Synthesis and antituberculosis activity of 5-methyl/trifluoromethoxy-1H-indole-2,3-dione 3-thiosemicarbazone derivatives

Article abstract of DOI:10.1016/j.bmc.2008.08.050

New series of 5-methyl/trifluoromethoxy-1H-indole-2,3-dione 3-thiosemicarbazones 3a-t, 1-methyl-5-methyl/trifluoromethoxy-1H-indole-2,3-dione 3-thiosemicarbazones 4a-y and 5-trifluoromethoxy-1-morpholinomethyl-1H-indole-2,3-dione 3-thiosemicarbazones 5a-m were synthesized. The structures of the synthesized compounds were confirmed by spectral data and elemental analysis. The new 5-methyl/trifluoromethoxy-1H-indole-2,3-dione derivatives, along with previously synthesized 5-methyl-1H-indole-2,3-dione 3-thiosemicarbazones 6a-l, were evaluated for in vitro antituberculosis activity against Mycobacterium tuberculosis H37Rv. 5-Methyl-1H-indole-2,3-dione 3-thiosemicarbazones (3b, 3d, 3f, 6c, 6d, and 6f), 5-trifluoromethoxy-1H-indole-2,3-dione 3-thiosemicarbazones (3q-s) and 5-trifluoromethoxy-1-morpholinomethyl-1H-indole-2,3-dione 3-thiosemicarbazones (5e and 5j-l) were found to be the most potent inhibitors of M. tuberculosis growth described in this study.

Full text of DOI:10.1016/j.bmc.2008.08.050

Bioorganic & Medicinal Chemistry 16 (2008) 8976–8987
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antituberculosis activity of 5-methyl/trifluoromethoxy-
1H-indole-2,3-dione 3-thiosemicarbazone derivatives
*
Özlen Güzel, Nilgün Karalı , Aydın Salman
Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 34116 Beyazıt, Istanbul, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
New series of 5-methyl/trifluoromethoxy-1H-indole-2,3-dione 3-thiosemicarbazones 3a-t, 1-methyl-
5-methyl/trifluoromethoxy-1H-indole-2,3-dione 3-thiosemicarbazones 4a-y and 5-trifluoromethoxy-
1-morpholinomethyl-1H-indole-2,3-dione 3-thiosemicarbazones 5a-m were synthesized. The structures
of the synthesized compounds were confirmed by spectral data and elemental analysis. The new
5-methyl/trifluoromethoxy-1H-indole-2,3-dione derivatives, along with previously synthesized
5-methyl-1H-indole-2,3-dione 3-thiosemicarbazones 6a-l, were evaluated for in vitro antituberculosis
activity against Mycobacterium tuberculosis H37Rv. 5-Methyl-1H-indole-2,3-dione 3-thiosemicarbazones
(3b, 3d, 3f, 6c, 6d, and 6f), 5-trifluoromethoxy-1H-indole-2,3-dione 3-thiosemicarbazones (3q–s) and
5-trifluoromethoxy-1-morpholinomethyl-1H-indole-2,3-dione 3-thiosemicarbazones (5e and 5j–l) were
found to be the most potent inhibitors of M. tuberculosis growth described in this study.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 30 April 2008
Revised 11 August 2008
Accepted 22 August 2008
Available online 27 August 2008
Keywords:
5-Methyl-1H-indole-2,3-diones
5-Trifluoromethoxy-1H-indole-2,3-diones
Thiosemicarbazones
Antituberculosis activity
1. Introduction
tion of thiacetazone and some structural analogues remain poorly
understood.7
Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a
growing global health problem because of lack of proper therapeu-
tic agents for its remedy.1 The human immunodeficiency virus
pandemic, which contributes substantially to the morbidity and
mortality from TB, and the emergence of multidrug-resistant
(MDR) strains of M. tuberculosis have compounded the problem.2
Although infections by drug-sensitive strains can be successfully
cured, the emergence of drug resistance has prompted new drug
research, particularly the search for new drug targets. When TB
cases cannot be treated by first-line protocols due to resistance is-
sues, the last resort for combating MDR infections relies on the ac-
tion of second-line anti-TB drugs.3 Among these second-line drugs
for the treatment of MDR-TB, thiacetazone is a thiosemicarbazone
antimicrobial that has been widely used in many developing con-
tries.4 Thiosemicarbazones have had a lengthy history as potential
prophylactic therapeutics for human disease.5 Recently, thiaceta-
zone-related compounds have been synthesized and described
for their activity against M. tuberculosis, M. avium and other myco-
bacterial species. Results indicate that SRI-224, SRI-286,6,7 oxazolyl
thiosemicarbazones8 and some S-alkylisothiosemicarbazones9,10
can be useful in the therapy and prophylaxis of mycobacteria infec-
tions and can represent a template for the development of novel
antimycobacterial drugs (Fig. 1). However, the mechanism of ac-
The discovery of the potent antimicrobial activity of 2-indoli-
nones led in the past decade to extensive synthesis of related com-
pounds and as a result, a variety of broad spectrum antimicrobial
agents were developed. In recent years, Schiff and Mannich bases
of 1H-indole-2,3-diones are reported to exhibit broad-spectrum
chemotherapeutic properties such as anti-TB, antiviral and antican-
cer activities.11–17 We described previously the anti-TB profile of 3-
thiosemicarbazone, 3-phenacylisothiosemicarbazone and 3-thia-
zolinehydrazone derivatives of some 1H-2-indolinones which have
exhibited a remarkable anti-TB activity.18,19 The results reflect the
effects of the various substituents introduced in the R1, R2 and R3.
Investigations regarding the structure–activity relationships of the
tested compounds revealed that halogenation in the R1, elongation
of alkyl chain in the R2, replacement of the alkyl in the R2 with cyclo-
hexyl or (non)substituted phenyl and the presence of substitution
in the R3 were efficient in increasing anti-TB activity. Moreover,
R1-nitro substituted derivatives were more active than the corre-
sponding R1-fluoro substituted derivatives in the 1H-indole-2,3-
dione 3-thiosemicarbazones. The presence of the morpholine ring
in some R1-nitro substituted N-Mannich bases also seems to have
a significant impact on the resultant anti-TB activity (Fig. 2).
In the light of these findings, new 5-methyl/trifluoromethoxy-
1H-indole-2,3-dione 3-thiosemicarbazone derivatives were syn-
thesized in order to obtain more potent and less toxic anti-TB com-
pounds. The structures of all the synthesized compounds were
determined by analytical and spectral (IR, 1H NMR, 13C NMR-APT,
HETCOR-2D, HSQC-2D and LCMS-APCI) methods. LogP (o/w)
* Corresponding author. Fax:+90 212 440 02 52.
E-mail address: karalin@istanbul.edu.tr (N. Karalı).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.08.050

Ö. Güzel et al. / Bioorg. Med. Chem. 16 (2008) 8976–8987
8977
CH3
NH NH2
S
NH NH2
N
CH3
N
H3C
S
O
O
NH
I
II
NH NH2
S
N
N
NH2
R
N
III
Ar
O
S
N
NH
N
R
N
NH
V
S
R1
IV
Figure 1. Structures of thiacetazone (I), SRI-224 (II), SRI-286 (III), oxazolyl thiosemicarbazones (IV) and S-alkylisothiosemicarbazones (V).
R3
R2 NH
NH
R3
S
A
N
V
A=
R2
R1
N
N
S
O
N
H
N
VI A=
VII A=
S
HN
R2
Figure. 2. Structures of 3-thiosemicarbazone (VI), 3-phenacylisothiosemicarbazone (VII) and 3-thiazolinehydrazone (VIII) derivatives of 1H-indole-2,3-diones.
values of the compounds were calculated with MOE. These new
compounds, along with previously reported compounds,20,21 were
tested against M. tuberculosis H37Rv to construct the structure–
anti-TB activity relationship.
O
O
R1
R1
i
O
N
O
N
H
CH3
1a,b
2a,b
NH2
HN
NH2
HN
ii
ii
S
S
R2 NH
R2 NH
R1
R1
R2
R2
NH
NH
N
N
NH
NH
N
NH
S
S
H3C
O
O
4a-y
3a-t
iii
OCF3
R2
NH
N
NH
S
N
O
N
O
5a-m
R1= CH3, F3CO
R2= CH3, C2H5, CH2-CH=CH2, n-C4H9, cyclo-C6H11, C6H5CH2, C6H5, 4-CH3C6H4, 4-CH3OC6H4, 4-FC6H4,
4-ClC6H4, 2-BrC6H4, 3-BrC6H4, 4-BrC6H4, 4-NO2C6H4
Scheme 1. Reagents and conditions: (i) NaH, CH3I, DMF, stir, room temperature, 30 min. and reflux, 4 h (ii) EtOH, reflux, 5 h (iii) morpholine, 37% HCHO, absolute EtOH, stir,
rt, 10 h.

8978
Ö. Güzel et al. / Bioorg. Med. Chem. 16 (2008) 8976–8987
Table 1
LogP values and primary in vitro antituberculosis activity screening results of 3–6
R1
OCF3
R2
R2
NH
NH
S
N
N
NH
NH
N
N
O
S
N
R
3
O
O
3, 4 and 6
5
Compound
R1
R2
R3
LogP (o/w)
IC90
(lg/mL)
IC50
(lg/mL)
Activity
3a
3b
3c
3d
3e
3f
3g
3h
3i
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CH3
CH2CH@CH2
C4H9
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1,6950
2,4300
2,8210
2,9740
3,6170
3,6560
2,7560
2,1338
2,4748
2,7958
3,5308
4,1128
3,9218
3,7878
4,0858
3,7438
3,9408
4,3798
4,5858
3,7228
1,2300
1,5710
1,8920
2,6270
3,0180
3,0370
3,6800
3,7190
2,8190
2,3308
2,6718
2,9928
3,7278
4,3098
4,1188
3,9848
4,2828
4,1378
4,5768
4,7828
3,9198
1,8838
2,2248
2,5458
3,2808
3,8628
3,6718
3,5378
3,8358
3,4938
3,6908
4,1298
4,3358
3,4728
1,0330
1,3740
3,0120
2,6870
2,9850
3,2790
3,4850
3,2090
2,8840
3,1820
3,4760
3,6820
>100
18.088
0.418
14.895
1.362
2.898
1.146
>100
1.311
0.767
0.635
5.476
33.267
1.110
14.211
3.472
3.339
1.333
1.259
1.341
3.77
>100
>100
>100
>100
>100
>100
>100
5.495
>100
Weakly active
Active
Weakly active
Active
Weakly active
Active
Inactive
Weakly active
Weakly active
Weakly active
Weakly active
Weakly active
Weakly active
Weakly active
Weakly active
Weakly active
Active
0.911
>100
1.587
>100
1.489
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
1.546
1.521
1.530
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
5.694
>100
>100
>100
>100
3.057
3.041
3.034
>100
>100
>100
0.795
1.433
>100
3.568
>100
>100
>100
>100
>100
>100
C6H5CH2
4-FC6H4
2-BrC6H4
3-BrC6H4
4-NO2C6H4
CH3
C2H5
CH2CH@CH2
C4H9
cycl-C6H11
C6H5CH2
C6H5
4-CH3C6H4
4-CH3OC6H4
4-FC6H4
4-ClC6H4
4-BrC6H4
4-NO2C6H4
CH3
C2H5
CH2CH@CH2
C4H9
C6H5CH2
4-FC6H4
2-BrC6H4
3-BrC6H4
4-NO2C6H4
CH3
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
4y
5a
5b
5c
5d
5e
5f
H
H
H
H
Active
Active
Weakly active
Inactive
Inactive
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
—
—
—
—
—
—
—
—
—
—
—
—
—
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Inactive
Inactive
Weakly active
Weakly active
Inactive
Weakly active
Inactive
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CH3
>100
Inactive
C2H5
CH2CH@CH2
C4H9
5.987
2.731
40.76
19.778
7.299
<0.2
66.434
0.865
1.706
0.612
>100
1.566
2.095
1.003
12.317
1.816
1.001
4.847
1.373
2.404
2.329
2.468
2.550
6.683
>100
6.962
0.705
1.076
1.422
2.526
22.669
>100
Weakly active
Weakly active
Weakly active
Weakly active
Weakly active
Weakly active
Weakly active
Weakly active
Weakly active
Weakly active
Inactive
Weakly active
Weakly active
Weakly active
Weakly active
Active
Weakly active
Weakly active
Weakly active
Weakly active
Active
Active
Active
Weakly active
Inactive
Weakly active
Active
cycl-C6H11
C6H5CH2
C6H5
4-CH3C6H4
4-FC6H4
4-ClC6H4
4-BrC6H4
4-NO2C6H4
CH3
C2H5
CH2CH@CH2
C4H9
cycl-C6H11
C6H5CH2
C6H5
4-CH3C6H4
4-CH3OC6H4
4-FC6H4
4-ClC6H4
4-BrC6H4
4-NO2C6H4
CH3
5g
5h
5i
5j
5k
51
5m
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C2H5
cycl-C6H11
C6H5
Active
Weakly active
Active
Weakly active
Inactive
Weakly active
Inactive
4-CH3C6H4
4-ClC6H4
4-BrC6H4
cycl-C6H11
C6H5
4-CH3C6H4
4-ClC6H4
4-BrC6H4
H
H
H
CH3
CH3
CH3
CH3
CH3
46.512
>100
>100
CH3
CH3
CH3
Inactive
Inactive
>100

Ö. Güzel et al. / Bioorg. Med. Chem. 16 (2008) 8976–8987
8979
determined from the dose-response curve as the IC90 using a curve
fitting program. Any IC90 value of 610 g/mL was considered ‘‘Ac-
2. Results and discussion
2.1. Chemistry
l
tive’’ for antitubercular activity. Compounds active in the initial
screen were tested for cytotoxicity (IC50) in VERO cells. Cytotoxic-
ity was determined from the dose–response curve as the IC50 using
a curve fitting program. Concurrent with the determination of
MICs, compounds were tested for cytotoxicity in VERO cells at con-
centrations 10Â the MIC for M. tuberculosis H37Rv (Table 1). The
selectivity index (SI) was defined as the ratio of the measured
IC50 in VERO cells to the MIC (Table 2). The MIC and SI values for
In this study, 5-methyl/trifluoromethoxy-1H-indole-2,3-dione
(1a,b) and 1-methyl-5-methyl/trifluoromethoxy-1H-indole-2,3-
dione (2a,b) reacted with different N-substituted thiosemicarbaz-
ides in ethanol containing a catalytic amount of sulphuric acid, to
give the corresponding 5-methyl/trifluoromethoxy-1H-indole-
2,3-dione 3-thiosemicarbazones (3a–t) and 1-methyl-5-methyl/
trifluoromethoxy-1H-indole-2,3-dione 3-thiosemicarbazones (4a-
y). 1-Morpholinomethyl-5-trifluoromethoxy-1H-indole-2,3-dione
3-thiosemicarbazones (5a–m) were synthesized from the consecu-
tive treatment of 3h-t with formaldehyde solution and morpholine
(Scheme 1).
