Synthesis and antituberculosis activity of novel mefloquine-isoxazole carboxylic esters as prodrugs

Article abstract of DOI:10.1016/j.bmcl.2009.11.105

5-(2,8-Bis(trifluoromethyl)quinolin-4-yloxymethyl)isoxazole-3-carboxylic acid ethyl ester (compound 3) was reported to have excellent antituberculosis activity against both replicating and non-replicating Mycobacterium tuberculosis, with a minimum inhibitory concentration (MIC) of 0.9 μM and 12.2 μM, respectively. In this study, the antituberculosis activity of compound 3 was further investigated. Its activity appeared to be very specific for organisms of the M. tuberculosis complex and it effected significant reductions of bacterial numbers in infected macrophages with an EC₉₀ of 4.1 μM. More importantly, the increased in vitro antituberculosis activity of the corresponding acid (compound 4) at pH 6.0 suggested that it may be active in vivo in an acidic environment produced as a consequence of inflammation in the lungs of TB patients. The fact that various ester bioisosteres of compound 3 lost anti-TB activity further suggested that the ester compound 3 may function as a prodrug. The detailed structure-activity relationships (SARs) from this study should facilitate our ultimate goal of improving the anti-TB potency of this isoxazole ester series.

Full text of DOI:10.1016/j.bmcl.2009.11.105

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1263–1268
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antituberculosis activity of novel mefloquine-isoxazole
carboxylic esters as prodrugs
Jialin Mao a, Hai Yuan b, Yuehong Wang a, Baojie Wan a, Dennis Pak a, Rong He b, Scott G. Franzblau a,
*
a Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612, United States
b Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
5-(2,8-Bis(trifluoromethyl)quinolin-4-yloxymethyl)isoxazole-3-carboxylic acid ethyl ester (compound 3)
Received 30 September 2009
Revised 18 November 2009
Accepted 19 November 2009
Available online 26 November 2009
was reported to have excellent antituberculosis activity against both replicating and non-replicating
Mycobacterium tuberculosis, with a minimum inhibitory concentration (MIC) of 0.9 lM and 12.2 lM,
respectively. In this study, the antituberculosis activity of compound 3 was further investigated. Its activ-
ity appeared to be very specific for organisms of the M. tuberculosis complex and it effected significant
reductions of bacterial numbers in infected macrophages with an EC90 of 4.1 lM. More importantly,
Keywords:
Antituberculosis
Quinoline
Isoxazole
SAR
the increased in vitro antituberculosis activity of the corresponding acid (compound 4) at pH 6.0 sug-
gested that it may be active in vivo in an acidic environment produced as a consequence of inflammation
in the lungs of TB patients. The fact that various ester bioisosteres of compound 3 lost anti-TB activity
further suggested that the ester compound 3 may function as a prodrug. The detailed structure–activity
relationships (SARs) from this study should facilitate our ultimate goal of improving the anti-TB potency
of this isoxazole ester series.
Ester
Ó 2009 Elsevier Ltd. All rights reserved.
Tuberculosis (TB) is a major cause of mortality and one of the
most frequent AIDS-associated diseases worldwide.1 The current
standard treatment for TB requires a combination regimen of iso-
niazid (INH), pyrazinamide (PZA), rifampin (RMP), and ethambutol
(EMB) for at least six months, which often leads to poor compli-
ance.2 The difficulty in treating drug-resistant TB, such as multi-
drug-resistant TB (MDR-TB) and extensively drug-resistant TB
(XDR-TB) also contributes to the increased morbidity and mortal-
ity.3 This demonstrates the urgency of the need for new anti-TB
agents.
