Triterpenic and monoterpenic esters from stems of Ichnocarpus frutescens and their drug likeness potential

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Medicinal Chemistry Research ISSN 1054-2523 Med Chem ResDOI 10.1007/s00044-014-1227-2

Triterpenic and monoterpenic esters fromstems of Ichnocarpus frutescens and theirdrug likeness potential

Babita Aggarwal, Rajeev K. Singla,Mohd. Ali, Vijender Singh, John O. Igoli,Rohit Gundamaraju & Kah Hwi Kim

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ORIGINAL RESEARCH

Triterpenic and monoterpenic esters from stems of Ichnocarpusfrutescens and their drug likeness potential

Babita Aggarwal • Rajeev K. Singla • Mohd. Ali • Vijender Singh •

John O. Igoli • Rohit Gundamaraju • Kah Hwi Kim

Received: 17 February 2014 / Accepted: 7 August 2014

� Springer Science+Business Media New York 2014

Abstract Phytochemical studies of ethanolic extract of

stems of Ichnocarpus frutescens resulted in the isolation of

tetracyclic triterpenic ester lanosteryl oleate (6), two new

monoterpenic esters, menth-1(7)-en-9-olyl dodecanoate

(7), and 2-(4-methylcyclohex-3-enyl)propyl dodecanoate

(8). Their structures were established on the basis of

extensive spectral data analysis and chemical methods.

From the literature survey, it was revealed that none of

these three compounds have been reported earlier from any

other parts of I. fructescens. Using StarDrop, drug metab-

olism and pharmacokinetic parameters have been studied,

while data of toxicological endpoints were generated using

Derek Nexus. 7 is metabolically stable as compared to 6

and 8. All three molecules are highly lipophilic so tendency

to get distributed in brain and adipose tissue is more. Based

on the current knowledge database, Derek Nexus predicted

that these molecules are not mutagenic, carcinogenic,

genotoxic, hepatotoxic, hERG channel inhibitor, nephro-

toxic, neurotoxic but StarDrop found them as probable

developmental toxic. So in a word, these phytoconstituents

have drug-likeness potential and can be evaluated for

possible targets.

Keywords Ichnocarpus frutescens � Apocyanaceae �Stem extract � Monoterpenic ester � Triterpenic ester

Introduction

Ichnocarpus frutescens R. Br. (Apocyanaceae), commonly

known as Black Sariva, is a climbing shrub found almost in

all parts of India, ascending to an altitude of 1,200 m.

(Medicinal Plants of India, 1987). Ichnocarpus frutescens

is considered as a substitute for Hemidesmus indicus, also

known as Indian Sarsaparilla (Chattejee and Pakrashi,

2003). Earlier studies on this plant in regards to the bio-

logical action on living organisms stood as a substantial

evidence for the present study. The potential of this plant in

attenuating tumor was a strong backbone in elucidating its

anti-neoplastic nature (Kumarappan and Mandal, 2007).

Anti microbial effects against some dreadful organisms

like Escherichia coli and Aspergillus flavus did add as

another evidence for its cytotoxic nature (Malathy and Sini,

2009). The plant did not only exhibit its potential in

attenuating microorganisms but also as an effective pro-

tective agent in the case of hepatotoxicity (Deepak Dash

et al., 2007). The plan could successfully exhibit protective

B. Aggarwal

Department of Pharmacognosy, HR Institute of Pharmacy,

Ghaziabad, Uttar Pradesh, India

R. K. Singla (&)

Division of Biotechnology, Netaji Subhas Institute of

Technology, University of Delhi, Azad Hind Fauz Marg,

Sector-3, Dwarka 110078, New Delhi, India

e-mail: [email protected]

Mohd. Ali

Department of Pharmacognosy and Phytochemistry, Faculty of

Pharmacy, Hamdard University, Hamdard Nagar 110062,

New Delhi, India

V. Singh

Department of Pharmacy, Ram-Eesh Institute of Vocational &

Technical Education, 3, Knowledge Park I, Greater Noida

Uttar Pradesh, India

J. O. Igoli

Natural Product Laboratories, SIPBS, University of Strathclyde,

161 Cathedral Street, Glasgow G4 0RE, Scotland, UK

R. Gundamaraju � K. H. Kim

Faculty of Medicine, University of Malaya,

50603 Kuala Lumpur, Malaysia

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Med Chem Res

DOI 10.1007/s00044-014-1227-2

MEDICINALCHEMISTRYRESEARCH

Author's personal copy

action toward the paracetamol-induced hepatotoxicity.

