Alkylsulfanyl-1,2,4-triazoles, a New Class of allosteric valosine containing protein inhibitors. Synthesis and structure-activity relationships

Article abstract of DOI:10.1021/jm3013213

Valosine containing protein (VCP), also known as p97, is a member of AAA ATPase family that is involved in several biological processes and plays a central role in the ubiquitin-mediated degradation of misfolded proteins. VCP is an ubiquitously expressed, highly abundant protein and has been found overexpressed in many tumor types, sometimes associated with poor prognosis. In this respect, VCP has recently received a great deal of attention as a potential new target for cancer therapy. In this paper, the discovery and structure-activity relationships of alkylsulfanyl-1,2,4-triazoles, a new class of potent, allosteric VCP inhibitors, are described. Medicinal chemistry manipulation of compound 1, identified via HTS, led to the discovery of potent and selective inhibitors with submicromolar activity in cells and clear mechanism of action at consistent doses. This represents a first step toward a new class of potential anticancer agents.

Full text of DOI:10.1021/jm3013213

Article
pubs.acs.org/jmc
Alkylsulfanyl-1,2,4-triazoles, a New Class of Allosteric Valosine
Containing Protein Inhibitors. Synthesis and Structure−Activity
Relationships
Paolo Polucci,† Paola Magnaghi,† Mauro Angiolini,† Daniela Asa,† Nilla Avanzi,† Alessandra Badari,†
Jay Bertrand,† Elena Casale,† Silvia Cauteruccio,† Alessandra Cirla,† Liviana Cozzi,† Arturo Galvani,†
Peter K. Jackson,‡ Yichin Liu,‡ Steven Magnuson,‡ Beatrice Malgesini,† Stefano Nuvoloni,†
Christian Orrenius,† Federico Riccardi Sirtori,† Laura Riceputi,† Simona Rizzi,† Beatrice Trucchi,†
Tom O’Brien,‡ Antonella Isacchi,† Daniele Donati,† and Roberto D’Alessio*,†
†Nerviano Medical Sciences S.r.l., Oncology, Viale Pasteur 10, 20014 Nerviano (MI), Italy
‡Genentech Inc., 1 DNA Way, South San Francisco, California 94080, United States
S
* Supporting Information
ABSTRACT: Valosine containing protein (VCP), also known as p97, is a member of AAA ATPase
family that is involved in several biological processes and plays a central role in the ubiquitin-mediated
degradation of misfolded proteins. VCP is an ubiquitously expressed, highly abundant protein and has
been found overexpressed in many tumor types, sometimes associated with poor prognosis. In this
respect, VCP has recently received a great deal of attention as a potential new target for cancer
therapy. In this paper, the discovery and structure−activity relationships of alkylsulfanyl-1,2,4-triazoles,
a new class of potent, allosteric VCP inhibitors, are described. Medicinal chemistry manipulation of
compound 1, identified via HTS, led to the discovery of potent and selective inhibitors with
submicromolar activity in cells and clear mechanism of action at consistent doses. This represents a first step toward a new class
of potential anticancer agents.
VCP is an essential and highly abundant protein, accounting
for more than 1% of total cellular protein content. Because of
its involvement in a wide variety of functions, altered expression
or mutation of VCP is expected to have pathological
consequences. Accordingly, genetic ablation of VCP in mice
was found to be embryonic lethal,5 while mutations in the VCP
gene are associated with neurodegenerative diseases.6 On the
other hand, elevated levels of VCP have been detected in many
cancer types, sometimes associated with poor prognosis.7,8 In
this respect, VCP has attracted a great deal of attention as a
potential new molecular target for cancer therapy. While VCP
involvement in a wide spectrum of cellular functions might raise
concerns regarding the tolerability of such therapy, it should be
noted that germline mutations in the human VCP gene are
associated with neurodegenerative diseases that are however of
late onset, suggesting that VCP inhibition might be sustainable
for a considerable time before inducing irreversible adverse
effects.
INTRODUCTION
■
Valosine-containing protein (VCP), also known as p97 (cdc48
in yeast), is a member of the type II ATPases associated with
various cellular activities (AAA) family of proteins, which are
characterized by the presence of two conserved ATPase
domains, also called AAA domains.1 As for other AAA proteins,
VCP is an enzymatic machine that transforms the chemical
energy produced by hydrolysis of ATP into mechanical work
necessary for a range of cellular processes including ubiquitin-
proteasome (Ub-Pr) mediated protein degradation, membrane
fusion, transcription activation, cell cycle control, apoptosis, and
general chaperone activity.2 Under physiologic conditions, VCP
forms a ring shaped homohexamer, with each protomer
containing an N-terminal domain (N) and two AAA domains
designated D1 and D2. The N domain has been shown to
interact with a variety of effector molecules. The D1 domain
mediates hexamerization and has very low hydrolytic activity,
while the D2 domain contributes to most of the ATPase
activity and it is thought to function as a mechanochemical
transducer. In the hexameric structure, D1 and D2 domains
form two stacked rings around a central pore with the N
domains protruding outward from the D1 domains. The VCP
hexamer is a highly flexible complex which undergoes global
conformational changes during ATP binding and hydrolysis
cycles as shown by comparison of the crystal structure
reconstructions of representative ATP hydrolysis states.3,4
Although inhibitors interfering with VCP function have been
previously described,9−11 biochemically potent, reversible, and
specific compounds with cellular activity and mechanism of
action clearly directly related to VCP inhibition have never
been reported.
Received: September 13, 2012
© XXXX American Chemical Society
A
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Here we describe the discovery and structure−activity
relationships (SAR) of a series of alkylsulfanyl-1,2,4-triazoles
representing a new class of VCP inhibitors, which were
identified through biochemical screening of a collection of
more than one million compounds and which inhibit the
enzyme through an allosteric mechanism not previously
described for this enzyme. The original hit of this class (1,
Figure 1) was able to inhibit VCP ATPase activity with an IC50
Starting from compound 1, a medicinal chemistry program
was initiated aimed at improving the biochemical and biological
profile of the class. Given the low resolution of available
structural data for VCP, attempts to dock compounds into the
site identified by PAL experiments did not yield a binding
model able to provide a sufficient rationalization of biochemical
results observed for initial analogues. This, together with the
difficulty encountered in attempts to obtain cocrystals suitable
for X-ray diffraction, led us to undertake medicinal chemistry
optimization through a traditional iterative SAR approach.
The synthesis of several analogues of 1, sequentially modified
at positions 3, 4, and 5,14 allowed us to optimize the key
features contributing to VCP inhibition and to obtain potent
derivatives with submicromolar antiproliferative activity and
with a dose-dependent mechanism of action.
Figure 1. Structure of alkylsulfanyltriazole 1.
Chemistry. Our strategy for the formation of the
substituted 4H-[1,2,4]triazole-3-thiol ring (general structure
C, Scheme 1) was based on the cyclization of intermediates B
in basic media, as reported in the literature.15
of 2.69 μM but was devoid of antiproliferative effects up to a
concentration of 20 μM. The biochemical potency of 1 was
unaffected by altering ATP concentration in assay conditions
(Supporting Information Table S1, p S26), suggesting a
mechanism of noncompetitive inhibition with respect to this
substrate. Moreover, serial dilution experiments and mass
spectrometric analysis of VCP upon incubation with molar
excess of the inhibitor indicated that it possesses reversible
binding properties (data not shown).
Although more extensive biochemical and biological
characterization will be described separately (Magnaghi et al.,
manuscript submitted), it is nevertheless worth noting within
the scope of this report that the putative binding site of the
compounds described herein has been identified by photo
affinity labeling (PAL) experiments and is located between the
D1 and D2 domains of adjacent protomers of the VCP
homohexamer in the lateral tunnel leading to the central pore
(Figure 2). On this basis, we currently hypothesize that
interference with the profound conformational changes which
this region undergoes during ATP binding and hydrolysis
cycles12,13 and a resulting impairment of the VCP molecular
machinery underlies the allosteric mechanism of this class of
inhibitors.
Intermediates B are easily accessible from hydrazides A and
the suitably substituted isothiocyanate. Compounds 1−9, 11,
12, 23−45, 48−51, and 53−57 were obtained by S-alkylation
directly from intermediates C, while compound 19 was
obtained upon oxidative cleavage with hydrogen peroxide and
acetic acid of the corresponding intermediate 18.16 Compounds
1 and 2 were then subjected to S-oxidation by reaction with 3-
chloroperbenzoic acid to provide the sufonyl derivatives 16 and
17.17 Compound 16 was in turn transformed into compounds
10 and 20−22 by nucleophilic displacement of the
methylsulfone with thiophenol, 2-propanol, cyclopentanol,
and cyclohexanol respectively.18 Compound 46 was obtained
upon treatment of 39 with iron and ammonium chloride in
methanol/water at reflux temperature.19 Compound 46 was
then converted to sodium azide 47 via diazonium salt.
Treatment of compounds 12 and 50 with boron trichloride
gave the hydroxyl derivatives 13 and 59 that were converted to
the activated chloro analogues 14 and 15 useful for the left-
hand expansion of the scaffold. Product 15 was transformed
into the N-derivatives 52 and 58 by reaction with the
corresponding aniline, while the O-derivatives 60 and 66
were obtained by treatment of 15 respectively with cyclo-
hexanol and 3-hydroxypyridine.20
N-Isosters 63−65 (Scheme 2) were obtained by cyclization
of methyl isothioureas E with phenoxyacetic acid hydrazide21
and subsequent removal of the benzylic protection of
intermediates F. Isothioureas E could be prepared by
methylation of the corresponding thioureas D readily obtained
from phenyl isothiocyanate and the appropriate benzylalkyl-
amine.22
C-Isosters 61 and 62 were synthesized similarly to previously
reported protocols,23 as depicted in Scheme 3. Amides G,
obtained by conventional methods, were treated with
phosphorus pentasulfide to give thioamides H. S-Alkylation
with methyl iodide yielded the alkyl thioimidate derivatives I,
which were subjected to cyclization to the final compounds 61
and 62 by heating with phenoxyacetic acid hydrazide.
Variations at the left-hand part of the scaffold were achieved
as described in Scheme 4. Triazoles 67−90 were obtained by
treatment of the chloro intermediate 14 with the suitable
substituted phenols J and potassium carbonate in dimethylfor-
mamide at 80−100 °C.24 Compounds 94−118 were
synthesized by Suzuki coupling, under microwave irradiation,
of derivatives 75 or 83 with the appropriate aryl boronic acids
Figure 2. A surface representation of the VCP homohexamer. The
approximate region of binding as indicated by photo affinity labeling
experiments is highlighted (red boundary). A blue/green/red color
scheme is used to distinguish the different VCP domains (see inset for
color code).
B
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a
Scheme 1. General Synthesis of 3-Alkylsulfanyl and 3-Alkoxy-1,2,4-triazoles
a
Reagents and conditions: (a) R2NCS, EtOH, reflux; (b) 1 M NaOH, reflux; (c) R3Br or R3I, K2CO3, DMF, rt; (d) from 18, H2O2, acetic acid,
CH2Cl2, rt; (e) from 1 or 2, 3-chloroperbenzoic acid, CH2Cl2, rt; (f) from 16, R4OH or R4SH, 60% NaH, THF, rt; (g) from 39, Fe, NH4Cl, MeOH/
H2O, reflux; (h) NaNO3, NaN3, 20% HCl, 0−5 °C; (i) from 12or 50, 1.0 M BCl3, CH2Cl2, 0−5 °C; (j) SOCl2, CH2Cl2, 0−5 °C; (k) from 15, for 58
and 52, appropriate aniline, K2CO3, DMF, 80 °C, for 60 and 66, cyclohexanol or 3-hydroxypyridine, 60% NaH, THF, reflux.
a
a
Scheme 2. Synthesis of 3-Alkylamino-1,2,4-triazoles
Scheme 3. Synthesis of 3-Alkyl-1,2,4-triazoles
a
Reagents and conditions: (a) P2S5, toluene, 70 °C; (b) MeI, Cs2CO3,
MeCN, 50 °C; (c) phenoxyacetic hydrazide, DMF, 120 °C.
a
Reagents and conditions: (a) MeI, t-BuOK, THF, 0−5 °C; (b)
phenoxyacetic hydrazide, TFA, THF, reflux; (c) formic acid, 5% Pd/C,
80 °C.