ring. Based on these similarities, compound 3 was designed using
chemical hybridization strategy.6 Initially, we expected to
a
develop potent anti-TB mefloquine-based ligands with an
isoxazolecarboxamide moiety. Instead, 5-(2,8-bis(trifluoro-
methyl)quinolin-4-yloxymethyl)isoxazole-3-carboxylic acid ethyl
ester (compound 3), was identified, with excellent antituberculo-
sis activity and devoid of detectable cytotoxicity. The minimum
inhibitory concentration (MIC) in the microplate Alamar Blue as-
say (MABA)7 and the low oxygen recovery assay (LORA)8 was
0.9
gesting that compound 3 was potent against both replicating
and non-replicating M. tuberculosis. The SI of compound
lM (0.4 lg/mL) and 12.2 lM (5.3 lg/mL), respectively, sug-
In our previous work,4,5 we have explored the SARs of meflo-
quine-based analogs as anti-TB agents. The modifications in-
cluded introduction of the hydrazone linker into mefloquine at
4-position, replacement of the piperdine with a piperazine, and
extension of the basic terminus of the piperazine ring at the 4-
position. Compound 7f, ( Fig. 1), was found to have improved
anti-TB activity and selectivity (SI: selectivity index). Moreover,
the isoxazole scaffold (Fig. 1) emerged as one of the most potent
hits in a high throughput screen of the ChemBridge NovaCore li-
brary against Mycobacterium tuberculosis in our laboratory. Both
compound 7f and the isoxazole hits consisted of an aromatic
ring, and a two-atom linker with either a five or six membered
3
(>142.2) was 158-fold higher than that of mefloquine, and it
was tolerated at a dosage of P400 mg/kg po in a vehicle of
carboxymethylcellulose.
In this study, the antituberculosis activity of compound 3 was
further investigated, and various modifications were performed
on the ester and the quinoline ring.
It is important to note that M. tuberculosis could reside in the
lung both intracellularly and extracellularly. While compound 3
was active against M. tuberculosis H37Rv and Erdman in axenic
medium (Table 1), it also effected a significant reduction in col-
ony forming units (CFU) in M. tuberculosis-infected macrophages
with an EC90 4.1
mL) (Fig. 2).
lM (1.7 lg/mL) and an EC99 18.3 lM (7.9 lg/
* Corresponding authors.
E-mail address: sgf@uic.edu (S.G. Franzblau).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.105

1264
J. Mao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1263–1268
HO
N
N
N
N
H
N
H
O
N
CF3
N
CF3
O
N
CF3
CF3
O
O
N
(+)-(S, R) Mefloquine
MIC: 13
7f
µ
µM
MIC: 4
IC50: 27 M
SI: 6.7
M
CF3
µ
µ
IC50: 11 M
SI: 0.9
CF3
3
R1
R1 = aryl, heteroaryl
2 = H, alkyl
R3 = alkyl
MABA MIC: 0.9
µ
M (0.4 g/mL)
µ
O
µ
µ
LORA MIC: 12.2 M (5.3 g/mL)
R
High throughput
screening of
ChemBridge's
NoVACore
O
IC50 > 128 M
µ
N
SI > 142.2 / > 10.5
No CYP3A4 inhibition
Maximum tolerated dose: 400 mg/kg
po in mice
O
R3
library in ITR
N
R2
Isoxazolecarboxamide
eg.
S
N
O
NH
O
O
N
MIC: 3.4
IC50 > 128
SI > 37.6
M
µ
O
µ
M
O
Figure 1. Summary of hybridization design of compound 3.
Table 1
bacteria. These findings bring to light possible penetration issues
Antimicrobial spectrum of compounds 3 and 4
of the acid. The acid may be able to penetrate the less hydrophobic
cell wall of Gram-positive bacteria but not Gram-negative bacteria
nor mycobacteria.
