Design and synthesis of mycobacterial pks13 inhibitors: Conformationally rigid tetracyclic molecules

Article abstract of DOI:10.1016/j.ejmech.2021.113202

We previously reported a series of coumestans?a naturally occurring tetracyclic scaffold containing a δ-lactone?that effectively target the thioesterase domain of polyketide synthase 13 (Pks13) in Mycobacterium tuberculosis (Mtb), resulting in superior an

Full text of DOI:10.1016/j.ejmech.2021.113202

European Journal of Medicinal Chemistry 213 (2021) 113202
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design and synthesis of mycobacterial pks13 inhibitors:
Conformationally rigid tetracyclic molecules
,
,
,
Wei Zhang a 1, Ling-ling Liu a 1, Shichun Lun b 1, Shuang-Shuang Wang a, Shiqi Xiao b,
Hendra Gunosewoyo c, Fan Yang a, Jie Tang d, William R. Bishai b **, Li-Fang Yu a
,
, *
a 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
b Center for Tuberculosis Research, Department of Medicine, Division of Infectious Disease, Johns Hopkins School of Medicine, Baltimore, MD, 21231-1044,
United States
c School of Pharmacy and Biomedical Sciences, Faculty of Health Sciences, Curtin University, Bentley, Perth, WA, 6102, Australia
d 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
a r t i c l e i n f o
a b s t r a c t
Article history:
We previously reported a series of coumestansꢀa naturally occurring tetracyclic scaffold containing a
d-
Received 12 August 2020
Received in revised form
3 November 2020
Accepted 12 January 2021
Available online 21 January 2021
lactoneꢀthat effectively target the thioesterase domain of polyketide synthase 13 (Pks13) in Mycobac-
terium tuberculosis (Mtb), resulting in superior anti-tuberculosis (TB) activity. Compared to the corre-
sponding ‘open-form’ ethyl benzofuran-3-carboxylates, the enhanced anti-TB effects seen with the
conformationally restricted coumestan series could be attributed to the extra
p-p stacking interactions
between the benzene ring of coumestans and the phenyl ring of F1670 residue located in the Pks13-TE
binding domain. To further probe this binding feature, novel tetracyclic analogues were synthesized and
evaluated for their anti-TB activity against the Mtb strain H37Rv. Initial comparison of the ‘open-form’
analogueues against the tetracyclic counterparts again showed that the latter is superior in terms of anti-
Keywords:
Polyketide synthase 13
conformationally rigid tetracyclics
Antitubercular agents
TB activity. In particular, the
promising results. Compound 65 demonstrated potent activity against Mtb H37Rv with MIC value be-
tween 0.0313 and 0.0625 g/mL, with high selectivity to Vero cells (64e128 fold). The thermal stability
d-lactam-containing 5H-benzofuro [3,2-c]quinolin-6-ones gave the most
m
analysis supports the notion that the tetracyclic compounds bind to the Pks13-TE domain as measured by
nano DSF, consistent with the observed SAR trends. Compound 65 also showed excellent selectivity
against actinobacteria and therefore unlikely to develop potential drug resistance to nonpathogenic
bacteria.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
in the 2019 World Health Organization Global Tuberculosis Report,
approximately 0.5 million people developed TB in 2018 that were
Tuberculosis (TB) which is caused by Mycobacterium tuberculosis
(Mtb) still poses a major threat to human health. Millions of people
continue to suffer from TB each year worldwide, with cases that can
be found in virtually all countries and age groups. The improper use
of TB drugs in combination with the long treatment regimen has
led to the emergence of drug-resistant strains of Mtb. As presented
resistant to rifampicin (1, Fig. 1), a first-line TB drug widely
considered to be the most effective [1]. Approximately 78% of these
patients had the multidrug-resistant TB (MDR-TB), which is defined
by resistance to at least isoniazid and rifampicin. Moreover, the TB-
HIV co-infection exacerbates the problem, adding a layer of
complexity with respect to designing an effective treatment
regimen that is devoid of unwanted drug-drug interactions. The
development of new drugs for TB has also been slow. For the past
decade, only two new drugs, bedaquiline (2) and delamanid (3)
have been introduced into the market (Fig. 1). Consequently, there
is still an urgent, unmet need for novel, more effective drugs that
are efficacious against drug-resistant strains of Mtb.
* Corresponding author.
** Corresponding author.
E-mail addresses: wbishai1@jhmi.edu (W.R. Bishai), lfyu@sat.ecnu.edu.cn
(L.-F. Yu).
1
Contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2021.113202
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

W. Zhang, L.-l. Liu, S. Lun et al.
European Journal of Medicinal Chemistry 213 (2021) 113202
Abbreviations used
THF
tetrahydrofuran
DMF
NMP
MABA
MIC
EWG
EDG
SI
dimethylformamide
N-methyl pyrrolidinone
TB
tuberculosis
Mycobacterium tuberculosis
multidrug-resistant
N terminus acyl carrier protein
C terminus acyl carrier protein
ketoacyl synthase
Mtb
MDR
N-ACP
C-ACP
KS
microplate alamar blue assay
minimum inhibitory concentration
electron-withdrawing group
electron-donating group
selectivity index
AT
TE
SIT
XDR
SARs
SIT
acyl transferase
thioesterase
serum inhibition titration
extensively drug-resistant
structure-activity relationships
serum inhibition titration
CC
TLC
LCMS
TMS
HRMS
column chromatography
thin layer chromatography
liquid chromatography mass spectrometry
tetramethylsilane
high-resolution mass spectra
nanoDSF Nano differential scanning fluorimetry
Fig. 1. Chemical structures of anti-TB drugs rifampicin, bedaquiline and delamanid; benzofuran-based Pks13 inhibitors TAM1, TAM16, and coumestan 6.
Mycolic acids are the essential building blocks used to assemble
the unique cell wall of Mtb, which is responsible for the pathogen’s
viability and virulence [2]. Inhibition of mycolic acid biosynthesis has
been widely accepted as a highly effective strategy in the anti-TB
drug discovery [3,4], with polyketide synthase 13 (Pks13) recently
emerging as an attractive and viable target. Briefly, Pks13 performs
the Claisen-type condensation step in the mycolic acid synthesis [5],
and this activity has been shown to be essential both in vitro and
in vivo for the bacteria survival [6]. Pks13 is encoded by the FadD32-
Pks13-AccD4 gene cluster and contains 1733 amino acid residues
divided into five distinct domains, including: two acyl carrier protein
domains located at the N terminus (N-ACP) and C terminus (C-ACP), a
ketoacyl synthase (KS), an acyl transferase (AT) and a thioesterase
domain (TE) located at the C terminus [7,8]. There has been an
increasing number of reported small molecule inhibitors for myco-
bacterial Pks13 in recent years [6,9e13]. In 2017, the Sacchettini
group reported the benzofuran compound TAM1 (4, Fig. 1) as an
initial lead molecule, and later solved an X-ray co-crystal structure of
Pks13 complexed with the benzofurans binding to the thioesterase
domain of Pks13 (Pks13-TE) [10]. The more refined compound
TAM16 (5, Fig. 1) emerged through a structure-guided approach as a
highly potent Pks13-TE inhibitor with good pharmacological prop-
erties. At the same time, our own group identified coumestan de-
rivatives represented by compound 6 (Fig. 1), a natural product-
the coumestans may be minimal, a head-to-head comparison
showed that the coumestans possess superior bioavailability in a
mouse serum inhibition titration (SIT) assay [14]. Whole genome
deep sequencing of the wild-type and coumestan-resistant mutants
identified mutation sites A1667V, D1644G, N1640K, and N1640S, all
of which are co-localized within the active site of the Pks13-TE,
consistent with the X-ray co-crystal structure reported by the Sac-
chettini group. Taken together, these observations substantiate
Pks13 as a druggable target and highlight its potential for the
development of new TB drugs.
1.1. Compound design
During our medicinal chemistry campaign on the coumestans,
one prominent trend that we observed was that the coumestans
generally exhibited enhanced anti-TB activity compared to the cor-
responding ethyl benzofuran-3-carboxylates [13,14]. Utilizing a
structure-based drug design approach, it is hypothesized that the
enhanced potency could be attributed to the extra
p-p stacking
between the benzene ring of coumestans and the phenyl ring of
F1670 residue located in the Pks13-TE domain as shown in Fig. 2. The
conformational rigidity brought about by the coumestan is predicted
to enhance the
p-p interactions between the tetracyclic hetero-
aromatic ring structure and the F1670 residue. To verify this hy-
pothesis, we proposed that the coumestan scaffold could be replaced
by other related tetracycles, which are aimed to further enhance the
inspired tetracyclic
d-lactone with modest activity against Mtb
strain H37Rv [13,14]. While the structural change from benzofuran to
2

W. Zhang, L.-l. Liu, S. Lun et al.
European Journal of Medicinal Chemistry 213 (2021) 113202
Fig. 2. Proposed binding modes of compounds 7 (colored cyan, left figure) and 6 (colored cyan, right figure). The key amino acid residues located in the active site of Pks13 (PDB ID
5V3Y) were colored green.
p
-
p
stacking interactions. Consequently, a series of various benzo-
The synthesis of compounds 41 and 45 were shown in Scheme 2.
2-Bromo-5-methoxybenzaldehyde, sulfur powder, and methyl 2-
(2-methoxyphenyl)acetate 36 were refluxed in DMF for 16 h to give
benzothiophene 37. Introduction of an aldehyde group at the 3-
position was achieved using dichloromethyl methyl ether and
TiCl4 to give intermediate 38. Pinnick oxidation afforded the car-
boxylic acid 39, which was subjected to sequential lactonization
and Mannich reaction to give the desired compound 41. Similarly,
compound 45 was synthesized in 6 steps utilizing the commercially
available starting material methyl phenylacetate 42.
thiophene-3-carboxylates, indole-3-carboxylates, benzofuran-3-
carboxylates, N-ethylbenzofuran-3-carboxamides and their corre-
sponding 6H-benzothieno [3,2-c] [1]benzopyran-6-ones [1],benzo-
pyrano [4,3-b]indol-6(11H)-ones, and 5H-benzofuro [3,2-c]quinolin-
6-ones were synthesized and evaluated for their anti-TB activity
against Mtb H37Rv.
2. Results and discussion
The synthesis of 5H-benzofuro [3,2-c]quinolin-6-one ana-
logueues 51e58 and 64e67 were achieved using a different route to
the synthesis of 26 which necessitates high pressure in a closed
vessel. As shown in Scheme 3, substituted o-iodoanilines 46 or 59
were subjected to double Sonogashira coupling employing a
palladium as well as copper co-catalyst to form alkyne 48 or 61.
Following a reported protocol, the 5H-benzofuro [3,2-c]quinolin-6-
one scaffolds were prepared utilizing Cs2CO3 as a source of both
carbonyl (CO) and ethereal oxygen in the presence of 5 mol%
Cu(OAc)2 and 1 equiv. of Ag2CO3 form 49 or 62 [19]. Demethylation
of the methoxy intermediate 49 followed by Mannich reaction gave
the desired compound 51. Whereas compound 62 was deprotected
under acidic conditions and subjected to sequential Mannich re-
action and demethylation to give the desired compound 65. Com-
pounds 52e58 and 66e67 were synthesized in a similar manner to
compounds 51 and 65 respectively. All final compounds (51e58,
66e67) were obtained as hydrochloride salts, except 64 (free
amine) and 65 (hydrobromide salt).
2.1. Chemistry
The synthetic routes for reference compounds 5e12 (structures
shown in Table 1) were described previously [13]. [10] For the syn-
thesis of compound 16 (Scheme 1), the key intermediate ethyl (Z)-3-
amino-3-(2-methoxyphenyl)acrylate 14 was obtained from ester 13
using ammonium formate in refluxing ethanol as described in the
literature [15]. Cyclization of acrylate 14 with 1,4-benzoquinone in
the presence of zinc bromide resulted in the formation of indole
derivative 15. Subsequent lactonization and Mannich reaction with
piperidine and 37% aqueous formaldehyde gave the desired product
16 [16]. The corresponding ‘open-form’ compound 18 was obtained
using ester 17 as the starting material in a similar fashion to the
synthesis of 16. Cyclization of the intermediate ethyl (Z)-3-amino-3-
phenylacrylate with 1,4-benzoquinone followed by Mannich reac-
tion gave compound 18. To access compounds 21 and 22, basic hy-
drolysis of esters 19 [17] and 20 was performed, followed by
sequential amide bond formation and Mannich reaction with
piperidine and formaldehyde.
Compound 24 was formed via a copper-catalyzed cyclization of
benzoquinone and ethyl 3-(2-bromophenyl)-3-oxopropanoate 23.
Lactam 25 was obtained via amination of 24 followed by a cycli-
zation reaction. The reaction took place in a closed vessel using
Cu2O as a catalyst in the presence of bromobenzene 24, N-methyl
pyrrolidinone (NMP) and excess of ammonia at 80 ꢁC according to a
reported protocol [18]. Compound 25 underwent a subsequent
Mannich reaction to yield compound 26. The synthesis of com-
pounds 31, 32, and 34 followed the methods similar to compound
12. Intermediate 27 was methylated and the aldehyde group was
installed using dichloromethyl methyl ether and TiCl4 to give iso-
mers 29 and 30, which could be readily separated using flash
chromatography. Each isomer was oxidized under Pinnick condi-
tion and sulfamic acid as hypochlorite scavenger to its corre-
sponding carboxylic acid. Amide bond formation with piperidine
gave the desired amides 31 and 32. Similarly, compound 34 was
synthesized using starting material 33 [13]. Compound 35 was
obtained via bromination of previously synthesized compound 7
using molecular bromine in dichloromethane at room temperature.
2.2. Structure activity relationships
All final compounds were evaluated in a microplate alamar
blue assay (MABA) [20] for anti-TB activity against Mtb H37Rv
expressed as the minimum inhibitory concentration (MIC) values.
As shown in Table 1, the coumestan 6 showed 16-fold increase in
activity compared to the corresponding ‘open-form’ 2-
phenylbenzofuran-3-carboxylate 7 (MIC values of 0.125 vs 2 mg/
mL). Installment of the 4-OH group on the benzene ring of 6
afforded a highly potent compound 9 with MIC value less than
0.0039
m
g/mL, being at least 8-fold more potent than TAM16
g/mL) [14]. The tetracyclic coume-
(compound 5, MIC ¼ 0.0313
m
stan 9 again resulted in at least a 64-fold increase of activity when
compared to ‘open-form’ ester 8. This trend is evident for most of
the synthesized compounds, notably upon the comparison of N-
ethylbenzofuran-3-carboxamides (10, 21, and 22) vs the corre-
sponding 5H-benzofuro [3,2-c]quinolin-6-ones (26, 57, and 64).
The trend is also observed upon comparison of the benzothio-
phene-3-carboxylate 45 vs the corresponding 6H-benzothieno
3