RESULTS AND DISCUSSION
■
Our initial work focused on exploring variants of the three
different arms branching off from the triazole system, namely
the thioalkylic chain at position 3, the aromatic ring at position
4, and the phenoxy moiety at the left-hand side, and studying
the effect these parts of the molecule have on potency. A first
set of analogues was designed in order to understand the
importance of the alkylsulfanyl group at position 3. Therefore,
K. Microwave irradiation was crucial for the outcome of the
reaction in order to overcome the poisoning effect of sulfur(II)
on palladium catalysts.25,26 Differently, biphenyl derivatives
91−93 were obtained by reaction of the pinacol-boronate 90
with the suitable p-bromophenyl derivatives L in analogous
conditions.
C
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a
while benzyl (11) was probably too large to be accommodated
in the hypothetical lipophilic pocket. Sulfur always represents a
possible liability from the metabolic point of view. Actually,
compound 17, one of the potential metabolites of 1, turned out
to be inactive. For this reason, we decided to investigate, early
on in the optimization stage, sulfur replacement by other
isosteric atoms such as oxygen, carbon, and nitrogen. As
evident from Table 1, none of the considered isosteric
replacements (20−22, 61−65) gave compounds as active as
the parent sulfanyl derivatives. In fact, structure−activity
relationship results indicate rather strict structural requirements
for this region of the molecule with the cyclopentylsulfanyl
group as the optimal substituent among the different
arrangements tried at this position.
Scheme 4. Variations at the Left-Hand Side
a
Table 2. SAR: Variations at Position 4
a
Reagents and conditions: (a) K2CO3, DMF, 80−100 °C; (b) from
75, 83, Pd(dppf)Cl2·CH2Cl2, Cs2CO3, MeCN/H2O, 100 °C, micro-
wave irradiation; (c) from 90, Pd(dppf)Cl2·CH2Cl2, Cs2CO3, MeCN/
H2O, 100 °C, microwave irradiation.
R2 = isopropyl
R2 = cyclopentyl
VCP IC50
(μM)
VCP IC50
(μM)
ID
R1
ID
R1
1
Ph
2.69
19.43
>30
6
Ph
0.72
0.74
0.96
2.78
2.81
>30
1.29
2.40
>30
>30
>30
0.47
>30
the isopropyl chain of the original hit was removed or replaced
with alkylic residues of different size. As can be seen from Table
23
24
25
26
27
28
29
30
31
32
33
34
35
Ph-o-F
36
37
38
39
40
46
47
41
42
43
44
45
Ph-m-Cl
Ph-o-OMe
Ph-m-Cl
Ph-m-F
Ph-m-Br
Ph-m-OMe
Ph-m-CF3
Ph-p-F
Ph-m-Br
1.88
2.06
2.32
16.22
>30
Ph-m-Me
Ph-m-NO2
Ph-m-CF3
Ph-m-NH2
Ph-m-N3
a
Table 1. SAR: Variations at Position 3
19.60
>30
Ph-3,5-Cl
cyclohexyl
2-pyridyl
Ph-p-Cl
Ph-p-OMe
Me
>30
VCP
IC50
(μM)
VCP
IC50
(μM)
>30
3-pyridyl
ID
R
ID
R
Bn
>30
CH2-2-furanyl
3-pyridyl
2.69
1
S-isopropyl
S-methyl
2.69
>30
9.92
1.29
0.96
0.72
0.96
1.15
>30
3.79
>30
17
18
19
20
21
22
61
62
63
64
65
SO2-isopropyl
SH
>30
>30
>30
>30
2.25
1.96
1.97
4.30
>30
2.85
3.45
a
2
Values are the means of two or more experiments.
3
S-ethyl
H
4
S-isobutyl
O-isopropyl
O-cyclopentyl
O-cyclohexyl
CH2-cyclopentyl
CH2-cyclohexyl
NH-isopropyl
NH-cyclopentyl
NH-cyclohexyl
5
S-cyclobutyl
S-cyclopentyl
S-cyclohexyl
S-CH2-cyclobutyl
S-CH2-cyclohexyl
S-Ph
Next we wanted to investigate the effect of the substituent at
6
position 4 of the triazolic ring (Table 2). This was initially done
with a set of compounds (23−35) bearing the isopropylsulfanyl
group of the original hit and then with a second set of
compounds (36−47) possessing the optimized cyclopentylsul-
fanyl group. The phenyl ring at position 4 emerged to be
mandatory for biochemical activity and seems to have an
optimal shape to interact with the protein. Indeed, compound
33, which possesses a methyl instead of phenyl, is inactive as is
compound 34, where the benzyl group is likely too large and
not sufficiently flat. This also applies to compounds 42 and 45
bearing cyclohexyl and furanyl-methyl at the same position.
Decoration of the phenyl ring with various substituents did not
prove to be efficacious in either series. Ortho- and para-
substitutions were definitely detrimental, while meta-substitu-
ents did not lead to significant improvements in activity. In
contrast, 3-pyridyl proved to be a profitable replacement for
phenyl. Although the activity was in the same order of
magnitude (35 vs 1) or slightly better (44 vs 6), the use of 3-
pyridyl allowed improvement of aqueous solubility which was
of borderline acceptability due to the lipophilic character of this
7
8
9
10
11
S-Bn
a
Values are the means of two or more experiments.
1, the presence of a lipophilic group in this position was
required. Compounds 18 and 19 that respectively lack the alkyl
chain or the whole alkylsulfanyl group were inactive at the
highest tested concentration (30 μM). Furthermore, the size of
the alkyl chain was found to be critical. Residues smaller than
the isopropyl led to less active compounds (2, 3 vs 1), while
larger groups (4−7) improved activity, reaching the optimal
size with cyclopentyl, as in compound 6. In this respect,
aliphatic rings directly linked to sulfur represented the best
arrangement and were preferred to aliphatic chains (5 vs 4) and
to rings with one carbon atom spacer (5, 7 vs 8, 9). Aromatic
rings at this position were found to be detrimental (10 vs 7),
D
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class of compounds. Interestingly, the 2-pyridyl isomer 43, was
inactive.
At the same time, we drew our attention to the left-hand side
of the molecule, examining the effect of modifications to the
heterocyclic system. Interestingly, these compounds maintained
some activity but were less active than the reference compound
6.
This preliminary expansion allowed us on one hand to
identify the basic structural requirements for this class in order
to maintain activity and on the other to optimize substituents at
position 3 and 4 of the central triazolic scaffold. In this respect,
compound 44 represented a good prototype to start further
chemical expansion. Compound 44, with a biochemical IC50 of
0.47 μM, was about 5-fold more potent than the original hit 1
but was still devoid of antiproliferative activity (IC50 > 20 μM
on HCT-116 cell line) despite good Caco-2 cell permeability
data (not shown). This clearly indicated the need to further
improve potency. To achieve this goal, an extensive expansion
of the left-hand part of the molecule was undertaken. This
region was particularly amenable to expansion by parallel
chemistry through the advanced intermediate 14 that was
reacted with a series of commercially available phenols (Scheme
4). More than 200 phenols were selected based on chemical
diversity, and results for the most representative compounds
are shown in Table 5. Even though a clear structure−activity
relationship could not be discerned, ortho-substituents on
phenyl, as for compound 67, were generally detrimental while
the overall activity of meta- and para-substituted derivatives
(68−79) was similar or slightly better (with the exception of
compound 72) than the reference compound 44, especially
when lipophilic substituents, such as halogens, were considered.
The combination of some of the most interesting groups
identified at meta- and para-positions resulted in a small series
of derivatives (Table 5, 80, 82−89) displaying, together with
compound 75, significant antiproliferative activity (<20 μM)
against HCT-116 cell lines for the first time in this class.
Interestingly, cell activity could be observed only for derivatives
with biochemical IC50 generally below 300 nM, thus fixing such
value as a necessary prerequisite for this class of compounds.
To unequivocally associate antiproliferative activity with VCP
inhibition, modulation of the most sensitive biomarkers was
assessed upon treatment of HCT-116 cells with increasing
doses of the representative compound 86. Previous siRNA
experiments (Supporting Information Figure S6, p S26 and
Magnaghi et al., manuscript submitted) had clearly shown that
VCP ablation induced accumulation of poly ubiquitinated
proteins and Cyclin E, as well as activation of the unfolded
protein response, as assessed by induction of C/EBP
homologous protein transcription factor (CHOP, also known
as Gadd153). Western blot analysis of HCT-116 cells treated
with compound 86 showed dose-dependent accumulation of
poly ubiquitinated proteins and of Cyclin E and clear induction
of CHOP at doses consistent with the antiproliferative IC50
(Figure 3).
a
Table 3. SAR: Effect of the Linker at Position 5
ID
R1
A−B
CH2O
R2
VCP IC50 (μM)
1
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
cyclopentyl
cyclopentyl
cyclopentyl
H
H
H
H
H
H
H
Br
Br
Br
2.69
7.73
48
49
50
51
52
58
53
54
55
CH2S
CH2CH2
CH2OCH2
CH2
11.78
26.72
>30
CH2N(Me)
CH2NH
CH2O
>30
11.17
0.44
CONH
18.64
2.86
CHCH
a
Values are the means of two or more experiments.
oxymethylene linker at position 5 between the phenyl and the
central scaffold (Table 3). Also in this case we tried various
isosteric replacements, moving from the original oxygen (1) to
sulfur (48), nitrogen (58, 52), and carbon (49). In none of
these cases did we observe any improvement of activity. A
similar outcome was obtained elongating the oxymethylene
linker by one carbon atom (50) or removing the oxygen (51).
In addition, the oxymethylene linker was replaced with other
spacers that should orientate substituents in the same direction,
such as an amidic bond (54) and a trans double bond (55),
without improvement of activity as compared to compound 53.
a
Table 4. SAR: Importance of the Aromatic Moiety at the
Left-Hand Side
ID
R1
R2
CH2OPh
VCP IC50 (μM)
1
isopropyl
isopropyl
isopropyl
isopropyl
cyclopentyl
cyclopentyl
cyclopentyl
2.70
>30
>30
5.56
0.72
1.94
8.72
59
60
66
6
CH2OH
CH2O-cyclohexyl
CH2O-3-pyridyl
CH2OPh
The real breakthrough of this expansion was however
represented by compound 81 characterized by a biphenyl
system with a cyano group at para-position. This compound,
despite a biochemical potency comparable to 86, possessed
significantly improved antiproliferative activity (IC50 = 4.07 μM
on the HCT-116 cell line). Considering the increased lipophilic
character of 81, this result may be simply explained in terms of
improved cell permeability. On the other hand, it is likely that
additional factors such as binding to adaptor proteins or post-
translational modifications27 may play a role in determining the
real activity in the cellular environment that cannot be
appreciated in a cell-free system. Alternatively, one could
postulate that the additional phenyl ring might lead to a better
56
57
2-benzofuranyl
2-furanyl
a
Values are the means of two or more experiments.