It has been reported that weak acids have antituberculosis
activity only at acid pH.9,10 As compound 4 is a weak acid with a
calculated pKa of 3.75, the MICs of compounds 3 and 4 were deter-
mined at pH 6.0. As anticipated, the MIC of compound 4 (Table 2)
O
O
OC
O
N
O
O
OH
O
2H5
N
N
CF3
CF3
N
3
4
CF3
Organism
CF3
MIC (lM)
decreased from >128
7.7 M in PZA medium (pH 6.0). At the latter pH, the positive con-
trol PZA had a reasonable MIC (829 M, 102.1 g/mL) comparable
lM in the normal pH 6.8 culture medium to
l
3
4
l
l
Mycobacterium tuberculosis Erdman
Mycobacterium bovis BCG
Mycobacterium smegmatis
Bacillus anthracis
Staphylococcus aureus
Escherichia coli
1.74
<0.5
>128
>128
>128
32
22
>128
>128
to the values reported in the literature.11 No changes in the MICs of
isoniazid (INH) and rifampin (RMP) were observed in response to
the acid pH environment. The increased in vitro antituberculosis
activity of compound 4 at pH 6.0 suggested that it may be active
in vivo as a localized acidic pH environment is likely produced in
the lungs of TB patients as a consequence of inflammation.10 Com-
pound 3 also appeared ninefold more potent in the acidic medium
than the neutral pH condition. The acid would be expected to be
pumped out by an efflux mechanism, as is the case for pyrazinoic
acid (POA, the active form of PZA).12,13 The charged form of the acid
4 may not be able to penetrate the cell wall at neutral pH. How-
ever, at acid pH, some of the neutral form of compound 4 will be
present, and thus it should be able to permeate the cell. The higher
accumulation of acid 4 may contribute to this ninefold decrease in
the MIC of compound 3 at acid pH. This well-correlated decrease in
MICs of both compound 3 and compound 4 suggests that com-
pound 3 may function as a prodrug, with the acid as the active
form.
>128
>128
>128
>128
>128
Candida albicans
Because of the length of TB treatment, it is preferable that anti-
TB agents possess a narrow antimicrobial spectrum. Although
compound 4 showed no activity against M. tuberculosis H37Rv in
our previously study, it may be caused by the poor penetration
of acid 4 through the wax-like cell wall of the TB bacterium.6 For
these reasons, compound 3 and its acid 4 were tested against
several mycobacteria (including M. tuberculosis Erdman, Mycobacte-
rium bovis BCG and Mycobacterium smegmatis), Gram-positive
bacteria (Bacillus anthracis and Staphylococcus aureus), a Gram-neg-
ative bacterium (Escherichia coli) and a fungus (Candida albicans)
(Table 1). The activity of compound 3 was very specific for organ-
isms of the M. tuberculosis complex, and it showed no activity
against the fast grower M. smegmatis, which is unusual for an
anti-TB agent. Interestingly, although the acid 4 was neither active
against M. tuberculosis nor the Gram-negative bacteria in vitro, it
did demonstrate modest activity against the Gram-positive
In order to further investigate whether compound 3 serves as a
prodrug, the ethyl ester was replaced by longer alkyl or aromatic
esters. Various esters were prepared from 5-(2,8-bis(trifluoro-
methyl)quinolin-4-yloxymethyl)isoxazole-3-carboxylic chloride
(6) with different alcohols at the presence of triethylamine
(Scheme 1). All the esters were active against M. tuberculosis in

J. Mao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1263–1268
1265
1.0E+06
1.0E+05
1.0E+04
1.0E+03
1.0E+02
1.0E+01
0 (T0)
0 (T7)
16
4
1
0.25
Concentration (µg/ml)
Figure 2. Compound 3 versus M. tuberculosis Erdman in J774A.1 macrophages.
were as potent as compound 3 against both replicating and non-
replicating M. tuberculosis (Table 3). When the methyl group in
compound 7a was replaced by a trifluoromethyl group, compound
Table 2
In vitro antituberculosis activity of compounds 3 and 4 at pH6.8 and pH6.0
Compd
pH 6.8 MIC (
lM)
pH 6.0 MIC (lM)
7b with a MIC of 25 lM, was 10-fold less active than that of 7a.
3
4
PZA
RMP
INH
0.9
0.1
7.7
829
0.2
0.1
This suggested that electronic effect may play a role in the antitu-
berculosis activity in this series. The putative binding site may
have a very limited space as it was observed that esters with a
longer alkyl chain (compound 7d) and an aromatic ring (compound
>128 (89%)
>4000
0.1
0.1
7e) had higher MICs (15.3
lM and 26.5 lM, respectively) com-
pared to that of compound 3 in the MABA.
