Synthesis and biological evaluation of 3-(1 H-imidazol- and triazol-1-yl)-2,2-dimethyl-3-[4-(naphthalen-2-ylamino)phenyl]propyl derivatives as small molecule inhibitors of retinoic acid 4-hydroxylase (CYP26)

Article abstract of DOI:10.1021/jm200695m

The synthesis and potent inhibitory activity of novel 3-(1H-imidazol- and triazol-1-yl)-2,2-dimethyl-3-(4-(naphthalen-2-ylamino)phenyl)propyl derivatives vs a MCF-7 CYP26A1 microsomal assay is described. This study focused on the effect of modifying the h

Full text of DOI:10.1021/jm200695m

ARTICLE
pubs.acs.org/jmc
Synthesis and Biological Evaluation of 3-(1H-Imidazol- and
Triazol-1-yl)-2,2-dimethyl-3-[4-(naphthalen-2-ylamino)phenyl]propyl
Derivatives as Small Molecule Inhibitors of Retinoic Acid
4-Hydroxylase (CYP26)
Mohamed S. Gomaa,† Caroline E. Bridgens,‡ Gareth J. Veal,‡ Christopher P. F. Redfern,‡ Andrea Brancale,†
Jane L. Armstrong,*,§ and Claire Simons*,†
†Medicinal Chemistry Division, Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3NB,
United Kingdom
‡Northern Institute for Cancer Research, Paul O'Gorman Building, Medical School, and §Institute of Cellular Medicine,
Framlington Place, Newcastle University, Newcastle upon Tyne, NE2 4HH, United Kingdom
S Supporting Information
b
ABSTRACT: The synthesis and potent inhibitory activity of novel 3-(1H-imidazol-
and triazol-1-yl)-2,2-dimethyl-3-(4-(naphthalen-2-ylamino)phenyl)propyl deriva-
tives vs a MCF-7 CYP26A1 microsomal assay is described. This study focused
on the effect of modifying the heme binding azole group and the flexible C3
chain on inhibitory activity and selectivity. The most promising inhibitor
2,2-dimethyl-3-[4-(naphthalen-2-ylamino)-phenyl]-3-[1,2,4]triazol-1-yl-propio-
nic acid methyl ester (17) (IC50 = 0.35 nM as compared with liarozole IC50
=
540 nM and R116010 IC50 = 10 nM) was evaluated for CYP selectivity and
hepatic stability. Compounds with CYP26 inhibitory IC50 values e50 nM
enhanced the biological activity of exogenous ATRA, as evidenced by a 3.7ꢀ
5.8-fold increase in CYP26A1 mRNA in SH-SY5Y neuroblastoma cells as
compared with ATRA alone. All compounds demonstrated an activity
comparable with or better than R116010, and the induction correlated well
with CYP26 inhibition data. These studies highlight the promising activity profile of this novel CYP26 inhibitor and suggest it as an
appropriate candidate for future development.
’ INTRODUCTION
RAMBAs that can be cleared from the body on cessation of therapy
may reduce the unwanted side effects associated with high-dose RA
treatment.13,14
Retinoic acid (RA) is a naturally occurring retinoid, the main
biologically active derivative of vitamin A (retinol), which
regulates cell growth and differentiation in a variety of cell types.
As a key signaling molecule, the intracellular concentrations of
RA are regulated by negative feedback controls tightly coupled to
requirements for signaling in relation to cell differentiation and
morphogenesis.1,2 Retinoids have long been major drugs used to
treat disorders of keratinization such as acne, psoriasis, and
ichthyosis3ꢀ6 and have been successful in the treatment of acute
promyelocytic leukemia7,8 and neuroblastoma.9 Unfortunately,
natural and induced resistance to RA as well as local and systemic
toxicity limit the potential of RA in the treatment of these diseases
and as a therapeutic strategy for a wider range of diseases.10,11
One approach to overcoming drug resistance is to increase
intracellular RA concentrations through inhibition of RA catabolic
enzymes. The major enzyme responsible for RA metabolism is the
RA-inducible cytochrome P450 (CYP) enzyme, CYP26.12 CYP26
therefore provides a good target for the development of RA
metabolism blocking agents (RAMBAs) to improve efficacy of
retinoid treatment. Reducing the rate of metabolism of RA using
Two RAMBAs have been developed clinically, the azoles liarozole
and talarozole (Figure 1), both for dermatological treatment. In
humans, topical application of liarozole exhibits similar clinical effects
to the RA analogue acitretin but with fewer side effects.15 However, it
lacks CYP26 specificity and inhibits steroidogenic CYP enzymes
including aromatase (CYP19).16 In humans, talarozole (formerly
R115866 and Rambazole) exhibits beneficial effects in both
psoriasis17 and acne,18 and the side effects are expected to be less
severe as compared with previous synthetic retinoids.19 N-(4-
((1R,2R)-2-(Dimethylamino)-1-(1H-imidazol-1-yl)propyl)phenyl)-
benzo[d]thiazol-2-amine (R116010)20 (Figure 1) is a potent and
selective inhibitor of RA metabolism, which inhibits all-trans-
retinoic acid (ATRA) metabolism in neuroblastoma both in vitro
and in vivo,21 although there are no further details regarding the
clinical development of this RAMBA.
Received: June 1, 2011
Published: August 15, 2011
r
2011 American Chemical Society
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In previous studies,22 we reported the development of the lead
CYP26 inhibitor, N-(4-((1H-imidazol-1-yl)(phenyl)methyl)phenyl)-
naphthalen-2-amine (1) (IC50 = 0.5 μM; as compared with
Liarozole, IC50 = 540 nM, and R116010, IC50 = 10 nM) by
modification of the aryl, NH linker, and phenyl to a C3 chain
(Figure 2).22,23 These studies indicated that a broad range of aryl
and heteroaryl substituents were tolerated, that the NH linker
was optimal with a noted reduction in inhibitory activity if re-
placed with either an ꢀOꢀ or a ꢀCH2ꢀ linker, and that the
dimethyl group was important in establishing hydrophobic
interactions and optimizing “fit” within the CYP26 active site.
