Discovery, synthesis, and biological evaluation of piperidinol analogs with anti-tuberculosis activity

Article abstract of DOI:10.1016/j.bmc.2009.04.005

Direct anti-tuberculosis screening of commercially available compound libraries identified a novel piperidinol with interesting anti-tuberculosis activity and drug like characteristics. To generate a structure activity relationship about this hit a 22 mem

Full text of DOI:10.1016/j.bmc.2009.04.005

Bioorganic & Medicinal Chemistry 17 (2009) 3588–3594
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery, synthesis, and biological evaluation of piperidinol analogs
with anti-tuberculosis activity
Dianqing Sun a, Michael S. Scherman b, Victoria Jones b, Julian G. Hurdle a, Lisa K. Woolhiser b,
Susan E. Knudson b, Anne J. Lenaerts b, Richard A. Slayden b, Michael R. McNeil b, Richard E. Lee a,
*
a Department of Pharmaceutical Sciences, The University of Tennessee Health Science Center, Memphis, TN 38163, USA
b Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Direct anti-tuberculosis screening of commercially available compound libraries identified a novel pipe-
ridinol with interesting anti-tuberculosis activity and drug like characteristics. To generate a structure
activity relationship about this hit a 22 member optimization library was generated using parallel syn-
thesis. Products of this library 1-((R)-3-(4-chlorophenoxy)-2-hydroxypropyl)-4-(4-chloro-3-(trifluoro-
methyl) phenyl)piperidin-4-ol and 1-((S)-3-(4-(trifluoromethyl) phenoxy)-2-hydroxypropyl)-4-(4-
chloro-3-(trifluoromethyl) phenyl) piperidin-4-ol demonstrated good anti-tuberculosis activity. Unfortu-
nately, side effects were observed upon in vivo anti-tuberculosis testing of these compounds precluding
their further advancement, which may be in part due to the secondary pharmacology associated with the
aryl piperidinol core.
Received 19 February 2009
Revised 31 March 2009
Accepted 2 April 2009
Available online 9 April 2009
Keywords:
Tuberculosis
Antibiotic
Piperidinol
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
to 1 is reported and the resulting anti-tuberculosis SAR is
discussed.
Tuberculosis continues to pose a serious world health threat for
which there is an urgent need to develop new therapeutic options.1
In our ongoing effort to discover novel anti-tuberculosis therapeu-
tics we have been performing primary anti-tuberculosis screening
of commercially available libraries. This effort produced a piperid-
inol hit (1 in Fig. 1). The anti-tuberculosis activity of 1 was con-
firmed by resynthesis and biological reevaluation showing good
2. Chemistry
The chemistry to make racemic and chiral aryl piperidinol ana-
logs is shown in Schemes 1 and 2, respectively.8,9 The synthesis of
racemic products 1 and 2 starts from commercially available pipe-
ridinol and racemic epoxide starting materials. Epoxide ring open-
ing by piperidine afforded diol product 1 and 2 in 75% and 79%
yield, respectively (Scheme 1). The synthesis of the chiral piperid-
inol compounds 4a–p was first performed by reacting optically ac-
tive epoxide intermediate (S)-(+)-epichlorohydrin or (R)-(ꢀ)-
epichlorohydrin with a substituted phenol or thiophenol in aceto-
nitrile under reflux in the presence of cesium carbonate to afford
chiral epoxide derivative 3.8 The yield of this reaction was limited
by a competing epoxide ring opening with the phenoxide nucleo-
phile.10,11 However, the bis-substituted byproducts12 (scheme 2)
were most easily removed by column chromatography purification
in the next step, therefore the crude product intermediate 3 was
used directly without further purification. The crude preparation
of 3 was reacted with 4-[4-chloro-3-(trifluoromethyl)-phenyl]-4-
piperidinol in ethanol under reflux to afford alcohol diols 4a–p in
19–50% two step overall yield after purification by silica gel col-
umn chromatography (Scheme 2).9 Based on the biological data
of the first series, a second set of compounds was designed and
synthesized to evaluate changes to the 4-Cl, 3-CF3 phenyl piperid-
inol core of 4b in an effort to gain further understanding of the A
anti-tuberculosis activity with an MIC 1.5 lg/mL. Compound 1
was then selected for further investigation because: (i) it is struc-
turally similar to existing orally bioavailable drugs haloperidol
and penfluridol2,3 shown in Figure 1; (ii) there has been reported
bioactivity of a number of compounds bearing 4-chloro-3-trifluo-
romethylphenyl-4-piperidinol scaffold as antivirals,4 antibacteri-
als,5 mu receptor agonists,6 and nociceptin (N/OFQ) receptor
ligands7 suggesting that it is a biologically useful scaffold for a vari-
ety of targets; (iii) there are no reports of similar compounds with
anti-tuberculosis activity and thus 1 represents a potentially novel
scaffold or pharmacophore for anti-tuberculosis activity, which is
unlikely to be cross resistant to existing tuberculosis therapies.
To this end, we became interested in the synthesis and evaluation
of a hit optimization library about compound 1 in an effort to ob-
tain a preliminary structure-activity relationship (SAR). In this
manuscript, the design and synthesis of a series of analogs related
* Corresponding author. Tel.: +1 901 448 6018; fax: +1 901 448 6828.
E-mail address: relee@utmem.edu (R.E. Lee).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.005

D. Sun et al. / Bioorg. Med. Chem. 17 (2009) 3588–3594
3589
3. Biological activity
F3C
HO
O
C
Cl
All derivatives were evaluated for anti-tuberculosis activity and
the results are described in Tables 1 and 2. In general, compounds
1, 2, and 4a–p demonstrated anti-tuberculosis activity with mini-
mum inhibitory concentrations (MICs) ranging from 1.4 to
A
Cl
B N
HO
HO
1 (MIC90: 1.5 µg/mL)
18.8
1.4
xyl group and chloro substitution at para position to the aryl C-
ring; and 4m (MIC 1.7 g/mL) with S stereochemistry at the central
l
g/mL (Table 1). The most active compounds are: 4b (MIC
O
F
l
g/mL) with R stereochemistry at the central secondary hydro-
Cl
N
F
l
secondary hydroxyl group and trifluoromethyl group at para posi-
tion of the aryl C-ring. In addition, among eight pairs of enantio-
mers synthesized, the R isomers 4b, 4j, and 4l of three pairs of
enantiomers showed more potent anti-tuberculosis activity than
their corresponding S isomers, whilst in the remaining five pairs
of enantiomers, the S isomers demonstrated approximately equal
(4c and 4o) or more than one fold active (4e, 4g, and 4m) anti-
tuberculosis activity compared with their corresponding R isomers.