As for the importance of the contiguous phenyl, the set of
compounds shown in Table 4 clearly indicate that its removal
(59) as well as its replacement with a cyclohexyl moiety (60)
caused a marked drop of activity. Changing phenyl with
pyridine did not prove to be effective in this case, and
compound 66 was actually 2-fold less active than its phenylic
counterpart 1. Compounds 56 and 57 were synthesized in the
attempt to incorporate an oxymethylene linker into a rigid
E
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a
Table 5. SAR: Expansion at the Left-Hand Side
ID
R
VCP IC50 (μM)
HCT-116 IC50(μM)
ID
R
VCP IC50 (μM)
HCT-116 IC50(μM)
44
67
68
69
70
71
72
73
74
75
76
77
78
79
H
0.468
1.277
0.279
0.283
0.379
0.524
3.129
0.375
0.140
0.168
0.604
0.220
0.609
0.587
>20
>20
>20
>20
>20
>20
>20
>20
>20
18.09
>20
>20
>20
>20
80
81
82
83
84
85
86
87
88
89
3-F-4-Cl
0.071
0.088
0.090
0.100
0.134
0.150
0.184
0.225
0.257
0.443
14.92
4.07
o-Cl
p-(Ph-p-CN)
3,4-Cl
m-Cl
m-Br
m-F
14.35
15.69
13.96
15.14
18.22
15.36
18.13
>20
3-Me-4-Br
3,5-Me-4-Cl
3-Cl-4-Me
3-Me-4-Cl
3-Br-5-F
m-Me
m-CN
m-OMe
p-Cl
3,5-Cl
p-Br
3-Me-4-F
p-F
p-Me
p-CN
p-OMe
a
Values are the means of two or more experiments.
important for activity, likely acting as a spacer and setting the
terminal group at position para at an optimal distance. Notably,
compound 106, without any substituent, was considerably less
active than 81, as well as compounds where the terminal group
was shifted to ortho- (107, 108 vs 81, 97) or meta-position
(109, 110, 111 vs 81, 97, 96).
As a next step, by analogy with compounds of Table 5, we
considered the opportunity to investigate the effect of
additional substituents on the proximal ring of the biphenyl
system. Given that substitutions at position ortho to the oxygen
(position 2′) did not prove to be effective, we attempted to
probe the effect of substitution at position 3′ by introducing a
methyl group in some of the most interesting previously
identified derivatives (Table 7). This might also provide insight
on the effect of disrupting planarity of the biphenyl system.
Interestingly, even though biochemical activity was generally
maintained in the same range (112−118 vs 81, 91, 93, 95−98),
this introduction resulted in an improvement of the
antiproliferative activity for some representatives and was
particularly significant for derivative 116 (IC50 = 0.38 μM on
HCT-116 cell line), which was 1 order of magnitude more
active in cells than the parent compound 96.
For this compound, we also observed a clear dose-dependent
modulation of key cellular VCP biomarkers starting from a
concentration of 0.5 μM (Figure 3). Compound 116 was also
profiled against other representative AAA ATPases (NSF,
SPATA5, VPS4B, and cdc6), HSP90, as well as a panel of 50
kinases and did not display any measurable cross-reactivity up
to the highest tested concentration of 10 μM (data not shown).
Similarly to the original hit 1, the potency of compound 116 in
standard assay conditions was unchanged at saturating ATP
concentration (Supporting Information, Table S1, p S26),
indicating a mechanism of noncompetitive inhibition for this
class. To assess the feasibility for an in vivo efficacy study,
pharmacokinetic parameters of compound 116 were evaluated
following intravenous (iv) and oral (po) administration in mice
(Table 8). After iv dosing, compound 116 was found to be well
distributed in tissues (volume of distribution about 10 times the
Figure 3. Analysis of biomarkers modulation upon treatment with
VCP inhibitors. HCT-116 cells were treated with increasing doses of
inhibitors for 8 h. Cells were then rinsed with PBS and lysed. Then 20
μg of lysate proteins were fractionated in SDS PAGE and subjected to
immunoblotting with the indicated antibodies. C stands for control;
MG stands for the proteasome inhibitor MG-132 used as a standard.
association with the endoplasmic reticulum membrane system
where VCP in part resides and to selective interference with the
function of membrane-associated VCP, as has been suggested
for the aromatic domain of Eeyarestatin.9
Indeed, the increased cellular potency of compound 81 was
accompanied by a VCP-related mechanism of action because
biomarker modulation could in this case be appreciated starting
from 5 μM, consistent with the antiproliferative IC50 of the
compound (Figure 3). To gain better understanding of the role
of the para-cyano biphenyl arrangement, a small set of
analogues (Table 6) was synthesized according to the same
procedure reported in Scheme 4. The results obtained showed
that the cyano group could conveniently be replaced by other
hydrogen-bond acceptor groups, such as amides (94, 95),
hydroxamates (91), ketones (92, 97), sulfones (96), and
sulfonamides (93), while other chemical functionalities (98−
105) were less effective. The biphenyl system proved to be
F
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Article
a
Table 6. SAR: Variations on the Biphenyl Series
ID
R
VCP IC50 (μM)
HCT-116 IC50(μM)
ID
R
VCP IC50 (μM)
HCT-116 IC50(μM)
81
91
92
93
94
95
96
97
98
99
100
p-CN
0.088
0.043
0.048
0.071
0.041
0.042
0.063
0.065
0.112
0.162
0.172
4.07
3.13
1.74
1.37
5.52
6.44
3.03
7.39
9.17
>10
>10
101
102
103
104
105
106
107
108
109
110
111
p-NH2
p-Br
0.262
0.523
0.683
0.976
1.236
2.031
0.536
0.379
1.394
0.451
0.908
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
p-CONHOMe
p-COCH2OH
p-SO2NH2
p-CONH2
p-CONHMe
p-SO2Me
p-F
p-SMe
p-CF3
H
o-CN
p-COMe
o-COMe
m-CN
m-COMe
m-SO2Me
p-CHO
p-NO2
p-OMe
a
Values are the means of two or more experiments.
a
Table 7. SAR: Effect of the ortho-Methyl on the Biphenyl
System
Further work to improve PK and ADME characteristics of
compound 116, particularly with respect to solubility and
metabolic stability issues, will be undertaken in order to identify
representatives with a PK profile more appropriate for in vivo
efficacy studies.
Compound 116 is the most potent VCP inhibitor disclosed
to date and thus represents a valuable tool for investigating the
biological consequences of inhibiting this enzyme in cells and
gather further insight into the rationale for VCP-targeted cancer
therapy.
R2 = H
R2 = Me
VCP
VCP
IC50
(μM)
IC50
HCT-116
HCT-116
CONCLUSIONS
R1
ID
(μM)
IC50(μM)
ID
IC50(μM)
■
CN
81
91
93
95
96
97
98
0.088
0.043
0.071
0.042
0.063
0.065
0.112
4.07
3.13
1.37
6.44
3.03
7.39
9.17
112
113
114
115
116
117
118
0.071
0.058
0.025
0.054
0.024
0.053
0.067
2.02
4.60
1.82
4.98
0.38
1.48
3.16
In this report, the first known allosteric inhibitors of VCP
ATPase activity are described. Starting from the basic
framework of alkylsulfanyltriazole 1, which emerged from
HTS as a marginally active hit and which was devoid of
antiproliferative activity, the structure−activity relationships
associated with this class of compounds were investigated. This
allowed determination of the major chemical features within
the class that are responsible for inhibition of VCP ATPase
activity, particularly:
CONHOMe
SO2NH2
CONHMe
SO2Me
COMe
CHO
a
Values are the means of two or more experiments.
• the cycloalkylic group, preferably cyclopentyl, directly
linked to sulfur at position 3 of the scaffold
• the 3-pyridyl group at position 4, able to confer potency
and solubility
total body water) but with modest systemic exposure, probably
due to high clearance (115 mL/min/kg), which was consistent
with the high in vitro intrinsic clearance observed during
stability testing in the presence of human liver microsomes
(Clint = 400 mL/min/kg). Following oral administration,
compound 116 showed modest bioavailability (F = 16.4%),
possibly due to extensive first-pass metabolism and/or solubility
issues (about 7 μM in aqueous ammonium acetate buffer at pH
7). On the other hand, cell permeability, measured in Caco-2
cells, was good (Papp = 21.6 cm 10−6/s).
• the aromatic ring, preferably phenyl, linked to the
oxymethylene linker at position 5 of the triazole
Further parallel expansion and optimization of the left-hand
part of the molecule following a traditional iterative medicinal
chemistry approach led to the identification of low nanomolar
inhibitors of VCP ATPase activity. Compound 116, the best
representative of this class to date, inhibits VCP ATPase with
Table 8. Pharmacokinetic Parameters of Compound 116 Following iv Bolus Single Dose and Oral Single Dose in Harlan nu/nu
a
Mice
b
c
PK data (iv), dose: 1 mg/kg
PK data (per os), dose: 10 mg/kg
d
Cmax (μM)
AUC∞ (μM h)
CL (mL/min/kg)
115 31
Vss (mL/kg)
5882 1298
t1/2 (h)
Cmax (μM)
tmax (h)
AUC∞ (μM h)
t1/2 (h)
F
(%)
16.4
0.30 0.08
2.26 0.07
0.9
0.06 0.02
0.8
0.43 0.11
6.0
a
b
c
d
n = 3 animals per study. Dosed in 10% Tween 80 in 5% dextrose. Dosed in 0.5% Methocel. Bioavailability.
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The ester (4.87 g, 25.09 mmol) was dissolved in EtOH (20 mL) and
hydrazine monohydrate added (2.44 mL, 50.18 mmol). The mixture
was heated at reflux for 3 h, then the solvent was evaporated under
vacuum. Water (50 mL) was added and the reaction was extracted
with DCM (5 × 20 mL). The organic phase was washed with brine,
dried with anhydrous sodium sulfate, and evaporated under vacuum.
Benzyloxyacetic acid hydrazide (4.24 g, 94% yield) was obtained as
white solid. A mixture of benzyloxyacetic acid hydrazide (3.56 g, 19.78
mmol) and 3-pyridyl isothiocyanate (2.69 g, 19.78 mmol) in EtOH
(40 mL), was heated at reflux for 1 h, then cooled at room
temperature. The suspension was filtered, and the solid was washed
with EtOH and dried. 2-[(Benzyloxy)acetyl]-N-(pyridin-3-yl)-
hydrazinecarbothioamide (5.82 g, 85% yield) was obtained as white
solid. Then 1 N NaOH (30 mL) was added to this solid and the
mixture was heated at reflux for 1 h. The resulting solution was cooled
at room temperature and acidified to pH 6 with 1 M HCl. The
precipitate was filtered, washed with water, and dried to obtain the title
compound (4.06 g, 74% yield) as white solid. 1H NMR (400 MHz), δ
(ppm, DMSO-d6): 14.06 (bs, 1H), 8.72 (m, 1H), 8.65 (d, J = 2.32 Hz,
1H), 7.94 (m, 1H), 7.61 (m, 1H), 7.30−7.25 (m, 3H), 7.08 (m, 2H),
4.40 (s, 2H), 4.35 (s, 2H). MS (ESI) m/z 299 [(M + H)+].
an IC50 of 24 nM and possesses antiproliferative activity in the
submicromolar range (IC50 = 0.38 μM on HCT-116 cell lines),
which is associated, at consistent concentrations, with
modulation of known VCP biomarkers.
Further work to optimize ADME and PK characteristics of
this class will next be undertaken for identification of candidates
suitable for in vivo efficacy studies and will be described in due
course.
EXPERIMENTAL SECTION
■
1. Chemistry. Unless otherwise noted, solvents and reagents were
obtained from commercial suppliers and used without further
purification. All reactions involving air- or moisture-sensitive reagents
were performed under an argon atmosphere. All final compounds were
purified to >95% purity as determined by high performance liquid
chromatography (HPLC). Purity was measured by HPLC on a Waters
X Terra RP18 (4.6 mm × 50 mm, 3.5 μm) column using a Waters
2790 HPLC system equipped with a 996 Waters PDA detector and
Micromass model A ZQ single quadrupole mass spectrometer,
equipped with an electrospray ion source (ESI). Mobile phase A
was an ammonium acetate 5 mM buffer (pH 5.5 with acetic acid/
acetonitrile 95:5), and mobile phase B was H2O/acetonitrile (5:95).