Several low nanomolar inhibitors were discovered through structureꢀ
activity relationship (SAR) studies with methyl 3-(1H-imidazol-1-yl)-
2,2-dimethyl-3-(4-(naphthalen-2-ylamino)phenyl)propanoate
(2) (IC50 = 3 nM, Figure 2) evaluated further for selectivity and
hepatic stability. Direct comparison of CYP selectivity profiles for
compound 2 and R116010 revealed markedly reduced activity as
compared with CYP26 for CYPs 1A2, 2C9, 2C19, and 2D6.
However, both compounds exhibited activity against CYP3A4, a
xenobiotic-metabolizing CYP with low substrate specificity and
which is known to have activity as an ATRA hydroxylase, with
IC50 values of <0.1 μM for compound 2 and 0.35 μM for R116010.
CYP3A4 liability will restrict further development of RAMBAs
for systemic use, although likely to be less of an issue for topical
administration in combination with ATRA; therefore, further
SAR studies were required to determine the effect on selectivity and
inhibitory activity. The two areas for SAR studies described here are
the C3 flexible chain and the heme binding azole (Figure 2).
temperature, with the ester products obtained in good yield after
purification by column chromatography (Scheme 1). The amide
derivatives (10ꢀ13) were prepared through the reaction of the
acid (4) with an amine in the presence of EDC and HOBt in
dichloromethane25,26 and obtained in good yield after purifica-
tion by flash column chromatography. The alcohol analogue
(15) of compound (14)22 was generated by reduction with
LiAlH4 in THF27 (Scheme 1).
The modification of the N-heterocycle moiety involved the
preparation of triazole derivatives. The triazole analogue was
prepared through the reaction of the alcohol precursor (16)22 with
1,10-carbonylditriazole and triazole in acetonitrile to give the
triazole derivative (17) in good yield (63%) (Scheme 2). Attempts
to prepare the triazole acid (18) derivative following the previously
described method for the imidazole acid (4) using KOHandEtOH
were unsuccessful with no reaction observed; however, hydrolysis
of the triazole ester (17) was accomplished with LiOH in THF and
H2O.26,28 The acid was obtained in good yield after acidification
(pH 3) and extraction with diethyl ether and used without further
purification. The triazole amide derivative (19) was obtained after
treatment of the acid (18) with methylamine in the presence of
EDC and HOBt in dichloromethane (Scheme 2).25,26
Enzyme Inhibition. The imidazole and triazole derivatives
were evaluated for their RA metabolism (CYP26) inhibitory
activity with a cell-free microsomal assay,23,29 using radiolabeled
[11,12-3H]ATRA as the substrate. Liarozole (a nonselective
CYP26 inhibitor15,16) and R11601020 were included in all experi-
ments as comparative standards.
In the ester series (2 and 5ꢀ9), a small alkyl substituent was
optimal with the methyl ester (2, IC50 = 3 nM) and ethyl ester
(5, IC50 = 0.35 nM) being preferred as compared with larger
chains such as butyl (6) and pentyl (7) (IC50 = 200 and 140 nM,
respectively), branched isopropyl (8, IC50 = 290 nM), and the
bulky aromatic benzyl ester (9, IC50 = 400 nM) (Table 1). It is
probable that the enhanced inhibitory activity observed for the
ethyl ester (5) is related to pharmacodynamic properties rather
than enhanced binding, whereas for the larger alkyl esters a
reduced fit has a direct impact on inhibitory activity. Replacing
the ester with an amide (10ꢀ13) was generally well tolerated
(IC50 range = 12ꢀ120 nM) with the methyl amide isostere (11)
of the lead methyl ester (2) displaying comparable inhibitory
activity with an IC50 of 12 nM. The order of preference for
inhibitory activity of the amides was NHMe > NH2 > NMe2 >
NHiPr. Removal of the alkyl group to give the free carboxylic acid
(4) resulted in a reduction in activity from 3 to 300 nM, whereas
reduction of the ester to the free alcohol (15) was tolerated
(IC50 = 40 nM).
’ RESULTS
Chemistry. The synthesis of several ester and amide analogues
of (2) was undertaken. The preparation of the acid (4) was
performed through saponification of the lead compound (2)
with KOH in EtOH under reflux.24 The acid was separated in
good yield after acidification of the potassium salt (3) with
aqueous 1 N HCl followed by extraction with dichloromethane
(Scheme 1).
Several esters (5ꢀ9) were prepared by stirring the potassium
salt (3) with the corresponding alkyl halides in EtOH at room
The triazole methyl ester (17) showed improved subnano-
molar inhibitory activity (IC50 = 0.35 nM) as compared with
the imidazole methyl ester lead (2, IC50 = 3 nM) (Table 2).
Figure 1. RAMBAs.
Figure 2. Development of compounds through SAR studies.
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Scheme 1a
a Reagents and conditions: (i) KOH, EtOH, reflux, 72 h. (ii) 1 N aqueous HCl, 64%. (iii) RBr, DMF, 80 °C, 2 h, 57ꢀ71%. (iv) EDC, HOBt, CH2Cl2,
0.5 h, room temperature and then R1R2NH, room temperature, overnight, 61ꢀ75%. (v) LiAlH4, THF, 0 °C, 1 h and then room temperature overnight, 79%.
Scheme 2a
a Reagents and conditions: (i) 1,10-Carbonylditriazole, triazole, CH3CN, reflux, 16 h, 63%. (ii) LiOH, THF, H2O, 20 h, reflux and then (iii) aqueous 1 N
HCl 77%. (iv) EDC, HOBt, CH2Cl2, 0.5 h, room temperature and then CH3NH2, room temperature, overnight, 63%.
However, a considerable reduction in activity was noted for the
triazole carboxylic acid derivative (18, IC50 > 1 μM) as compared
with the imidazole carboxylic acid derivative (4, IC50 = 300 nM),
and this same trend was observed for the triazole methyl amide
derivative (19, IC50 = 350 nM) as compared with imidazole
product (11, IC50 = 12 nM) (Table 2).
