Design, synthesis and structure - activity relationship of oxazolidinone derivatives containing novel S4 ligand as FXa inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.04.025

Abstract A novel series of potent and efficacious factor Xa inhibitors which possesses pyrrole/indole/thiazole moieties as S4 binding element was identified. Compound 7b showed strong human factor Xa inhibitory activity (IC₅₀ = 2.01 nM) and ant

Full text of DOI:10.1016/j.ejmech.2015.04.025

Accepted Manuscript
Design, Synthesis and Structure-Activity Relationship of oxazolidinone derivatives
containing novel S4 ligand as FXa inhibitors
Yanfang Zhao, Mingyan Jiang, Shunguang Zhou, Shasha Wu, Xiaolong Zhang,
Longsheng Ma, Kai Zhang, Ping Gong
PII:
S0223-5234(15)00274-3
10.1016/j.ejmech.2015.04.025
EJMECH 7840
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 January 2015
Revised Date: 7 April 2015
Accepted Date: 9 April 2015
Please cite this article as: Y. Zhao, M. Jiang, S. Zhou, S. Wu, X. Zhang, L. Ma, K. Zhang, P. Gong,
Design, Synthesis and Structure-Activity Relationship of oxazolidinone derivatives containing
novel S4 ligand as FXa inhibitors, European Journal of Medicinal Chemistry (2015), doi: 10.1016/
j.ejmech.2015.04.025.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical abstract
A novel series of potent and efficacious factor Xa inhibitors which possesses
pyrrole/indole/thiazole moieties as S4 binding element was identified. Compound 7b showed
potent human factor Xa inhibitory activity.

ACCEPTED MANUSCRIPT
Design, Synthesis and Structure-Activity Relationship of oxazolidinone derivatives
containing novel S4 ligand as FXa inhibitors
Yanfang Zhao, Mingyan Jiang, Shunguang Zhou, Shasha Wu, Xiaolong Zhang, Longsheng Ma, Kai Zhang, Ping
Gong*
Key Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical University), Ministry of
Education, 103 Wenhua Road, Shenhe District, Shenyang 110016, P. R. China.
Abstract
A novel series of potent and efficacious factor Xa inhibitors which possesses pyrrole/indole/thiazole moieties
as S4 binding element was identified. Compound 7b showed strong human factor Xa inhibitory activity (IC50
=
2.01 nM) and anticoagulant activities in both human (PTCT2 = 0.15 ꢀM, APPTCT2 = 0.30 ꢀM) and rabbit plasma
(PTCT2 = 0.46 ꢀM, APPTCT2 = 0.75 ꢀM). The SARs analyses indicated that the size and water solubility of
different alkylamino group at the position of S4 ligand were responsible for the anticoagulant activity.
Keywords: Factor Xa inhibitors; oxazolidinone derivatives; Anticoagulant activity; Synthesis.
1. Introduction
Thrombosis-related diseases including myocardial infarction, deep vein thrombosis, and unstable angina often
have life-threatening consequences. Anticoagulant therapy, the primary strategy for treatment and prevention of
thromboembolic diseases, requires parenteral administration or careful monitoring of the clotting time to achieve
the desired efficacy and dose titration to minimize excessive bleeding [1-2]. Several anticoagulants, such as
heparins and warfarin have proved to be effective in the prevention and treatment of these thrombotic diseases, but
many shortcomings restrict their clinical use. Thus, identifying novel oral anticoagulants with improved efficacy and
safety has become increasingly important.
Factor Xa (FXa), a trypsin-like serine protease, acts on the site of convergence between intrinsic and extrinsic
coagulation pathways and forms a prothrombinase complex with factor Va, calcium ion, and phospholipids to
generate thrombin via proteolysis of prothrombin [3]. Since this process involves signal amplification, with one
molecule of FXa activating many molecules of prothrombin to thrombin, FXa inhibitors are expected to be more
efficient in interrupting the coagulation cascade than direct thrombin inhibitors [4-5]. Furthermore, FXa inhibitors
are expected to exhibit a lower bleeding risk than thrombin inhibitors, since FXa inhibitors specifically affect the
coagulation pathway but not platelet function [6-7].
Many pharmaceutical companies have concentrated on exploring an orally active FXa inhibitor [8-11].
Nowadays, several oral FXa inhibitors such as Rivaroxaban, Apixaban, Edoxaban, Darexaban and Betrixaban have
been on the market or in clinical study (1–5, Fig. 1). These FXa inhibitors are used to treat the prevention of
Venous Thromboembolism (VTE) and stroke prophylaxis in patients with non-valvular Atrial Fibrillation (AF)
[12-14]. In recent years, Darexaban (4) and Betrixaban (5) are also used in the treatment of Deep Vein Thrombosis
(DVT) and Pulmonary Embolism (PE) [15-16].
(Figure 1. should be listed here)
As an extension of our work on the development of novel potent FXa inhibitors, we would like to obtain
another novel scaffold for further exploration. The X-ray crystal structure of rivaroxaban in complexes with human
*
Corresponding author. Tel./fax: +86 24 2398 6429.
E-mail address: gongpinggp@126.com (P. Gong).
1

ACCEPTED MANUSCRIPT
FXa demonstrated that the formation of two hydrogen bonds between the oxazolidinone ring and Gly219 has a
crucial role in its high affinity [27]. In addition, the overall structure of the FXa inhibitors has been explored by
many research groups, since they provide L- or V- like configurations to bind S1 ligand and S4 ligand with a proper
distance and angle, necessary for a strong inhibitory activity [17-22]. In the S1 pocket, there is a hydrogen bond
between the amide nitrogen of 5-chlorothiophene-2-carboxamide and the carbonyl oxygen of Gly216. FXa S4 bag,
made of Phe174, Tyr99 and Trp215, constitute a hydrophobic pocket. The pyrrole/indole/thiazole ring displayed a
multitude of biological activities, including antitumor, antibacterial, antihypertensive, antiplatelet, and
anti-inflammatory activities, however, these heterocyclic can insert a hydrogen bond between Tyr99 and Trp215.
According to the structure-activity relationships (SARs) of rivaroxaban, the oxazolidinone ring is the active
necessary fragment [23-26], and the 5-chlorothiophene-2-carboxamide group makes contact with the hydrophobic
side chains of Tyr228, Val213, Ala190, and Gly226. However, the morpholin-3-one moiety at the S4 ligand can be
replaced by several water-soluble groups.
Therefore, a novel series of potent and efficacious FXa inhibitors were designed and synthesized, wherein, the
5-chlorothiophene-2-carboxamide group (B moiety) as S1 ligand was retained and the pyrrole/indole/thiazole ring
(A moiety) as S4 ligand was introduced. In this paper, we describe the synthesis and structure-activity relationships
of compounds I, II, and III (Fig. 2) and identified compound 7b as a potent FXa inhibitor with excellent in vitro
efficacy, thus making it a promising drug candidate for further evaluation.
(Figure 2. should be listed here)
2. Chemistry
2.1. Synthesis of oxazolidinone derivatives containing pyrrole/indole pharmacophores
Scheme 1 shows the synthetic pathway for the target compounds 7a-7t. Aromatic nucleophilic substitution of
p-fluoronitrobenzene with pyrrole or indole under weak basic conditions afforded 1a and 1b, respectively.
Reduction of 1a or 1b with hydrazine hydrate produced aromatic amines, 2a and 2b, which were alkylated with
(S)-(+)-glycidyl phthalimide in 90% ethanol to yield 3a and 3b, respectively. Treatment with CDI resulted in
cyclization of 3a and 3b to form oxazolidinone 4a and 4b, respectively, which in turn produced a hydrazinolysis
reaction to afford intermediates 5a and 5b [28]. The condensation of corresponding 5a with
5-chlorothiophene-2-carboxylic acid in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI)
and hydroxybenzotriazole (HOBt) afforded 6a and 6b, respectively, which reacted with form aldehyde and
appropriate secondary amines undergoing Mannich-type condensation reactions to afford the target compounds
7a-7t, respectively.
(Scheme 1. should be listed here)
2.2. Synthesis of oxazolidinone derivatives containing thiazole pharmacophores
The synthesis of the target compounds 14a-14i is summarized in Scheme 2. 4-Aminobenzonitrile was
alkylated by (S)-(+)-glycidyl phthalimide with Mg(ClO4)2 as a catalyst in 1,4-dioxane to give 8, which underwent
the procedure described in section 2.2 to afford intermediates 9, 10, and 11, respectively. Treating 11 with sodium
hydrosulfide (NaSH) and ammonium chloride (NH4Cl) in DMF/H2O= (1:1) at room temperature afforded moderate
yields of 12 [29]. The thioacid amide part of 12 was cyclized with 1,3-dichloroacetone to yield 13 [30], which
underwent nucleophilic substitution with secondary amines to afford target compounds 14a-14i.
(Scheme 2. should be listed here)
2

ACCEPTED MANUSCRIPT
3. Results and discussion
3.1. In vitroFXa inhibitory, anticoagulant activity and structure–activity relationships
The compounds synthesized were evaluated for their in vitro inhibitory activity of human FXa, expressed as
IC50 or % inhibition at 0.1 ꢀM and their anticoagulant activity in human and rabbit plasma was measured as
prothrombin time (PT) and activated partial thromboplastin time (APTT). PT measures the effect of a compound on
the extrinsic pathway of coagulation, whereas APTT represents the effect on the intrinsic pathway and is expressed
as the concentration of the compound required to double the clotting time (PTCT2) in the PT assay. The results
were expressed as IC50 values and summarized in Table 1. The IC50 values in Table 1 are the average of at least
three independent experiments.
As illustrated in Table 1, all the target compounds displayed moderate to excellent inhibition of FXa activity
at 0.1 μM and in vitro anticoagulant activity. Most of the compounds showed similar sensitivity to PT and APTT,
which indicated that replacement of the morpholin-3-one (S4 ligand) framework in rivaroxaban with the
pyrrole/indole/thiazole ring maintained the anticoagulant activity. However, the introduction of the different S4
ligands resulted in different anticoagulant activity. For example, compounds 7a (98% inhibition), 7i (90%
inhibition), 7o (69% inhibition), and 14b (91% inhibition) exhibited comparable inhibition of FXa activity at 0.1
μM. Compound 7b was found to be detrimental to both FXa inhibitory activity (100% inhibition) and anticoagulant
activity [PTCT2 = 0.15 ꢀM, APPTCT2 = 0.30 ꢀM (human plasma); PTCT2 = 0.46 ꢀM, APPTCT2 = 0.75 ꢀM (rabbit
plasma)].
Preliminary SARs indicated that different anticoagulant properties were observed when various NR1R2 groups
were introduced into the S4 ligand (A moiety). Introduction of a methyl-ethyl amino group exhibited a positive
effect on the anticoagulant activity. However, the cyclic tertiary amino groups (CTAGs) exhibited a negative effect.
For example, compound 7a with a dimethyl amino group showed 98% inhibition of FXa activity at 0.1 ꢀM, and
displayed strong anticoagulant activity (PTCT2 = 0.61 ꢀM, APPTCT2 = 1.20 ꢀM [human plasma]). Derivatives with
a diethyl amino group improved the anticoagulant activity, such as compounds 7b (PTCT2 = 0.15 ꢀM, APPTCT2 =
0.30 ꢀM [human plasma]), 7j (PTCT2 = 0.35 ꢀM, APPTCT2 = 0.65 ꢀM [human plasma]), and 14d (PTCT2 = 0.51
ꢀM, APPTCT2 = 0.82 ꢀM [human plasma]), which showed potent inhibition of FXa activity at 0.1 ꢀM. However,
the ethyl amino groups were also well tolerated with comparable anticoagulant activity, such as in compound 14c
(PTCT2 = 0.65 ꢀM, APPTCT2 = 0.79 ꢀM [human plasma]) that showed 93% inhibition of FXa activity at 0.1 ꢀM.
In contrast, the introduction of CTAGs showed decreased anticoagulant activity, such as compounds 7d (PTCT2 =
1.42 ꢀM, APPTCT2 = 1.71 ꢀM [human plasma]), 7q (PTCT2 = 10.12 ꢀM, APPTCT2 = 11.23 ꢀM [human plasma]),
and 14i (PTCT2 = 4.06 ꢀM, APPTCT2 = 4.79 ꢀM [rabbit plasma]) that were reduced 3.55- to 16.04- fold compared
to rivaroxaban (PTCT2 = 0.40 ꢀM, APPTCT2 = 0.70 ꢀM [human plasma]), the same trend was observed for
compounds 7f, 7t, and 14g.
Further analysis revealed that the R3 group on the phenyl ring was closely related to the anticoagulant activity.
Compound 7c, with no substituent on the phenyl ring, displayed 92% inhibition at 0.1 ꢀM (PTCT2 = 0.70 ꢀM,
APPTCT2 = 1.31 ꢀM). However, the compounds with electron-withdrawing groups (EWGs) on the phenyl ring
showed an obvious decline, such as compounds 7k (82% inhibition at 0.1 ꢀM, PTCT2 = 2.45 ꢀM, APPTCT2 = 3.01
ꢀM [rabbit plasma]) and 7n (73% inhibition at 0.1 ꢀM, PTCT2 = 2.98 ꢀM, APPTCT2 = 3.67 ꢀM [rabbit plasma])
with a 3.5- to 5.8- fold reduction in the anticoagulant activity, respectively.
As shown in Table 1, the introduction of small alkylamino groups into the hydrophobic pocket (S4 ligand) can
improve the anticoagulant activity. However, the CTAGs showed significant reduction in anticoagulant activity.
The water solubility of the target compounds were measured (refer to the Chinese pharmacopoeia 2010 edition of
the second collection method). All of the target compounds showed limited to no solubility, such as compounds 7b
(R1R2 = diethyl, 6.9 ꢀg/mL), 7i (R1R2 = dimethyl, 10.2 ꢀg/mL), 7n (R1R2 = pyrrolidinyl, 20.4 ꢀg/mL), 7q (R1R2 =
3

