Design, synthesis, and evaluation of 4-(4′-aminobenzyl)-2-oxazolidinones as novel inhibitors of the cytochrome P-450 enzyme aromatase

Article abstract of DOI:10.1016/S0045-2068(02)00017-2

The synthesis of a series of N-alkylated 4-(4′aminobenzyl)-2-oxazolidinones is described using a synthetically useful scheme which avoids the use of phosgene - since the derivatization is undertaken with the oxazolidin-2-one ring intact. The compounds were tested for human placental aromatase (AR) inhibition in vitro, using [1β, 2β-³H]androstenedione as substrate for the AR enzyme. The compounds were found, in general, to be more potent than the standard compound, amino-glutethimide (AG), and as such proved to be good lead compounds in the search for more specific AR inhibitors.

Full text of DOI:10.1016/S0045-2068(02)00017-2

BIOORGANIC
CHEMISTRY
Bioorganic Chemistry 30 (2002) 315–331
www.elsevier.com/locate/bioorg
Design, synthesis, and evaluation of
4-(40-aminobenzyl)-2-oxazolidinones as
novel inhibitors of the cytochrome P-450
enzyme aromatase
Sabbir Ahmed,a,* Shaheen Adat,a Annabel Murrells,a
Caroline P. Owen,a and Yonas Amanuelb
a
School of Chemical and Pharmaceutical Sciences, Kingston University,
Penrhyn Road, Kingston upon Thames, Surrey KT1 2EE, UK
b
Novartis Pharama AG, CH-4002 Basel, Switzerland
Received 11 December 2001
Abstract
The synthesis of a series of N-alkylated 4-(40aminobenzyl)-2-oxazolidinones is
described using a synthetically useful scheme which avoids the use of phosgene—
since the derivatization is undertaken with the oxazolidin-2-one ring intact. The
compounds were tested for human placental aromatase (AR) inhibition in vitro,
using [1b; 2b-3H]androstenedione as substrate for the AR enzyme. The compounds
were found, in general, to be more potent than the standard compound, amino-
glutethimide (AG), and as such proved to be good lead compounds in the search for
more specific AR inhibitors.
Ó 2002 Elsevier Science (USA). All rights reserved.
1. Introduction
The estrogen synthetase enzyme aromatase (AR) consists of a cytochrome P-450
hemeoprotein and an NADPH-cytochrome P-450 reductase and is involved in the
* Corresponding author. Fax: +44-20-8547-7562.
E-mail address: s.ahmed@kingston.ac.uk (S. Ahmed).
0045-2068/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved.
PII: S0045-2068(02)00017-2

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S. Ahmed et al. / Bioorganic Chemistry 30 (2002) 315–331
final step of the steroidal cascade. The exact nature of the mechanism of the con-
version of the androgens (C19) to the estrogens (C18) (Scheme 1) is at present unclear,
however, some aspects have been elucidated [1]. It has been shown that three se-
quential oxidative steps occur on the C(19) methyl group of the substrate, resulting
in the loss of the methyl moiety as formic acid and the formation of the benzoid
A-ring (aromatization).
Recent work on the mechanism of AR, and the general P-450 family of enzymes,
has suggested that hydroxylations undertaken by the P-450 heme involve a ferroxy
radical (FeIV–OÅ) in an overall oxygen rebound process [1]. That is, the ferroxy ox-
ygen atom abstracts a hydrogen atom from the C(19) methyl of androstenedione,
resulting in the production of the FeIV–OH and substrate CH2Å radical, which is then
neutralized by the abstraction of the hydroxyl from the FeIV–OH, resulting in the
formation of FeIII and the mono-hydroxylated steroid (Fig. 1). The process is re-
peated resulting in the production of the dihydroxy steroid which then undergoes
non-enzymatic oxidation to the C(19) aldehyde. The final step involves the cleavage
of the C(10)–C(19) bond due to attack by the P-450 heme, however, the nature of the
attacking iron species is unclear. Although the experimental data have been utilized
in proposing probable mechanisms for the aromatization reaction, the specific
Scheme 1. Aromatization of androstenedione.

S. Ahmed et al. / Bioorganic Chemistry 30 (2002) 315–331
317
Fig. 1. Proposed mechanism for the hydroxylation steps in the aromatization of andro-
stenedione.
mechanism has eluded us since the orientation (or position) of the different reacting
species has not been considered as the exact structure of the active site is not clear.
The regulation of the enzymes involved in the biosynthesis of steroidal hormones
has proved to be useful in the control of hormone-dependent cancers such as breast
cancer [2]. In particular, AR inhibitors have been shown to be useful in the second
line therapy of estrogen-dependent breast cancer, as such, steroidal and non-
steroidal compounds have been synthesized as potential drugs. Non-steroidal AR
inhibitors (Fig. 2) include liarazole (1), an azolyl-substituted benzimidazole, and the
1-[(benzofuran-2-yl)phenylmethyl]-imidazoles (2). Compounds based upon pyrroli-
dine-2,5-dione [such as 3] and piperidine-2,6-dione [such as aminoglutethimide (4)
and pyridoglutethimide (5)] also exist, but are less potent than 1 or 2.
It was this latter series of compounds [3, 4, and 5 (Fig. 2)] which prompted us to
consider the oxazolidinone ring system within EvansÕ chiral auxiliary [3] (4-benzyl-
oxazolidin-2-one) as a potential mimic for the steroid A ring within the natural
substrate, androstenedione (AD), and therefore a possible inhibitor against P-450-
dependent AR. That is, initial modelling, involving the superimpositioning of the
two available enantiomers of 4-benzyl-oxazolidin-2-one onto AD [in a similar
manner to Banting et al. [4] (1988)], suggested that the carbonyl group of this
compound may be able to mimic the D-ring carbonyl moiety of the steroid backbone

