Synthesis, molecular modelling and CYP24A1 inhibitory activity of novel of (E)-N-(2-(1H-imidazol-1-yl)-2-(phenylethyl)-3/4-styrylbenzamides

Article abstract of DOI:10.1016/j.bmc.2017.05.055

CYP24A1 (25-hydroxyvitamin D-24-hydroxylase) is a useful enzyme target for a range of medical conditions including cancer, cardiovascular and autoimmune disease, which show elevated CYP24A1 levels and corresponding reduction of calcitriol (the biologically active form of vitamin D). A series of (E)-N-(2-(1H-imidazol-1-yl)-2-(phenylethyl)-3/4-styrylbenzamides have been synthesised using an efficient synthetic route and shown to be potent inhibitors of CYP24A1 (IC₅₀ 0.11–0.35?μM) compared with the standard ketoconazole. Molecular modelling using our CYP24A1 homology model showed the inhibitors to fill the hydrophobic binding site, forming key transition metal interaction between the imidazole nitrogen and the haem Fe³⁺ and multiple interactions with the active site amino acid residues.

Full text of DOI:10.1016/j.bmc.2017.05.055

Accepted Manuscript
Synthesis, molecular modelling and CYP24A1 inhibitory activity of novel of
(E)-N-(2-(1H-imidazol-1-yl)-2-(phenylethyl)-3/4-styrylbenzamides
Ismail M. Taban, Jinge Zhu, Hector F. DeLuca, Claire Simons
PII:
S0968-0896(17)30897-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.05.055
BMC 13774
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
28 April 2017
19 May 2017
26 May 2017
Please cite this article as: Taban, I.M., Zhu, J., DeLuca, H.F., Simons, C., Synthesis, molecular modelling and
CYP24A1 inhibitory activity of novel of (E)-N-(2-(1H-imidazol-1-yl)-2-(phenylethyl)-3/4-styrylbenzamides,
Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.05.055
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Synthesis, molecular modelling and CYP24A1 inhibitory activity of novel
of (E)-N-(2-(1H-imidazol-1-yl)-2-(phenylethyl)-3/4-styrylbenzamides
a
b
b
a*
Ismail M. Taban, Jinge Zhu, Hector F. DeLuca and Claire Simons
a
Medicinal Chemistry, School of Pharmacy & Pharmaceutical Sciences, Cardiff University,
b
King Edward VII Avenue, Cardiff CF10 3NB, UK; Department of Biochemistry, University
of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706-1544, USA.
Corresponding author: Tel.: +44 (0) 2920 876307; fax: +44 (0) 2920 874149.
E-mail address: simonsc@cardiff.ac.uk (C. Simons)
1

Key words:
Vitamin D
(E)-N-(2-(1H-imidazol-1-yl)-2-(phenylethyl)-3/4-styrylbenzamides
CYP24A1
Enzyme inhibition
Molecular modelling
Abstract
CYP24A1 (25-hydroxyvitamin D-24-hydroxylase) is a useful enzyme target for a range of medical
conditions including cancer, cardiovascular and autoimmune disease, which show elevated CYP24A1
levels and corresponding reduction of calcitriol (the biologically active form of vitamin D). A series
of (E)-N-(2-(1H-imidazol-1-yl)-2-(phenylethyl)-3/4-styrylbenzamides have been synthesised
using an efficient synthetic route and shown to be potent inhibitors of CYP24A1 (IC 0.11-
5
0
0
.35 M) compared with the standard ketoconazole. Molecular modelling using our
CYP24A1 homology model showed the inhibitors to fill the hydrophobic binding site,
3
+
forming key transition metal interaction between the imidazole nitrogen and the haem Fe
and multiple interactions with the active site amino acid residues.
2

1
. Introduction
Vitamin D is the starting compound of an endocrine system, and plays a principal role in the
maintenance of calcium and phosphate homeostasis and is essential for the maintenance of a
1
healthy skeleton . Vitamin D is produced in the epidermis from 7-dehydrocholestrol, by
sunlight or UV light. Vitamin D can also be obtained from dietary sources. Previtamin D is
3
produced by the opening of the steroid nucleus (B ring broken by UV light with spectrum 280
2
3
-
320 UV β) . Previtamin D isomerises into Vitamin D . Additionally the three most
3 3
important steps in vitamin D metabolism, 25-hydroxylation, 1 -hydroxylation, and 24-
4
hydroxylation are all performed by cytochrome P450 enzymes .
UVB
Vitamin D3
7- Dehydrocholesterol
Previtamin D
Skin
CYP2R liver
25- hydroxyvitamin D3
Complex with binding
protein in liver
CYP27B1
Renal tubles
Calcitriol
PTH
CYP24A1
Degradation
Calcitroic acid
inactive
=
stimulate the action
inhibit the action
=
Excreted in urine
Figure 1. Metabolic pathway of Vitamin D
The initial step in vitamin D metabolism is 25 hydroxylation (endoplasmic reticulum) by
CYP2R1 (25-hydroxylase),
(the circulatory form of
3
found in the liver to produce 25(OH)D
3

2
vitamin D)
. 1,25(OH) D (calcitriol), the active form of vitamin D, is produced in
2
3
(Figure 1)
the renal distal convoluted tubule at position 1α by CYP27B1 a cytochrome P450
monooxygenase which helps many reactions involved in drug metabolism and the synthesis
5
of cholesterol, steroids, and other lipids . CYP27B1 is stimulated by parathyroid hormone
6
(
PTH) and is indirectly deactivated by fibroblast growth factor (FGF-23) (Figure 1) .
The main function of calcitriol is mineralisation of the skeleton and maintaining levels of
7
,8
calcium . However, calcitriol also plays a role in many other organs with vitamin D
9
,10
11,12
deficiency linked to chronic kidney disease , cardiovascular disease
, autoimmune
1
3,14
15,16,17,18,19
disease
and cancer
. Reduction in calcitriol levels is linked with increased
2
0
CYP24A1 levels, which suggests that CYP24A1 may be a useful therapeutic target . Many
cancer cell lines display elevated levels of CYP24A1 expression as they progress to more
tumourigenic phenotypes, suggesting that these tumours have increased ability to catabolise
calcitriol. Subsequently, several CYP24A1 inhibitors have been designed for the treatment of
diseases associated with elevated vitamin D catabolism. Azole-based compounds can
inactivate a broad range of cytochrome P450 enzymes by binding to the haem moiety present
in the enzyme active site. Through the coordination of the azole nitrogen to the haem, it
blocks the catalytic cycle of the P450 and prevents oxygen activation required for substrate
oxidation. Despite their lack of specificity towards just CYP24A1, ketoconazole and liarazole
2
1
were shown to extend the half-life of calcitriol in prostate cancer cells in vitro and in vivo .
22,23,24,25
We have previously described azole CYP24A1 inhibitors
, of these the
2
4
styrylimidazole series was the most promising with potent CYP24A1 inhibitory activity
observed (Figure 2).The aim of this research was the design and synthesis of novel potent
inhibitors of CYP24A1 to enhance the endogenous levels of circulating calcitriol.
4

Figure 2. Lead styrylimidazoles and designed inhibitors
The lead styrylimidazoles (Figure 2) have been modified and developed, specifically
through (i) substitution of the phenyl ring on the imidazole side to explore structure
activity-relationships; (ii) substitution of the styrene benzene rings to explore structure
activity-relationships and (iii) variation of the alkene position to allow complete filling of
the CYP24A1 active site.
2
. Results and Discussion
2
.1 Chemistry
5

