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

Article abstract of DOI:10.1021/acs.jmedchem.7b01319

Inhibition of the mycolic acid pathway has proven a viable strategy in antitubercular drug discovery. The AccA3/AccD4/FadD32/Pks13 complex of Mycobacterium tuberculosis constitutes an essential biosynthetic mechanism for mycolic acids. Small molecules targeting the thioesterase domain of Pks13 have been reported, including a benzofuran-based compound whose X-ray cocrystal structure has been very recently solved. Its initial inactivity in a serum inhibition titration (SIT) assay led us to further probe other structurally related benzofurans with the aim to improve their potency and bioavailability. Herein, we report our preliminary structure-activity relationship studies around this scaffold, highlighting a natural product-inspired cyclization strategy to form coumestans that are shown to be active in SIT. Whole genome deep sequencing of the coumestan-resistant mutants confirmed a single nucleotide polymorphism in the pks13 gene responsible for the resistance phenotype, demonstrating the druggability of this target for the development of new antitubercular agents.

Full text of DOI:10.1021/acs.jmedchem.7b01319

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Article
Identification of Novel Coumestan Derivatives as Polyketide
Synthase 13 Inhibitors Against Mycobacterium Tuberculosis
Wei Zhang, Shichun Lun, Shu-Huan Wang, Xingwu Jiang, Fan Yang, Jie Tang, Abigail
L. Manson, Ashlee M. Earl, Hendra Gunosewoyo, William R. Bishai, and Li-Fang Yu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01319 • Publication Date (Web): 12 Jan 2018
Downloaded from http://pubs.acs.org on January 12, 2018
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Identification of Novel Coumestan Derivatives as Polyketide Synthase 13
Inhibitors against Mycobacterium Tuberculosis
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Wei Zhang,†, # Shichun Lun,§, # Shu-Huan Wang,† Xing-Wu Jiang,ǁ Fan Yang,† Jie Tang, Abigail L.
Manson,‡ Ashlee M. Earl,‡ Hendra Gunosewoyo,*, ┴ William R. Bishai,*, § and Li-Fang Yu*, †
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†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
ǁShanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of
Life Sciences East China Normal University, 500 Dongchuan Road, Shanghai 200241, China
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
┴School of Pharmacy, Faculty of Health Sciences, Curtin University, Bentley, Perth, WA 6102,
Australia
‡Broad Institute of MIT & Harvard, 415 Main St., Cambridge, MA 02142, United States
ABSTRACT
Inhibition of the mycolic acid pathway has proven a viable strategy in antitubercular drug discovery.
The AccA3/AccD4/FadD32/Pks13 complex of Mycobacterium tuberculosis constitutes an essential
biosynthetic mechanism for mycolic acids. Small molecules targeting the thioesterase domain of Pks13
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have been reported, including a benzofuran-based compound, whose X-ray co-crystal structure has been
very recently solved. Its initial inactivity in a serum inhibition titration (SIT) assay led us to further
probe other structurally-related benzofurans with the aim to improve their potency and bioavailability.
Herein we report our preliminary structure-activity relationship studies around this scaffold, highlighting
a natural products-inspired cyclization strategy to form coumestans that are shown to be active in SIT.
Whole genome deep sequencing of the coumestan-resistant mutants confirmed a single nucleotide
polymorphism in the pks13 gene responsible for the resistance phenotype, demonstrating the
druggability of this target for development of new antitubercular agents.
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INTRODUCTION
Tuberculosis (TB) is an insidious infectious disease caused by Mycobacterium tuberculosis (Mtb),
which has become a serious public health challenge globally.1, 2 According to the 2016 World Health
Organization (WHO) report, there were 10.4 million incident cases of TB worldwide in 2015,
accompanied with 1.8 million deaths. This has officially made TB the leading infectious disease killer,
surpassing HIV/AIDS. Global control efforts have been hampered by the widespread occurrence of
drug-resistant forms of TB, namely multidrug-resistant (MDR), extensively drug-resistant (XDR) and
totally drug-resistant (TDR) TB, as well as co-infection with HIV/AIDS in endemic areas. The WHO
reported that 400,000 out of the 1.8 million deaths caused by TB in 2015 were caused by TB-HIV co-
infection. These alarming figures clearly highlight the global burden caused by this pathogen,
necessitating the urgent development of novel chemical therapies. Drug discovery efforts towards this
goal have thus far resulted in the accelerated approval of two anti-TB drugs, bedaquiline3, 4 (1) and
delamanid5 (2), as part of the treatment regimen for MDR and XDR-TB (Figure 1). The diarylquinoline
bedaquiline specifically binds to subunit c of the mycobacterial ATP synthase and inhibits the energy
metabolism of Mtb. Delamanid belongs to the nitroimidazole derivatives that require bioactivation by an
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intracellular mycobacterial deazaflavin-dependent nitroreductase, resulting in the inhibition of mycolic
acid synthesis.6 Being the only two new drugs approved in the last five years, their use in MDR-TB
needs to be closely monitored for their safety and efficacy as per the WHO interim recommendations,
especially when administered in combination with antiretrovirals for HIV. Prolongations of QT interval
have been associated7 with the use of these two drugs in the clinic, urging discovery of more effective
agents with less serious side effects.
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Figure 1. Chemical structures of bedaquiline (1), delamanid (2), thiophene-based Pks13 inhibitors (3–
5), and a benzofuran-based Pks13 inhibitor TAM1 (6).
Mtb possesses a unique, thick cell wall complex composed of a variety of lipid components that aid in
survival and pathogenicity. In particular, mycolic acids are the characteristic, large 3-hydroxy, 2-alkyl
long chain fatty acids that form an integral component of the mycomembrane. They influence the cell
wall permeability and overall integrity of the cell.8 The biosynthesis of mycolic acid involves a complex
consisting of the acyl-AMP ligase (FadD32) and polyketide synthase 13 (Pks13), encoded by the
fadD32-pks13-accD4 cluster.9 Pks13 catalyzes a key Claisen condensation step between the long
meromycolic acid and saturated α-alkyl chain to form the α-alkyl β-ketoacids, the precursors for
mycolic acids biosynthesis.9 The Pks13 complex belongs to the type I polyketide synthase (PKS) gene
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family10 and is structurally made up of acyl carrier protein (ACP) domains located at the amino terminus
(N-ACP) and carboxy-terminus (C-ACP), a ketoacylsynthase (KS), an acyl transferase (AT) and a
thioesterase (TE) domain.11 The TE domain acts as a transacylase and transfers the ACP-bound
mycolates onto trehalose to provide the oxo form of trehalose monomycolate (TMM-o).12, 13 Further
reduction by the Mtb homolog of Corynebacterineae mycolate reductase A (CmrA) provides trehalose
monomycolate (TMM), which are then shuttled to the mycomembrane by the MmpL3 transporter.14, 15
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Inhibition of pathways related to mycolic acid synthesis has generally been accepted as a viable
approach for anti-TB drug discovery, as evidenced by the clinical use of the first-line drug isoniazid,
ethionamide, and more recently, preclinical efficacy of reported MmpL3 inhibitors, such as
indoleamides and adamantyl ureas.14, 15 The Alland group demonstrated the intracellular bactericidal
efficacy of thiophene-based Pks13 inhibitors, exemplified by compounds 3 and 4 (Figure 1), and that
sterilizing activity was obtained upon combination with isoniazid.16 Based on whole genome sequencing
of thiophene-resistant mutants, compounds 3 (MIC = 1.0 ꢀM) and 4 (MIC = 0.5 ꢀM) were found to
inhibit Pks13 complex by interfering with the function of the N-ACP domain, thereby compromising the
loading of meromycoloyl-AMP by FadD32.16 The Sucheck group reported a library of 42
aminothiophenes, with the most active compound 5 (Figure 1) having an MIC value of 0.2–0.4 ꢀM
against Mtb strains resistant to isoniazid, rifampin and fluoroquinolones.17
In 2013, compound 6 was discovered as a Pks13 inhibitor from high throughput screening efforts
combined with whole-genome sequencing of the resistant isolates.18 This compound possesses an MIC
value of 2.0 ꢀM, and the mutations observed (D1644G and D1607N) in the resistant colonies were
found in the carboxy terminus of the thioesterase domain of Pks13. However, our group herein
demonstrated that compound 6 was not active in the mouse serum inhibition titration assay, suggesting
its poor bioavailability. During the preparation of this manuscript, Sacchettini group reported their
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follow-up findings on this compound, culminating in a 2-phenol-3-amidobenzofuran analog TAM-16
that was shown to be efficacious in a murine model of TB, along with an X-ray co-crystal structure of
their initial compound 6 bound to the Pks13.19 In this article, we present our efforts in the SAR
optimization of compound 6 that led to the identification of coumestan analogues as potential novel anti-
TB compounds in vitro, and more importantly, showing activity in the mouse SIT assays.
