Synthesis and pharmacological studies of new pyrazole analogues of podophyllotoxin

Article abstract of DOI:10.1134/S106816201404013X

The pyrazole analogues of podophyllotoxin were synthesized by the chalcone route. This route attracts the attention because of its simple operating conditions and easy availability of the chemicals. Initially, benzylideneacetophenones (chalcones) were pre

Full text of DOI:10.1134/S106816201404013X

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2014, Vol. 40, No. 4, pp. 467–476. © Pleiades Publishing, Ltd., 2014.
Synthesis and Pharmacological Studies
of New Pyrazole Analogues of Podophyllotoxin1
B. Umesha and Y. B. Basavaraju2
Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore—570 006, Karnataka, India
Received December 20, 2013; in final form, February 10, 2014
Abstract—The pyrazole analogues of podophyllotoxin were synthesized by the chalcone route. This route
attracts the attention because of its simple operating conditions and easy availability of the chemicals. Iniꢀ
tially, benzylideneacetophenones (chalcones) were prepared in high yields by ClaisenꢀSchmidt reaction of
acetophenones with 4ꢀ(methylthio)benzaldehyde. The cyclopropyl ketones were prepared in good yields by
the reaction of chalcones with trimethylsulfoxonium iodide. Tetralones were prepared in good yields by the
FriedelꢀCraft’s intramolecular cyclization reaction of cyclopropyle ketones in the presence of anhyd. stannic
chloride and acetic anhydride. The tetralones on formylation to give substituted hydroxylmethylene tetralones.
Condensation of substituted hydroxylmethylene tetralones with hydrazine hydrate afforded target compounds.
The structures of the synthesized compounds were confirmed by IR, 1H NMR and Mass spectral technique. The
title compounds were screened for their antimitotic and antimicrobial activities. Among the synthesized comꢀ
pounds cyclopropyl ketones and pyrazole analogues of podophyllotoxin, compound 7ꢀ(methylthio)ꢀ5ꢀ(4ꢀ(methꢀ
ylthio)phenyl)ꢀ4,5ꢀdihydroꢀ2Hꢀbenzo[g]indazole is more active than 5ꢀ(4ꢀ(methylthio)phenyl)ꢀ4,5ꢀdihydroꢀ2H
ꢀ
benzo[g]indazole, 7ꢀmethylꢀ5ꢀ(4ꢀ(methylthio)phenyl)ꢀ4,5ꢀdihydroꢀ2Hꢀbenzo[g]indazole, 7ꢀmethoxyꢀ5ꢀ(4ꢀ
(methylthio)phenyl)ꢀ4,5ꢀdihydroꢀ2Hꢀbenzo[g]indazole and the key intermediate tetralones in 100, 200 and
400 ppm at 12, 18 and 24 h and also showed very good activity against screened bacteria and fungi compared
to their standard.
Keywords: acetophenones, chalcones, cyclopropylketones, tetralones, antimitotic activity, antimicrobial activity
DOI: 10.1134/S106816201404013X
2 1
INTRODUCTION
analgesic power. Among them, derivatives of 5ꢀisopyꢀ
razolone and pyrazolidineꢀ3,5ꢀdione are worth noting
due to their clinical interest. Several, substituted pyraꢀ
zolines are reported to possess moderate antibacterial
and antifungal activities. Besides, a number of nitrofuꢀ
rylpyrazoline derivatives were found to possess antiꢀ
bacterial activity. Pyrazole molecules are also exhibit
anticancer, antiꢀinflammatory, antidepressant, antiꢀ
convulsant [12]. The incorporation of pyrazole ring
into the podophyllotoxin enhances the biological
activities to a great extent and also presence of differꢀ
Podophyllotoxin (Fig. 1) is a most abundant natuꢀ
rally occurring antimitotic cyclolignan, derived from
variety of plants, such as podophyllum peltalum,
podophyllum emodi, podophyllum pleianthum,
podophyllum hexandrum anthriscus sylvertris and
juniperus Sabina [1–7]. Podophyllotoxin exhibits high
antimitotic and apoptotic activity, this is due to its
affinity for tubulin and mitotic spindles of dividing
cells at metaphase. Since it is having high toxicity, it
reduces the application of podophyllotoxin as an antiꢀ
cancer agent. The toxicity of podophyllotoxin liberꢀ
ates as diarrhea, nausea, vomiting and damages to the
healthy tissues [8, 9]. At the same time heterocyclic
ring systems have emerged as powerful scaffolds for
many biological evaluations [10]. Heterocyclic comꢀ
pounds provide scaffolds on which pharmacophores
can arrange to yield potent and selective drugs [11].
Pyrazoles and their derivatives play an important role
in medicinal chemistry research. Several derivatives of
pyrazole are of pharmaceutical interest due to their
OH
H
H
H
O
O
O
O
H
H3CO
OCH3
OCH3
1
The article is published in the original.
Corresponding author: phone: +91 948 044ꢀ18ꢀ04; eꢀmail:
araju_yb@yahoo.co.in; umesha.basaviah54@gmail.com.
2
Fig. 1. Podophyllotoxin.
467

468
UMESHA, BASAVARAJU
ent substituents on the new pyrazole analogues of the
βꢀcarbon atom of the chalcone which acts as
podophyllotoxin, well improve the biological properꢀ
ties. Hence these modifications of its structure are
required to reduce its toxicity and to enhance its bioꢀ
logical activity. The biologically active and less cytoꢀ
toxic new pyrazole analogues of podophyllotoxin have
been synthesized.
