ORIGINAL RESEARCH

Synthesis, evaluation and docking studies on steroidal pyrazolonesas anticancer and antimicrobial agents

Shamsuzzaman • Ashraf Mashrai • Anis Ahmad •

Ayaz Mahmood Dar • Hena Khanam •

Mohd Danishuddin • Asad U. Khan

Received: 15 February 2013 / Accepted: 20 May 2013

� Springer Science+Business Media New York 2013

Abstract A series of new steroidal pyrazolones have been

synthesized, characterised and evaluated for their in vitro

anticancer activity. They were tested against five cancer

(SW480, HepG2, A549, HeLa and HL-60) cell lines. The

synthesized compounds showed high selectivity and com-

pound 4 showed the strongest inhibitory activity against

human SW480 (IC50 = 11.67 lmol L-1). In addition, the

synthesized compounds were tested for their antimicrobial

activity by disc diffusion assay and MIC by broth micro

dilution method against Gram-positive, Gram-negative

strains of bacteria as well as fungus strains and we found a

correlation between the observed and predicted antimicro-

bial activities. Docking studies were performed to investi-

gate the hypothetical binding mode of the target compounds.

This study provided a new molecular scaffold for the further

development of anticancer as well as antimicrobial agents.

Keywords Steroid � Pyrazolone � Anticancer �Antimicrobial � Docking

Introduction

Steroids attract much attention in cell biology and patho-

physiology because of the wide range of biological

phenomena in which they are involved (Vejux and Lizard,

2009). The involvement of steroids in anticancer promotion

and suppression has been known for a long time. This

involvement goes far beyond the steroidal sex hormones

(Salvador et al., 2013). Many anticancer steroids are

enzyme inhibitors, such as aromatase and sulfatase inhib-

itors for breast cancer, 5 a-reductase inhibitors for the

treatment of benign prostatic hyperplasia and CYP 17

inhibitors for advanced prostate cancer therapy (Handratta

et al., 2005). A variety of steroids with unusual and

interesting structures have been synthesized and evaluated

for their antitumor activity (Krstic’a et al., 2007; Poza

et al., 2007; Bansal and Guleria, 2008; Koutsourea et al.,

2008; Thibeault et al., 2008). Among these steroids,

nitrogen containing steroid derivatives have been shown to

be more potent and have been used clinically for the

treatment of cancer (Guarna et al., 1999; Ling et al., 1997).

Among all the numerous antibiotics developed to date, few

compounds possessing a steroid nucleus have been studied

and the results clearly showed that these compounds

exhibited a good antibacterial activity against several

human pathogenic bacteria (Jayasinghe et al., 1998; Atta

et al., 1998). The potent mechanism of action of these

compounds described by the interactions of amine groups

with the negative phosphate groups of LPS displacing

divalent cations such as Ca2? and Mg2? (Nikaido, 1996;

Vaara, 1993). Pyrazolones are important structural cores in

many drug substances of medicinal fields. Heterocyclic

nucleus containing pyrazolones are useful antipyretic and

analgesic drugs (Himly et al., 2003), whilst edaravone

(MCI-186) has been used for treating the brain (Kawai

et al., 1997) and myocardial ischemia (Wu et al., 2002). In

addition, pyrazolones possess kinase inhibitory properties,

particularly of enzymes which catalyze the phosphoryla-

tion of serine and threonine in proteins and also used for

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00044-013-0636-y) contains supplementarymaterial, which is available to authorized users.

Shamsuzzaman (&) � A. Mashrai � A. M. Dar � H. Khanam

Department of Chemistry Aligarh Muslim University,

Aligarh 202 002, India

e-mail: shamsuzzaman9@gmail.com

A. Ahmad � M. Danishuddin � A. U. Khan

Interdisciplinary Unit of Biotechnology Aligarh Muslim

University, Aligarh 202 002, India

123

Med Chem Res

DOI 10.1007/s00044-013-0636-y

MEDICINALCHEMISTRYRESEARCH

treating diseases related to these enzymes and also act as

antifungal (Al-Haiza et al., 2001), antibacterial (Moreau

et al., 2008) and antitumor (Pasha et al., 2009) agent. A

molecular scaffold which assimilates steroid as well as

pyrazolone moieties might integrate the properties of both,

and the synergism of the heterocyclic moieties in a single

nucleus may result in the formation of some worthwhile

molecules from a biological point of view. Our group has

been interested in the study of the structural requirements

of steroids to display anticancer as well as antimicrobial

activities, specifically, on heterosteroids such studies

involving in vitro anticancer and in vitro antimicrobial

evaluation of newly synthesized steroids and analysis of

structure–activity relationships (SARs) (Shamsuzzaman

et al., 2010). In light of our interest in steroid chemistry

(Shamsuzzaman et al., 2013), we herein report the syn-

thesis, in silico study, anticancer, antimicrobial activities

and docking study of steroidal pyrazolones.

Results and discussion

Chemistry

The intensive research on steroidal compounds has focused

in recent years on the development of novel, potentially

bioactive heterocyclic molecules (Kaminskyy et al., 2012),

with the aim of obtaining new candidates that may be of

value in designing new, potent, selective and less toxic

anticancer and antimicrobial agent. We have envisioned a

one-step procedure for the preparation of 6-(50-amino-30-oxo-dihydro-4H-pyrazol-40-ylidine)-5a-cholestane 6, its

3b-acetoxy 4 and 3b-chloro 5 analogues from 3b-acet-

oxycholest-6-one (3a) (Callow and James, 1956), 3b-

chloro-cholest-6-one (3b) (Millurn and Truter, 1956) and

5a-cholest-6-one (3c) (Dauben and Takemura, 1953)

according to the synthetic pathway shown in Scheme 1.

First, it clearly appears that the yield of products is

highly solvent dependent. Thus, the expected pyrazolone

derivatives 4–6 were obtained in 75–80 % yield, per-

forming the reaction in EtOH (Table 1, entries 1–3)

whereas only moderate yields of 40–45 and 12–15 % were

encountered performing the reaction in MeOH and toluene

respectively (Table 1, entries 4–9).

Presumably, the reaction seems to proceed following the

mechanistic pathway presented in Scheme 2. A mecha-

nistic rationale includes the condensation between steroidal

ketone and cyanoacetohydrazide leading to the formation

of steroidal cyanoacetohydrazide, which later involves the

nucleophilic attack of nitrogen to the cyanide [C:N]

changing it to C=NH that causes the closure of the het-

erocyclic ring and hence, leading to the formation of

products 4–6 (Bondock et al., 2006).

The structures of the compounds were established by

means of their IR, 1H NMR, 13C NMR, MS and analytical

data. The selected diagnostic bands of IR spectra of synthe-

sized products provide useful information for determining the

structures of the pyrazolone derivatives. All the compounds

4–6 exhibited absorption bands at 3,380–3,390 cm-1 due to

N–H stretching and 1,685–1,691 cm-1 due to CONH while

the bands in the range of 1,650–1,657,1,622–1,628 and

1,371–1,378 cm-1 can be ascribed to (C=N), (C=C) and

(C–N) respectively. Moreover, the absence of sharp absorp-

tion band at 2,200–2,250 cm-1 ascribable to C:N group

revealed the conversion of C:N to C–NH2 which results in

the formation of cyclic products. The formation of steroidal

pyrazolones was further confirmed with the 1H NMR spectra.

The 1H NMR spectra of the compounds, besides the expected

signals for cholestane moiety, exhibited two singlets

(exchangeable with D2O) for one proton atd 8.6–8.9 (NH) and

for two protons at d 2.3–2.6 (NH2). 13C NMR spectrum,

besides the characteristic signals for the cholestane nucleus

showed d 169–173 corresponding to the CONH group, d149–154 indicating the C=N group and 120–147 were

attributed to C=C. These data confirmed the presence of

pyrazolone ring. The mass spectral data of compounds 4–6

showed molecular ion peaks [M?] at m/z 525, 501/499 and

467 respectively.

