Searching for new cures for tuberculosis: Design, synthesis, and biological evaluation of 2-methylbenzothiazoles

Article abstract of DOI:10.1021/jm901112f

The actual development and clinical use of new therapeutics for tuberculosis (TB) have remained stagnant for years because of the complexity of the disease process, the treatment of which at present requires the administration of drug combinations over a 6month period. There is thus an urgent need for the discovery and development of novel, more active, and less toxic anti-TB agents. In this study, we report on the chemistry and biology of a series of potent 5-(2-methylbenzothiazol-5-yloxymethyl) isoxazole-3-carboxamide derivatives, which proved to be active against replicating Mycobacterium tuberculosis (Mtb) H₃₇Rv. The most potent compounds 7j and 7s were found to inhibit Mtb growth at micromolar concentrations, with MICvalues of 1.4 and 1.9 μM, respectively. Impressively, all active compounds were nontoxic toward Vero cells (IC50 > 128 μM). Moreover, the best of these compounds were also tested against protozoan parasites, and some of these compounds were found to show activity, especially against Plasmodium falciparum. These studies thus suggest that certain 2-methylbenzothiazole based compounds may serve as promising lead scaffolds for further elaboration as anti-TB drugs and as possible antimalaria drugs. 2009 American Chemical Society.

Full text of DOI:10.1021/jm901112f

J. Med. Chem. 2009, 52, 6757–6767 6757
DOI: 10.1021/jm901112f
Searching for New Cures for Tuberculosis: Design, Synthesis, and Biological Evaluation
of 2-Methylbenzothiazoles
Qingqing Huang,† Jialin Mao,†,‡ Baojie Wan,‡ Yuehong Wang,‡ Reto Brun,§ Scott G. Franzblau,‡ and Alan P. Kozikowski*,†
†Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 South Wood Street,
Chicago, Illinois 60612, ‡Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street,
Chicago, Illinois 60612, and §Swiss Tropical Institute, Socinstrasse 57, CH-4002 Basel, Switzerland
Received July 27, 2009
The actual development and clinical use of new therapeutics for tuberculosis (TB) have remained
stagnant for years because of the complexity of the disease process, the treatment of which at present
requires the administration of drug combinations over a 6 month period. There is thus an urgent need for
the discovery and development of novel, more active, and less toxic anti-TB agents. In this study, we
report on the chemistry and biology of a series of potent 5-(2-methylbenzothiazol-5-yloxy-
methyl)isoxazole-3-carboxamide derivatives, which proved to be active against replicating Mycobac-
terium tuberculosis (Mtb) H37Rv. The most potent compounds 7j and 7s were found to inhibit Mtb
growth at micromolar concentrations, with MIC values of 1.4 and 1.9 μM, respectively. Impressively, all
active compounds were nontoxic toward Vero cells (IC50 > 128 μM). Moreover, the best of these
compounds were also tested against protozoan parasites, and some of these compounds were found to
show activity, especially against Plasmodium falciparum. These studies thus suggest that certain
2-methylbenzothiazole based compounds may serve as promising lead scaffolds for further elaboration
as anti-TB drugs and as possible antimalaria drugs.
Introduction
the two hit compounds 1 and 2 possessing a 5-(2-methylben-
zothiazol-5-yloxymethyl)isoxazole-3-carboxamide scaffold
(Figure 1).
Tuberculosis (TBa) represents a highly contagious, air-
borne disease that is caused by infection with Mycobacterium
tuberculosis (Mtb), and it currently represents one of the most
threatening health problems globally. According to the World
Health Organization (WHO) data (2009),1 Mtb has infected
one-third of the world’s population and results in the death
of approximately 1.3-1.75 million patients in 2007. Further-
more, the emergence of multidrug-resistant tuberculosis
(MDR-TB) and extensively drug-resistant tuberculosis
(XDR-TB) strains continues.2,3 Although TB is curable, all
of the aforementioned factors, along with the failure of some
patients to complete the prescribed treatment, result in the
burden of treating TB. Thus, the discovery and development
of novel, safe, and more effective anti-TB agents is now being
given more attention than ever before, in part because of the
attention brought to the field by the Bill and Melinda Gates
Foundation.4
Herein, we describe the synthesis, biological evaluation,
andanalysis of the structure-activityrelationships(SAR)ofa
series of 2-methylbenzothiazole derivatives as anti-TB agents.
Inordertoidentifyagentswithbetterpotency, lack of toxicity,
andreasonable physicochemicalparameters, systematicstruc-
tural modifications were made to the benzothiazole core, the
oxymethylene linker, and the isoxazolecarboxamide group
(Figure 1).
Chemistry
Modifications on the Isoxazolecarboxamide Group. Com-
pounds 7a-w possessing various amides were prepared
using the methods outlined in Scheme 1. Commercially
available benzothiazole 3 was reacted with 5-bromomethyl-
isoxazole-3-carboxylic acid ethyl ester (4)6 in the presence of
K2CO3 and a catalytic amount of TBAI to afford 5 in an
almost quantitative yield. After basic hydrolysis of 5 in
MeOH, carboxylic acid 6 was obtained in a quantitative
yield, and this intermediate was subsequently coupled with a
set of amines to generate the desired ligands 1, 2, 7a-k,
7n-q, 7s, 7t, and 7v according to one of four methods:
compounds 7c, 7g, 7n-q, 7t, and 7v-w were synthesized in
the presence of EDC/HOBt and a catalytic amount of
DMAP (method A) in yields of 14.1-93.9%; compound 7a
was obtained from the acyl chloride (method B) in 94.2%
yield; compounds 1, 2, 7b, 7d, 7h-k, and 7s were synthesized
in the presence of PyBOP and DIPEA (method C) in yields of
To identify new chemical entities that could be used as a
starting point in the development of new TB drugs, a 50000
compound library (NOVACore Chembridge) was screened
for activity against replicating Mtb using the microplate
Alamar blue Assay (MABA).5 Among the series of com-
pounds identified having encouraging anti-TB activities were
*To whom correspondence should be addressed. Phone: þ1-312-996-
7577. Fax: þ1-312-996-7107. E-mail: kozikowa@uic.edu.
a Abbreviations: INH, isoniazid; MABA, microplate Alamar blue
assay; MIC, minimum inhibitory concentration; MDR-TB, multidrug-
resistant tuberculosis; Mtb, Mycobacterium tuberculosis; RMP, rifam-
pin; SAR, structure-activity relationships; TB, tuberculosis; XDR-TB,
extensively drug-resistant tuberculosis.
r
2009 American Chemical Society
Published on Web 10/09/2009
pubs.acs.org/jmc

6758 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Huang et al.
45.1-96.6%; compounds 7e and 7f were produced with CDI
(method D) in 77.2-98.7% yield. Hydroxamate deriva-
tives 7l and 7m were obtained by a two-step procedure
consisting of generating the potassium salt of hydroxylamine
followed by the addition to the esters 5 and 7j in alcohol,
respectively. Methylation of compound 7o was carried out
by reaction with MeI in the presence of K2CO3/DMF to yield
compound 7r in 88.2%. The ester 7j was hydrolyzed by
treatment with LiOH in MeOH/H2O, and the resulting acid
7u was converted to amide 7w following method C discussed
for the synthesis of 7j from 6.
Modifications on the Oxymethylene Linker. Compounds
12a-c were synthesized from the appropriate methoxy-sub-
stituted anilines as shown in Scheme 2. Amide bond forma-
tion was achieved using basic acylating conditions to afford
8a-c, which were converted in turn to the corresponding
thioamides 9a-c by reaction with Lawesson’s reagent in
refluxing chlorobenzene. Subsequent cyclization to the cor-
responding methoxy-substituted 2-methylbenzothiazoles
10a-c was accomplished using a modified procedure as
described in the literature.7,8 Whereas the o- and p-methoxy-
thioacetanilides 9a and 9b gave only the single benzo-
thiazoles 10a and 10b, respectively, in the case of the
m-methoxythioacetanilide 9c, a mixture of both the 5- and
7-substituted benzothiazoles was obtained. This mixture of
isomers could be separated by silica gel chromatography
either at this stage or after the following set of reactions.
Subsequent demethylation with BBr3 afforded the hydroxyl
substituted 2-benzothiazoles 11a-c. Lastly, compounds
12a-c were obtained from compounds 11a-c, respectively,
following the same methodology as employed for the
preparation of 7j from 3.
