Identification of Novel Coumestan Derivatives as Polyketide Synthase 13 Inhibitors against Mycobacterium tuberculosis. Part II

Article abstract of DOI:10.1021/acs.jmedchem.9b00010

Our group recently reported the identification of novel coumestan derivatives as Mycobacterium tuberculosis (Mtb) Pks13-thioesterase (TE) domain inhibitors, with mutations observed (D1644G and N1640K) in the generated coumestan-resistant Mtb colonies. Herein, we report a further structure-activity relationships exploration exploiting the available Pks13-TE X-ray co-crystal structure that resulted in the discovery of extremely potent coumestan analogues 48 and 50. These molecules possess excellent anti-tuberculosis activity against both the drug-susceptible (MIC = 0.0039 μg/mL) and drug-resistant Mtb strains (MIC = 0.0078 μg/mL). Moreover, the excellent in vitro activity is translated to the in vivo mouse serum inhibitory titration assay, with administration of coumestan 48 at 100 mg/kg showing an 8-fold higher activity than that of isoniazid or TAM16 given at 10 or 100 mg/kg, respectively. Preliminary ADME-Tox data for the coumestans were promising and, coupled with the practicality of synthesis, warrant further in vivo efficacy assessments of the coumestan derivatives.

Full text of DOI:10.1021/acs.jmedchem.9b00010

Subscriber access provided by UNIV OF CAMBRIDGE
Article
Identification of Novel Coumestan Derivatives as Polyketide
Synthase 13 Inhibitors against Mycobacterium tuberculosis. Part II
Wei Zhang, Shichun Lun, Ling-Ling Liu, Shiqi Xiao, Guanfu Duan, Hendra
Gunosewoyo, Fan Yang, Jie Tang, William R. Bishai, and Li-Fang Yu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00010 • Publication Date (Web): 15 Mar 2019
Downloaded from http://pubs.acs.org on March 17, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 52
Journal of Medicinal Chemistry
1
2
3
Identification of Novel Coumestan Derivatives as Polyketide Synthase 13 Inhibitors
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
against Mycobacterium tuberculosis. Part II
Wei Zhang,†, # Shichun Lun,§, # Ling-ling Liu, Shiqi Xiao, Guanfu Duan, Hendra Gunosewoyo, Fan
†
§
†
┴
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Yang,*, † Jie Tang, William R. Bishai,

*, §
and Li-Fang Yu*, †
†Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development,
School of Chemistry and Molecular Engineering, East China Normal University, 3663 North
Zhongshan Road, Shanghai 200062, China
§Center for Tuberculosis Research, Department of Medicine, Division of Infectious Disease, Johns
Hopkins School of Medicine, Baltimore, Maryland 21231-1044, United States
┴School of Pharmacy and Biomedical Sciences, Faculty of Health Sciences, Curtin University, Bentley,
Perth, WA 6102, Australia

Shanghai Key Laboratory of Green Chemistry and Chemical Process, School of Chemistry and Molecular
Engineering, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China
ABSTRACT
Our group recently reported the identification of novel coumestan derivatives as Mycobaterium
tuberculosis Pks13 thioesterase (TE) domain inhibitors, with mutations observed (D1644G and N1640K)
in the generated coumestan-resistant Mtb colonies. Herein, we report a further SAR exploration exploiting
the available Pks13 TE X-ray co-crystal structure that resulted in the discovery of extremely potent
coumestan analogues 48 and 50. These molecules possess excellent anti-TB activity against both the drug
susceptible (MIC = 0.0039 μg/mL) and drug resistant Mtb strains (MIC = 0.0078 μg/mL). Moreover, the
excellent in vitro activity is translated to the in vivo mouse serum inhibitory titration (SIT) assay, with
administration of coumestan 48 given at 100 mg/kg showing an 8-fold higher activity than isoniazid or
ACS Paragon Plus Environment
1

Journal of Medicinal Chemistry
Page 2 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
TAM 16 given at 10 or 100 mg/kg, respectively. Preliminary ADME-Tox data for the coumestans were
promising, and coupled with the practicality of synthesis, warrant further in vivo efficacy assessments of
the coumestan derivatives.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
INTRODUCTION
Tuberculosis (TB) is an age-old infectious disease caused by Mycobacterium tuberculosis (Mtb), that
1
typically affects the lungs (pulmonary TB). TB is responsible for ill-health with approximately 10 million
2
incidence each year and is one of the top ten causes of death worldwide. According to the 2018 World
Health Organization (WHO) report, there were 10.0 million people suffering from TB worldwide in 2017,
accompanied with 1.6 million deaths, including 0.3 million co-infected with HIV. It is estimated that 40%
of HIV deaths were associated with TB, making it the leading infectious disease killer in HIV-positive
people. Of alarming global concern, the emergence of drug-resistant Mtb remains a public health crisis.
Approximately 558,000 new cases with resistance to rifampicin were reported, 82% of which had
multidrug-resistant (MDR) TB and 8.5% of MDR-TB classified as extensively drug-resistant (XDR) TB.3
The currently recommended treatment for drug-susceptible TB is a 6-month regimen containing four first-
line drugs: isoniazid, rifampicin, ethambutol, and pyrazinamide. Although treatment success rates of
>
85% is seen for cases of drug-susceptible TB, the front-line anti-TB drugs are still limited in terms of
4
available options and they need to be delivered in long treatment course. These observations, coupled
with patient non-compliance and over 50 years of overuse, render the development of drug-resistant Mtb
more problematic. Treatment for rifampicin-resistant TB (RR-TB), MDR-TB, and XDR-TB is longer (>20
months), and requires combination therapy with second-line drugs which are generally associated with
5
poor curative effect, higher toxicity, more expensive, and difficulty in administration. Only in the past
ACS Paragon Plus Environment
2

Page 3 of 52
Journal of Medicinal Chemistry
1
2
3
6
7
five years, two new anti-TB drugs, bedaquiline (1) and delamanid (2, Figure 1), are approved as
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
combination therapy against MDR-TB and XDR-TB infections when other effective treatment regimen is
8
deemed not responsive. However, their clinical use has been linked with various adverse side effects
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
especially irregular heart rhythms.9, 10 Therefore, it is imperative that the discovery and development of
safe, highly effective new anti-TB chemotherapeutics with novel mechanisms of action continue to push
forward.
O
O
O
OH
N
N
OCF3
F
F
F
F
O
NH
O
Br
O
N
S
F
N
N
O
2
O N
Bedaquiline, 1
Delamanid, 2
TP2, 3
O
O
O
OEt
O
O
5
F
F
F
F
F
F
F
H
N
NH
O
N
NH
N
S
F
7
H2N
S
O
F
F
Et
O
O
TP4, 4
5
EZ120, 6
N
O
N
O
N
O
O
NH
O
HO
HO
HO
OH
O
O
O
TAM1,7
TAM16,8
9
Figure 1. Chemical structures of bedaquiline (1), delamanid (2), thiophene-based Pks13 inhibitors (3–5),
β-lactones-based Pks13 inhibitor EZ120P (6), benzofuran-based Pks13 inhibitor TAM1 (7) and TAM16
(8), and coumestan-based Pks13 inhibitor (9).
Mycolic acids are major and specific lipid components of the mycobacterial cell envelope and are essential
for the survival of Mtb. Its biosynthesis occurs through the coordination of more than 20 enzymes.11, 12
The decades of anti-TB drug development have proven that the mycolic acid biosynthesis is a rich source
ACS Paragon Plus Environment
3

