Synthesis and CYP26A1 inhibitory activity of novel methyl 3-[4-(arylamino)phenyl]-3-(azole)-2,2-dimethylpropanoates

Article abstract of DOI:10.1016/j.bmc.2012.08.044

The role of all-trans-retinoic acid (ATRA) in the development and maintenance of many epithelial and neural tissues has raised great interest in the potential of ATRA and related compounds (retinoids) as pharmacological agents, particularly for the treatm

Full text of DOI:10.1016/j.bmc.2012.08.044

Bioorganic & Medicinal Chemistry 20 (2012) 6080–6088
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and CYP26A1 inhibitory activity of novel methyl
3-[4-(arylamino)phenyl]-3-(azole)-2,2-dimethylpropanoates
Mohamed S. Gomaa a, , Andrew S. T. Lim a, S. C. Wilson Lau a, Ann-Marie Watts a, Nicola A. Illingworth b,
Caroline E. Bridgens b, Gareth J. Veal b, Christopher P. F. Redfern b, Andrea Brancale a, Jane L. Armstrong c,
Claire Simons a,
⇑
a Medicinal Chemistry Division, Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3NB, UK
b Northern Institute for Cancer Research, Paul O’Gorman Building, Medical School, Framlington Place, Newcastle University, Newcastle Upon Tyne NE2 4HH, UK
c Institute of Cellular Medicine, Framlington Place, Newcastle University, Newcastle Upon Tyne NE2 4HH, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 July 2012
Revised 16 August 2012
Accepted 17 August 2012
Available online 30 August 2012
The role of all-trans-retinoic acid (ATRA) in the development and maintenance of many epithelial and
neural tissues has raised great interest in the potential of ATRA and related compounds (retinoids) as
pharmacological agents, particularly for the treatment of cancer, skin, neurodegenerative and autoim-
mune diseases. The use of ATRA or prodrugs as pharmacological agents is limited by a short half-life
in vivo resulting from the activity of specific ATRA hydroxylases, CYP26 enzymes, induced by ATRA in
liver and target tissues. For this reason retinoic acid metabolism blocking agents (RAMBAs) have been
developed for treating cancer and a wide range of other diseases.
Keywords:
CYP26A1
The synthesis, CYP26A1 inhibitory activity and molecular modeling studies of novel methyl 3-[4-
(arylamino)phenyl]-3-(azole)-2,2-dimethylpropanoates are presented. From this series of compounds
clear SAR can be derived for 4-substitution of the phenyl ring with electron-donating groups more
favourable for inhibitory activity. Both the methylenedioxyphenyl imidazole (17, IC50 = 8 nM) and tria-
zole (18, IC50 = 6.7 nM) derivatives were potent inhibitors with additional binding interactions between
the methylenedioxy moiety and the CYP26 active site likely to be the main factor. The 6-bromo-3-pyri-
dine imidazole 15 (IC50 = 5.7 nM) was the most active from this series compared with the standards
liarozole (IC50 = 540 nM) and R116010 (IC50 = 10 nM).
Methyl 3-[4-(arylamino)phenyl]-3-(azole)-
2,2-dimethylpropanoate derivatives
IC50 enzyme inhibition
MCF-7
Molecular modeling
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
The development of retinoids as pharmacological agents has pro-
gressed through two main routes—the design of synthetic retinoids
Differentiation and maintenance of many epithelial and neural
tissues is dependent on intracellular formation of retinoic acid
and its delivery to ligand-dependent transcription factors (retinoic
acid receptors or RARs) in the cell nucleus.1,2 Activity of this signal-
ling pathway in relation to differentiation is exquisitely controlled
by the expression of dehydrogenases and binding proteins to gen-
erate all-trans retinoic acid (ATRA) from retinol, and subsequent
negative feedback by the induction of metabolism to limit and re-
duce intracellular ATRA levels.3–5 These tight developmental con-
trols have raised great interest in the potential of ATRA and
related compounds (retinoids) as pharmacological agents, particu-
larly for the treatment of cancer, and skin, neurodegenerative and
autoimmune diseases.
which mimic ATRA by binding to and activating RARs, but which
have greater biological stability, and the administration of ATRA,
or pro-drugs such as 13-cis retinoic acid, at pharmacological doses.6
While synthetic retinoids have been successful in certain contexts,
some have suffered from high toxicity in vivo;7 moreover, the de-
sign of these compounds needs to take into consideration the ability
to bind to intracellular transport proteins (e.g., CRABPS) and to per-
mit the ligand-dependent conformational changes required for
RARs to function as transcriptional regulators.8 Conversely, the
use of ATRA or prodrugs as pharmacological agents is limited by a
short ATRA half-life of <1 h in vivo9 resulting from the activity of
specific ATRA hydroxylases induced by ATRA in target tissues.10
Although ATRA can be metabolised by several promiscuous cyto-
chrome p450 enzymes (CYP), of which CYP 3A4 and 2C8 may be
the most important, specific RA hydroxylases of the CYP26 family
(CYP26A1, 26B1 and 26C1) are central to regulating intracellular
ATRA concentrations as part of precise developmental schedules.11
Compounds that inhibit CYP26 have been developed; liarozole
(6-[(3-chlorophenyl)-imidazol-1-ylmethyl]-1H-benzimidazole)
⇑
Corresponding author. Tel.: +44 (0) 2920 876307; fax: +44 (0) 2920 874149.
E-mail address: SimonsC@Cardiff.ac.uk (C. Simons).
URL: http://www.cardiff.ac.uk/phrmy/staffinfo/CS/homepage/CS.html (C. Simons).
Present address: Pharmaceutical Chemistry Department, Faculty of Pharmacy,
Suez Canal University, Egypt.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.044

M. S. Gomaa et al. / Bioorg. Med. Chem. 20 (2012) 6080–6088
6081
and talarozole (N-[4-(2-ethyl-1-[1,2,4-triazol-1-yl]butyl)phenyl]-
1,3-benzothiazol-2-amine) (Fig. 1) have shown promise in clinical
trials for the treatment of dermatological disease.12,13 However, as
a strong aromatase inhibitor, liarozole lacks specificity leading to
unwanted side effects; talarozole is 750 times more potent than
liarozole and has greater specificity towards CYP26.12 In view of
the potential of retinoic acid metabolism blocking agents (RAMBAs)
for treating cancer and other diseases, identifying novel agents with
greater potency, selectivity for CYP26 and good pharmacological
profiles is a high priority.
yields. The coupled products were confirmed by the presence of an
NH singlet peak at dH 5.7–5.9 in 1H NMR (Scheme 1).
Introduction of the N-heterocycle involved reaction of the alco-
hol precursor (3) with 1,10-carbonyldiimidazole (CDI) and imidaz-
ole in acetonitrile16 to give the imidazole derivatives ( 1, 4–12)
(Scheme 1).
To explore additional hydrogen bonding interactions within the
CYP26 active site, the phenyl ring was replaced with a 6-bromo-3-
pyridine ring and a methylenedioxyphenyl ring. Using the same
Chan–Lam coupling methodology as described above, the alcohol
precursors 13 and 14 were prepared (Scheme 2). Introduction of
either imidazole or triazole ring, using CDI/imidazole or carbon-
ylditriazole (CDT)/triazole,16 gave the 6-bromo-3-pyridine deriva-
tives (15, 16) and the methylenedioxyphenyl derivatives (17, 18).
Exchanging the imidazole haem binding moiety for 2-methyl-
imidazole has been shown to improve CYP selectivity,20,21 there-
fore the unsubstituted phenyl 2-methylimidazole derivative (19)
and the methylenedioxyphenyl 2-methylimidazole derivative
(20) were prepared by reaction of the precursor alcohols (3a and
14, respectively) with 2-methyl imidazole and 1,10-carbonylbis(2-
methylimidazole) (Scheme 3).