ACCEPTED MANUSCRIPT
morpholinyl, 25.8 ꢀg/mL), and 14h (R1R2 = 4-methyl piperazinyl, 33.2 ꢀg/mL).
The pharmacological data suggested that the hydrophobic pocket (S4 ligand) played an important role in the
anticoagulant activity and was not sufficiently large enough to accommodate moiety A with its bulky groups. The
SARs based on the IC50 values (Table 1) showed that two main factors, the size of the substituent at N atom and the
water solubility of different tertiary amines, were responsible for the anticoagulant activity.
(Table 1. should be listed here)
3.2. In vitro anti-FXa inhibitory activity
The compounds selected based on their FXa inhibition and anticoagulant activities from Table 1 were
evaluated for their in vitro anticoagulant activity in human and rabbit plasma; In order to further research on
anti-FXa inhibitory activity, we selected five tested compounds had IC50 values less than 10 nM for human FXa
inhibition. As shown in Table 2, they all displayed relatively inferior anticoagulant activity in rabbit plasma,
compared to human plasma, as reflected by their PTCT2 values. However, compound 7b showed the most potent
activity with an IC50 value of 2.01 nM, which was comparable to that of the positive control, rivaroxaban (IC50
=
2.20 nM); this data indicates that compound 7b deserves further study with regard to its application in the treatment
of thrombosis-related diseases.
(Table 2. should be listed here)
3.3. Binding model analysis
To further elucidate the binding mode of compounds, a detail docking analysis was performed. The X-ray
crystal structure of rivaroxaban in complexes with human FXa was performed by Proteros Biostructures GmbH in
Planegg-Martinsried, Germany. Crystals of human FXa in complex with rivaroxaban were prepared as described
with small modifications. The co-crystal structure of rivaroxaban (BAY 59-7939) with human FXa was selected as
the docking model (PDB ID code: 1EQZ) [33]. The docking simulation was conducted using Glide XP
(Schrödinger 2014), since Glide uses a hierarchical series of filters to search for possible locations of the ligand in
the active-site region of the receptor. The shape and properties of the receptor are represented on a grid by several
different sets of fields that provide progressively more accurate scoring of the ligand poses. The image files were
generated using Accelrys DS visualizer 4.0 systems. The binding model was exemplified by the interaction of
compound 7b with human FXa. As shown in Figure 6, the model further suggested that the NH group of the amide
bond linked to the 5-chlorothiophene-2-carboxamide moiety forms H-bond with Gly219 and Gly216. Additionally,
carbonyl group of amide bond linked to the amide group forms a H-bond with Gln192. The S4 pocket in FXa is a
narrow hydrophobic channel defined by the aromatic rings of Tyr99, His57, and Trp215. The nonpolar aryl ring of
compound 7b extends across the face of Trp215, and the pyrrole moiety is sandwiched between Tyr99 and His57.
(Figure 3. should be listed here)
4. Conclusions
In an effort to find potent FXa inhibitors, we designed and synthesized a series of oxazolidinone derivatives
containing pyrrole/indole/thiazole ring moieties at the novel S4 ligand. The target compounds were evaluated for
their in vitro anticoagulant activity in human and rabbit plasma, which indicated that most of them exhibited
moderate- to- excellent anticoagulant activity and high inhibition of FXa activity at 0.1 ꢀM. Biological evaluation
of the synthesized compounds led to the identification of compound 7b (IC50 = 2.01 nM), which showed potent
human factor Xa inhibitory activity and anticoagulant activity in both human and rabbit plasma. The SARs analyses
indicated that the size and water solubility of different alkylamino groups at the position of S4 ligand were
4

ACCEPTED MANUSCRIPT
responsible for the anticoagulant activity. The hydrophobic pocket (S4 ligand) played an important role in the
anticoagulant activity. Furthermore, SARs studies and mechanism studies of these compounds are in progress. The
results will be reported in future.
5. Experimental
5.1. Chemistry
Unless otherwise specified, all melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi
Labortechnik, Flawil, Switzerland) and were uncorrected. Mass spectra (MS) were taken in ESI mode on Agilent
1100 LC-MS (Agilent, Palo Alto, CA, U.S.A.). 1H NMR and 13C NMR spectra were recorded on Bruker ARX-400,
400MHz (Bruker Bioscience, Billerica, MA, USA) with TMS as an internal standard. All materials were obtained
from commercial suppliers and were used without further purification. Reactions’ time and purity of the products
were monitored by TLC on FLUKA silica gel aluminum cards (0.2 mm thickness) with fluorescent indicator 254
nm. Column chromatography was run on silica gel (200–300 mesh) from Qingdao Ocean Chemicals (Qingdao,
Shandong, China). The IR spectra were recorded by means of the KBr pellet technique on a Bruker FTS 135
spectrometer. The elemental analysis of the compounds was performed on a Perkin Elmer 2400 Elemental Analyser
(In the mode of measurement C, H, and N, the sample into the combustion tube in pure oxygen atmosphere static
combustion and products by a specific reagent after formation of CO2, H2O, N2 and nitrogen oxides, uniform
mixing under the atmospheric pressure. The thermal conductivity detector is used for determining the content of C,
H and N from mixed gases.).
5.2. Preparation of oxazolidinone derivatives containing pyrrole/indole pharmacophores (7a-7t)
5.2.1 Preparation of 1-(4-nitrophenyl)-1H-pyrrole/indole (1a-1b)
A well-stirred mixture of pyrrole/indole (0.128 mol) and K2CO3 (35.4g, 0.257 mol) in DMF (150 mL) was
heated at 100 °C for 30 min. Then, p-fluoronitrobenzene (21.6g, 0.153 mol) was added and the mixture was stirred
for another 5h. Upon cooling to room temperature, the mixture was poured into water (300 mL) to stir for 30 min
and filtered. The residue was washed with water, dried to give crude product and then treated with diethyl ether to
afford 1a-1b.
5.2.1.1. 1-(4-nitrophenyl)-1H-pyrrole (1a)
Yellow solid; Yield: 95.6 %; M.p.: 160.9-162.1 °C; MS (ESI) m/z (%): 189.1 [M+H]+.
5.2.1.2. 1-(4-nitrophenyl)-1H-indole (1b)
Yellow solid; Yield: 95 %; M.p.: 159.9-161.3 °C; MS (ESI) m/z (%): 261.3 [M+H]+.
5.2.2 Preparation of 4-(1H-pyrrol/indol-1-yl)benzenamine (2a-2b)
A well-stirred mixture of 1a-1b (0.1064 mol), FeCl3·6H2O (4.3g, 0.016 mol) and activated carbon (0.38g,
0.0319 mol) in 1,4-dioxane (200 mL) was heated to 50 °C, and then hydrazine hydrate (66.5g, 1.0638 mol) was
added drop-wise at this temperature. After the completion of addition, the mixture was refluxed for 5h.The
precipitate was removed by hot filtration and the filtrate was concentrated to give a yellow viscous substance. The
residue was quenched with water (200 mL) to stir for 30 min, filtered, washed with a small amount of water and
dried to give 2a-2b.
5.2.2.1. 4-(1H-pyrrol-1-yl)benzenamine (2a)
1
Yellow solid; Yield: 86 %; M.p.: 77.4-78.8 °C; MS (ESI) m/z (%): 159.2 [M+H]+; H NMR (400 MHz,
DMSO) δ 7.17 (d, J = 8.7 Hz, 2H), 7.09 (t, J = 2.1 Hz, 2H), 6.62 (d, J = 8.7 Hz, 2H), 6.16 (t, J = 2.1 Hz, 2H), 5.13
(s, 2H).
5.2.2.2. 4-(1H-indol-1-yl)benzenamine (2b)
Yellow solid; Yield: 80 %; M.p.: 78.9-79.3 °C; MS (ESI) m/z (%): 209.2 [M+H]+.
5