Enhancement of RA Effects. The induction of CYP26A1
mRNA was used to determine the ability of the compounds to
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Table 1. IC50 Values for Imidazole Esters, Amides, and Alcohol
a IC50 values were derived from the best fit of a four point doseꢀresponse curve.
CYP26 inhibitory IC50 e 50 nM were selected, and CYP26A1
mRNA was not induced by treatment of SH-SY5Y cells with
these inhibitors alone at a concentration of 1 μM. Coincubation
of liarozole with 0.1 μM ATRA resulted in a 2.3-fold induction in
CYP26A1 mRNA as compared with 0.1 μM ATRA alone,
whereas coincubation with R116010 resulted in a 4.7-fold
induction of CYP26A1 mRNA, which was comparable to the
induction in response to a 10-fold higher concentration of ATRA
(1 μM) (Figure 3). Coincubation of 0.1 μM ATRA in combina-
tion with 1 μM compounds 2, 5, 10, 11, 15, and 17 for 72 h also
substantially increased expression of CYP26A1 as compared with
0.1 μM ATRA alone. Under these conditions, the two com-
pounds with the greatest CYP26 inhibitory activity, the ethyl
ester product 5 and the triazole methyl ester 17, enhanced the
expression of CYP26A1 mRNA by 5.8- and 5.2-fold, respectively
(Figure 3). Conversely, the two compounds with the lowest
CYP26 inhibitory activity, the reduced ester 15 and the amide 10,
enhanced CYP26A1 mRNA by 3.7- and 4.1-fold, respectively,
and were the least active (Figure 3).
Table 2. IC50 Values for Triazole Compounds as Compared
with Imidazole Compounds
no.
R
CYP26 IC50 (nM)a no.
R
CYP26 IC50 (nM)a
17 OCH3
18 OH
0.35
>1 μM
350
2
4
OCH3
OH
3
300
12
19 NHCH3
11 NHCH3
a IC50 values were derived from the best fit of a 4 point doseꢀresponse
curve. Liarozole, IC50 = 540 nM; R116010, IC50 = 10 nM.
enhance the biological effects of ATRA in retinoid-responsive
SH-SY5Y neuroblastoma cells. Compounds with a microsomal
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Table 4. CYP IC50 (μM) Profile of Triazole 17 as Compared
with Lead 2 and R116010
compd
1A2
2C9
2C19
3A4
2D6
26
17
10.9
1.2
21.4
8.0
3.14
1.5
0.33
<0.1
0.35
1.8
0.6
3.9
0.0003
0.003
0.01
2
R116010
8.3
11.0
5.9
Figure 3. Real-time PCR analysis of CYP26A1 mRNA expression after
treatment with ATRA in combination with liarozole (Liar.), R116010
(R116), 2, 5, 10, 11, 15, and 17. SH-SY5Y cells were treated with ATRA
alone (0.01ꢀ1 μM) or with ATRA (0.1 μM) in combination with
inhibitor (1 μM) for 72 h. Total RNA was isolated, reverse transcribed,
and subjected to real-time PCR using TaqMan probes for CYP26A1 and
β-actin. Values were normalized for β-actin levels and expressed as a fold
increase relative to CYP26A1 expression in SH-SY5Y cells treated with
0.01 μM ATRA. Data are mean values ( SDs (n = 6).
Table 3. Percent Inhibition Data against CYP Panel at
0.4 μM
Figure 4. Putative binding mode for 17. The Fe atom of the heme is
shown as a green sphere.
with imidazole (2), which was comparable with R116010
(CYP3A4 IC50 = 0.35 μM) (Table 4). Compound 17 exhibited
a low clearance rate (Cyprotex: t1/2 = 3 h and 44 min) in human
liver microsomes comparable with the imidazole lead 2
(Cyprotex: t1/2 = 5 h and 1 min), indicating minimal phase I
metabolism and good stability.30
Molecular Modeling. A series of molecular docking simula-
tions were performed on this series of compounds with ligands
docked within the active site of the CYP26A1 homology
model,31 following the methodology reported previously.22
Results obtained for 17 showed a putative binding mode very
similar to the one observed previously with the corresponding
imidazole analogue 2.22 In particular, the binding was dominated
by a series of hydrophobic interactions: The naphthyl group lay
in a channel formed, among others, by Phe222, Phe84, and
Phe374, while the ester moiety was in contact with Phe299
(Figure 4). The naphthylphenylamine moiety is not common in
drugs; however, in these inhibitors, it fulfills the role of binding
the molecule in the hydrophobic tunnel of the enzyme active site
and allows correct orientation of the imidazole for optimal
interaction with the heme.
Selectivity and Microsomal Stability. The imidazole ethyl
ester (5), imidazole methylamide (11), imidazole dimethylamide
(13), and triazole methyl ester (17) derivatives were evaluated
for their inhibitory activity against a panel of P450 isoforms
expressed in human liver microsomes (Table 3) in comparison
with the lead imidazole methyl ester (2). The imidazole ethyl
ester (5) had a CYP selectivity profile almost identical with the
methyl ester lead (2). The amides 11 and 13 were highly active
against CYP3A4 at concentrations of 0.4 μM, which would
discount these compounds for further evaluation. However, at
this concentration, the triazole methyl ester (17) was inactive
against the CYP panel (Table 3).
To determine the selectivity of the triazole methyl ester (17)
in comparison with R116010 and the lead imidazole methyl ester
(2), IC50 assays against CYP isoforms expressed in human liver
microsomes were performed. Micromolar concentrations of
compounds 2 or 17 or R116010 were needed to inhibit CYPs
1A2, 2C9, and 2C19.
