Discovery of potent, isoform-selective inhibitors of histone deacetylase containing chiral heterocyclic capping groups and a N-(2-aminophenyl)benzamide binding unit

Article abstract of DOI:10.1021/jm400634n

The synthesis of a novel series of potent chiral inhibitors of histone deacetylase (HDAC) is described that contain a heterocyclic capping group and a N-(2-aminophenyl)benzamide unit that binds in the active site. In vitro assays for the inhibition of HDAC1, HDAC2, HDAC3-NCoR1, and HDAC8 by the N-(2-aminophenyl)benzamide 24a gave respective IC₅₀ values of 930, 85, 12, and 4100 nM, exhibiting class I selectivity and potent inhibition of HDAC3-NCoR1. Both imidazolinone and thiazoline rings are shown to be effective replacements for the pyrimidine ring present in many other 2-(aminophenyl) benzamides previously reported, an example of each ring system at 1 μM causing an increase in histone H3K9 acetylation in the human cell lines Jurkat and HeLa and an increase in cell death consistent with induction of apoptosis. Inhibition of the growth of MCF-7, A549, DU145, and HCT116 cell lines by 24a was observed, with respective IC₅₀ values of 5.4, 5.8, 6.4, and 2.2 mM.

Full text of DOI:10.1021/jm400634n

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Article
Discovery of Potent, Isoform-Selective Inhibitors of Histone
Deacetylase containing Chiral Heterocyclic Capping
Groups and a N-(2-Aminophenyl)-benzamide Binding Unit
Charles Marson, Chris Matthews, Elena Yiannaki, Stephen Atkinson,
Peter Soden, Lena Shukla, Nermina Lamadema, and Shaun Thomas
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400634n • Publication Date (Web): 06 Jul 2013
Downloaded from http://pubs.acs.org on July 21, 2013
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Discovery of Potent, Isoform-Selective Inhibitors of Histone Deacetylase containing
Chiral Heterocyclic Capping Groups and a N-(2-Aminophenyl)-benzamide Binding
Unit
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,
a
a
a
b
Charles M. Marson,* Christopher J. Matthews, Elena Yiannaki, Stephen J. Atkinson,
b
c
d
d
Peter E. Soden, Lena Shukla, Nermina Lamadema and N. Shaun B. Thomas
a
Department of Chemistry, University College London,
Christopher Ingold Laboratories, 20 Gordon Street, London WC1H OAJ, U.K.
b
Epinova DPU, Immuno-Inflammation Therapy Area Unit, GlaxoSmithKline, Gunnels Wood
Road, Stevenage, Herts SG1 2NY, U.K.
c
Allergic Inflammation DPU, Respiratory Therapy Area Unit, GlaxoSmithKline, Gunnels
Wood Road, Stevenage, Herts SG1 2NY, U.K.
d
Department of Haematological Medicine, Leukemia Sciences Laboratories, Rayne Institute,
King’s College London, 123 Coldharbour Lane, London SE5 9NU, U.K.
Abstract: The synthesis of a novel series of potent chiral inhibitors of histone deacetylase
HDAC) is described that contain a heterocyclic capping group and a Nꢀ(2ꢀaminophenyl)ꢀ
(
benzamide unit that binds in the active site. In vitro assays for the inhibition of HDAC1,
HDAC2, HDAC3ꢀNCoR1 and HDAC8 by the Nꢀ(2ꢀaminophenyl)ꢀbenzamide 24a gave
respective IC50 values of 930, 85, 12 and 4100 nM, exhibiting Class I selectivity and potent
inhibition of HDAC3ꢀNCoR1. Both imidazolinone and thiazoline rings are shown to be
effective replacements for the pyrimidine ring present in many other 2ꢀ(aminophenyl)ꢀ
benzamides previously reported, an example of each ring system at 1 ꢁM causing an increase
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in histone H3K9 acetylation in the human cell lines Jurkat and HeLa, and an increase in cell
death consistent with induction of apoptosis. Inhibition of the growth of MCFꢀ7, A549,
DU145 and HCT116 cell lines by 24a was observed, with respective IC50 values of 5.4, 5.8,
6.4 and 2.2 mM.