Michael receptors to form an enolate ion, which unꢀ
dergoes nucleophilic attack on the methylene carbon
atom bearing the dimethylsulfoxonium cation inꢀ
tramolecularly finally to form the desired cyclopropyl
ketone. The structure of compounds (IIIa–d) was
confirmed by IR spectra. In its IR spectra exhibit C=O
stretching band in the range 1687–1671 cm–1 and
1H NMR showed the cyclopropane CH and CH2 peak
at the range 0.83–0.78 and 2.21–2.00 ppm respectiveꢀ
RESULTS AND DISCUSSION
The synthesis of new pyrazole analogues of podoꢀ
phyllotoxin has been carried out by chalcone route
(Scheme 1). The benzylideneacetophenones (chalꢀ
ly. Tetralones (IVa
the FriedelꢀCraft’s intramolecular cyclization reacꢀ
tion of cyclopropyle ketones (IIIa ) in the presence
–d) were prepared in good yields by
–d
cones) (IIa
enꢀSchmidt reaction of acetophenones (Ia
–
d
) were prepared in high yields by Claisꢀ
of anhyd. stannic chloride and acetic anhydride in dry
dichloromethane [17]. The cyclopropyl ketones unꢀ
dergo electrophilic ring opening in the presence of
Lewis acid to give benzylcarbocationic intermediate
–d) with
4ꢀ(methylthio)benzaldehyde in the presence of sodiꢀ
um hydroxide in waterꢀethanol mixture [13, 14]. The
structures of the chalcones were confirmed by IR and
1H NMR spectral studies. IR spectra of compounds
which is intramolecularly attacked by aryl ring πꢀelecꢀ
trons resulting in the formation of a six membered ring
with a pendant carbocation. This readily gives up proꢀ
ton to form tetralones. Acetic anhydride facilitates the
formation of desired tetralones. In its IR spectra apꢀ
peared absorption bands in the range 3133–2934 cm–1
and 1705–1685 cm–1 corresponds to aromatic C–H
(
IIa–d) showed the C=C stretching frequency in the
range 1667–1656 cm–1 and 1H NMR showed the abꢀ
sence of aldehyde proton at 9.83 ppm. The cycloproꢀ
pyl ketones (IIIa
–
d
) were prepared in good yields by
the reaction of chalcones (IIa
–d) with trimethylsulꢀ
foxonium iodide (TMSOI) in the presence of sodium
hydride in dry DMSO [15, 16]. The sodium hydride
acts as a base which abstracts a proton from the methyl
group in trimethylsulfoxonium iodide to form a dimeꢀ
1
and C=O stretching frequencies and H NMR of the
ring B proton appears in the range 2.65–2.18 ppm.
They are key intermediates for the preparation of the
thylsulfoxonium methylide. It attacks nucleophilically new pyrazole linked podophyllotoxin analogues.
O
H
O
O
O
TMSOI
DMSO
Ac O, Anhyd. SnCl
2 4
SCH3
Dry CH Cl
2
2
NaOH, EtOHꢀH O
2
R
R
R
SCH3
SCH3
(
Ia–d
)
(
IIa–d
)
(IIIa–d)
NH
O
O
N
HCOOC H
2
NH NH /EtOH
2
5
2
OH
A
B
A
B
A
B
NaH, dry C H , rt
6
reflux
6
R
R
R
C
C
C
SCH3
SCH3
SCH3
(
IVa–d
)
(Va – d
)
(VIa–d)
(Ia)–(VIa) R = H; (Ib)–(VIb) R = CH3; (Ic)–(VIc) R = OCH3; (Id)–(VId) R = SCH3
Scheme 1. Protocol for synthesis of tetralones (IVa–d).
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SYNTHESIS AND PHARMACOLOGICAL STUDIES
469
The tetralones on formylation to give substituted growth in 100, 200 and 400 ppm at 12, 18 and 24 h. It
hydroxylmethylene tetralones (Va ) [18]. Formylaꢀ can be said that compound VId has maximum inhibiꢀ
tion of the presently synthesized tetralones with ethyl tory effect compared to key intermediate (IVa ) and
formate using sodium hydride as the base at room temꢀ compounds VIa, VIb and VIc in 100, 200 and 400 ppm
perature gave single product in good yields. The at 12, 18 and 24 h exhibited inhibitory effect on the cell
–d
–d
β
ꢀ
dicarbonyl compounds which exist in the enol form division of the onion root.
show the carbonyl absorption in the region 1640–
From the above observations, it can be seen that
1610 cm–1. Generally 1,3ꢀdiketones absorption peak
with high intensity appeared at 1715 cm–1. Enols conꢀ
taining this grouping, but the shift that occurs in these
compounds is attributed to the intramolecular hydroꢀ
gen bonding.
At the same time, the true alcoholic hydroxyl band
near 3530 cm–1 is absent in enols, but there is a band
near 3258–3251 cm–1 (s) which is attributed to the
chelated hydroxyl group. 1H NMR showed the vinylic
proton absorption at 5.80–5.60 ppm. The new pyraꢀ
partialꢀcꢀmitosis, fullꢀcꢀmitosis with partially funcꢀ
tional spindles and comparatively normal mitotic cells
phases, chromosomal bridge and chromosomal breakꢀ
age were noticed in various cells of the same root tip
between 12, 18 and 24 h time duration (Figs. 2a–2f).
Therefore antimitotic ability of new pyrazole anaꢀ
logues of podophyllotoxin was remarkable in controlꢀ
ling the cell division and hence acts as a very good
antimitotic agent. The results of antimitotic activity
are given in (Tables 1 and 2).
zole analogues of podophyllotoxin (VIa–d) were synꢀ
thesized in high yields by the condensation of
hydroxymethylene tetralones and hydrazine hydrate
in absolute ethanol [19]. The products were purified by
Antimicrobial Activity
The antibacterial screening revealed that some of
the compounds showed good inhibition against variꢀ
ous tested microbial strains. The key intermediate
recrystallization from ethanol. The compounds (VIa–d)
exhibited NH stretching band at 3490–3360 cm–1 and
proton NMR showed singlet NH peak at 12.64–12.55
ppm. Based on this, the structures of the synthesized
compounds were confirmed.
compounds (IVa
and antifungal activities (Table 3). Further, hydrazine
was condensed with (Va ) gave new pyrazole anaꢀ
–d) possessed moderate antibacterial
–d
Biological Activities
Antimitotic Activity
logues of podophyllotoxin accounted for the
enhanced activities (Table 4). The results indicated
that VIa having thiomethyl substituent present at ring C
exhibited least activity and VIb and VIc possessing thiꢀ
omethyl substituent present at ring C and methyl and
methoxy group on ring A showed considerable activity
and at the same time VId possessing thiomethyl substitꢀ
uent present at ring A and C showed good activity
Allium cepa has been used to evaluate the antimiꢀ
totic activity of new pyrazole analogues of podophylꢀ
lotoxin. Root tip cells in (IVa–d) and (VIa–d) exhibꢀ
ited changes in cellular morphology such as slight
elongation in shape with many of them remain in the
earliest stages of mitosis called prophase stage. Onion
against all screened bacteria compared to VIa
and key intermediate compounds (IVa ) at concenꢀ
, VIb, Vic
roots in compound (IVa–d) and (VIa–d) of 100, 200
–
d
and 400 ppm at 12, 18 and 24 h exhibited changes in
chromosomes and shape of the cells with elongated
appearance. Using cytotoxic nature of new pyrazole
analogues of podophyllotoxin showed very less numꢀ
ber of dividing cells. Change in chromosomes and celꢀ
lular morphology were achieved in increasing time and
concentration. Treatment of root meristem with comꢀ
tration of 10.0 mg/mL compared to standard drug
Chloramphenicol.