Computational profiling

In order to study the biological behaviour of compounds 4–6,

it is gone to predict the activity by using pass (prediction of

activity spectra for substances) software. The drug likeness

of these compounds, type of molecular mechanism and Pa

and Pi values of each activity are presented in Table 2. The

high-drug likeness for the compounds 4 and 6 are determined

to be 0.912 and 0.887 respectively which proved the high

probability of these compounds to use as drug. The antihy-

pertensive activities of the compounds 4 and 6 predicted the

pharmacological effect of these compounds. Moreover, the

molecular mechanism of compound 4 was predicted as nitric

oxide agonist. The PASS software also predict the Pa:Pi

(active:inactive ratio) at prediction threshold of Pa [ 70 %,

30 % \ Pa \ 70 % and Pa \ 30 %. If Pa [ 0.7, the sub-

stance is very likely to exhibit the activity in experiment and

the chance of the substance being analogue with a known

pharmaceutical agent is high; and if 0.3 \ Pa \ 0.7, the

substance is likely to exhibit the activity in experiment, but

the probability is less and the substance is unlike the known

pharmaceutical agents while if Pa \ 0.3 like our case, the

substance is unlikely to exhibit the activity in the experiment,

However, if the presence of this is confirmed in the experi-

ment the substance might be a new entity (Pan et al., 2009).

The notion of chemical space in drug discovery has

received considerable attention since the pioneering work

Med Chem Res

123

of Lipinski (Lipinski et al., 1997). Following this work, we

studied the physicochemical parameters of the synthesized

compounds (4–6) in attempt to correlate the physico-

chemical properties with the predicted successful matric-

ulation of initial hits and subsequent late-stage leads. Good

intestinal absorption reduced the molecular flexibility

(measured by the number of rotatable bonds) and low-polar

surface area is important predictor of good oral bioavail-

ability (Veber et al., 2002; Refsgaard et al., 2005).

Molecular properties such as membrane permeability and

bioavailability are always associated with some basic

molecular descriptors such as log P (partition coefficient),

molecular weight (MW), hydrogen bond acceptors and

donors count in a molecule (Muegge, 2003). Lipinski used

these molecular properties in formulating his ‘‘Rule of

Five’’. This rule states that most molecules with good

membrane permeability have log P B 5, molecular weight

B500, number of hydrogen bond acceptors B10 and

number of hydrogen bond donors B5. This rule is widely

used as a filter for drug-like properties. As can be seen in

Table 3, the synthesized compounds show one violation of

Lipinski rules for compound 6 due to a calculated Clog

P value above the limit of 5 and two violations in com-

pounds 4 and 5 due to the same reason and the molecular

mass above 500. The results showed that all the compounds

having polar surface area less than 140 A2 moreover, the

Cholesterol

acetic anhydride pyridine thionyl chloride

H3CCOOH

H

H

ClH

H

HH

H

H

HNO3 Fuming HNO3Fuming HNO3

H3CCOOH

H

H

ClH

H

HH

H

HNO2 NO2 NO2

Acetic acid

H3CCOOH

H

H

ClH

H

H H

H

H

Zn dust Zn dust Zn dust

O

Acetic acid Acetic acid

OONH2NHCOCH2CN NH2NHCOCH2CN

NH2NHCOCH2CN

H3CCOOH

H

H

N NH

O

H

H

H

N NH

OH2N

Cl

H

H

H

N NH

OH2N

Na

(1a) (2a) (3a)

(1b) (2b) (3b)

(3a) (3b)(3c)

Et3N Et3NEt3N

(4) (5) (6)H2N

EtOH EtOH EtOH

Scheme 1 Showing the

formation of steroidal

pyrazolones 4–6

Med Chem Res

123

synthesized compound (4–6) got rotatable bonds less than

10. On the basis of the above results, we can say that the

synthesized compounds adhere to Lipinski’s ‘‘Rule of

Five’’ (Wani et al., 2011) and the exceptions to the

Lipinski’s rule of five are known and involve drugs that are

transported across the membranes by carrier proteins, such

as antibiotic erythromycin (Takano et al., 1998). The molar

refractivity (MR) represents size and the polarizability of a

molecule describing the steric effects to explain the activity

behaviour of the synthesized compounds. Acidity constant

(pKa) is a key parameter for understanding the chemical

interactions between the compound of interest and its

pharmacological target. The relationship between acidity

constant and structure may prove useful in drug design

studies and in explaining the biopharmaceutical properties

of substances. Many biologically active molecules are fully

or partially ionized at physiological pH, and it has often

been shown that the presence of ionizable groups is nec-

essary for biological activity and/or solubility. Moreover,

the acid–base property of a drug molecule is the key

parameter for drug development because it governs solu-

bility, absorption, distribution, metabolism and elimina-

tion. Particularly for developing new drug, the pKa has

become of great importance because the transport of drugs

into cells and across other membranes is a function of

physicochemical properties (Andrasi et al., 2007).

The bioactivity scores of the compounds 4–6 were also

calculated for six criterias, GPCR ligand activity, ion

channel modulation, kinase inhibition activity, protease

inhibitor, enzyme inhibitor and nuclear receptor ligand

activity. As we know for organic molecules, if the bioac-

tivity score is more than 0.00 then, the compound is active,

but if it is between -0.50 and 0.00 then the compound is

moderately active and if the compound has\-0.50 then it

is inactive compound (Uetrecht, 2001). As we can see in

Table 4, our synthesized compounds show good bioactivity

score.

From the (PASS), physicochemical properties and the

bioactivity score of the compounds 4–6, it could be con-

cluded that the synthesized compounds are suitable for

more study. However, the compound 4 showed high-drug-

like score compared to compound 6, but the molecular

surface area and topological surface area are lesser for the

compound 6 compared to compound 4 which help the

compound to be absorbed easily. Based on these facts,

compound 6 computationally is more active compared to

Table 1 Screening of different solvents for reaction between steroi-

dal ketones and cyanoacetohydrazide under reflux condition

Entrya Compound Solvent Yield (%)b,c

1 4 EtOH 80

2 5 EtOH 78

3 6 EtOH 75

4 4 MeOH 43

5 5 MeOH 40

6 6 MeOH 45

7 4 Toluene 12

8 5 Toluene 15

9 6 Toluene 13

a Reaction performed between steroidal ketones (1 mmol) and cya-

noacetohydrazide(1 mmol) under refluxing conditionsb completion of reaction was monitored by TLCc Isolated overall yield

Table 2 PASS predicted but not reported activities (hidden potential)

of all the synthesized compounds

Comp. Activity Drug

likeness

Pa Pi

4 Pharmacological

effect

Antihypertensive 0.912 0.226 0.098

Molecular

mechanism

Nitric oxide

agonist

0.266 0.235

5 Pharmacological

effect

0.821

Molecular

mechanism

6 Pharmacological

effect

Antihypertensive 0.887 0.251 0.080

Molecular

mechanism

OH

H

H

H

H

H

H

H

N NH

OH2N

NC-CH2-CO-NH-NH2

X

XCH

H

H

H

XH

H

H

HX

C CO

NHH2N

Et3N

N NH

OHN

H

N

Scheme 2 Plausible

mechanism for the formation of

steroidal pyrazolones 4–6

Med Chem Res

123

the rest of the synthesized compounds and reasonable

starting points for a drug discovery effort.

Pharmacological evaluation

Anticancer activity

On the basis of the encouraging theoretical results and

keeping in our mind the importance of presence of polar

substituent at ring B for the destabilization of cellular

membrane and for cytotoxicity (Massey and Pownall,

2006), we decided to study the anticancer activity of the

synthesized compounds. In the present study, steroidal

pyrazolones were evaluated for cytotoxicity in a panel of

selective human cancer cells using the MTT assay, as

already described by our group (Shamsuzzaman et al.,

2012).The panel of cancer cells encompassed HepG2 (from

hepatocellular carcinoma), A549 (from lung adenocarci-

noma epithelium), SW480 (from colon adenocarcinoma),

HeLa (from cervical carcinoma) and HL-60 from (pro-

myelocytic leukaemia). Doxorubicin and Cytarabine were

used as cytotoxic drugs of reference. The synthesized

compounds were firstly screened for (SW480) due to the

straight relation of sterols and colon tissues, where cho-

lesterol is both absorbed and synthesized (Cerda et al.,

1995; Ikonen, 2008). (HL60) was added basing on previous

studies that nitrogen containing steroids have the ability to

regulate a variety of biological processes, and thus, are

potential drug candidates for the treatment of leukaemia

(Roy et al., 2007). HepG2, A549 and HeLa were used

aiming at gaining new insights on the preferential cyto-

toxicity of the steroids tested against cancer cells and to

give an indication of selectivity for tumor cells. The

cytotoxicity of the synthesized steroids against cancer

(SW480, HepG2, A549, HeLa and HL-60) cell lines is

detailed in Table 5. A period of 48 h of drug exposure was

chosen to test the cytotoxicity.