Both the 4- and 3-substituted final compounds, (E)-N-(2-(1H-imidazol-1-yl)-2-(phenyl)ethyl)-4-
styrylbenzamides (9) and (E)-N-(2-(1H-imidazol-1-yl)-2-(phenyl)ethyl)-3-styrylbenzamides (15),
were obtained in an efficient three step synthesis from the precursor styrylbenzoic acids and 2-amino-
1
-phenylethanol derivatives.
.1.1. Synthesis of (E)-N-(2-(1H-imidazol-1-yl)-2-(phenyl)ethyl)-4-styrylbenzamides
2
The extension of the (E)-N-(2-(1H-imidazol-1-yl)-2-(phenyl)ethyl)-4-styrylbenzamide series
required coupling of the appropriate 2-amino-1-phenylethanol (5) with a 4-styrylbenzoic acid
(8). The 2-amino-1-phenylethanol derivatives (5) were prepared in two steps, first the ꢀ-
nitroalcohols (4) were prepared from the corresponding benzaldehyde (3) and nitromethane
by the Henry reaction employing either NaOH, Amberseep 900 (OH) or triethylamine as the
2
6,27,28
base
. Reduction of the ꢀ-nitroalcohols to the corresponding amino derivatives (5) was
achieved using a slurry of Raney nickel and formic acid under H atmosphere at room
2
2
9
temperature for 6 h (Scheme 1) . The 4-styrylbenzoic acids (8) were conveniently prepared
from the corresponding alkene (6) and 4-bromobenzoic acid (7) under Heck reaction
conditions (Scheme 1).
o
Scheme 1. Reagents and conditions: (i) Method A CH NO , MeOH, NaOH, 0 C, 24 h;
3
2
Method B CH NO , Ambersep 900 (OH), rt, o/n; Method C CH NO , Et N, rt, 12 h (ii)
3
2
3
2
3
o
Raney Ni/H , MeOH, HCO H, rt, 6 h (iii) Pd(OAc) , tri(o-tolylphosphine), Et N, 100 C, o/n.
2
2
2
3
6

Coupling of the appropriate 2-amino-1-phenylethanol (5) with a 4-styrylbenzoic acid (8)
using CDI as the coupling reagent gave the benzamides (9). The dihydrooxazoles (10) were
23
prepared following the procedure of Aboraia et al and involved the formation of the 1,3-
oxazole ring by reacting benzamide (9) with methansulfonyl chloride and triethylamine in dry
THF. In the last step of the reaction scheme, heating the oxazole compound (10) in the
o
presence of imidazole for 48 h at 125 C opened the oxazole ring by nucleophilic
2
4
displacement (Scheme 2) . A reasonable yield of the imidazole compounds (11) was
produced after recrystallisation from ethylacetate or acetonitrile (Table 1).
Scheme 2. Reagents and conditions: (i) Carbonyldiimidazole, DMF, rt, o/n (ii) (a)
o
o
CH SO Cl, THF, 0 C, 3 h (b) Et N, rt, o/n (iii) imidazole, 125 C, 48 h.
3
2
3
2
.1.2. Synthesis of (E)-N-(2-(1H-imidazol-1-yl)-2-(phenyl)ethyl)-3-styrylbenzamides
7

In a similar manner the (E)-N-(2-(1H-imidazol-1-yl)-2-(phenyl)ethyl)-3-styrylbenzamide series
(
17) were prepared in moderate yields (Table 1) from the appropriate 3-styrylbenzoic acid
14) and 2-amino-1-phenylethanol (5) (Scheme 3).
(
o
Scheme 3. Reagents and conditions: (i) Pd(OAc) , tri(o-tolylphosphine), Et N, 100 C, o/n
2
3
o
(
ii) Carbonyldiimidazole, DMF, rt, o/n (iii) (a) CH SO Cl, THF, 0 C, 3 h (b) Et N, rt, o/n
3 2 3
o
(
iv) imidazole, 125 C, 48 h.
Table 1. Substitutions, yields and melting points of final imidazole products
1
2
3
o
Cmpd
1a
R
R
R
R
Yield (%)
Mp ( C)
1
4-Cl
H
H
-
12
238-240
8

1
1b
1c
1d
1e
1f
4-CF
3
3
H
H
-
-
33
35
27
32
35
38
32
48
33
208-210
174-178
134-136
218-222
204-208
158-160
148-152
126-130
110-114
1
2-CF
H
H
1
3,4-diMe
H
H
-
1
4-OCH
4-Cl
H
3
H
H
-
1
OCH
3
OCH
3
-
1
7a
7b
-
-
-
-
-
-
-
-
H
H
H
CF
1
4-Cl
1
7c
4-CF
3
17d
H
3
2
.2 Molecular modelling
To investigate the possible binding mode of this series of compounds, we have performed a
24,30
set of molecular docking simulations, using our model of CYP24A1
. We have previously
shown the importance of the styrylphenyl structure for inhibitory activity and that the
imidazole was essential for metal-ligand interaction with the haem group of the
2
3,24,25
enzyme
. Compounds 11a - 11f have electron withdrawing and electron donating
substituents on the phenyl ring. All compounds were designed without substitution on styrene
group except compound 11f, which has a 4-Cl on the phenyl ring and 3,4-dimethoxy
substitution on the styrene group. All the compounds reached the active site through the
vitamin D access tunnel and interacted with multiple hydrophobic residues (Arg128, Glu130,
Ile131, Trp134, Leu148, Asn208, Phe212, Ile239, Ile242, Lys243, Met246, Ser247, Phe249,
Ala326, Val328, Glu329, The330, Val391 and Thr500). Compounds 11b - 11d, formed
hydrogen bonds between the nitrogen of the amide group with Leu325. For compound 11e an
additional hydrogen bond between the methoxy group and Arg128 was observed. The
9

distance between the imidazole ring and the haem iron was between 2.18 Å and 2.58 Å
Table 2).
(
Table 2. (A) Distance between N of heterocycle of compounds 11a-11f and iron of haem, (B)
3
D, (C) 2D with binding interactions and (D) binding energy (kcal/mol)
C
D
Cpd
A
B
-8.9
1
1a
2.56 Å
-7.1
1
1b
2.18 Å
-7.9
1
1c
2.41 Å
1
0

-7.4
1
1d
2.42 Å
-11.2
1
1e
2.58 Å
-11.8
1
1f
2.44 Å
Compounds 17a - 17d were modified by changing the position of the alkene group from the 4 to 3-
position. All compound showed good interaction with the active site and interaction between the
imidazole nitrogen and the iron of the haem with distances ranging between 2.04 Å and 2.29 Å.
Hydrogen bonds were observed between the amide group, through the C=O and/or NH, and amino
acids Glu329, Thr330 and Leu325 (Table 3).
1
1

Table 3. (A) Distance between N of heterocycle of compounds 17a-17d and iron of haem,
B) 3D, (C) 2D with binding interactions and(D) binding energy (kcal/mol)
(
Cpd
A
B
C
D
-
11.0
1
7a
2.04 Å
-
10.4
1
7b
2.29 Å
-
7.3
1
7c
2.22 Å
1
2

-
7.6
1
7d
2.28Å
2
.3 CYP24A1 enzyme inhibition
All the final compounds 11a - 11f and 17a - 17d were evaluated to determine
3
1
CYP24A1inhibitory activity. The assay was performed according to the method of Zhu et al
and in general, the compounds displayed potent inhibition of CYP24A1 activity when
2
4
compared with the ketoconazole standard and the lead compound 1 (IC 0.40 ꢁꢂ) . The
5
0
IC values ranged between 11 and 35 ꢁꢂwith the best IC value was obtained for the 3,4-
5
0
50
dimethoxy styrylbenzamide derivative 11e (0.11 µM) (Table 4). Interestingly, the 4-methoxy
derivative 11e was also found to interact with the iron of the haem via the methoxy group
(distance 2.19 Å), which may contribute to the optimal inhibitory activity in this series of
compounds (Figure 3).
1
3

Figure 3. Alternative binding mode of 11e interacting with the iron of the haem through the
methoxy group
The unsubstituted 3-styrylbenzamide showed an improvement in inhibitory activity compared with
the 4-styrylbenzamide lead compound 1 (17a IC 0.19 M compared with lead 1 IC 0.40 M),
5
0
50
whereas the 4-chloro (17b/11a) and 4-trifluoromethyl (17c/11b) substituted 3- and 4-
styrylbenzamides were comparable. In the 4-styrylbenzamide series substitution at the 2-position of
the phenyl ring is less favoured e.g. 2-CF 11c IC 0.35 M compared with 4-CF 11b IC 0.18 M.
3
50
3
50
Substitutions in the styryl group would suggest that introduction of substituents at the 3,4-position
11f IC50 0.12 M) are beneficial for inhibitory activity and comparable with 3,5-dimethoxy
substitution (lead 2 IC 0.11 M), while substitutions in the 2-position are less favourable (17d IC
(
50
50
0.34 M).
1
4