Based on these data, there appears to be no correlation or prefer-
ence between stereochemistry at the central secondary hydroxyl
group and anti-tuberculosis activity. The SAR with respect to the
phenoxy C ring was clearer with 4-chloro (4a,b) and 4-trifluoro-
methyl (4m,n) analogs being the most active and unsubstituted
phenyl (4c, d and 4e, f) and the 2,5-dimethyl substituted (4o,p)
analogs being the least active. The anti-tuberculosis activities of
the second set of compounds are shown in Table 2. Compared with
parent compound 4b, compounds 5a–c had significant decrease of
anti-tuberculosis activity. This demonstrated the importance of 4-
chloro and 3-CF3 groups. Removal of either of these groups is det-
rimental to anti-tuberculosis activity by comparing compound 5b,
5c, and 4b, removal of both groups in compound 5a led to the sig-
nificant loss of activity. The tertiary hydroxyl group also has a sig-
nificant effect on activity as well. Replacement of it with a cyano
Haloperidol
F3C
Cl
F
N
HO
Penfluridol
Figure 1. Chemical structures of piperidinol library hit 1, haloperidol, and
penfluridol.
and B-ring SAR, compound 4b was selected for further optimiza-
tion because it is the most active from the first series and has
the highest therapeutic index. As outlined in Scheme 3, the same
synthetic strategy was employed as in the synthesis of 4a-p except
that starting materials bearing different piperidine scaffolds were
used. Optically active epoxide (S)-(+)-epichlorohydrin was firstly
reacted with 4-chlorophenol in the presence of cesium carbonate
to give the chiral epoxide derivative, which was subsequently trea-
ted with a panel of 4-piperidines in ethanol under reflux to afford
alcohol 5a–e, in overall yield 37–57% after purification by silica gel
chromatography. (Scheme 3).
F3C
F3C
Cl
HO
O
R
EtOH, reflux
R
O
+
Cl
NH
N
Overnight
75-79%
O
HO
HO
1 R = Cl
2 R = H
Scheme 1. Synthesis of piperidinol racemate analogs 1 and 2.
F3C
F3C
HO
HO
X
F3C
Cl
X
X
(S)
R
R
R
R
Cl
(R)
(S)
Cl
Cl
N
NH
O
or
HO
Cs2CO3/CH3CN
Reflux, Overnight
HO
O
O
+
HX
(R)
or
or
EtOH, Reflux, Overnight
X
R
Cl
O
3
19-50%
OH
N
two step overall yield
+
X = S, O
X
X
HO
R
R
4a p
-
Bis-byproduct
Scheme 2. Synthesis of optically active piperidinol analogs 4a–p.
R2
R3
Cl
EtOH, reflux
NH
HO
O
Cl
Cl
(R)
O
R2
R3
N
Cs2CO3
(S)
(S)
HO
Cl
+
Overnight
37-57%
two step overall yield
MeCN
Reflux
O
O
5a-e
Scheme 3. Synthesis of optically active piperidinol analogs 5a–e.

3590
D. Sun et al. / Bioorg. Med. Chem. 17 (2009) 3588–3594
Table 1
In vitro anti-tuberculosis activity, cytotoxic LD50, and therapeutic indices of piperidinol analogs 1, 2, and 4 optimized at R1 position
F3C
Cl
R1
N
HO
g/mL)
Compd
R1
MIC (
l
Cytotoxic LD50
17.9
(l
g/mL)
Therapeutic index
11.9
HO
O
O
O
Cl
1
1.5
HO
HO
2
10.4
3.8
19.5
17.9
1.9
4.7
Cl
Cl
4a
(S)
HO
HO
O
S
4b
4c
1.4
5.2
18.2
35.7
13.3
6.8
(R)
(S)
HO
HO
HO
HO
S
O
O
O
4d
4e
4f
8.3
8.3
37.1
34.9
27.7
25.8
4.4
4.2
1.5
6.6
(R)
(S)
(R)
(S)
18.8
3.9
F
4g
HO
HO
HO
O
O
O
F
4h
4i
10.4
7.3
3.6
7.3
3.1
20.8
17.6
17.7
18.7
18.0
2.0
2.4
4.9
2.6
5.8
(R)
(S)
(R)
OCF3
OCF3
4j
Cl
Cl
4k
4l
HO
HO
HO
HO
O
O
O
O
Cl
(S)
(R)
(S)
(R)
Cl
CF3
CF3
4m
4n
4o
1.7
5.2
4.7
18.7
18.3
19.0
11.0
3.5
HO
4.1
O
O
(S)
(R)
HO
4p
5.9
19.3
3.3

D. Sun et al. / Bioorg. Med. Chem. 17 (2009) 3588–3594
3591
Table 2
In vitro anti-tuberculosis activity of piperidinol analogs 5 optimized at R4 position
5. Experimental
All solvents were purchased from Sigma-Aldrich and Fisher Sci-
entific and used as received, and remaining chemicals were from
Sigma–Aldrich. Thin layer chromatography (TLC) analysis was per-
formed on Merck Silica Gel 60F254 plates and the spots were visu-
alized under a UV lamp. Melting point (mp) was determined using
OptiMelt Automated Melting Point System (Stanford Research Sys-
tems) and is uncorrected. 1H NMR spectra were recorded at
500 MHz on a Varian Inova NMR instrument. Mass spectra were re-
corded on a Bruker Esquire LC/MS using ESI. Analytical RP-HPLC
was conducted on a Shimadzu HPLC system with a Phenomenex
HO
O
Cl
(R)
R4
Compd
R4
MIC (
lg/mL)
N
5a
100
HO
Cl
N
5b
5c
25
25
C18 column (100 Å, 3
l
m, 4.6 ꢁ 50 mm), flow rate 1.0 mL/min
HO
and a gradient of solvent A (water with 0.1% TFA) and solvent B
(acetonitrile): 0–2.00 min 100% A; 2.00–7.00 min 0–100% B (linear
gradient). UV detection at 254 and 218 nm.