’ DISCUSSION
Evaluation of CYP26 inhibitory activity revealed that in the
imidazole ester series, 2 and 5ꢀ9, a small alkyl group was optimal
with the methyl (2) and ethyl (5) esters displaying potent
inhibitory activity (2, IC50 = 3 nM; 5, IC50 = 0.35 nM) as
compared with the longer and branched chain esters and bulky
aromatic ester groups (6ꢀ9, IC50 range = 140ꢀ400 nM). A
similar pattern was observed in the amide imidazole series with
the methyl amide ꢀCONHMe (11) showing optimal activity
(IC50 = 12 nM) as compared with the dimethyl amide (13) and
Furthermore, compound 17 was g6000-fold more potent
against CYP26 as compared to CYPs 1A2, 2C9, 2C19, and 2D6,
with compound 2 and R116010 g 200- and g400-fold more
potent, respectively. However, although all three compounds
inhibited CYP3A4 with submicromolar activity, an improvement
was observed with the triazole methyl ester (17) showing
reduced potency against CYP3A4 (IC50 = 0.33 μM) as compared
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Figure 5. Optimal SAR for the 2,2-dimethyl-3-(4-(naphthalen-2-ylamino)phenyl) propyl azole derivatives.
branched isopropyl amide (12). These data would suggest that
one H-bond donor and a small alkyl group are preferred for
activity in the amide series. Although the esters (6ꢀ9) and
amides (10, 12, and 13) were less active than the lead compound
(2), they were more active than liarozole (IC50 = 540 nM). The
2,2-dimethyl-3-propanol derivative (15) also showed good in-
hibitory activity (IC50 = 40 nM), suggesting that a carbonyl
group is not essential for activity.
(United Kingdom). Acetic acid and ammonium acetate were obtained from
Fisher Scientific (United Kingdom). All solvents used for chromatography
were high-performance liquid chromatography (HPLC) grade from Fisher
Scientific.
1H and 13C NMR spectra were recorded with a Bruker Avance
DPX500 spectrometer operating at 500 and 125 MHz, with Me4Si as an
internal standard. Mass spectra were determined by the EPSRC mass
spectrometry center (Swansea, United Kingdom). Microanalyses were
determined by Medac Ltd. (Surrey, United Kingdom). CYP26 selec-
tivity assays were performed by Cyprotex (Cheshire, United Kingdom).
Flash column chromatography was performed with silica gel 60 (230ꢀ
400 mesh) (Merck), and thin-layer chromatography (TLC) was carried
out on precoated silica plates (kiesel gel 60 F254, BDH). Compounds
were visualized by illumination under UV light (254 nm). Melting points
were determined on an electrothermal instrument and are uncorrected.
All solvents were dried prior to use as described by the handbook
Purification of Laboratory Chemicals32 and stored over 4 Å molecular
sieves, under nitrogen. All compounds were more than 95% pure.
Cell Culture and Retinoid Treatment. SH-SY5Y or MCF-7
cells were cultured at 37 °C in RPMI 1640 medium containing fetal calf
serum (10%) and L-glutamine (2 mM) in a humidified atmosphere of 5%
CO2 in air. ATRA was dissolved in dimethyl sulfoxide and added to the
culture medium as described by Armstrong et al.21 Liarozole, R116010,
and imidazole/triazole derivatives were dissolved in ethanol and diluted
in cell culture medium. The final concentration of ethanol in all experi-
ments never exceeded 0.8%.
The triazole equivalents of the most potent compounds in the
imidazole series, the methyl ester (2) and methyl amide (11),
were prepared for evaluation. The triazole methyl ester (17) was
more potent with an IC50 of 0.35 nM, while the triazole methyl
amide (19) showed a reduction in activity with an IC50 of
350 nM as compared with the corresponding imidazole deriva-
tives. The triazole carboxylic acid derivative (18) showed a loss in
activity (IC50 > 1 μM) as compared with the imidazole carboxylic
acid (4, IC50 = 350 nM); this loss in activity may be related to
reduced uptake; however, the metabolic stability of the ester,
assessed previously,22 would suggest that the ester is the active
inhibitor rather than acting as a prodrug for the carboxylic acid.
Compounds with biochemical CYP26 inhibitory IC50 values
e50 nM also enhanced the biological activity of exogenous
ATRA, as evidenced by a 3.7ꢀ5.8-fold increase in CYP26A1
induction as compared to ATRA alone. All compounds demon-
strated an activity comparable with or better than R116010, and
the induction correlated well with CYP26 inhibition data. Direct
comparison of CYP selectivity profiles for triazole 17 and
R116010 revealed an almost identical CYP selectivity profile;
however, the greater CYP26 inhibitory activity of 17 as compared
with R116010 resulted in compound 17 being g6000-fold more
potent against CYP26 as compared with CYPs 1A2, 2C9, 2C19,
and 2D6, with R116010 g 400-fold more potent, respectively.
Importantly, the triazole (17) displayed improved CYP3A4
selectivity (1100-fold more selective for CYP26) as compared
with the imidazole (2) (<33-fold more selective for CYP26).
The studies have allowed evaluation of SAR with key func-
tional groups and optimal interactions noted (Figure 5), with the
promising biological activity profile of the triazole ester 17,
suggesting that it is an appropriate candidate for further evalua-
tion and development for use in the treatment of dermatological
diseases.
Microsomal CYP26 Inhibition Assay. MCF-7 cells were pre-
treated for 24 h with 1 μm ATRA to induce CYP26 expression.
Microsomes were prepared as described by Han and Choi.29 Briefly,
cells were homogenized in buffer A [10 mM Tris, pH 7.4, 1 mM EDTA,
0.5 M sucrose, and Complete Protease Inhibitor Cocktail (Roche,
United Kingdom)] using a Dounce homogenizer, diluted with an equal
volume of Tris/EDTA, and the diluted homogenate laid over a volume
of buffer A equal to the original volume. Microsomes were then isolated
by differential centrifugation (9000g, 10 min, 4 °C; 100000g, 60 min,
4 °C). The microsomal pellet was suspended in buffer B (10 mM Tris,
pH 7.4, 1 mM EDTA, 0.25 M sucrose, and Complete Protease Inhibitor
Cocktail) and stored at ꢀ70 °C. Cytochrome c reductase activity was
calculated at 5ꢀ15 U cyt c/μg protein, using the cytochrome c reductase
(NADPH) kit (Sigma) according to the manufacturer's instructions. For
ATRA metabolism, 50 μg of 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, and 0.1 μCi 3H-ATRA) in
amber eppendorfs in the absence or presence of CYP26 inhibitor
(1ꢀ1000 nM) in a final volume of 200 μL for 1 h at 37 °C with shaking.
