Novel retinoic acid 4-hydroxylase (CYP26) inhibitors based on a 3-(1H-imidazol- and triazol-1-yl)-2,2-dimethyl-3-(4-(phenylamino)phenyl)propyl scaffold

Article abstract of DOI:10.1016/j.bmc.2012.05.076

Retinoic acid (RA), the biologically active metabolite of vitamin A, is used medicinally for the treatment of hyperproliferative diseases including dermatological conditions and cancer. The antiproliferative effects of RA have been well documented as well as the limitations owing to toxicity and the development of resistance to RA therapy. RA metabolism inhibitors (RAMBAs or CYP26 inhibitors) are attracting increasing interest as an alternative method for enhancing endogenous levels of retinoic acid in the treatment of hyperproliferative disease. Here the synthesis and inhibitory activity of novel 3-(1H-imidazol- and triazol-1-yl)-2,2-dimethyl-3-(4-(phenylamino)phenyl)propyl derivatives in a MCF-7 CYP26A1 microsomal assay are described. The most promising inhibitor methyl 2,2-dimethyl-3-(4-(phenylamino)phenyl)-3-(1H-1,2,4- triazol-1-yl)propanoate (6) exhibited an IC₅₀ of 13 nM (compared with standards Liarozole IC₅₀ 540 nM and R116010 IC₅₀ 10 nM) and was further evaluated for CYP selectivity using a panel of CYP with >100-fold selectivity for CYP26 compared with CYP1A2, 2C9 and 2D6 observed and 15-fold selectivity compared with CYP3A4. The results demonstrate the potential for further development of these potent inhibitors.

Full text of DOI:10.1016/j.bmc.2012.05.076

Bioorganic & Medicinal Chemistry 20 (2012) 4201–4207
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel retinoic acid 4-hydroxylase (CYP26) inhibitors based
on a 3-(1H-imidazol- and triazol-1-yl)-2,2-dimethyl-
3-(4-(phenylamino)phenyl)propyl scaffold
Mohamed S. Gomaa a, , Caroline E. Bridgens b, Nicola A. Illingworth b, Gareth J. Veal b,
a,
Christopher P. F. Redfern b, Andrea Brancale a, Jane L. Armstrong c, , Claire Simons
⇑
⇑
a Medicinal Chemistry, 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:
Retinoic acid (RA), the biologically active metabolite of vitamin A, is used medicinally for the treatment of
hyperproliferative diseases including dermatological conditions and cancer. The antiproliferative effects
of RA have been well documented as well as the limitations owing to toxicity and the development of
resistance to RA therapy. RA metabolism inhibitors (RAMBAs or CYP26 inhibitors) are attracting increas-
ing interest as an alternative method for enhancing endogenous levels of retinoic acid in the treatment
of hyperproliferative disease. Here the synthesis and inhibitory activity of novel 3-(1H-imidazol- and
triazol-1-yl)-2,2-dimethyl-3-(4-(phenylamino)phenyl)propyl derivatives in a MCF-7 CYP26A1 micro-
somal assay are described. The most promising inhibitor methyl 2,2-dimethyl-3-(4-(phenylami-
no)phenyl)-3-(1H-1,2,4-triazol-1-yl)propanoate (6) exhibited an IC50 of 13 nM (compared with
standards Liarozole IC50 540 nM and R116010 IC50 10 nM) and was further evaluated for CYP selectivity
using a panel of CYP with >100-fold selectivity for CYP26 compared with CYP1A2, 2C9 and 2D6 observed
and 15-fold selectivity compared with CYP3A4. The results demonstrate the potential for further devel-
opment of these potent inhibitors.
Received 13 March 2012
Revised 24 May 2012
Accepted 30 May 2012
Available online 7 June 2012
Keywords:
CYP26A1
3-(1H-Imidazol- and triazol-1-yl)-2,2-
dimethyl-3-(4-(phenylamino)phenyl)propyl
derivatives
IC50 enzyme inhibition
MCF-7
CYP selectivity
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
alteration in gene expression, RA inhibits keratinocytes differenti-
ation and alters lipid production.9 Furthermore, RA induces the
Retinoic acid (RA) is one of several biologically active forms of
vitamin A1 and is the natural substrate to the three known retinoic
apoptosis of defective cells leading to the alleviation of symptoms.2
RA also has an important role in the treatment of several types of
cancer, including acute promyelocytic leukaemia (APL)10,11 and
neuroblastoma.12 In a high-risk neuroblastoma setting, the intro-
duction of 13-cis retinoic acid has led to a marked increase in
3 year event-free survival (EFS) rates when utilised after intensive
chemotherapy and myeloablative therapy.12
Unfortunately, retinoid use has limitations; due to the pleiotro-
pic actions of RA high pharmacological concentrations can lead to
serious side effects, such as multi-organ failure and hyperleukocy-
tosis.13,14 Additionally, retinoids cause auto-induction of their own
metabolism, through negative feedback mechanisms, leading to re-
duced plasma concentrations and a subsequent failure to respond
to therapy.1,15 These problems can limit the use of retinoids and
prevent their application in new areas.
acid receptors RARa . These receptors are ligand-
, RARb and RARc 2
dependent transcription factors which regulate gene expression as
a result of binding to retinoic acid response elements (RAREs)
within the DNA of retinoic acid-regulated genes. Once stimulated,
retinoic acid receptors induce the production of proteins that play
roles in the regulation of cell apoptosis, differentiation and prolif-
eration.3 Therefore, alteration of intracellular RA concentration
can influence signalling in these pathways and have important
consequences for cell function and survival.4
RA and other retinoids have long had a variety of clinical appli-
cations, including their utilisation for the treatment of hyperkera-
tinisation disorders such as acne and psoriasis.5–8 As a result of
RA metabolism inhibitors (RAMBAs) are attracting increasing
interest as an alternative method for enhancing endogenous levels
of retinoic acid in the treatment of hyperproliferative diseases with
notable success in the treatment of dermatological diseases.3,16
Two RAMBAs are currently licenced; liarozole17,18 for the treat-
ment of ichthyosis and talarozole (R115866) for psoriasis and acne
⇑
Corresponding authors. Tel.: +44 (0) 2920 876307; fax: +44 (0) 2920 874149
(C.S.); tel.: +44 (0) 191 222 5644; fax: +44 (0) 191 222 7179 (J.L.A.).
