Synthesis of potent inhibitors of ss-ketoacyl-acyl carrier protein synthase III as potential antimicrobial agents

Article abstract of DOI:10.3390/molecules17054770

Mycobacterium tuberculosis FabH, an essential enzyme in the mycolic acid biosynthetic pathway, is an attractive target for novel anti-tubercolosis agents. Structure-based design and synthesis of 1-(4-carboxybutyl)-4-(4- (substituted benzyloxy) phenyl)-1H-pyrrole-2-carboxylic acid derivatives 7a-h, a subset of eight potential FabH inhibitors, is described in this paper. The Vilsmeier-Haack reaction was employed as a key step. The structures of all the newly synthesized compounds were identified by IR, 1H-NMR, 13C-NMR, ESI-MS and HRMS. The alamarBlue microassay was employed to evaluate the compounds 7a-h against Mycobacterium tuberculosis H37Rv. The results demonstrate that the compound 7d possesses good in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv (Minimum Inhibitory Concentration value [MIC], 12.5 μg/mL).These compounds may prove useful in the discovery and development of new anti-tuberculosis drugs.

Full text of DOI:10.3390/molecules17054770

Molecules 2012, 17, 4770-4781; doi:10.3390/molecules17054770
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of Potent Inhibitors of β-Ketoacyl-Acyl Carrier
Protein Synthase III as Potential Antimicrobial Agents
Yan Liu 1,2, Wu Zhong 2,*, Rui-Juan Li 1,2 and Song Li 2
1
School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016,
China; E-Mail: liuyanbtnet@163.com (Y.L.)
2
Laboratory of Computer-Aided Drug Design & Discovery, Beijing Institute of Pharmacology and
Toxicology, Beijing 100850, China
* Author to whom correspondence should be addressed; E-Mail: zhongwu@nic.bmi.ac.cn;
Tel.: +86-10-66-931-634; Fax: +86-10-68-240-321.
Received: 26 December 2011; in revised form: 11 April 2012 / Accepted: 16 April 2012 /
Published: 25 April 2012
Abstract: Mycobacterium tuberculosis FabH, an essential enzyme in the mycolic
acid biosynthetic pathway, is an attractive target for novel anti-tubercolosis agents.
Structure-based design and synthesis of 1-(4-carboxybutyl)-4-(4-(substituted benzyloxy)
phenyl)-1H-pyrrole-2-carboxylic acid derivatives 7a–h, a subset of eight potential FabH
inhibitors, is described in this paper. The Vilsmeier-Haack reaction was employed as a key
step. The structures of all the newly synthesized compounds were identified by IR,
1H-NMR, 13C-NMR, ESI-MS and HRMS. The alamarBlue™ microassay was employed to
evaluate the compounds 7a–h against Mycobacterium tuberculosis H37Rv. The results
demonstrate that the compound 7d possesses good in vitro antimycobacterial activity against
Mycobacterium tuberculosis H37Rv (Minimum Inhibitory Concentration value [MIC],
12.5 µg/mL).These compounds may prove useful in the discovery and development of new
anti-tuberculosis drugs.
Keywords: tuberculosis; Mycobacterium tuberculosis FabH; structure based design;
synthesis; alamarBlue™ microassay

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1. Introduction
According to data of the World Health Organization [1], Tuberculosis (TB) caused by
Mycobacterium tuberculosis, is considered to be the most chronic communicable disease in the World
especially in Asia and Africa. This situation was made worse by the emergence of multi drug resistant
TB (MDR-TB) and the increasing number of HIV-positive TB cases [2]. Worldwide, TB accounts for
approximately one-fourth of HIV-related deaths and is the leading cause of death in HIV-infected adults
in developing countries [3–5], thus an urgent need exists for the development of new antimycobacterial
agents with a unique mechanism of action.
The mycobacterial cell wall, which is composed of mycolic acids (α-alkyl-β-hydroxy long chain
fatty acids) is known to be important for the growth, survival, and pathogenicity of mycobacteria.
Mycobacteria contain both type I (FAS I) and type II (FAS II) fatty acid biosynthetic pathways. FAS is
a single multifunctional polypeptide that catalyzes all the reactions in the elongation pathway [6,7]. On
the other hand, FAS II system is catalyzed by a series of small, soluble proteins that are each encoded
by a discrete gene existing as separate proteins. Mycobacterium tuberculosis β-ketoacyl-acyl carrier
protein synthase III (mtFabH) is a key condensing enzyme responsible for initiation of FAS II fatty
acid biosynthetic pathway, and has emerged as an attractive new target for novel anti-tuberculosis
agents in recent years. Structure-based design, synthesis of novel inhibitors of mtFabH was reported in
this paper.
2. Results and Discussion
We studied all available information about mtFabH, including known FabH inhibitors, substrates,
and the active site topology from available mtFabH crystal structures. According to the structures of
the anti-mycobacterial compounds FAS20013, SB418011 and the predicted binding mode of the other
known anti-tuberculosis agents [8–11], we designed a novel scaffold structurs. This structure was
predicted to possess improved binding affinity, as shown in Figure 1.
Figure 1. Predicted binding model of compound 7d in the active site tunnel of mtFabH.

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First, a substituted benzyloxy group is required to fill a complementary hydrophobic region at the
top of the active site tunnel. Second, the presence of halogen or alkyl substituent is needed in order to
form an ionic interaction with the arginine residues (Arg249) at the top of the active site tunnel. Third,
the substituted group acidic groups are required to fill the bottom of the active site tunnel, and
expected to form direct interaction (such as hydrogen bond) with the catalytic residues (Cys112,
His244, Ser276 and Asn274). The scaffold structure itself merely serves as a carrier on which these
three binding elements are arranged.