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S. Ahmed et al. / Bioorganic Chemistry 30 (2002) 315–331
Fig. 2. A selection of non-steroidal inhibitors of AR.
whilst allowing the derivatized phenyl ring to bind in a Type II manner to the cy-
tochrome P-450 heme (Fig. 3). That is, the derivatization of the phenyl ring with an
amine group would allow the phenylamine nitrogen atom to form a dative covalent
bond with the Fe atom of the P-450 heme. The oxazolidin-2-one ring was also ob-
served to be a good mimic of the pyrrolidine-2,5-dione and piperidine-2,6-dione
rings, in particular the C@O moieties (Fig. 3). EvansÕ chiral auxiliary has been
Fig. 3. Two dimensional consideration of the modes of superimpositioning of EvansÕ auxiliary
onto AD and a derivative of 3 (the asterisks show two sets of atoms on the substrate and the
atoms on the oxazolidineone ring used in the superimpositioning study).

S. Ahmed et al. / Bioorganic Chemistry 30 (2002) 315–331
319
extensively used in organic synthesis [5] and in particular stereochemical synthesis
[6], however, its use as a potential enzyme inhibitor has been ignored. Indeed, the
derivatization of the sterically important phenyl ring has not been considered ex-
tensively within the literature, instead work on the total synthesis of the chiral
auxiliary with a 4-substituted functionality (such as an OH group) has been inves-
tigated using phosgene in the production of the oxazolidin-2-one ring [7].
In this report, we consider the modelling of 4-amino derivatives of EvansÕ chiral
auxiliary (in designing the potential inhibitors), derivatization of the phenyl ring of
this highly useful chiral auxiliary, and the biochemical evaluation of some of the
synthesized compounds as potential anti-cancer compounds involving the inhibition
of the cytochrome P-450 enzyme, AR. Furthermore, we provide a general and ex-
tremely useful scheme which may be modified so as to allow the synthesis of the
highly desirable phenol derivative (via the diazotization of the amine), and which
would therefore allow the chiral auxiliary to be attached to solid support—the
synthetic scheme therefore does not require the synthesis of the oxazolidin-2-one ring
system. To the authorsÕ knowledge, this has not been previously reported, indeed,
workers have utilized severe conditions (such as the use of phosgene) to produce
alternative chiral auxiliaries to allow attachment to solid support for combinatorial
chemistry.
2. Experimental
2.1. Molecular modelling
The construction of the substrate–heme complex (SHC) and the theoretical as-
pects of this novel approach have been discussed at length elsewhere [8–10] and as
such, will not be considered here.
The proposed oxazolidin-2-one based inhibitors were constructed within the Al-
chemy III [11] molecular modelling software (using atoms/fragments/groups avail-
able within the Alchemy structure libraries). The completed molecules were then
subjected to an initial minimization using the conjugate-gradient algorithm available
within Alchemy until the gradient fell to below 10À6 (resulting, in general, in 500 or
more iterations per structure). Conformational analysis was then performed on
flexible parts of the inhibitors using Powersearch [11] (using the systematic search
method with energy window of 5–10 kcal/mol and bond rotations of 30–60°). The
low energy conformers produced were retained for further study.
The novel substrate–heme complex approach is based upon the mimicking of the
inhibition process involving the binding of the inhibitor directly to the Fe atom of
the P-450 heme. As such, the low energy conformers obtained were, in turn, bonded
to the heme of the substrate–heme complex and the iron–phenylamine nitrogen bond
rotated so as to obtain the distances between the carbonyl groups of the substrate–
heme complex and the C(2)@O and C(5)@O of the inhibitor—it is presumed that the
inhibitorÕs C@O group will mimic the substrate C@O and therefore hydrogen bond
with the part of the active site which is proposed to bind the C(3)@O or C(17)@O of
the substrate. The conformer displaying the smallest distance for each compound

