Arabian Journal of Chemistry (2015) xxx, xxx–xxx

King Saud University

Arabian Journal of Chemistry

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ORIGINAL ARTICLE

Design, synthesis of novel N prenylated indole-3-

carbazones and evaluation of in vitro cytotoxicity
and 5-LOX inhibition activities
Praveen Choppara a, Y.V. Prasad a, C.V. Rao b, K. Hari Krishna b,
G. Trimoorthulu b, G.U. Maheswara Rao b, J. Venkateswara Rao c,
M.S. Bethu c, Y.L.N. Murthy a,*

a
Department of Organic Chemistry, Foods, Drugs & Water, Andhra University, Visakhapatnam 530 003, India
b
Resaearch & Development Centre, Laila impex, Vijayawada 520 007, India
c
Biology Division, Indian Institute of Chemical Technology, Hyderabad 500 007, (AP), India

Received 23 August 2014; accepted 11 February 2015

KEYWORDS Abstract A series of novel N-1 and C-3 substituted indole derivatives (5a–f) were designed, synthe-
Brine shrimp lethality sized and evaluated for their cytotoxic properties, viz Brine Shrimp Lethality Bioassay (BSLB) besides
bioassay; 5-Lipoxygenase (5-LOX) inhibitory activities through in vitro assays. Structure Activity Relation
Cytotoxicity; (SAR) studies showed that compound 5d with an LC50 of 6.49 lM and 5c with an IC50 of 33.69 lM
5-Lipoxygenase inhibition; were found to be interesting for cytotoxicity and 5-LOX inhibitory activity respectively.
N prenylated ª 2015 Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access
indole-3-carbazones article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction activities (Diana et al., 2011a,b,c; Carbone et al., 2013, 2014)

and is widely used as a scaffold in agricultural and medicinal
Indole derivatives are an important class of heterocyclic com- chemistry. In particular, N-1 and C-3-substituted indole deriva-
pounds with a wide range of biological activities (Bandini and tives have been found to play an important role in many
Eichholzer, 2009). Indole is a substructural element of many biologically active compounds especially with anti inflammato-
natural products which is an important pharmacophore moiety ry (Hall et al., 2008; Singh et al., 2008), anti cancer (Madadi
of a large number of molecules with significant biological et al., 2014; Singh et al., 2008; Mashayekhi et al., 2013), anti
nociceptive (Adam et al., 2010; Moir et al., 2010) and anti psy-
* Corresponding author. Tel.: +91 9849229804; fax: +91 891 chotic activity (Madadi et al., 2013). On the other hand, car-
2713813. bazones (semicarbazones, thiosemicarbazones) are
E-mail address: murthyyln@gmail.com (Y.L.N. Murthy). compounds of considerable interest because of their important
Peer review under responsibility of King Saud University. chemical properties and potentially beneficial biological
activities.
Literature survey reveals that carbazone analogues attain a
wide range of biological activities. Thiosemicarbazones appear
Production and hosting by Elsevier to be a structural class with anti-pox virus activity (Katz, 1987;
http://dx.doi.org/10.1016/j.arabjc.2015.02.006
1878-5352 ª 2015 Production and hosting by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Choppara, P. et al., Design, synthesis of novel N prenylated indole-3-carbazones and evaluation of in vitro cytotoxicity and 5-LOX
inhibition activities. Arabian Journal of Chemistry (2015), http://dx.doi.org/10.1016/j.arabjc.2015.02.006
2 P. Choppara et al.