Newly synthesized compounds (IVa
) were also screened for their antifungal activity
–d) and (VIa–
d
against two tested microbial strains (Tables 5 and 6).
Among the tested compounds, compound VIa exhibꢀ
ited least activity and VIb and VIc showed moderate
activity where as VId showed very good activity against
two treated fungi compared to VIa, VIb, VIc and key
intermediate compounds (IVa–d) at concentration of
10.0 mg/mL compared to standard drug Nystatin.
pounds (IVa
cell shape with elongated appearance. (IVa
VIa ) compounds of 100, 200 and 400 ppm were
used for this experiment, pyrazole moiety present at
ring B of (VIa ) compounds showed highest antimiꢀ
totic activity when compared to (IVa ) compounds
–d
) and (VIa
–
d
) exhibited less change in
–d) and
(
–d
–d
–d
In summary, new pyrazole analogues of podophylꢀ
in 100, 200 and 400 ppm at 12, 18 and 24 h. Among lotoxin were synthesized in good yields by chalcone
VIa ), compound VId possessing thiomethyl substitꢀ route. The synthesized compounds were screened for
uent on ring A and C showed lowest mitotic index their antimitotic and antimicrobial activity. Among
there by indicating highest level of antimitotic activity the synthesized compounds (IVa ) and (VIa ),
in 400 ppm concentration at 24 h. Where as comꢀ compound VId is more active than VIa, VIb, VIc and
pounds VIb and VIc having methyl and methoxy group the key intermediates (IVa ) by displaying the antimiꢀ
present on ring A respectively and thiomethyl substituꢀ totic and antimicrobial activity at 10.0 mg/L concenꢀ
ent on ring C showed moderate activity and comꢀ tration. The compounds (VIa ) showed better antimꢀ
pound VIa having thiomethyl group exhibited highest itotic and antimicrobial activity than the compounds
mitotic index with lowest ability to inhibit the cell IVa ) in 100, 200, and 400 ppm at 12, 18 and 24 h.
(
–d
–d
–d
–d
–d
(
–d
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470
UMESHA, BASAVARAJU
(а)
(b)
(c)
(d)
(e)
(f)
Fig. 2. (a) Prophase, (b) anaphase, (c) metaphase, (d) telophase, (e) chromosomal bridge, and (f) chromosomal breakage.
EXPERIMENTAL
were stirred in water (40 mL) and ethanol (25 mL)
mixture in the presence of sodium hydroxide (2.00 g,
0.05 mol) at 15–30°C for 4 h. The reaction mixture
All reagents and chemicals were purchased from
Merck Chemicals used without further purification.
Melting points were taken in open capillary tubes and
are uncorrected. TLC is performed with E. Merck
precoated silica gel plates (60Fꢀ254) with iodine as a
developing agent. Acme, India silica gel, 60–120 mesh
is used for column chromatography. IR spectra in KBr
was kept overnight in an ice bath. The precipitated
products were filtered and recrystallized from ethanol.
General procedure for the synthesis of cyclopropyl
ketones analogues (IIIa–d). Sodium hydride (0.48 g of
0.02 mol) was added in portions to the stirred suspenꢀ
sions of trimethylsulfoxonium iodide (4.41 g of
0.02 mol) in dry DMSO (20 mL) under nitrogen gas
atmosphere. The reaction mixture was stirred for
(ν
, cm–1) were recorded on PerkinꢀElmer model 683
1
spectrometers.; H NMR (400 MHz) and 13C NMR
(100 MHz) spectra ( , ppm; , Hz) using trimethyl
δ
J
10 min at 25–30
ceased). Chalcones (0.02 mol) (IIa
°
C
(until the evolution of the H2 gas
) in dry DMSO
silane (TMS) as an internal reference were recorded in
CDCl3 on Bruker spectrometer, Elemental analyses
were performed on a PerkinꢀElmer 2400. Mass spectra
ESI were obtained by WaterꢀQꢀTOF ultima spectromꢀ
eter. Micro analytical data were obtained by elemenꢀ
talꢀVario ELꢀIII.
–d
(15 mL) were added drop wise during 30 min to the
above solution. The reaction mass was stirred at 26–
28°C for 2 h and raised the temperature to 50–60°C
for 1 h. The completion of the reaction was confirmed
by TLC and the reaction mixture was poured into
General procedure for the synthesis of chalcones water (20 mL). The precipitated gummy residue was
(IIa–d). Substituted acetophenones (0.05 mol) (Ia extracted into chloroform. The combined organic
and 4ꢀ(methylthio) benzaldehyde (6.64 g, 0.05 mol) layer was washed with water, dried over anhyd. Na2SO4
–d)
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SYNTHESIS AND PHARMACOLOGICAL STUDIES
471
Table 1. Antimitotic activity of the compounds (IVa–d) by onion root tip method
% Dividing cells
compared to control
% Inhibition compared
to control
% Dividing cells
ID50 in ppm
18 24
Comp. Conc.
No. in ppm
time, h
24 12
0.0
12
18
24
12
18
18
24
12
–
Control
–
32.91 24.72 21.74 100
(
(
(
(
IVa
)
100
200
400
100
200
400
100
200
300
100
200
400
22.24 20.03 14.50 67.57 81.02 66.69 32.43 18.98 33.31
19.91 17.02 13.23 60.49 68.85 60.35 39.51 31.15 39.15
15.85 11.99 10.77 48.16 48.50 49.54 51.84 51.50 50.46
19.02 17.18 17.01 57.79 69.49 78.24 42.21 30.51 21.76
17.47 12.01 13.13 53.08 48.58 60.39 46.92 51.42 39.61
15.51 10.00 10.56 47.12 40.45 48.57 52.88 59.55 51.43
19.99 18.48 15.45 60.74 74.75 71.06 39.26 25.25 28.94
16.66 15.56 13.00 50.63 62.94 59.79 49.37 37.06 40.21
14.56 11.45 10.01 44.24 46.31 46.04 55.76 53.69 53.96
17.56 13.01 13.20 53.35 52.62 60.71 46.65 47.38 39.29
15.69 09.56 09.21 45.85 39.88 42.36 54.15 60.12 57.64
11.78 07.09 05.45 35.79 28.68 25.06 64.21 71.32 74.94
380
320
220
140
390
190
350
120
400
370
350
160
IVb
)
IVc)
IVd
)
Table 2. Antimitotic activity of the compounds (VIa–d) by onion root tip method
% Dividing cells % Inhibition compared
% Dividing cells
ID50 in ppm
18
compared to control
time, h
24 12
0.0
to control
Comp. Conc.