Next, the ester group was replaced by various bioisosteres, such
as amides and the oxadiazole. While all the esters demonstrated
both MABA and LORA. The activities of the propyl ester 7a
(MIC = 1.8/26.1 M) and the butyl ester 7c (MIC = 0.9/9.9 M)
l
l
O
O
O
O
N
N
CF3
CF3
3
a
O
O
O
O
N
O
O
O
N
O
OH
O
N
O
R1
N
O
OH
b
O
N
N
N
e
d
R2
N
8
CF3
O
CF3
N
4
CF3
CF3
5b d
CF3
CF3
CF3
-
CF3
9
Cl
1
2
5b
5c
5d
: R , R = C2H5
f
1
2
: R , R = [1, 2] oxazinane
: R1 = NH2, R2 = H
N
O
c
O
N
N
O
N
O
OR
O
O
O
O
CF3
10
O
N
Cl
N
d
CF3
g
N
CF3
7a e
N
CF3
O
CF3
CF3
O
6
-
O
NH2
7a
7b
7c
7d
7e
: R = (CH2)2CH3
: R = (CH2)2CF3
: R = (CH2)3CH3
: R = (CH2)5CH3
: R = CH2C6H5
N
N
CF3
5a
CF3
Scheme 1. Synthesis of various esters and ester bioisosteres. Reagent and conditions: (a) LiOH, THF/H2O/MeOH = 3:1:1, rt, 4 h, 90%; (b) RNH2, EDC, HOBt, iPr2NH, CH2Cl2, rt,
overnight; (c) oxalyl chloride, DMF, 15 min, rt; (d) ROH/acetyl chloride, Et3N, CH2Cl2, reflux, overnight; (e) NaBH4, N2, anhydrous EtOH, overnight; (f) N-hydroxypropi-
onamidine, EDC, CH2Cl2, reflux, 3 d; (g) NH4OH, EtOAc, rt, overnight.

1266
J. Mao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1263–1268
Table 3
Compound 5d was prepared from the carboxylic acid 4 by reaction
with hydrazine monohydrate. However, neither 5a nor 5b was ac-
tive against M. tuberculosis (Table 3).
In vitro activity of esters and bioisosteres against M. tuberculosis H37Rv
O
R
Two tertiary carboxamides were prepared which included the
diethylamide 5b and the [1,2]oxazinane-2-carbonyl 5c as they
might be cleaved more easily. Both tertiary carboxamides were
prepared from the acid 4, which was reacted with the appropriate
amines in the presence of 1-ethyl-3-[3-dimethylaminopropyl]car-
bodiimide hydrochloride (EDC). Unfortunately, compound 5b and
5c failed to show any anti-TB activity. In summary, none of amides
were active against M. tuberculosis including primary (compound
5a), secondary (compounds in Ref. 6) and tertiary amides (com-
pounds 5b and 5c).
O
N
N
CF3
CF3
MIC (
MABA LORA
Compd Structure R=
l
M)
IC50
M)
Vero
SI
Clog P
(l
MABA
LORA
1
RMP
3
7a
7b
7c
7d
7e
5a
5b
Mefloquine
Rifampin
COOCH2CH3
COO(CH2)2CH3 1.8
COO(CH2)2CF3
COO(CH2)3CH3 0.9
COO(CH2)5CH3 15.3
COOCH2C6H5
CONH2
13
0.1
0.9
7
2
11
122
0.9
1220
1.6
61
3.6
—
12.1
26.1
29.9
9.9
26.7
50.7
>128
>128
>128
105.9
>128
>128
>142.2 >10.5
4.4
4.9
5.3
5.4
6.4
5.6
3.5
4.4
>71.2
>5.1
>4.9
>4.3
The acid 4 was reduced to its alcohol form 8 by sodium borohy-
dride, from which a new ester 9 was generated. The extended ester
9 failed to show any in vitro activity against M. tuberculosis as did
the intermediate alcohol 8.
25.0
>109.1 >10.6
>8.4
>4.8
—
>4.8
>2.5
—
26.5
>128
>128
>128 >128
>128 46.5
It is known that [1,2,4]oxadiazoles are able to serve as ester bio-
isosteres, and therefore this heterocycle was introduced into our
lead compound by the reaction of the acid 4 and N-hydroxypropi-
onamidine in the presence of EDC (Scheme 1). As with the other
bioisosteres, compound 10 also failed to show any anti-TB activity.