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S. Ahmed et al. / Bioorganic Chemistry 30 (2002) 315–331
was retained—the distance is considered to be inversely proportional to the strength
of any interaction between the inhibitor and the active site.
2.2. Chemistry
The reagents used were either general purpose or analytical grade and were ob-
tained from either Aldrich Chemical, Gillingham, Dorset; BDH, Poole, Dorset or
Lancaster Synthesis, Morecombe, Lancs. Melting points were determined with an
Electrothermal Instrument and infra-red spectra were determined using a Perkin–
Elmer 681 FT-infrared spectrophotometer. 1H (300 MHz) and 13C NMR (75.5 MHz)
were determined, using TMSas an internal standard, on a Brucker NMR spectro-
photometer. Mass Spectra were determined by SERC Mass Spectrometry Centre,
University of Wales College of Swansea. The purity of the compounds was checked
through the use of a HPLC system using different mobile phases; ½aŠ was deter-
D
mined at 20 °C, and at concentration of 0.25 M, using an AA-10 Automatic Polar-
imeter (from Optical Activity).
4(S)-(40-Nitrobenzyl)-oxazolidin-2-one (6): (S)-4-Benzyl-oxazolidin-2-one (1.0 g,
5.6 mmol) was dissolved in anhydrous dichloromethane (DCM) (15 ml). Nitric acid
(3.38 ml, 5 M) was added slowly and left to stir at room temperature. After 2 h, the
reaction mixture was quenched in an excess of ice-cold aqueous sodium hydrogen
carbonate. The organic layer was separated and the aqueous layer further extracted
with DCM (2 Â 20ml). The combined organic layers were dried over magnesium
sulfate (MgSO4). The crude product was purified by column chromatography (DCM
80: ethyl acetate 20) to give 6 as a yellow oil (yield, 65%, 0.82 g). m
(Film)/cmÀ1
:
ðmaxÞ
3279.6 (NH), 2918.1–2855.6 (CH-aliphatic), 1752 (C@O), 1519 (NO2), 1347.4 (C@C
aromatic), 1182.8–1163.2 (aromatic); dH ð300MHz; CDCl3): 8.13 (2H, d, J ¼ 8 Hz,
CH-aromatic), 7.37 (2 H, d, J ¼ 8 Hz, CH-aromatic), 6.26 (1 H, s, NH), 3.95 (1H, m,
N–CH), 3.81 (2H, m, O–CH2), 2.93 (1 H, d, J ¼ 8 Hz, CH-benzyllic), 2.55 (1 H, d,
J ¼ 8 Hz, CH-benzyllic); dc ð75:5MHz; CDCl3): 41.19 (CH2), 53.25 (CH), 69.34
(CH2), 124.18 (CH-aromatic), 130.06 (CH-aromatic), 143.47 (alternative C), 147.27
(alternative C), 159.41 (C@O); LRMS m=z (EIþ): 222 (Mþ þ 1); ½aŠ ¼ À63, (S)-4-
D
benzyl-oxazolidin-2-one was found to possess ½aŠ ¼ À62 (literature value [12]
D
½aŠ ¼ À62).
D
4(S)-(40-Aminobenzyl)-oxazolidin-2-one (7): 4(S)-(40-Nitrobenzyl)-oxazolidin-2-
one (0.2 g, 0.9 mmol) was dissolved in ethanol (7 ml). Saturated ammonium chloride
solution (2.5 ml) and indium powder (0.5 g) were added. The reaction mixture was
stirred under reflux for 4 h. After cooling, the reaction mixture was diluted with
water (15 ml) and filtered through celite. The aqueous filtrate was adjusted to pH ꢀ9
with sodium hydroxide (NaOH) (2 ml, 4 M) and extracted with DCM (3 Â 10ml).
The combined organic layers were dried over MgSO4. The crude product was pu-
rified by column chromatography (DCM 80: ethyl acetate 20) to give 7 as a dark
yellow oil (yield 47%, 0.082 g). mmax(Film)/cmÀ1: 3348.9 (C–N), 2923.1–2852.6 (CH-
aliphatic), 1745.5 (C@O), 1517.7 (C@C), 1408–1023.4 (aromatic); dH(300 MHz,
CDCl3): 6.88 (2H, d, J ¼ 8 Hz, CH-aromatic), 6.56 (2H, d, J ¼ 8 Hz, CH-aromatic),
5.81 (1H, m, NH), 4.09 (1H, m, N–CH), 3.92 (2H, m, O–CH2), 3.69 (2H, brs,
NH2), 3.05 (1H, d, J ¼ 8 Hz, CH-benzyllic), 2.49 (1H, d, J ¼ 8 Hz, CH-benzyllic);

S. Ahmed et al. / Bioorganic Chemistry 30 (2002) 315–331
321
dC ð75:5MHz; CDCl3): 159.45 (C@O), 145.53 (C), 129.89 (C), 125.59 (CH-aromatic),
115.55 (CH-aromatic),69.65 (CH2), 54.12 (CH), 40.59 (CH2); HRMS: Mþ ¼
192:0899 required C10H12N2O2Mþ ¼ 192:0899; ½aŠ ¼ À63.
D
2.3. Biochemistry
All non-radioactive steroids, NADPH (mono-sodium salt), and
phosphate (mono-sodium salt) were obtained from Sigma Chemical, Poole, Dorset.
-Glucose-6-phosphate dehydrogenase (suspension in ammonium sulfate) was
D-glucose-6-
D
purchased from Boehringer–Mannheim GmbH, Mannheim, Germany. [1,2-3H]An-
drostenedione (55.3 Ci/mmol) was purchased from Dupont (UK), Stevenage, Herts.
All unlabelled laboratory reagents were of analar grade and obtained from British
Drug House (BDH), Poole, Dorset. Radioactivity was determined on a KLB Wallac
1217 Rackbeta liquid scintillation counter. The scintillation fluid used was ÔCocktail
TÕ from Parkard Instrument, Illinois, USA. An MES Mistral 3000 was used for low
speed centrifugation (2000–3000g) and an MSB 65 M European ultracentrifuge for
high speed 4000–35,000 rpm (8500–105,000g).
For this study, the method of Thompson and Siiteri [13] was used, where the
aromatization reaction was quenched by mixing an aliquot of the incubation mixture
with activated charcoal suspended in mercuric chloride solution. The tritiated water
formed is thus separated from the unchanged radiolabelled steroid which is adsorbed
onto the activated charcoal. The charcoal was subsequently removed by centri-
fugation and aliquots of the supernatant were dispersed in ÔCocktail TÕ and radio-
activity counted by a scintillation counter.
The human placental aromatase enzyme was thawed under cold running water
and allowed to warm to 37 °C before use. Incubations, in triplicate, were carried out
in phosphate buffer (50 mM, pH 7.4) containing NADPH generating system (50ll,
substrate (0:84lM; 7ll), and inhibitor (10ll; 100lM final concentration), in a
shaking water bath. The incubation mixtures were warmed to 37 °C and the reaction
started by the addition of placental microsomal protein (50ll, 0.19 mg/ ml). After
incubation for 5 min, an aliquot (300ll) from each assay tube was removed and
added to tubes containing activated charcoal (1%; 900ll) and mercuric chloride
(1 mM, 300ll) and thoroughly mixed. The tubes were allowed to stand on ice for
20 min and then centrifuged at 3600 rpm for 15 min. Aliquots of the supernatant
(500ll) were removed and dispersed in scintillation fluid (2 ml) and the mixture
counted for 1 min.
3. Results and discussion
3.1. Molecular modelling
Using the novel SHC approach [8–10], we undertook a design process to deter-
mine whether the 40-aminophenyl derivatives would possess inhibitory activity
against AR. From the results of the molecular modelling study (Figs. 3 and 4) we
observe that after ligation to the P-450 heme and the rotation of the bond connecting

