Hemisynthesis, computational and molecular docking studies of novel nitrogen containing steroidal aromatase inhibitors: Testolactam and testololactam

Article abstract of DOI:10.1039/c8nj00063h

Testololactone (10) and testolactone (11) represent aromatase inhibitors containing lactone rings. We previously reported their hemisynthesis from the most common phytosterols, which are highly abundant in nature. Herein, we report the synthesis of their nitrogen congeners: testololactam (3) and testolactam (8). The reaction process involves the conversion of 4-androstene-3,17-dione to its corresponding oxime using hydroxylamine hydrochloride, whose Beckmann rearrangement under acid conditions yielded the desired testololactam (3). However, testolactam (8) was formed by the Beckmann rearrangement of the oxime (7) of 1,4-androstene-3,17-dienone (6). This expeditious reaction scheme may be exploited for the bulk production of testololactam (3) and testolactam (8). Theoretical DFT studies concerning the structural and electronic properties of all the end products were carried out using the Becke three-parameter Lee-Yang-Parr function (B3LYP) and 6-31G(d,p) level of theory. Molecular electrostatic potential map and frontier orbital analysis were carried out. The HOMO-LUMO energy gap was calculated, which allowed the calculation of relative reactivity descriptors like chemical hardness, chemical inertness, chemical potential, nucleophilicity and electrophilicity index of the synthesized products. The molecular docking studies of testololactam (3), testolactam (8) and testololactone (10), with aromatase (CYP19) revealed binding free energies of (ΔG_b) = -9.85, -9.62 and -10.14 kcal mol^-1 respectively, compared to the standard testolactone (11), a well-known aromatase inhibitor sold under the brand name TESLAC, which exhibited a binding free energy (ΔG_b) of -10.29 kcal mol^-1 with an inhibition constant K_i of 28.87 nM. The docking study revealed that the nitrogen congeners exhibit a relatively lower but appreciable therapeutic efficiency to be used as aromatase inhibitors.

Full text of DOI:10.1039/c8nj00063h

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Page 1 of 31
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Hemisynthesis, Computational and Molecular Docking studies of
novel Nitrogen containing steroidal aromatase inhibitors:
Testolactum and Testololactum
a,
b
c,1
c,1
Shabir H. Lone *, Muzzaffar A. Bhat , Rayees A. Lone , Salman Jameel , Javeed A.
c
, c
Lone , Khursheed A. Bhat*
a
Department of Chemistry, Govt Degree College Khanabal, Anantnag, Kashmir, India, 192101
b
Department of Chemistry, Islamic University of Science and Technology, Awantipora, Kashmir,
India, 192122.
c
Bioorganic Chemistry Division Indian Institute of Integrative Medicine, Sanatnagar, Srinagar,
Kashmir, India, 190005
*
Shabir Hussain Lone
Assistant Professor
Department of Chemistry
Govt Degree College Khanabal, Anantnag, Kashmir, India, 192101
Email: lone.shabir480@gmail.com
*
Khursheed Ahmad Bhat
Senior Scientist
Bioorganic Chemistry Division
Indian Institute of Integrative Medicine, Sanatnagar, Srinagar, Kashmir, India, 190005
Email: kabhat@iiim.ac.in
1
Both authors contributed equally
1

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DOI: 10.1039/C8NJ00063H
Abstract
Testololactone (10) and Testolactone (11) represent the aromatase inhibitors containing lactone
rings. We previously reported their hemisynthesis from the most common phytosterols which are
highly abundant in nature. Herein, we report the synthesis of their nitrogen congeners:
Testololactum (3) and Testolactum (8). The reaction process involves the conversion of 4ꢀ
androsteneꢀ3,17ꢀdione to its corresponding oxime using hydroxylamine hydrochloride whose
beckmann rearrangement under acid conditions yielded the desired Testololactum (3). However
Testolactum (8) was formed by the beckmann rearrangement of the oxime (7) of 1,4ꢀandrosteneꢀ
3
,17ꢀdienone (6). This expeditious reaction scheme may be exploited for the bulk production of
Testololactum (3) and Testolactum (8). Theoretical DFT studying structural and electronic
properties of all the end products was carried out using the Becke threeꢀparameter LeeꢀYangꢀ
Parr function (B3LYP) and 6ꢀ31G (d,p) basis set. Molecular electrostatic potential map and
frontier orbital analysis were carried out. HOMOꢀLUMO energy gap was calculated which
allowed the calculation of relative reactivity descriptors like chemical hardness, chemical
inertness, chemical potential, nucleophilicity and electrophilicity index of the synthesized
products. Molecular docking studies of Testololactum (3), Testolactum (8) and Testololactone
(
10), with aromatase (CYP19) displayed binding free energies of (ꢁG ) = ꢀ9.85, ꢀ9.62 and ꢀ10.14
b
kcal/mol respectively compared to the standard Testolactone (11), a wellꢀknown aromatase
inhibitor sold under the brand name TESLAC, which exhibited a binding free energy (ꢁG ) of ꢀ
b
1
0.29 kcal/mol with inhibition constant K of 28.87 nM. The docking study revealed that
i
nitrogen congeners exhibit relatively lower but appreciable therapeutic efficiency to be called as
aromatase inhibitors.
Keywords: Testolactone; Testololactone; Testololactum; Testolactum; DFT; Docking
2

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Introduction
Around the globe, cancer is the leading cause of death with an estimated 7.6 million deaths
during 2007 [1]. Apart from the cancers of skin, breast cancer is generally considered to be the
most prevalent cancer in women and ranks second as a cause of tumorꢀrelated death only after
lung cancer [2]. About twoꢀthirds of breast related tumors require estrogens to grow and hence
are called hormoneꢀdependent [3]. Approximately 60% of preꢀmenopausal and 75% of postꢀ
menopausal cancers are hormoneꢀdependant [4], implying that the endogenous estrogens are
essentially required for proliferation. Therefore the drugs used against treatment of estrogen
positive breast cancer involve obstruction of hormone production or hormone action.
Cytochrome P450 19 (CYP19) commonly known as aromatase or estrogen synthase, has always
been found to be the promising target for the treatment of breast cancer [5] because inhibition of
aromatase enzyme leads to decreased estrogen production, thereby stopping/reducing the tumorꢀ
growth. To block estrogen production, compounds used are known as aromatase inhibitors (AIs).
They represent the front line therapy for hormone dependent breast cancer [1] and are classified
as steroidal and nonꢀsteroidal aromatase inhibitors. Among the steroidal aromatase inhibitors
which exhibit this enzyme inhibition at nanoꢀmolar concentrations include Formestane,
Exemestane, Testololactone and Testolactone (Fig 1) etc. Formestane a steroidal analogue was
the first AI used in clinical trials and has been demonstrated to be effective as well as well
tolerated [3,6]. Testolactone (11), marketed under the trade name TESLAC is regarded as a
pioneer drug to treat breast cancer [7]. Similarly Exemestane sold under the brand name
®
AROMASIN is an irreversible aromatase inhibitor [8].
3