To gain an insight on how modifications on ring A can

affect the cytotoxicity, the human cancer cells were incu-

bated first with compound 6. Not surprisingly, the result

presented higher resistance to steroidal pyrazolones cyto-

toxicity. Cholesterol cell toxicity is known to be affected by

C-3 esterification (Tabas, 2002). Esterification is catalyzed

by acyl coenzyme A, cholesterol acyl transferase (ACAT)

intracellularly or by lecithin cholesterol acyl transferase

(LCAT) in plasma. Cholesteryl esters and oxysteryl esters of

long-chain fatty acids, particularly oleyl esters, have been

found in lipoproteins and oxidised lipoproteins, respec-

tively. Cholesteryl esters represent the way by which the

cholesterol can be accumulated in cells and included in lipid

droplets. On the other hand, esters can potentially act as

interesting hydrophilic weak acid transporters to target

cancer cells (Gerweck et al., 2006). Therefore, we decided to

study the effect of acetoxy group in ring A on cytotoxicity.

Table 3 Calculated physicochemical properties of steroidal pyrazolones 4–6

Comp. Chemical

formula

Lipinski rule of 5 MSAe TPSAd

(A2)

Rotatable

bonds

No.

violations

Gibbs Energy

(kj/mol)

MR

(cm3/mol)MiLog Pa Mw HBDb HBAc

4 C32H51N3O3 7.298 525.778 3 6 878.07 98.082 7 2 323.06 150.61

5 C30H48ClN3O 7.832 502.187 3 4 817.71 71.777 5 2 604.66 144.38

6 C30H49N3O 7.979 467.742 3 4 804.65 71.777 5 1 624.3 139.8

Comp. Henry’s law Heat of form (kJ/mol) Elemental analysis calculated (%) Boiling point (K) Melting point (K) LogD pKa

4 1.11 -608.72 C (73.10), H (9.78), N (7.99), O (9.13) 1,326.97 967.39 5.7 7.76

5 0.36 -265.9 C (71.75), H (9.63), N (8.37), O (3.19) 1,260.42 932.44 6.5 7.76

6 -0.1 -229.82 C (77.04), H (10.56), N (8.98), O (3.42) 1,227.66 906.76 6.5 7.76

a The log P value calculated using molinspiration serverb Hydrogen bond donor (expressed as the sum of OH and NH)c Hydrogen bond acceptor (expressed as the sum of O and N atoms)d Topological polar surface area (defined as a sum of surfaces of polar atoms in a molecule)e Molecular surface area

Table 4 Bioactivity score of steroidal pyrazolones 4–6

Comp. GPCR Ligand Ion channel Modulator kinase Protease inhibitor Nuclear receptor ligand Enzyme inhibitor

4 0.00 0.06 -0.12 0.09 -0.05 0.27

5 -0.00 -0.08 -0.12 -0.01 -0.07 0.19

6 -0.04 -0.02 -0.05 0.0 -0.05 0.21

Med Chem Res

123

Interestingly, the introduction of the 3b-acetoxy in the

cholestane moiety, as in compound 4, led to increase the

cytotoxic profile when compared to the compound 6. The

compound 4 showed minimum IC50 = 11.67 (SW480),

16.32 (HeLa) and 19.61 (A549) lmol L-1. Then, we moved

our attention to the influence of the chlorine group on

position C-3. The results indicate that the presence of

chlorine group has an impact on bioactivity which can be

explained by the effect of electron withdrawing group

(Seong et al., 2004). Compound 5 showed minimum

IC50 = 17.04 (HepG2) and 18.01 (SW480) lmol L-1.

Noteworthy, SW480 cell, derived from colon adenocarci-

noma is quite sensitive to the steroidal pyrazolones studied

herein, which is in agreement with the literature reports on

steroids (Carvalho et al., 2010). The results showed that for

all the synthesized compounds there was no linear rela-

tionship between in silico analysis and IC50 values.

Antimicrobial activity

In the context of our studies, 6-(50-amino-30-oxo-dihydro-

4H-pyrazol-40-ylidine)-5a-cholestane derivatives 4–6 were

screened for their in vitro antimicrobial activities against

Gram-positive and Gram-negative bacterial strains and

were found to possess activities against the microorganisms

listed in Tables 6 and 7.

Table 5 Summary of the screening data of steroidal pyrazolones 4–6 for the in vitro anticancer activity (in lg/mL)

Human tissue of origin Colon Lung Hepatic Cervical Leukaemia

Cell lines SW480 A549 HepG2 HeLa HL60

IC50 4 11.67 – 0.2 19.05 – 0.2 25.03 – 0.5 16.32 – 0.3 [50

5 18.01 – 0.4 29.21 – 0.3 17.04 – 0.2 [50 23.15 – 0.2

6 26.21 – 0.3 33.52 – 0.6 38.14 – 0.2 22.03 – 0.5 14.09 – 0.6

Doxorubicin 10.9 – 0.4 13.5 – 0.3 11.52 – 0.6 12.52 – 0.3 9.52 – 0.2

Cytarabine 15.01 – 0.3 16.05 – 0.2 13.04 – 0.4 14.32 – 0.2 10.09 – 0.2

Table 6 Antibacterial activity of (zones of inhibition) of steroidal pyrazolones 4–6

Comp. Diameter of zone of inhibition (mm)

Gram-positive bacteria Gram-negative bacteria

S. Pyogene MRSAa P. aeruginosa K. pneumoniae E. coli

4 15.7 ± 0.2 15.2 ± 0.5 17.1 ± 0.3 15.2 ± 0.4 15.1 ± 0.2

5 14.1 ± 0.5 14.7 ± 0.2 15.9 ± 0.6 13.1 ± 0.6 13.9 ± 0.4

6 16.9 ± 0.3 15.9 ± 0.4 19.2 ± 0.3 15.8 ± 0.3 16.3 ± 0.6

Standard 23.0 ± 0.2 22.0 ± 0.2 32.0 ± 0.3 19.0 ± 0.2 27.0 ± 0.2

DMSO – – – – –

Positive control (standard); Ciprofloxacin and negative control (DMSO) measured by the Halo Zone Test (Unit, mm)a Methicillin resistant Staphylococcus aureus (MRSA ?ve)

Table 7 MIC and MBC results of steroidal pyrazolones 4–6 against bacterial strains

Comp. Gram-positive bacteria Gram-negative bacteria

S. Pyogenes MRSAa P. aeruginosa K. pneumonia E. coli

MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC

4 50 100 50 100 50 100 50 [100 50 [100

5 50 100 50 [100 50 50 100 [100 100 [100

6 50 100 25 50 50 100 50 [100 50 [100

Standard 12.5 12.5 6.25 12.5 12.5 25 6.25 25 6.25 12.5

(Standard); ciprofloxacin; MIC (lg/ml) = minimum inhibitory concentration, i.e. the lowest concentration of the compound to inhibit the growth

of bacteria completely; MBC (lg/ml) = minimum bacterial concentration, i.e., the lowest concentration of the compound for killing the bacteria

completelya Methicillin resistant Staphylococcus aureus (MRSA ?ve)

Med Chem Res

123

All the compounds present excellent activities against

Gram-positive bacteria, exhibiting similar MIC values of

25–100 lg/mL suggesting that the presence of amino

group is necessary to lead to biologically active com-

pounds. On the other hand, all the derivatives possess

moderate to excellent antimicrobial activities against

Gram-negative strains. Since the antibacterial activity was

found almost same whatever the derivative used, suggest-

ing that the mechanism of action of these compounds is

depending on the class of bacteria considered. Among the

synthesized compounds, it was clear that compound 6

showed very good antibacterial activity nearly equivalent

to that of standard drug Ciprofloxacin. A potent mechanism

of action of amino steroids had been previously described

that this family of compounds act by disrupting the outer

membrane of Gram-negative bacteria in a detergent-like

mechanism of action and by depolarizing the bacterial

membrane of Gram-positive bacteria (Djouhri-Bouktab

et al., 2011).

The relationship between log P and activity has also

been studied in this paper and we found that compounds

having higher log P and lower MIC values are the most

active as shown in Fig. 1. Activity = m log P ? K0 (Lin-

ear) the compound with log P value close to the linear line

and have lower MIC values are the most active (Hansch

and Fujita, 1964).

