Synthesis, antimicrobial activity and QSAR studies of new 2,3-disubstituted-3,3a,4,5,6,7-hexahydro-2H-indazoles

Article abstract of DOI:10.1016/j.bmcl.2009.04.052

Antimicrobial activity of synthesized 2,3-disubstituted-3,3a,4,5,6,7-hexahydro-2H-indazole derivatives indicated that 3-(4-chlorophenyl)-2-(4-nitrophenylsulfonyl)-3,3a,4,5,6,7-hexahydro-2H-indazole (6) and 3-(4-fluorophenyl)-2-(4-nitrophenylsulfonyl)-3,3a,4,5,6,7-hexahydro-2H-indazole (20) were the most active compounds. Further, the results of QSAR studies indicated the importance of topological parameters ²χ and ²χ^v in defining the antimicrobial activity of hexahydroindazoles.

Full text of DOI:10.1016/j.bmcl.2009.04.052

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2960–2964
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, antimicrobial activity and QSAR studies of new
2,3-disubstituted-3,3a,4,5,6,7-hexahydro-2H-indazoles
a
a
a
Maninder Minu a, , Ananda Thangadurai , Sharad Ramesh Wakode , Shyam Sundar Agrawal ,
*
Balasubramanian Narasimhan b
a Delhi Institute of Pharmaceutical Sciences and Research, Pushp Vihar-3, M.B. Road, New Delhi 110 017, India
b Faculty of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak 124001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Antimicrobial activity of synthesized 2,3-disubstituted-3,3a,4,5,6,7-hexahydro-2H-indazole derivatives
indicated that 3-(4-chlorophenyl)-2-(4-nitrophenylsulfonyl)-3,3a,4,5,6,7-hexahydro-2H-indazole (6)
and 3-(4-fluorophenyl)-2-(4-nitrophenylsulfonyl)-3,3a,4,5,6,7-hexahydro-2H-indazole (20) were the
most active compounds. Further, the results of QSAR studies indicated the importance of topological
parameters 2v and 2vv in defining the antimicrobial activity of hexahydroindazoles.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 25 May 2008
Revised 28 March 2009
Accepted 15 April 2009
Available online 18 April 2009
Keywords:
Hexahydroindazoles
Antibacterial
Antifungal
QSAR
The usage of most antimicrobial agents is limited, not only by
the rapidly developing drug resistance, but also by the unsatisfac-
tory status of present treatments of bacterial and fungal infections
and drug side effects.1–5 Therefore, the development of new and
different antimicrobial drugs is a very important objective and
much of the research program efforts are directed towards the
design of new agents.
R2
O
O
NaOH/EtOH
+
R2
CHO
NH2NH2
MeOH
Hexahydroindazole, a novel heterocyclic compound has been
reported to have antimicrobial6,7 and anti-inflammatory
activities.8,9
QSAR models are mathematical equations relating chemical
structure to a wide variety of physical, chemical and biological
properties.
2 R1
H
SO
N
N
N
N
R1 SO2Cl
Pyridine
R2
R2
4-21
1-3
In view of above, and as a part our composite programme of ra-
tional drug design10–23 in the present study, we report the synthe-
sis, antimicrobial and QSAR studies of hexahydroindazole
derivatives.
The synthetic approach to obtain 3-substituted-3,3a,4,5,6,7-
hexahydro-2H-indazoles (1–3) and 2,3-disubstituted-3,3a,4,5,6,7-
hexahydro-2H-indazoles (4–21) followed the reaction shown in
Scheme 1.
1) R1 = H, R2 = Cl
2) R1= H, R2= F
12) R1= C10H7, R2= F
13) R1= 4-CH3C6H4, R2= F
14) R1= C6H5, R2= OCH3
15) R1= CH3, R2= OCH3
3 ) R1= H, R2= OCH3
4) R1= C6H5, R2= Cl
5) R1= CH3, R2= Cl
16) R1= 4-NO2C6H4, R2= OCH3
6) R1= 4-NO2C6H4, R2= Cl 17) R1= C10H7, R2= OCH3
7) R1= C10H7, R2= Cl
8) R1= 4-ClC6H4, R2= Cl
9) R1= 4-CH3C6H4, R2= Cl
10) R1= C6H5, R2= F
11) R1= CH3, R2= F
18) R1= 4-ClC6H4, R2= OCH3
19 ) R1= 4-CH3C6H4, R2= OCH3
20) R1= 4-NO2C6H4, R2= F
21) R1= 4-ClC6H4, R2 = F
We accomplished the synthesis of substituted benzylidene
cyclohexanones by the reaction of cyclohexanone with p-substi-
tuted benzaldehydes in the presence of ethanolic sodium hydrox-
* Corresponding author. Tel.: +91 11 29551321; fax: +91 11 29554503.