Journal of Medicinal Chemistry
Page 4 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
of druggable targets.13-16 Briefly, the biosynthesis of mycolic acids can be categorized into five distinct
steps, involving the initial syntheses of C -C saturated α-alkyl branch by FAS-I and C -C
60
2
4
26
50
1
7
meromycolate chain by FAS-II, followed by the subsequent incorporation of double bonds, cyclopropyl
and keto functionalities, before the final Pks13-catalyzed condensation18 of the two chains and
translocation19, 20 of the mature mycolic acid building blocks into the periplasm. Isoniazid (INH),
ethionamide (ETH), and delamanid all target various enzymatic machineries responsible for the mycolic
acid biosynthesis pathway. Of increasing interest in the past five years, the polyketide synthase 13 (Pks13),
a member of the type-I PKS family, was identified as the key enzyme for the final assembly step of mycolic
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
2
1
acid synthesis via Claisen-type condensation. Several studies have shown the importance of Pks13 for
mycobacterial viability,22, 23 and recently Pks13 inhibitors have been reported in succession.
The Alland group reported a class of thiophene compounds with whole-cell activity against Mtb, with the
most active compound, TP2 (3, MIC = 1.0 μM) and TP4 (4, MIC = 0.5 μM), when combined with isoniazid
24
exhibited sterilization of Mtb activity. These thiophene compounds were subsequently identified to
target Pks13 by interfering with the function of the N-ACP domain of Pks13, thus blocking the loading of
the meromycolyl-AMPs onto the P-pant attachment site (Ser-55) of Pks13 (Figure 1).24 The 2-
aminothiophenes-based compound 5 (Figure 1) with potency against Mtb H37Rv (MIC = 0.23 μM) was
2
5
also reported by the Sucheck group. However, the thiophene ring containing an ester group is prone to
hydrolysis, likely suggesting a metabolic liability. More recently, β-lactone EZ120 (Figure 1, compound
6
, MIC = 0.68 g/mL) was reported to effectively mimic mycolic acid and block serine hydrolases via
acylation of the active site located in the Pks13-thioesterase (TE) domain, thus exhibiting anti-
2
6
mycobacterial and bactericidal activity. Utilizing an activity-based protein profiling (ABPP) approach,
ser/Pks13 and ser/Ag85 enzymes were verified as major targets.26
ACS Paragon Plus Environment
4

Page 5 of 52
Journal of Medicinal Chemistry
1
2
3
From high-throughput screening, benzofuran derivative TAM1 (Figure 1, compound 7, MIC = 1.0 g/mL)
was active against Mtb H37Rv and identified to also target Pks13 through whole-genome sequencing and
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
7
recombineering of the resistance mutations. The mutations observed (D1644G and D1607N) in the
resistant colonies were located in the C-terminus of the TE domain of Pks13 which is in accordance with
the obtained X-ray co-crystal structure. However, TAM1 bearing an ester group is prone to hydrolysis to
the inactive carboxylic acid in mouse serum. To circumvent this issue, bioisosteric replacement of the
ester with amide strategy had significantly improved the metabolic stability. Benzofuran derivative
TAM16 (Figure 1, compound 8, MIC = 0.0313 g/mL) was efficacious in both acute and chronic mouse
TB infection models and has favorable drug-like properties, pharmacokinetic and safety profiles.28
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In our own previous work, coumestan was identified as a natural scaffold that showed antitubercular
2
9
activity in vitro and oral bioavailability in a mouse model of serum inhibition titration (SIT) assay. Whole
genome deep sequencing of the resistant mutants confirmed that these coumestans target the Pks13 as
shown by the N1640K, N1640S, D1644G, and A1667V mutations observed in the resistant mutants.
Herein, we report further SAR studies on the development of coumestan analogues, taking into
consideration the structural insights of the Pks13-TE domain. Together with microsomal stability analysis
and preliminarily evaluation for their in vivo bioavailability in the SIT assay head-to-head with compound
8
, herein we report the development of second-generation highly potent anti-TB coumestan analogues.
RESULTS AND DISCUSSION
Chemistry
The coumestan derivatives 12-15 and 17-24 were synthesized in 2-3 steps utilizing the synthetic routes
shown in Scheme 1. Compounds 12-15 were formed starting from the previously reported hydroxyl ester
ACS Paragon Plus Environment
5

Journal of Medicinal Chemistry
Page 6 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
0 29 through demethylation by boron tribromide and the subsequent Mannich reaction with 37% aqueous
formaldehyde and piperidine analogues. The hydroxyl ester 10 was also reacted with 37% aqueous
formaldehyde and a delta lactam using solid acid catalyst-silica-H SO (50% w/w) via Mannich-type
2
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
condensation to obtain compound 16. Compound 16 was demethylated using boron tribromide and
lactonization in refluxing ethanol. Next, alkylation of the phenolic group in the presence of K CO
2
3
afforded compounds 18 and 19. Following a method similar to the synthesis of compounds 17 and 18
utilising various lactams and alkyl iodides, compounds 20-24 were successfully formed starting from
hydroxyl ester 10.
Regioisomers 26 and 27 were obtained via formylation of compound 25 in the presence of dichloromethyl
30
methyl ether and TiCl . Pinnick oxidation of aldehyde 26 afforded the carboxylic acid intermediate
4
which was subsequently reacted with piperidine in the presence of 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDC·HCl) and 1-hydroxybenzotriazole (HOBt) as coupling agents and 3-
methylpyridine as a base to obtain compound 28. Subsequent demethylation by boron tribromide and
cyclization of the ester afforded compound 29, which underwent alkylation to give compound 30. The 6-
carboxaldehyde 27 was subjected to reductive amination using piperidine and sodium cyanoborohydride,
followed by demethylation/lactonization to afford compound 32. Synthesis of compounds 31 and 33 is
similar to compound 29 starting from intermediates 26 and 27.
The coumestan derivatives 34-37, 41-44, 48-51, 55, and 56 were synthesized utilizing the synthetic routes
shown in Scheme 2. Compound 9 underwent nitration or bromination to give 34 or the bromide 35.
Compound 35 next served as an Ullmann-type C-O coupling partner with sodium methoxide. The resultant
compound 36 was then demethylated using boron tribromide to give the hydroxyl compound 37.
ACS Paragon Plus Environment
6

Page 7 of 52
Journal of Medicinal Chemistry
1
2
3
Compound 39 was formed via a copper-catalyzed cyclization of 2-tert-butyl-1,4-benzoquinone and ethyl
-(2-methoxyphenyl)-3-oxopropanoate which was prepared as described in the literature from
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
3
1
commercially available 2-acetoanisole. Standard demethylation/lactonization afforded compound 40,
which then underwent Mannich reaction with 37% aqueous formaldehyde and piperidine to yield
coumestan derivative 41. To access the bis-phenolic analogues, compound 46 was formed via a copper-
catalyzed cyclization of 1,4-benzoquinone and ethyl 3-(2,4-dimethoxyphenyl)-3-oxopropanoate.
Compound 48 was obtained via intermediate 47 through Mannich reaction/demethylation/lactonization
sequence. Coumestan derivatives 42-44, 49-51 were synthesized in a similar manner from commercially
available substituted ethyl 3-(2-methoxyphenyl)-3-oxopropanoate and the substituted 1,4-benzoquinone.
Compounds 55 and 56 were formed via sequential bromination, nucleophilic substitution using sodium
methoxide and boron tribromide demethylation respectively from compounds 52 and 47.
Scheme 1. Synthesis of compounds 10−33 a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment
7