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Fig 1. Some wellꢀknown steroidal aromatase inhibitors
So far there are a few literature reports which highlight the synthetic routes for the preparation of
these aromatase inhibitors. [9ꢀ15]. Previously, we reported synthesis of Testololactone (10) and
Testolactone (11) (Fig 2) using the naturally known abundant phytosterols like βꢀsitosterol and
stigmasterol as precursors. Over the past decades few synthetic aromatase inhibitors have been
reported but their theoretical investigations of structural, molecular and electronic properties
using DFT or related computational methods still lack in the literature. However, in recent past a
number of docking studies have been carried out to examine the exact mechanistic and binding
mechanism of aromatase inhibitors [16ꢀ21]. Based on the aforementioned facts as well as our
ongoing research program to search for natural product based medicinal leads [15, 22ꢀ33],
particularly the development of new aromatase inhibitors, we turned our attention towards the
hemisynthesis of nitrogen congeners of the wellꢀknown aromatase inhibitors and studied their
electronic, structural and molecular properties along with their interaction mechanism with
aromatase enzyme using computational and molecular docking studies for the first time across
any part of the globe.
Results and discussion
4
ꢀandrosteneꢀ3,17ꢀdione (1) and 1,4ꢀandrostadienꢀ3,17ꢀdione (6) were synthesized from natural
precursors like βꢀsitosterol, stigmasterol as described previously [15]. 4ꢀAndrosteneꢀ3,17ꢀdione
4

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was then subjected to oxime formation at its 17ꢀketo moiety using hydroxylamine hydrochloride
13
to afford compound 2. Formation of 2 was easily confirmed by the close analysis of its Cꢀ
NMR. Disappearance of carbon resonance at 222 ppm (due to 17ꢀketo moiety in 1) and the
appearance of carbon signal at 175.33 ppm in 2 indicated its formation. Acidꢀcatalyzed
beckmann rearrangement of 2 resulted in an isomeric mixture of 3 and 4 in 4:1 ratio. Compound
3
which represents a nitrogen congener of Testololactone (10) (Fig 2) was hence forth named as
13
Testololactum. The two isomers 3 and 4 were differentiated on the basis of their CꢀNMR
spectral data. Carbon signals at 54.34 (due to quarternary carbonꢀ13) and 38.77 (due to CH at
2
position 16) of compound 3 indicated the NꢀꢀꢀC13 and CH (position 16)ꢀꢀꢀꢀꢀꢀC=O linkages
2
respectively. However in compound 4 carbon resonances at 44.89 and 48.21 ppm indicated C13ꢀ
ꢀ
ꢀꢀꢀC=O and NꢀꢀꢀꢀꢀꢀꢀCH (position 16) linkages respectively. To target the synthesis of better
2
congener (8) as shown in Fig 2 compound 1 was subjected to phenylselenation at position 2 of
ring A using phenylselenyl chloride in ethyl acetate to afford the desired αꢀphenylselenide (5)
whose degradation with hydrogen peroxide in dichloromethane afforded 1,4ꢀandrostadieneꢀ3,17ꢀ
dione (6). Compound 6 was then subjected to oxime formation at its 17ꢀketo moiety using
hydroxylamine hydrochloride to afford 7 whose acidꢀmediated beckmann rearrangement yielded
a mixture of compounds 8 and 9 in 4:1 ratio. Both 8 and 9 were characterized on the same
grounds as that of 3 and 4. Since 8 represents a nitrogen congener of Testolactone (11), it was
therefore named as Testolactum (8). After having synthesized the target compounds, we next
focussed on optimization of their structures using theoretical DFT. Optimization of structures is
needed for carrying out molecular docking study. The theoretical study allowed the calculation
of MEPS, FMO’s, HUMOꢀLUMO energy gap and related reactivity descriptors which depicted
the potential kinetic stability and reactivity of the target compounds.
5

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R
R
O
Cyclohexanone, Al(iopr)2
Mycobacterium sps
O
Reflux, Toluene
2 h
1
O
HO
1
Our Previous report
Steroids, 2015, 96,
Sitosterol, R =
Stigmasterol, R =
R =
164-168
R =
O
O
O
O
O
O
10
11
O
O
1
Our Previous report
Steroids, 2015, 96,
PhSeCl, LDA
EtOAc, rt, 2h
164-168
NOH
O
O
H O
2
2
H
PhSe
DCM, 6 h
O
O
2
6
O
5
NH OH. HCl,
2
H SO , Heat
2 4
EtOH
NOH
1
8
H
1
2
N
O
1
1
1
3
17
19
1
9
1
6
2
9
14
10
8
15
O
7
7
O
3
5
4
6
3
1
8
H
O
12
O
N
O
11
19
13
1
7
NH
1
4
9
NH
16
2
14
1
0
8
15
O
3
5
7
6
4
O
O
9
8
Fig 2. Synthesis of Testololactum (3) and Testolactum (8)
6

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MEP analysis
MEP is useful for predicting molecular reaction behavior. It is used to assess the molecular
reactivity towards charged reactants and depict the hydrogen bond interactions. Molecular
electrostatic surfaces reveal essential characteristics like size, shape and variation of electron
density while correlating it with dipole moment, partial charges, electronegativity, and chemical
reactivity sites located in the molecule. ESP is studied by computation organic chemists for
analyzing drugꢀreceptor and enzymeꢀsubstrate interactions along with Hꢀbonding interactions
[
(
34ꢀ38]. Fig 3 shows the MEP of compound 3, 8, 10 and 11, calculated using B3LYP/6ꢀ31G
d,p) basis set. A comparative view of molecular electrostatic maps is presented here which are
Fig 3. MEP of compounds 3, 8, 10 and 11 calculated using BL3YP/6ꢀ31G (d, p) basis set
7