No. in ppm
12
18
24
12
18
18
24
12
–
24
Control
–
41.16 35.04 31.32 100
(
(
(
(
VIa
)
100
200
400
100
200
400
100
200
300
100
200
400
25.43 28.33 20.34 61.78 80.86 64.94 38.22 19.14 35.05
23.61 16.81 18.09 57.36 47.97 57.75 42.64 52.02 42.25
19.03 14.56 15.58 46.23 41.55 49.74 53.77 58.44 50.26
22.90 21.66 19.87 55.63 61.81 63.44 44.37 38.18 36.56
20.01 15.72 21.10 48.61 44.86 67.36 51.38 55.13 32.64
17.81 13.32 14.74 43.27 38.01 47.06 56.72 61.98 52.94
23.41 22.23 22.52 56.87 63.44 71.90 43.12 36.55 28.10
20.01 18.41 14.90 48.61 52.53 47.57 51.38 47.46 52.43
16.66 14.03 12.83 40.47 40.03 40.96 59.52 59.96 59.04
22.70 21.41 19.29 55.15 61.11 61.59 44.84 38.89 38.41
19.68 12.24 13.32 47.81 34.93 42.52 52.18 65.07 57.48
15.57 07.92 06.27 37.82 22.60 20.01 62.17 77.40 79.99
330
180
180
170
190
170
240
150
400
370
190
160
VIb
)
VIc
)
VId
)
and concentrated under reduced pressure. The prodꢀ reaction mixture was stirred at 25–28°C for 3 h. The
completion of reaction was known by TLC. The reacꢀ
tion mixture was poured into 5% NaOH solution
(20 mL). The product was extracted into dichloꢀ
romethane. The organic layer was washed with
5% HCl followed by water, dried over anhyd. Na2SO4
and concentrated under vacuum using a rotary evaporaꢀ
tor to give brown residue. The product was purified by
ucts were recrystallized from ethanol.
General procedure for the synthesis of key intermediꢀ
ate tetralones (IVa–d). Cyclopropyl ketones (0.01 mol)
(
IIIa–d) were dissolved in dry dichloromethane
(50 mL). Acetic anhydride (0.94 mL, 0.01 mol) and
anhydrous stannic chloride (1.17 mL, 0.01 mol) were
added under nitrogen gas atmosphere. The resultant column chromatography using silica gel (60–120 mesh)
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472
UMESHA, BASAVARAJU
Table 3. Antibacterial activity of the compounds (IVa–d). Inhibitory zone (diameter) mm of the synthesized compounds
against tested bacterial strains by disc diffusion method
Comp. No.
E. faecalis
K. pneumonia
E. aerogenes
P. aeruginosa
A. faecalis
Conc. in mg/mL
10.0
10.0
10.0
10.0
10.0
Control
Chloramphenicol
00
00
00
00
00
18.4 0.08
14.2 0.02
12.4 0.03
15.6 0.04
16.0 0.03
18.8 0.06
12.2 0.04
14.1 0.06
14.1 0.02
15.8 0.04
19.2 0.04
11.1 0.06
12.2 0.06
12.8 0.02
13.2 0.0.2
18.6 0.06
12.4 0.02
14.1 0.04
12.9 0.02
13.8 0.04
18.4 0.03
12.1 0.08
13.1 0.02
13.1 0.02
14.3 0.04
(
(
(
(
IVa
)
IVb
)
IVc)
IVd
)
Values are means of triplicates.
Standard 10 mg/disc.
Table 4. Antibacterial activity of the compounds (VIa–d). Inhibitory zone (diameter) mm of the synthesized compounds
against tested bacterial strains by disc diffusion method
Comp. No.
E. faecalis
K. pneumonia
E. aerogenes
P. aeruginosa
A. faecalis
Conc. in mg/mL
10.0
10.0
10.0
10.0
10.0
Control
Chloramphenicol
00
00
00
00
00
18.4 0.08
16.4 0.02
13.4 0.02
16.4 0.04
17.2 0.02
18.8 0.06
15.2 0.04
15.8 0.03
15.9 0.02
18.0 0.04
19.2 0.04
14.1 0.02
13.5 0.03
14.1 0.04
16.2 0.02
18.6 0.06
15.4 0.04
15.6 0.02
14.6 0.03
16.9 0.04
18.4 0.03
14.8 0.02
14.4 0.04
15.1 0.02
17.4 0.04
(
(
(
(
VIa
)
)
VIb
VIc
)
VId
)
Values are Mean of triplicates.
Standard 10 mg/disc.
as adsorbent and benzene as eluent. The benzene soluꢀ
tion was concentrated to a small volume (20 mL) and
hexane (100 mL) was added drop wise to give products
in good yields. They were recrystallized from ethanol.
General procedure for the synthesis of substituted
hydroxyl methylene tetralones (Va–d). Sodium hydride
(1.2 g, 0.05 mol) was added to a mixture of absolute
ethanol (10 mL) and dry benzene (150 mL) and stirred
for 1 h. Ethyle formate (10 mL) was added dropwise to
the above reaction mixture and stirred for another 1 h,
followed by dropwise addition of tetralones (0.05 mol)
Table 5. Antifungal activity of the compounds (IVa–d). Inhibꢀ
itory zone (diameter) mm of the synthesized compounds
against tested bacterial strains by disc diffusion method
(
IVa–d), in dry benzene (100 mL) over a period of 1 h.
After stirring the red coloured mixture at room temꢀ
perature for 2 h, it was poured into 2 N H2SO4
(100 mL) in ice. The separated organic layer was
Comp. No.
F. verticillioides
A. niger
Conc. in mg/mL
10.0
10.0
washed with water (
sodium hydroxide solution (
3
×
50 mL) and extracted into 1%
50 mL). The sodium
3
×
Control
Nystatin
00
00
hydroxide extract was acidified with 2 N H2SO4 gave
products in good yields. They were recrystallized from
ethanol.
22.0 0.04
12.4 0.02
08.1 0.04
13.6 0.04
16.1 0.02
18.0 0.06
08.2 0.02
06.4 0.04
09.8 0.04
15.2 0.02
(
(
(
(
IVa
)
IVb
)
General procedure for the synthesis of new pyrazole
analogues of podophyllotoxin (VIa–d). A mixture of
hydroxymethylene tetralones (0.01 mol) (Va –d) and
IVc)
IVd
)
hydrazine (0.49 mL, 0.01 mol) in absolute ethanol was
refluxed for 3 h. The excess of solvent was removed
Values are Mean of triplicates.
Standard 10 mg/disc.
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SYNTHESIS AND PHARMACOLOGICAL STUDIES
473
Table 6. Antifungal activity of the compounds (VIa–d). Inhibꢀ
itory zone (diameter) mm of the synthesized compounds
against tested bacterial strains by disc diffusion method
under reduced pressure. The solid thus obtained were
collected and recrystallized from ethanol.