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RESULTS AND DISCUSSION
Chemistry
The benzofuran derivatives 6 and 8–36 were synthesized in 2–5 steps utilizing the synthetic routes
shown in Scheme 1. Compound 8 was formed via a copper-catalyzed cyclization of 1,4-benzoquinones
and ethyl 3-oxo-3-phenylpropanoate, which was prepared as described in the literature from
commercially available acetophenone 7.20 Compound 8 underwent Mannich reaction with 37% aqueous
formaldehyde and appropriate secondary amines to yield compounds 6 and 10–15. O-Methylation of the
phenolic group of 10 with methyl iodide afforded the methoxy derivative 20. When primary amines
were used in the Mannich reaction with intermediate 8, only the 1,3-oxazinanes 16–19 were formed and
no ring-opened product was observed. The structure of 1,3-oxazinane 16 was confirmed by X-ray
crystallography (Scheme 1). Hydrolysis of the ethyl ester 8 under basic condition afforded the
carboxylic acid 9, which was subjected to subsequent amide coupling and Mannich reaction with
piperidine to afford compounds 22–26. Similarly, compounds 27–33 and 34–36 were synthesized in a
similar manner to compounds 10 and 16 from commercially available substituted acetophenones.
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Scheme 1a
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a Reagents and conditions: (a) benzoquinone, Cu(OTf)2 (5 mol %), toluene, reflux, 7–12 hr; (b) LiOH,
1,4-dioxane, H2O, 60 ºC, 24 hr; (c) formaldehyde (37% aq), corresponding secondary amines, EtOH,
reflux, 8–12 hr; (d) CH3I, NaH, DMF, 0 ºC, 1 hr; (e) formaldehyde (37% aq), corresponding primary
amines, EtOH, reflux, 8–12 hr; (f) EDC·HCl, HOBt, 3-methylpyridine, corresponding amines, DMF, rt,
5–8 hr; (g) diethyl carbonate, NaH, toluene, reflux, 30 min.
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Scheme 2a
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aReagents and conditions: (a) diethyl carbonate, NaH, toluene, reflux, 30 min; (b) benzoquinone,
Cu(OTf)2 (5 mol %), toluene, reflux, 7–12 hr; (c) CH3I, K2CO3, THF, rt, 3–5 hr; (d) BBr3 in CH2Cl2 (1
M), CH2Cl2, rt, overnight, then EtOH, reflux, 1 hr; (e) formaldehyde (37% aq), piperidine, EtOH, reflux,
8–12 hr; (f) formaldehyde (37% aq), cyclopropylmethanamine, EtOH, reflux, 8–12 hr.
Following a similar procedure to synthesize compound 8, ethyl 5-hydroxy-2-(2-methoxyphenyl)
benzofuran-3-carboxylate 39 was formed starting from the commercially available 2-acetylphenol 37
(Scheme 2). Coumestan 40 was prepared via cyclization of ester 39 in refluxing ethanol.21 Subsequent
Mannich reaction afforded coumestan derivatives 41 and 47 (Scheme 2). Coumestans 42–46 and 48–57
were synthesized in a similar manner to compounds 41 and 47 respectively, from commercially
available substituted 2-acetylanisoles.
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Initial SAR for Benzofurans.
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All the final compounds were initially evaluated for their MIC values against Mtb strain H37Rv in a
microplate alamar blue assay (MABA).22 The original hit compound 6 (TAM1) had an MIC value of 1
ꢀg/mL, corresponding to 2.5 ꢀM, which was consistent with the reported activity.18 As shown in Table
1, we first examined the substitutions at position 4 of the benzofuran ring (the R1 group). Deletion of the
(methylpiperidin-1-yl)methyl to give compound 8 resulted in at least a 64-fold reduction of activity,
indicating the importance of R1 substitution, which is consistent with the SAR trend reported
previously.19 Piperidin-1-yl-methyl and pyrrolidin-1-yl-methyl (10 and 11) substituents at R1 generally
maintained activity, but the ring-open diisopropylamine analog 12 had an 8-fold reduction in activity.
Addition of polar atoms to the piperidine ring (13–15) were found to be deleterious for the activity, with
MIC values ranging from 16 to >64 ꢀg/mL. The SAR trends observed herein are consistent with those
reported by Sacchettini group. The X-ray co-crystal structure demonstrated the binding mode of these
benzofuran derivatives, with the piperidine and 5-hydroxy groups binding deeper into the pocket of the
Pks13 thioesterase domain’s active site, leaving the 2-phenyl and 3-ester groups more exposed to
solvents.19 The cyclization of 4-aminomethyl substituents with 5-hydroxyl group of the benzofurans
resulted in compounds 16–19 with MIC values ranging from 1–16 ꢀg/mL. Among this series, the
cycloaliphatic derivatives 16 and 18 were found to be more potent, with the cyclopropylmethyl analog
16 having an MIC value of 1 ꢀg/mL. Methylation of the 5-hydroxyl group of compound 10 to give the
methoxy analog 20 also resulted in an 8-fold decrease of activity (MIC = 16 ꢀg/mL), indicating that the
5-hydroxy group may provide an optimal hydrogen bond to the carboxylate group of D1644.19 Next, we
assessed the effects of substituting the benzene ring at 2-position of the benzofurans with various
electron-donating groups (EDG; OMe, Me) and electron-withdrawing groups (EWG; F, Br, CF3). At the
para position, a fluoro group (compound 27) resulted in a 4-fold increase of activity with MIC value of
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0.5 ꢀg/mL, while a trifluoromethyl or methoxy group (28 or 29) resulted in compounds with MIC values
4
5
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of 8 and 16 ꢀg/mL respectively. The most potent benzofuran derivative reported by Sacchettini group
was TAM16, which has a 4-hydroxyl substituent on the benzene ring.19 Various di- or tri-substitutions
on this benzene ring (30–33) generally resulted in compounds with reduced antitubercular activity (MIC
values of 4 to >64 ꢀg/mL), particularly with a 2-methoxy group being deleterious for activity. Within
the oxazine series (34–36), EDG substitutions were generally less tolerated with activities ranging from
2 to 8 ꢀg/mL.
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Compounds containing an ester group are prone to hydrolysis by various endogenous esterases.