Significant antioxidant potential of the polyphenols pre-

sents in the plant aided as an important and preliminary

reference to many biological activities (Kumarappan et al.,

2012).

On the other hand, investigation of the chemistry and

related studies on this plant led to the isolation of a-L-

rhamnopyranosyl-(1 ? 4)-b-D-glucopyranosyl-(1 ? 3)-a-

amyrin (Minchona and Tandon, 1980), 6,8,8-tri-

methylpentacosan-7-one (Minchona and Tandon, 1980), a-

amyrin and its acetates, lupeol and its acetates, friedelin,

epi-friedelinol, and b-sitosterol (Lakshmi et al., 1985) from

stems. Leaves mainly contain flavones and glycoflavones

(Daniel and Sabnis, 1978), Ursolic acid acetate, kaemferol,

kaemferol-3-galactoside (trifolin), and mannitol (Khan

et al., 1995). Flowers contain quercetin and quercetin-3-O-

b-D-glucopyranoside (Singh and Singh 1987). Previously

our team had isolated n-butyl oleate (1), n-octyl tertacon-

tane (2), tetratriacontadiene (3), n-nonadecanylbenzoate

(4), and benzocosanyl arachidate (5) from I. frutescens

(Aggarwal et al., 2010). Both the biological evidences and

chemistry affirmations, I. frutescens was employed in the

present study.

In the development of orally available drugs, significant

drug absorption and drug delivery are very important factors

to be considered. Oral bioavailability of a drug is primarily

affected by many factors like dissolution in GI tract, intes-

tinal membrane permeation, and intestinal/hepatic first-pass

metabolism. Thus, predictions of bioavailability and/or

bioavailability related properties, such as intestinal absorp-

tion (HIA in case of humans), solubility, the effect of

transporter proteins, metabolism based on cytochrome P450

enzymes, etc., are areas in need of progress to aid pharma-

ceutical drug development (Hou et al., 2007). Toxicity

accounts for approximately 30 % of expensive, late stage

failures in drug discovery. So, it would be advisable to in-

dentify and prioritize chemistries with lower toxicity risks,

as early as possible in the drug discovery process and it

would ultimately help researchers to address the high attri-

tion rate in pharmaceutical R&D (Segall and Barber, 2014).

Henceforth, in silico studies of drug likeness potential are

very important, both in the early stage of drug discovery to

select the most promising compounds for further optimiza-

tion and in the later stage to identify candidates for further

clinical development. This is how, we can approach an

economical boulevard to drug discovery.

In this study, we describe the isolation and structure

elucidation of lanosteryl oleate and two monoterpenic

esters from stems of the plant. None of these compounds

have been isolated earlier from any other parts of the plant.

Moreover, their drug-likeness potential has also been pre-

dicted by simulating P450 metabolism, toxicological end-

points, and other pharmacokinetic parameters.

Materials and methods

Collection and authentication of plant materials

The stems of I. fructescens R. Br. were procured from

Dehradun, Uttrakhand in September 2008 and were

authenticated by taxonomist Dr. Anjula Pandey, Dept. of

National Bureau of Plant Genetic Resources (NBPGR),

Pusa Campus, New Delhi. A voucher specimen (Specimen

No: NHCP/NBPGR/2009-13/889) is preserved (Dhorajiya

et al., 2011) in the Herbarium Section of the Taxonomy

Department NBPGR, New Delhi and also in the Phyto-

chemistry Laboratory, Faculty of Pharmacy, Ram-Eesh

Institute of Vocational and Technical Education, Greater

Noida, Uttar Pradesh (Aggarwal et al., 2010).