ACCEPTED MANUSCRIPT
5.2.3 Preparation of 2-(3-(4-(1H-pyrrol/indol-1-yl)phenylamino)-2-hydroxylpropyl)isoindoline-1,3- dione (3a-3b)
A mixture of 2a-2b (0.0633 mol) and (S)-N-glycidylphthalimide (12.9g, 0.0798 mol) in 90% ethanol (100
mL)was heated at 60 °C for 10h.The precipitate was collected by hot filtration, washed with ethanol and dried to
afford 3a-3b.
5.2.3.1. 2-(3-(4-(1H-pyrrol-1-yl)phenylamino)-2-hydroxylpropyl)isoindoline-1,3- dione (3a)
Pale yellow solid; Yield: 70 %; M.p.: 183.4-185.2 °C; MS (ESI) m/z (%): 362.0 [M+H]+; 1H NMR (400 MHz,
DMSO) δ 8.03 – 7.70 (m, 4H), 7.30 – 7.18 (m, 2H), 7.16 – 7.04 (m, 2H), 6.65 (dd, J = 24.7, 7.6 Hz, 2H), 6.16 (t, J
= 2.1 Hz, 2H), 5.69 (dd, J = 17.1, 11.0 Hz, 1H), 5.25 (s, 1H), 4.02 (s, 1H), 3.64 (qd, J = 13.7, 6.4 Hz, 2H), 3.23 –
3.11 (m, 1H), 3.09 – 2.96 (m, 1H).
5.2.3.2. 2-(3-(4-(1H-indol-1-yl)phenylamino)-2-hydroxylpropyl)isoindoline-1,3- dione (3b)
Yellow solid; Yield: 66 %; M.p.: 182.9-183.3 °C; MS (ESI) m/z (%): 412.5 [M+H]+.
5.2.4 Preparation of (S)-2-((3-(4-(1H-pyrrol/indol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-1,3-dione
(4a-4b)
A mixture of 3a-3b (0.0399 mol) and CDI (12.9g, 0.0798 mol) in THF (140 mL) was heated under reflux with
stirring for 7 h. Upon cooling to room temperature, the mixture was then poured into ice water. The separated solid
product was collected by filtration, washed with water and dried to afford 4a-4b.
5.2.4.1. (S)-2-((3-(4-(1H-pyrrol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-1,3-dione (4a)
Yellow solid; Yield: 79 %; M.p.: 196.9-198.7 °C; MS (ESI) m/z (%): 388.5 [M+H]+,410.1 [M+Na]+; 1H NMR
(400 MHz, DMSO) δ 8.07 – 7.72 (m, 4H), 7.70 – 7.44 (m, 4H), 7.30 (dt, J = 14.8, 4.8 Hz, 2H), 6.41 – 6.11 (m, 2H),
5.08 – 4.75 (m, 1H), 4.34 – 4.14 (m, 1H), 4.12 – 3.80 (m, 3H).
5.2.4.2. (S)-2-((3-(4-(1H-indol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-1,3-dione (4b)
White solid; Yield: 76 %; M.p.: 197.2-198.5 °C; MS (ESI) m/z (%): 438.5 [M+H]+,460.5 [M+Na]+.
5.2.5 Preparation of (S)-3-(4-(1H-pyrrol/indol-1-yl)phenyl)-5-(aminomethyl)oxazolidin-2-one (5a- 5b)
A mixture of 4a-4b (39.6 mmol) and hydrazine hydrate (14.4 mL, 0.2370 mol) in methanol (150 mL) was
heated at 40 °C for 3 h. Then, the solvent was removed and the residue treated with water (50 mL) and extracted
with CH2Cl2 (3 × 60 mL). The combined organic layer was washed with brine (2 × 20 mL), dried over anhydrous
MgSO4, and evaporated to afford 5a-5b.
5.2.5.1. (S)-3-(4-(1H-pyrrol-1-yl)phenyl)-5-(aminomethyl)oxazolidin-2-one (5a)
Yellow solid; Yield: 89 %; M.p.: 127.1-128.5 °C; MS (ESI) m/z (%): 258.0 [M+H]+,280.4 [M+Na]+; 1H NMR
(400 MHz, DMSO) δ 7.69 – 7.55 (m, 4H), 7.34 (t, J = 2.2 Hz, 2H), 6.25 (t, J = 2.2 Hz, 2H), 4.62 (td, J = 11.3, 5.0
Hz, 1H), 4.09 (t, J = 8.8 Hz, 1H), 3.90 (dd, J = 8.8, 6.5 Hz, 1H), 2.83 (qd, J = 13.7, 4.9 Hz, 2H), 1.69 (d, J = 30.9
Hz, 2H).
5.2.5.2. (S)-3-(4-(1H-indol-1-yl)phenyl)-5-(aminomethyl)oxazolidin-2-one (5b)
Yellow solid; Yield: 87 %; M.p.: 128.9-129.3 °C; MS (ESI) m/z (%): 308.4 [M+H]+,330.3 [M+Na]+.
5.2.6
Preparation
of
(S)-N-((3-(4-(1H-pyrrol/indol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)-5-chlorothiophene-2-carboxamide
(6a-6b)
To a well-stirred solution of 5-chlorothiophene-2-carboxylic acid (3.43g, 21.4 mmol) in DMF (50 mL) was
added EDCI (4.48g, 23.3 mmol) and HOBt (3.15g, 23.3 mmol).After stirring for 30 min, TEA (3.1 mL) and 5a-5b
(19.5 mmol) was added and the mixture was stirred for 12h at room temperature and then poured into water (200
mL). After stirring for 15 min, the separated solid was collected by filtration, washed with water and dried to afford
6a-6b.
5.2.6.1. (S)-N-((3-(4-(1H-pyrrol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)-5-chlorothiophene-2-carboxamide
(6a)
6

ACCEPTED MANUSCRIPT
1
Green solid; Yield: 66 %; M.p.: 218.3-220.3 °C; MS (ESI) m/z (%): 402.2 [M-H]+; H NMR (400 MHz,
DMSO) δ 9.11 (s, 1H), 7.75 (d, J = 3.9 Hz, 1H), 7.60 (s, 4H), 7.34 (d, J = 1.9 Hz, 2H), 7.20 (d, J = 4.0 Hz, 1H),
6.25 (d, J = 1.9 Hz, 2H), 4.84 (dt, J = 11.3, 5.6 Hz, 1H), 4.20 (t, J = 8.9 Hz, 1H), 3.90 (dd, J = 8.8, 6.3 Hz, 1H),
3.61 (t, J = 5.4 Hz, 2H).
5.2.6.2. (S)-N-((3-(4-(1H-indol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)-5-chlorothiophene-2-carboxamide (6b)
White solid; Yield: 69 %; M.p.: 217.9-218.7 °C; MS (ESI) m/z (%): 449.9 [M-H]+.
5.2.7 General procedure for preparation of oxazolidinone compounds containing pyrrolidine amine (7a-7t)
To a well-stirred glacial acetic acid (10 mL) was added drop-wise corresponding amine (1.9 mmol).After
well-distribution, 37% formaldehyde solution (0.08g, 0.83 mmol) was added drop-wise. After the completion of
addition and stirring for 0.5 h at room temperature, 6a-6b (0.747 mmol) was added. The mixture was stirred for
another 3 h, poured into ice-water and gently adjusted pH to be basic by 10 % NaOH in an ice bath. The separated
solid was collected by filtration and the residue was triturated with ethyl ether, filtrated and dried to afford 7a-7t.
5.2.7.1. (S)-5-chloro-N-((3-(4-(2-((dimethylamino)methyl)-1H-pyrrol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)th
iophene-2-carboxamide (7a)
Yield: 64.1%; M.p.: 142.2-144.0 °C; 1H NMR (400 MHz, DMSO) δ 9.29 (s, 1H, CONH), 7.76 (d, J = 3.3 Hz,
1H, thiophene-4-H), 7.60 (dd, J = 19.6, 8.4 Hz, 4H, phenyl-H), 7.19 (d, J = 3.3 Hz, 1H, thiophene-3-H), 6.93 (s, 1H,
pyrrole-H), 6.13 (s, 2H, pyrrole-H), 4.86 (m, 1H, O-CH), 4.21 (t, J = 8.7 Hz, 1H, CH2-NH), 3.99 – 3.87 (m, 1H,
CH2-NH), 3.62 (t, J = 10.8Hz, 2H, N-CH2), 3.20 (s, 2H, pyrrole-2-CH2), 2.09 (s, 6H, CH3);MS (ESI) m/z (%):
458.96 [M+H]+; Anal. calcd. for C22H23ClN4O3S (%): C, 57.57; H, 5.05; N, 12.21; Found (%): C,57.60 ; H,5.08 ;
N,12.19 .
5.2.7.2. (S)-5-chloro-N-((3-(4-(2-((diethylamino)methyl)-1H-pyrrol-1-yl)phenyl)-2-oxooxazolidin-5-yl)-methyl)thi
ophene-2-carboxamide (7b)
Yield: 63.6 %; M.p.: 142.5-143.2 °C; 1H NMR (400 MHz, DMSO) δ 9.29 (s, 1H, CONH), 7.76 (d, J = 3.3 Hz,
1H, thiophene-4-H), 7.60 (dd, J = 19.6, 8.4 Hz, 4H, phenyl-H), 7.19 (d, J = 3.3 Hz, 1H, thiophene-3-H), 6.93 (s, 1H,
pyrrole-H), 6.13 (s, 2H, pyrrole-H), 4.86 (m, 1H, O-CH), 4.21 (t, J = 8.7 Hz, 1H, CH2-NH), 3.99 – 3.87 (m, 1H,
CH2-NH), 3.62 (t, J = 10.8Hz, 2H, N-CH2), 3.20 (s, 2H, pyrrole-2-CH2), 2.09 (q, 4H, CH2)，1.09 (t, 6H, CH3);MS
(ESI) m/z (%): 487.01 [M+H]+; Anal. calcd. for C24H27ClN4O3S (%): C, 59.19; H, 5.59; N, 11.50; Found (%):
C,59.21 ; H,5.57 ; N, 11.52.
5.2.7.3. (S)-N-((3-(4-(2-(azetidin-1-ylmethyl)-1H-pyrrol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)-5-chlorothiop
hene-2-carboxamide (7c)
Yield: 58.4 %; M.p.: 143.0-144.7 °C; 1H NMR (400 MHz, DMSO) δ 9.04 (s, 1H, CONH), 7.72 (d, J = 4.0 Hz,
1H, thiophene-4-H), 7.64 (d, J = 8.8 Hz, 2H, phenyl-H), 7.55 (d, J = 8.9 Hz, 2H, pHenyl-2H), 7.20 (d, J = 4.0 Hz,
1H, thiophene-3-H), 6.90 (s, 1H, Pyrrole-H), 6.17 – 6.09 (m, 2H, pyrrole-H), 4.92 – 4.80 (m, 1H, O-CH), 4.22 (t, J
= 8.9 Hz, 1H, CH2-NH), 3.94 – 3.84 (m, 1H, CH2-NH), 3.62 (t, J = 5.3 Hz, 2H, N- CH2), 3.46 (s, J = 19.5 Hz, 2H,
pyrrole-2-CH2), 3.16 (m, 2H, azetidin-H), 1.95 (m, 2H, azetidin-H), 1.23 (m, 2H, azetidin-H);MS (ESI) m/z (%):
470.97 [M+H]+; Anal. calcd. for C23H23ClN4O3S (%): C, 58.65; H, 4.92; N, 11.90; Found (%): C,58.66; H,4.90;
N,11.88.
5.2.7.4. (S)-5-chloro-N-((2-oxo-3-(4-(2-(pyrrolidin-1-ylmethyl)-1H-pyrrol-1-yl)phenyl)oxazolidin-5-yl)-methyl)thi
ophene-2-carboxamide (7d)
Yield: 60.2 %; M.p.: 142.5-143.8 °C; 1H NMR (400 MHz, DMSO) δ 9.13 (t, J = 5.7 Hz, 1H, CONH), 7.77 (d,
J = 4.1 Hz, 1H, thiophene-4-H), 7.68 (d, J = 8.9 Hz, 2H, phenyl-H), 7.50 (d, J = 8.8 Hz, 2H, phenyl-H), 7.21 (d, J =
4.0 Hz, 1H, thiophene-3-H), 6.99 (s, 1H, pyrrole-H), 6.54 (s, 1H, pyrrole-H), 6.25 (s, 1H, pyrrole-H), 4.88 (dt, J =
11.4, 5.5 Hz, 1H, O-CH), 4.23 (t, J = 9.0 Hz, 1H, CH2-NH), 3.93 (dd, J = 9.1, 6.1 Hz, 1H, CH2-NH), 3.62 (t, J = 5.6
Hz, 2H, N- CH2), 3.48 – 3.37 (s, 2H, pyrrole-2- CH2), 2.83 (m, 4H, pyrrolidine-H), 1.78 (m, 4H, pyrrolidine-H);
7