E-mail address: maninderminu@rediffmail.com (M. Minu).
Scheme 1. Scheme for syntheses of hexahydroindazole derivatives.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.052

M. Minu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2960–2964
2961
IP31 have been employed as media for growth of bacterial and fun-
gal cells, respectively.
Table 1
Physicochemical characteristics of hexahydroindazole derivatives
Compd
Molecular formula
M.Wt.
mp (°C)
Rf value*
Yield (%)
The compounds 6 and 20 were found to be the most effective
ones against the representative fungal and bacterial species,
respectively (Table 2). Analysis of antimicrobial results (Table 2)
indicated that the electron withdrawing groups were generally
more active than other derivatives. The importance of electron
withdrawing groups in enhancing the antimicrobial activity is
supported by similar results observed by P. Sharma et al.32 From
the analysis of the structures of most active compounds 6 and
20, it may be concluded that among the electron withdrawing halo
groups, the presence of a p-fluorophenyl group at 3rd position of
hexahydroindazole (20) improved the antibacterial activity
whereas the presence of p-chlorophenyl group at 3rd position of
hexahydroindazole (6) improved the antifungal activity.
1
2
3
4
5
6
7
8
C13H15ClN2
C13H15FN2
C14H18N2O
C19H19ClN2O2S
C14H17ClN2O2S
C19H18ClN3O4S
C23H21ClN2O2S
C19H18Cl2N2O2S
234
218
230
374
312
419
424
409
388
358
296
408
372
370
308
415
420
404
384
403
392
124–126
94–96
0.46
0.44
0.40
0.68
0.65
0.73
0.60
0.76
0.70
0.80
0.59
0.74
0.69
0.80
0.73
0.55
0.61
0.67
0.50
0.66
0.84
73
79
87
60
73
57
71
80
65
60
68
72
59
64
71
59
60
51
62
62
56
106–108
162–164
180–182
185–187
158–160
170–172
154–156
172–174
186–188
190–192
179–181
150–152
188–190
199–201
196–198
176–178
174–176
193–195
164–166
9
C20H21ClN2O2S
10
11
12
13
14
15
16
17
18
19
20
21
C19H19FN2O2S
C14H17FN2O2S
C23H21FN2O2S
C20H21FN2O2S
C20H22N2O3S
C15H20N2O3S
In an attempt to determine the role of structural features, QSAR
studies were undertaken using the linear free energy relationship
model (LFER) of Hansch and Fujita.33 Biological activity data deter-
mined as MIC values was first transformed into pMIC values on
molar basis, which was used as dependent variable in QSAR stud-
ies. These were correlated with different molecular descriptors like
log of octanol-water partition coefficient (log P),34 molar refractiv-
C
20H21N3O5S
C24H24N2O3S
C20H21ClN2O3S
C21H24N2O3S
C19H18FN3O4S
C19H18ClFN2O2S
*
TLC mobile phase – CHCl3/CH3OH (9.8:0.2).
ity (MR),35 Kiers molecular connectivity (2 v) and shape (
v j, ja)
topological indices,36 Randic topological index (R),37 Balaban topo-
logical index (J),38 Wiener topological index (W),39 Total energy
(Te), energies of highest occupied molecular orbital (HOMO) and
lowest unoccupied molecular orbital (LUMO).40 The values of
selected descriptors are presented in Table 3.
ide.24 Benzylidene cyclohexanones, on heating with hydrazine
hydrate resulted in the formation of compounds 1–3.25 Further,
the sulfonation of 1–3 with appropriate sulfonyl chlorides in pyri-
dine yielded compounds 4–21.26,27 All compounds were character-
ized by IR, MS, elemental analysis and 1H NMR (Supplementary
data). The physicochemical parameters and molecular structures
of hexahydroindazole derivatives used in the present study are gi-
ven in Table 1.
In the present work, a training set consisting of 17 molecules
was used for linear regression model generation and a prediction
set consisting of 4 molecules was used for the evaluation of gener-
ated linear regression model. Firstly, correlation analysis of various
descriptors with antimicrobial activity was performed. The data
presented in Table 4 shows that most of the parameters are highly
correlated with antimicrobial activity. A correlation matrix (Table
5), constructed to find the interrelationship among the parameters
demonstrated that each parameter selected in the study is highly
correlated with the other (r > 0.8).