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S. Ahmed et al. / Bioorganic Chemistry 30 (2002) 315–331
Fig. 4. Modelling of compound 13 (bound to the heme via the phenylamine nitrogen) onto the
SHC for AR to show protrusion towards the C(17) and C(13) area of the steroid backbone.
the heme Fe atom to the inhibitor nitrogen atom, the molecules were able to undergo
conformational change (DE was found to be less than l0 kcal/mol) so as to allow the
inhibitorÕs C@O group to mimic the hydrogen bonding interaction which is pre-
sumed to occur between the C(17)@O or the C(3)@O of the steroid and the active
site.
From the initial observations, we concluded that the compounds based upon 3-
alkylated-(40-aminobenzyl)-2-oxazolidinone should possess inhibitory activity since
they were able to utilize either the steroid C(3) and/or C(17) carbonyl binding groups
within the enzyme active site. The effect of the alkyl chain could not be determined
easily from molecular modelling, however, from the modelling study we were able to
propose that the different enantiomers possessed different inhibitory profiles. That is,
from the modelling of the R- and S-enantiomers of 3-hexyl-(40-aminobenzyl)-2-
oxazolidinone (13 and 23, respectively), we observed that the hexyl moiety appeared
to be positioned such that in 13, the hexyl chain was found to be positioned below the
steroidal plane (Fig. 4). In 23, however, the hexyl chain was found to be positioned
beyond the C(13) and C(17) area of the steroid backbone, an area which is proposed
to be highly restricted in space (Fig. 5). The different binding profiles suggest that the
enantiomers, and therefore the observed inhibitory activity would differ considerably
between the R- and S-forms (since the steric interactions present in the latter form
would result in a lowered inhibitory activity whereas the R-form undergoes no such
interaction and would therefore be expected to possess greater inhibitory activity).
3.2. Chemistry
The general method employed to prepare the final compound from the starting
R- or S-enantiomer of EvanÕs chiral auxiliary is shown in Scheme 2. Overall, the
reactions proceeded without major difficulties and in good yield—as an example,

S. Ahmed et al. / Bioorganic Chemistry 30 (2002) 315–331
323
Fig. 5. Modelling of compound 23 (bound to the heme via the phenylamine nitrogen) onto the
SHC for AR to show protrusion towards the C(17) and C(13) area of the steroid backbone.
Scheme 2. Synthesis of novel AR inhibitors using the R- and S-enantiomers of the Evans
Chiral auxiliary (a ¼ NaH or K2CO3/DMF/D/R-X; b ¼ fuming HNO3/DCM/0 °C;
c ¼ In=NH4Cl=D) (R@H, Me, Et, Pr to dodecyl; X ¼ I, Br).
the synthesis of the 4(S)-(40-aminobenzyl)-oxazolidin-2-one (7) is given. The
remaining compounds were synthesized in a similar manner and the spectral data
for the N-alkylated compounds are reported in Table 1 together with the yields.

Table 1
1H and 13C NMR data for the 4-(R)-3-alkylated-(40-amminobenzyl)-2-oxazolidinone
R ¼ Compound
1H NMR
13C NMR
Yield
(%)
Number
Me
Et
8
6.89 (2H, d, J ¼ 8 Hz, Ar–H), 6.60 (2H, d, J ¼ 8 Hz, Ar–H),
3.95 (1H, m, N–CH), 3.81 (2H, m, O–CH2), 3.12 (2H, brs,
NH2), 2.93 (1H, d, J ¼ 6 Hz, CH-benzyllic), 2.83
158.73 (C@O), 145.54, 128.88, 124.80,
115.57 (C–Ar), 66.76 (O–CH2), 58.08
(N–CH), 37.41 (Ar–CH2), 18.26 (CH3)
88
(3H, s, CH3), 2.55 (1H, d, J ¼ 6 Hz,CH-benzyllic)
9
6.86 (2H, d, J ¼ 8 Hz, Ar–H), 6.58 (2H, d, J ¼ 8 Hz, Ar–H),
4.06 (1H, m, N–CH), 3.82 (2H, m, O–CH2), 3.65 (2H, brs,
NH2), 3.11 (1H, d, J ¼ 8 Hz, CH-benzyllic), 2.47 (1H, d,
J ¼ 8 Hz CH-benzylic), 1.11 (2H, m, CH2ðCH3Þ), 0.82 (3H, t,
J ¼ 7 Hz, CH3)
158.24 (C@O), 145.62, 128.82, 124.78,
115.56 (C–Ar), 66.85 (O–CH2), 55.74
(N–CH), 37.48 (Ar–CH2), 18.22 (CH2),
12.67(CH2)
90
Pr
Bu
Pe
10
11
12
6.89 (2H, d, J ¼ 8 Hz, Ar–H), 6.61 (2H, d, J ¼ 8 Hz, Ar–H),
4.13 (1H, m, N–CH), 3.97 (2H, m, O–CH2), 3.49 (2H, brs,
NH2), 3.02 (1H, d, J ¼ 8 Hz, CH-benzyllic), 2.51 (1H, d,
J ¼ 8 Hz CH-benzylic), 1.56 (2H, m, CH2CH2CH3), 1.08 (2H,
m, CH2CH2CH3), 0.83 (3H, t, J ¼ 7 Hz, CH3)
158.35 (C@O), 148.38, 129.86, 124.86,
115.50 (C–Ar), 66.76 (O–CH2), 56.12
(N–CH), 37.44 (Ar–CH2), 20.70 (CH2),
18.37 (CH2), 11.18 (CH3)
86
93
90
6.68 (2H, d, J ¼ 8 Hz, Ar–H), 6.41 (2H, d, J ¼ 8 Hz, Ar–H),
3.80 (1H, m, N–CH), 3.72 (2H, m, O–CH2), 3.36 (2H, brs,
NH2), 3.26 (1H, d, J ¼ 8 Hz, CH-benzyllic), 2.76 (1H, d,
158.36 (C@O), 146.65, 128.81, 124.74,
115.52 (C–Ar), 66.76 (O–CH2), 56.04
(N–CH), 37.37 (Ar–CH2), 28.40 (CH2),
19.17 (CH2), 18.23 (CH2), 13.71 (CH3)
J ¼ 8 Hz CH-benzylic), 1.27 (2H, m, CH2ðCH2Þ CH3), 1.08
2
(4H, m, CH2ðCH2Þ CH3), 0.96 (3H, t, J ¼ 7 Hz, CH3)
2
6.80 (2H, d, J ¼ 8 Hz, Ar–H), 6.61 (2H, d, J ¼ 8 Hz, Ar–H),
4.10 (1H, m, N–CH), 3.94 (2H, m, O–CH2), 3.62 (2H, brs,
NH2), 3.03 (1H, d, J ¼ 8 Hz, CH-benzyllic), 2.49 (1H, d,
158.25 (C@O), 145.62, 129.85, 124.84,
115.47 (C–Ar), 66.74 (O–CH2), 56.08
(N–CH), 37.45 (Ar–CH2), 28.82 (CH2),
27.20 (CH2), 27.07 (CH2), 22.33 (CH2),
14.00 (CH3)
J ¼ 8 Hz CH-benzylic), 1.50 (2H, m, CH2ðCH2Þ CH3), 1.28
3
(6H, m, CH2ðCH2Þ CH3), 0.91 (3H, t, J ¼ 7 Hz, CH3)
3