Katz et al., 1975; Rao et al., 1966). Methisazone (I) plays an
important role as prophylactic agent against several viral dis-
eases (Sethi, 2002). Thiosemicarbazone is the important phar-
macophore in the therapy and prophylaxis of mycobacterial
infections and can represent a template for the development
of novel antimycobacterial drugs such as SRI-224 (II), SRI-
286 (III) (Bermudez et al., 2003; Dover et al., 2007),
Oxazolyl thiosemicarbazones (IV) (Sriram et al., 2006) and
some S-alkylisothiosemicarbazones (V) (Cocco et al., 2002;
Logu et al., 2005). Thiacetazone (VI) is a thiosemicarbazone
analogue which has been widely used for the treatment of
Multi Drug Resistant-Tuberculosis (MDR-TB) in many devel-
oping countries (Houston and Fanning, 1994).
Thiosemicarbazide analogues possess a wide range of biologi-
cal activities including anticonvulsant (Tripathi et al., 2012),
antimicrobial (Zhong et al., 2011), antiviral (Garcial et al.,
2003), antitrypanosomal (Moreira et al., 2014), and mushroom
tyrosinase inhibitors (Yi et al., 2011). On the other hand,
Semicarbazide analogues exhibit anticonvulsant (Rajak et al.,
2013), antitubercular (Sriram et al., 2004), antitrypanosomal
(Cerecetto et al., 2000), anti inflammatory (Vieira et al.,
2012), anti amnesic, cognition enhancing and anti-
cholinesterase (Sinha and Shrivastava, 2013) and anticancer
activity (Qi et al., 2013).
and later at third position (C-3) of indole. The modifications
mainly include replacement of hydrogen at N–H by prenyla-
tion (2-methyl but-2-ene) and formation of carbazones at C-
3 with semicarbazide/thiosemicarbazide to increase its phar-
macological efficacy.
The target molecules (5a–f) were achieved by Vilsmeier–
Haack reaction of 5-substituted indoles (1a–c) yields 5-substi-
tuted 3-formyl indoles (2a–c), the modification at N–H posi-
tion was brought by the reaction with Prenyl bromide in
presence of NaH, and DMF at 0 C for 15 min yields N-preny-
lated 3-formyl indoles (3a–c). The formation of carbazones at
C-3 was accompanied by the reaction of compounds (3a–c)
with semicarbazide/thiosemicarbazide (4a, b). The synthetic
path involved in synthesis of 1-(5-substituted-1-(3-methylbut-
2-enyl)- 1H-indole-3-yl)methylene) carbazides (5a–f) was pre-
sented in Scheme 1. All the synthesized compounds were well
characterized by advanced spectroscopic techniques.
2.2. Bioevaluation
2.2.1. In vitro cytotoxic activity by Brine Shrimp Lethal
Bioassay (BSLB)
Brine Shrimp Lethal Bioassay (BSLB) (Meyer et al., 1982), i.e.
toxicity to the Artemia salina (brine shrimp) nauplii was per-

S
NH2
CH3
N NH H H
N NH2 N NH2
N N
O S S
N MeO N
CH3

I II III

H
N N NH2
H N NH2 CH3 N
N N R Ar N
O N S
H S O N
S R H
R1

IV V VI

Thus the importance of indole and carbazone nuclei in

medicinal chemistry prompted us to synthesize novel N preny-
lated indole carrying carbazones at C-3 position and in order
to extend the boundaries of pharmacological properties of
indole and carbazone moieties and the target compounds were
screened for cytotoxic (BSLB) and 5-LOX inhibitory activities.
2. Results and discussion
2.1. Chemistry
Due to various biological and pharmacological activities asso-
ciated with indole and carbazone skeletons, it is worthwhile to
incorporate the two moieties in a single molecular frame with
an assumption to obtain more potent biologically active com-
pounds, such as 1-(5-substituted-1-(3-methylbut-2-enyl)-1H-
indole-3-yl)methylene) carbazides (5a–f). The target molecules
have been accomplished by modifying first at N–H position
formed and has been used for establishing the fungal toxins,
plant extract toxicity, heavy metals, cyano bacteria toxins, pes-
ticides, cytotoxicity testing of dental materials (Carballo et al.,
2002), natural and synthetic organic compounds (Choudhary
and Thomsen, 2001). Toxicity to A. salina has a good correla-
tion with antitumor, pesticidal (Mc Laughlin, 1991), antitry-
panosomal activities (Zani et al., 1995), rodent and human
acute oral toxicity data. The brine shrimp larvae respond
similarly to the corresponding mammalian systems (Solis
et al., 1993). The DNA-dependent RNA polymerases of A.
salina have been shown to be similar to the mammalian type
(Birndorf et al., 1975). This test is not only used for predicting
cytotoxicity, but also used to predict antitumor, antibacterial
and pesticidal activities (Sanchez et al., 1993). The brine
shrimp lethality method is widely used in the bioassay for iden-
tification of the bioactive compounds (Venkateswara Rao
et al., 2007; Olowa and Nuneza, 2013). The bioassay determi-
nes lethal concentrations of newly synthesized 1-(5-substituted-

Please cite this article in press as: Choppara, P. et al., Design, synthesis of novel N prenylated indole-3-carbazones and evaluation of in vitro cytotoxicity and 5-LOX
inhibition activities. Arabian Journal of Chemistry (2015), http://dx.doi.org/10.1016/j.arabjc.2015.02.006
Design, synthesis of N prenylated indole-3-carbazones 3

CHO CHO
R R Br R
DMF, POCl3

N NaOH, H2O N
H NaH, DMF N
H
0oC, 15 min
1a-c 2a-c 3a-c

H
N NH2.HCl
H2N
X 4 a,b
R X
1a, 2a, 3a H - KOH, EtOH
1b, 2b, 3b Br - reflux
1c, 2c, 3c CN -
4a - O
4b - S
5a H O X
5b H S
5c Br O CH=N-NH-C-NH2
5d Br S R
5e CN O
5f CN S N

5a-f

Scheme 1 Synthetic scheme for the synthesis of 1-(5-substituted-1-(3-methylbut-2-enyl)- 1H-indole-3-yl)methylene) carbazides (5a-f).