Carboxylic acid derivative 21 was completely inactive (MIC value > 64 ꢀg/mL), suggesting that these 2-
phenylbenzofuran-3-carboxylates may be prone to metabolic liability, which was also reported by
Sacchettini group. Subsequently, we investigated the replacement of ethyl ester with substituted amides
22–26. The replacement of ethyl ester with an ethyl amide (22) resulted in a slight drop in activity (MIC
= 4 ꢀg/mL). The MIC values further dropped with increasing size of the amide substituents, ranging
from cyclopropyl to adamantyl (23–26). This is consistent with the obtained X-ray co-crystal structure
showing the ester group is partially solvent exposed.
Table 1. Antitubercular activity of benzofuran derivatives against the M. tuberculosis strain H37Rv.a
R1
H
R2
R3
H
MICa, ꢀg/mL
cLogPb
6.53
Compound
OCH2CH3
OCH2CH3
1
6
8
H
> 64
5.04
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OCH2CH3
H
H
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6.01
5.45
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OCH2CH3
OCH2CH3
H
8
6.94
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OCH2CH3
OCH2CH3
OCH2CH3
OCH2CH3
H
H
H
H
> 64
≥ 64
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4.73
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1
OCH2CH3
OCH2CH3
H
H
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2
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OCH2CH3
-
H
4
16
> 64
4
7.16
6.44
2.04
4.15
4.21
6.28
5.59
5.41
6.16
6.90
5.95
5.66
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-
-
OH
H
N-CH2CH3
N-cyclopropyl
N-1-adamantyl
N-Bn
H
H
8
H
H
> 64
16
8
N-Ph
H
OCH2CH3
OCH2CH3
OCH2CH3
OCH2CH3
4-F
0.5
8
4-CF3
4-OCH3
2-OCH3, 4-F
16
8
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OCH2CH3
2-OCH3, 4-Br
2,4-di-OCH3
2-OCH3, 4,5-di-CH3
4-OCH3
>64
16
4
6.38
5.47
6.34
6.09
6.65
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OCH2CH3
OCH2CH3
OCH2CH3
OCH2CH3
OCH2CH3
2
4-CH3
4
2-OCH3-5-CH3
8
aSee Experimental Section. The lowest concentration of drug leading to at least 90% inhibition of
bacterial growth signal by the MABA. MIC values are reported as an average of three individual
measurements. CLogP was calculated using ChemBioDraw Ultra 13.0.
Identification of the Coumestan Analogues.
Natural products have been long recognized as a rich source of lead compounds for drug development.23
More than half of the clinically available therapeutic agents were derived originally from natural
products and their related structures. Coumestan is a δ-lactone containing a tetracycle, which constitutes
the central core of various bioactive natural products such as coumestrol, wedelolactone, and psoralidin,
among others. A broad range of pharmacological activities of coumestans has been reported, including
anticancer, antioxidant, antibacterial and antidepressant effects.24-26 We envisaged that the ester oxygen
of the previously described benzofuran series could be connected to the adjacent ortho-position of the 2-
phenyl ring to form a coumestan framework (Figure 2). This approach is expected to circumvent the
ester liability issue that may arise in the original benzofuran series. Therefore, in the next iteration of
SAR studies, we employed the initial SAR obtained from the benzofuran series to the coumestans.
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Figure 2. From the benzofurans to the coumestans.
Gratifyingly, the coumestan derivatives 41–57 generally exhibited improved antitubercular activity
against the Mtb strain H37Rv as shown in Table 2. Compound 41, which is the coumestan derivative of
compound 10, had an 8- to 16-fold increase in activity with an MIC value of 0.125–0.25 ꢀg/mL.
Similarly, the antitubercular activity of the coumestans were either improved or maintained when
compared to the corresponding ethyl 2-phenylbenzofuran-3-carboxylates, e.g. 41 vs 10, 42 vs 29, and 43
vs 27. A bulkier 4-bromo derivative 44 and the smaller 5-methyl analog 45 also maintained activities
between 0.5 and 1 ꢀg/mL. Monosubstitutions on the right hand side benzene ring of the coumestans
with various EDG and EWG were generally tolerated, with the exception of 5-methoxy analog 46 (MIC
value of 4 ꢀg/mL). Among the oxazine-coumestans 47–57, the unsubstituted and the 4-methoxy analogs
47 and 48 were of similar activities compared to their direct benzofuran analogs 16 and 34, respectively.
However, EDG (methyl or methoxy) and EWG (halogen) substitutions at the 4- or 5-position were
generally less tolerated, with MIC values ranging from 4 ꢀg/mL to inactive (> 64 ꢀg/mL). Within the
oxazine-coumestans, the pyrrolidine analog 56 emerged as the most potent analog with an MIC value of
0.5 ꢀg/mL.
Table 2. Antitubercular activity of coumestan derivatives against the M. tuberculosis strain H37Rv a
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Compound
R1
-
R2
-
MICa, ꢀg/mL
CLogPb
3.19
4.17
4.24
4.31
5.03
4.67
4.24
4.37
10
11
12
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41
42
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57
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59
60
> 64
40
41
42
43
44
45
46
47
H
H
H
H
H
H
H
0.125–0.25
4-OCH3
4-F
0.25
1
4-Br
5-CH3
5-OCH3
H
1
0.5
4
2
4-OCH3
5-F
2
16
16
8
4.57
4.51
5.08
5.23
4.57
4.86
5.67
5.31
4.77
48
49
50
51
52
53
54
55
56
5-Cl
5-Br
5-OCH3
5-CH3
4,5-di-Cl
4,5-di-CH3
H
16
4
≥ 64
8
0.5
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H
16
5.33
57
5
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9
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
measurements. bCLogP was calculated using ChemBioDraw Ultra 13.0.
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11
12
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27
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41
42
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56
57
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Toxicity to Mammalian Cells.
All compounds showing MIC values lower than 1 µg/mL were next evaluated for their toxicity to Vero
cells, which are derived from African green monkey’s kidney. Comparison of the Vero cells toxicity
(IC50 values) and the MIC values for Mtb growth inhibition gives a measure of general toxicity profile of
the compound in interest. The selectivity index (SI) values were calculated from the ratio of IC50 (Vero)
/MIC
as shown in Table 3. Most selected compounds were found to possess low to moderate SI
(Mtb)
values of between 8–32 when compared to Vero cells, with the exception of compound 56, which
exhibited equal values of IC50 and MIC.
Table 3. Vero toxicity and Selectivity index (SI) of selected compounds.
Toxicity to Vero cells
Compound
MIC H37Rv, ꢀg/mL
SIa
IC50, ꢀg/mL
16
8
1
16
8
6
1
16
27
41
42
43
16
4
0.5
0.125–0.25
0.25
32
16–32
16
4
8
1
8
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8
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1
44
45
56
0.5
0.5
0.5
9
10
11
a SI = IC50 (Vero cells) / MIC (H37Rv)
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In addition to the Vero cells, the two most potent coumestans 41 and 42 were evaluated for their
cytotoxicity profiles against four human-derived cell lines: MRC-5 and HFL1 (human lung fibroblast
cells), QSG-7701 (human liver cell), and HEK-293 (human embryonic kidney cell). As shown in Table
4, both coumestans 41 and 42 were generally much less toxic to the human-derived cell lines, with SI
values between 155 and 400. The methoxy-substituted coumestan 42 was slightly more toxic to the
HFL-1 and QSG-7701 cells with IC50 values of 50.2 and 38.8 ꢀM, respectively. The observed trend
among the four human cell lines is consistent with that seen in the Vero cells, with compound 41
possessing slightly higher selectivity index compared to compound 42. Collectively, the coumestans are
shown to be non-cytotoxic to these human-derived cell lines at concentrations of more than 100-fold
their MIC values for M.tb.
