-one core. As shown in Table 2, analogues with various R1
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2
and R substitutions were prepared and evaluated for their antituberculosis activity, cytotoxicity
and MPS activity.
1
Among various R substitutions explored, the thiomorpholine substituted compound 19c
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
appeared to display the best antituberculosis activity with a MIC 0.03 μg/mL against the M.
tuberculosis H37Rv strain. Deletion of the fluorine on the phenyl ring of 19c to give compound
1
9t resulted in a 25-fold loss of activity, indicating the importance of fluorine substitution on the
activity. Compounds with other substitutions 21, 22 and 23a appeared less active with MICs 0.89,
0
.91 and 3.92 μg/mL respectively. The 3-thia-8-azabicyclo[3.2.1]octane substituted analogues
0a–i were designed with the intention to minimize the metabolism of thiomorpholine group.
2
However, these compounds were much less active against M. tuberculosis H37Rv strain with MIC
ranging from 0.92 to >32 μg/mL.
S-Oxidation of sulfur containing therapeutics is a well-known metabolic transformation
catalyzed by cytochrome P450s or by flavin monooxygenases.29 Pharmacokinetics and
metabolism study of sutezolid had shown that it is well absorbed orally but with significant first
8
pass metabolism to sulfoxide and to a lesser extent of sulfone metabolite. Therefore, the potential
metabolites of 19c were prepared and evaluated for antituberculosis activity against the H37Rv
strain. We found that the sulfoxide 19r and sulfone 19s of 19c maintained good antituberculosis
activity with MIC values 0.44 and 0.93 μg/mL, respectively, but less potent than 19c.
2
Further optimization of the R group around the thiomorpholine-substituted compound 19c
2
provided a series derivatives 18c and 19e–19q with a variety of substitutions at the R position.
2
When R was amino, compound 18c showed potent activity and low MPS inhibition consistent
with 19c. Several compounds, including the cyclopropanecarboxamido 19h,
cyclobutanecarboxamido 19i, methyl carbamate 19l and isoxazolamino 19n, all exhibited potent
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
antituberculosis activity with MICs similar to 19c and better than sutezolid. However, compound
2
1
9p with diacetylamine group at R had 10-fold decrease in antibacterial activity than 19c, and
2
when R was methylacetylamine or ester group, the related compounds 19q and 19e had almost
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
no activity.
1
2
The majority compounds with a variety of R and R groups exhibited low cytotoxicity against
the Vero cell line (Table 2). The 50% inhibitory concentrations (IC50) for most compounds
were >64 μg/mL. Compounds in the (3S,3aS)-3a,4-dihydro-1H,3H-benzo[b]oxazolo[3,4-
d][1,4]oxazin-1-one series appeared to have less mitochondrial protein synthesis (MPS) inhibition
compared to both linezolid and sutezolid. The MPS IC50 for compound 19c and many its analogues
were >100 μg/mL, compared to 7.98 μg/mL for linezolid and 8.17 μg/mL for sutezolid. This series
compounds appeared to have a major advantage in minimizing toxicity caused by inhibition of
mitochondrial protein synthesis.
Table 2. Antituberculosis Activity, Cytotoxicity and MPS Inhibition of Target Compounds
MICa
μg/mL)
Cytotoxicityb
IC50 (μg/mL)
MPSic
IC50 (μM)
Compds
R1
R2
X
F
F
(
1
1
8c
9c
0.03
0.03
36.05
>64
>100
>100
1
9t
19e
9f
19g
H
0.79
>64
F
F
>1
1
3.90
>64
>64
F
0.10
>100
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1
9h
F
F
0.03
0.03
>64
>64
>100
53.69
1
9i
1
9j
F
F
0.16
0.12
>64
>100
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
`19k
40.69
>100
>100
1
9l
F
F
0.03
>32
>64
19m
46.54
19n
F
F
0.03
0.23
>64
>64
>100
>100
19o
19p
F
0.34
>64
19q
F
F
F
>1
1
9r
0.44
0.93
>64
>64
60.57
25.97
19s
2
0a
0b
20c
F
F
F
0.92
1.68
1.48
58.5
>64
>64
2
20d
F
F
0.98
0.97
>64
2
0e
34.67
2
0f
F
F
2.83
>32
>64
2
0g
9.53
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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20h
0i
F
F
2.52
>32
>64
>64
2
2
1
2
F
F
F
F
0.89
0.91
3.92
3.09
>64
>64
>64
>64
51.2
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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8
