In silico-driven multicomponent synthesis of 4,5- and 1,5-disubstituted imidazoles as indoleamine 2,3-dioxygenase inhibitors

Article abstract of DOI:10.1039/c5md00317b

Indoleamine 2,3-dioxygenase is involved in pathological immune escape and has recently become an attractive target for anti-cancer therapy. 4-Phenylimidazole (4-PI) provides a promising starting point for the development of IDO1 inhibitors. With the aim o

Full text of DOI:10.1039/c5md00317b

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Sorba and T. Pirali, Med. Chem. Commun., 2015, DOI: 10.1039/C5MD00317B.
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DOI: 10.1039/C5MD00317B
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ARTICLE
In silico-driven multicomponent synthesis of 4,5- and 1,5-
disustituted imidazoles as indoleamine 2,3-dioxygenase inhibitors
Received 00th January 20xx,
Accepted 00th January 20xx
†
†
S. Fallarini, A. Massarotti, A. Gesù, S. Giovarruscio, G. Coda Zabetta, R. Bergo, B. Giannelli, A.
*
Brunco, G. Lombardi, G. Sorba and T. Pirali
DOI: 10.1039/x0xx00000x
www.rsc.org/
Indoleamine 2,3-dioxygenase is involved in pathological immune escape and has recently become an attractive target for
anti-cancer therapy. 4-Phenylimidazole (4-PI) provides a promising starting point for the development of IDO1 inhibitors.
With the aim of discovering more potent ligands, a virtual library of imidazoles synthesizable via the van Leusen
multicomponent reaction was created and filtered to afford a set of 4,5- and 1,5-disubstituted imidazoles as virtual lead
candidates. The compounds were selected according to their docking score and to their synthetic feasibility, synthesized
and biologically evaluated. This experimental approach yielded IDO1 inhibitors with enhanced potency compared to 4-PI:
the most active compounds displayed low micromolar potency, both in enzymatic and cellular assay, while showing no
detectable cellular toxicity. A 3D quantitative structure-activity relationship based on the electrostatic and steric ligand-
protein interactions was performed.
discovery has paved the way for computer-guided design and
the synthesis of novel inhibitors. In this regard, the crystal
structure of human IDO1 shows one binding pocket in the
Introduction
The kynurenine pathway is the main route for the oxidative
degradation of the amino acid tryptophan, and indoleamine
distal heme site (pocket A), connected to a second pocket
towards the entrance of the active site (pocket B).
2
,3-dioxygenase (IDO) catalyzes the first and rate-limiting step
1
In 1989 4-phenylimidazole (4-PI, 1, Fig. 1) was identified as a
7
along this pathway. Over the years, this enzyme has evolved
conceptually from a simple tryptophan-catabolizing enzyme
2
into an important player in tumor immune escape. IDO1
weak non-competitive inhibitor of IDO1 and in 2006 X-ray
crystal structure of human IDO1 complexed with this ligand
6
inhibitor was published, showing that the imidazole is
inhibitors have recently been identified as a priority in
translational research by the immune response modifier
3
pathway prioritization working group from the NIH. Indeed,
coordinated to the heme iron projecting its phenyl ring
towards pocket A (Fig. 2). No other specific interactions with
the enzyme were described, which explains the relative low
potency of this inhibitor.
several studies have offered evidences that IDO1 inhibition by
small molecule inhibitors can exert anti-tumor effects,
improving the efficacy of vaccination, chemotherapy or
OH
O
O
4
H
H
radiationtherapy. As a possible drug development target,
H
N
N
N
N
H
N
H
IDO1 has a number of appealing features. An Ido1 gene
5
knockout mouse has been reported to be viable and healthy,
N
N
N
indicating that IDO1 inhibitors will be unlikely to produce
severe mechanism-based side-effects; tryptophan and
kynurenine, the upstream substrate and downstream product
of the IDO1-catalyzed reaction, may serve as biomarkers to
monitor disease progression and response to therapy with
IDO1 modulators; small-molecule inhibitors of IDO1 offer
substantial cost advantages compared to biological or cell-
based therapies that aim at modulating immune response;
1 IC50 = 48
M
2 IC50 = 4.8
M
3 IC50 < 1
M
Br
OH
CN
O
N
N
S
N
N
N
H
H
6
last, crystal structure of IDO1 has been solved and this
N
4
IC50 < 1
M
5 IC50 = 0.077 M
Fig. 1 Representative reported imidazoles as IDO1 inhibitors.
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1
6
functionally rich imidazoles known to date.
This
View Article Online
DOI: 10.1039/C5MD00317B
transformation can be exploited in the synthesis of four
different series of compounds: 1,4,5-trisubstituted, 1,5- 1,4-
and 4,5-disubstituted imidazoles (Scheme 1).
Scheme 1 The van Leusen multicomponent reaction.
The van Leusen reaction represents an efficient and mild
protocol for preparing polysubstituted imidazoles in a single
Fig. 2 Compound 1 bound to heme iron of IDO1. Heme depicted as grey pot and in a completely regioselective manner from TosMIC
sticks, compound 1 as cyan sticks. Red mesh: pocket A; green mesh: pocket
B (PDB code: 2D0T).
reagents and imines generated in situ from an aldehyde and an
amine. The utility of this transformation in medicinal chemistry
is exemplified by the multi-kilogram scale synthesis of a potent
In 2008, Newlink Genetics described the generation of 4-PI
and selective p38 kinase inhibitor that was promoted to Phase
17
III clinical trials by Glaxo-Smith-Kline. Due to the availability
derivatives by introduction of substituents on the phenyl ring
of : interestingly, the introduction of a hydroxyl group in the
’-position led to 10-fold higher potency at IDO1 inhibition
compared with 4-PI, thanks to additional interactions with
1
of many α-substituted TosMIC compounds, aldehydes and
amines, the accessible imidazole chemical space is huge.
However, it is neither practical nor possible to synthesize all
interesting molecules and put them into a large compound
repository. A promising and complementary strategy which
leverages the strength of MCR chemistry and its exploratory
power is the use of computational screening, and in particular
of a structure-based virtual screening approach.
2
8
Ser167 in the interior of the IDO1 active site (2, Fig. 1). Next
year, the same company reported a large number of variously
substituted 4-arylimidazoles together with a small set of 1,5-
disubstituted imidazoles, showing at best IC50 values less than
9
0 µM. Taking advantage of these results, in a 2011
2
application an extensive series of compounds displaying O-
substituted 2’-hydroxyl-4-phenylimidazoles was described
featuring a long extension into the B pocket, with an IDO1
Results and Discussion
10
inhibition in the nanomolar range (
3
, Fig. 1). Finally, in 2012 a
, Fig.
), leading to the identification of NLG919 (undisclosed We have generated a virtual library of imidazoles synthesizable
structure, IC50 = 28 nM), an orally available IDO1 inhibitor that via the van Leusen MCR, developed a protocol for the
Virtual Screening
large number of fused phenyl-imidazoles was reported (
4
1
1
1
12
has recently entered Phase I clinical trials. Recently, the X-ray structure-based virtual screening of these compounds towards
structure of IDO1 bound to a novel imidazothiazole inhibitor IDO1, screened and filtered the virtual library, and identified a
(
PDB id: 4PK6) became available, showing that both pockets A set of virtual lead candidates. The virtual library has been
1
3
and B are occupied. The most active compound described created using an in house enumerating script built in perl. As
herein is (Fig. 1), which displays a nanomolar potency thanks starting materials, we have collected all the aldehydes, amines
5
to the generation of an induced fit and the resulting and TosMIC compounds available from chemical vendors. The
interaction with Phe226 and Arg231. All these reported data structures were downloaded using their web-based interface,
point out the phenylimidazole scaffold as a promising starting considering 300 Daltons the upper limit for the molecular
point for the development of IDO1 inhibitors; indeed, its weight. Using these criteria, we retrieved about 1,000, 1,000
binding mode to the active site is known through X-ray and 50 compounds for aldehydes, amines and TosMIC
crystallography, rational structural modifications of the reagents, respectively. The theoretical chemical space of this
imidazole ring have been shown to be feasible, and reasonable virtual library was 50,000,000. The library was filtered for
SAR studies are observed.