For assaying the antifungal activity, different fungal

strains like Candida albicans, Aspergillus fumigatus,

Penicillium marneffe and Trichophyton mentagrophytes

were chosen. The antifungal screening data showed mod-

erate to good fungal inhibition (Tables 8 and 9).

Among the screened compounds, 4 and 6 were found to have

good zones of inhibition. The compound 4 showed the maxi-

mum inhibition against C. albicans and T. mentagrophytes

strains, while the compound 6 is more effective by showing the

maximum inhibition against C. albicans, A. fumigatus and

P. marneffei strain. The MFC of all the compounds was two or

threefolds higher than the corresponding MIC results.

The deduced patterns of antimicrobial activity of the

newly synthesized steroidal pyrazolones are in the follow-

ing order: antifungal [ antibacterial. It is worthy to point

0

2

4

6

8

0 20 40 60

C L

og P

MIC

Fig. 1 Graph showing the correlation between log P and MIC

values. The compounds whose log P values are close to the linear line

and have lower MIC values are the most active

Table 8 Antifungal activities of steroidal pyrazolones 4–6

Comp. Diameter of zone of inhibition (mm)

CA AF TM PM

4 25.8 ± 0.2 20.7 ± 0.3 17.6 ± 0.6 14.5 ± 0.4

5 20.9 ± 0.4 16.9 ± 0.6 14.9 ± 0.3 12.8 ± 0.5

6 27.8 ± 0.2 22.7 ± 0.3 18.6 ± 0.6 16.5 ± 0.4

Standard 30.0 ± 0.2 27.0 ± 0.2 24.0 ± 0.3 20.0 ± 0.5

DMSO – – – –

Positive control (standard), Fluconazole and negative control (DMSO) measured

CA Candida albicans, AF Aspergillus fumigatus, TM Trichophyton mentagrophytes, PM Penicillium marneffei

Table 9 MIC and MFC of steroidal pyrazolones 4–6 against fungal strains

Comp. CA AF TM PM

MIC MFC MIC MFC MIC MFC MIC MFC

4 25 100 50 100 50 100 50 100

5 50 100 50 100 100 [100 100 [100

6 25 50 25 100 50 100 25 100

Standard 6.25 25 12.5 12.5 6.25 25 12.5 25

(standard); Fluconazole; CA; Candida albicans, AF; Aspergillus fumigatus, TM; Trichophyton mentagrophytes, PM; Penicillium marneffei. MIC

(lg/ml) = minimum inhibitory concentration, i.e. the lowest concentration of the compound to inhibit the growth of fungus completely; MFC

(lg/ml) = minimum fungicidal concentration, i.e., the lowest concentration of the compound for killing the fungus completely

Med Chem Res

123

out that we have found a correlation between the predicted

activities and observed in antimicrobial activity as com-

pound 6 is more active among the synthesized compounds.

Molecular docking

Molecular docking with the P 53 tumour suppressor

protein (PDB ID: 1TUP)

These compounds showed strong molecular interactions

within active site of P 53. Compounds 4, 5 and 6 form

stable complex with 61.04, 56.09 and 61.56 GOLD fitness

score which are reasonably good as compared to control

the compound having 46.90 fitness score. Fig. 2.

These novel compounds also found to have the highest

number of hydrophobic contacts as compared to the ref-

erence drug. Compounds 6 showed the best activity for p53

as compared to compound 4 and 5. This compound docked

within the active with one hydrogen bond and 28 hydro-

phobic contacts with the 3.1–3.8A (Table 10).

Molecular docking with S12 protein (PDB ID: 1FJG)

In order to know the exact interactions of the compounds

4–6, docking studies were carried out. (Fig. 3) depicts the

comparison of binding of these compounds with the Cip-

rofloxacin binding site. Although, the compounds 4, 5 and

6 had no hydrogen bonds with the S12 protein but have

hydrophobic interactions with the S12 protein.

The top score pose was selected for each compound and

their results are shown in Table 11 and compared with

ciprofloxacin, which was redocked with the target protein

using the same protocol. Among the synthesized three

compounds, compound 6 showed strong molecular inter-

actions within active site of S12 with 56.78 GOLD fitness

score as compared to other compounds 4, 5 and reference

drugs having 54.45, 52.62 and 55.45 fitness scores

respectively. Compound 6 also found to have the highest

number of hydrophobic contacts (26) as compared to other

novel compounds and reference drug.

It was evident from Fig. 3 that for each compound, the

binding site and hydrogen-bonding interactions were found

varied. It was interesting to observe that even though the

core structure of all the compounds was same, the degree

of interaction and binding location were found to be almost

similar. The binding sites of the compounds were found to

be in close proximity to the binding site of ciprofloxacin.

The variation in the bioactivity is mainly attributed to the

difference in their binding site. For instance, the activity

studies showed that the compounds 4, 5 and 6 showed

comparable results with ciprofloxacin in the case of P.

Fig. 2 a (Docked) all the three molecules docked in the binding site

of p53 protein. The protein is in cartoon representation and the ligand

molecules are in sticks coloured. b (Ligplot) the interaction plot of all

the three molecules with the binding site residues of P53 protein. The

hydrogen bonds and hydrophobic interactions are shown which are

holding the ligand within the binding site

Table 10 The ligand molecules with number of molecular interactions and the scores for strength of binding with P 53 tumour suppressor

protein

Compounds GOLD

fitness score

Residues involved in

hydrogen bonding

Residues involved in

hydrophobic interaction

H-bond

range (A)

Hydrophobic

interaction range

(A)

Control (cytarabine) 46.90 Thr8, Leu415 Thr6, Thr8, Leu292, Leu415, Gly418 2.7–3.11 3.45–3.73

Compound 4 61.04 NA Thr6, Leu292, Val293, Ile414, Leu415,

Gly418, Ser442

NA 3.31–3.80

Compound 5 56.09 NA Thr6, Leu292, Val293, Ile414, Leu415,

Gly418, Ser442

NA 3.12–3.87

Compound 6 61.56 Gly325 Thr6, Leu292, Val293, Ile414, Leu415,

Gly418, Ser442

2.0 3.37–3.85

The number of residues involved in the hydrogen and hydrophobic interactions are provided in braces with the total number of interactions the

molecule is experiencing

Med Chem Res

123

Fig. 3 a (Docked) all the three molecules docked in the binding site

of S12 protein. The protein is in cartoon representation and the ligand

molecules are in sticks coloured. b (Ligplot) The interaction plot of

all the three molecules with the binding site residues of S 12 protein.

The hydrogen bonds and hydrophobic interactions are shown which

are holding the ligand within the binding site

Table 11 The ligand molecules with number of molecular interactions and the scores for strength of binding with S 12 protein of E. coli

Compounds GOLD fitness

score

Residues involved in

hydrogen bonding

Residues involved in

hydrophobic interaction

H-bond

range (A)

Hydrophobic

interaction

range (A)

Ciprofloxacin 55.45 Lys47, Pro94 Val43, Lys46, Lys47, Leu93,

Val96

2.6–2.9 3.2–3.80

Compound 4 54.45 NA Val43, Pro45, Lys46, Pro94 NA 3.17–3.90

Compound 5 52.62 NA Val43, Lys46, Val55, Leu93,

Pro94, Val96

NA 3.12–3.87

Compound 6 56.78 NA Val43,Lys46, Val55, Leu93,

Pro94, Val96

NA 3.24–3.90

The number of residues involved in the hydrogen and hydrophobic interactions is provided in braces with the total number of interactions the

molecule is experiencing

Fig. 4 a (Docked) all the three molecules docked in the binding site

of Cytochrome P451 of C. albicans. The protein is in cartoon

representation and the ligand molecules are in sticks coloured.

b (Ligplot) the interaction plot of all the three molecules with the

binding site residues of Cytochrome P451 of C. albicans. The

hydrogen bonds and hydrophobic interactions are shown which are

holding the ligand within the binding site

Med Chem Res

123

aeruginosa. It may be due to the fact that their binding site

is close to the ciprofloxacin binding site when compared to

the other compounds as revealed by the docking studies

and these compounds had the maximum GOLD fitness

score as well. The binding energy and -log (Kd) values are

calculated using X-Score. The GOLD score and docking

data are summarized in the (Table 11).