E-mail addresses: j.l.armstrong@newcastle.ac.uk (J.L. Armstrong), SimonsC@
Cardiff.ac.uk (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.2012.05.076

4202
M. S. Gomaa et al. / Bioorg. Med. Chem. 20 (2012) 4201–4207
Figure 1. Retinoic acid metabolism blocking agents (RAMBAs).
(Fig. 1).19–21 Several promising RAMBAs have emerged including
N-(4-((1R,2R)-2-(dimethylamino)-1-(1H-triazol-1-yl)propyl)phenyl)-
benzo[d]thiazol-2-amine (R116010) (Fig. 1),22 a potent and selec-
tive inhibitor of RA metabolism, which inhibits all-trans-RA (ATRA)
metabolism in neuroblastoma both in vitro and in vivo.23 We have
previously described potent naphthalene derivatives which display
low to sub nM activity (Fig. 1).24,25 Here we report studies on the
comparative phenyl derivatives based on the lead phenyl CYP26
inhibitor (1) (Fig. 1), exploring the effect of modifying the hetero-
cycle, dimethyl propylester chain and substitution of the phenyl
ring to determine optimal structure for CYP26 inhibition.
belled [11,12-3H] all-trans retinoic acid as the substrate. Liarozole
(a non-selective CYP26 inhibitor17,18) and R11601022 were in-
cluded in all experiments as comparative standards.
The triazole methyl ester (6) showed inhibitory activity (IC50
13 nM) comparable with the imidazole methyl ester lead (1, IC50
10 nM) (Table 1), however a reduction was noted for the 4-carb-
oxymethylphenyl derivative (7, IC50 = 95 nM).
reduction in activity was observed for the imidazole carboxylic
acid derivative (8, IC50 >1 M) compared with the lead ester (1).
A considerable
l
In contrast, inhibitory activity for the dicarboxylic acid triazole
derivative (9, IC50 = 85 nM) was retained compared with the parent
diester (7). A reduced activity was observed for both the methyl
amide imidazole (10, IC50 = 200 nM) and ethyl amide imidazole
(11, IC50 = 250 nM) derivatives compared with the ester lead (1),
with introduction of either methoxy or chloro groups at the 4-po-
sition of the phenyl ring resulting in a substantial loss in activity
2. Chemistry
The N-arylation of the amine (2)24 using the Suzuki reaction,
followed described methodology26,27 employing a stoichiometric
amount of copper and a tertiary amine base, pyridine in the reac-
tion series. Using this method, reaction with the appropriate aryl
boronic acid gave the N-aryl products (3) in good yields. The cou-
pled products were confirmed by the presence of an NH singlet
peak at dH 5.7–5.9 in 1H NMR (Scheme 1). The preparation of the
acids (4, 8 and 9) was performed through saponification of the es-
ters (3, 1 and 7 respectively) with LiOH in THF and H2O.28,29 The
acid (4) was obtained in good yield after acidification (pH 3) and
extraction with diethyl ether and used without further purification
(Scheme 1).
The amide derivatives (5) were prepared through the reaction
of the acid (4) with an amine in the presence of EDC and HOBt in
dichloromethane,28,30 and obtained in good yield after purification
by flash column chromatography. Introduction of the N-heterocy-
cle involved reaction of the alcohol precursor (3 or 5) with either
1,10-carbonyldiimidazole (CDI) and imidazole or 1,10-carbonyldi-
triazole (CDT) and triazole in acetonitrile24 to give the imidazole
and triazole derivatives (1, 6–14) (Scheme 1).
(4-methoxy 12, IC50 >1
lM; 4-chloro 13, IC50 = 1
lM). The triazole
methyl amide (14, IC50 = 1
lM) showed a greater reduction in
activity compared with the imidazole methyl amide (10, IC50
200 = nM).
3.2. CYP selectivity
The triazole ester (6) and R116010 were evaluated for their
inhibitory activity against a panel of P450 isoforms expressed in
human liver microsomes (Table 2). The triazole ester (6) was bor-
derline against CYP3A4 and inactive against the other four CYP en-
zymes in the panel. This compared favourably with the standard
R116010, which showed high activity for CYP3A4 (Table 2).
To determine the selectivity of the triazole methyl ester (6) in
comparison with R116010, IC50 assays against P450 isoforms ex-
pressed in human liver microsomes were performed. Micromolar
concentrations of compounds 6 or R116010 were needed to inhibit
CYPs 1A2, 2C9, 2C19and 2D6. However, both compounds inhibited
CYP3A4 with sub-micromolar activity (triazole 6 IC50 0.2 lM com-
pared with R116010 IC50 0.35 lM) (Table 3).
3. Biology
4. Discussion
3.1. CYP26A1 inhibitory activity
Evaluation of CYP26 inhibitory activity revealed that the tria-
zole methyl ester (6, IC50 = 13 nM) displayed comparable activity
with the lead imidazole methyl ester (1, IC50 = 10 nM), both of
The imidazole and triazole derivatives were evaluated for their
retinoic acid metabolism (CYP26) inhibitory activity using a cell-
free microsomal assay as previously described,31,32 with radiola-

M. S. Gomaa et al. / Bioorg. Med. Chem. 20 (2012) 4201–4207
4203
Scheme 1. Reagents and conditions: (i) Aryl boronic acid, CuOAc, pyridine, 4 Å molecular sieves, CH2Cl2, rt, 2 days; (ii) LiOH, THF, H2O, 2 h, reflux then aqueous 1 N HCl; (iii)
EDC, HOBt, CH2Cl2, 0.5 h, rt then R0NH2, rt, overnight; (iv) 1,10-carbonyldiimidazole, imidazole, CH3CN, reflux or 1,10-carbonylditriazole, triazole, CH3CN, reflux, 24–48 h.