The following conditions were used: a gradient from 10 to 90% B in 8
min and held at 90% B for 2 min; UV detection at 220 and 254 nm; a
flow rate of 1 mL/min; an injection volume of 10 μL; full scan, mass
range from 100 to 800 amu. The capillary voltage was 2.5 kV; the
source temperature was 120 °C; the cone was 10 V. Masses are given
as an m/z ratio. When necessary, compounds were purified by
preparative HPLC on a Waters Symmetry C18 (19 mm × 50 mm, 5
μm) column using a Waters preparative HPLC 600 equipped with a
996 Waters PDA detector and a Micromass model A ZMD single
quadrupole mass spectrometer, with electrospray ionization, in the
positive mode, was used. Mobile phase A was water and 0.01%
trifluoroacetic acid and mobile phase B was acetonitrile. The following
conditions were used: a gradient from 10 to 90% B in 8 min and held
at 90% B for 2 min; a flow rate of 20 mL/min. Column
chromatography was conducted either under medium pressure on
silica (Merck silica gel 40−63 μm) or on prepacked silica gel cartridges
By analogous procedure, starting from the suitable carboxylic acid
and the appropriate isothiocyanate, all the compounds of general
formula C, Scheme 1 (see Supporting Information, p S2) and
compound 18 were prepared.
5-Phenoxymethyl-4-phenyl-4H-[1,2,4]triazole-3-thiol (18). Yield 2
1
g, 70%. H NMR (400 MHz), δ (ppm, DMSO-d6): 14.03 (bs, 1H),
7.55−7.47 (m, 3H), 7.43 (m, 2H), 7.23 (m, 2H), 6.94 (m, 1H), 6.82
(m, 2H), 4.96 (s, 2H). MS (ESI) m/z 284 [(M + H)+].
3-Isopropylsulfanyl-5-phenoxymethyl-4-phenyl-4H-[1,2,4]-
triazole (1). To a solution of 5-phenoxymethyl-4-phenyl-4H-[1,2,4]-
triazole-3-thiol (18) (0.10 g, 0.35 mmol) in anhydrous DMF (3 mL),
2-bromopropane (0.07 mL, 0.7 mmol) and potassium carbonate (0.12
g, 0.88 mmol) were added. The mixture was stirred at room
temperature for 15 h, then the reaction was poured in water and
extracted with EtOAc (2 × 10 mL). The organic phase was washed
with brine, dried with anhydrous sodium sulfate, and evaporated. The
crude residue was purified by silica gel column chromatography
(DCM/MeOH 97:3) to afford the title compound (0.08 g, 70% yield)
1
1
(Biotage) or on a Horizon system. H and 13C NMR spectra were
as white solid. H NMR (400 MHz), δ (ppm, DMSO-d6): 7.57−7.52
(m, 3H), 7.46−7.43 (m, 2H), 7.23 (m, 2H), 6.93 (m, 1H), 6.85 (m,
2H), 5.07 (s, 2H), 3.70 (m, 1H), 1.31 (d, J = 6.71 Hz, 6H). MS (ESI)
m/z 326 [(M + H)+]. HRMS (ESI) calculated for C18H20N3OS+ [(M
+ H)+] 326.1322, found 326.1321.
By analogous procedure, starting from the suitable 2,4-dihydro-
[1,2,4]triazole-3-thione (general formula C, Scheme 1) and the
appropriate alkyl bromide or alkyl iodide, derivatives 2−5, 7−9, 11,
23−43, 45, 48−51, 53−57 (see Supporting Information, p S6), and
the following compounds were prepared:
3-Cyclopentylsulfanyl-5-phenoxymethyl-4-phenyl-4H-[1,2,4]-
triazole (6). Yield 0.12 g, 70%. 1H NMR (400 MHz), δ (ppm, DMSO-
d6): 7.58−7.52 (m, 3H), 7.47−7.43 (m, 2H), 7.23 (m, 2H), 6.93 (m,
1H), 6.85 (m, 2H), 5.06 (s, 2H), 3.84 (m, 1H), 2.07 (m, 2H), 1.65−
1.53 (m, 6H). 13C NMR (100.7 MHz), δ (ppm, DMSO-d6): 157.3,
151.8, 151.4, 132.8, 129.8, 129.6, 129.3, 127.1, 121.4, 114.8, 59.8, 45.5,
33.1, 24.1. MS (ESI) m/z 352 [(M + H)+]. HRMS (ESI) calculated for
C20H22N3OS+ [(M + H)+] 352.1478; found 352.1494. Melting point
129−130 °C.
3-(3-Benzyloxymethyl-5-cyclopentylsulfanyl-[1,2,4]triazol-4-yl)-
pyridine (12). Yield 3.36 g, 67%. 1H NMR (400 MHz), δ (ppm,
DMSO-d6): 8.77 (m, 1H), 7.96 (m, 1H), 7.61 (m, 1H), 7.31−7.25 (m,
3H), 7.08 (m, 2H), 4.53 (s, 2H), 4.36 (s, 2H), 3.80 (m, 1H), 2.04 (m,
2H), 1.65−1.49 (m, 6H). MS (ESI) m/z 367 [(M + H)+].
3-(3-Cyclopentylsulfanyl-5-phenoxymethyl-[1,2,4]triazol-4-yl)-
pyridine (44). Yield 0.05 g, 50%. 1H NMR (400 MHz), δ (ppm,
DMSO-d6): 8.72−8.68 (m, 2H), 7.99 (m, 1H), 7.61 (m, 1H), 7.23 (m,
2H), 6.93 (m, 1H), 6.84 (m, 2H), 5.13 (s, 2H), 3.82 (m, 1H), 2.07 (m,
2H), 1.68−1.53 (m, 6H). 13C NMR (100.7 MHz), δ (ppm, DMSO-
d6): 157.1, 151.9, 151.7, 150.8, 147.7, 135.2, 130.0, 129.4, 124.3, 121.4,
114.7, 59.8, 46.0, 33.1, 24.0. MS (ESI) m/z 353 [(M + H)+]. HRMS
recorded on a Varian Inova 400 spectrometer operating respectively at
400.5 MHz for proton and 100.7 MHz for carbon and equipped with a
1
5 mm H{15N−31P} z-axis- PFG indirect detection probe. Residual
1
solvent signal was used as reference (DMSO-d6 at 2.50 ppm for H
and 39.5 ppm for 13C). Data are reported as follows: chemical shift δ
(in ppm), multiplicity (s = singlet, d = doublet, t = triplet, bs = broad
singlet, m = multiplet), coupling constants J (in Hz), and number of
protons. Low-resolution mass spectral (MS) data were determined on
a Finnigan MAT LCQ ion trap instrument equipped with ESI. High-
resolution mass spectra (HRMS) were obtained on a Waters Q-TOF
Ultima instrument equipped with ESI and using reserpine (MW
609.28065) for lock mass correction. Thin-layer chromatography was
performed on Merck silica gel 60 plates coated with a 250 μM layer
with a fluorescent indicator. Components were visualized by UV light
(λ = 254 and 366 nm) and iodine vapors.
The following abbreviations for solvents and reagents are used:
EtOH = ethanol, DCM = dichloromethane, EtOAc = ethyl acetate,
THF = tetrahydrofuran, DMF = dimethylformamide, Et2O = diethyl
ether, MeOH = methanol, MeCN = acetonitrile, TMOF = triethyl
orthoformate, Pd(dppf)Cl2·DCM = [1,1′-bis(diphenylphosphino)-
ferrocene]dichloro palladium(II) complex with dichloromethane,
DIPEA = N-ethyldiisopropylamine.
5-Benzyloxymethyl-4-pyridin-3-yl-4H-[1,2,4]triazole-3-thiol (Gen-
eral Formula C, Scheme 1). A solution of benzyloxyacetic acid (5 g,
30 mmol) and p-toluenesulfonic acid monohydrate in EtOH (25 mL)
was heated at reflux for 3 h. The solvent was evaporated under
vacuum, the residue dissolved in DCM (30 mL) and washed with satd
aq Na2CO3, water, and brine. The organic phase was dried with
anhydrous sodium sulfate and evaporated under vacuum. Benzyloxy-
acetic acid ethyl ester (4.87 g, 84% yield) was obtained as colorless oil.
H
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(ESI) calculated for C19H21N4OS+ [(M + H)+] 353.1431; found
353.1435. Melting point 83−84 °C.
added. The reaction was heated at reflux for 4 h, then cooled at room
temperature and filtered on Celite washing with MeOH. The solvent
was evaporated under vacuum, and the residue was dissolved in EtOAc
(20 mL) and washed with satd aq NaHCO3 (20 mL), water, and brine.
The organic phase was dried with anhydrous sodium sulfate and
evaporated. The title compound (0.18 g, 78% yield) was obtained as
3-Phenoxymethyl-4-phenyl-4H-[1,2,4]triazole (19). To a solution
of 18 (0.6 g, 2.12 mmol) in DCM (30 mL), cooled at 0−5 °C, a
solution of 30% aq H2O2 (0.6 mL, 5.3 mmol) in acetic acid (3.5 mL)
was added dropwise. The reaction was stirred at room temperature for
1 h, then was washed with 1 N NaOH (50 mL). The organic phase
was dried with anhydrous sodium sulfate and evaporated under
vacuum. The title compound (0.51 g, 95% yield) was obtained as
1
yellowish solid. H NMR (400 MHz), δ (ppm, DMSO-d6): 7.25 (m,
2H), 7.13 (m, 1H), 6.96−6.90 (m, 3H), 6.67 (m, 1H), 6.51 (m, 1H),
6.47 (m, 1H), 5.48 (bs, 2H), 5.01 (s, 2H), 3.87 (m, 1H), 2.09 (m,
2H), 1.67−1.50 (m, 6H). MS (ESI) m/z 367 [(M + H)+]. HRMS
(ESI) calculated for C20H23N4OS+ [(M + H)+] 367.1587; found
367.1596.
1
yellowish solid. H NMR (400 MHz), δ (ppm, DMSO-d6): 8.87 (s,
1H), 7.58−7.51 (m, 5H), 7.26 (m, 2H), 6.92 (m, 1H), 5.22 (s, 2H).
MS (ESI) m/z 252 [(M + H)+]. HRMS (ESI) calculated for
C15H14N3O+ [(M + H)+] 252.1132; found 252.1124
4-(3-Azido-phenyl)-3-cyclopentylsulfanyl-5-phenoxymethyl-4H-
[1,2,4]triazole (47). To a solution of 46 (0.17 g, 0.48 mmol) in 20% aq
HCl (5 mL), cooled at 0−5 °C, a solution of sodium nitrite (0.03 g,
0.48 mmol) in water (1 mL) was added dropwise while maintaining
the temperature below 5 °C. The reaction was stirred at 0−5 °C for 30
min, then a solution of sodium azide (0.03 g, 0.53 mmol) in water (1
mL) was added dropwise while maintaining the temperature below 5
°C. After 15 min, the mixture was diluted with EtOAc (20 mL) and
washed with water (20 mL) and brine. The organic phase was dried
with anhydrous sodium sulfate and evaporated under vacuum. The
title compound (0.14 g, 74% yield) was obtained as yellowish solid. 1H
NMR (400 MHz), δ (ppm, DMSO-d6): 7.57 (m, 1H), 7.30−7.22 (m,
5H), 6.94 (m, 1H), 6.87 (m, 2H), 5.10 (s, 2H), 3.84 (m, 1H), 2.07 (m,
2H), 1.67−1.52 (m, 6H). MS (ESI) m/z 393 [(M + H)+]. HRMS
(ESI) calculated for C20H21N6OS+ [(M + H)+] 393.1492; found
393.1482.
(5-Cyclopentylsulfanyl-4-pyridin-3-yl-4H-[1,2,4]triazol-3-yl)-
methanol (13). To a solution of 12 (7.41 g, 20 mmol) in anhydrous
DCM (50 mL), cooled at 0−5 °C, 1 M boron trichloride solution in
DCM (80 mL, 80 mmol) was added dropwise. The reaction was
stirred at 0−5 °C for 2 h, then 33% aq NH4OH (100 mL) was added
dropwise and the mixture was vigorously stirred for 1 h. The organic
phase was washed with brine, dried with anhydrous sodium sulfate,
and evaporated under vacuum. The crude residue was purified by silica
gel column chromatography (DCM/MeOH 95:5) to afford the title
compound (4.21 g, 76% yield) as white solid. 1H NMR (400 MHz), δ
(ppm, DMSO-d6): 8.75 (m, 1H), 8.67 (m, 1H), 7.97 (m, 1H), 7.64
(m, 1H), 5.43 (t, J = 5.61 Hz, 1H), 4.45 (d, J = 5.61 Hz, 2H), 3.77 (m,
1H), 2.03 (m, 2H), 1.65−1.51 (m, 6H). MS (ESI) m/z 277 [(M +
H)+].