9
0
1
2
3
4
5
6
7
8
9
0
2
10.12
23a
23b
23c
F
F
4.20
>32
>64
>64
23d
Linezolid
Sutezolid
INH
0.45
0.10
0.03
0.06
>64
>64
7.98
8.17
RFP
a
b
c
M. tuberculosis H37Rv; Vero cell lines; Mitochondrial protein synthesis inhibition.
Table 3. Microsome Stability of Selected Compounds
MLMa
Compds
Remaining%b
48.7
15.8
6.02
15.0
4.0
Stabilityc
stable
stable
stable
stable
stable
stable
stable
stable
stable
stable
stable
stable
stable
1
9c
18c
9g
19h
1
1
9i
9j
1
20.8
6.85
3.59
0
1
9k
9l
19n
1
1
9o
9r
21.7
100
1
Linezolid
Sutezolid
110.9
54.8
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a
b
Mouse Liver Microsome; Substrate concentrations were determined in incubations with
c
NADPH after 30 min and normalized to concentrations at time zero. Stability was determined
without NADPH cofactor.
Selective compounds with potent antituberculosis activities were further evaluated for in vitro
metabolic stability (Table 3). Compound 19c showed similar metabolic stability against mouse
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
liver microsomes compared with sutezolid. Compound 18c with an amino group at the R position
2
was less stable. Compounds 19g−l, and 19n−o, with a different R substituent such as aliphatic
amide, aryl amide or heterocycles, all displayed lower stability than 19c. While compound 19r,
the sulfoxide metabolite of 19c exhibited excellent metabolic stability.
Based on its potent activity against M. tuberculosis H37Rv, low MPS inhibition and good
microsome stability, compound 19c and its sulfoxide metabolite 19r were further evaluated against
drug-resistant TB strains (Table 4) using linezolid and sutezolid as comparators. Compound 19c
displayed a better potency than linezolid and a similar potency to sutezolid against MDR-TB
clinical isolated 12525 strain. Compound 19c was more potent than compound 19r, linezolid, and
sutezolid against the linezolid-resistant (L-R) strain, with MIC 0.24 μg/mL. It is worth noting that
there is about a 4- to10-fold increase in MIC value for linezolid-resistant (L-R) strain compared
with that for H37Rv for all four compounds 19c, 19r, linezolid and sutezolid, indicating that 19c
is likely have the same binding site as other oxazolidinones. Based on these results, compound 19c
was therefore identified as a potential development candidate for further investigation.
Table 4. In Vitro Activities of 19c and 19r against Drug-resistant TB
MIC (μg/mL)
Compds
H37Rv
12525a
L-Rb
0
0
0
0
.06
.67
.32
.12
0.03
0.24
1
9c
0.46
0.23
0.06
6.68
3.92
0.96
19r
Linezolid
Sutezolid
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
0
.03
.04
>10
0.04
0.03
INH
RFP
>10
a
b
Resistance to isoniazid (INH) and rifampicin (RFP); Resistance to linezolid.
