Design, synthesis and biological evaluation of novel 5-oxo-2-thioxoimidazolidine derivatives as potent androgen receptor antagonists

Article abstract of DOI:10.1016/j.ejmech.2015.05.039

A series of novel highly active androgen receptor (AR) antagonists containing spiro-4-(5-oxo-3-phenyl-2-thioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile core was designed based on the SAR studies available from the reported AR antagonists and in silico modeling. Within the series, compound (R)-6 (ONC1-13B) and its related analogues, including its active N-dealkylated metabolite, were found to be the most potent molecules with the target activity (IC₅₀, androgen-sensitive human PCa LNCaP cells) in the range of 59-80 nM (inhibition of PSA production). The disclosed hits were at least two times more active than bicalutamide, nilutamide and enzalutamide within the performed assay. Several compounds were classified as partial agonists. Hit-compounds demonstrated benefit pharmacokinetic profiles in rats. Comparative SAR and 3D molecular docking studies were performed for the hit compounds elucidating the observed differences in the binding potency.

Full text of DOI:10.1016/j.ejmech.2015.05.039

European Journal of Medicinal Chemistry 99 (2015) 51e66
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and biological evaluation of novel 5-oxo-2-
thioxoimidazolidine derivatives as potent androgen receptor
antagonists
,
b
,
, c, d, e,
Alexandre V. Ivachtchenko a c, Yan A. Ivanenkov b
*, Oleg D. Mitkin b,
Anton A. Vorobiev b, Irina V. Kuznetsova b, Natalia A. Shevkun b, Angela G. Koryakova b,
Ruben N. Karapetian b, Andrey S. Trifelenkov b, Dmitry V. Kravchenko b,
Mark S. Veselov d e, Nina V. Chufarova d
,
a Alla Chem LLC, 1835 East Hallandale Beach Boulevard 442, Hallandale Beach, FL 33009, United States
b Chemical Diversity Research Institute, Rabochaya Street 2a, Khimki, Moscow Region 141401, Russia
c ChemDiv, 6605 Nancy Ridge Drive, San Diego, CF 92121, USA
d Moscow Institute of Physics and Technology (State University), 9 Institutskiy Lane, Dolgoprudny City, Moscow Region 141700, Russia
e Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory, 1/3, Moscow 119991, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel highly active androgen receptor (AR) antagonists containing spiro-4-(5-oxo-3-phenyl-2-
thioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile core was designed based on the SAR studies
available from the reported AR antagonists and in silico modeling. Within the series, compound (R)-6
(ONC1e13B) and its related analogues, including its active N-dealkylated metabolite, were found to be
the most potent molecules with the target activity (IC50, androgen-sensitive human PCa LNCaP cells) in
the range of 59e80 nM (inhibition of PSA production). The disclosed hits were at least two times more
active than bicalutamide, nilutamide and enzalutamide within the performed assay. Several compounds
were classified as partial agonists. Hit-compounds demonstrated benefit pharmacokinetic profiles in rats.
Comparative SAR and 3D molecular docking studies were performed for the hit compounds elucidating
the observed differences in the binding potency.
Received 6 November 2014
Received in revised form
21 May 2015
Accepted 23 May 2015
Available online
Keywords:
Prostate cancer
Antiandrogen
Antagonist
© 2015 Elsevier Masson SAS. All rights reserved.
Androgen receptor
1. Introduction
(HSP70 and HSP90) release and subsequent receptor hyper-
phosphorylation leading to AR dimerization and binding to AR-
Prostate cancer (PC), following after skin cancer, is the most
common cancer occurred in American men, and the sixth cause of
cancer-related death among the male population worldwide [1].
More than 230K new cases of prostate cancer have been estimated
in the United States during 2014 by American Cancer Society and
about 29,480 deaths will be, actually, registered [2]. The vast ma-
jority of PCs is initially androgen dependent, and androgen receptor
(AR) is highly expressed throughout the various stages of disease
playing the crucial regulatory functions in cells [3,4]. The activation
of AR [5] strongly promotes prostate cancer growth and progres-
sion. Particularly, ligand binding triggers the heat-shock proteins
associated promotor area in target genes. This signaling pathway
can be efficiently blocked by competitive FDA-approved AR an-
tagonists (antiandrogens) such as 1 (flutamide) [6], 2 (bicaluta-
mide) [7], 3 (nilutamide) [8], 4 (enzalutamide) [9] or 5 (ARN-509)
[10] (Fig. 1). Therefore, androgen deprivation/withdrawal therapy
is currently regarded as the “first-line” therapeutic option for
localized, early stage prostate cancer. In spite of initial high effi-
ciency observed among a greater percentage of PCa patients, this
therapeutic route leads only to a temporary reduction of PC
resulting in a significant number of resistance outcomes. As a result,
in clinics, classical (steroidal) AR antagonists are frequently inef-
fective for the treatment of advanced stages of the disease while
nonsteroidal drugs are generally considered to be more potent in
terms of disease prevention and selectivity thereby minimizing
possible off-target as well as on-target side effects. Cells are able to
* Corresponding author. ChemDiv, 6605 Nancy Ridge Drive, San Diego, CF 92121,
USA.
E-mail address: yai@chemdiv.com (Y.A. Ivanenkov).
http://dx.doi.org/10.1016/j.ejmech.2015.05.039
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

52
A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 99 (2015) 51e66
Me
CN
F
O
H
CF
3
Me
S
CF
O
3
O
O
O N
2
Me
OH
1
2
O
NO
2
S
CONHMe
CN
HN
N
CF
3
N
N
Me
CF
F
3
O
Me
Me
O
Me
3
4
S
N
S
N
CONHMe
CN
CONHMe
CN
N
N
N
N
F
CF
3
CF
F
3
O
O
O
5
6
Fig. 1. Non-steroidal AR antagonists approved by FDA (1e4) and drug candidates currently evaluated in clinics (5,6).
regenerate and survive without the presence of high levels of
testosterone. It is known as hormone-resistant (AIPC) forms and
castration-resistant prostate cancer (CRPC) are commonly attrib-
uted to elevated AR gene expression or/and amplification [11,12],
AR gene mutation [13] as well as ligand-independent AR activation,
particularly through the related transcription factors and co-
activators [14,15]. A great number of painstaking efforts have
been made to develop novel AR antagonists with sufficient thera-
peutic potency against both types of PC cells with early and later
stages of androgen dependence and independence, respectively
[16]. In many cases the resistance is strongly associated with the
pull of point mutations occurred predominantly in androgen
binding site leading to the aberrant receptor up-regulation and
higher sensitivity rather than down-regulation and relaxation upon
antiandrogen therapy. It should be noted that AR agonists and AR
antagonists share different modes of action towards the related
transcriptional machinery inducing the “closed” and “open” con-
formations of AR-H12 helix [17e19]. Therefore, even minor modi-
fications in the structure of a small molecule AR ligand can lead to
dramatic alterations in the receptoreligand interaction thereby
providing opposite pharmacological responses. Accordingly, allo-
steric pockets in AR structure, including BF3 binding site [20], are
currently described as promising pathway to overcome the resis-
tance [for review see: [21e23]].
analogues (Fig. 2), including substituted 2-thioxo-7-oxa-1,3-diaza-
spiro[4.4]nonan-4-ones 6e9, heterocyclic analogues of 6-thioxo-
5,7-diaza-spiro[3.4]octan-8-ones 10 and 2-thioxo-1,3-diaza-spiro
[4.5]decan-4-ones 11, 2-thioxo-1,3-diaza-spiro[4.5]decan-4-ones
12e19, 2-thioxo-tetrahydro-pyrimidine-4-ones 20, and thioureas
21. We have also performed in silico modeling procedure for the
disclosed compounds using 3D-molecular docking approach to
estimate possible binding modes and diversity points.
2. Results and discussion
2.1. Chemistry
5-Methyl-2-thioxoimidazolidin-4-ones (R,S)-6, 11, (R,S)-14, (R)-
14, (S)-14, (R,S)-15, and (R,S)-16 were readily obtained from 4-
amino-2-fluoro-N-methylbenzamide (22) by reacting with the
appropriate ketone (23e28) and trimethylsilylcyanide in the
presence of ytterbium triflate (Scheme 1). The resulting product
(29e34) was then treated with 4-amino-2-(trifluoromethyl)ben-
zonitrile (35) and thiophosgene to generate the desired thio-
hydantoins mainly as racemic mixtures. Then, (R,S)-2-thioxo-7-
oxa-1,3-diazaspiro[4.4]nonan-4-one (6) and (R,S)-5-methyl-5-
(methoxymethyl)-2-thioxoimidazolidin-4-one (14) were success-
fully separated using chiral HPLC as optically pure compounds (R)-
6, (S)-6, (R)-14 and (S)-14. (R,S)-5-(Hydroxymethyl)-5-methyl-2-
thioxoimidazolidin-4-one (17) was synthesized by BBr3-mediated
Recently, we have developed novel AR antagonist, (R)-6 (Fig. 1),
containing 2-thioxo-7-oxo-1,3-diaza-spiro[4.4]nonan-4-one core
with sub-nanomolar activity [24] based on comprehensive SAR
studies reported for the related analogue 4 launched in 2012 by
Astellas Pharma and Medivation. Compound (R)-6 showed similar
cleavage
of
(R,S)-5-methyl-5-(methoxymethyl)-2-
thioxoimidazolidin-4-one (14), while (R,S)-(4-methyl-5-oxo-2-
thioxoimidazolidin-4-yl)acetic acid (18) was obtained by the hy-
drolysis of ethyl ester 16 in mild alkaline conditions.
to 4 and 5 mechanism of action and inhibited DHT (5a-dihy-
drotestosterone)-stimulated PSA expression and proliferation of
prostate cancer cells, prevented binding of androgens to the AR
ligand-binding domain, androgen-stimulated AR nuclear trans-
location and co-activator complex formation in vivo [25].
With the aim of obtaining more effective AR antagonists, we
have synthesized and tested an extended series of novel (R)-6
The
desired
(3R)-3-({3-fluoro-4-[(methylamino)carbonyl]
phenyl}amino)tetrahydrofuran-3-carboxylic acid ((R)-38) was
synthesized by the copper (I) catalysed reaction of 2-fluoro-4-iodo-
N-methylbenzamide (37), which in turn was obtained from inter-
mediate compound 22, and (R)-3-aminotetrahydrofuran-3-
carboxylic acid ((R)-36a) or its butyl ester (R)-36b. The reaction

A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 99 (2015) 51e66
53
S
S
S
R2
R1
R2
R1
R2
X
R1
N
N
N
N
N
N
O
O
O
X
O
6-9
10
11
S
S
S
R2
R3
R1
R2
R1
R 2
R1
O
N
N
N
H
N
H
N
N
R4
O
12-19
20
21
6-21: R1 = 6-cyano-5-trifluoromethylphenyl. 6, 10-21: R2 = 3-fluoro-4-(methylcarbamoyl)phenyl. (R)-7: R2 =
3-fluoro-4-(methoxycarbonyl)phenyl. (R)-8: R2 = 4-carboxy-3-fluorophenyl. (R)-9: R2 = 4-carbamoyl-3-fluorophenyl.
10a: X = NCHPh2. 10b (X = NMe) and 10c (X = O, synthesis was unsuccessful). 11a: X = O. 11b: X = NMe. 12: R3 =
R4 = H. (R,S)-13, (R)-13, (S)-13: R3 = H, R4 = Me. (R,S)-14, (R)-14, (S)-14,: R3 = Me, R4 = MeOCH2. (R,S)-15: R3 =
Me, R4 = PhCH2OCH2. (R,S)-16: R3 = Me, R4 = EtO2CCH2. (R,S)-17: R3 = Me, R4 = HOCH2. (R,S)-18: R3 = Me, R4 =
HO2CCH2. 19: R3 = R4 = MeOCH2.
Fig. 2. Structures of compounds described in the present work.
CN
S
+
R1
R3
R2
R3COR4
23-28
H2N
CF3
MeHNOC
F
N
N
MeHNOC
F
R3 R4
CN
35
R4
a
O
b
N
NH2
H
(R,S)-6, 11, (R,S)-14,
(R,S)-15, (R,S)-16
22
29-34
S
S
R1
HO2CCH2
R2
N
R1
HOCH2
R2
d
c
N
N
N
(R,S)-16
(R,S)-14
O
O
Me
(R,S)-18
c) - BBr3,
Me
(R,S)-17
Reagents and conditions: (a) - AcOH, MgSO4
b) - CSCl2
CH2Cl2
d) - EtOH, H2
Scheme 1.
proceeded smoothly under relatively mild conditions to provide
the desired molecule as well as a considerable amount of autoar-
ylation side product 39 (up to 50% by LC-MS). To overcome this
competitive by-reaction, we have protected the amide nitrogen in
compound 37 as 2-(trimethylsilyl)ethoxymethyl (SEM) ether
following the procedure depicted in Scheme 2. Then, the reaction of
protected amide 40 with aminoacid (R)-36a or its ester (R)-36b led
to individual arylated aminoacid (R)-41 which was further cyclized
upon the treatment with isothiocyanate 42 into the thiohydantoin
(R)-43. The final product (R)-6 was readily obtained after SEM-
deprotection.
better yield. This rational 3-step pathway is more adapted for the
synthesis of antagonist (R)-6 thereby providing a significantly
higher overall yield.
Methyl 4-{(5R)-3-[4-cyano-3-(trifluoromethyl)phenyl]-4-oxo-
2-thioxo-7-oxa-1,3-diazaspiro[4.4]non-1-yl}-2-fluorobenzoate (7)
was obtained starting from the aminolysis of 4-bromo-2-
fluorobenzoic acid (46) by aminoacid (R)-36a (Scheme 4). The
resulted diacid (R)-47 was then converted into the corresponding
diester (R)-48 which was further treated by isothiocyanate 42 to
afford compound (R)-7. Subsequent alkaline hydrolysis followed by
coupling with ammonia led to the final amide (R)-9.
It should be noted that in contrast to iodide 37 4-bromo-2-
fluoro-N-methylbenzamide 44 that was found to be less reactive
toward amines (R)-36a or (R)-36b produced aminoacid ((R)-38) as a
single product (Scheme 3). The intermediate compound 38 was
then readily converted into ester 45 using one of the classical
methylation procedures followed by the reaction with isothiocya-
nate 42. As a result the target compound (R)-6 was obtained in
Synthesis of 6-thioxo-2,5,7-triaza-spiro[3.4]octan-8-one (10a)
was performed following the route depicted in Scheme 5 below.
Initially, 1-benzhydrylazetidin-3-one (49) was smoothly converted
into 2-benzhydryl-2,5,7-triaza-spiro[3.4]octane-6,8-dione (50) by
the spiroejunction with KCN and (NH4)2CO3. The resulting product
was readily hydrolysed by KOH/H2O into 3-amino-1-
benzhydrylazetidine-3-carboxylic acid (51) which was then