Table 1
IC50 values for different R substituents
Compd
R
R0
X
CYP26 IC50 (l
M)a
1
6
7
8
9
10
11
12
13
14
Liarozole
R116010
H
H
—
—
—
—
CH
N
N
CH
N
CH
CH
CH
CH
N
0.01
0.013
0.095
>1.0
0.085
0.20
0.25
>1.0
1.0
COCH3
—
CO2H
H
H
OCH3
Cl
H
—
—
—
CH3
CH2CH3
CH3
CH3
CH3
—
1.0
0.54
0.01
—
—
—
a
IC50 values derived from the best fit of a 4 point dose–response curve.
which exhibited equal potency to the standard R116010 (IC50
=
Although the amides (10 and 11) were less active than the lead
compound (1) they were more active than Liarozole
(IC50 = 540 nM). This reduction in activity suggests that a H-bond
donor is less favourable for CYP26 inhibition and this finding is
10 nM). This contrasted with the comparable naphthalene imidaz-
ole and triazole inhibitors ( Fig. 1) where an enhancement in
activity was observed on going from the imidazole (IC50 = 3 nM)
to the triazole derivative (IC50 = 0.3 nM). Replacement of the
magnified for the triazole methyl amide (14, IC50 = 1 lM), with a
methyl ester group with a methyl amide isostere (10, IC50
=
substantial reduction in inhibitory activity. To explore the effect
200 nM) resulted in reduced activity for the imidazole derivative.
of substitution at the 4-position of the phenyl ring, compounds

4204
M. S. Gomaa et al. / Bioorg. Med. Chem. 20 (2012) 4201–4207
Table 2
Percent inhibition data against CYP panel at 0.4
l
M
Compound
6
R116010
1A2
10
6
3A4
47
83
2C9
19
3
2C19
19
17
2D6
13
27
Inactive
Borderline
Highly active
prior to use and stored over 4Å molecular sieves, under nitrogen.
All compounds were more than 95% pure.
Table 3
CYP IC50 (lM) profile of triazole 6 compared with R116010
Compd
1A2
2C9
2C19
3A4
2D6
26A1
5.1.1. General procedure for the Suzuki coupling preparation of
compounds 3a–3d
6
8.5
8.3
9.1
11.0
4.2
5.9
0.20
0.35
1.3
3.9
0.013
0.01
R116010
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 atmo-
sphere of air was added CH2Cl2 (15 mL) and the reaction stirred
under air atmosphere at ambient temperature for 2 days. The prod-
uct was isolated by direct flash column chromatography of the
crude reaction mixture (petroleum ether/EtOAc 70:30 v/v).
with an electron donating group (methoxy 12) and electron with-
drawing group (chloro 13) were prepared. The resulting enzyme
inhibition data (Table 1) showed that neither substituent was tol-
erated, with a marked loss in activity observed. The carboxylic acid
derivative (8) showed a loss in activity (8, IC50 >1 lM) compared
with the ester lead inhibitor (1), potentially related to reduced up-
take. However, the dicarboxylic acid triazole derivative (9) retained
activity compared to the parent ester (7), suggesting that binding
interaction rather than uptake is likely to account for the differ-
ences observed.
The triazole ester (6) was chosen for CYP selectivity as previous
research24 has shown improved CYP selectivity for triazole com-
pared with imidazole derivatives. Direct comparison of CYP selec-
tivity profiles for compound 6 and R116010 revealed a similar
profile, with decreased activity against CYPs 1A2, 2C9, 2C19 and
2D6 as compared with CYP26. However, both compounds exhib-
ited activity against the known RA hydroxylase CYP3A4, with
5.1.1.1. 3-Hydroxy-2,2-dimethyl-3-(4-phenylaminophenyl)pro-
pionic acid methyl ester (3a, R = H).
Prepared from the reac-
tion of phenylboronic acid and (2) in 79% yield as a light brown oil.
TLC (2:1 Petroleum ether/EtOAc, Rf = 0.53). 1H NMR (CDCl3): d 1.17
(s, 3H, CH3), 1.22 (s, 3H, CH3), 3.21 (s, 1H, OH), 3.75 (s, 3H,
COOCH3), 4.86 (s, 1H, CH-OH), 5.91 (s, 1H, NH), 6.96 (t, J = 7.2 Hz,
1H, Ar), 7.03 (d, J = 7.8 Hz, 2H, Ar), 7.09 (d, J = 7.9 Hz, 2H, Ar),
7.21 (d, J = 7.8 Hz, 2H, Ar), 7.29 (t, J = 7.4 Hz, 2H, Ar). 13C NMR
(CDCl3): d 19.19 (CH3), 22.95 (CH3), 47.96 (C, C-dimethyl), 52.08
(CH3-ester), 78.48 (CH, CH-OH), 116.56 (CH, Ar), 117.95 (CH, Ar),
121.04 (CH, Ar), 128.84 (CH, Ar), 129.56 (CH, Ar), 132.39 (C, Ar),
143.06 (C, Ar), 143.25 (C, Ar), 178.29 (C, COOCH3). HRMS (EI) m/z
Calcd for C18H21NO3 (M)+ 299.1516. Found 299.1521.
IC50 values of 0.2 lM for compound 6 and 0.35 lM for R116010.
CYP3A4 liability would discount the development of these inhibi-
tors for systemic use. However, as seen with Liarozole and
R116010, this is less likely to be an issue for topical administration
in combination with RA, suggesting that compound 6 represents an
appropriate candidate for further evaluation and development for
use in the treatment of dermatological diseases.
5.1.1.2. 3-Hydroxy-3-[4-(4-methoxy-phenylamino)phenyl]-2,2-
dimethyl-propionic acid methyl ester (3b, R = OCH3).
Pre-
pared from the reaction of 4-methoxyphenylboronic acid and (2)
in 81% yield as a light brown oil. TLC (2:1 Petroleum ether/EtOAc,
Rf = 0.45). 1H NMR (CDCl3): d 1.13 (s, 3H, CH3), 1.22 (s, 3H, CH3),
3.25 (s, 1H, OH), 3.75 (s, 3H, COOCH3), 3.81 (s, 3H, OCH3), 4.79 (s,
1H, CH-OH), 5.88 (s, 1H, NH), 6.87 (m, 4H, Ar), 7.05 (d, J = 7.8 Hz,
2H, Ar), 7.11 (d, J = 8.0 Hz, 2H, Ar). 13C NMR (CDCl3): d 19.13
(CH3), 22.87 (CH3), 47.97 (C, C-dimethyl), 52.00 (CH3-ester),
55.54 (CH3, OCH3), 78.62 (CH, CH-OH), 114.44 (CH, Ar), 117.87
(CH, Ar), 122.40 (CH, Ar), 128.33 (CH, Ar), 131.00 (C, Ar), 144.78
(C, Ar), 155.17 (C, Ar), 178.26 (C, COOCH3). HRMS (EI) m/z Calcd
for C19H24NO4 (M+H)+ 330.1700. Found 330.1700.