Modifications on the Benzothiazole Core. The coupling
reaction of m-anisidine (13) and 3-(trifluoromethyl)benzoic
acid (14) was carried out in the presence of EDC/HOBt to
yield intermediate 15, which was subsequently converted to
the corresponding benzothiazole 16 by employing the same
methodology as that used for the conversion of 8 to 11a
(Scheme 3). Target compounds 17, 19a, 19b and 23 were
synthesized from 16, 18a, and 18b, respectively, using the
same methodology as that used for the synthesis of 7j from 3.
Target compound 21 was synthesized from 20 via method C
from above.
Modifications on the Isoxazole Ring. For the prepa-
ration of 32, ethyl thiooxamate (24) was condensed
with 1,3-dichloroacetone (25) to provide 2,4-disubstituted
Figure 1. Systematic modifications to hits 1 and 2.
Scheme 1a
a Reagents and conditions: (a) K2CO3, TBAI, DMF, 50 °C; (b) LiOH H2O, MeOH-H2O. (c) Method A: R1R2NH, EDC, HOBt, DIPEA, DMAP, 4
A molecular sieves, DMF. Method B: (1) (COCl)2, CH2Cl2; (2) R1R2NH, pyridine. Method C: R1R2NH, PyBOP, DIPEA, DMF. Method D: R1R2NH,
CDI, DMF. (d) 1.76 M NH2OK, MeOH; (e) MeI, K2CO3, TBAI, DMF. For complete structures see Table 1.
3
˚

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6759
Scheme 2a
a Reagents and conditions: (a) Ac2O, pyridine; (b) Lawesson’s reagent, chlorobenzene, 135 °C, 3 h; (c) K3Fe(CN)6, KOH, H2O; (d) BBr3, CH2Cl2; (e)
˚
(1) 4, K2CO3, TBAI, DMF, 50 °C; (2) LiOH H2O, MeOH-H2O; (3) phenyl-Gly(OMe) HCl, PyBOP, DIPEA, 4 A molecular sieves, DMF.
3
3
Scheme 3a
a Reagents and conditions: (a) EDC, HOBt, DIPEA, DMAP, CH2Cl2; (b) (1) Lawesson’s reagent, chlorobenzene, 135 °C, 3 h; (2) K3Fe(CN)6, KOH,
˚
H2O; (3) BBr3, CH2Cl2; (c) (1) 4, K2CO3, TBAI, DMF, 50 °C; (2) LiOH H2O, MeOH-H2O; (3) phenyl-Gly(OMe) HCl, PyBOP, DIPEA, 4 A
3
molecular sieves, DMF; (d) phenyl-Gly(OMe) HCl, PyBOP, DIPEA, 4 A molecular sieves, DMF.
3
˚
3
Scheme 4a
a Reagents and conditions: (a) toluene, reflux 2 h; (b) (1) 3, K2CO3, TBAI, DMF, 50 °C; (2) LiOH H2O, MeOH-H2O; (3) phenyl-Gly(OMe) HCl,
3
3
˚
PyBOP, DIPEA, 4 A molecular sieves, DMF; (c) (1) Na2S 9H2O, H2O, 120 °C, 30 min; (2) Ac2O, HOAc, reflux 2 h; (d) phenyl-Gly(OMe) HCl, PyBOP,
3
3
˚
DIPEA, 4 A molecular sieves, DMF.
thiazole 26 in excellent yield (Scheme 4).9 The target com-
pounds 27 and 29 were synthesized from 26 and 28, respec-
tively, following the same methodology as that used in
the preparation of 7j from 4. 2-Methylbenzothiazole-5-car-
boxylic acid (31) was obtained from 4-chloro-3-nitrobenzoic
acid (30) in a one-pot reaction according to a modified
procedure reported in the literature,10,11 and this intermedi-
ate was then further converted to the final product 32 using
method C.
Results and Discussion
2-Methylbenzothiazole derivatives were prepared and eval-
uated for the ability to inhibit the growth of Mtb strain H37Rv
by using the microplate Alamar blue assay (MABA).5 All the
active compounds were further evaluated for their toxicity to
Vero cells. Initially, we introduced variously substituted amides
to the southern portion of the lead structure while keeping
intact the 5-(2-methylbenzothiazol-5-yloxymethyl)isoxazole
scaffold (Table 1). Replacement of the amide by an ester 5,

6760 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Huang et al.
Table 1. In Vitro Anti-TB Activity of Analogues Modified at the Isoxazolecarboxamide Group
a Mtb H37Rv. b ND: not determined. c RMP, rifampin. d INH, isoniazid.
an acid 6, and a hydroxamate acid 7l led to a loss of anti-TB
activity. Compounds 7a-w displayed a wide array of anti-TB
activity with MICs ranging from 1.4 to >128 μM. Since the hit
compound 1 showed good activity (MIC = 7.6 μM), the first
round of substitutions at the southern amide contained small
aliphatic and cycloaliphatic moieties (7a-f); unfortunately,
these were found to be inactive. Different amino acids were
then introduced to obtain compounds 7g-j. Surprisingly, this
slight modification of the alkyl appendage in the hit compound
2 (7g and 7i) led to a complete loss of activity. Upon replacing
the L-leucine methyl ester by an L-methionine methyl ester (7h),
we found that the inhibitory activity was retained (MIC = 3.7
μM). The introduction of a hydrophobic phenyl group as
an amide substituent (7j) enhanced activity to some extent
(MIC = 1.4 μM), making compound 7j an interesting starting
point for further optimization.
the phenyl-Gly(OMe) group (7o, 7q, and 7r) were well toler-
ated, although not leading to any improvement in potency
compared to 7j. Introducing an alkyl spacer between the
phenyl ring and the amide group (7k) was also well tolerated.
In contrast, removal of a methylene group between the phenyl
ring and the methyl ester (7p) had a detrimental effect on
inhibitory activity (MIC = 30.2 μM). Substitution of the
methyl ester with a hydroxamic acid (7m), hydrolysis to acid
(7u), reduction to alcohol (7n), as well as its removal (7t) led to
a complete loss of anti-TB potency in every case. Likely, these
negative results are a consequence of the poor permeability of
these compounds, indicating that the ester group may well
play an important role in penetration of the cell wall of the TB
bacteria.
As isoniazid (INH) is a first-line anti-TB medication,12 we
imagined that it might be valuable to incorporate this mole-
cule into our benzothiazoles, and thus the derivatives 7v and
7w were prepared. Compound 7v (MIC = 4.4 μM) retained
Thus, further modifications to compound 7j were carried
out. Introduction of various substituents on the phenyl ring of

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6761
good anti-TB potency, while the potency decreased consider-
ably for compound 7w (MIC = 47.5 μM), possibly because of
its steric bulkiness. It is worth mentioning that the enantiomer
7s of compound 7j was found to have an excellent anti-TB
activity with an MIC of 1.9 μM.
Toinvestigatethe roleofthe linker positionon activity, four
regioisomeric benzothiazoles (7j, 12a-c) varying in points of
attachment to the phenyl ring of the benzothiazole were
prepared as displayed in Table 2. Shifting the linker to the
4-position (12a) or to the 6-position (12b) resulted in about a
2- to 5-fold decrease in anti-TB activity when compared to the
5-position (7j). Attachment to the 7-position (12c) completely
abolished activity (MIC > 128 μM), showing that linkage to
the 5-position is preferable.
benzothiazole ring (17) led to a dramatic decrease in the anti-
TB activity (MIC = 59.5 μM). Relocation of the linker to the
thiazole ring of the benzothiazole, using either an oxygen
atom (19a) or a sulfur atom (19b) in the linker, also resulted in
compounds having higher MIC values of 15.2 and 13.0 μM,
respectively. On the other hand, removal of the benzothiazole
ring (21) resulted in a 20-fold decrease in potency compared to
7j. In contrast, the deletion of the thiazole ring alone (23)
maintained activity compared to hit compound 2, but this
analogue wasstill2-foldlesspotentthan7j, suggesting thatthe
benzothiazole ring might be the best choice at the northern
part.
Futhermore, replacement of the isoxazole ring by a thiazole
ring (27), an oxazole ring (29), or deletion of the isoxazole ring
(32) resulted in a loss of potency, suggesting a preference for
the isoxazole ring in maximizing anti-TB activity (Table 4).
Finally, Vero cells were used for the in vitro toxicity
evaluation of the active compounds. In general, the present
compounds failed to show any toxicity (IC50 > 128 μM), thus
suggesting that their anti-TB activity was not due to some
general cytotoxicity. Several of the present compounds were
also evaluated for their potency against four protozoan para-
sites as shownin Table 5.13 In this preliminary screening, some
of our compounds were found to show inhibitory activity
toward P. falciparum. Interestingly, hydroxamate 7m
showed on top of its excellent activity against T.b.rhodesiense
a good activity against the other three parasites, a result
that is not particularly surprising, as the hydroxamate group
of 7m could interact with any of a number of relevant
metalloproteases. Additionally, these data are suggestive of
both the selectivity of this compound series and the possible
commonality of their biological target between TB and
malaria.