Extraction and isolation

The stems of I. fructescens were carefully collected and air

dried under shade followed by pulverization to coarse

powder. The coarse powder (2.5 kg) of I. frutescens stem

was extracted with 95 % ethanol (ethanol/water, 19:1) for

2 days in a soxhlet apparatus. The combined extracts were

concentrated under reduced pressure to obtain dried dark

brown colored 200 g (8.0 %) residue (Aggarwal et al.,

2010). The residue was subjected to Silica gel column

chromatography using gradient solvent system of petro-

leum ether, CHCl3, and methanol. Elution of the column

with petroleum ether-chloroform (17:3) afforded colorless

crystals of compound Lanosteryl oleate {6, recrystallized

from diethyl ether: methanol (1:1), 120 mg (0.06 %

yield)}. Elution of the column with petroleum ether-chlo-

roform (1:1) gave colorless crystals of compound menth-

1(7)-en-9-olyl dodecanoate {7, recrystallized from diethyl

ether: methanol (1:1), 95 mg (0.0475 % yield)}. Elution of

the column with petroleum ether-chloroform (3:7) gave

brownish gummy product of compound 2-(4-methylcy-

clohex-3-enyl) propyl dodecanoate {8, recrystallized from

diethyl ether:methanol (1:1), 130 mg (0.065 % yield)}.

Drug metabolism and pharmacokinetics

StarDrop of Optibrium Ltd. was used for prediction of drug

metabolism and pharmacokinetics of these isolated phyto-

constituents (Bhaveshkumar Dhorajiya et al., 2012).

Parameters studied were LogS, [email protected], LogP,

LogD, 2C9 pKi, hERG pIC50, BBB Log ([brain]:[blood]),

BBB category, HIA category, P-gp category, 2D6 affinity

category, PPB90 category, developmental toxcity category,

and composite site lability of these molecules on three

isoforms of cytochrome P450, i.e., 3A4, 2D6, and 2C9

(Optibrium Ltd.).

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Toxicological studies

Derek Nexus module of LHASA ltd. was used to calculate

toxicological endpoints like carcinogenicity, photocarci-

nogenicity, chromosome damage in vitro, chromosome

damage in vivo, photo-induced chromosome damage

in vitro, genotoxicity in vitro, genotoxicity in vivo, pho-

togenotoxicity in vitro, photogenotoxicity in vivo, hepato-

toxicity, irritation (of the eye), irritation (of the

gastrointestinal tract), irritation (of the respiratory tract),

irritation (of the skin), lachrymation, HERG channel inhi-

bition in vitro, alpha-2-mu-globulin nephropathy, anaphy-

laxis, bladder urothelial hyperplasia, cardiotoxicity,

cerebral oedema, chloracne, cholinesterase inhibition,

cumulative effect on white cell count and immunology,

cyanide-type effects, high acute toxicity, methaemoglobi-

naemia, nephrotoxicity, neurotoxicity, oestrogenicity, per-

oxisome proliferation, phospholipidosis, phototoxicity,

pulmonary toxicity, uncoupler of oxidative phosphoryla-

tion, developmental toxicity, teratogenicity, testicular tox-

icity, ocular toxicity, mutagenicity in vitro, mutagenicity

in vivo, photomutagenicity in vitro, thyroid toxicity,

photoallergenicity, skin sensitization, occupational asthma

and respiratory sensitization (Lhasa Ltd.).

Results and discussion

Lanosteryl oleate (6)

Compound 6, named as lanosteryl oleate((10R,13R,14S,

17R)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-4,

4,10,13,14-pentamethyl-17-((R)-6-methylheptan-2-yl)-1H-

cyclopenta[a]phenanthren-3-yl oleate) was obtained as a

colorless crystalline mass from petroleum ether–chloroform

(17:3) eluents. It responded positively to Libermann Bur-

chard test. Molecular formula C48H84O2, Rf 0.85 (petroleum

ether:chloroform :: 1:1), m. p. 82–84 �C. IR tmax (KBr):

2918, 2850, 1728, 1640, 1467, 1380, 1268, 1175, 1103, 982,

719 cm-1; 1H NMR (CDCl3): Refer Table 1, 13C NMR

(CDCl3): Refer Table 1. TOF MS m/z (rel. int.): 692[M]?