The synthesized hexahydroindazole derivatives were evaluated
for their in vitro antimicrobial activity by tube dilution method28–
30
in duplicate, using ciprofloxacin and fluconazole as reference
compounds for antibacterial and antifungal activities, respectively.
Double strength nutrient broth-IP and Sabouraud dextrose broth-
The topological parameters, especially the molecular connectiv-
ity indices (2v and 2vv) have been found to exhibit best correlation
and high statistical significance (p < 0.01). The resulting best-fit
Table 2
Antimicrobial activity of hexahydroindazole derivatives
Compd
pMIC (lmol/mL)
S. aureus
B. subtilis
E. coli
P. aeruginosa
C. albicans
A. niger
Table 3
Training set
Value of selected descriptors used in linear regression analysis
1
2
3
5
6
7
9
10
11
12
14
15
16
17
19
20
21
SDa
2.07
2.07
2.24
2.01
2.54
2.72
2.67
2.64
2.65
2.57
2.66
2.47
2.53
2.62
2.62
2.63
2.76
2.69
0.22
2.19
2.24
2.06
2.40
2.67
2.63
2.59
2.65
2.47
2.71
2.57
2.39
2.66
2.62
2.58
2.76
2.75
0.20
2.19
2.24
2.19
2.50
2.78
2.67
2.64
2.71
2.52
2.77
2.61
2.49
2.72
2.67
2.58
2.81
2.79
0.20
2.27
2.16
2.19
2.59
2.82
2.73
2.59
2.60
2.47
2.61
2.61
2.59
2.77
2.62
2.63
2.65
2.59
0.19
2.27
2.24
2.19
2.50
2.82
2.67
2.64
2.55
2.57
2.66
2.57
2.53
2.66
2.62
2.68
2.76
2.64
0.18
Compd
logP
0v
0vv
1v
1vv
2v
2vv
ja1
2.04
2.01
2.40
2.67
2.53
2.49
2.55
2.37
2.61
2.57
2.31
2.66
2.53
2.49
2.70
2.64
0.22
Training set
1
2
3
5
6
7
9
10
11
12
14
15
16
17
19
20
21
4.4
4.0
3.6
3.7
5.3
6.4
5.8
5.0
3.3
6.0
4.6
2.9
4.5
5.6
5.1
4.9
5.1
11.0
11.0
11.7
14.3
19.9
20.0
18.3
17.4
14.3
20.0
18.2
15.0
20.6
20.7
19.0
19.9
18.3
9.8
7.8
7.8
8.4
6.2
5.8
6.2
7.0
7.0
7.1
4.9
10.8
10.4
11.4
14.4
20.2
19.2
18.1
16.8
14.1
18.8
17.8
15.1
19.6
19.8
18.7
20.2
17.7
9.0
10.0
13.0
16.6
17.5
16.3
14.6
12.2
16.7
15.6
13.2
16.8
17.7
16.5
15.8
14.9
4.4
4.6
7.9
9.4
10.1
9.5
8.5
7.5
9.7
8.7
7.7
9.1
9.9
9.2
8.9
8.6
9.5
9.2
9.5
13.4
14.0
12.5
12.1
9.5
14.0
12.6
10.0
13.9
14.6
13.0
13.4
12.5
11.0
11.9
10.9
10.1
8.8
11.5
10.5
9.2
11.0
11.9
10.9
10.6
10.2
12.8
13.3
11.9
11.3
9.5
13.3
11.5
9.7
13.0
13.5
12.1
12.8
11.9
Test set
4
8
13
18
Std.
2.57
2.61
2.47
2.61
2.03*
2.62
2.71
2.62
2.65
2.33*
2.57
2.66
2.62
2.65
2.03*
2.67
2.77
2.67
2.70
2.62*
2.62
2.77
2.57
2.61
3.16**
2.62
2.71
2.67
2.65
3.16**
Test set
4
8
13
18
5.4
5.9
5.4
4.3
17.4
18.3
18.3
19.7
15.4
16.5
15.5
16.9
12.1
12.5
12.5
13.5
10.5
11.0
10.5
11.0
11.3
11.9
11.9
12.3
9.0
9.6
9.0
9.1
17.1
18.4
17.8
19.7
Reference: * ciporfloxacin; ** fluconazole; a standard deviation.