With the above SAR data in hand, we combined the most
efficacious amide and the best regioisomer to generate com-
pounds 17, 19a, 19b, 21, and 23 in which modifications to the
northern portion of the starting scaffold were now explored
(Table 3). Attaching a phenyl group to the C-2 position of the
Table 2. In Vitro Anti-TB Activity of Analogues Modified by Point of
Attachment of Linker to the Benzothiazole Ring
compd linker position MABAa MIC (μM) Vero cells IC50 (μM)
12a
4
5
6
7
7.4
1.4
>128
>128
>128
>128
127
7j
12b
From the above in vitro results, it may be concluded that
5-(2-methylbenzothiazol-5-yloxymethyl)isoxazole-3-carbox-
amide represents a reasonable starting scaffold in generating
new chemical entities possessing anti-TB properties. In an
attempt to understand the possible biological target for our
3.4
12c
RMPb
INHc
>128
0.1
0.5
>128
a Mtb H37Rv. b RMP, rifampin. c INH, isoniazid.
Table 3. In Vitro Anti-TB Activity of Analogues Modified at the Benzothiazole Ring
a Mtb H37Rv. b ND: not determined. c RMP, rifampin. d INH, isoniazid.

6762 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Huang et al.
Table 4. In Vitro Anti-TB Activity of Analogues Modified at the Isoxazole Ring
a Mtb H37Rv. b ND: not determined. c RMP, rifampin. d INH, isoniazid.
Table 5. In Vitro Antiparasite Activity of Several Active Anti-TB Agents
IC50 (μM)
of these previously published results,14 we then utilized the
Protein Data Bank structure 1NH8, for which the active site is
known to be centered about Tyr116, to investigate the fit of
our compounds to this putative target. An automated
docking analysis of 2-methylbenzothiazoles reported in
Tables 1-4 was performed into the crystallographic struc-
ture of HisG, using the Surflex-Dock suite (Sybyl 8.0,
Tripos, Inc.). These molecules were found to fit reasonably
well into the active site, with one primary binding mode
being observed in which the ligands are located within the
ATP binding site. The predicted binding modes for two
compounds (7j and 7s) are shown in Figure 2. The ben-
zothiazole ring of both molecules was found to interact with
the hydrophobic pocket next to Leu12, Leu71, and Pro50.
For compound 7j (Figure 2a), a hydrogen bond formed
between the nitrogen atom present in the benzothiazole ring
and the hydrogen atom of the amino group of Lys51, while
a second hydrogen bond formed between the carboxylate
oxygen and the hydrogen of the hydroxyl group in Ser90. In
contrast, the terminal phenyl ring in the southern portion of
7s was predicted to be close to the P-loop (Gly157-Ser158,
Gly159)14 (Figure 2b); one hydrogen bond formed between
the carboxylate oxygen and the phenolic hydroxyl group in
Try116, and a second formed between the oxygen in the
linker and the hydrogen atom of the amino group of Ala11.
Figure 2c shows two previously identified HisG inhibitors
in complex with the HisG enzyme as taken from ref 14.
As is apparent, our compounds share similar binding sites
within this target protein with these reference compounds.
While these modeling studies are very preliminary, these studies
along with literature reports support the possibility that the
target of our 2-methylbenzothiazole series might be Mtb HisG.
compd
T.b. rhodesiense T. cruzi L. donovani P. falciparum
2
18.7
74.2
1.85
0.415
31.2
88.9
89.5
86.9
89.5
38
12.3
>90
15.9
6.2
2.79
27.5
6.06
3.25
>90
4.48
22.3
8.8
4.57
3.97
2.76
1.82
2.56
2.5
7j
7l
7m
7n
7o
>90
26.4
37.5
>90
>90
>90
18.8
25.4
14.7
26.8
16.9
>90
45.9
14.5
15.2
7s
3.33
3.43
>5
>5
>5
3.4
7t
7u
32.3
>90
4.12
4.27
4.89
4.99
6.03
56.3
13.5
4.44
5.01
7v
12a
>90
>90
66.8
88.6
88.9
>90
>90
37.5
73.8
0.004
12b
12c
4.89
>5
>5
>5
>5
4.86
4.65
19a
19b
21
23
27
29
melarsoprol
benznidazole
miltefosine
chloroquine
0.521
0.142
0.053
compounds, we made a search of the literature for structurally
related compounds. Wefound that several similar compounds
containing a nitrobenzothiazole fragment inhibit HisG,14
which is an ATP-phosphoribosyl transferase (ATP-PRTase)
that catalyzes the first step in the biosynthesis of histidine and
that represents a potential drug target for TB. Although our
compounds share a benzothiazole group, more careful ana-
lysis reveals common pharmacophoric elements including the
presence of an aryl group at either end of the molecule and a
hydrophobic aryl group in the middle. We thus hypothesized
that they may also share the same target in Mtb. On the basis
Conclusions
In this study a new class of 2-methylbenzothiazole analogues
showing good anti-TB properties was identified, with several
compounds found to possess MICs in the low micromolar

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6763
Figure 2. Docked structures of 7j (a) and 7s (b) with HisG as determined using the Surflex-Dock suite (Sybyl 8.0, Tripos, Inc.). (c) Docked
structures of two HisG inhibitors taken from ref 14.
range against replicating TB in the MABA. Variations in the
substitution of the southern amide site were well tolerated, with
groups ranging in size from the small dimethylamino group to
substituted amino acid esters providing compounds with good
anti-TB activity. The most potent compounds 7j and 7s exhi-
bited MICs of 1.4 and 1.9 μM, respectively, which amount to a
5-fold increase in potency compared to hit compound 1and a 2-
fold increase in potency compared to hit compound 2. Upon
migrating the linker around the phenyl ring of the benzothia-
zole, we found that substitution at the 5-position was preferred.
Attempts to replace either the benzothiazole or the isoxazole led
to a substantially reduced or complete loss in activity. As such,
the 2-methylbenzothiazole-isoxazole core appears essential to
retaining activity. In general, the active compounds failed to
show any toxicity to the Vero cells at 128 μM. Moreover,
several of the present compounds showed moderate inhibitory
activity toward P. falciparum. Preliminary molecular modeling
results along with other literature reports suggest that the target
of this 2-methylbenzothiazole series might be associated with
Mtb HisG. The overall results thus provide a useful starting
point in the quest for chemically distinctive classes of anti-TB
agents and as possible antiparasitic agents.
Experimental Section
Biology. MABA and Cytotoxicity Assays. These were carried
out according to the published protocols.15

6764 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Huang et al.
Antiprotozoal Assays. The in vitro activities against the pro-
tozoan parasites T.b. rhodesiense, T. cruzi, L. donovani, and
P. falciparum were determined as described earlier.16 The following
strains and parasite forms were used: T.b. rhodesiense, STIB900,
trypomastigote forms; T. cruzi, Tulahuen C2C4, amastigote forms
in L-6 rat myoblasts; L. donovani, MHOM/ET/67/L82, axenic
amastigote forms; P. falciparum, K1 (chloroquine and pyrimeth-
amine resistant), erythrocytic stages.
for 15 h and then diluted with EtOAc (40 mL), washed with 1 N
HCl (10 mL), saturated NaHCO3 (20 mL), H2O (20 mL), brine
(20 mL), dried over Na2SO4, and concentrated in vacuo. The
crude product was purified by column chromatography on silica
gel using CHCl3-MeOH (20:1) as the eluent to give 7a as a white
solid (10.3 mg, 94.2%).
Method C. To a solution of acid 6 (10 mg, 0.034 mmol) in
˚
DMF (1 mL) were added 4 A molecular sieves (10 mg), PyBOP
Chemistry. 1H NMR and 13C NMR spectra were recorded on
Bruker spectrometer at 400 and 100 MHz, respectively, with TMS
as an internal standard. Mass spectra were measured in the ESI
mode at an ionization potential of 70 eV with LCMS MSD
(Hewlett Packard); TLC was performed with Merck 60 F254 silica
gel plates. Column chromatography was performed using Merck
silica gel (40-60 mesh). Purity of compounds (>95%) was
established by HPLC, which was carried out with two methods:
(1) on an ACE AQ column (100 mm ꢀ 4.6 mm and 250 mm ꢀ 10
mm), with detection at 254 and 280 nm on a Shimadzu SPD-10A
VP detector, flow rate = 2.0-3.6 mL/min, from 10% acetonitrile
in water to 100% acetonitrile with 0.05% TFA; (2) on an Agilent
1100 HPLC system with a Synergi 4 μm Hydro-RP 80A column,
with detection at 254 on a variable wavelength detector G1314A,
flow rate = 1.4 mL/min, gradient elution over 20-29 min, from
30% CH3CN-H2O to 100% CH3CN with 0.05% TFA.