F3C
N
HO
5.1. General procedure for the synthesis of the piperidinols 1, 2,
4a–p, and 5a–e
N
5d
5e
25
N
Acetonitrile (15 mL) was added to (S)-(+)-epichlorohydrin or
(R)-(ꢀ)-epichlorohydrin (0.188 mL, 2.4 mmol, 1.2 equiv), substi-
tuted phenol (2 mmol, 1 equiv), followed by the addition of Cesium
carbonate (1.3 g, 4 mmol, 2 equiv). The reaction mixture was stir-
red under reflux overnight. The reaction was complete based on
TLC monitoring. The reaction mixture was filtered and washed
with acetonitrile. The filtrate was evaporated in vacuo to give
crude product 3, which was used directly for the next step without
further purification.
The crude product obtained above was dissolved in ethanol
(15 mL), followed by the addition of 4-[4-chloro-3-(trifluoro-
methyl)-phenyl]-4-piperidinol or the other 4-piperidines
(2 mmol). The reaction was stirred under reflux overnight and
the reaction was complete based on HPLC analysis. The resulting
solution was evaporated in vacuo and the residue was purified
by column chromatography on silica gel (hexane–50% ethyl acetate
in hexane in a volume of 255 mL, followed by 100% ethyl acetate in
a linear gradient in a volume of 204 mL). Evaporation of appropri-
ate fractions gave final product as white powder.
>200
Cl
N
(E)
group resulted in a more active compound 5d and replacement
with a dehydration product 5e results led to a complete loss of
anti-tuberculosis activity (Table 2).
The cytotoxicities of the compounds were next evaluated
against eukaryotic Vero cells (Table 1) to determine their suitabil-
ity for further advancement. In general cytotoxicities were ob-
served producing narrow therapeutic indices that ranged from
1.5 for 4f to 13.3 for 4b. However, the selectivity of 4b and 4m
was sufficient to advance for testing in vivo (>10). In subsequent
experiments for anti-tuberculosis efficacy of 4b in mice,13 severe
adverse effects were observed resulting in death at doses higher
than 50–100 mg/kg. These symptoms precluded further testing of
these compounds.
4. Conclusions
5.1.1. 1-(3-(4-Chlorophenoxy)-2-hydroxypropyl)-4-(4-chloro-3-
(trifluoromethyl)phenyl)piperidin-4-ol, 1 (racemate)
In summary, high-throughput screening of a commercial library
identified a piperidinol hit with anti-tuberculosis activities and an
unknown mechanism of action. Based on this hit compound, two
expanded sets of compounds 4a–p and 5a–e were subsequently
designed, synthesized, and evaluated toward anti-tuberculosis
activity. Two compounds 4b and 4m were identified with good
0.35 g (1 mmol scale), 75.4% yield. Mp: 147–149 °C. 1H NMR,
500 MHz (DMSO-d6): d 7.92 (d, J = 2.0 Hz, 1H), 7.76 (dd, J = 1.7
and 8.5 Hz, 1H), 7.66 (d, J = 8.3 Hz, 1H), 7.32 (dd, J = 3.3 and
8.8 Hz, 2H), 6.98 (dd, J = 3.3 and 8.8 Hz, 2H), 5.15 (s, 1H, OH),
4.85 (d, J = 4.2 Hz, 1H, OH), 4.01 (dd, J = 3.5 and 9.5 Hz, 1H, CHaO),
0
3.96 (m, 1H, CHOH), 3.88 (dd, J = 5.8 and 9.5 Hz, 1H, CHa O), 2.72
activity (MIC: 1.4–1.7 lg/mL) and acceptable therapeutic indices
(>10). We have showed that improvements in the therapeutic in-
dex can be made (Table 1), unfortunately these compounds pro-
duced significant side effects when tested in vivo for anti-
tuberculosis activity in mice likely due to secondary pharmacology
associated with 4-[4-chloro-3-(trifluoromethyl)-phenyl]-4-pipe-
ridinol core. This study may highlight some of the problems likely
to be encountered as more investigators switch to direct anti-
tuberculosis screening rather than single enzyme screening to dis-
cover novel anti-tuberculosis compounds from compound li-
braries. As such commercial libraries are rarely truly random and
are most often designed around pharmacologically active scaffolds
utilized in discovery of human non-infectious diseases, which
could compromise their use for infectious disease particularly
those requiring long-term treatment such as tuberculosis. As ob-
served in our study, this increases the risk of unwanted side effects
from residual secondary pharmacology associated with the chem-
ical scaffold that may preclude further advancement of the series.
(m, 2H), 2.50 (m, 3H, overlapped with DMSO peak), 2.42 (dd,
J = 6.5 and 12.6 Hz, 1H, NCHb), 1.95 (m, 2H), 1.57 (m, 2H). Mass
spectrum (ESI) m/z (MH)+ 464.1, (MꢀH)ꢀ 461.9. HPLC purity:
100% (254 nm), tR = 5.52 min.
5.1.2. 4-(4-Chloro-3-(trifluoromethyl)phenyl)-1-(2-hydroxy-3-
phenoxypropyl)piperidin-4-ol, 2 (racemate)
0.68 g (2 mmol scale), 79.1% yield. Mp: 127–130 °C. 1H NMR,
500 MHz (DMSO-d6): d 7.93 (d, J = 2.0 Hz, 1H), 7.77 (dd, J = 2.0
and 8.5 Hz, 1H), 7.66 (d, J = 8.5 Hz, 1H), 7.29 (m, 2H), 6.95 (m,
3H), 5.15 (s, 1H, OH), 4.82 (br s, 1H, OH), 3.99 (m, 2H, CHaO and
0
CHOH), 3.88 (dd, J = 5.4 and 9.0 Hz, 1H, CHa O), 2.73 (m, 2H), 2.50
(m, 3H, overlapped with DMSO peak), 2.43 (dd, J = 6.4 and
12.5 Hz, 1H, NCHb), 1.95 (m, 2H), 1.57 (m, 2H). Mass spectrum
(ESI) m/z (MH)+ 430.1, (MꢀH)ꢀ 428.0. HPLC purity: 100%
(254 nm), tR = 5.38 min.