The structures of the synthesized compounds were confirmed
by elemental analyses and spectral (IR, 1H NMR, 13C NMR-APT,
HETCOR-2D, HSQC-2D and LCMS-APCI) data. The IR spectra of 3,
4 and 5 showed absorption bands in the 3372–3160, 1703–1672
and 1238–1100 cmÀ1 regions resulting from the NH, C@O and
C@S functions, respectively. 13C NMR-APT of 3a, 3f, 3h, 3o, 4d,
4j, 4s, 5a and 5h, HETCOR spectra of 3k, 4m and 5d, and HSQC
spectra of 3b, 3s, 4b, 4h, 4p and 5l supported the IR findings and
displayed signals at d 140.45–143.28, 161.16–163.46 and 176.65–
178.06 ppm which showed that there was no 13C–1H connection
attributed to the indole C3, indole C2 and C@S functions. The 1H
NMR spectra of 3a-t displayed the NH protons of the thiosemicar-
bazone moiety (d 8.91–11.12 and 12.36–12.98 ppm) and the indole
NH proton (d 11.07–11.53 ppm) as three separate signals. The 1H
NMR and 13C NMR spectra of 4a–y showed two NH signals due
to the thiosemicarbazone moiety (d 8.93–11.17 and 12.27–
12.91 ppm) and a signal assigned to the N-CH3 resonance (d
3.07–3.29 and 26.11–26.62 ppm) confirming the 1-methyl-1H-in-
dole-2,3-dione 3-thiosemicarbazone structure. Observation of only
two NH signals assigned to the thiosemicarbazone moiety (d 8.86–
11.15 and 12.25–12.76 ppm) and of a singlet due to N–CH2–N
function (d 4.41–4.53 and 62.19–66.44 ppm) and of signals attrib-
uted to morpholine in the 1H and 13C NMR spectra of 5a–m pro-
vided support for N-Mannich base formation. LCMS-APCI of 3a,
3b, 3d, 3f, 3i, 3l, 3o, 3r, 3s, 4b, 4d, 4h, 4k, 4n, 4p, 4s, 5b, 5e, 5h,
5k and 5l chosen as prototypes displayed molecular ions with dif-
ferent intensities.
moving compounds into in vivo testing have to be 66.25
and P10 (occasionally lower), respectively.
lg/mL
5-Methyl-1H-indole-2,3-dione 3-thiosemicarbazones (3b, 3d,
3f, 6c, 6d and 6f), 5-trifluoromethoxy-1H-indole-2,3-dione 3-thio-
semicarbazones (3q-s) and 5-trifluoromethoxy-1-morpholinom-
ethyl-1H-indole-2,3-dione 3-thiosemicarbazones (5e and 5j–l)
were found to be the most potent inhibitors of M. tuberculosis
growth described in this study. MIC values (IC90) of these com-
pounds are 0.911, 1.587, 1.489, 0.795, 1.433, 3.568, 1.546, 1.521,
1.530, 5.694, 3.057, 3.041 and 3.034 lg/mL, respectively. However,
as can be seen from Table 2, the whole of the tested compounds
showed poor selectivity for mycobacteria. When these data are
examined, it is observed that the anti-TB activity was increased
due to elongation of the alkyl chain. Generally, replacement of
the alkyl in the R2 with cyclohexyl, phenyl, 4-fluorophenyl, 4-chlo-
rophenyl or 4-bromophenyl has been found to yield more active
compounds. The increase in activity against M. tuberculosis by alkyl
chain elongation is related to lipophilicity as confirmed by the logP
values of compounds (Table 1). In fact, in 5-methyl-1H-indole-2,3-
dione-3-thiosemicarbazones, the lowest MIC value was found for
the R2-cyclohexyl substituted 6c (0.795
tuted 3b (0.911 g/mL), R2-phenyl substituted 6d (1.433
and R2-(3-bromophenyl) substituted 3f (1.489 g/mL). In addition,
lg/mL), R2-butyl substi-
l
l
g/mL)
l
among 5-trifluoromethoxy-1H-indole-2,3-dione-3-thiosemicarba-
zones and its N-Mannich bases, the most active compounds were
3q-s and 5l-j incorporating 4-fluorophenyl, 4-chlorophenyl and
4-bromophenyl at R2. The presence of the morpholine ring in some
R1-trifluoromethoxy substituted N-Mannich bases also seems to
have a significant impact on the resultant anti-TB activity. Com-
pound 5e, a morpholine derivative incorporating a cyclohexyl
group at R2, was found to be one of active inhibitors, whereas li-
gand 3l showed weakly activity. It is interesting to note that, for
the R2-(3-bromophenyl) substituted 3f, 5-methyl-1H-indole-2,3-
dione 3-thiosemicarbazone derivative 3f was substantially more
active than both the R2-(2-bromophenyl) substituted 3e and the
R2-(4-bromophenyl) substituted 6g. In comparison with 5-
methyl/trifluoromethoxy-1H-indole-2,3-dione 3-thiosemicarba-
zones 3a–t and 6a–l, none of the 1-methyl-5-methyl/trifluoro-
methoxy-1H-indole-2,3-dione 3-thiosemicarbazones 4a-t were
LogP (o/w) values of the compounds were calculated with MOE
(Table 1).
2.2. Primary antituberculosis activity
The new 5-methyl/trifluoromethoxy-1H-indole-2,3-dione 3-
thiosemicarbazones 3a-t, 1-methyl-5-methyl/trifluoromethoxy-
1H-indole-2,3-dione 3-thiosemicarbazones 4a–y and 1-morpholi-
nomethyl-5-trifluoromethoxy-1H-indole-2,3-dione 3-thiosemicar-
bazones 5a-m, along with previously reported 5-methyl-1H-
indole-2,3-dione 3-thiosemicarbazones 6a–l, were evaluated
against Mycobacterium tuberculosis H37Rv (ATCC 27294) in BACTEC
12B medium using a broth microdilution assay, the Microplate Ala-
mar Blue Assay (MABA). The primary anti-TB screening was per-
formed in accordance with the protocol of the Tuberculosis
Antimicrobial Acquisition and Coordinating Facility (TAACF)
Southern Research Institute.22 Rifampin was used as the control
drug in the tests. Compounds demonstrating a percent inhibition
of bacterial growth of greater than or equal to 90% in the primary
screen were retested against M. tuberculosis H37Rv, to determine
the actual minimum inhibitory concentration (MIC) in the MABA.
The MIC was defined as the lowest concentration effecting a reduc-
tion in fluorescence of 90%, relative to controls. This value was
Table 2
The IC90, EC50 and SI values of some 3, 5 and 6 against M. tuberculosis H37Rv
Compound
R1
R2
R3
IC90
(lg/mL)
EC50
(lg/mL)
SI
3b
3d
3f
3q
3r
3s
5e
5j
5k
5l
CH3
CH3
CH3
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CF3O
CH3
CH3
CH3
—
C4H9
H
H
H
H
H
H
—
—
—
—
H
H
H
—
0.911
1.587
1.489
1.546
1.521
1.530
5.694
3.057
3.041
3.034
0.795
1.433
3.568
0.125
4.390
<0.195
0.704
2.536
6.391
5.767
23.437
2.396
9.713
7.453
1.572
1.063
12.361
>100
4.81888
<0.1228
0.4728
4-FC6H4
3-BrC6H4
4-FC6H4
4-ClC6H4
4-BrC6H4
cycl-C6H11
4-FC6H4
4-ClC6H4
4-BrC6H4
cycl-C6H11
C6H5
1.64036
4.20184
3.76928
4.11608
0.78377
3.19401
2.45649
1.97735
0.7418
6c
6d
6f
4-ClC6H4
—
3.4644
>800
Rifampin

8980
Ö. Güzel et al. / Bioorg. Med. Chem. 16 (2008) 8976–8987
active against M. tuberculosis. This modification was decreased the
anti-TB activity of new compounds.
formed after cooling was filtered and washed with ethanol or
recrystallized from ethanol.
4.3.1. 5-Methyl-1H-indole-2,3-dione 3-(N-allylthiosemicarba-
zone) (3a)
3. Conclusions
Yellow powder (78%): mp 220–221 °C; IR (KBr):
t 3335, 3289
New series of 5-methyl/trifluoromethoxy-1H-indole-2,3-dione-
3-thiosemicarbazones were synthesized, their structures were
confirmed by spectral data and elemental analysis. The new deriv-
atives, along with previously reported 5-methyl-1H-indole-2,3-
dione-3-thiosemicarbazones, were evaluated for in vitro anti-TB
activity. The anti-TB screening results evidenced that many of
the compounds from the series have emerged as potent anti-TB
agents. The aim of this work was to reveal the correlation between
structure and anti-TB activity in a series of 5-methyl/trifluoro-
methoxy-1H-indole-2,3-dione 3-thiosemicarbazones. The anti-TB
activity was increased due to elongation of the alkyl chain. Gener-
ally, replacement of the alkyl in the R2 with cyclohexyl and
(non)substituted phenyl have been found to yield more active
compounds. The absence of substitution at N1 of indole ring and
the increase of lipophilicity of the compounds were thought to
be responsible for their high activity against M. tuberculosis. In con-
clusion, structural modification may lead to new derivatives with
enhanced activity and high selectivity for mycobacteria.
(NH), 1696 (C@O), 1187 (C@S); 1H NMR (DMSO-d6/500 MHz): d
2.28 (s, 3H, 5-CH3), 4.24 (t, J = 5.61 Hz, 2H, allyl C1-H2), 5.12 (dd,
J = 10.49, 1.71 Hz, 1H, allyl C3-Hcis), 5.18 (dd, J = 17.32, 1.71 Hz, 1H,
allyl C3-Htrans), 5.88–5.93 (m, 1H, allyl C2-H), 6.80 (d, J = 7.81 Hz,
1H, indole C6-H), 7.13 (d, J = 7.81 Hz, 1H, indole C7-H), 7.48 (s, 1H, in-
dole C4-H), 9.39 (t, J = 5.61 Hz, 1H, N4-H), 11.07 (s, 1H, indole NH),
12.56 (s, 1H, N2-H); 13C NMR (APT, DMSO-d6/125 MHz): d 21.07
(5-CH3), 46.82 (allyl C1), 111.21 (indole C6), 116.65 (allyl C3),
120.42 (indole C3a), 121.67 (indole C4), 131.74 (indole C5), 131.99
(indole C7), 132.37 (indole C7a), 134.45 (allyl C2), 140.51 (indole
C3), 163.13 (indole C2), 177.77 (C@S); LCMS-APCI (À) m/z (%): 273
(MHÀ, 100). Anal. Calcd for C13H14N4OS (274.34): C, 56.91; H, 5.14;
N, 20.42. Found: C, 56.63; H, 5.38; N, 20.48.