5-(2-Methylbenzothiazol-5-yloxymethyl)isoxazole-3-carboxy-
lic Acid Ethyl Ester (5). A mixture of commercial 2-methylben-
zothiazol-5-ol 3 (165 mg, 1 mmol), 5-bromomethylisoxazole-
3-carboxylic acid ethyl ester 4 (304 mg, 1.3 mmol), anhydrous
K2CO3 (691 mg, 5 mmol), and TBAI (36.9 mg, 0.1 mmol) in
anhydrous DMF (2 mL) was heated overnight at 50 °C. After
cooling to room temperature, the mixture was diluted with
EtOAc (50 mL), washed with H2O (30 mL), dried over Na2SO4,
and concentrated in vacuo. The crude product was purified by
column chromatography on silica gel using hexane-EtOAc
(2:1) as the eluent to give 5 as a white solid (318 mg, 99.9%).
1H NMR (CDCl3) δ 7.73 (d, J = 12.0 Hz, 1H), 7.50 (s, 1H), 7.06
(d, J = 12.0 Hz, 1H), 6.80 (s, 1H), 5.29 (s, 2H), 4.46 (q, J = 8.0
Hz, 2H), 2.78 (s, 3H), 1.43 (t, J = 8.0 Hz, 3H).
(54 mg, 0.10 mmol), and DIPEA (22 mg, 0.17 mmol). After the
mixture was stirred at room temperature for 30 min, diethyl-
amine (4 mg, 0.051 mmol) was added. The mixture was stirred
overnight and diluted with EtOAc (40 mL), washed with 1 N
HCl (10 mL), saturated NaHCO3 (20 mL), H2O (20 mL), brine
(20 mL), dried over Na2SO4, and concentrated in vacuo. The
crude product was purified by column chromatography on silica
gel using CHCl3-MeOH (20:1) as the eluent to give 7b as a
yellow solid (11.5 mg, 96.6%).
Method D. To a solution of acid 6 (15 mg, 0.052 mmol) in
DMF (1 mL) was added CDI (13 mg, 0.078 mmol). After the
mixture was stirred at room temperature for 1 h, piperazine (9
mg, 0.10 mmol) was added. The mixture was stirred overnight
and diluted with EtOAc (40 mL), washed with saturated NaH-
CO3 (20 mL), H2O (20 mL), and brine (20 mL), dried over
Na2SO4, and concentrated in vacuo. The crude product was
purified by column chromatography on silica gel using
CHCl3-MeOH (20:1) as the eluent to give 7e as a yellow solid
(14.3 mg, 77.2%).
[5-(2-Methylbenzothiazol-5-yloxymethyl)isoxazol-3-yl][1,2]oxa-
zinan-2-yl-methanone (1). Method C was used. Yield: 70.8%
(white solid). 1H NMR (CDCl3 þ CD3OD) δ 7.72 (d, J=12.0
Hz, 1H), 7.50 (d, J = 2.0 Hz, 1H), 7.07 (dd, J = 12.0, 2.0 Hz,
1H), 6.71 (s, 1H), 5.28 (s, 2H), 4.05 (m, 2H), 3.94 (m, 2H), 2.83 (s,
3H), 1.89 (m, 4H); 13C NMR (CDCl3 þ CD3OD) δ 169.5, 168.2,
158.2, 157.0, 154.1, 128.6, 122.2, 115.1, 106.4, 104.5, 74.7, 61.5, 43.6,
24.1, 22.4, 20.0; HPLC purity 98.0%; MS (ESI) m/z 360 (M þ H)þ.
4-Methyl-2-{[5-(2-methylbenzothiazol-5-yloxymethyl)isoxa-
zole-3-carbonyl]-(S)-amino}pentanoic Acid Methyl Ester (2). Meth-
1
od C was used. Yield: 55.6% (white solid). H NMR (CDCl3) δ
5-(2-Methylbenzothiazol-5-yloxymethyl)isoxazole-3-carboxy-
lic Acid (6). The ester 5 (318 mg, 1 mmol) was dissolved into
7.71 (d, J = 8.8 Hz, 1H), 7.49 (d, J = 2.0 Hz, 1H), 7.14 (d, J = 8.4
Hz, 1H), 7.05 (dd, J = 8.8, 2.4 Hz, 1H), 6.81 (s, 1H), 5.27 (s, 2H),
4.79 (m, 1H), 3.77 (s, 3H), 2.83 (s, 3H), 1.71 (m, 3H), 0.97 (m, 6H);
13C NMR (CDCl3) δ 172.1, 169.0, 168.4, 157.9, 157.8, 156.2, 154.0,
128.5, 121.5, 114.5, 106.1, 103.1, 61.0, 52.1, 50.4, 41.1, 24.5, 22.4,
21.4, 19.8; HPLC purity 99.4%; MS (ESI) m/z 456 (M þ K)þ.
5-(2-Methylbenzothiazol-5-yloxymethyl)isoxazole-3-carboxylic
Acid Dimethylamide (7a). Method B was used. Yield: 94.2%
MeOH-H2O (4:1, 10 mL) and cooled to 0 °C. LiOH H2O (126
3
mg, 3.0 mmol) was added, and the reaction mixture was stirred
at room temperature for 5 h. After completion the reaction was
quenched with H2O (50 mL) and the mixture was acidified with
6 M HCl (pH ∼2), extracted with EtOAc (2 ꢀ 30 mL), washed
with brine (20 mL), and dried with Na2SO4. After filtration the
solvent was evaporated to give 6 in 100% yield as a white
1
(white solid). H NMR (CDCl3) δ 7.72 (d, J = 12.0 Hz, 1H),
1
powder. H NMR (CDCl3 þ CD3OD) δ 7.75 (d, J=12.0 Hz,
7.52 (s, 1H), 7.07 (d, J = 12.0 Hz, 1H), 6.69 (s, 1H), 5.27 (s, 2H),
3.30 (s, 3H), 3.14 (s, 3H), 2.84 (s, 3H); HPLC purity 96.1%; MS
(ESI) m/z 318 (M þ H)þ.
1H), 7.50 (s, 1H), 7.10 (d, J = 12.0 Hz, 1H), 6.83 (s, 1H), 5.32 (s,
2H), 2.84 (s, 3H).
General Procedures for the Synthesis of Compounds 1, 2,
7a-k, 7n-q,s,t,v. Method A. To a solution of acid 6 (24 mg,
0.083 mmol) in DMF (1 mL) was added 4 A molecular sieves (24
5-(2-Methylbenzothiazol-5-yloxymethyl)isoxazole-3-carboxylic
Acid Diethylamide (7b). Method C was used. Yield: 96.6% (yellow
solid). 1H NMR(CDCl3) δ 7.72 (d, J = 12.0 Hz, 1H), 7.52 (s, 1H),
7.06 (d, J = 12.0 Hz, 1H), 6.68 (s, 1H), 5.26 (s, 2H), 3.56 (m, 4H),
2.84 (s, 3H), 1.25 (m, 6H); HPLC purity 97.0%; MS (ESI) m/z 346
(M þ H)þ.
˚
mg), HOBt (34 mg, 0.25 mmol), and DMAP (cat.). After the
mixture was stirred at room temperature for 30 min, morpholine
(0.02 mL, 0.25 mmol) and EDC (48 mg, 0.25 mmol) were added.
The mixture was stirred overnight, diluted with EtOAc (40 mL),
washed with 1 N HCl (10 mL), saturated NaHCO3 (20 mL),
H2O (20 mL), brine (20 mL), dried over Na2SO4, and concen-
trated in vacuo. The crude product was purified by column
chromatography on silica gel using CHCl3-MeOH (20:1) as the
eluent to give 7c as a yellow solid (4.2 mg, 14.1%).