W. Zhang, L.-l. Liu, S. Lun et al.
European Journal of Medicinal Chemistry 213 (2021) 113202
Table 1
Antitubercular activity of compounds against the M. tuberculosis strain H37Rv.a
Conformationally relaxed ‘open-ring’ analogues
Corresponding rigid tetracyclic analogues
ID
Structure
MIC,
CLogPb
6.01
ID
Structure
MIC,
CLogP
4.17
mg/mL
m
g/mL
7c
2
6c
0.125
8c
0.25
32
5.38
5.57
9c
0.0039
32
4.11
4.00
18
16
10c
21
22
5c
4
4.15
4.34
4.10
2.98
5.24
5.24
6.70
6.56
26
57
64
65
12c
34
11c
41
0.25
3.41
3.70
3.60
3.14
3.46
3.46
4.86
6.79
4
0.5
2
0.25
3
5
0.0313
>64
>64
>32
16
0.0313e0.0625
31
32
35
45
>64
>64
>64
1
1
3
a
See 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 measurements.
b
CLogP was calculated using ChemBioDraw Ultra 14.0.
Compounds 5e12 [10,14] were reference compounds for comparision.
c
[3,2-c] [1]benzopyran-6-one 41. In these cases, at least an 8-fold
improvement in activity was observed in the tetracyclic ana-
logueues. The installment of 4-OH group on the benzene ring of
compound 26 gave an 8-fold more potent analogue 65, which is
equipotent with its ‘open-form’ analogue 5. However, replacing
the benzofuran nucleus with an indole resulted in compounds 18
‘open-form’ vs tetracyclic analogueues (31 vs 12, 32 vs 34, and 35
vs 11) revealed that the inactivity in the ‘open-form’ analogueues
were also mirrored in the corresponding tetracyclic analogues.
Molecular docking simulations suggested that the substituents at
6-Br of benzofurans may interfere with Arg 1641 residue causing
unfavorable ligand binding. Overall, encouraged by the observa-
tion that the 5H-benzofuro [3,2-c]quinolin-6-ones displayed
improved antimycobacterial properties compared to the acyclic
amides, we focused our attention on the SAR of the 5H-benzofuro
[3,2-c]quinolin-6-one derivatives.
and 16, both of which have MIC values of 32
the bioisosteric replacement is not favorable for activity. Six other
inactive compounds (MIC > 32 g/mL; compounds 11, 12, 31, 32,
34, and 35) were also displayed in Table 1. Comparison of the
mg/mL, indicating that
m
4

W. Zhang, L.-l. Liu, S. Lun et al.
European Journal of Medicinal Chemistry 213 (2021) 113202
a
Scheme 1. Synthesis of Compounds 16, 18, 21, 22, 26, 31, 32, 34 and 35.
.
a
Reagents and conditions: (a) ammonium formate, 4 Å molecular sieve, EtOH, reflux, 7 h; (b) benzoquinone, ZnBr2, THF, 3 h; (c) BBr3 in CH2Cl2 (1 M), CH2Cl2, rt, overnight, then
EtOH, reflux, 1 h; (d) formaldehyde (37% aq), piperidine, EtOH, reflux, 8e12 h; (e) KOH, MeOH, reflux, 3 h; (f) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, 1-
hydroxybenzotriazole, ethylamine or piperidine, pyridine, DMF, overnight; (g) benzoquinone, Cu(OTf)2 (5 mol %), toluene, reflux, 7e12 h; (h) Cu2O, ammonia, N-methylpyrrolidone,
80 ꢁC, 15 h; (i) K2CO3, CH3I, DMF, 3e5 h; (j) Cl2CHOCH3, TiCl4, CHCl3; (k) NaClO2, NH2SO3H, dioxane, H2O, 30 min; (l) Br2, CH2Cl2, rt, overnight.
In our previous work as well as Sacchettini group’s, it was
established that the 4’ -(methylpiperidin-1-yl)methyl and 5’ eOH
substituents play important roles for optimal anti-TB activity. The
X-ray cocrystal structure reported by the Sacchettini group [10]
revealed that both of these groups were completely buried in
Pks13-TE domain while the 30-ethyl ester and 20-phenyl group
(right-hand side benzene ring) were oriented towards the solvent.
In addition, substituents at the 60 or 70 position of the benzofuran
portion, whether electron-withdrawing groups (EWGs) or
electron-donating groups (EDGs) are generally tolerable [14]. With
these considerations in mind, various EWGs such as halogens (F, Cl,
Br e compounds 51e58) and EDGs (methoxy and hydroxyl e
compounds 64e67) were introduced at different positions of the
right-hand side benzene ring of the 5H-benzofuro [3,2-c]quinolin-
6-one (Table 2). In general, it was found that the 4-position
constitutes an ideal site of substitution for optimal potency, for
instance when comparing the bromides 51 vs 52, the chlorides
53e55 and the fluorides 56e58. Within this set of analogueues,
MIC values ranged from 0.5 to 1.0
mg/mL, with the exception of
bromide 52 (MIC ¼ 32
m
g/mL), being 64-fold less potent compared
to the 4-bromo derivative 51. Unfortunately, none of these eight
compounds demonstrated more potent anti-TB activities compared
to the unsubstituted analogueue 26 (MIC ¼ 0.25
duction of EDGs (OMe and OH) at the 5-position of the phenyl ring
g/mL)
mg/mL). Intro-
gave compounds 66 (MIC ¼ 4
m
g/mL) and 67 (MIC ¼ 1
m
respectively. Introduction of a hydroxyl group at the 4-position of
the phenyl ring dramatically increase the anti-TB activity (65) with
an MIC value of 0.0313e0.0625 mg/mL. This trend is consistent with
previous findings and the improved potency was likely mediated
through an optimal hydrogen bond to the carbonyl oxygen of
5

W. Zhang, L.-l. Liu, S. Lun et al.
European Journal of Medicinal Chemistry 213 (2021) 113202
a
Scheme 2. Synthesis of Compounds 41 and 45.
.
a Reagents and conditions: (a) 2-bromo-5-methoxybenzaldehyde, K2CO3, S, DMF, 110 ꢁC, 16 h; (b) Cl2CHOCH3, TiCl4, CHCl3, 0 ꢁC to rt, 30 min; (c) NaClO2, NH2SO3H, dioxane, H2O; (d)
BBr3 in CH2Cl2 (1 M), CH2Cl2, rt, overnight, then EtOH, reflux, 1 h; (e) formaldehyde (37% aq), piperidine, EtOH, reflux, 8e12 h; (f) EtOH, concentrated sulfuric acid, 70 ꢁC, 5 h; (g) BBr3
in CH2Cl2 (1 M), CH2Cl2, rt, overnight.
a
Scheme 3. Synthesis of Compounds 51e58, 64e67.
.
a
Reagents and conditions: (a) Pd(PPh3)2Cl2, CuI, Et3N, TMS-acetylene, N2, rt, 4 h; then K2CO3, MeOH, rt, 3 h; (b) Pd(PPh3)2Cl2, CuI, Et3N, 1-bromo-2-iodo-4-methoxybenzene or 1-
bromo-2-iodo-4-(methoxymethoxy)benzene, N2, rt, overnight; (c) Cu(OAc)2 (5 mol%), Ag2CO3, DMSO, Cs2CO3, 130 ꢁC, 24 h; (d) BBr3 in CH2Cl2 (1 M), CH2Cl2, rt, overnight; (e)
formaldehyde (37% aq), piperidine, EtOH, reflux, 8e12 h; (f) HCl, MeOH, reflux, 5 h.
6

W. Zhang, L.-l. Liu, S. Lun et al.
European Journal of Medicinal Chemistry 213 (2021) 113202
Table 2
Table 4
Vero toxicity and selectivity index of selected compounds.
Antitubercular activity of 5H-benzofuro [3,2-c]quinolin-6-ones 51e58 and 64e67
against the M. tuberculosis strain H37Rv.a.
ID
Toxicity to Vero cells IC50
,
m
g/mL
MIC H37Rv,
m
g/mL
SIa
51
54
57
58
64
65
67
64
32
16
16
64
4
0.5
0.5
0.5
1
128
64
32
16
0.25
0.0313e0.0625
1
256
64e128
16
4.
ID
R1
MIC,
m
g/mL
CLogPb
16
a
SI, selectivity index ¼ IC50 (Vero cells)/MIC
(H37Rv).
51
52
53
54
55
56
57
58
64
65
66
67
4-Br
5-Br
3-Cl
4-Cl
5-Cl
3-F
4-F
5-F
4-OMe
4-OH
5-OMe
5-OH
0.5
32
4.42
4.42
4.27
4.27
4.27
3.70
3.70
3.70
3.60
3.14
3.60
3.14
1.0
0.5
1.0
0.5
0.5
1
moderate SI value between 64 and 128 compared to 58 and 67,
which exhibited low SI values of 16.
2.4. Microbial selectivity
0.25
0.0313e0.0625
4
1
Following the cytotoxicity evaluation, antimicrobial selectivity
of the most promising compound 65 was further explored. When
tested against a selected panel of gram-positive and gram-negative
bacteria including S. aureus Newman, B. subtilis 168, E. coli AB1157,
and E. coli DH5
inhibition of growth (MIC values > 25
a
See 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 measurements.
a
(Table 5), compound 65 showed no appreciable
g/mL), suggesting that it
b
CLogP was calculated using ChemBioDraw Ultra 14.0.
m
was selective against mycobacteria and unlikely to develop po-
tential drug resistance to nonpathogenic bacteria.
Q1633. Comparison of analogues 64e65 vs the corresponding 5-
substituted analogues 66e67 further supported that the 4-
position of the benzene ring was optimal site for substitution.
Given the excellent MIC values of most benzofuranoquinolinone
derivatives, representative compound 65 was further evaluated its
antitubercular activity against clinical drug susceptible (DS, V4207),
MDR-TB (KZN494 and V2475), and XDR-TB (TF274 and R506)
strains (Table 3). To our delight, benzofuranoquinolinone 65
exhibited potent activities against all tested stains.
2.5. Thermal stability analysis and enzyme activity
As shown previously by our group, the results from whole
genome sequencing of the wild-type and coumestan-resistant
mutants demonstrated that these coumestans were likely to
target the Pks13-TE [13]. In order to demonstrate whether this
target engagement was responsible for their observed antituber-
cular effects, selected tetracyclic compounds were evaluated for
thermal shift assay in the presence of Pks13-TE using nano differ-
ential scanning fluorimetry (nanoDSF) method. Briefly, the binding
of ligands with high affinity to proteins generally leads to thermal
2.3. Toxicity to vero cells
In order to evaluate their general cytotoxicity profiles, selected
5H-benzofuro [3,2-c]quinolin-6-ones (Table 2) with MIC values
equal or less than 1 mg/mL were subsequently evaluated in Vero
Table 5
cells, which are derived from African green monkey kidney. The
selectivity index (SI) values were calculated from the ratio of IC50
(Vero)/MIC (Mtb) as shown in Table 4. Comparison of Vero cell
toxicity (IC50 value) and MIC value of Mtb growth inhibition gives a
measure of the general toxicity profile of the target compound.
Three of the selected seven compounds 51, 64 and 65 had SI values
of more than 100, with compound 64 having the highest SI value of
256. Compound 65 with excellent anti-TB activity exhibited a
Assessment of 65 against selected gram positive and gram-negative bacteria.
Compounds MIC,
mg/mL
S. aureus Newman B. subtilis 168 E. coli AB1157 E. coli DH5
a
Vancomycin 0.39e0.78
Tetracycline 0.2e0.39
>25
>25
9.39e18.75
>25
>25
>25
1.56e3.13
9.39e18.75
>25
0.78e1.56
2.34e4.68
>25
Kanamycin
2.34e4.68
>25
65
Table 3
Antitubercular activity of compound 65 against DS, MDR, and XDR strains of M. tuberculosis.
Mtb MICa
(
mg/mL)
strain/phenotype
V4207/DSb
V2475/MDRc
KZN494/MDR
TF274/XDRd
R506/XDR
Isoniazid
Rifampin
Levofloxacin
Ofloxacin
Kanamycin
65
0.04
0.0625
0.125
0.5
16
16
128
NT
0.5
2
8
8
4
>128
1
8
>128
0.0156e0.0313
>128
2
4
>128
0.0156e0.0313
NTe
0.5
2
2
0.0313
0.0625
0.0313
a
The 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.
b
Drug susceptible strain of M. tuberculosis.
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 kanamycin.
NT ¼ not tested.
c
d
e
7