Table 4. IC data against CYP24A1 of imidazole derivatives 11 and 17
5
0
1
2
3
Cmpd
1a
1b
1c
1d
1e
1f
Lead 1
R
R
R
IC (M)
Cmpd
R
R
IC (M)
50
50
1
4-Cl
H
H
H
H
0.21
0.18
0.35
0.31
0.11
0.12
0.40
17a
17b
17c
17d
H
H
H
0.19
0.16
0.15
0.34
0.5
1
4-CF
3
Cl
1
2-CF
3
H
H
CF
3
H
1
3,4-diMe
H
H
H
CF
3
1
4-OCH
4-Cl
H
3
H
H
Ketoconazole
1
OCH
H
3
OCH
H
3
In general, binding energies (Tables 2 and 3) and IC showed a good correlation with the exception
5
0
of compounds 11b and 17c. Substitution of these compounds with the more bulky trifluoromethyl
group in the para position of the phenyl ring results in a slightly less favourable conformer energy
compared with the other inhibitors, which affects the overall binding energy.
0
.4
.35
.3
.25
.2
.15
.1
11c
17d
0
0
0
0
11d
0
11a
17a
11b
0
17b
17c
11f
11e
0
.05
0
-
11.8 -11.2
-11
-10.4 -8.9
-7.9
-7.6
-7.4
-7.3
-7.1
Binding energy
kcal/mol
1
5

Figure 4. Correlation between CYP24A1 IC and binding energy for imidazole derivatives 11
5
0
and 17
3
. Conclusions
A series of 3- and 4-styrylbenzamide imidazole derivatives have been prepared using an
efficient synthetic route. All the derivatives showed very good inhibitory activity against
CYP24A1 and, although there were some small differences in IC50 values depending on
substituents, overall both 3- and 4-styrylbenzamide imidazoles and a range of substituents
were very well tolerated without any significant loss of inhibitory activity. This inhibitory
data would agree with the molecular docking where all the compounds fit well within the
active site and showed a good fill of the hydrophobic channel of the CYP24A1 active site as
well as transition metal interaction between the iron of the haem and the imidazole ring of the
derivatives.
4
. Experimental
4
.1. Chemistry
.1.1. General Experimental
,25(OH) D and 25(OH)D were purchased from SAFC-Pharma (Madison, WI). Human
4
1
2
3
3
MBP-CYP24A1, bovine adrenodoxin (Adx), and adrenodoxin reductase (AdR) were purified as
30
described previously . All solvents used for chromatography were HPLC grade from Fisher
1
13
Scientific (UK). H and C NMR spectra were recorded with a Bruker Avance DPX500 spectrometer
operating at 500 and 125 MHz, with Me Si as internal standard. Mass spectra and microanalysis were
4
determined by MEDAC (Chobham, UK). Flash column chromatography was performed with silica
gel 60 (230-400mesh) (Merck) and TLC was carried out on precoated silica plates (kiesel gel 60 F ,
254
BDH). Compounds were visualised by illumination under UV light (254 nm) or by the use of vanillin
stain followed by charring on a hotplate. Melting points were determined on an electrothermal
1
6

instrument and are uncorrected. All solvents were dried prior to use and stored over 4Å molecular
sieves, under nitrogen. All compounds were more than 95% pure.
4
.1.2. General procedure for the preparation of (E)-N-(2-(4-substitued-phenyl)-2-
hydroxyethyl)-4-styrylbenzamides
(9)
and
(E)-N-(2-hydroxy-2-phenylethyl)-3-
styrylbenzamides (15).
A suspension of (E)-styrylbenzoic acid (8 or 14) (2 mmol) in dry DMF (8 mL) was combined
with CDI (2.2 mmol). The reaction was stirred for 1 h at room temperature under nitrogen.
The mixture was cooled to 0 °C then added to a solution of 2-amino-1-phenylethan-1-ol (5)
(2 mmol) in dry DMF (2.5 mL). The resulting mixture was stirred at room temperature
overnight and on completion, ice was added into the flask and a white solid precipitated out.
The precipitate was then filtered, washed with ice-cold water and dried.
1
4
.1.2.1. (E)-N-(2-(4-chlorophenyl)-2-hydroxyethyl)-4-styrylbenzamide (9a, R = 4-Cl, R
2
=
R = H). White crystalline solid obtained after recrystallization from methanol. Yield: 67%;
1
Rf 0.86 (CH Cl -CH OH 9:1 v/v); mp 258 - 260 °C; H NMR (DMSO-d ):  8.52 (t, J = 5.3
2
2
3
6
Hz, 1H, NH), 7.85 (d, J = 8.2 Hz, 2H, Ar), 7.73 (d, J = 8.4 Hz, 2H, Ar), 7.74 (d, J = 8.2 Hz,
H, Ar), 7.42 (m, 5H, Ar) 7.24 (m, 4H, Ar), 5.64 (s, 1H, OH), 4.80 (m, 1H, CHOH), 3.49
2
13
(
m,1H, CH ), 3.40 (m, 1H, CH ). C NMR (DMSO–d ): 167.6 (CO), 140.9, 137.5, 135.2,
2
2
6
1
33.6, 133.2 (5ꢃꢄ C), 129.4, 128.5, 128.1, 127.3, 127.2, 127.1, 126.9 (15 ꢄ CH, Ar), 72.2
(
CH), 47.1 (CH ). Anal. Calcd for C H ClNO ꢅꢃ0.3H O (382.52): C 72.08 %, H 5.42 %, N
2
23 20
2
2
3
.65 %. Found C72.20 %, H 5.26 %, N 3.86 %.
.1.2.2. (E)-N-(2-hydroxy-2-(4-(trifluoromethyl)phenyl)ethyl)-4-styrylbenzamide (9b, R
4
1
2
=
4-CF , R = R = H). White crystalline solid obtained after recrystallization from methanol.
3
1
Yield: 35%; Rf 0.75 (petroleum ether-EtOAc 3:1 v/v); mp 220 - 224 °C; H NMR (DMSO-
d6): 8.58 (t, J = 5.7 Hz, 1H, NH), 7.84 (d, J = 8.4 Hz, 2H, Ar), 7.72 (ꢆt, J = 8.4, 10.5 Hz,
4
H, Ar), 7.65 (m, 4H, Ar), 7.42 (m, 3H, Ar) 7.33 (m, 2H, Ar), 5.77 (d, J = 4.5 Hz, 1H, OH),
1
7

1
3
4
.89 (t, J = 5.5 Hz, 1H, CHOH), 3.53 (m,1H, CH ), 3.43 (m, 1H, CH ). C NMR (DMSO–
2 2
d6): 166.6 (CO), 149.0, 140.3, 137.2, 133.6 (4ꢃꢄ C), 129.2, 128.5 (2ꢃꢄ CH, Ar), 128.3,
28.0 (2ꢃꢄ C), 127.9, 127.3, 127.2, 127.1, 126.7, 125.4 (13 ꢄ CH, Ar), 71.2 (CH), 47.9
CH ). Anal. Calcd for C H F NO (411.42): C 70.07 %, H 4.90 %, N 3.40 %. Found C
1
(
2
24 20
3
2
7
0.39 %, H 4.79 %, N 3.47 %.
.1.2.3. (E)-N-(2-hydroxy-2-(2-(trifluoromethyl)phenyl)ethyl)-4-styrylbenzamide (9c, R
4
1
2
=
2-CF , R = R = H). White crystalline solid obtained after recrystallization from methanol.
3
1
Yield: 58%; Rf 0.40 (petroleum ether-EtOAc 3:1 v/v); mp 160 - 166 °C; H NMR (DMSO-
d6): 8.58 (t, J = 6 Hz, 1H, NH), 7.87 (d, J = 8.5 Hz, 3H, Ar), 7.74 (m, 4H, Ar), 7.68 (d, J =
1
4
5.5 Hz, 2H, Ar), 7.51 (t, J = 7.7 Hz, 1H, Ar), 7.42 (m, 2H, Ar) 7.33 (m, 3H, Ar), 5.81 (d, J =
1
3
.0 Hz, 1H, OH), 5.14 (dd, J = 5.1, 11.9 Hz, 1H, CHOH), 3.51 (m, 2H, CH ). C NMR
2
(
DMSO–d ): 169.6 (CO), 141 (C), 140.3, 131.0 (CH), 128.8 (C), 128.2, 128.1, 127.8,
6
1
27.3, 127.0, 126.3, 126.6, 125.1, 126.7, 125.4 (14 ꢄCH, Ar), 124.3, 123.2 (5 ꢄC), 77.2
(
CH), 48.3 (CH ). Anal. Calcd for C H F NO (411.42): C 70.07 %, H 4.90 %, N 3.40 %.
2
24 20
3
2
Found C 70.22 %, H 4.92 %, N 3.38 %.
.1.2.4. (E)-N-(2-(3,4-dimethylphenyl)-2-hydroxyethyl)-4-styrylbenzamide (9d, R = 3,4-
4
1
2
diCH , R = R = H). White crystalline solid obtained after recrystallization from ethanol.
3
1
Yield: 60%; Rf 0.80 (CH Cl - CH OH 9.5:0.5 v/v); mp 150 - 154 °C; H NMR (CDCl ):
2
2
3
3
8.08 (s, 1H, Ar), 8.63 (d, J = 8.0 Hz, 1H, Ar), 7.57 (d, J = 7.5 Hz, 2H, Ar), 7.42 (t, J = 7.5
Hz, 2H, Ar), 7.33 (t, J = 7.5 Hz, 2H, Ar), 7.28 (s, 1H, Ar), 7.26 (s, 1H, Ar) 7.19 (d, J = 4.5
Hz, 2H, Ar), 7.15 (m, 3H, Ar), 5.88 (t, J = 10.0 Hz, 1H, OH), 4.56 (dd, J = 10.0, 15.0 Hz, 1H,
1
3
CHOH), 4.17 (m, 1H, CH ), 3.93 (dd, J = 8.5, 14.5 Hz, 1H, CH ), 3.5 (s, 6H, 2CH ) . C
2
2
3
NMR (CDCl ): 166.5 (CO), 142.1, 140.1, 137.3, 134.9, 133.7, 131.6 (6ꢃꢄC), 130.6, 130.3,
3
1
29.2, 128.4, 128.2, 128.0, 127.8, 127.2, 126.7 (14ꢄC, Ar), 68.6 (CH), 47.4 (CH ), 21.35,
2
1
8