Hex 13
6.82 (2H, d, J ¼ 8 Hz, Ar–H), 6.54 (2H, d, J ¼ 8 Hz, Ar–H),
4.05 (1H, m, N–CH), 3.87 (2H, m, O–CH2), 3.51 (2H, brs,
NH2), 2.96 (1H, d, J ¼ 8 Hz, CH-benzyllic), 2.43 (1H, d,
158.41 (C@O), 145.76, 128.73, 124.56,
115.47 (C–Ar), 66.74 (O–CH2), 57.62
(N–CH), 37.30 (Ar–CH2), 28.58 (CH2),
27.04 (CH2), 26.85 (CH2), 22.22 (CH2),
18.10 (CH2), 13.89 (CH3)
84
88
91
J ¼ 8 Hz CH-benzylic), 1.42 (2H, m, CH2ðCH2Þ CH3), 1.20
4
(8H, m, CH2ðCH2Þ CH3), 0.84 (3H, t, J ¼ 7 Hz, CH3)
4
He- 14
pt
6.73 (2H, d, J ¼ 8 Hz, Ar–H), 6.57 (2H, d, J ¼ 8 Hz, Ar–H),
4.02 (1H, m, N–CH), 3.87 (2H, m, O–CH2), 3.52 (2H, brs,
NH2), 3.30 (1H, d, J ¼ 8 Hz, CH-benzyllic), 2.81 (1H, d,
158.26 (C@O), 145.52, 129.87, 124.86,
115.53 (C–Ar), 66.75 (O–CH2), 56.10
(N–CH), 37.47 (Ar–CH2), 31.73 (CH2),
28.84(CH2), 27.84(CH2), 27.42 (CH2)
26.68 (CH2), 22.58 (CH2), 14.09 (CH3)
158.31 (C@O), 145.73, 129.81, 124.66,
115.47 (C–Ar), 66.75 (O–CH2), 55.06
(N–CH), 37.38 (Ar–CH2), 31.75 (CH2),
29.66 (CH2), 29.19 (CH2), 27.48 (CH2),
27.39 (CH2), 26.70 (CH2), 22.62 (CH2),
14.10 (CH3)
J ¼ 8 Hz CH-benzylic), 1.36 (2H, m, CH2ðCH2Þ CH3), 1.09
5
(10H, m, CH2ðCH2Þ CH3), 0.68 (3H, t, J ¼ 7 Hz, CH3)
5
Oct
15
6.66 (2H, d, J ¼ 8 Hz, Ar–H), 6.46 (2H, d, J ¼ 8 Hz, Ar–H),
4.03 (1H, m, N–CH), 3.89 (2H, m, O–CH2), 3.53 (2H, brs,
NH2), 2.23 (1H, d, J ¼ 8 Hz, CH-benzyllic), 2.28 (1H, d,
J ¼ 8 Hz CH-benzylic), 1.32 (2H, m, CH2ðCH2Þ CH3), 1.03
6
(12H, m, CH2ðCH2Þ CH3), 0.64 (3H, t, J ¼ 7 Hz, CH3)
6
Non 16
6.91 (2H, d, J ¼ 8 Hz, Ar–H), 6.46 (2H, d, J ¼ 8 Hz, Ar–H),
4.03 (1H, m, N–CH), 3.71 (2H, m, O–CH2), 3.50 (2H, brs,
NH2), 3.04 (1H, d, J ¼ 8 Hz, CH-benzyllic), 2.87 (1H, d,
159.19 (C@O), 145.47, 129.88, 125.03,
115.52 (C–Ar), 66.73 (O–CH2), 56.10
(N–CH), 42.04 (Ar–CH2), 37.47 (CH2),
31.88 (CH2), 29.54 (CH2), 29.30 (CH2),
29.22 (CH2), 27.43 (CH2), 26.73 (CH2),
22.68 (CH2), 14.14 (CH3)
91
91
93
J ¼ 8 Hz CH-benzylic), 1.34 (2H, m, CH2ðCH2Þ CH3), 1.05
7
(14H, m, CH2ðCH2Þ CH3), 0.68 (3H, t, J ¼ 7 Hz, CH3)
7
Dec 17
6.70 (2H, d, J ¼ 8 Hz, Ar–H), 6.42 (2H, d, J ¼ 8 Hz, Ar–H),
4.02 (1H, m, N–CH), 3.76 (2H, m, O–CH2), 3.56 (2H, brs,
NH2), 3.28 (1H, d, J ¼ 8 Hz, CH-benzyllic), 2.82 (1H, d,
158.43 (C@O), 145.36, 128.84, 125.05,
115.70 (C–Ar), 66.78 (O–CH2), 57.07
(N–CH), 37.39 (Ar–CH2), 31.88 (CH2),
29.55 (CH2), 29.26(CH2), 29.11 (CH2),
27.38 (CH2), 26.70 (CH2), 26.30 (CH2),
24.86 (CH2), 22.66 (CH2), 14.911 (CH3)
158.06 (C@O), 144.52, 129.88, 123.98,
115.05 (C–Ar), 66.71 (O–CH2), 54.38
(N–CH), 37.03 (Ar–CH2), 12.76 (CH3)
J ¼ 8 Hz CH-benzylic), 1.36 (2H, m, CH2ðCH2Þ CH3), 1.03
8
(16H, m, CH2ðCH2Þ CH3) 0.96 (3H, t, J ¼ 7 Hz, CH3)
8
Me
18
6.94 (2H, d, J ¼ 8 Hz, Ar–H), 6.65 (2H, d, J ¼ 8 Hz, Ar–H),
4.18 (1H, m, N–CH), 3.98 (2H, m, O–CH2), 3.41 (2H, brs,
NH2), 3.01 (1H, d, J ¼ 6 Hz, CH-benzyllic), 2.88
(3H, s, CH3), 2.58 (1H,d, J ¼ 6 Hz, CH-benzyllic)