54
A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 99 (2015) 51e66
O
F
O
O
H2N
CO2R
O
O
O
a
b
22
MeHN
+
CO2H
CO2H
+
MeHN
MeHN
N
N
N
H
O
H
I
Me
F
(R)-36a (R=H)
or
(R)-36b (R=Bu)
F
F
37
(R)-38
(R)-39
c
S
O
F
R1
N
O
O
O
N
e
d
SEM
b
+ R1NCS
(R)-6
N
N
SEM
+ 36a or 36b
N
SEM
CO2H
Me
42
O
N
I
Me
Me
F
H
F
O
40
(R)-41
(R)-43
42, (R)-43: R1 = 4-cyano-3-(trifluoromethyl)phenyl.
Reagents and conditions: (a) H2O, H2SO4, NaNO2
b) - CuI, K2CO3, H2O, DMF, Et3N,
e) - TFA,
c
3Si(CH2)2OCH2
d
CH2Cl2
Scheme 2.
O
O
O
b or c or d
a
42, e
MeHN
(R)-6
+ (R)-36a or (R)-36b
RHN
CO2Me
RHN
CO2H
F
Br
44
38
45
(R)-38, (R)-45: R = 3-fluoro-4-(methylcarbamoyl)phenyl.
Reagents and conditions: (a) - K2CO3, CuI, H2O, DMF, Et3
c) - MeCN, N,N
b) - DMF, K2CO3,
d) - MeOH, SOCl2, reflux, 4 h; (e) - DMSO,
Scheme 3.
O
O
F
HO2C
F
MeO2C
42, c
d
e
b
a
(R)-8
(R)-9
(R)-7
(R)-36a + Br
CO2H
CO2Me
N
CO2H
F
N
H
H
46
47
48
Reagents and conditions: (a) - CuI, K2CO3, H2O, DMF, 2-acetylcyclohexanone, 100 oC, 48 h; (b) - MeOH, SOCl2, reflux, 15 h; (c) - DMSO, EtOAc, 85 oC, 48
h, then MeOH, 85 oC, 30 min; (d) - MeOH, H2O, NaOH, 25 oC, 12 h; (e) - EDAC, HOBt, NH4Cl, Et3N, DMF, 25 oC, 24 h.
Scheme 4.
O
R1HN
R1HN
N
H2N
O
HN
42, e
a
b
NH
O
d
43, c
Ph2CH
CO2H
CO2Me
CO2H
10a
N
N
N
Ph2CH
N
Ph2CH
Ph2CH
49
51
52
53
Ph2CH
X
f, g
S
50
f
R1
R1HN
R1HN
R1HN
N
42, e
X
g
d
CO2Me
R2
N
CO2H
CO2H
N
N
Me
N
N
H
Me
Me
O
54
55
56
10b
10b, 52-56: R1 = 3-fluoro-4-(methylcarbamoyl)phenyl; 10b: R2 = 4-cyano-3-(trifluoromethyl)phenyl.
Reagents and conditions: (a) - KCN, (NH4)2CO3, H2 b) - KOH, H2O, reflux, 48 h; (c) - K2CO3, CuI,
2-acetylcyclohexanone, DMF, H2
d
e) - DMSO, EtOAc, 90
f
g) - H2
2
Scheme 5.

A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 99 (2015) 51e66
55
converted into 3-[3-fluoro-4-(methylcarbamoyl)phenylamino]-1-
methylazetidine-3-carboxylic acid (52) upon the conditions
described above. Esterification of 52 via CDI activation followed by
the cyclization with isothiocyanate 42 led to the desired 2-
benzhydryl-6-thioxo-2,5,7-triaza-spiro[3.4]octan-8-one 10a. Our
effort to convert 10a into2-methyl-6-thioxo-2,5,7-triazaspiro[3.4]
octan-8-one 10b has failed due to the destruction of the spiro-frame
upon the removal of benzhydryl protection group. We also did not
succeed to obtain 10b by sequential transformation of 1-
benzhydrylazetidine-3-carboxylic acid 52 into 1H-azetidine-3-
carboxylic acid 54, 1-methylazetidine-3-carboxylic acid 55 and its
methyl ester 56 finished with cyclisation to 10b in the last step.
3-[3-Fluoro-4-(methylcarbamoyl)phenylamino]oxetane-3-
followed by cyclization of the resulted [3-fluoro-4-(methyl-
carbamoyl)phenylamino]acetic acid (67) with isothiocyanatе 42
(Scheme 7).
5-Methyl-2-thioxoimidazolidin-4-ones (R,S)-13, (R)-13, (S)-13
were prepared generally following the same synthetic route
described above for 12 using corresponding alanines D,L-68, D-68
and L-68 with minor variations in reaction conditions (Scheme 8).
Compound 19 was synthesized following the route depicted in
Scheme 9. Thus, SEM-protected amide 40 was used instead of 37 to
construct the desired thiohydantoin core since 2-amino-3-
methoxy-2-(methoxymethyl)propionic acid was not sufficiently
reactive due to steric clashes and conditions required for the ary-
lation/autoarylation of 37 that also took place.
carboxylic acid (59) and its methyl ester 61 were obtained starting
from 3-аminooxetane-3-carboxylic acid (57) which was initially
arylated with 4-iodo-N-methylbenzamide 40 providing compound
58 (Scheme 6) which was readily deprotected upon the treatment
with acid or even during HPLC purification in the presence of TFA in
mobile phase to give acid 59. It should be noted that HPLC purifi-
cation of acid 58 with neutral mobile phase retained SEM-
protection group. Subsequent esterification of 58 followed by
deprotection particularly during HPLC purification with TFA led to
the formation of valuable intermediate 61. Interestingly, the reac-
tion of 3-aminooxetane-3-carboxylic acid 59 or its ester 61 with
isothiocyanate 42 afforded N-[4-cyano-3-(trifluoromethyl)phenyl]-
3-[3-fluoro-4-(methylcarbamoyl)phenylamino]oxetane-3-
carboxamide (62) as the main product instead of target 6-thioxo-2-
oxa-5,7-diazaspiro[3.4]octan-8-one 10c. Treatment of 62 with
CSCl2 did also not lead to any detectable amount of 10c. Our efforts
to construct thiohydantoin frame prior to 5-arylation in compound
66 by the reaction of aminoacid 57 with isothiocyanate 42 were
also unsuccessful.
Addition of aniline 22 to ethyl acrylate resulted in amino-
propionate 72 which then reacted with isothiocyanatе 42 to form
thioureidopropionic ester 73. Subsequent mild hydrolysis of 73
followed by cyclization with TBTU led to the formation of the final
4-oxo-2-thioxotetrahydropyrimidine 20 (Scheme 10). Thiourea 21
was synthesized by the microwave-assisted reaction of aniline 22
and isothiocyanatе 42 in DMF at 80 ꢀC.
2.2. In vitro biological activity
All the synthesized molecules have been thoroughly evaluated
in vitro using model LNCaP cells (see Experimental Section). As
shown in Table 1, the activity of the tested compounds varies in a
wide range. Among them, chiral methylamide (R)-6, ester (R)-7 and
amide (R)-9 were found to be the most active AR binders with a Ki
value in the range of 11.9e18.4 nM. It was certainly unexpected that
the parent spiro-acid did not show any detectable activity against
AR. 2-Thioxo-imidazolidin-4-one (R)-14 which can be regarded as
close structural analogue of the active triade was significantly less
active under the same conditions. Thus, bearing the same periph-
eral substituents compounds (R)-6 and (R)-14 demonstrated
dramatically different binding affinity and showed Ki values of 18.4
and 40.6 nM, respectively. It is especially important to note that (S)-
enantiomers of compounds (R)-6 and (R)-14 were 11.7- and 19.6-
times less active than the hit-compounds, respectively. Our pains-
taking efforts to synthesize and isolate 2-methyl-6-thioxo-2,5,7-
triaza-spiro[3.4]octan-8-one 10b and 6-thioxo-2-oxa-5,7-diaza-
spiro[3.4]octan-8-one 10c have failed ruling out any estimations of
their binding potency. Either 2-benzhydryl-6-thioxo-2,5,7-triaza-
In acidic media the reaction provided one main product with
molecular weight corresponding to compound 65 (LC-MS [MþH]þ
346), NMR spectra (13С NMR, DMSO-d6:
d 64.65 (СН2О) and 35.72
(СН2N)) fitted to 1-[4-cyano-3-(trifluoromethyl)phenyl]-4-
(hydroxymethyl)-2-thioxoimidazolidine-4-carboxylic acid (63).
The formation of the later presumably occurs through oxetane ring
opening in initially formed 65 followed by ring closure in the in-
termediate 64.
2-Thioxoimidazolidin-4-one 12 was prepared from 37 by copper
(I) catalysed reaction with glycine in microwave reactor at 140 ꢀC
O
O
R1HN
H2N
SEM
SEM
O
O
c
40, a
b
N
N
CO2Me
CO2H
Me
Me
O
O
F
N
CO2H
F
N
CO2Me
H
H
R1HN
57
58
60
61
c or d
CO2H
42, h
42, e or f
O
59
S
S
S
N
S
S
R2
R1HN
O
NHR2
O
g
R2
R2
R1
R2
R2
HN
OH
N
H
HN
O
N
H
HN
N
N
HN
O
N
CO2H
CO2H
O
O
HO
CO2H
O
OH
63
64
65
66
10c
62
Reagents and conditions: (a) - DMF, H2O, CuI, K2CO3, TEA, 2-acetylcyclohexanone; (b
(c) - TFA; (d) - HCl; (e
f) - DIPEA, MeCN; (g) - CSCl2; (h
Scheme 6.

56
A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 99 (2015) 51e66
MeHNOC
42, b
a
12
37 + H2NCH2CO2H
F
NHCH2CO2H
67
Reagents and conditions: (a) - DMF, K2CO3, CuI, MW, 140 oC, 18 min; (b) - DMF, 90 oC, 12 h.
Scheme 7.
MeHNOC
Me
42, b
a
(R,S)-13, (R)-13, (S)-13
37 + H2NCH(CH3)CO2H
DL-68, D-68, L-68
CO2H
(R,S)-69, (R)-69, (S)-69
N
F
H
Reagents and conditions: (a) - DMSO, Cs2CO3, CuI, Me2NCH2CO2H, 90 oC, 20 h; (b) - DMF, 80 oC, 18 h.
Scheme 8.
O
S
CN
O
N
SEM
H
N
N
CO2H
c
42, b
a
N
N
19
Me
40
SEM
F
CF3
Me
F
O
OMe
OMe OMe
OMe
70
71
Reagents and conditions: (a) - 3-methoxy-2-(methoxymethyl)alanine, CuI, 2-acetylcyclohexanone, K3PO4, DMF, 100
oC, 48 h; (b) - DMF, pyridine, 90 oC, 36 h; (c) - DCM, TFA, 25 oC, 2 h.
Scheme 9.
S
S
R2
R2
R1
d
R1
c
42, b
a
N
N
N
N
R1HN(CH2)2CO2Et
20
22 + CH2=CH-CO2Et
H
H
CO2Et
CO2H
72
73
74
72-74: R1 = 3-fluoro-4-(methylcarbamoyl)phenyl; 73, 74: R2 = 4-cyano-3-(trifluoromethyl)phenyl.
Reagents and conditions: (a c) - EtOH, H2
b
d) -
Scheme 10.
spiro[3.4]octan-8-one 10a or 8-methyl-2-thioxo-1,3,8-triaza-spiro
[4.5]decan-4-one 11b did not show a considerable binding affinity
toward AR. At the same time, oxygen-containing analogue, 8-
methyl-2-thioxo-1,3,8-triaza-spiro[4.5]decan-4-one 11a, demon-
strated almost nanomolar binding potency, Ki ¼ 126.1 nM. These
results clearly indicate that compounds containing “spiro-oxo-
cycloalkyl” moiety ((R)-6, 10с and 11a) are more promising AR li-
gands as compared to their “spiro-cycloalkyl” derivatives (4, 5,
10a,b and 11b).
Steric effect of the substituents in position 4 of 2-thioxo-imi-
dazolidin-4-one fragment of AR modulators is ambiguous. Thus, an
increase in their volume during the hopping from 4,4-dimethyl- 4
(Ki ¼ 28.2 nM) to 4-methyl-4-methoxymethyl- (R,S)-14, 4-methyl-
4-benzyloxymethyl-15 and 4,4-dimethoxymethyl-2-thioxo-imida-
zolidin-4-one (R,S)-19 is accompanied with a dramatic fold in the
target activity, by 2.4-, 18.2- and 20.7-times, respectively. Simul-
taneously, the transition from 4,4-dimethyl- 4 (IC50 ¼ 127.1 nM) to
4-methyl- (R,S)-13 (IC50 ¼ 944.4 nM) and unsubstituted (at position
4) 2-thioxo-imidazolidin-4-one 12 (IC50 > 10,000 nM) led not only
to insignificant loss of activity but also shifted the response speci-
ficity d antagonistic action was changed for partially agonistic
modulation (Fig. 3). Similar effect was observed in the case of
hopping from 4-methyl-4-methoxymethyl- (R,S)-14 to 4-
hydroxymethyl-4-methyl-2-thioxo-imidazolidin-4-one
(R,S)-17
and accompanied by 1.57-fold decrease in the target activity. The
replacement of 4-(ethoxycarbonylmethyl)-4-methyl- (R,S)-16
(Ki ¼ 235.9 nM) by 4-carboxymethyl-4-methyl-2-thioxo-imidazo-
lidin-4-one (R,S)-18 resulted in significant loss of activity. In
contrast to a relatively weak binding potency of 2-thioxo-imida-
zolidin-4-one 12, its related analogue, 2-thioxo-tetrahydro-pyr-
imidin-4-one 20, showed a moderate affinity (Ki ¼ 575.6 nM),
therefore this core can be reasonably regarded as promising start-
ing point for the development of novel highly potent AR ligands.
Finally, compound 21 was also found to be completely inactive
toward AR.
The second group of compounds includes partial (13.5%e29.2%)