1-(3-methylbut-2-enyl)-1H-indole-3-yl)methylene) carbazides synthesized compounds, unsubstituted analogues (5a, 5b)

(5a–f) in brine medium adopting the microplate assay devel-
oped by Meyer et al., 1982. The Median lethal concentration
(LC50) values of the target compounds were compared with
the LC50 value of the positive control, Podophyllotoxin. The
LC50 values (lM) for the test compounds (5a–f) obtained from
BSLB were tabulated in Table 1.
The results from Table 1 show that compound 5d exhibits
good cytotoxicity with LC50 of 6.49 lM and its relative active
(1.29) was close-at-hand to Podophyllotoxin (1.00). Among
the semicarbazides (5a,5c,5e), unsubstituted indole analogue
(5a) was found to be weak than the substituted analogues
(5c and 5e), bromo analogue (5c) was interesting candidate
with LC50 of 206.04 lM followed by cyano analogue (5e) with
LC50 274.02 lM and their relative activities were 39.29 and
44.32 respectively. The results of thiosemicarbazides
(5b,5d,5f) showed that bromo analogue (5d) was found to be
interesting with LC50 of 6.49 lM, then cyano analogue (5f)
with 205.27 lM and unsubstituted indole analogue (5b) was
weak with LC50 of 315.95 lM. In conclusion, among the
showed poor activity than the corresponding substituted ana-
logues (5c–f).
2.2.2. 5-Lipoxygenase inhibition assay
Lipoxygenases (LOXs), which are widely distributed in both
the plant and animal kingdoms, belong to a class of non-heme
iron-containing enzymes which catalyze the hydroperoxidation
reaction of fatty acids to peroxides (Peters-Golden and
Henderson, 2007). 5-Lipoxygenase (5-LOX) is the key enzyme
in the biosynthesis of leukotrienes (LTs) through catalyzing
the initial two steps in conversion of arachidonic acid to LTs
(Samuelson, 1983). LTB4 and the cysteinyl-leukotrienes are
potent constrictors of human airways, and have powerful
proinflammatory properties (Funk, 2001). Inhibition of 5-
LOX may lead to the development of new therapeutic treat-
ments for pathologies such as asthma, allergies, and other
inflammatory disorders (Samuelson, 1983, Young, 1999,
Drazen, 1998). Recent studies have implicated a role of
5-LOX products involved in a number of other diseases,

Table 1 In vitro cytotoxicity of title compounds (5a–f) by Brine Shrimp Lethal Bioassay.
*
Entry Compound LC50 ± SE (lM) Logarithmic Regression equation 95% Confidence fiducial limits Relative activity
Y = Ybar + b (x  xbar)
LCL UCL
1 5a 477.14 ± 44.66 Y = 0.61 + 2.66x 114.72 149.79 70.39
2 5b 315.95 ± 16.91 Y = 0.42 + 2.34x 81.45 105.66 49.55
3 5c 206.04 ± 11.57 Y = 0.71 + 2.31x 64.79 79.21 39.29
4 5d 6.49 ± 0.30 Y = 3.59 + 3.74x 2.158 2.599 1.29
5 5e 274.02 ± 11.08 Y = 0.83 + 3.05x 74.99 87.86 44.32
6 5f 205.27 ± 9.06 Y = 0.11 + 2.83x 58.39 69.42 34.88
7 Standard  4.42 ± 0.28 Y = 4.10 + 3.40x 1.63 2.02 1.00
48 h old nauplii of brine shrimp (A. salina) were treated with different concentrations of indole carbazides (5a-f) for 24 h and lethal concen-
tration was investigated through probit analysis.*Regression equation Y = Ybar + b (X  xbar) or (Ybar  b * xbar) + b * x). Ybar = weighted
avg. of working probit values; xbar = avg. of log 10 (concentration); b = slope of the line; X = x scale concentration in Log).  Podophyllotoxin
used as positive control. Three replicates for each concentration and the control (without test compound), were tested for lethal bio-efficacy and
the mean data were used to calculate the probit regression equation. *LC50 is defined as the concentration of extract that can cause 50%
mortality in the exposed population.