ACCEPTED MANUSCRIPT
MS (ESI) m/z (%): 485.00 [M+H]+; Anal. calcd. for C24H25ClN4O3S (%): C, 59.43; H, 5.20; N, 11.55; Found (%):
C,59.42; H,5.18; N,11.58.
5.2.7.5. (S)-5-chloro-N-((2-oxo-3-(4-(2-(piperidin-1-ylmethyl)-1H-pyrrol-1-yl)phenyl)oxazolidin-5-yl)-methyl)thi
ophene-2-carboxamide (7e)
Yield: 56.7 %; M.p.: 145.2-146.6 °C; 1H NMR (400 MHz, DMSO) δ 9.01 (t, J = 5.8 Hz, 1H, CONH), 7.70 (d,
J = 4.1 Hz, 1H, thiophene-4-H), 7.62 (s, 4H, phenyl-H), 7.20 (d, J = 4.0 Hz, 1H, thiophene-3-H), 6.93 (s, 1H,
pyrrole-H), 6.14 (s, 2H, pyrrole-H), 4.86 (dt, J = 11.6, 5.6 Hz, 1H, O-CH), 4.23 (t, J = 9.0 Hz, 1H, CH2-NH), 3.89
(dd, J = 9.1, 6.1 Hz, 1H, CH2-NH), 3.62 (t, J = 5.4 Hz, 2H, N-CH2), 3.22 (s, 2H, pyrrole-2-CH2), 2.30 (m, 4H,
piperidine-CH2), 1.46 (m, 4H, piperidine- CH2), 1.37 (m, 2H, piperidine-CH2); MS (ESI) m/z (%): 499.02 [M+H]+;
Anal. calcd. for C25H27ClN4O3S (%): C, 60.17; H, 5.45; N, 11.23; Found (%): C,60.18; H,5.44; N,11.23.
5.2.7.6. (S)-5-chloro-N-((3-(4-(2-(morpholinomethyl)-1H-pyrrol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)thioph
ene-2-carboxamide (7f)
Yield: 64.6 %; M.p.: 143.1-144.9 °C; 1H NMR (400 MHz, DMSO) δ 9.00 (t, J = 5.8 Hz, 1H, CONH), 7.69 (d,
J = 4.1 Hz, 1H, thiophene-4-H), 7.62 (t, J = 7.1 Hz, 4H, phenyl-H), 7.19 (d, J = 4.0 Hz, 1H, thiophene-3-H), 6.94 (s,
1H, pyrrole-H), 6.14 (s, 2H, pyrrole-H), 4.85 (dt, J = 11.3, 5.6 Hz, 1H, O-CH), 4.22 (t, J = 9.0 Hz, 1H, CH2-NH),
3.88 (dd, J = 9.1, 6.1 Hz, 1H, CH2-NH), 3.61 (t, J = 5.5 Hz, 2H, N-CH2), 3.53 (s, 4H, morphline-CH2), 3.26 (s, 2H,
pyrrole-2-CH2), 2.31 (s, 4H, morphline- CH2); MS (ESI) m/z (%): 501.00 [M+H]+; Anal. calcd. for C24H25ClN4O4S
(%): C, 57.54; H, 5.03; N, 11.18; Found (%): C,57.52; H,5.10; N,11.11.
5.2.7.7. (S)-5-chloro-N-((3-(4-(2-((4-methylpiperidin-1-yl)metHyl)-1h-pyrrol-1-yl)phenyl)-2-oxooxazolidin-5-yl)
methyl)thiophene-2-carboxamid (7g)
Yield: 67.9 %; M.p.: 144.3-145.1 °C; 1H NMR (400 MHz, DMSO) δ 9.00 (t, J = 5.8 Hz, 1H, CONH), 7.70 (d,
J = 4.1 Hz, 1H, thiophene-4-H), 7.62 (s, 4H, phenyl-H), 7.20 (d, J = 4.0 Hz, 1H, thiophene-3-H), 6.93 (s, 1H,
pyrrole-H), 6.15 – 6.06 (m, 2H, pyrrole-H), 4.86 (dt, J = 11.3, 5.5 Hz, 1H, O-CH), 4.22 (t, J = 9.0 Hz, 1H,
CH2-NH), 3.89 (dd, J = 9.1, 6.1 Hz, 1H, CH2-NH), 3.62 (t, J = 5.5 Hz, 2H, N-CH2), 3.21 (s, 2H, pyrrole-2-CH2),
2.78 (d, J = 10.4 Hz, 2H, 4-methylpiperdine-CH2), 1.84 (d, J = 10.4 Hz, 2H, 4-methylpiperdine-CH2), 1.56 (d, J =
11.8 Hz, 2H, 4-methylpiperdine-CH2), 1.26 (m, 1H, 4-methylpiperdine-CH), 1.07 (dd, J = 21.1, 11.4 Hz, 2H,
4-methylpiperdine-CH2), 0.87 (d, J = 6.5 Hz, 3H, CH3); MS (ESI) m/z (%): 513.05 [M+H]+; Anal. calcd. for
C26H29ClN4O3S (%): C, 60.87; H, 5.70; N, 10.92; Found (%): C,60.88; H,5.72; N,10.89.
5.2.7.8. (S)-5-chloro-N-((3-(4-(2-((4-methylpiperazin-1-yl)methyl)-1H-pyrrol-1-yl)phenyl)-2-oxooxazolidin-5-yl)
methyl)thiophene-2-carboxamide (7h)
Yield: 65.1 %; M.p.: 144.1-145.0 °C; 1H NMR (400 MHz, DMSO) δ 9.01 (t, J = 5.8 Hz, 1H, CONH), 7.70 (d,
J = 4.1 Hz, 1H, thiophene-4-H), 7.62 (s, 4H, phenyl-H), 7.20 (d, J = 4.0 Hz, 1H, thiophene-3-H), 6.97 – 6.89 (m,
1H, pyrrole-H), 6.16 – 6.09 (m, 2H, pyrrole-H), 4.86 (dt, J = 11.4, 5.6 Hz, 1H, O-CH), 4.22 (t, J = 9.0 Hz, 1H,
CH2-NH), 3.89 (dd, J = 9.1, 6.1 Hz, 1H, CH2-NH), 3.62 (t, J = 5.6 Hz, 2H, N-CH2), 3.25 (s, 2H, pyrrole-2-CH2),
2.47–2.19 (m, J= 61.1 Hz, 8H, 4-methylpiperzine-H), 2.18 (s, 3H, CH3); MS (ESI) m/z (%): 514.04 [M+H]+; Anal.
calcd. for C25H28ClN5O3S (%): C, 58.41; H, 5.49; N, 13.62; Found (%): C,58.40; H,5.47; N,13.60.
5.2.7.9. 5-chloro-N-(((S)-3-(4-(2-((dimethylamino)methyl)-1H-pyrrol-1-yl)-3-fluorophenyl)-2-oxooxazolidin-5-yl)
metyl)tiopene-2-carboxamide (7i)
Yield: 65.0%; M.p.: 142.9-143.3 °C; 1H NMR (400 MHz, DMSO) δ 9.07 (t, J = 5.7 Hz, 1H, CONH), 7.73 (d,
J = 4.1 Hz, 1H, thiophene-4-H), 7.65 (dd, J = 12.8, 2.3 Hz, 1H, Aryl-H), 7.54 (t, J = 8.8 Hz, 1H, Aryl-H), 7.39 (dd,
J = 8.8, 1.9 Hz, 1H, Aryl-H), 7.19 (d, J =4.0 Hz, 1H, thiophene-3-H), 6.79 (s, 1H, pyrrole-H), 6.13 (dt, J = 4.8, 3.3
Hz, 2H, pyrrole-H), 4.88 (dq, J = 11.4, 5.8 Hz, 1H, O-CH), 4.22 (t, J = 9.0 Hz, 1H, CH2-NH), 3.92 (dd, J = 9.2, 6.1
Hz, 1H, CH2-NH), 3.68 – 3.55 (m, 2H, N-CH2), 3.18 (s, 2H, pyrrole-2-CH2), 1.97 (s, 6H, CH3); MS (ESI) m/z (%):
8

ACCEPTED MANUSCRIPT
477.2 [M+H]+; Anal. calcd. for C22H22ClFN4O3S (%):C, 55.40; H, 4.65; N, 11.75; Found (%): C,55.38; H,4.63;
N,11.72.
5.2.7.10. 5-chloro-N-(((S)-3-(4-(2-((diethylamino)methyl)-1H-pyrrol-1-yl)-3-fluorophenyl)-2-oxooxazolidin-5-yl)m
ethyl)tHiophene-2-carboxamide (7j)
Yield: 61.2%; M.p.: 144.5-145.7 °C; 1H NMR (400 MHz, DMSO) δ 9.04 (t, J = 5.8 Hz, 1H, CONH), 7.72 (d,
J = 4.1 Hz, 1H, thiophene-4-H), 7.63 (dd, J = 12.7, 2.4 Hz, 1H, Aryl-H), 7.50 (t, J = 8.8 Hz, 1H, Aryl-H), 7.37 (dd,
J = 8.7, 2.1 Hz, 1H, Aryl-H), 7.19 (d, J = 4.0 Hz, 1H, thiophene-3-H), 6.77 (s, 1H, pyrrole-H), 6.12 (dt, J = 6.3, 3.3
Hz, 2H, pyrrole-H), 4.95 – 4.84 (m, 1H, O-CH), 4.22 (t, J = 9.0 Hz, 1H, CH2-NH), 3.90 (dd, J = 9.2, 6.1 Hz, 1H,
CH2-NH), 3.69 – 3.54 (m, 2H, N-CH2), 3.33 (s, 2H, pyrrole-2-CH2), 2.25 (q, J = 7.1 Hz, 4H, CH2CH3), 0.70 (t, J =
7.1 Hz, 6H, CH3); MS (ESI) m/z (%): 505.1 [M+H]+; Anal. calcd. for C24H26ClFN4O3S (%):C, 57.08; H, 5.19; N,
11.09; Found (%): C,57.06; H,5.20; N,11.08.
5.2.7.11. 5-chloro-N-(((S)-3-(3-fluoro-4-(2-(pyrrolidin-1-ylmethyl)-1H-pyrrol-1-yl)phenyl)-2-oxooxazolidin-5-yl)m
ethyl)thiopHene-2-carboxamide (7k)
Yield: 62.8%; M.p.: 145.8-146.2 °C; 1H NMR (400 MHz, DMSO) δ 9.12 (t, J = 5.7 Hz, 1H, CONH), 7.76 (d,
J = 4.1 Hz, 1H, thiophene-4-H), 7.65 (dd, J = 12.8, 2.2 Hz, 1H, Aryl-H), 7.56 (t, J = 8.8 Hz, 1H, Aryl-H), 7.39 (dd,
J = 8.8, 1.7 Hz, 1H, Aryl-H), 7.19 (d, J = 4.0 Hz, 1H, thiophene-3-H), 6.77 (s, 1H, pyrrole-H), 6.13 (dd, J = 7.7, 4.7
Hz, 2H, pyrrole-H), 4.89 (dq, J = 11.5, 5.8 Hz, 1H, O-CH), 4.22 (t, J = 9.0 Hz, 1H, CH2-NH), 3.94 (dd, J = 9.1, 6.1
Hz, 1H, CH2-NH), 3.69 – 3.54 (m, 2H, N-CH2), 3.33 (s, 2H, pyrrole-2-CH2), 2.27 (s, 4H, pyrrolidine-H), 1.58 (s,
4H, pyrrolidine-H); MS (ESI) m/z (%): 503.4[M+H]+; Anal. calcd. for C24H24ClFN4O3S (%):C, 57.31; H, 4.81; N,
11.14; O, 9.54; Found (%): C,57.29; H,4.79; N,11.16.
5.2.7.12. (S)-5-chloro-N-((3-(4-(2-((dimethylamino)methyl)-1H-pyrrol-1-yl)-3,5-difluorophenyl)-2-oxooxazolidin-5
-yl)methyl)thiophene-2-carboxamide (7l)
Yield: 57.6%; M.p.: 143.7-145.0 °C; 1H NMR (400 MHz, DMSO) δ 9.07 (t, J = 5.7 Hz, 1H, CONH), 7.73 (d,
J = 4.0 Hz, 1H, thiophene-4-H), 7.51 – 7.45 (m, 2H, Aryl-H), 7.19 (d, J = 4.0 Hz, 1H, thiophene-3-H), 6.79 (s, 1H,
pyrrole-H), 6.17 (dd, J = 15.2, 12.1 Hz, 2H, pyrrole-H), 4.91 (dq, J = 11.6, 5.9 Hz, 1H, O-CH), 4.22 (t, J = 9.1 Hz,
1H, CH2-NH), 3.91 (dd, J = 9.1, 6.1 Hz, 1H, CH2-NH), 3.68 – 3.57 (m, 2H, N-CH2), 3.15 (s, 2H, pyrrole-2-CH2),
1.92 (s, 6H, CH3); MS (ESI) m/z (%):495 [M+H]+; Anal. calcd. for C22H21ClF2N4O3S (%):C, 53.39; H, 4.28; N,
11.32; Found (%): C,53.37; H,4.28; N,11.33.
5.2.7.13. (S)-5-chloro-N-((3-(4-(2-((diethylamino)methyl)-1H-pyrrol-1-yl)-3,5-difluorophenyl)-2-oxooxazolidin-5-y
l)methyl)thiophene-2-carboxamide (7m)
Yield: 55.6%; M.p.: 142.9-144.1 °C; 1H NMR (400 MHz, DMSO) δ 9.04 (t, J = 5.7 Hz, 1H, CONH), 7.71 (d,
J = 4.0 Hz, 1H, thiophene-4-H), 7.46 (t, J = 12.8 Hz, 2H, Aryl-H), 7.19 (d, J = 4.0 Hz, 1H, thiophene-3-H), 6.77 (s,
1H, pyrrole-H), 6.14 (dd, J = 8.6, 5.6 Hz, 2H, pyrrole-H), 4.91 (td, J = 10.3, 5.8 Hz, 1H, O-CH), 4.21 (t, J = 9.1 Hz,
1H, CH2-NH), 3.88 (dd, J = 9.2, 6.1 Hz, 1H, CH2-NH), 3.70 – 3.53 (m, 2H, N-CH2), 3.28 (s, 2H, pyrrole-2-CH2),
2.19 (q, J = 7.0 Hz, 4H, CH2CH3), 0.66 (t, J = 7.1 Hz, 6H, CH3); MS (ESI) m/z (%):523.3[M+H]+; Anal. calcd. for
C24H25ClF2N4O3S (%):C, 55.12; H, 4.82; N, 10.71; Found (%): C,55.11; H,4.80; N,10.70.
5.2.7.14. (S)-5-chloro-N-((3-(3,5-difluoro-4-(2-(pyrrolidin-1-ylmethyl)-1H-pyrrol-1-yl)phenyl)-2-oxooxazolidin-5-y
l)methyl)thiophene-2-carboxamide (7n)
Yield: 58.9%; M.p.: 145.1-146.9 °C; 1H NMR (400 MHz, DMSO) δ 9.03 (t, J = 5.7 Hz, 1H, CONH), 7.72 (d,
J = 4.0 Hz, 1H, thiophene-4-H), 7.47 (t, J = 10.6 Hz, 2H, Aryl-H), 7.19 (d, J = 4.1 Hz, 1H, thiophene-3-H), 6.76 (s,
1H, pyrrole-H), 6.16 (dd, J = 14.2, 11.1 Hz, 2H, pyrrole-H), 4.93 – 4.87 (m, 1H, O-CH), 4.21 (t, J = 9.0 Hz, 1H,
CH2-NH), 3.89 (dd, J = 9.2, 6.1 Hz, 1H, CH2-NH), 3.68 – 3.56 (m, 2H, N-CH2), 3.32 (s, 2H, pyrrole-2-CH2), 2.20
(s, 4H, pyrrolidine-H), 1.53 (s, 4H, pyrrolidine-H); MS (ESI) m/z (%):523.3[M+H]+; Anal. calcd. for
C24H23ClF2N4O3S (%):C, 55.33; H, 4.45; N, 10.75; Found (%): C,55.34; H,4.48; N,10.77.
9