3-Methanesulfonyl-5-phenoxymethyl-4-phenyl-4H-[1,2,4]triazole
(16). To a solution of 2 (0.48 g, 1.61 mmol) in DCM (10 mL), 3-
chloroperbenzoic acid (1.11 g, 6.44 mmol) was added. The reaction
was stirred at room temperature for 15 h, then diluted with DCM (40
mL) and washed with 10% aq sodium thiosulfate (20 mL), satd aq
NaHCO3 (20 mL), water, and brine. The organic phase was dried with
anhydrous sodium sulfate and evaporated under vacuum. The crude
residue was ground with Et2O (10 mL) to obtain the title compound
1
(0.37 g, 70% yield) as white solid. H NMR (400 MHz), δ (ppm,
DMSO-d6): 7.60−7.53 (m, 5H), 7.24 (m, 2H), 6.94 (m, 1H), 6.87 (m,
2H), 5.12 (s, 2H), 3.46 (s, 3H). MS (ESI) m/z 330 [(M + H)+].
By analogous procedure, starting from 1 the following compound
was obtained:
3-Phenoxymethyl-4-phenyl-5-(propane-2-sulfonyl)-4H-[1,2,4]-
triazole (17). Yield 0.11 g, 68%. 1H NMR (400 MHz), δ (ppm,
DMSO-d6): 7.55−7.48 (m, 5H), 7.21 (m, 2H), 6.91 (m, 1H), 6.83 (m,
2H), 5.07 (s, 2H), 3.54 (m, 1H), 1.23 (d, J = 6.84 Hz, 6H). MS (ESI)
m/z 358 [(M + H)+]. HRMS (ESI) calculated for C18H20N3O3S+ [(M
+ H)+] 358.1220; found 358.1228.
3-Isopropoxy-5-phenoxymethyl-4-phenyl-4H-[1,2,4]triazole (20).
To a solution of 2-propanol (0.07 mL, 0.91 mmol) in anhydrous THF
(2 mL), 60% sodium hydride (0.03 g, 0.76 mmol) was added. The
mixture was stirred at room temperature for 15 min, then 16 (0.1 g,
0.30 mmol) was added in portions. The reaction was stirred at room
temperature for 5 h, then poured in water (20 mL) and extracted with
EtOAc (2 × 10 mL). The organic phase was dried with anhydrous
sodium sulfate and evaporated under vacuum. The crude residue was
purified by silica gel column chromatography (EtOAc/exane 7:3) to
afford the title compound (0.04 g, 42% yield) as white solid. 1H NMR
(400 MHz), δ (ppm, DMSO-d6): 7.54−7.44 (m, 5H), 7.24 (m, 2H),
6.93 (m, 1H), 6.88 (m, 2H), 5.09 (m, 1H), 4.99 (s, 2H), 1.32 (d, J =
6.10 Hz, 6H). MS (ESI) m/z 310 [(M + H)+]. HRMS (ESI)
By analogous procedure, starting from 50 the following compound
was prepared:
(5-Isopropylsulfanyl-4-phenyl-4H-[1,2,4]triazol-3-yl)-methanol
1
+
calculated for C18H20N3O2 [(M + H)+] 310.1550; found 310.1548.
(59). Yield 0.24 g, 77%. H NMR (400 MHz), δ (ppm, DMSO-d6):
7.60−7.53 (m, 3H), 7.43−7.39 (m, 2H), 5.38 (bs, 1H), 4.39 (s, 2H),
3.64 (m, 1H), 1.29 (d, J = 6.71 Hz, 6H). MS (ESI) m/z 250 [(M +
H)+]. HRMS (ESI) calculated for C12H16N3OS+ [(M + H)+]
250.1009; found 250.1005.
By analogous procedure, starting from 16 and the suitable alcohols
or thiophenol the following compounds were obtained:
3-Cyclopentyloxy-5-phenoxymethyl-4-phenyl-4H-[1,2,4]triazole
1
(21). Yield 0.07 g, 69%. H NMR (400 MHz), δ (ppm, DMSO-d6):
7.54−7.42 (m, 5H), 7.24 (m, 2H), 6.93 (m, 1H), 6.88 (m, 2H), 5.29
3-(3-Chloromethyl-5-cyclopentylsulfanyl-[1,2,4]triazol-4-yl)-pyri-
dine (14). To a solution of 13 (0.50 g, 1.81 mmol) in anhydrous DCM
(40 mL), cooled at 0−5 °C, thionyl chloride (0.20 mL, 2.72 mmol)
was added. The reaction was stirred at 0−5 °C for 2 h, then was
washed with satd aq NaHCO3, water, and brine. The organic phase
was dried with anhydrous sodium sulfate and evaporated under
vacuum. The title compound (0.52 g, 98% yield) was obtained as
white solid. 1H NMR (400 MHz), δ (ppm, DMSO-d6): 8.79 (m, 1H),
8.73 (m, 1H), 8.03 (m, 1H), 7.67 (m, 1H), 4.80 (s, 2H), 3.82 (m, 1H),
2.05 (m, 2H), 1.65−1.53 (m, 6H). MS (ESI) m/z 295 [(M + H)+].
By analogous procedure, starting from 59 the following compound
was prepared:
(m, 1H), 4.99 (s, 2H), 1.91 (m, 2H), 1.77 (m, 2H), 1.56 (m, 4H). MS
+
(ESI) m/z 336 [(M + H)+]. HRMS (ESI) calculated for C20H22N3O2
[(M + H)+] 336.1707; found 336.1704.
3-Cyclohexyloxy-5-phenoxymethyl-4-phenyl-4H-[1,2,4]triazole
1
(22). Yield 0.04 g, 38%. H NMR (400 MHz), δ (ppm, DMSO-d6):
7.54−7.44 (m, 5H), 7.24 (m, 2H), 6.93 (m, 1H), 6.88 (m, 2H), 4.99
(s, 2H), 4.86 (m, 1H), 1.95 (m, 2H), 1.65−1.20 (m, 8H). MS (ESI)
m/z 350 [(M + H)+]. HRMS (ESI) calculated for C21H24N3O2+ [(M +
H)+] 350.1863; found 350.1862.
3-Phenoxymethyl-4-phenyl-5-phenylsulfanyl-4H-[1,2,4]triazole
1
(10). Yield 0.08 g, 72%. H NMR (400 MHz), δ (ppm, DMSO-d6):
7.51−7.44 (m, 3H), 7.37−7.28 (m, 5H), 7.25−7.17 (m, 4H), 6.93 (m,
1H), 6.84 (m, 2H), 5.10 (s, 2H). MS (ESI) m/z 360 [(M + H)+].
HRMS (ESI) calculated for C21H18N3OS+ [(M + H)+] 360.1165;
found 360.1168.
3-Chloromethyl-5-isopropylsulfanyl-4-phenyl-4H-[1,2,4]triazole
1
(15). Yield 0.08 g, 75%. H NMR (400 MHz), δ (ppm, DMSO-d6):
7.59−7.55 (m, 3H), 7.45−7.38 (m, 2H), 4.72 (s, 2H), 3.75 (m, 1H),
1.32 (d, J = 6.70 Hz, 6H). MS (ESI) m/z 268 [(M + H)+].
3-(3-Cyclopentylsulfanyl-5-phenoxymethyl-[1,2,4]triazol-4-yl)-
phenylamine (46). To a solution of 39 (0.25 g, 0.63 mmol) in a
mixture of MeOH (10 mL) and water (3.5 mL), ammonium chloride
(0.17 g, 3.16 mmol), and iron powder (0.11 g, 1.89 mmol) were
(5-Isopropylsulfanyl-4-phenyl-4H-[1,2,4]triazol-3-ylmethyl)-phe-
nyl-amine (58). To a solution of 15 (0.08 g, 0.28 mmol) in anhydrous
DMF (2 mL), aniline (0.05 mL, 0.56 mmol) and potassium carbonate
(0.08 g, 0,56 mmol) were added. The reaction was stirred at 80 °C for
I
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1 h, then was cooled at room temperature, poured in water (20 mL),
and extracted with EtOAc (2 × 20 mL). The organic phase was
washed with brine, dried with anhydrous sodium sulfate, and
evaporated under vacuum. The crude residue was purified by silica
gel column chromatography (DCM/MeOH 97:3) to afford the title
compound (0.07 g, 70% yield) as yellowish oil. 1H NMR (400 MHz),
δ (ppm, DMSO-d6): 7.57−7.536 (m, 3H), 7.43−7.39 (m, 2H), 7.02
(m, 2H), 6.55−6.50 (m, 3H), 5.84 (t, J = 5.74 Hz, 1H), 4.20 (d, J =
5.74 Hz, 2H), 3.62 (m, 1H), 1.28 (d, J = 6.84 Hz, 6H). MS (ESI) m/z
325 [(M + H)+]. HRMS (ESI) calculated for C18H21N4S+ [(M + H)+]
325.1482; found 325.1472.
mixture was poured in water (100 mL) and extracted with EtOAc (2 ×
2o mL), then the organic phase was washed with brine, dried with
anhydrous sodium sulfate, and evaporated under vacuum. The title
compound (1.30 g, 87% yield) was obtained as yellowish oil. 1H NMR
(400 MHz), δ (ppm, DMSO-d6): 7.34−7.27 (m, 4H), 7.22−7.15 (m,
3H), 6.87 (m, 1H), 6.64 (m, 2H), 4.63 (m, 1H), 4.58 (s, 2H), 1.95 (s,
3H), 1.20 (d, J = 6.71 Hz, 6H). MS (ESI) m/z 299 [(M + H)+].
By analogous procedure, the following compounds were prepared:
1-Benzyl-1-cyclopentyl-2-methyl-3-phenyl-isothiourea (General
1
Formula E, Scheme 2). Yield 0.90 g, 80%. H NMR (400 MHz), δ
(ppm, DMSO-d6): 7.33 (m, 2H), 7.25 (m, 2H), 7.21−7.15 (m, 3H),
6.87 (m, 1H), 6.68 (m, 2H), 4.69 (m, 1H), 4.64 (s, 2H), 1.93 (s, 3H),
1.84 (m, 2H), 1.65−1.48 (m, 6H). MS (ESI) m/z 325 [(M + H)+].
1-Benzyl-1-cyclohexyl-2-methyl-3-phenyl-isothiourea (General
By analogous procedure, starting from 15 and N-methylaniline, the
following compound was prepared:
(5-Isopropylsulfanyl-4-phenyl-4H-[1,2,4]triazol-3-ylmethyl)-
1
1
methyl-phenyl-amine (52). Yield 0.06 g, 45%. H NMR (400 MHz),
Formula E, Scheme 2). Yield 0.75 g, 88%. H NMR (400 MHz), δ
δ (ppm, DMSO-d6): 7.58−7.53 (m, 3H), 7.37−7.34 (m, 2H), 7.07 (m,
2H), 6.61 (m, 1H), 6.56 (m, 2H), 4.51 (s, 2H), 3.62 (m, 1H), 2.57 (s,
3H), 1.28 (d, J = 6.84 Hz, 6H). MS (ESI) m/z 339 [(M + H)+].
HRMS (ESI) calculated for C19H23N4S+ [(M + H)+] 339.1570; found
339.1572.
(ppm, DMSO-d6): 7.32−7.25 (m, 4H), 7.21−7.14 (m, 3H), 6.86 (m,
1H), 6.65 (m, 2H), 4.64 (s, 2H), 4.22 (m, 1H), 1.92 (s, 3H), 1.77−
1.70 (m, 4H), 1.57−1.49 (m, 3H), 1.35−1.22(m, 2H), 1.07 (m, 1H).
MS (ESI) m/z 339 [(M + H)+].