drug-like compounds and a conformer library was generated
18
19
Driven by high atom and step economy, multicomponent with the program OMEGA2. The FRED software was used in
reactions (MCRs) have emerged in the last 15 years as order to dock all the compounds in the IDO1 binding site and
perfectly suited tools for the synthesis of collections of drug- the simulated binding energy was used to rank them all. Last,
14
like compounds to be used in hit to lead discovery. In this an experienced medicinal chemist selected a set of virtual lead
context, our research team has successfully applied MCRs to candidates according to their docking score and to their
1
5
the discovery of biologically active compounds. In this work, synthetic feasibility. The visual inspection of the ranked
we report the synthesis of 4-PI analogues via the van Leusen compounds revealed that all 1,4,5-trisubstituted and 1,4-
MCR, the most straightforward methodology to access to disubstituted imidazoles were discarded, suggesting that their
2
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substitution patterns suffer from steric hindrance compared to
,5- and 4,5-disubstituted imidazoles and are not suitable for
View Article Online
DOI: 10.1039/C5MD00317B
1
interaction with IDO1 binding site. 4,5-Disubstituted
imidazoles display better docking poses and fifteen structures
Scheme 2 Preparation of α-substituted TosMIC derivatives. Reagents and
were selected (Table 1,
6
-
20), while only ten 1,5-disubstituted
3
conditions: (a) TMSiCl, acetonitrile, toluene, 50 °C; (b) POCl , TEA, THF, -10
imidazoles were considered (Table 1, 21
-
30). Figure 3 depicts a °C.
docking pose of compound 15, which shows one of the lowest
score. 3-Bromophenyl ring is accommodated in the narrow
and hydrophobic pocket A (Tyr126, Cys129, Val130, Phe163
and Phe164), while the more steric hindered p-
acetamidophenyl substituent can fit in the extended pocket B
4
,5-Disubstituted imidazoles (
6-20, Table 1) were synthesized
replacing primary amines with aqueous ammonium hydroxide
21
(Scheme 3).
(Phe226 and Arg231). The docked pose of the selected
compounds shows that, while they all occupy both pockets A
and B, the imidazole ring is oriented very differently relative to
the heme group and is not always able to form a strong Scheme
3 Synthesis of 4,5-disubstituted imidazoles. Reagents and
nitrogen-iron bond. A figure depicting all docking results of conditions: (a) Piperazine, NH
4
OH aq. 30%, THF, rt.
the synthesized compounds is available in the Supplementary
Different functionalities could be accommodated in this mild
basic process (acetamido, hydroxyl, halogen, methoxy,
trifluoromethoxy groups). The reaction provided the desired
Information.
imidazoles in moderate (
7
,
10
16) yields, while in some cases the yields were poor
18). The transformation worked with both aryl- and
, 13, 15, 17, 19, 20) to excellent
(6
8
,
,
11
,
12
,
(
9
,
14
,
heteroaryl- (20) substituted TosMIC reagents. While o-
fluorophenylsubstituted TosMIC derivatives underwent clean
cycloaddition to form the corresponding imidazoles (10
methoxy- and o-chlorophenylsubstituted TosMIC reagents
gave lower yields (13 14 18), which can be attributed to the
increased congestion surrounding the benzylic carbon.
-12), o-
,
,
Regarding the carbonyl counterpart, both aryl and benzyl (14
)
)
aldehydes were used, while electron-poor aldehydes (
8, 9
reacted sluggishly.
1
,5-Disubstituted imidazoles (21-30, Table 1) were synthesized
using TosMIC itself, aryl aldehydes and primary aliphatic
amines in the presence of dimethylformamide as solvent and
potassium carbonate as mild base (Scheme 4). The reaction
was compatible with different functional groups (halogen,
hydroxyl, secondary amine, acetamido, methoxy moieties),
leading to the desired imidazoles in moderate yields under
Fig. 3 Docked pose of compound 15 (cyan sticks). Pocket A is depicted as red
lines, pocket B as green lines.
Chemistry
4
,5-Disubstituted imidazoles were synthesized starting from α- protecting group-free conditions.
substituted TosMIC derivatives h-n. Interestingly, even these
precursors were prepared by a three-component reaction,
where p-toluensulphinic acid, aldehyde and formamide
reacted in the presence of TMSiCl in toluene and acetonitrile
to yield formamides a-g (see Supplementary Information).
Subsequent dehydration performed with phosphorous
oxychloride and triethylamine in THF gave access to the α-
functionalized TosMIC derivatives h-n, stable crystalline and
easily-handled compounds (Scheme 2) (see Supplementary
Scheme
conditions: (a) K
4
Synthesis of 1,5-disubstituted imidazoles. Reagents and
CO , DMF, rt.
2
3
Biological Evaluation and Structure-Activity Relationships (SAR)
Findings
2
0
Information).
Cytotoxicity. The measurement of cell viability is mandatory
when reporting cellular IDO1 inhibitory activity, because the
observed reduction of tryptophan degradation could simply be
an effect of cytotoxicity. Thus, we decided to first investigate
the cytotoxicity of all the synthesized compounds on A375 cell
line. Cells were treated (48 h) with increasing concentrations
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0.01-100 μM) of each compound and cell viability was Compounds
Journal Name
(
6
-
11
,
15
-
18, 20 and 21 resulted active as IDO1
View Article Online
DOI: 10.1039/C5MD00317B
measured by MTT assay. As shown in Table 1, the compounds inhibitors; in detail, compounds
26
concentration tested (100 μM), while the other compounds 18 showed IC50 values greater than 5 μM. The most potent
resulted non-cytotoxic and biocompatible at all concentrations IDO1 inhibitor was compound with an IC50 of 1.5 μM. No
tested. These results prompted us to subject each compound inhibition was observed for compounds 12 14, 19 and 21 30
to further biological evaluation and to use them up to 30 μM, a up to the highest tested concentration of 30 μM. Surprisingly,
non-cytotoxic concentration for all compounds in this cell line. , that is reported to display a good inhibitory activity in
6, 8-11, 15-16, 20 and 21
2
4
-
, 29 and 30 affected cell viability at the highest showed IC50 values less than 5 μM, while compounds 7, 17 and
8
-
-
1
8
enzymatic assay, did not exhibit inhibitory activity on IDO1 in
Cellular IDO1 inhibitory activity
. The ability of each compound cellular assay at any tested concentration. These data has
to inhibit human IDO1 activity was first determined in an in shown the predictively limitation of our virtual screening
vitro cell-based assay, which evaluates not only the inhibitory protocol considering that several top ranked compounds have
effect of the compounds, but also their capacity to permeate not IDO1 inhibitory capability.
the cell membrane. IDO1 expression was induced in A375 cells Regarding 4,5-disubstituted imidazoles, all substitutions in
by IFN-
was determined by measuring the formation of the L- activity (10
kynurenine (L-KYN) product by high-performance liquid tolerated (
chromatography (HPLC) method. IFN- treatment induced the better IDO1 inhibitors (
γ
(500 U/mL) treatment (48 h) and enzymatic activity ortho position of the phenyl ring reduce the IDO1 inhibitory
14 18 19). Substitutions in meta positions are
15 17) and the presence of halogens yields
15 17). Substituents in para
-
,
,
6
-
9
,
-
γ
8
,
9
,
,
expression of functional IDO1 in A375 cell line, consequently position are compatible with the binding pocket (acetamido,
resulting in L-KYN release in the cell culture supernatant (data methoxy, hydroxyl, trifluoromethoxy groups), leading to a
not shown). The ability of increasing concentrations (0.01–30 wide variability of IC50 values, ranging from 2.9 µM (15) to no
μM) of each compound to inhibit IFN-
production was therefore determined. Results are shown in pattern of the other phenyl ring. 1,5-Disubstituted imidazoles
Table (see Supplementary Information for cellular are inactive, with the only exceptions of 21
concentration-response curves).
γ-mediated L-KYN detectable activity (12, 13, 19) according to the substitution
1
.
Table 1 Cytotoxicity and cellular IDO1 inhibition activity of 1 and the synthesized compounds.
Cell viability (%) at
Cpd Yield (%)
R
1
R
2
R
3
IC50 (µM)
1
00 µM ± S.E.M.
