Design, synthesis, and structure-activity and structure-pharmacokinetic relationship studies of novel [6,6,5] tricyclic fused oxazolidinones leading to the discovery of a potent, selective, and orally bioavailable FXa inhibitor

Article abstract of DOI:10.1021/jm501045e

The blood coagulation enzyme factor Xa (FXa) is a particularly promising target for anticoagulant therapy, and identification of oral small-molecule inhibitors of FXa remains a research focus. On the basis of the X-ray crystal structure of FXa and its inhibitor rivaroxaban, we designed and synthesized a series of conformationally restricted mimics containing a novel [6,6,5] tricyclic fused oxazolidinone scaffold. Intensive structure-activity relationship (SAR) and structure-pharmacokinetic relationship (SPR) studies on this new series led to the discovery of compound 11a: a highly potent, selective, direct, and orally bioavailable FXa inhibitor with excellent in vivo antithrombotic efficacy and preferable pharmacokinetic profiles. Druggability evaluation of compound 11a was undertaken and elicited positive outcomes. All results indicate that compound 11a is a promising drug candidate for the prevention and treatment of thromboembolic diseases in venous and arterial systems.

Full text of DOI:10.1021/jm501045e

Article
pubs.acs.org/jmc
Design, Synthesis, and Structure−Activity and Structure−
Pharmacokinetic Relationship Studies of Novel [6,6,5] Tricyclic Fused
Oxazolidinones Leading to the Discovery of a Potent, Selective, and
Orally Bioavailable FXa Inhibitor
Tao Xue,†,∥ Shi Ding,†,∥ Bin Guo,*,† Yuren Zhou,† Peng Sun,† Heyao Wang,† Wenjing Chu,†
Guoqing Gong,‡ Yinye Wang,§ Xiaoyan Chen,† and Yushe Yang*,†
†State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203,
China
‡Department of Pharmacology, School of Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu Province 210009, China
§Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
S
* Supporting Information
ABSTRACT: The blood coagulation enzyme factor Xa (FXa) is a particularly promising target for anticoagulant therapy, and
identification of oral small-molecule inhibitors of FXa remains a research focus. On the basis of the X-ray crystal structure of FXa
and its inhibitor rivaroxaban, we designed and synthesized a series of conformationally restricted mimics containing a novel
[6,6,5] tricyclic fused oxazolidinone scaffold. Intensive structure−activity relationship (SAR) and structure−pharmacokinetic
relationship (SPR) studies on this new series led to the discovery of compound 11a: a highly potent, selective, direct, and orally
bioavailable FXa inhibitor with excellent in vivo antithrombotic efficacy and preferable pharmacokinetic profiles. Druggability
evaluation of compound 11a was undertaken and elicited positive outcomes. All results indicate that compound 11a is a
promising drug candidate for the prevention and treatment of thromboembolic diseases in venous and arterial systems.
blood coagulation cascade has attracted great interest. Factor
Xa and thrombin (FIIa) are particularly promising targets. FXa
is located at the junction of the intrinsic and extrinsic pathways
of coagulation and catalyzes the conversion of prothrombin to
thrombin. Being different from thrombin inhibitors, selective
FXa inhibitors exhibit their antithrombotic effects by reducing
the further generation of thrombin while exerting less influence
on existing thrombin levels, which may result in a decreased
risk of bleeding and provide a much more favorable safety
profile.5
INTRODUCTION
■
Thromboembolic disorders such as deep venous thrombosis
(DVT), pulmonary embolism (PE), ischemic stroke, myocar-
dial infarction, and unstable angina remain the leading cause of
morbidity and mortality worldwide.1−3 Several anticoagulants
such as heparins (unfractionated and low-molecular-weight
heparins) and vitamin K antagonists (VKAs; e.g., warfarin) have
proved to be effective in the prevention and treatment of these
thrombotic diseases, but considerable shortcomings (e.g.,
inconvenient drug administration and unneglectable side effects
for heparins and extensive drug and food interactions for
VKAs) restrict their clinical use.4 Thus, identifying novel oral
anticoagulants with improved efficacy and safety has become
increasingly important.
Recently, three novel oral FXa inhibitors (Figure 1) have
been approved: rivaroxaban6−8 (1, Bayer) and apixaban9,10 (2,
Bristol-Myers Squibb/Pfizer) by the U.S. Food and Drug
After extensive studies, the discovery and development of
inhibitors that selectively target specific enzymes within the
Received: July 17, 2014
Published: September 2, 2014
© 2014 American Chemical Society
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Figure 1. Currently approved FXa inhibitors.
Administration (FDA) and edoxaban11 (3, Daiichi-Sankyo) in
Japan. In July 2011, rivaroxaban was the first FXa inhibitor to
be approved by FDA for prophylaxis of DVT in adults
undergoing hip and knee replacement surgery. Subsequently, in
November 2011, new indications for reducing the risk of stroke
and systemic embolism in patients with nonvalvular atrial
fibrillation were approved by the FDA.
The X-ray crystal structure of rivaroxaban in complex with
human FXa demonstrated that the formation of two hydrogen
bonds between rivaroxaban and Gly219 has a crucial role in its
high affinity.6,12 Additionally, the co-planar stereo conformation
between the aryl ring and oxazolidinone core also has an
indispensable effect on maintaining its high binding affinity. If
the co-planar conformation is disturbed into an unfavorable
twist arrangement by introducing a substitution at the 2-
position of the aryl ring, then the activity decreases sharply.6 In
light of the findings noted above, we bridged the benzene ring
and oxazolidinone ring by an additional linker to form a novel
[6,6,5] tricyclic fused oxazolidinone scaffold to maintain this
preferred conformation (Figure 2). With the intention of
and then reacted with potassium phthalimide to obtain
compounds 9a−j. Ammonolysis of the resulting compounds
9a−j using methylamine in ethanol afforded amines 10a−j. A
condensation reaction was carried out between 10a−j and 5-
chlorothiophene-2-carboxylic acid to yield compounds 11a−j.
TBDPS deprotection of 11h and 11i using tetrabutylammo-
nium fluoride (TBAF) gave alcohols 11k and 11m, which were
then converted to their corresponding fluorides 11l and 11n
using DAST as the fluorination reagent. Deprotection of the
Cbz group in 11j provided 11o. Subsequent reductive
amination with formaldehyde and acetaldehyde afforded 11p
and 11q, respectively. The benzyl analogue 11r was obtained by
a nucleophilic substitution reaction between 11o and benzyl
bromide. 11o was reacted with acetylchloride, mesyl chloride,
and benzenesulfonyl chloride to yield the corresponding
compounds 11s, 11t, and 11u, respectively.
The synthetic route for the preparation of compound 18, the
diastereomer of 11a, is depicted in Scheme 2, which is similar
to that described in Scheme 1. The starting material 12 was
obtained via published procedures with slight modifications.14
The synthesis of aromatic P4 moiety-modified benzoxazinyl-
oxazolidinones is outlined in Scheme 3. The first five steps in
this procedure for the synthesis of compound 23 are similar to
those in Scheme 1. Next, the Miyaura coupling reaction
between compound 23 and bis(pinacolato)diboron yielded
boric ester 24, which was then converted to biaryl compounds
25a and 25b by the Suzuki coupling reaction. Cleavage of the
N-tert-butyl group in compound 25b using trifluoroacetic acid
(TFA) afforded 25c.
Preparation of P1 moiety-modified benzoxazinyl-oxazolidi-
nones is depicted in Scheme 4. Compounds 26a−l or
compounds 28a−c were assembled by a condensation reaction
between intermediate 10a and various carboxylic acids or N-
substituted oxamic acids. 10a was reacted with different
isocyanates to afford compounds 27a and 27b. Dess−Martin
oxidation of intermediate 7a provided the corresponding
aldehyde, which, without further purification, was oxidized to
carboxylic acid 29 by NaClO2/H2O2. Compound 29 was then
converted to the reversed amide compounds 30a and 30b via a
condensation reaction.
As shown in Scheme 5, the tricyclic S-containing key
intermediate 41 was synthesized through a convergent process.
Nucleophilic substitution of 4-bromo-2-fluoro-1-nitrobenzene
31 with tert-butyl mercaptan afforded compound 32. Cleavage
of the t-butyl group by TFA and then reduction of the nitro
group using hydrazine hydrate/FeCl3 in MeOH provided
disulphide 34. Given the discrepant influence of different
substitutions in cis-2-butene-1,4-diol on the ee value of the
Sharpless asymmetric epoxidation reaction, trityl was chosen as
the protective group with an ee value ≥96% for compound 37.
Compound 37 was then converted to its corresponding
Figure 2. Design of [6,6,5] tricyclic fused oxazolidinones.
ascertaining if this new scaffold is a favorable framework for
FXa inhibitors, comprehensive structure−activity relationship
(SAR) studies were carried out. Aside from the traditional P1
and P4 surrogate optimizations, we were interested in
modification of the bridge linkers in the C ring, which provides
an opportunity to increase the activity and improve PK
properties. Herein, we describe the design, synthesis, and SAR
and structure−pharmacokinetic (SPR) studies of novel [6,6,5]
tricyclic fused oxazolidinone FXa inhibitors, which resulted in
the discovery of a promising anticoagulant candidate: 11a.
CHEMISTRY
■
The synthesis of the nonaromatic P4 moiety-modified
benzoxazinyl-oxazolidinones 11a−g, 11k−n, and 11o−u is
illustrated in Scheme 1. Key intermediate 5 was prepared using
published procedures with minor modifications.13 A Buch-
wald−Hartwig coupling reaction between compound 5 and a
series of amide analogues yielded compounds 6a, 6b, and 6d−j.
Because of the ineffectiveness of direct coupling of N-
methylacetamide with 5, acetamide was used, and then N-
methylation was undertaken to give the desired product 6c.
Cleavage of the O-TBS protective group gave products 7a−j.
Alcohol intermediates 7a−j were converted to their mesylates
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a
Scheme 1. Synthesis of Nonaromatic P4 Moiety-Modified Benzoxazinyl-oxazolidinones
a
Reagents and conditions: (a) for 6c: (i) acetamide, Pd2(dba)3, Xantphos, Cs2CO3, 1,4-dioxane, reflux, 72.8%; (ii) CH3I, NaH, THF, 0 °C to room
temp, 86.8%; for 6a, 6b, and 6d−j: amides, Pd2(dba)3, Xantphos, Cs2CO3, 1,4-dioxane, reflux, 51.7−76.2%; (b) TBAF, THF, 0 °C to room temp,
74.2−97.6%; (c) MsCl, Et3N, 0 °C to room temp, 82.3−95.9%; (d) potassium phthalimide, DMF, 80 °C, 63.8−87.8%; (e) MeNH2, EtOH, reflux;
(f) 5-chlorothiophene-2-carboxylic acid, HATU, Et3N, DMF, 0 °C to room temp, 61.7−84.2% (for two steps); (g) TBAF, THF, 0 °C to room temp,
83.9% for 11k and 86.0% for 11m; (h) DAST, CH2Cl2, 0 °C to room temp, 79.7% for 11l and 85.6% for 11n; (i) BF3·Et2O, Me2S, CH2Cl2, 0 °C to
room temp, 82.5%; (j) aldehyde, NaBH(OAc)3, MeOH, 0 °C to room temp, 69.7% for 11p and 61.3% for 11q; (k) benzyl bromide, Et3N, DMF, 0
°C to room temp, 78.1%; (l) acetyl chloride or sulfonyl chloride, Et3N, DMF, 0 °C to room temp, 61.9% for 11s, 85.6% for 11t, and 57.6% for 11u.
mesylate 38. Cleavage of the disulfide bond by 1,4-
dithiothreitol (DTT)15 and in situ alkylation with mesylate
38 afforded epoxide 39, which was protected further by Cbz to
yield compound 40. Next, treatment of compound 40 with
nBuLi afforded the desired intermediate 41.
The synthetic strategy toward compounds 47a−c, 48, 49a,
and 49b is summarized in Scheme 6. A Buchwald−Hartwig
coupling reaction between intermediate 41 and 3-morpholi-
none or 2-piperidinone yielded 42a or 42b, respectively.
Deprotection of the trityl group with TFA transformed
compounds 42a and 42b into alcohol 43a and 43b,
respectively. The subsequent four steps in this procedure for
the synthesis of 47a−c are similar to those depicted in Scheme
1. Finally, oxidation of the S-containing analogues 47a and 47b
proceeded smoothly upon exposure to 1 or 2 equiv of mCPBA
to give the corresponding sulfoxide 48 and sulphones 49a and
49b.
The preparation route for compounds 56a and 56b is
outlined in Scheme 7, and the reaction conditions were almost
the same as those used in Schemes 3 and 6.
Preparation of compounds 72a and 72b is outlined in
Scheme 8. Nucleophilic substitution of 4-bromo-2-fluoro-1-
nitrobenzene 31 with diethyl malonate followed by ester
hydrolyzation and then decarboxylation provided the single
ester 58. Compound 5916 was reacted with 58 to construct
intermediate 60, which was subjected to hydrolyzation and
decarboxylation to yield compound 61. Reduction of the nitro
group and then Cbz protection of the unpurified aniline
followed by O-TBS deprotection gave alcohol 63. A Sharpless
asymmetric epoxidation reaction was carried out to convert 63
into epoxide 64, and then the hydroxyl group was protected
with TBSCl to yield compound 65. Next, treatment of
compound 65 with nBuLi afforded the key tricyclic intermediate
66. The subsequent several steps for the synthesis of
compounds 72a and 72b were similar to the procedures
described in Scheme 1.
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a
Scheme 2. Synthesis of Compound 18
a
Reagents and conditions: (a) Pd2(dba)3, Xantphos, Cs2CO3, 1,4-dioxane, reflux, 73.1%; (b) TBAF, THF, 0 °C to room temp, 92.5%; (c) MsCl,
Et3N, 0 °C to room temp, 97.6%; (d) potassium phthalimide, DMF, 80 °C, 83.1%; (e) MeNH2, EtOH, reflux, 85.4%; (f) 5-chlorothiophene-2-
carboxylic acid, HATU, Et3N, DMF, 0 °C to room temp, 82.4%.
a
Scheme 3. Synthesis of Aromatic P4 Moiety-Modified Benzoxazinyl-oxazolidinones
a
Reagents and conditions: (a) TBAF, THF, 0 °C to room temp, 95.3%; (b) MsCl, Et3N, 0 °C to room temp, 94.4%; (c) potassium phthalimide,
DMF, 80 °C, 81.5%; (d) MeNH2, EtOH, reflux; (e) 5-chlorothiophene-2-carboxylic acid, HATU, Et3N, DMF, 0 °C to room temp, 61.3% (for two
steps); (f) bis(pinacolato)diboron, Pd(dppf)Cl2·CH2Cl2, KOAc, DMSO, 80 °C, 38.6%; (g) aryl bromide, Pd(PPh3)4, Cs2CO3, dioxane/H2O, 80 °C,
35.9% for 25a and 35.5% for 25b; (h) TFA, 40 °C, 84.0%.
The synthesis of compounds 85a and 85b was carried out
according to Scheme 9. Nucleophilic substitution between 4-
bromo-2-fluoro-1-nitrobenzene 31 and intermediate 7317
yielded compound 74. Boc-protection of 74 followed by
epoxidation with mCPBA as the oxidant gave epoxide 76. A
tandem reduction/ring-opening reaction was adopted to
provide bicyclic product 77. Cyclization of 77 yielded the key
intermediate 78. The following six steps employed for the
synthesis of 84a and 84b were similar to those depicted in
Scheme 1. Finally, Boc-deprotection of compounds 84a and
84b using TFA afforded the target compounds 85a and 85b,
respectively.
rivaroxaban in complex with human FXa in which the benzene
ring and oxazolidinone core are in a coplanar conformation, we
hypothesized that maintaining this preferred conformation by
introducing a C ring may enforce its good inhibitory activity
against FXa. Moreover, the additional fused C ring would
reduce the number of rotatable bonds and increase the rigidity
of the molecule, which would probably improve the PK/PD
profiles of these molecules.18−20 As an early exploration, 11a
and its diastereomer 18 were prepared to validate our tentative
idea and concurrently to probe the impact of chirality on FXa
inhibitory activity. To our delight, as shown in Table 1,
(3S,3aS) compound 11a exhibited activity superior to that of
rivaroxaban, whereas the (3S,3aR) diastereomer compound 18
displayed activity that was almost 20-fold less potent than that
seen for 11a. This result confirmed our hypothesis and
indicated that the (3S,3aS) absolute configuration was essential
for good in vitro potency for these new tricyclic FXa inhibitors.
RESULTS AND DISCUSSION
■
In Vitro FXa Inhibitory Activity and Selectivity.