That none of the ester bioisosteres were active against M. tuber-
culosis in vitro further supported the hypothesis that 3 functions as
a prodrug. The inactivity of the alcohol 8 and the isomeric ester 9
ruled out the possibility of the alcohol of compound 3 serving as
the active metabolite. Thus, in advancing the SAR work from this
point forward, we decided to maintain the ethyl ester moiety
and focus our further modifications on the quinoline ring.
Inspired by the SAR from the Novacore library (unpublished
data), the position of the oxymethylene linker on the quinoline ring
was investigated. Ideally, the SAR could be built around the 2,8-
bis(trifluoromethyl)quinoline as the key scaffold. Although the
hydroxyquinoline 14h was prepared by the traditional Combes
quinoline synthesis (Scheme 2), the route was somewhat lengthy
and the yield of quinoline ring formation was low. Therefore, in-
stead of employing 2,8-bis (trifluoromethyl)quinoline as the start-
CON(C2H5)2
—
—
O
N
O
5c
>128
>128 >128
—
—
4.6
5d
8
9
CONHNH2
CH2OH
CH2OCOCH3
>128
>128
>128
>128 >128
>128 >128
>128 >128
—
—
—
—
—
—
2.5
3.2
4.2
N
10
>128
>128 >128
—
—
4.6
O
N
some anti-TB activity, none of the ester bioisosteres (compounds
5a–d and 9–10) were active.
As INH and PZA are known to serve as prodrugs, the amide and
hydrazide moieties were introduced to the isoxazole carboxylic
acid. Compound 5a was prepared from 5-(2,8-bis(trifluoro-
methyl)quinolin-4-yloxymethyl)isoxazole-3-carboxylic acid chlo-
ride (6) by reaction with concentrated NH3 (aq) (Scheme 1).
O
O
O
O
a
OH
Br
O
N
N
O
O
12
11
O
b
Ar OH
O
Ar
O
N
13a-j
14a-j
13a: Ar = qunolin-4-ol
13b: Ar = qunolin-2-ol
13c: Ar = qunolin-5-ol
13d: Ar = qunolin-6-ol
13e: Ar = qunolin-7-ol
13f: Ar = qunolin-8-ol
14a: Ar = qunolin-4-ol
14b: Ar = qunolin-2-ol
14c: Ar = qunolin-5-ol
14d: Ar = qunolin-6-ol
14e: Ar = qunolin-7-ol
14f: Ar = qunolin-8-ol
13g: Ar = 2- trifluoromethylqunolin-7-ol
14g: Ar = 2- trifluoromethylqunolin-7-ol
14h: Ar = 2,8-bis(trifluoromethyl)qunolin-4,6-diol
14i: Ar = 1H-indol-6-ol
13h: Ar = 2,8-bis(trifluoromethyl)qunolin-4,6-diol
13i: Ar = 1H-indol-6-ol
13j: Ar = p-nitro-m-trifluoromethyl
14j: Ar = p-nitro-m-trifluoromethyl
OH
N
OH
N
O
O
HO
HO
O
c
d
e
f
NH
NO
2
NO
CF
3
2
CF
3
2
CF
CF
3
3
CF
3
CF
3
CF
3
15
13h
18
16
17
Scheme 2. Synthesis of compounds in Tables 4 and 5. Reagent and conditions: (a) CBr4/PPh3, CH2Cl2, À15 °C, 2 h; (b) 12, K2CO3, acetone, reflux, overnight; (c) MeI, K2CO3,
acetone, reflux, overnight, 95%; (d) Zn/HCl, MeOH, 90%; (e) ethyl 4,4,4-trifluoroacetoacetate, PPA, 40%; (f) 48% HBr, reflux, overnight, 95%.

J. Mao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1263–1268
1267
ing point of the SAR, the unsubstituted hydroxyquinoline was used
compound 14a (MIC = 25.1
l
M). However, the MICs of compounds
instead, as many of the starting materials are commercially
available.