ACCEPTED MANUSCRIPT
5.2.7.15. (S)-5-chloro-N-((3-(4-(2-(dimethylamino)-1H-indol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)thiophene-
2-carboxamide (7o)
1
Yield: 67.8%; M.p.: 140.2-141.0 °C; H NMR (400 MHz, DMSO) δ 9.13 (t, J = 5.7 Hz, 1H, CONH), 7.97 –
7.89 (m, 2H, Aryl-H), 7.82 – 7.74 (m, 2H, Aryl-H), 7.69 (d, J = 4.9 Hz, 1H, thiophene-4-H), 7.64 (d, J = 8.9 Hz, 2H,
Aryl-H), 7.53 (d, J = 8.2 Hz, 1H, Aryl-H), 7.27 (dd, J = 15.8, 7.4 Hz, 2H, Aryl-H), 7.21 (d, J = 4.9 Hz, 1H
thiophene-3-H), 4.90 (dt, J = 8.2, 5.6 Hz, 1H, O-CH), 4.49 (s, 2H, indole-2-CH2), 4.26 (t, J = 8.9 Hz, 1H, CH2-NH),
3.98 (dd, J = 9.0, 6.1 Hz, 1H, CH2-NH), 3.65 (s, 2H, N-CH2), 2.76 (s, 6H, CH3); MS (ESI) m/z (%): 508.8 [M+H]+;
Anal. calcd. for C25H23ClN4O3S(%): C, 60.66; H, 4.68; N, 11.32; Found (%): C,60.64; H,4.64; N,11.36.
5.2.7.16. (S)-5-chloro-N-((3-(4-(2-(diethylamino)-1H-indol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)thiophene-2-
carboxamide (7p)
Yield: 64.1%; M.p.: 135.4-136.1 °C; 1H NMR (400 MHz, DMSO) δ 9.14 (t, J = 5.7 Hz, 1H, CONH), 7.82 (d,
J = 7.5 Hz, 1H, Aryl-H), 7.78 (d, J = 4.1 Hz, 1H, Aryl-H), 7.74 (d, J = 8.9 Hz, 3H, , thiophene-4-H, Aryl-H), 7.61
(d, J = 8.9 Hz, 2H, Aryl-H), 7.51 (d, J = 8.2 Hz, 1H, Aryl-H), 7.27 – 7.12 (m, 3H, thiophene-3-H, Aryl-H), 4.89 (dt,
J = 11.3, 5.5 Hz, 1H, O-CH), 4.48 (s, 2H, indole-2-CH2), 4.25 (t, J = 9.0 Hz, 1H, CH2-NH), 3.97 (dd, J = 9.0, 6.1
Hz, 2H, CH2-NH), 3.73 – 3.55 (m, 2H, N-CH2), 2.81 (d, J = 63.5 Hz, 4H, CH2-CH3), 1.19 (d, J = 35.2 Hz, 6H,
CH3 ); MS (ESI) m/z (%): 536.4 [M+H]+; Anal. calcd. for C27H27ClN4O3S(%): C, 62.00; H, 5.20; N, 10.71;
Found (%): C,62.18; H,5.19; N,10.75.
5.2.7.17. (S)-5-chloro-N-((3-(4-(2-morpholino-1H-indol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)thiophene-2-car
boxamide (7q)
Yield: 67.9%; M.p.: 150.1-151.0 °C; 1H NMR (400 MHz, DMSO) δ 9.06 (t, J = 5.4 Hz, 1H, CONH), 7.77 (d,
J = 7.8 Hz, 1H, Aryl-H), 7.75 – 7.65 (m, 2H, Aryl-H), 7.69 (d, J = 7.3 Hz, 1H, thiophene-4-H), 7.60 (d, J = 8.8 Hz,
2H, Aryl-H), 7.56 – 7.45 (m, 2H, Aryl-H), 7.19 (t, J = 6.0 Hz, 2H, Aryl-H), 7.13 (d, J = 7.3 Hz, 1H, thiophene-3-H),
4.88 (dd, J = 8.3, 5.7 Hz, 1H, O-CH), 4.25 (t, J = 8.9 Hz, 1H, CH2-NH), 3.94 (dd, J = 8.9, 6.2 Hz, 1H, CH2-NH),
3.68 (s, 2H, indole-2-CH2), 3.63 (dd, J = 12.7, 7.3 Hz, 2H, N-CH2), 3.57 (s, 4H, morphline-H), 2.44 (s, 4H,
morphline-H); MS (ESI) m/z (%): 548.8 [M–H]–; Anal. calcd. for C27H25ClN4O4 S (%): C, 60.39; H, 4.69; N, 10.43;
Found (%): C,60.40; H,4.68; N,10.48.
5.2.7.18. (S)-5-chloro-N-((2-oxo-3-(4-(2-(piperidin-1-yl)-1H-indol-1-yl)phenyl)oxazolidin-5-yl)methyl)thiophene-2
-carboxamide (7r)
Yield: 59.9%; M.p.: 148.5-149.4 °C; 1H NMR (400 MHz, DMSO) δ 9.12 (t, J = 5.7 Hz, 1H, CONH), 7.80 –
7.74 (m, 2H, Aryl-H), 7.72 (d, J = 8.9 Hz, 1H, Aryl-H), 7.69 (d, J = 7.2 Hz, 1H, thiophene-4-H), 7.60 (d, J = 8.9 Hz,
2H, Aryl-H), 7.55 – 7.45 (m, 2H, Aryl-H), 7.19 (t, J = 6.7 Hz, 2H, Aryl-H), 7.12 (t, J = 7.2 Hz, 1H, thiophene-3-H),
4.88 (dd, J = 8.5, 5.6 Hz, 1H, O-CH), 4.25 (t, J = 9.0 Hz, 1H, CH2-NH), 3.96 (dd, J = 9.0, 6.1 Hz, 1H, CH2-NH),
3.71 (s, 2H, indole-2-CH2), 3.63 (d, J = 5.3 Hz, 2H, N-CH2), 2.94 (d, J = 10.1 Hz, 2H, piperidine-H), 2.01 (s, 2H,
piperidine-H), 1.58 (d, J = 11.9 Hz, 2H, piperidine-H), 0.88 (d, J = 6.4Hz, 4H, piperidine-H); MS (ESI) m/z (%):
548.7 [M+H]+; Anal. calcd. for C28H27ClN4O3S (%): C, 62.85; H, 5.09; N, 10.47; Found (%): C,62.86; H,5.12;
N,10.47.
5.2.7.19. (S)-5-chloro-N-((3-(4-(2-(4-methylpiperidin-1-yl)-1H-indol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)thi
ophene-2-carboxamide (7s)
Yield: 69.9%; M.p.: 160.5-161.2 °C; 1H NMR (400 MHz, DMSO) δ 9.20 (t, J = 5.7 Hz, 1H, CONH), 7.87 (d,
J = 7.6 Hz, 1H, Aryl-H), 7.81 (d, J = 4.0 Hz, 1H, Aryl-H), 7.75 (d, J = 8.9 Hz, 2H, Aryl-H), 7.69 (d, J = 7.0 Hz, 1H,
thiophene-4-H), 7.62 (d, J = 8.9 Hz, 2H, Aryl-H), 7.51 (d, J = 8.1 Hz, 1H, Aryl-H), 7.24 (d, J = 7.0 Hz, 1H,
thiophene-3-H), 7.22 – 7.13 (m, 2H, Aryl-H), 4.90 (dt, J = 11.4, 5.6 Hz, 1H, O-CH), 4.25 (t, J = 8.9 Hz, 1H,
CH2-NH), 4.14 (s, 2H, indole-2-CH2), 3.99 (dd, J = 8.9, 6.1 Hz, 1H, CH2-NH), 3.72 – 3.56 (m, 2H, N-CH2), 2.80 (d,
J = 51.1 Hz, 4H, 4-methylpiperdine-H), 2.15(m, 1H, CH) 1.67 (s, 4H, 4-methylpiperdine-H), 1.45 (s, 3H, CH3);
10