The mode of action of the synthesized compounds

with active site of CYP 51 of C. albicans (PDB ID: 1E9X)

The results show that the overall trend of the interaction

energies of all the derivatives is in good qualitative

agreement with the in vitro antifungal activities. It is

known that the regions in the active site of Cytochrome

P450 for noncovalent binding can be divided into four

subsites S1-S4, besides the site coordinating to the heme

(Ji et al., 2003) (Fig. 4).

The S1 subsite is a hydrophilic hydrogen-bonding

region; the S2 subsite is a hydrophobic region; the S3

subsite is a narrow hydrophobic cleft formed by the resi-

dues in the helix B0-Meander1 loop and the N terminus of

helix I; and the S4 subsite adjacent to the b6-1/b1-4 sheet is

another hydrogen-bonding region in the active site. Among

the synthesized three compounds, compound 6 showed

strong molecular interactions within active site of CYP 51

of C. albicans with 48.10 GOLD fitness score as compared

to other compounds 4, 5 and reference drugs having 38.37,

40.81 and 47.68 fitness scores respectively. Compound 6

also found to have the highest number of hydrophobic

contacts as compared to other novel compounds and ref-

erence drug Fluconazole. This compound docked within

the active with one hydrogen bond and 28 hydrophobic

contacts with the 3.2–3.9A (Table 12). In case of com-

pound 6, the pyrazolone ring forms H-bonding interactions

with the residues Glu133 in the S4 subsite. The binding

mode of these derivatives with the active site of Cyto-

chrome P450 provides reasonable explanation for their

antifungal activities. The antifungal activities of all the

compounds are comparable to the control Fluconazole.

This result suggests that the appropriate length of the

substituents on the derivatives is important for antifungal

activities. The S4 sub site is a hydrogen bond donor and

acceptor region, which interacts with the oxygen of the

pyrazolone ring. The binding mode of the docked molecules

to Cytochrome P450 and their corresponding antifungal

activities suggest that the compounds with hydrogen-

bonding donor or acceptor substituents.

Conclusion

A series of 6-(50-amino-30-oxo-dihydro-4H-pyrazol-40-yli-

dine)-5a-cholestanes derivatives 4–6 have been synthe-

sized and screened against five cancer (SW480, HepG2,

A549, HeLa and HL-60) cell lines in which the synthesized

compound showed a selective activity against SW480

(human colon adenocarcinoma cells) compared to the other

cancer cell lines. The antimicrobial studies of the synthe-

sized compounds showed a reasonable correspondence

between the experimental and predicted activities. The

results from the docking studies were found to be in good

agreement with the results from computational profiling

and pharmacological studies and suggested that compound

6 the most promising and can be explored in future as an

option for decreasing the pathogenic potential of infecting

cancer as well as microbial.

Experimental

Chemistry

All the chemicals used were purchased from Sigma Aldrich

and Merck as analytical grade. The solvents were purified

prior to use. Melting points were recorded on Buchi

Table 12 The ligand molecules with number of molecular interactions and the scores for strength of binding with Cytochrome P451 of C.albicans

Compounds GOLD

fitness score

Residues involved in

hydrogen bonding

Residues involved in hydrophobic

interaction

H-bond

range (A)

Hydrophobic

interaction range

(A)

Fuconazole 47.68 Arg446 Ala131, Gly132, Glu133, Glu416, Arg444, Arg446 2.96–3.90 3.43–3.82

Compound 4 38.37 NA Ala131, Gly132, Glu133, Glu416, Arg444, Arg446 NA 3.11–3.81

Compound 5 40.81 NA Ala131, Gly132, Glu133, Glu416, Phe417, Glu418,

Arg444, Arg446

NA 3.12–3.87

Compound 6 48.10 Glu133 Ala131, Gly132, Glu133, Glu416, Glu418, Arg444, Arg446 2.85 3.24–3.90

The number of residues involved in the hydrogen and hydrophobic interactions are provided in braces with the total number of interactions the

molecule is experiencing

Med Chem Res

123

melting point apparatus D-545; IR spectra (KBr discs) were

recorded on Interspec 2020 FT-IR Spectrometer Spectro-

Lab and only noteworthy absorptions were noted, its values

are given in cm-1. 1H and 13C NMR spectra in dilute

CDCl3 solutions at 303 K were run on a Bruker Avance

DRX 500 NMR spectrometer equipped with a 5-mm

diameter broad band inverse probehead working at

500 MHz for 1H and at 125 MHz for 13C, respectively. 1H

chemical shifts were referenced to the trace signal of

CHCl3 (7.26 ppm from int. TMS). Following abbreviations

were used to indicate the peak multiplicity s—singlet,

d—doublet, t—triplet, m—multiple and values are given

in parts per million (ppm) (d) and coupling constants

(J) are given in Hertz and 13C chemical shifts to the center

peak of the solvent signal (77.00 ppm from int. TMS).

Mass spectra were recorded on a JEOL D-300 mass spec-

trometer. Elemental analyses were carried out by the

instrumentation lab at the Department of Chemistry, Indian

Institute of Technology Roorkee, Roorkee, India within

0.04 % of the theoretical values. Follow-up of the reactions

and checking the homogeneity of the compounds were

made by ascending TLC run on silica gel G (Merck 60)

0.5-mm layer coated glass plates. The spots were visual-

ized by exposure to iodine vapour for few seconds. Sodium

sulphate (anhydrous) was used as a drying agent.

General method for the preparation of 6-(50-amino-30-oxo-

dihydro-4H-pyrazol-40-ylidine)-5a-cholestane

To a solution of steroidal ketones 1–3 (1 mmol) in absolute

ethanol (15 mL), cyanoacetohydrazide (1 mmol) was

added to it, followed by the addition of few drops of tri-

ethylamine in the same solvent (25 mL) then the reaction

mixture was refluxed for about 18–24 h. The progress of

reaction was monitored by TLC. After completion of

reaction, the excess solvent was removed to three-fourths

of the original volume under reduced pressure. The reac-

tion mixture was taken in ether, washed with water and

dried over anhydrous sodium sulphate. Removal of solvent

gave the crude product which was recrystallized from

methanol to furnish the corresponding 6-(50-amino-30-oxo-

dihydro-4H-pyrazol-40-ylidine)-5a-cholestane derivatives

4–6.

3b-Acetoxy 6-(50-amino-30-oxo-dihydro-4H-pyrazol-40-yli-

dine)-5a-cholestane (4) White solid; Yield 80 %. mp

159–160 �C; Anal. Calcd. for C32H51N3O3: C, 73.10, H,

9.78, N, 7.99; found; C, 73.12, H, 9.81, N, 8.0; IR (KBr, mcm-1): 3,390, 3,210 (NH, NH2), 1,736 (OAc), 1,689

(CONH), 1,655 (C=N), 1,628 (C=C), 1,376 (C–N), 1,240

(C–O); 1H NMR (500 MHz, CDCl3) d (ppm): 8.6 (s, 1H,

CONH, exchangeable with D2O), 4.7 (m, 1H, C3a-H, W

� = 15 Hz, axial), 2.5 (s, 2H, NH2 exchangeable with

D2O,), 2.3 (dd, 1H, J = 7.55, 4.52 Hz,C5-H), 2.03 (s, 3H,

OCOCH3), 1.18 (s, 3H, C10-CH3), 0.70 (s, 3H, C13-CH3),

0.97 and 0.83 (other methyl protons); 13C NMR (125 MHz,

CDCl3) d (ppm): 171 (CONH), 167.0 (OCOCH3), 154

(C=N), 145 (C6), 118 (C40), 75.1 (C3), 56.70, 56.04, 54.11,

42.86, 40.5, 39.89, 38.5, 37.3, 36.70, 36.1, 36.07, 34.2,

32.76, 30.73, 28.1, 28.08, 27.1, 26.57, 25.09, 24.8, 23.4,

23.1, 22.63, 19.9, 14.08, 13.21; MS(ESI): m/z 525 [M?.].