Inspired by the observation of the X-ray crystal structure of
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Scheme 4. Synthesis of P1 Moiety-Modified Benzoxazinyl-oxazolidinones
a
Reagents and conditions: (a) HATU, Et3N, DMF, 0 °C to room temp, 71.4−85.9% for 26a−l, 72.2−80.6% for 28a−c, 60.3% for 30a, and 52.6%
for 30b; (b) Et3N, DMF, room temp, 59.2% for 27a and 84.4% for 27b; (c) (i) DMP, DMSO, 0 °C to room temp; (ii) NaClO2, H2O2, NaH2PO4,
CH3CN/H2O, room temp, 42.9% for two steps.
a
Scheme 5. Synthesis of S-Containing Key Intermediate 41
a
Reagents and conditions: (a) t-butyl mercaptan, Cs2CO3, DMF, room temp, 94.2%; (b) TFA, CH2Cl2, room temp, 92.3%; (c) hydrazine hydrate,
FeCl3, MeOH, room temp, 87.3%; (d) trityl chloride, TEA, DMAP, CH2Cl2, room temp, 61.4%; (e) D-(−)DET, Ti(OiPr)4, TBHP, 4 Å molecular
sieves, extra dry CH2Cl2, −25 °C, 80.6%; (f) MsCl, TEA, THF, 0 °C to room temp, 97.9%; (g) DTT, THF, DMF, room temp, 60.7%; (h) CbzCl,
NaHCO3, THF/H2O = 2:1, 0 °C to room temp, 68.7%; (i) BuLi, THF, −78 °C to room temp, 75.9%.
n
As a result of the encouraging outcomes outlined above, a
systematic and comprehensive investigation was carried out to
explore the optimal P1 and P4 substituents in our new tricyclic
scaffold. Initially, replacement of the morpholinone ring of the
P4 group with various lactam-like moieties was explored. All
analogues showed decreased in vitro FXa inhibitory activity
compared with that of 11a (Table 2). Exchanging the
morpholinone to piperidinone resulted in a 6-fold loss of
potency (11b vs 11a), whereas ring-opening derivative 11c lost
potency by about 25-fold. Hydroxylation at position 2 of the
morpholinone ring in rivaroxaban is the main metabolic
pathway in humans,21 so compounds that could remove this
readily metabolized carbon atom were designed and assessed.
Unfortunately, this resulted in a significant loss of potency (11d
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a
Scheme 6. Synthesis of S-Containing Compounds 47a−c, 48, 49a, and 49b
a
Reagents and conditions: (a) 3-morpholinone or 2-piperidinone, Pd2(dba)3, Xantphos, Cs2CO3, 1,4-dioxane, reflux, 66.6% for 42a and 44.5% for
42b; (b) TFA, CH2Cl2, room temp, 89.8% for 43a and 62.6% for 43b; (c) MsCl, Et3N, 0 °C to room temp, 96.2% for 44a and 94.6% for 44b; (d)
potassium phthalimide, DMF, 80 °C, 89.0% for 45a and 93.0% for 45b; (e) MeNH2, EtOH, reflux, 83.3% for 46a and 74.5% for 46b; (f) carboxylic
acid, HATU, Et3N, DMF, 0 °C to room temp, 86.6% for 47a, 82.3% for 47b and 89.2% for 47c; (g) mCPBA (1 equiv for 48 and 2 equiv for 49a and
49b), THF, 0 °C, 91.9% for 48, 96.9% for 49a and 93.7% for 49b.
a
Scheme 7. Synthesis of S-Containing Compounds 56a and 56b
a
Reagents and conditions: (a) TFA, CH2Cl2, room temp, 87.1%; (b) MsCl, Et3N, 0 °C to room temp, 96.4%; (c) potassium phthalimide, DMF, 80
°C, 90.4%; (d) MeNH2, EtOH, reflux, 89.8%; (e) carboxylic acid, HATU, Et3N, DMF, 0 °C to room temp, 62.5% for 54a, 77.9% for 54b; (f)
bis(pinacolato)diboron, Pd(dppf)Cl2·CH2Cl2, KOAc, DMSO, 80 °C, 68.0% for 55a, 79.9% for 55b; (g) 2-bromo(methylsulfonyl)benzene,
Pd(PPh3)4, Cs2CO3, dioxane/H2O, 80 °C, 66.3% for 56a, 71.0% for 56b.
vs 11a). When an additional substituent at position 4 of the
piperidinone ring was introduced, a further loss of activity was
observed (11e−g and 11k−n vs 11b). The effect of the
absolute configuration at C4 was also explored. In general,
compounds with the R-configuration exhibited greater activity
than that of their S-configuration counterparts (11f vs 11g, 11k
vs 11m, and 11n vs 11l). Most of the unsubstituted or N-
substituted piperazinone derivatives (11o−u) showed similar
very low activity to that of piperidinone derivatives except the
N-acetyl-substituted piperazinone derivative 11s, which dis-
played moderate potency. It is well-demonstrated that three
aromatic amino-acid residues (Phe178, Trp215, and Tyr99)
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a
Scheme 8. Synthesis of C-Containing Compounds 72a and 72b
a
Reagents and conditions: (a) K2CO3, DMF, 45 °C, 67.2%; (b) LiCl, DMSO/H2O = 10:1, 110 °C, 82.5%; (c) Cs2CO3, DMF, 60 °C, 70.1%; (d) (i)
KOH, MeOH/H2O = 4:1, room temp, (ii) K2CO3, DMF, 60 °C, 87.0% for two steps; (e) (i) Zn powder, NH4Cl, THF, 0 °C to room temp, (ii)
CbzCl, NaHCO3, THF/H2O = 2:1, 0 °C to room temp, 66.7%; (f) TBAF, THF, room temp, 89.3%; (g) L-(+)DET, Ti(OiPr)4, TBHP, 4 Å
molecular sieves, extra-dry CH2Cl2, −25 °C, 83.2%; (h) TBSCl, imidazole, DMF, room temp, 93.1%; (i) nBuLi, THF, −78 °C to room temp, 78.2%;
(j) Pd2(dba)3, Xantphos, Cs2CO3, 1,4-dioxane, reflux, 63.9% for 67a, 52.7% for 67b; (k) TBAF, THF, 0 °C to room temp, 94.7% for 68a, 98.3% for
68b; (l) MsCl, Et3N, 0 °C to room temp, 82.1% for 69a, 98.7% for 69b; (m) potassium phthalimide, DMF, 80 °C, 83.9% for 70a, 84.9% for 70b; (n)
MeNH2, EtOH, reflux, 95.8% for 71a, 76.8% for 71b; (o) 5-chlorothiophene-2-carboxylic acid, HATU, Et3N, DMF, 0 °C to room temp, 80.6% for
72a, 82.3% for 72b.
endow the S4 pocket of FXa with an aromatic environment,
which would preferably accommodate an aromatic ligand by
face-to-face or edge-to-face π interactions.6,22,23 Thus, ana-
logues featuring a P4 aromatic ring were evaluated.
Unfortunately, neither the phenylsulfone derivative 25a nor
the benzsulfamide derivative 25c retained the excellent in vitro
activity of 11a. According to the investigation stated above,
morpholinone was found to be the optimal P4 pharmacophore
for our new tricyclic scaffold.
Further modifications were undertaken to explore favorable
P1 surrogates with morpholinone as the optimal P4 substituent,
but all efforts were unsuccessful (Table 3). Minor changes in
the molecular structure gave rise to a considerable loss of
potency. For example, closely related structures 4-chlorophenyl
derivative 26a and 3-chloropyridyl derivative 26b were 47- and
200-fold less potent than that of 11a, respectively. Replacement
of the chlorine atom in 26a with a methoxyl group gave
compound 26c, which exhibited a ≈30-fold loss of activity
compared with that of 26a. This finding suggested that the
Cl−π interaction in the S1 pocket is essential for activity.
Accordingly, the second chlorine was introduced at the 4-
position of the thiophene ring. However, this additional
chlorine caused a significant loss of activity (26d vs 11a).
Derivative 26e with 2-chlorothiazole as the P1 surrogate also
showed a sharply decreased FXa inhibitory activity compared
with that of 11a. To fine tune the spatial distance between Cl
and Tyr228,24 benzothiophene derivatives (26f−j) with one or
two chlorine atoms attached at different positions were
designed. Among compounds in this benzothiophene series,
only derivative 26g, which showed a similar orientation of the
chlorine like that in the chlorothiophene in 11a, exhibited
relatively higher potency, but it remained 500-fold less potent
than that of 11a. For derivatives bearing noncyclic side chains,
activity was almost completely lost (26k and 26l).
Modifications on the amide linker region were also
undertaken. It is well-established that the direct hydrogen-
bond interaction between Gly219 and the NH group of the
amide in rivaroxaban has a critical role in strong FXa inhibitory
potency.6 To mimic this interaction, urea, oxamide, and
reversed amide groups were introduced into the molecule as
a linker to find a more potent FXa inhibitor (Table 4).
Compared with amide derivatives, a loss of potency to varying
extents was observed for the urea, oxamide, and reversed amide
derivatives. Interestingly, unlike the amide series, the urea and
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a
Scheme 9. Synthesis of N-Containing Compounds 85a and 85b
a
Reagents and conditions: (a) Na2CO3, DMF, room temp, 76.7%; (b) (Boc)2O, K2CO3, DMF, 50 °C, 60.6%; (c) mCPBA, DCM, room temp,
75.6%; (d) hydrazine hydrate, FeCl3, MeOH, 50 °C, 32.9%; (e) CDI, DMAP, DMF, 100 °C, 84.6%; (f) Pd2(dba)3, Xantphos, Cs2CO3, 1,4-dioxane,
reflux, 58.6% for 79a, 67.6% for 79b; (g) TBAF, THF, 0 °C to room temp, 98.5% for 80a, 98.2% for 80b; (h) MsCl, Et3N, 0 °C to room temp,
91.3% for 81a, 95.6% for 81b; (i) potassium phthalimide, DMF, 80 °C, 80.2% for 82a, 82.2% for 82b; (j) MeNH2, EtOH, reflux, 88.7% for 83a,
91.1% for 83b; (k) 5-chlorothiophene-2-carboxylic acid, HATU, Et3N, DMF, 0 °C to room temp, 87.5% for 84a, 83.9% for 84b; (l) TFA, DCM,
room temp, 82.6% for 85a, 95.3% for 85b.
morpholinone with piperidinone, a greater or lesser decrease
of inhibition activity was observed for all series (11b vs 11a,
47b vs 47a, 72b vs 72a, and 85b vs 85a). This result indicated a
consistent SAR trend in the R4 group for these four series of
new tricyclic compounds, which would simplify further
optimization on other positions. Given the possibility of
oxidation of the S atom in 47a to the sulfoxide or sulphone in
vivo, its corresponding sulfoxide 48 and sulphone 49a
derivatives were prepared, respectively. Unlike the good
tolerance seen for replacement of O, S, C, and N for retaining
excellent in vitro activity, the sulfoxide 48 and sulphone 49a
displayed a 25- and 7-fold loss in potency compared with that
of their parent compound 47a, respectively. Similarly, the N-
Boc derivative 84b showed significantly decreased activity
compared with that of 85b. Due to 47a having the highest in
vitro activity, S-containing analogues were investigated further
by optimization of P1 and P4 substituents. Unfortunately,
compared with 47a, 18- or 8-fold decreased potency was
observed for the 4-chlorophenyl derivative 47c or phenyl-
sulfone derivative 56a, respectively. A further decrease was
displayed if the two substituents were combined in one
molecule, such as 56b.
Table 1. In Vitro FXa Inhibitory Activity of Chiral Center-
Changed Compounds 11a and 18
compd
ent
IC50 (nM)
11a
18
3aS
3aR
3.41 0.39
57.81 2.62
8.52 0.21
rivaroxaban (1)
oxamide series exhibited a different SAR at P1 position. As
shown in Table 4, chlorophenyl derivatives were more potent
than their corresponding chlorothiophene derivatives (27b vs
27a and 28c vs 28a). For the reversed amide series, FXa
inhibitory activity was lost completely (30a and 30b).
Further modification of the bridge linker in the C ring was
explored to evaluate its influence on in vitro FXa inhibitory
activity. Single-enantiomer analogues were accessed according
to different asymmetric synthetic methods except for the N-
containing series with respect to rapid preparation. Different
bridge linker analogues (47a, 72a, and 85a) of compound 11a
exhibited excellent potency, with the order being S > O > C >
N (Table 5). Upon replacement of the R4 substituent
As a result of their high potency against FXa, 11a, 47a, 72a,
and 85a were selected to investigate their selectivity versus
other related serine proteases. Differing from their excellent
FXa inhibitory potency, all test compounds showed no
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Table 2. In Vitro FXa Inhibitory Activity of P4-Substituted Derivatives
inhibitory activity against other related serine proteases (IC50
100 μM), with selectivity being more than 20 000-fold.
>
comparable with that of rivaroxaban. By contrast, a higher
concentration was required for 25a and 25c to double the PT
and aPTT, which was in accordance with their relatively lower
FXa inhibitory activities compared with that of 11a.
In Vitro Anticoagulant Activity. Prothrombin time (PT)
and activated partial thromboplastin time (aPTT) were
evaluated for selected compounds in rabbit plasma to
determine their in vitro anticoagulant activity. PT measures
the effect of a compound on the extrinsic pathway of
coagulation, whereas aPTT represents the effect on the
intrinsic pathway. Anticoagulant activity was defined as the
concentration at which the plasma clotting time (CT2) was
doubled (Table 6).
All test compounds displayed moderate-to-high in vitro
anticoagulant activity and showed similar sensitivity to PT and
aPTT. For 11a, PT and aPTT were doubled at 0.18 and 0.14
μM, respectively, which made it the most potent compound in
these two assays. With regard to its counterparts, 11b, 47a, 47b,
49a, 72a, and 85a exhibited good anticoagulant activity that was
PK Profile of Selected Compounds. As a result of the
excellent in vitro profiles, some compounds were selected to
evaluate their PK properties in rats and dogs, with the data for
rivaroxaban included for comparison. As summarized in Tables
7 and 8, different bridge linkers in the C ring had a significant
influence on the PK properties of compounds after oral
administration and intravenous injection. In rats, the O-
containing analogue 11a and C-containing analogue 72a,
which exhibited the best overall PK profiles, were comparable
or better than rivaroxaban (especially 11a). The PK of 11a in
rats was outstanding, with high maximal plasma concentration
(Cmax = 11 901 ng/mL), high plasma exposure (AUC0−∞ = 33
044 ng·h/mL), and better oral bioavailability (F = 63.7%) after
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Table 3. In Vitro FXa Inhibitory Activity of P1-Substituted Derivatives
Table 4. In Vitro FXa Inhibitory Activity of P1 Linker-Modified Derivatives
oral administration. These values were significantly higher than
those observed for rivaroxaban. When administered via the
intravenous route, 11a displayed lower clearance (CLp = 0.201
L/h/kg) compared with that of rivaroxaban. The P4 position-
modified derivatives such as 11b, 25a, and 25c displayed less
favorable overall PK properties compared with that of 11a,
including moderate Cmax, moderate-to-high AUC0−∞, and
relatively low oral bioavailability. However, 11b showed a
longer half-life (T1/2) compared with that of 11a. By contrast,
other analogues such as 47a, 49a, and 85a with S, SO2, or NH
as the bridge, respectively, showed unfavorable PK properties,
such as unacceptable oral bioavailability (F < 8%).
In beagle dogs, almost all aspects of the PK properties of 11a
were superior to those of rivaroxaban (Table 8). Detailed
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Table 5. In Vitro FXa Inhibitory Activity of Compounds Bearing Different Bridge Linkers in the C Ring
evaluation showed that the Cmax and AUC0−∞ of 11a were 1.5-
fold higher than those of rivaroxaban irrespective of the routes
they were administered. In addition, considerable improvement
was observed in terms of oral bioavailability (92% for 11a vs
68% for rivaroxaban).
In Vivo Antithrombotic Efficacy and Tail-Bleeding
Time Effect of 11a in Rats. Through comprehensive analyses
of in vitro potency, selectivity, and PK properties, 11a was
selected to evaluate its in vivo antithrombotic efficacy in three
models of thrombosis in rats: FeCl3-induced venous thrombosis
(VT), arteriovenous shunt (AV-SHUNT), and electrically
induced rat carotid artery thrombosis. Additionally, the
bleeding risk of 11a was evaluated in a rat tail-bleeding time
study. Rivaroxaban was chosen as the positive control.
As shown in Figure 3, 11a exhibited strong antithrombotic
activity in all three models. In VT and AV-SHUNT models,
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a
Table 6. In Vitro Anticoagulant Activity of Selected Compounds
compd
PT CT2 (μM)
aPTT CT2 (μM)
compd
PT CT2 (μM)
aPTT CT2 (μM)
11a
11b
25a
25c
26a
47a
0.18
0.42
5.88
4.58
1.06
0.58
0.14
1.63
4.65
4.86
0.93
0.33
47b
49a
72a
85a
0.64
0.47
0.37
0.48
0.40
0.62
0.76
0.33
0.43
0.32
rivaroxaban (1)
a
PT and aPTT in vitro clotting assays were carried out in rabbit plasma.
a
Table 7. PK Properties of Selected Compounds in Male Rats
compd
route
dose (mg/kg)
Cmax (ng/mL)
Tmax (h)
T1/2 (h)
AUC0−∞ (ng·h/mL)
CLp (L/h/kg)
Vss (L/kg)
F (%)
11a
p.o.
i.v.
10
3
11 901
0.75
1.33
1.0
33 044
15 339
28 527
33 562
24 547
43 935
12 277
12 226
1480
63.7
0.201
0.137
0.103
0.253
0.529
0.493
0.149
0.553
0.303
0.201
0.406
0.169
0.350
0.574
0.154
0.144
0.199
0.190
11b
25a
p.o.
i.v.
9
6927
6763
3626
167
0.67
1.0
2.10
1.95
1.43
0.88
1.65
1.38
3.15
0.89
2.14
0.54
3.54
0.74
1.52
0.86
2.90
1.27
42.0
27.3
29.0
7.4
4.5
9
p.o.
i.v.
4.5
10
3
25c
p.o.
i.v.
1.0
47a
p.o.
i.v.
10
3
4.0
5695
49a
p.o.
i.v.
10
3
917
0.38
2.3
1539
7.3
6122
72a
p.o.
i.v.
10
3
3020
565
14 200
20 450
1349
20.4
6.8
85a
p.o.
i.v.
10
3
0.44
0.88
5700
riv (1)
p.o.
i.v.