Table 4. Toxicity of 41 and 42 in human-derived cell lines.
MRC-5 cells
(IC50, ꢀg/mL)a
>50
HFL-1 cells
(IC50, ꢀg/mL)
>50
QSG-7701 cells
(IC50, ꢀg/mL)
>50
HEK-293 cells
(IC50, ꢀg/mL)
>50
Compound
41
42
>50
50.2 5.3
38.8 2.6
>50
a IC50 values were obtained as an average from three independent experiments and are expressed in
ꢀg/mL
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Serum Inhibition Titration (SIT) Assay.
8
9
The most potent benzofuran 27 and the coumestans 41 and 42, as well as the hit compound 6, were
evaluated in the SIT assay.22 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 of compounds 6,
27, 41, 42, or 10 mg/kg of INH, with blood collected at 30, 60 and 120 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, the original hit benzofuran 6 was inactive in the SIT assay, most likely due to the
hydrolysis of its ethyl ester into inactive carboxylic acid derivative. A similar trend was observed for the
most potent benzofuran derivative 27, supporting evidence for the ester metabolic liability. The
coumestans 41 and 42, however, showed activity in the SIT assay up to 1:32 / 1:64 dilution (Figure 3).
While the blood samples from 30, 60 and 120 min gave very comparable inhibitory effects for
coumestan 42, the blood sample from 30-min cohort gave the most potent inhibition for coumestan 41.
This result is consistent with the human microsomal stability data obtained for coumestans 41 and 42, in
which 14.5% and 45.5% remained non-metabolized, respectively, after incubation for 60 min at 10 ꢀM
concentration (see Supplementary Information).
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Figure 3. Serum inhibition titration data for compounds 6, 27, 41, and 42.
27
28
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Target Verification.
Nine resistant mutants of M. tuberculosis were selected from 7H11 agar plates containing compound 41,
compound 42 and compound 27, respectively (Table 5). MIC shifts of 8- to 128-fold were observed
between the mutated and wild-type strains. Again, the coumestan-resistant mutants showed more
dramatic MIC shifts (64- to 128-fold, for 41 and 42) compared to the benzofuran-resistant mutants (8-
fold, for 27). Genomic DNA was subsequently isolated from each mutant and the non-mutated parent
strain, and subjected to whole genome sequencing and comparative analysis (see Methods). As shown in
Table 6, all nine clones contained one of four distinct non-synonymous mutations in the pks13 gene.
Strikingly, six out of the nine clones contained only a pks13 gene mutation (A1667V, D1644G, N1640K
or N1640S). The other three resistant mutants contained a single additional non-synonymous mutation
within one of three genes encoding distinct hypothetical proteins. Given that pks13 was the only
consistently altered gene, we do not suspect a role for these other mutations in the resistant phenotype.
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Interestingly, the D1644G mutation observed for the coumestan 41-resistant mutant also conferred
resistance18 to the benzofuran 6 and is proximal to the more frequently observed N1640K in this study.
All the observed mutation sites (A1667V, D1644G, N1640K or N1640S) are co-localized within the
active site of the thioesterase domain of Pks13, consistent with the disruption of their binding as seen in
the obtained X-ray co-crystal structure.14
10
11
12
13
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26
27
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29
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
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59
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Table 5. Selection of resistant mutants.
Selection
WT MIC
Clones
Mutant MIC
MIC shift
(fold)
Compound
concentration
(ꢀg/mL)
selected
(ꢀg/mL)
(ꢀg/mL)
0.125-0.25
0.25
3
4
2
3
3
3
16
16
4
64-128
41
42
27
64
8
0.5
Table 6. Single nucleotide polymorphisms identified in resistant mutants.a
Compound 41
Compound 42 Compound 27
# of clones
with SNP
ORF
Gene
AA change
M95I
1
2
3
1
2
3
1
2
3
Conserved
hypothetical
Conserved
hypothetical
pks13
Rv1765c
Rv2015c
1
1
X
R58L
X
Rv3800c
Rv3800c
A1667V
D1644G
2
1
X
X
pks13
X
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Rv3800c
Rv3800c
pks13
pks13
N1640K
N1640S
5
1
X
X
X
X
X
X
7
8
9
10
11
12
Conserved
membrane
Rv3921c
A312V
1
X
aSee Experimental Section.
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Conclusions
Herein we reported the identification of coumestan analogs as novel antitubercular compounds targeting
Pks13, resulting from natural products-inspired structural modifications of the initial hit compound
benzofuran 6. These coumestans represent a natural scaffold that showed reasonably potent anti-TB
activity in vitro, as well as oral bioavailability in mouse. As Pks13 is intimately involved in the
biosynthesis of mycolic acids, which is also the target of well-known anti-tuberculosis drugs such as
isoniazid and ethionamide, this study and others support the notion that biochemical reactions on the
pathway to synthesis and processing of mycolic acids are viable targets for new anti-TB drug discovery
(target validation). In the present study, whole genome deep sequencing of the wild-type and resistant
mutants confirmed that these coumestans indeed inhibit pks13, since single SNPs in pks13 led to
resistance phenotypes, thus demonstrating the druggability for pks13. The mutation sites observed in our
resistant mutants correspond to the active site of the thioesterase domain of Pks13, consistent with the
reported X-ray co-crystal structure reported by Sacchettini group. Further investigation of the potential
use of these coumestans as anti-TB therapeutics is warranted and work is underway to improve their
general toxicity profile.
Experimental Section
General Methods: Starting materials, reagents, and solvents were purchased from commercial suppliers
and used without further purification, unless otherwise stated. Anhydrous THF and CH2Cl2 were
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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 reaction vessels
were oven-dried. The progress of reactions was monitored by TLC on SiO2 and LCMS. Silica gel for
column chromatography (CC) was of 200−300 mesh particle size, and an EtOAc/petroleum ether
10
11
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
mixture or gradient was used unless stated otherwise. H NMR spectra were recorded at a spectrometer
frequency of 400 MHz, and 13C NMR spectra were recorded at 101 MHz. Chemical shifts were reported
in δ (ppm) using the δ 0 signal of tetramethylsilane (TMS) 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 an Agilent
1260 HPLC system or a Waters HPLC system using either a Phenomenex Luna-C18 column (5 micron,
250 x 4.6 mm) or InertSustain-C18 column (5 micron, 250 x 4.6 mm) with detection at 280, 254 and 220
nm on a variable wavelength detector G1314F. Method A: Phenomenex Luna C18 column, flow rate =
1.0 mL/min, gradient elution of 30–90% acetonitrile in water (containing 0.05 vol% of HCO2H) over 20
min or Method B: InertSustain C18 column, flow rate = 1.0 mL/min, gradient of 30–90% acetonitrile in
water (containing 0.05 vol% of HCO2H) over 25 min. See Supplementary Information for analytical
HPLC purities and the melting points for all final compounds.
General Procedure for the Synthesis of 5-Hydroxy-2-phenyl Benzofurans from ethyl
Benzoylacetates. (Method A). To a solution of ethyl benzoylacetate (1.3 mmol) and Cu(OTf)2 (5
mol%) in toluene (5 mL) was added a solution of benzoquinone (1.0 mmol) in toluene (1.5 mL) under
N2. The reaction mixture was allowed to reflux for 8–12 hrs and then cooled to rt. The reaction mixture
was quenched with saturated NH4Cl solution and extracted with EtOAc (2×20 mL). The combined
organic phases were washed with water (3×10 mL), dried over anhydrous Na2SO4 and evaporated. The
residue was purified by flash chromatography to give the 5-hydroxy-2-phenyl benzofuran.