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). Liarozole was kindly donated by Janssen
(High Wycombe, UK) and R116010 was kindly donated by Barrier
Therapeutics Inc. (Princeton, NJ, USA)
5.1.1.3.
3-Hydroxy-3-[4-(4-chloro-phenylamino)phenyl]-2,2-
Prepared
dimethyl-propionic acid methyl ester (3c, R = Cl).
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
charring on a hotplate. Melting points were determined on an elec-
trothermal instrument and are uncorrected. All solvents were dried
from the reaction of 4-chlorophenylboronic acid and (2) in 70%
yield as a brown oil. TLC (2:1 Petroleum ether/EtOAc, Rf = 0.58).
1H NMR (CDCl3): d 1.09 (s, 3H, CH3), 1.16 (s, 3H, CH3), 3.22 (s,
1H, OH), 3.72 (s, 3H, COOCH3), 4.83 (s, 1H, CH-OH), 5.71 (s, 1H,
NH), 6.97 (d, J = 7.4 Hz, 4H, Ar), 7.23 (d, J = 7.3 Hz, 4H, Ar). 13C
NMR (CDCl3): d 19.42 (CH3), 22.90 (CH3), 47.89 (C, C-dimethyl),
52.08 (CH3-ester), 78.82 (CH, CH-OH), 116.89 (CH, Ar), 118.22
(CH, Ar), 122.87 (C, Ar), 128.55 (CH, Ar), 129.01 (CH, Ar), 132.80
(C, Ar), 141.54 (C, Ar), 142.34 (C, Ar), 178.27 (C, COOCH3). HRMS
(EI) m/z Calcd for C18H21ClNO3 (M+H)+ 334.1204. Found 334.1205.

M. S. Gomaa et al. / Bioorg. Med. Chem. 20 (2012) 4201–4207
4205
5.1.1.4.
phenyl]-2,2-dimethyl-propionic acid methyl ester (3d, R =
CO2CH3). Prepared from reaction of 4-methoxycarbonyl-
3-Hydroxy-3-[4-(4-methoxycarbonyl-phenylamino)-
1H NMR (DMSO-d6): d 1.22 (s, 6H, CH3), 5.88 (s, 1H, CH-OH),
6.82–6.85 (m, 1H, Ar), 7.03–7.08 (m, 5H, Ar), 7.23–7.26 (m, 2H,
Ar), 7.33–7.37 (m, 2H, Ar), 7.68 (s, 1H, Ar), 8.02 (s, 1H, Ar), 9.41
(s, 1H, NH), 15.05 (s, 1H, COOH). 13C NMR (DMSO-d6): d 22.62
(CH3), 23.26 (CH3), 46.35 (C, C-dimethyl), 68.67 (CH, CH-imd),
115.54 (CH, Ar), 117.64 (CH, Ar), 120.46 (CH, Ar), 124.90 (C, Ar),
129.17 (CH, Ar), 129.93 (CH, Ar), 142.49 (C, Ar), 144.20 (C, Ar),
176.01 (C, COOH). HRMS (EI) m/z Calcd for C20H21N3O2 (M+H)+
336.1712. Found 336.1710.
phenylboronic acid and (2) in 73% yield as a brown oil. TLC (2:1
Petroleum ether/EtOAc, Rf = 0.51). 1H NMR (CDCl3): d 1.08 (s, 3H,
CH3), 1.16 (s, 3H, CH3), 3.21 (s, 1H, OH), 3.65 (s, 3H, COOCH3),
3.82 (s, 3H, COOCH3-Ar), 4.83 (s, 1H, CH-OH), 5.72 (s, 1H, NH),
6.93 (d, J = 7.9 Hz, 2H, Ar), 7.07 (d, J = 7.8 Hz, 2H, Ar), 7.21 (d,
J = 7.8 Hz, 2H, Ar), 7.88 (d, J = 7.6 Hz, 2H, Ar). 13C NMR (CDCl3): d
19.16 (CH, CH3), 22.69 (CH, CH3), 47.88 (C, C-dimethyl), 51.88
(CH, CH3-ester), 52.06 (CH, CH3-ester-Ar), 78.23 (CH, CH-OH),
115.21 (CH, Ar), 119.23 (CH, Ar), 120.74 (C, Ar), 128.65 (CH, Ar),
130.82 (CH, Ar), 143.41 (C, Ar), 148.13 (C, Ar), 152.10 (C, Ar),
167.05 (C, COOCH3-Ar), 178.09 (C, COOCH3). HRMS (EI) m/z Calcd
for C20H24NO5 (M+H)+ 357.1576. Found 357.1578.
5.1.2.5. 4-(4-(2-Carboxy-2-methyl-1-(1H-1,2,4-triazol-1-yl)pro-
pyl)phenylamino)benzoic acid (9, R = CO2H, X = N).
Pre-
pared from (7) in 69% yield as a yellow solid; mp 136–140 °C.
TLC (97:3 EtOAC/MeOH, Rf = 0.31). 1H NMR (DMSO): d 1.16 (s,
3H, CH3), 1.27 (s, 3H, CH3), 5.99 (s, 1H, CH-tri), 7.02 (d, J = 8.0 Hz,
2H, Ar), 7.13 (d, J = 7.9 Hz, 2H, Ar), 7.48 (d, J = 7.9 Hz, 2H, Ar),
7.79 (d, J = 7.8 Hz, 2H, Ar), 8.03 (s, 1H, NH), 8.64 (s, 1H, Ar), 8.75
(s, 1H, Ar). 13C NMR (DMSO): d 21.80 (CH, CH3), 22.79 (CH, CH3),
47.16 (C, C-dimethyl), 67.00 (CH, CH-tri), 114.36 (CH, Ar), 117.93
(CH, Ar), 120.63 (C, Ar), 128.82 (C, Ar), 130.13 (CH, Ar), 131.12
(CH, Ar), 141.35 (C, Ar), 147.64 (C, Ar), 151.39 (CH, Ar), 167.06 (C,
COOH-Ar), 176.43 (C, COOH). EI-HRMS (MꢁH)+ found 379.1405,
Calcd for C20H19N4O4 379.1403.