The reaction was quenched with acetonitrile, mixed, and then centri-
fuged (18000g, 5 min, 4 °C). Resolution of retinoids was performed
with a Luna C18(2) column (3 μm, 50 mm ꢁ 2 mm) using a Waters
’ EXPERIMENTAL SECTION
General Procedures. [11,12-3H]-All trans-retinoic acid (37 MBq/mL)
and Ultima Flo M scintillation fluid were purchased from Perkin-Elmer
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2690 Separations Module and subsequent Radiomatic Series 500TR
Flow Scintillation Analyzer (Packard Biosciences), with Empower 2
Chromatography Data Software and Flow-ONE software, respectively,
Ar), 7.82 (m, 2H, Ar). EI-HRMS (M + H)+ found, 456.2637; calcd for
C29H33N3O2, 456.2632.
Isopropyl 3-(1H-Imidazol-1-yl)-2,2-dimethyl-3-[4-(naphthalen-2-yl-
amino)phenyl]propanoate (8). Compound 8 was prepared from reac-
tion of (2) and isopropylbromide in 68% yield as a white solid; mp
166ꢀ168 °C. TLC (97:3 CH2Cl2/MeOH, Rf = 0.65). 1H NMR
(CDCl3): δ 1.11 (d, J = 6.8 Hz, 6H, iPr), 1.29 (s, 6H, H-3, H-4), 4.93
3
3
for data acquisition. H-ATRA and H metabolites 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 between 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. The scintillant flow rate was 1 mL/min.
CYP26 inhibition was calculated as the percentage 3H ATRA metabolite
i
(sep, J = 6.8 Hz, 1H, Pr), 5.54 (s, 1H, H-5), 6.13 (s, 1H, NH), 7.07
(d, J = 8.1 Hz, 2H, Ar), 7.19 (m, 2H, Ar), 7.27 (m, 2H, Ar), 7.31 (s, 1H,
imid), 7.39 (m, 1H, Ar), 7.50 (m, 2H, Ar), 7.68 (d, J = 8.2 Hz, 2H, Ar),
7.76 (d, J = 8.3 Hz, 2H, Ar). EI-HRMS (M)+ found, 428.2337; calcd for
C27H29N3O2, 428.2338.
Benzyl 3-(1H-Imidazol-1-yl)-2,2-dimethyl-3-[4-(naphthalen-2-ylamino)-
phenyl]propanoate (9). Compound 9 was prepared from reaction of
(2) and benzylbromide in 57% yield as a yellow oil. TLC (97:3 CH2Cl2/
MeOH, Rf = 0.65). 1H NMR (CDCl3): δ 1.25 (s, 6H, H-3, H-4), 5.06
(s, 2H, benzyl), 5.55 (s, 1H, H-5), 5.94 (s, 1H, NH), 7.08 (d, J = 8.0 Hz,
2H, Ar), 7.15 (d, J = 7.8 Hz, 2H, Ar), 7.21 (m, 4H, Ar), 7.36 (m, 5H, Ar),
7.47 (m, 3H, Ar), 7.66 (d, J = 8.1 Hz, 1H, Ar), 7.71 (m, 2H, Ar). EI-
HRMS (M + H)+ found, 476.2323; calcd for C31H29N3O2, 476.2319.
3-(1H-Imidazol-1-yl)-2,2-dimethyl-3-[4-(naphthalen-2-
3
peak area formation (activity H metabolite(s)/total activity) as com-
pared to metabolite formation in the absence of inhibitor. IC50 values
were calculated by nonlinear regression analysis in SigmaPlot (Systat
Software Inc., United States) using an inhibition curve constructed from
a minimum of four data points. Compound 2 was used as a standard in
every assay.
Real-Time Polymerase Chain Reaction (PCR) for CYP26A1
Expression. RNA was isolated using an RNeasy Kit (Qiagen, Crawley,
United Kingdom), reverse-transcribed and real-time PCR performed on
20 ng of cDNA using TaqMan Gene Expression products for human
CYP26A1 in combination with the TaqMan Universal PCR master mix
(Applied Biosystems, Warrington, United Kingdom) on a GeneAmp
5700 Sequence Detection System as described previously.21
Chemistry. General Method—Synthesis of the Esters (5ꢀ9).
Methyl 3-(1H-imidazol-1-yl)-2,2-dimethyl-3-[4-(naphthalen-2-ylamino)-
phenyl]propanoate (2)22 (2.0 mmol) in EtOH (20 mL) was saponified
with KOH (4.0 mmol) under reflux for 72 h. The solvent was removed
underreducedpressure, then H2O(100 mL) wasadded, andtheunreacted
starting materials were extractedwith diethyl ether (3ꢁ 50 mL). The H2O
layer was reduced in vacuo to give the potassium salt (3), which was
dissolved in anhydrous DMF. Alkylbromide (2.4 mmol) was added, and
the mixture was heated at 80 °C for 2 h. H2O (70 mL) was then added,
and the aqueous layer was extracted with EtOAc (3 ꢁ 50 mL). The
organic layer was dried (MgSO4), filtered, and reduced in vacuo. The
product was purified by flash column chromatography (CH2Cl2ꢀ
MeOH 100:0 v/v increasing to 97:3 v/v).
Ethyl 3-(1H-Imidazol-1-yl)-2,2-dimethyl-3-[4-(naphthalen-2-ylamino)-
phenyl]propanoate (5). Compound 5 was prepared from reaction of
(2) and ethylbromide in 71% yield as a white solid; mp 156ꢀ158 °C.