Journal of Medicinal Chemistry
Page 8 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
R1
2
3
n
O
4
N
O
O
O
N
O
HO
b
1
a
O
13 R = 2-CH3
HO
HO
1
1
4 R = 3-CH3
O
O
1
O
15
O
R
= 4-CH3
HO
O
11
12 n = 1
n = 2
9
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
O
10
N
O
N
O
N
O
O
a
O
O
c
d
2
HO
HO
R O
O
O
O
O
18 R2 = CH3
9 R2 = CH CH OCH
3
1
6
17
1
2
2
O
O
O
O
N
O
20 R2 = H
N
O
HO
O
O
21 R2 = CH3
2
R O
HO
2 R2 = CH CH
2
O
2 3
23 R2 = CH CH OCH
O
O
2
2
3
O
24
10
H
O O
N
O O
O
N
O O
O
O
O
O
1
f, g
a
R O
O
O
O
O
O
O
O
2
6
28
29 R1 = H
0 R1 = OCH3
O
e
d
3
O
O
O
O
5
2
O
O
O
H
HO
h, a
O
N
O
O
O
2
7
32
O
O
HN
HO
O O
HO
O
26
27
N
O
O
O
31
33
a
Reagents and conditions: (a) BBr in CH Cl (1 M), CH Cl , rt, overnight, then EtOH, reflux, 1 h; (b)
3
2
2
2
2
formaldehyde (37% aq), corresponding primary amines, EtOH, reflux, 8–12 h; (c) formaldehyde (37%
aq), corresponding lactam, silica−H SO (50% w/w), acetonitrile, 50 °C for 2−5 h; (d)
2
4
CH I/CH OCH CH Br, K CO , THF, rt - 80 ºC, 4–6 h; (e) Cl CHOCH , TiCl , CHCl , rt, 40 min; (f)
3
3
2
2
2
3
2
3
4
3
ACS Paragon Plus Environment
8

Page 9 of 52
Journal of Medicinal Chemistry
1
2
3
NaClO , NH SO H, dioxane/H O (7/3), rt, 1 h; (g) EDC·HCl, HOBt, 3-methylpyridine, piperidine, DMF,
2
2
3
2
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
rt, 8 h; (h) piperidine, CH COOH, CH OH, rt, 10 min then NaBH CN, rt, 16 h.
3
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 2. Synthesis of compounds 34−56 a
N
O
N
O
N
O
N
O
O
O
O
O
HO
a
HO
b
HO
Br
c
HO
R
O
O
O
O
O2N
34
9
35
36 R = OCH3
37 R = OH
d
O
O
O
O
O
O
O
HO
HO
O
e
f
O
O
O
38
39
40
N
O
N
O
O
1
O
42 R = 7-CH3
g
HO
1
HO
43 R = 7-Cl
1
4
4 R = 7-Br
O
6
R1
O
7
41
O
O
N
O
O
O
N
O
O
O
HO
HO
O
e
g
f
HO
O
O
OH
O
O
O
O
O
O
O
45
46
47
48
O
O
R3
N
O
O
O
O
HO
O
49 R1
2
3
R3
R2
= H, R = H, R = OH
e
R2
HO
O
R2
50 R1 = 7- Bu, R = OH, R = H
t
2
3
O
51 R1 = 7- Bu, R = H, R = OH
t
2
3
R1
6
R1
O
O
R3
7
N
O
N
O
R3
N
O
O
O
O
HO
HO
R1
55 R3 = H
6 R3 = OH
R2
b
R2
c, f
HO
HO
OH
5
O
O
O
R3
O
O
2
53 R1 = Br, R2 = Br, R3 = H
54 R1 = Br, R2 = OCH , R3 = Br
5
2 R = Br
2
4
7 R = OCH
3
3
ACS Paragon Plus Environment
9

Journal of Medicinal Chemistry
Page 10 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Reagents and conditions: (a) HNO , AcOH, rt, 1 h; (b) Br , CH Cl , rt, overnight; (c) CuCl, NaOCH ,
3
2
2
2
3
t
DMF, 140 ºC, 1 h; (d) BBr in CH Cl (1 M), CH Cl , rt, overnight; (e) 1,4-benzoquinone or 2- Bu-1,4-
3
2
2
2
2
benzoquinone, Cu(OTf) (5 mol %), toluene, reflux, 7–12 h; (f) BBr in CH Cl (1 M), CH Cl , rt,
2
3
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
overnight, then EtOH, reflux, 1 h; (g) formaldehyde (37% aq), piperidine, EtOH, reflux, 8–12 h.
Structure−Activity Relationships.
All final compounds were initially evaluated for their MIC values against Mtb strain H37Rv in a standard
microplate alamar blue assay (MABA).32, 33 As shown in Table 1, benzofuran TAM16 potently inhibited
Mtb H37Rv with an MIC value of 0.0313 g/mL (0.09 M), consistent with data reported by the
28
1
Sacchettini group. We first examined the substitutions at position 4 of the coumestans (the R group).
Deletion of the (methylpiperidin-1-yl) methyl to give compound 11 resulted in a total loss of activity,
1
indicating the importance of R substitution, which is consistent with the SAR trend observed previously
in the benzofuran series of Pks13 inhibitors. A smaller pyrrolidin-1-yl-methyl (12) and various methyl
1
substituted piperidin-1-yl-methyl substituents at R decreased activity by 2-8 fold compared to compound
1
9
. Shifting the R substituent from position 4 to position 6 (32) resulted in loss of activity (MIC value >
1
64 μg/mL), further supporting the importance of R substitution at position 4 of coumestan.
The coumestan derivatives contain phenolic Mannich base substructure that were reported to be possible
3
4
Pan Assay Interference Compounds (PAINS). However, Sacchettini group reported that no adducts of
benzofuran derivatives were formed in mouse plasma and HLMs upon incubation with glutathione and
methoxylamine, suggesting that the Mannich substructure in benzofuran derivatives does not represent a
2
8
significant false-positive. Initially, deletion or alteration of the Mannich structure was attempted by
replacing the piperidin-1-yl-methyl with lactams (17-24) or amides (28-31 and 33), which are expected to
ACS Paragon Plus Environment
10

Page 11 of 52
Journal of Medicinal Chemistry
1
2
3
slightly lower the clogP while potentially maintaining the hydrogen bonding. However, these compounds
were found to be completely inactive (MIC value > 64 μg/mL), regardless of the ring size (5-7 membered
ring) or if the 5-hydroxyl group was free or masked with various alkyl groups such as methyl, ethyl, or
methoxyethyl. This is presumably due to the loss of intra-molecular hydrogen bond with the carbonyl
oxygen and the inter-molecular hydrogen bond with the side chain oxygen of Asn1640.
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. Antitubercular activity of coumestan derivatives against the M. tuberculosis strain H37Rv a
O
R1
O
3
R O
R2
O
9, 11-15, 17-24, 29-33
Compound
R1
-
R2
-
R3
-
MIC , μg/mL
a
cLogPb
6.53
7
8
1.0
-
-
-
0.0313
2.98
N
9
H
H
H
H
H
H
0.125-0.25
> 64
4.17
3.19
3.60
1
1
1
2
H
N
0.5
N
N
13
H
H
H
H
H
H
1.0
2.0
0.5
4.69
4.69
4.69
1
1
4
5
N
N
1
7
H
H
> 64
2.04
O
ACS Paragon Plus Environment
11

Journal of Medicinal Chemistry
Page 12 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N
18
H
H
H
H
H
H
H
CH3
> 64
2.52
2.41
2.60
3.08
3.08
2.97
3.16
O
N
1
2
2
9
0
1
CH CH OCH
3
> 64
> 64
> 64
>64
2
2
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
H
O
N
O
CH3
N
O
22
CH CH
2
3
N
O
2
3
CH CH OCH
3
> 64
> 64
2
2
N
2
2
4
9
H
H
O
N
N
H
H
H
> 64
> 64
3.21
3.46
O
O
30
CH3
NH
31
32
33
H
H
H
> 64
> 64
> 64
4.00
4.17
3.21
O
N
N
H
H
O
Isoniazidc
-
-
-
-
-
-
-
-
-
0.04
0.125
1
-0.67
4.25
0.12
-0.5
Rifampin
-
-
-
Ethambutol
Levofloxacin
0.25
ACS Paragon Plus Environment
12