(2.3), 427 (5.1), 410 (6.2), 282 (100). Elucidated structure of

Lanosteryl oleate is given in Fig. 1.

Table 1 1H NMR and 13C NMR spectral data of Lanosteryl Oleate

(6)

Position 1H NMR 13C NMR

Alpha Beta

1 1.47 m 2.03 m 37.38

2 1.98 m 1.86 m 27.96

3 4.49 dd (4.8, 10.3) – 80.60

4 – – 40.79

5 – – 142.70

6 5.18 m – 121.66

7 1.96 m 1.93 m 34.36

8 1.76 m – 43.35

9 1.56 m – 47.25

10 – – 37.16

11 1.27 m 1.39 m 21.14

12 1.52 m 1.42 m 33.35

13 – – 46.81

14 – – 55.60

15 1.63 m 1.23 m 31.36

16 1.61 m 1.29 m 31.10

17 1.86 m – 51.14

18 0.73 brs – 14.58

19 1.01 brs – 18.17

20 1.80 m – 38.62

21 0.94 d (7.8) – 16.79

22 1.71 m 1.63 m 34.89

23 1.16 m 1.46 m 23.74

24 1.63 m 1.32 m 41.73

25 2.01 m – 26.94

26 0.84 d (6.3) – 16.62

27 0.82 d (6.1) – 16.11

28 0.90 brs – 25.20

29 1.07 brs – 27.54

30 1.13 brs – 15.57

1’ – – 173.72

2’ 2.31 d (7.5) 2.26 d (7.2) 55.27

3’ 1.52 m – 31.95

4’ 1.25 brs – 31.10

5’ 1.25 brs – 29.72

6’ 1.25 brs – 29.72

7’ 1.25 brs – 32.38

8’ 1.76 m – 38.42

9’ 4.96 m – 129.79

10’ 4.94 m – 129.79

11’ 1.71 m – 37.73

12’ 1.25 brs – 32.51

13’ 1.25 brs – 29.72

14’ 1.25 brs – 29.39

15’ 1.25 brs – 29.30

16’ 1.23 brs – 26.19

Table 1 continued

Position 1H NMR 13C NMR

Alpha Beta

17’ 1.23 brs – 22.71

18’ 0.87 t (6.5) – 14.14

Coupling constant in Hertz are provided in parenthesis

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Its IR spectrum showed characteristic absorption bands

for ester groups (1728 cm-1), unsaturation (1640 cm-1),

and long aliphatic chain (719 cm-1). Its mass spectrum

exhibited a molecular ion peak at m/z 692 consistent with

the molecular formula C48H84O2. It indicated seven double

bond equivalents; four of them were adjusted in the tetra-

cyclic carbon framework, two in the vinylic linkage, and

the remaining one in the ester function.

The ion peaks arising at m/z 427 [M–CH3(CH2)7CH=

CH(CH2)7CO]?, 410 [M–CH3(CH2)7CH=CH(CH2)7COOH]?, and 282 [CH3(CH2)7CH=CH(CH2)7COOH]? that

oleic acid was esterified to lanosterol-type triterpene.

The 1H NMR spectrum of 6 showed three one-proton

multiplets at d 5.18, 4.96, and 4.94 assigned to vinylic H-6,

H-90 and H-100 protons, respectively. A one-proton doublet

at 4.49 (J = 4.8, 10.3 Hz) was ascribed to a-oriented H-3

carbinol proton. Three doublet at d 0.94 (J = 7.8 Hz), 0.84

(J = 6.3 Hz), and 0.82 (J = 6.1 Hz) and five broad signals

at d 0.73, 1.01, 0.90, 1.07, and 1.33, all integrated for three

protons each, were assigned correspondingly to secondary

C-21, C-26, and C-27 and tertiary C-18, C-19, C-28, C-29,

and C-30 methyl protons, all attached to saturated carbons.

A three proton triplet at d 0.87 (J = 6.5 Hz) was accounted

to C-180 primary methyl protons. The remaining methylene

and methane protons resonated between d 2.03–1.23.