4.3.2. 5-Methyl-1H-indole-2,3-dione 3-(N-butylthiosemicarba-
zone) (3b)
Yellow powder (72%): mp 179–180 °C; IR (KBr):
t 3252 (NH),
1695 (C@O), 1130 (C@S); 1H NMR (DMSO-d6/400 MHz): d 0.93 (t,
3H, J = 7.37 Hz, butyl C4-H), 1.34 (hex., J = 7.44 Hz, 2H, butyl C3-
H), 1.61 (quint., J = 7.32 Hz, 2H, butyl C2-H), 2.31 (s, 3H, 5-CH3),
3.61 (q, J = 6.87 Hz, 2H, butyl C1-H), 6.82 (d, J = 7.93 Hz, 1H, indole
C6-H), 7.17 (dd, J = 7.95, 0.89 Hz, 1H, indole C7-H), 7.50 (s, 1H, in-
dole C4-H), 9.26 (t, J = 5.82 Hz, 1H, N4-H), 11.10 (s, 1H, indole
NH), 12.53 (s, 1H, N2-H); 13C NMR (HSQC-2D, DMSO-d6/
125 MHz): d 14.22 (butyl C4), 20.06 (butyl C3), 21.06 (5-CH3),
31.03 (butyl C2), 44.33 (butyl C1), 111.21 (indole C6), 120.46 (indole
4. Experimental
Melting points were estimated with a Buchi 540 melting point
apparatus in open capillaries and are uncorrected. Elemental anal-
yses were performed on a Thermo Finnigan Flash EA 1112 elemen-
tal analyzer. IR spectra were recorded on KBr discs, using a Perkin-
Elmer Model 1600 FT-IR spectrometer. 1H NMR, 13C NMR APT,
HETCOR-2D and HSQC-2D spectra were obtained on Bruker Avance
DPX 400 and VarianUNITY INOVA 500 spectrophotometers using
DMSO-d6. Mass spectra were determined on a Mass-AGILENT
1100 MSD instruments. All chemicals and solvents were purchased
from Merck–Schuchardt, Aldrich and Fluka. LogP (o/w) values
were calculated with MOE (version 2009.05, Chemical Computing
Group, Montreal, Canada).
C3a), 121.61 (indole C4), 131.72 (indole C5), 131.93 (indole C7),
132.13 (indole C7a), 140.45 (indole C3), 163.14 (indole C2), 177.34
(C@S); LCMS-APCI (À/+) m/z (%): m/z 289 (MHÀ, 100), 291 (MH+,
58), 282 (100). Anal. Calcd for C14H18N4OS (290.38): C, 57.91; H,
6.25; N, 19.29. Found: C, 57.31; H, 6.26; N, 19.70.
4.3.3. 5-Methyl-1H-indole-2,3-dione 3-(N-benzylthiosemicarba-
zone) (3c)
4.1. General method for the synthesis of N-substituted
Orange crystals (93%): mp 236–237 °C; IR (KBr):
t 3367, 3160
thiosemicarbazides
(NH), 1686 (C@O), 1142 (C@S); 1H NMR (DMSO-d6/500 MHz): d
2.27 (s, 3H, 5-CH3), 4.86 (d, 2H, J = 6.34 Hz, benzyl CH2), 6.80 (d,
J = 8.29 Hz, 1H, indole C6-H), 7.14 (d, J = 7.81 Hz, 1H, indole C7-H),
7.24 (t, J = 7.32 Hz, 1H, benzyl C4-H), 7.31–7.36 (m, 4H, benzyl
To a solution of hydrazine hydrate (5 mmol) in ethanol (10 mL),
a suspension of an appropriate isothiocyanate (5 mmol) in ethanol
(10 mL) was added dropwise with vigorous stirring and cooling in
an ice bath. The mixture was allowed to stand overnight. The crys-
tals formed were recrystallized from ethanol.
C
2,3,5,6-H), 7.47 (s, 1H, indole C4-H), 9.77 (t, J = 6.34 Hz, 1H, N4-H),
11.08 (s, 1H, indole NH), 12.62 (s, 1H, N2-H). Anal. Calcd for
17H16N4OS (324.40): C, 62.94; H, 4.97; N, 17.27. Found: C,
C
62.12; H, 4.72; N, 17.15.
4.2. General method for the synthesis of 1-methyl-5-methyl/
trifluoromethoxy-1H-indole-2,3-dione (2a/2b)
4.3.4. 5-Methyl-1H-indole-2,3-dione 3-[N-(4-fluorophenyl)
thiosemicarbazone] (3d)
A
suspension of 5-methyl/trifluoromethoxy-1H-indole-2,3-
Orange powder (86%): mp 229–230 °C; IR (KBr):
t 3303, 3207
dione 1a/1b (5 mmol) and NaH (50% dispersion in mineral oil)
(0.2 g) in anhydrous DMF (5 mL) was stirred for 30 min at room
temperature. After addition of iodomethane (15 mmol), the mix-
ture was refluxed for 4 h. The product was poured onto ice and
water then filtered.
(NH), 1689 (C@O), 1163 (C@S); 1H NMR (DMSO-d6/400 MHz): d
2.31 (s, 3H, 5-CH3), 6.83 (d, J = 7.94 Hz, 1H, indole C6-H), 7.19 (dd,
J = 7.95, 0.95 Hz, 1H, indole C7-H), 7.27 (t, J = 7.74 Hz, 2H, phenyl
C2,6-H), 7.59–7.62 (m, 3H, indole C4-H and phenyl C3,5-H), 10.81 (s,
1H, N4-H), 11.16 (s, 1H, indole NH), 12.78 (s, 1H, N2-H); LCMS-APCI
(+) m/z (%): 329 (MH+, 100). Anal. Calcd for C16H13FN4OS (328.36): C,
58.52; H, 3.99; N, 17.06. Found: C, 58.32; H, 4.02; N, 17.23.
4.3. General method for the synthesis of 5-methyl/trifluorome-
thoxy-1H-indole-2,3-dione 3-thiosemicarbazones (3a–t)
A solution of N-substituted thiosemicarbazides (3.5 mmol) in
ethanol (10 mL) was added to a solution of 5-methyl/trifluoro-
methoxy-1H-indole-2,3-dione 1a/1b (3.5 mmol) in ethanol
(20 mL). After addition of a drop of concentrated sulfuric acid,
the mixture was refluxed on a water bath for 5 h. The product
4.3.5. 5-Methyl-1H-indole-2,3-dione 3-[N-(2-bromophenyl)
thiosemicarbazone] (3e)
Orange powder (99%): mp 249 °C; IR (KBr):
t 3207 (NH), 1689
(C@O), 1134 (C@S); 1H NMR (DMSO-d6/500 MHz): d 2.48 (s, 3H,
5-CH3), 6.82 (d, J = 7.81 Hz, 1H, indole C6-H), 7.17 (dd, J = 7.81,

Ö. Güzel et al. / Bioorg. Med. Chem. 16 (2008) 8976–8987
8981
0.97 Hz, 1H, indole C7-H), 7.29 (td, J = 7.56, 1.47 Hz, 1H, phenyl C4-
H), 7.45 (td, J = 7.56, 1.46 Hz, 1H, phenyl C5-H), 7.54–7.57 (m, 2H,
phenyl C3-H and indole C4-H), 7.73 (dd, J = 7.81, 0.97 Hz, 1H, phe-
nyl C6-H), 10.77 (s, 1H, N4-H), 11.13 (s, 1H, indole NH), 12.80 (s,
1H, N2-H). Anal. Calcd for C16H13BrN4OS (389.26): C, 49.37; H,
3.37; N, 14.39. Found: C, 49.51; H, 3.38; N, 14.36.
C3-Hcis), 5.19 (dd, J = 17.23, 1.67 Hz, 1H, allyl C3-Htrans), 5.87–5.93
(m, 1H, allyl C2-H), 7.00 (d, J = 8.54 Hz, 1H, indole C7-H), 7.33
(dd, J = 8.54, 1.53 Hz, 1H, indole C6-H), 7.66 (d, J = 1.53 Hz, 1H, in-
dole C4-H), 9.54 (t, J = 5.79 Hz, 1H, N4-H), 11.53 (s, 1H, indole
NH), 12.47 (s, 1 H, N2-H). Anal. Calcd for C13H11F3N4O2S (344.31):
C, 45.35; H, 3.22; N, 16.27. Found: C, 45.98; H, 2.95; N, 16.33.
4.3.6. 5-Methyl-1H-indole-2,3-dione 3-[N-(3-bromophenyl)
thiosemicarbazone] (3f)
4.3.11. 5-Trifluoromethoxy-1 H-indole-2,3-dione 3-(N-n-butyl-
thiosemicarbazone) (3k)
Yellow powder (72%): mp 236–238 °C; IR (KBr):
t 3310 (NH),
Yellow powder (87%): mp 234 °C; IR (KBr):
t 3257 (NH), 1695
1687 (C@O), 1145 (C@S); 1H NMR (DMSO-d6/400 MHz): d 2.31 (s,
3H, 5-CH3), 6.84 (d, J = 7.91 Hz, 1H, indole C6-H), 7.19 (d,
J = 8.08 Hz, 1H, indole C7-H), 7.39 (t, J = 8.00 Hz, 1H, phenyl C5-H),
7.47 (d, J = 8.27 Hz, 1H, phenyl C6-H), 7.61 (s, 1H, indole C4-H),
7.71 (d, J = 8.00 Hz, 1H, phenyl C4-H), 7.91 (s, 1H, phenyl C2-H),
10.86 (s, 1H, N4-H), 11.18 (s, 1H, indole NH), 12.84 (s, 1H, N2-H);
13C NMR (APT-DMSO-d6/125 MHz): d 21.10 (5-CH3), 111.34 (indole
C6), 120.28 (indole C3a), 121.21 (phenyl C3), 122.25 (indole C4),
124.82 (phenyl C6), 128.24 (phenyl C4), 129.08 (phenyl C2),
130.65 (phenyl C5), 131.87 (indole C5), 132.44 (indole C7), 133.24
(indole C7a), 140.52 (phenyl C1), 140.85 (indole C3), 163.22 (indole
C2), 176.65 (C@S); LCMS-APCI (+) m/z (%): 385, 387 (100, 17), 383,
385 (83, 100). Anal. Calcd for C16H13BrN4OS (389.26): C, 49.37; H,
3.37; N, 14.39. Found: C, 49.38; H, 3.04; N, 14.41.
(C@O), 1158 (C@S); 1H NMR (DMSO-d6/400 MHz): d 0.92 (t,
J = 7.30 Hz, 3H, butyl C4-H), 1.31–1.37 (m, 2H, butyl C3-H), 1.62
(p, J = 7.40 Hz, 2H, butyl C2-H), 3.62 (q, J = 6.80 Hz, 2H, butyl C1-
H), 7.00 (d, J = 8.50, Hz, 1H, indole C7-H), 7.34 (dd, J = 8.60,
2.40 Hz, 1 H, indole C6-H), 7.66 (d, J = 1.70 Hz, 1H, indole C4-H),
9.37 (t, J = 5.90 Hz, 1H, N4-H), 11.35 (s, 1H, indole NH), 12.44 (s,
1H, N2-H); 13C NMR (HETCOR-2D, DMSO-d6/125 MHz): d 14.19
(butyl C4), 20.05 (butyl C3), 30.98 (butyl C2), 44.38 (butyl C1),
112.55 (indole C7), 114.43 (indole C4), 120.68 (q, J = 258.10,
CF3O), 121.97 (indole C3a), 124.34 (indole C6), 131.00 (indole C7a),
141.56 (indole C3), 143.97 (indole C5), 163.16 (indole C2), 177.33
(C@S). Anal. Calcd for C14H15F3N4O2S (360.35): C, 46.66; H, 4.20;
N, 15.55. Found: C, 46.94; H, 3.82; N, 15.67.
4.3.12. 5-Trifluoromethoxy-1H-indole-2,3-dione 3-(N-cyclohe-
xylthiosemicarbazone) (3l)
4.3.7. 5-Methyl-1H-indole-2,3-dione 3-[N-(4-nitrophenyl)
thiosemicarbazone] (3g)
Yellow powder (75%): mp 249 °C; IR (KBr):
t 3262 (NH), 1696
Red powder (73%): mp 262–263 °C; IR (KBr):
t 3193 (NH), 1694
(C@O), 1162 (C@S); 1H NMR (DMSO-d6/400 MHz): d 1.13–1.94
(m, 10H, cycl. C2,3,4,5,6-H), 4.18–4.24 (m, 1H, cycl. C1-H), 7.01 (d,
J = 8.50 Hz, 1H, indole C7-H), 7.35, 7.37 (2xdd, J = 8.50, 2.40 Hz,
1H, indole C6-H), 7.76 (d, J = 2.40 Hz, 1H, indole C4-H), 8.91 (d,
J = 8.50 Hz, 1H, N4-H), 11.37 (s, 1H, indole NH), 12.49 (s, 1H, N2-
H); LCMS-APCI (À/+): m/z (%) 387 (MH+, 100), 385 (MHÀ, 6), 265
(100). Anal. Calcd for C16H17F3N4O2S (386.39): C, 49.73; H, 4.43;
N, 14.50. Found: C, 49.37; H, 4.30; N, 14.46.
(C@O), 1177 (C@S); 1H NMR (DMSO-d6/500 MHz): d 2.30 (s, 3H, 5-
CH3), 6.83 (d, J = 8.30 Hz, 1H, indole C6-H), 7.19 (d, J = 7.32 Hz, 1H,
indole C7-H), 7.60 (s, 1H, phenyl C4-H), 8.07 (d, J = 8.79 Hz, 2H, phe-
nyl C2,6-H), 8.28 (dd, J = 6.84, 1.95 Hz, 2H, phenyl C3,5-H), 11.06 (s,
1H, N4-H), 11.17 (s, 1H, indole NH), 12.98 (s, 1H, N2-H). Anal. Calcd
for C16H13N5O3S (355.37): C, 54.08; H, 3.69; N, 19.71. Found: C,
54.30; H, 3.73; N, 20.15.