5.1.2. General method for ester hydrolysis
To a solution of (1 or 3) (2 mmol) in THF (5 mL) was added H2O
(5 mL) containing LiOH (4 mmol) and the mixture was homoge-
nised with methanol and then heated under gentle reflux for 2 h.
The solvent was removed under reduced pressure and H2O
(100 mL) was added and the unreacted starting materials were ex-
tracted with diethyl ether (3 ꢀ 50 mL). The H2O layer was acidified
with 1 N HCl till pH 3 and extracted with diethyl ether (100 mL).
The organic layer was dried (MgSO4) filtered and the solvent re-
moved under reduced pressure. The product was used crude in
the following reaction for preparation of the amides (5).
5.1.3. General method for the synthesis of the amides (5)
EDC (0.18 mmol), (4) (0.18 mmol) and HOBt (0.18 mmol) in
CH2Cl2 (10 mL) were stirred at rt under N2 for 0.5 h. Amine
(0.56 mmol) was added and the reaction mixture was stirred over-
night at rt EtOAc (70 mL) was added and the organic layer was
washed with 1 M HCl (30 mL), saturated aqueous NaHCO3
(30 mL), brine (30 mL) and H2O (30 mL). The organic layer was
dried (MgSO4) filtered and reduced in vacuo. The residue was puri-
fied by flash column chromatography (CH2Cl2/MeOH 100:0 v/v
increasing to 98:2 v/v).
5.1.2.1. 3-Hydroxy-2,2-dimethyl-3-(4-phenylaminophenyl)-pro-
pionic acid (4a, R = H).
Prepared from (3a) in 76% yield as a
brown oil. TLC (97:3 CH2Cl2/MeOH, Rf = 0.32). 1H NMR (DMSO-
d6): d 0.87 (s, 3H, CH3), 1.01 (s, 3H, CH3), 4.72 (s, 1H, CH-OH),
5.77 (s, 1H, OH), 5.91 (s, 1H, NH), 6.76–6.79 (m, 1H, Ar), 7.03–
7.07 (m, 4H, Ar), 7.22–7.27 (m, 4H, Ar), 8.08 (s, 1H, NH). 13C NMR
(DMSO-d6): d 18.75 (CH3), 23.32 (CH3), 47.68 (C, C-dimethyl),
78.34 (CH, CH-OH), 116.81 (CH, Ar), 117.68 (CH, Ar), 121.29 (CH,
Ar), 128.72 (CH, Ar), 129.67 (CH, Ar), 131.79 (C, Ar), 142.82 (C,
Ar), 143.05 (C, Ar), 183.19 (C, COOH).
5.1.3.1. 3-Hydroxy-2,2,N-trimethyl-3-(4-phenylaminophenyl)-
propionamide (5a, R = H, R0 = CH3).
Prepared from the reac-
tion of (4a) and 33 % methylamine in absolute EtOH in 69% yield
as a brown oil. TLC (97:3 CH2Cl2/MeOH, Rf = 0.49). 1H NMR (CDCl3):
d 1.05 (s, 3H, CH3), 1.22 (s, 3H, CH3), 2.76 (d, J = 6.2 Hz, 3H, CH3-
amide), 4.40 (s, 1H, OH), 4.52 (s, 1H, CH-OH), 5.88 (s, 1H, NH),
6.23 (s, 1H, NH-amide), 6.91 (t, J = 7.1 Hz, 1H, Ar), 7.00 (d,
J = 7.6 Hz, 2H, Ar), 7.07 (d, J = 7.8 Hz, 2H, Ar), 7.16 (d, J = 7.8 Hz,
2H, Ar), 7.27 (t, J = 7.5 Hz, 2H, Ar). 13C NMR (CDCl3): d 20.66
(CH3), 24.44 (CH3), 26.40 (CH3-amide), 47.24 (C, C-dimethyl),
79.80 (CH, CH-OH), 116.90 (CH, Ar), 117.84 (CH, Ar), 121.31 (CH,
Ar), 128.75 (CH, Ar), 129.66 (CH, Ar), 133.20 (C, Ar), 142.70 (C,
Ar), 143.04 (C, Ar), 178.58 (C, CONH). EI-HRMS (M + Na)+ found
321.1575, Calcd for C18H22N2O2Na 321.1573.
5.1.2.2. 3-Hydroxy-3-[4-(4-methoxy-phenylamino)phenyl]-2,2-
dimethyl-propionic acid (4b, R = OCH3).
Prepared from (3b)
in 71% yield as a brown oil. TLC (95:5 CH2Cl2/MeOH, Rf = 0.39).
1H NMR (CDCl3): d 1.11 (s, 6H, CH3), 3.35 (s, 1H, OH), 3.82 (s, 3H,
OCH3), 4.88 (s, 1H, CH-OH), 6.21 (s, 1H, NH), 6.86–6.90 (m, 4H,
Ar), 7.06 (d, J = 7.7 Hz, 2H, Ar), 7.14 (d, J = 7.6 Hz, 2H, Ar). 13C
NMR (CDCl3): d 18.79 (CH3), 23.38 (CH3), 47.63 (C, C-dimethyl),
55.60 (CH3, OCH3), 78.47 (CH, CH-OH), 114.73 (CH, Ar), 114.81
(CH, Ar), 115.54 (CH, Ar), 121.81 (CH, Ar), 122.46 (CH, Ar), 128.69
(CH, Ar), 129.67 (CH, Ar), 130.40 (C, Ar), 135.45 (C, Ar), 145.08 (C,
Ar), 155.45 (C, Ar), 182.73 (C, COOH).
5.1.3.2. N-Ethyl-3-hydroxy-2,2-dimethyl-3-(4-phenylaminophe-
nyl)propionamide (5b, R = H, R0 = CH2CH3).
Prepared from
5.1.2.3.