The 13C NMR spectrum of 6 exhibited signals for ester

carbon at d 173.32 (C-10), vinylic carbons at d 142.70 (C-

5), 121.66 (C-6), 129.79 (C-90, C-100), carbinol carbon at d80.60 (C-3), and methyl carbons at d 14.58 (C-18), 18.17

(C-19), 16.79 (C-21), 16.62 (C-26), 16.11 (C-27), 25.20

(C-28), 27.54 (C-29), 15.57 (C-30), and 14.14 (C-18’). The13C NMR values of the lanosterol carbon framework were

compared with the reported values of Lanosterol-type

triterpenoids(Sharma and Ali, 1996). Alkaline hydrolysis

of 6 yielded oleic acid and lanosterol. Basis of these evi-

dences the structure of 6 has been elucidated as lanost-5-

en-3b-yl-octadec-90-enoate.

Menth-1(7)-en-9-olyl dodecanoate (7)

Elution of the column with petroleum ether–chloroform

(1:1) gave colorless crystals of compound 7 (2-(1-hy-

droxypropan-2-yl)-5-methylenecyclohexyl dodecanoate),

recrystallized from diethyl ether: methanol (1:1), 95 mg

(0.0475 % yield), Rf 0.44 (petroleum ether:chloroform ::

2:3), m.p. 62–64 �C. IR tmax (KBr): 3386, 2918, 2850,

1729, 1639, 1463, 1378, 1260, 1172, 1094, 1025, 802,

721 cm-1. 1H NMR (CDCl3): d 4.87 (1H, brs, H2-7a), 4.83

Fig. 1 Structure of 6

Fig. 2 Structure of 7

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(1H, brs, H2-7b), 3.56 (1H, dd, J = 3.2, 3.1 Hz, H-2b),

3.16 (1H, d, J = 11.6 Hz, H2-9a), 3.13 (1H, d,

J = 11.6 Hz, H2-9b), 2.21 (1H, d, J = 6.9 Hz, H2-20a),

2.19 (1H, d, J = 6.9 Hz, H2-20b), 1.97 (2H, m, H2-6), 1.79

(1H, m, H-4), 1.69 (1H, m, H-8), 1.61 (2H, m, CH2), 1.53

(2H, m, CH2), 1.40 (2H, m, CH2), 1.18 (16H, brs, 89 CH2),

0.90 (3H, d, J = 9.0 Hz, Me-10), 0.81 (3H, t, J = 6.5 Hz,

Me-120). 13C NMR (CDCl3): d 172.92 (C-1), 139.27 (C-1),

114.06 (C-7), 79.01 (C-2), 62.15 (C-9), 55.13 (C-4), 37.16

(C-8), 33.82 (CH2), 33.35 (CH2), 31.93 (CH2), 31.32

(CH2), 29.69 (49 CH2), 29.36 (CH2), 28.96 (CH2), 26.19

(CH2), 22.68 (CH2), 18.30 (Me-10), 14.10 (Me-120). ?ve

ion TOF MS m/z (rel. int.): 352 [M]? (C22H40O3) (15.2).

Elucidated structure is given in Fig. 2.

7 was obtained as a colorless crystalline mass from

petroleum ether–chloroform (1:1) eluents. Its IR spectrum

showed characteristic absorption bands for hydroxyl group

(3386 cm-1), ester groups (1729 cm-1), unsaturation

(1639 cm-1), and long aliphatic chain (802, 721 cm-1). On

the basis of mass spectrum, its molecular weight was

established at m/z 352 corresponding to the molecular

formula of a monoterpenic ester, C22H40O3. The 1H NMR

spectrum of 7 showed two one-proton doublets at d 4.87Fig. 3 Structure of 8

Fig. 4 Substrate specificity of 6 on CYP3A4

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Fig. 5 Substrate specificity of 6 on CYP2C9