1
8.71 (2ꢃꢄ CH ). Anal. Calcd for C H NO (371.48): C 80.83 %, H 6.78 %, N 3.77 %.
3
25 25
2
Found C 81.10 %, H 6.78 %, N 3.88 %
.1.2.5. (E)-N-(2-hydroxy-2-(4-methoxyphenyl)ethyl)-4-styrylbenzamide (9e, R = 4-
4
1
2
OCH , R = R = H). White crystalline solid obtained after recrystallization from methanol.
3
1
Yield: 35%; Rf 0.70 (petroleum ether-EtOAc 1:3 v/v); mp 218 - 220 °C; H NMR (DMSO-
d6): 8.50 (t, J = 5.5 Hz, 1H, NH), 7.87 (d, J = 8.3 Hz, 2H, Ar), 7.70 (ꢇ, 4H, Ar), 7.65 (m,
3
H, Ar), 7.33 (m, 2H, Ar), 7.33 (m, 2H, Ar), 6.92 (d, J = 8.3 Hz, 2H), 5.43 (d, J = 4.5 Hz, 1H,
13
OH), 4.76 (m, 1H, CHOH), 3.74 (s, 3H, OCH ), 3.50 (m,1H, CH ), 3.34 (m, 1H, CH ). C
3
2
2
NMR (DMSO-d ): 166.5 (CO), 158.8, 140.2, 137.3, 136.3, 133.8 (5ꢃꢄ C), 130.6, 129.2,
6
1
28.5, 128.1, 127.6, 127.2, 1114.3 (15 ꢄCH, Ar), 71.2 (CH), 55.4 (OCH ) 48.2 (CH ). Anal.
3
2
Calcd for C H NO (373.45): C 77.19 %, H 6.21 %, N 3.75 %. Found C 77.32 %, H 6.02 %,
2
4
23
3
N 3.70 %.
.1.2.6. (E)-N-(2-(4-chlorophenyl)-2-hydroxyethyl)-4-(3,4-dimethoxystyryl)benzamide
4
1
2
(
9f, R = 4-Cl, R = R = OCH ). White crystalline solid obtained after recrystallization from
3
1
acetonitrile. Yield: 69%; Rf 0.20 (petroleum ether-EtOAc 1:1 v/v); mp 168 - 172 °C; H
NMR (DMSO-d ):  8.53 (t, J = 6.0 Hz, 1H, NH), 7.84 (d, J = 8.0 Hz, 2H, Ar), 7.65 (d, J =
6
8
.5 Hz, 2H, Ar), 7.40 (s, 2H, Ar), 7.36 (m, 2H, Ar), 7.21 (m, 2H, Ar) 7.00 (m, 3H, Ar), 5.66
13
(
d, J = 4.5 Hz, 1H, OH), 4.81 (m, 1H, CHOH), 3.49 (m,2H, CH ). C NMR (DMSO-d ): 
2
6
1
66.5 (CO), 149.5, 149.4, 143.3, 133.2, 131.9, 128.2 (6 ꢄ C), 130.7 (CH, Ar), 130.2
(C),128.4, 128.3, 128.2, 126.3, 125.8, 120.7, 112.3, 109.7 (12 ꢄ CH, Ar), 71.0 (CH), 47.3
3
7Cl
+
35Cl
+
(
CH ). LRMS (ES-TOF) m/z: 440 [M
+ H] , 438 [M
+ H] . HRMS (ES-TOF)
2
+
+
Calculated mass: 438.1472 [M + H] , measured mass: 438.1463 [M + H]
3
4
.1.2.7. (E)-N-(2-hydroxy-2-phenylethyl)-3-styrylbenzamide (15a, R = R = H). White
crystalline solid obtained after recrystallization from methanol. Yield: 60%; Rf 0.68 (CH Cl -
2
2
1
9

1
CH OH 9:1 v/v); mp 128 - 132 °C; H NMR (DMSO-d ): 8.64 (t, J = 6.0 Hz, 1H, NH),
3
6
8
3
.11 (s, 1H, Ar), 7.73 (d, J = 8.0 Hz, 2H, Ar), 7.65 (d, J = 7.5 Hz, 3H, Ar), 7.49 (t, J = 8.0 Hz,
H, Ar) 7.42 (t, J = 7.5 Hz, 3H, Ar), 7.37 (m, 4H, Ar), 5.64 (s, 1H, OH), 4.80 (m, 1H,
1
3
CHOH), 3.49 (m, 1H, CH ), 3.40 (m, 1H, CH ). C NMR (DMSO-d ): 166.8 (CO), 144.3,
2
2
6
1
1
37.5, 137.3, 135.5 (4ꢃꢄ C), 129.8, 129.7, 129.3, 129.1, 127.9, 128.5, 128.4, 128.3, 127.5,
27.0, 126.9, 126.5, (16 ꢄCH, Ar), 71.6 (CH), 48.2 (CH ). Anal. Calcd for C H NO ꢃꢅꢃ0.4
2
23 21
2
H O (350.36): C 78.79 %, H 6.27 %, N 3.99 %. Found C 78.35 %, H 6.56 %, N 3.83 %.
2
3
4
.1.2.8. (E)-N-(2-(4-chlorophenyl)-2-hydroxyethyl)-3-styrylbenzamide (15b, R = 4-Cl, R
=
H). Cream crystalline solid obtained after recrystallization from methanol. Yield: 50%; Rf
1
0
5
7
.44 (petroleum ether-EtOAc 2:1 v/v); mp 138 - 142 °C; H NMR (DMSO-d ): 8.63 (t, J =
6
.0 Hz, 1H, NH), 8.09 (s, 1H, Ar), 7.63 (d, J = 7.5 Hz, 4H, Ar), 7.48 (t, J = 8.0 Hz, 4H, Ar),
.42 (t, J = 7.5 Hz, 3H, Ar) 7.36 (m, 3H, Ar), 5.69 (d, J = 4.0 Hz, 1H, OH), 4.83 (m, 1H,
1
3
CHOH), 3.51 (m, 1H, CH ), 3.39 (m, 1H, CH ). C NMR (DMSO-d ): 166.9 (CO), 138.6
2
2
6
1
1
37.5, 135.8, 134.2, 133.2 (5ꢃꢄ C), 131.9, 128.7,128.6, 127.9, 127.3, 127.4,127.1, 126.9,
26.7,123.4 (15 ꢄCH, Ar), 72.2 (CH), 48.9 (CH ). Anal. Calcd for C H ClNO ꢅꢃ0.2 H O
2
24 20
2
2
(381.47): C 72.42 %, H 5.39 %, N 3.67 %. Found C 72.11 %, H 5.36 %, N 3.77 %.
4
.1.2.9. (E)-N-(2-hydroxy-2-(4-(trifluoromethyl)phenyl)ethyl)-3-styrylbenzamide (15c, R
3
=
4-CF , R = H). White crystalline solid obtained after recrystallization from acetonitrile.
3
1
Yield: 31%; Rf 0.52 (petroleum ether-EtOAc 2:1 v/v); mp 184 - 186 °C; H NMR (DMSO-
d6): 8.66 (t, J = 5.6 Hz, 1H, NH), 8.08 (s, 1H, Ar), 7.74 (ꢆt, J = 6.6, 7.7 Hz, 4H, Ar), 7.64
(ꢆt, J = 8.3, 10.0 Hz, 4H, Ar), 7.49 (t, J = 7.7 Hz, 1H, Ar), 7.42 (t, J = 7.6 Hz, 2H, Ar) 7.33
(m, 3H, Ar), 5.80 (d, J = 4.5 Hz, 1H, OH), 4.93 (dd, J = 5.1, 11.9 Hz, 1H, CHOH), 3.54
1
3
(
m,1H, CH ), 3.44 (m, 1H, CH ). C NMR (DMSO-d ): 166.9 (CO), 149.0, 137.5, 137.2,
2
2
6
1
35.4 (4ꢃꢄC), 129.8, 129.7, 129,2, 129.1 (4ꢃꢄCH, Ar), 128.3, 128.0 (2ꢃꢄC), 127.7, 127.3,
2
0