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General Procedure for the Mannich Reaction of 5-Hydroxy-2-phenylbenzofuran or Coumestan
with Amine and Formaldehyde (Method B). To a solution of 5-hydroxy-2-phenylbenzofuran or
coumestan derivative (1 mmol) in ethanol (3 mL) were added formaldehyde (37% in water, 4 mmol) and
the appropriate amine (4 mmol) at rt under N2. The reaction mixture was allowed to reflux for 5–12 hr
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.
10
11
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60
General Procedure for the Amidation of 5-Hydroxy-2-phenylbenzofuran-3-carboxylic Acid
(Method C). To the solution of compound 9 (1 mmol) in anhydrous DMF (5 mL), EDC·HCl (1.3
mmol), HOBt (1.3 mmol), 3-methylpyridine (2.1 mmol) and appropriate amine (1.2 mmol) under N2
were added at rt. After stirring overnight at rt, the reaction mixture was quenched with saturated NH4Cl
solution and extracted with EtOAc (2×30 mL). The combined organic phases were washed with HCl
(5% aqueous solution), saturated aqueous NaHCO3 solution, brine, dried over anhydrous Na2SO4 and
evaporated. The residue was purified by flash chromatography to give the amide product.
General Procedure for the Preparation of Coumestans from Substituted Ethyl 5-hydroxy-2-(2-
methoxyphenyl)benzofuran-3-carboxylates (Method D). To a solution of ethyl 5-hydroxy-2-(2-
methoxyphenyl)benzofuran-3-carboxylate (1.0 mmol) in anhydrous CH2Cl2 (10 ml)BBr3 (1M in
CH2Cl2, 2.0 mmol) was added at rt under N2. After stirring overnight, the reaction mixture was
quenched with EtOH. The resulting mixture was allowed to reflux for 1 hr and evaporated. The residue
was purified by flash chromatography or by recrystallization in EtOH to obtain coumestans.
General Procedure for the Synthesis of Ethyl Benzoylacetates from Acetophenones. (Method E).
To a stirred solution of diethyl carbonate (2.0 mmol) in toluene (4 mL), NaH (60% dispersion in oil, 2.5
mmol) was added at rt. After stirring for 10 min, a solution of substituted acetophenone (1 mmol) in 1
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mL of toluene was added. The reaction mixture was allowed to reflux for 30 min and then cooled to rt.
The reaction was quenched with water under ice cooling, acidified with glacial AcOH and extracted
with EtOAc (3×10 mL). The combined organic phases were washed with water (3×10 mL), dried over
Na2SO4 and evaporated. The residue was purified by flash chromatography to give the substituted ethyl
benzoylacetate.
10
11
12
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60
Ethyl 5-hydroxy-4-((4-methylpiperidin-1-yl)methyl)-2-phenylbenzofuran-3-carboxylate (6). This
compound was obtained from 8 and 4-methylpiperidine employing Method B. Yield 82%; pale yellow
oil. 1H NMR (400 MHz, CDCl3) δ 7.74 (dd, J = 7.7, 1.7 Hz, 2H), 7.48–7.40 (m, 3H), 7.31 (d, J = 8.8 Hz,
1H), 6.86 (d, J = 8.8 Hz, 1H), 4.35 (q, J = 7.1 Hz, 2H), 3.97 (s, 2H), 3.03 (d, J = 10.9 Hz, 2H), 2.16 (t, J
= 11.0 Hz, 2H), 1.69 (d, J = 12.8 Hz, 2H), 1.45 (br s, 1H), 1.37–1.23 (m, 5H), 0.95 (d, J = 6.4 Hz, 3H).
13C NMR (101 MHz, CDCl3) δ 166.5, 156.9, 155.6, 148.3, 130.1, 129.7, 128.5, 128.0, 125.5, 115.4,
112.4, 110.8, 110.2, 61.6, 57.7, 53.5, 34.3, 30.6, 21.8, 14.1. HRMS (ESI) m/z: Calcd for C24H28NO4
(M+H)+ 394.2013, found 394.2014.
Ethyl 5-hydroxy-2-phenylbenzofuran-3-carboxylate (8). This compound was obtained from 7
employing Method A. Yield 46%; pale yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.12–7.75 (m, 2H),
7.52–7.41 (m, 4H), 7.35 (d, J = 8.8 Hz, 1H), 6.85 (dd, J = 8.8, 2.6 Hz, 1H), 4.35 (q, J = 7.1 Hz, 2H),
13
1.36 (t, J = 7.1 Hz, 3H). C NMR (101 MHz, CD3OD) δ 165.5, 162.5, 155.5, 149.7, 131.2, 131.2,
130.4, 129.2, 129.0, 115.2, 112.4, 109.8, 108.1, 61.7, 14.5. HRMS (ESI) m/z: Calcd for C17H14NaO4
(M+Na)+ 305.0784, found 305.0786.
5-Hydroxy-2-phenylbenzofuran-3-carboxylic Acid (9). To a solution of 8 (1.0 mmol) in 1,4-dioxane
(5 mL), LiOH (6.0 mmol) in H2O (1.0 mL) was added at rt. After stirring at 60 ºC for 24 hr the reaction
mixture was acidified with 2 M aqueous HCl to pH 5–6. The resultant solid was filtered off to give a
white solid with a 87.4% yield. 1H NMR (400 MHz, DMSO-d6) δ 12.95 (s, 1H), 9.38 (s, 1H), 7.98–7.92
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(m, 2H), 7.56–7.49 (m, 3H), 7.47 (d, J = 8.8 Hz, 1H), 7.40 (d, J = 2.3 Hz, 1H), 6.83 (dd, J = 8.8, 2.5 Hz,
4
5
6
13
1H). C NMR (101 MHz, DMSO-d6) δ 164.7, 159.7, 154.3, 147.4, 130.0, 129.5, 129.1, 128.1, 127.9,
7
8
114.2, 111.6, 109.4, 106.7.
9
10
11
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60
Ethyl 5-hydroxy-2-phenyl-4-(piperidin-1-ylmethyl)benzofuran-3-carboxylate (10). This compound
1
was obtained from 8 and piperidine employing Method B. Yield 89%; pale yellow solid. H NMR (400
MHz, CD3OD) δ 7.73–7.66 (m, 2H), 7.47–7.43 (m, 3H), 7.35 (d, J = 8.8 Hz, 1H), 6.83 (d, J = 8.9 Hz,
1H), 4.33 (q, J = 7.1 Hz, 2H), 4.04 (s, 2H), 2.67 (br s, 4H), 1.71–1.60 (m, 4H), 1.54 (brs, 2H), 1.24 (t, J
13
= 7.1 Hz, 3H). C NMR (101 MHz, CD3OD) δ 167.7, 158.6, 156.3, 149.6, 131.2, 130.9, 129.5, 129.0,
127.0, 116.0, 113.0, 112.3, 111.5, 62.8, 57.6, 54.8, 26.5, 24.7, 14.3. HRMS (ESI) m/z: Calcd for
C23H26NO4 (M+H)+ 380.1856, found 380.1860.
Ethyl 5-hydroxy-2-phenyl-4-(pyrrolidin-1-ylmethyl)benzofuran-3-carboxylate (11). This compound
was obtained from 8 and pyrrolidine employing Method B. Yield 67%; white solid. 1H NMR (400 MHz,
CD3OD) δ 7.76–7.66 (m, 2H), 7.50–7.41 (m, 3H), 7.32 (d, J = 8.8 Hz, 1H), 6.82 (d, J = 8.8 Hz, 1H),
4.34 (q, J = 7.1 Hz, 2H), 4.10 (s, 2H), 2.82–2.47 (m, 4H), 2.01–1.74 (m, 4H), 1.26 (t, J = 7.1 Hz, 3H).