TLC (97:3 CH2Cl2/MeOH, Rf = 0.62). 1H NMR (CDCl3): δ 1.21 (t, J =
7.0 Hz, 3H, CH2CH3), 1.37 (s, 6H, H-3, H-4), 4.13 (q, J = 7.0 Hz, 1H,
CH2CH3), 5.58 (s, 1H, H-5), 6.11 (s, 1H, NH), 7.08 (m, 3H, Ar), 7.19
(m, 2H, Ar), 7.25 (m, 2H, Ar), 7.31 (s, 1H, imid), 7.35 (m, 1H, Ar), 7.41
(m, 1H, Ar), 7.49 (d, J = 8.2 Hz, 1H, Ar), 7.77 (m, 1H, Ar), 7.85 (m, 2H,
Ar). EI-HRMS (M + H)+ found, 414.2179; calcd for C26H27N3O2,
414.2181.
Butyl 3-(1H-Imidazol-1-yl)-2,2-dimethyl-3-[4-(naphthalen-2-ylamino)-
phenyl]propanoate (6). Compound 6 was prepared from reaction of
(2) and butylbromide in 67% yield as a yellow oil. TLC (97:3 CH2Cl2/
MeOH, Rf = 0.65). 1H NMR (CDCl3): δ 0.96 (t, J = 6.9 Hz, 3H, butyl),
1.23 (m, 8H, CH2-butyl, H-3, H-4), 1.48 (m, 2H, butyl), 4.12 (t, J = 6.6
Hz, 2H, butyl), 5.08 (s, 1H, H-7), 5.93 (s, 1H, NH), 7.15 (d, J = 8.0 Hz,
2H, Ar), 7.22 (m, 2H, Ar), 7.41 (m, 3H, Ar), 7.50 (s, 1H, imid), 7.52
(s, 1H, imid), 7.74 (d, J = 8.2 Hz, 2H, Ar), 7.82 (m, 3H, Ar). EI-HRMS
(M + H)+ found, 442.2483; calcd for C28H31N3O2, 442.2489.
Pentyl 3-(1H-Imidazol-1-yl)-2,2-dimethyl-3-[4-(naphthalen-2-ylamino)-
phenyl]propanoate (7). Compound 7 was prepared from reaction of
(2) and pentylbromide in 69% yield as a yellow oil. TLC (97:3 CH2Cl2/
MeOH, Rf = 0.65). 1H NMR (CDCl3): δ 0.95 (t, J = 6.8 Hz, 3H, pentyl),
1.25 (m, 10H, 2 ꢁ CH2 pentyl, H-3, H-4), 1.52 (m, 2H, pentyl), 4.08 (t,
J = 6.5 Hz, 2H, pentyl), 5.55 (s, 1H, H-5), 6.01 (s, 1H, NH), 7.19 (m, 4H,
Ar), 7.33 (m, 3H, Ar), 7.42 (m, 1H, Ar), 7.48 (m, 2H, Ar), 7.74 (m, 2H,
ylamino)phenyl] Propanoic Acid (4). Methyl 3-(1H-imidazol-1-yl)-2,2-
dimethyl-3-[4-(naphthalen-2-ylamino)phenyl]propanoate (2)22 (2.0mmol)
in EtOH (20 mL) was saponified with KOH (4.0 mmol) under reflux for
72 h. The solvent was removed under reduced pressure, H2O (100 mL)
was added, and the unreacted starting materials were extracted with
ether (3 ꢁ 50 mL). The H2O layer was acidified with 1 N aqueous HCl
until pH 3 and extracted with CH2Cl2 (100 mL). The organic layer
was dried (MgSO4), and the solvent was removed under reduced
pressure to give the product in 64% yield as a brown solid; mp 148ꢀ
1
150 °C. TLC (97:3 CH2Cl2/MeOH, Rf = 0.21). H NMR (CDCl3):
δ 1.25 (s, 3H, H-3), 1.33 (s, 3H, H-4), 5.67 (s, 1H, H-NH), 7.07 (d, J =
8.0 Hz, 2H, Ar), 7.11 (s, 1H, H-5), 7.17 (s, 1H, Ar), 7.25 (m, 2H, Ar),
7.32 (m, 3H, Ar), 7.47 (s, 1H, imid), 7.50 (s, 1H, imid), 7.71 (d, J =
8.2 Hz, 1H, Ar), 7.83 (s, 1H, imid), 7.77 (m, 2H, Ar), 8.23 (s, 1H,
CO2H). EI-HRMS (M + H)+ found, 386.1865; calcd for C24H23N3O2,
386.1863.
General Method—Synthesis of the Amides. EDC (0.18 mmol),
carboxylic acid (4 or 18) (0.18 mmol), and HOBt (0.18 mmol) in
CH2Cl2 (10 mL) were stirred at room temperature under N2 for 0.5 h.
Amine (0.56 mmol) was added, and the reaction mixture was stirred
overnight. EtOAc (70 mL) was added, and the organic layer was washed
with 1 M aqueous 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 purified by
flash column chromatography (CH2Cl2ꢀMeOH 100:0 v/v increasing
to 98:2 v/v).
3-(1H-Imidazol-1-yl)-2,2-dimethyl-3-[4-(naphthalen-2-ylamino)phenyl]-
propanamide (10). Compound 10 was prepared from reaction of (4)
and methanolic NH3 in 71% yield as a brown oil. TLC (97:3 CH2Cl2/
MeOH, Rf = 0.47). 1H NMR (CDCl3): δ 1.27 (s, 3H, H-3), 1.29 (s, 3H,
H-4), 5.53 (s, 1H, H-5), 5.59 (s, 2H, NH2), 5.89 (s, 1H, NH), 7.05 (d, J =
8.2 Hz, 2H, Ar), 7.17 (s, 1H, imid), 7.22 (m, 4H, Ar), 7.29 (m, 1H, Ar),
7.35 (m, 1H, Ar), 7.46 (s, 1H, imid), 7.63 (d, J = 8.0 Hz, 1H, Ar), 7.73 (d,
J = 8.1 Hz, 2H, Ar), 7.81 (s, 1H, imid). EI-HRMS (M + H)+ found,
385.2026; calcd for C24H24N4O, 385.2028.