We chose mtFabH (PDB ID: 1HZP) [12] as the docking target, and used an automated molecular
docking and database screening program DOCK 4.0 [13,14]. A virtual compound library containing
7,560 diverse combinations of substituted benzyloxy and various lengths of fatty acid chains were
constructed using Project Library 2.0, a multifunctional combinatorial library construction program [15].
The constructed library was then virtually screened to identify the potential candidate compounds that
display favorable binding to the protein active site. The top 500 high free energy score of derivatives
from computer screening were carefully investigated. After taking drug-like properties and synthetic
accessibility into account, a subset of eight candidate compounds were chosen to be synthesized first.
The synthesis of a series of potential mtFabH inhibitors are reported in this paper.
Scheme 1. Synthesis of 4-(4-(substituted benzyloxy)phenyl)-1-(3-carboxybutyl)-1H-
pyrrole-2-carboxylic acid.
Reagents and conditions: (i) DMF POCl3, 90 °C, 5.5 h; (ii) H2O, NaClO4, r.t., 4 h, 76%; (iii) MeOH,
Na, methyl 2-aminoacetate hydrochloride reflux, 24 h, 76%; (iv) C2H5OOC(CH2)3Br, n-Bu4NBr,
K2CO3, acetone, 50 °C, 4 h, 82%; (v) PhNMe2, AlCl3, CH2Cl2, r.t., 0.5 h, 79%; (vi) RBr, n-Bu4NBr,
K2CO3, acetone, 50 °C, 2–4 h, 84%–96%; (vii) NaOH, CH3OH, H2O, 25 °C, 30 min, 67%–94%.

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Scheme 1. Cont.
Entry
R
Product
Yield (%)
1
2
3
4
5
6
7
8
4-chloro
7a
7b
7c
7d
7e
7f
67
90
66
94
89
88
97
75
4-methyl
4-methoxy
3,5-difluoro
3-methyl
3-chloro
2,6-dichloro
3,4-dimethyl
7g
7h
As shown in Scheme 1, commercially available 4-benzyloxybenzoic acid (1) was used to prepare
the vinamidinium salts 2 by the standard Vilsmeier-Haack reaction [16]. When compound 1 was
treated with a large excess of Vilsmeier reagent, followed by the addition of sodium perchlorate
solution, the 4-benzyloxyphenyl vinamidinium salt was isolated in 76% yield [17]. The cyclization of
4-benzyloxyphenyl vinamidinum salt 2 with methyl 2-aminoacetate hydrochloride in refluxing
methanol in the present of sodium methoxide afforded pyrrole-2-carboxylate 3 in 76% yield. Ethyl
4-bromobutanoate with n-Bu4NBr and anhydrous K2CO3 were successively added to a stirred
solution of 3 in acetone to afford pyrrole-2-carboxylate 4. Compound 4 was treated with AlC13 and
N,N-dimethylaniline in CH2Cl2 to obtain pyrrole-2-carboxylate 5 in 79% yield. To a DMF solution
were added compound 5, substituted benzyl halides and anhydrous K2CO3 to generate the
corresponding pyrrole-2-carboxylates 6a–h. Compounds 6a–h were hydrolyzed using sodium
hydroxide in a mixture of methanol and water to give compounds 7a–h in 60%–90% yield.
We determined the antimycobacterial activity of the compounds 7a–h utilizing a reference strain
sensitive to Mycobacterium tuberculosis first-line antibiotics (H37Rv), obtaining important activity,
with a MIC value 12.5 μg/mL. All of the compounds obtained were established unequivocally through
analysis of their spectroscopic data. We found no report in the literature on the antituberculosis
evaluation of these compounds. The results of their activity are presented in Table 1.
Table 1. Antimycobacterial activity (Minimum inhibitory concentration [MIC] μg/mL) of
the compounds 7a–h.
Comp. No.
R
MIC
>25
>25
>25
12.5
>25
25
7a
7b
7c
4-chloro
4-methyl
4-methoxy
3,5-difluoro
3-methyl
7d
7e
7f
3-chloro
7g
2,6-dichloro
3,4-dimethyl
25
7h
INH
>25
0.046
Compounds 7a, 7b, 7e and 7h have alkyl substituted benzyloxy, which did not demonstrate
antituberculosis activity in this study. With regard to the antituberculous activity of the compounds 7f

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and 7g, they demonstrated little such activity against the assayed strains (H37Rv), with MIC values
ranging between 12.5 and 25 μg/mL. The compound 7d’s MIC value was found to be 12.5 μg/mL.
Further evaluation such as the activity of anti XDR-Mtb strains is still in progress.
3. Experimental
3.1. Materials and Reagents
All the reagents for synthesis were commercially available and either used without further
purification or purified by standard methods prior to use. Melting points were determined using a
RY-1 apparatus, and the thermometer was uncorrected. 1H-NMR and 13C-NMR spectra were measured
using an ARX400 multinuclear Fourier transform (FT)-NMR spectrometer (Bruker). Mass spectra
were obtained from Micromass ZabSpec 1000 and API3000 instruments.