Method B. To a solution of 6 (10 mg, 0.034 mmol) in
anhydrous CH2Cl2 (1 mL) was added (COCl)2 (2 M in CH2Cl2,
0.05 mL, 0.102 mmol) and 1 drop of DMF at 0 °C. The resulting
mixture was allowed to warm to room temperature and stirred
for 2 h. After evaporation of the solvent, the residue was
dissolved in pyridine (1 mL) and then dimethylamine hydro-
chloride (28 mg, 0.34 mmol) was added at 0 °C. The resulting
mixture was allowed to warm to room temperature and stirred
[5-(2-Methylbenzothiazol-5-yloxymethyl)isoxazol-3-yl]-mor-
pholin-4-ylmethanone (7c). Method A was used. Yield: 14.1%
(yellow solid). 1H NMR (CDCl3) δ 7.72 (d, J = 12.0 Hz, 1H),
7.52 (s, 1H), 7.06 (d, J = 12.0 Hz, 1H), 6.70 (s, 1H), 5.25 (s, 2H),
3.94 (m, 2H), 3.80 (m, 4H), 3.74 (m, 2H), 2.84 (s, 3H); HPLC
purity 99.0%; MS (ESI) m/z 360 (M þ H)þ.
[5-(2-Methylbenzothiazol-5-yloxymethyl)isoxazol-3-yl]piperi-
din-1-ylmethanone (7d). Method C was used. Yield: 73.6%
(yellow solid). 1H NMR (CDCl3) δ 7.76 (d, J = 12.0 Hz, 1H),
7.75 (s, 1H), 7.17 (d, J = 12.0 Hz, 1H), 6.70 (s, 1H), 5.30 (s, 2H),
3.74 (m, 4H), 2.96 (s, 3H), 1.68 (m, 6H); HPLC purity 99.7%;
MS (ESI) m/z 358 (M þ H)þ.
[5-(2-Methylbenzothiazol-5-yloxymethyl)isoxazol-3-yl]piperazin-
1-ylmethanone (7e). Method D was used. Yield: 77.2% (yellow

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6765
solid). 1H NMR (CDCl3) δ 7.75 (d, J = 8.0 Hz, 1H), 7.66 (s, 1H),
7.14 (d, J = 8.0 Hz, 1H), 6.80 (s, 1H), 5.32 (s, 2H), 4.37 (m, 2H),
4.13(m, 2H), 3.43(br, 1H), 4.34(m, 4H), 2.92(s, 3H);HPLCpurity
99.4%; MS (ESI) m/z 359 (M þ H)þ.
Method A was used. Yield: 93.9% (white solid). 1H NMR
(CDCl3) δ 7.87 (br, 1H), 7.82 (d, J = 6.8 Hz, 1H), 7.69 (d,
J = 8.8 Hz, 1H), 7.45 (d, J = 2.4 Hz, 1H), 7.24 (d, J = 8.4 Hz,
2H), 7.03 (dd, J = 8.8, 2.8 Hz, 1H), 6.80 (d, J = 8.4 Hz, 2H), 6.76
(s, 1H), 5.61 (d, J = 7.2 Hz, 1H), 5.20 (s, 2H), 3.74 (s, 3H), 2.82
(s, 3H); 13C NMR (CDCl3) δ 171.0, 170.4, 169.0, 157.7, 157.6,
156.7, 156.3, 153.5, 128.4, 128.2, 126.5, 121.5, 115.7, 114.8,
105.7, 103.1, 60.9, 55.7, 52.6, 19.6; HPLC purity 96.8%; MS
(ESI) m/z 454 (M þ H)þ.
[5-(2-Methylbenzothiazol-5-yloxymethyl)isoxazol-3-yl]-(4-meth-
ylpiperazin-1-yl)methanone (7f). Method D was used. Yield:
1
98.7% (yellow solid). H NMR (CDCl3) δ 7.71 (d, J = 8.0 Hz,
1H), 7.51 (d, J = 2.0 Hz, 1H), 7.06 (dd, J = 8.0, 2.0 Hz, 1H),
6.69 (s, 1H), 5.26 (s, 2H), 3.91 (m, 4H), 2.82 (s, 3H), 2.55 (m, 4H),
2.35 (s, 3H); 13C NMR (CDCl3) δ 168.5, 167.6, 158.6, 158.1,
156.4, 154.0, 128.4, 121.6, 114.5, 106.0, 104.6, 61.0, 54.7, 53.9,
46.2, 45.2, 41.8, 19.8; HPLC purity 95.4%; MS (ESI) m/z 373
(M þ H)þ.
2-{[5-(2-Methylbenzothiazol-5-yloxymethyl)isoxazole-3-carbo-
nyl]amino}benzoic Acid Methyl Ester (7p). Method A was used.
Yield: 14.9% (white solid). 1H NMR (CDCl3) δ 12.4 (s, 1H), 8.82
(d, J = 8.0 Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 8.0 Hz,
1H), 7.63 (t, J = 8.0 Hz, 1H), 7.52 (s, 1H), 7.18 (t, J = 8.0 Hz,
1H), 7.07 (d, J = 8.0 Hz, 1H), 6.81 (s, 1H), 5.31 (s, 2H), 4.00 (s,
3H), 2.83 (s, 3H); 13C NMR (CDCl3) δ 169.3, 168.0, 159.1, 156.7,
156.3, 140.0, 134.3, 130.7, 123.0, 121.6, 120.3, 115.5, 114.5, 106.2,
103.1, 61.1, 52.3, 19.8; HPLC purity 98.5%; MS (ESI) m/z 424
(M þ H)þ.
(3S)-Methyl-2-{[5-(2-methylbenzothiazol-5-yloxymethyl)isoxa-
zole-3-carbonyl]-(S)-amino}pentanoic Acid Methyl Ester (7g).
Method A was used. Yield: 30.6% (white solid). 1H NMR
(CDCl3) δ 7.72 (d, J = 12.0 Hz, 1H), 7.50 (s, 1H), 7.26 (d, J =
8.0 Hz, 1H), 7.05 (d, J = 12.0 Hz, 1H), 6.81 (s, 1H), 5.28 (s, 2H),
4.77 (dd, J = 8.0, 5.2 Hz, 1H), 3.78 (s, 3H), 2.84 (s, 3H), 2.02 (m,
1H), 1.53 (m, 1H), 1.28 (m, 1H), 0.97 (m, 6H); 13C NMR (CDCl3)
δ 171.7, 169.6, 169.0, 158.5, 156.9, 154.7, 129.1, 122.3, 115.2,
106.7, 103.7, 61.6, 56.8, 52.5, 38.2, 25.4, 20.4, 15.8, 11.7; HPLC
purity 99.0%; MS (ESI) m/z 418 (M þ H)þ.
(2-Chlorophenyl){[5-(2-methylbenzothiazol-5-yloxymethyl)iso-
xazole-3-carbonyl]-(S)-amino}acetic Acid Methyl Ester (7q).
Method A was used. Yield: 59.4% (yellow solid). 1H NMR
(CDCl3) δ 7.86 (d, J=7.2 Hz, 1H), 7.72 (d, J=8.8 Hz, 1H),
7.58 (s, 1H), 7.44 (m, 2H), 7.30 (m, 2H), 7.07 (d, J=8.8 Hz, 1H),
6.80(s, 1H), 6.10(d, J=7.2 Hz, 1H), 5.27 (s, 2H), 3.79 (s, 3H), 2.87
(s, 3H); 13C NMR (CDCl3) δ 170.0, 169.4, 168.8, 158.0, 157.9,
156.6, 154.4, 134.0, 133.7, 130.2, 130.1, 130.0, 127.4, 122.0, 114.9,
106.5, 103.5, 61.3, 54.7, 53.2, 20.2; HPLC purity 97.3%; MS (ESI)
m/z 510 (M þ K)þ.
2-{[5-(2-Methylbenzothiazol-5-yloxymethyl)isoxazole-3-carbo-
nyl]-(S)-amino}-4-methylsulfanylbutyric Acid Methyl Ester (7h).
Method C was used. Yield: 51.7% (white solid). 1H NMR
(CDCl3) δ 7.71 (d, J = 8.8 Hz, 1H), 7.49 (d, J = 2.4 Hz, 1H),
7.41 (d, J = 8.0 Hz, 1H), 7.05 (dd, J = 8.8, 2.4 Hz, 1H), 6.81 (s,
1H), 5.28 (s, 2H), 4.90 (m, 1H), 3.80 (s, 3H), 2.83 (s, 3H), 2.58 (t,
J = 7.2 Hz, 2H), 2.28 (m, 1H), 2.11 (m, 4H); 13C NMR (CDCl3) δ
171.1, 169.1, 168.4, 157.9, 157.7, 156.2, 154.0, 128.5, 121.7, 114.5,
106.1, 103.0, 61.0, 52.3, 51.1, 31.2, 29.5, 19.8, 15.1; HPLC purity
98.6%; MS (ESI) m/z 436 (M þ H)þ.