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more or less similar. The pictorial representation with rainbow colour scheme of electrostatic
potential lies in the range of ꢀ6.138eꢀ2 to 6.138eꢀ2, ꢀ6.315eꢀ2 to +6.315eꢀ2, ꢀ6.018eꢀ2 to
+6.108eꢀ2 and ꢀ6.082eꢀ2 to 6.082eꢀ2 for compounds 3, 8, 10 and 11 respectively. The darkest
red regions indicate regions with high electron density (negative potential) while as the darkest
blue regions indicate regions with low electron density (positive potential). As can be seen in the
MEP for all the four compounds the regions around carbonyl oxygens at positions 3 and 17 of
the steroidal skeletons are red and hence possess high electron density or in other words a site
more vulnerable to electrophilic attack. This is quite consistent with the basic organic chemistry
because carbonyl oxygen is electron rich and carbonyl carbon is electron deficient.
Frontier Molecular analysis
Frontier molecular orbitals (FMO) (Fig 4) help us to determine the electric and optical properties
electronic transitions and kinetic stability [39]. FMO’s of all four target compounds (3, 8, 10 and
1
1) were calculated using using BL3YP/6ꢀ31G (d, p) level of theory. As can be seen from Fig. 4,
HOMO and LUMO are located over ring A of all the four steroid skeletons. The HOMOꢀLUMO
energy gaps are 0.18571, 0.17798, 0.18527 and 0.17791 e. v for compounds 3, 8, 10 and 11
respectively. (Table 1). The small difference among the HOMOꢀLUMO energies of all the
compounds depicts their similar nature of reactivity. This HOMOꢀLUMO energy gap was
explored to evaluate the important chemical reactivity descriptors like softness, hardness,
electronegativity, chemical potential, electronꢀaffinity and ionization energy. Chemical hardness
(η) and softness is basically the measurement of chemical reactivity to which the addition of
charge stabilizes the system [40, 41] and chemical potential µ gives an idea about the transfer of
charge from higher
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Fig 4 HOMOꢀLUMO surfaces of compounds 3, 8, 10 and 11 simulated using BL3YP /6ꢀ31 G (d,
p) level of theory.
potential to lower potential. Another important descriptor electronegativity (χ) represents the
tendency to attract electrons. These properties have been defined as follows [42ꢀ44]:
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η =
µ =
χ =
where I and A represent ionization potential and electron affinity of the compound, which are
actually obtained from HOMO and LUMO energies as I = ꢀEHOMO and A = ꢀELUMO as per Janak
theorem [45] and Perdew et al. [46]. Using above equations these descriptors were calculated.
Hardness (η) which is directly related to stability was found to be 0.09285, 0.08899, 0.09263 and
0
.08895 e.v. for 3, 8, 10 and 11 respectively. Chemical potential ‘µ’ which is actually the
escaping ability of electrons from an equilibrium system was found to be ꢀ0.140875, ꢀ0.14378, ꢀ
.140695 and ꢀ0.145295 for 3, 8, 10 and 11 respectively. The global electrophilicity index (ω), a
0
global reactivity index that is related to chemical hardness and chemical potential and first
introduced by Parr et al [44] represents the measure of the stabilization in energy achieved when
the system acquires an additional electronic charge from the environment and is given by ω =
.
The corresponding values for compounds 3, 8, 10 and 11 are 0.10686, 0.11615, 0.10684
and 0.11865 e.v respectively. All these parameters have been calculated for the target
compounds using B3LYP/6ꢀ31G (d, p) basis set and are depicted in Table 1.
Table 1: Calculated energy values for compounds 3, 8, 10 and 11 using B3LYP/6ꢀ31G (d, p)
basis set
Parameter
Energy (au)
Compound 3
ꢀ945.53034
1.4785
Compound 8
ꢀ944.31317
7.8287
Compound 10
ꢀ965.39716
6.7148
Compound 11
ꢀ964.17202
7.6207
Dipole moment (Debye)
EHOMO (eV)
ꢀ0.23373
ꢀ0.04802
0.18571
ꢀ0.23277
ꢀ0.05479
0.17798
ꢀ0.23333
ꢀ0.04806
0.18527
ꢀ0.23425
ꢀ0.05634
0.17791
ELUMO (eV)
EHOMOꢀLUMO (eV)
EHOMOꢀ1 (eV)
ꢀ0.23760
ꢀ0.23889
ꢀ0.25770
ꢀ0.25846
1
0