3-(1H-Imidazol-1-yl)-N,2,2-trimethyl-3-[4-(naphthalen-2-ylamino)-
phenyl]propanamide (11). Compound 11 was prepared from reaction
of (4) and 33% methylamine in absolute EtOH in 75% yield as a brown
1
solid; mp 120ꢀ124 °C. TLC (97:3 CH2Cl2/MeOH, Rf = 0.50). H
NMR (CDCl3): δ 1.26 (s, 3H, H-3), 1.29 (s, 3H, H-4), 2.75 (d, J =
2.5 Hz, 3H, NHCH3), 5.62 (s, 1H, H-5), 5.77 (d, J = 2.5 Hz, 1H,
NHCH3), 6.11 (s, 1H, NH), 7.04 (s, 1H, imid), 7.09 (d, J = 8.2 Hz, 1H,
Ar), 7.15 (s, 1H, imid), 7.22 (m, 4H, Ar), 7.30 (m, 1H, Ar), 7.39 (m, 1H,
6809
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Journal of Medicinal Chemistry
ARTICLE
Ar), 7.45 (d, J = 2.6 Hz, 1H, Ar), 7.65 (m, 2H, Ar), 7.76 (d, J = 8.1 Hz,
2H, Ar). Anal. C, H, N.
1H, Ar), 7.34 (ddd, J = 1.1, 7.0, 8.1 Hz, 1H, Ar), 7.40ꢀ7.45 (m, 3H, Ar),
7.49 (d, J = 2.1 Hz, 1H, Ar), 7.68 (d, J = 8.1 Hz, 1H, Ar), 7.76 (d, J =
8.8 Hz, 2H, Ar), 8.00 (s, 1H, H-triaz), 8.14 (s, 1H, triaz). EI-HRMS
(M + Na)+ found, 423.1786; calcd for C24H24N4O2Na, 423.1789.
2,2-Dimethyl-3-(4-(naphthalen-2-ylamino)phenyl)-3-(1H-1,2,4-
triazol-1-yl)propanoic Acid (18). To a solution of (17) (2 mmol) in
THF (5 mL) was added an aqueous solution of LiOH (4 mmol in 5 mL
H2O), and the mixture was homogenized with methanol and heated
under gentle reflux for 2 h. The solvent was removed under reduced
pressure, H2O (100 mL) was added, and the unreacted starting materials
were extracted with ether (3 ꢁ 50 mL). The H2O layer was acidified with
1 N HCl until pH 3 and extracted with ether (100 mL). The organic
layer was dried (MgSO4), and the solvent was removed under reduced
pressure to give the product as a brown oil in 77% yield. TLC (97:3
CH2Cl2/MeOH, Rf = 0.36). 1H NMR (CDCl3): δ 1.23 (s, 3H, H-3),
1.31 (s, 3H, H-4), 5.73 (s, 1H, H-5), 6.27 (s, 1H, NH), 7.13 (d, J =
7.3 Hz, 2H, Ar), 7.32ꢀ7.42 (m, 6H, Ar), 7.69 (d, J = 7.4 Hz, 1H, Ar),
7.76 (d, J = 7.5 Hz, 2H, Ar), 8.01 (s, 1H, triaz), 8.22 (s, 1H, COOH),
8.31 (s, 1H, triaz). EI-HRMS (M + H)+ found, 387.1816; calcd for
C23H23N4O2, 387.1818.
Molecular Modeling. All molecular modeling studies were per-
formed on a MacPro dual 2.66 GHz Xeon running Ubuntu 9. Ligand
structures were built in MOE33 minimized using the MMFF94x force-
field until a rmsd gradient of 0.05 kcal molꢀ1 Åꢀ1 was reached. Docking
simulations were performed using PLANTS34 (aco_ants 20; aco_evap
0.15; aco_sigma 5.0), with ligands docked within the active site of the
CYP26A1 homology model,31 and the results were visualized in MOE.
Molecular dynamics simulations were performed with GROMACS35,36
and the Gromos96 force field in a NPT environment. Individual ligand/
protein complexes obtained from the docking results were soaked in a
triclinic water box and minimized using a steepest descent algorithm to
remove unfavorable van der Waals contacts. The system was then
equilibrated via a 100 ps MD simulation at 300 K with restrained
ligand/protein complex atoms. Finally, a 5 ns simulation was performed
at 300 K with a time step of 2 fs and hydrogen atoms constrained with a
LINCS algorithm. Visualization of the dynamics trajectories was per-
formed with the VMD software package, version 1.8.3.37
3-(1H-Imidazol-1-yl)-N-isopropyl-2,2-dimethyl-3-[4-(naphthalen-
2-ylamino)phenyl]propanamide (12). Compound 12 was prepared
from reaction of (4) and isopropylamine in 64% yield as a pale yellow
oil. TLC (97:3 CH2Cl2/MeOH, Rf = 0.56). 1H NMR (CDCl3): δ 1.08
(d, J = 6.6 Hz, 6H, NHiPr), 1.25 (s, 3H, H-3), 1.29 (s, 3H, H-4), 4.13
(sep, J = 6.6 Hz, 1H, NHiPr), 5.41 (d, J = 6.6 Hz, 1H, NHiPr), 5.63
(s, 1H, NH), 6.16 (s, 1H, H-5), 7.08 (m, 3H, Ar), 7.23 (m, 4H, Ar), 7.30
(m, 1H, Ar), 7.44 (m, 2H, Ar), 7.65 (d, J = 8.2 Hz, 2H, Ar), 7.74 (d, J =
8.3 Hz, 2H, Ar). EI-HRMS (M + H)+ found, 427.2492; calcd for
C27H30N4O, 427.2492.