{[5-(2-Methylbenzothiazol-5-yloxymethyl)isoxazole-3-carbo-
nyl]-(R)-amino}phenylacetic Acid Methyl Ester (7s). Method C
was used. Yield: 61.9% (white solid). 1H NMR (CDCl3) δ 7.78
(d, J = 7.2 Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.48 (d, J = 2.4 Hz,
1H), 7.39 (m, 5H), 7.04 (dd, J=8.8, 2.4 Hz, 1H), 6.79 (s, 1H),
5.72 (d, J=7.6 Hz, 1H), 5.26 (s, 2H), 3.77 (s, 3H), 2.82 (s, 3H);
13C NMR (CDCl3) δ 170.1, 169.1, 168.4, 157.7, 157.5, 156.2,
154.0, 135.3, 128.7, 128.5, 128.4, 127.0, 121.6, 114.5, 106.1,
103.1, 61.0, 56.1, 52.6, 19.8; HPLC purity 97.7%; MS (ESI)
m/z 438 (M þ H)þ.
3-Methyl-2-{[5-(2-methylbenzothiazol-5-yloxymethyl)isoxa-
zole-3-carbonyl]-(S)-amino}butyric Acid Methyl Ester (7i).
Method C was used. Yield: 45.1% (yellow solid). 1H NMR
(CDCl3) δ 7.70 (d, J = 8.0 Hz, 1H), 7.48 (s, 1H), 7.23 (d, J = 8.0
Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H), 6.80 (s, 1H), 5.26 (s, 2H), 4.70
(m, 1H), 3.76 (s, 3H), 2.82 (s, 3H), 2.25 (m, 1H), 0.98 (m, 6H); 13C
NMR (CDCl3) δ 171.2, 169.0, 168.4, 158.0, 157.9, 156.3, 154.0,
128.5, 121.6, 114.5, 106.1, 103.1, 61.0, 56.8, 51.9, 31.0, 19.8, 18.6,
17.4; HPLC purity 97.6%; MS (ESI) m/z 404 (M þ H)þ.
{[5-(2-Methylbenzothiazol-5-yloxymethyl)isoxazole-3-carbo-
nyl]-(S)-amino}phenylacetic Acid Methyl Ester (7j). Method C was
used. Yield: 54.6% (white solid). 1H NMR (CDCl3) δ 7.80 (d, J =
7.2 Hz, 1H), 7.70 (d, J= 8.8 Hz, 1H), 7.49 (d, J= 2.4 Hz, 1H), 7.37
(m, 5H), 7.04 (dd, J = 8.4, 2.0 Hz, 1H), 6.79 (s, 1H), 5.72 (d, J =
7.2 Hz, 1H), 5.26 (s, 2H), 3.77 (s, 3H), 2.82 (s, 3H); 13C NMR
(CDCl3) δ 170.1, 169.1, 168.4, 157.7, 157.5, 156.2, 154.0, 135.3,
128.7, 128.5, 127.0, 121.6, 114.5, 106.1, 103.1, 61.0, 56.1, 52.7, 19.8;
HPLC purity 99.4%; MS (ESI) m/z 460 (M þ Na)þ.
5-(2-Methylbenzothiazol-5-yloxymethyl)isoxazole-3-carboxylic
Acid Benzylamide (7t). Method A was used. Yield: 70.6% (white
solid). 1H NMR (CDCl3) δ 7.70 (d, J = 8.8 Hz, 1H), 7.48 (d, J =
2.4 Hz, 1H), 7.32 (m, 6H), 7.04 (dd, J = 8.8, 2.4 Hz, 1H), 6.83 (s,
1H), 5.25 (s, 2H), 4.61 (d, J = 5.6 Hz, 2H), 2.82 (s, 3H); 13C NMR
(CDCl3) δ 169.3, 168.8, 158.6, 158.4, 156.4, 154.4, 137.3, 128.8,
128.6, 127.8, 122.0, 114.9, 106.4, 103.5, 61.4, 43.5, 20.2; HPLC
purity 99.4%; MS (ESI) m/z 380 (M þ H)þ.
Isonicotinic acid N0-[5-(2-methylbenzothiazol-5-yloxymethyl)-
isoxazole-3-carbonyl]hydrazide (7v). Method A was used. Yield:
74.4% (white solid). 1H NMR (CDCl3þCD3OD) δ 8.63 (d, J =
4.0 Hz, 2H), 7.76 (d, J = 5.6 Hz, 2H), 7.67 (d, J = 8.8 Hz, 1H),
7.41 (d, J = 1.5 Hz, 1H), 7.02 (dd, J = 8.8, 2.0 Hz, 1H), 6.82
(s, 1H), 5.26 (s, 2H), 2.74 (s, 3H); 13C NMR (CDCl3þCD3OD) δ
169.8, 169.5, 165.1, 158.4, 157.1, 156.9, 153.6, 149.7, 140.1,
128.4, 122.1, 121.9, 115.0, 106.0, 103.4, 61.1, 19.3; HPLC purity
99.1%; MS (ESI) m/z 432 (M þ Na)þ.
2-{[5-(2-Methylbenzothiazol-5-yloxymethyl)isoxazole-3-carbo-
nyl]-(S)-amino}-3-phenylpropionic Acid Methyl Ester (7k). Method
C was used. Yield 54.7% (white solid). 1H NMR (CDCl3) δ 7.71
(d, J = 8.4 Hz, 1H), 7.49 (s, 1H), 7.29 (M, 3H), 7.16 (d, J = 6.8 Hz,
2H), 7.04 (d, J = 8.8 Hz, 1H), 6.79 (s, 1H), 5.26 (s, 2H), 5.07 (m,
1H), 3.75 (s, 3H), 3.21 (m, 2H), 2.83 (s, 3H); 13C NMR (CDCl3) δ
170.7, 169.0, 168.5, 157.7, 156.3, 154.0, 135.3, 128.8, 128.5, 128.3,
126.9, 121.7, 114.5, 106.0, 103.0, 61.0, 52.9, 52.1, 37.6, 19.8; HPLC
purity 97.3%; MS (ESI) m/z 474 (M þ Na)þ.
5-(2-Methylbenzothiazol-5-yloxymethyl)isoxazole-3-carboxylic
Acid Hydroxyamide (7l). A suspended solution of hydroxylamine
hydrochloride (2.35 g, 0.034 mol) in MeOH (12 mL) was refluxed
under argon until a clear solution was obtained. To this hot and
clear solution was added a solution of KOH (3.35 g, 0.05 mol) in
MeOH (7 mL). After refluxing 30 min, the mixture was cooled to
room temperature and the upper clean solution (about 1.76 M
NH2OK in methanol17) was used directly. To a suspension of
ester 5 (100mg, 0.31 mmol) inMeOH (2 mL) was added the above
NH2OK solution (1.8 mL, 3.14 mmol). The mixture was stirred at
room temperature overnight and then acidified to pH 5-6 with 1
N HCl at 0 °C. The resulting mixture was diluted with EtOAc (40
mL), washed with brine (20 mL), dried over Na2SO4, and
concentrated in vacuo. The crude product was purified by short
5-(2-Methylbenzothiazol-5-yloxymethyl)isoxazole-3-carboxylic
Acid (2-Hydroxy-1-phenylethyl)-(S)-amide (7n). Method A was
used. Yield: 90.3% (white solid). 1H NMR (CDCl3 þ CD3OD) δ
7.73 (d, J = 8.0 Hz, 1H), 7.48 (s, 1H), 7.33 (m, 5H), 7.07 (d, J = 8.0
Hz, 1H), 6.82 (s, 1H), 5.30 (s, 2H), 5.18 (m, 1H), 3.89 (m, 2H), 2.82
(s, 3H);13CNMR(CDCl3) δ172.7, 172.4, 162.0, 161.5, 159.7, 156.7,
141.7, 131.5, 131.4, 130.6, 129.6, 125.1, 118.0, 109.2, 106.3, 67.8, 64.2,
58.7, 22.6; HPLC purity 98.9%; MS (ESI) m/z 410 (M þ H)þ.
(4-Hydroxyphenyl){[5-(2-methylbenzothiazol-5-yloxymethyl-
isoxazole-3-carbonyl]-(S)-amino}acetic Acid Methyl Ester (7o).

6766 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Huang et al.
column chromatographyonsilica gelusing EtOAc asthe eluent to
give 7l as a white solid (49.2 mg, 51.3%). 1H NMR (DMSO) δ
11.6 (s, 1H), 9.42 (s, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.63 (s, 1H),
7.13 (d, J = 8.0 Hz, 1H), 6.94 (s, 1H), 5.44 (s, 2H), 2.77 (s, 3H);
HPLC purity 97.5%; MS (ESI) m/z 306 (M þ H)þ.
methodology as employed for the preparation of 7j from 3.