Page 13 of 52
Journal of Medicinal Chemistry
1
2
3
Moxifloxacin
-
-
-
-
-
-
-
-
-
-
-
-
0.0625-0.125
-0.08
-3.88
-8.40
-4.12
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Kanamycin
Capreomycin
Amikacin
2
1
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
aSee Experimental Section. The lowest concentration of compounds leading to at least 90% inhibition of
bacterial growth signal by the MABA. MIC values are reported as an average of three individual
b
c
measurements. cLogP was calculated using ChemBioDraw Ultra 14.0. MIC values for reference
compounds isoniazid, rifampin, ethambutol, levofloxacin, moxifloxacin, kanamycin, capreomycin, and
amikacin were obtained from our previous publication.14
Next, keeping the piperidin-1-yl-methyl (position 4) and hydroxyl group (position 5) intact, we then
investigated the SAR at positions 6 and 7 of the coumestan derivative (Table 2). It is suspected that the
hydroxyl groups play significant roles for the observed pharmacological activities of coumestans such as
those found in the naturally occurring coumestans, examplified by coumestrol, wedelolactone, and
plicadin.35-37 We speculated that an introduction of additional hydroxyl group to the phenyl rings would
possibly increase the antitubercular activity. Initially, hydroxyl group along with other electron-donating
t
groups (EDGs; CH , Bu, OCH ) and various electron-withdrawing groups (EWGs; F, Br, Cl, NO ) were
3
3
2
examined. Consistent with the SAR in Table 1, substituents at position 6 were generally found to be
detrimental to the activity, as seen in the nitro (34) and bromo compounds (35) that were completely
inactive (MIC value > 64 μg/mL). EDG substitutions (36 and 37) had at least 16-32 fold increase in activity
with an MIC value of 2-4 μg/mL, whereas in contrast, EDGs substituents (41, 42) at position 7 favored
the antitubercular activity with MIC values of 0.125 μg/mL and 0.0625-0.125 μg/mL, which are very
similar to the parent coumestan 9. Halogen substituents (43 and 44), however, resulted in 16-fold reduction
ACS Paragon Plus Environment
13

Journal of Medicinal Chemistry
Page 14 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
of antitubercular activity (MIC = 2 μg/mL), suggesting the importance of EDG at this position. Molecular
docking studies suggested that the substituents at 6-position may interfere with Arg1641 resulting in
t
unfavorable ligand binding (data not shown), while CH and Bu substituents at 7-position (41 and 42)
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
seem to accommodate well with the adjacent amino acid Tyr1637.
2
Investigating the right-hand side benzene ring of the coumestans (R substitutions), we further probed
positions 4’ or 5’ of with various EDGs and EWGs. In our previous findings, 4’-Br, 4’-OCH , 4’-F, 5’-
3
2
9
CH , 5’-OCH were reported to possess MIC values ranging from 0.25 to 4 μg/mL. We found that
3
3
monosubstitutions on the right-hand side benzene ring of the coumestans with EDG and EWG were
generally tolerated. Utilizing the structural information obtained from the co-crystal structure of the
Pks13-TE-TAM16 complex and the vastly improved activity of TAM16, we speculated that an
introduction of hydroxyl group to the phenyl ring would increase the anti-TB activity, likely through an
optimal hydrogen bond to the carboxylate group of D1644. To our delight, introduction of hydroxyl group
at the 4’-position to give compound 48 resulted in an extremely potent analogue with an MIC value of
0
.0039 μg/mL, whereas a hydroxyl walk to the 5’-position (49) resulted in significantly decreased MIC
value of 0.5 μg/mL. Similarly, the tert-butyl analogue 50 showed similar activities with 48, whereas 51
showed a 4-fold increase in activity compared to 49. Introduction of poly-hydroxyl group on the benzene
ring of the coumestans to give compounds 55 and 56 resulted in reduction of activity with MIC values of
2
and 32 μg/mL.
Bactericidal activity of 48 on Mtb H37Rv was evaluated by minimum bactericidal concentration assay
(MBC) using broth dilution and colony forming unit determination methods. The minimum concentration
ACS Paragon Plus Environment
14

Page 15 of 52
Journal of Medicinal Chemistry
1
2
3
that killed 99% of the inoculum was 0.0039-0.0078 μg/mL, demonstrating 48 to be a potent bactericidal
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
compound for Mtb.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Antitubercular activity of coumestan derivatives against the M. tuberculosis strain H37Rv. a
N
O
O
HO
4'
R
2
6
O
R1
5'
7
3
4-37, 41-44,
48-51, 55, 56
Compound R1
R2
MIC , μg/mL cLogP
a
b
3
3
4
5
6-NO2
6-Br
H
H
H
H
H
H
H
H
> 64
> 64
2.0
4.36
4.86
3.87
3.63
36
6-OCH3
6-OH
3
4
7
1
4.0
t
7- Bu
0.0625-0.125 5.94
42
7-CH3
7-Cl
7-Br
H
0.125
2.0
4.67
4.99
5.14
4.11
4.11
5.89
5.89
3.52
3.08
4
4
3
4
2.0
48
4’-OH
5’-OH
4’-OH
5’-OH
4’-OH
0.0039
0.5
4
5
5
5
9
0
1
5
H
t
7- Bu
0.0039
0.125
2.0
t
7- Bu
6-OH
6-OH
56
4’,5’-di-OH 32
ACS Paragon Plus Environment
15

Journal of Medicinal Chemistry
Page 16 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
aSee Experimental Section. The lowest concentration of compounds leading to at least 90% inhibition of
bacterial growth signal by the MABA. MIC values are reported as an average of three individual
b
measurements. cLogP was calculated using ChemBioDraw Ultra 14.0.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Molecular Docking
For the molecular docking of compounds 9 and 48, we used the solved crystal structures of Pks13-TE
(PDB ID 5V3Y). The protein structure was prepared by Protein Preparation Wizard. In this step, missing
atoms and all hydrogen atoms were added to the protein according to their local environment. All
compounds were flexibly docked into the binding pocket defined by residues Q1633, F1670, Y1674, and
N1640 using Glide with XP scoring function. Top scored poses were used for visualization analysis. Pks13
interactions with each compound were pictured using Pymol. All the molecular docking work was
performed using the Maestro software.
The proposed binding modes are shown in Figure 2, demonstrating the aminophenol part of coumestan
binding favorably to the TE domain and the coumarin portion relatively exposed to the solvent. Interaction
of the 4’-hydroxyl group with the side chain amide of Q1633 is proposed to be responsible for the
significant improvement in the MIC value. This is also consistent with the proposed binding mode of the
compound 8 (TAM16).28
Figure 2. Proposed binding modes of compounds 9 (colored cyan) (A) and 48 (colored cyan) (B). The
key amino acid residues are colored green in the active site of Pks13 (PDB ID 5V3Y). Relative Binding
Affinity Estimated by Glide-XP scoring Function. 9 (-8.7), 48 (-10.7).
ACS Paragon Plus Environment
16

Page 17 of 52
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Encouraged by the vast improvement of the MIC values of our most potent coumestans (48 and 50) as
well as their bactericidal activity thus far, their activities against clinical drug sensitive (DS, V4207),
3
8
MDR-TB (KZN494 and V2475), and XDR-TB (TF274 and R506) strains (Table 3) were evaluated. The
MDR and XDR strains are resistant to the first-line anti-TB drugs isoniazid and rifampin, and second-line
anti-TB drugs levofloxacin, ofloxacin and kanamycin were used as positive controls. Again, to our delight,
coumestans 48 and 50 demonstrated potent activities against susceptible clinical isolates of Mtb (MICs
0.0039-0.0078 μg/mL) as well as all the MDR and XDR-TB strains examined.
Table 3. Antitubercular activity of compounds 48 and 50 against susceptible, MDR, and XDR strains of
M. tuberculosis.
a
Mtb MIC (μg/mL)
strain/phenotype V4207/DSb
V2475/MDRc KZN494/MDR TF274/XDRd R506/XDR
Isoniazid
Rifampin
0.04
16
4
16
8
8
0.0625
128
>128
>128
ACS Paragon Plus Environment
17