3
850
2264
21.3
3
9985
a
Abbreviations: Cmax, peak plasma concentration of a drug after administration; Tmax, time to reach Cmax; T1/2, elimination half-life; AUC, area under
the concentration−time curve; CLp, plasma clearance; Vss, volume of distribution at steady state; F, bioavailability; p.o., per oral; i.v., intravenous; riv,
rivaroxaban.
a
Table 8. PK Properties of 11a and Rivaroxaban in Male Beagle Dogs
compd
route
dose (mg/kg)
Cmax (ng/mL)
Tmax (h)
T1/2 (h)
AUC0−∞ (ng·h/mL)
CLp (L/h/kg)
Vss (L/kg)
F (%)
11a
p.o.
i.v.
1
530.0
0.5
2.0
3.1
1.8
2.4
1525.8
1316.6
1001.6
915.7
92
0.5
1
0.4
0.6
1.7
2.8
riv (1)
p.o.
i.v.
332.6
0.7
68
0.5
a
Abbreviations: Cmax, peak plasma concentration of a drug after administration; Tmax, time to reach Cmax; T1/2, elimination half-life; AUC, area under
the concentration−time curve; CLp, plasma clearance; Vss, volume of distribution at steady state; F, bioavailability; p.o., per oral; i.v., intravenous; riv,
rivaroxaban.
11a and rivaroxaban, which were administered orally 60 min
before the induction of thrombosis, reduced thrombus
formation in a dose-dependent manner. The in vivo efficacy
of 11a was similar to that of rivaroxaban, with ED50 values of
5.63 and 5.28 mg/kg for 11a compared with 4.86 and 3.72 mg/
kg for rivaroxaban, respectively. In the model of arterial
thrombosis in which 11a and rivaroxaban were given via the
oral route, a dose-dependent antithrombotic effect against
thrombus formation was observed. Moreover, 11a exhibited
superior antithrombotic efficacy compared to that of rivarox-
aban, with ED50 values of 2.97 and 4.53 mg/kg, respectively.
These results suggested that 11a would be beneficial in the
prevention and treatment of thromboembolic diseases in
venous and arterial systems.
procedure. As depicted in Figure 4, 11a and rivaroxaban
prolonged the bleeding time in a dose-dependent manner to
the same extent. This outcome suggested that 11a and
rivaroxaban have a similar safety profile in terms of bleeding
risk.
Liability Profiling. Parameters of liability profiling were
tested to evaluate the druggability of 11a. 11a showed almost
no inhibition of the hERG K+ channel (IC50 = 79.75 μM, patch
clamp assay) and various CYP450 isozymes (IC50 > 40 μM,
summarized in Table 9). In addition, 11a gave no genetic
toxicity in the Ames mutagenicity test and micronucleus
formation assay. Additionally, 11a was administered orally once
a day for 2 weeks at medium (50 mg/kg) and high (100 mg/
kg) doses to evaluate its subacute toxicity in rats. We found that
11a was well-tolerated at both doses with no obvious toxic
reaction in terms of behavior, body weight, food intake, organ
coefficients, and survival rate.
To evaluate the bleeding risk of compound 11a, a rat tail-
bleeding time study was carried out. 11a and rivaroxaban were
administered via the oral route 60 min before the surgical
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CONCLUSIONS
■
In an effort to find potent FXa inhibitors, we focused on the
development of a series of conformationally constrained
rivaroxaban analogues. Rigidification of the benzene ring and
oxazolidinone moiety by introduction of an additional bridge
linker yielded the [6,6,5] tricyclic fused scaffold. Extensive
modifications focused on the P1 group, P4 group, and bridge
linker in the C ring resulted in a novel series of FXa inhibitors
showing high in vitro FXa inhibitory activity and anticoagulant
potency. Further SPR studies in rats and dogs demonstrated
that analogues bearing different bridges represented various PK
properties. Among these, compounds carrying a methylene-
oxide bridge showed the most favorable PK profiles. On the
basis of excellent in vitro anticoagulant potency and favorable
PK profiles, compound 11a was selected to evaluate its in vivo
antithrombotic efficacy in models of VT, AV-SHUNT, and
arterial thrombosis. Importantly, compound 11a displayed
excellent efficacy in all three models, especially in the model of
arterial thrombosis, with ED50 = 2.97 mg/kg, which is superior
to that of rivaroxaban (4.53 mg/kg). Additionally, 11a showed
no significant inhibitory activity against hERG K+ channel and
various CYP450 isozymes and had no genetic toxicity in the
Ames mutagenicity test and micronucleus formation assay.
Compound 11a also exhibited significant selectivity against
other related serine proteases (IC50 > 100 μM). Furthermore,
in a 2 week subacute toxicity study in rats, 11a was well-
tolerated and showed no obvious toxic reactions at medium and
high doses. These data suggest that compound 11a is a highly
potent and selective FXa inhibitor and warrants further
evaluation as a potential candidate for the prevention and
treatment of thromboembolic disease in venous and arterial
systems. Studies on 11a are ongoing in our research team.
EXPERIMENTAL SECTION
■
Chemistry. Compounds not described below were purchased from
commercial vendors and were used as supplied unless it is stated
otherwise. Inert atmosphere operations were conducted under argon
in flame-dried glassware. All reaction mixtures were monitored using
thin-layer chromatography (TLC) on silica gel F-254 TLC plates.
Column chromatography was carried out using silica gel (200−300
Figure 3. (A) Antithrombotic effect of 11a and rivaroxaban in a rat
FeCl3-induced venous thrombosis model. (B) Antithrombotic effect of
11a and rivaroxaban in a rat arteriovenous thrombosis model. (C)
Antithrombotic effect of 11a and rivaroxaban in a rat arterial
thrombosis model. Average thrombus weight or thrombus formation
time are the mean SD. Statistical significance compared with the
vehicle group is *P < 0.05, **P < 0.01, and ***P < 0.001.
1
mesh). H and 13C NMR spectra were recorded on a Bruker 300
NMR or a Bruker 400 NMR or a Bruker 500 NMR spectrometer using
solvent as an internal standard. Chemical shifts (δ) are reported in
parts per million (ppm), and coupling constants (J) are reported in
Hertz (Hz). EI-MS spectra were obtained on a Finnigan MAT95
spectrometer, and ESI-MS spectra were obtained on a Krats MS 80
mass spectrometer. All final compounds were purified to >95% purity
as determined by an Agilent 1100 series LC system (PLATISIL ODS 5
μm 250 × 4.6 mm) with two solvent systems (acetonitrile/water or
acetonitrile/buffer (0.1% CF3COOH in water)). Chiral LC analysis
was performed on an Shimadzu LC-10AT VP LC system (Chiralpak
AS-H 5 μm 250 × 4.6 mm) with a two solvent system (nhexane/2-
propanol = 90:10 (v/v)), UV detection (λ = 210 nm) at 35 °C.
For detailed synthesis and characterization of all intermediates, see
the Supporting Information.
5-Chloro-N-(((3S,3aS)-1-oxo-7-(3-oxomorpholino)-1,3,3a,4-
tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)methyl)-
thiophene-2-carboxamide (11a). The crude amine 10a together
with 5-chlorothiophene-2-carboxylic acid (1.56 g, 9.59 mmol), TEA
(1.62 g, 16.04 mmol), and HATU (4.56 g, 11.99 mmol) was dissolved
in 50 mL of dry DMF. The resulting solution was stirred for 4 h at
room temperature. Water was added, and it was extracted with ethyl
acetate. The organic phase was washed with saturated NH4Cl solution
and brine, dried over anhydrous Na2SO4, and filtered, and then the
filtrate was concentrated in vacuo. The residue was purified by
chromatography on silica gel with dichloromethane/methanol (30:1)
Figure 4. Influence of 11a and rivaroxaban on rat tail-bleeding time.
Results are the mean SD. Statistical significance compared with the
vehicle group is *P < 0.05, **P < 0.01, and ***P < 0.001.
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Table 9. Inhibition of hERG K+ Channel and CYP450 Isozymes by 11a
CYP inhibition IC50 (μM)
compd
hERG K+ inhibition IC50 (μM)
1A2
2C8
2C9
2C19
>200
2D6
3A4
11a
79.75
>200
99.73
43.23
>200
85.12
to give 3.13 g (84.2%, for two steps) of 11a as a white solid. 1H NMR
(400 MHz, DMSO-d6) δ 9.00 (t, J = 5.8 Hz, 1H), 7.85 (d, J = 8.7 Hz,
1H), 7.71 (d, J = 4.1 Hz, 1H), 7.21 (d, J = 4.0 Hz, 1H), 7.05 (d, J = 2.3
Hz, 1H), 7.01 (dd, J = 8.7, 2.3 Hz, 1H), 4.63−4.52 (m, 2H), 4.18 (s,
2H), 4.10−4.01 (m, 2H), 3.97−3.92 (m, 2H), 3.73 (t, J = 5.5 Hz, 2H),
3.71−3.66 (m, 2H). 13C NMR (126 MHz, DMSO-d6) δ 166.41,
161.32, 153.74, 144.52, 138.79, 138.05, 133.85, 129.06, 128.70, 122.15,
118.88, 118.81, 114.68, 75.01, 68.18, 66.24, 63.90, 53.42, 49.46, 41.76.
MS (ESI) m/z: 486.3 (M + 23)+. HRMS (EI): Anal. Calcd for
C20H18SClN3O6, 463.0605; found, 463.0623.
5-Chloro-N-(((3S,3aS)-1-oxo-7-(2-oxopiperidin-1-yl)-1,3,3a,4-
tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)methyl)-
thiophene-2-carboxamide (11b). Compound 11b (0.72 g, 74.2%,
for two steps) was prepared from crude 10b and 5-chlorothiophene-2-
carboxylic acid (0.41 g, 2.52 mmol) in the same manner as that
described for 11a. 1H NMR (400 MHz, CDCl3) δ 7.98 (d, J = 9.3 Hz,
1H), 7.34 (d, J = 4.0 Hz, 1H), 6.90 (d, J = 4.0 Hz, 1H), 6.87 (dt, J =
4.5, 2.3 Hz, 3H), 4.54−4.41 (m, 2H), 3.96 (ddd, J = 10.1, 7.0, 3.1 Hz,
1H), 3.90−3.70 (m, 3H), 3.59 (s, 2H), 2.55 (d, J = 6.3 Hz, 2H), 1.97−
1.89 (m, 4H). 13C NMR (126 MHz, DMSO-d6) δ 169.24, 161.31,
153.75, 144.48, 140.15, 138.79, 133.85, 129.05, 128.69, 121.83, 119.68,
118.76, 115.40, 74.96, 66.23, 53.45, 51.35, 41.78, 33.05, 23.43, 21.34.
MS (EI) m/z: 461 (M+). HRMS (EI): Anal. Calcd for
C21H20SClN3O5, 461.0812; found, 461.0817.
118.83, 115.25, 106.14, 74.97, 66.24, 64.56 (overlap), 53.43, 47.61,
43.35, 41.77, 32.11. MS (ESI) m/z: 542.0 (M + 23)+. HRMS (ESI):
Anal. Calcd for C23H22O7N3ClNaS, 542.0765; found, 542.0752.
5-Chloro-N-(((3S,3aS)-7-((R)-4-methoxy-2-oxopiperidin-1-
yl)-1-oxo-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]-
oxazin-3-yl)methyl)thiophene-2-carboxamide (11f). Compound
11f (0.48 g, 75.3%) was prepared from 10f (0.45 g, 1.30 mmol) and 5-
chlorothiophene-2-carboxylic acid (0.31 g, 1.91 mmol) in the same
1
manner as that described for 11a. H NMR (400 MHz, DMSO-d6) δ
9.01 (t, J = 5.8 Hz, 1H), 7.83 (d, J = 9.4 Hz, 1H), 7.72 (d, J = 4.1 Hz,
1H), 7.21 (d, J = 4.0 Hz, 1H), 6.89 (s, 1H), 6.87 (d, J = 2.4 Hz, 1H),
4.59 (q, J = 5.5 Hz, 1H), 4.55 (d, J = 8.1 Hz, 1H), 4.12−4.00 (m, 2H),
3.78−3.70 (m, 3H), 3.63 (ddd, J = 12.5, 8.2, 4.8 Hz, 1H), 3.48 (dt, J =
11.6, 5.7 Hz, 1H), 3.29 (s, 3H), 2.68 (dd, J = 17.3, 4.7 Hz, 1H), 2.38
(dd, J = 17.3, 5.5 Hz, 1H), 2.09 (dt, J = 13.3, 4.0 Hz, 1H), 1.94 (dt, J =
12.6, 6.0 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) δ 167.92, 161.31,
153.71, 144.46, 139.63, 138.77, 133.83, 129.04, 128.65, 121.84, 119.44,
118.76, 115.16, 74.96, 73.16, 66.25, 55.74, 53.46, 46.88, 41.78, 38.89,
27.40. MS (ESI) m/z: 514.1 (M + 23)+. HRMS (ESI): Anal. Calcd for
C22H22O6N3ClNaS, 514.0810; found, 514.0808.
5-Chloro-N-(((3S,3aS)-7-((R)-4-methoxy-2-oxopiperidin-1-
yl)-1-oxo-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]-
oxazin-3-yl)methyl)thiophene-2-carboxamide (11g). Com-
pound 11g (0.50 g, 72.0%) was prepared from 10g (0.49 g, 1.41
mmol) and 5-chlorothiophene-2-carboxylic acid (0.34 g, 2.09 mmol)
5-Chloro-N-(((3S,3aS)-7-(N-methylacetamido)-1-oxo-
1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)-
methyl)thiophene-2-carboxamide (11c). Compound 11c (0.32 g,
77.4%, for two steps) was prepared from crude 10c and 5-
chlorothiophene-2-carboxylic acid (0.19 g, 1.17 mmol) in the same
1
in the same manner as that described for 11a. H NMR (400 MHz,
DMSO-d6) δ 9.01 (t, J = 5.6 Hz, 1H), 7.83 (d, J = 8.7 Hz, 1H), 7.72
(d, J = 4.1 Hz, 1H), 7.21 (d, J = 3.9 Hz, 1H), 6.89 (s, 1H), 6.88 (s,
1H), 4.68−4.52 (m, 2H), 4.13−3.96 (m, 2H), 3.83−3.70 (m, 3H),
3.63 (p, J = 6.4, 5.9 Hz, 1H), 3.48 (dt, J = 11.9, 5.9 Hz, 1H), 3.29 (s,
3H), 2.68 (dd, J = 17.3, 4.7 Hz, 1H), 2.38 (dd, J = 17.1, 5.6 Hz, 1H),
2.18−2.03 (m, 1H), 1.95 (dq, J = 14.0, 6.9, 6.4 Hz, 1H). 13C NMR
(151 MHz, DMSO-d6) δ 167.92, 161.30, 153.71, 144.45, 139.62,
138.77, 133.83, 129.04, 128.65, 121.85, 119.43, 118.76, 115.20, 74.96,
73.17, 66.26, 55.74, 53.46, 46.89, 41.78, 38.90, 27.42. MS (ESI) m/z:
514.1 (M + 23)+. HRMS (ESI): Anal. Calcd for C22H22O6N3ClNaS,
514.0810; found, 514.0800.
1
manner as that described for 11a. H NMR (300 MHz, DMSO-d6) δ
7.91 (ddd, J = 17.8, 10.0, 7.3 Hz, 5H), 7.09−6.91 (m, 2H), 4.70 (dd, J
= 18.5, 8.4 Hz, 2H), 4.21 (s, 1H), 4.09 (dd, J = 18.0, 7.4 Hz, 3H), 3.11
(s, 3H), 1.79 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 169.49,
161.33, 153.73, 144.97, 140.73, 138.78, 133.86, 129.07, 128.70, 123.08,
120.57, 119.39, 116.10, 75.09, 66.20, 53.30, 41.72, 36.97, 22.61. MS
(EI) m/z: 435 (M+). HRMS (EI): Anal. Calcd for C19H18SClN3O5,
435.0656; found, 435.0651.
5-Chloro-N-(((3S,3aS)-7-((R)-4-hydroxy-2-oxopiperidin-1-yl)-
1-oxo-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-
3-yl)methyl)thiophene-2-carboxamide (11k). To a solution of
11h (0.25 g, 0.35 mmol) in THF (10 mL) was added nBu4NF (1 M in
THF, 0.7 mL, 0.7 mmol) dropwise at 0 °C. The solution was stirred
for 3 h and then concentrated in vacuo. The residue was purified by
chromatography on silica gel with dichloromethane/methanol (30:1)
5-Chloro-N-(((3S,3aS)-1-oxo-7-(2-oxooxazolidin-3-yl)-
1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)-
methyl)thiophene-2-carboxamide (11d). Compound 11d (0.17 g,
63.3%, for two steps) was prepared from crude 10d and 5-
chlorothiophene-2-carboxylic acid (0.12 g, 0.74 mmol) in the same
1
manner as that described for 11a. H NMR (400 MHz, DMSO-d6) δ
9.01 (t, J = 5.9 Hz, 1H), 7.83 (d, J = 8.9 Hz, 1H), 7.72 (d, J = 4.0 Hz,
1H), 7.28 (d, J = 2.4 Hz, 1H), 7.21 (d, J = 4.0 Hz, 1H), 7.14 (dd, J =
9.0, 2.5 Hz, 1H), 4.62−4.57 (m, 1H), 4.55 (d, J = 7.6 Hz, 1H), 4.42 (t,
J = 8.0 Hz, 2H), 4.13−3.94 (m, 4H), 3.73 (t, J = 5.5 Hz, 2H). 13C
NMR (151 MHz, DMSO-d6) δ 161.30, 155.29, 153.72, 144.72, 138.77,
135.36, 133.84, 129.04, 128.65, 119.59, 119.05, 111.53, 107.13, 74.94,
66.42, 61.87, 53.45, 45.30, 41.79. MS (ESI) m/z: 472.0 (M + 23)+.
HRMS (ESI): Anal. Calcd for C19H16O6N3ClNaS, 472.0346; found,
472.0350.