3b-Chloro 6-(50-amino-30-oxo-dihydro-4H-pyrazol-40-yli-

dine)-5a-cholestane (5) Off white solid; Yield 78 % mp

147–148 �C; Anal. Calcd. for C30H48N3ClO: C, 71.75, H,

9.63, N, 8.37; found; C, 71.79, H, 9.59, N, 8.34; IR (KBr,

m cm-1): 3,380, 3,226 (NH, NH2), 1,685 (CONH), 1,650

(C=N), 1,625 (C=C), 1,371 (C–N), 756 (C–Cl); 1H NMR

(500 MHz, CDCl3) d (ppm): 8.7 (s, 1H, CONH,

exchangeable with D2O), 3.9 (m, 1H, C3a-H, W � =

17 Hz, axial), 2.6 (s, 2H, NH2, exchangeable with D2O),

2.3 (dd, 1H, J = 7.55, 4.52 Hz,C5-H), 1.19 (s, 3H,

C10-CH3), 0.75 (s, 3H, C13-CH3), 0.97 and 0.80 (other

methyl protons); 13C NMR (125 MHz, CDCl3) d (ppm):

169 (CONH), 149 (C=N), 142 (C6), 118 (C40), 60.2 (C3),

56.70, 56.04, 54.11, 42.86, 40.5, 39.89, 38.5, 37.3, 36.70,

36.1, 36.07, 34.2, 32.76, 30.73, 28.1, 28.08, 27.1, 26.57,

25.09, 24.8, 23.8, 23.2, 19.9, 14.08, 13.21; MS (ESI):

m/z 501/499 [M?.]..

6-(50-Amino-30-oxo-dihydro-4H-pyrazol-40-ylidine)-5a-choles-

tane (6) Yellow solid; Yield 75 % mp 134-135 �C; Anal.

Calcd. for C30H49N3O: C, 77.04, H, 10.56, N, 8.98; found;

C, 77.05, H, 10.52, N, 8.95; IR (KBr, m cm-1): 3,385, 3,230

(NH, NH2), 1,691 (CONH), 1,657 (C=N), 1,622 (C=C),

1,378 (C–N); 1H NMR (500 MHz, CDCl3) d (ppm): 8.9

(s, 1H, CONH, exchangeable with D2O), 2.3 (s, 2H, NH2,

exchangeable with D2O), 2.0 (dd, 1H, J = 7.55, 4.52 Hz,

C5-H), 1.19 (s, 3H, C10-CH3), 0.75 (s, 3H, C13-CH3), 0.96

& 0.83 (other methyl protons); 13C NMR (125 MHz,

CDCl3) d (ppm): 173 (CONH), 150 (C=N), 140 (C6), 119.8

(C40), 56.70, 56.04, 54.11, 42.86, 40.5, 39.89, 38.5, 37.3,

36.70, 36.1, 36.07, 34.2, 32.76, 30.73, 28.1, 28.08, 27.1,

27.6, 26.57, 25.09, 24.8, 23.7, 23.8, 19.9, 14.08, 13.21;

MS(ESI): m/z 467 [M?.].

Computational profiling

Prediction of biological activity spectra

This computer system can predict the biological activity

based on structural formula of a chemical compound. The

PASS approach is based on the suggestion, Activ-

ity = function (structure). Molecule activity prediction is

done by ‘‘comparing’’ the structure of query compound

with the structure of well-known biological active substrate

Med Chem Res

123

existing in database of the freely available PASS web

service. Molecule activity prediction is done by ‘‘compar-

ing’’ the structure of query compound with the structure of

well-known biological active substrate existing in database

of PASS web service.

External files of the substance

Activity of the molecule was predicted, using PASS (Pre-

diction of Activity Spectra for Substances) which estimates

the probable biological activity profiles for compounds

under study based on their structural formulae presented

in.MOLfile or .SDfile format using Marvin applet. SD file

is can be exported either from ISIS/Base 2.0? (MDL

Information system, Inc.) or from ISIS/Base molecular

editor which has the option of SD file’s export. MOL file

can be prepared by ISIS/Draw. Molecular properties and

3D structure of a compound were determined by using sdf

format which is obtained from Pubchem database (NCBI).

The.mol generates 3D images using ArgusLab.

Algorithm of activity spectrum estimation

It is based on Bayesian approach that estimates the proba-

bilities of a molecule belonging to the classes of active and

inactive compounds, respectively. Comparison of PASS

prediction results with the experimental reported literature

provides independent validation of the approach versus

compounds in query with various kinds of biological activ-

ity. Average accuracy of prediction of online PASS is about

95 % according to the leave-one-out cross validation (LOO

CV) estimation. Accuracy of PASS prediction depends on

comprehensive information about biological activity spec-

trum for each compound available in PASS training set

which is regularly updated. Therefore, the estimate of bio-

logical activity tends to be more correct (Bhandarkar and

Khan, 2004).

Physicochemical properties

The physicochemical parameters including octanol parti-

tion coefficients (miLogP), Mw, HBD, HBA, TPSA and

Rotatable bonds were calculated using molinspiration

server (http://www.molinspiration.com/cgi-bin/properties)

and ChemAxon (chemicalize.org).

Pharmacological evaluation

Anticancer activity

Cell lines and culture conditions Human cancer cell lines

SW480 (human colon adenocarcinoma cells), HeLa (human

cervical cancer cells), A549 (human lung carcinoma cells),

HepG2 (human hepatic carcinoma cells) and HL-60 (human

leukaemia) were taken for the study. SW480, A549, HL60

and HepG2 cells were grown in RPMI 1640 supplemented

with 10 % foetal bovine serum (FBS), 10U penicillin and

100 lg/mL streptomycin at 37 �C with 5 % CO2 in a

humidified atmosphere. HeLa cells were grown in Dul-

becco’s modified Eagle’s medium (DMEM) supplanted

with FCS and antibiotics as described above for RPMI

1640. Fresh medium was given every second day. Cells

were passaged at preconfluent densities using a solution

containing 0.05 % trypsin and 0.5 mM EDTA.

Cell viability assay (MTT)

The anticancer activity in vitro was measured using the MTT

assay. The assay was carried out according to known pro-

tocol (Slater et al., 1963; Mosmann, 1983). Exponentially

growing cells were harvested and plated in 96-well plates at a

concentration of 1 9 104 cells/well. After 24 h incubation at

37 �C under a humidified 5 % CO2 to allow cell attachment,

the cells in the wells were respectively treated with target

compounds at various concentrations for 48 h. The con-

centration of DMSO was always kept below 1.25 %, which

was found to be non-toxic to the cells. A solution of 3-(4,5-

dimethylthizao1-2-y1)-2,5-diphenyltetrazolium bromide

(MTT) was prepared at 5 mg/mL in phosphate buffered

saline (PBS; 1.5 mM KH2PO4, 6.5 mM Na2HPO4, 137 mM

NaCl, 2.7 mM KCl; pH 7.4). 20 lL of this solution was

added to each well. After incubation for 4 h at 37 �C in a

humidified incubator with 5 % CO2, the medium/MTT

mixtures were removed, and the formazan crystals formed by

the mitochondrial dehydrogenase activity of vital cells were

dissolved in 100 lL of DMSO per well. The absorbance of

the wells was read with a microplate reader (Bio-Rad

Instruments) at 570 nm. Effects of the drug cell viability

were calculated using the cell treated with DMSO as control.

Data analysis

Cell survival was calculated using the formula: Survival

(%) = [(absorbance of treated cells - absorbance of cul-

ture medium)/(absorbance of untreated cells - absorbance

of culture medium)] 9 100 (Woerdenbag et al., 1993;

Saxena et al., 2007). The experiment was done in triplicate

and the inhibitory concentration (IC) values were calcu-

lated from a dose response curve. IC50 is the concentration

in ‘lM’ required for 50 % inhibition of cell growth as

compared to that of untreated control. IC50 values were

determined from the linear portion of the curve by calcu-

lating the concentration of agent that reduced the absor-

bance in treated cells compared to control cells by 50 %.

Evaluation is based on the mean values from three

Med Chem Res

123

independent experiments, each comprising at least six

microcultures per concentration level.