3ꢀ(4ꢀ(Methylthio)phenyl)ꢀ1ꢀphenylpropꢀ2ꢀenꢀ1ꢀone
(IIa). Color: light yellow solid. Yield: 85.90%. mp:
Comp. No.
F. verticillioides
A. niger
95–97
°
C. IR: 3150–2972 (ArꢀCH), 1677 (C=O),
= 7.8, ꢀCH),
= 7.8, ꢀCH),
Conc. in mg/mL
10.0
10.0
1543 (C=C); 1H NMR: 8.05 (d, 1H,
J
J
β
α
7.80–7.28 (9 H, m, ArꢀH), 7.62 (1 H, d,
Control
Nystatin
00
00
2.52 (3 H, s, SCH3); 13C NMR: 189.6, 145.3, 139.5,
137.9, 135.6, 134.6, 130.8, 129.3, 128.7, 125.8, 124.9,
22.0 0.04
13.6 0.04
10.1 0.02
16.6 0.02
20.8 0.02
18.0 0.06
09.8 0.04
09.2 0.02
14.2 0.04
17.6 0.04
(
(
(
(
VIa
)
)
124.8, 121.5, 14.9; MS, m/z: 254.10 (M
+). Anal. calcd.
for C17H14OS: C, 86.40; H, 6.82. Found: C, 86.32; H,
6.78%.
VIb
VIc
)
VId
)
3ꢀ(4ꢀ(Methylthio)phenyl)ꢀ1ꢀ
(IIb). Color: light yellow solid. Yield: 79.25%. mp:
111–113 C. IR: 3158–2968 (ArꢀCH), 1683 (C=O),
1558 (C=C); 1H NMR: 8.09 (1 H, d,
= 7.5, ꢀCH),
7.85–7.32 (8 H, m, ArꢀH), 7.58 (1 H, d, = 7.3, ꢀCH),
2.54 (3 H, s, SCH3), 2.32 (3 H, s, CH3); 13C NMR:
189.5, 145.5, 144.5, 139.5, 135.6, 134.9, 130.8, 129.9, s, SCH3), 2.32 (3 H, s, CH3), 2.18–2.02 (2 H, m, cyꢀ
129.6, 125.3, 124.9, 124.7, 121.5, 21.6, 14.3; MS,
268.15 (
+). Anal. calcd. for C17H16OS: C, 76.08; H,
6.01. Found: C, 76.05; H, 6.03%.
1ꢀ(4ꢀMethoxyphenyl)ꢀ3ꢀ(4ꢀ(methylthio)phenyl)propꢀ
2ꢀenꢀ1ꢀone (IIc). Color: light yellow solid. Yield: 80.10%.
mp: 98–100 C. IR: 3162–2953 (ArꢀCH), 1675 (C=O),
pꢀtolylpropꢀ2ꢀenꢀ1ꢀone
Values are means of triplicates.
Standard 10 mg/disc.
°
J
J
β
α
(C–C); 1H NMR: 7.97–7.35 (8 H, m, ArꢀH), 2.58 (3 H,
m/z:
cloproꢀCH ), 0.81 (2 H, d,
13C NMR: 192.5, 143.1, 142.5, 138.1, 133.3, 128.7,
128.5, 128.1. 125.9. 121.5, 120.4, 27.6, 24.8, 21.1,
14.8, 14.5; MS,
C18H18OS: C, 76.56; H, 6.42. Found: C, 76.55; H,
J = 6.6, cycloproꢀCH2);
M
m/z: 282.10 (M
+). Anal. calcd. for
6.44%.
°
1
1549 (C=C); H NMR: 7.98 (1 H, d,
7.91–7.32 (8 H, m, ArꢀH), 7.61 (1 H, d,
J
J
= 8.2,
= 8.2,
β
ꢀCH),
(4ꢀMethoxyphenyl)(2ꢀ(4ꢀ(methylthio)phenyl)cyꢀ
clopropyl)methanone (IIIc). Color: dark brown semiꢀ
solid. Yield: 76.90%. IR: 3183–2975 (ArꢀCH), 1689
(C=O), 1269 (C–C); H NMR: 7.98–7.31 (8 H, m,
ArꢀH), 3.82 (3 H, s, OCH3), 2.57 (3 H, s, SCH3),
αꢀCH),
13
3.82 (3 H, s, OCH3), 2.56 (3 H, s, SCH3); C NMR:
189.4, 166.5, 145.3, 139.5, 135.6, 130.9, 130.8, 130.5,
130.3, 125.9, 124.9, 124.7, 121.5, 114.9, 55.5, 14.3;
MS, m/z: 284.14 (M
+). Anal. calcd. for C17H16O2S: C,
71.80; H, 5.67. Found: C, 71.83; H, 5.66%.
1
2.15–2.01 (2 H, m, cycloproꢀCH ), 0.84 (2 H, d, J = 6.3,
cycloproꢀCH2); 13C NMR: 192.1, 165.1, 143.6, 138.3,
129.9, 129.0, 128.2, 125.9, 121.4, 120.4, 114.5, 55.5,
1,3ꢀbis(4ꢀ(Methylthio)phenyl)propꢀ2ꢀenꢀ1ꢀone (IId).
Color: light yellow solid. Yield: 76.03%. mp: 120– 27.4, 24.9, 14.6, 14.5; MS,
m/z: 298.13 (M
+). Anal.
calcd. for C18H18O2S: C, 72.45; H, 6.08. Found: C,
72.42; H, 6.07%.
122
°C. IR (KBr, ν
, cmꢀ1): 3158–2962 (ArꢀCH), 1680
= 8.2,
= 8.2,
ꢀCH), 2.54 (6 H, s, SCH3); 13C NMR: 189.3, 145.6,
(C=O), 1553 (C=C); 1H NMR: 8.02 (1 H, d,
J
β
ꢀCH), 7.85–7.35 (8 H, m, ArꢀH), 7.58 (1 H, d, J
(4ꢀ(Methylthio)phenyl)ꢀ(2ꢀ(4ꢀ(methylthio)pheꢀ
nyl)cyclopropyl)methanone (IIId). Color: dark brown
semisolid. Yield: 65.35%. IR: 3181–2976 (ArꢀCH),
1691 (C=O), 1273 (CꢀC); 1H NMR: 7.96–7.33 (8 H,
m, ArꢀH), 2.55 (6 H, s, SCH3), 2.11–2.00 (2 H, m,
α
145.2, 139.5, 135.6, 135.4, 134.4, 130.7, 128.9, 127.5,
125.9, 124.9, 124.7, 121.5, 14.5; MS, : 300.15
+). Anal. calcd. for C17H16OS2: C, 67.96; H, 5.37.
found: C, 67.95; H, 5.39%.
m/z
(M
cycloproꢀCH ), 0.87 (2 H, d, J = 6.8, cycloproꢀCH2);
13C NMR: 192.3, 143.6, 143.5, 138.4, 133.3, 132.0,
128.5, 126.6, 125.4, 121.3, 120.3, 27.4, 24.8, 14.9,
(2ꢀ(4ꢀ(Methylthio)phenyl)cyclopropyl)(phenyl)methꢀ
anone (IIIa). Color: dark brown semisolid. Yield:
71.40%. IR: 3183–2972 (ArꢀCH), 1685 (C=O),
14.4; MS,
m/z: 314.13 (M
+). Anal. calcd. for
C18H18OS2: C, 68.75; H, 5.77. Found: C, 68.72; H,
5.78%.