A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 99 (2015) 51e66
57
Table 1
Table 2
In vitro activity of the tested compounds in LNCaP cells.
PK parameters for the selected compounds after single po and iv doses in rats
(n ¼ 3).
ꢀ
N
Type
IC50, nM
Ki SD, nM
Compound
(R)-6
(R)-7
(R)-8
(R)-9
2
4
5
antagonist
antagonist
antagonist
antagonist
antagonist
antagonist
antagonist
antagonist
antagonist
antagonist
antagonist
antagonist
partial agonist
partial agonist
partial agonist
partial agonist
antagonist
antagonist
antagonist
antagonist
antagonist
partial agonist
antagonist
antagonist
antagonist
antagonist
732.7
127.1
174.0
195.6
79.2
160.2 95.7
28.2 12.5
34.7 12.6
33.8 6.3
18.4 10.4
215.4 18.6
11.9 10.9
NA
Administration
Dose, mg/kg
po
5
0.13
5.2
2
iv
1
po
5
iv
1
po
5
iv
1
po
5
iv
1
(R,S)-6
(R)-6
(S)-6
(R)-7
(R)-8
(R)-9
10a
11a
11b
12
(R,S)-13
(R)-13
(S)-13
(R,S)-14
(R)-14
(S)-14
(R,S)-15
(R,S)-16
(R,S)-17
(R,S)-18
19
K
el, 1/h
0.167 0.18 0.051 0.19 0.25
4.1 4.3 17.1 4.0 3.3
0.083 1.67 0.083 0.42 0.083 2.0
531 104 518 231 1177 261
0.39 0.46
1.8
T , h
1.5
0.083
185
½
2000.0
59.0
T
C
max, h
max, ng/ml
1062
NAa
AUC(0et), h,ng/mL 13,001 2572 570 599
AUC(0e∞), h,ng/mL 13,105 2624 576 712
Vd, L/kg
Cl, L/h/kg
MRTlast, h
MRTINF, h
716 497
743 509
38.4 8.0
7.0
3.5
4.2
e
1280 295
1389 303
9.4
3.6
3.1
3.7
e
71.4
NA
14.1 8.0
NA
e
2.3
0.4
4.8
5.3
2.0
e
53.0 46
7.0
3.3
1.6
1.8
6.1
e
539.8
>10,000
>10,000
944.4
1511.1
398.1
344.0
184.8
3166.7
2300.4
1378
126.1 23.6
e
8.8
4.6
5.0
e
1.7
4.3
12.8
22.3
e
2.0
0.5
0.8
1.6
e
NCb
6.9
7.2
e
NC
257 334.1
481.8 222.3
95.3 66.5
68.0 34.6
40.6 16.1
797.6 270.6
514.5 312.5
235.9 57.0
43.3 18.9
NA
V
ss, L/kg
F, %
100
19
29
87
the reported AR ligands 4 and 5 (Table 1), main pharmacokinetic
(PK) features have been investigated exclusively for this triade in
rats (Table 2). During the study amide (R)-9 and acid (R)-8 were
revealed as basic Phase I metabolites of methylamide (R)-6 (N-
dealkylation) and ester (R)-7 (O-dealkylation), respectively.
Bioavailability of the hit-compounds was also calculated and pre-
sented in Table 2. Upon oral (po) administration of ester (R)-7 only
its metabolite (R)-8 was detected in plasma, therefore PK param-
eters were calculated based on the concentration of this metabolite.
As shown in Table 2, the max bioavailability (F ~ 100%) was revealed
for methylamide (R)-6, while the min value was observed for acid
(R)-8 (F ¼ 19%) upon po administration of ester precursor (R)-7. The
principal claim is in very favourable PK profile of methylamide (R)-
6 as compared to other compounds tested. Thus, the hit compound
showed a relatively low clearance expressed in Kel and Cl values,
212.2
NAb
3374.0
3005.3
>10,000
584.6 386.2
575.6 437.9
NC
20
21
a
NA e not active.
b
NC e not calculated.
agonists: 12 without any substituents in position 4 of 2-thioxo-
imidazolidin-4-one fragment core as well as (R,S)-13, (R)-13, and
(S)-13 with one substituent in the same position (Fig. 1). It should
be noted that in contrast to methoxymethyl- and benzyloxymethyl-
containing derivatives (R,S)-14, (R)-14, (S)-14 and (R,S)-15 hydrox-
ymethyl analogue (R,S)-17 can be classified as partial AR agonist
(37.7%) with a Ki value of 43.3 18.9 nM that is 1.57-fold higher
than that observed for compound (R,S)-14 (Ki ¼ 68.0 34.6 nM).
The hopping from poorly active partial (13.5%) agonist 12
extended half-life time (T ) and, that seems to be the most
½
important, d significantly higher exposition in plasma expressed in
terms of Сmax and AUC. Considering a prolonged stability and high
activity of (R)-6 and its active metabolite (R)-9 in rats this com-
pound (methylamide) was unambiguously selected for further
evaluation as promising clinical candidate.
(IС50 > 10 mM) without any substituents in position 4 of 2-thioxo-
imidazolidin-4-one fragment to 4- and 5-unsubstituted 2-thioxo-
tetrahydro-pyrimidin-4-one 20 led to a drastic increase in the
target antagonistic activity (IС50 ¼ 3
mM) with only a trace amount
2.4. In silico modeling
of agonistic action. It should also be mentioned that 3-(3-fluoro-4-
methylcarbamoyl-phenylamino)-oxetane-3-carboxylic acid (4-
cyano-3-trifluoromethyl-phenyl)-amide (62) was formally attrib-
uted to partial (16.2%) agonist class with a relatively low activity
(IС50 ¼ 1425 nM, Ki ¼ 310.3 nM).
Androgen receptors share similar to other nuclear receptors
(e.g., estrogen receptor) three-dimensional structure containing 11
a-helices and two
b-turns that forms three layers packed in the
antiparallel “ -helical sandwich” cluster [26,27]. Upon agonist
a
binding, the carboxylterminal helix 12 (H12) undergoes a confor-
mational change thereby forming a cap that results in ligand sta-
2.3. Pharmacokinetics
bilization and retaining. Simultaneously, the second
b-turn is
Considering higher antagonistic activity of the most potent
compounds from this series (R)-6, (R)-7, (R)-9, as compared with
formed to lock the active conformation of H12. As a result, the
activation function 2 (AF2) site is immediately formed. It plays a
Fig. 3. Agonistic activity of the tested compounds.

58
A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 99 (2015) 51e66
crucial role in the recruitment of co-activators thereby controlling
the activity of down-stream transcription factors related to AR [28].
membrane permeability of polar (R)-8 containing the negatively
charged carboxylic acid moiety through the membrane of the
model cells. It is not surprising, that only single binding mode (with
H874) was predicted for ester (R)-7. Under the applied assay con-
ditions compounds 4 and 5 were approximately two times less
active than (R)-6. It may be due to the synergetic effect achieved by
alternative binding mode and additional weak H-bond observed
between oxygen atom in tetrahydrofuran spiro-moiety and T877
(not shown here).
To elucidate three-dimensional structure of the selected hit-
compound (R)-6 we have performed crystallographic analysis
(Fig. 5a,b). As shown in Fig. 5, two conformations were observed in
the crystal cell providing two different locations of the terminal
amide bond (Fig. 5a). Two torsion angles (ta) of ꢁ 125ꢀ and ꢁ 49.3ꢀ
(HNeC(O)-Car-Car(F)) are fixed by rotation of the amide group. The
active conformation of (R)-6 outputted from the docking study has
the invert orientation realized by the rotation of benzamide frag-
ment by 180ꢀ (ta ¼ ꢁ66.6ꢀ). Conformational analysis (in vacuo)
provided 15 possible conformations of compound (R)-6 with the
values of potential energy close to the calculated minimum
(Fig. 5b). One of the most fitted conformations is presented in
Fig. 5a (ta ¼ 153ꢀ).
Several small-molecule antagonists, e.g. 2, induce
a partial
unfolding of H12 resulting in functionally disabled AF2 region
similar to that previously observed for estrogen receptor com-
plexed with the selective antagonist raloxifene [29].
To elucidate the possible mechanisms of action and the antag-
onistic potencies of the evaluated compounds, we initially per-
formed a thorough computational analysis of the target structure
based on available protein data (PDB codes: 2IHQ, 1Z95, 3V49 and
3V4A) and SAR evaluations. The results of several recently reported
studies were taken into consideration [30,31]. At least one residue
His874 was found to be conformationally flexible. In addition, three
aminoacid residues positioned close to His874 were also suggested
to be not strictly rigid in their native location. In silico modeling
procedure was then performed for compounds (R)-6e9 in ICM-Pro
software [32] using the crystallographic structures published pre-
viously by Nique and colleagues [33] (PDB codes: 3V49 and 3V4A)
as the major 3D template. Binding site was then completely
reconstructed with flexible points assigned to His874, W741, M742
and I899. Reference compounds 4-[(4R)-4-(4-hydroxyphenyl)-3,4-
dimethyl-2,5-dioxoimidazolidin-1-yl]-2-(trifluoromethyl)benzoni-
trile (from 3V49) and
(5R)-3-(3,4-dichlorophenyl)-5-(4-
hydroxyphenyl)-1,5-dimethyl-2-thioxoimidazolidin-4-one (from
3V4A) were docked into the active binding site as 2D structures
with a predefined stereospecifity. Docking procedure with all the
force-field components available classified the compounds as high-
score AR ligands. The predicted active conformations correlated
well with that observed in X-Ray structures (RMSD < 0.2). Then,
compounds (R)-6e9 were docked into the same binding site
applying the semi-static mode using the same software settings. 3D
representations of the supramolecular interfaces predicted for the
active conformations, related scorings and correlation plot are
presented in the Supporting Information (SI). Fig. 4 shows the
resulting 2D supramolecular frame reconstructed based on the
obtained docking results between crucial binding points (BPs) of AR
antagonists 4, 5, (R)-6, (R)-7, (R)-8, (R)-9 and key aminoacids of AR.
As shown in Fig. 3, the binding mode predicted for the known AR
antagonists 4 and 5 is absolutely identical to that observed for the
reference compounds. The key conservative interactions were
identified for all the compounds tested between nitrile moiety and
R752 (H-bonding) as well as (trifluoromethyl)phenyl ring and
W741/T877 (hydrophobic contacts). The aminoacid surrounding
covering the spiro-moiety in 5, (R)-6e9 and di-methyl joint in 4 is
also very similar and includes F876, L701 and M780. Considerable
differences in binding of the reference molecules, known ligands
and compounds (R)-6e9 were observed within the ensemble of
aminoacids surrounding the substituted (trifluoromethyl)phenyl
ring in the opposite side of the molecules. Thus, the hydrogen bond
is readily formed between the phenyl OH group in the structures of
the reference compounds and H874, whereas for known amides 4
and 5 this contact was not predicted within the pull of generated
conformations. However, these molecules provide the alternative
H-bond observed between NH-group of methylamide fragment
and the oxygen of peptide chain formed by M895. Contrariwise,
two alternative binding modes were revealed for amide (R)-6. The
first binding mode is similar to that predicted for compounds 4 and
5 (contact with M895 or Asn705), while the second one is stabilized
by H-bonding with H847 as has been revealed for the template
compounds. The analogous binding mode was predicted for the N-
demethylated derivative (R)-9 and acid (R)-8. However, the H-bond
between acid OH group and M895 is less possible than with H874.
Interestingly, acid (R)-8 was completely inactive in the biological
assay performed but showed virtual score comparative to active
analogues. It can be reasonably elucidated by a drastically poor
3. Conclusions
A small-sized library of novel substituted spiro-4-(5-oxo-3-
phenyl-2-thioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzoni-
triles were synthesized and evaluated in vitro against androgen-
sensitive LNCaP cell line. Three compounds from the described
series, (R)-6, (R)-7 and (R)-9, were identified as the most promising
hits with IC50 values of: 79.2, 59.0 and 71.4 nM (PSA expression/
production inhibition), respectively. These molecules were 1.6-,
2.15- and 1.78-fold more active than 4. On the basis of extensive PK
study performed in rats compound (R)-6 that showed an appro-
priate profile was selected for further clinical evaluation. This in-
class small molecule antiandrogen can be reasonably regarded as
the pro-drug of its N-dealkylated active metabolite (R)-9. SAR study
performed for the evaluated compounds has revealed the key role
of spiro-moiety and functionalities in para-position of fluoro-
phenyl ring. Possible binding modes of the active compounds
were predicted using 3D molecular docking approach and
compared with the available X-Ray data for analogue compounds.
Several distinctive aspects in binding were observed elucidating
differences in the target activity.
4. Experimental section
4.1. Chemistry general procedures
Reference substance 2 was purchased from Tocris, catalogue no.
3389, 4 from Selleck Chemicals, catalogue no. S1250 and 5 from
Selleck Chemicals, catalogue no. S2840. All chemicals, starting
materials/reagents and solvents were used as received from the
suppliers without further purification. The crude reaction mixtures
were concentrated under reduced pressure by removing the
organic solvents in a rotary evaporator. Nuclear magnetic reso-
nance (NMR) spectra were recorded using a Bruker DPX-400
spectrometer at room temperature (rt) with tetramethylsilane as
an internal standard. The chemical shifts (d) are reported in parts
per million (ppm) and the signals are reported as s (singlet),
d (doublet), t (triplet), q (quartet), m (multiplet), or br s (broad
singlet). The purities of the final compounds were determined by
HPLC and were greater than 98%. The HPLC conditions for assessing
purity were as follows: Shimadzu HPLC, XBridge™ C18,
4.6 mm ꢂ 250 mm (3.5
mm); gradient of 0.1% TFA in 5% acetonitrile/

A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 99 (2015) 51e66
59
Fig. 4. 2D supramolecular interface predicted for AR antagonists 4, 5, (R)-6, (R)-7, (R)-8 and (R)-9.
Fig. 5. 3D Representation of (R)-6: (a) rigid alignment of two X-Ray structures and active conformation resulted from the docking study; (b) rigid alignment of two X-Ray structures
and conformation generated in vacuo.
water (A) and 0.1% TFA acetonitrile (B); flow rate, 0.5 mL/min;
acquisition time, 20 min; wavelength, UV 214 and 254 nm. The
preparative HPLC system included two sets of Shimadzu LC-8A
pumps, a Shimadzu controller SCL 10Avp, and a Shimadzu detec-
XBridge C18 3.5
m
m column (4.6 mm ꢂ 150 mm). High resolution
mass spectra (HRMS) were acquired on an Orbitrap Elite mass
spectrometer (Thermo, Bremen, Germany) equipped with an HESI
ion source.
tor SPD 10Avp. A Reprosil-Pur C-18-AQ 10
m
m, 250 mm ꢂ 20 mm
column was used. The mobile phase was a gradient of 0.1% TFA in
water (A) and 0.1% TFA in acetonitrile (B). LC/MS was conducted on
a PE Sciex API 165 system using electrospray in positive ion mode,
[MþH]þ, and a Shimadzu HPLC system equipped with a Waters
4.1.1. General procedure 1
4.1.1.1. 4-{3-[4-cyano-3-(trifluoromethyl)phenyl]-5-substitute-4-
oxo-2-thioxoimidazolidin-1-yl}-2-fluoro-N-methylbenzamides (R,S)-
6, 11a, 11b, (R,S)-14, (R)-14, (S)-14, (R,S)-15, and (R,S)-16.