W. Zhang, L.-l. Liu, S. Lun et al.
European Journal of Medicinal Chemistry 213 (2021) 113202
Table 7
stability shift of proteins, which can be detected using nanoDSF.
IC50 and MIC values of compounds 65 and 7.
Stabilization of the Pks-TE protein at 30
mM concentration upon
a
binding of high affinity ligands (at 300
mM concentration) was
Compounds
Pks13-TE IC50
,
m
M
H37Rv MICb,
mg/mL
evaluated by comparing the melting temperatures (Tm) of Pks13-TE
65
7
0.58 0.03
4.60 0.20
0.0313e0.0625
2
(56.2 0.03 ꢁC) in the absence or presence of the compounds and
calculating the shift in the Tm (
thermal stability of Pks13-TE was observed following the addition
of the 10-fold tetracycle-containing compounds (
Tm > 3.0 ꢁC,
DTm). A significant increase in
a
IC50 values were tested as an average of three individual measurements.
The lowest concentration of compounds leading to at least 90% inhibition of
b
D
bacterial growth signal by the MABA. MIC values are reported as an average of three
individual measurements.
Table 6), indicating high-affinity binding of the compounds to the
Pks13-TE. Although caution must be exercised upon correlating
D
Tm and the observed antitubercular phenotype, in our case a
nonpathogenic bacteria strains. Selected tetracyclic compounds
were also demonstrated to bind with high affinities to the recom-
binant Pks13-TE protein as measured by nanoDSF assay, indicating
their target engagement.
consistent trend was evident: the extent of the antitubercular ac-
tivity of the compounds tend to be accompanied by an increase in
the DTm values. Collectively, the observed thermal stabilization was
consistent with the notion that the tetracyclic analogues bind to the
Pks13-TE, leading to the antimycobacterial effects.
Compounds 65 and 7 were next selected for enzymatic potency
evaluation using a protocol reported by Sacchettini group [10]. As
shown in Table 7, compound 65 inhibited the Pks13-TE activity with
4. Experimental section
General. Starting materials, reagents, and solvents were pur-
chased from commercial suppliers and used without further puri-
fication, unless otherwise stated. Anhydrous toluene and CH2Cl2
were obtained by distillation over sodium wire or calcium hydride
respectively. All non-aqueous reactions were run under a nitrogen
atmosphere with exclusion of moisture from reagents, and all re-
action vessels were oven-dried. The progress of reactions was
monitored by thin layer chromatography on SiO2 and LCMS.
Products were purified by column chromatography on 200e300
mesh SiO2, and an EtOAc/petroleum ether mixture or gradient was
used unless stated otherwise. 1H NMR and 13C NMR spectra were
recorded at 400 and 101 MHz respectively. NMR chemical shifts
an IC50 value of 0.58
mM while the IC50 value of compound 7 was
found to be 4.6
mM, which agreed with their MIC data.
3. Conclusions
In this report, we demonstrated that targeting Pks13 is a valid
strategy when designing antitubercular compounds that work via
inhibition of the synthesis of Mtb cell wall. Our work on the cou-
mestans and Sacchettini’s group on the benzofuran esters revealed
novel scaffolds that are highly amenable for SAR and further
physiochemical parameters optimization. Consequently, a total of
23 compounds were synthesized and evaluated for their antitu-
bercular activity against the M. tuberculosis H37Rv. The observed
SAR comparison between the conformationally relaxed ‘open-form’
benzofuran esters and the corresponding rigid tetracyclic ana-
logues showed that the latter tend to be superior anti-TB com-
pounds. This trend is consistent with our hypothesis that the
were reported in
d
(ppm) using the
d
0 signal of tetramethylsilane,
2.50 signal of
d
7.26 signal of CDCl3,
d
3.31 signal of CD3OD or d
(CD3)2SO as internal standards. High resolution mass spectra
(HRMS) were performed using a Bruker ESI-TOF high-resolution
mass spectrometer. Purities of final compounds (>95%) were
established by analytical HPLC, which was carried out on a Waters
HPLC system using InertSustain-C18 column (5 micron,
250 ꢂ 4.6 mm) with detection at 280 and 254 nm on a variable
wavelength detector 2998 PDA. The melting points were recorded
using a WRR-Y melting point instrument. See Supporting Infor-
mation for NMR spectra (1H and 13C) of all final compounds and
analytical HPLC traces of compound 65.
General procedure for the synthesis of substituted 5-hydroxy-2-
phenyl benzofuran from substituted ethyl benzoylacetates (method
A) was reported previously [13].
General procedure for the preparation of coumestan de-
rivatives, 8-hydroxychromeno[4,3-b]indol-6(11H)-one or 8-
additional
p-p stacking interactions between the benzene ring of
the tetracyclic compounds and the phenyl ring of F1670 in the
Pks13-TE domain is important for binding. We also explored bio-
sisosteric replacements of the benzofuran nucleus to the benzo-
thiophene and the indole ring, which unfortunately proved to be
detrimental for their anti-TB activities. Lactam 65 possessed
outstanding anti-TB activity, improved selectivity to Vero cells
(SI ¼ 128), and excellent selectivity to mycobacteria against
Table 6
hydroxy-6H-benzo[4,5]thieno[3,2-c]chromen-6-one
substituted ethyl 5-hydroxy-2-(2-methoxyphenyl)benzocyclo-
heterocycle-3-carboxylate (method B). To solution of
from
Thermal stabilization of Pks13-TE upon binding with compounds measured by
nanoDSF.
a
a
ID
D
Tm ꢁC
MICb,
mg/mL
,
substituted ethyl 5-hydroxy-2-(2-methoxyphenyl)benzocyclohe-
terocycle-3-carboxylate (1.0 mmol) in anhydrous CH2Cl2 (4 mL),
BBr3 (1 M in CH2Cl2, 4.0 mmol) was added at room temperature
under N2. After being stirred overnight, the reaction mixture was
quenched with EtOH. The resulting mixture was allowed to reflux
for 1 h and evaporated. The residue was purified by flash chro-
matography or by recrystallization in EtOH to obtain target
compound.
5X
10X
DMSO
16
26
41
45
64
65
67
ꢀ1.3 0.03
0.8 0.14
9.6 0.03
7.9 0.16
3.2 0.04
8.7 0.15
7.5 0.10
7.4 0.00
e
3.1 0.09
10.2 0.64
9.1 0.14
3.3 0.15
9.0 0.39
9.4 0.25
8.5 0.06
32
0.25
1
16
0.25
0.0313e0.0625
1
General procedure for the Mannich reaction of substituted
ethyl 2-(2-methoxyphenyl)-5-hydroxybenzocyclo-3-carboxylate
or coumestan analogueue with amine and formaldehyde
a
D
Tm was calculated as Tm (Pks13-TE) in the presence of compounds (3% DMSO
final concentration) minus Tm (Pks-TE) with DMSO only. Final concentration ratio of
Pks-TE and compounds is 1:5 and 1:10. Tm values are tested as an average of three
individual measurements.
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 measurements.
(method C). To
a
solution of substituted ethyl 2-(2-
methoxyphenyl)-5-hydroxybenzocyclo-3-carboxylate or coume-
stan analogueues (1 mmol) in ethanol (3 mL) were added formal-
dehyde (37% in water, 4 mmol) and the appropriate amine (4 mmol)
b
8