Benzyl-isopropyl-(5-phenoxymethyl-4-phenyl-4H-[1,2,4]triazol-3-
yl)-amine (General Formula F, Scheme 2). To a solution of 1-benzyl-
1-isopropyl-2-methyl-3-phenyl-isothiourea (1.29 g, 4.33 mmol) and
phenoxy-acetic acid hydrazide (0.72 g, 4.33 mmol) in THF (20 mL),
trifluoroacetic acid (0.17 mL, 2.17 mmol) was added. The mixture was
heated at reflux for 48 h, then cooled to room temperature and poured
in satd aq NaHCO3. The reaction was extracted with EtOAc (2 × 20
mL), the organic phase was washed with brine and dried with
anhydrous sodium sulfate. After evaporation under vacuum, the crude
residue was purified by silica gel column chromatography (DCM/
MeOH 97:3) to afford the title compound (0.97 g, 56% yield) as white
solid. 1H NMR (400 MHz), δ (ppm, DMSO-d6): 7.54−7.47 (m, 3H),
7.33−7.25 (m, 5H), 7.23−7.19 (m, 4H), 6.91 (m, 1H), 6.80 (m, 2H),
4.87 (s, 2H), 4.23 (s, 2H), 3.21 (m, 1H), 0.97 (d, J = 6.71 Hz, 6H).
MS (ESI) m/z 399 [(M + H)+].
3-Cyclohexyloxymethyl-5-isopropylsulfanyl-4-phenyl-4H-[1,2,4]-
triazole (60). To a solution of cyclohexanol (0.09 mL, 0.89 mmol) in
anhydrous THF (2 mL), cooled at 0−5 °C, 60% sodium hydride (0.05
g, 1.34 mmol) was added. The mixture was stirred at 0−5 °C for 15
min, then 15 (0.12 g, 0.45 mmol) was added in portions. The reaction
was stirred at reflux for 1 h, then poured in water (20 mL) and
extracted with EtOAc (2 × 10 mL). The organic phase was dried with
anhydrous sodium sulfate and evaporated under vacuum. The crude
residue was purified by silica gel column chromatography (DCM/
MeOH 97:3) to afford the title compound (0.10 g, 68% yield) as
1
colorless oil. H NMR (400 MHz), δ (ppm, DMSO-d6): 7.60−7.54
(m, 3H), 7.44−7.39 (m, 2H), 4.43 (s, 2H), 3.66 (m, 1H), 3.09 (m,
1H), 1.68−1.06 (m, 16H). MS (ESI) m/z 332 [(M + H)+]. HRMS
(ESI) calculated for C18H26N3OS+ [(M + H)+] 332.1791; found
332.1797.
By analogous procedure, starting from the appropriate substituted
2-methyl-3-phenyl-isothiourea, the following compounds were pre-
pared:
By analogous procedure, starting from 15 and 3-hydroxypyridine,
the following compound was prepared:
3-(5-Isopropylsulfanyl-4-phenyl-4H-[1,2,4]triazol-3-ylmethoxy)-
pyridine (66). Yield 0.03 g, 31%. 1H NMR (400 MHz), δ (ppm,
DMSO-d6): 8.16−8.14 (m, 2H), 7.58−7.52 (m, 3H), 7.47−7.45 (m,
2H), 7.34 (m, 1H), 7.28 (m, 1H), 5.02 (s, 2H), 3.85 (m, 1H), 1.45 (d,
J = 6.71 Hz, 6H). MS (ESI) m/z 327 [(M + H)+]. HRMS (ESI)
calculated for C17H19N4OS+ [(M + H)+] 327.4211; found 327.4215.
1-Benzyl-1-isopropyl-3-phenyl-thiourea (General Formula D,
Scheme 2). A solution of phenyl isothiocyanate (0.89 mL, 7.40
mmol) and benzylisopropyl amine (1.24 mL, 7.40 mmol) in EtOH (10
mL) was stirred at room temperature for 2 h. The solid precipitated
was filtered and washed with EtOH. After drying under vacuum, the
Benzyl-cyclopentyl-(5-phenoxymethyl-4-phenyl-4H-[1,2,4]triazol-
1
3-yl)-amine (General Formula F, Scheme 2). Yield 0.75 g, 61%. H
NMR (400 MHz), δ (ppm, DMSO-d6): 7.51−7.46 (m, 3H), 7.29 (m,
2H), 7.25−7.19 (m, 5H), 7.09 (m, 2H), 6.91 (m, 1H), 6.83 (m, 2H),
4.90 (s, 2H), 4.13 (s, 2H), 3.52 (m, 1H), 1.60−1.53 (m, 6H), 1.35−
1.30 (m, 2H). MS (ESI) m/z 425 [(M + H)+].
Benzyl-cyclohexyl-(5-phenoxymethyl-4-phenyl-4H-[1,2,4]triazol-
1
3-yl)-amine (General Formula F, Scheme 2). Yield 0.62 g, 51%. H
NMR (400 MHz), δ (ppm, DMSO-d6): 7.55−7.46 (m, 3H), 7.34 (m,
2H), 7.27−7.18 (m, 5H), 6.91 (m, 1H), 6.79 (m, 2H), 4.87 (s, 2H),
4.28 (s, 2H), 2.80 (m, 1H), 1.58 (m, 2H), 1.45−1.24 (m, 3H), 1.00−
0.80 (m, 5H). MS (ESI) m/z 439 [(M + H)+].
1
title compound (1.42 g, 68% yield) was obtained as white solid. H
NMR (400 MHz), δ (ppm, DMSO-d6): 8.93 (bs, 1H), 7.34 (m, 2H),
7.26−7.16 (m, 7H), 7.09 (m, 1H), 5.50 (m, 1H), 4.97 (s, 2H), 1.14 (d,
J = 6.71 Hz, 6H). MS (ESI) m/z 285 [(M + H)+].
By analogous procedure, starting from phenyl isothiocyanate and
the appropriate amine, the following compounds were prepared:
1-Benzyl-1-cyclopentyl-3-phenyl-thiourea (General Formula D,
Scheme 2). Yield 1.20 g, 71%. 1H NMR (400 MHz), δ (ppm, DMSO-
d6): 9.98 (bs, 1H), 7.34 (m, 2H), 7.29−7.16 (m, 7H), 7.10 (m, 1H),
5.45 (m, 1H), 4.99 (s, 2H), 1.85 (m, 2H), 1.63−1.44 (m, 6H). MS
(ESI) m/z 311 [(M + H)+].
1-Benzyl-1-cyclohexyl-3-phenyl-thiourea (General Formula D,
Scheme 2). Yield 1.10 g, 77%. 1H NMR (400 MHz), δ (ppm,
DMSO-d6): 8.96 (bs, 1H), 7.33 (m, 2H), 7.28−7.16 (m, 7H), 7.10 (m,
1H), 5.11 (m, 1H), 5.01 (s, 2H), 1.74−1.68 (m, 3H), 1.58 (m, 2H),
1.43−1.26 (m, 3H), 1.07 (m, 2H). MS (ESI) m/z 325 [(M + H)+].
1-Benzyl-1-isopropyl-2-methyl-3-phenyl-isothiourea (General
Formula E, Scheme 2). To a solution of 1-benzyl-1-isopropyl-3-
phenyl-thiourea (1.42 g, 5.00 mmol) in anhydrous THF (20 mL),
cooled at 0−5 °C, potassium tert-butoxide (0.67 g, 6.00 mmol) was
added in portions. After a few minutes, methyl iodide (0.37 mL, 6.00
mmol) was added and the reaction was stirred at 0−5 °C for 1 h. The
Isopropyl-(5-phenoxymethyl-4-phenyl-4H-[1,2,4]triazol-3-yl)-
amine (63). To a solution of benzyl-isopropyl-(5-phenoxymethyl-4-
phenyl-4H-[1,2,4]triazol-3-yl)-amine (0.20 g, 0.50 mmol) in formic
acid (2 mL), 5% palladium on carbon (50% w/w, 0.10 g) was added.
The mixture was heated at 75 °C for 6 h, then cooled at room
temperature. The reaction was diluted with MeOH (10 mL), filtered
on Celite, and evaporated under vacuum. The crude residue was
purified by silica gel column chromatography (DCM/MeOH 96:4) to
afford the title compound (0.07 g, 46% yield) as white solid. 1H NMR
(400 MHz), δ (ppm, DMSO-d6): 7.52−7.43 (m, 3H), 7.38−7.35 (m,
2H), 7.19 (m, 2H), 6.88 (m, 1H), 6.85 (m, 2H), 5.38 (d, J = 7.81 Hz,
1H), 4.83 (s, 2H), 3.77 (m, 1H), 1.11 (d, J = 6.47 Hz, 6H). MS (ESI)
m/z 309 [(M + H)+]. HRMS (ESI) calculated for C18H21N4O+ [(M +
H)+] 309.1710; found 309.1715.
By analogous procedure, the following compounds were prepared:
Cyclopentyl-(5-phenoxymethyl-4-phenyl-4H-[1,2,4]triazol-3-yl)-
amine (64). Yield 0.05 g, 39%. 1H NMR (400 MHz), δ (ppm, DMSO-
d6): 7.54−7.45 (m, 3H), 7.41−7.39 (m, 2H), 7.22 (m, 2H), 6.91 (m,
1H), 6.85 (m, 2H), 5.61 (bs, 1H), 4.86 (s, 2H), 3.95 (m, 1H), 1.89
(m, 2H), 1.60 (m, 2H), 1.50−1.43 (m, 4H). MS (ESI) m/z 335 [(M +
J
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H)+]. HRMS (ESI) calculated for C20H23N4O+ [(M + H)+] 335.1867;
found 335.1862.
methyl ester (0.30 g, 1.29 mmol) and phenoxy-acetic acid hydrazide
(0.21 g, 1.29 mmol) in DMF (5 mL) was heated at 120 °C for 7 h,
then was cooled at room temperature, poured in water (50 mL), and
extracted with EtOAc (2 × 20 mL). The organic phase was washed
with brine, dried with anhydrous sodium sulfate, and evaporated under
vacuum. The crude residue was purified by silica gel column
chromatography (DCM/MeOH 95:5) to afford the title compound
Cyclohexyl-(5-phenoxymethyl-4-phenyl-4H-[1,2,4]triazol-3-yl)-
amine (65). Yield 0.06 g, 41%. 1H NMR (400 MHz), δ (ppm, DMSO-
d6): 7.54−7.45 (m, 3H), 7.40−7.38 (m, 2H), 7.22 (m, 2H), 6.91 (m,
1H), 6.85 (m, 2H), 5.40 (d, J = 7.69 Hz, 1H), 4.86 (s, 2H), 3.44 (m,
1H), 1.92 (m, 2H), 1.86−1.46 (m, 3H), 1.29−0.99 (m, 5H). MS (ESI)
m/z 349 [(M + H)+]. HRMS (ESI) calculated for C21H25N4O+ [(M +
H)+] 349.2023; found 349.2019.
2-Cyclopentyl-N-phenyl-acetamide (General Formula G, Scheme
3). A solution of cyclopentylacetic acid (3.30 mL, 26.31 mmol) in
thionyl chloride (10 mL) was heated at reflux for 2 h, then
concentrated under vacuum and dried. The acyl chloride, as obtained,
was dissolved in anhydrous THF (10 mL) and added dropwise to a
solution of aniline (2 mL, 22 mmol) in pyridine (25 mL) cooled at 0−
5 °C. The mixture was stirred at 0−5 °C for 3 h, then poured in 2 M
HCl (200 mL) and extracted with EtOAc (100 mL). The organic
phase was washed with satd aq Na2CO3 (50 mL), water and brine,
dried with anhydrous sodium sulfate, and concentrated under vacuum.
The title compound (3.75 g, 84% yield) was obtained as white solid.
1H NMR (400 MHz), δ (ppm, DMSO-d6): 9.80 (bs, 1H), 7.59 (m,
2H), 7.27 (m, 2H), 7.01 (m, 1H), 2.33−2.20 (m, 3H), 1.78−1.48 (m,
6H), 1.21−1.16 (m, 2H). MS (ESI) m/z 204 [(M + H)+].