Please cite this article in press as: Choppara, P. et al., Design, synthesis of novel N prenylated indole-3-carbazones and evaluation of in vitro cytotoxicity and 5-LOX
inhibition activities. Arabian Journal of Chemistry (2015), http://dx.doi.org/10.1016/j.arabjc.2015.02.006
4 P. Choppara et al.
including cancer (Romano and Claria, 2003), atherosclerosis
(Spanbroek and Habenicht, 2003), stroke (Helgadottir et al.,
2004) and osteoporosis (Werz and Steinhilber, 2005).
Therapeutic potential of 5-LOX inhibition has been widely
highlighted in recent years (Balkan and Berk, 2003; Funk,
2005; Yoshimura et al., 2005). Literature survey reveals N-1
and C-3 substituted indoles are identified as 5-LOX inhibitors
(Prasher et al., 2014; Singh and pooja, 2013; Zheng et al.,
2007). Based on the literature survey the novel 1-(5-substitut-
ed-1-(3-methylbut-2-enyl)-1H-indole-3-yl) methylene) car-
bazides (5a–f) were screened for their inhibitory properties
against 5-LOX enzyme employing the assay described by
Reddanna et al., 1990. 5-LOX inhibition of the tested com-
pounds was determined by measuring the Inhibition
Concentration; IC50 (IC50 represents the concentration of a
drug that is required for 50% inhibition) expressed in lM.
The results for test compounds (5a–) were compared with
the positive control Curcumin. The IC50 values for the test
compounds (5a–f) obtained from 5-LOX inhibition assay were
tabulated in Table 2.
The results obtained from Table 2 shows that the unsubsti-
tuted (5a, 5b) and cyano (5e, 5f) analogues were weak with an
IC50 value of >100 lM and bromo analogues (5c, 5d) were the
engrossing compounds compared with the standard ‘‘curcum-
in’’ with an IC50 of 33.69 and 36.65 lM respectively.
2.2.3. Structure–Activity Relationship Study (SARS)
In 1-(5-substituted-1-(3-methylbut-2-enyl)-1H-indole-3-
yl)methylene) carbazides (5a–f), substituent plays an important
role to make difference in the activity. From the results of
Brine Shrimp Lethal Bioassay as shown in Table 1, compound
5d (Table 1 entry-4) is more potent among the series, in which
bromine is present on the 5th position of indole and thiosemi-
carbazone at 3rd position. From the results of 5-LOX inhibi-
tion assay as shown in Table 2, compound 5c (Table 2 entry-
3) is more potent 5-LOX inhibitor. In compound 5c bromine
is present on the 5th position of indole and semicarbazone at
3rd position.
Bioevaluation assays reveal that among the target com-
pounds (5a–f), compounds 5c and 5d exhibit potent activity.
In both the compounds 5c and 5d bromine is present on the
5th position of indole. The 3rd position was occupied by semi-
carbazone and thiosemicarbazone in compounds 5c and 5d
respectively. In the SAR studies of novel 1-(5-substituted-1-
(3-methylbut-2-enyl)-1H-indole-3-yl)methylene) carbazides
(5a–f), it was observed that the presence of hydrophobic sub-
stituent (bromine) at 5th position of indole moiety makes the
Table 2 IC50 values obtained from in vitro 5-Lipoxygenase
inhibition assay for the compounds (5a–f).
Entry Compound
1 5a
2 5b
3 5c
4 5d
5 5e
6 5f
7 Standarda
IC50 represents the concentration of a drug that is required for 50%
inhibition expressed in lM.
a
Curcumin as positive control.
Please cite this article in press as: Choppara, P. et al., Design, synthesis of novel N prenylated indole-3-carbazones and evaluation of in vitro cytotoxicity and 5-LOX
inhibition activities. Arabian Journal of Chemistry (2015), http://dx.doi.org/10.1016/j.arabjc.2015.02.006
compounds 5c and 5d more potent than unsubstituted ana-
logues (5a, 5b) and cyano analogues (5e, 5f) which lack
hydrophobic substituent.