We have recently synthesised and characterised a novel series
of arylamine CYP26 inhibitors, with a naphthyl imidazole ([4-
(imidazol-1-yl-phenyl-methyl)-phenyl]-naphthalen-2-yl-amine)
(MCC147, Fig. 1) being the most potent with an IC50 against CYP26
of 0.5
chain resulted in
l
M.14 Replacement of the phenyl ring with a flexible C3
a
compound with a CYP26 IC50 of 3 nM
(MCC154, Fig. 1), comparable with that of talarozole; retention of
the 2-naphthyl and NH linker is important for maximal activity.15
Although showing good selectivity for CYP26, this compound
showed some activity against CYP3A4, a relatively promiscuous
xenobiotic metabolising CYP.15 Selectivity to CYP26 was increased
from <33 to 1100-fold by replacing the imidazole with a triazole,
with a concomitant increase in potency to an IC50 of 0.35 nM
(MCC219, Fig. 1).16 Further studies to assess the potency and selec-
tivity of triazole and imidazole derivatives containing modifica-
tions to the flexible side-chain and phenyl substituents in place
of the 2-naphthyl led to the identification of new potent CYP26
inhibitors.17 Therefore, we have now explored the possibility that
4-substituted and 3,4,5-substituted phenyl derivatives in combina-
tion with the imidazole, triazole and 2-methylimidazole would im-
prove activity and CYP26 selectivity while optimising the drug-like
properties of these compounds.
2.2. CYP26A1 enzyme inhibition
The imidazole and triazole derivatives were evaluated for their
ability to inhibit CYP26-mediated retinoic acid metabolism
(CYP26) inhibitory activity using a cell-free microsomal assay as
previously described,14,22 with radiolabelled [11,12-3H] all-trans
retinoic acid as the substrate. Liarozole (a non-selective CYP26
inhibitor23,24) and R11601025 (Fig. 1) were included in all experi-
ments as comparative standards.
Introduction of an electron-withdrawing group in the 4-posi-
tion of the phenyl ring for example F (4), Cl (5), CN (9) or NO2
(10) reduced inhibitory activity (IC50 range from 250 nM to
2. Results and discussion
2.1. Chemistry
>1 lM) compared with the unsusbtituted phenyl derivative (1,
IC50 = 10 nM), whereas introduction of an electron-donating group,
for example CH3 (6), CF3 (7) retained activity compared with
unsubstituted phenyl (1), with the exception the larger 4-methoxy
derivative (8) for which a large loss in activity was observed. Multi-
ple substitutions were well tolerated with comparable inhibitory
activity observed for the 3,4,5-trimethoxy substituted phenyl
derivative (11, IC50 = 45 nM) and the 3,5-dimethyl-4-methoxy
The N-arylation of the amine (2)15 using the Chan–Lam cou-
pling reaction, followed described methodology18,19 employing a
stoichiometric amount of copper and a tertiary amine base, pyri-
dine in this reaction series. Using this method, reaction with the
appropriate aryl boronic acid gave the N-aryl products (3) in good
Figure 1. Examples of reported CYP26 inhibitors and inhibitory activity.

6082
M. S. Gomaa et al. / Bioorg. Med. Chem. 20 (2012) 6080–6088
Scheme 1. Reagents and conditions: (i) aryl boronic acid, CuOAc, pyridine, 4 Å molecular sieves, CH2Cl2, rt, 2 days; (ii) 1,10-carbonyldiimidazole, imidazole, CH3CN, reflux, 24–
48 h.
Scheme 2. Reagents and conditions: (i) 6-bromo-3-pyridinyl boronic acid or 3,4-(methylenedioxy)phenyl boronic acid, CuOAc, pyridine, 4 Å molecular sieves, CH2Cl2, rt,
2 days; (ii) 1,10-carbonyldiimidazole, imidazole, CH3CN, reflux, 24–48 h; (iii) 1,10-carbonyditriazole, triazole, CH3CN, reflux, 24–48 h.
Scheme 3. Reagents and conditions: (i) phenyl boronic acid or 3,4-(methylenedioxy)phenyl boronic acid, CuOAc, pyridine, 4 Å molecular sieves, CH2Cl2, rt, 2 days; (ii) 2-
methylimidazole, 1,10-carbonylbis(2-methylimidazole), CH3CN, reflux, 24–48 h.
substituted phenyl derivative (12, IC50 = 14 nM) (Table 1). The
most interesting results were observed with the 6-bromo-3-pyri-
dine and methylenedioxyphenyl derivatives with improved
CYP26 inhibitory activity observed for both imidazole derivatives
(15, IC50 = 5.7 nM and 17, IC50 = 8 nM, respectively). Activity was
lost for the 6-bromo-3-pyridine triazole derivative (16,
IC50 = 500 nM) but retained for the methylenedioxyphenyl triazole
derivative (18, IC50 = 6.7 nM) (Table 1). Reduction in inhibitory
activity was observed for the 2-methylimidazole derivatives (19
and 20) activity compared with the unsubstituted phenyl imidaz-
ole (1) (Table 1).
ported previously.15 Results obtained for the phenyl derivatives
4–12 and methylenedioxyphenyl derivatives 17 and 18 showed a
putative binding mode similar to the one observed previously for
the corresponding phenyl imidazole analogue 1. In particular,
binding was dominated by a series of hydrophobic interactions:
for the methylenedioxyphenyl inhibitor 18 the methylenedioxy-
phenyl group lay in a channel formed, among others, by Pro338,
Phe53 and Phe343 while the ester moiety was in contact with
Phe268 (Fig. 2). Furthermore, Ser192 is within hydrogen bond dis-
tance to the methylenedioxyphenyl ring.
The 6-bromo-3-pyridine imidazole derivative 15 was also posi-
tioned within the hydrophobic pocket (Fig. 3) with an additional
hydrogen bond interaction between Ser84 and the pyridine nitrogen.
It should also be noted that the 2-methylimidazole compounds
19 and 20 did not dock that favourably, as the substitution induces
a change in the compound conformations that do not allow coordi-
nation between the imidazole ring and the Fe atom (Fig. 4).
2.3. Molecular modelling
A series of molecular docking simulations were performed on
this series of compounds with ligands docked within the active site
of the CYP26A1 homology model,26 using the methodology re-

M. S. Gomaa et al. / Bioorg. Med. Chem. 20 (2012) 6080–6088
6083
Table 1
IC50 values for imidazole, triazole and 3-methylimidazole derivatives
3. Discussion
Compd
R1
X
aIC50 (nM)
A clear SAR was observed for the 4-substituted and 3,4,5-substi-
tuted phenyl derivatives 4–12 with electron-withdrawing groups
detrimental to inhibitory activity whereas electron-donating
groups generally resulted in inhibitory activity comparable with
the unsubstituted phenyl imidazole 1. The methylenedioxyphenyl
inhibitors 18 and 19 were able to interact within the active site in a
manner similar to the phenylimidazole 1 with additional hydrogen
bonding interaction between the methylenedioxy ring and active
site residues favourable for inhibitory activity. Both the imidazole
and triazole displayed potent CYP26 inhibitory activity (18,
IC50 = 8 nM; 19, IC50 = 6.7 nM) comparable with the phenylimida-
zole 1 (IC50 = 10 nM) and the R116010 standard (IC50 = 10 nM)
and was considerably more active than Liarozole (IC50 = 540 nM).
The 6-bromo-3-pyridine imidazole derivative 15 was the most po-
tent CYP26 inhibitor from this series (IC50 = 5.7 nM) however
replacing the imidazole group with a triazole to give compound
16 resulted in a substantial loss in inhibitory activity. From our
1
4
5
6
7
8
9
10
11
12
15
16
17
18
19
20
H
4-F
4-Cl
4-CH3
4-CF3
4-OCH3
4-CN
4-NO2
—
—
—
—
—
—
—
—
—
—
CH
N
CH
N
—
—
—
—
10
250
450
16
9.4
700
>1 lM
100
45
14
5.7
500
8
6.7
90
>10 nM
540
10
3,4,5-triOCH3
3,5-diCH3, 4-OCH3
—
—
—
—
—
—
—
—
Liarozole
R116010
a
IC50 values derived from the best fit of a 4 point dose–response curve.
Figure 2. Putative binding mode for 18. The Fe atom of the heme is shown as a green sphere.
Figure 3. Putative binding mode for 15. The Fe atom of the heme is shown as a green sphere.

6084
M. S. Gomaa et al. / Bioorg. Med. Chem. 20 (2012) 6080–6088
Figure 4. Different binding mode between 18 (gray) and 20 (cyan). The Fe atom of the heme is shown as a green sphere.
previous studies changing the imidazole for a triazole results in
comparable15,17 or improved16 CYP26 inhibitory activity. At this
stage, molecular modelling studies do not suggest any specific
structural justification for this difference in activity. Finally, for
both the phenyl and methylenedioxyphenyl derivates replacing
the imidazole with 2-methylimidazole was unfavourable as a re-
sult of a different orientation of the imidazole ring in the binding
pocket (Fig. 4).
charring on a hotplate. Melting points were determined on an elec-
trothermal instrument and are uncorrected. All solvents were dried
prior to use and stored over 4 Å molecular sieves, under nitrogen.