W. Zhang, L.-l. Liu, S. Lun et al.
European Journal of Medicinal Chemistry 213 (2021) 113202
at room temperature under N2. The reaction mixture was allowed
to reflux for 5e12 h and then cooled to rt. The reaction mixture was
evaporated and the residue was purified by flash chromatography
or recrystallized in EtOH to give the product.
General procedure for the hydrolysis of ester to afford car-
boxylic acid (method D). To a solution of ester (1.0 mmol) in 5 mL
MeOH was added KOH (6.0 mmol) in 1 mL H2O at rt. The reaction
mixture was allowed to reflux for 3 h and then acidified with HCl
(2 M) to pH 5e6. The resultant solid was filtered off to give car-
boxylic acid without further purification.
J ¼ 8.7 Hz, 1H), 6.73 (d, J ¼ 8.7 Hz, 1H), 4.17e4.08 (m, 4H), 2.75e2.48
(m, 4H), 1.66e1.61 (m, 4H), 1.51e1.46 (m, 2H), 1.06 (t, J ¼ 7.1 Hz, 3H).
13C NMR (101 MHz, CD3OD)
d 169.4, 154.8, 144.3, 134.4, 132.1, 130.0
(2C), 129.4, 129.1 (2C), 127.1, 114.7, 112.5, 111.9, 106.2, 61.4, 59.2, 54.7
(2C), 26.9 (2C), 24.9, 14.2. HRMS (ESI) m/z: Calcd for C23H27N2O3
(M þ H)þ 379.2016, found 379.2002.
N-ethyl-2-(4-fluorophenyl)-5-hydroxy-4-(piperidin-1-
ylmethyl)benzofuran-3-carboxamide (21). This compound was
obtained from 19 by employing methods D, E, and C. Yields 47%, 91%
and 77%; pale yellow solid; m.p 152.6e154.9 ꢁC. 1H NMR (400 MHz,
General procedure for the amidation of carboxylic acid
(method E). To a solution of carboxylic acid to anhydrous DMF
(5 mL), EDC$HCl (1.3 mmol), HOBt (1.3 mmol), pyridine (2.1 mmol),
and ethylamine or piperidine (1.2 mmol) under N2 were added at rt.
After being stirred overnight, the reaction mixture was quenched
with saturated NH4Cl solution and extracted with EtOAc
(2 ꢂ 30 mL). The combined organic phases were washed with HCl
(5% aqueous solution), saturated aqueous NaHCO3 solution
(1 ꢂ 30 mL), dried over anhydrous Na2SO4 and evaporated. The
residue was purified by flash chromatography to give the amide
product.
CDCl3)
d
7.78e7.73 (m, 2H), 7.24 (d, J ¼ 8.9 Hz, 1H), 7.13e7.09 (m,
2H), 6.79 (d, J ¼ 8.8 Hz, 1H), 6.07 (t, J ¼ 5.5 Hz, 1H), 3.86 (s, 2H),
3.53e3.41 (m, 2H), 3.20e2.06 (m, 4H), 1.63e1.54 (m, 6H), 1.21 (t,
J ¼ 7.2 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 166.4, 163.3 (d, JC-
¼ 250.2 Hz), 155.2, 151.8, 148.0, 128.7 (d, JC-F ¼ 8.3 Hz, 2C), 126.0,
F
126.0 (d, JC-F ¼ 3.3 Hz), 116.0 (d, JC-F ¼ 21.9 Hz, 2C), 115.1, 113.2 (d, JC-
¼ 0.8 Hz), 112.2, 110.6, 57.1, 53.9 (2C), 35.2, 25.9 (2C), 24.0, 14.5.
F
HRMS(ESI) m/z: Calcd for C23H26N2O3 (M þ H)þ 397.1922, found
397.1906.
N-ethyl-5-hydroxy-2-(4-methoxyphenyl)-4-(piperidin-1-
ylmethyl)benzofuran-3-carboxamide (22). This compound was
obtained from 20 by employing methods D, E, and C. Yields 93%,
91%, and 61%; pale yellow solid; m.p 182.0e184.4 ꢁC. 1H NMR
Ethyl (Z)-3-amino-3-(2-methoxyphenyl)acrylate (14).
A
mixture of commercially available 13 (1.0 mmol), ammonium
formate (5.0 mmol) and molecular sieves (4 Å, 0.1 g) in 10 mL of
ethanol were refluxed for 7 h under N2 and then cooled to rt. The
reaction mixture was filtered through celite, and the filtrate was
evaporated and the residue was extracted with ethyl acetate
(3 ꢂ 20 mL). Combined organic layers were dried over anhydrous
Na2SO4 and evaporated to give product without further purifica-
(400 MHz, CD3OD)
d
7.74 (d, J ¼ 8.8 Hz, 2H), 7.30 (d, J ¼ 8.8 Hz, 1H),
7.02 (d, J ¼ 8.8 Hz, 2H), 6.75 (d, J ¼ 8.8 Hz, 1H), 3.92 (s, 2H), 3.85 (s,
3H), 3.43 (q, J ¼ 7.3 Hz, 2H), 2.72e2.44 (m, 4H), 1.71e1.60 (m, 4H),
1.58e1.50 (m, 2H), 1.23 (t, J ¼ 7.3 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d
166.7, 160.5, 155.2, 153.1, 147.9, 128.4 (2C), 126.3, 122.3, 114.6, 114.3
(2C), 112.2, 112.1, 110.5, 57.2, 55.5, 54.0 (2C), 35.2, 26.0 (2C), 24.1,
14.6. HRMS(ESI) m/z: Calcd for C24H28N2O4 (M þ H)þ 409.2122,
found 409.2108.
tion. Yield 97%; yellow oil. 1H NMR (400 MHz, CDCl3)
d 7.35 (d,
J ¼ 7.6 Hz, 1H), 7.28 (t, J ¼ 8.0 Hz, 1H), 6.92e6.83 (m, 2H), 4.76 (s,
1H), 4.09 (q, J ¼ 6.9 Hz, 2H), 3.78 (s, 3H), 1.21 (t, J ¼ 7.1 Hz, 3H). 13
C
Ethyl
2-(2-bromophenyl)-5-hydroxybenzofuran-3-
NMR (101 MHz, CDCl3)
d
169.5, 158.7, 155.6, 129.9, 128.6, 124.9,
carboxylate (24). This compound was obtained from 23 employ-
120.0, 110.5, 84.4, 57.7, 54.7, 13.6.
Ethyl 5-hydroxy-2-(2-methoxyphenyl)-1H-indole-3-
ing method A. Yield 32%; yellow solid. 1H NMR (400 MHz,
DMSO‑d6)
d
9.51 (s,1H), 7.80 (dd, J ¼ 7.6, 1.2 Hz,1H), 7.64 (dd, J ¼ 7.3,
carboxylate (15). To a solution of 14 (1.0 mmol) and ZnBr2
(1.0 mol) in 3 mL anhydrous THF was added a solution of benzo-
quinone (1.0 mmol) in THF (2 mL) under N2. After stirring for 3 h at
room temperature. The reaction mixture was quenched with
saturated NH4Cl solution and extracted with EtOAc (3 ꢂ 10 mL). The
combined organic phases were dried over anhydrous Na2SO4 and
evaporated. The residue was purified by flash chromatography to
give 15. Yield 58%; pale yellow solid. 1H NMR (400 MHz, DMSO‑d6)
1.9 Hz, 1H), 7.55e7.48 (m, 3H), 7.39 (d, J ¼ 2.4 Hz, 1H), 6.88 (dd,
J ¼ 8.9, 2.5 Hz, 1H), 4.16 (q, J ¼ 7.1 Hz, 2H), 1.11 (t, J ¼ 7.1 Hz, 3H). 13
C
NMR (101 MHz, DMSO‑d6)
d 162.3, 159.7, 154.6, 147.9, 132.4, 132.4,
131.9, 131.5, 127.4, 126.2, 123.0, 114.6, 112.0, 110.8, 106.1, 60.1, 13.7.
8-Hydroxybenzofuro[3,2-c]quinolin-6(5H)-one (25). To a so-
lution of 24 (1.0 mmol), Cu2O (0.05 mmol) and 6 mL NH3$H2O/NMP
(v/v ¼ 1:1) in a vessel with thick wall, the reaction mixture was
stirring at 80 ꢁC for 15 h under closed condition. Then cooled to
room temperature. The reaction mixture was evaporated. The res-
idue was purified by flash chromatography using silica gel to give
product. Yield 21%; pale yellow solid. 1H NMR (400 MHz, DMSO‑d6)
d
11.65 (s,1H), 8.95 (s,1H), 7.46e7.39 (m, 2H), 7.34 (d, J ¼ 7.2 Hz,1H),
7.20 (d, J ¼ 8.5 Hz, 1H), 7.10 (d, J ¼ 8.2 Hz, 1H), 7.02 (t, J ¼ 7.2 Hz, 1H),
6.69 (d, J ¼ 8.1 Hz, 1H), 4.06 (q, J ¼ 6.8 Hz, 2H), 3.72 (s, 3H), 1.09 (t,
J ¼ 6.9 Hz, 3H). 13C NMR (101 MHz, DMSO‑d6)
d
164.6, 157.2, 152.4,
d
11.88 (s, 1H), 9.58 (s, 1H), 8.02 (d, J ¼ 7.9 Hz, 1H), 7.63 (d, J ¼ 9.0 Hz,
141.2, 131.4, 130.2, 129.8, 128.0, 121.8, 119.7, 112.2, 112.1, 111.1, 105.1,
103.7, 58.4, 55.4, 14.1.
1H), 7.59 (d, J ¼ 7.4 Hz,1H), 7.52 (d, J ¼ 8.2 Hz,1H), 7.47 (d, J ¼ 2.3 Hz,
1H), 7.33 (t, J ¼ 7.5 Hz, 1H), 6.93 (dd, J ¼ 8.9, 2.3 Hz, 1H). 13C NMR
8-Hydroxy-7-(piperidin-1-ylmethyl)chromeno[4,3-b]indol-
6(11H)-one (16). This compound was obtained from 15 employing
methods B and C. Yields 64% and 80%; yellow solid; m.p > 250 ꢁC. 1H
(101 MHz, DMSO‑d6) d 159.1, 158.2, 154.7, 148.7, 138.3, 130.6, 124.6,
122.3, 121.0, 116.1, 114.6, 112.1, 110.9, 110.1, 105.9.
8-Hydroxy-7-(piperidin-1-ylmethyl)benzofuro[3,2-c]quino-
lin-6(5H)-one (26). This compound was obtained from 25 by
employing method C. Yield 89%; white solid; m.p 243.8e245.3 ꢁC.
NMR (400 MHz, DMSO‑d6)
d
8.27 (br, 1H), 8.21 (d, J ¼ 7.6 Hz, 1H),
7.61 (t, J ¼ 7.6 Hz, 1H), 7.53e7.41 (m, 3H), 6.93 (d, J ¼ 8.6 Hz, 1H),
4.79 (s, 2H), 2.80e2.66 (m, 4H), 1.67e1.55 (m, 4H), 1.54e1.45 (m,
1H NMR (400 MHz, CDCl3/CD3OD ¼ 4:1)
d
8.04 (d, J ¼ 8.0 Hz, 1H),
2H). 13C NMR (101 MHz, DMSO‑d6)
d
158.3,154.5,152.3,142.3,132.2,
7.52e7.46 (m, 1H), 7.41e7.35 (m, 2H), 7.27 (t, J ¼ 7.2 Hz, 1H),
6.90e6.87 (m, 1H), 4.83 (s, 2H), 2.93e2.36 (m, 4H), 1.71e1.46 (m,
130.7, 124.5, 124.2, 122.5, 116.7, 115.1, 112.9, 112.8, 111.5, 99.9, 56.1,
52.7 (2C), 24.5 (2C), 23.0. HRMS (ESI) m/z: Calcd for C21H21N2O3
(M þ H)þ 349.1547, found 349.1539.
6H). 13C NMR (101 MHz, CDCl3/CD3OD ¼ 4:1)
d 160.6, 159.9, 156.4,
149.6, 137.7, 130.7, 123.5, 122.8, 121.8, 115.8, 115.7, 114.9, 111.9, 111.0,
Ethyl
5-hydroxy-2-phenyl-4-(piperidin-1-ylmethyl)-1H-
110.9, 59.1, 53.6 (2C), 25.8 (2C), 24.0. HRMS (ESI) m/z: Calcd for
indole-3-carboxylate (18). This compound was obtained from
commercially available 17 by employing amination, lactonization
and Mannich reaction according to the methods described for 14,
15, and method C. Overall yield 38%; pale yellow semisolid. 1H NMR
C
21H21N2O3 (M þ H)þ 349.1547, found 349.1531.
Ethyl 5-methoxy-2-phenylbenzofuran-3-carboxylate (28). To
a stirred solution of 27 (1.0 mmol) and K2CO3 (2.1 mmol) in anhy-
drous DMF (20 mL) was added CH3I (1.2 mmol) under N2. After
being stirred overnight at rt, the reaction mixture was quenched
(400 MHz, CD3OD)
d 7.50e7.48 (m, 2H), 7.42e7.39 (m, 3H), 7.22 (d,
9