1
1273 (C–C); H NMR: 7.90–7.32 (9 H, m, ArꢀH),
2.58 (3 H, s, SCH3), 2.16–2.00 (2 H, m, cycloproꢀCH),
0.80 (2 H, d,
192.3, 143.6, 138.1, 136.5, 133.2, 128.9, 126.6, 128.1,
125.9, 121.5, 120.3, 27.1, 24.6, 14.6, 14.5; MS,
268.12 (
+). Anal. calcd. for C17H16OS: C, 76.08; H,
J
= 6.8, cycloproꢀCH2); 13C NMR:
4ꢀ(4ꢀ(Methylthio)phenyl)ꢀ1,2,3,4ꢀtetrahydronaphꢀ
thalenꢀ1(2H)ꢀone (IVa). Color: dark brown gummy
solid. Yield: 78.81%. IR: 3128–2935 (ArꢀCH), 1691
m/z:
1
M
(C=O); H NMR : 7.83–7.33 (9 H, m, ArꢀH), 4.22
(1 H, t,
6.01. Found: C, 76.07; H, 6.03%.
J = 4.6, CH), 2.64–2.26 (4 H, tt, J = 6.3,
CH2), 2.55 (3 H, s, SCH3), 13C NMR: 198.3, 141.6,
140.9, 140.5, 134.7, 133.6, 129.5, 128.5, 128.2, 126.5,
(2ꢀ(4ꢀ(Methylthio)phenyl)cyclopropyl)(pꢀtolyl)methꢀ
anone (IIIb). Color: dark brown semisolid. Yield:
69.65%. IR: 3186–2974 (ArꢀCH), 1688 (C=O), 1270 45.5, 37.6, 31.4, 14.9; MS,
m/z: 268.07 (M
+). Anal.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
No. 4
2014

474
UMESHA, BASAVARAJU
calcd. for C17H16OS: C, 76.08; H, 6.01. Found: C, 144.9, 141.2, 140.6, 139.5, 130.5, 130.0, 129.5, 128.5,
128.4, 127.2, 117.2, 42.8, 35.3, 21.8, 14.6 ; MS,
310.12 (
+). Anal. calcd. for C19H18O2S: C, 73.52; H,
5.84. Found: C, 73.50; H, 5.85%.
2ꢀ(Hydroxymethylene)ꢀ6ꢀmethoxyꢀ4ꢀ(4ꢀ(methꢀ
ylthio)phenyl)ꢀ1,2,3,4ꢀtetrahydronaphthalenꢀ1(2 )ꢀ
one (Vc). Color: dark brown solid. Yield: 85.95%. mp:
m/z:
76.09; H, 6.03%.
M
6ꢀMethylꢀ4ꢀ(4ꢀ(methylthio)phenyl)ꢀ1,2,3,4ꢀtetrahyꢀ
dronaphthalenꢀ1(2H)ꢀone (VIb). Color: dark brown
gummy solid. Yield: 71.94%. IR: 3125–2938 (ArꢀCH),
1695 (C=O); 1H NMR: 7.89–7.32 (7 H, m, ArꢀH), 4.26
H
(1 H, t,
J = 4.8, CH), 2.65–2.28 (4 H, tt, J = 6.5,
CH2), 2.54 (3 H, s, SCH3), 2.34 (3 H, s, CH3);
162–164°C. IR: 3267 (O–H), 3175–2969 (ArꢀCH),
1695 (C=O); 1H NMR: 7.76–7.18 (7 H, m, ArꢀH), 5.7
(1 H, bs, OH vinyl), 5.39 (1 H, s, CHOH), 4.07 (1 H,
13C NMR: 198.1, 143.6, 141.4, 140.6, 140.1, 131.0,
129.6, 128.5, 128.1, 126.1, 125.4, 45.9, 37.6, 31.6,
21.5, 14.5; MS,
m/z: 282.14 (M
+). Anal. calcd. for
C18H18OS: C, 76.56; H, 6.42. Found: C, 76.58; H,
6.45%.
t,
J
J
= 5.2, CH), 3.84 (3 H, s, OCH3), 2.76 (2 H, d,
= 6.4, CH2), 2.52 (3 H, s, SCH3); 13C NMR: 183.3,
172.4, 166.8, 142.8, 140.2, 139.5, 129.5, 128.6, 125.1,
117.5, 112.5, 105.3, 55.6, 42.6, 35.5, 14.2; MS,
326.14 (
+). Anal. calcd. for C19H18O3S: C, 69.91; H,
5.56. Found: C, 69.93; H, 5.55%.
2ꢀ(Hydroxymethylene)ꢀ6ꢀ(methylthio)ꢀ4ꢀ(4ꢀ(methꢀ
ylthio)phenyl)ꢀ1,2,3,4ꢀtetrahydronaphthalenꢀ1(2 )ꢀone
(Vd). Color: dark brown solid. Yield: 61.87%. mp:
m/z:
6ꢀMethoxyꢀ4ꢀ(4ꢀ(methylthio)phenyl)ꢀ1,2,3,4ꢀtetꢀ
M
rahydronaphthalenꢀ1(2H)ꢀone (IVc). Color: dark
brown gummy solid. Yield: 75.18%. IR: 3128–2939
(ArꢀCH), 1697 (C=O); 1H NMR: 7.88–7.35 (7 H, m,
H
ArꢀH), 4.24 (1 H, t, J = 4.7, CH), 2.66–2.25 (4 H, tt,
J
= 6.4, CH2), 3.84 (3 H, s, OCH3), 2.53 (3 H, s,
175–177°C. IR: 3265 (O–H), 3175–2972 (ArꢀCH),
SCH3); 13C NMR: 198.0, 165.9, 141.7, 140.1, 130.5,
129.5, 128.5, 126.5, 111.9, 104.7, 55.7, 45.6, 37.4,
1696 (C=O); 1H NMR: 7.71–7.21 (7 H, m, ArꢀH), 5.5
(1 H, bs, OH vinyl), 5.37 (1 H, s, CHOH), 4.09 (1 H, t,
31.4, 14.5; MS,
m/z: 298.15 (M
+). Anal. calcd. for
C18H18O2S: C, 72.45; H, 6.08. Found: C, 72.40; H,
6.09%.