13C NMR (101 MHz, CD3OD) δ 167.7, 157.8, 156.4, 149.4, 131.2, 130.7, 129.6, 128.8, 126.4, 116.1,
114.7, 111.8, 111.5, 62.7, 54.7, 54.3, 24.6, 14.3. HRMS (ESI) m/z: Calcd for C22H24NO4 (M+H)+
366.1700, found 366.1688.
Ethyl
4-((dipropylamino)methyl)-5-hydroxy-2-phenylbenzofuran-3-carboxylate
(12).
This
compound was obtained from 8 and dipropylamine employing Method B. Yield 53%; pale yellow solid.
1H NMR (400 MHz, CDCl3) δ 7.78–7.72 (m, 2H), 7.48–7.40 (m, 3H), 7.31 (d, J = 8.8 Hz, 1H), 6.85 (d,
J = 8.8 Hz, 1H), 4.35 (q, J = 7.1 Hz, 2H), 4.06 (s, 2H), 2.54 (t, J = 7.5 Hz, 4H), 1.66–1.56 (m, 4H), 1.29
(t, J = 7.2 Hz, 3H), 0.91 (t, J = 7.4 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 166.4, 156.6, 155.6, 148.1,
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130.0, 129.5, 128.3, 127.8, 125.3, 115.2, 112.9, 110.6, 110.0, 61.4, 55.7, 54.2, 19.6, 13.9, 11.8. HRMS
(ESI) m/z: Calcd for C24H30NO4 (M+H)+ 396.2169, found 396.2168.
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8
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Ethyl 5-hydroxy-4-(morpholinomethyl)-2-phenylbenzofuran-3-carboxylate (13). This compound
was obtained from 8 and morpholine employing Method B. Yield 89%; pale yellow solid. 1H NMR (400
MHz, CD3OD) δ 7.79–7.59 (m, 2H), 7.52–7.39 (m, 3H), 7.32 (d, J = 8.8 Hz, 1H), 6.84 (d, J = 8.8 Hz,
1H), 4.33 (q, J = 7.1 Hz, 2H), 3.92 (s, 2H), 3.66 (t, J = 4.6 Hz, 4H), 2.50 (t, J = 4.0 Hz, 4H), 1.26 (t, J =
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7.2 Hz, 3H). C NMR (101 MHz, CD3OD) δ 167.6 , 157.8, 155.2, 149.7, 131.1, 130.7, 129.5, 128.8,
127.1, 115.8, 114.3, 111.9, 111.9, 67.9, 62.7, 56.5, 54.0, 14.3. HRMS (ESI) m/z: Calcd for C22H24NO5
(M+H)+ 382.1649, found 382.1629.
Ethyl 5-hydroxy-4-((4-methylpiperazin-1-yl)methyl)-2-phenylbenzofuran-3-carboxylate (14). This
compound was obtained from 8 and 1-methylpiperazine employing Method B. Yield 68%; pale yellow
1
oil. H NMR (400 MHz, CD3OD) δ 7.72–7.70 (m, 2H), 7.47–7.45 (m, 3H), 7.33 (d, J = 8.8 Hz, 1H),
6.84 (d, J = 8.8 Hz, 1H), 4.34 (q, J = 7.1 Hz, 2H), 3.95 (s, 2H), 2.57 (br, 8H), 2.28 (s, 3H), 1.27 (t, J =
13
7.1 Hz, 3H). C NMR (101 MHz, CD3OD) δ 167.6, 157.8, 155.4, 149.7, 131.1, 130.8, 129.6, 128.7,
127.0, 115.8, 114.3, 111.9, 111.8, 62.7, 56.2, 55.8, 53.0, 45.9, 14.3. HRMS (ESI) m/z: Calcd for
C23H27N2O4 (M+H)+ 395.1965, found 395.1938.
Ethyl 5-hydroxy-4-((4-(hydroxymethyl)piperidin-1-yl)methyl)-2-phenylbenzo-furan-3- carboxylate
(15). This compound was obtained from 8 and piperidin-4-ylmethanol employing Method B. Yield 56%;
pale yellow oil. 1H NMR (400 MHz, CD3OD) δ 7.73–7.67 (m, 2H), 7.51–7.45 (m, 3H), 7.39 (d, J = 8.9
Hz, 1H), 6.88 (d, J = 8.9 Hz, 1H), 4.33 (q, J = 7.1 Hz, 2H), 4.16 (s, 2H), 3.43 (d, J = 6.3 Hz, 2H), 3.33–
3.30 (m, 1H), 3.19 (d, J = 11.9 Hz, 2H), 2.45 (t, J = 11.3 Hz, 2H), 1.84 (d, J = 13.0 Hz, 2H), 1.63 (brs,
13
1H), 1.45–1.30 (m, 2H), 1.23 (t, J = 7.1 Hz, 3H). C NMR (101 MHz, CD3OD) δ 167.6, 159.3, 156.1,
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149.7, 131.2, 130.9, 129.5, 129.2, 127.2, 115.8, 112.7, 112.5, 111.4, 67.3, 62.7, 56.5, 53.8, 38.9, 29.1,
14.2. HRMS (ESI) m/z: Calcd for C24H28NO5 (M+H)+ 410.1962, found 410.1980.
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Ethyl 2-(cyclopropylmethyl)-8-phenyl-2,3-dihydro-1H-benzofuro[4,5-e][1,3]oxaz-ine-9-carboxylate
(16). This compound was obtained from 8 and cyclopropylmethanamine employing Method B. Yield
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87%; white solid. H NMR (400 MHz, CDCl3/CD3OD=4/1) δ 7.75–7.70 (m, 2H), 7.49–7.43 (m, 3H),
7.31 (d, J = 8.9 Hz, 1H), 6.81 (d, J = 8.9 Hz, 1H), 4.94 (s, 2H), 4.43– 4.29 (m, 4H), 2.64 (d, J = 6.7 Hz,
2H), 1.28 (t, J = 7.1 Hz, 3H), 1.03–0.92 (m, 1H), 0.60–0.51 (m, 2H), 0.15 (q, J = 4.7 Hz, 2H). 13C NMR
(101 MHz, CDCl3/CD3OD = 4/1) δ 166.5, 159.1, 151.5, 149.8, 130.8, 130.7, 129.2, 129.0, 125.4, 116.1,
112.8, 111.3, 111.0, 82.0, 62.3, 57.1, 49.9, 14.2, 9.9, 4.1. HRMS (ESI) m/z: Calcd for C22H24NO4
(M+H)+ 366.1700, found 366.1707.
Ethyl 2-cyclopropyl-8-phenyl-2,3-dihydro-1H-benzofuro[4,5-e][1,3]oxazine-9-carboxylate (17).
This compound was obtained from 8 and cyclopropylamine employing Method B. Yield 79%; pale
yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.78 (dd, J = 6.3, 1.6 Hz, 2H), 7.50–7.43 (m, 3H), 7.31 (d, J
= 8.9 Hz, 1H), 6.87 (d, J = 8.9 Hz, 1H), 4.91 (s, 2H), 4.44–4.31 (m, 4H), 2.42 (t, J = 4.7 Hz, 1H), 1.31 (t,
13
J = 7.1 Hz, 3H), 0.58 (d, J = 4.8 Hz, 4H). C NMR (101 MHz, CDCl3) δ 165.3, 158.0, 150.7, 148.7,
129.9, 129.7, 128.3, 128.3, 124.5, 115.4, 112.9, 110.3, 110.2, 82.0, 61.3, 49.8, 32.9, 13.9, 7.0. HRMS
(ESI) m/z: Calcd for C22H22NO4 (M+H)+ 364.1543, found 364.1555.