Table 1 (continued)
R ¼ Compound
1H NMR
13C NMR
Yield
(%)
Number
Et
Pr
Bu
Pe
19
20
21
22
6.87 (2H, d, J ¼ 8 Hz, Ar–H), 6.57 (2H, d, J ¼ 8 Hz, Ar–H),
4.11 (1H, m, N–CH), 3.99 (2H, m, O–CH2), 3.33 (2H, brs,
NH2), 3.04 (1H, d, J ¼ 8 Hz, CH-benzyllic), 2.51 (1H, d,
J ¼ 8 Hz CH-benzylic), 1.18 (2H, m, CH2CH3), 1.11 (3H, t,
J ¼ 7 Hz, CH3)
158.05 (C@O), 145.51, 129.88, 124.88,
115.50 (C–Ar), 66.81 (O–CH2), 55.78
(N–CH), 37.63 (Ar–CH2), 34.21 (CH2),
12.74 (CH2)
86
86
90
93
88
6.73 (2H, d, J ¼ 8 Hz, Ar–H), 6.45 (2H, d, J ¼ 8 Hz, Ar–H),
3.97 (1H, m, N–CH), 3.79 (2H, m, O–CH2), 3.24 (2H, brs,
NH2), 2.98 (1H, d, J ¼ 8 Hz, CH-benzyllic), 2.32 (1H, d,
158.45 (C@O), 145.55, 129.84, 124.88,
115.55 (C–Ar), 66.79 (O–CH2), 56.04
(N–CH), 37.41 (Ar–CH2), 20.16 (CH2),
J ¼ 8 Hz CH-benzylic), 1.36 (2H, m, CH2CH2CH3), 1.11 (3H, t, 18.25(CH2), 11.13 (CH3)
J ¼ 7 Hz, CH3)
6.94 (2H, d, J ¼ 8 Hz, Ar–H), 6.65 (2H, d, J ¼ 8 Hz, Ar–H),
4.15 (1H, m, N–CH), 3.79 (2H, m, O–CH2), 3.46 (2H, brs,
NH2), 3.05 (1H, d, J ¼ 8 Hz, CH-benzyllic), 2.55 (1H, d,
157.93 (C@O), 146.54, 128.88, 123.88,
115.51 (C–Ar), 66.74 (O–CH2), 57.54
(N–CH), 37.51 (Ar–CH2), 22.17 (CH2),
20.73 (CH2), 18.37 (CH2), 11.20 (CH3)
J ¼ 8 Hz CH-benzylic), 1.60 (2H, m, CH2ðCH2Þ CH3), 1.24
2
(4H, m, CH2ðCH2Þ CH3), 0.83 (3H, t, J ¼ 7 Hz, CH3)
2
6.92 (2H, d, J ¼ 8 Hz, Ar–H), 6.71 (2H, d, J ¼ 8 Hz, Ar–H),
4.15 (1H, m, N–CH), 4.05 (2H, m, O–CH2), 3.48 (2H, brs,
NH2), 3.01 (1H, d, J ¼ 8 Hz, CH-benzyllic), 2.51 (1H, d,
158.22 (C@O), 145.51, 129.88, 125.01,
115.50 (C–Ar), 66.73 (O–CH2), 56.11
(N–CH), 37.48 (Ar–CH2), 28.84
(CH2), 27.10 (CH2), 23.35 (CH2),
14.01 (CH3)
J ¼ 8 Hz CH-benzylic), 1.54 (2H, m, CH2ðCH2Þ CH3), 1.24
3
(6H, m, CH2ðCH2Þ CH3), 0.92 (3H, t, J ¼ 7 Hz, CH3)
3
Hex 23
6.71 (2H, d, J ¼ 8 Hz, Ar–H), 6.42 (2H, d, J ¼ 8 Hz, Ar–H),
4.14 (1H, m, N–CH), 3.93 (2H, m, O–CH2), 3.35 (2H, brs,
NH2), 2.87 (1H, d, J ¼ 8 Hz, CH-benzyllic), 2.33 (1H, d,
158.24 (C@O), 145.58, 129.86, 124.90,
115.48 (C–Ar), 66.74 (O–CH2), 56.10
(N–CH), 37.48 (Ar–CH2), 34.08
(CH2), 27.08(CH2), 22.35 (CH2),
14.21 (CH3)
J ¼ 8 Hz CH-benzylic), 1.34 (2H, m, CH2ðCH2Þ CH3), 1.01
4
(8H, m, CH2ðCH2Þ CH3), 0.69 (3H, t, J ¼ 7 Hz, CH3)
4