To better understand the druggability of compound 19c, a battery of in vitro ADME assays were
performed. As shown in Table 5, compound 19c showed good membrane permeability and
excellent metabolic stability against human liver microsomes. Compound 19c also showed
excellent metabolic stability in hepatocytes from different species. Compound 19c was also tested
against several isoforms of the cytochrome P450 (CYP450) enzymes, CYP1A2, CYP2D6,
CYP2C9, CYP2C19, and CYP3A4. The IC50 values against all these CYP450 isoforms were all >
45 μM, indicating their low potential for drug-drug interactions.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 5. ADME Profiles of Compound 19c
a
CYP Inhibitionc
(IC50, µM)
Metabolic stability of hepatocytes
Caco-2
HLM
%
Species
-6
b
T
1/2 (min)
> 500
315
Clint (mL/min/million cell) Papp×10 cm/s
Human
Monkey
Dog
< 1.39
2.20
1A2 (>50)
2C9 (>50)
2C19 (48.2)
68.0
10.2
67.58
81.5
Rat
100
76.2
6.90
9.10
2D6 (>50)
3A4 (>50)
Mouse
a
Human Liver Microsome, Positive control: Dextromethorphan (T1/2 26.7 min), Midazolam
Phenacetin (T1/2 53.3 min), Diclofenac (T1/2 6.1 min), Omeprazole
(T1/2 47.8 min); Substrate concentrations were determined in incubations with NADPH after 30
min and normalized to concentrations at time zero. Positive control, for 1A2, Naphthoflavone
IC50): 0.018 µM; for 2C9, Sulfaphenazole (IC50): 0.77 µM; for 2C19, Tranylcypromine (IC50):
(T1/2 3.65 min),
b
c
(
5
.86 µM; for 2D6, Quinidine (IC50): 0.044 µM; for 3A4, Ketoconazole(IC50): 0.069 µM
Subsequently, compound 19c was further progressed into in vivo pharmacokinetic study (Table
). Compound 19c was quickly absorbed after oral administration at 25 mg/kg dose in mice, with
6
T
max value of 0.25 h, Cmax of 3 μg/mL and half-life (t1/2) of 14.7 h. The exposure of compound 19c
after oral administration was high with area under the curve (AUC) value of > 6000 ng·h/mL. As
anticipated, sulfoxide 19r and sulfone 19s were identified as the metabolites of 19c with oral
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exposure in the order of 19r>19c>19s. After intravenous administration, compound 19c and its
metabolite 19r were detected in the whole blood samples, with 19c>19r in AUC. The
concentration of metabolite 19s was extremely low (<10 ng/mL). The oral bioavailability of
compound 19c reached 55.9%, supporting the further evaluation of its in vivo efficacy via oral
administration.
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Table 6. Mouse PK Properties of Compound 19c
a
b
po (25 mg/kg)
iv (2.5 mg/kg)
19c
Parameters
19c
19r
19s
19r
t
1/2β
h
h
14.7
6.28
7.64
0.923
3.81
T
max
max
0.25
3000
6478
7621
7.59
15.2
0.50
1518
8752
9021
9.83
1.00
118
0.70
115
C
ng/mL
ng/mL *h
ng/mL *h
h
4202
1159
1194
0.540
0.691
2189
2722
AUC(0-t)
AUC(0-∞)
MRT(0-t)
MRT(0-∞)
CL
1254
1475
10.3
14.3
443
592
3.52
6.22
h
10.8
mL/h/kg
mL/kg
V
F%
55.9%
a
b
Five mice for po. Five mice for iv.
The in vivo efficacy of compound 19c was assessed in BALB/c mice in an acute TB infection
model (Table 7). Compound 19c was orally administered at 100 mg/kg/day, whereas the positive
controls linezolid and isoniazid (INH) were given at 100 mg/kg/day and 25 mg/kg/day,
respectively. After three weeks of treatment, compound 19c showed better efficacy than linezolid
at the same dose level (19c, log10CFU/lung = 1.954 vs linezolid, log10CFU/lung = 4.136).
Subsequently, a dose-response study was conducted in an acute TB model in BALB/c mice (Figure
4
). In this study, sutezolid was added as the positive control besides INH and linezolid, the efficacy
of linezolid dosed at 50 and 100 mg/kg/day, sutezolid at 25 and 50 mg/kg/day and 19c at 25, 50
and 100 mg/kg/day was evaluated. CFUs were enumerated in lungs following 3 weeks of treatment
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by oral gavage dosing 5 days of one week. We were pleased to find that 19c exhibited better
antitubercular activity than linezolid at all doses. Compound 19c also showed better efficacy than
sutezolid at the same dose levels.