4.3.8. 5-Trifluoromethoxy-1H-indole-2,3-dione 3-(N-methyl-
thiosemicarbazone) (3h)
4.3.13. 5-Trifluoromethoxy-1H-indole-2,3-dione-3-(N-benzyl-
thiosemicarbazone) (3m)
Yellow powder (80%): mp 234 °C; IR (KBr):
t 3295 (NH), 1699
(C@O), 1142 (C@S); 1H NMR (DMSO-d6/400 MHz): d 3.10 (d,
J = 4.6 Hz, 3H, CH3), 7.02 (d, J = 8.50 Hz, 1H, indole C7-H), 7.36
(dd, J = 8.50, 1.80 Hz, 1H, indole C6-H), 7.63 (d, J = 1.40 Hz, 1H, in-
dole C4-H), 9.39 (q, J = 4.50 Hz, 1H, N4-H), 11.37 (s, 1H, indole
NH), 12.46 (s, 1H, N2-H); 13C NMR (APT, DMSO-d6,/125 MHz): d
31.73 (CH3), 112.52 (indole C7), 114.18 (indole C4), 118.11 (q,
J = 255.72, CF3O), 121.93 (indole C3a), 124.26 (indole C6), 130.88
(indole C7a), 141.50 (indole C3), 143.97 (indole C5), 163.12 (indole
C2), 178.06 (C@S). Anal. Calcd for C11H9F3N4O2S (318.27): C,
41.51; H, 2.85; N, 17.60. Found: C, 42.28; H, 2.53; N, 17.97.
Orange crystals (90%): mp 215–216 °C; IR (KBr):
t 3221 (NH),
1702 (C@O), 1238 (C@S); 1H NMR (DMSO-d6/500 MHz): d 4.88
(d, J = 4.10 Hz, 2H, benzyl CH2), 7.00 (d, J = 8.54 Hz, 1H, indole C7-
H), 7.25 (t, J = 6.40 Hz, 1H, benzyl C4-H), 7.33–7.36 (m, 5H, indole
C6-H, benzyl C2,3,4,5,6-H), 7.64 (d, J = 1.83 Hz, 1H, indole C4-H),
9.90 (t, J = 6.25 Hz, 1H, N4-H), 11.35 (s, 1H, indole NH), 12.53 (s,
1H, N2-H). Anal. Calcd for C17H13F3N4O2S.1/2H2O (403.38): C,
50.61; H, 3.49; N, 13.89. Found: C, 50.72; H, 3.36; N, 13.61.
4.3.14. 5-Trifluoromethoxy-1H-indole-2,3-dione-3-(N-phenyl-
thiosemicarbazone) (3n)
4.3.9. 5-Trifluoromethoxy-1H-indole-2,3-dione 3-(N-ethylthio-
semicarbazone) (3i)
Orange powder (99%): mp 229–232 °C; IR (KBr):
t 3316, 3282
(NH), 1698 (C@O), 1171 (C@S); 1H NMR (DMSO-d6/500 MHz): d
7.02 (d, J = 8.54 Hz, 1H, indole C7-H), 7.28 (t, J = 7.32 Hz, 1H, phenyl
C4-H), 7.35 (dd, J = 8.54, 2.44 Hz, 1H, indole C6-H), 7.43 (t,
J = 7.32 Hz, 2H, phenyl C3,5-H), 7.58 (d, J = 7.32 Hz, 2H, phenyl
Yellow crystals (67%): mp 235–236 °C; IR (KBr):
t 3324, 3182
(NH), 1696 (C@O), 1155 (C@S); 1H NMR (DMSO-d6/400 MHz): d
1.21 (t, J = 7.10 Hz, 3H, ethyl CH3), 3.65 (p, J = 7.0 Hz, 2H, ethyl
CH2), 7.02 (d, J = 8.50, Hz, 1H, indole C7-H), 7.36 (dd, J = 8.50,
2.30 Hz, 1H, indole C6-H), 7.66 (d, J = 1.70 Hz, 1H, indole C4-H),
9.41 (t, J = 5.60 Hz, 1H, N4-H), 11.36 (s, 1H, indole NH), 12.43 (s,
1H, N2-H); LCMS-APCI (À/+): m/z (%) 333 (MH+, 100), 331 (MHÀ,
87), 230 (100). Anal. Calcd for C12H11F3N4O2S (332.30): C, 43.37;
H, 3.34; N, 16.86. Found: C, 43.34; H, 2.81; N, 16.82.
C
2,6-H), 7.79 (d, J = 1.83 Hz, 1H, indole C4-H), 10.87 (s, 1H, N4-H),
11.38 (s, 1H, indole NH), 12.64 (s, 1H, N2-H). Anal. Calcd for
16H11F3N4O2S (380.34): C, 50.53; H, 2.92; N, 14.73. Found: C,
C
50.38; H, 2.83; N, 14.76.
4.3.15. 5-Trifluoromethoxy-1H-indole-2,3-dione 3-[N-(4-methyl-
phenyl)thiosemicarbazone] (3o)
4.3.10. 5-Trifluoromethoxy-1H-indole-2,3-dione 3-(N-allylthio-
semicarbazone) (3j)
Orange crystals (88%): mp 230–233 °C; IR (KBr):
t 3307 (NH),
1697 (C@O), 1153 (C@S); 1H NMR (DMSO-d6/400 MHz): d 2.34 (s,
3H, CH3), 7.03 (d, J = 8.50 Hz, 1H, indole C7-H), 7.24 (d,
J = 8.10 Hz, 2H, phenyl C3,5-H), 7.37 (dd, J = 8.50, 1.70 Hz, 1H, indole
C6-H), 7.46 (d, J = 8.30 Hz, 2H, phenyl C2,6-H), 7.67 (d, J = 1.40 Hz,
Orange crystals (74%): mp 224–226 °C; IR (KBr):
t 3228 (NH),
1698 (C@O), 1172 (C@S); 1H NMR (DMSO-d6/500 MHz): d 4.26 (t,
J = 5.49 Hz, 2H, allyl C1-Y), 5.14 (dd, J = 10.22, 1.37 Hz, 1H, allyl

8982
Ö. Güzel et al. / Bioorg. Med. Chem. 16 (2008) 8976–8987
1H, indole C4-H), 10.66 (s, 1H, N4-H), 11.20 (s, 1H, indole NH),
12.36 (s, 1H, N2-H); 13C NMR (APT DMSO-d6/125 MHz): d 21.32
(CH3), 112.86 (indole C7), 115.17 (indole C4), 120.91 (q,
J = 255.45 Hz, CF3O), 122.16 (indole C3a), 124.82 (indole C6),
126.38 (phenyl C3, C5), 129.61 (phenyl C2, C6),131.75 (indole C7a),
136.30 (phenyl C4), 136.44 (phenyl C1), 142.10 (indole C3),
144.29 (indole C5), 163.46 (indole C2), 177.10 (C@S); LCMS-APCI
(À/+): m/z (%) 395 (MH+, 100), 393 (MHÀ, 100). Anal. Calcd for
dole C6-H), 7.77 (br s, 1H, indole C4-H), 8.06 (d, J = 9.15 Hz, 2H, phe-
nyl C2, 6-H), 8.30 (d, J = 9.15 Hz, 2H, phenyl C3, 5-H), 11.12 (s, 1H,
N4-H), 11.44 (s, 1H, indole NH), 12.85 (s, 1H, N2-H). Anal. Calcd
for C16H10F3N5O4S (425.34): C, 45.18; H, 2.37; N, 16.47. Found: C,
45.08; H, 2.26; N, 16.39.
4.4. General method for the synthesis of 1-methyl-5-methyl/
trifluoromethoxy-1H-indole-2,3-dione 3-thiosemicarbazones
(4a–y)
C17H13F3N4O2S (394.37): C, 51.77; H, 3.32; N, 14.21. Found: C,
51.71; H, 3.06; N, 14.39.
A solution of N-substituted thiosemicarbazides (3.5 mmol) in
ethanol (10 mL) was added to a solution of 1-methyl-5-methyl/tri-
fluoromethoxy-1H-indole-2,3-dione 2a/2b (3.5 mmol) in ethanol
(20 mL). After addition of a drop of concentrated sulfuric acid,
the mixture was refluxed on a water bath for 5 h. The product
formed after cooling was filtered and washed with ethanol or
recrystallized from ethanol.
4.3.16. 5-Trifluoromethoxy-1H-indole-2,3-dione 3-[N-(4-
methoxyphenyl)thiosemicarbazone] (3p)
Orange powder (89%): mp 228–229 °C; IR (KBr):
t 3266 (NH),
1701 (C@O), 1154 (C@S); 1H NMR (DMSO-d6/500 MHz): d 3.77 (s,
3H, OCH3), 6.98 (d, J = 9.16 Hz, 2H, phenyl C3,5-H), 7.01 (d,
J = 8.24 Hz, 1H, indole C7-H),), 7.35 (dd, J = 8.54, 2.30 Hz, 1H, indole
C6-H), 7.44 (d, J = 9.15 Hz, 2H, phenyl C2, 6-H), 7.78 (br s, 1H, indole
C4-H), 10.78 (s, 1H, N4-H), 11.37 (s, 1H, indole NH), 12.59 (s, 1H,
N2-H). Anal. Calcd for C17H13F3N4O3S (410.37): C, 49.76; H, 3.19;
N, 13.65. Found: C, 49.40; H, 3.31; N, 13.88.
4.4.1. 1,5-Dimethyl-1H-indole-2,3-dione 3-(N-
methylthiosemicarbazone) (4a)
Yellow powder (88%): mp 259–60 °C; IR(KBr):
t 3238 (NH),
1683 (C@O), 1100 (C@S). 1H NMR (DMSO-d6/500 MHz): d 2.31 (s,
3H, 5-CH3), 3.07 (d, J = 4.39 Hz, 3H, N-CH3), 3.17 (s, 3H, N-CH3),
7.00 (d, J = 8.29 Hz, 1H, indole C6-H), 7.23 (d, J = 7.80 Hz, 1H, indole
C7-H), 7.48 (s, 1H, indole C4-H), 9.22 (s, 1H, N4-H), 12.47 (s, 1H, N2-
H). Anal. Calcd for C12H14N4OS (262.33): C, 54.94; H, 5.38; N, 21.36.
Found: C, 54.73; H, 5.07; N, 21.28.
4.3.17. 5-Trifluoromethoxy-1H-indole-2,3-dione 3-[N-(4-
fluorophenyl)thiosemicarbazone] (3q)
Orange powder (84%): mp 224–227 °C; IR (KBr):
t 3291 (NH),
1690 (C@O), 1154 (C@S); 1H NMR (DMSO-d6/500 MHz): d 7.01
(d, J = 8.54 Hz, 1H, indole C7-H), 7.26 (t, J = 8.84 Hz, 2H, phenyl
C3,5-H), 7.35 (dd, J = 8.54, 2.44 Hz, 1H, indole C6-H), 7.58 (dd,
J = 8.84, 4.88 Hz, 2H, phenyl C2,6-H), 7.76 (br s, 1H, indole C4-H),
10.87 (s, 1H, N4-H), 11.39 (s, 1H, indole NH), 12.65 (s, 1H, N2-H).
Anal. Calcd for C16H10F4N4O2S (398.33): C, 48.24; H, 2.53; N,
14.07. Found: C, 48.86; H, 2.45; N, 14.21.
4.4.2. 1,5-Dimethyl-1H-indole-2,3-dione 3-(N-
ethylthiosemicarbazone) (4b)
Yellow powder (80%): mp 250–251 °C; IR(KBr):
t 3301 (NH),
1686 (C@O), 1100 (C@S); 1H NMR (DMSO-d6/400 MHz): d 1.20 (t,
J = 7.14 Hz, 3H, ethyl C2-H), 2.33 (s, 3H, 5-CH3), 3.20 (s, 3H, N-
CH3), 3.65 (quin., J = 6.61 Hz, 2H, ethyl C1-H), 7.02 (d, J = 8.00 Hz,
1H, indole C6-H), 7.25 (dd, J = 7.98, 0.88 Hz, 1H, indole C7-H),
7.52 (s, 1H, indole C4-H), 9.31 (t, J = 5.80 Hz, 1H, N4-H), 12.45 (s,
1H, N2-H); 13C NMR (HSQC-2D, DMSO-d6/125 MHz): d 14.51 (ethyl
C2), 21.08 (5-CH3), 26.13 (N-CH3), 39.36–40.61 (DMSO, ethyl C1),
110.00 (indole C6), 119.69 (indole C3a), 121.33 (indole C4), 131.32
(indole C5), 131.77 (indole C7), 132.39 (indole C7a), 141.78 (indole
C3), 161.19 (indole C2), 177.09 (C@S); LCMS-APCI (+) m/z (%): 277
(MH+, 100). Anal. Calcd for C13H16N4OS (276.35): C, 56.50; H,
5.84; N, 20.27. Found: C, 56.46; H, 5.41; N, 20.48.