3-Hydroxy-3-[4-(4-chloro-phenylamino)phenyl]-2,2-
Prepared from (3c) in
the reaction of (4a) and 2 M ethylamine in THF in 66% yield as a
brown oil. TLC (97:3 CH2Cl2/MeOH, Rf = 0.51). 1H NMR (CDCl3): d
1.04 (s, 3H, CH3), 1.13 (t, J = 6.1 Hz, 3H, CH3-amide), 1.27 (s, 3H,
CH3), 3.21–3.27 (m, 2H, CH2-amide), 4.39 (s, 1H, OH), 4.53 (s, 1H,
CH-OH), 5.31 (s, 1H, NH), 6.11 (s, 1H, NH-amide), 6.90 (t,
J = 7.2 Hz, 1H, Ar), 7.03 (d, J = 7.8 Hz, 2H, Ar), 7.09 (d, J = 7.8 Hz,
2H, Ar), 7.17 (d, J = 7.7 Hz, 2H, Ar), 7.27 (t, J = 7.6 Hz, 2H, Ar). 13C
NMR (CDCl3): d 14.80 (CH3-amide), 20.76 (CH3), 24.50 (CH3),
34.58 (CH2-amide), 46.06 (C, C-dimethyl), 79.88 (CH, CH-OH),
116.61 (CH, Ar), 117.80 (CH, Ar), 121.33 (CH, Ar), 128.55 (CH, Ar),
129.64 (CH, Ar), 133.34 (C, Ar), 142.63 (C, Ar), 143.08 (C, Ar),
177.78 (C, CONH). EI-HRMS (M + Na)+ found 335.1532, Calcd for
dimethyl-propionic acid (4c, R = Cl).
61% yield as a brown oil. TLC (95:5 CH2Cl2/MeOH, Rf = 0.40). 11H
NMR (CDCl3): d .16 (s, 3H, CH3), 1.18 (s, 3H, CH3), 3.25 (s, 1H,
OH), 4.90 (s, 1H, CH-OH), 6.19 (s, 1H, NH), 6.97–7.01 (m, 4H, Ar),
7.22–7.26 (m, 4H, Ar). 13C NMR (CDCl3): d 18.76 (CH3), 23.33
(CH3), 47.65 (C, C-dimethyl), 78.28 (CH, CH-OH), 117.97 (CH, Ar),
119.10 (CH, Ar), 125.77 (C, Ar), 128.81 (CH, Ar), 129.32 (CH, Ar),
132.25 (C, Ar), 141.51 (C, Ar), 142.55 (C, Ar), 183.10 (C, COOH).
5.1.2.4.
nyl)propionic acid (8, R = H, X = CH).
in 62% yield as a brown oil. TLC (95:5 CH2Cl2/MeOH, Rf = 0.29).
3-Imidazol-1-yl-2,2-dimethyl-3-(4-phenylaminophe-
24
Prepared from (1)
C19H24N2O2Na 321.1573.

4206
M. S. Gomaa et al. / Bioorg. Med. Chem. 20 (2012) 4201–4207
5.1.3.3. 3-Hydroxy-3-[4-(4-methoxy-phenylamino)phenyl]-2,2,
N-trimethyl-propionamide (5c, R = OCH3, R0 = CH3).
Pre-
(CH, CH3-ester-Ar), 52.33 (CH, CH3-ester), 68.72 (CH, CH-imd),
115.28 (CH, Ar), 118.75 (CH, Ar), 121.66 (C, Ar), 128.79 (C, Ar),
130.51 (CH, Ar), 131.76 (CH, Ar), 141.67 (C, Ar), 147.26 (C, Ar),
166.87 (C, COOCH3-Ar), 176.16 (C, COOCH3). EI-HRMS (M+H)+
found 409.1872, Calcd for C22H25N4O4 409.1870.
pared from the reaction of (4b) and 33 % methylamine in absolute
EtOH in 67% yield as a brown oil. TLC (97:3 CH2Cl2/MeOH,
Rf = 0.44). 1H NMR (CDCl3): d 1.03 (s, 3H, CH3), 1.23 (s, 3H, CH3),
2.77 (d, J = 6.4 Hz, 3H, CH3-amide), 3.81 (s, 3H, OCH3), 4.41 (s, 1H,
OH), 4.52 (s, 1H, CH-OH), 5.58 (s, 1H, NH), 6.23 (s, 1H, NH-amide),
6.84–6.90 (m, 4H, Ar), 7.02–7.07 (m, 4H, Ar). 13C NMR (CDCl3): d
20.62 (CH3), 24.47 (CH3), 26.41 (CH3-amide), 46.25 (C, C-dimethyl),
55.59 (CH3, OCH3), 79.86 (CH, CH-OH), 114.66 (CH, Ar), 114.85 (CH,
Ar), 122.11 (CH, Ar), 128.48 (CH, Ar), 131.79 (C, Ar), 135.65 (C, Ar),
144.66 (C, Ar), 155.22 (C, Ar), 178.60 (C, CONH). EI-HRMS (M + Na)+
found 351.1681, Calcd for C19H24N2O3Na 351.1682.
5.1.4.3. 3-(1H -Imidazol-1-yl)-N,2,2-trimethyl-3-(4-(phenylami-
no)phenyl)propanamide (10).
Prepared by the reaction of 5a
with CDI and imidazole. After 48 h reflux column chromatography
(EtOAc/MeOH 100:0 v/v increasing to 90:10 v/v) gave this product
in 46 % yield as a colourless oil. TLC (95:5 EtOAC/MeOH, Rf = 0.41).
1H NMR (CDCl3): d 1.22 (s, 3H, CH3), 1.26 (s, 3H, CH3), 2.71 (d,
J = 6.0 Hz, 3H, CH3-amide), 5.53 (s, 1H, NH), 5.62 (s, 1H, CH-imid),
5.89 (s, 1H, NH-amide), 6.94–7.01 (m, 4H, Ar), 7.12 (d, J = 7.7 Hz,
2H, Ar), 7.19 (s, 1H, Ar), 7.26 (d, J = 7.8 Hz, 2H, Ar), 7.29–7.32 (m,
2H, Ar), 7.71 (s, 1H, Ar). 13C NMR (CDCl3): d 22.37 (CH3), 23.60
(CH3), 26.80 (CH3-amide), 47.82 (C, C-dimethyl), 68.04 (CH, CH-
imid), 116.52 (CH, Ar), 118.75 (CH, Ar), 121.83 (CH, Ar), 127.93
(C, Ar), 129.41 (CH, Ar), 129.87 (CH, Ar), 142.18 (C, Ar), 143.65 (C,
Ar), 176.04 (C, CONH). EI-HRMS (M+H)+ found 349.2028, Calcd
for C21H25N4O 349.2023.