1
to give 0.14 g (83.9%) of 11k as a white solid. H NMR (400 MHz,
DMSO-d6) δ 9.01 (t, J = 5.7 Hz, 1H), 7.82 (d, J = 8.9 Hz, 1H), 7.72
(d, J = 4.1 Hz, 1H), 7.21 (d, J = 4.0 Hz, 1H), 6.89 (s, 1H), 6.87 (d, J =
3.0 Hz, 1H), 5.08 (d, J = 3.7 Hz, 1H), 4.62−4.52 (m, 2H), 4.17−3.99
(m, 3H), 3.73 (t, J = 5.5 Hz, 2H), 3.66 (dd, J = 13.3, 7.2 Hz, 1H), 3.48
(dt, J = 11.7, 5.2 Hz, 1H), 2.60 (dd, J = 17.2, 4.7 Hz, 1H), 2.27 (dd, J =
17.2, 6.0 Hz, 1H), 2.08−1.94 (m, 1H), 1.80 (dt, J = 13.6, 6.7 Hz, 1H).
13C NMR (126 MHz, DMSO-d6) δ 169.89, 162.72, 155.14, 145.87,
5-Chloro-N-(((3S,3aS)-1-oxo-7-(7-oxo-1,4-dioxa-8-azaspiro-
[4.5]decan-8-yl)-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d]-
[1,4]oxazin-3-yl)methyl)thiophene-2-carboxamide (11e). Com-
pound 11e (0.15 g, 66.3%, for two steps) was prepared from crude 10e
and 5-chlorothiophene-2-carboxylic acid (0.10 g, 0.62 mmol) in the
same manner as that described for 11a. 1H NMR (400 MHz, DMSO-
d6) δ 9.02 (t, J = 5.9 Hz, 1H), 7.84 (d, J = 9.3 Hz, 1H), 7.72 (d, J = 4.1
Hz, 1H), 7.21 (d, J = 4.1 Hz, 1H), 6.91−6.86 (m, 2H), 4.60 (q, J = 5.5
Hz, 1H), 4.57−4.53 (m, 1H), 4.11−4.02 (m, 2H), 3.99−3.93 (m, 4H),
3.73 (t, J = 5.6 Hz, 2H), 3.60 (t, J = 6.3 Hz, 2H), 2.64 (s, 2H), 2.05 (t,
J = 6.3 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) δ 167.35, 161.30,
153.71, 144.51, 139.20, 138.77, 133.83, 129.04, 128.66, 122.01, 119.51,
141.25, 140.19, 135.24, 130.46, 130.08, 123.22, 120.95, 120.17, 116.69,
76.36, 67.66, 64.96, 54.87, 48.54, 43.45, 43.19, 32.62. MS (ESI) m/z:
500.1 (M + 23)+. HRMS (ESI): Anal. Calcd for C21H20O6N3ClNaS,
500.0654; found, 500.0647.
5-Chloro-N-(((3S,3aS)-7-((S)-4-fluoro-2-oxopiperidin-1-yl)-1-
oxo-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-
yl)methyl)thiophene-2-carboxamide (11l). To a solution of
compound 11k (5.0 g, 32.7 mmol) in anhydrous DCM (5 mL) was
added DAST (33 mg, 0.20 mmol) dropwise at 0 °C under an argon
atmosphere. After addition, the reaction mixture was stirred for 3 h at
room temperature and then concentrated in vacuo. The resulting
residue was purified by silica gel chromatography with DCM/MeOH
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1
(50:1) to afford 11l (40 mg, 79.7%) as a white solid. H NMR (400
MHz, DMSO-d6) δ 9.01 (t, J = 6.0 Hz, 1H), 7.89−7.79 (m, 1H), 7.72
(d, J = 4.0 Hz, 1H), 7.21 (d, J = 4.0 Hz, 1H), 7.02−6.84 (m, 2H),
5.32−5.07 (m, 1H), 4.63−4.57 (m, 1H), 4.55 (d, J = 7.9 Hz, 1H),
4.18−3.96 (m, 2H), 3.79−3.67 (m, 3H), 3.54 (dt, J = 11.6, 5.4 Hz,
1H), 2.85 (ddd, J = 34.4, 17.8, 4.3 Hz, 1H), 2.61 (td, J = 17.4, 4.3 Hz,
1H), 2.32−1.93 (m, 2H). 13C NMR (151 MHz, DMSO-d6) δ 166.69
(d, J = 4.5 Hz), 161.31, 153.72, 144.50, 139.34, 138.76, 133.83, 129.04,
128.66, 122.04, 119.50, 118.82, 115.22, 87.36 (d, J = 167.8 Hz), 74.96,
66.24, 53.44, 46.01 (d, J = 6.8 Hz), 41.78, 39.11 (d, J = 22.9 Hz), 28.39
(d, J = 19.9 Hz). MS (ESI) m/z: 502.1 (M + 23)+. HRMS (ESI): Anal.
Calcd for C21H19O5N3ClFNaS, 502.0610; found, 502.0612.
5-Chloro-N-(((3S,3aS)-7-(4-methyl-2-oxopiperazin-1-yl)-1-
oxo-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-
yl)methyl)thiophene-2-carboxamide (11p). To a solution of 11o
(78 mg, 0.17 mmol) and HCHO (37% aq., 21 mg, 0.26 mmol) in
MeOH (5 mL) at room temperature was added NaBH(OAc)3 (143
mg, 0.67 mmol). After being stirred at room temperature overnight,
the solution was concentrated in vacuo. The residue was partitioned
between 5% aq. NaHCO3 and ethyl acetate. The organic phase was
separated, concentrated in vacuo, and purified on silica gel
chromatography with dichloromethane/methanol (50:1) to give 11p
1
(56 mg, 69.7%) as a white solid. H NMR (400 MHz, CDCl3) δ 7.99
(d, J = 8.6 Hz, 1H), 7.34 (d, J = 4.0 Hz, 1H), 6.90 (dd, J = 6.2, 4.0 Hz,
3H), 6.79 (t, J = 6.1 Hz, 1H), 4.49 (ddd, J = 16.7, 10.4, 4.5 Hz, 2H),
4.04−3.94 (m, 1H), 3.92−3.74 (m, 3H), 3.69 (s, 2H), 3.31 (s, 2H),
2.84 (s, 2H), 2.44 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 166.42,
161.31, 153.74, 144.52, 138.79, 138.58, 133.84, 129.05, 128.70, 122.06,
119.28, 118.80, 115.00, 74.99, 66.23, 59.76, 53.42, 51.82, 49.80, 44.93,
41.77. MS (EI) m/z: 476 (M+). HRMS (EI): Anal. Calcd for
C21H21SClN4O5, 476.0921; found, 476.0932.
5-Chloro-N-(((3S,3aS)-7-((S)-4-hydroxy-2-oxopiperidin-1-yl)-
1-oxo-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-
3-yl)methyl)thiophene-2-carboxamide (11m). To a solution of
11i (0.68 g, 0.95 mmol) in THF (10 mL) was added nBu4NF (1 M in
THF, 1.8 mL, 1.8mmol) dropwise at 0 °C. The solution was stirred for
3 h and then concentrated in vacuo. The residue was purified by
chromatography on silica gel with dichloromethane/methanol (30:1)
5-Chloro-N-(((3S,3aS)-7-(4-ethyl-2-oxopiperazin-1-yl)-1-oxo-
1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)-
methyl)thiophene-2-carboxamide (11q). To a solution of 11o
(40 mg, 0.086 mmol) and acetaldehyde (5.8 mg, 0.13 mmol) in
MeOH (5 mL) at room temperature was added NaBH(OAc)3 (74 mg,
0.35 mmol). After being stirred at room temperature overnight, the
solution was concentrated in vacuo. The residue was partitioned
between 5% aq. NaHCO3 and ethyl acetate. The organic phase was
separated, concentrated in vacuo, and purified on silica gel
chromatography with dichloromethane/methanol (50:1) to give 11q
1
to give 0.39 g (86.0%) of 11m as a white solid. H NMR (400 MHz,
DMSO-d6) δ 9.01 (t, J = 6.0 Hz, 1H), 7.82 (d, J = 9.4 Hz, 1H), 7.72
(d, J = 4.0 Hz, 1H), 7.21 (d, J = 4.0 Hz, 1H), 6.89 (s, 1H), 6.87 (d, J =
2.3 Hz, 1H), 5.08 (d, J = 3.7 Hz, 1H), 4.59 (q, J = 6.5, 5.9 Hz, 1H),
4.56−4.53 (m, 1H), 4.12−3.97 (m, 3H), 3.73 (t, J = 5.7 Hz, 2H), 3.67
(td, J = 7.7, 4.0 Hz, 1H), 3.52−3.44 (m, 1H), 2.60 (dd, J = 17.2, 4.7
Hz, 1H), 2.28 (dd, J = 17.4, 5.6 Hz, 1H), 2.08−1.94 (m, 1H), 1.80 (dt,
J = 13.3, 6.5 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 168.02,
160.84, 153.27, 143.99, 139.38, 138.32, 133.37, 128.59, 128.22, 121.34,
119.08, 118.30, 114.80, 74.49, 65.78, 63.08, 52.99, 46.65, 41.57, 41.32,
30.72. MS (ESI) m/z: 500.1 (M + 23)+. HRMS (ESI): Anal. Calcd for
C21H20O6N3ClNaS, 500.0654; found, 500.0655.
5-Chloro-N-(((3S,3aS)-7-((R)-4-fluoro-2-oxopiperidin-1-yl)-1-
oxo-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-
yl)methyl)thiophene-2-carboxamide (11n). To a solution of
compound 11m (5.0 g, 32.7 mmol) in anhydrous DCM (5 mL)
was added DAST (33 mg, 0.20 mmol) dropwise at 0 °C under an
argon atmosphere. After addition, the reaction mixture was stirred for
3 h at room temperature and then concentrated in vacuo. The
resulting residue was purified by silica gel chromatography with DCM/
MeOH (50:1) to afford 11n (43 mg, 85.6%) as a white solid. 1H NMR
(400 MHz, DMSO-d6) δ 9.01 (t, J = 5.8 Hz, 1H), 7.84 (d, J = 8.9 Hz,
1H), 7.72 (d, J = 4.1 Hz, 1H), 7.21 (d, J = 4.0 Hz, 1H), 6.98−6.87 (m,
2H), 5.29−5.10 (m, 1H), 4.65−4.51 (m, 2H), 4.13−4.00 (m, 2H),
3.82−3.66 (m, 3H), 3.55 (dt, J = 11.6, 5.4 Hz, 1H), 2.85 (ddd, J =
34.2, 17.8, 4.4 Hz, 1H), 2.61 (td, J = 17.8, 4.7 Hz, 1H), 2.30−1.92 (m,
2H). 13C NMR (151 MHz, DMSO-d6) δ 166.69 (d, J = 4.9 Hz),
161.30, 153.72, 144.50, 139.34, 138.77, 133.83, 129.04, 128.66, 122.04,
119.50, 118.82, 115.25, 87.36 (d, J = 167.8 Hz), 74.97, 66.26, 53.44,
46.02 (d, J = 6.8 Hz), 41.78, 39.12 (d, J = 22.8 Hz), 28.41 (d, J = 19.9
Hz). MS (ESI) m/z: 502.1 (M + 23)+. HRMS (ESI): Anal. Calcd for
C21H19O5N3ClFNaS, 502.0610; found, 502.0608.
1
(26 mg, 61.3%) as a white solid. H NMR (300 MHz, CDCl3) δ 7.97
(d, J = 8.8 Hz, 1H), 7.36 (d, J = 3.4 Hz, 1H), 7.17 (s, 1H), 6.90 (d, J =
6.4 Hz, 3H), 4.45 (dd, J = 13.7, 8.2 Hz, 2H), 3.97 (s, 1H), 3.88−3.72
(m, 3H), 3.66 (s, 2H), 3.30 (s, 2H), 2.81 (s, 2H), 2.53 (d, J = 7.1 Hz,
2H), 1.14 (t, J = 6.9 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ
166.56, 161.31, 153.74, 144.51, 138.79, 138.59, 133.84, 129.06, 128.70,
122.04, 119.25, 118.79, 114.97, 74.99, 66.23, 57.69, 53.42, 51.06,
49.85, 49.55, 41.77, 12.27. MS (EI) m/z: 490 (M+). HRMS (EI): Anal.
Calcd for C22H23SClN4O5, 490.1078; found, 490.1071.
N-(((3S,3aS)-7-(4-Benzyl-2-oxopiperazin-1-yl)-1-oxo-
1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)-
methyl)-5-chlorothiophene-2-carboxamide (11r). To a solution
of 11o (30 mg, 0.065 mmol) and TEA (13 mg, 0.13 mmol) in DMF
(5 mL) was added benzyl bromide (11.1 mg, 0.065 mmol). The
resulting solution was stirred at room temperature for 3 h. Then, the
solution was poured into 10 mL of water and extracted with ethyl
acetate. The combined organic phase was washed with water and
brine, dried over anhydrous Na2SO4, and filtered, and then the filtrate
was concentrated in vacuo. The residue was purified on silica gel
chromatography with dichloromethane/methanol (50:1) to give 28
1
mg (78.1%) of 11r as a white solid. H NMR (400 MHz, CDCl3) δ
7.97 (d, J = 8.4 Hz, 1H), 7.34 (t, J = 4.2 Hz, 5H), 7.32−7.27 (m, 1H),
7.04 (t, J = 5.9 Hz, 1H), 6.94−6.83 (m, 3H), 4.45 (ddd, J = 17.0, 10.9,
4.3 Hz, 2H), 4.00−3.91 (m, 1H), 3.86−3.69 (m, 3H), 3.63 (d, J = 8.7
Hz, 4H), 3.31 (s, 2H), 2.80 (t, J = 5.3 Hz, 2H). 13C NMR (126 MHz,
DMSO-d6) δ 166.39, 161.31, 153.74, 144.51, 138.79, 138.52, 137.63,
133.84, 129.48 (overlap), 129.05, 128.80 (overlap), 128.70, 127.75,
122.05, 119.25, 118.77, 114.97, 74.99, 66.22, 61.02, 57.78, 53.42,
49.80, 49.43, 41.76. MS (EI) m/z: 552 (M+). HRMS (EI): Anal. Calcd
for C27H25SClN4O5, 552.1234; found, 552.1222.
N-(((3S,3aS)-7-(4-Acetyl-2-oxopiperazin-1-yl)-1-oxo-1,3,3a,4-
tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)methyl)-5-
chlorothiophene-2-carboxamide (11s). To a cooled solution of
11o (40 mg, 0.086 mmol) and TEA (13.1 mg, 0.13 mmol) in dry
DMF (5 mL) was added acetyl chloride (7.1 mg, 0.090 mmol)
dropwise. The resulting mixture was allowed to warm to room
temperature and stirred for 3 h. Then, the reaction was quenched by
addition of cold water and extracted with ethyl acetate. The organic
phase was washed with water and brine, dried over anhydrous Na2SO4,
and filtered, and then the filtrate was concentrated in vacuo. The
residue was purified on silica gel chromatography with dichloro-
methane/methanol (30:1) to yield 11s (27 mg, 61.9%) as a white
5-Chloro-N-(((3S,3aS)-1-oxo-7-(2-oxopiperazin-1-yl)-
1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)-
methyl)thiophene-2-carboxamide (11o). To a cooled (0 °C)
solution of 11j (0.86 g, 1.44 mmol) in DCM were added Me2S (0.45 g,
7.24 mmol) and BF3·Et2O (1.3 g, 9.15 mmol) dropwise. The resulting
solution was allowed to warm to room temperature and stirred for 8 h.
The solution was concentrated in vacuo and purified on silica gel
chromatography with dichloromethane/methanol (30:1) to give 0.55 g
1
(82.5%) of 11o as a white solid. H NMR (400 MHz, DMSO-d6) δ
9.00 (t, J = 5.7 Hz, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 4.1 Hz,
1H), 7.21 (d, J = 4.1 Hz, 1H), 6.99−6.91 (m, 2H), 5.76 (s, 1H), 4.65−
4.50 (m, 2H), 4.13−3.98 (m, 2H), 3.72 (t, J = 5.5 Hz, 2H), 3.55 (t, J =
5.7 Hz, 2H), 3.37 (s, 2H), 3.00 (t, J = 5.4 Hz, 2H). 13C NMR (126
MHz, DMSO-d6) δ 166.53, 161.32, 153.74, 144.52, 138.80 (overlap),
133.84, 129.08, 128.70, 122.12, 119.21, 118.89, 114.97, 74.99, 66.24,
53.42, 50.10, 49.53, 42.54, 41.75. MS (ESI) m/z: 463.3 (M+1)+.
HRMS (EI): Anal. Calcd for C20H19SClN4O5, 462.0765; found,
462.0774.
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solid. 1H NMR (400 MHz, CDCl3) δ 7.99 (t, J = 9.2 Hz, 1H), 7.35 (d,
J = 3.8 Hz, 1H), 7.04−6.82 (m, 4H), 4.59−4.44 (m, 2H), 4.34 (d, J =
38.9 Hz, 2H), 4.03−3.92 (m, 2H), 3.85 (d, J = 10.5 Hz, 4H), 3.71 (dd,
J = 20.5, 15.1 Hz, 2H), 2.16 (s, 3H). 13C NMR (126 MHz, DMSO-d6)
δ 168.88, 165.59, 165.02, 161.32, 153.74, 144.50, 138.79, 133.85,
129.06, 128.70, 122.20, 119.20, 119.09, 115.00, 75.00, 66.24, 53.42,
49.91, 49.18, 43.55, 41.76, 21.78. MS (EI) m/z: 504 (M+). HRMS
(EI): Anal. Calcd for C22H21SClN4O6, 504.0870; found, 504.0868.