4.3.18. 5-Trifluoromethoxy-1H-indole-2,3-dione 3-[N-(4-
chlorophenyl)thiosemicarbazone] (3r)
Orange crystals (77%): mp 233–234 °C; IR (KBr):
t 3300 (NH),
1698 (C@O), 1157 (C@S); 1H NMR (DMSO-d6/400 MHz): d 6.94
(d, J = 8.50 Hz, 1H, indole C7-H), 7.28 (dd, J = 8.50, 1.70 Hz, 1H, in-
dole C6-H), 7.41 (d, J = 8.70 Hz, 2H, phenyl C3,5-H), 7.56 (d,
J = 8.70 Hz, 2H, phenyl C2,6-H), 7.69 (d, J = 1.50 Hz, 1H, indole C4-
H), 10.83 (s, 1H, N4-H), 11.33 (s, 1H, indole NH), 12.61 (s, 1H, N2-
H); LCMS-APCI (À/+): m/z (%) 415, 417 [(MH+, MH++2) 100, 39],
413, 415 [(MHÀ, MHÀ+2) 100, 51], 230 (79). Anal. Calcd for
C15H10ClF3N4O2S (414.78): C, 46.33; H, 2.43; N, 13.51. Found: C,
46.49; H, 2.07; N, 13.76.
4.4.3. 1,5-Dimethyl-1H-indole-2,3-dione 3-(N-
4.3.19. 5-Trifluoromethoxy-1H-indole-2,3-dione 3-[N-(4-
allylthiosemicarbazone) (4c)
bromophenyl)thiosemicarbazone] (3s)
Orange powder (67%): mp 208–209 °C; IR(KBr):
t 3266 (NH),
1687 (C@O), 1186 (C@S). 1H NMR (DMSO-d6/500 MHz): d 2.31 (s,
3H, 5-CH3), 3.17 (s, 3H, N-CH3), 4.24 (td, J = 5.80, 1.47 Hz, 2H, allyl
C1-Y), 5.13 (dd, J = 7.32, 1.46 Hz, 1H, allyl C3-Hcis), 5.19 (dd,
J = 15.61, 1.46 Hz, 1H, allyl C3-Htrans), 5.89–5.94 (m, 1H, allyl C2-
H), 6.99 (d, J = 7.57 Hz, 1H, indole C6-H), 7.22 (d, J = 8.29 Hz, 1H, in-
dole C7-H), 7.50 (s, 1H, indole C4-H), 9.42 (t, J = 5.80 Hz, 1H, N4-H),
12.49 (s, 1H, N2-H). Anal. Calcd for C14H16N4OS (288.36): C, 58.31;
H, 5.59; N, 19.43. Found: C, 58.37; H, 5.42; N, 19.60.
Orange crystals (95%): mp 238–239 °C; IR (KBr):
t 3302 (NH),
1698 (C@O), 1161 (C@S); 1H NMR (DMSO-d6/500 MHz): d 7.02
(d, J = 8.29 Hz, 1H, indole C7-H), 7.35 (dd, J = 8.29, 2.44 Hz, 1H, in-
dole C6-H), 7.57–7.62 (m, 4H, phenyl C2,3,5,6-H), 7.77 (d,
J = 1.95 Hz, 1H, indole C4-H), 10.88 (s, 1H, N4-H), 11.39 (s, 1H, in-
dole NH), 12.69 (s, 1H, N2-H); 13C NMR (HSQC-2D, DMSO-d6/
125 MHz): d 112.94 (indole C7), 117.19 (indole C4), 119.31 (phenyl
C4), 120.91 (q, J = 255.93 Hz, CF3O), 122.03 (indole C3a), 124.99 (in-
dole C6), 128.42 (phenyl C3, C5), 132.02 (phenyl C2, C6), 132.21 (in-
dole C7a), 138.41 (phenyl C1), 142.12 (indole C3), 144.31 (indole C5),
163.46 (indole C2), 177.10 (C@S); LCMS-APCI (À): m/z (%) 457, 459
[(MHÀ, MHÀ+2) 97, 100]. Anal. Calcd for C16H10BrF3N4O2S (459.24):
C, 41.85; H, 2.19; N, 12.20. Found: C, 42.38; H, 2.06; N, 12.73.
4.4.4. 1,5-Dimethyl-1H-indole-2,3-dione 3-(N-
butylthiosemicarbazone) (4d)
Orange crystals (69%): mp 201–203 °C; IR(KBr):
t 3281 (NH),
1685 (C@O), 1173 (C@S); 1H NMR (DMSO-d6/400 MHz): d 0.93 (t,
J = 7.36 Hz, 3H, butyl C4-H), 1.34 (hex., J = 7.45 Hz, 2H, butyl C3-
H), 1.61 (pent, J = 7.45 Hz, 2H, butyl C2-H), 2.34 (s, 3H, 5-CH3),
3.20 (s, 3H, N-CH3), 3.61 (q, J = 6.86 Hz, 2 H, butyl C1-H), 7.04 (d,
J = 8.00 Hz, 1H, indole C6-H), 7.26 (dd, J = 8.04, 0.81 Hz, 1H, indole
C7-H), 7.54 (s, 1H, indole C4-H), 9.29 (t, J = 5.84 Hz, 1 H, N4-H),
12.47 (s, 1H, N2-H); 13C NMR (APT, DMSO-d6/125 MHz): d 14.24
4.3.20. 5-Trifluoromethoxy-1H-indole-2,3-dione-3-[N-(4-
nitrophenyl)thiosemicarbazone] (3t)
Orange powder (60%): mp 246–247 °C; IR (KBr):
t 3283 (NH),
1698 (C@O), 1151 (C@S); 1H NMR (DMSO-d6/500 MHz): d 7.03
(d, J = 8.54 Hz, 1H, indole C7-H), 7.38 (dd, J = 8.54, 1.83 Hz, 1H, in-

Ö. Güzel et al. / Bioorg. Med. Chem. 16 (2008) 8976–8987
8983
(butyl C4), 20.06 (butyl C3), 21.07 (5-CH3), 26.11 (N-CH3), 31.02
(butyl C2), 44.37 (butyl C1), 109.97 (indole C6), 119.69 (indole
H), 8.07 (d, J = 8.78 Hz, 2H, phenyl C2,6-H), 8.28 (d, J = 8.78 Hz,
2H, phenyl C3,5-H), 11.17 (s, 1H, N4-H), 12.91 (s, 1H, N2-H). Anal.
Calcd for C17H15N5O3S (369.39): C, 55.27; H, 4.09; N, 18.96. Found:
C, 54.66; H, 3.69; N, 18.81.
C3a), 121.34 (indole C4), 131.29 (indole C5), 131.75 (indole C7),
132.37 (indole C7a), 141.76 (indole C3), 161.18 (indole C2), 177.32
(C@S); LCMS-APCI (+) m/z (%): 305 (MH+, 56), 282 (96), 79 (99),
65 (100). Anal. Calcd for C15H20N4OS (304.41): C, 59.18; H, 6.62;
N, 18.41. Found: C, 59.44; H, 6.79; N, 18.70.
4.4.10. 1-Methyl-5-trifluoromethoxy-1H-indole-2,3-dione 3-(N-
methylthiosemicarbazone) (4j)
Yellow powder (82%): mp 218–20 °C; IR(KBr):
t 3355, 3279
4.4.5. 1,5-Dimethyl-1H-indole-2,3-dione 3-(N-benzylthiosemi-
carbazone) (4e)
(NH), 1689 (C@O), 1124 (C@S); 1H NMR (DMSO-d6/400 MHz): d
3.09 (d, J = 4.60 Hz, 3H, NHCH3), 3.23 (s, 3H, N-CH3), 7.25 (d,
J = 8.50 Hz, 1H, indole C7-H), 7.46 (dd, J = 8.50, 1.70 Hz, 1H, indole
C6-H), 7.66 (d, J = 1.30 Hz, 1H, indole C4-H), 9.41 (q, J = 4.50 Hz,
1H, N4-H), 12.38 (s, 1H, N2-H); 13C NMR APT (DMSO-d6,/
125 MHz): d 26.35 (N-CH3), 31.81 (NHCH3), 111.52 (indole C7),
113.92 (indole C4), 120.67 (q, J = 255.85, CF3O), 121.28 (indole
Orange powder (79%): mp 191–192 °C; IR(KBr):
t 3287 (NH),
1687 (C@O), 1172 (C@S); 1H NMR (DMSO/500 MHz): 2.29 (s, 3H,
5-CH3), 3.16 (s, 3H, N-CH3), 4.87 (d, J = 5.85 Hz, 2H, benzyl CH2),
6.96 (d, J = 7.81 Hz, 1H, indole C6-H), 7.20 (d, J = 8.30 Hz, 1H, indole
C7-H), 7.25 (t, J = 7.32 Hz, 1H, benzyl C4-H), 7.32–7.37 (m, 4H, ben-
zyl C2,3,5,6-H), 7.47 (s, 1H, indole C4-H), 9.78 (t, J = 5.86 Hz, 1H, N4-
H), 12.53 (s, 1H, N2-H). Anal. Calcd for C18H18N4OS (338.42): C,
63.88; H, 5.36; N, 16.56. Found: C, 63.23; H, 5.48; N, 16.48.
C3a), 124.24 (indole C6), 130.08 (indole C7a), 142.78 (indole C3),
144.41 (indole C5), 161.30 (indole C2), 178.05 (C@S). Anal. Calcd
for C12H11F3N4O2S (332.30): C, 43.37; H, 3.34; N, 16.86. Found: C,
44.09; H, 3.00; N, 17.08.
4.4.6. 1,5-Dimethyl-1H-indole-2,3-dione 3-[N-(4-fluorophenyl)
thiosemicarbazone] (4f)
4.4.11. 1-Methyl-5-trifluoromethoxy-1H-indole-2,3-dione 3-(N-
Orange powder (81%): mp 236 °C; IR(KBr):
t
3208 (NH), 1675
ethylthiosemicarbazone) (4k)
(C@O), 1161 (C@S); 1H NMR (DMSO/400 MHz): 2.34 (s, 3H, 5-
CH3), 3.21 (s, 3H, N-CH3), 7.05 (d, J = 8.00 Hz, 1H, indole C6-H),
7.25–7.29 (m, 3H, indole C7-H and phenyl C2,6-H), 7.59–7.63 (m,
3H, indole C4-H and phenyl C3,5-H), 10.84 (s, 1H, N4-H), 12.71 (s,
1H, N2-H). Anal. Calcd for C17H15FN4OS (342.39): C, 59.63; H,
4.42; N, 16.36. Found: C, 59.66; N, 4.36; N, 16.54.
Yellow powder (81%): mp 217–219 °C; IR(KBr): t 3372, 3320
(NH), 1685 (C@O), 1176 (C@S); 1H NMR (DMSO-d6/400 MHz): d
1.11 (t, J = 7.1 Hz, 3H, ethyl CH3), 3.14 (s, 3H, N-CH3), 3.56 (p,
J = 6.20 Hz, 2H, ethyl CH2), 7.16 (d, J = 8.50 Hz, 1H, indole C7-H),
7.37 (dd, J = 8.50, 1.70 Hz, 1H, indole C6-H), 7.60 (d, J = 1.40 Hz,
1H, indole C4-H), 9.34 (t, J = 5.70 Hz, 1H, N4-H), 12.27 (s, 1H, N2-
H); LCMS-APCI (- / +) m/z (%): 347 (MH+, 100), 345 (MHÀ, 100).
Anal. Calcd for C13H13F3N4O2S (346.32): C, 45.08; H, 3.78; N,
16.18. Found: C, 45.85; H, 3.72; N, 16.47.
4.4.7. 1,5-Dimethyl-1H-indole-2,3-dione 3-[N-(2-bromophenyl)
thiosemicarbazone] (4g)
Orange powder (85%): mp 231–232 °C; IR(KBr):
t 3234 (NH),
1682 (C@O), 1162 (C@S). 1H NMR (DMSO-d6/500 MHz): d 2.32 (s,
3H, 5-CH3), 3.20 (s, 3H, N-CH3), 7.04 (d, J = 7.81 Hz, 1H, indole C6-
H), 7.26 (d, J = 7.32 Hz, 1H, indole C7-H), 7.30 (td, J = 7.81,
1.46 Hz, 1H, phenyl C4-H), 7.45 (t, J = 7.81 Hz, 1H, phenyl C5-H),
7.54 (d, J = 7.81 Hz, 1H, phenyl C3-H), 7.58 (s, 1H, indole C4-H),
7.74 (d, J = 8.29 Hz, 1H, phenyl C6-H), 10.80 (s, 1H, N4-H), 12.73
(s, 1H, N2-H). Anal. Calcd for C17H15BrN4OS.1/2H2O (412.29): C,
49.52; H, 3.91; N, 13.58. Found: C, 49.62; N, 3.92; N, 14.17.
4.4.12. 1-Methyl-5-trifluoromethoxy-1H-indole-2,3-dione 3-(N-
allylthiosemicarbazone) (4l)
Yellow crystals (79%): mp 181–182 °C; IR(KBr):
t 3344, 3210
(NH), 1682 (C@O), 1155 (C@S); 1H NMR (DMSO-d6/500 MHz): d
3.29 (s, 3H, N-CH3), 4.26 (t, J = 5.86 Hz, 2H, allyl C1-Y), 5.14 (dd,
J = 10.25, 1.46 Hz, 1H, allyl C3-Hcis), 5.20 (dd, J = 17.09, 1.46 Hz,
1H, allyl C3-Htrans), 5.88–5.96 (m, 1H, allyl C2-H), 7.23 (d,
J = 8.78 Hz, 1H, indole C7-H), 7.44 (dd, J = 7.52, 1.95 Hz, 1H, indole
C6-H), 7.70 (s, 1H, indole C4-H), 9.56 (t, J = 5.85 Hz, 1H, N4-H),
12.41 (s, 1H, N2-H). Anal. Calcd for C14H13F3N4O2S (358.33): C,
46.92; H, 3.66; N, 15.64. Found: C, 46.71; H, 3.77; N, 15.61.