100 ± 5.1
97 ± 5.6
1
6
7
8
9
-
Phenyl
H
H
>30
3.5 ± 0.6
6.3 ± 1.2
1.5 ± 0.1
1.7 ± 0.1
4.7 ± 0.8
4.9 ± 1.1
>30
84
53
35
32
54
98
80
58
19
44
95
64
32
48
40
49
35
44
30
35
47
55
36
26
50
Phenyl
4-Hydroxy-3-methoxyphenyl
3,4-Dihydroxyphenyl
3-Bromophenyl
H
Phenyl
H
100 ± 8.4
96 ± 8.6
Phenyl
H
Phenyl
3-Chlorophenyl
H
97 ± 10.5
100 ± 2.5
100 ± 4.3
98 ± 3.6
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2-Fluorophenyl
4-Acetamidophenyl
H
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
2-Fluorophenyl
3-Hydroxy-4-methoxyphenyl
4-Hydroxyphenyl
H
2-Fluorophenyl
H
2-Chlorophenyl
4-Methoxyphenyl
H
100 ± 8.6
98 ± 10.4
100 ± 9.8
98 ± 9.5
>30
2-Chlorophenyl
Benzyl
H
>30
3-Bromophenyl
4-Acetamidophenyl
H
2.9 ± 0.5
3.8 ± 1.2
5.4 ± 1.1
7.2 ± 1.2
>30
3-Methylphenyl
4-Hydroxy-3-methoxyphenyl
3-Bromo-4-Hydroxyphenyl
3-Hydroxy-4-methoxyphenyl
3-Hydroxy-4-methoxyphenyl
4-Trifluoromethoxyphenyl
5-Bromo-2-hydroxyphenyl
4-Methoxynaphthalen-1-yl
4-Phenoxyphenyl
H
3-Methylphenyl
H
100 ± 7.2
99 ± 7.4
2-Methoxyphenyl
H
2-Methylphenyl
H
100 ± 3.2
98 ± 6.8
Thiophen-3-yl
H
2.1 ± 0.8
3.4 ± 0.9
>30
H
H
H
H
H
H
H
H
H
H
2-(Naphthalen-1-ylamino)ethyl
2-(Naphthalen-1-ylamino)ethyl
2-(Naphthalen-1-ylamino)ethyl
2-(Naphthalen-1-ylamino)ethyl
98 ± 10.3
97 ± 7.2
100 ± 10.2
22 ± 4.2
>30
4-Bromophenoxy)phenyl
>30
2,6-Difluoro-3-hydroxyphenyl 2-(Naphthalen-1-ylamino)ethyl
3,5-Dibromo-2-hydroxyphenyl 2-(Naphthalen-1-ylamino)ethyl
53 ± 9.8
>30
39 ± 8.9
>30
2-Chloro-4-hydroxyphenyl
2-Hydroxy-5-methoxyphenyl
4-Phenoxyphenyl
2-(Naphthalen-1-ylamino)ethyl
Cyclopropyl
99 ± 1.5
>30
96 ± 3.7
>30
2-Acetamidoethyl
Propyl
87 ± 6.8
>30
2-Hydroxy-5-methoxyphenyl
89 ± 2.3
>30
4
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Fig. 4 (a) Different views of IDO1 active site (PDB id: 2D0T). Depicted compound: 15, as cyan sticks. Red lines: pocket A, green lines: pocket B. (b) Different
views of interaction fields derived from the 3D-QSAR analysis. Depicted compound: 15 (cyan sticks). Electrostatic effects: favourable (blue) and
unfavourable (red). Steric effects: favourable (green) and unfavourable (yellow). Iron-nitrogen distance for the heme-bound nitrogen: 2.4 Å.
Enzymatic IDO1 inhibition. To evaluate whether the inhibitory 3D-Quantitative Structure-Activity Relationship (QSAR)
activity of tested compounds could be influenced by their Analysis.
ability to permeate cell membranes,
compounds (IC50 < 3 µM) were also tested in an enzymatic
assay. Compounds 15 and 20 resulted active as IDO1
1 and the most potent
As a strong correlation between docking scores and biological
activity was not found, a different approach was used to
analyze the experimental data. Exploiting the results obtained
in the cellular IDO1 inhibitory assay, we attempted to find a
consistent relationship between biological activity and 3D
structures of the synthesized 4,5-disubstituted imidazoles. As
the 1,5-disubstituted compounds displayed poor inhibition
8, 9,
inhibitors showing an IC50 comparable with that obtained in
the cellular assay (see Supplementary Information for
enzymatic concentration-response curves and computed Hill
slope values). In enzymatic assay 4-PI inhibited IDO1 activity
with a potency similar to that reported by Kumar and
8
colleagues, confirming that
activity together with a narrow range of pIC , they were not
50
1
is active as IDO1 inhibitor. These
included in our study. The molecules were aligned using
Open3DAlign, which generates one structure-and-field based
alignment of the data set using each compound as a template.
During visual inspection, the best alignment was selected to
perform the analysis, according to alignment scores and
results also suggest that the data obtained in the cellular assay
could be due to an intrinsic inability of 4-PI in crossing the cell
membrane.
Table 2 Enzymatic IDO1 inhibition activity of
20
1 and compounds 8, 9,
22
superposition of imidazole substituents. Open3DQSAR and
1
5
,
.
23
Forge software were used, with the aim to provide the
regions in space where interactive fields may influence the
activity. The 3D-QSAR models were derived for a data set of 15
compounds using 75% of the compounds in the training set
and 25% in the test set. The compounds of both training and
test sets were carefully selected in order to cover the entire
range of activity values. The model was evaluated by
measuring its accuracy in predicting the activity using Forge
Cpd
1
Enzymatic IC50 (μM)
48.0 ± 4.5
1.9 ± 0.6
8
9
4.7 ± 1.1
1
2
5
0
2.8 ± 0.5
4.5 ± 1.2
23
software. The results confirmed the reliability of the 3D-
QSAR model, considering the statistical values obtained for the
2
2
left-one-out (LOO) analyses (R = 0.998, Q = 0.555). We
manually placed the 3D-QSAR model and its Molecular
Interaction Fields (MIFs) inside IDO1 active site according to
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13
the pockets shapes and the surrounding aminoacids using 6.78 (d, Ј = 7.9 Hz, 1 H), 3.66 (s, 3 H). C NMR δ (75 MHz; DMSO-
C
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2
4
DOI: 10.1039/C5MD00317B
PyMOL software. In Figure 4 MIFs and compound 15 are d ) 172.7, 170.9, 147.9, 146.5, 135.5, 134.6, 128.7, 127.8, 127.1,
6
+
shown. It is possible to appreciate favorable steric contribution 124.5, 120.9, 116.1, 112.5, 55.9. m/z 267 (M ).
(green) to the predicted activity nearby the acetamido group, 4-(5-Phenyl-1H-imidazol-4-yl)benzene-1,2-diol (7). Light brown
that protrudes into the almost unoccupied pocket B, and solid. Yield: 53%. mp 81-82 °C. Found: C, 71.48; H, 4.83; N, 11.05.
-1
unfavorable steric effects (yellow) surrounding the C H N O requires C, 71.42; H, 4.79; N, 11.10. νmax/cm 3129,
15 12 2 2
1
bromophenyl substituent, that is placed in the narrow and 3060, 2295, 1874, 1514, 1274, 1116, 769, 697. H NMR δ (300
H
hydrophobic pocket A. Interestingly, compound 15 displays MHz; CD OD) 7.75 (s, 1 H), 7.45 (d, Ј = 7.9 Hz, 2 H), 7.29-7.17 (m, 3
3
13
good electrostatic interactions (blue) at the rear of pocket A; H), 6.88 (s, 1 H), 6.77-6.75 (m, 2 H) . C NMR δ (75 MHz; CD OD)
C
3
this field points to Ser167, suggesting that a hydrogen bond 145.1 (2 C), 132.9, 131.2, 131.0, 128.9, 128.2, 127.5, 126.9, 125.5,
+
between the NH of the imidazole ring and this residue of the 123.7, 119.8, 115.2. m/z 253 (M ).
protein can be formed. This interaction is peculiar of our 4,5- 4-(3-Bromophenyl)-5-phenyl-1H-imidazole (8). White solid. Yield:
disubstituted imidazoles. Indeed, 4-substituted imidazoles are 35%. mp 178-179 °C. Found: C, 60.30; H, 3.80; N, 9.45. C H BrN
2
1
5
11
-1
not prone to form this hydrogen-bond, as demonstrated by requires C, 60.22; H, 3.71; N, 9.36. νmax/cm 2816, 2642, 1598,
1
6
the X-ray crystal structure of IDO1 complexed with 1. Further 1481, 958, 769. H NMR δ (300 MHz; DMSO-d ) 7.81 (s, 1 H), 7.68
H 6
13
exploration of this interaction near pocket A might improve (m, 1 H), 7.49-7.20 (m, 7H), 7.22 (m, 1 H). C NMR δ (75 MHz;
C
the activity of 4,5-disubstituted imidazoles, while in pocket B DMSO-d ) 137.0, 136.6, 131.0 (2C), 130.0, 129.9 (2 C), 129.2, 128.4
6
+
electrostatic interactions are found to have a negative effect (2C), 128.1, 126.4, 122.2. m/z 300 (M ).
(red) and should be carefully considered in the next generation 4-(3-Chlorophenyl)-5-phenyl-1H-imidazole (9). White solid. Yield:
of compounds.