He- 24
pt
6.76 (2H, d, J ¼ 8 Hz, Ar–H), 6.60 (2H, d, J ¼ 8 Hz, Ar–H),
4.28 (1H, m, N–CH), 4.12 (2H, m, O–CH2), 3.04 (2H, brs,
NH2), 2.84 (1H, d, J ¼ 8 Hz, CH-benzyllic), 2.63 (1H, d,
158.31 (C@O), 145.57, 129.85, 124.86,
115.48 (C–Ar), 66.75 (O–CH2), 56.08
(N–CH), 37.42 (Ar–CH2), 31.70 (CH2),
28.91 (CH2), 27.39 (CH2), 26.65 (CH2),
20.43 (CH2), 15.33 (CH2), 14.05 (CH3)
158.43 (C@O), 145.35, 129.46, 125.03,
114.84 (C–Ar), 66.68 (O–CH2), 55.98
(N–CH), 42.01 (Ar–CH2), 37.45 (CH2),
31.74 (CH2), 29.18 (CH2), 27.40 (CH2),
26.68 (CH2), 22.61 (CH2), 18.23 (CH2),
14.09 (CH3)
91
88
J ¼ 8 Hz CH-benzylic), 1.25 (2H, m, CH2ðCH2Þ CH3), 1.10
5
(10H, m, CH2ðCH2Þ CH3), 0.87 (3H, t, J ¼ 7 Hz, CH3)
5
Oct
25
6.91 (2H, d, J ¼ 8 Hz, Ar–H), 6.61 (2H, d, J ¼ 8 Hz, Ar–H),
4.08 (1H, m, N–CH), 3.92 (2H, m, O–CH2), 3.40 (2H, brs,
NH2), 2.98 (1H, d, J ¼ 8 Hz, CH-benzyllic), 2.58 (1H, d,
J ¼ 8 Hz CH-benzylic), 1.52 (2H, m, CH2ðCH2Þ CH3),
6
1.19 (12H, m, CH2ðCH2Þ CH3), 0.84 (3H, t, J ¼ 7 Hz,
6
CH3)
Non 26
6.92 (2H, d, J ¼ 8 Hz, Ar–H), 6.73 (2H, d, J ¼ 8 Hz, Ar–H),
4.09 (1H, m, N–CH), 3.94 (2H, m, O–CH2), 3.44 (2H, brs,
NH2), 3.04 (1H, d, J ¼ 8 Hz, CH-benzyllic), 2.53 (1H, d,
158.34 (C@O), 145.07, 129.96, 126.63,
116.67 (C–Ar), 66.67 (O–CH2), 55.95
(N–CH), 37.28 (Ar–CH2), 33.99 (CH2),
31.82 (CH2), 29.48 (CH2), 29.26 (CH2)
29.22 (CH2), 27.42 (CH2), 26.70 (CH2),
22.64 (CH2), 14.11 (CH3)
91
91
J ¼ 8 Hz CH-benzylic), 1.52 (2H, m, CH2ðCH2Þ CH3),
7
1.23 (14H, m, CH2ðCH2Þ CH3), 0.84 (3H, t, J ¼ 7 Hz,
7
CH3)
Dec 27
6.60 (2H, d, J ¼ 8 Hz, Ar–H), 6.34 (2H, d, J ¼ 8 Hz, Ar–H),
4.10 (1H, m, N–CH), 3.92 (2H, m, O–CH2), 3.40 (2H, brs,
NH2), 3.03 (1H, d, J ¼ 8 Hz, CH-benzyllic), 2.54 (1H, d,
158.51 (C@O), 145.53, 129.67, 12.68,
115.67 (C–Ar), 66.74 (O–CH2), 57.40
(N–CH),37.21 (Ar–CH2), 31.72 (CH2),
30.69 (CH2), 29.38 (CH2), 29.14 (CH2)
27.22 (CH2), 26.55 (CH2), 22.51 (CH2),
21.71 (CH2), 17.82 (CH2), 13.90 (CH3)
J ¼ 8 Hz CH-benzylic), 1.22 (2H, m, CH2ðCH2Þ CH3),
8
1.07 (16H, m, CH2ðCH2Þ CH3), 0.87 (3H, t, J ¼ 7 Hz,
8
CH3)