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Introduction
Epigenetic therapy represents a paradigm shift in terms of the selection of the biology
1
,2
to drug targets. Of the postꢀtranslational modifications relevant to disease, histone protein
acetylation status has received much attention in regard to potential for epigenetic cancer
therapy. Deacetylation of histone protein at the eꢀamino group of lysine residues in the Nꢀ
terminal tails of core histones in the nucleosome through the action of histone deacetylases
(HDACs) increases the number of protonated lysine termini; those bind closely to the
negatively charged DNA phosphate groups, leading to chromatin compaction that reduces
3
4ꢀ6
accessibility of transcription factors to DNA, and hence to transcriptional repression. Such
hypoacetylation has been associated with precancerous or malignant states, and relief of
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,8
transcriptional repression present in various leukemias has been achieved using HDAC
1
,2
inhibitors. In addition, the inappropriate recruitment of HDAC enzymes by oncogenic
proteins may alter gene expression in favor of arrested differentiation and/or unregulated
9
proliferation. HDAC inhibitors reduce cancer cell proliferation by induction of cell cycle
1
0ꢀ13
arrest, differentiation and/or apoptosis.
HDAC inhibitors have been shown to affect
and JAKꢀ
several pathways involving proliferation and differentiation, including the TGFꢀ
β
1
4
STAT pathways, and myc and Bclꢀ6 expression in lymphoid cells.
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O
N
H
N
N
OH
N
N
H
NH
2
H
H
N
O
N
Vorinostat
O
(
SAHA)
Mocetinostat
(MGCD0103)
O
O
O
N
H
O
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H
NH
O
N
H
NH
2
O
S
H
N
S
N
H
NH
HN
O
O
O
Entinostat
MS-275)
(
Romidepsin
Figure 1. Examples of clinical HDAC inhibitors.
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,2
HDAC inhibitors are a promising class of antiꢀcancer agents of which those
currently approved for clinical use (Fig. 1) comprise three structural classes: hydroxamic
acids, including Vorinostat (suberoylanilide hydroxamic acid), Merck & Co., for the
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5
treatment of cutaneous Tꢀcell lymphoma), the aminoanilides Mocetinosat (MethylGene
Inc., for the treatment of myelodysplastic syndromes and leukemia, especially acute
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6
myelogenous leukemia) and MSꢀ275 (Syndax Pharmaceuticals/Schering AG, for the
1
7
treatment of metastatic melanoma), and the cyclic disulfide Romidepsin (cyclodepsipeptide
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8
or FKꢀ228, Gloucester Pharmaceuticals, for the treatment of cutaneous Tꢀcell lymphoma).
With the wide range of known hydroxamic acids available, mainly with limitations of
efficacy and toxicity, and the limited number of potential disulfide inhibitors, we sought
alternative structures to Mocetinosat that were also aminoanilides, and could be expected to
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9
show similar slow, tightꢀbinding to HDACs, the slow decomplexation also leading to
1
9ꢀ21
prolonged inhibitory effects and longer intervals between dosing.
In addition,
Mocetinostat did not show acquired resistance, at least to HCT116 colon tumor cells, unlike
2
2
hydroxamic acid HDAC inhibitors. We describe here the synthesis and preliminary
evaluation of new classes of aminoanilides containing chirality as part of a heterocyclic ring.
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Chemistry
A series of analogs having in common the linker and cap region of Mocetinostat was
prepared by coupling carboxylic acid 7 with a set of arylamines (Schemes 1 and 2). In order
to ensure regiocontrolled acylation, routes via the corresponding oꢀnitroanilines were
developed (Scheme 1).
0
1
2
3
4
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7
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9
0
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0
Scheme 1.a
NO
2
NO
2
NO
2
NO
2
H
2
N
H
2
N
H
2
N
2
H N
a or b
c
d
OH
OR
Ph
OH
OTBS
O
O
1
2a R=Me
R=CH
2
3
4
2
b
NO
2
NO
2
H
2
N
2
H N
e
Br
5
6
a
Reagents and conditions: (a) SOCl
(c) 2a, DIBAL, THF-CH Cl , 0 1C, 1 h, then 20 1C, 3 h; (d) TBSCl, imidazole, DMF, 20 1C, 16 h;
e) PhB(OH) , (Ph P) Pd, Na CO , aq. 1,2-dimethoxyethane, 80 1C, 48 h.
2 2 2 3
, MeOH, reflux,16 h ; (b) PhCH Br, Na CO , DMF, 20 1C, 18 h.
2
2
(
2
3
4
2
3
2
3
Methyl 4ꢀ(guanidinomethyl)benzoate
and (E)ꢀ3ꢀ(dimethylamino)ꢀ1ꢀ(pyridinꢀ3ꢀ
2
4
yl)propꢀ2ꢀenꢀ1ꢀone were heated in propanꢀ2ꢀol for 20 h at reflux to give methyl 4ꢀ((4ꢀ
2
4
(pyridinꢀ3ꢀyl)pyrimidinꢀ2ꢀylamino)methyl)benzoate as previously described
(67%);
hydrolysis of this ester to the carboxylic acid 7 was achieved using aqueous lithium hydroxide
2
4
(24 h, 20 °C, 97%).