Journal of Medicinal Chemistry
Page 18 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Levofloxacin
Ofloxacin
0.125
0.5
NTe
0.5
NT
1
2
0.5
8
4
Kanamycin
2
2
2
>128
0.0078
0.0039
>128
0.0078
0.0039
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
5
8
0
0.0078
0.0156
0.0039
0.0039-0.0078 0.0156
0.0010-0.0020
aThe lowest concentration of drug leading to at least a 90% reduction of bacterial growth signal by
microplate alamar blue assay (MABA); MIC values are reported as an average of three individual
measurements. MIC values for reference compounds isoniazid, rifampin, levofloxacin, ofloxacin, and
kanamycin were obtained from our previous publication.39 bDrug susceptible strain of M. tuberculosis.
c
d
Multidrug resistant strain of M. tuberculosis, resistance to isoniazid and rifampin. Extensively drug
resistant strain of M. tuberculosis, resistance to isoniazid, rifampin, levofloxacin, ofloxacin, and
e
kanamycin. NT = not tested.
The follow-up investigation was then to examine if these compounds possess improved selectivity index
(
SI) against normal cells by examining their cytotoxicity profiles. In our previous work, we have evaluated
the potential cytotoxicity of the coumestans on the human-derived cell lines and found that the coumestans
29
were not cytotoxic to these at concentrations of more than 500-fold their MIC values for Mtb. As seen
in Tables 4 and 5, the cytotoxicity of both coumestans 48 and 50 were tested against Vero cells as well as
4 human derived cell lines. Gratifyingly, coumestans 48 and 50 were non-cytotoxic to these mammalian
cells at concentrations of more than 1000-fold their MIC values for Mtb, unlike the cytotoxic profile
towards breast cancer cells displayed by wedelolactone for example.40, 41
Table 4. Vero toxicity and selectivity index (SI) of selected compounds.
ACS Paragon Plus Environment
18

Page 19 of 52
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Toxicity to Vero cells MIC
IC , μg/mL μg/mL
H37Rv,
Compd
SI a
5
0
12
8
0.5
16
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
3
5
16
16
16
8
1
16
0.5
32
41
0.0625-0.125
0.125
0.0039
0.5
128-256
64
4
4
2
8
4
1024
8
49
4
5
5
0
1
8
0.0039
0.125
2048
64
8
a
SI = IC50 (Vero cells) / MIC (H37Rv)
Table 5. Toxicity of selected coumestans 48 and 50 in 4 human-derived cell lines.
IC50 a, M
Compound MRC-5 cells HFL-1 cells
QSG-7701 cells
HEK-293 cells
> 50
33.6 ±3.4
> 50
> 50
40.1±3.5
4
5
8
0
30.6 ±4.5 22.3 ± 3.9
34.1 ±1.7
a
IC values were obtained as an average from three independent experiments and are expressed in M.
5
0
To further probe their microbial selectivity, coumestans 48 and 50 were tested in a panel of gram positive
and gram negative bacteria. As shown in Table 6, no appreciable inhibition of growth was observed in the
ACS Paragon Plus Environment
19

Journal of Medicinal Chemistry
Page 20 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
tested strains, suggesting that these coumestans were selective against actinobacteria and development
of potential drug resistance to nonpathogenic bacteria is unlikely.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 6. Assessment of coumestans 48 and 50 against a panel of gram positive and gram negative bacteria.
Compounds
MIC, μg/mL
S. aureus Newman B. subtilis 168
E. coli AB1157
> 25
E. coli DH5α
> 25
Vancomycin
Tetracycline
Kanamycin
0.39-0.78
0.2-0.39
2.34-4.68
> 25
> 25
> 25
1.56-3.13
9.39-18.75
> 25
0.78-1.56
2.34-4.68
> 25
9.39-18.75
> 25
4
5
8
0
6.25-12.5
> 25
> 25
> 25
Microsomal stability profile of the coumestan 48 was improved compared to the benzofuran counterpart
(Table 7), with a half-life of 120 min. Introduction of hydroxyl group at the 4-position therefore not only
9
resulted in drastic improvement of anti-TB potency but also improved metabolic stability.
Table 7. Microsomal stability of compounds 57, 58, 9, and 48 on human liver microsomes.
N
O
N
O
O
O
HO
HO
R
R
O
O
57 R = H
8 R = OH
9 R = H
48 R = OH
5
Compound T1/2a, min CLint
mL/min/kg
(mic)b, CLint
mL/min/kg
(liver)c, Remaining (T = 60 min)
ACS Paragon Plus Environment
20

Page 21 of 52
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
7 d
8 d
5
18.8
53.0
21.4
120.1
73.7
26.2
64.9
11.5
69.8
116.4
184.5
66.3
23.6
58.4
10.4
62.8
104.8
10.8%
5
46.6%
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
9
4
14.5%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
73.6%
Testosterone 19.9
Diclofenac 11.9
12.5%
3.2%
Propafenone 7.5
166.1
0.4%
a
b
b
T1/2 is half life, CL
is the intrinsic clearance. CL
= 0.693/half life/mg microsome protein
int (mic)
int (mic)
c
per mL. CL
= CLint(mic) * mg microsomal protein/g liver weight * g liver weight/kg body weight;
int(liver)
d
mg microsomal protein /g liver weight: 45 mg/g; liver weight: 20 g/kg. See reference 28 and 29 for
synthetic details.
Serum Inhibition Titration (SIT) Assay.
Selected coumestans 48 and 50, as well as benzofuran 8 for head-to-head comparison, were evaluated for
3
3
their in vivo bioavailability in the serum inhibition titration (SIT) assay. Carboxymethylcellulose (0.5%)
was used as vehicle and isoniazid included as a positive control. In this assay, BALB/c mice were orally
gavaged with 100 mg/kg (carboxymethylcellulose as vehicle) and 50 mg/kg (Propylene glycol and Tween
8
0 at 4:1 as vehicle) of compounds 48, 50, or 10 mg/kg of INH, with blood collected at 30 min. The sera
were then separated and prepared in 2-fold dilutions and incubated with a bacterial suspension for 7 days.
Growth inhibition of serially diluted serum on H37Rv was determined using the microplate alamar Blue
assay. As shown in Figure 3, compound 8 (TAM16, 100 mg/kg) displayed a very similar profile to
isoniazid (10 mg/kg). On the other hand, coumestan 48 showed improved bioavailability up to 8-fold
higher than that of TAM16 when tested at a dose of 100 mg/kg. The tert-butyl analogue 50 was
ACS Paragon Plus Environment
21