All compounds were more than 95% pure.
Compound 3a (R = H), 3c (R = Cl), 3f (R = OCH3), 14 and 17 were
prepared as previously described.15,17
5.1.1. General procedure for the Chan–Lam coupling
preparation of compounds 3a–3j and 13
4. Conclusions
To the appropriate aryl boronic acid (4.0 mmol), 3-(4-amino-
phenyl)-3-hydroxy-2,2-dimethylpropionic acid methyl ester (2)
(2.2 mmol), anhydrous Cu(II)(OAc)2 (3.0 mmol), pyridine
(4.0 mmol) and 250 mg activated 4 Å molecular sieves under an
atmosphere of air was added CH2Cl2 (15 mL) and the reaction stir-
red under air atmosphere at ambient temperature for 2 days. The
product was isolated by direct flash column chromatography of
the crude reaction mixture (Petroleum ether–EtOAc 70:30 v/v).
From this series of compounds clear SAR can be derived for 4-
substitution of the phenyl ring, with electron-donating groups
more favourable for inhibitory activity. Both the methylenedioxy-
phenyl imidazole (17) and triazole (18) derivatives were potent
inhibitors with additional binding interactions between the meth-
ylenedioxy moiety and the active site likely to be the main factor.
The 6-bromo-3-pyridine imidazole 15 was the most active from
this series and has the additional potential benefit of improved
drug like properties, most notably the option to prepare as the
hydrochloride salt to enhance aqueous solubility. Introduction of
the pyridine ring does have limitations with replacement of the
imidazole ring with a weakly basic azole resulting in loss of activ-
ity. Extention and further exploration of the pyridine series is war-
ranted to develop SAR and optimise drug properties.
5.1.1.1.
dimethyl-propionic acid methyl ester (3b, R = 4-F).
pared from the reaction of 4-fluorophenylboronic acid and (2) in
65% yield as brown oil. TLC (2:1 Petroleum ether/EtOAc,
3-Hydroxy-3-[4-(4-fluoro-phenylamino)-phenyl]-2,2-
Pre-
a
Rf = 0.55). 1H NMR (CDCl3): d 1.11 (s, 3H, CH3), 1.17 (s, 3H, CH3),
3.17 (s, 1H, OH), 3.70 (s, 3H, COOCH3), 4.81 (s, 1H, CH-OH), 5.66
(s, 1H, NH), 6.88 (d, J = 7.0 Hz, 2H, Ar), 6.99 (d, J = 6.9 Hz, 2H, Ar),
7.04–7.08 (m, 2H, Ar), 7.15 (d, J = 7.2 Hz, 2H, Ar). 13C NMR (CDCl3):
d 19.14 (CH, CH3), 22.92 (CH, CH3), 47.90 (C, C-dimethyl), 52.06
(CH, CH3-ester), 78.46 (CH, CH-OH), 115.80 (CH, Ar), 115.98 (CH,
Ar), 120.57 (CH, Ar), 128.43 (CH, Ar), 131.94 (C, Ar), 138.49 (C,
Ar), 143.60 (C, Ar), 147.58 (C, Ar), 178.29 (C, COOCH3). EI-HRMS
(M+Na)+ found 340.1321, calculated for C18H20FNO3Na 340.1319.
5. Experimental
5.1. Materials and methods: chemistry
[11,12-3H] All trans-retinoic acid (37 MBq/mL) and Ultima Flo M
scintillation fluid were purchased from Perkin Elmer (UK). Acetic
acid and ammonium acetate were obtained from Fisher Scientific
(UK). All solvents used for chromatography were HPLC grade from
Fisher Scientific (UK).
5.1.1.2. 3-Hydroxy-3-[4-(4-methyl-phenylamino)-phenyl]-2,2-
dimethyl-propionic acid methyl ester (3d, R = 4-CH3).
Prepared
from the reaction of 4-methylphenylboronic acid and (2) in 74%
yield as a brown oil. TLC (2:1 Petroleum ether/EtOAc, Rf = 0.62).
1H NMR (CDCl3): d 1.09 (s, 3H, CH3), 1.18 (s, 3H, CH3), 2.32 (s,
3H, CH3-Ar), 3.13 (s, 1H, OH), 3.71 (s, 3H, COOCH3), 4.82 (s, 1H,
CH-OH), 5.69 (s, 1H, NH), 6.89 (d, J = 7.6 Hz, 2H, Ar), 7.04 (d,
J = 7.7 Hz, 2H, Ar), 7.15 (d, J = 7.1 Hz, 2H, Ar), 7.22 (d, J = 7.2 Hz,
2H, Ar). 13C NMR (CDCl3): d 19.12 (CH, CH3), 23.04 (CH, CH3),
26.54 (CH, CH3-Ar), 47.90 (C, C-dimethyl), 52.07 (CH, CH3-ester),
78.54 (CH, CH-OH), 115.92 (CH, Ar), 119.04 (CH, Ar), 125.12 (CH,
1H and 13C NMR spectra were recorded with a Bruker Avance
DPX500 spectrometer operating at 500 and 125 MHz, with Me4Si
as internal standard. Mass spectra were determined by the EPSRC
mass spectrometry centre (Swansea, UK). Flash column chroma-
tography was performed with silica gel 60 (230–400 mesh)
(Merck) and TLC was carried out on precoated silica plates (kiesel
gel 60 F254, BDH). Compounds were visualised by illumination un-
der UV light (254 nm) or by the use of vanillin stain followed by

M. S. Gomaa et al. / Bioorg. Med. Chem. 20 (2012) 6080–6088
6085
Ar), 128.61 (CH, Ar), 129.87 (C, Ar), 131.03 (C, Ar), 140.16 (C, Ar),
143.63 (C, Ar), 178.32 (C, COOCH3). EI-HRMS (M+H)+ found
314.1752, calculated for C19H24NO3 314.1751.
reddish brown oil. TLC (2:1 Petroleum ether/EtOAc, Rf = 0.53). 1H
NMR (CDCl3): d 1.08 (s, 3H, CH3), 1.14 (s, 3H, CH3), 2.25 (s, 6H,
CH3-Ar), 3.21 (s, 1H, OH), 3.70-3.74 (m, 6H, OCH3-COOCH3), 4.79
(s, 1H, CH-OH), 5.66 (s, 1H, NH), 6.72 (s, 2H, Ar), 6.91 (d,
J = 7.6 Hz, 2H, Ar), 7.15 (d, J = 7.8 Hz, 2H, Ar). 13C NMR (CDCl3):
d16.30 (CH, CH3-Ar), 16.50 (CH, CH3-Ar), 19.23 (CH, CH3), 22.91
(CH, CH3), 47.93 (C, C-dimethyl), 52.11 (CH, CH3-ester), 59.93
(CH, OCH3), 78.81 (CH, CH-OH), 116.27 (CH, Ar), 119.63 (CH, Ar),
126.80 (CH, Ar), 131.52 (C, Ar), 137.80 (C, Ar), 143.82 (C, Ar),
157.91 (C, Ar), 178.26 (C, COOCH3). EI-HRMS (M+H)+ found
358.2013, calculated for C21H28NO4 358.2013.
5.1.1.3.
phenyl]-2,2-dimethyl-propionic acid methyl ester (3e,
R = CF3). Prepared from the reaction of 4-trifluoromethylphen-
3-Hydroxy-3-[4-(4-trifluoromethyl-phenylamino)-
ylboronic acid and (2) in 71% yield as a brown oil. TLC (2:1 Petroleum
ether/EtOAc, Rf = 0.58). 1H NMR (CDCl3): d 1.06 (s, 3H, CH3), 1.18 (s,
3H, CH3), 3.16 (s, 1H, OH), 3.71 (s, 3H, COOCH3), 4.89 (s, 1H, CH-
OH), 6.04 (s, 1H, NH), 7.06 (d, J = 7.6 Hz, 2H, Ar), 7.14 (d, J = 7.8 Hz,
2H, Ar), 7.38 (d, J = 7.1 Hz, 2H, Ar), 7.51 (d, J = 7.0 Hz, 2H, Ar). 13C
NMR (CDCl3): d19.17 (CH, CH3), 22.95 (CH, CH3), 47.83 (C, C-di-
methyl), 52.19 (CH, CH3-ester), 78.38 (CH, CH-OH), 115.78 (CH, Ar),
119.31 (CH, Ar), 125.10 (CH, Ar), 127.84 (CH, Ar), 129.12 (C, Ar),
140.84 (C, Ar), 146.64 (C, Ar), 178.27 (C, COOCH3). EI-HRMS
(M+Na)+ found 390.1289, calculated for C19H20F3NO3Na 390.1287.