32%. mp 175-176 °C. Found: C, 70.78; H, 4.40; N, 11.12. C H ClN
15 11 2
-1
requires C, 70.73; H, 4.35; N, 11.00. νmax/cm 3055, 2985, 1600,
1
Experimental
1509, 1470, 1273, 971, 767, 699, 652. H NMR δ (300 MHz; DMSO-
H
13
d6) 7.80 (s, 1 H), 7.51-7.26 (m, 9 H). C NMR δ (75 MHz; DMSO-d )
C
6
Chemistry
1
1
36.6, 133.7, 132.5, 130.8, 129.2, 129.0, 128.4, 128.1, 128.0, 127.5,
+
27.2, 127.0, 126.1. m/z 255 (M )
Materials and Instrumentation. 1 and commercially available
reagents and solvents were purchased from Sigma-Aldrich and Alfa
Aesar and used without further purification. When needed, the
reactions were performed in flame- or oven-dried glassware under
N-(4-(5-(2-Fluorophenyl)-1H-imidazol-4-yl)phenyl)acetamide (10).
Yellow solid. Yield: 54%. mp 214-215 °C. Found: C, 69.28; H, 4.76; N,
-1
1
3
4.24. C H FN O requires C, 69.14; H, 4.78; N, 14.23. νmax/cm
17 14 3
1
450, 2382, 2152, 1583, 1151, 955, 577, 412. H NMR δ (300 MHz;
H
a positive pressure of dry N . Melting points were determined in
2
CD OD) 7.80 (s, 1 H), 7.48 (d, Ј = 8.5 Hz, 2 H), 7.42-7.33 (m, 2 H),
3
open glass capillary with a Stuart scientific SMP3 apparatus. All the
target compounds were checked by IR (FT-IR Thermo-Nicolet
13
7
.30 (d, Ј = 8.5 Hz, 2 H), 7.23-7.12 (m, 2 H), 2.10 (s, 3 H). C NMR δC
1
13
(75 MHz; DMSO-d ) 168.9, 159.6 (d, Ј = 245.0 Hz), 138.7, 136.4,
6
Avatar), H and C APT (Jeol ECP 300 MHz), and mass spectrometry
Thermo Finningan LCQ-deca XP-plus) equipped with an ESI source
1
2
5
32.3, 131.0 (2 C), 130.9, 129.5, 126.9, 125.2, 119.9, 119.5, 116.6,
(
+
4.4. m/z 296 (M )
and an ion trap detector. Chemical shifts are reported in parts per
million (ppm). Column chromatography was performed on silica gel
Merck Kieselgel 70-230 mesh ASTM. Thin layer chromatography
-(5-(2-Fluorophenyl)-1H-imidazol-4-yl)-2-methoxyphenol
(11).
Yellow solid. Yield: 98%. mp 109-110 °C. Found: C, 67.65; H, 4.71; N,
-1
9
3
.91. C H FN O requires C, 67.60; H, 4.61; N, 9.85. ν /cm
16 13 2 2 max
(
TLC) was carried out on 5 cm × 20 cm plates with a layer thickness
1
528, 3002, 2838, 1590, 1515, 1254, 1216, 1129, 884, 816, 761. H
of 0.25 mm (Merck silica gel 60 F254). When necessary, they were
NMR δ (300 MHz; DMSO-d ) 8.90 (br s, 1 H), 7.75 (s, 1 H), 7.46-7.38
H
6
visualized with KMnO . The purity of the target compounds (> 95%)
13
4
(
m, 2 H), 7.25-7.21 (m, 2 H), 6.84-6.72 (m, 3 H), 3.73 (s, 3 H).
C
was determined via elemental analysis and was within ± 0.4% of the
calculated value.
NMR δ (75 MHz; DMSO-d ) 159.9 (d, Ј = 245.0 Hz), 147.4, 147.2 (d,
C
6
Ј = 38.4 Hz), 146.7, 138.2, 136.0, 132.4, 130.0, 125.0, 117.8, 116.3
+
(
d, Ј = 21.1 Hz), 114.4 (2 C), 112.6 (2 C), 56.0. m/z 285 (M )
-(5-(2-Fluorophenyl)-1H-imidazol-4-yl)phenol (12). Yellow solid.
General procedure for the synthesis of 4,5-disubstituted
imidazoles. To a solution of aldehyde (1.5 equiv) in THF an
ammonium hydroxide (4 equiv) solution (30% in water) is added.
After stirring for one hour at room temperature,
tosylmethylisocyanide (1 equiv) and piperazine (1.5 equiv) are
added. The reaction is stirred overnight at room temperature. The
solvent is evaporated and the crude material is purified by column
chromatography.
4
Yield: 80%. mp 99-100 °C. Found: C, 70.90; H, 4.35; N, 11.12.
-1
C H FN O requires C, 70.86; H, 4.36; N, 11.02. νmax/cm 3637,
1
5
11
2
1
3
057, 2666, 2289, 1612, 1516, 1451, 1263, 1171, 836, 760. H NMR
δ (300 MHz; CD OD) 7.75 (s, 1H), 7.36-7.29 (m, 2H), 7.19 (d, Ј = 8.5
H
3
13
Hz, 2H), 7.14-7.09 (m, 2H), 6.72 (d, Ј = 8.5 Hz, 2H). C NMR δ (75
C
MHz; CD OD) 160.0 (d, Ј = 245.6 Hz), 156.7, 133.0, 131.5, 129.4 (d, Ј
3
=
8.0 Hz), 128.1, 125.1, 124.0 (d, Ј = 3.4 Hz), 123.5, 121.3 (d, Ј = 14.9
2
-Methoxy-4-(5-phenyl-1H-imidazol-4-yl)phenol (6). Brown solid.
+
Hz), 115.7 (d, Ј = 21.7 Hz), 115.0 (2C). m/z 255 (M )
Yield: 84%. mp 113-114 °C. Found: C, 72.34; H, 5.35; N, 10.71.
-1
5-(2-Chlorophenyl)-4-(4-methoxyphenyl)-1H-imidazole (13). White
solid. Yield: 58%. mp 239-240 °C. Found: C, 67.59; H, 4.65; N, 9.88.
C H N O requires C, 72.16; H, 5.30; N, 10.52. ν /cm 3064,
16
14
2
2
max
1
2
814, 2345, 1731, 1519, 1260, 1215, 1125, 769, 698. H NMR δ
-1
H
C H ClN O requires C, 67.49; H, 4.60; N, 9.84. νmax/cm 2835,
1
6
13
2
(
300 MHz; DMSO-d ) 7.75 (s, 1 H), 7.53 (d, Ј = 6.8 Hz, 2 H), 7.32 (t, Ј
1
6
1
7
616, 1470, 1246, 1178, 834, 762. H NMR δ (300 MHz; CD OD)
H 3
=
6.8 Hz, 2 H), 7.21 (m, 1 H), 7.03 (s, 1 H), 6.89 (d, Ј = 7.9 Hz, 1 H),
.75 (s, 1 H), 7.48 (d, Ј = 7.1 Hz, 1 H), 7.39-7.30 (m, 3 H), 7.20 (d, Ј =
6
| J. Name., 2015, 00, 1-3
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Pleas eM de od Cn oh te am d Cj uos mt mm argins
Journal Name
ARTICLE
1
3
8
.5 Hz, 2 H), 6.80 (d, Ј = 8.5 Hz, 2 H), 3.75 (s, 3 H). C NMR δ (75 5-(Thiophen-3-yl)-4-(4-(trifluoromethoxy)phenyl)-1H-imidazole
C
View Article Online
DOI: 10.1039/C5MD00317B
MHz; DMSO-d ) 158.5, 146.1, 135.8, 135.7, 133.9, 133.4, 133.3, (20). Light pink solid. Yield: 40%. mp 180-181 °C. Found: C, 54.28; H,
6
+
1
4
1
30.4, 130.2, 127.8, 127.5, 127.4, 114.3, 55.6. m/z 285 (M )
3.00; N, 9.13. C H F N OS requires C, 54.19; H, 2.92; N, 9.03.