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Table 7. In Vivo Efficacy of Compound 19c
Group
CMC
19c
Dosage (mg/kg)
Weight (g)
21.29±0.84
21.25±0.88
20.83±0.43
21.30±0.66
log10CFU/lung
7.102±0.152
1.954±0.159
4.136±0.498
1.929±0.213
-
100
100
25
Linezolid
INH
7
6
5
4
3
2
1
0
Figure 4. Efficacy in the Mouse Model of Acute M. tuberculosis Infection
As a potential candidate, the preliminary toxicity of compound 19c and its main metabolite 19r
were further evaluated and the results were summarized in Table 8. Both compounds were not
cytotoxic with high IC50 values >64 µg/mL against HepG2 cell line. Both compounds showed no
+
inhibition of the hERG K , hCav1.2 and Nav1.5 channels at the highest concentration tested (>30
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µM), indicating that 19c and 19r had low potential for cardiotoxicity. As the main adverse effect
of peripheral neuropathy related to inhibition of MAOs during the long-term use of oxazolidinone,
the in vitro MAO-A and MAO-B inhibition activity was conducted to assess the risk of 19c for
peripheral neuropathy toxicity. The inhibitory activity of compound 19c against MAOs was
significantly lower, with a 4-fold improvement over sutezolid, while the metabolite 19r had an
IC50 > 100 µM . These results suggested that 19c might have an improved safety profile in terms
of the risk for peripheral neuropathy. In addition, genotoxicity studies of compound 19c and its
metabolite 19r were all negative.
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1
2
2
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3
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3
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0
1
2
3
4
5
6
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9
0
1
2
3
4
5
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9
0
1
2
3
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8
9
0
1
2
3
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0
Table 8. Preliminary Toxicity Properties of Compound 19c and 19r
Cytotoxicity
HepG2
MAO- MAO-
hERG K+ hCav1.2 Nav1.5
IC50, µM
Mice Bone Marrow Chromosome Mini-
A
B
Compds
a
b
c
Micronucleus
Aberration
Ames
IC50, µg/mL
> 64
19c
Negative
Negative
> 30
> 30
> 30
> 30
> 30
> 30
44.8
> 100
13
3.2
> 100
0.7
1
9r
> 64
Sutezolid
> 64
a
b
ICR mice, at dose levels of 100, 200, 500, 1000 and 2000 mg/kg. Compounds 19c and 19r
were tested at concentrations from 20 to 150 μg/mL with or without metabolic activation (+/- S9)
c
using CHO-WBL cell. Compounds 19c and 19r were tested at concentrations from 0.005 to 1000
μg/well with or without metabolic activation (+/- S9) using the two tester strains TA98 and TA100.
To further assess the risk of compound 19c for myelosuppression, a repeated dose toxicity study
in SD rat (12 female and 12 male/group) was performed. Compound 19c was administered orally
once daily at doses of 50, 150, and 450 mg/kg/day for 4 weeks, followed by a two-week recovery
period. No mortality and significant body weight loss was observed in any of the compound 19c
groups. Compound 19c had no significant effect on food consumption, eye examination, urine
examination and neuropsychic symptoms of SD rats. The histopathology revealed no changes in
main organs after 28-day administration and 14-day recovery period in any groups. More
importantly, there were no bone marrow histopathological changes found in the highest 450
mg/kg/day dosing group. Furthermore, a hematologic analysis further confirmed that 19c had less
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myelosuppressive effect (Table 9). No significant reductions in white blood cell (WBC) and red
blood cells (RBC) counts were observed in any of the compound 19c dosing groups compared to
the vehicle control. Although the platelet (PLT) count in the 150 mg/kg/day and 450 mg/kg/day
female rat groups didn’t show statistically significant differences (P≥0.05) compared to the control
group, the drop in the mean value was observed. The decline of PLT count should be evaluated in
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0
GLP toxicity study. According to the reported data, there was a pronounced drop in WBC, RBC
and PLT counts in 100 mg/kg/day rat group of linezolid in a repeated dose toxicity study. The
results of MPS assay and repeated dose toxicity in rat demonstrated that compound 19c displayed
lower potential risk of myelosuppressive toxicity compared to linezolid.