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ELUMO+1 (eV)
E(HOMOꢀ1)ꢀ(LUMO+1) (eV)
Hardness (η)
0.03033
ꢀ0.00192
0.23697
0.08899
ꢀ0.14378
0.14378
0.11615
0.14433
0.40203
0.09263
ꢀ0.140695
0.140695
0.10684
0.01289
0.27135
0.08895
ꢀ0.145295
0.145295
0.11865
0.26793
0.09285
Chemical Potential (µ)
Electronegativity (χ)
Electrophilicity index (ω)
ꢀ0.140875
0.140875
0.10686
From Table 1, it is clear that corresponding HOMOꢀLUMO energy gap and reactivity
descriptors like hardness, potential and global electrophilicity of all the four compounds are more
or less similar to each other, implying their nearly similar behaviour in enzymeꢀprotien
interaction study.
PASS and Molecular Docking
After having successfully optimized and calculated various reactivity descriptors for all the four
compounds using DFT employing the wellꢀknown B3LYP/6ꢀ31G (d,p) basis function, we next
studied their behaviour towards CYP19 enzyme. Before carrying out the actual analysis, it was
envisaged to predict the pharmacological properties of these four compounds, particularly their
performance towards CYP19 enzyme, using an available online PASS [47]. PASS is an
important tool that evaluates the biological activity of a molecule in relation to its structure. It
assesses the druglikeness and toxicities of molecules like teratogenicity, carcinogenicity
embroyogenecity etc. The average accuracy of prediction is about 95 % according to leaveꢀoneꢀ
outꢀcross validation (LOOCV) estimation and the probabilities, Pa (Probable activity) and Pi
(Probable inactivity), are values that vary from 0.000 to 1.000, and generally Pa + Pi = 1 as these
probabilities are calculated independently [48]. PASS analysis results of the desired compounds
1
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3
, 8, 10 and 11 have been enlisted in Table 2 below. A thorough analysis of PASS result shows
that the target compounds 3, 8, 10 and 11 are highly active as expected by the authors against
Table 2.
Pa
PASS (Prediction Activity Spectra) of compounds 3, 8, 10 and 11 with Pa >0. 4.
Testololactum (3)
Pi
Activity
Pa
Pi
Activity
Testosterone 17betaꢀ
dehydrogenase
0
.971 0.002
0.587
0.013
Anesthetic general
(NADP+) inhibitor
0
0
0
.964 0.003 CYP2C12 substrate 0.616
0.042
0.015
0.040
Antineoplastic
CYP3A3 substrate
CYP3A4 substrate
.925 0.003
.918 0.003
CYP2J substrate
CYP2J2 substrate
0.578
0.602
Morphine 6ꢀdehydrogenase
inhibitor
0
.911 0.003
Lysase inhibitor
0.564
0.004
0
0
0
.881 0.003
.844 0.003
.840 0.003 CYP2A11 substrate 0.552
7ꢀ
CYP2B5 substrate
Ovulation inhibitor
0.583
0.560
0.027
0.007
0.004
CYP3A5 substrate
Indanol dehydrogenase inhibitor
CYP2C8 inducer
2
Hydroxycholesterol
7alphaꢀ
0
.836 0.004
0.557
0.009
Menopausal disorders treatment
monooxygenase
inhibitor
0
0
0
.820 0.003
.798 0.002
.794 0.003
CYP2A2 substrate
CYP17 inhibitor
CYP2A1 substrate
Oxidoreductase
inhibitor
0.550
0.554
0.620
0.003
0.010
0.077
Testosterone agonist
Interleukin 2 agonist
Membrane permeability inhibitor
0
.797 0.007
.790 0.003
0.566
0.573
0.028
Alopecia treatment
0
0
CYP2A4 substrate
0.044
0.007
CYP3A substrate
RELA expression inhibitor
Estradiol 17alphaꢀdehydrogenase
inhibitor
.787 0.004 CYP2C11 substrate 0.532
0
0
.768 0.003 Androgen antagonist 0.527
0.003
0.012
.747 0.007
.738 0.004
CYP3A1 substrate
Acetylcholine
0.536
0.546
CYP2C18 substrate
0
neuromuscular
blocking agent
CYP3A4 inducer
CYP3A inducer
CYP2C9 inducer
CYP3A2 substrate
Prostate disorders
treatment
0.024
Antipruritic, allergic
0
0
0
0
.737 0.010
.718 0.011
.706 0.004
.711 0.010
0.524
0.528
0.518
0.539
0.004
0.008
0.003
0.026
Lipocortins synthesis antagonist
CYP3A7 substrate
CYP2A10 substrate
Erythropoiesis stimulant
DELTA14ꢀsterol reductase
inhibitor
0
.704 0.006
.695 0.004
0.523
0.539
0.012
0.030
0
Prostate cancer
Respiratory analeptic
1
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treatment
ProstaglandinꢀE2 9ꢀ
reductase inhibitor
0
.696 0.016
.651 0.004
0.511
0.509
0.004
0.003
Steroid synthesis inhibitor
Cholestenone 5alphaꢀreductase
inhibitor
0
CYP2C6 substrate
0
0
0
.656 0.011
.647 0.003
.645 0.003 CYP2B11 substrate 0.515
Dermatologic
CYP7 inhibitor
0.507
0.534
0.004
0.033
0.014
CYP19 inhibitor
CYP2C9 substrate
Gonadotropin antagonist
Steroid Nꢀ
0
.634 0.004
CYP2G1 substrate
0.519
0.023
acetylglucosaminyltransferase
inhibitor
0
0
0
0
.631 0.004
.603 0.009
.597 0.009 Gestagen antagonist 0.517
.606 0.027
CYP2A5 substrate
UGT1A4 substrate
0.502
0.558
0.006
0.062
0.026
0.005
Antiacne
Antiseborrheic
CYP2C19 substrate
Cholesterol oxidase inhibitor
CYP2C substrate
Prostatic (benign)
hyperplasia treatment
0.491
0
.579 0.005
0.524
0.047
JAK2 expression inhibitor
Testolactum (8)
Pa
Pi
Activity
Pa
Pi
Activity
Testosterone 17betaꢀ
dehydrogenase
0
.926 0.004
0.600
0.041
CYP3A4 substrate
(NADP+) inhibitor
0
0
0
.919 0.008 CYP2C12 substrate 0.586
0.029
0.017
0.006
CYP2C substrate
Antipruritic, allergic
CYP7 inhibitor
.822 0.016
.801 0.004
CYP2J substrate
CYP2B5 substrate
0.571
0.560
Prostatic (benign) hyperplasia
treatment
0
.796 0.014
CYP2J2 substrate
0.558
0.005
0
0
0
0
0
.788 0.011
.738 0.003
.719 0.011
.710 0.007 CYP2C11 substrate 0.528
.703 0.012
Lysase inhibitor
CYP2C9 inducer
CYP3A4 inducer
0.587
0.550
0.571
0.041
0.007
0.044
0.003
0.005
Oxidoreductase inhibitor
Indanol dehydrogenase inhibitor
CYP3A substrate
Lipocortins synthesis antagonist
CYP2C8 inducer
CYP3A inducer
Prostate disorders
treatment
0.526
0
.696 0.006
0.514
0.007
CYP2B11 substrate
0
0
.691 0.007
.686 0.004
CYP2A2 substrate
CYP17 inhibitor
Acetylcholine
0.521
0.541
0.014
0.036
Muscular dystrophy treatment
Alopecia treatment
0
.686 0.005
neuromuscular
blocking agent
Ovulation inhibitor
Antiinflammatory
0.519
0.015
Gestagen antagonist
0
0
.686 0.007
.691 0.017
0.529
0.512
0.029
0.015
Antipruritic
Gonadotropin antagonist
2
7ꢀ
Hydroxycholesterol
alphaꢀ
monooxygenase
0
.680 0.015
0.487
0.004
CYP19 inhibitor
7
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inhibitor
Dermatologic
Prostate cancer
treatment
0
.652 0.011
.645 0.005
0.479
0.477
0.005
0.007
Growth stimulant
0
CYP2C6 substrate
0
0
0
.635 0.004 Androgen antagonist 0.484
.629 0.009 CYP2A11 substrate 0.474
0.015
0.005
0.039
CYP2G1 substrate
Thyroxine 5ꢀdeiodinase inhibitor
CYP2C9 substrate
.625 0.010
CYP2A1 substrate
0.504
Mannan endoꢀ1,4ꢀbetaꢀ
mannosidase inhibitor
0
.618 0.012
CYP2A4 substrate
0.496
0.031
0.639 0.037
0.604 0.028
0.589 0.022
Antineoplastic
CYP3A2 substrate
CYP3A1 substrate
0.528
0.509
0.477
0.067
0.051
0.021
Antiseborrheic
JAK2 expression inhibitor
Menopausal disorders treatment
Testololactone (10)
Pa
Pi
Activity
Pa
Pi
Activity
0
0
.970 0.001
CYP19 inhibitor
Testosterone 17betaꢀ
dehydrogenase
0.723
0.006
CYP2C11 substrate
.961 0.002
0.708
0.002
CYP2B11 substrate
(NADP+) inhibitor
0
0
0
.956 0.004 CYP2C12 substrate 0.689
0.003
0.009
0.010
CYP2G1 substrate
Dermatologic
Cholesterol antagonist
Acetylcholine neuromuscular
blocking agent
.948 0.001
.929 0.003
CYP2B5 substrate
CYP2J substrate
0.684
0.682
0
.922 0.003
CYP2J2 substrate
0.674
0.006
0
0
0
0
0
.907 0.001
.907 0.003
.882 0.002 CYP2A11 substrate 0.661
.880 0.002
.878 0.002
CYP2A4 substrate
CYP3A1 substrate
0.670
0.667
0.014
0.012
0.009
0.004
0.014
CYP3A4 inducer
HMOX1 expression enhancer