14 9 3 2
-1
1
-Benzyl-5-(2-chlorophenyl)-1H-imidazole (14). Yellow solid. Yield: νmax/cm 2830, 1509, 1460, 1258, 837, 768, 653, 547. H NMR δ
H
9%. mp 166-167 °C (dec). Found: C, 71.69; H, 4.96; N, 10.45. (300 MHz; DMSO-d ) 7.78 (s, 1 H), 7.68-7.55 (m, 3 H), 7.33 (m, 1 H),
6
-1
13
C H ClN requires C, 71.51; H, 4.88; N, 10.42. νmax/cm 2632, 7.12 (d, Ј = 5.0 Hz, 1 H). C NMR δ (75 MHz; DMSO-d ) 147.6,
16
13
2
C
6
1
1
7
483, 1430, 1255, 1061, 966, 758. H NMR δ (300 MHz; DMSO-d ) 144.5, 136.2, 134.5, 129.4, 127.9, 127.7, 127.2, 126.5, 122.9, 121.5,
H 6
1
3
+
.85 (s, 1 H), 7.50 (m, 1 H), 7.34-7.15 (m, 8 H), 3.09 (s, 2 H). C NMR 120.7 (q, Ј = 254.2 Hz, 1 H). m/z 311 (M )
δ (75 MHz; DMSO-d ) 140.3, 139.9, 136.2, 132.9, 132.5, 130.2,
C
6
+
1
29.8, 129.7, 129.1, 128.9, 128.8, 127.5, 124.2, 48.0. m/z 270 (M )
General procedure for the synthesis of 1,5-disubstituted
N-(4-(5-(3-Bromophenyl)-1H-imidazol-4-yl)phenyl)acetamide (15). imidazoles. To a solution of aldehyde (1 equiv) in dry DMF, amine (1
Yellow solid. Yield: 44%. mp 234-235 °C (dec). Found: C, 57.40; H, equiv) is added. The reaction is stirred at room temperature under
3
.96; N, 11.85. C H BrN O requires C, 57.32; H, 3.96; N, 11.80. a nitrogen atmosphere for one hour. K CO (3 equiv) and TosMIC
17 14 3 2 3
-1
1
νmax/cm 2360, 2057, 1650, 1463, 1401, 984, 841, 686. H NMR δ
(1.5 equiv) are added. When the reaction is complete (typically
H
(
300 MHz; CD OD) 7.76 (s, 1 H), 7.63 (s, 1 H), 7.56 (d, Ј = 7.7 Hz, 2 after 24 hours), the reaction is diluted with EtOAc and washed with
3
13
H), 7.40-7.34 (m, 4 H), 7.20 (t, Ј = 7.9 Hz, 1 H), 2.13 (s, 3 H). C NMR water (x 1). The organic layer is dried over sodium sulfate and
δ (75 MHz; DMSO-d ) 169.1, 139.6, 138.4, 136.2, 134.4, 130.9, evaporated. The crude material is purified by column
C
6
1
29.6, 129.3, 129.2, 128.3, 125.9, 122.2,119.6,119.4, 24.6. m/z 358 chromatography.
+
(
M )
-Methoxy-4-(5-(m-tolyl)-1H-imidazol-4-yl)phenol (16)
4-Bromo-2-(1-(2-(naphthalen-1-ylamino)ethyl)-1H-imidazol-5-
2
.
White yl)phenol (21). Light green solid. Yield: 49%. mp 190-191 °C (dec).
solid. Yield: 95%. mp 145-146 °C. Found: C, 72.90; H, 5.79; N, 9.99. Found: C, 61,85; H, 4.49; N, 10.33. C H BrN O requires C, 61.78; H,
2
1
18
3
-1
-1
C H N O requires C, 72.84; H, 5.75; N, 9.99. ν /cm 3508, 2831, 4.44; N, 10.29. ν /cm 2852, 1731, 1581, 1494, 1411, 1278, 1110,
17
16
2
2
max
max
1
1
1
733, 1599, 1463, 1261, 1214, 1123, 1034, 788. H NMR δ (300 771. H NMR δ (300 MHz; CD OD) 7.81 (m, 1 H), 7.71-7.68 (m, 2 H),
H H 3
MHz; CDCl ) 7.62 (s, 1 H), 7.36 (s, 1 H), 7.28 (d, Ј = 7.4 Hz, 1 H), 7.19 7.37-7.34 (m, 3 H), 7.25 (m, 1 H), 7.14-7.18 (m, 2 H), 6.90 (s, 1 H),
3
(
(
t, Ј = 7.4 Hz, 1 H), 7.09-7.06 (m, 2 H), 6.98 ( d, Ј = 8.2 Hz, 1 H), 6,83 6.81 (d, Ј = 8.8 Hz, 1 H), 6.23 (d, Ј = 7.1 Hz, 1 H), 4.26 (q, Ј = 6.3 Hz, 2
13
13
d, Ј = 8.2 Hz, 1 H), 3.73 (s, 3 H), 2.30 (s, 3 H). C NMR δ (75 MHz; H), 3.64 (t, Ј = 6.3 Hz, 1 H), 3.50 (t, Ј = 6.3 Hz, 2 H). C NMR δ (75
C
C
DMSO-d ) 153.7, 148.8, 147.9, 146.4, 142.6, 138.1, 135.5, 134.3, MHz; CDCl ) 155.0, 142.4, 139.9, 139.8, 139.7, 139.6, 134.0, 133.3,
6
3
1
28.7, 128.5, 127.8, 125.1, 120.8, 116.0, 112.4, 55.9, 21.6. m/z 281 133.2, 132.7, 128.6, 128.5, 126.8, 126.2, 125.5, 123.4, 117.7, 111.0,
+
+
(
M )
103.5, 60.3, 43.9. m/z 409 (M )
2
-Bromo-4-(5-(m-tolyl)-1H-imidazol-4-yl)phenol (17). White solid. N-(2-(5-(4-methoxynaphthalen-1-yl)-1H-imidazol-1-
Yield: 64%. mp 213-214 °C. Found: C, 58,43; H, 4.00; N, 8.55. yl)ethyl)naphthalen-1-amine (22). Brown oil. Yield: 35%. Found: C,
-1
C H BrN O requires C, 58.38; H, 3.98; N, 8.51. νmax/cm 3399, 79.41; H, 5.95; N, 10.68. C H N O requires C, 79.36; H, 5.89; N,
1
6
13
2
26 23 3
1
-1
1
3
145, 1573, 1507, 1428, 1286, 1156, 828, 624. H NMR δ (300 10.68. νmax/cm 3341, 2935, 1838, 1671, 1586, 1080, 765. H NMR
H
MHz; DMSO-d ) 7.79 (s, 1 H), 7.58 (s, 1 H), 7.32 (s, 1 H), 7.25-7.21 δ (300 MHz; CDCl ) 8.35 (d, Ј = 8.0 Hz, 1 H), 7.75-7-72 (m, 2 H),
6
H
3
13
(
m, 3 H), 7.08 (m, 1H), 6.91(d, Ј = 8.2 Hz, 1 H), 2.28 (s, 3 H). C NMR 7.56-7.33 (m, 7 H), 7.15-7.07 (m, 2 H), 7.15 (t, Ј = 8.0 Hz, 1 H), 6.75
δ (75 MHz; DMSO-d ) 153.8, 138.1, 135.8, 133.2, 132.3, 130.8, (d, Ј = 8.0 Hz, 1 H), 5.98 (d, Ј = 8.0 Hz, 1 H), 4.07-4.03 (m, 5 H), 3.34
C
6
1
3
1
30.7, 128.9, 128.6, 128.4, 128.3, 126.3, 125.2, 116.8, 109.8, 21.6 . (t, Ј = 5.8 Hz, 2 H), 2.27 (br s, 1 H). C NMR δ (75 MHz; DMSO-d )
C 6
+
m/z 329 (M )
156.1, 143.5, 138.8, 134.5, 134.0, 131.6, 130.6, 130.4, 129.0, 128.4,
2
-Methoxy-5-(5-(2-methoxyphenyl)-1H-imidazol-4-yl)phenol (18). 127.9, 126.9, 126.1, 125.5, 125.4, 124.5, 123.5, 122.5, 122.0, 119.5,
+
Yellow solid. Yield: 32%. mp 208-209 °C. Found: C, 68.99; H, 5.46; N, 116.2, 104.5, 103.0, 60.3, 44.5, 44.3. m/z 394 (M )
-1
9
.55. C H N O requires C, 68.91; H, 5.44; N, 9.45. ν /cm 3009, N-(2-(5-(4-phenoxyphenyl)-1H-imidazol-1-yl)ethyl)naphthalen-1-
17 16 2 3 max
1
2
932, 1513, 1483, 1287, 1244, 1023, 752. H NMR δ (300 MHz; amine (23). Brown oil. Yield: 44%. Found: C, 79.95; H, 5.70; N,
H
-1
CD OD) 7.70 (s, 1 H), 7.30 (t, Ј = 8.2 Hz, 1 H), 7.22 (d, Ј = 7.4 Hz, 1 H), 10.35. C H N O requires C, 79.97; H, 5.72; N, 10.36. νmax/cm
3
27 23 3
1
7
.02 (d, Ј = 7.4 Hz, 1 H), 6.96-6.78 (m, 4 H), 3.80 (s, 3 H), 3.67 (s, 3 3050, 1903, 1582, 1482, 1237, 770. H NMR δ (300 MHz; CDCl )
H 3
13
H). C NMR δC (75 MHz; DMSO-d ) 157.5, 156.5, 146.7, 146.4, 7.76 (d, Ј = 7.9 Hz, 1 H), 7-63-7.58 (m, 2 H), 7.47-7.35 (m, 8 H), 7.16
6
1
1
2
35.1, 131.9, 129.9, 128.8, 120.9, 120.5, 117.7, 114.4, 112.4, 112.2, (t, Ј = 7.1 Hz, 1 H), 7.07-7.04 (m, 3 H), (6.99 (d, Ј = 7.7 Hz, 2 H), 6.37
+
13
11.7. m/z 297 (M )
(m, 1 H), 4.31 (t, Ј = 6.0 Hz, 2 H), 3.55-3.53 (m, 2 H). C NMR δ (75
C
-Methoxy-5-(5-(o-tolyl)-1H-imidazol-4-yl)phenol (19). Yellow MHz; DMSO-d ) 157.2, 156.7, 143.7, 139.4, 134.6, 132.4, 130.9,
6
solid. Yield: 48%. mp 204-205 °C. Found: C, 72.90; H, 5.79; N, 9.99. 130.7, 128.5, 127.9, 127.2, 126.2, 125.3, 124.6, 124.4, 123.6, 122.0,
-
1
+
C H N O requires C, 72.84; H, 5.75; N, 9.99. ν /cm 3550, 3359, 119.7, 119.0, 116.5, 103.5, 44.3, 43.7. m/z 406 (M )
17
16
2
2
max
1
2
931, 2495, 2238, 1928, 1440, 1268, 1126, 772. H NMR δ (300 N-(2-(5-(4-(4-bromophenoxy)phenyl)-1H-imidazol-1-
H
MHz; DMSO-d ) 8.81 (s, 1 H), 7.69 (s, 1 H), 7.42-7.13 (m, 4 H), 6.88 yl)ethyl)naphthalen-1-amine (24). Green oil. Yield: 30%. Found: C,
6
(
s, 1 H), 6.75 (d, Ј = 8.2 Hz, 1 H), 6.65 (d, Ј = 8.2 Hz, 1 H), 3.70 (s, 3 67.03; H, 4.64; N, 8.73. C H BrN O requires C, 66.95; H, 4.58; N,
27 22 3
13
-1
H), 2.04 (s, 3 H). C NMR δ (75 MHz; DMSO-d ) 153.9, 147.5, 8.67. ν /cm 3049, 2859, 2359, 1894, 1732, 1581, 1289, 770, 420.