Ethyl 2-cyclopentyl-8-phenyl-2,3-dihydro-1H-benzofuro[4,5-e][1,3]oxazine-9-carboxylate (18).
This compound was obtained from 8 and cyclopentylamine employing Method B. Yield 65%; pale
yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.84–7.72 (m, 2H), 7.50–7.42 (m, 3H), 7.29 (d, J = 8.9 Hz,
1H), 6.82 (d, J = 8.9 Hz, 1H), 4.96 (s, 2H), 4.49–4.22 (m, 4H), 3.37–3.12 (m, 1H), 2.11–1.84 (m, 2H),
13
1.84–1.69 (m, 2H), 1.65–1.46 (m, 4H), 1.31 (t, J = 7.2 Hz, 3H). C NMR (101 MHz, CDCl3) δ 165.4,
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157.8, 151.2, 148.6, 129.9, 129.7, 128.3, 128.2, 124.4, 115.1, 112.7, 110.2, 110.2, 81.1, 61.3, 59.8, 48.0,
31.5, 23.7, 14.0. HRMS (ESI) m/z: Calcd for C24H25NNaO4 (M+Na)+ 414.1676, found 414.1679.
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Ethyl 2-isopentyl-8-phenyl-2,3-dihydro-1H-benzofuro[4,5-e][1,3]oxazine-9-carboxylate (19). This
compound was obtained from 8 and 3-methylbutan-1-amine employing Method B. Yield 91%; pale
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yellow oil. H NMR (400 MHz, CDCl3) δ 7.71–7.66 (m, 2H), 7.39–7.32 (m, 3H), 7.20 (d, J = 8.9 Hz,
1H), 6.74 (d, J = 8.9 Hz, 1H), 4.80 (s, 2H), 4.27 (q, J = 7.1 Hz, 2H), 4.19 (s, 2H), 2.68 (t, J = 7.6 Hz,
2H), 1.59–1.52 (m, 1H), 1.40 (dd, J = 14.9, 7.1 Hz, 2H), 1.21 (t, J = 7.1 Hz, 3H), 0.82 (d, J = 6.6 Hz,
6H).13C NMR (101 MHz, CDCl3) δ 165.3, 157.9, 150.7, 148.6, 129.9, 129.6, 128.2, 128.2, 124.5, 115.2,
112.6, 110.2, 110.2, 81.7, 61.2, 49.7, 49.2, 37.1, 26.1, 22.6, 13.9. HRMS (ESI) m/z: Calcd for
C24H28NO4 (M+H)+ 394.2013, found 394.2014.
Ethyl 5-methoxy-2-phenyl-4-(piperidin-1-ylmethyl)benzofuran-3-carboxylate (20). To a solution of
10 (1 mmol) in anhydrous DMF (5 mL), NaH (60% dispersion in oil, 1.2 mmol) and CH3I were added at
0 ºC under N2. After stirring for 1 hr at rt, the reaction was quenched with water and extracted with
EtOAc (2×30 mL). The combined organic phases were washed with water (3×15 mL), dried over
Na2SO4, and evaporated. The residue was purified by flash chromatography to give methyl ether. Yield
33%; pale yellow oil. 1H NMR (400 MHz, CD3OD) δ 7.80–7.66 (m, 2H), 7.49–7.42 (m, 3H), 7.40 (d, J
= 9.0 Hz, 1H), 7.04 (d, J = 9.0 Hz, 1H), 4.31 (q, J = 7.2 Hz, 2H), 3.83 (s, 3H), 3.79 (s, 2H), 2.25 (brs,
13
4H), 1.50–1.31 (m, 6H), 1.22 (t, J = 7.2 Hz, 3H). C NMR (101 MHz, CD3OD) δ 167.2, 158.0, 156.2,
150.7, 131.2, 130.7, 129.5, 129.0, 127.9, 121.1, 113.2, 111.7, 111.2, 62.4, 57.6, 55.0, 53.1, 26.8, 25.6,
14.2. HRMS (ESI) m/z: Calcd for C24H28NO4 (M+H)+ 394.2013, found 394.2004.
5-Hydroxy-2-phenyl-4-(piperidin-1-ylmethyl)benzofuran-3-carboxylic acid (21). This compound
1
was obtained from 9 and piperidine employing Method B. Yield 44%; pale yellow solid. H NMR (400
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MHz, CD3OD) δ 7.81–7.74 (m, 2H), 7.57 (d, J = 8.9 Hz, 1H), 7.53–7.46 (m, 3H), 7.03 (d, J = 8.9 Hz,
1H), 4.71 (s, 2H), 3.56 (d, J = 12.0 Hz, 2H), 3.16 (t, J = 11.0 Hz, 2H), 1.97 (d, J = 14.2 Hz, 2H), 1.86–
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1.71 (m, 3H), 1.65–1.54 (m, 1H). C NMR (101 MHz, CD3OD) δ 169.0, 162.8, 155.8, 150.0, 131.4,
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131.2, 130.3, 129.2, 128.6, 115.3, 115.1, 111.6, 108.7, 54.1, 53.3, 24.1, 22.8.
N-Ethyl-5-hydroxy-2-phenyl-4-(piperidin-1-ylmethyl)benzofuran-3-carboxamide
(22).
This
compound was obtained from 9, ethylamine, and piperidine employing Methods C and B. Yields 92%
and 38%; pale yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.81 (d, J = 8.0 Hz, 2H), 7.58–7.29 (m, 4H),
6.86 (d, J = 8.8 Hz, 1H), 4.10 (s, 2H), 3.42 (q, J = 7.3 Hz, 2H), 2.83 (br s, 4H), 1.83–1.68 (m, 4H), 1.59
(br s, 2H), 1.20 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz, CD3OD) δ 168.7, 155.7, 154.8, 149.5, 130.8,
130.6, 129.9, 127.9, 127.8, 115.5, 114.4, 112.9, 111.3, 54.8, 54.6, 36.0, 26.1, 24.3, 14.2. HRMS (ESI)
m/z: Calcd for C23H27N2O3 (M+H)+ 379.2025, found 379.2016.
N-Cyclopropyl-5-hydroxy-2-phenyl-4-(piperidin-1-ylmethyl)benzofuran-3-carboxamide (23). This
compound was obtained from 9, cyclopropylamine, and piperidine employing Methods C and B. Yields
1
74% and 56%; yellow oil. H NMR (400 MHz, CD3OD) δ 7.79–7.65 (m, 2H), 7.56–7.40 (m, 4H), 6.96
(d, J = 8.9 Hz, 1H), 4.32 (s, 2H), 3.12 (br s, 4H), 3.05–2.89 (m, 1H), 1.91–1.75 (m, 4H), 1.67 (br s, 2H),
13
0.90–0.68 (m, 2H), 0.59–0.45 (m, 2H). C NMR (101 MHz, CD3OD) δ 169.9, 156.2, 155.5, 149.6,
131.0, 130.5, 129.8, 128.3, 128.3, 115.2, 114.3, 113.9, 109.1, 54.3, 54.1, 25.1, 24.2, 23.4, 5.8. HRMS
(ESI) m/z: Calcd for C24H27N2O3 (M+H)+ 391.2016, found 391.2011.