By analogous procedure, starting from cyclohexylacetic acid, the
following compound was prepared:
2-Cyclohexyl-N-phenyl-acetamide (General Formula G, Scheme
3). Yield 2.38 g, 90%. 1H NMR (400 MHz), δ (ppm, DMSO-d6): 9.80
(bs, 1H), 7.59 (m, 2H), 7.29 (m, 2H), 7.01 (m, 1H), 2.18 (d, J = 7.08
Hz, 2H), 1.76 (m, 1H), 1.74−1.65 (m, 4H), 1.22−1.12 (m, 4H),
1.02−0.92 (m, 2H). MS (ESI) m/z 218 [(M + H)+].
1
(0.11 g, 25% yield) as white solid. H NMR (400 MHz), δ (ppm,
DMSO-d6): 7.57−7.52 (m, 3H), 7.48−7.45 (m, 2H), 7.22 (m, 2H),
6.92 (m, 1H), 6.85 (m, 2H), 5.02 (s, 2H), 2.55 (d, J = 7.45 Hz, 2H),
2.06 (m, 1H), 1.63 (m, 2H), 1.52−1.40 (m, 6H). MS (ESI) m/z 334
[(M + H)+]. HRMS (ESI) calculated for C21H24N3O+ [(M + H)+]
334.1914; found 334.1916.
By analogous procedure, starting from 2-cyclohexyl-N-phenyl-
thioacetimidic acid methyl ester, the following compound was
prepared:
3-Cyclohexylmethyl-5-phenoxymethyl-4-phenyl-4H-[1,2,4]-
triazole (62). Yield 0.11 g, 26%. 1H NMR (400 MHz), δ (ppm,
DMSO-d6): 7.57−7.52 (m, 3H), 7.46−7.44 (m, 2H), 7.22 (m, 2H),
6.92 (m, 1H), 6.85 (m, 2H), 5.02 (s, 2H), 2.44 (d, J = 6.71 Hz, 2H),
1.63−1.47 (m, 6H), 1.12−1.05 (m, 3H), 0.87−0.79 (m, 2H). MS
(ESI) m/z 348 [(M + H)+]. HRMS (ESI) calculated for C22H26N3O+
[(M + H)+] 348.2071; found 348.2069.
3-[3-(2-Chloro-phenoxymethyl)-5-cyclopentylsulfanyl-[1,2,4]-
triazol-4-yl]-pyridine (67). To a solution of 14 (0.05 g, 0.17 mmol) in
anhydrous DMF (2 mL), 2-chlorophenol (0.02 g, 0.18 mmol) and
potassium carbonate (0.05 g, 0.34 mmol) were added. The mixture
was heated at 80 °C for 1 h, then was cooled at room temperature and
poured in water (20 mL). The suspension was extracted EtOAc (2 ×
20 mL), the organic phase was washed with brine, dried with
anhydrous sodium sulfate, and evaporated under vacuum. The crude
residue was purified by silica gel column chromatography (DCM/
MeOH 98:2) to afford the title compound (0.05 g, 76% yield) as white
solid. 1H NMR (400 MHz), δ (ppm, DMSO-d6): 8.72−8.70 (m, 2H),
8.00 (m, 1H), 7.60 (m, 1H), 7.36 (m, 1H), 7.28−7.19 (m, 2H), 6.96
(m, 1H), 5.26 (s, 2H), 3.83 (m, 1H), 2.07 (m, 2H), 1.66−1.54 (m,
6H). MS (ESI) m/z 387 [(M + H)+]. HRMS (ESI) calculated for
C19H20ClN4OS+ [(M + H)+] 387.1041; found 387.1042.
By analogous procedure, starting from 14 and the appropriate
substituted phenol, derivatives 68−74, 76−80, 82, 84, 85, 87−89 (see
Supporting Information, p S13), and the following compounds were
prepared:
3-[3-(4-Bromo-phenoxymethyl)-5-cyclopentylsulfanyl-[1,2,4]-
triazol-4-yl]-pyridine (75). Yield 0.07 g, 96%. 1H NMR (400 MHz), δ
(ppm, DMSO-d6): 8.70 (m, 1H), 8.67 (m, 1H), 7.98 (m, 1H), 7.59
(m, 1H), 7.25 (m, 2H), 6.85 (m, 2H), 5.13 (s, 2H), 3.80 (m, 1H), 2.07
(m, 2H), 1.65−1.51 (m, 6H). MS (ESI) m/z 431 [(M + H)+]. HRMS
(ESI) calculated for C19H20BrN4OS+ [(M + H)+] 431.0536; found
431.0534.
2-Cyclopentyl-N-phenyl-thioacetamide (General Formula H,
Scheme 3). To a solution of 2-cyclopentyl-N-phenyl-acetamide (3.70
g, 18.23 mmol) in toluene (80 mL), phosphorus pentasulfide (4.46 g,
20.05 mmol) was added. The mixture was heated at 70 °C for 2 h,
then was cooled at room temperature and filtered on Celite. After
evaporation under vacuum, the crude residue was purified by silica gel
column chromatography (exane/EtOAc 85:15) to afford the title
1
compound (1.54 g, 39% yield) as yellow oil. H NMR (400 MHz), δ
(ppm, DMSO-d6): 11.43 (bs, 1H), 7.77 (m, 2H), 7.41 (m, 2H), 7.23
(m, 1H), 2.75 (d, J = 7.45 Hz, 2H), 2.43 (m, 1H), 1.77−1.52 (m, 6H),
1.26−1.23 (m, 2H). MS (ESI) m/z 220 [(M + H)+].
By analogous procedure, starting from 2-cyclohexyl-N-phenyl-
acetamide, the following compound was prepared:
2-Cyclohexyl-N-phenyl-thioacetamide (General Formula H,
Scheme 3). Yield 0.96 g, 38%. 1H NMR (400 MHz), δ (ppm,
DMSO-d6): 11.41 (bs, 1H), 7.77 (m, 2H), 7.39 (m, 2H), 7.23 (m,
1H), 2.64 (d, J = 7.20 Hz, 2H), 1.96 (m, 1H), 1.75−1.68 (m, 4H),
1.24−1.15 (m, 4H), 1.01−0.90 (m, 2H). MS (ESI) m/z 234 [(M +
H)+].
2-Cyclopentyl-N-phenyl-thioacetimidic Acid Methyl Ester (Gen-
eral Formula I, Scheme 3). To a solution of 2-cyclopentyl-N-phenyl-
thioacetamide (1.54 g, 7.03 mmol) in MeCN (25 mL), methyl iodide
(0.86 mL, 14.06 mmol) and cesium carbonate (4.60 g, 14.06 mmol)
were added. The mixture was heated at 50 °C for 1 h, then was cooled
at room temperature and poured in water (100 mL). The suspension
was extracted with EtOAc (2 × 30 mL), then the organic phase was
washed with brine, dried with anhydrous sodium sulfate, and
evaporated under vacuum. The title compound (1.51g, 92% yield)
was obtained as yellow oil. 1H NMR (400 MHz), δ (ppm, DMSO-d6):
7.30 (m, 2H), 7.03 (m, 1H), 6.70 (m, 2H), 2.40−2.32 (m, 5H), 1.96
(m, 1H), 1.63−1.12 (m, 6H), 1.04−0.89 (m, 2H). MS (ESI) m/z 234
[(M + H)+].
4′-(5-Cyclopentylsulfanyl-4-pyridin-3-yl-4H-[1,2,4]triazol-3-ylme-
thoxy)-biphenyl-4-carbonitrile (81). Yield 0.06 g, 79%. 1H NMR (400
MHz), δ (ppm, DMSO-d6): 8.73−8.71 (m, 2H), 8.03 (m, 1H), 7.88
(m, 2H), 7.82 (m, 2H), 7.68−7.60 (m, 3H), 7.00 (m, 2H), 5.22 (s,
2H), 3.82 (m, 1H), 2.06 (m, 2H), 1.66−1.54 (m, 6H). 13C NMR
(100.7 MHz), δ (ppm, DMSO-d6): 157.7, 152.0, 151.5, 150.9, 147.7,
143.9, 135.2, 132.7, 131.4, 129.9, 128.2, 126.9, 124.3, 118.8, 115.3,
109.3, 60.0, 46.0, 33.1, 24.0. MS (ESI) m/z 454 [(M + H)+]. HRMS
(ESI) calculated for C26H24N5OS+ [(M + H)+] 454.1696; found
454.1688. Melding point 144−145 °C.
3-[3-(4-Bromo-3-methyl-phenoxymethyl)-5-cyclopentylsulfanyl-
1
[1,2,4]triazol-4-yl]-pyridine (83). Yield 0.07 g, 86%. H NMR (400
MHz), δ (ppm, DMSO-d6): 8.73 (m, 1H), 8.69 (m, 1H), 8.00 (m,
1H), 7.61 (m, 1H), 7.41 (d, J = 8.79 Hz, 1H), 6.86 (d, J = 2.81 Hz,
1H), 6.65 (m, 1H), 5.12 (s, 2H), 3.81 (m, 1H), 2.25 (s, 3H), 2.06 (m,
2H), 1.62−1.54 (m, 6H). MS (ESI) m/z 445 [(M + H)+]. HRMS
(ESI) calculated for C20H22BrN4OS+ [(M + H)+] 445.0692; found
445.0683.
By analogous procedure, starting from 2-cyclohexyl-N-phenyl-
thioacetamide, the following compound was prepared:
2-Cyclohexyl-N-phenyl-thioacetimidic Acid Methyl Ester (General
1
Formula I, Scheme 3). Yield 0.85 g, 85%. H NMR (400 MHz), δ
(ppm, DMSO-d6): 7.30 (m, 2H), 7.03 (m, 1H), 6.69 (m, 2H), 2.33−
2.25 (m, 5H), 1.92 (m, 1H), 1.63−1.45 (m, 4H), 1.24−1.00 (m, 4H),
0.75−0.64 (m, 2H). MS (ESI) m/z 248 [(M + H)+].
3-Cyclopentylmethyl-5-phenoxymethyl-4-phenyl-4H-[1,2,4]-
triazole (61). A mixture of 2-cyclopentyl-N-phenyl-thioacetimidic acid
3-[3-(4-Chloro-3-methyl-phenoxymethyl)-5-cyclopentylsulfanyl-
1
[1,2,4]triazol-4-yl]-pyridine (86). Yield 0.06 g, 88%. H NMR (400
MHz), δ (ppm, DMSO-d6): 8.73 (m, 1H), 8.69 (m, 1H), 8.00 (m,
K
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1H), 7.62 (m, 1H), 7.26 (d, J = 8.79 Hz, 1H), 6.85 (d, J = 3.05 Hz,
1H), 6.72 (m, 1H), 5.13 (s, 2H), 3.82 (m, 1H), 2.24 (s, 3H), 2.05 (m,
2H), 1.66−1.50 (m, 6H). 13C NMR (100.7 MHz), δ (ppm, DMSO-
d6): 155.9, 151.9, 151.4, 150.8, 147.7, 136.4, 135.2, 129.9, 129.4, 125.5,
124.3, 117.4, 113.9, 60.1, 46.0, 33.1, 24.0, 19.6. MS (ESI) m/z 401 [(M
+ H)+]. HRMS (ESI) calculated for C20H22ClN4OS+ [(M + H)+]
401.1198; found 401.1202. Melting point 116−117 °C.
pyruvate kinase and 3 mM phosphoenolpyruvate by Sigma-Aldrich)
converted the ADP produced by VCP activity back to ATP, keeping
constant the substrate concentration and preventing product
inhibition. In the second step, after quenching VCP enzymatic
reaction with 30 mM EDTA and 250 μM NADH, the stoicometric
amount of pyruvate produced during the previous step was reduced by
40 U/mL lactic dehydrogenase (Sigma-Aldrich), resulting in the
oxidation of an equivalent amount of NADH. The decrease of NADH
concentration was then measured at 340 nm using Tecan Safire 2
reader plate. Compounds were assayed in 96- or 384-well UV-plates
(Corning) using a reaction buffer containing 50 mM Hepes at pH 7.5,
0.2 mg/mL BSA, 10 mM MgCl2, and 2 mM DTT. After 20 min
preincubation with 150 nM VCP, 60 μM ATP was added to the
reaction mixture that was then allowed to proceed for 90 min before
quenching
3-{3-Cyclopentylsulfanyl-5-[4-(4,4,5,5-tetramethyl-[1,3,2]-
dioxaborolan-2-yl)-phenoxymethyl]-[1,2,4]triazol-4-yl}-pyridine
1
(90). Yield 0.14 g, 58%. H NMR (400 MHz), δ (ppm, DMSO-d6):
8.71 (m, 1H), 8.68 (m, 1H), 7.99 (m, 1H), 7.60 (m, 1H), 7.55 (m,
2H), 6.85 (m, 2H), 5.17 (s, 2H), 3.81 (m, 1H), 2.06 (m, 2H), 1.66−
1.51 (m, 6H), 1.26 (s, 12H). MS (ESI) m/z 478 [(M + H)+]. HRMS
(ESI) calculated for C25H32BN4O3S+ [(M + H)+] 478.2319; found
478.2319.