3592
D. Sun et al. / Bioorg. Med. Chem. 17 (2009) 3588–3594
5.1.3. 1-((S)-3-(4-Chlorophenoxy)-2-hydroxypropyl)-4-(4-chlo-
ro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 4a
7.77 (dd, J = 1.2 and 8.5 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.30 (t,
J = 7.6 Hz, 2H), 6.94 (m, 3H), 5.15 (s, 1H, OH), 4.81 (d, J = 4.4 Hz,
0.39 g (2 mmol scale), 42.0% 2-step overall yield. Mp: 146–
149 °C. 1H NMR, 500 MHz (DMSO-d6): d 7.92 (d, J = 2.0 Hz, 1H),
7.76 (dd, J = 1.7 and 8.5 Hz, 1H), 7.66 (d, J = 8.5 Hz, 1H), 7.32 (dd,
J = 3.5 and 8.9 Hz, 2H), 6.98 (dd, J = 3.4 and 8.9 Hz, 2H), 5.15 (s,
1H, OH), 4.84 (d, J = 4.6 Hz, 1H, OH), 4.01 (dd, J = 3.5 and 9.5 Hz,
1H, OH), 3.99 (m, 2H, CHOH and CHaO), 3.89 (dd, J = 5.6 and
0
9.3 Hz, 1H, CHa O), 2.74 (m, 2H), 2.51 (m, 3H), 2.43 (dd, J = 6.4
and 12.5 Hz, 1H, NCHb), 1.96 (m, 2H), 1.57 (m, 2H). Mass spectrum
(ESI) m/z (MH)+ 430.1, (MNa)+ 452.1, (MꢀH)ꢀ 427.9. HPLC purity:
100% (254 nm), tR = 5.41 min; 99.4% (218 nm), tR = 5.42 min.
1H, CHaO), 3.96 (m, 1H, CHOH), 3.88 (dd, J = 5.9 and 9.5 Hz, 1H,
0
CHa O), 2.72 (m, 2H), 2.50 (m, 3H, overlapped with DMSO peak),
5.1.9. 1-((S)-3-(4-Fluorophenoxy)-2-hydroxypropyl)-4-(4-chloro-
3-(trifluoromethyl)phenyl)piperidin-4-ol, 4g
2.41 (dd, J = 6.5 and 12.6 Hz, 1H, NCHb), 1.95 (m, 2H), 1.56 (m,
2H). Mass spectrum (ESI) m/z (MH)+ 464.1, (MꢀH)ꢀ 461.9. HPLC
purity: 99.1% (218 nm), tR = 5.59 min.
0.35 g (2 mmol scale), 39.1% 2-step overall yield. Mp: 114–
115 °C. 1H NMR, 500 MHz (DMSO-d6): d 7.93 (d, J = 2.0 Hz, 1H),
7.77 (dd, J = 1.7 and 8.5 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.12 (m,
2H), 6.97 (m, 2H), 5.15 (s, 1H, OH), 4.81 (d, J = 4.2 Hz, 1H, OH),
5.1.4. 1-((R)-3-(4-Chlorophenoxy)-2-hydroxypropyl)-4-(4-chlo-
ro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 4b
0.30 g (2 mmol scale), 32.3% 2-step overall yield. Mp: 144–
146 °C. 1H NMR, 500 MHz (DMSO-d6): d 7.93 (d, J = 2.0 Hz, 1H),
7.76 (dd, J = 2.0 and 8.5 Hz, 1H), 7.66 (d, J = 8.3 Hz, 1H), 7.32 (dd,
J = 3.5 and 9.0 Hz, 2H), 6.98 (dd, J = 3.4 and 9.0 Hz, 2H), 5.14 (s, 1H,
3.98 (m, 2H, CHOH and CHaO), 3.87 (dd, J = 5.4 and 9.0 Hz, 1H,
0
CHa O), 2.72 (m, 2H), 2.50 (m, 3H, overlapped with DMSO peak),
2.42 (dd, J = 6.4 and 12.7 Hz, 1H, NCHb), 1.95 (m, 2H), 1.56 (m,
2H). Mass spectrum (ESI) m/z (MH)+ 448.1, (MNa)+ 470.1, (MꢀH)ꢀ
445.9. HPLC purity: 99.5% (254 nm), tR = 5.44 min; 99.5% (218 nm),
tR = 5.45 min.
OH), 4.84 (d, J = 4.4 Hz, 1H, OH), 4.01 (dd, J = 3.7 and 9.8 Hz, 1H,
0
CHaO), 3.96 (m, 1H, CHOH), 3.88 (dd, J = 5.9 and 9.5 Hz, 1H, CHa O),
2.72 (m, 2H), 2.50 (m, 3H, overlapped with DMSO peak), 2.41 (dd,
J = 6.4 and 12.5 Hz, 1H, NCHb), 1.95 (m, 2H), 1.56 (m, 2H). Mass spec-
trum (ESI) m/z (MH)+ 464.1, (MꢀH)ꢀ 461.9. HPLC purity: 100%
(254 nm), tR = 5.58 min; 98.8% (218 nm), tR = 5.59 min.
5.1.10. 1-((R)-3-(4-Fluorophenoxy)-2-hydroxypropyl)-4-(4-chlo-
ro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 4h
0.38 g (2 mmol scale), 42.4% 2-step overall yield. Mp: 114–
116 °C. 1H NMR, 500 MHz (DMSO-d6): d 7.93 (d, J = 1.7 Hz, 1H),
7.77 (dd, J = 1.7 and 8.5 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.12 (m,
2H), 6.98 (m, 2H), 5.15 (s, 1H, OH), 4.81 (d, J = 4.4 Hz, 1H, OH),
5.1.5. 4-(4-Chloro-3-(trifluoromethyl)phenyl)-1-((S)-2-hydroxy-
3-(phenylthio)propyl)piperidin-4-ol, 4c
0.17 g (2 mmol scale), 19.1% 2-step overall yield. Mp: 117–
119 °C. 1H NMR, 500 MHz (CDCl3): d 7.84 (s, 1H), 7.56 (d,
J = 8.1 Hz, 1H), 7.45 (d, J = 8.3 Hz, 1H), 7.37 (d, J = 7.8 Hz, 2H),
7.28 (t, J = 7.8 Hz, 2H), 7.18 (t, J = 7.3 Hz, 1H), 3.89 (m, 1H, CHOH),
3.98 (m, 2H, CHOH and CHaO), 3.87 (dd, J = 5.4 and 9.0 Hz, 1H,
0
CHa O), 2.72 (m, 2H), 2.50 (m, 3H, overlapped with DMSO peak),
2.42 (dd, J = 6.4 and 12.5 Hz, 1H, NCHb), 1.96 (m, 2H), 1.57 (m,
2H). Mass spectrum (ESI) m/z (MH)+ 448.1, (MꢀH)ꢀ 445.9. HPLC
purity: 99.2% (254 nm), tR = 5.43 min; 98.2% (218 nm),
tR = 5.44 min.