5-Chloro-N-(((3S,3aS)-7-(4-(methylsulfonyl)-2-oxopiperazin-
1-yl)-1-oxo-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]-
oxazin-3-yl)methyl)thiophene-2-carboxamide (11t). To a
cooled solution of 11o (40 mg, 0.086 mmol) and TEA (13.1 mg,
0.13 mmol) in dry DMF (5 mL) was added mesyl chloride (11.9 mg,
0.10 mmol). The resulting mixture was allowed to warm to room
temperature and stirred for 3 h. Then, the reaction was quenched by
addition of cold water and extracted with ethyl acetate. The organic
phase was washed with water and brine, dried over anhydrous Na2SO4,
and filtered, and then the filtrate was concentrated in vacuo. The
residue was purified on silica gel chromatography with dichloro-
methane/methanol (50:1) to yield 11t (40 mg, 85.6%) as a white
solid. 1H NMR (400 MHz, DMSO-d6) δ 9.00 (t, J = 5.9 Hz, 1H), 7.86
(d, J = 8.7 Hz, 1H), 7.71 (d, J = 4.1 Hz, 1H), 7.21 (d, J = 4.1 Hz, 1H),
7.02 (d, J = 2.3 Hz, 1H), 6.98 (dd, J = 8.7, 2.3 Hz, 1H), 4.58 (dd, J =
15.7, 7.0 Hz, 2H), 4.13−4.01 (m, 2H), 3.93 (s, 2H), 3.73 (q, J = 4.9
Hz, 4H), 3.57−3.51 (m, 2H), 3.05 (s, 3H). 13C NMR (126 MHz,
DMSO-d6) δ 164.13, 161.32, 153.74, 144.58, 138.79, 138.14, 133.85,
129.06, 128.70, 122.47, 119.45, 118.88, 115.24, 75.02, 66.23, 53.41,
49.75, 49.24, 43.10, 41.76, 35.13. MS (EI) m/z: 540 (M+). HRMS
(EI): Anal. Calcd for C21H21S2ClN4O7, 540.0540; found, 540.0532.
5-Chloro-N-(((3S,3aS)-1-oxo-7-(2-oxo-4-(phenylsulfonyl)-
piperazin-1-yl)-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d]-
[1,4]oxazin-3-yl)methyl)thiophene-2-carboxamide (11u). To a
cooled solution of 11o (40 mg, 0.086 mmol) and TEA (16.7 mg, 0.16
mmol) in dry DMF (5 mL) was added benzenesulfonyl chloride (18.3
mg, 0.10 mmol) dropwise. The resulting mixture was allowed to warm
to room temperature and stirred for 3 h. Then, water was added, and
the solution was extracted with ethyl acetate. The organic phase was
washed with water and brine, dried over anhydrous Na2SO4, and
filtered, and then the filtrate was concentrated in vacuo. The resulting
residue was purified on silica gel chromatography with dichloro-
methane/methanol (50:1) to afford 30 mg (57.6%) of 11u as a white
mixture was heated at 80 °C overnight. Dichloromethane (15 mL) was
added, and the mixture was filtered through Celite, washed with brine,
dried over anhydrous Na2SO4, and evaporated. The residue was
purified by chromatography on silica gel with petroleum/ethyl acetate
(2:1) to afford 19 mg (35.9%) of 25a as a white solid. 1H NMR (400
MHz, DMSO-d6) δ 9.01 (t, J = 5.8 Hz, 1H), 8.08 (dd, J = 7.9, 1.2 Hz,
1H), 7.93 (d, J = 8.2 Hz, 1H), 7.80−7.70 (m, 2H), 7.70−7.63 (m,
1H), 7.39 (dd, J = 7.5, 1.2 Hz, 1H), 7.22 (d, J = 4.0 Hz, 1H), 7.07−
6.99 (m, 2H), 4.67−4.54 (m, 2H), 4.19−4.06 (m, 2H), 3.73 (d, J = 5.6
Hz, 2H), 2.86 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 161.33,
153.72, 143.66, 140.51, 139.68, 138.80, 134.85, 133.86, 133.76, 133.21,
129.08, 128.83, 128.71, 128.38, 123.73, 123.34, 118.78, 118.01, 75.09,
66.18, 53.47, 44.04, 41.73. MS (EI) m/z: 518 (M+). HRMS (EI): Anal.
Calcd for C23H19S2ClN2O6, 518.0373; found, 518.0371.
5-Chloro-N-(((3S,3aS)-1-oxo-7-(2-sulfamoylphenyl)-1,3,3a,4-
tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)methyl)-
thiophene-2-carboxamide (25c). Compound 25b (0.12 g, 0.21
mmol) was dissolved in TFA (2 mL) and heated at 40 °C for 2 h.
Then, the solution was evaporated, and the residue was purified by
chromatography on silica gel with petroleum/ethyl acetate (2:1) to
1
afford 91 mg (84.0%) of 25c as a white solid. H NMR (300 MHz,
DMSO-d6) δ 9.03 (t, J = 5.5 Hz, 1H), 8.01 (d, J = 7.6 Hz, 1H), 7.87
(d, J = 8.6 Hz, 1H), 7.73 (d, J = 4.1 Hz, 1H), 7.64−7.52 (m, 2H), 7.30
(d, J = 7.3 Hz, 1H), 7.27−7.18 (m, 3H), 7.00 (d, J = 7.5 Hz, 2H), 4.60
(dd, J = 16.6, 6.9 Hz, 2H), 4.20−4.01 (m, 2H), 3.75 (d, J = 5.0 Hz,
2H). 13C NMR (126 MHz, DMSO-d6) δ 161.34, 153.75, 143.67,
142.65, 139.45, 138.80, 136.42, 133.86, 132.83, 131.92, 129.08, 128.72,
128.12, 127.73, 123.12, 122.91, 118.22, 118.02, 75.01, 66.20, 53.51,
41.75. MS (EI) m/z: 519 (M+). HRMS (EI): Anal. Calcd for
C22H18S2ClN3O6, 519.0326; found, 519.0339.
4-Chloro-N-(((3S,3aS)-1-oxo-7-(3-oxomorpholino)-1,3,3a,4-
tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)methyl)-
benzamide (26a). Compound 26a (72 mg, 83.7%) was prepared
from 10a (60 mg, 0.19 mmol) and 4-chlorobenzoic acid (35 mg, 0.22
1
mmol) in the same manner as that described for 11a. H NMR (300
MHz, DMSO-d6) δ 8.97 (t, J = 5.7 Hz, 1H), 7.87 (dd, J = 13.0, 8.6 Hz,
3H), 7.56 (d, J = 8.6 Hz, 2H), 7.11−6.96 (m, 2H), 4.67−4.52 (m,
2H), 4.17 (s, 2H), 4.08 (dt, J = 19.5, 6.2 Hz, 2H), 3.98−3.91 (m, 2H),
3.76 (t, J = 5.5 Hz, 2H), 3.72−3.64 (m, 2H). 13C NMR (126 MHz,
DMSO-d6) δ 166.50, 166.41, 153.82, 144.56, 138.06, 136.78, 133.10,
129.77 (overlap), 128.95 (overlap), 122.17, 118.89 (overlap), 114.69,
74.94, 68.18, 66.31, 63.90, 53.52, 49.47, 41.95. MS (ESI) m/z: 480.1
(M + 23)+. HRMS (EI): Anal. Calcd for C22H20N3O6, 457.1041;
found, 457.1032.
1
solid. H NMR (300 MHz, DMSO-d6) δ 8.98 (s, 1H), 7.77 (dd, J =
27.8, 19.6 Hz, 7H), 7.19 (s, 1H), 6.80 (s, 2H), 4.53 (s, 2H), 4.02 (s,
2H), 3.73 (s, 5H), 3.60 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ
163.55, 161.31, 153.71, 144.52, 138.78, 137.92, 135.47, 134.23, 133.84,
130.21 (overlap), 129.05, 128.69, 128.16 (overlap), 122.51, 119.41,
118.82, 115.22, 75.00, 66.19, 53.37, 49.44, 49.17, 43.47, 41.74. MS
(EI) m/z: 490 (M+). HRMS (EI): Anal. Calcd for C26H23S2ClN4O7,
602.0697; found, 602.0691.
5-Chloro-N-(((3S,3aS)-1-oxo-7-(3-oxomorpholino)-1,3,3a,4-
tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)methyl)-
picolinamide (26b). Compound 26b (74 mg, 85.8%) was prepared
from 10a (60 mg, 0.19 mmol) and 5-chloropicolinic acid (36 mg, 0.23
1
mmol) in the same manner as that described for 11a. H NMR (300
MHz, DMSO-d6) δ 9.20 (s, 1H), 8.75 (s, 1H), 8.16 (d, J = 9.1 Hz,
1H), 8.08 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 8.7 Hz, 1H), 7.09−6.99 (m,
2H), 4.66 (d, J = 5.4 Hz, 1H), 4.52 (d, J = 10.2 Hz, 1H), 4.19 (s, 3H),
4.05 (t, J = 9.9 Hz, 1H), 3.95 (d, J = 4.6 Hz, 2H), 3.79 (s, 2H), 3.70 (s,
2H). 13C NMR (126 MHz, DMSO-d6) δ 166.40, 164.28, 153.75,
148.50, 147.70, 144.55, 138.14, 138.06, 134.66, 124.10, 122.14, 118.91,
118.86, 114.67, 74.79, 68.17, 66.32, 63.90, 53.53, 49.46, 41.72. MS
(EI) m/z: 458 (M+). HRMS (EI): Anal. Calcd for C21H19N4O6,
458.0993; found, 458.1000.
4-Methoxy-N-(((3S,3aS)-1-oxo-7-(3-oxomorpholino)-
1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)-
methyl)benzamide (26c). Compound 26c (61 mg, 85.9%) was
prepared from 10a (50 mg, 0.16 mmol) and 4-methoxybenzoic acid
(29 mg, 0.19 mmol) in the same manner as that described for 11a. 1H
NMR (500 MHz, DMSO-d6) δ 8.76 (t, J = 5.8 Hz, 1H), 7.90−7.83
(m, 3H), 7.03 (dt, J = 8.9, 2.4 Hz, 4H), 4.62 (dd, J = 12.0, 5.3 Hz, 1H),
4.54 (dd, J = 10.1, 2.8 Hz, 1H), 4.18 (s, 2H), 4.13 (ddd, J = 9.8, 6.9,
2.8 Hz, 1H), 4.06 (t, J = 10.1 Hz, 1H), 3.95 (t, J = 5.0 Hz, 2H), 3.80
(d, J = 14.1 Hz, 3H), 3.74 (dd, J = 10.6, 5.3 Hz, 2H), 3.71−3.67 (m,
2H). 13C NMR (126 MHz, DMSO-d6) δ 166.95, 166.41, 162.22,
153.85, 144.53, 138.02, 129.67 (overlap), 126.49, 122.18, 118.90,
118.86, 114.69, 114.04 (overlap), 75.09, 68.16, 66.32, 63.89, 55.83,
5-Chloro-N-(((3S,3aR)-1-oxo-7-(3-oxomorpholino)-1,3,3a,4-
tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)methyl)-
thiophene-2-carboxamide (18). Compound 18 (73 mg, 82.4%)
was prepared from 17 (61 mg, 0.19 mmol) and 5-chlorothiophene-2-
carboxylic acid (47 mg, 0.29 mmol) in the same manner as that
1
described for 11a. H NMR (500 MHz, DMSO-d6) δ 8.94 (s, 1H),
7.87 (d, J = 8.7 Hz, 1H), 7.73 (d, J = 3.6 Hz, 1H), 7.22 (d, J = 3.6 Hz,
1H), 7.06 (s, 1H), 7.02 (d, J = 8.6 Hz, 1H), 5.02 (d, J = 3.3 Hz, 1H),
4.66 (d, J = 10.4 Hz, 1H), 4.40 (t, J = 8.5 Hz, 1H), 4.17 (d, J = 14.1
Hz, 3H), 3.95 (d, J = 4.6 Hz, 2H), 3.72−3.62 (m, 3H), 3.55−3.45 (m,
1H). 13C NMR (126 MHz, DMSO-d6) δ 166.35, 160.97, 153.64,
144.53, 138.86, 138.02, 133.70, 129.00, 128.61, 122.53, 119.00, 118.76,
114.63, 73.82, 68.12, 63.85, 63.62, 52.05, 49.42, 39.32. MS (EI) m/z:
463 (M+). HRMS (EI): Anal. Calcd for C20H18SClN3O6, 463.0605;
found, 463.0608.
5-Chloro-N-(((3S,3aS)-7-(2-(methylsulfonyl)phenyl)-1-oxo-
1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)-
methyl)thiophene-2-carboxamide (25a). A mixture of 24 (50 mg,
0.10 mmol), cesium carbonate (83 mg, 0.26 mmol), 1-bromo-2-
(methylsulfonyl)benzene (29 mg, 0.12 mmol), and tetrakis-
(triphenylphosphine)palladium(0) (12 mg, 0.01 mmol) in dioxane
(5 mL) and H2O (0.5 mL) was degassed and flushed with argon. The
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Journal of Medicinal Chemistry
Article
53.56, 49.46, 41.84. MS (EI) m/z: 453 (M+). HRMS (EI): Anal. Calcd
for C23H23N3O7, 453.1536; found, 453.1531.
d6) δ 9.31−9.16 (t, J = 5.7 Hz, 1H), 8.13−8.03 (m, 3H), 7.90−7.77 (d,
J = 8.7 Hz, 1H), 7.54−7.43 (dd, J = 8.7, 2.1 Hz, 1H), 7.09−7.02 (d, J =
2.3 Hz, 1H), 7.02−6.94 (dd, J = 8.7, 2.3 Hz, 1H), 4.66−4.59 (q, J = 5.9
Hz, 1H), 4.59−4.48 (dd, J = 9.4, 2.2 Hz, 1H), 4.19−4.13 (s, 2H),
4.13−4.00 (m, 2H), 3.97−3.88 (dd, J = 6.0, 4.1 Hz, 2H), 3.81−3.72 (t,
J = 5.6 Hz, 2H), 3.70−3.61 (dd, J = 6.1, 4.2 Hz, 2H). 13C NMR (126
MHz, DMSO-d6) δ 166.40, 162.40, 153.74, 144.52, 141.88, 141.03,
139.18, 138.03, 130.42, 126.88, 126.61, 125.08, 124.97, 122.16, 118.87,
118.82, 114.68, 74.90, 68.17, 66.27, 63.90, 53.47, 49.46, 41.96. MS
(ESI) m/z: 536.2 (M + 23)+. HRMS (ESI): Anal. Calcd for
C24H20O6N3ClNaS, 536.0654; found, 536.0640.
4,5-Dichloro-N-(((3S,3aS)-1-oxo-7-(3-oxomorpholino)-
1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)-
methyl)thiophene-2-carboxamide (26d). Compound 26d (76
mg, 81.2%) was prepared from 10a (60 mg, 0.19 mmol) and 4,5-
dichlorothiophene-2-carboxylic acid (44 mg, 0.22 mmol) in the same
1
manner as that described for 11a. H NMR (400 MHz, DMSO-d6) δ
9.10 (t, J = 5.8 Hz, 1H), 7.91 (s, 1H), 7.86 (d, J = 8.7 Hz, 1H), 7.06 (d,
J = 2.3 Hz, 1H), 7.02 (dd, J = 8.7, 2.4 Hz, 1H), 4.58 (ddd, J = 9.3, 6.4,
3.4 Hz, 2H), 4.18 (s, 2H), 4.11−4.02 (m, 2H), 3.95 (dd, J = 5.9, 4.2
Hz, 2H), 3.75 (t, J = 6.0 Hz, 2H), 3.72−3.67 (m, 2H). 13C NMR (151
MHz, DMSO-d6) δ 166.38, 160.47, 153.66, 144.50, 138.04, 136.99,
129.10, 128.36, 123.80, 122.14, 118.85, 118.77, 114.66, 74.88, 68.18,
66.23, 63.90, 53.36, 49.46, 41.79. MS (ESI) m/z: 520.0 (M + 23)+.
HRMS (ESI): Anal. Calcd for C20H17O6N3Cl2NaS, 520.0107; found,
520.0112.
4-Chloro-N-(((3S,3aS)-1-oxo-7-(3-oxomorpholino)-1,3,3a,4-
tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)methyl)-
benzo[b]thiophene-2-carboxamide (26i). Compound 26i (76 mg,
78.7%) was prepared from 10a (60 mg, 0.19 mmol) and 4-
chlorobenzo[b]thiophene-2-carboxylic acid (48 mg, 0.22 mmol) in
1
the same manner as that described for 11a. H NMR (400 MHz,
DMSO-d6) δ 9.39 (t, J = 5.8 Hz, 1H), 8.34 (s, 1H), 8.04 (d, J = 8.0 Hz,
1H), 7.87 (d, J = 8.7 Hz, 1H), 7.56 (dd, J = 7.7, 0.9 Hz, 1H), 7.48 (t, J
= 7.9 Hz, 1H), 7.06 (d, J = 2.3 Hz, 1H), 7.02 (dd, J = 8.7, 2.4 Hz, 1H),
4.66 (q, J = 5.6 Hz, 1H), 4.60 (dd, J = 10.0, 2.7 Hz, 1H), 4.18 (s, 2H),
4.16−4.12 (m, 1H), 4.12−4.05 (m, 1H), 3.95 (dd, J = 6.0, 4.1 Hz,
2H), 3.84−3.78 (m, 2H), 3.72−3.67 (m, 2H). 13C NMR (151 MHz,
DMSO-d6) δ 166.38, 162.22, 153.76, 144.53, 142.04, 141.33, 138.05,
137.64, 129.01, 127.83, 125.33, 123.19, 122.54, 122.15, 118.84-
(overlap), 114.66, 74.94, 68.17, 66.28, 63.90, 53.50, 49.46, 42.02.