5.1.3.4. 3-Hydroxy-3-[4-(4-chloro-phenylamino)phenyl]-2,2,N-
trimethyl-propionamide(5d, R = Cl, R0 = CH3).
Prepared from
the reaction of (4c) and 33 % methylamine in absolute EtOH in 60%
yield as a brown oil. TLC (97:3 CH2Cl2/MeOH, Rf = 0.48). 1H NMR
(CDCl3): d 1.07 (s, 3H, CH3), 1.29 (s, 3H, CH3), 2.82 (d, J = 6.6 Hz,
3H, CH3-amide), 4.47 (s, 1H, OH), 4.63 (s, 1H, CH-OH), 5.74 (s,
1H, NH), 6.03 (s, 1H, NH-amide), 6.94–6.99 (m, 4H, Ar), 7.12–7.20
(m, 4H, Ar). 13C NMR (CDCl3): d 20.60 (CH3), 24.44 (CH3), 26.43
(CH3-amide), 46.19 (C, C-dimethyl), 79.82 (CH, CH-OH), 117.19
(CH, Ar), 118.85 (CH, Ar), 125.55 (C, Ar), 128.61 (CH, Ar), 129.27
(CH, Ar), 133.74 (C, Ar), 141.69 (C, Ar), 142.19 (C, Ar), 178.49 (C,
5.1.4.4. N-Ethyl-3-(1H-imidazol-1-yl)-2,2-dimethyl-3-(4-(phe-
nylamino)phenyl)propanamide (11).
Prepared by the reac-
tion of 5b with CDI and imidazole. After 48 h reflux column
chromatography (EtOAc/MeOH 100:0 v/v increasing to 90:10 v/v)
gave this product in 38 % yield as a yellow oil. TLC (95:5 EtOAc/
MeOH, Rf = 0.42). 1H NMR (CDCl3): d 1.25–1.35 (m, 9H, CH3, CH3-
amide), 3.26 (q, J = 7.2 Hz, 2H, CH2-amide), 5.51 (s, 1H, NH), 5.62
(s, 1H, CH-imid), 5.84 (s, 1H, NH-amide), 6.95–7.01 (m, 3H, Ar),
7.11 (d, J = 7.8 Hz, 2H, Ar), 7.22 (s, 1H, Ar), 7.29 (d, J = 7.8 Hz, 2H,
Ar), 7.29–7.35 (m, 3H, Ar), 7.72 (s, 1H, Ar). 13C NMR (CDCl3): d
14.49 (CH3-amide), 22.50 (CH3), 23.50 (CH3), 34.81 (CH2-amide),
47.78 (C, C-dimethyl), 67.90 (CH, CH-imid), 116.62 (CH, Ar),
118.66 (CH, Ar), 121.80 (CH, Ar), 128.22 (C, Ar), 129.42 (CH, Ar),
129.93 (CH, Ar), 142.19 (C, Ar), 143.49 (C, Ar), 175.11 (C, CONH).
EI-HRMS (M+H)+ found 363.2184, Calcd for C22H27N4O 363.2184.
CONH). EI-HRMS (M
+
Na)+ found 355.1186, Calcd for
C18H21ClN2O2Na 355.1186.
5.1.4. General method for addition of the N-heterocyclic ring to
prepare compounds 1, 6, 8–12
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). The mixture was then heated
under reflux for 24–48 h. The reaction 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.
5.1.4.1. Methyl 2,2-dimethyl-3-(4-(phenylamino)phenyl)-3-(1H-
5.1.4.5. 3-(4-(4-Methoxyphenylamino)phenyl)-3-(1H-imidazol-
1,2,4-triazol-1-yl)propanoate (6, R = H, X = N).
Prepared by
1-yl)-N,2,2-trimethylpropanamide (12).
Prepared by the
the reaction of 3a with CDT and triazole. After 24 h reflux, column
chromatography (Petroleum Ether/EtOAc 70:30 v/v increasing to
50:50 v/v) gave this product in 68 % yield as a pale yellow oil.
TLC (50:50 petroleum ether/EtOAc, Rf = 0.58). 1H NMR (CDCl3): d
1.22 (s, 3H, CH3), 1.31 (s, 3H, CH3), 3.63 (s, 3H, COOCH3), 5.71 (s,
1H, CH-tri), 6.10 (s, 1H, NH), 6.91–7.01 (m, 3H, Ar), 7.11 (d,
J = 8.0 Hz, 2H, Ar), 7.29–7.33 (m, 2H, Ar),7.38 (d, J = 7.9 Hz, 2H,
Ar), 7.93 (s, 1H, Ar), 8.15 (s, 1H, Ar). 13C NMR (CDCl3): d 20.99
(CH3), 23.95 (CH3), 48.06 (C, C-dimethyl), 52.30 (CH3-ester),
68.88 (CH, CH-tri), 116.23 (CH, Ar), 118.78 (CH, Ar), 121.76 (CH,
Ar), 126.57 (C, Ar), 129.39 (CH, Ar), 130.44 (CH, Ar), 142.23 (C,
Ar), 143.94 (C, Ar), 144.40 (CH, Ar), 151.46 (CH, Ar), 176.35 (C,
COOCH3). EI-HRMS (M+H)+ found 351.1815, Calcd for C20H23N5O2
351.1816.
reaction of 5c with CDI and imidazole. After 48 h reflux column
chromatography (EtOAc/MeOH 100:0 v/v increasing to 90:10 v/v)
gave this product in 40 % yield as a yellow oil. TLC (95:5 EtOAc/
MeOH, Rf = 0.38). 1H NMR (CDCl3): d 1.27 (s, 3H, CH3), 1.29 (s,
3H, CH3), 2.89 (s, 3H, CH3-amide), 4.79 (s, 1H, CH-imid), 5.52 (s,
1H, NH), 5.83 (s, 1H, NH-amide), 6.63 (d, J = 7.9 Hz, 2H, Ar), 6.81–
6.90 (m, 3H, Ar), 7.05–7.11 (m, 1H, Ar), 7.28 (s, 3H, Ar). 13C NMR
(CDCl3): d 19.01 (CH3), 23.08 (CH3), 26.45 (CH3-amide), 46.27 (C,
C-dimethyl), 55.55 (CH3, OCH3), 67.88 (CH, CH-imid), 114.43 (CH,
Ar), 114.67 (CH, Ar), 114.72 (CH, Ar), 123.11 (CH, Ar), 128.63 (CH,
Ar), 131.88 (C, Ar), 134.72 (C, Ar), 144.15 (C, Ar), 155.16 (C, Ar),
177.99 (C, CONH). EI-HRMS (M+H)+ found 379.2133, Calcd for
C22H27N4O2 379.2134.