4.4.8. 1,5-Dimethyl-1H-indole-2,3-dione 3-[N-(3-bromophenyl)
thiosemicarbazone] (4h)
Orange powder (79%): mp 226–229 °C; IR(KBr):
t 3304, 3217
(NH), 1682 (C@O), 1173 (C@S); 1H NMR (DMSO-d6/400 MHz): d
2.34 (s, 3H, 5-CH3), 3.21 (s, 3H, N-CH3), 7.06 (d, J = 8.01 Hz, 1H, in-
dole C6-H), 7.29 (dd, J = 7.56, 0.84 Hz, 1H, indole C7-H), 7.40 (t,
J = 8.00 Hz, 1H, phenyl C5-H), 7.48 (d, J = 7.68 Hz, 1H, phenyl C6-
H), 7.64 (s, 1H, indole C4-H), 7.71 (d, J = 7.51 Hz, 1H, phenyl C4-
H), 7.91 (t, J = 1.90 Hz, 1H, phenyl C2-H), 10.89 (s, 1H, N4-H),
12.77 (s, 1H, N2-H); 13C NMR (HSQC-2D, DMSO-d6/125 MHz): d
21.09 (5-CH3), 26.22 (N-CH3), 110.13 (indole C6), 119.53 (indole
4.4.13. 1-Methyl-5-trifluoromethoxy-1H-indole-2,3-dione 3-(N-
butylthiosemicarbazone) (4m)
Yellow powder (76%): mp 179 °C; IR(KBr):
t 3364, 3336 (NH),
1685 (C@O), 1168 (C@S); 1H NMR (DMSO-d6/400 MHz): d 0.93 (t,
J = 7.30 Hz, 3H, butyl CH3), 1.32–1.38 (m, 2H, butyl C3-H2), 1.63
(p, J = 7.40 Hz, 2H, butyl C2-H2), 3.23 (s, 1H, N-CH3), 3.61 (q,
J = 6.90 Hz, 2H, butyl C1-H2), 7.18 (d, J = 8.50 Hz, 1H, indole C7-H),
7.39 (dd, J = 8.50, 1.60 Hz, 1H, indole C6-H), 7.65 (d, J = 1.60 Hz,
1H, indole C4-H), 9.34 (t, J = 5.90 Hz, 1H, N4-H), 12.31 (s, 1H, N2-
H); 13C NMR (HETCOR-2D, DMSO-d6/125 MHz): d 14.16 (CH3),
20.07 (butyl C3), 26.23 (N-CH3), 30.96 (butyl C2), 44.43 (butyl C1),
111.27 (indole C7), 114.09 (indole C4), 120.66 (q, J = 255.70,
CF3O), 121.19 (indole C3a), 124.05 (indole C6), 129.88 (indole C7a),
142.59 (indole C3), 144.41 (indole C5), 161.16 (indole C2), 177.29
(C@S). Anal. Calcd for C15H17F3N4O2S (374.38): C, 48.12; H, 4.58;
N, 14.97. Found: C, 48.37; H, 4.50; N, 15.03.
C3a), 121.22 (phenyl C3), 121.98 (indole C4), 124.89 (phenyl C4),
128.31 (phenyl C2), 129.16 (phenyl C6), 130.70 (phenyl C5),
132.25 (indole C7), 132.42 (indole C5), 132.53 (indole C7a), 140.48
(phenyl C1), 142.15 (indole C3), 161.29 (indole C2), 176.69 (C@S);
LCMS-APCI (+) m/z (%): 403, 405 [(MH+, MH++2) 36, 33], 401
(100). Anal. Calcd for C17H15 BrN4OS (403.29): C, 50.63; H, 3.75;
N, 13.89. Found: C, 51.12; H, 3.57; N, 13.95.
4.4.9. 1,5-Dimethyl-1H-indole-2,3-dione 3-[N-(4-nitrophenyl)
thiosemicarbazone] (4i)
4.4.14. 1-Methyl-5-trifluoromethoxy-1H-indole-2,3-dione 3-(N-
Orange powder (66%): mp 255–256 °C; IR(KBr):
t
3285 (NH),
cyclohexylthiosemicarbazone) (4n)
1672 (C@O), 1168 (C@S). 1H NMR (DMSO-d6/500 MHz): d 2.33 (s,
3H, 5-CH3), 3.20 (s, 3H, N-CH3), 7.04 (d, J = 7.81 Hz, 1H indole C6-
H), 7.28 (d, J = 7.80 Hz, 1H, indole C7-H), 7.63 (s, 1H, phenyl C4-
Yellow powder (78%): mp 228–229 °C; IR(KBr): t 3364, 3319
(NH), 1684 (C@O), 1167 (C@S); 1H NMR (DMSO-d6/400 MHz): d
1.29–1.94 (m, 10H, cyclohexyl C2,3,4,5,6-H), 3.23 (s, 3H, N-CH3),

8984
Ö. Güzel et al. / Bioorg. Med. Chem. 16 (2008) 8976–8987
4.09–4.32 (m, 1H, cyclohexyl C1-H), 7.25 (d, J = 8.60 Hz, 1H, indole
C7-H), 7.45, 7.48 (2xdd, J = 8.50, 2.30 Hz, 1H, indole C6-H), 7.79 (d,
J = 2.10 Hz, 1H, indole C4-H), 8.93 (d, J = 8.50 Hz, 1H, N4-H), 12.43
(s, 1H, N2-H); LCMS-APCI (+) m/z (%): 401 (MH+, 100). Anal. Calcd
for C17H19F3N4O2S (400.41): C, 50.99; H, 4.78; N, 13.99. Found: C,
51.41; H, 4.85; N, 14.05.
C
C
2,6), 129.96 (phenyl C3,5), 131.06 (phenyl C4), 131.24 (indole
7a), 137.94 (phenyl C1), 143.28 (indole C3), 144.74 (indole C5),
161.59 (indole C2), 177.15 (C@S); LCMS-APCI (À/+) m/z (%): 429
(MH+, 26), 79 (100), 427 (MHÀ, 100). Anal. Calcd for
C17H12ClF3N4O2S.½ H2O (437.82): C, 46.63; H, 2.99; N, 12.79.
Found: C, 46.68; H, 2.76; N, 13.16.
4.4.15. 1-Methyl-5-trifluoromethoxy-1H-indole-2,3-dione 3-(N-
4.4.20. 1-Methyl-5-trifluoromethoxy-1H-indole-2,3-dione 3-[N-
benzylthiosemicarbazone) (4o)
(4-bromophenyl) thiosemicarbazone] (4t)
Yellow powder (79%): mp 200–201 °C; IR(KBr):
t
3349, 3211
Yellow powder (77%): mp 223 °C; IR(KBr): t 3317, 3230 (NH),
(NH), 1685 (C@O), 1167 (C@S); 1H NMR (DMSO-d6/500 MHz): d
3.22 (s, 3H, N-CH3), 4.89 (d, J = 6.35 Hz, 2H, benzyl CH2), 7.23 (d,
J = 8.30 Hz, 1H, indol C7-H), 7.25–7.27 (m, 1H, benzyl C4-H),
7.32–7.34 (m, 4H, benzyl C2,3,5,6-H), 7.44 (dd, J = 8.78, 1.95 Hz,
1H, indole C6-H), 7.67 (d, J = 1.95 Hz, 1H, indole C4-H), 9.92 (t,
J = 6.34 Hz, 1H, N4-H), 12.47 (s, 1H, N2-H). Anal. Calcd for
C18H15F3N4O2S (408.39): C, 52.94; H, 3.70; N, 13.72. Found: C,
52.82; H, 3.66; N, 13.65.
1685 (C@O), 1150 (C@S); 1H NMR (DMSO-d6/500 MHz): d 3.23 (s,
3H, N-CH3), 7.23 (d, J = 8.79 Hz, 1H, indole C7-H), 7.45 (d,
J = 7.81 Hz, 1H, indole C6-H), 7.60 (q, J = 8.78 Hz, 4H, phenyl
C3,4,5,6-H), 7.79 (s, 1H, indole C4-H), 10.89 (s, 1H, N4-H), 12.61 (s,
1H, N2-H). Anal. Calcd for C17H12BrF3N4O2S (473.26): C, 43.14; H,
2.56; N, 11.84. Found: C, 42.69; H, 2.36; N, 11.78.
4.4.21. 1-Methyl-5-trifluoromethoxy-1H-indole-2,3-dione 3-[N-
(4-nitrophenyl) thiosemicarbazone] (4y)
4.4.16. 1-Methyl-5-trifluoromethoxy-1H-indole-2,3-dione 3-(N-
phenylthiosemicarbazone) (4p)
Yellow powder (94%): mp 249 °C; IR(KBr): t 3335, 3290 (NH),
1679 (C@O), 1163 (C@S); 1H NMR (DMSO-d6/500 MHz): d 3.24 (s,
3H, N-CH3), 7.26 (d, J = 8.30 Hz, 1H, indole C7-H), 7.48 (dd,
J = 8.54, 1.46 Hz, 1H, indole C6-H), 7.81 (d, J = 1.47 Hz, 1H, indole
C4-H), 8.06 (dd, J = 6.83, 1.95 Hz, 2H, phenyl C2,6-H), 8.29 (dd,
J = 7.32, 1.95 Hz, 2H, phenyl C3,5-H), 11.14 (s, 1H, N4-H), 12.78 (s,
1H, N2-H). Anal. Calcd for C17H12F3N5O4S (439.36): C, 46.47; H,
2.75; N, 15.94. Found: C, 46.17; H, 2.70; N, 16.75.
Orange powder (75%): mp 203–205 °C; IR(KBr):
t 3322, 3226
(NH), 1686 (C@O), 1157 (C@S); 1H NMR (DMSO-d6/500 MHz): d
3.23 (s, 3H, N-CH3), 7.23 (d, 1H, indol C7-H), 7.29 (t, J = 7.32 Hz,
1H, phenyl C4-H), 7.43 (t, 3H, phenyl C3,5-H, indole C6-H), 7.58 (d,
J = 7.80 Hz, 1H, phenyl C2,6-H), 7.81 (s, 1H, indole C4-H), 10.88 (s,
1H, N4-H), 12.56 (s, 1H, N2-H); 13C NMR (HSQC-2D, DMSO-d6/
125MHz): d 26.61 (N-CH3), 111.71 (indole C7), 114.00 (indole C4),
119.89 (OCF3), 121.42 (indole C3a), 124.67 (indole C6), 126.51 (phe-
nyl C2,6), 127.04 (phenyl C4), 129.19 (phenyl C3,5), 130.96 (indole
4.5. General method for the synthesis of 5-trifluoromethoxy-1-
morpholinomethyl-1H-indole-2,3-dione 3-thiosemicarbazones
(5a–m)
C7a), 138.97 (phenyl C1), 143.20 (indole C3), 144.75 (indole C5),
161.59 (indole C2), 177.11 (C@S); LCMS-APCI (-) m/z (%): 393
(MHÀ, 100). Anal. Calcd for C17H13F3N4O2S (394.37): C, 51.77; H,
3.32; N, 14.21. Found: C, 51.84; H, 3.08; N, 14.32.
To a suspension of 3h–t (2 mmol) in absolute ethanol (20 mL),
37% formaldehyde solution (0.5 mL) and morpholine (2 mmol)
were added dropwise with vigorous stirring. After combining all
reagents, the reaction mixture was stirred at room temperature
for 10 h. The solid product was filtered and washed with petroleum
ether.
4.4.17. 1-Methyl-5-trifluoromethoxy-1H-indole-2,3-dione 3-[N-
(4-methylphenyl)thiosemicarbazone] (4q)
Orange crystals (74%): mp 216–217 °C; IR(KBr):
t 3322, 3232
(NH), 1686 (C@O), 1149 (C@S); 1H NMR (DMSO-d6/400 MHz): d
2.34 (s, 3H, CH3), 3.26 (s, 3H, N-CH3), 7.24–7.48 (m, 6H, indole
C7-H, C6-H, C6H4), 7.85 (d, J = 1.20 Hz, 1H, indol C4-H), 10.87 (s,
1H, N4-H), 12.57 (s, 1H, N2-H). Anal. Calcd for C18H15F3N4O2S
(408.39): C, 52.94; H, 3.70; N, 13.72. Found: C, 53.00; H, 3.67; N,
13.86.
4.5.1. 5-Trifluoromethoxy-1-(morpholin-4-ylmethyl)-1H-
indole-2,3-dione 3-(N-methylthiosemicarbazone) (5a)
Yellow powder (82%): mp 201–202 °C; IR (KBr):
t 3305, 3239
(NH), 1694 (C@O), 1155 (C@S); 1H NMR (DMSO/d6, 400 MHz): d
2.56 (br t, J = 4.00 Hz, 4H, morph. C3,5-H), 3.10 (d, J = 4.57 Hz, 3H,
CH3), 3.54 (br t, J = 4.30 Hz, 4H, morph. C2,6-H), 4.51 (s, 2H, N-CH2-
N), 7.38 (d, J = 8.60 Hz, 1H, indole C7-H), 7.44 (dd, J = 8.60, 1.80 Hz,
1H, indole C6-H), 7.69 (d, J = 1.20 Hz, 1H, indole C4-H), 9.43 (q,
J = 4.50 Hz, 1H, N4-H), 12.37 (s, 1H, N2-H); 13C NMR (APT, DMSO/
d6, 125 MHz): d 31.78 (NHCH3), 50.93 (morph. C3, 5), 61.89 (morph.