3.2. Synthesis of Starting Compounds 2–5
4-Benzyloxyphenyl vinamidinium salt (2). The Vilsmeier-Haack reagent was prepared by slow addition
of anhydrous DMF (44 g, 0.6 mol) to phosphorus oxychloride (18.4 g, 0.12 mol) with stirring at 0 °C
under a nitrogen atmosphere. The reaction mixture was left at room temperature for 1 h and then
4-benzyloxyphenylacetic acid (1, 96.8 g, 0.4 mol) was added and the reaction mixture was stirred for
4.5 h at 90 °C. An aqueous solution (100 mL) of sodium perchlorate (6.2 g, 0.044 mol) was added and
the resulting mixture was stirred for 1 h at room temperature. The 4-benzyloxyphenyl vinamidinium salt
was isolated in 76% yield.
4-(4-(Benzyloxy)phenyl)-1H-pyrrole-2-carboxylate (3). A dry, three-necked, round-bottomed flask
(500 mL) was equipped with a reflux condenser and magnetic stirrer. Under a nitrogen atmosphere
sodium (1.75 g, 0.08 mol) was charged to the flask and then dry methanol (200 mL) was added and the
resulting mixture was allowed to react for several minutes while stirring. Methyl 2-aminoacetate
hydrochloride (6.4 g, 0.046 mol) was added and then compound 2 (12.5 g, 0.031 mol) was added. The
resulting mixture was refluxed for 24 h, and the solvent was removed in vacuo. To the residue was
added water (100 mL) and then the aqueous solution was extracted by EtOAc (3 × 50 mL). Combined
organic phase was dried with anhydrous sodium sulfate for 2 h. After filtering out sodium sulfate, the
solvent was removed in vacuo. The crude product was crystallized twice with the mixture of EtOAc
and hexane to give 7.2 g (76% yield) of pure compound 3 as a white solid which had the following
properties: Mp 207–208 °C; Rf = 0.2 (hexanes-EtOAc, 3:1). IR (KBr): 3,282, 3,117, 1,678, 1,617,
1,581, 1,570, 1,523, 1,477, 1,465, 1,440, 1,382, 1,297, 1,254, 1,192, 1,180, 1,053, 1,041, 1,026, 994,
926, 809, 769, 728, 692 cm–1. 1H-NMR (DMSO-d6): δ = 11.98 (s, 1H), 7.521–7.543 (d, 2H, J = 8.5 Hz),
7.306–7.460 (m, 6H), 7.101–7.111 (t, 1H, J = 4 Hz), 6.958–6.980 (d, 2H, J = 8.8 Hz), 5.11 (s, 2H),
3.78 (s, 3H). 13C-NMR (DMSO-d6): δ = 160.8, 156.6, 137.2, 128.4, 127.7, 127.6, 127.4, 125.9, 124.9,
122.4, 120.5, 115.0, 111.6, 69.1, 51.0. ESI-MS m/z = 308.1 [M+H]+.
Methyl 1-(4-ethoxy-4-oxobutyl)-4-(4-(benzyloxy)phenyl)-1H-pyrrole-2-carboxylate (4). Ethyl 4-bromo-
butanoate (4.73 g, 24.4 mol) with n-Bu4NBr (10.5 g, 32.6 mmol) and anhydrous K2CO3 (4.5 g,
32.6 mmol) were successively added to a stirred solution of intermediate 3 (5 g, 16.3 mmol) in acetone

Molecules 2012, 17
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(100 mL). The mixture was stirred at 50 °C for 4 h and then the aqueous layer was separated and was
acidified with 1 N HCl to pH 4. The aqueous layer was extracted with EtOAc (3 × 50 mL). The
combined organic layer was dried (Na2SO4), filtered, and concentrated under reduced pressure, the
remaining residue was purified by column chromatography [silica gel, hexane-EtOAc (v/v) = 10:1] to
afford 5.6 g (13.3 mmol, 82% yield) of intermediate 4 as a white solid which had the following
properties: Mp 70–71 °C; Rf = 0.3 (hexanes-EtOAc, 8:1). IR (KBr): 3,442, 2,955, 1,728, 1,698, 1,618,
1,567, 1,513, 1,449, 1,392, 1,277, 1,258, 1,192, 1,102, 1,069, 1,041, 1,025, 829, 800, 759, 735,
697 cm–1. 1H-NMR (DMSO-d6): δ = 7.326–7.537 (m, 8H), 7.172–7.177 (d, 1H, J = 2 Hz), 6.976–6.997
(d, 2H, J = 8.4 Hz), 5.107 (s, 2H), 4.302–4.336 (t, 2H, J = 13.6 Hz), 3.989–4.042 (q, 2H, J = 21.2 Hz),
3.758 (s, 3H), 2.235–2.272 (t, 2H, J = 14.8 Hz), 1.959–1.995 (m, 2H), 1.132–1.168 (t, 3H, J = 14.4 Hz).
13C-NMR (DMSO-d6): δ = 172.1, 160.6, 156.7, 137.2, 128.4, 127.7, 127.6, 126.8, 126.1, 125.8, 122.9,
121.4, 115.1, 114.3, 69.2, 59.9, 51.0, 47.5, 30.5, 26.2, 14.0. ESI-MS m/z = 422.2 [M+H]+. HRMS-FAB:
m/z [M+H]+ calcd for C25H28N1O5: 422.19620; found: 422.19754.