Prostate disorders treatment
Androgen antagonist
CYP3A inducer
CYP2A1 substrate
CYP2A2 substrate
0.656
0.665
3
ꢀOxosteroid 1ꢀdehydrogenase
0
.878 0.004
Lysase inhibitor
Antineoplastic
0.645
0.002
inhibitor
Estradiol 17alphaꢀdehydrogenase
inhibitor
0
0
0
.869 0.005
.824 0.004
.823 0.008
0.640
0.642
0.638
0.002
0.005
0.005
CYP3A2 substrate
ProstaglandinꢀE2 9ꢀ
reductase inhibitor
Oxidoreductase
inhibitor
Gestagen antagonist
Menopausal disorders treatment
JAK2 expression inhibitor
0
.818 0.005
0.647
0.024
2
7ꢀ
Hydroxycholesterol
7alphaꢀ
0
.792 0.006
0.623
0.011
CYP3A3 substrate
monooxygenase
inhibitor
Transꢀ1,2ꢀdihydrobenzeneꢀ1,2ꢀ
diol dehydrogenase inhibitor
Prostate cancer treatment
0
0
.798 0.015
.784 0.004
CYP3A4 substrate
Ovulation inhibitor
0.618
0.616
0.005
0.005
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0
.781 0.002 Aromatase inhibitor 0.613
0.004
0.013
0.009
CYP2A3 substrate
Antiprotozoal (Leishmania)
UGT2B substrate
0
.793 0.015
.779 0.008
CYP3A substrate
CYP3A5 substrate
0.622
0.613
0
Testosterone 17betaꢀ
dehydrogenase inhibitor
Steroid Nꢀ
0
.764 0.003
CYP17 inhibitor
0.606
0.003
0
0
0
.756 0.002
.772 0.024
.744 0.004
Androgen agonist
0.611
0.604
0.610
0.012
0.005
0.018
acetylglucosaminyltransferase
inhibitor
UGT1A8 substrate
Antiseborrheic
Indanol
dehydrogenase
inhibitor
Analeptic
0
0
.747 0.012 Respiratory analeptic 0.603
0.012
0.013
Antipruritic, allergic
UGT1A substrate
.733 0.003
CYP2A5 substrate
0.604
Testolactone (11)
Pa Pi
Pa
Pi
Activity
Activity
0
0
.965 0.001
.913 0.002
CYP19 inhibitor
CYP2B5 substrate
Testosterone 17betaꢀ
dehydrogenase
0.701 0.005
0.697 0.002
CYP2A1 substrate
Androgen agonist
0
.900 0.006
0.696 0.009
Antiprotozoal (Leishmania)
(NADP+) inhibitor
0
0
0
0
0
0
0
0
0
0
0
0
0
.900 0.012 CYP2C12 substrate 0.699 0.015
CYP3A5 substrate
Lysase inhibitor
Dermatologic
Prostate disorders treatment
Immunosuppressant
Growth stimulant
CYP3A4 inducer
CYP3A inducer
Ovulation inhibitor
CYP17 inhibitor
Antipruritic, allergic
CYP2C11 substrate
JAK2 expression inhibitor
Acetylcholine neuromuscular
blocking agent
.877 0.005
.832 0.014
.804 0.003
.805 0.012
.788 0.005
.797 0.015
.783 0.004
.792 0.015
.765 0.009
Antineoplastic
CYP2J substrate
CYP2A4 substrate
CYP2J2 substrate
CYP3A1 substrate
CYP3A4 substrate
CYP2A2 substrate
CYP3A substrate
Antiinflammatory
0.706 0.022
0.681 0.009
0.651 0.010
0.661 0.022
0.642 0.004
0.649 0.015
0.647 0.015
0.634 0.012
0.627 0.007
.745 0.005 CYP2A11 substrate 0.624 0.009
.739 0.002 Aromatase inhibitor 0.624 0.013
.741 0.008
CYP3A2 substrate
0.633 0.026
0
.758 0.027
Antiseborrheic
0.620 0.017
Indanol
dehydrogenase
inhibitor
0
.733 0.004
0.609 0.015
Antipruritic
CYP group of enzymes. Since we were interested in exploring their potential against aromatase
enzyme (CYP19 group) we turned our attention toward the same. PASS results showed that
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compounds 3, 8, 10 and 11 have Pa values of 0.507, 0.487, 0.970 and 0.965 respectively. The
support for PASS results of compound 11 comes from the fact that the same is sold under the
brand name TESLAC as a wellꢀestablished drug having high power of aromatase inhibition.
Taking cue from the pass results we resorted to the docking studies of target compounds against
CYP19 enzyme to gain insights in to their possible modes of binding with the active sites of
CYP19. A thorough analysis of the binding site of enzyme aromatase using PDBsum [49]
˚
3
reveals that it occupies a volume of 1525.92 A . Also the entrance to the binding site as
˚
measured by AutoDock 4.2 was found to be 3.2 A in diameter. Because of the small entrance
cavity in aromatase bigger molecules find it difficult to reach the binding site and hence only
rigid and small molecules easily diffuse inside to bind the active site as suggested by Suvannang
et al. [50]. All the four compounds 3, 8, 10 and 11 were docked to the aromatase binding site
though compound 11 is a marketed drug for aromatase inhibition under the brand name
TESLAC. Xꢀray crystal structure of human aromatase enzyme (PDBꢀID 3EQM) was obtained
from the Protien Data Bank web site (http://www.rrscb.org/pdb/). The preparation of target,
ligand, grid and docking parameter files of all the target compounds 3, 8, 10 and 11 was done
employing AutoDock 4.2 using the Lamarkian Genetic Algorithm [51]. A grid box size of 50 x
˚
6
4 x 78 A˚ with a grid spacing of 0.375 A was generated using AutoGrid [52] and the grid
positioned at coordinates of 83.35, 49.60, 50.60 A˚ which are almost reported to be the binding
site residues [53]. The results obtained here provided information on the binding interactions
(
Fig 5 and 6) along with the binding free energies (ꢁG ) and inhibition constants (Ki). Table 3
b
summarizes the free energies of binding and inhibition constants along with the key amino acid
residues interacting with CYP19 enzyme.
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Table 3. Approximate mean binding free energies, inhibition constants and bonding interactions.
Compound
No of
Best Binding Inhibition Amino acid residues of CYP19 involved
Rotational energy (ꢁGb) Constant
in interaction with compounds
bonds
(kcal/mol)
ꢀ9.85
(Ki) (nM)
60.09
(
(
3)
8)
0
0
Hꢀbond: LEU477, SER478
Hꢀbonds: ARG115, LEU372, LEU477
ꢀ9.62
88.77
Charge repulsion with both NH protons
2
of ARG115
Steric Bump: LEU372
(
10)
11)
0
0
ꢀ10.14
ꢀ10.29
37.10
28.87
Hꢀbonds: THR310, LEU372, SER478,
TRP224
Electrostatic attraction: ASP309
Hꢀbonds: ARG115, PRO429, LEU372,
LEU477, VAL373, PHE430
(
Charge repulsion with one NH proton of
2
ARG115
It is seen that Testolactum (8) engages in four favaourable hydrogenꢀbond interactions with
ARG115, LEU372, PRO429, LEU477 and unfavourable interactions which include a charge
repulsion with two NH protons of ARG115 and a steric bump of lactum NH proton with
2
LEU372 giving it a relatively low ꢁG of ꢀ9.62 kcal/mol and K of 88.77 nM. However its close
b
i
associate, Testololactum (3) engages only in two favourable hydrogen bond interactions with
LEU477 and SER478, strong enough to give it a relatively higher ꢁG of ꢀ9.85 kcal/mol and K
b
i
of 60.09 nM. The slightly lower value of binding energy and inhibition constant in 8 is possibly
because of unfavourable interactions which decrease the stability of the compound. On the other
hand, Testololactone (11) was found to engage itself in five favourable interactions which
include four hydrogen bond interactions with TRP224, THR310, LEU372, SER478 and an
electrostatic attractive interaction with ASP309 giving a ꢁG of ꢀ10.14 kcal/mol and K of 37.10
b
i
nM. However Testolactone (11) displayed potent inhibition as it exhibits six favourable
hydrogen bond interactions with ARG115, ASP309, THR310, VAL373, LEU372, LEU477 and
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Fig 5 Best docked conformations of compounds 3, 8, 10 and 11.
one unfavourable repulsive interaction with one proton of NH of ARG115 sufficient enough
2
giving it a binding free energy (ꢁG ) of ꢀ10.29 kcal and inhibition constant K of 28.87 nM. From
b
i
the above results it is clear that compounds which possess more number of favourable hydrogen
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8