Antimicrobial activity

In the context of our studies, all of the synthesized com-

pounds were screened for their in vitro antibacterial

activities against the culture of Streptococcus pyogenes

(ATCC-29213), Staphylococcus aureus (ATCC-25923),

Pseudomonas aeruginosa (ATCC-27853), Escherichia coli

(ATCC-25922) and Klebsiella pneumoniae (Clinical iso-

late) by disc diffusion method (Shamsuzzaman et al.,

2010). The minimum inhibitory concentration (MIC) of all

the compounds was determined. Ciprofloxacin (30 mg)

was used as positive control, whereas DMSO poured disc

was used as negative control and then minimum inhibitory

concentration (MIC) was evaluated by the macrodilution

test using standard inoculums of 1–2 9 107c.f.u. mL-1

(0.5 McFarland standards). Serial dilutions of the test

compounds, previously dissolved in dimethyl sulfoxide

(DMSO) were prepared to final concentrations of 512, 256,

128, 64, 32, 16, 8, 4, 2 and 1 mg mL-1. To each tube was

added 100 mL of 24 h old inoculums. The MIC, defined as

the lowest concentration of the test compound which

inhibits the visible growth after 18 h and it was determined

visually after incubation for 18 h, at 37 �C. The suscepti-

bility of the bacteria to the test compounds was determined

by the formation of an inhibitory zone after 18 h of incu-

bation at 36 �C. The tests use DMSO and Ciprofloxacin as

negative and positive controls. The in vitro antifungal

activities of synthesized compounds were carried out using

C. albicans, A. fumigatus, T. mentagrophytes and Penicil-

lium marneffei (recultured) in DMSO by agar diffusion

method (Shamsuzzaman et al., 2010). The minimum

inhibitory concentration (MIC) was determined by broth

dilution technique as in antibacterial activity. The Inhibi-

tion zones of compounds were compared with Fluconazole

used as standard drug. The nutrient broth which contained

logarithmic serially twofold diluted amount of test com-

pound and controls was inoculated with approximately

1.6–6 9 104 c.f.u. mL-1. The cultures were incubated for

48 h at 35 �C and the growth was monitored.

Docking study

Protein and ligand preparation

The three dimensional structures of the targets were down-

loaded from protein databank. Hydrogen atom and MMFF

partial charge were added to the enzyme. Potential steric

clashes and added hydrogen atoms were relaxed by using the

minimization procedure. The minimization was performed

by using a CHARMm force field (Brooks et al., 1983) with

dependent dielectric implicit solvent model along and con-

jugates gradient method. This process was carried out until

the average absolute derivative of co-ordinates with respect

to energy fell below the 0.1 kcal A-1.The two dimensional

structures of ligands were prepared by using the ChemDraw

Ultra 11.0 software integrated with Cambridgesoft Software

(Cambridgesoft Corporation) (Lagunin et al., 2010).Further

refinement of compounds was performed by using energy

minimization protocol with cvff force field.

Molecular docking

GOLD (Genetic Optimisation for Ligand Docking) 5.0

(Jones et al., 1995) was used for docking of the compounds

dataset against the selected targets in present study.

Docking annealing parameters for van der Walls and

hydrogen bonding were set to 5.0 and 2.5 respectively. The

parameters used for genetic algorithm were population size

100, selection pressure 1.2, number of operations 1,00,000,

number of islands 5, niche size 2, migrate 10, mutate 100

and cross-over 100. Interaction analyses were performed

by using Ligplot (Wallace et al., 1995). Figures of the

complexes were prepared by using discovery studio visu-

alizer (Brooks et al., 1983).

Acknowledgments Authors would like to thank the Chairman,

Department of Chemistry, A.M.U., Aligarh, for providing the nec-

essary research facilities. Facilities provided by SAP (DRS-I) for their

generous research support are also gratefully acknowledged. We are

also thankful to the Department of Biochemistry, JNMC and Inter-

disciplinary Unit of Biotechnology, A.M.U., Aligarh, for biological

studies. A.A. would like to thank in part CSIR-RA (File No. 09/112

(0487) 2K2-EMR-1) for providing the fellowship.

References

Al-Haiza MA, El-Assiery SA, Sayed GH (2001) Synthesis and

potential antimicrobial activity of some new compounds con-

taining the pyrazol-3-one moiety. Acta Pharm 51:251–261

Andrasi M, Buglyo P, Zekany L, Gaspar A (2007) A comparative

study of capillary zone electrophoresis and pH-potentiometry for

determination of dissociation constants. J Pharm Biomed Anal

44:1040–1047

Atta UR, Anjum S, Farooq A, Khan MR, Parveen Z, Choudhary MI

(1998) Antibacterial steroidal alkaloids from Sarcococca salig-na. J Nat Prod 61:202–206

Bansal R, Guleria S (2008) Synthesis of 16E-[3-methoxy-4-(2-

aminoethoxy) benzylidene] androstene derivatives as potent

cytotoxic agents. Steroids 73:1391–1399

Bhandarkar MR, Khan A (2004) Antihepatotoxic effect of Nymphaea

stellata willd., against carbon tetrachloride-induced hepatic

damage in albino rats. J Ethnopharmacol 91:61–64

Bondock S, Tarhoni A El-G, Fadda A A (2006) Utility of cyanoacetic

acid hydrazide in heterocyclic synthesis. ARKIVOC (ix)

113-156

Brooks BR, Bruccoleri RE, Olafson BD, States DJ, Swaminathan S,

Karplus M (1983) CHARMM: a program for macromolecular

Med Chem Res

123

energy minimization and dynamics calculations. J Comp Chem

4:187–217

Callow RK, James VHT (1956) By-ways of synthesis of cortisone

from hecogenin. Part II. A route to 11-oxygenated compounds

through 3b: 20b-diacetoxy-11-bromoallopregnan-12-one. J Chem

Soc 4744–4749

Carvalho JFS, Silva MMC, Moreira JN, Simoes S, Melo MLS (2010)

Sterols as anticancer agents: synthesis of ring-B oxygenated

steroids, cytotoxic profile, and comprehensive SAR analysis.

J Med Chem 53:7632–7638

Cerda S, Wilkinson J, Broitman S (1995) Regulation of cholesterol

synthesis in four colonic adenocarcinoma cell lines. Lipids

30:1083–1092

Dauben GW, Takemura KH (1953) A study of the mechanism of

conversion of acetate to cholesterol via squalene. J Am Chem

Soc 75:6302–6304

Djouhri-Bouktab L, Vidal N, Rolain JM, Brunel JM (2011) Synthesis

of new 3, 20 bispolyaminosteroid squalamine analogues and

evaluation of their antimicrobial activities. J Med Chem 54:

7417–7421

Gerweck LE, Vijayappa S, Kozin S (2006) Tumor pH controls the

in vivo efficacy of weak acid and base chemotherapeutics. Mol

Cancer Ther 5:1275–1279

Guarna A, Occhiato EG, Macheeti F, Giacomelli V (1999) A

synthetic strategy from the preparation of (?)-17- (3-pyridyl)-5

b-10-azaestra-1, 16-dien-3-one, a novel potential inhibitor for

human cytochrome P45017a. J Org Chem 64:4985–4989

Handratta VD, Vasaitis TS, Njar VCO, Gediya LK, Kataria R, Chopra

P, Newman D Jr, Farquhar R, Guo Z, Qiu Y, Brodie AMH

(2005) Novel C-17-heteroaryl steroidal CYP17 inhibitors/an-

tiandrogens: synthesis, in vitro biological activity, pharmacoki-

netics, and antitumor activity in the LAPC4 human prostate

cancer xenograft model. J Med Chem 48:2972–2984

Hansch C, Fujita T (1964) p-r-p analysis. A method for the

correlation of biological activity and chemical structure. J Am

Chem Soc 86:1616–1626

Himly M, Jahn-Schmid B, Pittertschatscher K, Bohle B, Grubmayr K,

Ferreira F, Ebner H, Ebner C (2003) IgE-mediated immediate-

type hypersensitivity to the pyrazolone drug propyphenazone.

J Allergy Clin Immunol 111:882–888

Ikonen E (2008) Cellular cholesterol trafficking and compartmental-

ization. Nat Rev Mol Cell Biol 9:125–138

Jayasinghe UL, Nadeem M, Atta R, Choudhary MI, Ratnayake HD,

Amtul Z (1998) New antibacterial steroidal alkaloids from

Sarcococca brevifolia. Nat Prod Lett 12:103–109

Ji H, Zhang W, Zhang M, Kudo M, Aoyama Y, Yoshida Y, Sheng C,

Song Y, Yang S, Zhou Y, Lu J, Zhu J (2003) Structure based de

novo design, synthesis, and biological evaluation of nonazole

inhibitors specific for lanosterol 14R-demethylase of fungi.

J Med Chem 46:474–485

Jones G, Willett P, Glen RC (1995) Molecular recognition of receptor

sites using a genetic algorithm with a description of desolvation.