1
27.2,127.1, 126.8, 125.9 (11 ꢄ CH, Ar), 71.2 (CH), 47.9 (CH ). Anal. Calcd for
2
C H F NO ꢅꢃ0.1H O (413.23): C 69.76 %, H 4.93 %, N 3.39 %. Found C 69.51%, H 4.94
24
20
3
2
2
%, N 3.42 %.
4
.1.2.10. (E)-N-(2-hydroxy-2-phenylethyl)-3-(2-(trifluoromethyl)styryl)benzamide (15d,
3
R = H, R = CF ). White solid obtained after purification by flash column chromatography,
3
product eluted with petroleum ether-EtOAc 1:1 v/v. Yield: 75%; Rf 0.70 (petroleum ether-
1
EtOAc 1:1 v/v); mp 88 - 90 °C; H NMR (CDCl ): 7.89 (s, 1H, Ar), 7.75 (d, J = 7.7 Hz,
3
1
1
3
1
H, Ar), 7.68 (m, 3H, Ar), 7.56 (t, J = 7.7 Hz, 2H, Ar), 7.50 (m, 5H, Ar), 7.41 (t, J = 8 Hz,
H, Ar), 7.36 (t, J = 7.7 Hz, 1H, Ar) 7.29 (m, 1H, Ar), 4.97 (dd, J = 3.2, 8.1 Hz, 1H, CHOH),
1
3
.93 (m, 1H, CH ), 3.55 (m, 1H, CH ). C NMR (DMSO-d ): 167.5 (CO), 141.5, 137.3,
2
2
6
36.3 (3ꢃꢄC), 133.6, 129.1, 128.7, 128.4, 127.9, 127.6, 125.7 (14ꢃꢄ CH, Ar), 122.6, 122.2
2ꢄC), 114.3 (CH, Ar), 71.2 (CH), 48.2 (CH ). Anal. Calcd for C H F NO (411.42): C
(
2
24 20
3
2
7
0.07%, H 4.90 %, N 3.40 %. Found C 69.99 %, H 5.05 %, N 3.53 %.
.1.3. General procedure for the preparation of (E)-5-(4-substitued-phenyl)-2-(4-
4
styrylphenyl)-4,5-dihydrooxazoles
(10) and
(E)-5-phenyl-2-(3-styrylphenyl)-4,5-
dihydrooxazoles (16).
A solution of (E)-N-(2-hydroxy-2-phenylethyl)-3/4-styrylbenzamide (9 or 15) (3.4 mmol) in
dry THF (30 mL) was cooled to 0 °C. Then methansulfonylchloride (2.2 mL, 27 mmol) was
added and the resulting mixture stirred at 0 °C for 3 h. Triethylamine (6.2 mL, 40.8 mmol)
was added dropwise and the solution was stirred overnight at room temperature. The mixture
was quenched by the addition of NH OH (28 %, 3 mL) and the reaction stirred at room
4
temperature for 30 min. Then THF was removed under reduce pressure and the residue was
extracted by EtOAc (100mL) and water (2 ꢄ100 mL). The organic layer was collected, dried
2
1

(
MgSO ) and concentrated under vacuum. The product was pure enough for use in the next
4
reaction.
1
4
.1.3.1. (E)- 5-(4-chlorophenyl)-2-(4-styrylphenyl)-4,5-dihydrooxazole (10a, R = 4-Cl, R
2
=
R = H). Light brown solid obtained. Yield: 40%; Rf 0.47 (CH Cl -CH OH 9:1 v/v); mp
2
2
3
1
1
28 - 130 °C; H NMR (DMSO-d ): 7.94 (d, J = 8.3 Hz, 2H, Ar), 7.74 (d, J = 8.3 Hz, 2H,
6
Ar), 7.66 (d, J = 7.4 Hz, 2H, Ar), 7.48 (d, J = 8.4 Hz, 2H, Ar), 7.42 (m, 5H, Ar), 7.32 (dd, J =
.6, 7.3 Hz, 2H, Ar), 5.82 (dd, J = 3.0, 10.0 Hz, 1H, CH), 4,48 (dd, J = 10, 14.9 Hz, 1H,
CH ), 3.85 (dd, J = 7.4, 15.0 Hz,1H, CH ).
2
2
2
4
.1.3.2. (E)- 2-(4-styrylphenyl)-5-(4-(trifluoromethyl)phenyl)-4,5-dihydrooxazole (10b, R
1
2
=
4-CF , R = R = H). Yellow solid obtained. Yield: 90%; Rf 0.70 (petroleum ether-EtOAc
3
1
1
7
:1 v/v); H NMR (DMSO-d ): 7.97 (d, J = 8.3 Hz, 2H, Ar), 7.80 (m, 4H, Ar), 7.65 (d, J =
6
.4 Hz, 4H, Ar), 7.61 (d, J = 8.3 Hz, 2H, Ar), 7.34 (m, 3H, Ar), 5.93 (dd, J = 7.4, 10 Hz, 1H,
CH), 4,53 (dd, J = 10.0, 15 Hz, 1H, CH ), 3.87 (dd, J = 7.2, 1.00 Hz, 1H, CH ).
2
2
4
.1.3.3. (E)- 2-(4-styrylphenyl)-5-(2-(trifluoromethyl)phenyl)-4,5-dihydrooxazole (10c, R
1
2
=
2-CF , R = R = H). Yellow solid obtained. Yield: 70%; Rf 0.65 (petroleum ether-EtOAc
3
1
1
:1 v/v); mp 112 - 118 °C; H NMR (DMSO-d ): 8.10 (m, 4H, Ar), 7.89 (m, 3H, Ar), 7.85
6
(m, 4H, Ar), 7.82 (d, J = 16.2 Hz, H, alkene), 7.59 (m, 3H, Ar), 5.71 (dd, J = 8.0, 10.1 Hz,
1
H, CH), 4.51 (dd, J = 10.3, 15.0 Hz, 1H, CH ), 4.01 (dd, J = 8.0, 15.0 Hz, 1H, CH ).
2 2
4
.1.3.4. (E)-5-(3,4-dimethylphenyl)-2-(4-styrylphenyl)-4,5-dihydrooxazole (10d, R = 3,4-
1
2
diCH , R = R = H). Light brown solid obtained. Yield: 75%; Rf 0.40 (petroleum ether-
3
1
EtOAc 1:1 v/v); H NMR (DMSO-d ): 7.97 (d, J = 8.3 Hz, 2H, Ar), 7.76 (d, J = 8.3 Hz,
6
2
H, Ar), 7.66 (d, J = 7.4 Hz, 2H, Ar), 7.42 (t, J = 7.4 Hz, 3H, Ar), 7.36 (s, 1H, Ar), 7.33 (t, J
7.4 Hz, 2H, Ar), 7.13 (d, J = 7.4 Hz, 2H, Ar), 5.92 (dd, J = 8.2, 10.1 Hz, 1H, CH), 4.52 (dd,
=
2
2