W. Zhang, L.-l. Liu, S. Lun et al.
European Journal of Medicinal Chemistry 213 (2021) 113202
with H2O. The mixture was extracted with EtOAc (2 ꢂ 20 mL), and
the combined organic phases were washed with water (2 ꢂ 20 mL),
dried over Na2SO4, and evaporated. The residue was purified by
flash chromatography to give the product. Yield 98%; pale yellow
2H), 3.23e3.19 (m, 2H), 1.71e1.68 (m, 4H), 1.47e1.43 (m, 2H). 13C
NMR (101 MHz, CDCl3)
d 166.6, 161.1, 158.3, 153.8, 153.7, 149.9,
132.3, 126.1, 124.9, 124.8, 122.1, 117.7, 112.7, 111.1, 106.0, 102.9, 56.5,
48.2, 42.9, 26.5, 25.7, 24.7. HRMS (ESI) m/z: Calcd for C22H19NNaO5
(M þ Na)þ 400.1155, found 400.1165.
solid. 1H NMR (400 MHz, CDCl3)
d 7.93e7.90 (m, 2H), 7.49 (d,
J ¼ 2.6 Hz, 1H), 7.43e7.38 (m, 3H), 7.35 (d, J ¼ 8.9 Hz, 1H), 6.89 (dd,
Ethyl 6-bromo-5-hydroxy-2-phenyl-4-(piperidin-1-ylmethyl)
benzofuran-3-carboxylate (35). To the solution of 7 (1.0 mmol) in
CH2Cl2 (5 mL), Br2 (1.2 mmol) under N2 was added at room tem-
perature. After stirring overnight at room temperature, the reaction
mixture was quenched with saturated aqueous Na2S2O3 solution and
extracted with CH2Cl2 (2 ꢂ 30 mL). The combined organic phases
were washed saturated aqueous NaHCO3 solution (1 ꢂ 20 mL), brine
(1 ꢂ 20 mL), dried over anhydrous Na2SO4 and evaporated. The
residue was purified by flash chromatography to give the product.
Yield 38%; yellow solid; m.p 134.5e135.7 ꢁC. 1H NMR (400 MHz,
J ¼ 8.9, 2.7 Hz, 1H), 4.33 (q, J ¼ 7.1 Hz, 2H), 3.82 (s, 3H), 1.32 (t,
J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 164.2, 161.5, 156.9,
149.0, 130.3, 129.9, 129.6 (2C), 128.2 (2C), 128.05, 114.3, 111.8, 109.1,
104.9, 60.7, 56.0, 14.4.
Ethyl
carboxylate
phenylbenzofuran-3-carboxylate (30). To
4-formyl-5-methoxy-2-phenylbenzofuran-3-
(29) and ethyl 6-formyl-5-methoxy-2-
solution of 28
a
(1 mmol) and Cl2CHOCH3 (5.0 mmol) in CHCl3 (5 mL) were added
slowly TiCl4 (2.5 mmol) at rt. The reaction mixture was being stirred
at rt for 40 min and quenched with H2O. The mixture was extracted
with EtOAc (2 ꢂ 20 mL), washed with saturated aqueous NaHCO3
solution (1 ꢂ 20 mL), dried over Na2SO4, and evaporated. The res-
idue was purified by silica gel column chromatography to give the
products 29 and 30.29: Yield 27%; pale yellow solid. 1H NMR
CDCl3)
d 7.72e7.71 (m, 2H), 7.64 (s, 1H), 7.45e7.44 (m, 3H), 4.35 (q,
J ¼ 7.1 Hz, 2H), 4.01 (s, 2H), 2.60e2.27 (m, 4H), 1.70e1.50 (m, 6H),
1.28 (t, J ¼ 7.2 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 166.1, 157.5, 152.7,
147.7, 130.0, 129.8, 128.5 (2C), 128.1 (2C), 125.1, 114.3, 113.0, 110.0,
109.2, 61.7, 58.2, 53.9 (2C), 25.8 (2C), 23.9,14.1. HRMS (ESI) m/z: Calcd
for C23H25NO4Br (M þ H)þ 458.0967, found 458.0980.
(400 MHz, CDCl3)
d
10.58 (s, 1H), 7.91 (dd, J ¼ 8.0, 1.7 Hz, 2H), 7.69
(d, J ¼ 9.0 Hz, 1H), 7.50e7.42 (m, 3H), 7.01 (d, J ¼ 9.0 Hz, 1H), 4.53 (q,
5-Methoxy-2-(2-methoxyphenyl)benzo[b]thiophene (37). A
mixture of 36 (1.0 mmol), 2-bromo-5-methoxybenzaldehyde
(1.5 mmol), K2CO3 (3.0 mmol), S (4.0 mmol) were dissolved in
3 mL of dimethylformamide and stirred for 16 h at 110 ꢁC under
nitrogen. The mixture was filtered and the filtrate was evaporated
to remove solvent. The residue was purified by flash chromatog-
raphy using silica gel to give product. Yield 43%; red oil. 1H NMR
J ¼ 7.2 Hz, 2H), 3.98 (s, 3H), 1.37 (t, J ¼ 7.2 Hz, 3H). 13C NMR
(101 MHz, CDCl3)
d 189.4, 166.0, 160.3, 157.5, 149.0, 130.2, 129.3,
128.9 (2C), 127.4 (2C), 125.6, 117.9, 117.2, 112.3, 109.6, 61.9, 57.0,
14.2.30: Yield 49%; pale yellow solid. 1H NMR (400 MHz, CDCl3)
d
10.54 (s, 1H), 8.03e7.95 (m, 3H), 7.65 (s, 1H), 7.55e7.48 (m, 3H),
4.41 (q, J ¼ 7.1 Hz, 2H), 4.02 (s, 3H), 1.40 (t, J ¼ 7.1 Hz, 3H). 13C NMR
(101 MHz, CDCl3)
d
189.7,164.8,163.6,159.2,148.4,134.1,131.1,129.9
(400 MHz, CDCl3) d 7.72e7.65 (m, 3H), 7.34e7.27 (m, 1H), 7.24 (d,
(2C), 129.2, 128.3 (2C), 123.0, 110.6, 109.2, 104.2, 61.0, 56.2, 14.3.
Ethyl 5-methoxy-2-phenyl-4-(piperidine-1-carbonyl)benzo-
furan-3-carboxylate (31). To a solution of 29 (1 mmol) in 10 mL of
dioxane/H2O (v/v ¼ 7/3) was added NaClO2 (6 mmol) and NH2SO3H
(5.7 mmol) at rt. The reaction mixture was stirred at rt for 1 h and
quenched with saturated aqueous Na2S2O3 solution. The mixture
was extracted with EtOAc (2 ꢂ 20 mL), washed with saturated
aqueous NaCl solution, dried over Na2SO4, and evaporated. The
residue and piperidine employing method E to give the product 31.
Yields 83% and 70%; white solid; m.p 121.5e123.6 ꢁC. 1H NMR
J ¼ 2.1 Hz, 1H), 7.05e6.98 (m, 2H), 6.95 (dd, J ¼ 8.8, 2.5 Hz, 1H), 3.95
(s, 3H), 3.87 (s, 3H).
5-Methoxy-2-(2-methoxyphenyl)benzo[b]thiophene-3-
carbaldehyde (38). This compound was obtained from 37 accord-
ing to the method described for 29. Yield 47%; white solid. 1H NMR
(400 MHz, DMSO‑d6)
d
9.73 (s, 1H), 8.12 (d, J ¼ 2.5 Hz, 1H), 7.98 (d,
J ¼ 8.9 Hz, 1H), 7.60e7.54 (m, 1H), 7.52 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.25
(d, J ¼ 8.4 Hz, 1H), 7.18e7.11 (m, 2H), 3.86 (s, 3H), 3.81 (s, 3H). 13
C
NMR (101 MHz, DMSO‑d6)
d 186.4, 158.5, 157.0, 156.4, 137.3, 132.1,
132.0, 130.3, 129.5, 123.2, 120.9, 119.4, 115.5, 112.2, 106.1, 55.8, 55.3.
5-Methoxy-2-(2-methoxyphenyl)benzo[b]thiophene-3-
(400 MHz, CDCl3)
d 7.84e7.78 (m, 2H), 7.48e7.41 (m, 4H), 6.97 (d,
J ¼ 9.0 Hz, 1H), 4.39e4.21 (m, 2H), 4.05e3.95 (m, 1H), 3.87 (s, 3H),
carboxylic acid (39). To a solution of 38 (1 mmol) in 10 mL of
dioxane/H2O (v/v ¼ 7:3) was added NaClO2 (6 mmol) and NH2SO3H
(5.7 mmol) at rt. The reaction mixture was stirred at rt for 1 h and
quenched with saturated aqueous Na2S2O3 solution. The mixture
was extracted with EtOAc (2 ꢂ 20 mL), washed with saturated
aqueous NaCl solution, dried over Na2SO4, and evaporated to give
3.59e3.48 (m, 1H), 3.41e3.28 (m, 1H), 3.23e3.11 (m, 1H), 1.83e1.48
(m, 6H), 1.26 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 165.4,
164.0, 158.8, 152.3, 149.2, 130.0, 129.8, 128.6 (2C), 128.3 (2C), 125.1,
118.5, 111.8, 110.2, 110.0, 61.2, 57.1, 48.0, 42.4, 26.1, 25.4, 24.9, 14.1.
HRMS (ESI) m/z: Calcd for C24H25NNaO5 (M þ Na)þ 430.1625, found
430.1630.
39. Yield 57%; white solid. 1H NMR (400 MHz, CDCl3)
d 7.93 (d,
Ethyl 5-methoxy-2-phenyl-6-(piperidine-1-carbonyl)benzo-
furan-3-carboxylate (32). This compound was obtained from 30
according to the methodology described for 31. Yields 90% and 70%;
white solid; m.p 141.3e142.8 ꢁC. 1H NMR (400 MHz, CDCl3)
J ¼ 2.4 Hz, 1H), 7.68 (d, J ¼ 8.8 Hz, 1H), 7.43e7.39 (m, 2H), 7.08e7.01
(m, 2H), 6.97 (d, J ¼ 8.6 Hz, 1H), 3.91 (s, 3H), 3.79 (s, 3H).
8-Hydroxy-6H-benzo[4,5]thieno[3,2-c]chromen-6-one (40).
This compound was obtained from 39 employing method B. Yield
d
7.99e7.97 (m, 2H), 7.58 (s, 1H), 7.52e7.45 (m, 3H), 7.40 (s, 1H), 4.40
85%; yellow solid. 1H NMR (400 MHz, DMSO‑d6)
d 9.93 (s, 1H),
(q, J ¼ 7.1 Hz, 2H), 3.92 (s, 3H), 3.84e3.69 (m, 2H), 3.26e3.17 (m,
8.06e7.91 (m, 3H), 7.68 (t, J ¼ 7.6 Hz, 1H), 7.57 (d, J ¼ 8.3 Hz, 1H),
2H), 1.76e1.59 (m, 6H), 1.39 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz,
7.46 (t, J ¼ 7.5 Hz, 1H), 7.07 (dd, J ¼ 8.7, 2.2 Hz, 1H).
CDCl3)
d
167.2, 164.0, 162.1, 153.0, 148.4, 130.5, 129.7 (2C), 129.6,
8-Hydroxy-7-(piperidin-1-ylmethyl)-6H-benzo[4,5]thieno
[3,2-c]chromen-6-one (41). This compound was obtained from 40
by employing method C. Yield 84%; white solid; m.p
128.6, 128.2 (2C), 124.9, 110.4, 109.0, 103.8, 60.8, 56.2, 48.2, 42.8,
26.5, 25.8, 24.8, 14.3. HRMS (ESI) m/z: Calcd for C24H25NNaO5
(M þ Na)þ 430.1625, found 430.1598.
214.9e216.1 ꢁC. 1H NMR (400 MHz, CDCl3/CD3OD ¼ 4/1)
d 7.89e7.80
8-Methoxy-9-(piperidine-1-carbonyl)-6H-benzofuro[3,2-c]
chromen-6-one (34). This compound was obtained from 33 ac-
cording to the methodology described for 31, then by employing
methods E, B, and methylation described for 28. Overall yield 19%;
(m, 2H), 7.61 (t, J ¼ 7.8 Hz,1H), 7.48e7.36 (m, 2H), 7.33 (d, J ¼ 8.9 Hz,
1H), 5.04 (s, 2H), 3.59e3.56 (m, 2H), 3.13e3.07 (m, 2H), 1.97e1.94
(m, 2H), 1.86e1.80 (m, 3H), 1.59e1.48 (m, 1H). 13C NMR (101 MHz,
CDCl3/CD3OD ¼ 4/1)
d 159.2, 157.8, 155.5, 151.1, 137.3, 132.7, 130.5,
white solid; m.p 244.2e245.1 ꢁC. 1H NMR (400 MHz, CDCl3)
d
8.02
126.2, 125.6, 124.6, 118.8, 117.1 (2), 116.8, 110.8, 53.4, 53.2 (2C), 23.3
(2C), 22.04. HRMS (ESI) m/z: Calcd for C21H20NO3S (M þ H)þ
366.1158, found 366.1148.
(dd, J ¼ 7.8, 0.9 Hz, 1H), 7.64e7,59 (m, 7.1 Hz, 2H), 7.55 (s, 1H), 7.50
(d, J ¼ 8.3 Hz, 1H), 7.42 (t, J ¼ 7.5 Hz, 1H), 3.95 (s, 3H), 3.87e3.67 (m,
10