J Mol Biol 245:43–53

Kaminskyy D, Bednarczyk-Cwynar B, Vasylenko O, Kazakova O,

Zimenkovsky B, Zaprutko L, Lesyk R (2012) Synthesis of new

potential anticancer agents based on 4-thiazolidinone and

oleanane scaffolds. Med Chem Res 21:3568–3580

Kawai H, Nakai H, Suga M, Yuki S, Watanabe T, Saito KI (1997)

Effects of a novel free radical scavenger, MCl-186, on ischemic

brain damage in the rat distal middle cerebral artery occlusion

model. J Pharmacol Exp Ther 281:921–927

Koutsourea AI, Fousteris MA, Arsenou ES, Papageorgiou A, Pairas

GN, Nikolaropoulos SS (2008) Rational design, synthesis, and

in vivo evaluation of the antileukemic activity of six new

alkylating steroidal esters. Bioorg Med Chem 16:5207–5215

Krstic’a NM, Bjelakovic0 MS, Zizakb Z, Pavlovic MD NM, Juranic0

ZD, Pavlovic0 VD (2007) Synthesis of some steroidal oximes,

lactams, thiolactams and their antitumor activities. Steroids

72:406–414

Lagunin A, Filimonov DA, Borodina YV, Lagunin AA, Kos A (2010)

Multitargeted natural products evaluation based on biological

activity prediction with PASS. Cur pharm des 16:1703–1717

Ling YZ, Li JS, Liu Y, Kato K, Klus GT, Brodie A (1997)

17-Imidazolyl, pyrazolyl, and isoxazolyl androstene derivatives.

Novel steroidal inhibitors of human Cytochrome C17, 20-Lyase

P45017a. J Med Chem 40:3297–3304

Lipinski CA, Lombardo F, Dominy BW, Feeney PJ (1997) Exper-

imental and computational approaches to estimate solubility and

permeability in drug discovery and development settings. Adv

Drug Delivery Rev 23:3–25

Massey JB, Pownall HJ (2006) Structures of biologically active

oxysterols determine their differential effects on phospholipid

membranes. Biochemistry 45:10747–10758

Millurn AH, Truter EV (1956) The components of wool wax. Part III.

7-Oxocholesterol and the alleged presence of cholestanol.

J Chem Soc 341:1736–1739

Moreau F, Desroy N, Genevard JM, Vongsouthi V, Gerusz V, Fralliec

GL, Oliveira C, Floquet S, Denis A, Escaich S, Wolf K,

Busemann M, Aschenbsenner A (2008) Discovery of new Gram-

negative antivirulence drugs: structure and properties of novel

E. coli WaaC inhibitors. Bioorg Med Chem Lett 18:4022–4026

Mosmann T (1983) Rapid colorimetric assay for cellular growth and

survival: application to proliferation and cytotoxicity assays.

J Immunol Methods 65:55–63

Muegge I (2003) Selection criteria for drug-like compounds. Med Res

Rev 23:302–321

Nikaido H (1996) Outer Membrane in Escherichia coli and Salmo-nella. In: Neidhardt FC, Curtis R III, Ingraham JL (eds) Cellular

and molecular biology. ASM, Washington, DC, pp 29–47

Pan Y, Cai B, Wang K, Wang S, Zhou S, Yu X, Xu B, Chen L (2009)

Neferine enhances insulin sensitivity in insulin resistant rats.

J Ethnopharmacol 124:98–102

Pasha FA, Muddassar M, Neaz MM, Cho SJ (2009) Pharmacophore

and docking-based combined in silico study of KDR inhibitors.

J Mol Graph Model 28:54–61

Poza J, Rega M, Paz V, Alonso B, Rodrı’guez J, Salvador N,

Ferna’ndez A, Jime’nez C (2007) Synthesis and evaluation of

new 6-hydroximinosteroid analogs as cytotoxic agents. Bioorg

Med Chem 15:4722–4740

Refsgaard HH, Jensen BF, Brockhoff PB, Padkjaer SB, Guldbrandt

M, Christensen MS (2005) In silico prediction of membrane

permeability from calculated molecular parameters. J Med Chem

48:805–811

Roy J, DeRoy P, Poirier D (2007) 2b-(N-substituted piperazino)- 5a-

androstane-3a17b-diols: parallel solid-phase synthesis and anti-

proliferative activity on human leukemia HL-60 cells. J Comb

Chem 9:347–358

Salvador JAR, Carvalho JFS, Neves MAC, Silvestre SM, Leitao AJ,

Silva MMC, Melo MLS0 (2013) Anticancer steroids: linking

natural and semi-synthetic compounds. Nat Prod Rep 30:

324–374

Saxena HO, Faridi U, Kumar JK, Luqman S, Darokar MP, Shanker K,

Chanotiya CS, Gupta MM, Negi AS (2007) Synthesis of

chalcone derivatives on steroidal framework and their anticancer

activities. Steroids 72:892–900

Seong BC, Yon K, Yun MY, Byung ZA (2004) 5-Arylidene-2(5H)-

Furanone derivatives: synthesis and structure-activity relation-

ship for cytotoxicity. Arch Pharm Res 5:485–494

Shamsuzzaman, Khan MS, Alam M, Tabassum Z, Ahmad A, Khan

AU (2010) Synthesis, antibacterial and antifungal activities of 6,

Med Chem Res

123

5 fused steroidal oxazoles in cholestane series. Eur J Med Chem

45:1094–1097

Shamsuzzaman, Khanam H, Dar AM, Siddiqui N, Rehman S (2012)

Synthesis, characterization, antimicrobial and anticancer studies

of steroidal pyrazolines. J Saudi Chem Soc. doi:10.1016/j.jscs.

2012.05.004

Shamsuzzaman, Khanam H, Mashrai A, Siddiqui N (2013) Construc-

tion of novel steroidal isoxazolidinone derivatives under Vils-

meier-Haack conditions. Tetrahedron Lett 54:874–877

Slater TF, Swyer B, Strauli U (1963) Studies on succinate-tetrazolium

reductase systems. III. Points of coupling of four different

tetrazolium salts. Biochim Biophys Acta 77:383–393

Tabas I (2002) Consequences of cellular cholesterol accumulation:

basic concepts and physiological implications. J Clin Invest

110:905–911

Takano M, Hasegawa R, Fukuda T, Yumoto R, Nagai J, Murakami T

(1998) Interaction with P-glycoprotein and transport of erythro-

mycin, midazolam and ketoconazole in Caco-2 cells. Eur J

Pharmacol 358:289–294

Thibeault D, Roy J, Roy PD, Poirier D (2008) Chemical synthesis of

2bamino-5 a- androstane-3a17 b-diol N-derivatives and their

antiproliferative effect on HL-60 human leukemia cells. Bioorg

Med Chem 16:5062–5077

Uetrecht J (2001) Prediction of a new drug’s potential to cause

idiosyncratic reactions. Curr Opin Drug Discov Devel 4:55–59

Vaara M (1993) Outer membrane permeability barrier to azithromy-

cin, clarithromycin in Gram-negative enteric bacteria. Antimic-

rob Agents Chemother 37:354–356

Veber DF, Johnson SR, Cheng HY, Smith BR, Ward KW, Kapple KD

(2002) Molecular properties that influence the oral bioavailabil-

ity of drug candidates. J Med Chem 45:2615–2623

Vejux A, Lizard G (2009) Cytotoxic effects of oxysterols associated

with human diseases: induction of cell death (apoptosis and/or

oncosis), oxidative and inflammatory activities, and phospholip-

idosis. Mol Aspects Med 30:153–170

Wallace AC, Laskowski RA, Thornton JM (1995) LIGPLOT: a

program to generate schematic diagrams of protein-ligand

interactions. Protein Eng 8:127–134

Wani MY, Athar F, Salauddin A, Agarwal SM, Azam A, Choi I, Bhat

AR (2011) Novel terpene based 1,4,2-dioxazoles: synthesis,

characterization, molecular properties and screening against

Entamoeba histolytica. Eur J Med Chem 46:4742–4752

Woerdenbag HJ, Moskal TA, Pras N, Malingre TM, El-Feraly FS,

Kampinga HH, Konings AWT (1993) Cytotoxicity of artemis-

inin-related endoperoxides to ehrlich ascites tumor-cells. J Nat

Prod 56:849–856

Wu TW, Zeng LH, Wu J, Fung KP (2002) Myocardial protection of

MCI-186 in rabbit ischemia–reperfusion. Life Sci 71:2249–2255

Med Chem Res

123