60
A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 99 (2015) 51e66
Thiophosgene (0.473 g, 4.15 mmol) was added dropwise to the
solution of 4-[(cyanomethyl)amino]-2-fluoro-N-methylbenzamide
29e34 (3.77 mmol, procedure below) and 4-amino-2-(tri-
fluoromethyl)benzonitrile (35) (0.772 g, 4.15 mmol, Apollo Scien-
tific) in DMF (15 mL) keeping the temperature below 25 ꢀС. The
resulting mixture was stirred for 0.5 h at the ambient temperature
and then for 12 h at 80 ꢀС. After the stirring was completed, the
mixture was mildly cooled down. The analogous procedure was
applied for compound 35. DMF was evaporated in vacuo, the
methylbenzamide ((R,S)-15). Yield 17%. MS (ESI) [MþH]þ 571. 1H
NMR (400 MHz, CDCl3)
d
8.22 (t, J ¼ 8.4 Hz, 1H), 7.96 (d, J ¼ 8.0 Hz,
1H), 7.86 (s, 1H), 7.70 (dd, J1 ¼ 8.0 Hz, J2 ¼ 1.2 Hz, 1H), 7.39 (m, 3H),
7.29 (m, 2H), 7.25 (dd, J1 ¼ 8.4 Hz, J2 ¼ 1.6 Hz, 1H), 7.18 (dd,
J1 ¼8.4 Hz, J2 ¼ 1.6 Hz,1H), 6.71 (q, J ¼ 4.8 Hz,1H), 4.59 (m, 2H), 3.79
(d, J ¼ 10.2 Hz, 1H), 3.45 (d, J ¼ 10.2 Hz, 1H), 3.08 (d, J ¼ 4.8 Hz, 3H),
1.51 (s, 3H).
4.1.1.7. Ethyl {1-[4-cyano-3-(trifluoromethyl)phenyl]-3-[3-fluoro-4-
(methylcarbamoyl)phenyl]-4-methyl-5-oxo-2-thioxoimidazolidin-4-
yl]acetate ((R,S)-16). Yield 14%. MS (ESI) [MþH]þ 536. 1H NMR
formed residue was dissolved in 15 mL of MeOH and 1.5 mL of 3
М
HCl, then the resulting mixture was refluxed for 3 h. The solvent
was removed under reduced pressure, the formed residue was
treated with water, filtered off, washed with water and dried in
vacuo. The desired product was then purified by HPLC.
(400 MHz, CDCl3)
d
8.26 (t, J ¼ 8.4 Hz, 1H), 8.01 (d, J ¼ 8.0 Hz, 1H),
8.00 (s,1H), 7.90 (dd, J1 ¼8.0 Hz, J2 ¼ 1.6 Hz,1H), 7.18 (dd, J1 ¼8.0 Hz,
J2 ¼ 1.6 Hz, 1H), 7.10 (dd, J1 ¼ 8.0 Hz, J2 ¼ 1.6 Hz, 1H), 6.78 (q,
J ¼ 4.8 Hz, 1H), 4.26 (m, 1H), 3.13 (d, J ¼ 18.0 Hz, 1H), 3.09 (d,
J ¼ 4.8 Hz, 3H), 2.64 (d, J ¼ 18.0 Hz, 1H), 1.67 (s, 3H), 1.31 (t,
J ¼ 7.0 Hz, 3H).
4.1.1.2. 4-{3-[4-Cyano-3-(trifluoromethyl)phenyl]-4-oxo-2-thioxo-7-
oxa-1,3-diazaspiro[4.4]non-1-yl}-2-fluoro-N-methylbenzamide
((R,S)-6). Yield 21%. MS (ESI) [MþH]þ 493. 1H NMR (400 MHz,
CDCl3)
d
8.30 (t, J ¼ 8.4 Hz, 1H), 8.02 (d, J ¼ 8.4 Hz, 1H), 7.98 (d,
4.1.1.8. (R)-3-Aminotetrahydrofuran-3-carboxylic acid ((R)-36a).
NaOH (1N, 150 mL) was added to the solution of compound (R)-36b
(16 g, 85 mmol) [36] in fresh methanol (150 mL). The resulting
mixture was vigorously stirred for 15 h, then acidified with HCl to
pH ¼ 3e4 and rotovapped. The obtained product can be purified by
the extraction with methanol. MS (ESI) [MþH]þ 132. 1H NMR
J ¼ 1.6 Hz, 1H), 7.85 (dd, J1 ¼ 8.4 Hz, J2 ¼ 1.6 Hz, 1H), 7.34 (dd,
J1 ¼ 8.4 Hz, J2 ¼ 1.6 Hz, 1H), 7.25 (dd, J1 ¼ 11.8 Hz, J2 ¼ 1.6 Hz, 1H),
6.78 (q, J ¼ 4.4 Hz, 1H), 4.43 (d, J ¼ 10.0 Hz, 1H), 4.16 (d, J ¼ 10.0 Hz,
1H), 3.96 (m, 1H), 3.75 (m, 1H), 3.09 (d, J ¼ 4.4 Hz, 3H), 2.74 (m, 1H),
2.48 (m, 1H). ESIHRMS m/z calcd for C22H17F4N4O3S [MþH]þ
493.0952; found 493.0949. Separation of individual enantiomers
(R)-6 and (S)-6 from racemic mixture was performed by HPLC with
(400 MHz, D2O)
d 4.06 (m, 1Н), 3.97 (m, 3Н), 2.55 (m, 1Н), 2.18 (m,
1Н).
chiral column Phenomenex Lux Amylose-2 (4 5
m
m, 250 ꢂ 20 mm).
Isocratic 15/85 system of MeOH/EtOH (A) and n-hexane (B) was
used as a mobile phase, flow rate was 20 mL/min. Retention times
for (S)-6 and (R)-6 were 19 and 22 min, respectively.
4.1.1.9. 4-{(R)-3-[4-cyano-3-(trifluoromethyl)phenyl]-4-oxo-2-
thioxo-7-oxa-1,3-diazaspiro[4.4]non-1-yl}-2-fluoro-N-methyl-
benzamide ((R)-6)
4.1.1.9.1. Procedure
А. The mixture of 4-amino-2-fluoro-N-
4.1.1.3. 4-{3-[4-Cyano-3-(trifluoromethyl)phenyl]-4-oxo-2-thioxo-8-
oxa-1,3-diazaspiro[4.5]dec-1-yl}-2-fluoro-N-methylbenzamide (11a).
methylbenzamide (22) (1.68 g, 10 mmol) [35], H2SO4 (0.68 mL) and
water (13 mL) was gently heated until all the components were
completely dissolved. The mixture was cooled to 0e5ꢀС under
stirring, then the solution of NaNO2 (0.7 g,10 mmol) in water (2 mL)
was added dropwise. The resulting mixture was stirred at 0e5ꢀС
for 0.5 h and then slowly poured into the solution of KI (5 g) in cold
water (20 mL). The solution was then heated up to 80ꢀС and stirred
for 0.5 h. After cooling it was treated with 30 mL of chloroform and
filtered. The organic layer was washed with 5% Na2SO3 solution,
dried over Na2SO4 and rotovapped. Column chromatography on
silica gel (hexane/EtOAc ¼ 6:1) afforded 2.33 g (83%) of 2-fluoro-4-
iodo-N-methylbenzamide (37). MS (ESI) [MþH]þ 280. 1H NMR
Yield 10%. MS (ESI) [MþH]þ 507. 1H NMR (400 MHz, CDCl3)
d 8.32 (t,
J ¼ 8.4 Hz,1H), 8.01 (d, J ¼ 8.0 Hz,1H), 7.95 (s,1H), 7.83 (d, J ¼ 8.0 Hz,
1H), 7.20 (d, J ¼ 8.4 Hz, 1H), 7.10 (d, J ¼ 8.0 Hz, 1H), 6.73 (m, 1H), 4.18
(m, 2H), 3.94 (m, 2H), 3.09 (d, J ¼ 4.4 Hz, 3H), 2.07 (m, 4H). ESIHRMS
m/z calcd for C23H19F4N4O3S [MþH]þ 507.1109; found 507.1110.
4.1.1.4. 4-{3-[4-Cyano-3-(trifluoromethyl)phenyl]-8-methyl-4-oxo-
2-thioxo-1,3,8-triazaspiro[4.5]dec-1-yl}-2-fluoro-N-methyl-
benzamide (11b). Yield 3%. MS (ESI) [MþH]þ 520. 1H NMR
(400 MHz, DMSO-d6)
d
10.09 (brs, 1H), 8.48 (q, J ¼ 4.4 Hz, 1H), 8.43
(d, J ¼ 8.4 Hz, 1H), 8.29 (s, 1H), 8.11 (d, J ¼ 8.4 Hz, 1H), 7.84 (t,
J ¼ 8.0 Hz, 1H), 7.42 (d, J ¼ 10.4 Hz, 1H), 7.30 (d, J ¼ 8.0 Hz, 1H), 3.50
(m, 4H), 2.80 (d, J ¼ 4.4 Hz, 3H), 2.78 (s, 3H), 2.72 (d, J ¼ 14.0 Hz,1H),
2.16 (m, 2H).
(400 MHz, DMSO-d6)
d
8.25 (m, 1H), 7.73 (d, J ¼ 10.0 Hz,1H), 7.65 (d,
J ¼ 7.8 Hz, 1H), 7.37 (t, J ¼ 7.8 Hz, 1H), 2.75 (d, J ¼ 4.4 Hz, 3H). The
solution of compound 37 (9 g, 32 mmol) in DMF (25 mL) was added
to the ice cooled suspension of NaH (1.48 g, 36.8 mmol, 60% in oil,
washed with hexane) in DMF (50 mL). The resulting mixture was
vigorously stirred for 0.5 h in an ice bath, then SEM-chloride (6.46 g,
39 mmol) was added and the mixture was continuously stirred
overnight at the ambient temperature. After the reaction was
completed, the mixture was poured into water (400 mL) and the
obtained product was extracted with benzene (2 ꢂ 200 mL),
washed with water, dried over Na2SO4 and then filtered through
3 cm layer of silica gel washing with 5:1 hexane/EtOAc. The solvent
was evaporated in vacuo providing 11 g (84%) of 2-fluoro-4-iodo-N-
methyl-N-[2-(trimethylsilyl)ethoxymethyl]benzamide (40). MS
4.1.1.5. 4-{3-[4-Cyano-3-(trifluoromethyl)phenyl]-5-(methox-
ymethyl)-5-methyl-4-oxo-2-thioxoimidazolidin-1-yl}-2-fluoro-N-
methylbenzamide ((R,S)-14). Yield 16%. MS (ESI) [MþH]þ 495. 1H
NMR (400 MHz, CDCl3)
d
8.28 (t, J ¼ 8.4 Hz, 1H), 7.99 (d, J ¼ 8.0 Hz,
1H), 7.92 (s, 1H), 7.80 (d, J ¼ 8.0 Hz, 1H), 7.29 (dd, J1 ¼ 8.8 Hz,
J2 ¼ 1.2 Hz, 1H), 7.21 (dd, J1 ¼ 11.6 Hz, J2 ¼ 1.2 Hz, 1H), 6.72 (q,
J ¼ 4.4 Hz, 1H), 3.71 (d, J ¼ 10.0 Hz, 1H), 3.43 (s, 3H), 3.35 (d,
J ¼ 10.0 Hz, 1H), 3.09 (d, J ¼ 4.4 Hz, 3H), 1.52 (s, 3H). ESIHRMS m/z
calcd for C22H19F4N4O3S [MþH]þ 495.1109; found 495.1107. Enan-
tiomers were readily separated following the procedure described
above. Isocratic 20/80 system of 0.02% Et2NH in 20/80 MeOH/i-
PrOH (A) and n-hexane (B) was used as a mobile phase, flow rate
was the same. Retention times for (S)-14 and (R)-14 were 16 and
19 min, respectively.
(ESI) [MþH]þ 520. 1H NMR (400 MHz, CDCl3)
d 7.58 (m,1H), 7.52 (m,
1H), 7.12 (m, 1H), 5.01 (s, 0.8H), 4.58 (s, 1.2H), 3.63 (t, J ¼ 8.2 Hz,
0.8H), 3.31 (t, J ¼ 8.2 Hz, 1.2H), 3.14 (s, 1.8H), 2.92 (d, J ¼ 0.8 Hz,
1.2H), 0.99 (t, J ¼ 8.2 Hz, 0.8H), 0.82 (t, J ¼ 8.2 Hz, 1.2H), 0.04 (s,
3.6Н), ꢁ0.01 (s, 5.4Н). The mixture of 6.69 g (51 mmol) of aminoacid
(R)-36a or 9.55 g of (R)-36b, 17.4 g (42.5 mmol) of 40, 1.62 g
(8.5 mmol) of CuI, 23.5 g (0.17 mol) of K2CO3, 36 mL of water,145 mL
of DMF and 3e5 drops of Et3N was stirred for 10 min, then 6.56 g
4.1.1.6. 4-{5-(Benzyloxymethyl)-3-[4-cyano-3-(trifluoromethyl)
phenyl]-5-methyl-4-oxo-2-thioxoimidazolidin-1-yl]-2-fluoro-N-