3. Experimental
3.1. General
All chemical reagents were obtained from Sigma Aldrich and
were used without further purification. Melting points were
determined in open capillaries and are uncorrected. Infrared
(IR) spectra were recorded using FT-IR Bruker Alpha spec-
trometer, ESI-Mass spectra were recorded on Finnigan Matt
Mass spectrometer and NMR (1H and 13C) spectra were record-
ed with a Bruker Ascend-400 and Jeol JNM EX-90 spectrometer.
3.2. General procedure for the synthesis of substituted 1H-
indole-3-carbaldehyde (2a–c)
To a solution of substituted indoles (1a–c) (42.6 mmol) in dry
DMF (187.4 mmol) in an ice-salt bath and POCl3 (47.1 mmol)
is subsequently added with stirring over a period of 30 min.
After completion of addition, raise the temperature to 40 C
and stir the syrup for 1.5 h at that temperature. At the end
of the reaction (as indicated by TLC) 25 gms crushed ice was
added to the reaction mixture. The obtained solution is trans-
ferred into 250 mL RB flask, added with NaOH (470 mmol)
dissolved in 50 mL water with constant stirring and the resul-
tant suspension is heated rapidly to the boiling point and
allowed to cool to room temperature, after which it is placed
in refrigerator overnight. The precipitate was filtered off and
washed thrice with 100 mL water, yielding substituted 1H-in-
dole-3-carbaldehyde (2a–c).
3.2.1. 1H-indole-3-carbaldehyde (2a): (James and Snyder,
1959)
Yield: 92%; Mp: 196–198 C. 1H NMR (DMSO-d6,
400 MHz): d 7.14 (t, J = 7.2 Hz, 1H), 7.22 (t, J = 7.2 Hz,
1H), 7.34 (d, J = 8 Hz, 1H), 7.52 (s, 1H), 7.62 (d, J = 8 Hz,
1H), 8.12 (s, 1H), 9.52 (s, 1H). 13C NMR (DMSO-d6,
22.4 MHz): d 111.4, 118.0, 119.4, 120.5, 122.4, 127.7, 131.8,
137.2, 182.7. ESI-MS: m/z 146.20 [M+H]+. IR (KBr, cm1):
1224, 1632, 3442. Anal. Calcd. for C9H7NO: C, 74.47, H,
4.86, N, 9.65%; Found: C, 74.44, H, 4.91, N, 9.64%.
3.2.2. 5-bromo-1H-indole-3-carbaldehyde (2b)
Yield: 90%; Mp: 192 C. 1H NMR (DMSO-d6, 400 MHz): d
7.34 (d, J = 8.8 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.75 (s,
1H), 8.25 (s, 1H), 8.32 (s, 1H), 9.94 (s, 1H). 13C NMR
IC50 (lM) (DMSO-d6, 22.4 MHz): d 113.0, 114.8, 117.3, 123.1, 125.6,
135.2, 136.7, 144.4, 183.9. ESI-MS: m/z 245.95 [M+Na]+;
>100
>100 247.95 [M+Na+2]+. IR (KBr, cm1): 1229, 1643, 3312.
33.69 Anal. Calcd. for C9H6BrNO: C, 48.25, H, 2.70, N, 6.25%;
36.65 Found: C, 48.22, H, 2.76, N, 6.24%.
>100
>100 3.2.3. 3-formyl-1H-indole-5-carbonitrile (2c)
27.58
Yield: 84%; Mp: 220–224 oC. 1H NMR (DMSO-d6,
400 MHz): d 7.27 (d, J = 8.4 Hz, 1H), 7.41 (d, J = 8.4 Hz,
1H), 7.69 (s, 1H), 8.21 (s, 1H), 8.43 (s, 1H), 10.23 (s, 1H).
13
C NMR (DMSO-d6, 100 MHz): d 103.4, 111.3, 113.6,
Design, synthesis of N prenylated indole-3-carbazones
116.1, 117.2, 123.8, 126.1, 134.8, 137.1, 139.8, 185.8. ESI-MS:
m/z 171. [M+H]+. IR (KBr, cm1): 1221, 1650, 3345. Anal.
Calcd. for C10H6N2O: C, 70.58, H, 3.55, N, 16.46%; Found:
C, 70.60, H, 3.52, N, 16.45%.
3.3. General procedure for the synthesis of substituted 1-(3-
methylbut-2-enyl)- 1H-indole-3-carbaldehyde (3a–)
To a solution of substituted 1H-indole-3-carbaldehyde (2a–c)
(2.20 mmol) in dry DMF (5 mL) was added NaH (2.64 mmol,
60% oil dispersion) and the resulting mixture was stirred for
10 min in an ice bath. 3,3-dimethylallyl bromide (2.20 mmol)
was added and the resulting mixture stirred for 15 min at
0 C. The mixture was diluted with EtOAc (20 mL) and
washed five times with distilled water (50 mL). The organic
layer was dried over anhydrous Na2SO4, filtered and the sol-
vent was removed under reduced pressure to get crude residue
which was purified by silica gel column chromatography (100–