J = 10.3, 14.9 Hz, 1H, CH ), 3.73 (dd, J = 8.0, 14.9 Hz, 1H, CH ), 2.26 (s, 1H, CH ), 2.24 (s,
2
2
3
1
H, CH3).
.1.3.5. (E)-5-(4-methoxyphenyl)-2-(4-styrylphenyl)-4,5-dihydrooxazole (10e, R = 4-
4
1
2
OCH , R = R = H). Light brown solid obtained. Yield: 90%; Rf 0.55 (petroleum ether-
3
1
EtOAc 1:1 v/v); mp 144 - 146 °C; H NMR (DMSO-d ): 7.92 (d, J = 8.3 Hz, 2H, Ar), 7.73
6
(d, J = 8.3 Hz, 2H, Ar), 7.65 (d, J = 7.5 Hz, 2H, Ar), 7.42 (m, 2H, Ar), 7.34 (m, 5H, Ar), 6.98
(
d, J = 8.3 Hz, 2H, Ar), 5.74 (ꢆꢈ, J = 7.8, 9.8 Hz, 1H, CH), 4.26 (m, 1H, CH ), 4.03 (m, 1H,
2
CH ), 3.76 (s, 3H, OCH ).
2
3
4
.1.3.6.
(E)-5-(3-chlorophenyl)-2-(3-(3,4-dimethoxystyryl)phenyl)-4,5-dihydrooxazole
1
2
(
(
(
10f, R = 4-Cl, R = R = OCH ). Light brown solid obtained. Yield: 63%; Rf 0.71
3
1
petroleum ether-EtOAc 1:1 v/v); H NMR (DMSO-d ): 7.59 (d, J = 8.4 Hz, 3H, Ar), 7.43
6
d, J = 7.5 Hz, 2H, Ar), 7.17 (m, 2H, Ar), 7.15 (m, 3H, Ar), 6.73 (m, 3H, Ar), 5.68 (m, 1H,
CH), 4.52 (m, 1H, CH ), 4.1 (m, 1H, CH ), 3.86 (s, 3H, OCH ), 3.72 (s, 3H, OCH ).
2
2
3
3
3
4
.1.3.7. (E)-5-phenyl-2-(3-styrylphenyl)-4,5-dihydrooxazole (16a, R = R = H). Cream
1
solid obtained. Yield: 85%; Rf 0.55 (petroleum ether-EtOAc 3:1 v/v); H NMR (DMSO-d ):
6
8.01 (d, J = 7.3 Hz, 2H, Ar), 7.87 (m, 5H, Ar), 7.52 (d, J = 7.3 Hz, 2H, Ar), 7.44 (m, 5H,
Ar), 7.38 (d, 7.3 Hz, 2H, Ar), 5.66 (dd, J = 3.0, 9.7 Hz, 1H, CH), 4.56 (dd, J = 9.7, 15.0 Hz,
1
H, CH ), 4.51 (dd, J = 7.3, 15.0 Hz,1H, CH ).
2 2
3
4
.1.3.8. (E)-5-(4-chlorophenyl)-2-(3-styrylphenyl)-4,5-dihydrooxazole (16b, R = 4-Cl, R
1
=
H). Cream solid obtained. Yield: 80%; Rf 0.40 (petroleum ether-EtOAc 1:1 v/v); H NMR
(
(
(
DMSO-d ): 8.19 (d, J = 8.5 Hz, 2H, Ar), 7.99 (d, 8.5 Hz, 2H, Ar), 7.50 (m, 2H, Ar), 7.41
6
m, 4H, Ar), 7.32 (m, 3H, Ar) 7.18 (m, 3H, Ar), 5.79 (dd, J = 8.2, 10.2 Hz, 1H, CH), 4.50
dd, J = 10.2, 15.3 Hz, 1H, CH ), 4.31 (dd, J = 8.2, 15.3 Hz,1H, CH ).
2
2
2
3

4
.1.3.9. (E)-2-(3-styrylphenyl)-5-(4-(trifluoromethyl)phenyl)-4,5-dihydrooxazole (16c, R
3
=
4-CF , R = H). Light brown solid obtained. Yield: 92%; Rf 0.57 (petroleum ether-EtOAc
3
1
1
7
1
:1 v/v); H NMR (DMSO-d ): 8.02 (d, J = 8.5 Hz, 2H, Ar), 7.56 (d, J = 8.5 Hz, 2H, Ar),
6
.36 (m, 5H, Ar), 7.17 (m, 3H, Ar), 7.08 (m, 2H, Ar) 7.97 (m, 1H, Ar), 5.51 (dd, J = 10.1,
5.0 Hz, 1H, CH), 4.09 (dd, J = 8.5, 15.0 Hz, 1H, CH ), 3.99 (dd, J = 8.2, 15.3 Hz,1H, CH ).
2
2
4
.1.3.10. (E)-5-phenyl-2-(3-(2-(trifluoromethyl)styryl)phenyl)-4,5-dihydrooxazole (16d,
3
R = H, R = CF ). Light brown solid obtained. Yield: 56%; Rf 0.60 (petroleum ether-EtOAc
3
1
1
7
1
:1 v/v); H NMR (DMSO-d ): 7.95 (d, J = 8.3 Hz, 2H, Ar), 7.66 (d, J = 8.3 Hz, 2H, Ar),
6
.58 (d, J = 8.3 Hz, 2H, Ar), 7.52 (d, J = 8.3 Hz, 2H, Ar), 7.32 (m, 7H, Ar), 5.80 (dd, J = 7.7,
0.1 Hz, 1H, CH), 4.45 (dd, J = 10.1, 14.9 Hz, 1H, CH ), 3.89 (dd, J = 7.5, 14.9 Hz,1H,
2
CH2).
4
.1.4. General procedure for the preparation of (E)-N-(2-(1H-imidazol-1-yl)-2-
phenylethyl)-4-styrylbenzamides (11) and (E)-N-(2-(1H-imidazol-1-yl)-2-(phenylethyl)-
-styrylbenzamides (17).
(
3
A mixture of (E)-5-(4-chlorophenyl)-2-(3/4-styrylphenyl)-4,5-dihydrooxazole (10 or 16) (3
mmol) and imidazole (7.4 g, 110 mmol) was refluxed at 125 °C for 48 h. On completion, the
mixture was extracted between EtOAc (100 mL) and brine (2ꢃꢄ100 mL). The organic layer
was dried (MgSO ) and concentrated under vacuum.
4
4
.1.4.1. (E)-N-(2-(4-chlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-styrylbenzamide (11a, R
1
2
=
4-Cl, R = R = H). White solid obtained after recrystallization from EtOAc. Yield: 12%;
1
Rf 0.85 (CH Cl -CH OH 9:1 v/v); mp 238 - 240 °C; H NMR (DMSO-d ): 8.73 (t, J = 5.2
2
2
3
6
Hz, 1H, NH), 7.86 (s, 1H, imid), 7.77 (d, J = 8.2 Hz, 4H, Ar), 7.68 (d, J = 8.2 Hz, 3H, Ar),
.64 (d, J = 7.5 Hz, 1H, imid), 7.46 (m, 8H, Ar), 6.92 (s, 1H, imid), 5.70 (dd, J = 6.5, 8.2 Hz,
7
2
4