Table 9. Repeated Dose Toxicity of 19c: Hematology Parameters in Rata
Groupb
Parameter
5
0 mg/kg/day
150 mg/kg/day
3.04±1.16
6.11±0.88
6.61±1.43
7.62±0.44
787±411
998±57
450 mg/kg/day
2.82±1.40
5.06±0.86
6.53±1.20
7.46±0.52
735±418
1038±45
7.5±1.2
vehicle
Fc 4.2±1.44
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
3.61±0.75
6.12±1.10
7.32±0.20
7.86±0.32
1051±122
1077±117
7.1±0.5
9
WBC(10 /L)
d
6.22±2.17
6.85±0.58
7.29±0.54
1094±91
955±300
7.1±0.4
1
2
RBC(10 /L)
9
PLT(10 /L)
7.4±1.1
7.0±0.4
MPV(fL)
MCV (fL)
MCH(pg)
HGB(g/L)
HCT(%)
7.0±0.6
6.9±0.6
7.0±0.4
56.5±1.3
59.7±1.5
18.5±0.5
18.7±0.6
126±10
56.6±0.6
58.5±1.3
18.8±0.5
18.5±0.5
124±27
56.5±1.3
58.7±1.0
18.7±0.5
18.7±0.4
122±22
56.1±0.8
57.5±2.0
18.5±0.6
18.3±0.4
136±7
136±10
141±11
139±9
144±5
38.7±3.1
43.5±3.5
3.25±0.72
2.97±0.49
14.8±4.5
13.0±2.6
80.7±5.2
82.0±2.9
37.4±8.0
44.5±2.9
2.89±0.53
3.15±0.49*
12.2±4.8
17.0±6.3
83.0±6.0
77.3±6.4
36.9±6.5
43.8±2.7
2.93±0.73
2.99±0.40
11.8±3.6
13.7±3.5
83.7±4.0*
80.7±5.3
41.1±1.4
45.2±1.7
2.85±0.53
2.48±0.34
18.5±5.3
15.9±3.4
76.9±4.6
77.4±3.7
%
Retic(%)
Neut(%)
%
%Lymph(%)
a
WBC, white blood cells; RBC, red blood cells; PLT, platelets; MPV, mean platelet volume;
MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; HGB, hemoglobin; HCT,
hematocrit; %Neut, neutrophils percentage; %Lymph, lymphocytes percentage; %Retic,
b
c
reticulocytes percentage. Results are expressed as the mean ± SD (n = 12 for each group). F,
d
Female rat. M, Male rat. *P<0.05 vs vehicle.
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CONCLUSION
A novel conformationally constrained oxazolidinone 19c has been identified through a focused
lead optimization effort. Compound 19c displayed potent activity against susceptible M.
tuberculosis H37Rv and drug-resistant MDR-TB isolated and linezolid-resistant strain. Further
evaluation of compound 19c revealed that this compound possesses favorable mouse and human
microsomal stability, low cytotoxicity, low liability for drug-drug interactions and an excellent PK
profile. Compound 19c exhibited better in vivo efficacy in a mouse TB model than linezolid and
sutezolid. Compound 19c exhibited an excellent safety profile in a panel of safety assessments,
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0
+
including inhibition of MPS, inhibition of hERG K , hCav1.2 and Nav1.5 channels, inhibition of
MAO-A and MAO-B enzymes, and genotoxicity assays. Compound 19c showed reduced potential
for myelosuppression in the 4-week repeated dose toxicity study. Compound 19c was therefore
selected as an antituberculosis drug candidate and currently under preclinical development.
EXPERIMENTAL SECTION
Chemistry. General. All solvents used were of analytical grade. Inert atmosphere operations
were conducted under argon in flame-dried glassware. All melting points were measured with a
1
13
micro melting point apparatus (MP-J3, Yanaco) and were uncorrected. H and C NMR spectra
were recorded on a Varian 400 NMR or 500 NMR or 600 NMR spectrometer using CDCl or
DMSO-d as a solvent and tetramethylsilane (TMS) as an internal standard. Chemical shift (δ) are
3
6
reported in parts per million (ppm) and coupling constants (J) are reported in Hertz (Hz). Splitting
patterns are indicated as follows: s, singlet; d, doublet; t, triplet; m, multiplet; brs; broad singlet.