3.58 (br s, 2H, OH and OH), 3.03 (m, 2H, CH2S), 2.81 (d, J = 11.0 Hz,
0
1H, NCHb ), 2.71 (m, 2H), 2.58 (dd, J = 2.9 and 12.5 Hz, 1H, NCHb),
2.45 (t, J = 10.3 Hz, 2H), 2.09 (m, 1H), 2.01 (m, 2H), 1.70 (m, 2H).
Mass spectrum (ESI) m/z (MH)+ 446.1, (MꢀH)ꢀ 443.9. HPLC purity:
99.4% (254 nm), tR = 5.48 min; 99.5% (218 nm), tR = 5.48 min.
5.1.11. 1-((S)-3-(4-(Trifluoromethoxy)phenoxy)-2-hydroxypro-
pyl)-4-(4-chloro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 4i
0.43 g (2 mmol scale), 41.8% 2-step overall yield. Mp: 123–
126 °C. 1H NMR, 500 MHz (DMSO-d6): d 7.93 (d, J = 1.7 Hz, 1H),
7.77 (dd, J = 1.7 and 8.5 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.29 (d,
J = 9.0 Hz, 2H), 7.06 (d, J = 9.0 Hz, 2H), 5.15 (s, 1H, OH), 4.85 (d,
5.1.6. 4-(4-Chloro-3-(trifluoromethyl)phenyl)-1-((R)-2-hydroxy-
3-(phenylthio)propyl)piperidin-4-ol, 4d
0.22 g (2 mmol scale), 24.7% 2-step overall yield. Mp: 116–
119 °C. 1H NMR, 500 MHz (DMSO-d6): d 7.93 (d, J = 2.0 Hz, 1H),
7.77 (dd, J = 2.0 and 8.3 Hz, 1H), 7.68 (d, J = 8.3 Hz, 1H), 7.37 (d,
J = 7.6 Hz, 2H), 7.31 (t, J = 7.8 Hz, 2H), 7.17 (t, J = 7.3 Hz, 1H), 5.14
(s, 1H, OH), 4.88 (d, J = 4.6 Hz, 1H, OH), 3.80 (m, 1H, CHOH), 3.19
J = 4.6 Hz, 1H, OH), 4.04 (dd, J = 3.4 and 9.5 Hz, 1H, CHaO), 3.98
0
(m, 1H, CHOH), 3.92 (dd, J = 5.9 and 9.5 Hz, 1H, CHa O), 2.72 (m,
2H), 2.50 (m, 3H, overlapped with DMSO peak), 2.42 (dd, J = 6.4
and 12.7 Hz, 1H, NCHb), 1.95 (m, 2H), 1.57 (m, 2H). Mass spectrum
(ESI) m/z (MH)+ 514.2, (MꢀH)ꢀ 511.9. HPLC purity: 99.0%
(254 nm), tR = 5.70 min; 98.3% (218 nm), tR = 5.71 min.
(dd, J = 4.6 and 13.2 Hz, 1H, CHaS), 2.96 (dd, J = 6.7 and 13.2 Hz,
00
1H, CHa S), 2.65 (m, 2H), 2.46 (m, 3H), 2.40 (dd, J = 6.4 and
12.7 Hz, 1H, NCHb), 1.94 (m, 2H), 1.55 (m, 2H). Mass spectrum
(ESI) m/z (MH)+ 446.1, (MꢀH)ꢀ 443.9. HPLC purity: 99.3%
(254 nm), tR = 5.48 min; 99.3% (218 nm), tR = 5.48 min.
5.1.12. 1-((R)-3-(4-(Trifluoromethoxy)phenoxy)-2-hydroxypro-
pyl)-4-(4-chloro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 4j
0.43 g (2 mmol scale), 41.8% 2-step overall yield. Mp: 123–
125 °C. 1H NMR, 500 MHz (DMSO-d6): d 7.93 (d, J = 2.0 Hz, 1H),
7.77 (dd, J = 2.0 and 8.5 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.29 (d,
J = 8.8 Hz, 2H), 7.06 (d, J = 9.0 Hz, 2H), 5.15 (s, 1H, OH), 4.85 (d,
5.1.7. 4-(4-Chloro-3-(trifluoromethyl)phenyl)-1-((S)-2-hydroxy-
3-phenoxypropyl)piperidin-4-ol, 4e
0.40 g (2 mmol scale), 46.5% 2-step overall yield. Mp: 124–
126 °C. 1H NMR, 500 MHz (DMSO-d6): d 7.93 (d, J = 1.7 Hz, 1H),
7.77 (dd, J = 1.7 and 8.3 Hz, 1H), 7.67 (d, J = 8.3 Hz, 1H), 7.29 (m,
2H), 6.94 (m, 3H), 5.15 (s, 1H, OH), 4.81 (d, J = 4.4 Hz, 1H, OH),
3.99 (m, 2H, CHOH and CHaO), 3.89 (dd, J = 5.5 and 9.2 Hz, 1H,
CHa’O), 2.74 (m, 2H), 2.51 (m, 3H), 2.43 (dd, J = 6.4 and 12.7 Hz,
1H, NCHb), 1.96 (m, 2H), 1.57 (m, 2H). Mass spectrum (ESI) m/z
(MH)+ 430.1, (MNa)+ 452.1, (MꢀH)ꢀ 427.9. HPLC purity: 100%
(254 nm), tR = 5.42 min; 100% (218 nm), tR = 5.43 min.
J = 4.6 Hz, 1H, OH), 4.04 (dd, J = 3.4 and 9.8 Hz, 1H, CHaO), 3.98
0
(m, 1H, CHOH), 3.92 (dd, J = 6.1 and 9.8 Hz, 1H, CHa O), 2.72 (m,
2H), 2.50 (m, 3H, overlapped with DMSO peak), 2.42 (dd, J = 6.4
and 12.7 Hz, 1H, NCHb), 1.95 (m, 2H), 1.57 (m, 2H). Mass spectrum
(ESI) m/z (MH)+ 514.2, (MꢀH)ꢀ 511.9. HPLC purity: 98.9%
(254 nm), tR = 5.71 min; 97.5% (218 nm), tR = 5.72 min.