W. Zhang, L.-l. Liu, S. Lun et al.
European Journal of Medicinal Chemistry 213 (2021) 113202
5-Methoxy-2-phenylbenzo[b]thiophene-3-carboxylic
acid
3-Bromo-8-methoxybenzofuro[3,2-c]quinolin-6(5H)-one
(49). To a solution of 48 (1.0 mmol), Cu(OAc)2 (10 mol%), Ag2CO3
(1.0 mmol) and Cs2CO3 (4.0 mmol) in 5 mL of DMSO was stirred in a
preheated oil bath at 130 ꢁC for 24 h. After cooled to rt, the reaction
mixture was diluted with EtOAc (10 mL) and filtered through a
celite pad. Filtrate was washed with water (2 ꢂ 10 mL) and water
layer was further extracted with ethyl acetate (2 ꢂ 10 mL). After
evaporation of the solvent, the crude mixture was purified by col-
umn chromatography to give product. Yield 13%; pale yellow solid.
(43). This compound was obtained from 42 according to the
methodology described for 39. Yields 30%, 20%, and 42%; white
solid. 1H NMR (400 MHz, CDCl3)
J ¼ 8.8 Hz, 1H), 7.58e7.48 (m, 2H), 7.45e7.34 (m, 3H), 7.07 (dd,
J ¼ 8.8, 2.4 Hz, 1H), 3.92 (s, 3H).
d
7.99 (d, J ¼ 2.2 Hz, 1H), 7.69 (d,
Ethyl 5-methoxy-2-phenylbenzo[b]thiophene-3-carboxylate
(44). To a solution of 43 (1.0 mmol) in 20 mL EtOH followed by a
dropwise addition of concentrated sulfuric acid (6 mL). The mixture
was further stirred for 5 h under reflex condition, the mixture was
diluted with water (20 mL) and extracted with 200 mL ethyl ace-
tate. The organic layer was washed with water (3 ꢂ 20 mL) and
evaporated. The residue was purified by flash chromatography to
1H NMR (400 MHz, DMSO‑d6)
d
12.03 (s, 1H), 7.92 (d, J ¼ 8.4 Hz, 1H),
7.72 (d, J ¼ 9.0 Hz, 1H), 7.64 (s, 1H), 7.50 (s, 1H), 7.46 (d, J ¼ 8.5 Hz,
1H), 7.07 (dd, J ¼ 9.0, 0.9 Hz, 1H), 3.85 (s, 3H). 13C NMR (101 MHz,
DMSO‑d6)
d 159.0, 157.8, 156.8, 149.5, 139.2, 125.3, 124.3, 123.7,
give 44. Yield 95%; transparent oil. 1H NMR (400 MHz, CDCl3)
d
7.96
123.0, 118.4, 115.0, 112.6, 110.5, 110.0, 103.3, 55.7.
(d, J ¼ 2.3 Hz, 1H), 7.66 (d, J ¼ 8.7 Hz, 1H), 7.51e7.46 (m, 2H),
7.44e7.40 (m, 3H), 7.00 (dd, J ¼ 8.7, 2.4 Hz, 1H), 4.20 (q, J ¼ 7.1 Hz,
2H), 1.05 (t, J ¼ 7.1 Hz, 3H).
3-Bromo-8-hydroxybenzofuro[3,2-c]quinolin-6(5H)-one
(50). This compound was obtained from 49 according to the
methodology described for 45. Yield 75%; white solid. 1H NMR
Ethyl 5-hydroxy-2-phenyl-4-(piperidin-1-ylmethyl) benzo[b]
thiophene-3-carboxylate (45). To a solution of 44 (1.0 mmol) in
4 mL anhydrous dichloromethane, BBr3 (1 M in CH2Cl2, 4.0 mmol)
was added at room temperature under N2. After stirring overnight,
the reaction mixture was quenched with EtOH. After evaporation of
the solvent, the crude mixture then employing method C to give 45.
(400 MHz, DMSO‑d6)
d
11.96 (s, 1H), 9.61 (s, 1H), 7.95 (d, J ¼ 8.5 Hz,
1H), 7.68 (s, 1H), 7.64 (d, J ¼ 8.8 Hz, 1H), 7.48 (d, J ¼ 8.4 Hz, 1H), 7.44
(s,1H), 6.94 (d, J ¼ 8.9 Hz,1H). 13C NMR (101 MHz, DMSO‑d6)
d 159.1,
157.7, 154.8, 148.8, 139.2, 125.3, 124.4, 123.6, 123.0, 118.4, 115.0,
112.3, 110.5, 110.1, 105.9.
3-Bromo-8-hydroxy-7-(piperidin-1-ylmethyl)benzofuro[3,2-
c]quinolin-6(5H)-one (51). This compound was obtained from 50
by employing method C. Yield 67%; white solid; m.p > 250 ꢁC. 1H
Overall yield 71%; yellow oil. 1H NMR (400 MHz, CDCl3)
d 7.58 (d,
J ¼ 8.7 Hz, 1H), 7.53e7.51 (m, 2H), 7.43e7.40 (m, 3H), 6.94 (d,
J ¼ 8.7 Hz, 1H), 4.25 (q, J ¼ 7.1 Hz, 2H), 3.87 (s, 2H), 1.70e1.59 (m,
4H), 1.27e1.22 (m, 6H), 1.20 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz,
NMR (400 MHz, CDCl3/CD3OD ¼ 1/1)
d
7.99 (d, J ¼ 8.5 Hz, 1H), 7.70
(s, 1H), 7.63 (d, J ¼ 8.9 Hz, 1H), 7.49 (d, J ¼ 8.6 Hz, 1H), 7.11 (d,
J ¼ 8.9 Hz, 1H), 4.90 (s, 2H), 3.70e3.48 (m, 2H), 3.15e2.98 (m, 2H),
2.05e1.91 (m, 2H), 1.85e1.76 (m, 3H), 1.63e1.52 (m, 1H). 13C NMR
CDCl3)
d 168.4, 157.4, 145.4, 136.5, 133.7, 130.8, 129.0 (2C), 128.9,
128.7 (2C), 125.6, 122.0, 116.6, 114.1, 61.8, 58.6, 54.0 (2C), 25.9 (2C),
24.0, 14.0. HRMS(ESI) m/z: Calcd for C23H26NO3S (M þ H)þ
396.1628, found 396.1649.
(101 MHz, DMSO‑d6) d 159.8, 158.3, 154.8, 148.7, 139.0, 125.8, 125.6,
124.3, 123.3, 118.4, 115.4, 114.1, 111.8, 109.8, 109.0, 52.5, 52.1 (2C),
22,4 (2C), 21.4. HRMS (ESI) m/z: Calcd for C21H20N2O3Br (M þ H)þ,
427.0657, found 427.0646.
2-Bromo-8-hydroxy-7-(piperidin-1-ylmethyl)benzofuro[3,2-
c]quinolin-6(5H)-one (52). This compound was obtained from 4-
bromo-2-ethynylaniline according to the methodology described
for 51. Overall yield 2.2%; white solid; m.p > 250 ꢁC. 1H NMR
5-Bromo-2-ethynylaniline (47). To a solution of 46 (1.0 mmol),
Pd(PPh3)2Cl2 (0.02 mmol) and CuI (0.04 mmol) in 5 mL triethyl-
amine followed by a dropwise addition of trimethylsilylacetylene
(1.5 mmol) under an atmosphere of nitrogen. The resultant reaction
mixture was stirred at rt about 4 h when 46 was consumed, the
mixture was diluted with ethyl acetate (10 mL) and filtered through
the celite and washed with ethyl acetate (2 ꢂ 5 mL). The filtrate was
evaporated. The TMS-ethynylaniline so obtained was dissolved in
5 mL MeOH followed by addition of anhydrous K2CO3 (2.0 mmol)
and the reaction mixture was stirred at rt for 5 h. Upon completion
of the reaction MeOH was evaporated. The residue was dissolved in
20 mL of ethyl acetate and was washed with water (3 ꢂ 20 mL).
Organic phase washed with saturated aqueous NaCl solution, dried
over Na2SO4 then evaporated and the compound was purified using
silica column. Yield 66%; yellow solid. 1H NMR (400 MHz, CDCl3)
(400 MHz, CD3OD)
d
8.24 (d, J ¼ 1.2 Hz, 1H), 7.75 (dd, J ¼ 9.0, 1.9 Hz,
1H), 7.72 (d, J ¼ 9.0 Hz,1H), 7.43 (d, J ¼ 8.8 Hz,1H), 7.13 (d, J ¼ 8.9 Hz,
1H), 4.97 (s, 2H), 3.70e3.57 (m, 2H), 3.21e3.12 (m, 2H), 2.02e1.91
(m, 2H), 1.84e1.71 (m, 3H), 1.65e1.56 (m, 1H). 13C NMR (101 MHz,
CD3OD)
d 162.0, 150.0, 156.2, 151.2, 138.4, 135.3, 126.6, 125.1, 119.1,
116.8, 116.3, 115.6, 114.1, 113.1, 109.8, 54.1, 53.8 (2C), 24.1 (2C), 22.9.
HRMS (ESI) m/z: Calcd for C21H20N2O3Br (M þ H)þ, 427.0657, found
427.0646.
4-Chloro-8-hydroxy-7-(piperidin-1-ylmethyl)benzofuro[3,2-
c]quinolin-6(5H)-one (53). This compound was obtained from 2-
chloro-6-ethynylaniline according to the methodology described
for 51. Overall yield 0.6%; white solid; m.p > 250 ꢁC. 1H NMR
d
7.16 (d, J ¼ 8.2 Hz, 1H), 6.85 (s, 1H), 6.79 (dd, J ¼ 8.2, 1.1 Hz, 1H),
4.30 (s, 2H), 3.42 (s, 1H). 13C NMR (101 MHz, CDCl3)
124.1, 120.9, 117.0, 105.6, 83.6, 79.8.
d 149.6, 133.8,
5-Bromo-2-((2-bromo-5-methoxyphenyl)ethynyl)aniline
(48). To solution of 1-bromo-2-iodo-4-methoxybenzene
(1.2 mmol), Pd(PPh3)2Cl2 (0.02 mmol) and CuI (0.04 mmol) in
mL triethylamine followed by a dropwise addition of 47
(400 MHz, CD3OD)
d
8.01 (d, J ¼ 7.8 Hz, 1H), 7.71 (d, J ¼ 7.5 Hz, 1H),
a
7.64 (d, J ¼ 8.9 Hz, 1H), 7.35 (t, J ¼ 7.9 Hz,1H), 7.09 (d, J ¼ 8.9 Hz,1H),
4.91 (s, 2H), 3.64e3.61 (m, 2H), 3.19e3.14 (m, 2H), 1.98e1.95 (m,
2H), 1.79e1.75 (m, 3H), 1.61e1.58 (m, 1H). 13C NMR (101 MHz,
3
(1.0 mmol) under N2. The resultant reaction mixture was being
stirred at rt overnight. The mixture was diluted with ethyl acetate
(10 mL) and filtered through the celite and washed with ethyl ac-
etate (2 ꢂ 5 mL). The organic phase was added 20 mL water then
extracted with EtOAc (2 ꢂ 20 mL), washed with saturated aqueous
NaCl solution, dried over Na2SO4 then evaporated. The residue was
purified by silica gel column chromatography to give the product.
CD3OD) d 161.7, 160.6, 156.2, 151.2, 135.7, 132.5, 126.4, 124.8, 121.8,
121.3, 116.4, 115.6, 114.1, 112.9, 109.7, 54.0, 53.8 (2C), 24.1 (2C), 22.8.
HRMS (ESI) m/z: Calcd for C21H20N2O3Cl (M þ H)þ, 383.1162, found
383.1138.
3-Chloro-8-hydroxy-7-(piperidin-1-ylmethyl)benzofuro[3,2-
c]quinolin-6(5H)-one (54). This compound was obtained from 5-
chloro-2-ethynylaniline according to the methodology described
for 51. Overall yield 2.8%; white solid; m.p > 250 ꢁC. 1H NMR
Yield 64%; white solid. 1H NMR (400 MHz, CDCl3)
d 7.47 (d,
J ¼ 8.9 Hz,1H), 7.23 (d, J ¼ 8.2 Hz,1H), 7.07 (d, J ¼ 2.3 Hz,1H), 6.89 (s,
(400 MHz, CD3OD)
d
8.01 (d, J ¼ 8.5 Hz, 1H), 7.64 (d, J ¼ 8.9 Hz, 1H),
1H), 6.83 (d, J ¼ 8.2 Hz,1H), 6.77 (dd, J ¼ 8.8, 2.3 Hz,1H), 4.57 (s, 2H),
7.47 (d, J ¼ 1.2 Hz, 1H), 7.35 (dd, J ¼ 8.6, 1.4 Hz, 1H), 7.09 (d,
J ¼ 8.9 Hz, 1H), 4.91 (s, 2H), 3.72e3.47 (m, 2H), 3.26e3.03 (m, 2H),
2.04e1.88 (m, 2H), 1.86e1.69 (m, 3H), 1.67e1.55 (m, 1H). 13C NMR
3.81 (s, 3H). 13C NMR (101 MHz, CDCl3)
d 158.7, 149.6, 133.2, 133.1,
125.9, 124.3, 120.9, 117.5, 117.0, 116.7, 115.8, 106.2, 94.4, 90.0, 55.7.
11