200 mesh) using 8:2 (Hexane:EtOAc) as eluents affording 3a–c.
3.3.1. 1-(3-methylbut-2-enyl)-1H-indole-3-carbaldehyde (3a)
Yield: 87%; Mp: 79–81 C. 1H NMR (CDCl3, 400 MHz): d
1.85 (s, 3H), 1.86 (s, 3H), 4.74 (d, J = 7.2 Hz, 2H), 5.44 (t,
J = 7.2 Hz, 1H), 7.28–7.41 (m, 4H), 7.76 (s, 1H), 9.99 (s,
1H). 13C NMR (CDCl3, 22.4 MHz): d 17.9, 25.4, 44.6, 110.0,
117.7, 117.9, 121.8, 122.7, 123.6, 125.5, 137.3, 137.5, 138.7,
184.3, ESI-MS: m/z 214.20 [M+], IR (KBr, cm1): 1161,
1640. Anal. Calcd. for C14H15NO: C, 78.84, H, 7.09, N,
6.57%; Found: C, 78.80, H, 7.16, N, 6.55%.
3.3.2. 5-bromo-1-(3-methylbut-2-enyl)-1H-indole-3-
carbaldehyde (3b)
Yield: 89%; Mp: 95–98 C. 1H NMR (CDCl3, 400 MHz): d
1.85 (s, 3H), 1.86 (s, 3H), 4.71 (d, J = 7.2 Hz, 2H), 5.42 (t,
J = 7.2 Hz, 1H), 7.25 (d, J = 9.6 Hz, 1H), 7.43 (dd, J = 2,
8.8 Hz, 1H), 7.73 (s, 1H), 8.47 (d, J = 2 Hz, 1H), 9.96 (s,
1H). 13C NMR (CDCl3, 22.4 MHz): d 12.7, 20.1, 39.4, 106.0,
110.8, 111.7, 119.0, 121.1, 121.4, 130.3, 132.6, 132.8, 133.8,
178.6, ESI-MS: m/z 292.03 [M]+; 294.03 [M+2]+; 314.01
[M+Na]+; 316.01 [M+Na+2]+. IR (KBr, cm1): 1162,
1654. Anal. Calcd. for C14H14BrNO: C, 57.55, H, 4.83, N,
4.79%; Found: C, 57.53, H, 4.90, N, 4.77%.
3.3.3. 3-formyl-1-(3-methylbut-2-enyl)-1H-indole-5-carbonitrile
(3c)
Yield: 79%; Mp: 90–92 C 1H NMR (CDCl3, 400 MHz): d
1.73 (s, 3H), 1.75 (s, 3H), 4.68 (d, J = 7.2 Hz, 2H), 5.44 (t,
J = 7.2 Hz, 1H), 7.27 (d, J = 8.8 Hz, 1H), 7.51 (d, J = 2,
8.4 Hz, 1H), 7.81 (s, 1H), 8.32 (d, J = 2 Hz, 1H), 10.03 (s,
1H). 13C NMR (CDCl3, 100 MHz): d 17.7, 25.8, 40.2, 109.1,
111.4, 112.5, 116.7, 120.3, 122.2, 122.6, 129.5, 132.2, 133.0,
133.7, 179.1, ESI-MS: m/z 239 [M+H]+; IR (KBr, cm1):
1134, 1649. Anal. Calcd. for C15H14N2O: C, 75.61, H, 5.92,
N, 11.76%; Found: C, 75.62, H, 5.93, N, 11.77%.
3.4. General procedure for the synthesis of 1-(5-substituted-1-
(3-methylbut-2-enyl)- 1H-indole-3 yl)methylene) semicarbazide
(5a, 5c, 5e)
To the solution of substituted 1-(3-methylbut-2-enyl)-1H-in-
dole-3-carbaldehyde (3a–c) (3 mmol) and KOH (4 mmol) in
Please cite this article in press as: Choppara, P. et al., Design, synthesis of novel N prenylated indole-3-carbazones and evaluation of in vitro cytotoxicity and 5-LOX
inhibition activities. Arabian Journal of Chemistry (2015), http://dx.doi.org/10.1016/j.arabjc.2015.02.006
5
10 mL absolute ethanol, solution of semicarbazide hydrochlo-
ride (4a) (4 mmol) in absolute ethanol and water (9:1 mL) was
added slowly and the reaction mixture was refluxed for 1.5 h.
Removal of the solvent in vacuo furnished a thick liquid which
produced precipitate on addition of water. The precipitate was
filtered off, washed with hot water, and dried to afford as a
solid, which was purified by silica gel column chromatography
(100–200 mesh) using 6:4 (Hexane:EtOAc) as eluents to afford
target compounds 5a, 5c and 5e.
3.4.1. 1-((1-(3-methylbut-2-enyl)- 1H-indol-3-yl)methylene)
semicarbazide (5a)
Yield: 79%; Mp: 148 C. 1H NMR (DMSO-d6, 400 MHz): d
1.70 (s, 3H), 1.81 (s, 3H), 4.76 (d, J = 7.2 Hz, 2H), 5.32 (t,
J = 7.2 Hz, 1H), 6.25 (s, 2H), 7.34–7.45 (m, 4H), 7.79 (s,
1H), 8.09 (s, 1H), 8.94 (s, 1H). 13C NMR (DMSO-d6,
22.4 MHz): d 17.8, 25.4, 44.1, 109.8, 111.0, 118.8, 120.8,
121.8, 122.7, 125.2, 130.7, 137.0, 137.3, 138.8, 158.7. ESI-
MS: m/z 271.29 [M+H]+. IR (KBr, cm1): 1165, 1694,
3148, 3469. Anal. Calcd. for C15H18N4O: C, 66.64, H, 6.71,
N, 20.73%; Found: C, 66.63, H, 6.75, N, 20.71%.
3.4.2. 1-(5-bromo-1-(3-methylbut-2-enyl)-1H-indole-3-yl)
methylene) semicarbazide (5c)
Yield: 82%; Mp: 188–190 C. 1H NMR (DMSO-d6,
400 MHz): d 1.71 (s, 3H), 1.81 (s, 3H), 4.76 (d, J = 6.8 Hz,