5.1.1.8. Methyl
hydroxy-2,2-dimethylpropanoate (13).
3-(4-(6-bromopyridin-3ylamino)phenyl)-3-
Prepared from the
reaction of 6-bromo-3-pyridinylboronic acid and (2) in 58% yield
as pale orange crystals. Mp 120–124 °C. TLC (2:1 Petroleum
ether/EtOAc, Rf = 0.55). 1H NMR (DMSO-d6): d 0.94 (s, 3H, CH3),
1.06 (s, 3H, CH3), 3.61 (s, 3H, OCH3), 4.77 (d, J = 4.4 Hz, 1H, CH-
OH), 5.43 (d, J = 4.5 Hz, 1H, CH-OH), 7.06 (d, J = 8.4 Hz, 2H, Ar),
7.20 (d, J = 8.4 Hz, 2H, Ar), 7.41 (s, 2H, Py), 8.11 (d, J = 0.9 Hz, 1H,
Py), 8.48 (s, 1H, NH). 13C NMR (DMSO-d6): d19.54 (CH3, C-4),
21.38 (CH3, C-5), 47.80 (C-dimethyl, C-3), 51.45 (OCH3, C-1),
76.38 (CH(OH), C-6), 116.72 (2 ꢁ C, Ar), 125.42 (CH, Py), 127.82
(CH, Py), 128.96 (C, Py/Ar), 134.53 (C, Py/Ar), 138.24 (CH, Py),
140.21 (C, Py/Ar), 140.49 (C, Py/Ar), 176.60 (CO, C-2). EI-HRMS
(M+H)+ found 379.0654, calculated for C17H20BrN2O3 379.0657.
5.1.1.4.
3-Hydroxy-3-[4-(4-cyano-phenylamino)-phenyl]-2,2-
Pre-
dimethyl-propionic acid methyl ester (3g, R = 4-CN).
pared from the reaction of 4-cyanophenylboronic acid and (2) in
62% yield as a reddish brown oil. TLC (2:1 Petroleum ether/EtOAc,
Rf = 0.50). 1H NMR (CDCl3): d 1.07 (s, 3H, CH3), 1.17 (s, 3H, CH3),
3.22 (s, 1H, OH), 3.71 (s, 3H, COOCH3), 4.87 (s, 1H, CH-OH), 6.44 (s,
1H, NH), 6.95 (d, J = 7.6 Hz, 2H, Ar), 7.11 (d, J = 7.6 Hz, 2H, Ar), 7.26
(d, J = 7.5 Hz, 2H, Ar), 7.47(d, J = 7.6 Hz, 2H, Ar). 13C NMR (CDCl3): d
19.21 (CH, CH3), 22.87 (CH, CH3), 47.87 (C, C-dimethyl), 52.14 (CH,
CH3-ester), 78.25 (CH, CH-OH), 101.33 (C, C-CN), 114.75 (CH, Ar),
116.40 (C, CN), 117.49 (CH, Ar), 130.90 (CH, Ar), 135.37 (CH, Ar),
139.71 (C, Ar), 143.23 (C, Ar), 152.18 (C, Ar), 178.17 (C, COOCH3). EI-
HRMS (MꢀH)+ found 323.1396, calculated for C19H19N2O3 323.1396.
5.1.2. General method for addition of the N-heterocyclic ring to
prepare compounds 1, 6, 8–12, 15, 16, 18–20
To a solution of alcohol (3 or 5) (1.5 mmol) in anhydrous CH3CN
(20 mL) was added imidazole (4.5 mmol) and CDI (2.25 mmol) or
triazole (6 mmol) and CDT (3 mmol) or 2-methylimidazole
(4.5 mmol) and 1,10-carbonylbis(2-methylimidazole) (2.3 mmol).
The mixture was then heated under reflux for 24–48 h. The reac-
tion mixture was allowed to cool and then extracted with EtOAc
(150 mL) and H2O (3 ꢁ 100 mL). The organic layer was dried
(MgSO4) filtered and reduced in vacuo. The product was purified
by flash column chromatography.
5.1.1.5.
3-Hydroxy-3-[4-(4-nitro-phenylamino)-phenyl]-2,2-
Pre-
dimethyl-propionic acid methyl ester (3h, R = 4-NO2).
pared from the reaction of 4-nitrophenylboronic acid and (2) in
72% yield as a reddish brown oil. TLC (2:1 Petroleum ether/EtOAc,
Rf = 0.53). 1H NMR (CDCl3): d 1.19 (s, 3H, CH3), 1.20 (s, 3H, CH3),
3.15 (s, 1H, OH), 3.72 (s, 3H, COOCH3), 4.85 (s, 1H, CH-OH), 6.73
(s, 1H, NH), 6.93 (d, J = 7.8 Hz, 2H, Ar), 7.13 (d, J = 7.4 Hz, 2H, Ar),
7.28 (d, J = 7.1 Hz, 2H, Ar), 8.09 (d, J = 7.0 Hz, 2H, Ar). 13C NMR
(CDCl3): d 19.23 (CH, CH3), 22.87 (CH, CH3), 47.86 (C, C-dimethyl),
52.35 (CH, CH3-ester), 78.32 (CH, CH-OH), 113.77 (CH, Ar), 121.14
(CH, Ar), 126.20 (CH, Ar), 128.41 (CH, Ar), 136.04 (C, Ar), 139.23 (C,
Ar), 139.56 (C, Ar), 150.22 (C, Ar), 178.22 (C, COOCH3). EI-HRMS
(MꢀH)+ found 343.1291, calculated for C18H19N2O5 343.1299.
5.1.2.1. Methyl 3-(1H-imidazol-1-yl)-3-(4-((4-fluorophenyl)
amino)phenyl)-2,2-dimethylpropanoate (4).
Prepared by the
reaction of 3b with CDI and imidazole. After 48 h reflux column
chromatography (EtOAc–MeOH 100:0 v/v increasing to 99:1 v/v)
gave this product in 44% yield as a yellow oil. TLC (99:1 EtOAc/
MeOH, Rf = 0.58). 1H NMR (CDCl3): d 1.23 (s, 3H, CH3), 1.25 (s,
3H, CH3), 3.63 (s, 3H, COOCH3), 5.49 (s, 1H, CH-imid), 5.96 (s, 1H,
NH), 6.88 (d, J = 7.8 Hz, 2H, Ar), 6.95–7.00 (m, 2H, Ar), 7.03–7.09
(m, 4H, Ar), 7.13 (d, J = 7.9 Hz, 2H, Ar), 7.62 (s, 1H, Ar). 13C NMR
(CDCl3): d 22.89 (CH3), 23.41 (CH3), 47.63 (C, C-dimethyl), 52.46
(CH3-ester), 67.52 (CH-imid), 115.54 (CH, Ar), 115.95 (CH, Ar),
116.13 (CH, Ar), 121.55 (CH, Ar), 127.54 (C, Ar), 129.65 (CH, Ar),
138.00 (C, Ar), 138.02 (C, Ar), 144.39 (C, Ar), 176.27 (C, COOCH3).
EI-HRMS (M)+ found 368.1768, calculated for C21H22N3O2F
368.1769.
5.1.1.6. 3-Hydroxy-3-[4-(3,4,5-trimethoxy-phenylamino)-phe-
nyl]-2,2-dimethyl-propionic acid methyl ester (3i, R = 3,4,
5-tri-OCH3).