MS (ESI) m/z: 536.1 (M + 23)+. HRMS (ESI): Anal. Calcd for
C24H20O6N3ClNaS, 536.0654; found, 536.0653.
2-Chloro-N-(((3S,3aS)-1-oxo-7-(3-oxomorpholino)-1,3,3a,4-
tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)methyl)-
thiazole-5-carboxamide (26e). Compound 26e (72 mg, 82.4%)
was prepared from 10a (60 mg, 0.19 mmol) and 2-chlorothiazole-5-
carboxylic acid (37 mg, 0.22 mmol) in the same manner as that
1
described for 11a. H NMR (400 MHz, DMSO-d6) δ 9.23 (t, J = 5.8
Hz, 1H), 8.31 (s, 1H), 7.86 (d, J = 8.7 Hz, 1H), 7.06 (d, J = 2.3 Hz,
1H), 7.02 (dd, J = 8.7, 2.4 Hz, 1H), 4.64−4.55 (m, 2H), 4.18 (s, 2H),
4.11−4.02 (m, 2H), 3.95 (t, J = 5.0 Hz, 2H), 3.76 (t, J = 5.2 Hz, 2H),
3.73−3.65 (m, 2H). 13C NMR (151 MHz, DMSO-d6) δ 166.38,
159.98, 154.44, 153.67, 144.51, 142.64, 138.05, 138.01, 122.14, 118.86,
118.78, 114.67, 74.96, 68.18, 66.23, 63.90, 53.36, 49.46, 41.75. MS
(ESI) m/z: 462.8 (M−1)−. HRMS (ESI): Anal. Calcd for
C19H17O6N4ClNaS, 487.0450; found, 487.0443.
7-Chloro-N-(((3S,3aS)-1-oxo-7-(3-oxomorpholino)-1,3,3a,4-
tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)methyl)-
benzo[b]thiophene-2-carboxamide (26f). Compound 26f (74
mg, 76.6%) was prepared from 10a (60 mg, 0.19 mmol) and 7-
chlorobenzo[b]thiophene-2-carboxylic acid (48 mg, 0.22 mmol) in the
same manner as that described for 11a. 1H NMR (400 MHz, DMSO-
d6) δ 9.30 (t, J = 5.8 Hz, 1H), 8.27 (s, 1H), 7.99 (dd, J = 8.0, 1.0 Hz,
1H), 7.87 (d, J = 8.7 Hz, 1H), 7.62 (dd, J = 7.7, 0.9 Hz, 1H), 7.51 (t, J
= 7.8 Hz, 1H), 7.06 (d, J = 2.3 Hz, 1H), 7.02 (dd, J = 8.7, 2.4 Hz, 1H),
4.66 (q, J = 5.6 Hz, 1H), 4.60 (dd, J = 9.9, 2.7 Hz, 1H), 4.18 (s, 2H),
4.16−4.12 (m, 1H), 4.12−4.04 (m, 1H), 3.95 (t, J = 5.0 Hz, 2H),
3.85−3.78 (m, 2H), 3.71−3.66 (m, 2H). 13C NMR (151 MHz,
DMSO-d6) δ 166.38, 162.25, 153.73, 144.53, 141.03, 140.67, 139.47,
138.04, 127.22, 126.99, 126.61, 126.39, 124.95, 122.16, 118.86, 118.81,
114.67, 74.89, 68.17, 66.28, 63.90, 53.48, 49.46, 41.99. MS (ESI) m/z:
511.7 (M − 1)−. HRMS (ESI): Anal. Calcd for C24H20O6N3ClNaS,
536.0654; found, 536.0668.
6-Chloro-N-(((3S,3aS)-1-oxo-7-(3-oxomorpholino)-1,3,3a,4-
tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)methyl)-
benzo[b]thiophene-2-carboxamide (26g). Compound 26g (81
mg, 83.9%) was prepared from 10a (60 mg, 0.19 mmol) and 6-
chlorobenzo[b]thiophene-2-carboxylic acid (48 mg, 0.22 mmol) in the
same manner as that described for 11a. 1H NMR (400 MHz, DMSO-
d6) δ 9.29 (t, J = 5.9 Hz, 1H), 8.22 (d, J = 1.9 Hz, 1H), 8.19 (s, 1H),
7.99 (d, J = 8.6 Hz, 1H), 7.86 (d, J = 8.7 Hz, 1H), 7.48 (dd, J = 8.6, 2.0
Hz, 1H), 7.06 (d, J = 2.3 Hz, 1H), 7.01 (dd, J = 8.7, 2.3 Hz, 1H),
4.68−4.62 (m, 1H), 4.59 (dd, J = 10.2, 2.8 Hz, 1H), 4.18 (s, 2H),
4.16−4.12 (m, 1H), 4.07 (t, J = 10.0 Hz, 1H), 3.95 (t, J = 5.0 Hz, 2H),
3.79 (t, J = 5.6 Hz, 2H), 3.69 (dd, J = 6.2, 4.1 Hz, 2H). 13C NMR (151
MHz, DMSO-d6) δ 166.38, 162.46, 153.74, 144.52, 141.96, 140.64,
138.33, 138.04, 131.65, 127.20, 126.05, 125.40, 122.88, 122.16, 118.86,
118.82, 114.66, 74.89, 68.17, 66.29, 63.90, 53.49, 49.46, 41.91. MS
(ESI) m/z: 536.1 (M + 23)+. HRMS (ESI): Anal. Calcd for
C24H20O6N3ClNaS, 536.0654; found, 536.0650.
3,6-Dichloro-N-(((3S,3aS)-1-oxo-7-(3-oxomorpholino)-
1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)-
methyl)benzo[b]thiophene-2-carboxamide (26j). Compound
26j (86 mg, 83.4%) was prepared from 10a (60 mg, 0.19 mmol)
and 3,6-dichlorobenzo[b]thiophene-2-carboxylic acid (56 mg, 0.23
1
mmol) in the same manner as that described for 11a. H NMR (400
MHz, DMSO-d6) δ 8.92 (t, J = 5.9 Hz, 1H), 8.34 (d, J = 1.9 Hz, 1H),
7.91 (d, J = 8.7 Hz, 1H), 7.85 (d, J = 8.7 Hz, 1H), 7.63 (dd, J = 8.7, 1.9
Hz, 1H), 7.06 (d, J = 2.3 Hz, 1H), 7.03 (dd, J = 8.7, 2.4 Hz, 1H), 4.69
(q, J = 5.2 Hz, 1H), 4.62−4.59 (m, 1H), 4.18 (s, 2H), 4.13−4.06 (m,
2H), 3.95 (t, J = 5.0 Hz, 2H), 3.83 (q, J = 5.0 Hz, 2H), 3.69 (dd, J =
6.3, 4.3 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) δ 166.39, 161.24,
153.83, 144.62, 138.37, 138.13, 135.20, 133.16, 132.84, 127.14, 124.56,
123.56, 122.09, 119.44, 119.10, 118.89, 114.68, 74.64, 68.17, 66.32,
63.90, 53.28, 49.47, 41.93. MS (ESI) m/z: 570.0 (M + 23)+. HRMS
(ESI): Anal. Calcd for C24H19O6N3Cl2NaS, 570.0264; found,
570.0255.
2-((2-Chloroethyl)thio)-N-(((3S,3aS)-1-oxo-7-(3-oxomorpho-
lino)-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-
yl)methyl)acetamide (26k). Compound 26k (72 mg, 84.0%) was
prepared from 10a (60 mg, 0.19 mmol) and 2-((2-chloroethyl)thio)-
acetic acid (35 mg, 0.23 mmol) in the same manner as that described
1
for 11a. H NMR (400 MHz, DMSO-d6) δ 8.50 (t, J = 5.9 Hz, 1H),
7.87 (d, J = 8.7 Hz, 1H), 7.06 (d, J = 2.3 Hz, 1H), 7.02 (dd, J = 8.7, 2.4
Hz, 1H), 4.57 (dd, J = 9.6, 2.4 Hz, 1H), 4.55−4.49 (m, 1H), 4.18 (s,
2H), 4.09−3.98 (m, 2H), 3.97−3.93 (m, 2H), 3.76 (t, J = 7.6 Hz, 2H),
3.69 (dd, J = 6.1, 4.1 Hz, 2H), 3.59 (dt, J = 10.1, 5.4 Hz, 2H), 3.24 (s,
2H), 2.95−2.88 (m, 2H). 13C NMR (151 MHz, DMSO-d6) δ 170.59,
166.39, 153.69, 144.52, 138.03, 122.12, 118.86, 118.83, 114.64, 74.95,
68.18, 66.31, 63.90, 53.22, 49.47, 43.65, 41.12, 34.37, 34.32. MS (ESI)
m/z: 456.1 (M
+
1)+. HRMS (ESI): Anal. Calcd for
C19H22O6N3ClNaS, 478.0816; found, 478.0812.
2,2-Dichloro-N-(((3S,3aS)-1-oxo-7-(3-oxomorpholino)-
1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)-
methyl)acetamide (26l). Compound 26l (65 mg, 80.4%) was
prepared from 10a (60 mg, 0.19 mmol) and 2,2-dichloroacetic acid
(29 mg, 0.22 mmol) in the same manner as that described for 11a. 1H
NMR (400 MHz, DMSO-d6) δ 9.05 (t, J = 5.9 Hz, 1H), 7.84 (d, J =
8.7 Hz, 1H), 7.06 (d, J = 2.3 Hz, 1H), 7.02 (dd, J = 8.7, 2.4 Hz, 1H),
6.54 (s, 1H), 4.57 (d, J = 6.9 Hz, 2H), 4.18 (s, 2H), 4.08−3.99 (m,
2H), 3.95 (dd, J = 6.0, 4.2 Hz, 2H), 3.72−3.63 (m, 4H). 13C NMR
(151 MHz, DMSO-d6) δ 166.39, 164.93, 153.63, 144.54, 138.10,
122.05, 118.94, 118.88, 114.67, 74.48, 68.17, 67.06, 66.19, 63.90,
5-Chloro-N-(((3S,3aS)-1-oxo-7-(3-oxomorpholino)-1,3,3a,4-
tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)methyl)-
benzo[b]thiophene-2-carboxamide (26h). Compound 26h (69
mg, 71.4%) was prepared from 10a (60 mg, 0.19 mmol) and 5-
chlorobenzo[b]thiophene-2-carboxylic acid (48 mg, 0.22 mmol) in the
same manner as that described for 11a. 1H NMR (300 MHz, DMSO-
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53.22, 49.46, 41.90. MS (ESI) m/z: 452.0 (M + 23)+. HRMS (ESI):
Anal. Calcd for C17H17O6N3Cl2Na, 452.0387; found, 452.0384.
1-(5-Chlorothiophen-2-yl)-3-(((3S,3aS)-1-oxo-7-(3-oxomor-
pholino)-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]-
oxazin-3-yl)methyl)urea (27a). To a solution of 10a (98 mg, 0.31
mmol) and TEA (47 mg, 0.46 mmol) in DCM (10 mL) was added 2-
chloro-5-isocyanatothiophene (59 mg, 0.37 mmol). The solution was
stirred at room temperature overnight and then concentrated. The
resulting residue was purified by chromatography on silica gel with
dichloromethane/methanol (100:1) to give 27a (87 mg, 59.2%) as a
white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.83 (s, 1H), 7.85 (d, J
= 8.7 Hz, 1H), 7.08−6.99 (m, 2H), 6.81−6.73 (m, 2H), 6.25 (d, J =
4.1 Hz, 1H), 4.54 (dd, J = 12.1, 6.2 Hz, 2H), 4.17 (s, 2H), 4.08−3.98
(m, 2H), 3.97−3.91 (m, 2H), 3.72−3.65 (m, 2H), 3.58 (t, J = 5.4 Hz,
2H). 13C NMR (126 MHz, DMSO-d6) δ 166.40, 154.89, 153.80,
144.56, 140.26, 138.07, 123.70, 122.12, 118.90, 118.86, 117.75, 114.67,
107.00, 75.35, 68.18, 66.31, 63.90, 53.14, 49.45, 42.00. MS (ESI) m/z:
501.1 (M + 23)+. HRMS (ESI): Anal. Calcd for C20H19O6N4ClNaS,
501.0606; found, 501.0610.
(m, 3H), 3.70 (t, J = 5.1 Hz, 4H). 13C NMR (126 MHz, DMSO-d6) δ
166.41, 161.09, 158.78, 153.71, 144.58, 138.07, 137.08, 129.11
(overlap), 128.74, 122.49 (overlap), 122.14, 118.96, 118.84, 114.67,
74.49, 68.18, 66.28, 63.90, 53.41, 49.46, 41.78. MS (EI) m/z: 500
(M)+. HRMS (EI): Anal. Calcd for C23H21ClN4O7, 500.1099; found,
500.1098.
(3R,3aS)-N-(5-Chlorothiophen-2-yl)-1-oxo-7-(3-oxomorpho-
lino)-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazine-
3-carboxamide (30a). Compound 30a (73 mg, 60.3%) was prepared
from 29 (90 mg, 0.27 mmol) and 5-chlorothiophen-2-amine (43 mg,
1
0.32 mmol) in the same manner as that described for 11a. H NMR
(300 MHz, DMSO-d6) δ 11.97 (s, 1H), 7.87 (d, J = 8.6 Hz, 1H), 7.10
(d, J = 2.1 Hz, 1H), 7.06 (dd, J = 8.6, 2.3 Hz, 1H), 6.95 (d, J = 4.2 Hz,
1H), 6.77 (d, J = 4.2 Hz, 1H), 5.15 (d, J = 6.9 Hz, 1H), 4.64 (dd, J =
10.6, 3.2 Hz, 1H), 4.49−4.34 (m, 1H), 4.30−4.13 (m, 3H), 4.04−3.91
(m, 2H), 3.80−3.65 (m, 2H). 13C NMR (126 MHz, DMSO-d6) δ
166.44, 164.53, 153.04, 144.65, 138.37, 136.98, 124.09, 121.72, 120.71,
119.07, 118.96, 114.78, 112.28, 73.14, 68.18, 66.44, 63.91, 53.20,
49.45. MS (EI) m/z: 449 (M+). HRMS (EI): Anal. Calcd for
C19H16SClN3O6, 449.0448; found, 449.0452.
1-(4-Chlorophenyl)-3-(((3S,3aS)-1-oxo-7-(3-oxomorpholino)-
1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)-
methyl)urea (27b). Compound 27b (75 mg, 84.4%) was prepared
from 10a (60 mg, 0.19 mmol) and 1-chloro-4-isocyanatobenzene (35
(3R,3aS)-N-(5-Chloropyridin-2-yl)-1-oxo-7-(3-oxomorpholi-
no)-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazine-3-
carboxamide (30b). Compound 30b (63 mg, 52.6%) was prepared
from 29 (90 mg, 0.27 mmol) and 5-chloropyridin-2-amine (41 mg,
1
mg, 0.23 mmol) in the same manner as that described for 27a. H
1
NMR (300 MHz, DMSO-d6) δ 8.75 (s, 1H), 7.84 (d, J = 8.6 Hz, 1H),
7.44−7.37 (m, 2H), 7.29−7.21 (m, 2H), 7.06−6.96 (m, 2H), 6.58 (s,
1H), 4.55 (d, J = 7.2 Hz, 2H), 4.16 (s, 2H), 4.06−3.98 (m, 2H), 3.96−
3.89 (m, 2H), 3.70−3.62 (m, 2H), 3.57 (m, 2H). 13C NMR (126
MHz, DMSO-d6) δ 166.41, 155.71, 153.82, 144.56, 139.64, 138.06,
128.99 (overlap), 125.25, 122.14, 119.73 (overlap), 118.87 (overlap),
114.69, 75.56, 68.18, 66.34, 63.90, 53.14, 49.46, 41.60. MS (ESI) m/z:
495.1 (M + 23)+. HRMS (ESI): Anal. Calcd for C22H21O6N4ClNa,
495.1042; found, 495.1043.
0.32 mmol) in the same manner as that described for 11a. H NMR
(3400 MHz, CDCl3) δ 8.86 (s, 1H), 8.31 (d, J = 2.2 Hz, 1H), 8.18 (d,
J = 8.9 Hz, 1H), 8.04 (d, J = 8.7 Hz, 1H), 7.73 (dd, J = 8.8, 2.5 Hz,
1H), 7.06−6.97 (m, 2H), 4.81−4.72 (m, 2H), 4.40−4.29 (m, 3H),
4.06−3.96 (m, 3H), 3.77−3.71 (m, 2H). 13C NMR (126 MHz,
DMSO-d6) δ 166.68, 166.43, 153.89, 150.31, 147.07, 144.89, 138.70,
138.51, 126.53, 121.75, 119.91, 118.84, 115.67, 114.74, 73.31, 68.18,
66.17, 63.91, 53.13, 49.46. MS (EI) m/z: 444 (M+). HRMS (EI): Anal.
Calcd for C20H17ClN4O6, 444.0837; found, 444.0845.