A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 99 (2015) 51e66
61
(46.8 mmol) of 2-acetylcyclohexanone was added and stirring
continued at 100ꢀС for 24 h. After cooling the mixture was roto-
vapped, the residue was treated with 200 mL of water and acidified
with hydrochloric acid to рН 2e3 (~30 mL). Then 200 mL of ether
was added, the mixture was stirred for 0.5 h and the formed pre-
cipitate was filtered off, washed with 50 mL of ether and dried in
vacuo to give 10.7 g (65%) of (R)-3-(3-fluoro-4-{methyl[2-(trime-
thylsilyl)ethoxymethyl]carbamoyl}phenylamino)-tetrahydrofuran-
3-carboxylic acid ((R)-41) (55% from ester (R)-36b). MS (ESI)
the alternative synthetic route, CDI (6.89 g, 42.5 mmol) was added
to the solution of acid (R)-38 (10 g, 35.4 mmol) in acetonitrile
(100 mL) and the resulting mixture was stirred at 60ꢀС for 1 h then
methanol (20 mL) was added. Stirring was continued for 2 h, then
the solvent was removed under reduced pressure and the formed
residue was treated with water, filtered off, washed with cold water
and dried in vacuo furnishing the desired product (R)-45 in good
yield (7.5 g, 71%). Following the third approach, thionyl chloride
(4.81 mL, 65.9 mmol) was added dropwise (approximately for
10 min) to the ice cooled solution of acid (R)-38 (15.5 g, 54.9 mmol)
in methanol (150 mL). The resulting mixture was refluxed for 4 h.
The solvent was removed under reduced pressure and the formed
residue was dissolved in chloroform (200 mL), washed with satu-
rated NaHCO3 solution (50 mL), brine (50 mL), dried over Na2SO4,
then the solvent was evaporated in vacuo and the residue was
treated with ether. The precipitate was filtered off, washed with
ether and dried in air to afford 12.5 g (77%) of (R)-45. MS (ESI)
[MþH]þ 413. 1H NMR (400 MHz, DMSO-d6)
d 12.97 (brs, 1H), 7.07
(m, 2H), 6.31 (brs, 1H), 6.17 (brs, 1H), 4.85 (brs, 0.67H), 4.60 (brs,
1.33H), 4.10 (brs, 1H), 3.87 (brs, 3H), 3.51 (brs, 0.67H), 3.24 (brs,
1.33H), 2.93 (s, 2H), 2.86 (brs, 1H), 2.56 (brs, 1H), 2.16 (brs, 1H), 0.89
(brs, 0.67H), 0.73 (brs, 1.33H), ꢁ0.03 (m, 9H). The mixture of 10.7 g
(26 mmol) of (R)-41 and 8.9 g (39 mmol) of 4-isothiocyanato-2-
(trifluoromethyl)benzonitrile (42) [34] in pyridine (100 mL) was
stirred at 80ꢀС for 48 h. The residue formed after cooling and
rotovapping was then treated with ethyl acetate and filtered
through 2 cm layer of silica gel. The solvent was removed under
reduced pressure and the desired product was crystallized from
[MþH]þ 297. 1H NMR (400 MHz, CDCl3)
d
7.94 (t, J ¼ 8.8 Hz, 1Н),
6.59 (brs, 1Н), 6.39 (dd, J1 ¼ 8.8 Hz, J1 ¼ 2.4 Hz, 1Н), 6.20 (dd,
J1 ¼ 14.4 Hz, J1 ¼ 2.4 Hz, 1Н), 4.80 (brs, 1Н), 4.19 (d, J ¼ 9.6 Hz, 1Н),
4.06 (m, 2Н), 4.00 (d, J ¼ 9.6 Hz, 1Н), 3.77 (s, 3Н), 3.01 (d, J ¼ 4.4 Hz,
3Н), 2.70 (m, 1Н), 2.29 (m, 1Н).
ethanol to obtain 4.85
g (30%) of 4-{(R)-3-[4-cyano-3-(tri-
fluoromethyl)phenyl]-4-oxo-2-thioxo-7-oxa-1,3-diazaspiro[4.4]
non-1-yl}-2-fluoro-N-methyl-N-[2-(trimethylsilyl)ethoxymethyl]
benzamide ((R)-43). MS (ESI) [MþH]þ 623. 1H NMR (400 MHz,
The mixture of acid (R)-45 (4.44 g, 15 mmol), isothiocyanate 42
(6.85 g, 30 mmol), DMSO (1.1 mL) and ethyl acetate (5.9 mL) was
stirred in a closed vessel at 85 ꢀС for 48 h (till the absence of initial
(R)-45 by LC-MS control). After the reaction was completed, the
solution was cooled to rt, the solvent was removed under reduced
pressure and the product was subjected to column chromatography
on silica gel (CH2Cl2/MeOH ¼ 60:1) providing 5.09 g (69%) of pure
CDCl3)
d
8.01 (d, J ¼ 8.0 Hz, 1Н), 7.98 (s, 1Н), 7.85 (d, J ¼ 8.0 Hz, 1Н),
7.57 (m, 1Н), 7.28 (m, 1Н), 7.21 (m, 1Н), 5.06 (s, 0.8Н), 4.63 (s, 1.2Н),
4.41 (d, J ¼ 10.4 Hz, 1Н), 4.16 (d, J ¼ 10.4 Hz, 1Н), 3.97 (q, J ¼ 7.6 Hz,
1Н), 3.78 (m, 1Н), 3.66 (t, J ¼ 8.2 Hz, 0.8Н), 3.66 (t, J ¼ 8.2 Hz, 1.2Н),
3.20 (s, 1.8Н), 2.99 (s, 0.8Н), 2.72 (m, 1Н), 2.47 (m, 1Н), 1.01 (t,
J ¼ 8.2 Hz, 0.8Н), 0.83 (t, J ¼ 8.2 Hz, 1.2Н), 0.06 (s, 3.6Н), ꢁ0.01 (s,
5.4Н). TFA (15 mL) was added to the solution of compound (R)-43
(4.8 g, 7.7 mmol) in DCM (30 mL), then the resulting mixture was
stirred for 3 h. The solvent was removed under reduced pressure
and the formed residue was subjected to column chromatography
on silica gel (CHCl3/MeOH ¼ 60:1) to give 2.96 g (78%) of desired
product (R)-6.
(R)-6. MS (ESI) [MþH]þ 493. 1H NMR (400 MHz, CDCl3)
d 8.30 (t,
J ¼ 8.4 Hz, 1Н), 8.02 (d, J ¼ 8.8 Hz, 1Н), 7.97 (d, J ¼ 1.6 Hz, 1Н), 7.85
(dd, J1 ¼ 8.4 Hz, J1 ¼1.6 Hz, 1Н), 7.33 (dd, J1 ¼ 8.4 Hz, J1 ¼1.6 Hz, 1Н),
7.24 (dd, J1 ¼ 11.6 Hz, J1 ¼ 2.4 Hz, 1Н), 6.73 (m, 1Н), 4.42 (d,
J ¼ 10.4 Hz, 1Н), 4.16 (d, J ¼ 10.4 Hz, 1Н), 3.96 (m, 1Н), 3.75 (m, 1Н),
3.09 (d, J ¼ 4.8 Hz, 3Н), 2.73 (m, 1Н), 2.48 (m, 1Н). ESIHRMS m/z
calcd for C22H17F4N4O3S [MþH]þ 493.0952; found 493.0947. A brief
X-Ray description of (R)-6: monoclinic syngony, space group P21,
4.1.1.9.2. Procedure В. The mixture of compound (R)-36b
(3.29 g, 17.6 mmol), 4-bromo-2-fluoro-N-methylbenzamide 44
(3.40 g, 14.7 mmol) [41], CuI (0.53 g, 2.79 mmol), K2CO3 (7.97 g,
58.6 mmol) and Et3N (0.2 mL) in water (6 mL) and DMF (27.5 mL)
was stirred for 10 min, then 2-acetylcyclohexanone (0.41 g,
2.9 mmol) was added and stirring was continued at 100ꢀС for 48 h.
The mixture was cooled to rt, the solvent was removed under
reduced pressure, then the formed residue was treated with water
and acidified with hydrochloric acid to рН 2e3. The resulting so-
lution was stirred for 0.5 h, the formed precipitate was filtered off,
washed with cold water, dried in air, then washed with ether and
dried again to provide 3.35 g (81%) of (R)-3-[3-fluoro-4-(methyl-
a ¼ 8.4706 (17) Å, b ¼ 14.507 (3) Å, c ¼ 17.925 (3) Å,
b
¼ 92.191 (5)ꢀ,
V ¼ 2201.0 (8) Å3, Z ¼ 4, T ¼ 120 K,
m
(MoK
a
) ¼ 0.214 mmꢁ1
,
Dcalc ¼ 1.486 g/mm3, 19,657 reflections measured (2
Q
ꢃ 54), 9431
independent (Rint ¼ 0.0637). Final divergence factors: R1 ¼ 0.0603
(I > 2
s
(I)), wR2 ¼ 0.1271 (all data). Detailed structural information,
including tables of bond lengths and 3D-representations is pre-
sented in SI.
4.1.1.10. Methyl 4-{(5R)-3-[4-cyano-3-(trifluoromethyl)phenyl]-4-
oxo-2-thioxo-7-oxa-1,3-diazaspiro[4.4]non-1-yl}-2-fluorobenzoate
((R)-7). The mixture of (R)-36a (85 mmol), 4-bromo-2-
fluorobenzoic acid 46 (15.5 g, 71 mmol) [40], K2CO3 (39.3 g,
284 mmol), CuI (2.02 g,10.6 mmol) and 2-acetylcyclohexanone (2 g,
14.3 mmol) dissolved in DMF (150 mL) and water (35 mL) was
stirred for 48 h at 100ꢀС. After the reaction was completed the
mixture was rotovapped, the residue was treated with water,
acidified to pH 2e3 with hydrochloric acid and the solvent was
removed under reduced pressure. The obtained crude (3R)-3-[(4-
carboxy-3-fluorophenyl)amino]-tetrahydrofuran-3-carboxylic
acid ((R)-47) (MS (ESI) 270 [MþH]þ) was further used without any
purification. Thionyl chloride (13 mL, 177 mmol) was added drop-
wise to a cooled solution of (R)-47 in methanol (150 mL) and the
resulting mixture was refluxed for 15 h. After the reaction was
completed, the solution was cooled to rt, solvent was evaporated in
vacuo, the formed residue was treated with ethyl acetate (200 mL)
and water (100 mL), then the solution was basified with NaHCO3
(8.95 g, 107 mmol). The organic layer was separated, rotovapped
and subjected to column chromatography on silica gel (CH2Cl2) to
carbamoyl)phenylamino]-tetrahydrofuran-3-carboxylic
acid
((R)-38). MS (ESI) [MþH]þ 283. 1H NMR (400 MHz, DMSO-d6)
d
12.96 (brs, 1Н), 7.65 (t, J ¼ 4.4 Hz, 1Н), 7.47 (t, J ¼ 8.8 Hz, 1Н), 7.11
(brs, 1Н), 6.32 (dd, J1 ¼ 8.8 Hz, J1 ¼ 2.0 Hz, 1Н), 6.13 (dd, J1 ¼14.4 Hz,
J1 ¼ 2.0 Hz, 1Н), 4.11 (d, J ¼ 8.8 Hz, 1Н), 3.88 (m, 3Н), 2.73 (d,
J ¼ 4.4 Hz, 3Н), 2.54 (m, 1Н), 2.15 (m, 1Н). Iodomethane (301 mg,
2.12 mmol) was added under vigorous stirring to the mixture of
compound (R)-38 (500 mg, 1.77 mmol) and K2CO3 (293 mg,
2.12 mmol) dissolved in DMF (4 mL) at 30ꢀС. The resulting mixture
was heated up to 40ꢀС and stirred keeping the predefined tem-
perature for 1 h, then diluted with 40 mL of water, heated up to
60ꢀС and filtered. The filtrate was extracted with chloroform (2 Ч
50 mL), organic phase was washed with water, dried over Na2SO4
and the solvent was evaporated using a vacuo pump. The formed
residue was treated with ether, filtered off and dried to afford
372 mg (70%) of methyl (R)-3-[3-fluoro-4-(methylcarbamoyl)
phenylamino]-tetrahydrofuran-3-carboxylate ((R)-45). Following