Methyl 1-(4-ethoxy-4-oxobutyl)-4-(4-(benzyloxy)phenyl)-1H-pyrrole-2-carboxylate (5). To a mixture
of compound 4 (5.6 g, 13.2 mmol) and N,N-dimethylaniline (12.7 mL, 100 mmol) in anhydrous
CH2Cl2 (30 mL) was added powdered AlC13 (5.2 g, 39.6 mmol) at room temperature. The reaction
mixture was stirred for 30 min and then quenched by addition of 1N HCl. The aqueous layer was
separated and extracted with EtOAc (3 × 50 mL). The combined organic phase was successively
washed with 5% NaHCO3 solution and brine, and then dried over anhydrous Na2SO4 for 1 h. After
filtered out Na2SO4, the organic solvent was removed under reduced pressure. The remaining residue
was purified by column chromatography [silica gel, hexanes-EtOAc (v/v) = 4:1] to afford compound 5
3.48 g (10.5 mmol) as a colorless, oily liquid in 79% yield which had the following properties: Rf = 0.3
(hexanes-EtOAc, 3:1). 1H-NMR (DMSO-d6): δ = 9.110 (s, 1H), 7.347–7.483 (m, 3H), 7.094–7.099 (d,
1H, J = 2 Hz), 6.711–6.740 (m, 2H), 4.312–4.346 (t, 2H, J = 13.6 Hz), 3.997–4.050 (q, 2H, J = 21.2 Hz),
3.768 (s, 3H), 2.245–2.282 (t, 2H, J = 14.8 Hz), 1.967–2.002 (m, 2H), 1.121–1.177 (t, 3H, J = 14.4 Hz).
13C-NMR (DMSO-d6): δ = 172.1, 160.6, 155.8, 125.8, 125.0, 123.4, 121.2, 115.5, 114.1, 59.9, 51.0,
47.5 30.5, 26.3, 14.0. ESI-MS m/z = 322.1 [M+H]+.
3.3. General Procedure for the Synthesis of Compounds 6a–h
General Procedure
Methyl 1-(4-ethoxy-4-oxobutyl)-4-(4-(substituted-benzyloxy)phenyl)-1H-pyrrole-2-carboxylate. To a
solution of intermediate 5 (1 mmol, 0.331 g) and n-Bu4NBr (0.64 g, 2 mmol) in acetone (20 mL) were
added anhydrous K2CO3 (0.28 g, 2 mmol) and substituted benzyl halides (1.5 mmol). After stirring for
4 h at room temperature, to the reaction mixture 1 N HCl (10 mL) was added. The organic portion was
extracted with EtOAc (3 × 20 mL). The combined organic phase was dried for a while and then
Na2SO4 was filtered off and the filtrate was concentrated in vacuo. The residue was purified by column
chromatography [silica gel, gradient elution with hexane-EtOAc (v/v) = 1:10–1:5] to give the
corresponding product.

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Methyl
1-(4-ethoxy-4-oxobutyl)-4-(4-(4-chlorobenzyloxy)phenyl)-1H-pyrrole-2-carboxylate (6a).
1
Colorless, oily liquid; yield: 513 mg (93%); Rf = 0.3 (hexanes-EtOAc, 8:1). H-NMR (DMSO-d6):
δ = 7.403–7.558 (m, 6H), 7.176–7.226 (m, 2H), 6.972–7.009 (m, 2H), 5.111–5.152 (t, 2H, J = 16.4 Hz),
4.306–4.339 (t, 2H, J = 13.2 Hz), 3.988–4.042 (q, 2H, J = 21.6 Hz), 3.761 (s, 3H), 2.238–2.276 (t, 2H,
J = 15.2 Hz), 1.962–2.014 (m, 2H), 1.132–1.168 (t, 3H, J = 14.4 Hz). 13C-NMR (DMSO-d6):
δ = 172.5, 161.0, 156.8, 136.6, 132.7, 130.7, 129.8, 127.6, 126.4, 126.1, 121.7, 115.4, 114.7, 110.8,
68.5, 60.2, 51.3, 47.9, 30.9, 26.6, 14.3. ESI-MS m/z = 456.2 [M+H]+.
Methyl 1-(4-ethoxy-4-oxobutyl)-4-(4-(4-methylbenzyloxy)phenyl)-1H-pyrrole-2-carboxylate (6b).
1
Colorless, oily liquid; yield: 501 mg (95%); Rf = 0.3 (hexanes-EtOAc, 6:1). H-NMR (DMSO-d6):
δ = 7.476–7.498 (m, 3H), 7.319–7.339 (d, 2H, J = 8 Hz), 7.170–7.201 (m, 3H), 6.957–6.980 (d, 2H,
J = 9.2 Hz), 5.050 (s, 2H), 4.302–4.336 (t, 2H, J = 13.6 Hz), 3.990–4.043 (q, 2H, J = 21.2 Hz), 3.758
(s, 3H), 2.235–2.303 (m, 5H), 1.961–1.996 (m, 2H), 1.133–1.168 (t, 3H, J = 14 Hz).13C-NMR
(DMSO-d6): δ = 172.1, 160.1, 156.8, 136.9, 134.1, 128.9, 127.7, 126.7, 125.1, 122.9, 121.4, 115.1,
114.3, 69.1, 59.5, 51.0, 47.5, 30.5, 26.3, 20.7, 14.0. ESI-MS m/z = 436.2 [M+H]+.
Methyl 1-(4-ethoxy-4-oxobutyl)-4-(4-(4-methoxybenzyloxy)phenyl)-1H-pyrrole-2-carboxylate (6c).