Yield: 71.1% for three steps (white solid). 1H NMR (CDCl3) δ
7.79 (d, J = 7.2 Hz, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.40 (m, 6H),
6.83 (d, J = 8.4 Hz, 1H), 6.81 (s, 1H), 5.72 (d, J = 7.2 Hz,
1H), 5.35 (s, 2H), 3.77 (s, 3H), 2.83 (s, 3H); 13C NMR (CDCl3)
δ 170.1, 168.7, 167.5, 157.7, 157.4, 155.0, 151.5, 135.3,
128.7, 128.5, 127.0, 126.3, 124.4, 116.1, 105.8, 103.1, 61.0,
56.1, 52.6, 19.7; HPLC purity 97.7%; MS (ESI) m/z 460
(M þ Na)þ.
5-(2-Methylbenzothiazol-5-yloxymethyl)isoxazole-3-carboxylic
Acid (Hydroxycarbamoylphenylmethyl)-(S)-amide (7m). The syn-
thesis involved starting with 7j and following the same methodol-
ogy as employed for the preparation of 7l from 5. Yield: 90.7%
(white solid). 1H NMR (CD3OD) δ 7.78 (d, J = 8.0 Hz, 1H), 7.48
(m, 3H), 7.36 (m, 3H), 7.11 (d, J = 8.0 Hz, 1H), 6.85 (s, 1H), 5.57
(s, 1H), 5.35 (s, 2H), 2.78 (s, 3H); HPLC purity 96.3%; MS (ESI)
m/z 439 (M þ H)þ.
Phenyl({5-[2-(3-trifluoromethylphenyl)benzothiazol-5-yloxy-
methyl]isoxazole-3-carbonyl}-(S)-amino)acetic Acid Methyl Es-
ter (17). The synthesis involved starting with 16 and following
the same methodology as employed for the preparation of 7j
1
from 3. Yield: 24.4% for three steps (white solid). H NMR
(4-Methoxyphenyl)-{[5-(2-methylbenzothiazol-5-yloxymethyl)iso-
xazole-3-carbonyl]-(S)-amino}acetic Acid Methyl Ester (7r). To a
solution of 7o (22 mg, 0.05 mmol) in DMF (1 mL) was added
K2CO3 (20 mg, 0.15 mmol), n-Bu4NI (5 mg) and MeI (0.02 mL, 0.24
mmol). The mixture was stirred at room temperature for 2 h and
then diluted with EtOAc (40 mL), washed with brine (20 mL), dried
over Na2SO4, and concentrated in vacuo. The crude product was
purified by column chromatography on silica gel using hexa-
ne-EtOAc (1:2) as the eluent to give 7r as a yellow solid (20 mg,
88.2%). 1H NMR (CDCl3) δ 7.72 (d, J = 8.8 Hz, 2H), 7.55 (s, 1H),
7.35 (d, J = 8.0 Hz, 2H), 7.06 (d, J = 8.0 Hz, 1H), 6.91 (d, J = 8.4
Hz, 2H), 6.80 (s, 1H), 5.65 (d, J = 7.2 Hz, 1H), 5.27 (s, 2H), 3.81 (s,
3H), 3.78 (s, 3H), 2.86 (s, 3H); 13C NMR (CDCl3) δ 170.4, 169.0,
168.4, 159.5, 157.7, 157.4, 156.2, 154.0, 128.5, 128.3, 127.4, 121.6,
114.5, 114.1, 106.1, 103.1, 61.0, 56.2, 55.6, 52.5, 19.8; HPLC purity
99.1%; MS (ESI) m/z 468 (M þ K)þ.
(CDCl3) δ 8.37 (s, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.79 (m, 3H),
7.63 (m, 2H), 7.42 (m, 5H), 7.13 (d, J = 8.8 Hz, 1H), 6.84 (s, 1H),
5.74 (d, J = 7.2 Hz, 1H), 5.32 (s, 2H), 3.79 (s, 3H); 13C NMR
(CDCl3) δ 170.5, 169.3, 167.8, 158.1, 157.8, 157.1, 155.0, 135.7,
134.3, 131.8(q, J = 32.6 Hz), 130.5, 129.6, 129.1, 128.8, 128.4,
127.4, 127.3, 124.2, 124.1, 122.4, 116.4, 106.9, 103.5, 61.3, 56.5,
53.0; HPLC purity 96.3%; MS (ESI) m/z 606 (M þ K)þ.
{[5-(Benzothiazol-2-yloxymethyl)isoxazole-3-carbonyl]-(S)-ami-
no}phenylacetic Acid Methyl Ester (19a). The synthesis involved
starting with 18a and following the same methodology as em-
ployed for the preparation of 7j from 3. Yield: 49.8% for three steps
(white solid). 1HNMR(CDCl3) δ7.73(d, J= 7.2 Hz, 1H), 7.45 (d,
J = 7.6 Hz, 1H), 7.36 (m, 6H), 7.21 (m, 1H), 7.06 (d, J = 8.0 Hz,
1H), 6.63 (s, 1H), 5.69 (d, J = 7.2 Hz, 1H), 5.27 (s, 2H), 3.75 (s,
3H); 13C NMR (CDCl3) δ 170.1, 169.3, 167.5, 157.9, 157.2, 135.4,
135.3, 128.7, 128.5, 126.9, 126.4, 123.6, 122.6, 122.0, 110.1, 102.8,
56.1, 52.6, 37.2; HPLC purity 95.8%; MS (ESI) m/z446 (M þ Na)þ.
{[5-(Benzothiazol-2-ylsulfanylmethyl)isoxazole-3-carbonyl]-(S)-
amino}phenylacetic Acid Methyl Ester (19b). The synthesis in-
volved starting with 18b and following the same methodology as
employed for the preparation of 7j from 3. Yield: 53.8% for three
steps (white solid). 1H NMR (CDCl3) δ 7.89 (d, J = 8.4 Hz, 1H),
7.75 (d, J = 8.0 Hz, 2H), 7.39 (m, 7H), 6.75 (s, 1H), 5.69 (d, J = 7.2
Hz, 1H), 4.72 (s, 2H), 3.76 (s, 3H); 13C NMR (CDCl3) δ 170.1,
169.8, 163.1, 157.8, 157.6, 152.3, 135.4, 135.1, 128.7, 128.4, 127.0,
125.9, 124.3, 121.5, 120.8, 103.0, 56.1, 52.6, 26.6; HPLC purity
95.8%; MS (ESI) m/z 478 (M þ K)þ.
{[5-(2-Methylbenzothiazol-5-yloxymethyl)isoxazole-3-carbo-
nyl]-(S)-amino}phenylacetic Acid (7u). The synthesis involved
starting with 7j and following the same methodology as em-
ployed for the preparation of 6 from 5. Yield: 54.4% (white
solid). 1H NMR (CD3OD) δ 7.87 (d, J = 8.8 Hz, 1H), 7.54 (d,
J = 2.4 Hz, 1H), 7.48 (m, 2H), 7.35 (m, 3H), 7.19 (dd, J = 8.8,
2.4 Hz, 1H), 6.88 (s, 1H), 5.65 (s, 1H), 5.40 (s, 2H), 2.88 (s, 3H);
HPLC purity 98.0%; MS (ESI) m/z 446 (M þ Na)þ.
5-(2-Methylbenzothiazol-5-yloxymethyl)isoxazole-3-carboxylic
Acid {2-Oxo-1-phenyl-2-[N0-(pyridine-4-carbonyl)hydrazino]ethyl}-
(S)-amide (7w). The synthesis involved starting with 7u and follow-
ing the method C. Yield: 29.9% (white solid). 1H NMR
(CDCl3þCD3OD) δ 8.76 (m, 2H), 7.87 (m, 2H), 7.67 (d, J = 8.8
Hz, 1H), 7.50 (d, J = 7.2 Hz, 2H), 7.45 (s, 1H), 7.36 (m, 3H), 7.01
(d, J = 8.8 Hz, 1H), 6.78 (s, 1H), 5.78 (s, 1H), 5.24 (s, 2H), 2.78 (s,
3H); HPLC purity 96.6%; MS (ESI) m/z 543 (M þ H)þ.
[(5-Methylisoxazole-3-carbonyl)-(S)-amino]phenylacetic Acid
Methyl Ester (21). The synthesis involved starting with 20 and
following method C. Yield: 84.7% (white solid). 1H NMR
(CDCl3) δ 7.79 (d, J = 6.8 Hz, 1H), 7.41 (d, J = 6.8 Hz, 2H),
7.34 (m, 3H), 6.38 (s, 1H), 5.70 (d, J = 7.2 Hz, 1H), 3.72 (s, 3H),
2.41 (s, 3H); 13C NMR (CDCl3) δ 170.9, 170.2, 158.1, 157.7,
135.5, 128.6, 128.3, 127.0, 101.0, 56.0, 52.5, 11.8; HPLC purity
96.7%; MS (ESI) m/z 297 (M þ Na)þ.