62
A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 99 (2015) 51e66
afford 12.2
g
(58%) of methyl (3R)-3-{[3-fluoro-4-(methox-
75 mmol) and KOH (21 g, 0.375 mol) dissolved in water (115 mL)
was refluxed for 48 h. The cooled solution was neutralized with
citric acid, the formed precipitate was filtered off, washed with cold
water, with ether and dried in vacuo to afford 18 g (85%) of 3-
amino-1-benzhydrylazetidine-3-carboxylic acid (51). MS (ESI)
ycarbonyl)phenyl]amino}-tetrahydrofuran-3-carboxylate ((R)-
48). MS (ESI) [MþH]þ 298. 1H NMR (400 MHz, CDCl3)
d 7.78 (t,
J ¼ 8.8 Hz, 1Н), 6.32 (dd, J1 ¼ 8.8 Hz, J2 ¼ 2.4 Hz, 1Н), 6.21 (dd,
J1 ¼ 13.6 Hz, J2 ¼ 2.4 Hz, 1Н), 4.82 (brs, 1Н), 4.18 (d, J ¼ 9.4 Hz, 1Н),
4.07 (m, 2Н), 4.00 (d, J ¼ 9.4 Hz, 1Н), 3.88 (s, 3Н), 3.77 (s, 3Н), 2.71
(m, 1Н), 2.29 (m, 1H).
[MþH]þ 283. 1H NMR (400 MHz, DMSO-d6)
7.43 (d, J ¼ 7.6 Hz, 4Н),
d
7.27 (t, J ¼ 7.2 Hz, 4Н), 7.17 (t, J ¼ 7.2 Hz, 2Н), 4.56 (s, 1Н), 3.45 (d,
J ¼ 8.0 Hz, 2Н), 3.13 (d, J ¼ 8.0 Hz, 2Н). The mixture of compound 51
(2.54 g, 9 mmol), compound 44 (2.32 g, 10 mmol), K2CO3 (3.45 g,
25 mmol), CuI (0.19 g, 1 mmol) and 2-acetylcyclohexanone (0.14 g,
1 mmol) dissolved in DMF (40 mL) and water (6 mL) was stirred for
48 h in a closed vessel at 105ꢀС. The cooled mixture was rotovap-
ped, diluted with water and extracted with ethyl acetate. The water
layer was acidified with citric acid (10%), the formed precipitate was
filtered off, washed with water and dried in vacuo to provide 2.73 g
(70%) of 1-benzhydryl-3-[3-fluoro-4-(methylcarbamoyl)phenyl-
amino]azetidine-3-carboxylic acid (52). MS (ESI) [MþH]þ 434. 1H
The mixture of compound (R)-48 (12.2 g, 41 mmol), 42 (18.7 g,
82 mmol), DMSO (2.9 mL) and ethyl acetate (16 mL) was stirred for
48 h at 85 ꢀС. After cooling the mixture was rotovapped and sub-
jected to column chromatography on silica gel (CH2Cl2). After
crystallization from methanol the final product (R)-7 was obtained
in good yield (6.9 g, 34%); MS (ESI) [MþH]þ 494. 1H NMR (400 MHz,
CDCl3)
d
8.14 (t, J ¼ 8.0 Hz, 1Н), 8.01 (d, J ¼ 8.4 Hz, 1Н), 7.97 (d,
J ¼ 2.9 Hz, 1Н), 7.85 (dd, J1 ¼ 8.4 Hz, J2 ¼ 2.0 Hz, 1Н), 7.27 (m, 2Н),
4.42 (d, J ¼ 10.2 Hz, 1Н), 4.15 (d, J ¼ 10.2 Hz, 1Н), 3.99 (s, 3Н), 3.96
(m, 1Н), 3.76 (m, 1Н), 2.73 (m, 1Н), 2.47 (m, 1Н). ESIHRMS m/z calcd
for C22H16F4N3O4S [MþH]þ 494.0792; found 494.0792.
NMR (400 MHz, DMSO-d6)
d
7.63 (t, J ¼ 6.4 Hz, 1Н), 7.44 (m, 6Н),
7.28 (t, J ¼ 9.6 Hz, 4Н), 7.19 (m, 2Н), 6.21 (d, J ¼ 12.0 Hz, 1Н), 6.02 (d,
J ¼ 18.8 Hz, 1Н), 4.58 (s, 1Н), 3.60 (d, J ¼ 10.4 Hz, 2Н), 3.22 (d,
J ¼ 10.4 Hz, 2Н), 2.72 (d, J ¼ 5.6 Hz, 3Н). The mixture of compound
52 (2.17 g, 5 mmol) and CDI (973 mg, 6 mmol) dissolved in aceto-
nitrile (20 mL) was stirred for 1 h, then methanol (0.43 g, 15 mmol)
was added and the solution was stirred for 24 h at 50ꢀС. The solvent
was evaporated under reduced pressure, the formed residue was
treated with К2CO3 (5%), the precipitate was filtered off, washed
with cold water and dried in vacuo to afford 1.79 g (80%) of methyl
1-benzhydryl-3-[3-fluoro-4-(methylcarbamoyl)phenylamino]
azetidine-3-carboxylate (53). MS (ESI) [MþH]þ 448. 1H NMR
4.1.1.11. 4-{(5R)-3-[4-cyano-3-(trifluoromethyl)phenyl]-4-oxo-2-
thioxo-7-oxa-1,3-diazaspiro[4.4]non-1-yl}-2-fluorobenzoic acid ((R)-
8). NaOH (16.5 mg, 0.41 mmol) in water (2 mL) was added to the
solution of compound (R)-7 (200 mg, 0.41 mmol) in methanol
(2 mL). The resulting mixture was stirred for 12 h and then the
solvent was evaporated under reduced pressure. The residue was
treated with water (10 mL) and acidified to pH ¼ 3 with hydro-
chloric acid. The formed precipitate was filtered off, washed with
water and dried in vacuo to give 179 mg (92%) of (R)-8. MS (ESI)
[MþH]þ 490. 1H NMR (400 MHz, DMSO-d6)
d 13.53 (brs, 1H), 8.40
(d, J ¼ 8.4 Hz, 1Н), 8.26 (s, 1Н), 8.08 (dd, J1 ¼ 8.4 Hz, J2 ¼ 1.2 Hz, 1Н),
8.03 (t, J ¼ 8.4 Hz,1Н), 7.57 (dd, J1 ¼11.2 Hz, J2 ¼ 1.2 Hz,1Н), 7.48 (dd,
J1 ¼ 8.4 Hz, J2 ¼ 1.2 Hz, 1Н), 4.43 (d, J ¼ 10.8 Hz, 1Н), 3.94 (d,
J ¼ 10.8 Hz, 1Н), 3.75 (q, J ¼ 8.0 Hz, 1Н), 3.53 (q, J ¼ 8.0 Hz, 1Н), 2.58
(t, J ¼ 7.2 Hz, 2Н).
(400 MHz, DMSO-d6) d 7.44 (m, 6Н), 7.29 (m, 5Н), 7.19 (m, 2Н), 6.20
(d, J ¼ 11.6 Hz, 1Н), 6.04 (d, J ¼ 18.8 Hz, 1Н), 4.59 (s, 1Н), 3.67 (s, 3Н),
3.65 (d, J ¼ 10.6 Hz, 2Н), 3.25 (d, J ¼ 10.6 Hz, 2Н), 2.74 (d, J ¼ 6.0 Hz,
3Н). The mixture of compound 53 (224 mg, 0.5 mmol) and com-
pound 42 (228 mg, 1 mmol) dissolved in DMSO (2 mL) and ethyl
acetate (5 mL) was stirred for 20 h at 90ꢀС. The cooled mixture was
diluted with ethyl acetate (20 mL), washed with saturated NH4Cl
solution, dried over Na2SO4, rotovapped and subjected to HPLC to
obtain 32 mg (10%) of 10a. MS (ESI) [MþH]þ 644. 1H NMR
4.1.1.12. 4-{(5R)-3-[4-cyano-3-(trifluoromethyl)phenyl]-4-oxo-2-
thioxo-7-oxa-1,3-diazaspiro[4.4]non-1-yl}-2-fluorobenzamide ((R)-
9). The mixture of compound (R)-8 (179 mg, 0.37 mmol), EDAC
(107 mg, 0.55 mmol), HOBt (75 mg, 0.55 mmol), ammonium
(400 MHz, CDCl3)
d
8.44 (t, J ¼ 8.0 Hz, 1Н), 7.99 (d, J ¼ 8.4 Hz, 1Н),
chloride (26 mg, 0.48 mmol) and TEA (52
mL, 0.37 mmol) dissolved
7.94 (d, J ¼ 12.0 Hz, 1Н), 7.81 (dd, J1 ¼8.4 Hz, J2 ¼ 1.2 Hz, 1Н), 7.42 (d,
J ¼ 8.0 Hz, 1Н), 7.32 (d, J ¼ 8.0 Hz, 1Н), 7.25 (m, 4Н), 7.20 (m, 6Н),
6.85 (q, J ¼ 4.4 Hz,1Н), 4.40 (brs,1Н), 3.76 (m, 2Н), 3.60 (m, 2H), 3.15
(d, J ¼ 4.4 Hz, 3Н). ESIHRMS m/z calcd for C34H26F4N5O2S [MþH]þ
644.1738; found 644.1730.
in dry DMF (2 mL) was stirred for 24 h. The solvent was removed in
vacuo, the residue was dissolved in DCM, washed with 10% sodium
carbonate solution, dried over Na2SO4, the solvent was removed
and the obtained product was subjected to HPLC to afford 86 mg
(48%) of (R)-9. MS (ESI) [MþH]þ 489. 1H NMR (400 MHz, DMSO-d6)
d
8.40 (d, J ¼ 8.0 Hz, 1Н), 8.26 (d, J ¼ 1.6 Hz, 1Н), 8.08 (dd, J1 ¼8.0 Hz,
4.1.1.14. 4-{7-[4-Cyano-3-(trifluoromethyl)phenyl]-2-methyl-8-oxo-
6-thioxo-2,5,7-triazaspiro[3.4]oct-5-yl}-2-fluoro-N-methyl-
benzamide (10b, unsuccessful attempts to synthesize)
J2 ¼ 1.6 Hz, 1Н), 7.92 (s, 1Н), 7.79 (t, J ¼ 8.0 Hz, 1Н), 7.76 (s, 1Н), 7.53
(dd, J1 ¼ 10.8 Hz, J2 ¼ 1.6 Hz, 1Н), 7.43 (dd, J1 ¼ 8.0 Hz, J2 ¼ 1.6 Hz,
1Н), 4.42 (d, J ¼ 10.6 Hz, 1Н), 3.94 (d, J ¼ 10.6 Hz, 1Н), 3.75 (m, 1Н),
3.53 (m, 1Н), 2.58 (m, 2Н). ESIHRMS m/z calcd for C21H15F4N4O3S
[MþH]þ 479.0796; found 479.0793.
4.1.1.14.1. Procedure
А. The solution of compound 53 (32 mg,
0.05 mmol) in methanol (15 mL) was stirred under hydrogen at-
mosphere with 100 mg of 10% Pd/C. No reaction was observed, as
well as upon hydrogenation with hydrochloric acid or in acetic acid.
4.1.1.14.2. Procedure B. The mixture of compound 52 (1.734 g,
4 mmol), acetic acid (0.48 mL, 8 mmol) and Pd/C (0.1 g, 10%) dis-
solved in methanol (50 mL) was stirred for 12 h under hydrogen
atmosphere. The solution was filtered through celite and rotovap-
ped. The residue was treated with ether, filtered off and dried to
afford 855 mg (80%) of 3-[3-fluoro-4-(methylcarbamoyl)phenyl-
amino]azetidine-3-carboxylic acid (54). MS (ESI) [MþH]þ 268. 1H
4.1.1.13. 4-{2-Benzhydryl-7-[4-cyano-3-(trifluoromethyl)phenyl]-8-
oxo-6-thioxo-2,5,7-triazaspiro[3.4]oct-5-yl}-2-fluoro-N-methyl-
benzamide (10a). The mixture of 1-benzhydrylazetidin-3-one (49)
(21.4 g, 90 mmol) [37], KCN (6.5 g, 0.1 mol) and (NH4)2CO3 (19.2 g,
0.2 mol) dissolved in water (80 mL) and ethanol (150 mL) was
stirred for 72 h in a closed vessel at 80ꢀС. After cooling the mixture
was rotovapped, the residue was treated with water, filtered off,
washed with cold water, then with methanol and dried in air to
provide 23.5 g (85%) of 2-benzhydryl-2,5,7-triaza-spiro[3.4]oc-
tane-6,8-dione (50). MS (ESI) [MþH]þ 308. 1H NMR (400 MHz,
NMR (400 MHz, D2O)
d
7.51 (t, J ¼ 8.6 Hz, 1Н), 6.26 (dd, J1 ¼ 8.6 Hz,
J2 ¼ 2.2 Hz, 1Н), 6.15 (dd, J1 ¼ 13.8 Hz, J2 ¼ 2.2 Hz, 1Н), 4.56 (d,
J ¼ 11.4 Hz, 2Н), 4.11 (d, J ¼ 11.4 Hz, 2Н), 2.84 (s, 3Н). The mixture of
compound 54 (802 mg, 3 mmol) and formalin (0.5 mL) dissolved in
aqueous methanol (50 mL, 50% v/v) was stirred for 0.5 h, then Pd/C
(0.3 g, 10%) was added and stirring was continued for 12 h. The
DMSO-d6)
d
10.46 (s, 1Н), 8.57 (s, 1Н), 7.43 (d, J ¼ 7.6 Hz, 4Н), 7.29 (t,
J ¼ 7.6 Hz, 4Н), 7.18 (t, J ¼ 7.2 Hz, 2Н), 4.48 (s, 1Н), 3.40 (d, J ¼ 8.2 Hz,
2Н), 3.20 (d, J ¼ 8.2 Hz, 2Н). The mixture of compound 50 (23 g,