Fig. 6 Substrate specificity of 6 on CYP2D6

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and 4.83 assigned to exocyclic methylene H2-7 protons. A

one-proton double doublet at 3.56 (J = 3.2, 3.1 Hz) was

ascribed to oxygenated methine H-2b proton. Two one-

proton doublets at d 3.16 (J = 11.6 Hz) and 3.13

(J = 11.6 Hz) were accounted to hydroxymethylene H2-9

protons. Two one-proton doublet at d 2.21 (J = 6.9 Hz)

and 2.19 (J = 6.9 Hz) were ascribed to methylene H2-2

protons adjacent to ester group. Two three-proton signals

as a doublet at d 0.90 (J = 9.0 Hz) and as a triplet at d 0.81

(J = 6.5 Hz) were associated with the secondary C-10 and

primary C-12 methyl protons. The remaining methine and

methylene protons appeared between d 1.97 and 1.18.

The 13C NMR spectrum of 7 exhibited signals for ester

carbon at d 172.92 (C-10), vinylic carbons at d 139.27 (C-1)

and 114.06 (C-7), oxygenated methine carbon at d 79.01

(C-2), hydroxymethylene carbon at d 62.15 (C-9), methyl

carbons at d 18.30 (C-10) and 14.10 (C-120), and methylene

and methine carbons between d 55.13 and 22.68. The

appearance of oxygenated methine carbon in the de-

shielded region at d 79.01 (C-2) indicated the location of

the ester function at this carbon. Alkaline hydrolysis of 7

yielded lauric acid, m.p. 43–44 �C (Co TLC comparable).

On the basis of these evidences, the structure of 7 has been

elucidated as menth-1(7)-en-9-olyl dodecanoate. This is a

Fig. 7 Substrate specificity of 7on CYP3A4

Fig. 8 Substrate specificity of 7on CYP2D6

Fig. 9 Substrate specificity of 7on CYP2C9

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new menthene-type monoterpenic ester isolated from a

plant source.

2-(4-Methylcyclohex-3-enyl)propyl dodecanoate (8)

Elution of the column with petroleum ether–chloroform

(3:7) gave brownish gummy product of compound 8, re-

crystallized from diethyl ether: methanol (1:1), 130 mg

(0.065 % yield), Rf 0.71 (petroleum ether:chloro-

form:methanol :: 2:8:1 drop). IR tmax (KBr): 2925, 2853,

1735, 1635, 1464, 1376, 1245, 1180, 1081, 1029, 972,

721 cm-1. 1H NMR (CDCl3): d 5.36 (1H, brs, H-2), 4.13

(1H, t, J = 6.9 Hz, H2-9a), 4.08 (1H, t, J = 6.6 Hz, H2-

9b), 2.28 (2H, m, H2-20), 2.12 (2H, m, H2-3), 2.04 (2H, m,

H2-6), 1.87 (1H, m, H-4), 1.83 (1H, m, H-8), 1.68 (2H, m,

H2-3), 1.60 (3H, brs, Me-7), 1.25 (18H, brs, 99 CH2), 0.97

(3H, d, J = 6.1 Hz, Me-10), 0.87 (3H, t, J = 6.5 Hz, Me-

120). 13C NMR (CDCl3): d 169.13 (C-10), 139.83 (C-1),

122.05 (C-2), 67.53 (C-9), 55.96 (C-4), 33.92 (C-8), 31.93

(CH2), 31.16 (CH2), 29.70 (69 CH2), 29.35 (CH2), 29.17

(CH2), 27.99 (CH2), 24.93 (CH2), 22.69 (CH2), 19.31 (Me-

7), 18.18 (Me-10), 14.11 (Me-120). ?ve ion TOF MS m/

z (rel. int.): 336 [M]? (C22H40O2) (1.1), 137 (23.8). Elu-

cidated structure is given in Fig. 3.

8 was obtained as a gummy product from petroleum

ether–chloroform (3:7) eluents. Its IR spectrum showed

characteristic absorption bands for ester groups

(1735 cm-1), unsaturation (1635 cm-1), and long aliphatic

chain (721 cm-1). The mass spectrum of 8 exhibited a

molecular ion peak at m/z 336 corresponding to the

molecular formula of a monoterpenic ester, C22H40O2. An

ion peak arising at m/z 137 [C10H17]? indicated that p-

menthene-type of molecule was esterified with a C12-fatty

acid.