2962
M. Minu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2960–2964
Table 4
2.7
Correlation of different molecular descriptors with antimicrobial activity of hexa-
hydroindazole derivatives
2.6
2.5
2.4
2.3
2.2
pMICsa
pMICbs
pMICec
pMICpa
pMICca
pMICan
log P
MR
0v
0.586
0.859
0.932
0.893
0.897
0.910
0.936
0.913
0.936
0.943
0.897
ꢀ0.307
0.513
0.782
0.856
0.837
0.807
0.888
0.882
0.909
0.855
0.873
0.807
ꢀ0.168
0.658
0.850
0.919
0.872
0.894
0.894
0.932
0.898
0.917
0.917
0.894
ꢀ0.373
0.608
0.848
0.923
0.878
0.891
0.904
0.936
0.909
0.923
0.929
0.891
ꢀ0.331
0.498
0.851
0.894
0.902
0.849
0.920
0.898
0.929
0.900
0.915
0.849
ꢀ0.211
0.560
0.847
0.909
0.893
0.862
0.913
0.920
0.926
0.913
0.940
0.862
ꢀ0.243
0vv
1v
1vv
2v
2vv
j1
ja1
R
B
2.1
2.0
2.0
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
Observed pMICsa
Table 5
Correlation matrix for pMICsa with selected molecular descriptors
Figure 1. Plot of predicted pMICsa values against the experimental pMICsa values
for the linear regression developed model by Eq. (1).
pMICsa 0v
0vv
1vv
2v
2vv
j1
ja1
pMICsa
0v
1.000
0.932
0.893
0.910
0.936
0.913
0.936
0.943
1.000
0vv
1vv
2v
0.979
0.964
0.994
0.943
0.999
0.995
1.000
0.986
0.978
0.973
0.976
0.984
increases with an increase in magnitude of ja1. This is evidenced
by the values of ja1 in Table 3. The values of ja1 for compounds
6 and 20 were 20.22 and 20.24, respectively. These are higher than
the ja1 value of other compounds, which make them to be the
most active compounds against S. aureus. Similarly the compounds
1 and 2 having the minimum ja1 values of 10.78 and 10.44 have
minimum activity against S. aureus. Similar trend was observed
in case of B. subtilis, C. albicans and A. niger with 2vv, E. coli and P.
1.000
0.976
0.995
0.958
0.967
1.000
0.962
0.988
0.985
2vv
j1
ja1
1.000
0.936
0.950
1.000
0.997
1.000
models are reported in Eqs. (1)–(6) together with statistical param-
eters of regression. It is important to note that all these models
were developed by using the entire set of hexahydroindazoles
(n = 21), since no outliers were identified.
aeruginosa with 2v
.
In order to confirm our results, we have constituted a test set
consisting of 4 hexahydroindazole derivatives viz. 4, 8, 13 and 18
and predicted their activities using the model expressed by Eqs.
(1)–(6) and compared them with the observed values. We have
also applied the same model to predict the activity of training set
and the prediction results of QSAR models (Supplementary Table
1) indicated that the observed and the estimated activities are very
close to each other evidenced by low values of residual activity.
Further the plot of linear regression predicted pMICsa values
against the observed pMICsa values also favors the model
expressed by Eq. (1) (Fig. 1). In Figure 2, the propagation of the
residuals on both sides of zero indicates that no systemic error
exists in the development of linear regression model as suggested
by Jalali-Heravi and Kyani.41 The cross-validation of the models
(q2 > 0.5) was also done by leave one out (LOO) technique.42
Generally for QSAR studies, the biological activities of com-
pounds should span 2–3 orders of magnitude. But in the present
study the range of antimicrobial activities of the synthesized com-
pounds is within one order of magnitude. But it is important to
note that the predictability of the QSAR models developed in the
present study is highly evidenced by the low residual values
(Supplementary Table 1). This is in accordance with results sug-
gested by the Bajaj et al.,43 who stated that the reliability of the
QSAR model lies in its predictive ability even though the activity
data are in the narrow range. Further, recent literature reveals that
the QSAR has been applied to describe the relationship between
narrow range of biological activity and physicochemical properties
of the molecules.5,21,44,45 When biological activity data lies in the
narrow range, the presence of minimum standard deviation
observed between the entire antimicrobial data against a particu-
lar species justifies its use in QSAR studies.16,21 The minimum
standard deviation (Table 2) observed in the antimicrobial activity
data justifies it use in QSAR studies.