C
6
max
1
1
1
46.8, 146.7, 137.5 (2 C), 131.2, 130.7, 130.4, 128.5, 126.3, 124.9,
H NMR δ (300 MHz; DMSO-d ) 8.02 (d, Ј = 6.9 Hz, 1 H), 7.73 (d, Ј =
H
6
+
17.2, 114.0, 112.6, 56.2, 20.2. m/z 281 (M )
7.1 Hz, 1 H), 7.57 (d, Ј = 8.5 Hz, 2 H), 7.54-7.40 (m, 4 H), 7.20-7.09
(
m, 3 H), 7.08-7.00 (m, 4 H), 6.31-6.29 (m, 2 H), 4.31-4.29 (m, 2 H),
This journal is © The Royal Society of Chemistry 20xx
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Pleas eM de od Cn oh te am d Cj uos mt mm argins
Page 8 of 11
ARTICLE
Journal Name
1
3
-1
3
1
1
4
2
.41-3.39 (m, 2 H). C NMR δ (75 MHz; DMSO-d ) 156.0, 155.7, C, 67.22; H, 6.94; N, 12.06. ν /cm 3143, 2964, 1807, 1556, 1497,
C 6 max
View Article Online
1
DOI: 10.1039/C5MD00317B
43.2, 139.0, 134.1, 133.0, 131.5, 130.5, 128.0, 127.4, 126.7, 125.7, 1449, 1209, 801. H NMR δ (300 MHz; DMSO-d ) 7.70 (s, 1 H), 6.87-
H
6
25.3, 124.1, 123.1, 121.5, 121.0, 118.8, 116.0, 115.5, 103.0, 43.8, 6.70 (m, 3 H), 6.68 (s, 1 H), 3.84 (t, Ј = 7.1 Hz, 2 H), 3.68 (s, 3 H), 1.49
+
13
3.1. m/z 485 (M )
(sex, Ј = 7.4 Hz, 2 H), 0.68 (t, Ј = 7.4 Hz, 3 H). C NMR δ (75 MHz;
C
,4-Difluoro-3-(1-(2-(naphthalen-1-ylamino)ethyl)-1H-imidazol-5-
DMSO-d ) 152.1, 149.1, 137.9, 129.9, 127.6, 117.8, 116.7, 116.5,
6
+
yl)phenol (25). Brown solid. Yield: 35%. mp 190-191 °C. Found: C, 115.3, 55.5, 46.7, 23.6, 11.0. m/z 233 (M )
6
1
1
1
6
4
9.18; H, 4.73; N, 11.57. C H F N O requires C, 69.03; H, 4.69; N,
21 17 2 3
-1
1.50. νmax/cm 3371, 2866, 2359, 1732, 1582, 1470, 1252, 1109, Biology
1
092, 879, 404. H NMR δ (300 MHz; DMSO-d ) 8.03 (d, Ј = 7.9 Hz,
H
6
H), 8.00 (s, 1 H), 7.73 (d, Ј = 7.7 Hz, 1 H), 7-45-7.36 (m, 2 H), 7.17- Cell culture. Human A375 cells were cultured in DMEM medium
.98 (m, 5 H), 6.30 (br t, Ј = 5.2 Hz, 1 H), 6.17 (d, Ј = 6.9 Hz, 1 H), with high glucose (4.5 g/L), containing 10% heat inactivated fetal
13
.12 (t, Ј = 6.6 Hz, 2 H), 3.40 (m, 2 H). C NMR δ (75 MHz; DMSO- bovine serum (FBS), 2 mM L-glutamine 100 U/mL of penicilline and
C
d6) 152.8 (d, Ј = 232.4 Hz), 149.2 (d, Ј = 229.3), 143.6, 142.4 (d, Ј = 10 µ/mL of streptomycine (GE Healthcare, Milan, Italy). Cells were
1
1
1
2
2.0 Hz), 140.0, 134.5, 130.2, 128.6, 127.1, 126.3, 124.7, 123.5, cultured in a humidified atmosphere (5% CO , 37 °C).
2
21.9, 119.7, 118.8 (m), 116.7, 111.6 (d, Ј = 27.0 Hz), 107.4 (dd, Ј =
+
2.0, 11.5 Hz), 103.0, 44.2, 43.7. m/z 366 (M )
Cell cytotoxicity. Cell viability was measured by the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)
,4-Dibromo-6-(1-(2-(naphthalen-1-ylamino)ethyl)-1H-imidazol-5-
25
5
yl)phenol (26). Green solid. Yield: 47%. mp 130-131 °C. Found: C, assay, as previously described. A375 cells were seeded (0.5 x 10
5
8
1.79; H, 3.58; N,8.62. C H Br N O requires C, 51.77; H, 3.52; N, cells/well) in 24-well plates and treated with increasing
21 17 2 3
-1
1
.63. νmax/cm 3440, 3066, 2921, 1739, 1584, 1225, 1109, 768. H concentrations (0.01–100 μM) of each compound for 48 h at 37 °C
NMR δ (300 MHz; DMSO-d ) 7.87-7.72 (m, 3 H), 7.97 (m, 1H), 7.42- in a 5% CO humidified incubator. The percentage of cell viability
H
6
2
7
.32 (m, 3 H), 7.18-7.07 (m, 2 H), 6.94 (s, 1 H), 6.15 (d, Ј = 7.4 Hz, 1 was calculated as [100 (x−y)/ (z−y)], where x, y, and z were the
13
H), 4.48 (br s, 1 H), 4.11 (t, Ј = 6.3 Hz, 2 H), 3.36 (m, 2 H). C NMR δC absorbance read in compound-treated, resting and compound-