N-((3S,5S,7S)-Adamantan-1-yl)-5-hydroxy-2-phenyl-4-(piperidin-1-ylmethyl)benzofuran-3-
carboxamide (24). This compound was obtained from 9, amantadine, and piperidine employing
1
Methods C and B. Yields 86% and 40%; pale yellow solid. H NMR (400 MHz, CD3OD) δ 7.84 (d, J =
7.3 Hz, 2H), 7.53–7.44 (m, 3H), 7.40 (d, J = 8.8 Hz, 1H), 6.86 (d, J = 8.8 Hz, 1H), 4.20 (s, 2H), 2.88 (br
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s, 4H), 2.15–2.11 (m, 11H), 1.66 (s, 10H). 13C NMR (101 MHz, CDCl3) δ 164.4, 153.9, 151.1, 146.9,
128.6, 128.1, 127.6, 125.6, 125.1, 114.0, 113.5, 111.1, 109.5, 56.0, 53.0, 51.7, 40.2, 35.3, 28.4, 24.7,
22.9. HRMS (ESI) m/z: Calcd for C31H37N2O3 (M+H)+ 485.2799, found 485.2805.
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N-Benzyl-5-hydroxy-2-phenyl-4-(piperidin-1-ylmethyl)benzofuran-3-carboxamide
(25).
This
compound was obtained from 9, benzylamine, and piperidine employing Methods C and B. Yields 96%
1
and 87%; white solid. H NMR (400 MHz, CDCl3) δ 7.65 (d, J = 3.5 Hz, 2H), 7.34–7.10 (m, 8H), 6.71
(d, J = 8.8 Hz, 1H), 6.26 (t, J = 6.4 Hz, 1H), 4.51 (d, J = 6.0 Hz, 2H), 3.75 (s, 2H), 2.92–1.85 (br s, 4H),
13
1.63–1.26 (m, 6H). C NMR (101 MHz, CDCl3) δ 165.2, 154.1, 151.7, 146.9, 136.3, 128.4, 128.1,
127.8, 127.7, 127.4, 126.7, 125.6, 124.9, 114.0, 112.0, 111.1, 109.5, 56.1, 52.6, 43.3, 24.8, 22.8. HRMS
(ESI) m/z: Calcd for C28H29N2O3 (M+H)+ 441.2173, found 441.2188.
5-Hydroxy-N,2-diphenyl-4-(piperidin-1-ylmethyl)benzofuran-3-carboxamide (26). This compound
was obtained from 9, aniline, and piperidine employing Methods C and B. Yields 74% and 79%; brown
1
solid. H NMR (400 MHz, CDCl3) δ 7.91 (s, 1H), 7.73 (d, J = 6.5 Hz, 2H), 7.49 (d, J = 7.4 Hz, 2H),
7.36–7.30 (m, 4H), 7.19 (s, 1H), 7.12 (t, J = 6.3 Hz, 1H), 6.78 (d, J = 8.3 Hz, 1H), 3.89 (s, 2H), 2.79–
2.25 (br s, 4H), 1.55 (br s, 6H). 13C NMR (101 MHz, CDCl3) δ 164.4, 155.2, 153.2, 148.0, 137.7, 129.4,
129.4, 129.2, 128.9, 126.7, 125.9, 125.0, 120.1, 115.3, 113.5, 111.9, 110.8, 57.0, 53.8, 25.6, 23.7.
HRMS (ESI) m/z: Calcd for C27H27N2O3 (M+H)+ 427.2016, found 427.2029.
Ethyl 2-(4-fluorophenyl)-5-hydroxy-4-(piperidin-1-ylmethyl)benzofuran-3-carboxylate (27). This
compound was obtained from ethyl 4-fluorobenzoylacetate and piperidine employing Methods A and B.
Yields 56% and 84%; pale yellow solid. 1H NMR (400 MHz, CDCl3/CD3OD = 5/1) δ 7.75 (dd, J = 8.6,
5.4 Hz, 2H), 7.33 (d, J = 8.8 Hz, 1H), 7.19 (t, J = 8.7 Hz, 2H), 6.83 (d, J = 8.8 Hz, 1H), 4.35 (q, J = 7.1
13
Hz, 2H), 4.02 (s, 2H), 2.64 (br s, 4H), 1.73–1.63 (m, 4H), 1.54 (br s, 2H), 1.29 (t, J = 7.2 Hz, 3H). C
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NMR (101 MHz, CDCl3/CD3OD = 5/1) δ 167.2, 164.7 (d, JC-F = 249.8 Hz), 157.4, 156.2, 149.3, 131.1
(d, JC-F = 8.5 Hz), 127.3 (d, JC-F = 3.3 Hz), 126.5, 116.3 (d, JC-F = 22.2 Hz), 116.0, 113.0, 111.9, 111.1,
62.6, 58.1, 54.6, 26.5, 24.6, 14.3. HRMS (ESI) m/z: Calcd for C23H25FNO4 (M+H)+ 398.1762, found
398.1778.
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Ethyl 5-hydroxy-4-(piperidin-1-ylmethyl)-2-(4-(trifluoromethyl)phenyl)benzo-furan-3-carboxylate
(28). This compound was obtained from ethyl 4-trifluoromethylbenzoylacetate and piperidine
employing Methods A and B. Yields 46% and 79%; white solid. 1H NMR (400 MHz, CDCl3/CD3OD =
3/1) δ 7.87 (d, J = 8.2 Hz, 2H), 7.73 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 8.9 Hz, 1H), 6.84 (d, J = 8.9 Hz,
1H), 4.37 (q, J = 7.1 Hz, 2H), 3.95 (s, 2H), 2.58 (br s, 4H), 1.70–1.61 (m, 4H), 1.52 (br s, 2H), 1.29 (t, J
13
= 7.1 Hz, 3H). C NMR (101 MHz, CDCl3/CD3OD = 3/1) δ 167.4, 156.6, 155.9, 149.8, 134.7, 134.7,
133.3 (q, JC-F = 32.8 Hz), 129.3, 126.5 (q, JC-F = 3.8 Hz), 125.4 (q, JC-F = 272.6 Hz), 117.0, 113.7, 112.9,
112.2, 63.1, 58.6, 55.0, 26.9, 25.0, 14.6. HRMS (ESI) m/z: Calcd for C24H25F3NO4 (M+H)+ 448.1730,
found 448.1748.
Ethyl 5-hydroxy-2-(4-methoxyphenyl)-4-(piperidin-1-ylmethyl)benzofuran-3-carboxylate (29).
This compound was obtained from ethyl 4-methoxybenzoylacetate and piperidine employing Methods A
and B. Yields 67% and 86%; white solid. 1H NMR (400 MHz, CD3OD) δ 7.62 (d, J = 8.7 Hz, 2H), 7.25
(d, J = 8.8 Hz, 1H), 6.95 (d, J = 8.7 Hz, 2H), 6.75 (d, J = 8.8 Hz, 1H), 4.31 (q, J = 7.1 Hz, 2H), 3.91 (s,
2H), 3.80 (s, 3H), 2.53 (br s, 4H), 1.67–1.55 (m, 4H), 1.48 (br s, 2H), 1.26 (t, J = 7.1 Hz, 3H). 13C NMR
(101 MHz, CD3OD) δ 167.8, 162.3, 158.2, 156.2, 149.2, 130.4, 126.9, 123.4, 115.5, 114.9, 113.3, 111.7,
110.1, 62.6, 58.3, 55.8, 54.8, 26.8, 24.9, 14.4. HRMS (ESI) m/z: Calcd for C24H28NO5 (M+H)+
410.1962, found 410.1973.
ACS Paragon Plus Environment
29