Journal of Medicinal Chemistry
Page 22 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
significantly less effective in this assay, most likely due to increased lipophilicity (clogP difference of 1.8)
by introduction of tert-butyl group on the benzene ring. Administration of the coumestan 48 at 50 mg/kg
using PG ether still maintained inhibitory activity up to 1:1024 dilution, but this was not the case for the
tert-butyl analogue 50.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
0000
0000
5
Vehicle
INH_CMC_10
40000
8_CMC_100
4
8_CMC_100
3
0000
0000
50_CMC_100
48_PG_50
2
50_PG_50
10000
0
4
8
6
2
4
8
6
2
4
8
6
2
9
:
:
1
3
6
2
5
1
2
4
9
1
1
:
:
:
1
2
5
0
0
0
1
8
1
1
1
:
:
:
1
2
4
1
1
1
:
:
:
:
1
1
1
1
Serum titer
Figure 3. Serum inhibition titration for compounds 8, 48, and 50.
Mannich base is essential for
activity, piperidine ring is best,
amides lost activity.
Lactone is beneficial to potency
and metabolic stability compared
to the open ester.
N
O
H-bond donor
O
Monosubstitutions are
generally tolerated,
contributes to
activity.
HO
B
4
A
7
4-position favorable for
6
O
substitution, OH is best
6-Position unfavorable
for substitution, EWGs
cause inactivity
EDGs substituents at
-position are favorable
7
Figure 4. SAR summary on the coumestans.
Conclusions
In summary, rational SAR studies on the coumestan series (Figure 4) as mycobacterial Pks13 inhibitors
resulted in the discovery of highly potent compound 48, with MIC value of 0.0039 μg/mL. Preliminary
ADME-Tox evaluation of this compound revealed favorable human microsomal stability and selectivity
against normal cells as shown in the cytotoxicity data. More importantly, coumestan 48 maintained potent
ACS Paragon Plus Environment
22

Page 23 of 52
Journal of Medicinal Chemistry
1
2
3
activities against the clinical isolates of MDR and XDR-TB and showed excellent microbial selectivity to
nonpathogenic bacteria strains. Furthermore, compound 48 is orally bioavailable as shown by the serum
inhibition titration assay. In the SIT assay, coumestan 48 given at 100 mg/kg showed 8-fold higher activity
than INH or 8 given at 10 or 100 mg/kg, respectively. Taken together, these data strongly suggest the
translational utility of coumestan-based Pks13 inhibitors as novel anti-TB medications.
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Experimental Section
General Methods: Starting materials, reagents, and solvents were purchased from commercial suppliers
and used without further purification, unless otherwise stated. Anhydrous CH Cl and tetrahydrofuran
2
2
(THF) were obtained by distillation over calcium hydride or sodium wire, respectively. All non-aqueous
reactions were run under a nitrogen atmosphere with exclusion of moisture from reagents. The progress
of reactions was monitored by liquid chromatography mass spectrometry (LCMS) and thin layer
chromatography (TLC) on SiO . Silica gel for column chromatography (CC) was of 200-300 mesh particle
2
size, and an EtOAc/petroleum ether mixture or gradient was used unless stated otherwise. High resolution
1
mass spectra (HRMS) were performed using a Bruker ESI-TOF high-resolution mass spectrometer. H
1
3
NMR spectra were recorded at a spectrometer frequency of 400 MHz, and C NMR spectra were recorded
at 101 MHz. Chemical shifts were reported in δ (ppm) using the δ 0 signal of tetramethylsilane (TMS), δ
7
.26 signal of CDCl , δ 3.31 signal of CD OD or δ 2.50 signal of (CD ) SO as internal standards. Purities
3 3 3 2
of all final compounds were established by analytical HPLC (> 95%), which was carried out on a Waters
HPLC system using InertSustain-C18 column (5micron, 250×4.6 mm) with detection at 280, 254, and 220
nm on a variable wavelength detector 2998 PDA. InertSustain-C18 column, flow rate = 1.0 mL/min,
gradient of 30-95% acetonitrile in water or 10-95% acetonitrile in water (containing 0.05 vol% of HCO H)
2
ACS Paragon Plus Environment
23

Journal of Medicinal Chemistry
Page 24 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
over 25 min. Melting points and analytical HPLC purities for all final compounds were included in the
Supporting Information.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
General procedure for the synthesis of substituted 5-hydroxy-2-phenyl benzofurans from
substituted ethyl benzoylacetates (method A) were reported previsouly. 29
General procedure for the Mannich reaction of hydroxy-2-phenyl benzofuran or coumestan with
amine and formaldehyde (method B) were reported previsouly. 29
General procedure for the preparation of coumestan derivatives from substituted ethyl 5-hydroxy-
2
-(2-methoxyphenyl)benzofuran-3-carboxylate or ethyl 2-(2,4-dimethoxyphenyl)-5-hydroxy-4-
(
piperidin-1-ylmethyl)benzofuran-3-carboxylate (method C) were reported previously.29
General procedure for the synthesis of Mannich-type products from ethyl 5-hydroxy-2-(2-
methoxyphenyl)benzofuran-3-carboxylate (method D). To a solution of ethyl 5-hydroxy-2-(2-
methoxyphenyl)benzofuran-3-carboxylate (1 mmol) in acetonitrile (5 mL) was added formaldehyde (3
mmol), lactam (1.5 mmol) and silica-H SO (50% w/w). Reaction mixture was then stirred at 50 °C for
2
4
2
-5 h. Reaction mixture was cooled to rt and filtered. Filtrate was evaporated. The residue was purified by
flash chromatography to get Mannich-type products.
8
-Hydroxy-7-(pyrrolidin-1-ylmethyl)-6H-benzofuro[3,2-c]chromen-6-one (12). This compound was
1
obtained from 11 and pyrrolidine employing method B. Yield 71%; pale yellow solid. H NMR (400 MHz,
CDCl /CD OD = 2/1) δ 8.02 (d, J = 7.8 Hz, 1H), 7.65 (t, J = 7.8 Hz, 1H), 7.58 (d, J = 8.9 Hz, 1H), 7.50-
3
3
ACS Paragon Plus Environment
24

Page 25 of 52
Journal of Medicinal Chemistry
1
2
3
13
7
.42 (m, 2H), 7.10 (d, J = 8.9 Hz, 1H), 5.03 (s, 2H), 3.38 (s, 4H), 2.11 (s, 4H). C NMR (101 MHz,
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CDCl /CD OD = 2/1) δ 162.1, 160.0, 155.8, 153.9, 150.0, 133.2, 125.7, 124.6, 122.6, 117.6, 116.2, 114.7,
3
3
+
112.6, 110.5, 106.2, 54.2, 52.2, 23.5. HRMS (ESI) m/z: Calcd for C H NO (M+H) 336.1230, found
2
0
18
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
36.1217.
8
-Hydroxy-7-((2-methylpiperidin-1-yl)methyl)-6H-benzofuro[3,2-c]chromen-6-one
(13).
This
compound was obtained from 11 and 2-methylpiperidine employing method B. Yield 61%; white solid.
1
H NMR (400 MHz, DMSO-d ) δ 8.05 (d, J = 8.0 Hz, 1H), 7.72 (t, J = 7.1 Hz, 1H), 7.63-7.56 (m, 2H),
6
7
.50 (t, J = 7.5 Hz, 1H), 6.88 (d, J = 8.9 Hz, 1H), 4.77-4.64 (m, 2H), 2.83 (d, J = 12.4 Hz, 1H), 2.68 (br,
1
3
1H), 2.34 (t, J = 9.2 Hz, 1H), 1.82-1.28 (m, 6H), 1.16 (d, J = 6.1 Hz, 3H). C NMR (101 MHz, DMSO-
d6) δ 160.2, 157.3, 156.8, 152.8, 148.6, 132.4, 124.9, 121.9, 121.8, 116.7, 116.0, 115.0, 111.7, 111.0,
+
1
05.4, 55.4, 53.7, 50.4, 33.0, 25.1, 21.9, 16.9. HRMS (ESI) m/z: Calcd for C H NO (M+H) 364.1543,
2
2
22
4
found 364.1538.
8
-Hydroxy-7-((3-methylpiperidin-1-yl)methyl)-6H-benzofuro[3,2-c]chromen-6-one
(14).
This
compound was obtained from 11 and 3-methylpiperidine employing method B. Yield 61%; pale yellow
1
solid. H NMR (400 MHz, DMSO-d ) δ 8.05 (d, J = 8.0 Hz, 1H), 7.72 (t, J = 7.3 Hz, 1H), 7.63-7.57 (m,
6
2
H), 7.49 (t, J = 7.5 Hz, 1H), 6.92 (d, J = 8.9 Hz, 1H), 4.63-4.52 (m, 2H), 2.89 (br, 2H), 2.14 (t, J = 11.0
Hz, 1H), 1.87 (t, J = 10.6 Hz, 1H), 1.68 (br, 3H), 1.54-1.44 (m, 1H), 1.02-0.93 (m, 1H), 0.85 (d, J = 6.1
1
3
Hz, 3H). C NMR (101 MHz, DMSO-d ) δ 160.3, 157.2, 156.5, 152.8, 148.6, 132.4, 124.9, 122.3, 121.8,
6
1
16.7, 115.9, 114.4, 111.7, 111.2, 105.4, 60.1, 57.1, 52.6, 31.9, 30.6, 24.7, 19.1. HRMS (ESI) m/z: Calcd
+
for C H NO (M+H) 364.1543, found 364.1550.
2
2
22
4
ACS Paragon Plus Environment
25