W. Zhang, L.-l. Liu, S. Lun et al.
European Journal of Medicinal Chemistry 213 (2021) 113202
(101 MHz, CD3OD)
d
162.1, 160.6, 156.1, 151.0, 140.2, 138.3, 126.5,
2.3 Hz, 1H), 6.25 (d, J ¼ 2.2 Hz, 1H), 5.17 (s, 2H), 4.52 (s, 2H), 3.79 (s,
124.7, 124.2, 116.7, 116.1, 115.4, 112.3, 111.2, 109.7, 54.1, 53.8 (2C), 24.1
(2C), 22.8. HRMS (ESI) m/z: Calcd for C21H20N2O3Cl (M þ H)þ,
383.1162, found 383.1138.
3H), 3.48 (s, 3H). 13C NMR (101 MHz, CDCl3)
d 161.7, 156.3, 150.2,
133.5, 133.0, 126.7, 119.9, 117.8, 116.9, 104.8, 100.2, 99.4, 94.6, 92.5,
91.5, 56.2, 55.3.
2-Chloro-8-hydroxy-7-(piperidin-1-ylmethyl)benzofuro[3,2-
c]quinolin-6(5H)-one (55). This compound was obtained from 4-
chloro-2-ethynylaniline according to the methodology described
for 51. Overall yield 0.6%; white solid; m.p > 250 ꢁC. 1H NMR
3-Methoxy-8-(methoxymethoxy)benzofuro[3,2-c]quinolin-
6(5H)-one (62). This compound was obtained from 61 according to
the methodology described for 49. Yield 30%; brown solid. 1H NMR
(400 MHz, DMSO‑d6)
d
11.93 (s, 1H), 7.91 (d, J ¼ 8.6 Hz, 1H), 7.70 (d,
(400 MHz, CD3OD)
d
8.03 (d, J ¼ 2.0 Hz, 1H), 7.68 (d, J ¼ 8.9 Hz, 1H),
J ¼ 8.8 Hz, 1H), 7.65 (s, 1H), 7.11e7.07 (m, 2H), 6.94 (d, J ¼ 8.3 Hz,
7.59 (dd, J ¼ 8.9, 2.2 Hz, 1H), 7.45 (d, J ¼ 8.9 Hz, 1H), 7.10 (d,
J ¼ 8.9 Hz, 1H), 4.93 (s, 2H), 3.62e3.59 (m, 2H), 3.18e3.12 (m, 2H),
1.98e1.94 (m, 2H), 1.84e1.70 (m, 3H), 1.63e1.57 (m, 1H). 13C NMR
1H), 5.26 (s, 2H), 3.83 (s, 3H), 3.42 (s, 3H). 13C NMR (101 MHz,
DMSO‑d6) d 161.5,159.3,159.0,154.1,149.8,140.4,124.8,122.6,115.5,
112.2, 111.4, 107.8, 106.9, 104.7, 99.2, 94.7, 55.6, 55.5.
(101 MHz, CD3OD)
d
162.0, 160.0, 156.2, 151.1, 138.0, 132.6, 129.7,
8-Hydroxy-3-methoxybenzofuro[3,2-c]quinolin-6(5H)-one
(63). Treat a solution of 62 (1.0 mmol) in 5 mL MeOH dropwise with
HCl (3 M in aqueous, 8.0 mmol) at rt. Heat the mixture to reflux for
5 h. After cooled to rt, the reaction mixture was evaporated of the
solvent, the crude mixture was purified by column chromatography
to give products. Yield 30%; brown solid. 1H NMR (400 MHz,
126.5, 121.9, 118.9, 116.3, 115.6, 113.6, 113.1, 109.8, 54.0, 53.8 (2C),
24.1 (2C), 22.8. HRMS (ESI) m/z: Calcd for C21H20N2O3Cl (M þ H)þ,
383.1162, found 383.1138.
4-Fluoro-8-hydroxy-7-(piperidin-1-ylmethyl)benzofuro[3,2-
c]quinolin-6(5H)-one (56). This compound was obtained from 2-
ethynyl-6-fluoroaniline according to the methodology described
for 51. Overall yield 0.4%; white solid; m.p > 250 ꢁC. 1H NMR
DMSO‑d6)
d
11.76 (s, 1H), 9.54 (s, 1H), 7.94 (d, J ¼ 8.7 Hz, 1H), 7.59 (d,
J ¼ 8.8 Hz, 1H), 7.41 (d, J ¼ 2.1 Hz, 1H), 7.03 (d, J ¼ 1.6 Hz, 1H), 6.96
(400 MHz, CD3OD)
d
7.91 (d, J ¼ 8.0 Hz, 1H), 7.69 (d, J ¼ 8.9 Hz, 1H),
(dd, J ¼ 8.9, 1.8 Hz, 1H), 6.87 (dd, J ¼ 8.8, 2.2 Hz, 1H), 3.86 (s, 3H). 13
C
7.51e7.43 (m, 1H), 7.39e7.34 (m, 1H), 7.12 (d, J ¼ 9.0 Hz, 1H), 4.96 (s,
2H), 3.65e3.62 (m, 2H), 3.20e3.14 (m, 2H), 1.98e1.95 (m, 2H),
1.79e1.72 (m, 3H), 1.64e1.58 (m, 1H). 13C NMR (101 MHz, CD3OD)
NMR (101 MHz, DMSO‑d6) d 161.3, 159.5, 158.8, 154.6, 148.5, 140.3,
124.8, 122.6, 113.8, 111.9, 111.3107.8, 105.8, 104.8, 99.1, 55.5.
8-Hydroxy-3-methoxy-7-(piperidin-1-ylmethyl)benzofuro
d
161.9, 160.6 (d, JC-F ¼ 4.2 Hz), 156.2, 151.3 (d, JC-F ¼ 247.7 Hz), 151.1,
[3,2-c]quinolin-6(5H)-one (64). This compound was obtained
from 63 by employing method C. Yield 29%; white solid;
128.3 (d, JC-F ¼ 14.6 Hz), 126.6, 124.5 (d, JC-F ¼ 7.2 Hz), 118.5 (d, JC-
¼ 3.9 Hz), 117.4 (d, JC-F ¼ 17.8 Hz), 116.3, 115.6, 114.7 (d, JC-
m.p > 250 ꢁC. 1H NMR (400 MHz, CDCl3/CD3OD ¼ 1/3)
d 7.96 (d,
F
¼ 2.02 Hz), 1132, 109.8, 54.0, 53.8 (2C), 24.1 (2C), 22.8. HRMS (ESI)
J ¼ 8.0 Hz, 1H), 7.39 (dd, J ¼ 8.8, 2.7 Hz, 1H), 6.93e6.90 (m, 2H), 6.85
(d, J ¼ 8.8 Hz, 1H), 4.84 (s, 2H), 3.89 (s, 3H), 2.87e2.56 (m, 4H),
F
m/z: Calcd for C21H20N2O3F (M þ H)þ, 367.1458, found 367.1470.
3-Fluoro-8-hydroxy-7-(piperidin-1-ylmethyl)benzofuro[3,2-
c]quinolin-6(5H)-one (57). This compound was obtained from 2-
ethynyl-5-fluoroaniline according to the methodology described
for 51. Overall yield 3.0%; white solid; m.p > 250 ꢁC. 1H NMR
1.66e1.53 (m, 6H). 13C NMR (101 MHz, CDCl3/CD3OD ¼ 1/3)
d 162.3,
161.0, 160.7, 156.3, 149.4, 139.8, 123.9, 123.4, 115.2, 114.0, 112.5, 111.1,
108.8, 105.9, 98.6, 58.7, 55.7, 53.6 (2C), 25.7 (2C), 23.9. HRMS (ESI)
m/z: Calcd for C22H23N2O4 (M þ H)þ, 379.1658, found 379.1653.
3,8-Dihydroxy-7-(piperidin-1-ylmethyl)benzofuro[3,2-c]qui-
nolin-6(5H)-one (65). This compound was obtained from 64
demethylation according to the methodology described for 45.
Yield 57%; yellow solid; m.p 226.3e227.5 ꢁC. 1H NMR (400 MHz,
(400 MHz, CD3OD)
d 8.10e8.07 (m, 1H), 7.68e7.56 (m, 1H),
7.25e7.12 (m, 2H), 7.12e6.99 (m, 1H), 4.91 (s, 2H), 3.72e3.47 (m,
2H), 3.25e2.98 (m, 2H), 2.00e1.62 (m, 6H). 13C NMR (101 MHz,
CD3OD)
d
165.8 (d, J ¼ 250.0 Hz), 162.3, 161.0, 156.1, 150.9, 141.1 (d,
J ¼ 12.3 Hz), 126.6, 125.3 (d, J ¼ 10.6 Hz), 115.8, 115.3, 112.7 (d,
J ¼ 24.3 Hz), 111.4, 109.7, 109.5, 103.3 (d, J ¼ 26.4 Hz), 54.1, 53.8 (2C),
24.1 (2C), 22.9. HRMS (ESI) m/z: Calcd for C21H20N2O3F (M þ H)þ,
367.1458, found 367.1470.
CD3OD)
d
7.86 (d, J ¼ 9.1 Hz, 1H), 7.57 (d, J ¼ 8.9 Hz, 1H), 7.01 (d,
J ¼ 8.9 Hz, 1H), 6.87e6.85 (m, 2H), 4.87 (s, 1H), 3.71e3.43 (m, 2H),
3.23e3.01 (m, 2H), 1.93e1.60 (m, 6H). 13C NMR (101 MHz, CD3OD)
d
162.6, 162.5, 162.3, 155.8, 150.6, 141.7, 126.8, 124.3, 115.0, 114.9,
2-Fluoro-8-hydroxy-7-(piperidin-1-ylmethyl)benzofuro[3,2-
c]quinolin-6(5H)-one (58). This compound was obtained from 2-
ethynyl-4-fluoroaniline according to the methodology described
for 51. Overall yield 5.0%; white solid; m.p > 250 ꢁC. 1H NMR
114.4, 109.4, 108.9, 105.6, 101.9, 53.9, 53.6 (2C), 24.2 (2C), 22.9.
HRMS (ESI) m/z: Calcd for C21H21N2O4 (M þ H)þ, 365.1501, found
365.1489.
8-Hydroxy-2-methoxy-7-(piperidin-1-ylmethyl)benzofuro
[3,2-c]quinolin-6(5H)-one (66). This compound was obtained
from 2-ethynyl-4-methoxyaniline according to the methodology
described for 64. Overall yield 4.0%; white solid. 1H NMR (400 MHz,
(400 MHz, CD3OD)
d
7.73 (dd, J ¼ 8.3, 2.3 Hz, 1H), 7.65 (d, J ¼ 8.9 Hz,
1H), 7.49 (dd, J ¼ 9.0, 4.4 Hz, 1H), 7.43e7.39 (m, 1H), 7.09 (d,
J ¼ 8.9 Hz, 1H), 4.91 (s, 2H), 3.61e3.58 (m, 2H), 3.17e3.12 (m, 2H),
2.01e1.94 (m, 2H), 1.84e1.69 (m, 3H), 1.60e1.55 (m, 1H). 13C NMR
CD3OD)
d
7.66 (d, J ¼ 8.9 Hz, 1H), 7.47 (d, J ¼ 2.2 Hz, 1H), 7.42 (d,
(101 MHz, DMSO‑d6)
d
159.6, 158.0 (d, JC-F ¼ 3.2 Hz), 157.5 (d, JC-
J ¼ 9.0 Hz, 1H), 7.25 (dd, J ¼ 9.0, 2.2 Hz, 1H), 7.09 (d, J ¼ 8.9 Hz, 1H),
4.91 (s, 2H), 3.93 (s, 3H), 3.61e3.58 (m, 2H), 3.17e3.12 (m, 2H),
2.01e1.95 (m, 2H), 1.85e1.70 (m, 3H), 1.61e1.58 (m, 1H). 13C NMR
¼ 240.5 Hz),154.7,148.7,134.8,125.5,119.5 (d, JC-F ¼ 23.7 Hz),118.3
F
(d, JC-F ¼ 8.2 Hz), 115.5, 114.1, 112.2, 111.2, 108.8, 106.5 (d, JC-
¼ 24.8 Hz), 52.3, 51.9 (2C), 22.2 (2C), 21.2. HRMS (ESI) m/z: Calcd
(101 MHz, CD3OD) d 161.7, 161.1, 157.3, 156.0, 151.0, 134.0, 126.6,
F
for C21H20N2O3F (M þ H)þ, 367.1458, found 367.1470.
122.4, 118.9, 115.9, 115.4, 113.1, 112.2, 109.7, 103.3, 56.3, 53.9, 53.7
(2C), 24.2 (2C), 22.9. HRMS (ESI) m/z: Calcd for C22H23N2O4
(M þ H)þ, 379.1658, found 379.1653.
2-Ethynyl-5-methoxyaniline (60). This compound was ob-
tained from 59 according to the methodology described for 47.
Yield 67%; reddish solid. 1H NMR (400 MHz, CDCl3)
d
7.24 (d,
2,8-Dihydroxy-7-(piperidin-1-ylmethyl)benzofuro[3,2-c]qui-
nolin-6(5H)-one (67). This compound was obtained from 66
demethylation according to the methodology described for 45.
J ¼ 8.5 Hz, 1H), 6.26 (dd, J ¼ 8.5, 2.4 Hz, 1H), 6.22 (d, J ¼ 2.3 Hz, 1H),
4.26 (s, 2H), 3.76 (s, 3H), 3.32 (s, 1H).
2-((2-Bromo-5-(methoxymethoxy)phenyl)ethynyl)-5-
methoxyaniline (61). This compound was obtained from 60 with
1-bromo-2-iodo-4-(methoxymethoxy)benzene according to the
methodology described for 48. Yield 44%; yellow solid. 1H NMR
Yield 37%; white solid. 1H NMR (400 MHz, CD3OD)
d 7.70 (d,
J ¼ 9.0 Hz,1H), 7.45 (d, J ¼ 1.2 Hz,1H), 7.41 (d, J ¼ 8.9 Hz, 1H), 7.18 (d,
J ¼ 8.6 Hz, 1H), 7.11 (d, J ¼ 8.8 Hz, 1H), 4.97 (s, 2H), 3.67e3.52 (m,
2H), 3.17e3.14 (m, 2H), 2.05e1.92 (m, 2H), 1.89e1.73 (m, 3H),
(400 MHz, CDCl3)
7.23 (d, J ¼ 2.9 Hz, 1H), 6.85 (dd, J ¼ 8.8, 2.9 Hz, 1H), 6.31 (dd, J ¼ 8.5,
d
7.47 (d, J ¼ 8.8 Hz, 1H), 7.31 (d, J ¼ 8.5 Hz, 1H),
1.66e1.57 (m, 1H). 13C NMR (101 MHz, CD3OD)
d
161.7, 161.2, 156.0,
154.9, 151.0, 133.1, 126.6, 122.4, 118.7, 115.9, 115.3, 113.4, 112.1, 109.8,
12