2H), 5.34 (t, J = 7.2 Hz, 1H), 6.25 (s, 2H), 7.34 (d,
J = 8.8 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.75 (s, 1H), 8.02
(s, 1H), 8.25 (s, 1H), 9.94 (s, 1H). 13C NMR (DMSO-d6,
100 MHz): d 18.3, 25.8, 44.3, 111.0, 112.8, 113.7, 119.9,
124.2, 125.4, 126.6, 133.0, 135.8, 137.0, 137.9, 157.2. ESI-
MS: m/z 349.06 [M+]; 351.06 [M+2]+; 387.02 [M+K]+;
389.02 [M+K+2]+. IR (KBr, cm1): 1196, 1623, 3142,
3473. Anal. Calcd. for C15H17BrN4O: C, 51.59, H, 4.91, N,
16.04%; Found C, 51.57, H, 4.99, N, 16.01%.
3.4.3. 1-(5-cyano-1-(3-methylbut-2-enyl)-1H-indole-3-yl)
methylene) semicarbazide (5e)
Yield: 84%; Mp: 178–180 C. 1H NMR (DMSO-d6,
400 MHz): d 1.69 (s, 3H), 1.74 (s, 3H), 4.69 (d, J = 7.2 Hz,
2H), 5.41 (t, J = 7.2 Hz, 1H), 6.51 (s, 2H), 7.21 (d,
J = 8.4 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.75 (s, 1H), 8.19
(s, 1H), 8.43 (s, 1H), 10.37 (s, 1H). 13C NMR (DMSO-d6,
100 MHz): d 17.7, 26.9, 43.9, 105.1, 111.3, 113.2, 113.7,
116.9, 120.1, 125.1, 125.9, 132.6, 136.9, 137.3, 138.8, 160.1.
ESI-MS: m/z 296 [M+H]+. IR (KBr, cm1): 1254, 1642,
3135, 3489. Anal. Calcd. for C16H17N5O: C, 65.07, H, 5.80,
N, 23.71%; Found C, 65.08, H, 5.81, N, 23.70%.
3.5. General procedure for the synthesis of 1-(5-substituted-1-
(3-methylbut-2-enyl)- 1H-indole-3 yl)methylene)
thiosemicarbazide (5b, 5d, 5f)
To the solution of substituted 1-(3-methylbut-2-enyl)- 1H-in-
dole-3-carbaldehyde (3a–c) (3 mmol) and KOH (3 mmol) in
10 mL absolute ethanol, solution of thiosemicarbazide (4b)
(4 mmol) in absolute ethanol and water (9:1 mL) was added
slowly, and the reaction mixture was refluxed for 1 h.
Removal of the solvent in vacuo furnished a thick liquid which
produced precipitate on addition of water. The precipitate was
6 P. Choppara et al.
filtered off, washed with excess of hot water and dried to afford
crude products, which was purified by silica gel column chro-
matography (100–200 mesh) using 6:4 (Hexane:EtOAc) as elu-
ents to afford target compounds 5b, 5d and 5f.
3.5.1. 1-((1-(3-methylbut-2-enyl)- 1H-indol-3-
yl)methylene)thiosemicarbazide (5b)
Yield: 85%; Mp: 162 C. 1H NMR (DMSO-d6, 400 MHz): d
1.75 (s, 3H), 1.78 (s, 3H), 4.51(d, J = 7.2 Hz, 2H), 5.26 (t,
J = 7.2 Hz 1H), 5.90 (s, 2H), 7.20–7.29 (m, 4H), 7.78 (s,
1H), 8.04 (s, 1H), 9.99 (s, 1H). 13C NMR (DMSO-d6,
22.4 MHz): d 17.9, 25.5, 44.0, 109.9, 110.6, 118.6, 121.1,
121.8, 122.8, 125.0, 131.8, 137.0, 137.5, 140.8, 177.0. ESI-
MS: m/z 287.25 [M]+. IR (KBr, cm1): 1161, 1613, 3148,
3413. Anal. Calcd. for C15H18N4S: C, 62.91, H, 6.33, N,
19.56%; Found: C, 62.88, H, 6.39, N, 19.55%.
3.5.2. 1-(5-bromo-1-(3-methylbut-2-enyl)-1H-indole-3-yl)
methylene) thiosemicarbazide (5d)
Yield: 83%; Mp: 220 C. 1H NMR (DMSO-d6, 400 MHz):
1.71 (s, 3H), 1.81 (s, 3H), 4.76(d, J = 6.8 Hz, 2H), 5.32 (t,
J = 1.2 Hz, 1H), 6.26 (s, 2H), 7.34 (d, J = 8.4 Hz, 1H), 7.43
(d, J = 8.4 Hz, 1H), 7.75 (s, 1H), 8.02 (s, 1H), 8.25 (s, 1H),
9.95 (s, 1H). 13C NMR (DMSO-d6, 22.4 MHz): d 18.4, 26.0,
44.2, 110.9, 112.7, 114.2, 120.0, 124.3, 124.5, 126.7, 133.1,
135.5, 137.5, 138.0, 179.3. ESI-MS: m/z 365.04 [M]+; 367.04
[M+2]+; 387.03 [M+Na]+; 389.02 [M+Na+2]+. IR (KBr,
cm1): 1187, 1621, 3160, 3387. Anal. Calcd. for
C15H17BrN4S: C, 49.32, H, 4.69, N, 15.34%; Found: C,
49.31, H, 4.72, N, 15.31%.
3.5.3. 1-(5-cyano-1-(3-methylbut-2-enyl)-1H-indole-3-yl)
methylene) thiosemicarbazide (5f)
Yield: 91%; Mp: 192–194 C. 1H NMR (DMSO-d6,
400 MHz): 1.75 (s, 3H), 1.79 (s, 3H), 4.75 (d, J = 7.2 Hz,
2H), 5.21 (t, J = 1.6 Hz, 1H), 6.84 (s, 2H), 7.29 (d,
J = 8.0 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.72 (s, 1H), 8.14
(s, 1H), 8.46 (s, 1H), 10.41 (s, 1H). 13C NMR (DMSO-d6,