1
3
1
1
1
H, CH), 4.06 (m, 1H, CH ), 3.44 (m, 1H, CH). C NMR (DMSO–d ): 166.8 (CO), 140.5,
2
6
38.7,133.2, 133.1 (5ꢃꢄ C), 137.2, 130.7, 129.3, 129.2, 129.1, 128.5, 127.9,127.1, 126.7,
37Cl
+
18.7 (18 ꢄCH, Ar), 59.1 (CH), 43.7 (CH ). LRMS (ES-TOF) m/z: 430 [M
+ H] , 428
2
35Cl
+
-imid
+
+
[
M
+ H] , 362 [M
+ H] . HRMS (ES-TOF) Calculated mass: 428.1523 [M + H] ,
+
measured mass: 428.1524 [M + H] .
4
.1.4.2.
(E)-N-(2-(1H-imidazol-1-yl)-2-(4-(trifluoromethyl)phenyl)ethyl)-4
1
2
styrylbenzamide (11b, R = 4-CF , R = R = H). Cream solid obtained after recrystallization
3
1
from EtOAc. Yield: 33%; Rf 0.32 (CH Cl -CH OH 9:1 v/v); mp 208 - 210 °C; H NMR
2
2
3
(
DMSO-d ): 8.80 (t, J = 5.0 Hz, 1H, NH), 7.91 (s, 1H, imid), 7.78 (dd, J = 3.0, 8.5 Hz, 4H,
6
Ar), 7.69 (d, J = 8.5 Hz, 2H, Ar), 7.64 (d, J = 7.5 Hz, 2H, Ar), 7.60 (s, 1H, imid), 7.42 (m,
4
2
H, Ar), 7.32 (m, 3H, Ar), 6.95 (s, 1H, imid), 5.83 (t, J = 7.5 Hz, 1H, CH), 4.13 (m, 12 Hz,
1
3
H, CH ).. C NMR (DMSO–d ): 166.8 (CO), 144.4, 140.5,137.4, 137.1, 135.4, 133.1
2
6
(6ꢃꢄC), 130.8, 129.2, 128.5, 128.2, 128.1, 127.9, 127.1,126.7, 126.1, 118.9 (18 ꢄCH, Ar),
5
9.3 (CH), 43.7 (CH ). Anal. Calcd for C H F N Oꢃꢅꢃ0.15H O (463.87): C 69.86 %, H 4.84
2
27 22
3
3
2
%, N 9.05 %. Found C 69.56 %, H 4.79 %, N 8.98 %.
4
.1.4.3.
(E)-N-(2-(1H-imidazol-1-yl)-2-(2-(trifluoromethyl)phenyl)ethyl)-4
1
2
styrylbenzamide (11c , R = 2-CF , R = R = H). Cream solid obtained after
3
recrystallization from EtOAc. Yield: 33%; Rf 0.32 (CH Cl -CH OH 9:1 v/v); mp 208 - 210
2
2
3
1
°
C; H NMR (DMSO-d ): 8.80 (t, J = 5.0 Hz, 1H, NH), 7.91 (s, 1H, imid), 7.78 (dd, J =
6
3
.0, 8.5 Hz, 4H, Ar), 7.69 (d, J = 8.5 Hz, 2H, Ar), 7.64 (d, J = 7.5 Hz, 2H, Ar), 7.60 (s, 1H,
imid), 7.42 (m, 4H, Ar), 7.32 (m, 3H, Ar), 6.95 (s, 1H, imid), 5.83 (t, J = 7.5 Hz, 1H, CH),
1
3
4
1
.13 (m, 12 Hz, 2H, CH ). C NMR (DMSO–d ): 166.8 (CO), 144.4, 140.5, 137.4, 137.1,
2
6
35.4, 133.1 (6ꢃꢄC), 130.8, 129.2, 128.5, 128.2, 128.1, 127.9, 127.1,126.7, 126.1, 118.9 (18
ꢄCH, Ar), 59.3 (CH), 43.7 (CH ). Anal. Calcd for C H F N Oꢃꢅꢃ0.15H O (463.87): C 69.86
2
27 22
3
3
2
%, H 4.84 %, N 9.05 %. Found C 69.56 %, H 4.79 %, N 8.98 %.
2
5

4
.1.4.4.
(E)-N-(2-(3,4-dimethylphenyl)-2-(1H-imidazol-1-yl)ethyl)-4-styrylbenzamide
1
2
(
11d, R = 3,4-diCH , R = R = H). Yellow solid obtained after recrystallization from
3
1
EtOAc. Yield: 27%; Rf 0.65 (CH Cl -CH OH 9:1 v/v); mp 134 - 136 °C; H NMR (DMSO-
2
2
3
d6): 8.73 (t, J = 5.5 Hz, 1H, NH), 7.82 (s, 1H, imid), 7.78 (d, J = 8.5 Hz, 2H, Ar), 7.69 (m,
H, Ar), 7.41 (m, 2H, Ar), 7.33 (m, 5H, Ar), 7.18 (s,1H, imid), 7.14 (m, 2H, Ar), 7.02 (s, 1H,
imid), 6.89 (s, 1H, Ar), 5.59 (dd, J = 5.5, 9.0 Hz, 1H, CH), 4.09 (m,1H, CH ), 3.94 (m, 1H,
2
2
1
3
CH ), 2.18 (s, 6H, 2 x CH ). C NMR (DMSO–d ): 167.8 (CO), 157.5, 140.9, 138.7,
2
3
6
1
1
+
37.5, 136.7, 133.4 (6ꢃꢄ C), 133.2, 130.7, 129.3, 129.2, 129.1, 128.5, 127.9, 127.1, 126.7,
18.7 (17 ꢄCH, Ar), 59.9 (CH), 43.7 (CH ), 25.7 (2ꢃꢄꢃCH ). LRMS (ES-TOF) m/z: 422 [M
2
3
+
+
H] , 214 [M – C H -CH=CH-C H -C=O] . HRMS (ES-TOF) Calculated mass: 422.2251
6
5
6
4
+
+
[
M + H] , measured mass: 422.2232 [M + H] .
.1.4.5. (E)-N-(2-(1H-imidazol-1-yl)-2-(4-methoxyphenyl)ethyl)-4-styrylbenzamide (11e,
4
1
2
R = 4-OCH , R = R = H). White solid obtained after recrystallization from EtOAc. Yield:
3
1
3
2%; Rf 0.41 (CH Cl -CH OH 9:1 v/v); mp 218 - 222 °C; H NMR (CDCl ): 8.06 (s, 1H,
2
2
3
3
imid), 7.78 (d, J = 7.7 Hz, 2H, Ar), 7.52 (d, J = 7.5 Hz, 2H, Ar), 7.36 (t, J = 7.0 Hz, 2H, Ar),
7
5
.31 (m, 5H, Ar), 7.24 (d, J = 8 Hz, 2H, Ar), 717 (m, 2H, Ar), 6.94 (d, J = 8.0 Hz, 2H, Ar),
.70 (dd, J = 4.0, 9.5 Hz, 1H, CH), 4.34 (m, 1H, CH ), 4.04 (m, 1H, CH), 3.81 (s, 3H,
2
1
3
OCH ). C NMR (DMSO–d ): 167.8 (CO), 157.5, 140.9, 138.7, 137.5, 136.7, 133.4 (6ꢃꢄ
3
6
C), 133.2, 130.7, 129.3, 129.2, 129.1, 128.5, 127.9, 127.1, 126.7, 118.7 (18 ꢄCH, Ar), 59.9
+
+
(
CH), 55.1 (s, 3H, OCH ), 43.7 (CH ). LRMS (ES-TOF) m/z: 424 [M + H] , 356 [M-imid] .
3 2
+
+
HRMS (ES-TOF) Calculated mass: 424.2020 [M + H] , measured mass: 424.2016 [M + H] .
4
.1.4.6. (E)-N-(2-(4-chlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(3,4-dimethoxystyryl)
1
2
benzamide (11e, R = 4-Cl, R = R = OCH ). White solid obtained after recrystallization
3
1
from acetonitrile. Yield: 35%; Rf 0.66 (CH Cl -CH OH 9:1 v/v); mp 204 - 208 °C; H NMR
2
2
3
(
DMSO–d ): 8.74 (t, J = 5.4 Hz, 1H, NH), 7.86 (s, 1H, imid), 7.75 (d, J = 8.4 Hz, 2H, Ar),
6
2
6