1
Colorless, oily liquid; yield: 525 mg (96%); Rf = 0.3 (hexanes-EtOAc, 5:1). H-NMR (DMSO-d6):
δ = 7.479–7.530 (m, 3H), 7.364–7.386 (d, 2H, J = 8.8 Hz), 7.173–7.239 (m, 1H), 6.867–6.981 (m, 4H),
5.015 (s, 2H), 4.304–4.338 (t, 2H, J = 13.6 Hz), 3.991–4.044 (q, 2H, J = 21.2 Hz), 3.731–3.759 (m, 6H),
13
2.237–2.274 (t, 2H, J = 14.8 Hz), 1.963–2.016 (m, 2H), 1.134–1.169 (t, 3H, J = 14.4 Hz). C-NMR
(DMSO-d6): δ = 172.1, 160.5, 158.9, 156.8, 129.4, 127.9, 126.7, 126.1, 125.7, 122.9, 115.1, 114.3,
113.8, 113.4, 68.9, 59.8, 55.0, 50.9, 47.6, 30.5, 26.2, 14.0. ESI-MS m/z = 452.2 [M+H]+.
Methyl 1-(4-ethoxy-4-oxobutyl)-4-(4-(3,5-difluorobenzyloxy)phenyl)-1H-pyrrole-2-carboxylate (6d).
1
Colorless, oily liquid; yield: 519 mg (94%); Rf = 0.3 (hexanes-EtOAc, 6:1). H-NMR (DMSO-d6):
δ = 7.514–7.542 (m, 3H), 7.189–7.201 (m, 4H), 6.994–7.016 (d, 2H, J = 8.8 Hz), 5.149 (s, 2H),
4.318–4.352 (t, 2H, J = 13.6 Hz), 3.773 (s, 3H), 2.249–2.287 (t, 2H, J = 15.2 Hz), 1.978–2.032
13
(m, 2H), 1.141–1.177 (t, 3H, J = 14.4 Hz). C-NMR (DMSO-d6): δ = 172.1, 163.6, 160.6, 156.3,
142.0, 127.2, 126.1, 125.8, 122.8, 121.5, 115.1, 114.3, 110.2, 103.0, 67.8, 59.8, 50.9, 47.6, 30.5, 26.2,
14.0. ESI-MS m/z = 458.2 [M+H]+.
Methyl
1-(4-ethoxy-4-oxobutyl)-4-(4-(3-methylbenzyloxy)phenyl)-1H-pyrrole-2-carboxylate
(6e).
1
Colorless, oily liquid; yield: 489 mg (93%); Rf = 0.3 (hexanes-EtOAc, 6:1). H-NMR (DMSO-d6):
δ = 7.488–7.529 (m, 3H), 7.126–7.291 (m, 5H), 6.972–6.994 (m, 2H), 5.054 (s, 2H), 4.307–4.342 (t,
2H, J = 14 Hz), 3.993–4.047 (q, 2H, J = 21.6 Hz), 3.762 (s, 3H), 2.239–2.317 (m, 5H), 1.986–2.004
13
(m, 2H), 1.135–1.171 (t, 3H, J = 14.4 Hz). C-NMR (DMSO-d6): δ = 172.1, 160.6, 137.5, 137.1,
128.3, 126.8, 126.1, 125.8, 124.7, 122.9, 121.4, 115.0, 114.3, 69.2, 59.9, 50.9, 47.5, 30.5, 26.2, 20.9,
14.0. ESI-MS m/z = 436.2 [M+H]+.
Methyl
1-(4-ethoxy-4-oxobutyl)-4-(4-(3-chlorobenzyloxy)phenyl)-1H-pyrrole-2-carboxylate
(6f).
1
Colorless, oily liquid; yield: 402 mg (84%); Rf = 0.3 (hexanes-EtOAc, 8:1). H-NMR (DMSO-d6):
δ = 7.420–7.552 (m, 6H), 7.173–7.201 (m, 2H), 6.987–7.009 (m, 2H), 5.107–5.149 (t, 2H,

Molecules 2012, 17
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J = 16.8 Hz), 4.311–4.345 (t, 2H, J = 13.6 Hz), 3.993–4.046 (q, 2H, J = 21.2 Hz), 3.766 (s, 3H),
13
2.243–2.280 (t, 2H, J = 14.8 Hz), 1.988–2.005 (m, 2H), 1.135–1.171 (t, 3H, J = 14.4 Hz). C-NMR
(DMSO-d6): δ = 72.5, 160.9, 156.8, 140.1, 133.4, 130.6, 129.7, 128.7, 128.0, 127.5, 126.1, 123.2,
121.7, 115.4, 114.6, 110.7, 68.5, 60.2, 51.3, 47.9, 30.9, 26.6, 14.3. ESI-MS m/z = 456.2 [M+H]+.
Methyl 1-(4-ethoxy-4-oxobutyl)-4-(4-(2,6-dichlorobenzyloxy)phenyl)-1H-pyrrole-2-carboxylate (6g).
1
Colorless, oily liquid; yield: 493 mg (95%); Rf = 0.3 (hexanes-EtOAc, 10:1). H-NMR (DMSO-d6):
δ = 7.451–7.580 (m, 6H), 7.196–7.201 (d, 1H, J = 2 Hz), 7.032–7.054 (d, 2H, J = 8.8 Hz), 5.232 (s,
2H), 4.313–4.347 (t, 2H, J = 13.6 Hz), 3.995–4.048 (q, 2H, J = 21.2 Hz), 3.766 (s, 3H), 2.245–2.282 (t,
2H, J = 14.8 Hz), 1.970–2.005 (m, 2H), 1.139–1.174 (t, 3H, J = 14 Hz). 13C-NMR (DMSO-d6):
δ = 172.5, 160.9, 158.9, 141.7, 133.8, 129.7, 129.2, 128.0, 127.3, 122.5, 115.3, 114.9, 111.1, 60.2,
51.3, 47.5 30.9, 26.3, 14.0. ESI-MS m/z = 490.1 [M+H]+.