75 MHz; DMSO-d ) 152. 5, 143.7, 135.5, 134.6, 134.2, 133.6, 129.5, untreated cells, respectively.
(
6
1
1
3
28.8, 127.2, 126.2, 124.7, 123.5, 122.0, 117.8, 116.5, 115.3, 113.5,
+
11.4, 103.1, 42.3, 42.1. m/z 488 (M )
Cellular IDO1 enzymatic assay. The enzymatic activity of IDO1 was
-Chloro-4-(1-(2-(naphthalen-1-ylamino)ethyl)-1H-imidazol-5-
evaluated by measuring the levels of L-KYN into A375 cell media, as
2
6
4
yl)phenol (27). Green solid. Yield: 55%. mp 103-104 °C. Found: C, previously described. A375 cells (5X10 ) were seeded in a 24-well
6
1
9.37; H, 5.02; N, 11.60. C H ClN O requires C, 69.32; H, 4.99; N, culture plate (500 μL/well) and grown overnight. Serial DMSO
21 18 3
-1
1
1.55. νmax/cm 2755, 2561, 1728, 1581, 1439, 1286, 1109, 770. H dilutions (0.01–30 μM) of each compounds in a total volume of 500
NMR δ (300 MHz; DMSO-d ) 8.03 (d, Ј = 7.1 Hz, 1 H ), 7.86 (s, 1 H), μL culture medium including a 100 μM L-tryptophan (Sigma-Aldrich)
H
6
7
7
8
.72 (d, Ј = 6.9 Hz, 1 H), 7.44-7.35 (m, 2 H), 7.24 (d, Ј = 8.2 Hz, 1 H), and human IFN-γ (500 U/mL final concentration) per well were
.13-7.07 (m, 2 H), 7.00 (d, Ј = 2.2 Hz, 1H), 6.88 (s, 1 H), 6.82 (dd, Ј = added into wells containing the cells. All compounds were dissolved
.2, 2.2 Hz, 1 H), 6.31 (t, Ј = 5.5 Hz, 1 H), 6.01 (dd, Ј = 6.3, 2.5 Hz, 1 in DMSO (Sigma–Aldrich). The DMSO final concentration in cell
13
H), 4.04 (m, 2 H), 3.34 (m, 2 H). C NMR δ (75 MHz; DMSO-d ) culture medium was always 0.1%. Equivalent amount of DMSO was
C
6
1
1
4
2
64.0, 148.0, 143.0, 139.5, 139.0, 138.9, 133.9, 133.0, 132.9, 131.4, always added to drug untreated controls. After 48h incubation, cell
30.5, 129.0, 127.9, 126.3, 123.7, 121.0, 120.7, 119.5, 107.3, 48.5, medium was collected, deproteinized by 20% (v/v) aqueous
+
5.2. m/z 364 (M )
CCl COOH, centrifuged at 13,200 rpm for 10 minutes, and the
3
-(1-Cyclopropyl-1H-imidazol-5-yl)-4-methoxyphenol (28). Orange amounts of L-KYN in A375 cell media were quantified with HPLC. 20
solid. Yield: 36%. mp 204-205 °C. Found: C, 67.85; H, 6.21; N, 12.17. µL of supernatants were injected by a multi-sampler (Beckman
-1
C H N O requires C, 67.81; H, 6.13; N, 12.17. νmax/cm 2828, Coulter, Milan, Italy) into a HPLC-UV-VIS system (System Gold,
1
3 14 2 2
1
1
814, 1600, 1513, 1279, 1214, 1113, 937. H NMR δ (300 MHz; Beckman Coulter), equipped with a C-18 sphereClone ODS
H
CDCl ) 7.63 (s, 1 H), 7.01 (s, 1 H), 6.96 (d, Ј = 8.8 Hz, 1 H), 6.87 (d, Ј = analytical column (5 µm particle size, 250 mm x 4.0 mm;
3
8
.8 Hz, 1 H), 6.77 (s, 1 H), 3.77 (s, 3 H), 3.31 (m, 1 H), 0.86-0.81 (m, 4 Phenomenex, Torrance, CA, USA). The mobile phase (50 mM
13
H). C NMR δC (75 MHz; DMSO-d ) 152.3, 149.7, 138.1, 131.6, potassium dihydrogen phosphate, 10% v/v acetonitrile; pH 4.8) was
6
+
1
28.3, 118.3, 116.8, 116.6, 115.5, 55.9, 27.4, 6.7. m/z 231 (M )
N-(2-(5-(4-phenoxyphenyl)-1H-imidazol-1-yl)ethyl)acetamide (29). absorbance was measured at 330 nm. Amounts of L-KYN in A375
Yellow oil. Yield: 26%. Found: C, 71.00; H, 5.98; N, 13.10. C H N O cell media were quantified on the basis of a calibration curve
requires C, 71.01; H, 5.96; N, 13.08. νmax/cm 3064, 2031, 1658, obtained using the same HPLC-UV-VIS experimental setting.
delivered at a flow-rate of 1 mL/min at room temperature, and the
19
19
3
2
-1
1
1
488, 1231, 756. H NMR δ (300 MHz; CD OD) 7.74 (s, 1 H), 7.40- IC50 values were calculated from concentration-response curves
H 3
7
.32 (m, 4 H), 7.13 (t, Ј = 7.4 Hz, 1 H), 7.09-6.97 (m, 4 H), 6.96 (s, 1 obtained in at least three different experiments run in triplicate.
13
H), 4.15 (t, Ј = 6.0 Hz, 2 H), 3.30 (t, Ј = 6.0 Hz, 2 H), 1.79 (s, 3 H).
C
NMR δC (75 MHz; DMSO-d ) 170.2, 157.0, 156.8, 139.3, 131.7, rhIDO1 enzymatic assay. rhIDO1 activity was determined as follow.
6
1
3
4
30.8, 130.7, 128.0, 125.4, 124.4, 119.6, 119.1, 55.4, 42.6, 22.9. m/z In brief, the standard reaction mixture (200 μL) contained 50 mM
+
22 (M )
potassium phosphate buffer (KPB) (pH 6.5), 20 mM ascorbic acid
-Methoxy-2-(1-propyl)-1H-imidazol-5-yl)phenol (30). Yellow solid. (neutralized with NaOH and HCl) (Sigma Aldrich), 100 μg/mL
Yield: 50%. Found: C, 67.29; H, 6.99; N, 12.05. C H N O requires catalase (Sigma Aldrich), 10 μM methylene blue (Alfa Aesar,
13 16 2 2
8
| J. Name., 2015, 00, 1-3
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Page 9 of 11
Pleas eM de od Cn oh te am d Cj uos mt mm argins
Journal Name
ARTICLE
Heysham, Lancashire, United Kingdom), 100 µM L-tryptophan constraint: if an atom of the molecule was within aVinewaAcrtcicelepOtanblinlee
DOI: 10.1039/C5MD00317B
(
Sigma Aldrich), 50 nM rhIDO1 (Origene, Bologna, Italy), and distance to it, the docking pose was retained; if not, the compound
dimethyl sulfoxide (DMSO) solution of the compound (4 μL). The was discarded.
reaction was carried out at 37 °C for 60 min and stopped by the
addition of 40 μL of 30% (w/v) CCl COOH. After heating at 50 °C for Virtual screening procedure. Conformations were generated for all
3
1
5 min, the reaction mixture was centrifuged at 1500 g for 10 min. structures using OMEGA2 (maximum number of conformations:
18
The supernatant (150 μL) was transferred into a well of a 96-well 350, rmsd threshold: 1.0 Å), and charges were assigned according
31
19
microplate and mixed with 150 μL of 2% (w/v) p- to MMFF94 scheme. Docking was performed with FRED. Docked
dimethylaminobenzaldehyde (Ehrlich’s reagent) in acetic acid. The conformations were scored using Chemgauss4 (see Supporting
3
2
yellow pigment derived from kynurenine was measured at 490 nm Information for the docked pose of the synthesized compounds).
using an Ultramark Microplate Imaging System (Bio-Rad). IC50
values were calculated from concentration-response curves 3D-QSAR conformer generation. 4-PI (1) and 4,5-disubstituted
obtained in at least three different experiments run in triplicate.
imidazoles 6-20 were taken into account. The sixteen compounds
used to generate the 3D-QSAR model were processed using
Statistical analysis. Results are expressed as means ± SEM of at OpenEye software suite according to the following scheme:
33
least three different experiments run in triplicate. The software FixPKA to assign correct protonation at pH 7, tautomers in order
GraphPad 5.0 for Windows (GraphPad Software, La Jolla, California, to generate the most aromatic tautomer for each compound and
18
USA) was used as a nonlinear regression model for analysis of the finally OMEGA2 to generate one minimized 3D conformation for
concentration–response data to obtain the 50% inhibitory each compound in sdf file format. As we wanted to focus on 4,5-
concentration value (IC ).
diarylimidazoles, the inactive compound 14, that displays a benzyl
substituent, was removed from the list of molecules used to build
the 3D-QSAR model.