Prepared from the reaction of 3,4,5-trimethoxy-
phenylboronic acid and (2) in 69% yield as a brown oil. TLC (2:1
Petroleum ether/EtOAc, Rf = 0.41). 1H NMR (CDCl3): d 1.07 (s, 3H,
CH3), 1.15 (s, 3H, CH3), 3.24 (s, 1H, OH), 3.59 (s, 3H, COOCH3),
3.72 (s, 9H, OCH3), 4.82 (s, 1H, CH-OH), 5.82 (s, 1H, NH), 6.37 (s,
2H, Ar), 6.95 (d, J = 7.5 Hz, 2H, Ar), 7.13 (d, J = 7.4 Hz, 2H, Ar). 13C
NMR (CDCl3): d19.16 (CH, CH3), 22.88 (CH, CH3), 47.88 (C, C-di-
methyl), 52.01 (CH, CH3-ester), 56.03 (CH, OCH3), 56.43 (CH,
OCH3), 60.98 (CH, OCH3), 78.40 (CH, CH-OH), 107.34 (CH, Ar),
116.39 (CH, Ar), 119.94 (CH, Ar), 128.92 (CH, Ar), 132.07 (C, Ar),
139.16 (C, Ar), 143.26 (C, Ar), 153.78 (C, Ar), 178.22 (C, COOCH3).
EI-HRMS (M+H)+ found 390.1912, calculated for C21H28NO6
390.1911.
5.1.2.2. Methyl 3-(1H-imidazol-1-yl)-3-(4-((4-chlorophenyl)
amino)phenyl)-2,2-dimethylpropanoate (5).
Prepared by the
reaction of 3c with CDI and imidazole. After 48 h reflux column
chromatography (EtOAc–MeOH 100:0 v/v increasing to 99:1 v/v)
gave this product in 44% yield as a yellow oil. TLC (99:1 EtOAc/
MeOH, Rf = 0.59). 1H NMR (CDCl3): d 1.23 (s, 3H, CH3), 1.25 (s,
3H, CH3), 3.63 (s, 3H, COOCH3), 5.48 (s, 1H, CH-imid), 5.93 (s, 1H,
NH), 6.95 (d, J = 7.9 Hz, 2H, Ar), 6.99 (d, J = 7.7 Hz, 2H, Ar), 7.04
(s, 1H, Ar), 7.07 (s, 1H, Ar), 7.17 (d, J = 7.8 Hz, 2H, Ar), 7.22 (d,
J = 7.8 Hz, 2H, Ar), 7.63 (s, 1H, Ar). 13C NMR (CDCl3): d 22.97
(CH3), 23.41 (CH3), 47.64 (C, C-dimethyl), 52.41 (CH3-ester),
5.1.1.7. 3-Hydroxy-3-[4-(3,5-dimethyl-4-methoxy-phenylami-
no)-phenyl]-2,2-dimethyl-propionic acid methyl ester (3j,
R = 3,5-di-OCH3, 4-CH3).
Prepared from the reaction of 3,5-di-
methyl-4-methoxyphenylboronic acid and (2) in 70% yield as a

6086
M. S. Gomaa et al. / Bioorg. Med. Chem. 20 (2012) 6080–6088
67.56 (CH-imid), 116.78 (CH, Ar), 119.78 (CH, Ar), 126.39 (C, Ar),
128.56 (C, Ar), 129.36 (CH, Ar), 129.66 (CH, Ar), 140.91 (C, Ar),
143.19 (C, Ar), 176.18 (C, COOCH3). EI-HRMS (M)+ found
384.1471, calculated for C21H22N3O2Cl 384.1470.
5.1.2.7.
Methyl
3-(1H-imidazol-1-yl)-3-(4-((4-nitro-
Pre-
phenyl)amino)phenyl)-2,2-dimethylpropanoate (10).
pared by the reaction of 3h with CDI and imidazole. After 48 h
reflux column chromatography (EtOAc–MeOH 100:0 v/v increasing
to 98:2 v/v) gave this product in 36% yield as a yellow oil. TLC (99:1
EtOAc/MeOH, Rf = 0.35). 1H NMR (CDCl3): d 1.32 (s, 6H, CH3), 3.63
(s, 3H, COOCH3), 5.54 (s, 1H, CH-imid), 6.54 (s, 1H, NH), 6.95 (d,
J = 7.9 Hz, 2H, Ar), 7.04 (s, 1H, Ar), 7.09 (s, 1H, Ar), 7.16 (d,
J = 7.9 Hz, 2H, Ar), 7.22 (d, J = 7.9 Hz, 2H, Ar), 7.70 (s, 1H, Ar), 8.12
(d, J = 7.7 Hz, 2H, Ar). 13C NMR (CDCl3): d 23.13 (CH3), 23.36
(CH3), 47.56 (C, C-dimethyl), 52.52 (CH3-ester), 67.51 (CH, CH-
imid), 114.30 (CH, Ar), 120.55 (CH, Ar), 126.16 (CH, Ar), 129.77
(CH, Ar), 131.78 (C, Ar), 140.13 (C, Ar), 140.24 (CH, Ar), 149.45 (C,
Ar), 176.00 (C, COOCH3). EI-HRMS (M)+ found 395.1709, calculated
for C21H22N4O4 395.1714.
5.1.2.3.
Methyl
3-(1H-imidazol-1-yl)-3-(4-((4-methyl-
(6). Pre-
phenyl)amino)phenyl)-2,2-dimethylpropanoate
pared by the reaction of 3d with CDI and imidazole. After 48 h
reflux column chromatography (EtOAc–MeOH 100:0 v/v increasing
to 99:1 v/v) gave this product in 56% yield as a yellow oil. TLC (99:1
EtOAc/MeOH, Rf = 0.70). 1H NMR (CDCl3): d 1.23 (s, 3H, CH3), 1.26
(s, 3H, CH3), 2.32 (s, 3H, CH3-Ar), 3.65 (s, 3H, COOCH3), 5.51 (s,
1H, CH-imid), 5.90 (s, 1H, NH), 6.87 (d, J = 7.8 Hz, 2H, Ar), 7.03 (d,
J = 7.9 Hz, 2H, Ar), 7.11–7.18 (m, 6H, Ar), 7.72 (s, 1H, Ar). 13C
NMR (CDCl3): d 20.78 (CH3-Ar), 22.80 (CH3), 23.59 (CH3), 47.61
(C, C-dimethyl), 52.50 (CH3-ester), 67.77 (CH-imid), 115.61 (CH,
Ar), 119.80 (CH, Ar), 116.92 (C, Ar), 129.62 (CH, Ar), 129.94 (CH,
Ar), 131.81 (C, Ar), 139.25 (C, Ar), 144.47 (C, Ar), 176.27 (C,
COOCH3). EI-HRMS (M)+ found 364.2022, calculated for
5.1.2.8. Methyl 3-(1H-imidazol-1-yl)-3-(4-((3,4,5-trimethoxy-
phenyl)amino)phenyl)-2,2-dimethylpropanoate (11).
Pre-
pared by the reaction of 3i with CDI and imidazole. After 48 h
reflux column chromatography (EtOAc–MeOH 100:0 v/v increasing
to 98:2 v/v) gave this product in 39% yield as a colourless oil. TLC
(99:1 EtOAc/MeOH, Rf = 0.51). 1H NMR (CDCl3): d 1.25 (s, 3H,
CH3), 1.27 (s, 3H, CH3), 3.64 (s, 3H, COOCH3), 3.81–3.86 (m, 9H,
OCH3), 5.52 (s, 1H, CH-imid), 5.84 (s, 1H, NH), 6.32 (s, 2H, Ar),
6.93 (d, J = 8.1 Hz, 2H, Ar), 7.02 (s, 1H, Ar), 7.08 (s, 1H, Ar), 7.13
(d, J = 8.0 Hz, 2H, Ar), 7.60 (s, 1H, Ar). 13C NMR (CDCl3): d 22.95
(CH3), 23.39 (CH3), 47.69 (C, C-dimethyl), 52.37 (CH3-ester),
56.13 (OCH3), 61.02 (OCH3), 67.56 (CH, CH-imid), 97.55 (CH, Ar),
116.02 (CH, Ar), 127.77 (C, Ar), 129.67 (CH, Ar), 133.56 (C, Ar),
138.14 (C, Ar), 144.19 (C, Ar), 153.87 (C, Ar), 176.21 (C, COOCH3).
EI-HRMS (M)+ found 440.2180, calculated for C24H29N3O5
440.2180.
C
22H25N3O2 364.2020.