Journal of Medicinal Chemistry
Page 26 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
8
-Hydroxy-7-((4-methylpiperidin-1-yl)methyl)-6H-benzofuro[3,2-c]chromen-6-one
(15).
This
compound was obtained from 11 and 4-methylpiperidine employing method B. Yield 89%; pale yellow
1
solid. H NMR (400 MHz, CDCl /CD OD = 3/1) δ 7.96 (d, J = 7.7 Hz, 1H), 7.56 (t, J = 7.5 Hz, 1H), 7.47-
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
.28 (m, 3H), 6.89 (d, J = 8.8 Hz, 1H), 4.64 (s, 2H), 2.98 (d, J = 10.5 Hz, 2H), 2.29 (t, J = 10.3 Hz, 2H),
1
3
1
.66 (d, J = 13.0 Hz, 2H), 1.44 (s, 1H), 1.30-1.20 (m, 2H), 0.91 (d, J = 6.2 Hz, 3H). C NMR (101 MHz,
CDCl /CD OD = 3/1) δ 161.0, 158.8, 157.0, 153.4, 149.6, 132.0, 124.8, 122.6, 122.0, 117.0, 116.5, 114.8,
3
3
+
1
3
12.5, 111.2, 106.0, 58.0, 53.1, 34.1, 30.4, 21.6. HRMS (ESI) m/z: Calcd for C H NO (M+H)
22 22 4
64.1543, found 364.1532.
Ethyl 5-hydroxy-2-(2-methoxyphenyl)-4-((2-oxopyrrolidin-1-yl)methyl)benzofuran-3-carboxylate
(16). This compound was obtained from ethyl 5-hydroxy-2-(2-methoxyphenyl)benzofuran-3-carboxylate
1
and pyrrolidin-2-one employing method D. Yield 24%; light yellow solid. H NMR (400 MHz, CDCl ) δ
3
9.65 (s, 1H), 7.59 (d, J = 7.4 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 7.38 (d, J = 8.8 Hz, 1H), 7.08 (t, J = 7.4
Hz, 1H), 7.02 (d, J = 8.8 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 5.12 (s, 2H), 4.15 (q, J = 7.1 Hz, 2H), 3.81 (s,
1
3
3
H), 3.49 (t, J = 7.1 Hz, 2H), 2.42 (t, J = 8.1 Hz, 2H), 2.03-1.93 (m, 2H), 1.04 (t, J = 7.1 Hz, 3H). C
NMR (101 MHz, CDCl ) δ 177.6, 166.1, 157.1, 156.9, 153.9, 148.8, 131.4, 130.4, 125.9, 120.7, 120.2,
3
1
16.8, 114.5, 112.4 (2), 110.9, 61.0, 55.7, 48.2, 38.4, 30.4, 18.0, 13.8.
1
-((8-Hydroxy-6-oxo-6H-benzofuro[3,2-c]chromen-7-yl)methyl)pyrrolidin-2-one
(17).
This
1
compound was obtained from 16 employing method C. Yield 71%; pale yellow solid. H NMR (400 MHz,
CDCl ) δ 10.06 (s, 1H), 8.04 (d, J = 7.7 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.54-7.48 (m, 2H), 7.43 (t, J =
3
7
.5 Hz, 1H), 7.14 (d, J = 8.9 Hz, 1H), 5.44 (s, 2H), 3.65 (t, J = 7.1 Hz, 2H), 2.45 (t, J = 8.2 Hz, 2H), 2.07-
1
3
1.92 (m, 2H). C NMR (101 MHz, CDCl ) δ 178.0, 161.4, 158.8, 155.4, 153.5, 149.7, 132.3, 124.9, 124.0,
3
ACS Paragon Plus Environment
26

Page 27 of 52
Journal of Medicinal Chemistry
1
2
3
1
22.2, 118.6, 117.2, 115.9, 112.9, 112.6, 106.0, 48.3, 39.6, 30.6, 18.0. HRMS (ESI) m/z: Calcd for
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
C H NNaO (M+Na) 372.0842, found 372.0858.
2
0
15
5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
-((8-Methoxy-6-oxo-6H-benzofuro[3,2-c]chromen-7-yl)methyl)pyrrolidin-2-one (18). To a stirred
solution of 17 (1.0 mmol) and K CO (2.1 mmol) in anhydrous DMF (20 mL) was added CH I (1.2 mmol)
2
3
3
under N . After stirring overnight at rt, the reaction mixture was quenched with H O. The mixture was
2
2
extracted with EtOAc (2×20 mL), and the combined organic phases were washed with water (2×20 mL),
dried over Na SO , and evaporated. The residue was purified by flash chromatography to give the product
2
4
1
1
8. Yield 52%; white solid. H NMR (400 MHz, CDCl ) δ 8.00 (d, J = 7.2 Hz, 1H), 7.64-7.54 (m, 2H),
3
7.47 (d, J = 8.3 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.39(t, J = 7.5 Hz, 1H), 7.05(d, J = 9.0 Hz, 1H), 5.40
13
(
s, 2H), 3.88 (s, 3H), 3.16 (t, J = 7.0 Hz, 2H), 2.37 (t, J = 8.1 Hz, 2H), 1.90-1.78 (m, 2H). C NMR (101
MHz, CDCl ) δ 174.1, 161.8, 158.2, 156.7, 153.8, 150.0, 132.4, 125.3, 124.8, 122.1, 118.9, 117.2, 112.4,
3
+
111.5, 110.9, 106.1, 56.9, 46.94 39.0, 31.3, 17.9. HRMS (ESI) m/z: Calcd for C H NNaO (M+Na)
2
1
17
5
3
86.0999, found 386.0985.
1
-((8-(2-Methoxyethoxy)-6-oxo-6H-benzofuro[3,2-c]chromen-7-yl)methyl)pyrrolidin-2-one (19). To
a stirred solution of 17 (1.0 mmol) and K CO (2.1 mmol) in anhydrous DMF (20 mL) was added 1-
2
3
bromo-2-methoxyethane (1.2 mmol) under N . After stirring 5 h at 80 ℃ , the reaction mixture was
2
quenched with H O. The mixture was extracted with EtOAc (2×20 mL), and the combined organic phases
2
were washed with brine (1×20 mL), dried over Na SO , and evaporated. The residue was purified by flash
2
4
1
chromatography to give the product 19. Yield 51%; white solid. H NMR (400 MHz, CDCl ) δ 8.04 (d, J
3
= 7.8 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.57 (d, J = 9.0 Hz, 1H), 7.49 (d, J = 8.3 Hz, 1H), 7.42 (t, J = 7.6
Hz, 1H), 7.10 (d, J = 9.0 Hz, 1H), 5.44(s, 2H), 4.16 (t, J = 4.8 Hz, 2H), 3.82 (t, J = 4.8 Hz, 2H), 3.47 (s,
ACS Paragon Plus Environment
27