Methyl 1-(4-ethoxy-4-oxobutyl)-4-(4-(3, 4-dimethylbenzyloxy)phenyl)-1H-pyrrole-2-carboxylate (6h).
1
Colorless, oily liquid; yield: 449 mg (95%); Rf = 0.3 (hexanes-EtOAc, 6:1). H-NMR (DMSO-d6):
δ = 7.483–7.527 (m, 3H), 7.067–7.262 (m, 4H), 6.957–7.020 (m, 2H), 5.003–5.062 (d, 2H, J = 23.6 Hz),
4.311–4.339 (t, 2H, J = 11.2 Hz), 4.009–4.030 (m, 2H), 3.762–3.801 (m, 3H), 2.203–2.266 (m, 8H),
13
1.968–2.003 (m, 2H), 1.134–1.169 (m, 3H). C-NMR (DMSO-d6): δ = 172.5, 160.9, 157.2, 136.5,
136.0, 130.6, 134.7, 129.9, 129.7, 129.2, 127.0, 126.4, 126.1, 125.5, 123.3, 121.7, 115.3, 114.6, 68.8,
60.2, 51.3, 47.9, 30.9, 26.6, 19.7, 19.4, 14.3. ESI-MS m/z = 450.2 [M+H]+.
3.4. General Procedure for the Synthesis of Compounds 7a–h
General Procedure
1-(4-carboxybutyl)-4-(4-(substituted benzyloxy)phenyl)-1H-pyrrole-2-carboxylic acid. To a stirred
solution of intermediate 6a–h in MeOH (50 mL) was added 1 N NaOH (10 mL) at 25 °C. The mixture
was stirred at the same temperature for 30 min. The aqueous layer was separated and was acidified
with 1N HCl to pH-4. The aqueous layer was extracted with EtOAc (3 × 50 mL). The combined
organic layer was dried (Na2SO4), filtered, and crystallized twice with EtOAc to give pure 7a–h.
1-(3-Carboxypropyl)-4-(4-(4-chlorobenzyloxy)phenyl)-1H-pyrrole-2-carboxylic acid (7a). White solid;
yield: 313 mg (67%); Mp: 163–164 °C; IR (KBr): 2,958, 1,690, 1,597, 1,512, 1,489, 1,454, 1,434,
1
1,376, 1,291, 1,178, 1,107, 1,062, 931, 827, 802 cm–1; H-NMR (DMSO-d6): δ = 12.193 (s, 2H),
7.464–7.514 (m, 6H), 7.123–7.198 (m, 2H), 6.965–7.001 (m, 2H), 5.109 (s, 2H), 4.290–4.324 (t, 2H,
J = 13.6 Hz), 2.147–2.185 (t, 2H, J = 15.2 Hz), 1.922–1.958 (m, 2H); 13C-NMR (DMSO-d6):
δ = 172.5, 160.9, 156.8, 140.1, 133.4, 130.6, 129.7, 128.7, 128.0, 127.5, 126.1, 123.2, 121.7, 115.4,
114.6, 110.7, 68.5, 60.2, 51.3, 47.9, 30.9, 26.6, 14.3; ESI-MS: m/z = 416.5 [M+H]+; HRMS-FAB: m/z
[M+H]+ calcd for C22H21Cl1N1O5: 414.11028; found: 414.10991.
1-(3-Carboxypropyl)-4-(4-(4-methylbenzyloxy)phenyl)-1H-pyrrole-2-carboxylic acid (7b). White
solid; yield: 409 mg (90%); Mp: 186–187 °C; IR (KBr): 2,936, 1,702, 1,616, 1,568, 1,486, 1,291,
1,266, 1,255, 1,203, 1,178, 815, 802 cm–1; 1H-NMR (DMSO-d6): δ = 12.174 (s, 2H), 7.107–7.482 (m,
8H), 6.951–6.973 (d, 2H, J = 8.8 Hz), 5.049 (s, 1H), 4.287–4.321 (t, 2H, J = 13.6 Hz), 2.304 (s, 3H),

Molecules 2012, 17
4778
13
2.145–2.183 (t, 2H, J = 15.2 Hz), 1.902–1.975 (m, 2H); C-NMR (DMSO-d6): δ = 173.8, 161.7,
156.6, 137.0, 134.1, 128.9, 127.7, 127.1, 125.7, 125.5, 122.5, 122.5, 115.1, 114.3, 69.1, 47.5, 30.7,
26.4, 20.7; ESI-MS m/z = 394.4 [M+H]+; HRMS-FAB: m/z [M+H]+ calcd for C23H24N1O5: 394.16490;
found: 394.16551.