3-(1H-Imidazol-1-yl)-N,N,2,2-tetramethyl-3-[4-(naphthalen-2-yla-
mino)phenyl]propanamide (13). Compound 13 was prepared from
reaction of (4) and dimethylamine (2 M in THF) in 61% yield as a
brown oil. TLC (97:3 CH2Cl2/MeOH, Rf = 0.59). 1H NMR (CDCl3):
δ 1.37 (s, 3H, H-3), 1.41 (s, 3H, H-4), 2.95 (s, 6H, N(CH3)2), 5.80
(s, 1H, H-5), 6.23 (s, 1H, NH), 7.08 (m, 4H, Ar), 7.25 (m, 3H, Ar), 7.36
(m, 1H, Ar), 7.41 (m, 1H, Ar), 7.45 (d, J = 2.6 Hz, 1H, Ar), 7.66 (d, J =
7.8 Hz, 1H, Ar), 7.72 (m, 3H, Ar). 13C NMR (CDCl3): δ 24.34 (CH3,
C-3), 25.16 (CH3, C-4), 38.75 (2 ꢁ CH3, N(CH3)2), 47.93 (C, C-2),
68.69 (CH, C-5), 112.91, 116.91, 120.50, 123.86, 123.92, 126.52, 127.65
(10 ꢁ CH), 128.11 (C), 129.24 (CH, imid), 129.51 (C), 129.93 (CH,
imid), 129.99 (2 ꢁ CH), 134.49, 139.86, 143.67, 175.05 (4 ꢁ C). EI-
HRMS (M + H)+ found, 413.2335; calcd for C26H28N4O, 413.2336.
N,2,2-Trimethyl-3-[4-(naphthalen-2-ylamino)phenyl]-3-(1H-1,2,4-
triazol-1-yl)propanamide (19). This compound was prepared from
(18) and 33% methylamine in absolute EtOH in 63% yield as a pale
yellow oil. TLC (98:2 CH2Cl2/MeOH, Rf = 0.51). 1H NMR (CDCl3):
δ 1.33 (s, 6H, H-3, H-4), 2.71 (d, J = 2.7 Hz, 3H, NHCH3), 5.78 (d, J =
2.8 Hz, 1H, NHCH3), 5.87 (s, 1H, NH), 7.08 (d, J = 7.5 Hz, 2H, Ar),
7.16ꢀ7.19 (m, 1H, Ar), 7.26ꢀ7.29 (m, 1H, Ar), 7.37ꢀ7.40 (m, 1H, Ar),
7.45ꢀ7.50 (m, 3H, Ar), 7.65 (d, J = 7.6 Hz, 1H, Ar), 7.74 (d, J = 7.5 Hz,
2H, Ar), 7.99 (s, 1H, triaz), 8.19 (s, 1H, triaz). EI-HRMS (M)+ found,
400.2133; calcd for C24H25N5O, 400.2132.
3-(1H-Imidazol-1-yl)-2,2-dimethyl-3-[4-(naphthalen-2-ylamino)-
phenyl]propan-1-ol (15). A solution of (14)22 (0.23 mmol) in dry
THF (10 mL) under N2 was cooled to 0 °C. LiAlH4 (1 M in THF,
1.2 mmol) was added dropwise, and the reaction mixture was stirred at
0 °C for 1 h and then at room temperature overnight. The reaction
was quenched by the addition of EtOAc (70 mL). The organic layer
was washed with H2O (50 mL), dried (MgSO4), and reduced in vacuo.
The residue was purified by flash column chromatography (CH2Cl2ꢀ
MeOH 100:0 v/v increasing to 97:3 v/v) to give the product as a light
brown oil in 79% yield. TLC (97:3 CH2Cl2/MeOH, Rf = 0.60).
1H NMR (CDCl3): δ 1.07 (s, 3H, H-3), 1.71 (s, 3H, H-4), 2.48
(s, 1H, OH), 3.26 (d, J = 8.0 Hz, 1H, CH2OH), 3.30 (d, J = 8.0 Hz, 1H,
CH2OH), 3.91 (s, 3H, OCH3), 5.38 (s, 1H, H-5), 5.91 (s, 1H, H-NH),
7.03 (d, J = 7.8 Hz, 2H, Ar), 7.11 (m, 3H, Ar), 7.22 (m, 2H, Ar), 7.33 (d, J =
7.7 Hz, 2H, Ar), 7.42 (d, J = 2.8 Hz, 1H, Ar), 7.57 (d, J = 8.1 Hz, 1H, Ar),
7.69 (d, J = 8.2 Hz, 1H, Ar), 7.73 (s, 1H, Ar). EI-HRMS (M)+ found,
402.2176; calcd for C25H27N3O2, 402.2176.
’ ASSOCIATED CONTENT
S
Supporting Information. Numbering of compounds for
b
1H and 13C NMR characterization and 13C NMR spectroscopic
data for compounds 4ꢀ 12, 15, and 17ꢀ19. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Authors
*Tel: +44(0)191-222 5644. Fax: +44(0)191-222 7179. E-mail: j.l.
armstrong@newcastle.ac.uk (J.L.A.). Tel: +44(0)2920-876307.
Fax: +44(0)2920-874149. E-mail: SimonsC@Cardiff.ac.uk (C.S.).
Methyl 2,2-Dimethyl-3-[4-(naphthalen-2-ylamino)phenyl]-3-(1H-
1,2,4-triazol-1-yl)propanoate (17). To a solution of alcohol (16)22
(1.5 mmol) in anhydrous CH3CN (20 mL) were added triazole
(6 mmol) and CDT (3 mmol). The mixture was then heated under
reflux for 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 (CH2Cl2ꢀMeOH 100:0 v/v
increasing to 98:2 v/v) to give the product as a brown oil. Yield, 63%.
’ ACKNOWLEDGMENT
We acknowledge Cancer Research UK for funding (M.S.G. and
C.E.B., Grant ref. C7735/A9612) and the EPSRC Mass Spectro-
metry Centre, Swansea, United Kingdom, for mass spectroscopy data.
’ ABBREVIATIONS USED
1
TLC (99:1 CH2Cl2/MeOH, Rf = 0.57). H NMR (CDCl3): δ 1.37
ATRA, all-trans-retinoic acid; RA, retinoic acid; RAMBAs, reti-
noic acid metabolism blocking agents
(s, 3H, H-3), 1.40 (s, 3H, H-4), 3.67 (s, 3H, OCH3), 6.16 (s, 1H, CH),
6.25 (s, 1H, NH), 7.10 (d, J = 8.7 Hz, 2H, Ar), 7.25 (dd, J = 2.3, 8.7 Hz,
6810
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Journal of Medicinal Chemistry
ARTICLE
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