5.1.13. 1-((S)-3-(3,4-Dichlorophenoxy)-2-hydroxypropyl)-4-
(4-chloro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 4k
0.41 g (2 mmol scale), 41.1% 2-step overall yield. Mp: 121–
124 °C. 1H NMR, 500 MHz (DMSO-d6): d 7.93 (d, J = 2.0 Hz, 1H),
7.77 (dd, J = 2.0 and 8.5 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.52 (d,
J = 8.8 Hz, 1H), 7.27 (d, J = 2.9 Hz, 1H), 7.00 (dd, J = 2.9 and 9.0 Hz,
5.1.8. 4-(4-Chloro-3-(trifluoromethyl)phenyl)-1-((R)-2-hydroxy-
3-phenoxypropyl)piperidin-4-ol, 4f
0.43 g (2 mmol scale), 50.0% 2-step overall yield. Mp: 124–
126 °C. 1H NMR, 500 MHz (DMSO-d6): d 7.93 (d, J = 1.2 Hz, 1H),

D. Sun et al. / Bioorg. Med. Chem. 17 (2009) 3588–3594
3593
1H), 5.15 (s, 1H, OH), 4.87 (d, J = 4.2 Hz, 1H, OH), 4.08 (m, 1H), 3.95
(m, 2H), 2.72 (m, 2H), 2.50 (m, 3H, overlapped with DMSO peak),
2.41 (dd, J = 5.9 and 12.5 Hz, 1H, NCHb), 1.96 (m, 2H), 1.57 (m,
2H). Mass spectrum (ESI) m/z (MH)+ 500.0. HPLC purity: 96.1%
(254 nm), tR = 5.69 min; 95.8% (215 nm), tR = 5.69 min.
2H, overlapped with DMSO peak), 2.44 (dd, J = 6.6 and 12.5 Hz,
1H, NCHb), 2.26 (s, 6H, CH3 and CH3), 1.95 (m, 2H), 1.59 (m,
2H). Mass spectrum (ESI) m/z (MH)+ 458.1, (MNa)+ 480.1,
(MꢀH)ꢀ 455.9. HPLC purity: 100% (254 nm), tR = 5.58 min; 100%
(218 nm), tR = 5.58 min.
5.1.14. 1-((R)-3-(3,4-Dichlorophenoxy)-2-hydroxypropyl)-4-
(4-chloro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 4l
0.41 g (2 mmol scale), 41.1% 2-step overall yield. Mp: 136–
138 °C. 1H NMR, 500 MHz (DMSO-d6): d 7.93 (d, J = 1.7 Hz, 1H),
7.77 (dd, J = 2.0 and 8.5 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.52 (d,
J = 9.0 Hz, 1H), 7.27 (d, J = 2.9 Hz, 1H), 7.00 (dd, J = 2.9 and
9.0 Hz, 1H), 5.15 (s, 1H, OH), 4.87 (d, J = 3.9 Hz, 1H, OH), 4.08
(m, 1H), 3.95 (m, 2H), 2.71 (m, 2H), 2.50 (m, 3H, overlapped with
DMSO peak), 2.41 (dd, J = 5.9 and 12.5 Hz, 1H, NCHb), 1.96 (m,
2H), 1.56 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 500.0. HPLC
purity: 95.8% (254 nm), tR = 5.68 min; 95.2% (218 nm),
tR = 5.69 min.
5.1.19. 1-((R)-3-(4-Chlorophenoxy)-2-hydroxypropyl)-4-phenyl-
piperidin-4-ol, 5a
0.41 g (2 mmol scale), 56.7% 2-step overall yield. Mp: 108–
111 °C. 1H NMR, 500 MHz (DMSO-d6): d 7.48 (d, J = 7.6 Hz, 2H),
7.32 (m, 4H), 7.20 (t, J = 7.3 Hz, 1H), 6.99 (d, J = 9.0 Hz, 2H), 4.84
(d, J = 4.6 Hz, 1H, OH), 4.77 (s, 1H, OH), 4.02 (dd, J = 3.4 and
9.5 Hz, 1H, CHaO), 3.97 (m, 1H, CHOH), 3.88 (dd, J = 6.1 and
0
9.5 Hz, 1H, CHa O), 2.69 (m, 2H), 2.50 (m, 3H, overlapped with
DMSO peak), 2.41 (dd, J = 6.6 and 12.7 Hz, 1H, NCHb), 1.93 (m,
2H), 1.57 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 362.3, (MNa)+
384.1. HPLC purity: 100% (254 nm), tR = 5.16 min; 100% (215 nm),
tR = 5.16 min.
5.1.15. 1-((S)-3-(4-(Trifluoromethyl)phenoxy)-2-hydroxypro-
pyl)-4-(4-chloro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 4m
0.35 g (2 mmol scale), 35.2% 2-step overall yield. Mp: 135–
137 °C. 1H NMR, 500 MHz (DMSO-d6): d 7.93 (d, J = 2.0 Hz, 1H),
7.77 (dd, J = 2.0 and 8.5 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.65 (d,
J = 8.5 Hz, 2H), 7.15 (d, J = 8.8 Hz, 2H), 5.15 (s, 1H, OH), 4.89 (d,
J = 4.2 Hz, 1H, OH), 4.12 (m, 1H), 4.00 (m, 2H), 2.73 (m, 2H), 2.50
(m, 3H, overlapped with DMSO peak), 2.44 (dd, J = 5.9 and
12.5 Hz, 1H, NCHb), 1.95 (m, 2H), 1.57 (m, 2H). Mass spectrum
(ESI) m/z (MH)+ 498.1. HPLC purity: 98.4% (218 nm), tR = 5.68 min.
5.1.20. 1-((R)-3-(4-Chlorophenoxy)-2-hydroxypropyl)-4-(4-
chlorophenyl)piperidin-4-ol, 5b
0.35 g (2 mmol scale), 44.2% 2-step overall yield. Mp: 146–
148 °C. 1H NMR, 500 MHz (DMSO-d6): d 7.50 (d, J = 8.5 Hz, 2H),
7.36 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 8.8 Hz, 2H), 6.99 (d, J = 8.8 Hz,
2H), 4.89 (s, 1H, OH), 4.84 (d, J = 4.4 Hz, 1H, OH), 4.01 (dd, J = 3.7
and 9.8 Hz, 1H, CHaO), 3.97 (m, 1H, CHOH), 3.88 (dd, J = 6.1 and
0
9.8 Hz, 1H, CHa O), 2.69 (m, 2H), 2.50 (m, 3H, overlapped with
DMSO peak), 2.41 (dd, J = 6.4 and 12.5 Hz, 1H, NCHb), 1.90 (m,
2H), 1.56 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 396.2, (MNa)+
418.1, (MꢀH)ꢀ 393.9. HPLC purity: 100% (254 nm), tR = 5.33 min;
99.3% (215 nm), tR = 5.33 min.