ACCEPTED MANUSCRIPT
MS (ESI) m/z (%): 562.8 [M+H]+; Anal. calcd. for C29H29ClN4O3S (%):C, 63.43; H, 5.32; N, 10.20; Found (%):
C,63.47; H,5.38; N,10.16.
5.2.7.20. (S)-5-chloro-N-((3-(4-(2-(4-methylpiperidin-1-yl)-1H-indol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)thi
ophene-2-carboxamide (7t)
1
Yield: 69.0%; M.p.: 159.8-160.5 °C; H NMR (400 MHz, DMSO) δ 9.15 (t, J = 5.3 Hz, 1H, CONH), 7.83 –
7.76 (m, 2H, Aryl-H), 7.73 (d, J = 8.9 Hz, 2H, Aryl-H), 7.64 – 7.54 (m, 2H, Aryl-H), 7.58 (d, J = 7.3 Hz, 1H,
thiophene-4-H),7.50 (d, J = 8.2 Hz, 1H, Aryl-H), 7.26 – 7.17 (m, 2H, Aryl-H), 7.14 (d, J = 7.3 Hz, 1H,
thiophene-3-H), 4.88 (dt, J = 11.4, 5.6 Hz, 1H, O-CH), 4.25 (t, J = 8.9 Hz, 1H, CH2-NH), 3.97 (dd, J = 8.9, 6.1 Hz,
1H, CH2-NH), 3.73 (d, J = 34.9 Hz, 2H, indole-2-CH2), 3.62 (d, J = 16.1 Hz, 2H, N-CH2), 2.90 – 2.55 (m, 8H,
4-methylpiperzine-H), 2.35 (d, J = 16.8 Hz, 3H, CH3); MS (ESI) m/z (%): 563.8 [M+H]+; Anal. calcd. for
C29H29ClN4O3S (%):C, 63.43; H, 5.32; N, 10.20; Found (%): C,63.39; H,5.29; N,10.25.
5.3 Preparation of oxazolidinone compounds containing thiazole pharmacophores (14a-14i)
5.3.1 Preparation of 4-(3-(1,3-dioxoisoindolin-2-yl)-2-hydroxypropylamino)benzonitrile (8)
A mixture of 4-aminobenzonitrile (9.7 g, 82.1 mmol), (S)-(+)-glycidyl phthalimide (20.0 g, 98.5 mmol) and
Mg(ClO4)2 (0.2 g, 0.82 mmol) in 1,4-dioxane (150 mL) was refluxed for 10 h. After the completion of reaction, the
solvent was removed and the residue was treated with ethyl acetate (100 mL). The organic layer was washed with
water (2 × 30 mL) and evaporated to afford 8 (20.5g, 78% yield) as a yellow solid; MS (ESI) m/z (%): 322.1
[M+H]+.
5.3.2 Preparation of (S)-4-(5-((1,3-dioxoisoindolin-2-yl)methyl)-2-oxooxazolidin-3-yl)benzonitrile (9)
A mixture of 8 (20.0 g, 62.3 mmol), CDI (20.2 g, 124.6 mmol) and N,N-dimethylpyridin-4-amine (3.8 g, 31.2
mmol) in THF (200 mL) was refluxed for 8h. After the completion of reaction, the solvent was removed and the
residue was treated with isopropyl alcohol and stirred for 30 min. The precipitate was collected by filtration and
dried to afford 9 (15.7 g, 73% yield) as a white solid; MS (ESI) m/z (%): 348.3 [M+H]+.
5.3.3 Preparation of (S)-4-(5-(aminomethyl)-2-oxooxazolidin-3-yl)benzonitrile (10)
A well-stirred mixture of 9 (15.0 g, 43.2 mmol) in a mixed solvent of methanol and CH2Cl2 (150 mL,
MeOH:DCM=1:1) was added drop-wise hydrazine hydrate (13.0 g, 259.4 mmol) at room temperature. After the
completion of addition, the mixture was heated at 40 °C for 10 h. The solvent was removed and the residue was
quenched with water (100 mL) and extracted with CH2Cl2 (3 × 50 mL). The combined organic layer was washed
with water (2 × 20 mL), brine (2 × 30 mL) and dried with MgSO4. Filtration for removal of MgSO4 and
evaporation of CH2Cl2 to afford 10 (7.8 g, 83% yield) as a white solid; MS (ESI) m/z (%): 218.1 [M+H]+.
5.3.4 Preparation of (S)-5-chloro-N-((3-(4-cyanophenyl)-2-oxooxazolidin-5-yl)methyl)thiophene-2-carboxamide
(11)
To a well-stirred solution of 5-chlorothiophene-2-carboxylic acid (6.7 g, 41.5 mmol) in DMF (100 mL) was
added EDCI (13.3 g, 69.1mmol) and HOBt (7.0 g, 51.8 mmol). After stirring for 30 min, TEA (4.0 mL, 69.1 mmol)
and 10 (7.5 g, 34.6 mmol) was added and the mixture was stirred for 12 h at room temperature and then poured into
ice-water (300 mL). After stirring for 15 min, the separated solid was collected by filtration, washed with water and
dried to afford 11 (11.4 g, 91.2% yield) as a white solid; MS (ESI) m/z (%): 362.2 [M+H]+.
5.3.5
Preparation
of
(S)-N-((3-(4-carbamothioylphenyl)-2-oxooxazolidin-5-yl)methyl)-5-chlorothiophene-2-carboxamide (12)
A well-stirred mixture of 11 (10.0 g, 27.7 mmol), NaSH (11.1 g, 138.5 mmol) and ammonium chloride (7.4 g,
138.5 mmol) in a mixed solvent of DMF and H2O (100 mL, DMF:H2O = 1:1) was stirred for 1 h at room
temperature and then poured into ice-water (200 mL). After stirring for 15 min, the precipitate was filtered and
washed with a small amount of water and dried to give 12 (9.7 g, 89% yield) as a white solid; MS (ESI) m/z (%):
396.2 [M+H]+.
11

ACCEPTED MANUSCRIPT
5.3.6
Preparation
of
(S)-5-chloro-N-((3-(4-(4-(chloromethyl)thiazol-2-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)thiophene-2-carboxamide
(13)
A mixture of 12 (9.0 g, 22.8 mmol) and 1,3-dichloropropan-2-one (5.7 g, 45.6 mmol) in DMF (90 mL) was
heated at 100 °C for 2 h. Upon cooling to room temperature, the mixture was poured into ice-water (250 mL). After
stirring for 15 min, the precipitate was filtered and washed with water and dried to give 13 (7.6 g, 72% yield) as a
white solid; MS (ESI) m/z (%): 467.9[M+H]+.
5.3.7 General procedure for preparation of (S)-N-((3-(4-(4-(substituted aminomethyl)thiazol-2-
yl)phenyl)-2-oxooxazolidin-5-yl)methyl)-5-chlorothiophene-2-carboxamide (14a-14i)
To a well-stirred solution of 13 (0.3 g, 0.64 mmol) in DMF (10 mL) was added K2CO3 (0.18 g, 1.28 mmol)
and corresponding amine (1.28 mmol). The mixture was stirred for 4-5 h at room temperature and then poured into
ice-water (30 mL). After stirring for 15 min, the precipitate was filtered and washed with water and dried to give
crude product, which was purified by column to afford 14a-14i.
5.3.7.1. (S)-5-chloro-N-((3-(4-(4-((methylamino)methyl)thiazol-2-yl)phenyl)-2-oxooxazolidin-5-yl)-methyl)thioph
ene-2-carboxamide (14a)
Pale yellow solid; Yield: 46.7%; M.p.: 202.2-205.6 °C; 1H NMR (400 MHz, DMSO) δ 9.00 (t, J = 5.6 Hz, 1H,
CH2NHCO), 7.94 (d, J = 8.8 Hz, 2H, phenyl-3,5-H), 7.70 (d, J = 4.0 Hz, 1H, thiophene-3-H), 7.67 (d, J = 8.8 Hz,
2H, phenyl-2,6-H), 7.45 (s, 1H, thiazole-H), 7.20 (d, J = 4.0 Hz, 1H, thiophene-4-H), 4.89-4.82 (m, 1H, OCHCH2),
4.22 (t, J = 8.8 Hz, 1H, NCH2CH), 3.89 (dd, J =9.2Hz, 6.4 Hz, 1H, NCH2CH), 3.82 (s, 2H, methylene), 3.62 (t, J =
5.6 Hz, 2H, CHCH2NH), 2.37 (s, 3H, CH3); MS (ESI) m/z (%): 462.8[M+H]+, 924.7[2M+H]+; Anal. calcd. for
C20H19ClN4O3S2 (%): C, 51.89; H, 4.14; N, 12.10; Found (%): C,51.80; H,4.22; N,12.13.
5.3.7.2. (S)-5-chloro-N-((3-(4-(4-((dimethylamino)methyl)thiazol-2-yl)phenyl)-2-oxooxazolidin-5-yl)-methyl)thiop
hene-2-carboxamide (14b)
Pale yellow solid; Yield: 53.3%; M.p.: 215.2-217.1 °C; 1H NMR (400 MHz, DMSO) δ 9.01 (t, J = 5.6 Hz, 1H,
CH2NHCO), 7.93 (d, J = 8.8 Hz, 2H, phenyl-3,5-H), 7.70 (d, J = 4.0 Hz, 1H,thiophene-3-H), 7.67 (d, J = 8.8 Hz,
2H, phenyl-2,6-H), 7.46 (s, 1H, thiazole-H), 7.19 (d, J = 4.0 Hz, 1H, thiophene-4-H), 4.89-4.83 (m, 1H, OCHCH2),
4.22 (t, J = 9.2 Hz, 1H, NCH2CH), 3.90 (dd, J = 9.2, 6.4 Hz, 1H, NCH2CH), 3.62 (t, J = 5.6 Hz, 2H, CHCH2NH),
3.58 (s, 2H, methylene-H), 2.23 (s, 6H, CH3); MS (ESI) m/z (%): 477.0[M+H]+; Anal. calcd. for C21H21ClN4O3S2
(%): C, 52.88; H, 4.44; N, 11.75; Found (%): C,52.80; H,4.50; N,11.78.
5.3.7.3. (S)-5-chloro-N-((3-(4-(4-((ethylamino)methyl)thiazol-2-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)thiophene
-2-carboxamide (14c)
Pale yellow solid; Yield: 60.0%; M.p.: 195.7-199.1 °C; 1H NMR (400 MHz, DMSO) δ 9.01 (t, J = 5.6 Hz, 1H,
CH2NHCO), 7.94 (d, J = 8.8 Hz, 2H, phenyl-3,5-H), 7.70 (d, J = 4.0 Hz, 1H, thiophene-3-H), 7.67 (d, J = 8.8 Hz,
2H, phenyl-2,6-H), 7.46(s, 1H, thiazole-H), 7.19 (d, J = 4.0 Hz, 1H, thiophene-4-H), 4.89-4.83 (m, 1H, OCHCH2),
4.22 (t, J = 8.8 Hz, 1H, NCH2CH), 3.92 (m, 1H, NCH2CH), 3.88 (s, 2H, methylene-H), 3.62 (t, J = 5.6 Hz, 2H,
CHCH2NH), 2.66 (q, J = 7.2 Hz, 2H, CH2CH3), 1.07 (t, J = 7.2 Hz, 3H, CH3) ; MS (ESI) m/z (%): 476.7 [M + H]+,
952.4 [2M+H]+; Anal. calcd. for C21H21ClN4O3S2 (%): C, 52.88; H, 4.44; N, 11.75; Found (%): C, 52.89; H,4.47;
N,11.73.
5.3.7.4. (S)-5-chloro-N-((3-(4-(4-((diethylamino)methyl)thiazol-2-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)thiophe
ne-2-carboxamide (14d)
Pale yellow solid; Yield: 53%; M.p.: 211.3-215.1 °C; 1H NMR (400 MHz, DMSO) δ 9.02 (t, J = 5.6 Hz, 1H,
CH2NHCO), 7.92 (d, J = 8.8 Hz, 2H, phenyl-3,5-H), 7.71 (d, J = 4.0 Hz, 1H, thiophene-3-H), 7.66 (d, J = 8.8 Hz,
2H, phenyl-2,6-H), 7.42 (s, 1H, thiazole-H), 7.19 (d, J = 4.0 Hz, 1H, thiophene-4-H), 4.89-4.83 (m, 1H, OCHCH2),
4.22 (t, J = 8.8 Hz, 1H, NCH2CH), 3.90 (dd, J = 8.8 Hz, 6.5 Hz, 1H, NCH2CH), 3.74 (s, 2H, methylene-H), 3.62 (t,
12