W. Zhang, L.-l. Liu, S. Lun et al.
European Journal of Medicinal Chemistry 213 (2021) 113202
106.0, 53.9, 53.6 (2C), 24.2 (2C), 22.9. HRMS (ESI) m/z: Calcd for
C
for each compound was obtained by nonlinear regression curve
fitting of a three-parameter variable slope equation to the dose-
response data using Prism software (GraphPad).
21H21N2O4 (M þ H)þ, 365.1501, found 365.1523.
4.1. General procedures for biological studies
Declaration of competing interest
Antitubercular activity of compounds against the M. tuberculosis
strain H37Rv was determined using the MABA assay as reported in
the literature [21]. Cytotoxicity of selected compounds was deter-
mined using Vero cells and the MABA assay as reported previously
[22].
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Growth inhibition of compound 65 against S. aureus Newman,
B. subtilis 168, E. coli AB1157, and E. coli DH5a was determined using
Acknowledgements
a previously reported protocol [23]. All strains were grown at 37 ꢁC
overnight in 10 mL TSB without antibiotics. Overnight cultures
diluted 1000-fold were grown at 37 ꢁC for 2e3 h until A600 0.6.
Then bacteria were diluted 1:400 into fresh TSB medium. Com-
pound 65 were prepared in DMSO and diluted serially by two-fold
to final concentrations in the range of 0.20e25 mg/mL. Equal vol-
ume of bacteria and compound were added to 96 well plates and
mixed well by shaking. After incubating in 37 ꢁC for 18 h, the MIC of
compound 65 was observed. Vancomycin, Tetracycline and Kana-
mycin was used as positive control. Experiments were performed
three times for each condition.
This work has been supported by the National Natural Science
Foundation of China (21778019). This project has also been funded
in part with Federal funds from the National Institutes of Health
and National Institute of Allergy and Infectious Diseases, Depart-
ment of Health and Human Services under grants AI37856 and
HL133190. H.G. is the recipient of an Australian Research Council
Discovery Early Career Researcher Award (DE160100482) funded
by the Australian Government. The authors thank Prof. Cai-Guang
Yang at Shanghai Institute of Material Medica for performing the
nonpathogenic bacteria selectivity assays and guidance on nanoDSF
and enzymatic activity assay.
Tm of purified Pks13-TE was determined using nanoDSF assay. As
reported in NCBI database, the nucleotide sequence of the Mtb
Pks13 gene (Rv3800c) was obtained. The wild-type Pks13-TE
domain construct gene was cloned and inserted into PMCSG-19
plasmid, which was synthesized by Generay Biotechnology
(Shanghai, China). The expression of the N-Mbp-His tagged Pks13-
TE protein was induced with 0.5 mM IPTG in E. coli BL21 (DE3)pLysS
strains (Shanghai Institute of Materia Media, China), and the cells
were harvested at 20 ꢁC after 18 h of growth. Similar to the pro-
cedures reported previously, the Pks13 TE protein was purified with
>95% purity monitored by SDS-PAGE, and stored at ꢀ80 ꢁC in gel
filtration buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 1 mM
DTT). To a solution of 1 mM of Pks13-TE protein in buffer (100 mM
NaCl, 20 mM Tris-HCl pH 8.0) was incubated with different con-
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113202.
References
[1] Who, Global Tuberculosis Report 2018, World Health Organization, 2018.
[2] J.A. Verschoor, M.S. Baird, J. Grooten, Towards understanding the functional
diversity of cell wall mycolic acids of Mycobacterium tuberculosis, Prog. Lipid
Res. 51 (2012) 325e339.
[3] K.A. Abrahams, G.S. Besra, Mycobacterial cell wall biosynthesis: a multifaceted
antibiotic target, Parasitology 145 (2018) 116e133.
[4] K. Takayama, C. Wang, G.S. Besra, Pathway to synthesis and processing of
mycolic acids in Mycobacterium tuberculosis, Clin. Microbiol. Rev. 18 (2005)
81e101.
[5] S. Gavalda, F. Bardou, F. Laval, C. Bon, W. Malaga, C. Chalut, C. Guilhot,
L. Mourey, M. Daffe, A. Quemard, The polyketide synthase Pks13 catalyzes a
novel mechanism of lipid transfer in mycobacteria, Chem. Biol. 21 (2014)
1660e1669.
[6] R. Wilson, P. Kumar, V. Parashar, C. Vilcheze, R. Veyron-Churlet, J.S. Freundlich,
S.W. Barnes, J.R. Walker, M.J. Szymonifka, E. Marchiano, S. Shenai, R. Colangeli,
W.R. Jacobs Jr., M.B. Neiditch, L. Kremer, D. Alland, Antituberculosis thio-
phenes define a requirement for Pks13 in mycolic acid biosynthesis, Nat.
Chem. Biol. 9 (2013) 499e506.
[7] F. Bergeret, S. Gavalda, C. Chalut, W. Malaga, A. Quemard, J.D. Pedelacq,
M. Daffe, C. Guilhot, L. Mourey, C. Bon, Biochemical and structural study of the
atypical acyltransferase domain from the mycobacterial polyketide synthase
Pks13, J. Biol. Chem. 287 (2012) 33675e33690.
[8] S. Gavalda, M. Leger, B. van der Rest, A. Stella, F. Bardou, H. Montrozier,
C. Chalut, O. Burlet-Schiltz, H. Marrakchi, M. Daffe, A. Quemard, The Pks13/
FadD32 crosstalk for the biosynthesis of mycolic acids in Mycobacterium
tuberculosis, J. Biol. Chem. 284 (2009) 19255e19264.
[9] T.R. Ioerger, T. O’Malley, R. Liao, K.M. Guinn, M.J. Hickey, N. Mohaideen,
K.C. Murphy, H.I. Boshoff, V. Mizrahi, E.J. Rubin, C.M. Sassetti, C.E. Barry 3rd,
D.R. Sherman, T. Parish, J.C. Sacchettini, Identification of new drug targets and
resistance mechanisms in Mycobacterium tuberculosis, PloS One 8 (2013),
e75245.
centrations of tested compounds (1e10 mM in DMSO) in a 30
reaction volume for 30 min, and drug-free proteins containing
DMSO solution served as a blank control. Approximately 10 L of
mL
m
the supernatant fraction was loaded to each capillary, which was
then placed on the holder in the sample rack. The thermal dena-
turation curves were determined by the measurement of the pro-
tein intrinsic fluorescence on label-free native nanoDSF
(NanoTemper, Prometheus NT.48). The temperature was increased
from 30 to 90 ꢁC at a rate of 2 ꢁC$minꢀ1. The fluorescence intensity
was recorded at 330 and 350 nm, respectively. Changes in the
fluorescence ratio (F350/F330) was monitored to determine the
apparent melting temperature (Tm).
Enzyme activity of Pks13-TE were assessed based on previously
reported protocol [10]. IC50 values were measured using 4-
methylumbelliferyl heptanoate (4-MUH) as a fluorogenic sub-
strate. The concentrations of the tested compounds were diluted to
20
0.156
(1 M) in 0.1 M Tris-HCl, pH 7.0 buffer, with 1
of the compound or DMSO in a total volume of 99
in a 96-well plate format. The reaction was initiated by addition of
L of 2 mM 4-MUH in DMSO (20 M final concentration) to the
m
M, 10
m
M, 5
m
M, 2.5
m
M, 1.25
m
M, 0.625
M respectively. Pks13-TE
L of each dilution
L was incubated
mM, 0.3125 mM,
mM, 0.078
m
M, 0.039
mM, 0.0195
m
[10] A. Aggarwal, M.K. Parai, N. Shetty, D. Wallis, L. Woolhiser, C. Hastings,
N.K. Dutta, S. Galaviz, R.C. Dhakal, R. Shrestha, S. Wakabayashi, C. Walpole,
D. Matthews, D. Floyd, P. Scullion, J. Riley, O. Epemolu, S. Norval, T. Snavely,
G.T. Robertson, E.J. Rubin, T.R. Ioerger, F.A. Sirgel, R. van der Merwe, P.D. van
Helden, P. Keller, E.C. Bottger, P.C. Karakousis, A.J. Lenaerts, J.C. Sacchettini,
Development of a novel lead that targets M. tuberculosis polyketide synthase
13, Cell 170 (2017) 249e259 e225.
[11] S. Thanna, S.E. Knudson, A. Grzegorzewicz, S. Kapil, C.M. Goins, D.R. Ronning,
M. Jackson, R.A. Slayden, S.J. Sucheck, Synthesis and evaluation of new 2-
aminothiophenes against Mycobacterium tuberculosis, Org. Biomol. Chem.
14 (2016) 6119e6133.
m
m
m
1
m
m
reaction mix. The fluorescence of the hydrolyzed product 4-
methylumbelliferone was read (excitation at 360 nm and emis-
sion at 465 nm) using Spectra Max Flex Station 3 reader (Molecular
Device) at 10 min intervals over 60 min. The data points were
collected in triplicate and the averaged value was used to generate
concentration-response plots for tested compounds. The IC50 value
[12] J. Lehmann, T.Y. Cheng, A. Aggarwal, A.S. Park, E. Zeiler, R.M. Raju, T. Akopian,
O. Kandror, J.C. Sacchettini, D.B. Moody, E.J. Rubin, S.A. Sieber, An antibacterial
13

W. Zhang, L.-l. Liu, S. Lun et al.
European Journal of Medicinal Chemistry 213 (2021) 113202
beta-lactone kills Mycobacterium tuberculosis by disrupting mycolic acid
biosynthesis, Angew Chem. Int. Ed. Engl. 57 (2018) 348e353.
[13] W. Zhang, S. Lun, S.H. Wang, X.W. Jiang, F. Yang, J. Tang, A.L. Manson,
A.M. Earl, H. Gunosewoyo, W.R. Bishai, L.F. Yu, Identification of novel cou-
mestan derivatives as polyketide synthase 13 inhibitors against Mycobacte-
rium tuberculosis, J. Med. Chem. 61 (2018) 791e803.
[14] S.L. Wei Zhang, Ling-Ling Liu, Shiqi Xiao, Guanfu Duan, Hendra Gunosewoyo,
Yang Fan, Jie Tang, William R. Bishai, Li-Fang Yu, Identification of novel cou-
mestan derivatives as polyketide synthase 13 inhibitors against Mycobacte-
rium tuberculosis. Part II, J. Med. Chem. 61 (2019) 3575e3589.
[15] X. Li, Y. Du, Z. Liang, X. Li, Y. Pan, K. Zhao, Simple conversion of enamines to
2H-azirines and their rearrangements under thermal conditions, Org. Lett. 11
(2009) 2643e2646.
[18] H. Xu, C. Wolf, Efficient copper-catalyzed coupling of aryl chlorides, bromides
and iodides with aqueous ammonia, Chem. Commun. (2009) 3035e3037.
[19] W. Ali, A. Modi, A. Behera, P.R. Mohanta, B.K. Patel, Cs2CO3 as a source of
carbonyl and ethereal oxygen in a Cu-catalysed cascade synthesis of benzo-
furan [3,2-c] quinolin-6[5-H]ones, Org. Biomol. Chem. 14 (2016) 5940e5944.
[20] M. Pieroni, S.K. Tipparaju, S. Lun, Y. Song, A.W. Sturm, W.R. Bishai,
A.P. Kozikowski, Pyrido[1,2-a]benzimidazole-based agents active against
tuberculosis (TB), multidrug-resistant (MDR) TB and extensively drug-
resistant (XDR) TB, ChemMedChem 6 (2011) 334e342.
[21] L. Collins, S.G. Franzblau, Microplate alamar blue assay versus BACTEC 460
system for high-throughput screening of compounds against Mycobacterium
tuberculosis and Mycobacterium avium, Antimicrob. Agents Chemother. 41
(1997) 1004e1009.
[16] U.A. Kshirsagar, R. Parnes, H. Goldshtein, R. Ofir, R. Zarivach, D. Pappo, Aerobic
iron-based cross-dehydrogenative coupling enables efficient diversity-
oriented synthesis of coumestrol-based selective estrogen receptor modula-
tors, Chemistry 19 (2013) 13575e13583.
[22] S. Lun, H. Guo, O.K. Onajole, M. Pieroni, H. Gunosewoyo, G. Chen,
S.K. Tipparaju, N.C. Ammerman, A.P. Kozikowski, W.R. Bishai, Indoleamides
are active against drug-resistant Mycobacterium tuberculosis, Nat. Commun.
4 (2013) 2907.
[17] K. Chen, S.C. Kuo, M.C. Hsieh, A. Mauger, C.M. Lin, E. Hamel, K.H. Lee, Anti-
tumor agents. 174. 2’,3’,4’,5,6,7-Substituted 2-phenyl-1,8-naphthyridin-4-
ones: their synthesis, cytotoxicity, and inhibition of tubulin polymerization,
J. Med. Chem. 40 (1997) 2266e2275.
[23] J. Wang, T. Yang, H. Chen, Y.N. Xu, L.F. Yu, T. Liu, J. Tang, Z. Yi, C.G. Yang,
W. Xue, F. Yang, The synthesis and antistaphylococcal activity of 9, 13-
disubstituted berberine derivatives, Eur. J. Med. Chem. 127 (2017) 424e433.
14