Condensation of acid 7 with a range of arylamines (Scheme 2) afforded the
corresponding aminoanilides. The nitroaniline derivatives (e.g. Scheme 1) could not be
directly acylated; however, deprotonation of the aromatic amino group using NaH permitted
coupling
yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) or, for the preparation of
h and 8i, with the acid chloride of 7. The aminoanilides were further modified by standard
with
carboxylic
acids
activated
by
(benzotriazolꢀ1ꢀ
8
functional group manipulations, to afford 8aꢀ8i.
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N
Scheme 2.a
N
N
NH
2
H
H
N
N
N
N
8b
O
N
N
NH
2
N
N
NH2
H
H
H
H
N
N
N
N
8a
O
8c
O
Br
b,c
d,e
a
10
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23
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N
N
N
n
f
N
N
H
NH
2
N
N
NH
2
N
N
H
H
H
H
N
N
OH
N
N
N
7
Mocetinostat
O
O
8d R=Me
8e R=H
O
g
OR
k and
l or m
h and
i or j
N
N
N
N
NH
2
N
N
NH2
H
H
H
H
N
N
N
N
O
O
But
2
O
8
8
h
R=CH
i R=H
2
Ph
8f R=SiMe
8g R=H
OR
OR
a
Reagents and conditions: (a) (i) SOCl
pyridine, then NaH, 20 1C, 7 h; (c) H , 10% Pd/C, MeOH, CH
aq. NH , EtOH, 60 1C, 4 h; (f) 3-methoxyphenylene-1,2-diamine, BOP, Et
then NaH, 20 1C, 2.5 h; (i) H , 10% Pd/C, MeOH, CHCl , 20 1C, 7 h; (j) SnCl
k) NaH and 2b, anhyd. pyridine, -10 1C, 5 min; then add acid chloride (7, SOCl
0 1C, 5.5 h; (m) H , 10% Pd/C, MeOH, CH Cl , 20 1C, 45 h; (n) phenylene-1,2-diamine, BOP, DMF, 20 1C, 24 h.
2
, reflux, 3 h; (ii) naphthalene-2,3-diamine, Et
Cl , 20 1C, 6 h; (d) 5, NaH, BOP, pyridine, Et
N, DMF, 20 1C, 27 h; (g) BBr , CH
.2H O, NH OAc, 1:1:1 THF:MeOH:H
, toluene, reflux, 3 h), 20 1C, 2 h; (l) H
3
N, CH
2
Cl
2
, DMF, 0 1C, 30 min then 20 1C, 1 h; (b) BOP, 6,
N,20 1C, 2.5 h; (e) Fe SO .7H O,
Cl , 20 1C, 24 h; (h) BOP, 4, pyridine,
O, 20 1C, 21 h then 50 1C, 1 h;
, 10% Pd/C, MeOH, CH Cl
2
2
2
3
2
4
2
3
3
3
2
2
2
3
2
2
4
2
(
2
2
2
2
,
2
2
2
2
Substituted imidazolinꢀ4ꢀones (Schemes 3 and 4) were prepared from the corresponding
25
26
thiohydantoins 10 and 15. During Sꢀmethylation to give the corresponding methylthio
derivatives 11 and 16, racemisation occurred, presumably because of enolate formation in the
presence of K
2 3
CO . Those 2ꢀ(methylthio)imidazolinꢀ4ꢀones were reacted with 4ꢀ
(
methylamino)benzoic acid in ethanol at reflux to give the corresponding carboxylic acids 12
and 17 which could then be converted into the aminoanilides 13 and 18 by BOPꢀmediated
amide formation, and subsequent deprotection of 18 to give 19.
Scheme 3.a
O
O
CO
2
H
R
H
H
NH
N
c
a
b
R
R
NH
2
N
N
SMe
Ac
S
Ac
9a R=Ph
10a R=Ph
10b R=3-indolyl
11a R=Ph
11b R=3-indolyl
9b
R=3-indolyl
O
O
R
R
N
N
d
N
N
N
Ac
H
Ac
N
H
NH
2
H
OH
N
12a R=Ph
2b R=3-indolyl
13a R=Ph
O
O
1
13b R=3-indolyl
a
Reagents and conditions: (a) NH
(b) MeI, K CO
reflux, 16 h; (d) BOP, Et
4
2
SCN, Ac O, 100 1C (1 h, 9a; 10 min, 9b);
2
3
, MeCN, 20 1C, 2.5 h; (c) 4-(methylamino)benzoic acid, ethanol,
3
N, phenylene-1,2-diamine, DMF, 20 1C, 3.5 h.