5.1.2.4. Methyl 3-(1H-imidazol-1-yl)-3-(4-((4-trifluoromethyl-
phenyl)amino)phenyl)-2,2-dimethylpropanoate (7). Pre-
pared by the reaction of 3e with CDI and imidazole. After 48 h
reflux column chromatography (EtOAc–MeOH 100:0 v/v increasing
to 99:1 v/v) gave this product in 40% yield as a yellow oil. TLC (99:1
EtOAc/MeOH, Rf = 0.68). 1H NMR (CDCl3): d 1.27 (s, 6H, CH3), 3.62
(s, 3H, COOCH3), 5.55 (s, 1H, CH-imid), 6.48 (s, 1H, NH), 7.05 (d,
J = 7.9 Hz, 6H, Ar), 7.17 (d, J = 7.8 Hz, 2H, Ar), 7.44 (d, J = 7.8 Hz,
2H, Ar), 7.68 (s, 1H, Ar). 13C NMR (CDCl3): d 23.04 (CH3), 23.36
(CH3), 47.61 (C, C-dimethyl), 52.43 (CH3-ester), 67.56 (CH-imid),
116.15 (CH, Ar), 118.58 (CH, Ar), 125.71 (CH, Ar), 129.67 (CH, Ar),
129.97 (C, Ar), 141.78 (C, Ar), 145.94 (C, Ar), 176.12 (C, COOCH3).
EI-HRMS (M)+ found 418.1734, calculated for C22H22N3O2F3
418.1734.
5.1.2.9. Methyl 3-(1H-imidazol-1-yl)-3-(4-((3,5-dimethyl-4-
methoxyphenyl)amino)phenyl)-2,2-dimethylpropanoate
(12).
Prepared by the reaction of 3j with CDI and imidaz-
5.1.2.5.
Methyl
3-(1H-imidazol-1-yl)-3-(4-((4-methoxy-
(8). Pre-
ole. After 48 h reflux column chromatography (EtOAc–MeOH
100:0 v/v increasing to 99:1 v/v) gave this product in 42% yield
as a yellow oil. TLC (99:1 EtOAc/MeOH, Rf = 0.57). 1H NMR
(CDCl3): d 1.23 (s, 3H, CH3), 1.26 (s, 3H, CH3), 2.22 (s, 6H,
CH3-Ar), 3.61 (s, 3H, COOCH3), 3.71 (s, 3H, OCH3), 5.52 (s, 1H,
CH-imid), 5.71 (s, 1H, NH), 6.74 (s, 2H, Ar), 6.91 (d, J = 8.0 Hz,
2H, Ar), 7.02 (s, 1H, Ar), 7.10 (s, 1H, Ar), 7.13 (d, J = 8.0 Hz,
2H, Ar), 7.61 (s, 1H, Ar). 13C NMR (CDCl3): d 16.19 (CH, CH3-
Ar), 22.89 (CH3), 23.44 (CH3), 47.69 (C, C-dimethyl), 52.36 (CH,
CH3-ester), 59.88 (OCH3), 67.55 (CH, CH-imid), 115.56 (CH, Ar),
120.27 (CH, Ar), 127.22 (C, Ar), 129.57 (CH, Ar), 131.75 (C, Ar),
137.40 (C, Ar), 144.62 (C, Ar), 152.35 (C, Ar), 176.30 (C,
COOCH3). EI-HRMS (M)+ found 408.2275, calculated for
C24H29N3O3 408.2282.
phenyl)amino)phenyl)-2,2-dimethylpropanoate
pared by the reaction of 3f with CDI and imidazole. After 48 h
reflux column chromatography (EtOAc–MeOH 100:0 v/v increasing
to 99:1 v/v) gave this product in 53% yield as a yellow oil. TLC (99:1
EtOAc/MeOH, Rf = 0.55). 1H NMR (CDCl3): d 1.23 (s, 3H, CH3), 1.25
(s, 3H, CH3), 3.66 (s, 3H, COOCH3), 3.81 (s, 3H, OCH3), 5.43 (s, 1H,
CH-imid), 5.79 (s, 1H, NH), 6.83 (d, J = 7.8 Hz, 2H, Ar), 6.87 (d,
J = 7.8 Hz, 2H, Ar), 7.01–7.09 (m, 6H, Ar), 7.61 (s, 1H, Ar). 13C
NMR (CDCl3): d 22.83 (CH3), 23.29 (CH3), 47.68 (C, C-dimethyl),
52.35 (CH3-ester), 55.55 (OCH3), 67.61 (CH, CH-imid), 114.71
(CH, Ar), 123.03 (CH, Ar), 125.71 (CH, Ar), 126.64 (C, Ar), 127.68
(CH, Ar), 129.59 (CH, Ar), 134.82 (C, Ar), 145.57 (C, Ar), 155.76 (C,
Ar), 176.28 (C, COOCH3). EI-HRMS (M)+ found 380.1964, calculated
for C22H25N3O3 380.1960.
5.1.2.6.
Methyl
3-(1H-imidazol-1-yl)-3-(4-((4-cyano-
(9). Pre-
5.1.2.10. Methyl 3-(4-(6-bromopyridin-3-ylamino)phenyl)-3-
phenyl)amino)phenyl)-2,2-dimethylpropanoate
(1H-imidazol-1-yl)-2,2-dimethylpropanoate (15).
Prepared
pared by the reaction of 3g with CDI and imidazole. After 48 h
reflux column chromatography (EtOAc–MeOH 100:0 v/v increasing
to 99:1 v/v) gave this product in 40% yield as a pale yellow oil. TLC
(99:1 EtOAc/MeOH, Rf = 0.39). 1H NMR (CDCl3): d 1.31 (s, 6H, CH3),
3.65 (s, 3H, COOCH3), 5.54 (s, 1H, CH-imid), 6.54 (s, 1H, NH), 7.02
(d, J = 7.9 Hz, 6H, Ar), 7.08–7.14 (m, 4H, Ar), 7.22 (d, J = 7.8 Hz,
2H, Ar), 7.50 (d, J = 7.9 Hz, 2H, Ar), 7.69 (s, 1H, Ar). 13C NMR
(CDCl3): d 23.12 (CH3), 23.34 (CH3), 47.57 (C, C-dimethyl), 52.49
(CH3-ester), 67.49 (CH-imid), 102.34 (C, Ar), 115.60 (CH, Ar),
119.66 (C, Ar), 119.90 (CH, Ar), 129.74 (CH, Ar), 131.19 (C, Ar),
133.78 (CH, Ar), 140.59 (C, Ar), 147.15 (C, Ar), 176.03 (C, COOCH3).
EI-HRMS (M)+ found 375.1812, calculated for C22H22N4O2
375.1816.
by the reaction of 13 with CDI and imidazole. After 48 h reflux col-
umn chromatography (Petroleum ether–EtOAc 100:0 v/v increas-
ing to CH2Cl2–MeOH 90:10 v/v) gave this product in 30% yield as
a yellow oil. TLC (EtOAc, Rf = 0.15). 1H NMR (DMSO-d6): 1.20 (s,
6H, 2 ꢁ CH3), 3.54 (s, 3H, OCH3), 5.64 (s, 1H, CH-imid), 7.07 (d,
J = 8.3 Hz, 2H, Ar), 7.34 (d, J = 8.4 Hz, 2H, Ar), 7.59 (m, 4H, 2Ar/
2Py), 8.13 (d, J = 02.3 Hz, 1H, Py), 8.31 (s, 1H, Ar), 8.60 (s, 1H,
NH). 13C NMR (DMSO-d6): d 22.74 (CH3), 22.86 (CH3), 47.24 (C, C-
dimethyl), 51.93 (OCH3), 116.69 (2 ꢁ CH, Ar), 126.23 (CH, Py),
127.83 (CH, Py), 129.10 (C, Ar/Py), 129.67 (C, Ar/Py), 129.92
(2CH, Ar), 138.98 (CH, Py), 139.69 (C, Ar/Py), 141.71 (C, Ar/Py),
176.07 (COOCH3). EI-HRMS (M + H)+ found 429.0924, calculated
for C20H22BrN4O2 429.0921.

M. S. Gomaa et al. / Bioorg. Med. Chem. 20 (2012) 6080–6088
6087
5.1.2.11. Methyl 3-(4-(6-bromopyridin-3-ylamino)phenyl)-3-
(1H-1,2,4-triazol-1-yl)-2,2-dimethylpropanoate (16). Pre-
22.04 (CH3), 24.21 (CH3), 47.26 (C-dimethyl), 51.92 (CH3), 64.81
(CH), 100.73 (CH2), 101.32 (CH, Ar), 108.46 (CH, Ar), 111.47 (CH,
Ar), 114.44 (2 ꢁ CH, Ar), 117.85 (CH, imid), 126.26 (C, Ar), 126.50
(CH, imid), 129.63 (2 ꢁ CH, Ar), 137.13 (C, Ar), 141.60 (C, Ar),
144.49 (C, Ar), 144.65 (C, Ar), 147.69 (C, Ar), 175.44 (COOCH3).