1-(3-Carboxypropyl)-4-(4-(4-methoxybenzyloxy)phenyl)-1H-pyrrole-2-carboxylic acid (7c). White
solid; yield: 313 mg (66%); Mp: 177–178 °C; IR (KBr): 3,412, 1,749, 1,731, 1,690, 1,612, 1,567,
1
1,487, 1,402, 1,385, 1,305, 1,284, 1,177, 1,149, 1,094, 832, 810 cm–1; H-NMR (DMSO-d6):
δ = 12.182 (s, 2H), 7.464–7.486 (m, 3H), 7.367–7.388 (d, 2H, J = 8.4 Hz), 7.109–7.114 (d, 1H,
J = 2.0 Hz), 6.933–6.974 (m, 4H), 5.013 (s, 1H), 4.287–4.322 (t, 2H, J = 14.0 Hz), 3.754 (s, 3H),
13
2.145–2.183 (t, 2H, J = 15.2 Hz), 1.902–1.956 (m, 2H); C-NMR (DMSO-d6): δ = 173.8, 161.7,
158.9, 156.7, 129.4, 129.0, 127.0, 125.7, 125.5, 122.6, 122.5, 115.1, 114.2, 113.8, 68.9, 55.1, 47.5,
30.7, 26.4; ESI-MS m/z = 410.2 [M+H]+; HRMS-FAB: m/z [M+H]+ calcd for C23H24N1O6: 410.15981;
found: 410.16113.
1-(3-Carboxypropyl)-4-(4-(3,5-difluorobenzyloxy)phenyl)-1H-pyrrole-2-carboxylic acid (7d). White
solid; yield: 445 mg (94%); Mp: 178–179 °C; IR (KBr): 3,113, 1,732, 1,689, 1,666, 1,615, 1,567,
1,513, 1,451, 1,431, 1,404, 1,383, 1,307, 1,272, 1,255, 1,247, 1,201, 1,094, 1,057, 1,017, 833, 801,
778, 699 cm–1; 1H-NMR (DMSO-d6): δ = 12.203 (s, 2H), 7.488–7.516 (m, 3H), 7.127–7.198 (m, 4H),
6.980–7.002 (d, 2H, J = 8.8 Hz), 5.151 (s, 2H), 4.292–4.326 (t, 2H, J = 13.6 Hz), 2.148–2.168 (t, 2H,
13
J = 15.2 Hz), 1.924–1.960 (m, 2H); C-NMR (DMSO-d6): δ = 173.8, 161.7, 156.1, 127.5, 125.8,
125.6, 122.5, 122.4, 115.1, 114.3, 110.4, 110.4, 110.2, 103.1, 67.8, 47.5, 30.7, 26.5; ESI-MS
m/z = 416.3 [M+H]+; HRMS-FAB: m/z [M+H]+ calcd for C22H19F2N1O5: 416.13041; found: 416.13095.
1-(3-Carboxypropyl)-4-(4-(3-methylbenzyloxy)phenyl)-1H-pyrrole-2-carboxylic acid (7e). White solid;
yield: 381 mg (89%); Mp: 181–182 °C; IR (KBr): 3,113, 2,912, 1,732, 1,689, 1,666, 1,615, 1,567,
1,513, 1,451, 1,431, 1,404, 1,383, 1,307, 1,272, 1,255, 1,247, 1,201, 1,180, 1,094, 1,057, 1,017, 833,
801, 778 cm–1; 1H-NMR (DMSO-d6): δ = 12.217 (s, 2H), 7.474–7.497 (m, 3H), 7.224–7.296 (m, 3H),
7.119–7.149 (m, 2H), 6.964–6.986 (d, 2H, J = 8.8 Hz), 5.060 (s, 2H), 4.290–4.323 (t, 2H, J = 13.2 Hz),
2.321 (s, 3H), 2.147–2.184 (t, 2H, J = 14.8 Hz), 1.903–1.975 (m, 2H); 13C-NMR (DMSO-d6):
δ = 173.8, 161.7, 156.7, 137.5, 137.1, 128.4, 128.3, 128.2, 127.1, 125.7, 125.5, 124.7, 122.5, 122.4,
115.0, 114.2, 113.8, 69.2, 47.5, 30.7, 26.4, 21.0; ESI-MS m/z = 394.4 [M+H]+; HRMS-FAB: m/z
[M+H]+ calcd for C23H24N1O5: 394.16490; found: 394.16600.
1-(3-Carboxypropyl)-4-(4-(3-chlorobenzyloxy)phenyl)-1H-pyrrole-2-carboxylic acid (7f). White solid;
yield: 322 mg (88%); Mp: 173–174 °C; IR (KBr): 2,972, 1,716, 1,668, 1,599, 1,567, 1,513, 1,449,
1,436, 1,379, 1,287, 1,245, 1,215, 1,102, 1,055, 932, 830, 810 cm−1; 1H-NMR (DMSO-d6): δ = 12.217
(s, 2H), 7.424–7.519 (m, 6H), 7.127–7.178 (m, 2H), 6.977–7.000 (m, 2H), 5.110 (s, 2H), 4.291–4.324
13
(t, 2H, J = 13.2 Hz), 2.148–2.186 (t, 2H, J = 15.2 Hz), 1.923–1.976 (m, 2H); C-NMR (DMSO-d6):
δ = 174.4, 162.3, 156.9, 140.4, 133.6, 130.9, 130.0, 129.0, 128.2, 127.8, 126.6, 126.3, 126.1, 115.7,
114.9, 111.0, 68.8, 48.1, 31.2, 27.0; ESI-MS m/z = 414.6 [M+H]+; HRMS-FAB: m/z [M+H]+ calcd for
C22H21Cl1N1O5: 414.11028; found: 414.10996.