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bond interactions with the binding site of CYP19, give more negative values of binding free
energy (ꢁG ) and lowest values of inhibition constant (K ). Also it is seen that nitrogen
b
i
Fig 6 Amino acid residues of CYP19 enzyme binding with the compounds 3, 8, 10 and 11.
congeners have relatively less therapeutic efficiency compared to oxygenated ones. This is
possibly because in lactone containing compounds, Testololactone (10) and Testolactone (11)
both Oꢀatoms of lactone ring are involved in bonding interactions as against Testololactum (3)
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and Testolactum (8) in which only Oꢀatom of lactam ring is involved in hydrogen bond
interaction. Further Testolactum (8) is involved in unfavourabe interactions sufficient enough to
impart it a relatively lower therapeutic efficiency as CYP19 inhibitor.
Conclusion
Testololactum (3) and Testolactum (8) were synthesized and characterized using spectral data
analysis. Theoretical DFT calculations using the Becke threeꢀparameter LeeꢀYangꢀParr function
(B3LYP) and 6ꢀ31G (d,p) basis set were performed. MEP maps revealed that the regions with
high electron density or more negative potential are concentrated over the regions spanning the
carbonyl oxygen atoms of all the four compounds. FMO analysis helped to evaluate the HOMOꢀ
LUMO energy gap of all the compounds and indicates a similar nature of reactivity of the target
molecules. PASS prediction revealed that all the four compounds are highly active against
CYP19 enzyme. Molecular docking studies established that the newly synthesized steroidal
lactums have the potential to be developed as aromatase inhibitors, although with relatively
lower therapeutic efficiency than their corresponding oxygen containing congeners.
Experimental
General
All the solvents and reagents for the chemical synthesis were purchased from Sigma Aldrich.
The chemical reactions were monitored using F254 silica gel TLC plates (E. Merck) using ceric
ammonium sulphate as charring agent and UV chamber (366 and 254 nm) for the detection of
spots. The synthesized products were purified using column chromatography on silica gel (60–
1
13
1
20 mesh). H NMR and C NMR spectra (chemical shifts expressed in ppm and coupling
constants in Hertz) were recorded on Bruker DPX 400 MHz spectrometer using CDCl as the
3
2
0