{[5-(2-Methylbenzothiazol-4-yloxymethyl)isoxazole-3-carbonyl]-
(S)-amino}phenylacetic Acid Methyl Ester (12a). The synthesis
involved starting with 11a and following the same methodology
as employed for the preparation of 7j from 3. Yield: 63.0% for three
steps (white solid). 1H NMR (CDCl3) δ 7.74 (d, J = 7.2 Hz, 1H),
7.45 (d, J = 8.0 Hz, 1H), 7.41 (m, 5H), 7.26 (m, 1H), 6.90 (d, J =
8.0 Hz, 1H), 6.79 (s, 1H), 5.70 (d, J = 7.2 Hz, 1H), 5.55 (s, 2H), 3.76
(s,3H),2.85(s, 3H);13CNMR(CDCl3) δ170.1, 169.4, 165.8, 157.7,
157.5, 150.1, 143.3, 137.5, 135.3, 128.7, 128.4, 126.9, 125.0, 114.8,
108.9, 103.3, 61.6, 56.1, 52.6, 19.8; HPLC purity 97.7%; MS (ESI)
m/z 476 (M þ K)þ.
[(5-Phenoxymethylisoxazole-3-carbonyl)-(S)-amino]phenylace-
tic Acid Methyl Ester (23). The synthesis involved starting with 22
and following the same methodology as employed for the pre-
paration of 7j from 3. Yield: 59.1% for three steps (white solid).
1H NMR (CDCl3) δ 7.81 (d, J = 7.2 Hz, 1H), 7.46 (m, 2H), 7.39
(m, 3H), 7.32 (m, 2H), 7.03 (m, 1H), 6.96 (d, J = 8.8 Hz, 2H), 6.78
(s, 1H), 5.74 (d, J = 7.2 Hz, 1H), 5.19 (s, 2H), 3.78 (s, 3H), 2.41 (s,
3H); 13C NMR (CDCl3) δ 170.5, 169.8, 158.1, 158.0, 157.5, 135.8,
129.7, 129.1, 128.8, 127.4, 122.0, 114.8, 103.3, 60.9, 56.5, 53.0;
HPLC purity 98.4%; MS (ESI) m/z 389 (M þ Na)þ.
{[5-(2-Methylbenzothiazol-6-yloxymethyl)isoxazole-3-carbo-
nyl]-(S)-amino}phenylacetic Acid Methyl Ester (12b). The syn-
thesis involved starting with 11b and following the same
methodology as employed for the preparation of 7j from 3.
Yield: 79.0% for three steps (yellow solid). 1H NMR (CDCl3) δ
7.84 (d, J = 8.8 Hz, 1H), 7.79 (d, J = 7.2 Hz, 1H), 7.38 (m, 6H),
7.08 (dd, J = 8.8, 2.4 Hz, 1H), 6.78 (s, 1H), 5.72 (d, J = 7.2 Hz,
1H), 5.26 (s, 2H), 3.77 (s, 3H), 2.79 (s, 3H); 13C NMR (CDCl3) δ
170.1, 169.0, 165.0, 157.7, 157.4, 154.8, 148.4, 136.5, 135.3,
128.7, 128.5, 127.0, 122.7, 114.9, 105.6, 103.0, 61.3, 56.1, 52.6,
19.6; HPLC purity 96.2%; MS (ESI) m/z 438 (M þ H)þ.
{[5-(2-Methylbenzothiazol-7-yloxymethyl)isoxazole-3-carbo-
nyl]-(S)-amino}phenylacetic Acid Methyl Ester (12c). The syn-
thesis involved starting with 11c and following the same
{[4-(2-Methylbenzothiazol-5-yloxymethyl)thiazole-2-carbonyl]-
(S)-amino}phenylacetic Acid Methyl Ester (27). The synthesis
involved starting with 26 and following the same methodology as
employed for the preparation of 7j from 4. Yield: 56.9% for three
steps (white solid). 1H NMR (CDCl3) δ 8.17 (d, J = 7.2 Hz, 1H),
7.70 (d, J= 8.8 Hz, 1H), 7.60 (s, 1H), 7.53 (d, J= 2.0 Hz, 1H), 7.47
(m, 2H), 7.36 (m, 3H), 7.08 (dd, J = 8.8, 2.0 Hz, 1H), 5.76 (d, J =
7.2 Hz, 1H), 5.28 (s, 2H), 3.79 (s, 3H), 2.82 (s, 3H); 13C NMR
(CDCl3) δ 170.3, 168.2, 162.4, 158.3, 156.9, 154.1, 153.1, 135.5,
128.7, 128.4, 127.1, 121.9, 121.5, 114.6, 106.0, 65.9, 56.2, 52.6, 19.8;
HPLC purity 97.4%; MS (ESI) m/z 476 (M þ Na)þ.

Article
{[2-(2-Methylbenzothiazol-5-yloxymethyl)oxazole-4-carbonyl]-
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6767
resistance to second-line drugs;Worldwide, 2000-2004. JAMA,
J. Am. Med. Assoc.. 2006, 295, 2349–2351.
(S)-amino}phenylacetic Acid Methyl Ester (29). The synthesis
involved starting with 28 and following the same methodology
as employed for the preparation of 7j from 4. Yield: 34.6% for
three steps (white solid). 1H NMR (CDCl3) δ 8.22 (s, 1H), 7.85 (d,
J = 7.2 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.54 (d, J = 2.0 Hz,
1H), 7.44 (m, 2H), 7.34 (m, 3H), 7.06 (dd, J = 8.8, 2.0 Hz, 1H),
5.73 (d, J = 7.2 Hz, 1H), 5.19 (s, 2H), 3.76 (s, 3H), 2.80 (s, 3H);
13C NMR (CDCl3) δ 170.8, 168.7, 159.5, 159.3, 156.8, 154.4,
142.4, 136.0, 129.0, 128.8, 128.7, 127.4, 122.0, 114.9, 106.5, 62.4,
56.2, 52.9, 20.1; HPLC purity 100%; MS (ESI) m/z 438 (M þ H)þ.
[(2-Methylbenzothiazole-5-carbonyl)-(S)-amino]phenylacetic
Acid Methyl Ester (32). The synthesis involved starting with 31
and following method C. Yield: 50.9% (white solid). 1H NMR
(CDCl3) δ 8.37 (s, 1H), 7.79 (s, 2H), 7.80 (d, J = 6.8 Hz, 1H),
7.44 (d, J = 7.2 Hz, 2H), 7.34 (m, 3H), 5.80 (d, J = 6.8 Hz, 1H),
3.74 (s, 3H), 2.78 (s, 3H); 13C NMR (CDCl3) δ 171.1, 168.2,
166.0, 152.7, 138.8, 136.1, 131.4, 128.6, 128.2, 127.0, 123.2,
121.1, 120.7, 56.6, 52.5, 19.8; HPLC purity 99.5%; MS (ESI)
m/z 341 (M þ H)þ.
(5) Falzari, K.; Zhu, Z. H.; Pan, D. H.; Liu, H. W.; Hongmanee, P.;
Franzblau, S. G. In vitro and in vivo activities of macrolide
derivatives against Mycobacterium tuberculosis. Antimicrob.
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Westwell, A. D.; Stevens, M. F. G. Antitumor benzothiazoles. 8.
Synthesis, metabolic formation, and biological properties of the
C- and N-oxidation products of antitumor 2-(4-aminophe-
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M. L.; Klunk, W. E. Synthesis and evaluation of C-11-labeled
6-substituted 2-arylbenzothiazoles as amyloid imaging agents.
J. Med. Chem. 2003, 46, 2740–2754.
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range structural effects on the stereochemistry of aldol condensa-
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Acknowledgment. The authors thank the Global Alliance
for Tuberculosis Drug Development for financial support
through a collaborative research agreement.
(10) Klar, U.; Buchmann, B.; Schwede, W.; Skuballa, W.; Hoffinann,
J.; Lichtner, R. B. Total synthesis and antitumor activity of
ZK-EPO: the first fully synthetic epothilone in clinical develop-
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Supporting Information Available: Details for the synthesis of
compounds 4, 11a-c, 15, 16, 26, and 31. This material is
available free of charge via the Internet at http://pubs.acs.org.
(12) Timmins, G. S.; Deretic, V. Mechanisms of action of isoniazid.
Mol. Microbiol. 2006, 62, 1220–1227.
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nitrobenzothiazole inhibitors for Mycobacterium tuberculosis
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