HR-MS spectra were obtained on a ThermoFisher Exactive Plus mass spectrometer (ThermoFisher
Scientific, Bremen, Germany). Optical rotations were measured with a Rudolph Research
Analytical (Autopol IV-T). Reactions were monitored by TLC (silica gel GF254). Column
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chromatography was carried out using silica gel (200-300 mesh), and the solvent proportions were
expressed on a volume: volume basis. The purity of all final compounds (≥95%) was established
by HPLC, which was carried out on a ThermoFisher Accela HPLC system (ThermoFisher
Scientific, Bremen, Germany) with an agilent Zorbax SB-C18 column (5 μm, 2.1 × 50 mm),
column temperature 40°C; detection wavelength at 254 nm; flow rate = 0.3 mL/min; and gradient
of 5−95% MeCN in water (both containing 0.1 vol % of HCOOH) in 10 min.
Synthesis of compounds 9a-d
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0
(Z)-4-(Trityloxy)but-2-en-1-ol (8a). To a mixture of (Z)-1,4-butenediol 7a (30 g, 340 mmol),
Et
3
N (24 ml, 170 mmol) and DMAP (1 g) in DCM (250 mL) cooled with ice-water bath was slowly
added a solution of triphenylchloromethane (31 g, 113.6 mmol) in DCM (100 mL). The reaction
mixture was stirred overnight at room temperature. The mixture was washed with water and brine,
dried over anhydrous Na
2
SO , filtered and then the filtrate was concentrated in vacuo. The residue
4
was purified by column chromatography (10%~25% EtOAc in PE) to give compound 8a (27 g,
1
7
3.0%) as an off-white solid. H NMR (400 MHz, CDCl
3
) δ: 7.49−7.41 (m, 6 H), 7.36−7.18 (m, 9
H), 5.87−5.64 (m, 2 H), 4.03 (d, J = 6.0 Hz, 2 H), 3.71 (d, J = 5.6 Hz, 2 H).
E)-4-(Trityloxy)but-2-en-1-ol (8b). To a mixture of (E)-1,4-butenediol 7b (0.88 g, 10 mmol),
,4,6-Collidine (0.4 mL, 3 mmol) and TBAI (50 mg) in DCM (10 mL) cooled with ice-water bath
(
2
was added triphenylchloromethane (0.56 g, 2 mmol). The reaction mixture was stirred overnight
at room temperature. The mixture was washed with 0.5 N HCl, water and brine, dried over
anhydrous Na
2
SO , filtered and then the filtrate was concentrated in vacuo. The residue was
4
purified by column chromatography (15% EtOAc in PE) to give compound 8b (0.32 g, 48.3%) as
1
an off-white solid. H NMR (400 MHz, CDCl
3
) δ: 7.49−7.44 (m, 6 H), 7.32−7.27 (m, 6 H),
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.25−7.19 (m, 3 H), 6.10−5.69 (m, 2 H), 4.17 (d, J = 4.8 Hz, 2 H), 3.71−3.56 (m, 2 H), 1.34 (brs,
1
H).
cis-(3-((Trityloxy)methyl)oxiran-2-yl)methanol (9a). To a solution of 8a (10 g, 30.4 mmol) in
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9
0
DCM (80 mL) was added m-CPBA (6.4 g, 31.8 mmol). The reaction mixture was stirred overnight.