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solvent with TMS as the internal standard. Mass spectra were carried out on LC–MS 8030
tandem mass spectrometer manufactured by Shimadzu Corporation, Kyoto, Japan. The
compounds were analysed in full scan mode with nitrogen serving as interface gas. Detection
was done in ESI mode having probe voltage of 180.0 V, with probe temperature of 400 ˚C. HR
ESI MS was done on Agilent HRMS instrument (Model No. 6540) and all the compounds were
tested for their purity using HPLC (Agilent 1260 affinity).
Synthesis
Synthesis of compound 2: To a solution of compound (1) (2 g) in ethanol, 0.5 g of hydoxylamine
hydrochloride was added in a round bottomed flask. 10 ml of deionised water and 10 ml of 10 %
by wieght of aqueous NaOH solution was added and the reaction mixture heated under reflux for
2
h. The completion of reaction was monitored using TLC. The reaction mixture was evaporated
under vaccuo using rotary evaporater. The residue obtained was worked up in ethyl acetate:water
and washed with NaHCO and brine. The organic layers were collected and then combined and
3
concentrated under vaccum to yield crude residue which was purified using column
chromatography to produce pure compound 2:
Spectral data of (10R,13S)ꢀ17ꢀ(hydroxyimino)ꢀ10,13ꢀdimethylꢀ6,7,8,9,10,11,12,13,14,15,16,17ꢀ
1
dodecahydroꢀ1Hꢀcyclopenta[a]phenanthrenꢀ3(2H)ꢀone (2): H NMR (400 MHz, CDCl ) δ 6.02
3
(s, 1H), 2.48 (s, broad, 1H, OH), 2.24 (m 5H), 2.00ꢀ1.74 (m, 3H), 1.68 (d, J= 15.7 HZ, 1H), 1.62ꢀ
1
3
1
1
2
.14 (m, 10H), 1.10ꢀ1.01 (m, 3H), 0.91 (s, 3H). C NMR (101 MHz, CDCl ) δ 199.21, 175.33,
3
71.28, 123.18, 52.63, 46.51, 45.12, 38.17, 35.89, 35.50, 34.12, 31.62, 31.09, 30.16, 29.69,
+
6.21, 22.12, 19.13, 14.16. ESIꢀMS at m/z = 302 for [M+1] .
Synthesis of compound 3 and 4: To a solution of compound 2 (600 mg) in acetonitrile, a few
drops of concentrated sulfuric acid were added and the reaction mixture heated under reflux for 3
2
1