ACCEPTED MANUSCRIPT
J = 5.6 Hz, 2H, CHCH2NH), 2.53 (q, J = 7.2 Hz, 4H, CH2CH3), 1.02 (t, J = 7.2 Hz, 6H, CH3); MS (ESI) m/z (%):
505.2 [M+H]+; Anal. calcd. for C23H25ClN4O3S2 (%): C, 54.70; H, 4.99; N, 11.09; Found (%): C,54.74; H,4.94;
N,11.12.
5.3.7.5. (S)-5-chloro-N-((2-oxo-3-(4-(4-(pyrrolidin-1-ylmethyl)thiazol-2-yl)phenyl)oxazolidin-5-yl)methyl)thiophe
ne-2-carboxamide (14e)
1
Pale yellow solid; Yield: 65.6%; M.p.: 223.6-227.1 °C; H NMR (400 MHz, DMSO) δ 9.06 (t, J = 5.6
Hz,1H,CH2NHCO), 7.93 (d, J = 8.8 Hz, 2H, phenyl-H-3,5), 7.71 (d, J = 4.0 Hz, 1H, thiophene-H-3), 7.66 (d, J =
8.8 Hz, 2H, phenyl-H-2,6), 7.43 (s, 1H, thiazole-H), 7.19 (d, J = 4.0 Hz, 1H, thiophene-H-4), 4.89-4.83 (m, 1H,
OCHCH2), 4.22 (t, J = 9.0 Hz, 1H, NCH2CH), 3.90 (dd, J = 8.8Hz, 6.4 Hz, 1H, NCH2CH), 3.72 (s, 2H,
methylene-H), 3.62 (t, J = 5.2 Hz, 2H, CHCH2NH), 2.52 (m ， 4H, pyrrolidine-2,5-H), 1.70 (s, 4H,
pyrrolidine-3,4-H); MS (ESI) m/z (%):502.8 [M+H]+; Anal. calcd. for C23H23ClN4O3S2 (%): C, 54.92; H, 4.61; N,
11.14; Found (%): C,54.88; H,4.61; N,11.20.
5.3.7.6. (S)-5-chloro-N-((2-oxo-3-(4-(4-(piperidin-1-ylmethyl)thiazol-2-yl)phenyl)oxazolidin-5-yl)methyl)thiophen
e-2-carboxamide (14f)
Pale yellow solid; Yield: 51.5%; M.p.: 243.6-245.9 °C; 1H NMR (400 MHz, DMSO) δ 9.05 (t, J = 5.6 Hz ,1H,
CH2NHCO), 7.93 (d, J = 8.8 Hz, 2H, phenyl-3,5-H), 7.70 (d, J = 4.0 Hz, 1H, thiophene-3-H), 7.66 (d, J = 8.8 Hz,
2H, phenyl-2,6-H), 7.42 (s, 1H, thiazole-H), 7.19 (d, J = 4.0 Hz, 1H, thiophene-4-H), 4.89-4.83(m, 1H, OCHCH2),
4.22 (t, J = 8.8 Hz, 1H, NCH2CH), 3.90 (dd, J = 9.2 Hz, 6.4 Hz, 1H, NCH2CH), 3.62 (t, J = 5.3 Hz, 2H,
CHCH2NH), 3.58 (s, 2H, methylene-H), 2.41 (s ,4H, piperidine-2,6-H), 1.56-1.46 (m, 4H, piperidine-3,5-H),
1.38-1.37 (m, 2H, piperidine-4-H); MS (ESI) m/z (%): 516.7[M+H]+; Anal. calcd. for C24H25ClN4O3S2 (%):C,
55.75; H, 4.87; N, 10.84; Found (%): C,55.79; H,4.81; N,10.93.
5.3.7.7. (S)-5-chloro-N-((3-(4-(4-((4-methylpiperidin-1-yl)methyl)thiazol-2-yl)phenyl)-2-oxooxazolidin
-5-yl)methyl)thiophene-2-carboxamide (14g)
Pale yellow solid; Yield: 61.8%; M.p.: 241.2-244.4 °C; 1H NMR (400 MHz, DMSO) δ 9.05 (t, J = 5.6 Hz ,1H,
CH2NHCO), 7.93 (d, J = 8.8 Hz, 2H, phenyl-3,5-H), 7.70 (d, J = 4.0 Hz, 1H, thiophene-3-H), 7.66 (d, J = 8.8 Hz,
2H, phenyl-2,6-H), 7.42 (s, 1H, thiazole-H), 7.19 (d, J = 4.0 Hz, 1H, thiophene-4-H), 4.89-4.83 (m, 1H, OCHCH2),
4.22 (t, J = 9.2 Hz, 1H, NCH2CH), 3.90 (dd, J = 9.2 Hz, 6.4 Hz, 1H, NCH2CH), 3.62 (t, J = 5.2 Hz, 2H,
CHCH2NH), 3.59 (s, 2H, methylene-H), 2.87 (t, J = 10.8 Hz, 2H, piperidine-2-H), 1.99 (t, J = 10.8 Hz, 2H,
piperidine-H-6), 1.55 (s, 3.6 Hz, 2H, piperidine-3-H), 1.36-1.25 (m, 1H, piperidine-H-4), 1.19-1.09 (m, 2H,
piperidine-5-H), 0.88 (d, J = 6.4 Hz, 3H, CH3); MS (ESI) m/z (%): 530.8 [M+H]+; Anal. calcd. for C25H27ClN4O3S2
(%): C, 56.54; H, 5.12; N, 10.55; Found (%): C,56.55; H,5.18; N,10.53.
5.3.7.8. 5.3.7.8(S)-5-chloro-N-((3-(4-(4-((4-methylpiperazin-1-yl)methyl)thiazol-2-yl)phenyl)-2-oxooxazolidin-5-y
l)methyl)thiophene-2-carboxamide (14h)
Pale yellow solid; Yield: 58.8%; M.p.: 243.6-245.3 °C; 1H NMR (400 MHz, DMSO) δ 9.00 (t, J = 5.6 Hz, 1H,
CH2NHCO), 7.93 (d, J = 8.8 Hz, 2H, phenyl-3,5-H), 7.70 (d, J = 4.0 Hz, 1H, thiophene-3-H), 7.66 (d, J = 8.8 Hz,
2H, phenyl-2,6-H), 7.44 (s, 1H, thiazole-H), 7.19 (d, J = 4.0 Hz, 1H, thiophene-4-H), 4.89-4.83 (m, 1H, OCHCH2),
4.22 (t, J = 9.2 Hz, 1H, NCH2CH), 3.89 (dd, J = 9.2Hz, 6.4 Hz, 1H, NCH2CH), 3.63 (t, J = 5.6 Hz, 2H, CHCH2NH),
3.61 (s, 2H, methylene-H), 2.47(t, J = 10.8 Hz, 4H, piperazine-2,6-H),2.33 (t, J = 10.8 Hz, 4H, piperazine-3,5-H),
2.15 (s, 3H, CH3); MS (ESI) m/z (%):531.7 [M+H]+; Anal. calcd. for C24H26ClN5O3S2 (%): C, 54.18; H, 4.93; N,
13.16; Found (%): C,54,20; H,4.94; N,13.13.
5.3.7.9. 5.3.7.9(S)-5-chloro-N-((3-(4-(4-(morpholinomethyl)thiazol-2-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)
thiophene-2-carboxamide (14i)
Pale yellow solid; Yield: 69.7%; M.p.: 231.5-236.6 °C; 1H NMR (400 MHz, DMSO) δ 8.99 (t, J = 5.6 Hz, 1H,
CH2NHCO), 7.94 (d, J = 8.8 Hz, 2H, phenyl-3,5-H), 7.69 (d, J = 4.4 Hz, 1H, thiophene-3-H), 7.67 (d, J = 8.8 Hz,
13

ACCEPTED MANUSCRIPT
2H, phenyl-2,6-H), 7.48 (s, 1H, thiazole-H), 7.19 (d, J = 4.0 Hz, 1H, thiophene-4-H), 4.92-4.80 (m, 1H, OCHCH2),
4.22 (t, J = 9.2 Hz, 1H, NCH2CH), 3.89 (dd, J = 8.9 Hz, 6.4 Hz, 1H, NCH2CH), 3.63 (t, J = 5.6 Hz, 2H,
CHCH2NH),3.62 (s, 2H, methylene-H), 3.61 - 3.56 (m, 4H, morpholine-3,5-H), 2.46 (s, 4H,morpholine-2,6-H); MS
(ESI) m/z (%): 518.7 [M+H]+ ; Anal. calcd. for C23H23ClN4O4S2 (%): C, 53.22; H, 4.47; N, 10.79; Found (%):
C,53.19; H,4.52; N,10.78.
5.4. Pharmacology
5.4.1. In vitro Coagulation Assays
Prothrombin time (PT) and activated partial thromboplastin time (APTT) were measured using commercially
available kits. Blood was obtained from healthy volunteers or rabbit and anticoagulated with 3.8% sodium citrate.
Plasma was obtained after centrifugation at 2000 g for 10 min. An initial stock solution of the inhibitor was
prepared in DMSO. Subsequent dilutions were done in plasma. Clotting time was determined on control plasma
and plasma containing five to seven different concentrations of inhibitor. Prothrombin time (PT) measurement was
performed in a temperature-controlled automated coagulation device (Sysmex CA50, Dade-Behring) using
Thromborel-S (Dade Behring) kit according to the reagent instructions. Determinations at each plasma
concentration were done in duplicate. The concentration of inhibitors required to double the clotting time (PTCT2)
was estimated from the concentration-response curve by a regression analysis [31].
5.4.2. Anti-FXa activity in vitro
The inhibitory activity of different compounds against purified serine proteases was measured using
chromogenic substrates in 96-well microtiter plates at RT. The enzymes were incubated with test compound or its
solvent, dimethyl sulfoxide (DMSO). The plate was incubated at 37 °C for 45 min. At the end of incubation the
plate was read at 405 nm using a Spectra Max. The following buffer (final concentrations) was used in the assay:
human FXa (10 ng), 50 mM Tris-HCl buffer pH 7.5, 150
substrate (S-2765) (Hyphen Biomed), 2.5% DMSO with varying concentrations of test compound [15,32].
Anti-FXa activity (inhibition %) was calculated as follows: Anti-FXa activity = [1 (reaction velocity of sample)
ꢀM NaCl, and 1 mM calcium chloride and 500 ꢀM
–
–
(reaction velocity of control)] × 100. The IC50 value was obtained by plotting the anti-FXa activity against the
inhibitor concentration.
Acknowledgments
The work was supported by Program for Innovative Research Team of the Ministry of Education of the
People’s Republic of China and Program for Liaoning Innovative Research Team in University (IRT1073).
References
[1] A. Mochizuki, T. Nagata, H. Kanno, M. Suzuki, T. Ohta, Bioorg. Med. Chem. 19 (2011) 1623–1642.
[2] (a) P.D. Stein, D. Grandison, T.A. Hua, Postgrad. Med. 70 (1994) S72; (b) J. Hirsh, L. Poller, Arch. Int. Med. 154 (1994) 282.
[3] E.W. Davie, K. Fujikawa, W. Kisiel, Biochemistry. 30 (1991) 10363–10370.
[4] S. Elodi, K. Varadi, Thromb. Res. 15 (1979) 617–620.
[5] T. Hara, A. Yokoyama, K. Tanabe, H. Ishihara, M. Iwamoto, Thromb. Haemost. 74 (1995) 635–638.
[6] Y. Morishima, K. Tanabe, Y. Terada, T. Hara, S. Kunitada, Thromb. Haemost. 78 (1997) 136–140.
[7] B. Kaiser, Drugs Future. 23 (1998) 423–426.
[8] D.J.P. Pinto, J.M. Smallheer, D.L. Cheney, R. M. Knabb, R.R. Wexler, J. Med. Chem. 53 (2010) 6243-6247.
[9] G. Lippi, M. Franchini, G. Targher, Nat. Rev Cardiol. 8 (2011) 502–512.
14