After filtration, the filtrate was washed with saturated sodium bicarbonate solution and brine, dried
over anhydrous Na
2
SO , filtered and concentrated. To the residue was added diethyl ether and
4
stirred for 3 h. After filtration, the filter cake was washed with diethyl ether and dried to afford 9a
1
(7.3 g, 69.2%) as a white solid. H NMR (400 MHz, CDCl
3
) δ: 7.47−7.41 (m, 6 H), 7.38−7.17 (m,
9
H), 3.65−3.47 (m, 3 H), 3.30−3.16 (m, 2 H), 3.07 (dd, J = 10.4, 5.6 Hz, 1 H) 1.87 (brs, 1 H). HR-
+
MS (ESI): m/z [M+Na] calcd for C23
H
22NaO : 369.1461, found: 369.1445.
3
trans-(3-((Trityloxy)methyl)oxiran-2-yl)methanol (9b). To a solution of 8b (2.2 g, 6.7 mmol)
in DCM (25 mL) was added m-CPBA (75%, 1.7 g, 7 mmol) in portionwise. The reaction mixture
was stirred overnight. After filtration, the filtrate was washed with saturated sodium bicarbonate
solution and brine, dried over anhydrous Na
2
SO , filtered and concentrated. To the residue was
4
added EtOAc/PE (1/5, 10 mL) and stirred for 2.5 h. After filtration, the filter cake was washed
1
with the mixture solution and dried to afford 9b (1.1 g, 47.4%) as a white solid. H NMR (400
MHz, CDCl
3
) δ: 7.48−7.42 (m, 6 H), 7.34−7.28 (m, 6 H), 7.26−7.21 (m, 3 H), 3.94 (dd, J = 12.8,
2
.4 Hz, 1 H), 3.64 (dd, J = 12.4, 4.0 Hz, 1 H), 3.38 (dd, J = 10.4, 2.4 Hz, 1 H), 3.25−3.15 (m, 2 H),
+
3
.12−3.11 (m, 1 H). HR-MS (ESI): m/z [M+Na] calcd for C23
H
22NaO : 369.1461, found:
3
369.1455.
(2R,3S)-(3-((Trityloxy)methyl)oxiran-2-yl)methanol (9c). To a four-necked flask were added
o
4
Å molecular sieve (12 g) and anhydrous DCM (330 mL). The mixture was cooled to -40 C under
argon. D-(-)-diethyl tartrate (13.6 mL, 79.2 mmol) was added, followed by titanium
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tetraisopropanolate (18.8 mL, 63.4 mmol), and the reaction mixture turned to yellow. After stirring
for 0.5 h, a solution of 8a (26.1 g, 79.2 mmol) in DCM (120 mL) was added to the mixture and
stirred for 0.5 h. A solution of tert-butyl hydroperoxide in toluene (3.8 M, 50 mL, 190 mmol) was
o
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0
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9
0
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9
0
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9
0
added and stirred overnight at -20 C. After the reaction completed monitored by TLC, tartaric acid
o
solution (10%, 200 mL) containing FeSO
4
·7H
2
O (30 g) was added and stirred at 0 C for 1 h before
layering. The organic layer was separated, and the water layer was extracted with DCM for twice.
The organic phase was combined and washed with brine for twice, filtered, and evaporated to give
a solid, which was stirred in n-hexane and filtered, followed by recrystallizing with petroleum
ether/ethyl acetate to afford 9c (15 g, 57.5%) as an off-white solid. The corresponding alcohol was
analyzed by Chiral HPLC (OD-H, 5 μm, 4.6 mm×250mmL; eluent: hexane/ethanol, 90/10; flow
o
rate: 1 mL/min; λ = 215 nm; T = 20 C): t
R
= 6.46 min (96.49%), t
R
= 12.58 min (3.51%). The
2
4
1
enantiomeric purity of 9c was determined to be 92.98% ee. [α]
D
= +31.9 (c = 1, CHCl
3
). H
NMR (400 MHz, CDCl
3
) δ: 7.48−7.41 (m, 6 H), 7.35−7.19 (m, 9 H), 3.64−3.50 (m, 3 H),
3
.30−3.15 (m, 2 H), 3.07 (dd, J = 10.4, 5.6 Hz, 1 H), 1.90 (brs, 1 H). HR-MS (ESI): m/z [M+Na]+
calcd for C23
H22NaO
3
: 369.1461, found: 369.1427.