5.1.16. 1-((R)-3-(4-(Trifluoromethyl)phenoxy)-2-hydroxypro-
pyl)-4-(4-chloro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 4n
0.21 g (2 mmol scale), 21.1% 2-step overall yield. Mp: 138–
141 °C. 1H NMR, 500 MHz (DMSO-d6): d 7.92 (d, J = 1.7 Hz, 1H),
7.77 (dd, J = 1.5 and 8.3 Hz, 1H), 7.67 (d, J = 8.3 Hz, 1H), 7.65
(d, J = 8.5 Hz, 2H), 7.15 (d, J = 8.5 Hz, 2H), 5.15 (s, 1H, OH), 4.89
(d, J = 4.2 Hz, 1H, OH), 4.12 (m, 1H), 4.00 (m, 2H), 2.73 (m,
2H), 2.50 (m, 3H, overlapped with DMSO peak), 2.44 (dd,
J = 5.9 and 12.5 Hz, 1H, NCHb), 1.95 (m, 2H), 1.57 (m, 2H). Mass
spectrum (ESI) m/z (MH)+ 498.1. HPLC purity: 96.8% (218 nm),
tR = 5.69 min.
5.1.21. 1-((R)-3-(4-Chlorophenoxy)-2-hydroxypropyl)-4-(3-
(trifluoromethyl)phenyl)piperidin-4-ol, 5c
0.32 g (2 mmol scale), 37.2% 2-step overall yield. Mp: 101–
104 °C. 1H NMR, 500 MHz (DMSO-d6): d 7.83 (s, 1H), 7.77 (d,
J = 7.3 Hz, 1H), 7.57 (m, 2H), 7.33 (dd, J = 3.4 and 8.2 Hz, 2H),
6.99 (dd, J = 3.4 and 8.8 Hz, 2H), 5.06 (s, 1H, OH), 4.85 (d,
J = 4.4 Hz, 1H, OH), 4.02 (dd, J = 3.7 and 9.8 Hz, 1H, CHaO), 3.97
0
(m, 1H, CHOH), 3.89 (dd, J = 5.9 and 9.5 Hz, 1H, CHa O), 2.72
(m, 2H), 2.50 (m, 3H, overlapped with DMSO peak), 2.42 (dd,
J = 6.6 and 12.7 Hz, 1H, NCHb), 1.97 (m, 2H), 1.57 (m, 2H). Mass
spectrum (ESI) m/z (MH)+ 430.2, (MNa)+ 452.1, (MꢀH)ꢀ 427.9.
HPLC purity: 98.9% (254 nm), tR = 5.45 min; 98.7% (215 nm),
tR = 5.45 min.
5.1.17. 1-((S)-3-(2,6-Dimethylphenoxy)-2-hydroxypropyl)-4-(4-
chloro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 4o
0.37 g (2 mmol scale), 40.4% 2-step overall yield. Mp: 138–
141 °C. 1H NMR, 500 MHz (DMSO-d6): d 7.93 (d, J = 2.0 Hz, 1H),
7.76 (dd, J = 1.7 and 8.3 Hz, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.01 (d,
J = 7.3 Hz, 2H), 6.91 (t, J = 7.3 Hz, 1H), 5.16 (s, 1H, OH), 4.80 (d,
5.1.22. 1-((R)-3-(4-Chlorophenoxy)-2-hydroxypropyl)-4-phenyl-
piperidine-4-carbonitrile, 5d
J = 4.6 Hz, 1H, OH), 3.99 (m, 1H, CHOH), 3.76 (dd, J = 3.7 and
0.30 g (2 mmol scale), 40.4% 2-step overall yield. Mp: 74–76 °C.
1H NMR, 500 MHz (DMSO-d6): d 7.54 (d, J = 7.6 Hz, 2H), 7.45 (t,
J = 7.3 Hz, 2H), 7.37 (t, J = 7.3 Hz, 1H), 7.33 (d, J = 8.8 Hz, 2H), 6.99
0
9.3 Hz, 1H, CHaO), 3.70 (dd, J = 5.6 and 9.3 Hz, 1H, CHa O), 2.75
0
(m, 2H), 2.57 (dd, J = 5.9 and 12.5 Hz, 1H, NCHb ), 2.50 (m, 2H,
overlapped with DMSO peak), 2.44 (dd, J = 6.6 and 12.5 Hz, 1H,
NCHb), 2.26 (s, 6H, CH3 and CH3), 1.95 (m, 2H), 1.59 (m, 2H). Mass
spectrum (ESI) m/z (MH)+ 458.1, (MNa)+ 480.1, (MꢀH)ꢀ 455.9.
HPLC purity: 100% (254 nm), tR = 5.58 min; 100% (218 nm),
tR = 5.58 min.
(d, J = 8.8 Hz, 2H), 4.94 (d, J = 3.4 Hz, 1H, OH), 3.99 (m, 2H, CHaO
0
and CHOH), 3.89 (dd, J = 7.1 and 10.7 Hz, 1H, CHa O), 3.05 (m,
2H), 2.50 (m, 3H, overlapped with DMSO peak), 2.42 (m, 1H),
2.04 (m, 4H). Mass spectrum (ESI) m/z (MH)+ 371.2, (MNa)+
393.1. HPLC purity: 100% (254 nm), tR = 5.38 min; 98.5%
(215 nm), tR = 5.38 min.
5.1.18. 1-((R)-3-(2,6-Dimethylphenoxy)-2-hydroxypropyl)-4-(4-
chloro-3-(trifluoromethyl)phenyl)piperidin-4-ol, 4p
0.43 g (2 mmol scale), 47.0% 2-step overall yield. Mp: 138–
141 °C. 1H NMR, 500 MHz (DMSO-d6): d 7.94 (d, J = 1.7 Hz, 1H),
7.76 (dd, J = 1.5 and 8.3 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.01
(d, J = 7.6 Hz, 2H), 6.91 (t, J = 7.6 Hz, 1H), 5.16 (s, 1H, OH), 4.80
5.1.23. (R)-1-(4-Chlorophenoxy)-3-(4-(4-chlorophenyl)-5,6-
dihydropyridin-1(2H)-yl)propan-2-ol, 5e
0.28 g (2 mmol scale), 37.0% 2-step overall yield. Mp: 124–
126 °C. 1H NMR, 500 MHz (DMSO-d6): d 7.46 (d, J = 8.5 Hz, 2H),
7.38 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 9.0 Hz, 2H), 6.98 (d,
J = 8.8 Hz, 2H), 6.20 (br t, 1H, =CH), 4.94 (d, J = 4.2 Hz, 1H, OH),
(d, J = 4.4 Hz, 1H, OH), 3.99 (m, 1H, CHOH), 3.76 (dd, J = 3.9
0
and 9.3 Hz, 1H, CHaO), 3.70 (dd, J = 5.6 and 9.3 Hz, 1H, CHa O),
0
4.00 (m, 2H, CHaO and CHOH), 3.89 (dd, J = 6.8 and 10.7 Hz,
0
2.75 (m, 2H), 2.57 (dd, J = 6.1 and 12.5 Hz, 1H, NCHb ), 2.50 (m,
1H, CHa O), 3.17 (m, 2H), 2.71 (m, 2H), 2.58 (dd, J = 5.6 and

3594
D. Sun et al. / Bioorg. Med. Chem. 17 (2009) 3588–3594
0
12.5 Hz, 1H, NCHb ), 2.50 (m, 1H, overlapped with DMSO peak,
NCHb), 2.46 (m, 2H). Mass spectrum (ESI) m/z (MH)+ 378.1,
(MNa)+ 400.1. HPLC purity: 99.7% (254 nm), tR = 5.57 min;
99.7% (215 nm), tR = 5.56 min.