ACCEPTED MANUSCRIPT
[10] Y. Morishima, K. Tanabe, Y. Terada, T. Hara, S. Kunitada, Thromb. Haemostasis. 78 (1997) 1366–1371.
[11] M. Sanford, G.L. Plosker, Dabigatran etexilate, Drugs. 68 (2008) 1699–1709.
[12] E. Perzborn, S. Roehrig, A. Straub, D. Kubitza, F. Misselwitz, Nat. Rev. Drug. Discov. 10 (2011) 61–75.
[13] D.J. Pinto, M.J. Orwat, S. Koch, K.A. Rossi, R.S. Alexander, A. Smallwood, P.C. Wong, A.R. Rendina, J.M. Luettgen, R.M.
Knabb, K. He, B. Xin, R.R. Wexler, P.Y. Lam, J. Med. Chem. 50 (2007) 5339–5356.
[14] T. Furugohri, K. Isobe, Y. Honda, C. Kamisato-Matsumoto, N. Sugiyama,T. Nagahara, Y. Morishima, T.
Thromb. Haemostasis. 6 (2008) 1542–1549.
Shibano, DU-176b, J.
[15] F. Hirayama, H. Koshio, T. Ishihara, S. Hachiya, K. Sugasawa, Y. Koga, N. Seki, R. Shiraki, T. Shigenaga, Y. Iwatsuki, Y.
Moritani, K. Mori, T. Kadokura, T. Kawasaki, Y. Matsumoto, S. Sakamoto, S. Tsukamoto, J. Med. Chem. 54 (2011) 8051–8065.
[16] A.G. Turpie, K.A. Bauer, B.L. Davidson, W.D. Fisher, M. Gent, M.H. Huo, U. Sinha, D.D. Gretler, Thromb. Haemostasis. 101
(2009) 68–76.
[17] Y.L. Chou, D.D. Davey, K.A. Eagen, B.D. Griedel, R. Karanjawala, G.B. Phillips, K.L. Sacchi, K.J. Shaw, S.C. Wu, D. Lentz,
A.M. Liang, L. Trinh, M.M. Morrissey, M.J. Kochanny, Bioorg. Med. Chem. Lett. 13 (2003) 507–511.
[18] D. Mendel, A.L. Marquart, S. Joseph, P. Waid, Y.K. Yee, A.L. Tebbe, A.M. Ratz, D.K. Herron, T. Goodson, J.J. Masters, J.B.
Franciskovich, J.M. Tinsley, M.R. Wiley, L.C. Weir, J.A. Kyle, V.J. Klimkowski, G.F. Smith, R.D. Towner, L.L. Froelich, J.
Buben, T.J. Craft, Bioorg. Med. Chem. Lett. 17 (2007) 4832–4836.
[19] T. Nagata, T. Yoshino, N. Haginoya, K. Yoshikawa, Y. Isobe, T. Furugohri, H. Kanno, Bioorg. Med. Chem. Lett. 17 (2007) 4683–
4688.
[20] J.X. Qiao, C.H. Chang, D.L. Cheney, P.E. Morin, G.Z. Wang, S.R. King, T.C. Wang, A.R. Rendina, J.M. Luettgen, R.M. Knabb,
R.R. Wexler, P.Y. Lam, Bioorg. Med. Chem. Lett. 17 (2007) 4419–4427.
[21] Y.K. Yee, A.L. Tebbe, J.H. Linebarger, D.W. Beight, T.J. Craft, D. Gifford-Moore, T. Goodson Jr., D.K. Herron, V.J. Klimkowski,
J.A. Kyle, J.S. Sawyer, G.F. Smith, J.M. Tinsley, R.D. Towner, L. Weir, M.R. Wiley, J. Med. Chem. 43 (2000) 873–882.
[22] P. Zhang, L. Bao, J.F. Zuckett, E.A. Goldman, Z.J. Jia, A. Arfsten, S. Edwards, U. Sinha, A. Hutchaleelaha, G. Park, J.L. Lambing,
S.J. Hollenbach, R.M. Scarborough, B.Y. Zhu, Bioorg. Med. Chem. Lett. 14 (2004) 983–987.
[23] S.
Roehrig,
A.
Straub,
J.
Pohlmann,
Discovery
of
the
novel
antithrombotic
agent
5-chloro-N-({(5S)-2-oxo-3-[4-(3-oxomorpholin-4-yl)phenyl]-1,3-oxazolidin-5-yl}methyl)thiophene-2-carboxamide(BAY
59-7939): an oral, direct factor Xa inhibitor. J. Med. Chem, 19 (2005) 5900–5908.
[24] S. Alexander, L. Thomas, Substituted oxazolidinones and their use in the field of blood coagulation. US7157456, 2007.
[25] T. Zhu, S.O. Friedrich, A. Diacon, R.S. Wallis, Anti. Agents. Chem. 58 (2014) 3306–3311.
[26] M.F. Gordeev, Z.Y. Yuan, J. Med. Chem. 57 (2014) 4487–4497.
[27] T. Xue, S. Ding, B. Guo, Y.R. Zhou, P. Sun, H.Y. Wang, W.J. Chu, G.Q. Gong, Y.Y. Wang, X.Y. Chen, Y.S. Yang, J. Med. Chem.
57 (2014) 7770–7791.
[28] A. Straub, T. Lampe, J. Pohlmann Substituted oxazolidinones and their use in the field of blood coagulation [P]. WO:
2001047919, 2001. 07. 05.
[29] Sugiura, Satoshi et al. Preparation of 4-substituted benzothioamide derivatives [P].WO2010143735, 2010.
[30] Thomas, J. Russel, Preparation of aryl dicarboxamides as protein-tyrosine phosphatase inhibitors. WO2005011685, 2005.
[31] V. Pandya, M. Jain, G. Chakrabart, H. Soni, B. Parmar, B. Chaugule, J. Patel, T. Jarag, J. Joshi, A. Rath, V. Unadkat, B. Sharma,
H. Ajani, J. Kumar, K.V.V.M. Sairam, H. Patel, P. Patel, Eur. J. Med. Chem. 58 (2012) 136–152.
[32]E. Perzborn, J. Strassburger, A. Wilmen, J. Pohlmann, S. Roehrig, K.H. Schlemmer, A. Straub, Thromb. Haemost. 3 (2005) 514–
521.
[33]S. Roehrig, A.Straub, J. Pohlmann, T. Lampe, J. Pernerstorfer, K.H. Schlemmer, P. Reinemer, E. Perzborn, J. Med. Chem. 48
(2005) 5900–5908.
15

ACCEPTED MANUSCRIPT
Legends
Fig. 1. Structures of oral factor Xa inhibitors.
Fig. 2. Structure of the target compounds.
Scheme 1. Reagents and conditions: (1) K2CO3, DMF, 100 °C, 5h; (2) hydrazine hydrate, 1,4-dioxane, reflux, 2h;
(3) 90% EtOH, reflux, 20h; (4) CDI, THF, reflux, 5h; (5) hydrazine hydrate, 40 °C, 3h; (6) EDCI, HOBt, TFA,
DMF, r.t., 12h; (7) HCHO, HNR1R2, HOAc, r.t..
Scheme 2. Reagents and conditions: (1) Mg(ClO4)2, 1,4-dioxane, reflux, 10h; (2) CDI, DMAP, THF, reflux, 8h; (3)
DCM:MeOH=1:1, hydrazine hydrate, 40 °C, 10h; (4) EDCI, HOBt, TFA, DMF, r.t., 12h; (5) NaSH, NH4Cl, DMF,
H2O, r.t., 1h; (6) 1,3-dichloroacetone, DMF, 100 °C, 1.5h; (7) K2CO3, DMF, r.t., 5h.
Table 1
In vitro FXa inhibitory and anticoagulant activity data of compounds 7a–7n, 7o–7t and 14a–14i.
Table 2
In vitro FXa inhibitory activity of compounds 7a, 7b, 7i, 7j, 14d and Rivaroxaban.
Fig. 3. The human FXa active site in complex with compound 7b and their hydrophobic surface.
16

ACCEPTED MANUSCRIPT
Table 1
In vitro FXa inhibitory and anticoagulant activity data of compounds 7a
–
7n, 7o
–7t and 14a
–
14i.
Compd.
NR1R2
R3
IC50 (μM)
b
b
b
b
%Inhibitiona
at 0.1μM
98
PT CT2
APTT CT2
Human
1.20
PT CT2
Rabbit
0.78
APTT CT2
Rabbit
1.39
Human
0.61
7a
7b
7c
7d
7e
7f
H
H
100
92
89
82
76
70
71
90
93
82
88
89
73
69
61
0.15
0.70
1.42
2.62
2.93
3.22
3.80
0.65
0.35
2.21
1.83
1.51
2.79
5.78
5.32
0.30
1.31
1.71
2.85
3.31
3.53
4.01
1.33
0.65
2.92
2.31
1.74
3.06
6.02
5.45
0.46
0.81
1.58
2.89
3.19
3.45
3.90
0.91
0.82
2.45
1.98
1.78
2.98
5.92
6.02
0.75
1.52
1.98
3.23
3.52
3.78
4.14
1.58
1.13
3.01
2.56
1.96
3.67
6.23
6.11
H
H
H
H
7g
7h
7i
H
H
2-F
2-F
2-F
2,6-(F)2
2,6-(F)2
2,6-(F)2
H
7j
7k
7l
7m
7n
70
7p
H

ACCEPTED MANUSCRIPT
7q
H
H
H
H
H
H
H
H
H
H
H
H
H
58
52
56
45
89
91
93
100
79
72
68
56
62
100
10.12
11.02
10.45
11.29
1.65
0.87
0.65
0.51
2.42
2.72
3.03
3.85
3.60
0.40
11.23
ND
11.12
11.45
12.15
13.32
1.66
0.92
0.89
0.72
2.78
3.12
3.78
4.23
4.06
0.51
ND
ND
7r
7s
7t
11.89
ND
13.11
ND
14a
1.87
0.98
0.79
0.82
2.71
2.95
3.39
4.12
3.81
0.70
1.91
1.24
1.16
1.03
3.04
3.46
4.02
5.12
4.79
0.86
14b
14c
14d
14e
14f
14g
14h
14i
Rivaroxabanc
Bold values show the IC50 values of the target compounds lower than the values of the positive
control. ND: Not determined.
a Inhibitory activity against human FXa. Values shown are the mean of duplicate measurements.
b Concentration of the compound required to double the clotting time in the PT assay using human
plasma. PTCT2 values shown are the mean of duplicated measurements unless otherwise
indicated.
c Used as a positive control.

Table 2
In vitro FXa inhibitory activity of compouAndCs 7CaE, 7PbT, 7Ei,D7j,M14AdNanUd SRiCvaRroIxPaTban.
IC50 (nM)a
Compd.
7a
7.24
2.01
6.58
3.25
9.46
2.20
7b
7i
7j
14d
Rivaroxabanb
a Inhibitory activity against human FXa. IC50 values shown are the mean of duplicate measurements.
b Used as a positive control.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
ꢀ
A series of oxazolidinone derivatives containing pyrrole/indole/thiazole moiety were
designed and synthesized.
ꢀ
ꢀ
ꢀ
The target compounds showed potent anticoagulant activity.
Five compounds were further examined for their anti-FXa inhibitory activity.
Compound 7b showed an IC50 value of 2.01 nM against human FXa inhibition.

ACCEPTED MANUSCRIPT
Spectrum of intermediates and the target compounds
1H-NMR spectrum of compound 2a
MS spectrum of compound 2a

ACCEPTED MANUSCRIPT
1H-NMR spectrum of compound 3a
MS spectrum of compound 3a

ACCEPTED MANUSCRIPT
1H-NMR spectrum of compound 4a
MS spectrum of compound 4a