C2,6), 66.44 (N-CH2-N), 112.77 (indole C7), 113.83 (indole C4), 121.37
(indole C3a), 121.93 (q, J = 256.10, CF3O), 124.04 (indole C6), 129.77
(indole C7a), 142.56 (indole C3), 144.50 (indole C5), 162.06 (indole
C2), 178.00 (C@S). Anal. Calcd for C16H18F3N5O3S (417.40): C,
46.04; H, 4.35; N, 16.78. Found: C, 45.81; H, 4.41; N, 16.74.
4.4.18. 1-Methyl-5-trifluoromethoxy-1H-indole-2,3-dione 3-[N-
(4-fluorophenyl) thiosemicarbazone] (4r)
Yellow powder (77%): mp 214–215 °C; IR(KBr):
t 3314, 3250
(NH), 1687 (C@O), 1151 (C@S).); 1H NMR (DMSO-d6/500 MHz): d
3.24 (s, 3H, N-CH3), 7.25 (d, J = 8.79 Hz, 1H, indole C7-H), 7.26 (t,
2H, J = 8.78 Hz, phenyl C3,5-H), 7.46 (d, J = 8.05 Hz, 1H, indole C6-
H), 7.58 (dd, 2H, J = 8.78, 1.95 Hz, phenyl C2,6-H), 7.80 (s, 1H, indole
C4-H), 10.89 (s, 1H, N4-H), 12.58 (s, 1H, N2-H). Anal. Calcd for
C17H12F4N4O2S (412.36): C, 49.52; H, 2.93; N, 13.59. Found: C,
49.36; H, 2.85; N, 13.61.
4.5.2. 5-Trifluoromethoxy-1-(morpholin-4-ylmethyl)-1H-
indole-2,3-dione 3-(N-ethylthiosemicarbazone) (5b)
4.4.19. 1-Methyl-5-trifluoromethoxy-1H-indole-2,3-dione 3-[N-
(4-chlorophenyl) thiosemicarbazone] (4s)
Yellow powder (78%): mp 205–207 °C; IR (KBr): t 3260 (NH),
Yellow powder (89%): mp 221–222 °C; IR(KBr):
t
3353, 3200
1695 (C@O), 1165 (C@S); 1H NMR (DMSO/d6, 400 MHz): d 1.11 (t,
J = 7.10 Hz, 3H, ethyl CH3), 2.47 (br t, J = 4.30 Hz, 4H, morph. C3,5-
H), 3.45 (br t, J = 4.40 Hz, 4H, morph. C2,6-H), 3.56 (p, J = 6.20 Hz,
2H, ethyl CH2), 4.41 (s, 2H, N–CH2–N), 7.28 (d, J = 8.60 Hz, 1H, in-
dole C7-H), 7.35 (dd, J = 8.60, 1.80 Hz, 1H, indole C6-H), 7.62 (d,
J = 1.40 Hz, 1H, indole C4-H), 9.35 (t, J = 5.80 Hz, 1H, N4-H), 12.25
(s, 1H, N2-H); LCMS-APCI (-/+): m/z (%) 432 (MH+, 14), 333 (100),
265 (99), 430 (MHÀ, 8), 265 (100). Anal. Calcd for C17H20F3N5O3S
(NH), 1678 (C@O), 1168 (C@S); 1H NMR (DMSO-d6/400 MHz): d
3.26 (s, 3H, N-CH3), 7.28 (d, J = 8.60 Hz, 1H, indol C7-H), 7.49–
7.53 (m, 3H, indol C6-H, phenyl C3,5-H), 7.66 (d, J = 8.70 Hz, 2H,
phenyl C2,6-H), 7.83 (d, J = 1.50 Hz, 1H, indol C4-H), 10.95 (s, 1H,
N4-H), 12.64 (s, 1H, N2-H); 13C NMR (APT, DMSO-d6/125 MHz): d
26.62 (N-CH3), 111.77 (indole C7), 114.89 (indole C4), 121.33
(OCF3), 121.92 (indole C3a), 124.80 (indole C6), 128.11 (phenyl

Ö. Güzel et al. / Bioorg. Med. Chem. 16 (2008) 8976–8987
8985
(431.43): C, 47.33; H, 4.67; N, 16.23. Found: C, 47.48; H, 4.35; N,
16.27.
C2,6-H), 4.51 (s, 2H, N–CH2–N), 7.29 (t, J = 7.32 Hz, 1H, phenyl C4-
H), 7.37 (d, J = 8.55 Hz, 1H, indole C7-H), 7.43 (d, J = 8.23 Hz, 1H, in-
dole C6-H), 7.44 (d, J = 7.63 Hz, 2H, phenyl C3,5-H), 7.58 (d,
J = 7.62 Hz, 2H, phenyl C2, 6-H), 7.85 (br s, 1H, indole C4-H), 10.91
(s, 1H, N4-H), 12.55 (s, 1H, N2-H). Anal. Calcd for C20H20F3N5O3SÁ½-
H2O (488.48): C, 51.63; H, 4.33; N, 14.33. Found: C, 51.73; H, 4.25;
N, 14.54.
4.5.3. 5-Trifluoromethoxy-1-(morpholin-4-ylmethyl)-1H-
indole-2,3-dione 3-(N-allylthiosemicarbazone) (5c)
Dark yellow powder (84%): mp 153 °C; IR (KBr):
t 3250 (NH),
1694 (C@O), 1162 (C@S); 1H NMR (DMSO-d6/500 MHz): d 2.55 (t,
J = 4.27 Hz, 4H, morph. C3,5-H), 3.53 (t, J = 4.42 Hz, 4H, morph. C2,
6-H), 4.26 (t, J = 5.64 Hz, 2H, allyl C1-Y), 4.49 (s, 2H, N-CH2-N),
5.15 (dd, J = 10.07, 1.52 Hz, 1H, allyl C3-Hcis), 5.20 (dd, J = 17.38,
1.52 Hz, 1H, allyl C3-Htrans), 5.87–5.95 (m, 1H, allyl C2-H), 7.36 (d,
J = 8.54 Hz, 1H, indole C7-H), 7.42 (dd, J = 8.54, 1.83 Hz, 1H, indole
C6-H), 7.72 (d, J = 1.52 Hz, 1H, indole C4-H), 9.59 (t, J = 5.95 Hz,
1H, N4-H), 12.38 (s, 1H, N2-H). Anal. Calcd for C18H20F3N5O3SÁ½H2O
(452.45): C, 47.78; H, 4.67; N, 15.47. Found: C, 48.20; H, 5.05; N,
15.49.
4.5.8. 5-Trifluoromethoxy-1-(morpholin-1-ylmethyl)-1H-
indole-2,3-dione 3-[N-(4-methylphenyl) thiosemicarbazone)
(5h)
Dark yellow crystals (71%): mp 169oC; IR (KBr):
t 3303, 3231
(NH), 1697 (C@O), 1156 (C@S); 1H NMR (DMSO-d6/400 MHz): d
2.34 (s, 3H, CH3), 2.59 (br t, J = 4.20 Hz, 4H, morph. C3,5-H),
3.56 (br t, J = 4.30 Hz, 4H, morph. C2,6-H), 4.53 (s, 2H, N–CH2–
N), 7.25 (d, J = 8.30 Hz, 2H, phenyl C3,5-H), 7.40 (d, J = 8.60 Hz,
1H, indole C7-H), 7.46 (d, J = 8.30 Hz, 1 H, indole C6-H, phenyl
4.5.4. 5-Trifluoromethoxy-1-(morpholin-4-ylmethyl)-1H-
indole-2,3-dione 3-(N-butylthiosemicarbazone) (5d)
C
2,6-H), 7.87 (br s, 1H, indole C4-H), 10.88 (s, 1H, N4-H), 12.54
(s, 1H, N2-H); 13C NMR (APT DMSO-d6/125 MHz):
d
21.32
Yellow powder (78%): mp 129–132 °C; IR (KBr):
t
3302 (NH),
(CH3), 51.20 (morph. C3, C5), 62.19 (N–CH2–N), 66.69 (morph.
C2, C6), 113.10 (indole C7), 114.77 (indole C4), 120.91 (q,
J = 255.93 Hz, CF3O), 121.59 (indole C3a), 124.53 (indole C6),
126.38 (phenyl C3, C5), 129.63 (phenyl C2, C6),130.69 (indole
1692 (C@O), 1162 (C@S); 1H NMR (DMSO-d6/400 MHz): d 0.93 (t,
J = 7.30 Hz, 3H, butyl C4-H), 1.32–1.39 (m, 2H, butyl C3-H), 1.63
(p, J = 7.40 Hz, 2H, butyl C2-H), 2.56 (t, J = 3.80 Hz, 4H, morph.
C3,5-H), 3.54 (t, J = 4.20 Hz, 4H, morph. C2,6-H), 3.63 (q,
C7a), 136.35 (phenyl C4), 136.41 (phenyl C1), 143.08 (indole C3),
J = 6.80 Hz, 2H, butyl C1-H), 4.50 (s, 2H, N–CH2–N), 7.37 (d,
J = 8.60, Hz, 1H, indole C7-H), 7.42 (dd, J = 8.60, 1.80 Hz, 1H, indole
C6-H), 7.72 (d, J = 1.60 Hz, 1H, indole C4-H), 9.42 (t, J = 5.90 Hz, 1H,
N4-H), 12.34 (s, 1H, N2-H); 13C NMR (HETCOR-2D, DMSO/d6,
100 MHz): 14.19 (butyl C4), 20.05 (butyl C3), 30.97 (butyl C2),
44.43 (butyl C1), 50.93 (morph. C3,5), 61.86 (morph. C2,6), 66.44
(N–CH2–N), 112.79 (indole C7), 114.06 (indole C4), 120.67 (q,
J = 258.10, CF3O), 121.39 (indole C3a), 124.08 (indole C6), 129.90
(indole C7a), 142.63 (indole C3), 144.48 (indole C5), 162.10 (indole
C2), 177.28 (C@S). Anal. Calcd for C19H24F3N5O3S (459.48): C,
49.66; H, 5.26; N, 15.24. Found: C, 49.44; H, 5.08; N, 15.01.
144.79 (indole C5), 162.43 (indole C2), 177.06 (C@S); LCMS-APCI
(+): m/z (%) 494 (MH+, 2), 439 (100). Anal. Calcd for
C22H22F3N5O3S (493.50): C, 53.54; H, 4.49; N, 14.19. Found: C,
53.88; H, 4.36; N, 14.19.
4.5.9. 5-Trifluoromethoxy-1-(morpholin-1-ylmethyl)-1H-
indole-2,3-dione 3-[N-(4-methoxyphenyl) thiosemicarbazone)
(5i)
Orange powder (90%): mp 171–173 oC; IR (KBr):
t 3316, 3210
(NH), 1689 (C@O), 1154 (C@S); 1H NMR (DMSO-d6/500 MHz): d
2.57 (t, J = 4.39 Hz, 4H, morph. C3,5-H), 3.54 (t, J = 4.39 Hz, 4H,
morph. C2,6-H), 3.77 (s, 3H, OCH3), 4.51 (s, 2H, N–CH2–N), 6.98
(d, J = 8.79 Hz, 2H, phenyl C3,5-H), 7.37 (d, J = 8.30 Hz, 1H, indole
C7-H), 7.42–7.45 (m, 3H, indole C6-H, phenyl C2,6-H), 7.84 (br s,
1H, indole C4-H), 10.83 (s, 1H, N4-H), 12.51 (s, 1H, N2-H). Anal.
Calcd for C22H22F3N5O4S (509.50): C, 51.86; H, 4.35; N, 13.75.
Found: C, 51.92; H, 4.04; N, 13.78.
4.5.5. 5-Trifluoromethoxy-1-(morpholin-4-ylmethyl)-1H-
indole-2,3-dione 3-(N-cyclohexylthiosemicarbazone) (5e)
Orange crystals (93%): mp 178–180 °C; IR (KBr):
t 3281 (NH),
1696 (C@O), 1160 (C@S); 1H NMR (DMSO-d6/400 MHz): d 1.03–
1.86 (m, 10H, cycl. C2,3,4,5,6-H), 2.46 (br t, J = 4.30 Hz, morph. C3,5-
H), 3.45 (br t, J = 4.40 Hz, 4H, morph. C2,6-H), 4.10–4.13 (m, 1H, cycl.