J
= 5.7, CH), 2.76 (2 H, d, J = 6.3, CH2), 2.56 (6 H, s,
SCH3); 13C NMR: 183.3, 172.5, 145.3, 141.9, 140.2,
139.6, 130.5, 129.4, 129.1, 128.5, 124.9, 123.6, 117.3,
6ꢀ(Methylthio)ꢀ4ꢀ(4ꢀ(methylthio)phenyl)ꢀ1,2,3,4ꢀ
42.2, 35.1, 14.8; MS (ESI),
m/z: 342.09 (M
+). Anal.
calcd. for C19H18O2S2: C, 66.63; H, 5.30. Found: C,
66.60; H, 5.33%.
tetrahydronaphthalenꢀ1(2
brown gummy solid. Yield: 64.71%. IR (KBr,
3125–2938 (ArꢀCH), 1688 (C=O); 1H NMR: 7.84–7.21
(7 H, m, ArꢀH), 4.21 (1 H, t, = 4.6, CH), 2.61–2.10
(4 H, tt,
= 5.9, CH2), 2.56 (6 H, s, SCH3); 13C NMR:
198.2, 1439, 141.5, 140.6, 140.0, 130.5, 129.4, 129.3,
128.6, 124.4, 123.2, 44.7, 37.4, 31.3, 14.8; MS,
314.12 (
+). Anal. calcd. for C18H18OS2: C, 68.75; H,
5.77. Found: C, 68.73; H, 5.74%.
2ꢀ(Hydroxymethylene)ꢀ4ꢀ(4ꢀ(methylthio)pheꢀ
nyl)ꢀ1,2,3,4ꢀtetrahydronaphthalenꢀ1(2 )ꢀone (Va).
Color: dark brown solid. Yield: 81.63%. mp: 151–153 C.
IR (KBr,
, cm–1): 3268 (O–H), 3170–2964 (Arꢀ
CH), 1695 (C=O); H NMR (CDCl3 400 MHz)
ppm: 7.74–7.17 (8 H, m, ArꢀH), 5.8 (1 H, bs, OH viꢀ
nyl), 5.37 (1 H, s, CHOH), 4.05 (1 H, t, = 5.4, CH),
2.76 (2 H, d,
= 6.1, CH2), 2.54 (3 H, s, SCH3); 13C
NMR (CDCl3 100 MHz) ppm: 183.4, 172.6, 141.6,
140.3, 139.5, 135.2, 133.4, 129.3, 128.7, 128.6, 127.9,
126.4, 117.3, 42.5, 35.5, 14.6; MS, : 296.11 (
+).
H
)ꢀone (IVd). Color: dark
ν
, cm–1):
5ꢀ(4ꢀ(Methylthio)phenyl)ꢀ4,5ꢀdihydroꢀ2
zo[g]indazole (VIa). Color: dark brown solid. Yield:
85.90%. mp: 153–155 C. IR: 3265 (N–H), 3153ꢀ
2965 (ArꢀCH); H NMR: 12.48 (1 H, s, NH), 7.69–
7.21 (8 H, m, ArꢀH), 7.55 (1 H, s, pyrazoleꢀCH), 4.36
Hꢀbenꢀ
J
J
°
1
m/z:
M
(1 H, t,
J = 6.7, CH), 3.29 (2 H, dd, J = 4.3, CH2),
2.54 (3 H, s, SCH3); 13C NMR: 144.2, 140.9, 140.1,
139.5, 133.4, 129.3, 128.9, 128.6, 126.8, 126.3, 123.5,
H
120.6, 114.8, 45.9, 37.3, 14.5; MS, m/z: 292.13 (M
+).
Anal. calcd. for C18H16N2S: C, 73.94; H, 5.52; N,
9.58. Found: C, 73.95; H, 5.50; N, 9.59%.
°
ν
1
δ
7ꢀMethylꢀ5ꢀ(4ꢀ(methylthio)phenyl)ꢀ4,5ꢀdihydroꢀ
2
H
ꢀbenzo[g]indazole (VIb). Color: dark brown solid.
Yield: 75.95%. mp: 148–150 C. IR: 3269 (N–H),
3155–2963 (ArꢀCH); H NMR: 12.47 (1 H, s, NH),
7.73–7.20 (7 H, m, ArꢀH), 7.52 (1 H, s, pyrazoleꢀ
J
°
J
1
δ
CH), 4.38 (1 H, t, J = 6.5, CH), 3.28 (2 H, dd, J = 4.1,
CH2), 2.83 (3 H, s, CH3), 2.52 (s, 3H, SCH3); 13C
NMR: 144.3, 140.5, 140.1, 139.5, 138.9, 133.4, 130.6,
129.4, 128.3, 127.9, 127.3. 125.9, 114.6, 46.2, 37.5,
m/
z
M
Anal. calcd. for C18H16O2S: C, 72.94; H, 5.44. Found:
C, 72.93; H, 5.42%.
21.7, 14.9; MS,
m/z: 306.14 (M
+). Anal. calcd. for
C19H18N2S: C, 74.47; H, 5.92; N, 9.14. Found: C,
74.49; H, 5.90; N, 9.15%.
2ꢀ(Hydroxymethylene)ꢀ6ꢀmethylꢀ4ꢀ(4ꢀ(methylthio)ꢀ
phenyl)ꢀ1,2,3,4ꢀtetrahydronaphthalenꢀ1(2
Color: dark brown solid. Yield: 84.48%. mp: 149–
151 C. IR: 3269 (O–H), 3177–2968 (ArꢀCH), 1699
(C=O); H NMR: 7.73–7.15 (7 H, m, ArꢀH), 5.8
(1 H, bs, OH vinyl), 5.38 (1 H, s, CHOH), 4.06 (1 H, t,
= 5.6, CH), 2.84 (3 H, s, CH3), 2.77 (2 H, d, = 6.3,
CH2), 2.55 (3 H, s, SCH3); 13C NMR: 183.6, 172.4,
H)ꢀone (Vb).