Molecules 2012, 17
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1-(3-Carboxypropyl)-4-(4-(2,6-dichlorobenzyloxy)phenyl)-1H-pyrrole-2-carboxylic acid (7g). White
solid; yield: 394 mg (87%); Mp: 208–209 °C; IR (KBr): 3,433, 2,956, 1,706, 1,657, 1,564, 1,510,
1,453, 1,437, 1,383, 1,302, 1,288, 1,263, 1,237, 1,208, 1,190, 1,175, 1,106, 1,055, 1,016, 830,
1
791 cm–1; H-NMR (DMSO-d6): δ = 12.232 (s, 2H), 7.456–7.586 (m, 6H), 7.142–7.148 (d, 1H,
J = 2.4 Hz), 7.030–7.052 (d, 2H, J = 8.8 Hz), 5.230 (s, 2H), 4.300–4.334 (t, 2H, J = 13.6 Hz),
13
2.157–2.195 (t, 2H, J = 15.2 Hz), 1.913–1.991 (m, 2H); C-NMR (DMSO-d6): δ = 173.8, 161.7,
156.8, 136.0, 131.8, 131.5, 128.8, 127.6, 125.8, 125.6, 122.5, 122.4, 115.0, 114.3, 65.0, 40.1, 30.7,
26.4; ESI-MS m/z = 448.3 [M+H]+; HRMS-FAB: m/z [M+H]+ calcd for C22H20Cl2N1O5: 448.07130;
found: 448.07186.
1-(3-Carboxypropyl)-4-(4-(3,4-dimethylbenzyloxy)phenyl)-1H-pyrrole-2-carboxylic acid (7h). White
solid; yield: 306 mg (75%); Mp: 157–158 °C; IR (KBr): 2,947, 1,716, 1,668, 1,567, 1,512, 1,451,
1,436, 1,364, 1,286, 1,263, 1,178, 1,103, 1,053, 931, 829, 811 cm–1; 1H-NMR (DMSO-d6): δ = 12.228
(s, 2H), 7.465–7.508 (m, 3H), 7.069–7.262 (m, 4H), 6.948–7.012 (m, 2H), 5.013 (s, 2H), 4.293–4.321
(t, 2H, J = 11.2 Hz), 2.212–2.271 (m, 6H), 2.146–2.166 (t, 2H, J = 8 Hz), 1.922–1.974 (m, 2H);
13C-NMR (DMSO-d6): δ = 173.8, 161.7, 156.7, 135.7, 134.8, 134.5, 129.4, 128.9, 127.1, 125.7, 125.5,
125.2, 122.5, 122.5, 115.1, 115.0, 114.3, 68.5, 47.5, 30.7, 26.5, 19.4, 19.1; ESI-MS m/z = 408.4
[M+H]+; HRMS-FAB: m/z [M+H]+ calcd for C24H26N1O5: 408.18055; found: 408.18128.
3.5. Antimycobacterial Activity Determination by Fluorometric Microplate Alamar Blue Assay
The methodology was fully described by Collins and Luna-Herrera [18,19], but some modifications
were made in the present study. The test was performed in 96-well sterile microplates (Falcon3072;
Becton Dickinson, Lincoln Park, NJ, USA). A solution of isoniazid (Sigma cooperation) was prepared
in sterile water at a concentration of 5 mg/mL; Pure compounds 7a–h were dissolved in DMSO at a
concentration of 5 mg/mL. The highest concentration wells received 7H9 broth (199 μL).Then the
solution of compound (1 μL) was added to the well. The H37Rv was grown in 7H9 broth (Difco,
Detroit, MI, USA) supplemented with 10% (vol/vol) OADC (oleic acid, albumin, dextrose, catalase;
Difco), and 0.05% (vol/vol) Tween 80 (Sigma) at 37 °C in one or two weeks until their turbidity
reached McFarland 1 (107 CFU/mL). Simultaneously a 1:20 diluted control was prepared from the
bacterial suspension, which represented the growth of 10% of the bacterial population tested.
Final testing concentrations are 0.2–0.0125 μg/mL for the isoniazid and from 0.2–25 μg/mL for
pure compounds.
Next, serial two-fold dilutions were prepared and an aliquot of the bacterial suspension (100 μL)
was added to these wells. The plates were incubated at 37 °C after 7 days of incubation, one control
was developed with 20 μL of 10× alamarBlue™ and 5% Tween80 50 μL solution. The plates were
reincubated at 37 °C for 24 h. After this incubation, if the well turned pink, all wells received
alamarBlue™ solution and were incubated for an additional 24 h. Fluorescence was measured in a
plate fluorometer at a 530-nm excitation wavelength and a 590-nm emission wavelength and Relative
fluorescence units (RFU) were recorded. Minimum inhibitory concentration (MIC) was defined as the
lowest extract of pure compounds concentration that presented RFU values lower than those presented

Molecules 2012, 17
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by the 10% growth control. There was always a correlation between fluorometric and visual observation,
i.e., pink wells presented high RFU values. The assay results are summarized in Table 1.
4. Conclusions
The present study supports the fact that 1-(4-carboxybutyl)-4-(4-(substituted-benzyloxy)phenyl)-
1H-pyrrole-2-carboxylic acid derivatives display good activity against resistant Mycobacterium
tuberculosis. The compound 7d’s MIC value was found 12.5 μg/mL. Further evaluationg such as the
activity of anti XDR-Mtb strains is still in progress. These products may be useful for the development
of anti-tuberculosis drugs.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/17/5/4770/s1.
Acknowledgements
This work was financially supported by the National High Technology Research and Development
Program of China (No. 2006AA020601).
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Sample Availability: Samples of the compounds 7a–h are available from the authors.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).