A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 99 (2015) 51e66
63
solution was filtered through celite and rotovapped. The residue
was treated with ether, filtered off and dried to afford 717 mg (85%)
phenylamino]oxetane-3-carboxylate (61). MS (ESI) [MþH]þ 283.
1H NMR (400 MHz, CDCl3)
d
7.96 (t, J ¼ 9.2 Hz, 1Н), 6.60 (brs, 1Н),
of
3-[3-fluoro-4-(methylcarbamoyl)phenylamino]-1-
6.31 (dd, J1 ¼ 8.8 Hz, J2 ¼ 2.0 Hz, 1Н), 6.12 (dd, J1 ¼ 14.4 Hz,
J2 ¼ 2.0 Hz, 1Н), 5.13 (d, J ¼ 6.4 Hz, 2Н), 5.03 (brs, 1Н), 4.76 (d,
J ¼ 6.4 Hz, 2Н), 3.84 (s, 3Н), 3.01 (d, J ¼ 4.4 Hz, 3Н). Compound 42
(63 mg, 0.276 mmol) was added to the solution of compound 59 or
61 (0.25 mmol) in DMF (1 mL). The resulting mixture was stirred for
12 h at 80ꢀС under argon atmosphere. After the reaction was
completed, the solution was poured into cold water, extracted with
ethyl acetate, dried over Na2SO4 and rotovapped. Separation by
HPLC afforded 17 mg (16%) of N-[4-cyano-3-(trifluoromethyl)
phenyl]-3-[3-fluoro-4-(methylcarbamoyl)phenylamino]oxetane-3-
carboxamide (62). MS (ESI) [MþH]þ 437. 1H NMR (400 MHz, DMSO-
methylazetidine-3-carboxylic acid (55). MS (ESI) [MþH]þ 282. 1H
NMR (400 MHz, D2O)
d
7.51 (t, J ¼ 8.6 Hz, 1Н), 6.27 (dd, J1 ¼ 8.6 Hz,
J2 ¼ 2.0 Hz, 1Н), 6.20 (dd, J1 ¼ 13.8 Hz, J2 ¼ 2.0 Hz, 1Н), 4.68 (d,
J ¼ 11.4 Hz, 2Н), 4.23 (d, J ¼ 11.4 Hz, 2Н), 3.78 (s, 3Н), 2.82 (s, 3Н).
The mixture of compound 55 (281 mg, 1 mmol) and CDI (195 mg,
1.2 mmol) dissolved in DMF (5 mL) was stirred for 1 h at rt. Then,
methanol (0.405 mL, 10 mmol) was added and the mixture was
stirred for 24 h at 50ꢀС. The solvent was removed in vacuo, the
precipitate was dissolved in ethyl acetate, washed with К2CO3 (5%),
dried over Na2SO4 and the solvent was removed providing methyl
3-[3-fluoro-4-(methylcarbamoyl)phenylamino]-1-
d6)
d
10.59 (s, 1Н), 8.30 (s, 1Н), 8.16 (d, J ¼ 8.8 Hz, 1Н), 8.08 (d,
methylazetidine-3-carboxylate (56) with good yield (236 mg,
J ¼ 8.4 Hz, 1Н), 7.73 (m, 1Н), 7.55 (s, 1Н), 7.49 (t, J ¼ 8.4 Hz, 1Н), 6.23
(d, J ¼ 10.0 Hz, 1Н), 6.10 (d, J ¼ 13.6 Hz, 1Н), 5.09 (d, J ¼ 6.8 Hz, 2Н),
4.59 (d, J ¼ 6.4 Hz, 2Н), 2.70 (d, J ¼ 4.0 Hz, 3Н).
80%). MS (ESI) [MþH]þ 296. 1H NMR (400 MHz, CDCl3)
d 7.92 (t,
J ¼ 8.0 Hz, 1Н), 6.60 (m, 1Н), 6.29 (dd, J1 ¼ 11.2 Hz, J2 ¼ 2.4 Hz, 1Н),
6.09 (dd, J1 ¼ 15.6 Hz, J2 ¼ 2.4 Hz, 1Н), 5.04 (s, 1Н), 3.85 (d,
J ¼ 10.6 Hz, 2Н), 3.77 (s, 3Н), 3.42 (d, J ¼ 10.6 Hz, 2Н), 3.00 (d,
J ¼ 6.4 Hz, 3Н), 2.46 (s, 3Н). The same procedure was applied for the
reaction of compound 56 with isothiocyanate 42 as for conversion
of (R)-45 to (R)-6 described above, there was no product 10b
detected in the reaction mixture.
4.1.1.16. 4-{3-[4-Cyano-3-(trifluoromethyl)phenyl]-4-oxo-2-
thioxoimidazolidin-1-yl}-2-fluoro-N-methylbenzamide
(12). The
mixture of compound 37 (279 mg, 1 mmol), glycine (80 mg,
1.07 mmol), К2CO3 (207 mg, 1.5 mmol) and CuI (19 mg, 0.1 mmol)
dissolved in DMF (3 mL) was kept in microwave reactor for 20 min
at 140 ꢀC. After cooling the mixture was diluted with 10 mL of ethyl
acetate and 10 mL of water, neutralized with hydrochloric acid to
pH ¼ 2e3 and the product was extracted with 5 ꢂ 20 mL of ethyl
acetate. The extracts were washed with brine, dried over Na2SO4
and the solvent was evaporated in vacuo. The product was then
subjected to HPLC providing 113 mg (50%) of [3-fluoro-4-(meth-
ylcarbamoyl)phenylamino]acetic acid (67). MS (ESI) [MþH]þ 227.
The mixture of compound 67 (113 mg, 0.5 mmol) and 42 (174 mg,
1.0 mmol) dissolved in DMF (2 mL) was stirred for 12 h at 90 ꢀC. The
cooled solution was then rotovapped and subjected to HPLC to give
86 mg (39%) of compound 12. MS (ESI) [MþH]þ 437. 1H NMR
4.1.1.15. 4-{7-[4-Cyano-3-(trifluoromethyl)phenyl]-8-oxo-6-thioxo-
2-oxa-5,7-diazaspiro[3.4]oct-5-yl]-2-fluoro-N-methylbenzamide
(10c, synthesis was unsuccessful). The mixture of compound 40
(500 mg, 1.22 mmol), 3-аminooxetane-3-carboxylic acid (57)
(215 mg, 1.84 mmol) [38], K2CO3 (590 mg, 4.27 mmol), CuI (47 mg,
0.24 mmol) and 1 drop of Et3N dissolved in DMF (4 mL) and water
(1 mL) was stirred for 15 min, then 2-acetylcyclohexanone (190 mg,
1.35 mmol) was added and the mixture was stirred for 15 h at
100ꢀС. After cooling the mixture was rotovapped and subjected to
HPLC with neutral mobile phase to afford 340 mg (70%) of 3-(3-
fluoro-4-{methyl-[2-(trimethylsilyl)ethoxymethyl]carbamoyl}
phenylamino)oxetane-3-carboxylic acid (58). MS (ESI) [MþH]þ
(400 MHz, DMSO-d6)
d
8.39 (d, J ¼ 8.4 Hz, 1H), 8.32 (m, 1H), 8.17 (s,
1H), 7.99 (dd, J1 ¼ 8.0 Hz, J2 ¼ 1.4 Hz, 1H), 7.86 (dd, J1 ¼ 12.0 Hz,
J2 ¼ 1.4 Hz, 1H), 7.75 (m, 2H), 4.95 (s, 2H), 2.79 (d, J ¼ 4.4 Hz, 3H).
ESIHRMS m/z calcd for C19H13F4N4O2S [MþH]þ 437.0690; found
437.0686.
399. 1H NMR (400 MHz, DMSO-d6)
d
7.61 (m, 1H), 7.53 (t, J ¼ 8.8 Hz,
1H), 6.21 (d, J ¼ 8.4 Hz, 1H), 5.96 (d, J ¼ 13.6 Hz, 1H), 4.97 (d,
J ¼ 6.0 Hz, 2H), 4.55 (d, J ¼ 6.0 Hz, 2Н), 2.73 (d, J ¼ 4.4 Hz, 3Н), 2.21
(m, 2Н), 1.12 (m, 2Н), 0.14 (s, 9Н). The mixture of compound 40
(500 mg, 1.22 mmol), 3-аminooxetane-3-carboxylic acid (57)
(215 mg, 1.84 mmol),38 K2CO3 (590 mg, 4.27 mmol), CuI (47 mg,
0.24 mmol) and 1 drop of Et3N dissolved in DMF (4 mL) and water
(1 mL) was stirred for 15 min, then 2-acetylcyclohexanone (190 mg,
1.35 mmol) was added and the mixture was stirred for 15 h at
100ꢀС. After cooling the mixture was acidified with hydrochloric
acid to pH ¼ 3 and extracted with ethyl acetate. The organic layer
was washed with cold water, with brine, dried over Na2SO4 and the
solvent evaporated under reduced pressure to give 287 mg (88%) of
3-[3-fluoro-4-(methylcarbamoyl)phenylamino]oxetane-3-
4.1.1.17. 4-{3-[4-Cyano-3-(trifluoromethyl)phenyl]-5-methyl-4-oxo-
2-thioxoimidazolidin-1-yl}-2-fluoro-N-methylbenzamides ((R,S)-13,
(R)-13, (S)-13). D,L-, D- or -alanine (69) (638 mg, 7.16 mmol),
L
Cs2CO3 (2.337 g, 7.17 mmol), CuI (68 mg, 0.358 mmol) and N,N-
dimethylglycine (74 mg, 0.717 mmol) were added to the solution of
compound 37 (1 g, 3.58 mmol) in DMSO (2.5 mL). The resulting
mixture was stirred for 20 h at 90ꢀС. After cooling the mixture was
freeze-dried, the residue was extracted with ethanol and the
extract was subjected to HPLC to obtain 413e450 mg (24e26%) of
2-[3-fluoro-4-(methylcarbamoyl)phenylamino]propionic
acid
carboxylic acid (59). MS (ESI) [MþH]þ 269. 1H NMR (400 MHz,
((R,S)-69, (R)-69, (S)-69). (R,S)-69: MS (ESI) [MþH]þ 241. 1H NMR
DMSO-d6),
d
7.69 (m, 1H), 7.48 (t, J ¼ 8.8 Hz, 1H), 6.15 (d, J ¼ 8.4 Hz,
(400 MHz, DMSO-d6) d 12.66 (brs, 1H), 7.62 (m, 1H), 7.45 (t,
1H), 5.96 (d, J ¼ 13.6 Hz, 1H), 4.97 (d, J ¼ 6.0 Hz, 2H), 4.55 (d,
J ¼ 6.0 Hz, 2Н), 2.73 (d, J ¼ 4.4 Hz, 3Н). Sodium hydride (66 mg,
1.65 mmol, 60% in oil) was added at 0ꢀС to the solution of com-
pound 58 (600 mg, 1.5 mmol) in DMF (6 mL). The resulting mixture
was stirred for 0.5 h keeping same temperature. After the reaction
was completed, iodomethane (0.14 mL, 2.25 mmol) was added to
the mixture and stirring was continued for 6 h at 0ꢀС. Finally, the
solution was accurately poured into cold water, extracted with
ethyl acetate, dried over Na2SO4 and rotovapped. The obtained
crude methyl 3-(3-fluoro-4-{methyl-[2-(trimethylsilyl)ethox-
J ¼ 8.8 Hz, 1H), 6.67 (d, J ¼ 7.2 Hz, 1H), 6.42 (dd, J1 ¼ 8.4 Hz,
J2 ¼ 2.0 Hz, 1H), 6.29 (dd, J1 ¼14.8 Hz, J2 ¼ 2.0 Hz, 1H), 4.03 (m, 1H),
2.73 (d, J ¼ 4.4 Hz, 3H), 1.37 (d, J ¼ 7.2 Hz, 3H). Compound 42
(104 mg, 0.456 mmol) was added to the solution of (R,S)-69, (R)-69
or (S)-69 (100 mg, 0.416 mmol) in DMF (1 mL). The resulting
mixture was stirred for 18 h at 80ꢀС, then the solvent was removed
under reduced pressure and the product was subjected to HPLC to
afford 28e37 mg (15e20%) of (R,S)-13, (R)-13 or (S)-13. (R,S)-13: MS
(ESI) [MþH]þ 451. 1H NMR (400 MHz, CDCl3)
d
8.28 (t, J ¼ 8.6 Hz,
1H), 8.01 (d, J ¼ 8.0 Hz, 1H), 7.94 (d, J ¼ 1.2 Hz, 1H), 7.81 (dd,
J1 ¼ 8.0 Hz, J2 ¼ 1.2 Hz, 1H), 7.48 (dd, J1 ¼ 12.4 Hz, J2 ¼ 1.6 Hz, 1H),
7.36 (dd, J1 ¼ 8.4 Hz, J2 ¼ 1.6 Hz, 1H), 6.72 (m, 1H), 4.83 (q, J ¼ 7.2 Hz,
1H), 3.08 (d, J ¼ 4.8 Hz, 3H), 1.60 (d, J ¼ 7.2 Hz, 3H). ESIHRMS m/z
ymethyl]carbamoyl}phenylamino)oxetane-3-carboxylate
(60)
was subjected to HPLC with mobile phase containing 0.1% of TFA to
afford 46 mg (11%) of methyl 3-[3-fluoro-4-(methylcarbamoyl)

64
A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 99 (2015) 51e66
calcd for C20H15F4N4O2S [MþH]þ 451.0846; found 451.0844.
4.1.1.21. 4-{3-[4-Cyano-3-(trifluoromethyl)phenyl]-4-oxo-2-thioxo-
tetrahydropyrimidin-1(2H)-yl}-2-fluoro-N-methylbenzamide (20).
DBU (0.81 g, 5.4 mmol) was added to the solution of compound 22
(9 g, 53.6 mmol) and ethyl acrylate (8 g, 80 mmol) in DMSO (90 mL).
The resulting mixture was stirred for 24 h at 70ꢀС, then freeze-
dried and the residue was crystallized from 1:1 ethanol/water to
obtain 5.46 g (38%) of ethyl 3-[3-fluoro-4-(methylcarbamoyl)
phenylamino]propionate (72). MS (ESI) [MþH]þ 269. 1H NMR
4.1.1.18. 4-{3-[4-Cyano-3-(trifluoromethyl)phenyl]-5-(hydrox-
ymethyl)-5-methyl-4-oxo-2-thioxo-imidazolidin-1-yl}-2-fluoro-N-
methylbenzamide ((R,S)-17). BBr3 (53 mL, 0.55 mmol) was added to
the solution of compound (R,S)-14 (55 mg, 0.11 mmol) in CH2Cl2
(1.5 mL) under argon atmosphere at ꢁ 78ꢀС. The resulting mixture
was stirred for 3 h at ꢁ 78ꢀС then for 3 h at rt, quenched with 10 mL
of 5% sodium carbonate solution, extracted with ethyl acetate, dried
over Na2SO4 and rotovapped. HPLC afforded 15 mg (28%) of (R,S)-
(400 MHz, DMSO-d6)
d
7.57 (brs, 1Н), 7.48 (t, J ¼ 8.8 Hz, 1Н), 6.47
(brs, 1Н), 6.42 (d, J ¼ 8.8 Hz, 1Н), 6.33 (d, J ¼ 14.8 Hz, 1Н), 4.07 (q,
J ¼ 7.2 Hz, 2Н), 3.32 (m, 2Н), 2.73 (d, J ¼ 4.4 Hz, 3Н), 2.55 (t,
J ¼ 6.4 Hz, 2Н), 1.18 (t, J ¼ 7.2 Hz, 3Н). The mixture of compound 42
(320 mg, 1.51 mmol) and 72 (404 mg, 1.51 mmol) dissolved in DMF
(8 mL) was kept in microwave reactor for 8 h at 60ꢀС. The solvent
was evaporated in vacuo, and the residue was subjected to column
chromatography on silica gel (hexane/ethyl acetate ¼ 1:2) to give
200 mg (27%) of ethyl 3-{3-[4-cyano-3-(trifluoromethyl)phenyl]-
1-[3-fluoro-4-(methylcarbamoyl)phenyl]-thioureido}propionate
(73). MS (ESI) [MþH]þ 497. The solution of NaOH (32 mg, 0.8 mmol)
in water (0.25 mL) was added to the solution of compound 73
(200 mg, 0.4 mmol) in ethanol (1 mL). The resulting mixture was
stirred for 2 h at 80ꢀС. The cooled solution was neutralized with
17 MS (ESI) [MþH]þ 481. 1H NMR (400 MHz, DMSO-d6)
d 8.43 (m,
1H), 8.39 (d, J ¼ 8.4 Hz, 1H), 8.13 (s, 1H), 7.98 (d, J ¼ 8.4 Hz, 1H), 7.78
(t, J ¼ 8.0 Hz, 1H), 7.42 (d, J ¼ 10.8 Hz, 1H), 7.37 (d, J ¼ 8.0 Hz, 1H),
5.93 (t, J ¼ 4.4 Hz, 1H), 3.81 (dd, J1 ¼ 11.6 Hz, J2 ¼ 4.4 Hz, 1H), 3.45
(dd, J1 ¼11.6 Hz, J2 ¼ 5.0 Hz, 1H), 2.79 (d, J ¼ 4.0 Hz, 3H), 1.38 (s, 3H).
ESIHRMS m/z calcd for C21H17F4N4O3S [MþH]þ 481.0952; found
481.0951.
4.1.1.19. {1-[4-Cyano-3-(trifluoromethyl)phenyl]-3-[3-fluoro-4-
(methylcarbamoyl)phenyl]-4-methyl-5-oxo-2-thioxoimidazolidin-4-
yl}acetic acid ((R,S)-18). NaOH (7 mg, 0.172 mmol) in water (0.5 mL)
was added to the solution of compound (R,S)-16 (46 mg,
0.086 mmol) in ethanol (2.5 mL). The resulting mixture was stirred
for 12 h and rotovapped. Then isopropanol (2 mL) and hydrochloric
hydrochloric acid (69 mL, 0.8 mmol) and rotovapped. The residue
was extracted with hot isopropanol and the solvent was removed
under reduced pressure to afford 110 mg (59%) of 3-{3-[4-cyano-3-
(trifluoromethyl)phenyl]-1-[3-fluoro-4-(methylcarbamoyl)
acid (15
mL, 0.172 mmol) were added, the solution was filtered,
phenyl]-thioureido}-propionic acid (74), MS (ESI) [MþH]þ 469.
TBTU (57 mg, 0.33 mmol) and DIPEA (96 mg, 0.74 mmol) were
added to the solution of compound 74 (100 mg, 0.21 mmol) in DMF
(1.5 mL). The resulting mixture was stirred for 15 h at 45ꢀС. After
the reaction was completed (LC-MS control), the mixture was
poured into water and extracted with ethyl acetate. The organic
layer was dried over Na2SO4 and the solvent was removed by the
standard procedure. Separation using HPLC afforded 17 mg (17%) of
rotovapped and subjected to HPLC to give 15 mg (38%) of (R,S)-18.
MS (ESI) [MþH]þ 469. 1H NMR (400 MHz, DMSO-d6)
d 13.31 (brs,
1H), 8.44 (m, 2H), 8.10 (s, 1H), 7.95 (d, J ¼ 7.6 Hz, 1H), 7.81 (t,
J ¼ 8.0 Hz, 1H), 7.25 (d, J ¼ 10.8 Hz, 1H), 7.19 (d, J ¼ 8.0 Hz, 1H), 3.16
(d, J ¼ 17.6 Hz, 1H), 2.79 (d, J ¼ 3.6 Hz, 3H), 2.70 (d, J ¼ 17.6 Hz, 1H),
1.59 (s, 3H). ESIHRMS m/z calcd for C22H17F4N4O4S [MþH]þ
509.0901; found 509.0897.
20. MS (ESI) [MþH]þ 451. 1H NMR (400 MHz, DMSO-d6)
d 8.35 (q,
4.1.1.20. 4-{3-[4-Сyano-3-(trifluoromethyl)phenyl]-5,5-
bis(methoxymethyl)-4-oxo-2-thioxoimidazolidin-1-yl}-2-fluoro-N-
methylbenzamide (19). The mixture of 2-fluoro-4-iodo-N-methyl-
benzamide (37) (371 mg, 0.91 mmol), 3-methoxy-2-(methox-
ymethyl)alanine (222 mg, 1.36 mmol) [39], CuI (35 mg,
J ¼ 4.4 Hz, 1H), 8.27 (d, J ¼ 8.0 Hz, 1H), 8.06 (d, J ¼ 1.6 Hz, 1H), 7.83
(dd, J1 ¼ 8.0 Hz, J2 ¼ 1.6 Hz, 1H), 7.71 (t, J ¼ 8.2 Hz, 1H), 7.42 (dd,
J1 ¼11.0 Hz, J2 ¼ 1.8 Hz,1H), 7.33 (dd, J1 ¼8.2 Hz, J2 ¼ 1.8 Hz,1H), 4.13
(t, J ¼ 6.8 Hz, 2H), 3.17 (t, J ¼ 6.8 Hz, 2H), 2.78 (d, J ¼ 4.4 Hz, 3H).
ESIHRMS m/z calcd for C20H15F4N4O2S [MþH]þ 451.0846; found
451.0843.
0.184 mmol), 2-acetylcyclohexanone (24 mL, 0.184 mmol) and dry
K3PO4 (577 mg, 2.72 mmol) in DMF (3 mL) was stirred for 48 h at
100ꢀС. After cooling the mixture was neutralized with acetic acid
(163 mL, 2.72 mmol), diluted with water and extracted with DCM.
4.1.1.22. 4-{3-[4-Cyano-3-(trifluoromethyl)phenyl]-thioureido}-2-
fluoro-N-methylbenzamide (21). The solution of compound 22
(0.34 g, 20 mmol) and 42 (0.46 g, 20 mmol) in DMF (1 mL) was
stirred for 0.5 h in microwave reactor at 80 ꢀC. The mixture was
poured into water, the formed precipitate was filtered off, washed
with water and dried in vacuo. Purification using column chroma-
tography on silica gel (CH2Cl2/Et3N ¼ 20:1) afforded 0.3 g (44%) of
The organic layer was dried over Na2SO4 and the solvent was
removed under reduced pressure. The obtained 2-(3-fluoro-4-
{methyl[2-(trimethylsilyl)ethoxymethyl]carbamoyl}phenyl-
amino)-3-methoxy-2-(methoxymethyl)propanoic acid (40) (MS
(ESI) 445 [MþH]þ) was dissolved in DMF (3 mL) and pyridine
(3 mL), then compound 42 (414 mg, 0.276 mmol) was added and
the resulting mixture was stirred for 36 h at 90ꢀС. After cooling the
solution was rotovapped and 4-{3-[4-cyano-3-(trifluoromethyl)
phenyl]-5,5-bis(methoxymethyl)-4-oxo-2-thioxoimidazolidin-1-
yl}-2-fluoro-N-methyl-N-[2-(trimethylsilyl)ethoxymethyl]benza-
mide (71) was subjected to HPLC. The isolated product was dis-
solved in DCM (3 mL) and TFA (3 mL), the solution was then stirred
for 2 h and rotovapped. Column chromatography on silica gel
(CHCl3/MeCN ¼ 15:1) afforded 46 mg of 19. MS (ESI) [MþH]þ 525.
21. MS (ESI) [MþH]þ 397. 1H NMR (400 MHz, CDCl3)
d 10.67 (s, 1H),
10.65 (s, 1H), 8.29 (s, 1H), 8.15 (m, 1H), 8.09 (d, J ¼ 8.4 Hz, 1H), 7.99
(d, J ¼ 8.0 Hz, 1H), 7.62 (m, 2H), 7.32 (d, J ¼ 8.4 Hz, 1H), 2.77 (d,
J ¼ 4.0 Hz, 3H). ESIHRMS m/z calcd for C17H13F4N4OS [MþH]þ
397.0741; found 397.0737.
4.1.2. General procedure 2. 4-(Cyanomethylamino)-2-fluoro-N-
methylbenzamides 29e34
The mixture of compound 22 (336 mg, 2 mmol), corresponding
ketone 23e28 (4 mmol), trimethylsilylcyanide (0.5 mL, 4 mmol)
and ytterbium(III) (62 mg, 0.1 mmol) was stirred in a closed vessel
for 12 h at 80 ꢀC (36 h at 90 ꢀC for 1-methylpiperidin-4-one). The
cooled mixture was diluted with ethyl acetate, washed with cold
water, dried over Na2SO4 and the solvent was removed under
reduced pressure. Purification was performed using column chro-
matography on silica gel (hexane/EtOAc ¼ 1:1).
1H NMR (400 MHz, CDCl3)
d
8.27 (t, J ¼ 8.4 Hz, 1Н), 7.98 (d,
J ¼ 8.4 Hz, 1Н), 7.90 (d, J ¼ 1.2 Hz, 1Н), 7.79 (dd, J1 ¼ 8.0 Hz,
J2 ¼ 1.2 Hz, 1Н), 7.33 (dd, J1 ¼ 8.4 Hz, J2 ¼ 1.2 Hz, 1Н), 7.25 (dd,
J1 ¼8.0 Hz, J2 ¼ 1.2 Hz,1Н), 6.71 (q, J ¼ 4.4 Hz,1Н), 3.70 (d, J ¼ 9.8 Hz,
2Н), 3.43 (s, 6H), 3.35 (d, J ¼ 9.8 Hz, 2Н), 3.08 (d, J ¼ 4.4 Hz, 3Н).
ESIHRMS m/z calcd for C23H21F4N4O4S [MþH]þ 525.1214; found
525.1211.