°
7ꢀMethoxyꢀ5ꢀ(4ꢀ(methylthio)phenyl)ꢀ4,5ꢀdihydroꢀ
1
2
H
ꢀbenzo[g]indazole (VIc). Color: dark brown solꢀ
id. Yield: 81.05%. mp: 163–165 C. IR: 3267 (N–
H), 3151–2960 (ArꢀCH); H NMR: 12.51 (1 H, s,
NH), 7.75–7.25 (7 H, m, ArꢀH), 7.54 (1 H, s, pyraꢀ
°
1
J
J
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
No. 4
2014

SYNTHESIS AND PHARMACOLOGICAL STUDIES
475
zoleꢀCH), 4.39 (1 H, t,
OCH3), 3.29 (2 H, dd,
J
J
= 6.3, CH), 3.83 (3 H, s, The slides were observed under microscope and phoꢀ
tographed.
= 4.6, CH2), 2.52 (3 H, s,
SCH3); 13C NMR: 161.3, 144.3, 141.7, 140.2, 139.5,
133.2, 129.6, 129.3, 128.6, 121.3, 114.5, 112.9, 112.4,
Total number of dividing cells
Total number of cells examined
M.I. = ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ × 100.
55.9, 46.0, 37.5; MS, m/z: 322.12 (M
+). Anal. calcd.
for C19H18N2OS: C, 70.78; H, 5.63; N, 8.69. Found:
C, 70.76; H, 5.65; N, 8.67%.
The percentage of the number of dividing cells
compared to the control and the percent inhibition of
mitosis by antimitotic agent at a different concentraꢀ
tion such as 100, 200, and 400 ppm against a control
were calculated. The concentration needed for 50%
inhibition (ID50) was extrapolated from the graph of
the concentration verses percentage inhibition. ID50
values for new pyrazole analogues of podophyllotoxin
for antimitotic activity were calculated individually.
7ꢀ(Methylthio)ꢀ5ꢀ(4ꢀ(methylthio)phenyl)ꢀ4,5ꢀdiꢀ
hydroꢀ2Hꢀbenzo[g]indazole (VId). Color: dark brown
solid. Yield: 65.91%. mp: 168–170
1
°
C. IR: 3269 (N–
H), 3155–2963 (ArꢀCH); H NMR: 12.55 (1 H, s,
NH), 7.76–7.23 (7 H, m, ArꢀH), 7.53 (1 H, s, pyraꢀ
zoleꢀCH), 4.38 (1 H, t,
= 4.5, CH2), 2.54 (6 H, s, SCH3); 13C NMR: 144.3,
140.9, 140.3, 139.6, 139.1, 133.4, 129.4, 128.5, 128.4,
125.5, 124.9, 123.9, 114.8, 45.3, 37.2, 14.9; MS,
338.12 (
+). Anal. calcd. for C19H18N2S2: C, 67.42;
J = 6.3, CH), 3.23 (2 H, dd,
Antimicrobial Studies
Antibacterial Activity
J
m/z:
The antimicrobial assay was performed by agar disc
diffusion method [22, 23]. For antibacterial activity,
the molten Mueller Hinton Agar (HiMedia) was inocꢀ
M
H, 5.36; N, 8.28. Found: C, 67.40; H, 5.39; N, 8.25%.
ulated with the 100 μL of the inoculum (1 ×
108 Cfu)
Testing of Biological Activities
and poured into the sterile Petri plates (HiMedia). For
agar disc diffusion method, the disc (0.7 cm) (HiMeꢀ
dia) was saturated with 100 μL of 10.0 mg/mL of the
Antimitotic Studies
test compound in the dimethylformamide (DMF),
allowed to dry and was introduced on the upper layer
of the seeded agar plate. The plates were incubated
The antimitotic activity of synthesized new pyraꢀ
zole analogues of podophyllotoxin (VIa–d) were
examined using onion root tip method and the ID50
was determined. Materials required are acetoorcein
solution, compound microscope, glass slides, cover
slips, hydrochloric acid (0.1 N), Carney’s solution II
70% ethanol and tested samples (100, 200, and 400 ppm).
To study the effect of new pyrazole analogues of podoꢀ
phyllotoxin on somatic cells, onion base was
immersed to an extent of about half a centimeter in a
overnight at 37°C for 24 h. Antibacterial activity of all
the new pyrazole analogues of podophyllotoxin was
evaluated by measuring the zone of growth inhibition
against the test organisms with zone reader (Hi antibiꢀ
otic zone scale). The medium with dimethylformaꢀ
mide (DMF) as solvent was used as a negative control
whereas media with Chloromphenicol (standard antiꢀ
bacterial drug) was used as positive control. The experꢀ
iments were performed in triplicates.
sample tube and control solution tube compounds (7 × 3)
after removing the old root from it and immersion is
continued for 12 h, 18 h and 24 h. intervals respectively
for germination. After different time intervals, the gerꢀ
minated root tips were removed and were fixed in Carꢀ
ney’s solution II (alcohol and acetic acid in 3 : 1 ratio
respectively) for 24 h. After 24 h Carney’s solution II
was decanted carefully and the root tips were washed
with preserving solvent (70% ethanol). The fixed root
tips were persevered in 70% ethanol in refrigerator.
The root tips were taken in watch glass and stained
with a drop of acetoorcein stain and a drop of 1 N HCl
(7 : 1). The glasses were warmed and kept for 1 h. The
roots were taken on a clean glass slide and squashed
using 45% acetic acid following the method of Levan
[20]. A microscope cover glass was placed on the
material and then pressure was applied on a cover glass
to ensure uniform spreading. The cover glass was
shield with molten paraffin wax and slide was observed
under microscope. Mitotic index was calculated by
following method of Fissceja [21]. The mitotic index
was determined by examination minimum of zone
Antifungal Activity
New pyrazole analogues of podophyllotoxin were
dissolved in dimethylformamide (DMF) and evaluꢀ
ated for their antifungal activity by disc diffussion
method. For agar disc diffusion method, one week old
culture of the mold was used as inoculums for evaluatꢀ
ing antifungal activity of chemical compounds. The
molten Mueller Hinton Agar (HiMedia) was inocuꢀ
lated with the 100
and poured into the sterile Petri plates (HiMedia) and
the disc (0.7 cm) (HiMedia) was saturated with 100
μL of the inoculum (1 ×
108 CFU)
μL
of 10.0 mg/mL of the test compound in the dimethylꢀ
formamide (DMF), allowed to dry and was introꢀ
duced on the upper layer of the seeded agar plate. The
plates were incubated overnight at 25°C for 7 days.
Antifungal activity of all the new pyrazole analogues of
podophyllotoxin was evaluated by measuring the zone
of growth inhibition against the test organisms with
zone reader (Hi antibiotic zone scale). The medium
with dimethylformamide (DMF) as solvent was used
cells. Three replicates were made for each calculation. as a negative control where as media with Nystatin
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
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2014

476
UMESHA, BASAVARAJU
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