7
.64 (d, J = 8.4 Hz, 2H, Ar), 7.46 (m, 2H, Ar), 7.37 (s, 1H, imid), 7.32 (s, 2H, Ar), 7.29 (d, J
5.1 Hz, 2H, Ar), 7.17 (s, 1H, imid), 7.14 (dd, J = 1.8, 8.4 Hz, 1H, Ar), 6.98 (d, J = 8.4 Hz,
H, Ar), 6.92 (s, 1H, Ar), 5.71 (dd, J = 6.3, 8.7 Hz, 1H, CH), 4.08 (m, 2H, CH ), 3.83 (s, 3H,
=
2
2
1
3
OCH ), 3.78 (s, 3H, OCH ). C NMR (DMSO–d ): 166.8 (CO), 149.5, 149.4, 140.9, 138.8
3
3
6
(4ꢃꢄ C), 137.3 (CH, Ar), 133.2, 132.6 (ꢉꢄC), 130.8 (CH, Ar), 130.1 (C), 129.3, 129.2,
1
29.1, 128.1, 126.3, 120.8, 118.7, 112.1, 109.6 (14 ꢄCH, Ar), 59.2 (CH), 43.7 (CH ). LRMS
2
3
7Cl
+
35Cl
+
(ES-TOF) m/z: 490 [ M
+ H] , 488 [M
+ H] . HRMS (ES-TOF) Calculated mass:
+
+
4
4
88.1743 [M + H] , measured mass: 488.1741 [M + H] .
3
.1.4.7. (E)-N-(2-(1H-imidazol-1-yl)-2-phenylethyl)-3-styrylbenzamide (17a, R = R = H).
White solid obtained after recrystallization from EtOAc. Yield: 38%; Rf 0.27 (CH Cl -
2
2
1
CH OH 9:1 v/v); mp 158 - 160 °C; H NMR (DMSO–d ): 8.82 (t, J = 5.7 Hz, 1H, NH),
3
6
7
7
4
.97 (s, 1H, Ar), 7.75 (d, J = 8.0 Hz, 1H, Ar), 7.64 (d, J = 8.0 Hz, 4H, Ar), 7.47 (s, 1H, Ar),
.42 (m, 7H, Ar), 7.35 (m, 4H, Ar), 6.92 (s,1H, imid), 5.71 (dd, J = 6.0, 9.5 Hz, 1H, CH),
1
3
.13 (m,1H, CH ), 4.02 (m, 1H, CH ). C NMR (DMSO–d ): 167.2 (CO), 139.7, 137.6
2
2
6
(2ꢃꢄ C), 137.3 (CH, Ar), 137.2, 135.2 (2ꢃꢄ C), 129.9, 129.8, 129.6, 129.2, 128.5, 128.3,
1
28.1, 127.7, 127.4, 126.7, 126.3, 125.5, 118.9 (18 ꢄCH, Ar), 59.9 (CH), 43.9 (CH ). Anal.
2
Calcd for C H N Oꢃꢅ0.25 H O (397.99): C 78.47 %, H 5.95 %, N 10.56 %. Found C 78.17
2
6
23
3
2
%, H 5.95 %, N 10.56.
4
.1.4.8. (E)-N-(2-(4-chlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-3-styrylbenzamide (17b, R
3
=
4-Cl, R = H). White solid obtained after recrystallization from EtOAc. Yield: 32%; Rf 0.8
1
(
CH Cl -CH OH 9:1 v/v); mp 148 - 152 °C; H NMR (DMSO–d ): 8.82 (t, J = 5.5 Hz, 1H,
2
2
3
6
NH), 7.95 (s, 1H, imid), 7.88 (s, 1H, Ar), 7.75 (d, J = 7.5 Hz, 2H, Ar), 7.47 (m, 5H, Ar), 7.31
(m, 8H, Ar), 6.93 (s, 1H, imid), 5.71 (dd, J = 6.0, 8.5 Hz, 1H, CH), 4.11 (m, 1H, CH), 3.91
13
(
m,1H, CH ). C NMR (DMSO–d ): 167.2 (CO), 138.7, 137.6 (2ꢃꢄ C), 137.2 (CH, Ar),
2
6
1
37.1, 135.1, 133.2 (3ꢃꢄ C), 129.9, 129.8, 129.4, 192.2, 129.1, 129.0, 128.4, 128.4, 128.1,
2
7

1
27.1, 126.7, 125.5, 118.8 (17 ꢄCH, Ar), 59.1 (CH), 43.7 (CH ). Anal. Calcd for Calculated
2
C H ClN O (427.93): C 72.98 %, H 5.18 %, N 9.81 %. Found C 72.77 %, H 5.36 %, N
26
22
3
9
.40.
.1.4.9.
4
(E)-N-(2-(1H-imidazol-1-yl)-2-(4-(trifluoromethyl)phenyl)ethyl)-3-
3
styrylbenzamide (17c, R = 4-CF , R = H). White solid obtained after recrystallization from
3
1
EtOAc. Yield: 48%; Rf 0.6 (CH Cl -CH OH 9:1 v/v); mp 126 - 130 °C; H NMR (DMSO–
2
2
3
d6): 8.84 (t, J = 5.5 Hz, 1H, NH), 7.96 (m, 2H, Ar), 7.78 (m, 4H, Ar), 7.64 (m, 5H, Ar),
7
.49 (m, 4H, Ar), 7.33 (m, 2H, Ar), 6.96 (s,1H, imid) 5.84 (m, 1H, CH), 4.15 (m, 2H, CH2).
1
3
C NMR (DMSO–d ): 166.9 (CO), 149.0, 144.4, 137.6, 137.5, 135.4, 135.1, 134.9 (7ꢃꢄC),
6
1
1
1
29.9, 129.8, 129.7, 129.2, 129.1, 128.4, 128.3, 128.2, 128.1, 127.3, 127.0, 126.1, 125.5,
+
25.4, 118.9 (18 ꢄCH, Ar), 59.3 (CH), 43.7 (CH ). LRMS (ES-TOF) m/z: 462 [M + H] ,
2
+
+
+
20 [C H CONH + H] . HRMS (ES-TOF) Calculated mass: 462.1793 [M + H] , measured
6
5
+
mass: 462.1799 [M + H] .
4
.1.4.10.
(E)-N-(2-(1H-imidazol-1-yl)-2-phenylethyl)-3-(2-(trifluoromethyl)styryl)
3
benzamide (17d, R =H, R = CF ). White solid obtained after recrystallization from EtOAc.
3
1
Yield: 33%; Rf 0.5 (CH Cl -CH OH 9:1 v/v); mp 110 - 114 °C; H NMR (DMSO–d ): 8.87
2
2
3
6
(t, J = 5.5 Hz, 1H, NH), 8.02 (d, J = 7.5 Hz, 2H, Ar), 7.86 (s, 1H, IMID), 7.78 (m, 4H, Ar),
7
.54 (m, 5H, Ar), 7.40 (m, 5H, Ar), 6.91 (s,1H, Ar) 5.71 (dd, J = 6.0, 9.0 Hz, 1H, CH), 4.12
1
3
(
m, 2H, CH ). C NMR (DMSO–d ): 167.9 (CO), 149.0, 144.4, 138.6, 138.5, 136.4, 134.9
2
6
(
6ꢃꢄC), 129.9, 129.8, 129.7, 129.2, 129.1, 128.4, 128.3, 128.2, 128.1, 127.3, 127.0, 126.1,
25.5, 125.4, 12.4 (18 ꢄCH, Ar), 59.3 (CH), 43.7 (CH ). LRMS (ES-TOF) m/z: 462 [M +
1
2
+
+
+
H] , 394 [M-imidazole] . HRMS (ES-TOF) Calculated mass: 462.1793 [M + H] , measured
+
mass: 462.1795 [M + H] .
2
8

4
.2. CYP24A1 inhibition assay
Inhibition of CYP24A1 was performed as previously described . Briefly, reaction
mixture containing 0.1 μM each of Adx and AdR, 0.075 μM MBP-CYP24A1, 2.5 μM
,25(OH) D , varying concentrations of inhibitors, and 0.5 mM NADPH was incubated at 37
3
1
1
2
3
°
C for 25 min in a buffer of 20 mM Tris (pH 7.5) and 125 mM NaCl. All inhibitors were
dissolved in ethanol (>10 mM) or DMSO (>50 mM) and further diluted in ethanol to make
working stock (<1 mM). The reaction was extracted with CH Cl and analysed by HPLC.
2
2
The IC values were determined by fitting the relative activity (V/V ) against the inhibitor
5
0
0
concentration [I] using the equation V/V = IC /(IC + [I]), where V and V are the reaction
0
50
50
0
rates in the presence and absence of inhibitors. The assay for each compound was performed
in at least duplicate and in triplicate for compounds with good inhibitory properties.
4
.3. Molecular modelling
3
2
Docking studies were performed using LeadIT2.1.2 docking program by BioSolve.IT . The
important amino acid residues of the active pocket (Gln82, Ile131, Trp134, Met246, Ala326,
3
0
Glu329, Thr330, Val391, Phe393, Thr394, Ser498, Gly499, Tyr500) were selected and then
the selection was extended to 12 Å in order to include in the docking site the haem iron
region and the access tunnel to the catalytic site. A ligands database in mol2 format, prepared
3
3
using MOE , was used as input for the docking calculations. The iron atom of the catalytic
site was set as essential pharmacophoric feature. Ligand docking was performed using the
default values and no water molecules were considered. Ten output solutions were obtained
from each compound and visual inspection in MOE was used to identify the interaction
between ligand and protein.
2
9