N1-(5-Chlorothiophen-2-yl)-N2-(((3S,3aS)-1-oxo-7-(3-oxo-
morpholino)-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]-
oxazin-3-yl)methyl)oxalamide (28a). Compound 28a (71 mg,
74.5%) was prepared from 10a (60 mg, 0.19 mmol) and 2-((5-
chlorothiophen-2-yl)amino)-2-oxoacetic acid (47 mg, 0.23 mmol) in
5-Chloro-N-(((3S,3aR)-1-oxo-7-(3-oxomorpholino)-1,3,3a,4-
tetrahydrobenzo[b]oxazolo[3,4-d][1,4]thiazin-3-yl)methyl)-
thiophene-2-carboxamide (47a). Compound 47a (62 mg, 86.6%)
was prepared from 46a (50 mg, 0.15 mmol) and 5-chlorothiophene-2-
carboxylic acid (29 mg, 0.18 mmol) in the same manner as that
1
1
the same manner as that described for 11a. H NMR (400 MHz,
described for 11a. H NMR (500 MHz, DMSO-d6) δ 9.00 (t, J = 5.9
DMSO-d6) δ 12.35 (s, 1H), 9.41 (t, J = 6.2 Hz, 1H), 7.84 (d, J = 8.7
Hz, 1H), 7.05 (d, J = 2.3 Hz, 1H), 7.01 (dd, J = 8.7, 2.3 Hz, 1H), 6.95
(d, J = 4.2 Hz, 1H), 6.90 (d, J = 4.2 Hz, 1H), 4.63−4.52 (m, 2H), 4.18
(s, 2H), 4.16−4.11 (m, 1H), 4.03 (t, J = 10.2 Hz, 1H), 3.99−3.89 (m,
2H), 3.75−3.64 (m, 4H). 13C NMR (126 MHz, DMSO-d6) δ 166.40,
160.01, 157.10, 153.70, 144.59, 138.07, 137.07, 124.37, 122.14, 121.43,
118.97, 118.83, 114.66, 113.57, 74.41, 68.18, 66.28, 63.90, 53.40,
49.46, 41.73. MS (ESI) m/z: 529.1 (M + 23)+. HRMS (ESI): Anal.
Calcd for C21H19O7N4ClNaS, 529.0555; found, 529.0562.
Hz, 1H), 7.95 (d, J = 8.9 Hz, 1H), 7.70 (d, J = 4.1 Hz, 1H), 7.29 (d, J
= 2.4 Hz, 1H), 7.22 (d, J = 4.1 Hz, 1H), 7.15 (dd, J = 8.9, 2.4 Hz, 1H),
4.58 (dd, J = 10.2, 5.5 Hz, 1H), 4.18 (s, 2H), 4.14−4.09 (m, 1H),
3.98−3.92 (m, 2H), 3.70 (dt, J = 10.3, 5.1 Hz, 4H), 3.34−3.27 (m,
2H). 13C NMR (126 MHz, DMSO-d6) δ 166.46, 161.29, 154.04,
138.83, 137.84, 133.80, 130.99, 128.99, 128.70, 123.84, 122.57, 122.53,
121.02, 77.15, 68.15, 63.88, 55.56, 49.37, 41.84, 28.16. MS (EI) m/z:
479 (M+). HRMS (EI): Anal. Calcd for C20H18S2ClN3O5, 479.0376;
found, 479.0356.
N1-(5-Chloropyridin-2-yl)-N2-(((3S,3aS)-1-oxo-7-(3-oxomor-
pholino)-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]-
oxazin-3-yl)methyl)oxalamide (28b). Compound 28b (76 mg,
80.6%) was prepared from 10a (60 mg, 0.19 mmol) and 2-((5-
chloropyridin-2-yl)amino)-2-oxoacetic acid (45 mg, 0.22 mmol) in the
same manner as that described for 11a. 1H NMR (300 MHz, DMSO-
d6) δ 10.37 (s, 1H), 9.46 (t, J = 6.1 Hz, 1H), 8.51−8.45 (m, 1H),
8.13−7.99 (m, 2H), 7.86 (dd, J = 6.0, 4.3 Hz, 1H), 7.10−6.98 (m,
2H), 4.66−4.55 (m, 2H), 4.19 (s, 2H), 4.17−4.11 (m, 1H), 4.10−4.04
(m, 1H), 3.96 (dd, J = 5.9, 4.2 Hz, 2H), 3.71 (dd, J = 11.2 Hz, 7.5,
4H). 13C NMR (126 MHz, DMSO-d6) δ 166.41, 160.66, 158.80,
153.68, 149.21, 147.34, 144.58, 138.79, 138.07, 127.14, 122.14, 118.93,
118.84, 115.70, 114.67, 74.38, 68.18, 66.28, 63.90, 53.35, 49.46, 41.85.
MS (EI) m/z: 501 (M)+. HRMS (EI): Anal. Calcd for C22H20ClN5O7,
501.1051; found, 501.1059.
5-Chloro-N-(((3S,3aR)-1-oxo-7-(2-oxopiperidin-1-yl)-
1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]thiazin-3-yl)-
methyl)thiophene-2-carboxamide (47b). Compound 47b (59
mg, 82.3%) was prepared from 46b (50 mg, 0.15 mmol) and 5-
chlorothiophene-2-carboxylic acid (29 mg, 0.18 mmol) in the same
1
manner as that described for 11a. H NMR (500 MHz, DMSO-d6) δ
8.99 (t, J = 5.9 Hz, 1H), 7.91 (d, J = 8.9 Hz, 1H), 7.70 (d, J = 4.1 Hz,
1H), 7.22 (d, J = 4.0 Hz, 1H), 7.15 (d, J = 2.3 Hz, 1H), 7.03 (dd, J =
8.9, 2.3 Hz, 1H), 4.58 (dd, J = 10.2, 5.4 Hz, 1H), 4.10 (ddd, J = 9.4,
5.4, 4.0 Hz, 1H), 3.76−3.65 (m, 2H), 3.55 (t, J = 5.5 Hz, 2H), 3.34−
3.25 (m, 2H), 2.36 (t, J = 6.3 Hz, 2H), 1.89−1.77 (m, 4H). 13C NMR
(126 MHz, DMSO-d6) δ 169.31, 161.28, 154.04, 139.92, 138.83,
133.79, 130.68, 128.98, 128.70, 124.62, 123.32, 122.33, 121.03, 77.11,
55.56, 51.26, 41.85, 33.02, 28.21, 23.40, 21.33. MS (EI) m/z: 477
(M+). HRMS (EI): Anal. Calcd for C21H20S2ClN3O4, 477.0584; found,
477.0589.
N1-(4-Chlorophenyl)-N2-(((3S,3aS)-1-oxo-7-(3-oxomorpholi-
no)-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-
yl)methyl)oxalamide (28c). Compound 28c (68 mg, 72.2%) was
prepared from 10a (60 mg, 0.19 mmol) and 2-((4-chlorophenyl)-
amino)-2-oxoacetic acid (45 mg, 0.22 mmol) in the same manner as
4-Chloro-N-(((3S,3aR)-1-oxo-7-(3-oxomorpholino)-1,3,3a,4-
tetrahydrobenzo[b]oxazolo[3,4-d][1,4]thiazin-3-yl)methyl)-
benzamide (47c). Compound 47c (63 mg, 89.2%) was prepared
from 46a (50 mg, 0.15 mmol) and 4-chlorobenzoic acid (28 mg, 0.18
1
1
that described for 11a. H NMR (300 MHz, DMSO-d6) δ 10.88 (s,
mmol) in the same manner as that described for 11a. H NMR (500
1H), 9.34 (s, 1H), 7.93−7.83 (m, 3H), 7.44 (d, J = 8.7 Hz, 2H), 7.10−
7.00 (m, 2H), 4.60 (dd, J = 14.2, 8.4 Hz, 2H), 4.19 (s, 3H), 4.11−3.91
MHz, DMSO-d6) δ 8.95 (t, J = 5.8 Hz, 1H), 7.93 (d, J = 8.9 Hz, 1H),
7.89−7.84 (m, 2H), 7.59−7.54 (m, 2H), 7.28 (d, J = 2.4 Hz, 1H), 7.14
7787
dx.doi.org/10.1021/jm501045e | J. Med. Chem. 2014, 57, 7770−7791

Journal of Medicinal Chemistry
Article
(dd, J = 8.9, 2.4 Hz, 1H), 4.60 (q, J = 5.1 Hz, 1H), 4.17 (s, 2H), 4.14
(ddd, J = 9.3, 5.3, 4.0 Hz, 1H), 3.94 (dd, J = 5.7, 4.4 Hz, 2H), 3.73 (t, J
= 5.4 Hz, 2H), 3.70−3.65 (m, 2H), 3.30 (dd, J = 6.4, 4.3 Hz, 2H). 13C
NMR (126 MHz, DMSO-d6) δ 166.52, 166.46, 154.12, 137.86, 136.75,
133.17, 130.99, 129.73 (overlap), 128.95 (overlap), 123.84, 122.64,
122.52, 121.13, 77.14, 68.15, 63.88, 55.57, 49.38, 42.01, 28.25. MS
(EI) m/z: 473 (M+). HRMS (EI): Anal. Calcd for C22H20SClN3O5,
473.0812; found, 473.0798.
7.74 (td, J = 7.5, 1.3 Hz, 1H), 7.69 (d, J = 4.1 Hz, 1H), 7.66 (dd, J =
7.8, 1.2 Hz, 1H), 7.38 (dd, J = 7.5, 1.1 Hz, 1H), 7.23 (d, J = 2.0 Hz,
1H), 7.20 (d, J = 4.0 Hz, 1H), 7.15 (dd, J = 8.5, 2.1 Hz, 1H), 4.58 (dd,
J = 10.5, 5.7 Hz, 1H), 4.20−4.11 (m, 1H), 3.77−3.65 (m, 2H), 3.35
(d, J = 12.5 Hz, 2H), 2.88 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ
161.28, 154.01, 140.23, 139.62, 138.82, 134.70, 133.78, 133.20, 132.66,
132.49, 131.90, 128.93, 128.68, 128.50, 127.98, 126.86, 121.57, 119.77,
77.15, 55.68, 44.22, 41.81, 28.09. MS (EI) m/z: 534 (M+). HRMS
(EI): Anal. Calcd for C23H19S3ClN2O5, 534.0145; found, 534.0152.
4-Chloro-N-(((3S,3aR)-7-(2-(methylsulfonyl)phenyl)-1-oxo-
1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]thiazin-3-yl)-
methyl)benzamide (56b). Compound 56b (75 mg, 71.0%) was
prepared from 55b (100 mg, 0.20 mmol) and 1-bromo-2-
(methylsulfonyl)benzene (56 mg, 0.24 mmol) in the same manner
as that described for 25a. 1H NMR (500 MHz, DMSO-d6) δ 8.95 (t, J
= 5.8 Hz, 1H), 8.06 (dd, J = 8.0, 1.2 Hz, 1H), 8.01 (d, J = 8.5 Hz, 1H),
7.89−7.85 (m, 2H), 7.74 (td, J = 7.5, 1.4 Hz, 1H), 7.68−7.64 (m, 1H),
7.58−7.54 (m, 2H), 7.38 (dd, J = 7.6, 1.2 Hz, 1H), 7.23 (d, J = 2.0 Hz,
1H), 7.15 (dd, J = 8.6, 2.1 Hz, 1H), 4.61 (dd, J = 10.6, 5.3 Hz, 2H),
4.24−4.16 (m, 2H), 3.74 (dd, J = 8.7, 5.3 Hz, 2H), 2.88 (s, 3H). 13C
NMR (126 MHz, DMSO-d6) δ 166.49, 154.09, 140.24, 139.61, 136.75,
134.72, 133.78, 133.21, 133.17, 132.66, 129.73 (overlap), 128.93
(overlap), 128.50, 127.96, 126.85, 121.63, 119.89, 77.13, 55.70, 44.23,
41.99, 28.19. MS (EI) m/z: 528 (M+). HRMS (EI): Anal. Calcd for
C23H19S3ClN2O5, 528.0580; found, 528.0580.
5-Chloro-N-(((3S,3aS)-1-oxo-7-(3-oxomorpholino)-3,3a,4,5-
tetrahydro-1H-oxazolo[3,4-a]quinolin-3-yl)methyl)thiophene-
2-carboxamide (72a). Compound 72a (88 mg, 80.6%) was prepared
from 71a (75 mg, 0.24 mmol) and 5-chlorothiophene-2-carboxylic
acid (46 mg, 0.28 mmol) in the same manner as that described for
11a. 1H NMR (500 MHz, DMSO-d6) δ 8.99 (t, J = 5.8 Hz, 1H), 8.10−
8.05 (m, 1H), 7.70 (d, J = 4.1 Hz, 1H), 7.19 (dd, J = 7.1, 3.0 Hz, 3H),
4.47 (dt, J = 8.2, 5.2 Hz, 1H), 4.16 (s, 2H), 3.94 (t, J = 5.0 Hz, 3H),
3.66 (dt, J = 6.7, 3.3 Hz, 4H), 2.88−2.82 (m, 2H), 2.18−2.11 (m, 1H),
1.78 (dt, J = 21.9, 10.5 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ
166.37, 161.15, 153.70, 138.89, 136.94, 133.76, 133.56, 128.94, 128.67,
126.94, 126.24, 124.34, 117.74, 78.18, 68.16, 63.92, 57.21, 49.57,
41.45, 26.62, 25.26. MS (EI) m/z: 461 (M+). HRMS (EI): Anal. Calcd
for C21H20SClN3O5, 461.0812; found, 461.0804.
5-Chloro-N-(((3S,3aR)-5-oxido-1-oxo-7-(3-oxomorpholino)-
1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]thiazin-3-yl)-
methyl)thiophene-2-carboxamide (48). To a cooled (0 °C)
solution of 47a (20 mg, 0.042 mmol) in THF (5 mL) was added
mCPBA (75%, 9.6 mg, 0.042 mmol). The solution was stirred at 0 °C
for 1 h, and then ethyl acetate was added. The organics were washed
with saturated sodium thiosulfate and a 10% NaOH aqueous solution,
dried over anhydrous Na2SO4, and filtered, and then the filtrate was
concentrated in vacuo. The residue was purified by chromatography
on silica gel with dichloromethane/methanol (30:1) to afford 48 (19
1
mg, 91.9%) as a white solid. H NMR (500 MHz, DMSO-d6) δ 9.05
(t, J = 5.7 Hz, 1H), 8.39 (d, J = 9.1 Hz, 1H), 7.95 (d, J = 2.5 Hz, 1H),
7.71 (d, J = 4.1 Hz, 1H), 7.67 (dd, J = 9.1, 2.5 Hz, 1H), 7.19 (d, J = 4.1
Hz, 1H), 4.72 (dt, J = 7.4, 5.0 Hz, 1H), 4.55 (ddd, J = 12.4, 7.5, 2.0 Hz,
1H), 4.21 (s, 2H), 3.97 (dd, J = 5.8, 4.3 Hz, 2H), 3.80−3.71 (m, 4H),
3.50 (dd, J = 13.9, 2.0 Hz, 1H), 3.39−3.35 (m, 1H). 13C NMR (126
MHz, DMSO-d6) δ 166.67, 161.26, 153.23, 138.82, 137.20, 133.79,
132.08, 130.83, 129.67, 129.05, 128.66, 127.70, 119.33, 76.72, 68.15,
63.87, 49.27, 47.49, 45.33, 41.28. MS (ESI) m/z: 518.1 (M + 23)+.
HRMS (ESI): Anal. Calcd for C20H18O6N3ClNaS2, 518.0218; found,
518.0221.
5-Chloro-N-(((3S,3aR)-5,5-dioxido-1-oxo-7-(3-oxomorpholi-
no)-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]thiazin-3-
yl)methyl)thiophene-2-carboxamide (49a). To a cooled (0 °C)
solution of 47a (30 mg, 0.062 mmol) in THF (5 mL) was added
mCPBA (75%, 36 mg, 0.16 mmol). The solution was stirred at 0 °C
for 1 h, and then ethyl acetate was added. The organics were washed
with saturated sodium thiosulfate and a 10% NaOH aqueous solution,
dried over anhydrous Na2SO4, and filtered, and then the filtrate was
concentrated in vacuo. The residue was purified by chromatography
on silica gel with dichloromethane/methanol (30:1) to afford 49a (31
1
5-Chloro-N-(((3S,3aS)-1-oxo-7-(2-oxopiperidin-1-yl)-3,3a,4,5-
tetrahydro-1H-oxazolo[3,4-a]quinolin-3-yl)methyl)thiophene-
2-carboxamide (72b). Compound 72b (120 mg, 82.3%) was
prepared from 71b (100 mg, 0.32 mmol) and 5-chlorothiophene-2-
carboxylic acid (62 mg, 0.38 mmol) in the same manner as that
mg, 96.9%) as a white solid. H NMR (500 MHz, DMSO-d6) δ 8.99
(t, J = 5.7 Hz, 1H), 8.31 (d, J = 9.2 Hz, 1H), 7.96 (d, J = 2.5 Hz, 1H),
7.74−7.68 (m, 2H), 7.21 (d, J = 4.1 Hz, 1H), 4.74 (ddd, J = 11.8, 7.3,
2.6 Hz, 1H), 4.71−4.65 (m, 1H), 4.22 (s, 2H), 4.20−4.08 (m, 2H),
3.98 (t, J = 5.0 Hz, 2H), 3.82−3.71 (m, 4H). 13C NMR (126 MHz,
DMSO-d6) δ 166.79, 161.31, 153.06, 138.78, 137.70, 133.81, 132.07,
130.88, 129.07, 128.66, 128.07, 120.82, 119.82, 76.41, 68.13, 63.82,
55.76, 50.62, 49.15, 40.97. MS (ESI) m/z: 534.1 (M + 23)+. HRMS
(ESI): Anal. Calcd for C20H18O7N3ClNaS2, 534.0167; found,
534.0169.