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h in a round bottomed flask. The reaction progress was continously monitered using TLC. After
completion, the reaction mixture was concentrated under vaccuo and the residue was worked up
with ethyl acetate and washed with brine and NaHCO solutions. The organic layers were
3
collected and concentrated under vaccuo and the residue obtained was subjected to column
chromatography using hexane:ethyl acetate as the eluent. A mixture of compounds (3 and 4) in
the ratio 4 :1 was obtained.
Spectral data of (10aR,12aS)ꢀ10a,12aꢀdimethylꢀ4,4a,4b,5,6,9,10,10a,10b,11,12,12aꢀdodeca
1
hydronaphtho[2,1ꢀf]quinolineꢀ2,8(1H,3H)ꢀdione (3): H NMR (400 MHz, CDCl ) δ 6.03 (S,
3
NH), 5.73 (s, 1H), 2.47ꢀ2.34 (m, 5H), 2.29ꢀ1.87 (m, 4H), 1.71ꢀ1.68 (m, 3H), 1.53ꢀ1.45 (m, 4H),
1
3
1
4
.39ꢀ0.85 (m, 9H). C NMR (101 MHz, CDCl ) δ 199.31, 171.85, 169.87, 124.17, 54.34, 53.33,
3
7.32, 40.00, 38.77, 36.28, 36.19, 35.71, 34.04, 32.65, 32. 60, 22.32, 21.97, 20.50, 17.59. HRꢀ
+
ESIꢀMS at m/z = 302.2110 for [M+1] .
Spectral data of 10aR,12aS)ꢀ10a,12aꢀdimethylꢀ2,3,4,4a,5,6,10,10a,10b,11,12,12aꢀdodecahydro
1
naphtho[2,1ꢀf]isoquinolineꢀ1,8(4bH,9H)ꢀdione (4): H NMR (400 MHz, CDCl ) δ 6.45 (s, NH),
3
5
1
1
3
.73 (s, IH), 3.50ꢀ3.09 (m, 2H), 2.54ꢀ2.23 (m, 3H), 2.18ꢀ2.09 (m, 1H), 2.09ꢀ1.85 (m, 3H), 1.82ꢀ
1
3
.41 (m, 5H), 1.39ꢀ0.92 (m, 9H), 0.90ꢀ0.68 (m, 2H). C NMR (101 MHz, CDCl ) δ 200.01,
3
70.57, 160.85, 119.47, 53.55, 51.22, 48.21, 44.89, 42.18, 36.85, 35.84, 35.17, 34.87, 32.85,
+
1.87, 22.45, 21.77, 21.18, 14.10. HRꢀESIꢀMS at m/z = 302.2099 for [M+1]
Synthesis of 2ꢀphenylselenoꢀ4ꢀandrosteneꢀ3,17ꢀdione (5): A solution of 4ꢀandrosteneꢀ3, 17ꢀdione
(1) (220 mg, 0.768 mmol) and phenylselenyl chloride (330 mg, 3 equivalents) in ethyl acetate
(20 ml) using LDA as base was stirred at room temperature for 2 h. After the reaction was
complete the solvent was removed under vaccuo and the residue was subjected to flash
2
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chromatography in Hex:EtOAc (75:25) to furnish 2ꢀphenylselenoꢀ4 androstenedione (5) in 75%
yields.
1
Spectral data of 5: H NMR (400 MHz, CDCl ) δ 7.30–7.24 (m, 3H), 7.11–6.87 (m,1H), 6.20 (s,
3
1
1
1
3
H), 6.13 (s, 1H), 4.88 (s, 1H), 2.48 (m, 2H), 2.29 (m, 2H), 2.09 (m, 3H), 2.00 1.74 (m, 3H),
1
3
.69 (s, 3H), 1.40–1.19 (m, 34H), 0.96 (s, 3H). C NMR (101 MHz, CDCl ) d 219.11, 185.82,
3
61.32, 156.32, 132.52, 129.34, 128.07, 126.85, 50.92, 49.98, 48.72, 48.41, 47.13, 43.35, 40.58,
+
5.83, 31.47, 30.21, 22.65, 21.73, 21.47, 13. 39. LCꢀESIꢀMS at m/z = 441 for [M+1] and 459
+
for [M+1+H O] .
2
Synthesis of compound 6: To a solution of compound 5 (134 mg, 0.474 mmol) in CH Cl was
2
2
added 30% H O solution and the reaction mixture was stirred for 4 h until complete as indicated
2
2
by TLC profiling. After completion the mixture was worked up in CH Cl and the organic layers
2
2
collected. The organic layers were then combined and concentrated under vaccum to yield crude
residue which was purified using column chromatography to produce pure androstenedienone (6)
in 90% yields.
1
Spectral data of 1,4ꢀandrostadienꢀ3,17ꢀdione (6): H NMR (400 MHz, CDCl ) δ 7.00 (d, J = 9.8
3
Hz, 1H), 6.23 (s, 1H), 6.17 (d, J = 9.6 Hz, 1H), 2.54–2.39 (m, 2H), 2.37–2.27 (m, 2H), 2.15 (s,
1
3
2
1
3
H), 1.97–1.81 (m, 2H), 1.48 (s, 5H), 1.30–0.88 (m, 8H). C NMR (101 MHz, CDCl ) d 221.23,
3
86.08, 161.29, 155.98, 127.98, 127.13, 50.99, 50.31, 48.31, 47.85, 43.65, 40.04, 35.80, 31.37,
+
0.53, 30.33, 22.71, 22.09, 21.79, 14.09. LCꢀESIꢀMS at m/z = 285 for [M+1] and 326 for
+
[
M+1+ACN] .
Synthesis of compound 7: To a solution of compound (6) (100 mg) in ethanol was added 25 g of
Hydoxylaminehydrochloride in a round bottomed flask. 2 ml of deionised water and 2 ml of 10
%
by wieght of aqueous NaOH solution was added and the reaction mixture heated under reflux
2
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for 1.5 h. The completion of reaction was monitored using TLC. The reaction mixture was
evaporated under vaccuo using rotary evaporater. The residue obtained was worked up in ethyl
acetate:water and washed with NaHCO and brine. The organic layers were collected and then
3
combined and concentrated under vaccum to yield crude residue which was purified using
column chromatography to produce pure compound 7:
Spectral data of (10R,13S)ꢀ17ꢀ(hydroxyimino)ꢀ10,13ꢀdimethylꢀ6,7,8,9,10,11,12,13,14,15,16,17ꢀ
1
dodecahydroꢀ3Hꢀcyclopenta[a]phenanthrenꢀ3ꢀone (7): H NMR (400 MHz, CDCl ) δ 7.10 (d, J
3
=
9.8 Hz, 1H), 6.21 (s, 1H), 6.10 (d, J = 9.6 Hz, 1H), 2.58 (br s, OH), 2.51ꢀ2.36 (m, 2H), 2.31 ꢀ
1
3
2
1
3
.25 (m, 2H), 2.12 (s, 2H), 1.97ꢀ1.48 (m, 6H), 1.36ꢀ0.90 (m, 9H). C NMR (101 MHz, CDCl ) δ
3
85.32, 174.18, 167.39, 155.31, 127.21, 127.16, 50.86, 48.76, 48.10, 44.33, 42.70, 40.18, 31.67,
+
0.02, 26.14, 23.63, 22.97, 20.51, 14.06. LCꢀESIꢀMS at m/z = 300 for [M+1] .
Synthesis of compound 8 and 9: A solution of compound 7 (80 mg) in acetonitrile, was added
few drops of concentrated sulfuric acid and the reaction mixture heated under reflux for 3 h in a
round bottomed flask. The reaction progress was continously monitered using TLC. After
completion, the reaction mixture was concentrated under vaccuo and the residue was worked up
with ethyl acetate and washed with brine and NaHCO solutions. The organic layers were
3
collected and concentrated under vaccuo and the residue obtained was subjected to column
chromatography using hexane:ethyl acetate as the eluent. A mixture of compounds (8 and 9) in
the ratio 4 :1 was obtained.
1
Spectral data of Testolactum (8): H NMR (400 MHz, CDCl ) δ 7.00 (d, J = 8 Hz, 1H), 6.24ꢀ
3
6
.21 (m, 2H), 6.06 (s, 1H), 2.51ꢀ2.28 (m, 2H), 2.25 ꢀ1.75 (m, 6H), 1.72ꢀ1.52 (m, 2H), 1.48ꢀ1.26
1
3
(
m, 8H). C NMR (101 MHz, CDCl ) δ 186.24, 171.93, 167.96, 154.82, 128.23, 124.18, 54.40,
3
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DOI: 10.1039/C8NJ00063H
5
1.77, 47.14, 43.35, 39.80, 36.16, 32.57, 30.62, 22.94, 22.74, 22.32, 20.45, 18.93. HRꢀESIꢀMS at
+
m/z = 300.1955 for [M+1] .
Density Functional Theory study
All the computations are carried out using GAUSSIAN 09 software [54]. The DFT modeling
method, using the hybrid B3LYP [55] functional was used to calculate theoretical parameters for
target compounds with the basis set combination 6ꢀ31 G (d, p) [56]. Geometry optimization was
carried out until global minima were achieved.
Acknowledgement
SHL thanks DSTꢀSERB India for financial grant supporting this work in the form of a project
under the scheme National Postdoctoral Fellowship (NPDF, File No. PDF/2016/001690/CS).
Conflict of interest
The authors declare no conflict of interest.
Supplementary Information
1
13
H, C NMR and Mass spectra are available in the form of Supplementary information with this
article and can be availed free of cost from the online version.
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