5
0
Molecular modeling
22
All molecular modeling studies were performed on a Tesla 3D-QSAR model generation. According to the Open3DTools
workstation equipped with two Intel Xeon X5650 2.67 GHz workflow the sdf containing all data set was processed with
22
processors and Ubuntu 10.04 (www.ubuntu.com). Different Open3DAlign, which generates one structure-and-field based
27
software was used to elaborate the chemical information: PerlMol
alignment of the data set using each compound in the file as a
and OpenEye (www.eyesopen.com) applications. The protein template and leading to fifteen different alignments. Following
2
4
structures and 3D chemical structures were generated in PyMol.
visual inspection, the alignment 0012 based on compound 13 was
selected to perform the analysis, taking into account alignment
Compounds source. The collection of reactants was retrieved from scores and superposition of imidazole substituents. In particular, in
28
ZINC. This searchable database includes chemical structures this alignment the smaller substituents were all identically oriented,
available from chemical vendors. The structures were downloaded as well as the more hindered ones (See Supplementary Information
29
using the web-based interface and filtered using FILTER software
for a pocket-binding hypothesis of compounds 1 and 6-20). IC50
from OpenEye to select compounds containing aldehyde, amine or values were converted to pIC50 and added to the sdf file. In this step
TosMIC moieties. Additionally, we made 300 Daltons the upper limit we set to an arbitrary value (150 µM) the IC50 for compounds 1 and
for the molecular weights for the structures, facilitating 13, while the IC50 values of 12 and 19 were extrapolated as these
compatibility with synthetic drugs. Using these criteria, 932 compounds inhibited IDO activity without reaching the maximal
aldehydes, 983 amines and 54 TosMIC were retrieved. The inhibition at the highest concentration tested (30 µM). The data set
reactants were saved as canonical SMILES.
was split into a training set and a test set (containing respectively
5% and 25% of the total compounds). In order to generate a
consistent statistical model we selected the compounds of the
7
27
Generation of database. An in house perl script based on PerlMol
was used to retrieve each reactant and perform a virtual training and test sets so that they span across the entire range of
combinatorial reaction with all downloaded compounds. The pIC50 values (see Supplementary Information). The analysis was
30
resulting structures were filtered using Lipinski’s rule of 5, the performed with Open3DQSAR software and then the model was
23
most stable tautomers were calculated and the retained SMILES for evaluated again using command line version of Forge skipping in-
,4,5-trisubstituted (49,472,424 compounds), 1,5-disubstituted software alignment and conformational hunt, and using LOO type
1
2
2
(
916,156 compounds), 1,4-disubstituted (53,082 compounds) and cross validation (R = 0.998, Q = 0.555) (see Supplementary
4
,5-disubstituted (50,328 compounds) imidazoles were saved. Each Information).
18
structure was rebuilt as a 3D structure using OMEGA2.
Conclusions
Preparation of protein. The X-ray structure of the 4-
phenylimidazole-IDO1 complex was used in this study, entry code 4-PI is a non-competitive inhibitor of IDO1 and represents a
6
D0T. Water molecules were removed, all hydrogen atoms and promising starting point for the development of more potent
2
MMFF94 charges were added. Then, the complex was transferred compounds, as demonstrated by the amount of patents
19
into fred_receptor and prepared for docking with FRED. The displaying imidazoles as IDO1 inhibitors and by the recent
interaction with iron moiety of heme group was used as a introduction of the fused imidazole NLG919 into Phase I
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2015, 00, 1-3 | 9
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Pleas eM de od Cn oh te am d Cj uos mt mm argins
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ARTICLE
Journal Name
1
1
2 NCT02048709, https://clinical trials.gov, (acVcieewsAsreticdle OJnulinlye
015); S. Crunkhorn, Nat. Rev. Drug Dis. 2014, 13, 879.
3 S. Tojo, T. Kohno, T. Tanaka, S. Kamioka, Y. Ota, T. Ishii, K.
clinical trials. The van Leusen multicomponent reaction has the
potential to generate large and diverse libraries of imidazoles:
thus, in order to reduce the number of theoretical possibilities
to a practical level for synthesis and testing, a virtual screening
2
DOI: 10.1039/C5MD00317B
Kamimoto, S. Asano and Y. Isobe, ACS Med. Chem. Lett.
2
014, 5, 1119-1123.
approach was used and afforded a series of 4,5- and 1,5- 14 Dömling A, W. Wang and K. Wang, Chem. Rev. 2012, 112
,
3
5 M. Guasconi, X. Lu, A. Massarotti, A. Caldarelli, E. Ciraolo, G.
083-3135.
disubstituted imidazoles as putative IDO1 inhibitors. These
compounds were synthesized and tested, leading to the
identification of four 4,5-disubstituted imidazoles that display
a 10-fold improvement in potency compared to 4-PI in
enzymatic assay, are able to permeate the cell membrane, in
1
C. Tron, E. Hirsch, G. Sorba and T. Pirali, Org. Biomol. Chem.
011, 9, 4144-4149; T. Pirali, V. Faccio, R. Mossetti, A. A.
2
Grolla, S. Di Micco, G. Bifulco, A. A. Genazzani and G. C. Tron,
Mol. Div. 2010, 14, 109-121; A. A. Grolla, V. Podestà, M. G.
Chini, S. Di Micco, A. Vallario, A. A. Genazzani, P. L. Canonico,
G. Bifulco, G. C. Tron, G. Sorba and T. Pirali, J. Med. Chem.
contrast to 1, and show no detectable toxicity in cells. A 3D-
QSAR analysis applicable to 4,5-diaryl imidazoles was
performed. A putative hydrogen bond between the NH of the
imidazole ring and Ser167 of the protein might be responsible
for the improvement of potency, together with favorable
interactions with the partially occupied pocket B.
2
009, 52, 2776-2785.
1
6 A. M. van Leusen, J. Wildeman and O. H. Oldenziel, J. Org.
Chem. 1977, 42, 1153-1159; J. Sisko, A. J. Kassick, M.
Mellinger, J. J. Fiulan, A. Allen and M. A. Olsen, J. Org. Chem.
2
000, 65, 1516-1524; D. van Leusen and A. M. van Leusen, In
Organic Reactions, Overman, L. E., Ed.; John Wiley & Sons:
New York, 2001, vol. 57. pp. 417.
The information obtained in this study, together with the
performed 3D-QSAR analysis, demonstrate that the 4,5-
disubstituted imidazole scaffold might provide a new direction
for future design of IDO1 inhibitors. Further efforts to discover
promising candidates are in progress and aim to optimize
molecular recognition by pocket B: this would help to increase
potency together with selectivity for IDO1 over other targets
and especially heme proteins.
1
1
7 J. Sisko and M. Mellinger, Pure Appl. Chem. 2002, 74, 1349.
8 OMEGA, version 2.4.6; OpenEye Scientific Software: Santa
Fe, NM. http://www.eyesopen.com; P. C. D Hawkins, A.
Skillman, G. L. Warren, B. A. Ellingson and M. T. Stahl, J.
Chem. Inf. Model. 2010, 50, 572−584; P. C. D. Hawkins and A.
Nicholls, J. Chem. Inf. Model. 2012, 52, 2919-2936.
9 FRED, version 3.0.0; OpenEye Scientific Software: Santa Fe,
NM. http://www.eyesopen.com; McGann, M. J. Chem. Inf.
Model. 2011, 51, 578−596.
1
2
0 J. Sisko, M. Mellinger, P. W. Sheldrake P. W. and N. H. Baine,
Acknowledgements
Tetrahedron Lett. 1996, 37
Mellinger, P. W. Sheldrake and N. H. Baine, Org. Synth. 2000,
, 198-202.
1 J. Sisko, A. J. Kassick, M. Mellinger, J. J. Fiulan, A. Allen and
M. A. Olsen, J. Org. Chem. 2000, 65, 1516-1524.
2 P. Tosco, T. Balle and F. Shiri J. Comput. Aided Mol. Des.
, 8113-8116; J. Sisko, M.
This study was financially supported by Associazione Italiana
per la Ricerca sul Cancro (AIRC) (Grant 15918), Italy. We also
gratefully acknowledge support from Ministero dell’Istruzione,
dell’Università e della Ricerca (MIUR) (PRIN 2010-2011; Grant
7
7
2
2
2
2
2
2
011, 25, 777-783.
3 Cresset
group.com/products/forge/. Forge V.10
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010W7YRLZ_002), Italy.
Group.
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4 The PyMOL Molecular Graphics System, version 1.3;
Schrödinger LLC: 2010.
5 S. Fallarini , T. Paoletti, C. O. Battaglini, P. Ronchi, L. Lay, R.
Bonomi, S. Jha, F. Mancin, P. Scrimin and G. Lombardi,
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MedChemComm
View Article Online
DOI: 10.1039/C5MD00317B
A virtual library of 50,000,000 compounds synthesizable via the van Leusen MCR was created, screened and
filtered to afford a series of disubstituted imidazoles with improved properties compared to the parent
compound 4-phenylimidazole.
1
15
IC (rhIDO) = 2.8 µM
IC (rhIDO) = 48.0 µM
5
0
50
IC (A375) > 30 µM
IC (A375) = 2.9 µM
5
0
50