Journal of Medicinal Chemistry
Page 28 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
3
H), 3.19 (t, J = 7.1 Hz, 2H), 2.38 (t, J = 8.1 Hz, 2H), 1.92-1.82 (m, 2H). C NMR (101 MHz, CDCl ) δ
3
1
73.8, 161.6, 158.0, 155.9, 153.6, 150.1, 132.4, 125.2, 124.7, 122.0, 119.3, 117.1, 112.3, 112.2, 111.5,
+
105.9, 71.2, 69.3, 59.2, 46.2, 39.0, 31.2, 17.8. HRMS (ESI) m/z: Calcd for C H NNaO (M+Na)
2
3
21
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
30.1261, found 430.1254.
1
-((8-Hydroxy-6-oxo-6H-benzofuro[3,2-c]chromen-7-yl)methyl)piperidin-2-one
(20).
This
compound was obtained from 10 and piperidin-2-one according to the methodology described for 17.
1
Yield 45%, 92%; white solid. H NMR (400 MHz, CDCl ) δ 8.04 (dd, J = 7.8, 1.0 Hz, 1H), 7.65-7.59 (m,
3
1
H), 7.54-7.47 (m, 2H), 7.42 (t, J = 7.6 Hz, 1H), 7.10 (d, J = 8.9 Hz, 1H), 5.62 (s, 2H), 3.47 (t, J = 5.2 Hz,
1
3
2H), 2.46 (t, J = 6.4 Hz, 2H), 1.81-1.63 (m, 4H). C NMR (101 MHz, CDCl ) δ 172.9, 161.5, 158.8,
3
1
2
55.6, 153.4, 149.4, 132.3, 124.9, 124.6, 122.1, 118.0, 117.2, 115.2, 112.9, 112.5, 106.0, 47.5, 43.3, 31.9,
+
2.8, 20.6. HRMS (ESI) m/z: Calcd for C H NNaO (M+Na) 386.0999, found 386.0980.
2
1
17
5
1
-((8-Methoxy-6-oxo-6H-benzofuro[3,2-c]chromen-7-yl)methyl)piperidin-2-one
(21).
This
compound was obtained from 20 according to the methodology described for 18. Yield 52%; white solid.
1
H NMR (400 MHz, CDCl ) δ 8.02 (d, J = 7.6 Hz, 1H), 7.65-7.54 (m, 2H), 7.47 (d, J = 8.3 Hz, 1H), 7.40
3
(
t, J = 7.5 Hz, 1H), 7.06 (d, J = 8.9 Hz, 1H), 5.60 (s, 2H), 3.89 (s, 3H), 3.16 (t, J = 5.3 Hz, 2H), 2.40 (t, J
1
3
=
5.9 Hz, 2H), 1.77-1.62 (m, 4H). C NMR (101 MHz, CDCl ) δ 169.2, 161.8, 158.2, 156.8, 153.8, 150.1,
3
1
32.4, 126.0, 124.7, 122.1, 120.2, 117.2, 112.5, 111.3, 111.0, 106.4, 56.9, 46.9, 42.9, 32.7, 23.5, 21.5.
+
HRMS (ESI) m/z: Calcd for C H NNaO (M+Na) 400.1155, found 400.1161.
2
2
19
5
1
-((8-Ethoxy-6-oxo-6H-benzofuro[3,2-c]chromen-7-yl)methyl)piperidin-2-one (22). This compound
was obtained from 20 and CH CH I according to the methodology described for 18. Yield 63%; pale
3
2
ACS Paragon Plus Environment
28

Page 29 of 52
Journal of Medicinal Chemistry
1
2
3
1
yellow solid. H NMR (400 MHz, CDCl ) δ 8.01 (d, J = 7.7 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.53 (d, J
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
=
8.9 Hz, 1H), 7.46 (d, J = 8.3 Hz, 1H), 7.39 (t, J = 7.5 Hz, 1H), 7.03 (d, J = 9.0 Hz, 1H), 5.60 (s, 2H),
4.07 (q, J = 6.9 Hz, 2H), 3.15 (t, J = 5.7 Hz, 2H), 2.40 (t, J = 6.4 Hz, 2H), 1.76-1.62 (m, 4H), 1.43 (t, J =
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
6
1
.9 Hz, 3H). C NMR (101 MHz, CDCl ) δ 168.9, 161.7, 158.2, 156.1, 153.8, 150.0, 132.3, 125.9, 124.7,
3
22.1, 120.2, 117.2, 112.5, 111.7, 111.2, 106.4, 65.1, 46.8, 42.9, 32.8, 23.4, 21.6, 15.2. HRMS (ESI) m/z:
+
Calcd for C H NNaO (M+Na) 414.1312, found 414.1319.
23 21
5
1
-((8-(2-Methoxyethoxy)-6-oxo-6H-benzofuro[3,2-c]chromen-7-yl)methyl)piperidin-2-one
(23).
This compound was obtained from 20 according to the methodology described for 19. Yield 45%; white
1
solid. H NMR (400 MHz, CDCl ) δ 7.99 (d, J = 7.7 Hz, 1H), 7.58 (t, J = 7.7 Hz, 1H), 7.52 (d, J = 8.9 Hz,
3
1
H), 7.44 (d, J = 8.3 Hz, 1H), 7.37 (t, J = 7.5 Hz, 1H), 7.05 (d, J = 8.9 Hz, 1H), 5.59 (s, 2H), 4.13 (t, J =
.7 Hz, 2H), 3.78 (t, J = 4.8 Hz, 2H), 3.44 (s, 3H), 3.14 (t, J = 5.7 Hz, 2H), 2.38 (t, J = 6.4 Hz, 2H), 1.77-
4
1
3
1.60 (m, 4H). C NMR (101 MHz, CDCl ) δ 168.9, 161.7, 158.1, 156.1, 153.7, 150.2, 132.3, 125.9, 124.7,
3
1
22.1, 120.5, 117.1, 112.4, 112.3, 111.3, 106.3, 71.3, 69.2, 59.2, 46.7, 42.7, 32.7, 23.4, 21.5. HRMS (ESI)
+
m/z: Calcd for C H NNaO (M+Na) 444.1418, found 444.1433.
2
4
23
6
1
-((8-Hydroxy-6-oxo-6H-benzofuro[3,2-c]chromen-7-yl)methyl)azepan-2-one (24). This compound
1
was obtained from 10 according to the methodology described for 17. Yields 17 and 81%; white solid. H
NMR (400 MHz, CDCl ) δ 10.16 (s, 1H), 8.03 (d, J = 7.7 Hz, 1H), 7.62 (t, J = 7.5 Hz, 1H), 7.52-7.46 (m,
3
2H), 7.42 (t, J = 7.4 Hz, 1H), 7.09 (d, J = 8.9 Hz, 1H), 5.54 (s, 2H), 3.46 (s, 2H), 2.60 (s, 2H), 1.70 (s,
13
4
H), 1.60 (s, 2H). C NMR (101 MHz, CDCl ) δ 178.1, 160.3, 157.8, 154.5, 152.3, 148.3, 131.1, 123.8,
3
1
23.0, 121.0, 116.5, 116.0, 114.4, 111.6, 111.4, 104.7, 47.5, 42.6, 35.4, 28.7, 26.3, 22.2. HRMS (ESI)
+
m/z: Calcd for C H NNaO (M+Na) 400.1155, found 400.1148.
2
2
19
5
ACS Paragon Plus Environment
29