(2S,3R)-(3-((Trityloxy)methyl)oxiran-2-yl)methanol (9d). Compound 9d (6 g, 51.3%) was
prepared from 8a in the same manner as described for 9c by replacing D-(-)-diethyl tartrate with
24
L-(+)-diethyl tartrate. The enantiomeric purity of 9d was determined to be 96.84% ee. [α]
D
= -
1
3
1.1 (c = 1, CHCl
3
). H NMR (400 MHz, CDCl
3
) δ: 7.49−7.20 (m, 15 H) 3.65−3.50 (m, 3 H),
3.28−3.17 (m, 2 H), 3.06 (dd, J = 10.4, 5.6 Hz, 1 H), 1.85 (t, J = 6.4 Hz, 1 H). HR-MS (ESI): m/z
+
[
M+Na] calcd for C23
H
22NaO : 369.1461, found: 369.1448.
3
Synthesis of compounds 12a-f
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Page 29 of 86
Journal of Medicinal Chemistry
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-Fluoro-2-nitro-5-thiomorpholinophenol (11a). To a solution of 4,5-difluoro-2-nitrophenol
1
0a (1.75 g, 10 mmol) in MeCN (20 mL) was added N-methylmorpholine (1.5 mL), followed by
o
thiomorpholine (11 mL, 11 mmol). The reaction mixture was heated at 80 C for 3 h. After cooling,
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water (20 mL) was added to form precipitate which was filtered and washed with water, dried to
1
afford 2.53 g of 11a as an orange solid (98.1%). H NMR (400 MHz, CDCl
3
) δ: 10.86 (s, 1 H),
7
.73 (d, J = 13.2 Hz, 1 H), 6.42 (d, J = 7.6 Hz, 1 H), 3.70−3.61 (m, 4 H), 2.81−2.74 (m, 4 H). HR-
+
MS (ESI): m/z [M+H] calcd for C10
H12FN
2
3
O S: 259.0547, found: 259.0543.
Benzyl (5-fluoro-2-hydroxy-4-thiomorpholinophenyl)carbamate (12a). To a suspension of 11a
(
4 g, 15.5 mmol) in THF (40 mL) was added Raney nickel (1 g). The mixture was hydrogenated
at 20-50 psi for 2 h. The reaction mixture was filtered into a flask containing sodium bicarbonate
2.6 g, 31 mmol) and water (10 mL) in an ice bath under argon. Benzyl chloroformate (2.0 mL,
4.4 mmol) was added in dropwise, and the mixture was stirred for 20 minutes. The organic phase
was washed with brine, dried over anhydrous Na SO , filtered and concentrated to give a red solid.
(
1
2
4
The crude product was purified by column chromatography eluted with petroleum ether/ethyl
1
acetate = 7/3 to give 12a (4.5 g, 80.4%) as a pink solid. H NMR (400 MHz, DMSO-d
6
) δ: 9.51 (s,
1
H), 8.45 (s, 1 H), 7.37−7.28 (m, 6 H), 6.48 (d, J = 8.8 Hz, 1 H), 5.07 (s, 2 H), 3.10 (t, J = 4.8 Hz,
+
4
H), 2.68 (t, J = 4.8 Hz, 4 H). HR-MS (ESI): m/z [M+H] calcd for C18
H20FN
2
3
O S: 363.1173,
found: 363.1170.
2
-Nitro-5-thiomorpholinophenol (11b). Compound 11b (13.1 g, 94.9%) was prepared from 5-
1
fluoro-2-nitrophenol 10b (9 g, 57.3 mmol) in the same manner as described for 11a. H NMR (400
MHz, CDCl
3
) δ: 11.24 (s, 1 H), 7.96 (d, J = 10.0 Hz, 1 H), 6.38 (dd, J = 10.0, 2.8 Hz, 1 H), 6.27
+
(
d, J = 2.8 Hz, 1 H), 4.00−3.72 (m, 4 H), 2.82−2.56 (m, 4 H). HR-MS (ESI): m/z [M+H] calcd for
C
10
H
13
N
2
3
O S: 241.0641, found: 241.0638.
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