5.1.4.6.
3-(4-(4-Chlorophenylamino)phenyl)-3-(1H-imidazol-
Prepared by the
5.1.4.2. Methyl 4-((4-(3-methoxy-2,2-dimethyl-3-oxo-1-(1H-
1-yl)-N,2,2-trimethylpropanamide (13).
1,2,4-triazol-1-yl)propyl)phenyl)amino)benzoate (7, R = CO2CH3,
reaction of 5d with CDI and imidazole. After 48 h reflux column
chromatography (EtOAc/MeOH 100:0 v/v increasing to 90:10 v/v)
gave this product in 35 % yield as a yellow oil. TLC (95:5 EtOAc/
MeOH, Rf = 0.44). 1H NMR (CDCl3): d 1.25 (s, 3H, CH3), 1.28 (s,
3H, CH3), 2.79 (d, J = 6.1 Hz, 3H, CH3-amide), 5.56 (s, 1H, NH),
5.65 (s, 1H, CH-imid), 5.79 (s, 1H, NH-amide), 6.93–6.99 (m, 3H,
Ar), 7.05 (s, 1H, Ar), 7.11 (d, J = 7.9 Hz, 2H, Ar), 7.19–7.24 (m, 2H,
Ar), 7.26–7.31 (m, 3H, Ar). 13C NMR (CDCl3): d 19.25 (CH3), 23.61
(CH3), 26.48 (CH3-amide), 46.19 (C, C-dimethyl), 67.34 (CH, CH-
imid), 114.43 (CH, Ar), 114.89 (CH, Ar), 119.72 (CH, Ar), 123.25
X = N).
Prepared by the reaction of 3d with CDT and triazole.
After 24 h reflux, column chromatography (EtOAC/MeOH 100:0 v/v
increasing to 98:2 v/v) gave this product in 63 % yield as a brown
oil. TLC (99:1 EtOAC/MeOH, Rf = 0.48). 1H NMR (CDCl3): d 1.23 (s,
3H, CH3), 1.28 (s, 3H, CH3), 3.64 (s, 3H, COOCH3), 5.73 (s, 1H, CH-
imd), 6.52 (s, 1H, NH), 6.96 (d, J = 8.0 Hz, 2H, Ar), 7.11 (d,
J = 7.8 Hz, 2H, Ar), 7.43 (d, J = 7.7 Hz, 2H, Ar), 7.92 (d, J = 7.8 Hz,
2H, Ar), 7.95 (s, 1H, Ar), 8.11 (s, 1H, Ar). 13C NMR (CDCl3): d
21.62 (CH, CH3), 23.77 (CH, CH3), 48.08 (C, C-dimethyl), 51.75

M. S. Gomaa et al. / Bioorg. Med. Chem. 20 (2012) 4201–4207
4207
(CH, Ar), 125.38 (C, Ar), 128.72 (CH, Ar), 133.76 (C, Ar), 142.28 (C,
Ar), 143.05 (C, Ar), 177.83 (C, CONH). EI-HRMS (M+H)+ found
383.1636, Calcd for C21H24N4OCl 383.1638.
tween the specified 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 calcu-
lated as the percentage 3H ATRA metabolite peak area formation
(activity 3H metabolite(s)/total activity) compared to metabolite
formation in the absence 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.
5.1.4.7.
N,2,2-trimethyl-3-(4-(phenylamino)phenyl)-3-
Prepared by the
(1H-1,2,4-triazol-1-yl)propanamide (14).
reaction of 5a with CDT and triazole. After 24 h reflux column chro-
matography (EtOAc/MeOH 100:0 v/v increasing to 99:1 v/v) gave
this product in 67 % yield as a white solid; mp 106–110 °C. TLC
(99:1 EtOAc/MeOH, Rf = 0.37). 1H NMR (CDCl3): d 1.23 (s, 3H,
CH3), 1.25 (s, 3H, CH3), 2.66 (d, J = 6.0 Hz, 3H, CH3-amide), 5.80
(s, 1H, CH-tri), 5.83 (s, 1H, NH), 5.97 (s, 1H, NH-amide), 6.90–
6.99 (m, 3H, Ar), 7.09 (d, J = 8.0 Hz, 2H, Ar), 7.25–7.29 (m, 2H,
Ar), 7.46 (d, J = 7.9 Hz, 2H, Ar), 7.92 (s, 1H, Ar), 8.17 (s, 1H, Ar).
13C NMR (CDCl3): d 21.33 (CH3), 23.36 (CH3), 26.62 (CH3-amide),
48.41 (C, C-dimethyl), 68.66 (CH, CH-tri), 116.26 (CH, Ar), 118.58
(CH, Ar), 121.65 (CH, Ar), 127.04 (C, Ar), 129.38 (CH, Ar), 130.36
(CH, Ar), 142.34 (C, Ar), 143.54 (C, Ar), 145.06 (CH, Ar), 151.66
(CH, Ar), 176.07 (C, CONH). Anal. C, H, N. EI-HRMS (M+H)+ found
350.1980, Calcd for C20H24N5O 350.1980.
Acknowledgements
We acknowledge Cancer Research UK for funding (MSG and
CEB, Grant Ref. C7735/A9612) and the EPSRC Mass Spectrometry
Centre, Swansea, UK for mass spectroscopy data.
References and notes
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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.32 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
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5–15 U cyt c/
(NADPH) 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 eppendorfs 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 shak-
lg protein, using the cytochrome c reductase
l
l
l
ing. The reaction was quenched with acetonitrile, mixed, then cen-
trifuged (18,000g, 5 min, 4 °C). Resolution of retinoids was
performed with a Luna C18(2) column (3
lm, 50 ꢀ 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 respectively for data acquisition. 3H ATRA and 3H metab-
olites were separated 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 be-
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