22.4 MHz): d 17.2, 26.4, 45.1, 105.2, 111.1, 112.9, 114.7,
115.7, 119.5, 125.6, 126.2, 133.7, 134.8, 136.3, 138.7, 180.1.
ESI-MS: m/z 312 [M+H]+; IR (KBr, cm1): 1192, 1626,
3178, 3421. Anal. Calcd. for C16H17N5S: C, 61.71, H, 5.50,
N, 22.49%; Found: C, 61.72, H, 5.51, N, 22.48%.
3.6. Procedure for in vitro cytotoxic activity of 1-(5-substituted-
1-(3-methylbut-2-enyl)-1H-indole-3-yl) methylene) carbazides
(5a–f) by Brine Shrimp Lethal Bioassay (BSLB)
Brine Shrimp Lethal Bioassay was used according to the
method of Meyer et al., 1982. The lethality experiments were
conducted in a 12 well microplate, each well consisting of
5 mL artificial seawater with required test concentration. The
derivatives were dissolved in dimethylsulfoxide (DMSO) and
diluted with artificial sea salt water to achieve the required test
concentrations, and maintained final concentration of DMSO,
which did not exceed 0.05%. The control experiments were
also performed simultaneously with an addition of carrier sol-
vent alone (without sample). Ten numbers of 48 h old nauplii
were added into each well and exposed for 24 h to different test
concentrations under illumination with a minimum of three
replicates. A drop of dry east suspension (3 mg in 5 mL
Please cite this article in press as: Choppara, P. et al., Design, synthesis of novel N prenylated indole-3-carbazones and evaluation of in vitro cytotoxicity and 5-LOX
inhibition activities. Arabian Journal of Chemistry (2015), http://dx.doi.org/10.1016/j.arabjc.2015.02.006
artificial seawater) was added as food to each well. The plates
were then examined under magnification (10·) and the number
of dead nauplii in each well was counted. Percent mortalities
were corrected for natural mortality in controls using Abbots
formula (Abbot, 1925), i.e., corrected percent mortality =
(PI – C/100 – C) * 100, where PI denotes the observed mor-
tality rate, and C is natural mortality. The median lethal con-
centration (LC50 for 24 h value) for each assay was calculated
by taking average of three experiments using statistical soft-
ware, BioStat-2008 based on the Finney Probit analysis
(Finney, 1971). The results for test compounds were compared
with the positive control Podophyllotoxin. All samples were
done in triplicate. The LC50 values for the test compounds
(5a–f) obtained from BSLB were expressed in lM.
3.7. Procedure for 5-Lipoxygenase inhibition assay for 1-(5-
substituted-1-(3-methylbut-2-enyl)-1H-indole-3-yl) methylene)
carbazides (5a–f)
5-Lipoxygenase inhibition assay mixture contained 2.97 mL of
50 mmol phosphate buffer pH at 6.3. 5 lL of 80 mmol Linoleic
acid and sufficient amount of potato 5-Lipoxygenase enzyme.
The reaction was started by the addition of substrate (Linoleic
acid) and the increase in UV absorption at 234 nm was followed
at 25 C. The activities were measured at 2 min. The reaction was
linear during this time period. In the inhibition studies, the activ-
ities were measured in the presence of various concentrations of
target molecules (5a–f). All the assays were performed in tripli-
cate. The IC50 values for the test compounds (5a–f) obtained
from 5-LOX inhibition assay were expressed in lM.
4. Conclusion
In conclusion, we report a novel series of six N prenylated
indole-3-carbazones, in which five compounds were interesting
in cytotoxic and two compounds in 5-LOX inhibition assay.
Acknowledgments
We are grateful to DRDO, New Delhi, India for providing
financial assistance through the Project ERIP/ER/1003916/
M/01/1354 and UGC, New Delhi, India for the award of
UGC-BSR JRF to the author P.C.
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