328
S. Ahmed et al. / Bioorganic Chemistry 30 (2002) 315–331
It should be noted, however, that with the increase in chain length (N-alkylation),
the nitration step required an increase in the reaction time. That is, with the more
hydrophobic chains, a greater length of time was required for the nitration reaction
to proceed to completion, with the result that the N-dodecyl derivative required
some 7 h (yield 80%) whereas the smaller alkyl chain containing compounds took 3 h.
The reduction of the nitro to the amine (using a mixture of indium and ammonium
chloride) also proceeded in good yield and without any major problems.
In an effort to ensure that the chiral centre had not undergone any kind of al-
teration/racemization, the ½aŠ values of the compounds were determined and were
D
found to be consistent with the starting enantiomer. For example, the starting
compound in the synthesis of the S-enantiomer [namely (S)-4-benzyl-oxazolidin-2-
one] was found to possess (under the given conditions) a ½aŠD value of À62. Although
the value of the number may be expected to change, the direction of rotation of the
plane of polarized light would not. The amino compounds [containing the (S)-4-
benzyl-oxazolidin-2-one] were all found to possess a negative ½aŠ value (ranging
D
from À62 to À84, where R@H to decyl respectively). The derivatives of (R)-4-benzyl-
oxazolidin-2-one were found to possess a similar range, from þ63 to þ85, where
R@H to decyl, respectively.
3.3. Inhibition of human placental aromatase
The novel inhibitors, in general, were found to possess a good range of inhibitory
activity (Tables 2 and 3) [for example, N-nonyl-4(S)-(40-aminobenzyl)-oxazolidin-
2-one (26) possessed 10% inhibition whilst N-nonyl-4(R)-(40-aminobenzyl)-oxazoli-
din-2-one (16) possessed 73% inhibition] compared to the standard compound
aminoglutethimide (AG) which possessed 53% inhibition at ½IŠ ¼ 100 lM. A detailed
consideration of the inhibitory activity (initial screening and IC50 values) of the
N-alkylated derivatives of the two enantiomers shows that, in general, the R-form
appears to be more potent than the inhibitors based upon the S-enantiomer (Tables
1 and 2). Indeed, further consideration of the IC50 values shows that the trend in
inhibitory activity between the S- and the R-forms is very different. For example,
within the first series of compounds (8–17), inhibitory activity increases and is ob-
served to reach a maximum with the pentyl (12) and hexyl (13) derivatives (Fig. 6);
within the series of inhibitors of the S-enantiomer, however, the inhibitory activity
decreases with an increase in alkyl chain length (Fig. 7). As such, the inhibitory data
correlate very closely with the observations from the molecular modelling studies
and therefore gives strong support for the novel SHC approach.
The biochemical evaluation of the synthesized compounds also adds further
support for the existence of a large hydrophobic pocket. Consideration of the in-
hibitory activity of the R-enantiomer based compounds shows that the C10 (17)
possesses greater inhibitory activity than the majority of the compounds based upon
the S-enantiomer—only the methyl-substituted compound (18) possessed greater
inhibitory activity than 17 (Table 2). We have previously reported an extensive
overall model for the active site of AR where the existence of a protrusion has been
proposed to exist below the steroidal plane, which is able to accommodate alkyl
chains of up to C12 in length [10].

S. Ahmed et al. / Bioorganic Chemistry 30 (2002) 315–331
329
Table 2
Initial screening data for R- and S-enantiomers of EvansÕ chiral auxiliary and AG (values are
mean of triplicate determinations)
Compound (R ¼ )
R-enantiomer
Compound number
Percentage of inhibition (½IŠ ¼ 100 lM)
Me
Et
8
9
38
53
68
86
93
87
86
76
73
62
Pr
Bu
10
11
12
13
14
15
16
17
Pe
Hex
Hept
Oct
Non
Dec
S-enantiomer
Me
Et
18
19
20
21
22
23
24
25
26
27
65
60
50
42
37
21
15
12
10
10
Pr
Bu
Pe
Hex
Hept
Oct
Non
Dec
AG
–
53
Table 3
IC50 of some of the synthesised inhibitors (values are mean of triplicate determinations)
Compound
IC50 (lM)
Relative potency
10
11
12
13
14
20
21
22
23
24
25
26
27
4:2 ꢁ 0:2
22.67
63.45
114.70
79.33
26.44
7.52
1:5 ꢁ 0:2
0:83 ꢁ 0:05
1:2 ꢁ 0:1
3:6 ꢁ 0:06
12:66 ꢁ 0:3
15:00 ꢁ 0:3
16:82 ꢁ 0:2
35:00 ꢁ 0:5
71:26 ꢁ 0:25
77:0 ꢁ 0:15
85:8 ꢁ 0:05
125:0 ꢁ 0:4
6.35
5.66
2.72
1.34
1.24
1.11
0.76
AG
95:2 ꢁ 0:5
1

330
S. Ahmed et al. / Bioorganic Chemistry 30 (2002) 315–331
Fig. 6. Plot of chain length versus IC50 of the R-enantiomer based inhibitors.
Fig. 7. Plot of IC50 versus chain length to show the decrease in the potency of the S-enan-
tiomer based compounds with an increase in alkyl chain length.
Modelling of compounds based on the S-enantiomer (e.g., 23, Fig. 5) onto the SHC
showed that the alkyl chain protruded towardsthe C(17) and C(13) area of the steroidal
backbone, an area which is proposed to be sterically constrained due to the protein
backbone, resulting in greatly reduced inhibitory activity. As such, the current study
therefore adds further support to the previous studies. Rotation of the benzyllic bond
(so as to produce a conformer whereby the alkyl chains do not protrude towards the
sterically constrained area of the protein backbone) results in an unfavourable con-
formational change which gives rise to a large increase in energy (DE > 20kcal=mol),
and therefore a decrease in the inhibitory activity.
4. Conclusion
In conclusion, the results of the biochemical evaluation of the two sets of
enantiomers of 3-alkylated-(40-aminobenzyl)-2-oxazolidinone support the molecular
modelling study and in particular provides further support for the SHC approach

S. Ahmed et al. / Bioorganic Chemistry 30 (2002) 315–331
331
which has previously been used to rationalize the inhibitory activity of a wide range
of inhibitors of AR and other cytochrome P-450 enzymes.
Acknowledgment
The authors thank the National Mass Spectrometry Centre at University of Wales
College of Swansea for the accurate mass of synthesized inhibitors.
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