Compounds 14a–j were prepared from various hydroxyquino-
lines by reaction with ethyl 5-(bromomethyl)-3-isoxazolecarboxy-
late (12), which is the bromination product of 5-hydroxy
methylisoxazole-3-carboxylic acid ethyl ester (Scheme 2). Com-
14d (7.4 M) and 14e (10.8 lM) with oxymethylene linkers at-
l
tached to the 6- and 7-positions, respectively, decreased 2–3-fold
compared with that of compound 14a. We expected that introduc-
tion of a trifluoromethyl group into the quinoline of 14e to improve
its MIC value. Indeed, the 2-trifluoromethyl substituted compound
14g (MABA MIC = 3.6 lM) with an oxymethylene linker attached
pound 14a (MABA MIC = 25.1
tive than compound 3, although it had similar MIC in the LORA
assay (11.3 vs 12.2 M, respectively) (Table 4). This suggested that
the 2- and 8-trifluoromethyl groups on the quinoline ring were
essential for the antituberculosis activity against replicating
l
M) was more than 26-fold less ac-
to the 7-position was threefold more active than the unsubstituted
compound 14e.
l
As the 4-, 6-, and 7-positions of quinoline were promising posi-
tions for attachment of the oxymethylene linker, the capacity of
the binding site was further explored. We assume that for the ac-
tive compounds 14a, 14d and 14e, the interaction between the
isoxazole ester moiety and the active site of the putative target is
important for activity. And it is likely that the isoxazole carboxylic
ester groups in these compounds bind to the same binding pocket
M. tuberculosis. In the MABA, compound 14b (MIC = 103.3
lM),
14c (MIC = 35.1 M), and 14f (MIC = 39.8 M) with the oxymethyl-
l
l
ene linker attached to the 2-, 5-, and 8-positions of the quinoline
ring, respectively, were less active than the 4-substituted
Table 4
In vitro activity of various quinolines against M. tuberculosis H37Rv
Compd
Structure
MIC (
MABA
13
0.1
l
M)
IC50
(l
M) Vero
SI
Clog P
LORA
MABA
LORA
1
RMP
Mefloquine
Rifampin
7
2
11
122
0.9
1220
1.6
61
3.6
—
O
O
O
O
N
3
0.9
12.2
>128
>144
>10.5
4.4
N
CF3
CF3
O
O
O
O
N
14a
14b
14c
25.1
103.3
35.1
11.3
64
>128
>128
>128
>5.1
>1.2
>3.6
>11.3
>2
2.5
2.5
2.5
N
N
O
O
O
O
N
O
O
O
O
N
31.1
>4.1
N
O
O
14d
7.4
12.8
>128
>17.3
>10
2.5
N
O
O
N
O
14e
14f
14g
10.8
39.8
3.6
40.3
125.1
21.6
107.6
>128
>128
>9.9
>3.2
>25.6
>2.7
> 1.0
>5.9
2.5
2.5
3.8
N
O
O
O
O
N
O
N
N
O
O
O
O
O
N
CF3
O
N
O
O
O
O
N
N
O
O
O
14h
17.6
15.6
>128
>7.3
>8.2
5.6
N
O
CF3
CF3

1268
J. Mao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1263–1268
while the orientations of the quinoline rings may be different. To
confirm this assumption, compound 14h which contains two such
oxymethylene linked isoxazole groups at 4- and 6- positions was
designed and synthesized.
* small ester substitutents were favored,
such as propyl and butyl ester
O
O
N
* carboxylic ester along with the isoxazole
moiety may facilitate the orientation of
ligands in the putative binding site
O
O
N
CF3
Compound 14h was prepared in five steps from 4-hydroxy-
1-nitro-2-trifluoromethylbenzene (15) (Scheme 2). The hydroxyl
group was protected by a methyl group, and then compound 16
was reduced using Zn–HCl to afford 4-methoxy-2-trifluoromethyl-
phenylamine (17). The Combes quinoline synthesis was carried out
by reacting compound 17 with ethyl 4,4,4-trifluoroacetoacetate in
the presence of polyphosphoric acid (PPA). The product 18 was
demethylated to give compound 13h, which was reacted in turn
with ethyl 5-(bromomethyl)-3-isoxazolecarboxylate to afford 2,8-
bis(trifluoromethyl)quinolin-4,6-bis(5-methoxyl-isoxazole-3-car-
boxylic acid ethyl ester) (14h). The MABA MIC of compound 14h
CF3
* 2,8-bis(trifluoromethyl) groups were
favored on the quinoline ring
* 6/7-position > 4-position
* phenyl ring in quinoline was
important for antituberculosis activity
Figure 3. SARs on compound 3.