Supplementary data
Supplementary data (1H NMR spectra and HPLC profiles of all
target molecules) associated with this article can be found, in the
online version, at doi:10.1016/j.bmc.2009.04.005.
5.1.24. MIC determination
References and notes
MIC values were determined against Mycobacterium tuberculosis
(H37Rv) by the microbroth dilution method.14 A broth culture of
M. tuberculosis was grown in Middlebrook 7H9 medium with 10%
OADC supplement to an OD600 of 0.4–0.6 and aliquots were frozen.
The CFU/mL of these aliquots were determined. The tested com-
1. Sacchettini, J. C.; Rubin, E. J.; Freundlich, J. S. Nat. Rev. Microbiol. 2008, 6, 41.
2. Williams, D. A.; Lemke, T. L. Foye’s Principles of Medicinal Chemistry, 5th ed.;
Lippincott Williams & Wilkins, 2002.
3. Gallant, D. M.; Mielke, D. H.; Spirtes, M. A.; Swanson, W. C.; Bost, R. Am. J.
Psychiat. 1974, 131, 699.
4. Manikowski, A.; Verri, A.; Lossani, A.; Gebhardt, B. M.; Gambino, J.; Focher, F.;
Spadari, S.; Wright, G. E. J. Med. Chem. 2005, 48, 3919.
5. Zhi, C.; Long, Z. Y.; Gambino, J.; Xu, W. C.; Brown, N. C.; Barnes, M.; Butler, M.;
LaMarr, W.; Wright, G. E. J. Med. Chem. 2003, 46, 2731.
6. Chen, Z.; Davies, E.; Miller, W. S.; Shan, S.; Valenzano, K. J.; Kyle, D. J. Bioorg.
Med. Chem. Lett. 2004, 14, 5275.
7. Chen, Z.; Miller, W. S.; Shan, S.; Valenzano, K. J. Bioorg. Med. Chem. Lett. 2003, 13,
3247.
8. Yang, W.; Wang, Y.; Roberge, J. Y.; Ma, Z.; Liu, Y.; Michael Lawrence, R.; Rotella, D. P.;
Seethala, R.; Feyen, J. H.; Dickson, J. K., Jr. Bioorg. Med. Chem. Lett. 2005, 15, 1225.
9. Shagufta; Srivastava, A. K.; Sharma, R.; Mishra, R.; Balapure, A. K.; Murthy, P. S.;
Panda, G. Bioorg. Med. Chem. 2006, 14, 1497.
pounds were serially diluted in DMSO and 4
l
L added to microtier
L of 7H9 medium. Subsequently,
lL of 7H9 medium were added to each
plate wells containing 100
l
2 ꢁ 104 TB bacteria in 100
well. The plates were incubated at 37 °C for 9–11 days. The MIC90
was determined by visual inspection and defined as the concentra-
tion that inhibited 90% of growth. For each experiment, controls of
INH were run; the MIC of INH under these conditions was between
0.032 and 0.064 lg/mL.
10. Surendra, K.; Krishnaveni, N. S.; Nageswar, Y. V.; Rao, K. R. J. Org. Chem. 2003,
68, 4994.
11. Das, B.; Krishnaiah, M.; Thirupathi, P.; Laxminarayana, K. Tetrahedron Lett.
2007, 48, 4263.
5.1.25. Cytotoxicity study
The LD50 for each drug was determined using a microplate dilu-
tion method.15 African green monkey kidney cells (Vero cells) were
grown in L15 medium without phenol (Invitrogen). The media was
supplemented with glutamine, 10 mL/L penicillin-streptomycin
12. Representative characterization of Bis-byproduct (R = 4-Cl, X = O):
OH
solution (10,000 IU/10,000 lg/mL), and 10% bovine calf serum at
O
O
37 °C. The microtiter plates were sealed and testing was conducted
for 72 h at 37 °C, alamarBlueÒ was added to each well, and plates
were incubated overnight at 37 °C. Plates were read at 570 and
600 nm using a spectrophotometric plate reader, and the absor-
bance readings were used to calculate the 50% lethal concentration.
Cl
Cl
1H NMR, 500 MHz (DMSO-d6): 7.33 (d, J = 8.8 Hz, 4H), 6.99 (d, J = 9.0 Hz, 4H),
5.44 (d, J = 4.6 Hz, 1H, OH), 4.14 (m, 1H), 4.07 (dd, J = 4.6 and 9.8 Hz, 2H), 4.01
(dd, J = 5.9 and 10.0 Hz, 2H). Mass spectrum (ESI) m/z (MH)+ 335.1. HPLC
purity: 100% (254 nm and 215 nm), tR = 7.00 min.
13. Lenaerts, A. J. M.; Veronica, G.; Brooks, J. V.; Orme, I. M. Antimicrob. Agents
Chemother. 2003, 47, 783.
14. Hurdle, J. G.; Lee, R. B.; Budha, N. R.; Carson, E. I.; Qi, J.; Scherman, M. S.; Cho, S.
H.; McNeil, M. R.; Lenaerts, A. J.; Franzblau, S. G.; Meibohm, B.; Lee, R. E.
J. Antimicrob. Chemother. 2008, 62, 1037.
Acknowledgment
We thank National Institutes of Health grant AI057836 for
financial support.
15. Collins, L.; Franzblau, S. G. Antimicrob. Agents Chemother. 1997, 41, 1004.