The 1H NMR spectrum of 8 showed a one-proton

multiplet at d 5.36 assigned to vinylic H-2 proton. Two

one-proton doublet at d 4.13 (J = 6.9 Hz) and 4.08

(J = 6.6 Hz) were ascribed to oxygenated methylene H2-9

proton. Three two-proton multiplets at d 2.28, 2.12, and

2.04 were ascribed to methylene H2-20 nearby ester func-

tion and H2-3 and H2-6, respectively. The remaining

methylene and methine protons resonated between d1.87–1.25.

The 13C NMR spectrum of 8 exhibited signals for vinylic

carbons at d 139.83 (C-1), and 122.05 (C-2), ester carbon at d169.13 (C-10), oxygenated methylene carbon at d 67.53 (C-

9), methyl carbons at d 19.31 (C-7), 18.18 (C-10), and 14.11

Fig. 10 Substrate specificity of 8 on CYP3A4 Fig. 11 Substrate specificity of 8 on CYP2D6

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(C-120) and methylene and methine carbons between d55.96–22.69. Alkaline hydrolysis of 8 yielded lauric acid,

m.p. 43-44 �C (Co TLC comparable). On the basis of the

foregoing account, the structure of 8 has been elucidated as

2-(4-methylcyclohex-3-enyl)propyl dodecanoate.

When 6, 7, and 8 were subjected to predict the most likely

sites of metabolism for CYP3A4, CYP2D6, and CYP2C9, it

was found that basic scaffold of 6, i.e., lanost-5-en-3b-yl is

stable and does not possess any potential and labile sites of

metabolism. All the labile positions are there on its sub-

stituent, i.e., oleic acid (Refer Figs. 4, 5, 6). In case of 7, most

likely sites of metabolism for CYP3A4, CYP2D6, and

CYP2C9 are present in 2-(1-hydroxypropan-2-yl)-5-meth-

ylenecyclohexyl ring and the terminal 2� methyl group

(Refer Figs. 7, 8, 9), while in case of 8, most likely sites of

metabolism for these three isoforms of cytochrome P450

enzymes are present in the 4-methylcyclohex-3-enyl ring

(Refer Figs. 10, 11, 12). Composite site lability (CSL) is

lowest in case of 7, assuming it as the most stable out of these

three phytoconstituents and 6 and 8 is having high risk of

rapid metabolism by cytochrome P450 enzymes. As can be

seen in Table 2 (Refer Table 2), only 7 and 8 is passing

Lipinski rule of five. By referring Table 2, it can be observed

that except equivocal chances of skin sensitization in case of

Fig. 12 Substrate specificity of 8 on CYP2C9

Table 2 In silico prediction of drug metabolism, pharmacokinetics

and specificity to toxicological end points

Properties 6 7 8

MW 693.2 352.6 336.6

HBD 0 1 0

HBA 2 3 2

TPSA 26.3 46.53 26.3

Flexibility 0.4151 0.56 0.5833

Rotatable Bonds 22 14 14

Lipinski Rule of Five Score 0.25 0.5017 0.5

P450_3A4_CSL 0.9723 0.842 0.96

LogS -1.028 1.575 0.9789

logS @ pH7.4 -1.028 1.575 0.9789

LogP 9.9 6.175 7.039

LogD 9.9 6.175 7.039

2C9 pKi 4.871 4.489 4.593

hERG pIC50 7.219 5.896 6.198

BBB log([brain]:[blood]) 0.4174 0.191 0.6962

BBB category ? ? ?

HIA category ? ? ?

P-gp category Yes Yes Yes

2D6 affinity category High High High

PPB90 category High High High

Ames mutagenicity

category

Non-

mutagenic

Non-

mutagenic

Non-

mutagenic

Bioconcentration Factor

GPOpt

1.012 1.894 2.331

Caco-2_log(Papp)_PLS -4.1 -4.553 -4.275

Daphnia Magna-log(LC50) M 9.012 5.708 6.304

Developmental tox.