Anal. Calcd for C23H25N3O4ꢂ0.5H2O (416.4758): C, 65.73, H, 6.39,
N, 10.10. Found: C, 66.33, H, 6.29, N, 10.09. EI-HRMS (M+H)+ found
408.1923, calculated for C23H26N3O4 408.1921.
pared by the reaction of 13 with CDT and triazole. After 48 h reflux
column chromatography (Petroleum ether–EtOAc 100:0 v/v
increasing to 100% CH2Cl2) gave this product in 54% yield as a yel-
low oil. TLC (EtOAc, Rf = 0.68). 1H NMR (DMSO-d6): 1.20 (s, 3H,
CH3), 1.24 (s, 3H, CH3), 3.55 (s, 3H, OCH3), 5.92 (s, 1H, CH-triazole),
7.08 (d, J = 8.7 Hz, 2H, Ar), 7.44 (m, 4H, 2Ar/2Py), 8.02 (s, 1H, tria-
zole), 8.13 (dd, J = 0.7 Hz, 2.8 Hz, 1H, Py), 8.59 (s, 1H, NH), 8.64 (s,
1H, triazole). 13C NMR (DMSO-d6): d 21.72 (CH3), 22.71 (CH3),
47.52 (C-dimethyl), 51.92 (CH), 67.17 (CH), 116.48 (2 ꢁ CH, Ar),
126.23 (CH, Py), 127.83 (CH, Py), 129.65 (C, Py/Ar), 130.25
(2 ꢁ CH, Ar), 138.97 (CH, Py), 139.71 (CH, Py/Ar), 141.82 (CH, Py/
Ar), 151.43 (2 ꢁ CH, triazole), 175.03 (CO, C-2). EI-HRMS (M+H)+
found 430.0876, calculated for C19H21BrN5O2 430.0873.
5.2. MCF-7 (CYP26A1) assay for inhibition of metabolism of
ATRA
MCF-7 cells were cultured at 37 °C in RPMI 1640 medium con-
taining foetal calf serum (10%) and L-glutamine (2 mM) in a humid-
ified atmosphere of 5% CO2 in air. ATRA was dissolved in dimethyl
sulfoxide and added to the culture medium as described by Arm-
strong et al.10 Liarozole, R116010 and imidazole derivatives were
dissolved in ethanol and diluted in cell culture medium. The final
concentration of ethanol in all experiments never exceeded 0.8%.
5.1.2.12. Methyl 3-(4-benzo[d][1,3]dioxol-5-ylamino)phenyl)-3-
(1H-1,2,4-triazol-1-yl)-2,2-dimethylpropanoate (18).
Pre-
pared by the reaction of 14 with CDT and triazole. After 48 h reflux
column chromatography (Petroleum ether–EtOAc 100:0 v/v
increasing to CH2Cl2–MeOH 95:5 v/v) gave this product in 51%
yield as a brown oil. TLC (CH2Cl2–MeOH 95:5 v/v, Rf = 0.55). 1H
NMR (DMSO-d6): 1.19 (s, 3H, CH3), 1.23 (s, 3H, CH3), 3.69 (s, 3H,
OCH3), 5.85 (s, 1H, CH-triazole), 5.96 (s, 2H, methylene CH2), 6.55
(d, J = 7.2 Hz, 2H, Ar), 6.69 (s, 1H, Ar), 6.81 (d, J = 7.8 Hz, 1H, Ar),
6.90 (d, J = 7.3 Hz, 2H, Ar), 7.33 (d, J = 7.3 Hz, 2H, Ar), 8.00 (s, 1H,
triazole), 8.03 (s, 1H, triazole), 8.62 (s, 1H, NH). 13C NMR (DMSO-
d6): d 21.63 (CH3), 22.76 (CH3), 47.56 (C-dimethyl), 51.88 (CH3),
67.38 (CH), 100.73 (CH2), 101.34 (CH, Ar), 108.46 (CH, Ar), 111.52
(CH, Ar), 114.20 (2 ꢁ CH, Ar), 125.28 (C, Ar), 130.06 (2 ꢁ CH, Ar),
137.11 (C, Ar), 141.61 (C, Ar), 144.71 (C, Ar), 147.68 (C, Ar),
151.33 (2 ꢁ CH, triaz), 175.13 (COOCH3). EI-HRMS (M+H)+ found
395.1708, calculated for C21H23N4O4 395.1714.
MCF-7 cells were pretreated for 24 h with 1 lM RA to induce
CYP26 expression. Microsomes were prepared as described by
Han and Choi.22 Briefly, cells were homogenised in Buffer A
(10 mM Tris, pH 7.4, 1 mM EDTA, 0.5 M sucrose, and Complete Pro-
tease Inhibitor Cocktail (Roche, UK)) using a Dounce homogeniser,
diluted with an equal volume of Tris/EDTA, and the diluted homog-
enate laid over a volume of Buffer A equal to the original volume.
Microsomes were then isolated by differential centrifugation
(9000g, 10 min, 4 °C; 100,000g, 60 min, 4 °C). The microsomal pel-
let was suspended in Buffer B (10 mM Tris, pH 7.4, 1 mM EDTA,
0.25 M sucrose, Complete Protease Inhibitor Cocktail) and stored
at ꢀ70 °C. Cytochrome c reductase activity was calculated at 5–
15 U cyt c/
kit (Sigma) according to the manufacturer’s instructions. For ATRA
metabolism, 50 g microsomal protein was incubated in Assay
buffer (50 mM Tris, pH 7.4, 150 mM KCl, 10 mM MgCl2, 0.02% w/
v BSA, 2 mM NADPH, 10 nM ATRA, 0.1
Ci 3H ATRA) in amber ep-
pendorfs in the absence or presence of CYP26 inhibitor (1–
1000 nM) in a final volume of 200 L for 1 h at 37 °C with shaking.
lg protein, using the cytochrome c reductase (NADPH)
l
5.1.2.13. Methyl 3-((4-phenylamino)phenyl)-3-(2-methyl-1H-
imidazol-1-yl)-2,2-dimethylpropanoate (19).
Prepared by
l
the reaction of 3a with 1,10-carbonylbis(2-methylimidazole) and
2-methylimidazole. After 24 h reflux column chromatography
(Petroleum ether–EtOAc 100:0 v/v increasing to CH2Cl2–MeOH
95:5 v/v) gave this product in 73% yield as a light brown crystalline
solid. Mp 66–70 °C. TLC (CH2Cl2–MeOH 9:1 v/v, Rf = 0.50). 1H NMR
(DMSO-d6): 1.25 (s, 6H, C(CH3)2), 2.30 (s, 3H, 2-CH3-imid), 3.52 (s,
3H, OCH3), 5.43 (s, 1H, CH), 6.78 (s, 1H, Ar), 6.84 (s, 1H, Ar), 7.05 (m,
4H, Ar), 7.23 (s, 4H, Ar), 7.45 (s, 1H, imid), 8.25 (s, 1H, NH). 13C NMR
(DMSO-d6): d 13.35 (CH3, 2-CH3-imid), 22.07 (CH3), 24.22 (CH3),
39.52 (C-dimethyl), 51.93 (CH3), 64.80 (CH), 115.67 (CH, Ar),
117.22 (CH, Ar), 117.48 (CH, Ar), 120.07 (CH, Ar), 126.60 (CH, imid),
127.19 (2 ꢁ C, Ar), 129.13 (CH, Ar), 129.62 (CH, Ar), 142.85 (C, Ar),
l
The reaction was quenched with acetonitrile, mixed, then centri-
fuged (18,000g, 5 min, 4 °C). Resolution of retinoids was performed
with a Luna C18(2) column (3
l
m, 50 mm ꢁ 2 mm) using a Waters
2690 Separations Module and subsequent Radiomatic Series 500TR
Flow Scintillation Analyzer (Packard Biosciences), with Empower 2
Chromatography Data Software and Flow-ONE software, respec-
tively for data acquisition. 3H ATRA and 3H metabolites were sep-
arated by gradient reversed-phase chromatography, using mobile
phase A (50% acetonitrile, 50% (0.2%) acetic acid, w/w) and mobile
phase B (acetonitrile, 0.1% acetic acid, w/w). A flow rate of 0.3 mL/
min was used with linear gradients employed between the speci-
fied times as follows: 0, 100% A; 5 min, 100% A; 5.5 min, 40% A,
60% B; 12 min, 40% A, 60% B; 12.5 min, 20% A, 80% B; 17.5 min,
20% A, 80% B; 18 min, 100%A; 25 min, 100% A. Scintillant flow rate
was 1 mL/min. CYP26 inhibition was calculated as the percentage
3H ATRA metabolite peak area formation (activity 3H metabo-
lite(s)/total activity) compared to metabolite formation in the ab-
sence of inhibitor. IC50 values were calculated by non-linear
regression analysis in SigmaPlot (Systat Software Inc., USA) using
an inhibition curve constructed from a minimum of four data
points.