C1-H), 4.41 (s, 2H, N–CH2–N), 7.28 (d, J = 8.60 Hz, 1H, indole C7-H),
7.36 (dd, J = 8.70, 1.5 Hz, 1H, indole C6-H), 7.72 (d, J = 2.10 Hz, 1H,
indole C4-H), 8.86 (d, J = 8.40 Hz, 1 H, N4-H), 12.29 (s, 1H, N2-H);
LCMS-APCI (À/+): m/z (%) 486 (MH+, 1), 387 (100), 484 (MHÀ, 5),
4.5.10. 5-Trifluoromethoxy-1-(morpholin-4-ylmethyl)-1H-
indole-2,3-dione 3-[N-(4-fluorophenyl) thiosemicarbazone) (5j)
Orange powder (76%): mp 162–163 °C; IR (KBr):
t 3289, 3215
(NH), 1703 (C@O), 1110 (C@S); 1H NMR (DMSO-d6/500 MHz): d
2.57 (t, J = 4.27 Hz, 4H, morph. C3,5-H), 3.54 (t, J = 4.27 Hz, 4H,
morph. C2,6-H), 4.51 (s, 2H, N–CH2–N), 7.27 (t, J = 8.84 Hz, 2H, phe-
nyl C3,5-H), 7.38 (d, J = 8.55 Hz, 1H, indole C7-H), 7.44 (dd, J = 8.54,
2.13 Hz, 1H, indole C6-H), 7.58 (dd, J = 8.85, 4.88 Hz, 2H, phenyl
420 (100). Anal. Calcd for
51.00; H, 5.50; N, 14.16. Found: C, 50.65; H, 5.57; N, 14.00.
C
21H26F3N5O3SÁ½H2O (494.53): C,
4.5.6. 5-Trifluoromethoxy-1-(morpholin-4-ylmethyl)-1H-
indole-2,3-dione 3-(N-benzylthiosemicarbazone) (5f)
C2,6-H), 7.82 (br s, indole C4-H), 10.91 (s, 1H, N4-H), 12.56 (s, 1H,
Yellow powder (91%): mp 168–169 °C; IR (KBr):
t
3270 (NH),
N2-H). Anal. Calcd for C21H19F4N5O3S (497.46): C, 50.70; H, 3.85;
N, 14.08. Found: C, 50.06; H, 3.53; N, 13.82.
1694 (C@O), 1153 (C@S); 1H NMR (DMSO-d6/500 MHz): d 2.55 (t,
J = 4.27 Hz, 4H, morph. C3,5-H), 3.53 (t, J = 4.27 Hz, 4H, morph.
C
2,6-H), 4.49 (s, 2H, N–CH2–N), 4.89 (d, J = 6.10 Hz, 2H, benzyl
4.5.11. 5-Trifluoromethoxy-1-(morpholin-4-ylmethyl)-1H-
indole-2,3-dione 3-[N-(4-chlorophenyl) thiosemicarbazone)
(5k)
CH2), 7.25 (t, J = 6.41 Hz, 1H, benzyl C4-H), 7.32–7.36 (m, 5H, indole
C7-H, benzyl C2,3,5,6-H), 7.42 (dd, J = 8.84, 1.83 Hz, 1H, indole C6-H),
7.69 (br s, 1H, indole C4-H), 9.95 (t, J = 6.25 Hz, 1H, N4-H), 12.44 (s,
1H, N2-H). Anal. Calcd for C22H22F3N5O3S (493.50): C, 53.54; H,
4.49; N, 14.19. Found: C, 53.79; H, 4.30; N, 14.31.
Yellow powder (75%): mp 180–182 °C; IR (KBr):
t 3305, 3214
(NH), 1698 (C@O), 1164 (C@S); 1H NMR (DMSO-d6/400 MHz): d
2.59 (br t, J = 4.20 Hz, 4H, morph. C3,5-H), 3.56 (br t, J = 4.30 Hz,
4H, morph. C2,6-H), 4.53 (s, 2H, N–CH2–N), 7.40 (d, J = 8.60 Hz,
1H, indole C7-H), 7.48 (dd, J = 8.80, 1.50 Hz, 1H, indole C6-H),
7.51 (d, J = 8.70 Hz, 2H, phenyl C3,5-H), 7.66 (d, J = 8.70 Hz, 2H,
phenyl C2,6-H), 7.85 (d, J = 1.50 Hz, indole C4-H), 10.97 (s, 1H,
N4-H), 12.61 (s, 1H, N2-H); LCMS-APCI (À): m/z (%) 512 (MHÀ,
4.5.7. 5-Trifluoromethoxy-1-(morpholin-4-ylmethyl)-1H-
indole-2,3-dione 3-(N-phenylthiosemicarbazone) (5g)
Yellow powder (68%): mp 182–183 °C; IR (KBr):
t 3227 (NH),
1697 (C@O), 1159 (C@S); 1H NMR (DMSO-d6/500 MHz): d 2.57 (t,
J = 4.27 Hz, 4H, morph. C3,5-H), 3.54 (t, J = 4.27 Hz, 4H, morph.
2), 413, 415 (100, 37). Anal. Calcd for
C21H19ClF3N5O3S

8986
Ö. Güzel et al. / Bioorg. Med. Chem. 16 (2008) 8976–8987
(513.92): C, 49.08; H, 3.73; N, 13.63. Found: C, 49.12; H, 3.43; N,
13.67.
deionized water, and subsequent twofold dilutions were per-
formed in 0.1 mL of 7H9GC (no Tween 80) in the microplates. BAC-
TEC 12B-passaged inocula were initially diluted 1:2 in 7H9GC, and
0.1 mL was added to wells. The determination of bacterial titer
yielded 1 Â 106 CFU/mL in plate well for H37Rv. Frozen inocula
were initially diluted 1:20 in BACTEC 12B medium followed by a
1:50 dilution in 7H9GC. Addition of 1/10 mL to wells resulted in fi-
nal bacterial titer of 20 Â 105 CFU/mL for H37Rv. Wells containing
drug only were used to detect autofluorescence of compounds.
Additional control wells consisted of bacteria only (B) and medium
only (M). Plates were incubated at 37 oC. Starting at day 4 of incu-
4.5.12. 5-Trifluoromethoxy-1-(morpholin-4-ylmethyl)-1H-
indole-2,3-dione 3-[N-(4-bromophenyl) thiosemicarbazone)
(5l)
Yellow powder (91%): mp 180oC; IR (KBr):
t 3307, 3217 (NH),
1699 (C@O), 1158 (C@S); 1H NMR (DMSO-d6/500 MHz): d 2.57 (t,
J = 4.39 Hz, 4H, morph. C3,5-H), 3.54 (t, J = 4.39 Hz, 4H, morph.
C2,6-H), 4.51 (s, 2H, N–CH2–N), 7.37 (d, J = 8.78 Hz, 1H, indole C7-
H), 7.44 (dd, J = 8.78, 1.47 Hz, 1H, indole C6-H), 7.58 (d,
J = 8.78 Hz, 2H, phenyl C3,5-H), 7.62 (d, J = 8.78 Hz, 2H, phenyl
bation, 20
lL of 10 Â alamarBlue solution (Alamar Biosciences/Acc-
C2,6-H), 7.83 (d, J = 1.46 Hz, indole C4-H), 10.92 (s, 1H, N4-H),
umed, Westlake, Ohio) and 12.5 lL of 20% Tween 80 were added to
12.60 (s, 1H, N2-H); 13C NMR (HSQC-2D, DMSO-d6/125 MHz): d
51.19 (morph. C3, C5), 62.22 (N–CH2–N), 66.69 (morph. C2, C6),
113.18 (indole C7), 114.80 (indole C4), 119.37 (phenyl C4), 120.90
(q, J = 255.93 Hz, CF3O), 121.48 (indole C3a), 124.72 (indole C6),
128.42 (phenyl C3, C5), 131.16 (indole C7a), 132.05 (phenyl C2,
C6), 138.38 (phenyl C1), 143.20 (indole C3), 144.79 (indole C5),
162.44 (indole C2), 177.07 (C@S). LCMS-APCI (À): m/z (%) 556,
558 [(MHÀ,MHÀ+2) 85, 100]. Anal. Calcd for C21H19BrF3N5O3S
(558.37): C, 45.17; H, 3.43; N, 12.54. Found: C, 45.10; H, 3.30; N,
12.98.
one B well and one M well, and plates were reincubated at 37 oC.
Wells were observed 12 and 24 h later for a color change from blue
to pink and for a reading of P50,000 fluorescence units (FU). Fluo-
rescence was measured in a Cytofluor II microplate fluorometer
(PerSeptive Biosystems, Framingham, Mass.) in bottom-reading
mode with excitation at 530 nm and emission at 590 nm. If the B
wells become pink after 24 h, the reagent is being added to the en-
tire plate. If the well-remain blue or P50,000 FU is measured, addi-
tional M and B wells are tested daily until a color change occurred,
at which time reagents are added to all remaining wells. Plates
were then incubated at 37 °C, and results were recorded at 24 h
post-reagent addition. Visual MICs were defined as the lowest con-
centration of drug that prevented a color change. For fluorometric
MICs, a background subtraction was performed on all wells with a
mean of triplicate M wells. Percent inhibition was defined as
(1 À (test well FU/mean FU of triplicate B wells) Â 100). The lowest
drug concentration effecting an inhibition of P90% was considered
the MIC.
4.5.13. 5-Trifluoromethoxy-1-(morpholin-4-ylmethyl)-1H-
indole-2,3-dione 3-[N-(4-nitrophenyl) thiosemicarbazone) (5m)
Dark yellow powder (73%): mp 169–171 °C; IR (KBr):
t 3238
(NH), 1701 (C@O), 1154 (C@S); 1H NMR (DMSO-d6/500 MHz): d
2.58 (t, J = 4.27 Hz, 4H, morph. C3,5-H), 3.54 (t, J = 4.57 Hz, 4H,
morph. C2,6-H), 4.52 (s, 2H, N–CH2–N), 7.39 (d, J = 8.85 Hz, 1H, in-
dole C7-H), 7.47 (dd, J = 8.84, 1.52 Hz, 1H, indole C6-H), 7.84 (d,
J = 1.53 Hz, 1H, indole C4-H), 8.21 (d, J = 9.15 Hz, 2H, phenyl C2,6
-
H), 8.30 (d, J = 9.15 Hz, 2H, phenyl C3,5-H), 11.15 (s, 1H, N4-H),
12.76 (s, 1H, N2-H). Anal. Calcd for C21H19F3N6O5S (524.47): C,
48.09; H, 3.65; N, 16.02. Found: C, 48.08; H, 3.32; N, 15.74.
4.6.2. BACTEC radiometric assay
A total of 1/10 ml of BACTEC 12B-passaged inoculum was deliv-
ered without prior dilution into 4 mL of test medium. The determi-
nation of bacterial titer yielded average titer of 1 Â 105 CFU/ml of
BACTEC 12B medium for H37Rv. Frozen inocula were initially di-
luted 1:20 in BACTEC 12B medium, and then 0.1 mL was delivered
to the test medium. This yielded 5.0 Â 105 CFU per BACTEC vial for
4.6. In vitro evaluation of antituberculosis activity
The primary screen was conducted against Mycobacterium
tuberculosis H37Rv (ATCC 27294) in BACTEC 12B medium using a
broth microdilution assay, the Microplate Alamar Blue Assay
(MABA).22 Compounds were tested in 10 twofold dilutions, usually
H
37Rv. Twofold drug dilutions were prepared in either dimethyl
sulfoxide (DMSO) or distilled deionized water and delivered via a
0.5-mL insulin syringe in a 50- L volume. Drug-free control vials
l
from 100 lg/mL to 0.19 lg/mL. Compounds demonstrating a per-
consisted of solvent with bacterial inoculum and solvent with a
1:100 dilution of bacterial inoculum (1:100 controls). Vials were
incubated at 37 oC, and the GI was determined in a BACTEC 460
instrument (Becton–Dickinson) until the GI of the 1:100 controls
reached at least 30. All vials were read the following day, and the
cent inhibition of bacterial growth of greater than or equal to
90% in the primary screen were retested against M. tuberculosis
H37Rv to determine the actual minimum inhibitory concentration
(MIC) in the MABA. The MIC was defined as the lowest concentra-
tion effecting a reduction in fluorescence of 90% relative to con-
trols. This value was determined from the dose-response curve as
GI and daily change in GI (
tion. The MIC was defined as the lowest concentration for which
the GI was less than the GI of the 1:100 control. If the GI of
the test sample was greater than 100, the sample was scored as
DGI) were recorded for each drug dilu-
the IC90 using a curve fitting program. Any IC90 value of 610 lg/mL
D
D
was considered ‘‘Active’’ for antitubercular activity. Compounds
active in the initial screen were tested for cytotoxicity in VERO
cells. After 72 h exposure, viability was assessed using the CellTiter
96Ò Non-Radioactive Cell Proliferation Assay (MTT) reagent from
Promega. Cytotoxicity was determined from the dose-response
curve as the IC50 using a curve fitting program. Concurrent with
the determination of MICs, compounds were tested for IC50 in
VERO cells at concentrations 10x the MIC for M. tuberculosis H37Rv.
resistant even if the
DGI was less than the DGI of the 1:100 control.
Acknowledgments
We thank Dr. Joseph A. Maddry from the Tuberculosis Antimi-
crobial Acquisition and Coordinating Facility (TAACF), National
Institute of Allergy and Infectious Diseases Southern Research
Institute, Alabama, USA for the evaluation of anti-TB activity.
This work was supported by Istanbul University Research Fund,
Project Nos.: 167/15012004 and BYP-809/07112005.
4.6.1. Microplate Alamar Blue Assay (MABA)
Antimicrobial susceptibility testing was performed in black,
clear-bottomed, 96-well microplates (black view plates; Packard
Instrument Company, Meriden, Conn.) in order to minimize back-
ground fluorescence. Outer perimeter wells were filled with sterile
water to prevent dehydration in experimental wells. Initial drug
dilutions were prepared in either dimethyl sulfoxide or distilled
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