5-Chloro-N-(((3S,3aR)-5,5-dioxido-1-oxo-7-(2-oxopiperidin-
1-yl)-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]thiazin-3-
yl)methyl)thiophene-2-carboxamide (49b). Compound 49b (30
mg, 93.7%) was prepared from 47b (30 mg, 0.063 mmol) and mCPBA
(75%, 32 mg, 0.14 mmol) in the same manner as that described for
49a. 1H NMR (500 MHz, DMSO-d6) δ 9.01 (t, J = 5.8 Hz, 1H), 8.28
(d, J = 9.1 Hz, 1H), 7.80 (d, J = 2.5 Hz, 1H), 7.72 (d, J = 4.1 Hz, 1H),
7.60 (dd, J = 9.1, 2.5 Hz, 1H), 7.22 (d, J = 4.1 Hz, 1H), 4.74 (ddd, J =
11.9, 7.3, 2.6 Hz, 1H), 4.70−4.66 (m, 1H), 4.20−4.08 (m, 2H), 3.82−
3.74 (m, 2H), 3.62 (td, J = 11.6, 5.8 Hz, 2H), 2.41 (t, J = 6.3 Hz, 2H),
1.92−1.80 (m, 4H). 13C NMR (126 MHz, DMSO-d6) δ 169.70,
161.32, 153.09, 139.77, 138.80, 133.82, 131.95, 131.82, 129.08, 128.69,
128.00, 121.54, 119.76, 76.38, 55.76, 51.05, 50.66, 40.99, 33.03, 23.32,
21.23. MS (ESI) m/z: 532.1 (M + 23)+. HRMS (ESI): Anal. Calcd for
C21H20O6N3ClNaS2, 532.0374; found, 532.0379.
1
described for 11a. H NMR (500 MHz, DMSO-d6) δ 8.99 (t, J = 5.8
Hz, 1H), 8.07−8.02 (m, 1H), 7.70 (d, J = 4.1 Hz, 1H), 7.19 (d, J = 4.1
Hz, 1H), 7.09−7.05 (m, 2H), 4.47 (dt, J = 8.1, 5.2 Hz, 1H), 3.94 (ddd,
J = 11.1, 8.2, 2.6 Hz, 1H), 3.70−3.62 (m, 2H), 3.57−3.49 (m, 2H),
2.88−2.79 (m, 2H), 2.34 (t, J = 6.3 Hz, 2H), 2.17−2.09 (m, 1H),
1.88−1.72 (m, 5H). 13C NMR (126 MHz, DMSO-d6) δ 169.19,
161.14, 153.68, 139.02, 138.90, 133.75, 133.22, 128.93, 128.66, 127.63,
126.08, 125.00, 117.70, 78.13, 57.22, 51.43, 41.47, 33.00, 26.59, 25.35,
23.46, 21.39. MS (EI) m/z: 459 (M+). HRMS (EI): Anal. Calcd for
C22H22SClN3O4, 459.1020; found, 459.1017.
tert-Butyl 3-((5-chlorothiophene-2-carboxamido)methyl)-1-
oxo-7-(2-oxopiperidin −1-yl)-3a,4-dihydro-1H-oxazolo[3,4-a]-
quinoxaline-5(3H)-carboxylate (84b). Compound 84b (0.26 g,
83.9%) was prepared from 83b (0.23 g, 0.55 mmol) and 5-
chlorothiophene-2-carboxylic acid (0.11 g, 0.68 mmol) in the same
1
manner as that described for 11a. H NMR (600 MHz, DMSO-d6) δ
9.03 (t, J = 5.8 Hz, 1H), 8.10 (d, J = 8.9 Hz, 1H), 7.72 (d, J = 4.1 Hz,
1H), 7.60 (d, J = 1.5 Hz, 1H), 7.22 (d, J = 4.1 Hz, 1H), 7.04 (dd, J =
8.9, 2.4 Hz, 1H), 4.62 (dt, J = 7.6, 5.3 Hz, 1H), 4.54 (dd, J = 12.8, 3.0
Hz, 1H), 3.94 (ddd, J = 10.7, 7.7, 3.2 Hz, 1H), 3.77−3.70 (m, 2H),
3.55 (dq, J = 24.3, 5.9 Hz, 2H), 3.19 (dd, J = 12.7, 10.6 Hz, 1H), 2.37
(t, J = 6.4 Hz, 2H), 1.85 (ddd, J = 17.7, 7.5, 5.0 Hz, 4H), 1.45 (s, 9H).
13C NMR (151 MHz, DMSO-d6) δ 169.23, 161.33, 153.81, 152.20,
138.74, 138.67, 133.88, 129.04, 128.68, 127.16, 125.26, 122.69, 122.54,
117.70, 82.05, 75.43, 55.38, 51.55, 41.39, 32.99, 31.17, 28.16 (overlap),
23.46, 21.39. MS (ESI) m/z: 583.0 (M + 23)+. HRMS (ESI): Anal.
Calcd for C26H30SClN4O6, 561.1569; found, 561.1570.
5-Chloro-N-(((3S,3aR)-7-(2-(methylsulfonyl)phenyl)-1-oxo-
1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]thiazin-3-yl)-
methyl)thiophene-2-carboxamide (56a). Compound 56a (56 mg,
66.3%) was prepared from 55a (80 mg, 0.16 mmol) and 1-bromo-2-
(methylsulfonyl)benzene (45 mg, 0.19 mmol) in the same manner as
that described for 25a. 1H NMR (500 MHz, DMSO-d6) δ 8.98 (t, J =
5.9 Hz, 1H), 8.06 (dd, J = 8.0, 1.2 Hz, 1H), 8.03 (d, J = 8.6 Hz, 1H),
7788
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Journal of Medicinal Chemistry
Article
5-Chloro-N-((1-oxo-7-(3-oxomorpholino)-3,3a,4,5-tetrahy-
dro-1H-oxazolo[3,4-a]quinoxalin-3-yl)methyl)thiophene-2-car-
boxamide (85a). To a solution of 84a (0.53 g, 0.94mmol) in DCM
(5 mL) was added TFA (0.3 mL). The solution was stirred at room
temperature for 2 h and then evaporated under reduced pressure. The
residue was purified by chromatography on silica gel with dichloro-
methane/methanol (30:1) to give 85a (0.36 g, 82.6%) as a white solid.
1H NMR (500 MHz, DMSO-d6) δ 8.98 (t, J = 5.9 Hz, 1H), 7.70 (d, J
= 4.1 Hz, 1H), 7.64 (d, J = 8.6 Hz, 1H), 7.19 (d, J = 4.0 Hz, 1H), 6.65
(d, J = 2.3 Hz, 1H), 6.56 (dd, J = 8.6, 2.3 Hz, 1H), 6.34 (d, J = 4.0 Hz,
1H), 4.50 (dd, J = 11.6, 5.3 Hz, 1H), 4.14 (s, 2H), 3.95−3.90 (m, 2H),
3.84 (ddd, J = 9.8, 6.6, 3.3 Hz, 1H), 3.71−3.65 (m, 2H), 3.63−3.59
(m, 2H), 3.53 (dt, J = 7.5, 4.0 Hz, 1H), 3.10−3.02 (m, 1H). 13C NMR
(126 MHz, DMSO-d6) δ 166.17, 161.25, 154.05, 138.83, 138.35,
136.65, 133.80, 128.96, 128.68, 120.00, 119.37, 113.49, 112.08, 76.07,
68.17, 63.92, 54.10, 49.66, 43.30, 41.87. MS (EI) m/z: 462 (M+).
HRMS (EI): Anal. Calcd for C20H19SClN4O5, 462.0765; found,
462.0767.
5-Chloro-N-((1-oxo-7-(2-oxopiperidin-1-yl)-3,3a,4,5-tetrahy-
dro-1H-oxazolo[3,4-a]quinoxalin-3-yl)methyl)thiophene-2-car-
boxamide (85b). Compound 85b (0.18 g, 95.3%) was prepared from
84b (0.23 g, 0.41 mmol) in the same manner as that described for 85a.
1H NMR (600 MHz, DMSO-d6) δ 9.00 (t, J = 5.9 Hz, 1H), 7.71 (d, J
= 4.1 Hz, 1H), 7.62 (d, J = 8.6 Hz, 1H), 7.21 (d, J = 4.0 Hz, 1H), 6.54
(d, J = 2.2 Hz, 1H), 6.47 (dd, J = 8.6, 2.2 Hz, 1H), 6.28 (d, J = 3.8 Hz,
1H), 4.52 (dd, J = 11.6, 5.4 Hz, 1H), 3.85 (ddd, J = 9.8, 6.6, 3.3 Hz,
1H), 3.69 (td, J = 5.7, 2.3 Hz, 2H), 3.54 (dt, J = 11.0, 3.6 Hz, 1H),
3.50 (t, J = 5.6 Hz, 2H), 3.10−3.05 (m, 1H), 2.34 (t, J = 6.4 Hz, 2H),
1.82 (dt, J = 10.9, 7.1 Hz, 4H). 13C NMR (151 MHz, DMSO-d6) δ
168.97, 161.26, 154.07, 140.45, 138.85, 136.61, 133.80, 128.98, 128.68,
119.75, 119.34, 114.40, 112.84, 76.03, 54.17, 51.54, 43.40, 41.91,
33.04, 23.50, 21.40. MS (EI) m/z: 460 (M+). HRMS (EI): Anal. Calcd
for C21H21SClN4O4, 460.0972; found, 460.0988.
AV-SHUNT Model. An arteriovenous (AV) shunt in anesthetized
rats (n = 10 per dose group) was undertaken as described previously
with slight modifications.8,26 The left jugular vein and right carotid
artery were each cannulated with 8 cm polyethylene tubing. A saline-
filled arteriovenous shunt was assembled by inserting a 6 cm long
polyethylene tube containing 4-0 silk thread (60 × 0.15 mm) between
the jugular and carotid cannulas. 11a or rivaroxaban suspended in 0.5%
carboxymethylcellulose sodium (CMC-Na) solution or vehicle was
given via the oral route 60 min before the shunt was opened for 15
min. The thread with its associated thrombus was withdrawn, cleaned
in saline, blotted dry, and weighed. The thrombus weight formed on
the thread was calculated by subtracting the average weight of the silk
thread. The protocol for this study was reviewed and approved by the
Animal Care and Use Committee of China Pharmaceutical University
(Nanjing, China).
Arterial Thrombosis Model. Antithrombotic activity in a model of
arterial thrombosis in anesthetized rats was determined as described
previously with minor modifications.27 11a or rivaroxaban dissolved in
PEG400/ethanol/H2O [40:10:50 (v/v/v)] or vehicle was given via the
oral route 45 min before the induction of anesthesia. The left carotid
artery was exposed and fitted on the proximal end with a stimulating
electrode of an experimental intracorporeal thrombosis surveyor
(BT87-4; BaoTou Medical College, Inner Mongolia, China). Sixty
minutes after oral administration of the test compound, electrical
stimulation (3 mA) was started and continued for 2 min. After the end
of stimulation, we removed the electrode and fitted a temperature
probe on the distal end of the artery. The time taken for thrombus
formation was recorded as long as the stimulation began and lasted
until the alarm started. If compounds displayed antithrombotic effects,
the thrombus-formation time would be prolonged compared with that
of the control. The protocol for this study was reviewed and approved
by the Animal Care and Use Committee of Peking University (Beijing,
China).
In Vitro Enzyme Assays..8,25 Enzymatic activity against human
FXa and related serine proteases (FIIa, FVIIa, FIXa, FXIa, and trypsin)
was measured using chromogenic substrates. Reactions were initiated
by addition of different substrates (200 μM of L-pyro-Glu-Pro-Arg-
pNA, S-2366 for FIIa; 2 mM of S-2366 for FVIIa and FXIa; 3.2 mM of
CH3SO2-(D)CHG-Gly-Arg-pNA, Biophen-CS51 for FIXa; 300 μM of
N-Z-D-Arg-Gly-Arg-pNA, S-2765 for FXa and trypsin) to the wells of
96-well microplates to which a premixed and preincubated enzyme/
inhibitor mixture was added. Generation of pNA product was
monitored continuously at 405 nm using a microplate reader
(FlexStation 3; Molecular Devices, Silicon Valley, CA, USA) for 20
min at 37 °C. Percent inhibition and IC50 were calculated by nonlinear
regression using GraphPad v5.0.
Tail-Bleeding Model. Bleeding risk was determined in anesthetized
rats as described previously with minor modifications.8 11a or
rivaroxaban suspended in 0.5% carboxymethylcellulose sodium
(CMC-Na) solution or vehicle was given via the oral route 60 min
before the tails of anesthetized rats were transected 4 mm from the tip
and immersed vertically immediately into saline at 37 °C. The time
until continuous blood flow ceased for >30 s was recorded with a
maximum observation time of 30 min (longer bleeding times were
assigned a value of 30 min). The protocol for this study was approved
by the Animal Care and Use Committee of China Pharmaceutical
University (Nanjing, China).
Pharmacokinetic Studies. Compounds 11a, 11b, 25a, 25c, 47a,
49a, 72a, and 85a (5% DMSO + 5% Tween-80 in 90% saline) and
rivaroxaban (10% DMSO + 10% Tween-80 + 80% PEG400 (40%
solution in saline)) were subjected to PK studies on male Sprague−
Dawley rats (200−220 g) with four animals in each group. Test
compounds were administered via the oral route at 10 and 9 mg/kg or
administered via the intravenous route at 3 and 4.5 mg/kg. Serial
specimens (0.3 mL) were collected via the retrobulbar vein and
quantified by liquid chromatography−mass spectrometry (LC-MS).
PK parameters were calculated from the mean plasma concentration
by noncompartmental analyses. The protocol for this study was
reviewed and approved by the Institutional Animal Care and Use
Committee of the Shanghai Institute of Materia Medica, Chinese
Academy of Sciences (Shanghai, China).
In male dogs, 11a (5% DMSO + 5% Tween-80 in 90% saline) and
rivaroxaban (10% DMSO + 10% Tween-80 + 80% PEG400) were
administered via the oral route at 1 mg/kg and intravenous infusion at
0.5 mg/kg with three dogs in each group. Blood samples (1 mL) were
collected via the foreleg and quantified by LC-MS. PK parameters
were calculated from the mean plasma concentration by non-
compartmental analyses. The protocol for this study was reviewed
and approved by the Institutional Animal Care and Use Committee of
C&O Pharmaceutical Technology (Holdings) Limited (Nanjing,
China).
In Vitro Coagulation Assays.8 Prothrombin time (PT) and
activated partial thromboplastin time (aPTT) were measured using
commercially available kits. Compounds 11a, 11b, 25a, 25c, 26a, 47a,
47b, 49a, 72a, and 85a and rivaroxaban or DMSO (3 μL) were added
to 100 μL citrated rabbit plasma [3.8% sodium citrate, 10:90 (v/v)]
and preincubated for 10 min at 37 °C. Clotting times were measured
using a coagulometer (C2000-1 Single Channel Coagulation Analyzer;
Precil, Beijing, China) in accordance with the manufacturer’s
instructions. Anticoagulant activity was defined as the concentration
required to double the plasma clotting times [CT2 (μM)].
In Vivo Antithrombotic Efficacy Assays. Venous Thrombosis
Model. Thrombosis formation was induced in anesthetized rats (n =
10 per dose group) as described previously with slight modifica-
tions.8,26 The vena cava was isolated through a midline abdominal
incision, and the surface was cleared by blunt dissection between the
renal and iliolumbar veins. 11a or rivaroxaban suspended in 0.5%
carboxymethylcellulose sodium (CMC-Na) solution or vehicle was
given via the oral route 60 min before thrombus formation. A strip of
filter paper saturated with 20% FeCl3 in water was placed on the vena
cava for 1 min. Sixty minutes after application of the filter paper, the
vena cava was dissected free. The thrombus was removed, cleaned in
saline, blotted dry, and weighed. The protocol for this study was
reviewed and approved by the Animal Care and Use Committee of
China Pharmaceutical University (Nanjing, China).
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Journal of Medicinal Chemistry
Article
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Schlemmer, K.-H.; Reinemer, P.; Perzborn, E. Discovery of the novel
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4-yl)phenyl]-1,3-oxazolidin-5-yl}methyl)thiophene-2-carboxamide
(BAY 59−7939): an oral, direct factor Xa inhibitor. J. Med. Chem.
2005, 48, 5900−5908.
ASSOCIATED CONTENT
* Supporting Information
Synthesis and characterization of all intermediates. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
S
(7) Perzborn, E.; Roehrig, S.; Straub, A.; Kubitza, D.; Misselwitz, F.
The discovery and development of rivaroxaban, an oral, direct factor
Xa inhibitor. Nat. Rev. Drug Discovery 2011, 10, 61−75.
AUTHOR INFORMATION
Corresponding Authors
*(B.G.) Tel: 86-21-5080 6600-3405. Fax: 86-21-5080 6786. E-
mail: hemera@163.com.
*(Y.Y.) Tel: 86-21-5080 6786. Fax: 86-21-5080 6786. E-mail:
ysyang@mail.shcnc.ac.cn.
■
(8) Perzborn, E.; Strassburger, J.; Wilmen, A.; Pohlmann, J.; Roehrig,
S.; Schlemmer, K. H.; Straub, A. In vitro and in vivo studies of the
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highly selective factor Xa inhibitor: in vitro, antithrombotic and
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Author Contributions
∥Tao Xue and Shi Ding contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants from the National Natural
Science Foundation of China (no. 21372236), the National
Science and Technology Major Project “Key New Drug
C r e a t i o n a n d Ma n u f a c t u r i n g P r o g r a m ” ( n o .
2012ZX09301001-001), and the Science and Technology
Commission of Shanghai Municipality (no. 13431900402).
ABBREVIATIONS USED
■
VT, venous thrombosis; AV-SHUNT, arteriovenous thrombo-
sis; SAR, structure−activity relationship; SPR, structure−
pharmacokinetic relationship; hERG, human ether-a-go-go
related gene; CYP450, cytochrome P450; DTT, 1,4-dithio-
threitol; TBSCl, tert-butyldimethylsilyl chloride; CbzCl, benzyl
chloroformate; HATU, o-(7-azabenzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium hexafluorophosphate
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