143.24
(C,
Ar),
175.43
(COOCH3).
Anal.
Calcd
for
C22H25N3O2ꢂ0.7H2O (376.06904): C, 70.26, H, 7.08, N, 11.17. Found:
C, 70.57, H, 6.97, N, 10.56. EI-HRMS (M+H)+ found 364.2025, calcu-
lated for C22H26N3O2 364.2024.
5.1.2.14. Methyl 3-(4-benzo[d][1,3]dioxol-5-ylamino)phenyl)-3-
(2-methyl-1H-imidazol-1-yl)-2,2-dimethylpropanoate
(20).
Prepared by the reaction of 14 with 1,10-carbonylbis(2-
methylimidazole) and 2-methylimidazole. After 48 h reflux
column chromatography (Petroleum ether–EtOAc 100:0 v/v
increasing to CH2Cl2–MeOH 95:5 v/v) gave this product in 47%
yield as
a light brown crystalline solid. Mp 62–64 °C. TLC
(CH2Cl2–MeOH 95:5 v/v, Rf = 0.48). 1H NMR (DMSO-d6): 1.24 (s,
6H, C(CH3)2), 2.28 (s, 3H, 2-CH3-imid), 3.52 (s, 3H, OCH3), 5.40 (s,
1H, CH), 5.96 (s, 2H, methylene CH2), 6.55 (d, J = 7.6 Hz, 1H, Ar),
6.69 (s, 1H, Ar), 6.77 (s, 1H, imid), 6.81 (d, J = 8.1 Hz, 1H, Ar), 6.89
(d, J = 7.8 Hz, 2H, Ar), 7.19 (d, J = 7.7 Hz, 2H, Ar), 7.44 (s, 1H, imid),
8.00 (s, 1H, NH). 13C NMR (DMSO-d6): d 13.32 (CH3, 2-CH3-imid),
5.3. Molecular modeling
All molecular modeling studies were performed on a MacPro
dual 2.66 GHz Xeon running Ubuntu. Ligand structures were built
in MOE27 minimised using the MMFF94x forcefield until a RMSD
gradient of 0.05 kcal molꢀ1 Åꢀ1 was reached. Docking simulations

6088
M. S. Gomaa et al. / Bioorg. Med. Chem. 20 (2012) 6080–6088
were performed using PLANTS28 (aco_ants 20; aco_evap 0.15;
aco_sigma 5.0), with ligands docked within the active site of the
CYP26A1 homology model26 and the results were visualised in
MOE.
12. Njar, V. C.; Gediya, L.; Purushottamachar, P.; Chopra, P.; Vasaitis, T. S.;
Khandelwal, A.; Mehta, J.; Huynh, C.; Belosay, A.; Patel, J. Bioorg. Med. Chem.
2006, 14, 4323.
13. van Steensel, M. A. M. Expert Opin. Emerg. Drugs 2007, 12, 647–656.
14. Gomaa, M. S.; Armstrong, J. L.; Bobillon, B.; Veal, G. J.; Brancale, A.; Redfern, C.
P. F.; Simons, C. Bioorg. Med. Chem. 2008, 16, 8301.
15. Gomaa, M. S.; Bridgens, C. E.; Aboraia, A. S.; Veal, G. J.; Redfern, C. P. F.;
Brancale, A.; Armstrong, J. L.; Simons, C. J. Med. Chem. 2011, 54, 2778.
16. Gomaa, M. S.; Bridgens, C. E.; Veal, G. J.; Redfern, C. P. F.; Brancale, A.;
Armstrong, J. L.; Simons, C. J. Med. Chem. 2011, 54, 6803.
17. Gomaa, M. S.; Bridgens, C. E.; Illingworth, N. A.; Veal, G. J.; Redfern, C. P. F.;
Brancale, A.; Armstrong, J. L.; Simons, C. Bioorg. Med. Chem. 2012, 20, 4201.
18. Chan, D. M. T.; Monaco, K. L.; Wang, R.; Winters, M. P. Tetrahedron Lett. 1998,
39, 2933.
19. Lam, P. Y. S.; Clark, C. G.; Saubern, S.; Adams, J.; Winters, M. P.; Chan, D. M. T.;
Combs, A. Tetrahedron Lett. 1998, 39, 2941.
20. Tang, C.; Chiba, M.; Nishime, J.; Hochman, J. H.; Chen, I.; Williams, T. M.; Lin, J.
H. Drug Metab. Dispos. 2000, 28, 680.
Acknowledgments
We acknowledge Cancer Research UK (M.S.G. and C.E.B., Grant
Ref. C7735/A9612) and the Nuffield Foundation (A.S.T.L.) for fund-
ing and the EPSRC Mass Spectrometry Centre, Swansea, UK for
mass spectroscopy data.
References and notes
21. Chiba, M.; Tang, C.; Neway, W. E.; Williams, T. M.; Desholms, S. J.; Dinsmore, C.
J.; Wai, J. S.; Lin, J. H. Biochem. Pharmacol. 2001, 62, 773.
22. Han, I. S.; Choi, J. H. J. Clin. Endocrinol. Metab. 1996, 81, 2069.
23. Verfaille, C. J.; Vanhoutte, F. P.; Blanchet-Bardon, C.; van Steensel, M. A.;
Steijlen, P. M. Br. J. Dermatol. 2007, 156, 965.
1. Clagett-Dame, M.; Plum, L. A. Crit. Rev. Eukaryot. Gene Expr. 1997, 7, 299.
2. Napoli, J. L. Biochim. Biophys. Acta 1821, 2012, 152.
3. Linney, E.; Donerly, S.; Mackey, L.; Dobbs-McAuliffe, B. Neurotoxicol. Teratol.
2011, 33, 631.
4. Rhinn, M.; Dolle, P. Development 2012, 139, 843.
24. Bryson, H. M.; Wagstaff, A. J. Drugs Aging 1996, 9, 478484. discussion 485.
25. Van Heusden, J.; Van Ginckel, R.; Bruwiere, H.; Moelans, P.; Janssen, B.; Floren,
W.; van der Leede, B. J.; van Dun, J.; Sanz, G.; Venet, M.; Dillen, L.; Van Hove, C.;
Willemsens, G.; Janicot, M.; Wouters, W. Br. J. Cancer 2002, 86, 605.
26. Gomaa, M. S.; Yee, S. W.; Milbourne, C. E.; Barbera, M. C.; Simons, C.; Brancale,
A. J. Enzyme Inhib. Med. Chem. 2006, 21, 361.
27. Molecular Operating Environment (MOE). Chemical Computing Group, Inc.
Montreal, Quebec, Canada. http://www.chemcomp.com.
28. Korb, O.; Stützle, T.; Exner, T. E. Lect. Notes Comput. Sci. 2006, 4150, 247.
5. Armstrong, J. L.; Redfern, C. P. F.; Veal, G. J. Biochem. Pharmacol. 2005, 69, 1299.
6. Brtko, J.; Thalhamer, J. Curr. Pharm. Design. 2003, 9, 2067.
7. Benbrook, D. M. Mini. Rev. Med. Chem. 2002, 2, 277.
8. Alvarez, S.; Bourguet, W.; Gronemeyer, H.; de Lera, A. R. Exp. Opin. Ther. Pat.
2011, 21, 55.
9. Muindi, J. R. F.; Frankel, S. R.; Huselton, C.; DeGrazia, F.; Garland, W. A.; Young,
C. W.; Warrell, R. P., Jr. Cancer Res. 1992, 52, 2138.
10. Armstrong, J. L.; Taylor, G. A.; Thomas, H. D.; Boddy, A. V.; Redfern, C. P. F.; Veal,
G. J. Brit. J. Cancer 2007, 96, 1675.
11. Ross, A. C.; Zolfaghari, R. Ann. Rev. Nutr. 2011, 31, 65.