QSAR model for antibacterial activity against Staphylococcus
aureus
pMICsa ¼ 0:063
j
a1 þ 1:403
ð1Þ
ð2Þ
ð3Þ
n = 17; r = 0.943; F = 119.93; s = 0.076; q2 = 0.860
QSAR model for antibacterial activity against Bacillus subtilis
pMICbs ¼ 0:1092vv þ 1:650
n = 17; r = 0.909; F = 70.97; s = 0.094; q2 = 0.760
QSAR model for antibacterial activity against Escherichia coli
pMICec ¼ 0:0832
v þ 1:609
n = 17; r = 0.932; F = 98.60; s = 0.076; q2 = 0.823
QSAR model for antibacterial activity against Pseudomonas
aeruginosa
pMICpa ¼ 0:0842
v
þ 1:662
ð4Þ
ð5Þ
ð6Þ
n = 17; r = 0.936; F = 105.62; s = 0.074; q2 = 0.843
QSAR model for antifungal activity against Candida albicans
pMICca ¼ 0:0942vv þ 1:791
n = 17; r = 0.929; F = 94.64; s = 0.071; q2 = 0.830
QSAR model for antifungal activity against Aspergillus niger
pMICan ¼ 0:0892vv þ 1:840
n = 17; r = 0.926; F = 90.16; s = 0.068; q2 = 0.823
The coefficient of ja1 in the mono-parametric model in Eq. (1)
is positive indicating thereby that antibacterial activity of hexa-
hydroindazoles derivatives against S. aureus is directly propor-
In conclusion, the present study revealed that the compounds 6
and 20 exhibited appreciable antimicrobial activity. Further, the
tional to the magnitude of ja1
. The antibacterial activity

M. Minu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2960–2964
2963
.2
.1
tion was determined by the absence of growth. MIC was deter-
mined by the lowest concentration of sample that prevented the
development of turbidity. From the MIC values observed, the inter-
mediate concentrations between MIC values were prepared and
the accurate MIC values were determined.
QSAR analysis. The calculation34–40 of molecular descriptors of
hexahydroindazole derivatives as well as the regression analysis
was carried out by using the molecular package TSAR 3D version
3.3.46
0.0
-.1
-.2
Acknowledgments
Authors are grateful to the All India Council of Technical Educa-
tion, New Delhi, India for the financial support and University of
Delhi, India, for providing necessary research facilities to carry
out this work.
2.0
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
Observed pMICsa
Supplementary data
Figure 2. Plot of residual pMICsa values against the experimental pMICsa values for
the linear regression developed model by Eq. (1).
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.04.052.
results of QSAR studies indicated that the topological parameters,
the second order molecular connectivity index and valence second
order molecular connectivity index, 2v and 2vv can be used
successfully for modeling antimicrobial activity of hexahydroin-
dazoles. The validity of models obtained by linear and multiple
linear regressions are clearly evidenced by the high q2 values
obtained for the developed QSAR models.
References and notes
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153.
3. Hong, C. Y. J. Farmaco 2001, 56, 41.
4. Macchiarulo, A.; Constantino, G.; Fringuelli, D.; Vecchiarelli, A.; Schiaffella, F.;
Fringuelli, R. Bioorg. Med. Chem. 2002, 10, 3415.
Melting points were determined in open glass capillary using
Bells India melting point apparatus and are uncorrected. The infra-
red (IR) spectra were recorded with Shimadzu 8400S-FTIR spectro-
photometer in KBr discs. The 1H NMR spectra in CDCl3 were
recorded on Bruker-DPX 300 NMR spectrophotometer using TMS
as an internal standard. Mass spectra were measured on a Shima-
dzu 2010A spectrophotometer. Elemental analysis was performed
on a Perkin–Elmer 2400 C, H, N analyzer and values were within
the acceptable limits of the calculated values (within 0.4%). The
homogeneity of the compounds was monitored by ascending thin
layer chromatography (TLC) on Silica Gel-G coated aluminum
plates, visualized by iodine vapour and UV light. Developing
solvent was chloroform/methanol (9.8:0.2).
General procedure for synthesis of 3-substituted-3,3a,4,5,6,7-hexa-
hydro-2H-indazoles (1–3). The solution of hydrazine hydrate
(0.03 mol) and appropriate p-substituted benzylidene cyclohexa-
nones (0.01 mol) in methanol (200 ml) was refluxed for 2–3 h.
The above reaction mixture was cooled and kept at 0 °C for 24 h.
The precipitated product was filtered, washed with methanol and
recrystallized from a mixture of methanol–hydrazine hydrate.
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