Category

Toxic Toxic Toxic

Fathead minnow-log(LC50)

M GP2DSearch

6.486 6.05 6.305

HTS promiscuity alerts 0 3 3

log(VDss) RBF -0.2439 0.07152 0.2169

Oral rat-log(LD50) M/kg 2.882 1.641 1.63

RBF_T_Half_Life 0.1932 0.512 0.4462

Tetrahymena pyriformis-

log(IGC50) M

10.21 6.407 6.893

Carcinogenicity No report No report No report

Photocarcinogenicity No report No report No report

Chromosome damage

in vitro

No report No report No report

Chromosome damage

in vivo


Photo-induced chromosome

damage in vitro


Genotoxicity in vitro No report No report No report

Genotoxicity in vivo No report No report No report

Photogenotoxicity in vitro No report No report No report

Photogenotoxicity in vivo No report No report No report

Hepatotoxicity No report No report No report

Med Chem Res

123


6 and 8, all these three phytomolecules are not having any

predictable toxicity like carcinogenicity, photocarcinoge-

nicity, chromosome damage in vitro, chromosome damage

in vivo, photo-induced chromosome damage in vitro,

genotoxicity in vitro, genotoxicity in vivo, photogenotoxic-

ity in vitro, photogenotoxicity in vivo, hepatotoxicity, irri-

tation (of the eye), irritation (of the gastrointestinal tract),

irritation (of the respiratory tract), irritation (of the skin),

lachrymation, HERG channel inhibition in vitro, alpha-2-

mu-globulin nephropathy, anaphylaxis, bladder urothelial

hyperplasia, cardiotoxicity, cerebral oedema, chloracne,

cholinesterase inhibition, cumulative effect on white cell

count and immunology, cyanide-type effects, high acute

toxicity, methaemoglobinaemia, nephrotoxicity, neurotox-

icity, oestrogenicity, peroxisome proliferation, phospholip-

idosis, phototoxicity, pulmonary toxicity, uncoupler of

oxidative phosphorylation, developmental toxicity, terato-

genicity, testicular toxicity, ocular toxicity, mutagenicity

in vitro, mutagenicity in vivo, photomutagenicity in vitro,

thyroid toxicity, photoallergenicity, occupational asthma,

and respiratory sensitization.

This toxicological end points prediction was done on the

basis of up-to-date knowledge of LHASA-Derek Nexus

and StarDrop. Reports are influencing and suggested that

these phytoconstituents can be the basis of derivatizing

new chemical entities with targeted pharmacological

activities. These compounds might be the key constituents

in I. frutescens responsible for its biological activity.

Conclusion

Thus summing up of biological, Pharmacological, and

chemical affirmations, the present study was performed on

the plant. The revealing of tetracyclic triterpenic & monot-

erpenic esters and their drug likeness potential adds a great

mile stone in the field of medicinal chemistry and natural

products. The targeting of the pharmacological activities by

the endorsement of chemical studies would lead to devel-

opment of novel and flawless chemical molecules.

Acknowledgments The authors owe their gratitude to Ramesh

Group of institutions for providing platform to perform this research

work. RK Singla is thankful to Science & Engineering Research

Board for providing young scientist fellowship vide project notifica-

tion SR/FT/LS-149/2011. Authors are thankful to Optibrium Ltd and

LHASA Ltd for providing StarDrop and Derek Nexus package,

respectively.

Conflict of interest The authors declare no conflict of interest.

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Table 2 continued

Properties 6 7 8

Irritation (of the eye) No report No report No report

Irritation (of the

gastrointestinal tract)


Irritation (of the respiratory

tract)


Irritation (of the skin) No report No report No report

Lachrymation No report No report No report

HERG channel inhibition

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Alpha-2-mu-Globulin

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Anaphylaxis No report No report No report

Bladder urothelial

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Cardiotoxicity No report No report No report

Cerebral oedema No report No report No report

Chloracne No report No report No report

Cholinesterase inhibition No report No report No report

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Cyanide-type effects No report No report No report

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Peroxisome proliferation No report No report No report

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Triterpenic and monoterpenic esters from stems of Ichnocarpus frutescens and their drug likeness potential