may be acting as a prodrug. The well-correlated improved antitu-
berculosis activity of the acid 4 and compound 3 at acid pH further
support this novel hypothesis. As the acidic pH environment is pos-
sibly produced as a consequence of inflammation in the lungs of TB
patients, the acid may be active in vivo. In this context, our modi-
fications were focused on the quinoline ring and positions of the
oxymethylene linker substitution. 2,8-Bis(trifluoromethyl) substi-
tutions on the quinoline were found to be favored, and the phenyl
ring in the quinoline was important for its antituberculosis activity.
The 6- and 7-positions were better than the 4-position for location
of the oxymethylene linker. It is also suggested that the isoxazole
carboxylic acid ester moiety forms a key interaction with the active
site of the putative target, which is crucial for activity, while the
quinoline ring may be less important (Fig. 3). As the SAR results
in the present study support the idea that compound 3 may func-
tion as a prodrug, the metabolism of compound 3 has been studied,
and these results then used to design and synthesize a new series
of mefloquine-isoxazole carboxylic acid esters with better in vitro
and in vivo metabolic stability.
was 17.6 lM; 18-fold higher than that of compound 3. If there
were more than one binding pockets for isoxazole carboxylic es-
ters, the 4,6-disubstituted compound 14h should show comparable
activity with that of 3. The remarkably decreased activity of 14h is
consistent with our assumption that the isoxazole carboxylic acid
ester groups of different active compounds bind to the same bind-
ing pocket.
As discussed above, compared to the isoxazole carboxylic ester
part, the quinoline ring may not be crucial for activity. To further
explore the importance of the quinoline ring, it was replaced by
1H-indole and phenyl rings, and compounds 14i–j were prepared
by the same synthetic route as compound 14a (Scheme 2).
Both compounds 14i (MABA MIC = 8.6
MIC = 3.5 M) were shown to be more active in vitro than the cor-
responding quinoline compound 14e (MABA MIC = 10.8 M).
lM) and 14j (MABA
l
l
When 14e and 14i–j were compared structurally, it was found that
the phenyl part in the quinoline group of 14e was conserved in
14i–j while the pyridine part in the quinoline group was
downsized (14i) or completely removed (14j). This suggests that
among the two rings in the quinoline moiety, the phenyl ring
may play a more important role than the pyridine ring against
replicating M. tuberculosis.
As a summary, compound 3 was found to be active against M.
tuberculosis both intracellularly and extracellularly, and its activity
was specific for members of the M. tuberculosis complex. Various
ester analogs were active against M. tuberculosis in both MABA
and LORA. The compounds with small ester substituents, such as
propyl and butyl esters (compound 7a and 7c), were as active as
the lead compound 3. However, none of the ester bioisosteres,
including the isomeric ester, amides, and the oxadiazole analogs,
showed any activity. These observations suggested that the ester
Acknowledgments
We thank the Global Alliance for TB Drug Development for fund-
ing support, and thank Dr. Zhenkun Ma and Dr. Takushi Kaneko for
theirhelpfuldiscussions. We thankDr. SanghyunChofor performing
the spectrum of activity assay. We thank Dr. Andrew D. Mesecar for
the Bacillus anthracis MICs.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.11.105.
References and notes
Table 5
In vitro activity of various aryl substituents against M. tuberculosis H37Rv
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14i
14j
8.6
42.9
>128
>128
>14.9
>36.6
>2.9
2.4
N
H
3.5
62.2
>2.1
3.6
F3C
NO2