4′-(5-Cyclopentylsulfanyl-4-pyridin-3-yl-4H-[1,2,4]triazol-3-ylme-
thoxy)-biphenyl-4-carboxylic Acid Methoxyamide (91). To a
solution of 90 (0.07 g, 0.15 mmol) and 4-bromo-N-methoxy-
benzamide (0.04 g, 0.15 mmol) in a mixture of dioxane (3 mL) and
water (1 mL), cesium carbonate (0.14 g, 0.44 mmol) and
Pd(dppf)Cl2·DCM (10% mol, 0.012 g, 0.015 mmol) were added.
The mixture was heated, under argon, by microwave irradiation at 100
°C for 3 h, then cooled at room temperature and poured in water (20
mL). The suspension was extracted with EtOAc (2 × 20 mL), the
organic phase was washed with brine, dried with anhydrous sodium
sulfate, and evaporated under vacuum. The crude residue was purified
by silica gel column chromatography (DCM/MeOH 95:5) to afford
4. Cell Culture. HCT-116 cell line was purchased from the
American Type Culture Collection (ATCC, Manassas, VA). HCT-116
cells were cultured in McCoy’s 5A medium (Gibco BRL, Gaithersburg,
MD, USA) at 37 °C in a humidified atmosphere with 5% CO2 . Media
were supplemented with 10% (v/v) heat-inactivated fetal bovine
serum (Gibco BRL), 100 units/mL penicillin, and 100 μg/mL
streptomycin.
5. Inhibition of Cell Proliferation. Cells were seeded at 1600/
well in 384-well white clear-bottom plates (Greiner). Twenty-four
hours after seeding, cells were treated with the compounds to be tested
and incubated for additional 72 h at 37 °C under a 5% CO2 atm. At
the end of incubation, cells were lysed and the ATP content in the well
was used as a measure of viable cells. This was determined using a
thermostable firefly luciferase based assay (CellTiter-Glo) from
Promega. IC50 values were calculated using the percentage of growth
versus the untreated control.
6. Assessment of the Mechanism of Action of the
compounds. First, 1.2−1.5 million of exponentially growing HCT-
116 cells were seeded in 35 mm plates 24 h before treatment. Then 3
μL of compound diluted in DMSO were added to 3 mL of the
appropriate medium in the well with (0.1% DMSO final
concentration) and the plates incubated for 8 h. Cells were washed
twice with ice cold PBS, lysed in RIPA buffer (50 mM Hepes pH 7.5,
150 mM NaCl, 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS,
10 mM EDTA, protease inhibitors Cocktail (Sigma P8340),
phosphatase inhibitors Cocktails I and II (Sigma P2850 and P5725),
and the lysates clarified by centrifugation for 10 min at 16000g.
Then 20 μg of each sample were fractionated in 4−12% SDS-PAGE
and blotted onto a nitrocellulose transfer membrane (Whatman-
Protan nitrocellulose transfer membrane 10401196). Immunoblot was
performed with the described antibodies in TBS 1× (Biorad) with 5%
milk and 0.1% Tween 80. HRP-conjugated secondary antibodies were
used 1:10000 (Immunopure Goat Anti-Mouse and Immunopure Goat
Antirabbit from Thermo-Scientific). The detection was performed
using SuperSignal west Pico Chemiluminescent substrate from
Thermo Scientific.
7. In Vivo Pharmacokinetics. The pharmacokinetic profile of the
compounds was investigated in overnight fasted male nude mice
following a single dose given intravenously (iv) or orally (po). The
compound was formulated in 10% Tween 80 in 5% dextrose (iv) or in
0.5% Methocel (os) as vehicle. A total of six mice were treated (three
for each leg). Blood samples of each mouse were collected from the
saphenous vein at predose, 0.083, 0.5, 1, 6, and 24 h postdosing
following iv dosing, and at predose, 0.25, 0.5, 1, 6, and 24 h following
oral dosing. Samples were centrifuged at 10000 rpm for 3 min at 4 °C,
and the plasma was stored at −80 °C until analysis. Samples were
analyzed byLC/MS/MS technique.
1
the title compound (0.03 g, 40% yield) as white solid. H NMR (400
MHz), δ (ppm, DMSO-d6): 11.73 (bs, 1H), 8.74−8.71 (m, 2H), 8.02
(m, 1H), 7.80 (m, 2H), 7.71 (m, 2H), 7.64−7.60 (m, 3H), 6.97 (m,
2H), 5.21 (s, 2H), 3.82 (m, 1H), 3.71 (s, 3H), 2.06 (m, 2H), 1.65−
1.52 (m, 6H). MS (ESI) m/z 502 [(M + H)+]. HRMS (ESI)
calculated for C27H28N5O3S+ [(M + H)+] 502.1908; found 502.1911.
By analogous procedure, starting from 90 or 75 or 83 and the
appropriate aryl bromide or aryl boronic acid, derivatives 92−115,
117, 118 (see Supporting Information, p S17), and the following
compound were prepared:
3-[3-Cyclopentylsulfanyl-5-(4′-methanesulfonyl-2-methyl-bi-
phenyl-4-yloxymethyl)-[1,2,4]triazol-4-yl]-pyridine (116). Yield 0.06
1
g, 40%. H NMR (400 MHz), δ (ppm, DMSO-d6): 8.75−8.71 (m,
2H), 8.03 (m, 1H), 7.95 (m, 2H), 7.63 (m, 1H), 7.57 (m, 2H), 7.14
(d, J = 8.30 Hz, 1H), 6.83−6.79 (m, 2H), 5.18 (s, 2H), 3.83 (m, 1H),
3.25 (s, 3H), 2.18 (s, 3H), 2.07 (m, 2H), 1.67−1.53 (m, 6H). 13C
NMR (100.7 MHz), δ (ppm, DMSO-d6): 156.9, 152.0, 151.6, 150.9,
147.8, 145.9, 139.0, 136.3, 135.2, 133.1, 130.6, 129.9, 129.9,126.8,
124.3, 116.7, 112.4, 59.9, 46.0, 43.5, 33.1, 24.0, 20.2. MS (ESI) m/z
+
521 [(M + H)+]. HRMS (ESI) calculated for C27H29N4O3S2 [(M +
H)+] 521.1676; found 521.1678. mp 134−135 °C.
2. Protein Expression and Purification. Human full length VCP
(residues 2−806) was expressed in High5 insect cells as His-Gst-
tagged proteins, using Baculovirus expression vector based on
pVL1393 (Invitrogen).
Cells were lysed by sonication in lysis buffer (50 mM Tris-HCl, pH
7.6, 150 mM NaCl, 10% glycerol, 0.2% CHAPS, 20 mM DTT, and
protease inhibitors), and the cleared lysates were loaded on a
Glutathione Sepharose 4B (Amersham Biosciences) or Nickel
Sepharose column. N-Terminal tags were removed by on-column
cleavage by addition of PreScission protease (Amersham Biosciences),
and the resulting cleaved recombinant proteins were eluted in a final
buffer containing 50 mM Tris-HCl, pH 7.0, 150 mM NaCl, 10%
glycerol, 1 mM DTT, 1 mM EDTA.
Size exclusion chromatography (Superdex200 16/60 column) and
NativePAGE Novex gel were used to analyze VCP proteins
oligomerization status.
3. Biochemical Assay. The ATPase activity of recombinant VCP
wt was evaluated by monitoring ADP formation, using a modified
version of NADH coupled assay.28 Because ADP and NADH are two
ATP competitive inhibitors of VCP ATPase activity, the standard
protocol of NADH coupled assay was splitted into a two-step
procedure. In the first step, an ATP regenerating system (40 U/mL
Pharmacokinetic Noncompartmental Data Analysis. The
pharmacokinetic parameters were derived by noncompartmental
methods using the WinNonlin software program. The highest
concentration Cmax and the time to peak tmax were read as the
coordinates of the highest observed concentration. The terminal half-
life (t1/2,z) was calculated by the formula t1/2,z = ln(2)/λz, where λz is
the slope of the terminal linear phase of natural-log concentrations vs
time curve. The choice of the points on the terminal phase was based
L
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on visual inspection of the data. The area under the plasma level vs
time curve, AUC∞, was calculated by the linear trapezoidal rule up to
the last detectable concentration C(tz) and beyond that time by
extrapolation from C(tz) assuming monoexponential decay, using the
following formula: AUC = AUC(0 − tz) + C(tz)/λz. The following
formulas were applied for the estimate of plasma clearance (CL) and
volume of distribution at steady state (Vss). CL = dose/AUC∞; MRT
= AUMC/AUC∞; Vss = CL MRT; AUMC = AUMC(0 − tz) + C(tz)
× tz/λz + C(tz)/λz2, with AUMC(0 − tz) calculated using the linear
trapezoidal rule on C·t vs t plots. The oral bioavailability (expressed as
percent) was estimated by the ratio of dose-normalized AUC∞ values
after oral and iv dose.
8. In Vitro ADME and PK. High-throughput solubility, Caco-2 cell
permeability, and metabolic stability (human liver microsomes) data
were acquired according to previously reported procedures.29
9. Visualization of the Structure. The VCP biological assembly,
i.e., the functional form of the molecule, was shown to be an hexamer.
Such a protein complex can be derived from standard symmetry
operations in, for example, PyMOL (the PyMOL Molecular Graphics
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ASSOCIATED CONTENT
■
S
* Supporting Information
Analytical data for additional compounds; 13C NMR spectra for
representative compounds; biological data. This material is
available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +39-0331-581523. Fax: +39-0331-581347. E-mail:
roberto.dalessio@nervianoms.com.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to the personnel of the Biochemical and
Cellular Screening Department of Nerviano Medical Sciences,
particularly to Antonella Leone and Dario Ballinari. We thank
Daniela Borghi for skillful assistance in NMR spectra recording
and interpretation and to Marco Guanci and Daniele Pezzetta
for ADME characterization. Thanks are also due to Eduard
Felder, Barbara Salom, Michele Caruso, and Enrico Pesenti for
helpful discussion and support, and to Marcella Nesi for
proofreading.
ABBREVIATIONS USED
■
VCP, valosine containing protein; cdc48, cell division cycle 48;
AAA, ATPases associated with various cellular activities; Ub-Pr,
ubiquitin-proteasome; PAL, photo affinity labeling; HCT-116,
human colorectal carcinoma-116; Caco-2, adenocarcinoma of
the colon-2; C/EBP, CCAAT-enhancer-binding proteins;
CHOP, CCAAT/enhancer-binding protein homologous pro-
tein; gadd153, growth arrest and DNA damage induced gene-
153; MG-132, N-(benzyloxycarbonyl)-leucinyl-leucinyl-leuci-
nal; NSF, N-ethylmaleimide sensitive fusion protein;
SPATA5, spermatogenesis-associated protein 5; VPS4B,
vacuolar protein sorting-associated protein 4B; Cdc6, cell
division cycle 6; Hsp90, heat shock protein 90
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