A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 99 (2015) 51e66
65
4.1.2.1. 4-(2-Cyano-1-methoxypropan-2-ylamino)-2-fluoro-N-meth-
ylbenzamide (29). Yield: 413 mg (78%). MS (ESI) [MþH]þ 266. 1H
corresponded to EC80 value, while the final concentration of DMSO
was 0.1%. Model cells were then incubated for 1 day at 37 ꢀC. The
related concentration of PSA (prostate-specific antigen) was then
measured in the culture medium using ELISA (Vector-Best, cata-
logue no. Т-8458) in full accordance with the procedure provided
by manufacturer.
NMR (400 MHz, CDCl3)
d
7.99 (t, J ¼ 8.8 Hz,1H), 6.89 (dd, J1 ¼8.8 Hz,
J2 ¼ 2.4 Hz, 1H), 6.63 (m, 1H), 6.62 (dd, J1 ¼14.4 Hz, J2 ¼ 2.4 Hz, 1H),
4.77 (s,1H), 3.69 (AB sys, J ¼ 9.4 Hz,1H), 3.62 (AB sys, J ¼ 9.4 Hz,1H),
3.51 (s, 3H), 3.01 (dd, J1 ¼ 4.8 Hz, J2 ¼ 1.2 Hz, 3H), 1.70 (s, 3H).
4.1.2.2. 4-[1-(Benzyloxy)-2-cyanopropan-2-ylamino]-2-fluoro-N-
methylbenzamide (30). Yield: 506 mg (76%). MS (ESI) [MþH]þ 342.
4.3. Pharmacokinetics
1H NMR (400 MHz, DMSO-d6)
d
7.78 (m, 1H), 7.54 (t, J ¼ 8.8 Hz, 1H),
Male Sprague Dawley rats (250e350 g) obtained from the Ani-
mal Breeding Facility, Branch of Shemyakin-Ovchinnikov Institute
of Bioorganic Chemistry of the Russian Academy of Sciences,
Moscow Region were used for pharmacokinetics studies. Com-
pounds (R)-6, (R)-7, (R)-8 and (R)-9 were administered to animals at
the dose of 1 mg/kg (iv) and at the dose of 5 mg/kg (po). For iv
administration the compounds were dissolved in 20% HP-b-CD
(hydroxypropylbetacyclodextran), while for po treatment d in
0.5% HPMC. Blood samples were taken in EDTA containing tubes 5,
15, 30 min, 1, 2, 4, 8 and 24 h after iv administration and 15, 30 min,
7.37 (m, 5H), 7.32 (m, 1H), 6.82 (s, 1H), 6.70 (d, J ¼ 8.4 Hz, 1H), 6.60
(d, J ¼ 14.0 Hz, 1H), 4.64 (m, 2H), 3.85 (d, J ¼ 9.2 Hz, 1H), 3.60 (d,
J ¼ 9.2 Hz, 1H), 2.75 (d, J ¼ 4.4 Hz, 1H), 1.67 (s, 3H).
4.1.2.3. Ethyl
3-cyano-3-[3-fluoro-4-(methylcarbamoyl)phenyl-
amino]butanoate (31). Yield: 547 mg (89%). MS (ESI) [MþH]þ 308.
1H NMR (400 MHz, CDCl3)
d
8.01 (t, J ¼ 8.8 Hz, 1H), 6.73 (dd,
J1 ¼ 8.4 Hz, J2 ¼ 1.6 Hz, 1H), 6.67 (dd, J1 ¼ 14.4 Hz, J2 ¼ 2.0 Hz, 1H),
6.63 (m, 1H), 5.14 (s, 1H), 4.23 (q, J ¼ 7.2 Hz, 2H), 3.01 (d, J ¼ 4.4 Hz,
3H), 3.00 (AB sys, J ¼ 15.6 Hz, 1H), 2.95 (AB sys, J ¼ 15.6 Hz, 1H), 1.84
(s, 3H), 1.29 (t, J ¼ 7.2 Hz, 3H).
1, 2, 4, 8 and 24 h after po administration. Plasma samples (45
were prepared by centrifugation of blood, then mixed with 5
acetonitrile:H2O (1:1 v/v) and precipitated with 150 L of aceto-
m
L of
L)
m
m
4.1.2.4. 4-(3-Cyano-tetrahydrofuran-3-ylamino)-2-fluoro-N-methyl-
benzamide (32). Yield: 305 mg (58%). MS (ESI) [MþH]þ 264.
nitrile containing internal standard tolbutamide (50 ng/mL). Sam-
ples were incubated at ꢁ 20 ꢀC for 15 min followed by
centrifugation at 11,000 g for 10 min, then supernatants (130 mL)
4.1.2.5. 4-(4-Cyano-tetrahydro-2H-pyran-4-ylamino)-2-fluoro-N-
methylbenzamide (33). Yield: 338 mg (61%). MS (ESI) [MþH]þ 278.
were transferred into a new plate for LC-MS/MS analysis. The
analysis was performed using Agilent 1290 UPLC coupled with
Qtrap5500 mass spectrometer (ABSciex, USA). The corresponding
m/z values were: (R)-6 d 493.1/380.0, (R)-7 d 493.482/237.900,
(R)-8 d 480.2/224.0, (R)-9 d 479/366. Pharmacokinetic parame-
ters were calculated using non-compartmental analysis provided
by the software WinNonlin Professional 5.2 (Pharsight
Corporation).
1H NMR (400 MHz, DMSO-d6)
d
7.79 (m, 1Н), 7.55 (t, J ¼ 8.8 Hz, 1Н),
6.92 (s, 1Н), 6.70 (d, J ¼ 8.8 Hz, 1Н), 6.60 (d, J ¼ 14.4 Hz, 1Н), 3.88 (m,
2Н), 3.58 (m, 2Н), 2.75 (d, J ¼ 4.0 Hz, 3Н), 2.35 (m, 2Н), 1.86 (m, 2Н).
4.1.2.6. 4-(4-Cyano-1-methylpiperidin-4-ylamino)-2-fluoro-N-meth-
ylbenzamide (34). Yield: 340 mg (59%). MS (ESI) [MþH]þ 291. 1H
NMR (400 MHz, DMSO-d6)
d
7.76 (m, 1Н), 7.55 (t, J ¼ 8.8 Hz, 1Н),
6.83 (s, 1Н), 6.69 (dd, J1 ¼ 8.8 Hz, J2 ¼ 2.0 Hz, 1Н), 6.58 (dd,
J1 ¼ 14.0 Hz, J2 ¼ 1.6 Hz, 1Н), 2.75 (d, J ¼ 4.4 Hz, 3Н), 2.34 (m, 2Н),
2.22 (m, 2Н), 2.20 (s, 3Н), 1.94 (m, 1Н), 1.83 (m, 2Н), 1.73 (m, 1Н).
ESIHRMS m/z calcd for C15H19FN4O [MþH]þ 291.1611; found
291.1616.
4.4. In silico modeling
The 3D molecular docking study was performed in ICM Pro
software (MolSoft).30 All the force-field (FF) components available in
the software, including H-bond capacity, dipoleedipole in-
teractions and Van der Waals contacts, hydrophobic andep-
4.1.2.7. 1-[4-Сyano-3-(trifluoromethyl)phenyl]-4-(hydroxymethyl)-
2-thioxoimidazolidine-4-carboxylic acid (63). Compound 42
(228 mg, 1 mmol) was added to the solution of compound 57
(117 mg, 1 mmol) in acetonitrile (3 mL). The resulting mixture was
stirred for 18 h (LC-MS control). The solvent was removed following
the standard procedure and the product was subjected to HPLC to
give 159 mg (46%) of 63. MS (ESI) [MþH]þ 346. 1H NMR (DMSO-d6)
cationic interactions, were used for the model construction (grid
size ¼ 0.50 Å, map features: grid step ¼ 0.50, dimensions:
55 ꢂ 51 ꢂ 43, size ¼ 120,615) and further modeling. More than 10
different three-dimensional conformations were generated within
the defined binding site with the potential energy close to the
theoretical minimum. Binding energy (scoring) was calculated
automatically for all the conformations predicted using the integral
internal FF equation. Each “active” conformation within the pull
was visually inspected and got the priority following the expert
opinion. 2D supramolecular interface was constructed using MOE
Software (Molecular Operating Environment, CCG) [42].
d
12.73 (brs, 1Н), 10.35 (brs, 1Н), 8.17 (s, 1Н), 8.02 (d, J ¼ 8.0 Hz, 1Н),
5.17 (brs, 2Н), 3.72 (t, J ¼ 11.2 Hz, 2Н), 3.62 (d, J ¼ 10.0 Hz, 1Н), 3.46
(d, J ¼ 11.6 Hz, 1Н). 13С NMR (90 MHz, DMSO-d6)
d 136.42, 132.40,
131.98, 131.56, 131.15, 124.37, 120.74, 116.13, 99.71, 64.65, 35.72.
4.2. In vitro biological activity
Acknowledgements
LNCaP cells (ATCC, catalogue no. CRL-1740) were propagated in
RPMI-1640 (Invitrogen, catalogue no. 11,835-030) þ 10% FCS (Fetal
Calf Serum, Hyclone, catalogue no. SH300070.03) þ 1%AAS (Sigma,
catalogue no. 5955). Cultivated culture were plated into 96-well
We sincerely thank Kathy Brendza and Lorendana Serafini for
the HRMS data provided.
Appendix A. Supplementary data
plates (BD, catalogue no. 354620) at 10,000 cells per well in 200 mL
of RPMI 1640 þ 10% CSS (Charcoal-stripped serum, Invitrogen,
catalogue no. 12,676-011) þ 1% AAS. Patterns were incubated for 3
days at 37 ꢀC. After 3 days the culture medium was substituted with
new one containing serial dilutions of the evaluated compounds in
the absence (to estimate agonism) or presence (to estimate antag-
onism) of DHT. The final concentration of DHT was 1 nM and
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.05.039.
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