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2
2
2
2
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Scheme 4.a
O
O
CO
2
H
a
NH
b
N
c
R
R
R
NHBoc
N
N
SMe
Boc
S
Boc
14a R=CH
2
Ph
15a R=CH Ph
15b R=3-indolylmethyl
15c R=Ph
2
16a R=CH Ph
16b R=3-indolylmethyl
16c R=Ph
2
1
1
4b R=3-indolylmethyl
4c R=Ph
0
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0
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0
1
2
3
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7
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9
0
1
2
3
4
5
6
7
8
9
0
O
O
N
N
R
R
d
N
N
N
N
NH
2
Boc
X
H
H
H
N
OH
O
O
X=Boc
X=H
e
17a R=CH
2
Ph
18a R=CH Ph
2
19a R=CH Ph
2
1
1
7b R=3-indolylmethyl
7c R=Ph
18b R=3-indolylmethyl 19b R=3-indolylmethyl
18c R=Ph
19c R=Ph
a
Reagents and conditions: (a) ethoxycarbonyl isothiocyanate, pyridine, MeCN, 20 1C,
8 h; (b) MeI, K CO , MeCN, 20 1C, 2.5 h; (c) 4-(methylamino)benzoic acid, ethanol,
reflux, 4 h (18 h for 17a); (d) BOP, Et N, phenylene-1,2-diamine, DMF, 20 1C, 3 h;
e)TFA, CH Cl , 20 1C, 3 h.
1
2
3
3
(
2
2
In view of the lack of stereointegrity in the imidazolinꢀ4ꢀone series, an unsaturated fiveꢀ
membered ring system that lacked a carbonyl group was sought; a set of 2ꢀaminothiazoline
derivatives 24 was prepared (Scheme 5). Several approaches were attempted, the first by
analogy with Scheme 4. Addition of 4ꢀ(methylamino)benzoic acid to 2ꢀ(methylthio)ꢀ4ꢀ
substituted thiazolines failed, although addition of the corresponding methyl ester was
successful. However, this could not be progressed because phenyleneꢀ1,2ꢀdiamine could not
be satisfactorily reacted with the carboxylic acids derived from latter adducts. In the second
approach, reaction of Nꢀ(2ꢀaminophenyl)ꢀ4’ꢀ(aminomethyl)benzamide, or its Boc derivative
2
1b, with 2ꢀ(methylthio)ꢀ4ꢀsubstituted thiazolines was also unsuccessful.
Since displacement of the 2ꢀthiomethyl group by substituted benzylamines could not be
achieved, advantage was taken of the Sꢀnucleophilic nature of thioureas (Scheme 5). By
2
7
analogy with previous work the isothiocyanate 22 was reacted with enantiopure 1,2ꢀ
aminoalcohols to give the corresponding thioureas 23 which underwent acidꢀcatalyzed ringꢀ
closure to the desired 2ꢀaminothiazolines 24 (Scheme 5). However, ringꢀclosure to the
thiazoline was unsuccessful for 24 where R=(3ꢀindolyl)methyl and for 24c. Consequently,
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Mitsunobu conditions were applied to 23c and afforded the desired thiazoline which was
then deprotected to 24c.
Scheme 5.a
F
3
COCHN
RHN
NHBoc
a
H
c
OH
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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2
0
O
O
2
1a R=COCF
3
b
21b R=H
R4
HO
R2
R3
S
S
C
N
NHBoc
R1
N
N
H
NHBoc
H
N
d
H
H
N
O
23
O
2
2
R3
R4
R2
S
R1
N
N
NH
2
e or
f
H
H
N
24
O
R1
R2
R3
R4
2
3/24 a
Ph
H
H
H
H
H
H
H
H
b
c
Ph
H
6 4
p-HOC H
d
e
f
PhCH
2
H
H
H
H
PhCH
2
H
H
H
H
H
Ph
H
H
g
h
i
H
Ph
H
H
Ph
H
Ph
H
Ph
Ph
a
Reagents and conditions: (a) Et
3 2 2 2
N, (COCl) , DMF, CH Cl ; then N-Boc-
phenylene-1,2-diamine, 20 1C, 20 h; (b) K CO , aq. MeOH, 70 1C, 4 h;
2
3
(
c) CS
amino alcohol, MeCN, 20 1C, 16 h; (e) conc. HCl, 90 1C (for all 23 except
3c); (f) diethyl azodicarboxylate, Ph P, THF, 20 1C, 75 min, then HCl in
Et O, 20 1C, 5 h (for 23c).
2 3 2 2
, Et N,CH Cl , 20 1C, 10 min, then 0 1C and TsCl, 10 min; (d) 1,2-
2
3
2
The sulfurꢀlinked thiazolines 30 (Scheme 6) were prepared by Sꢀalkylation of the
2
6
corresponding thiazolineꢀ2ꢀthiones. The method was established by reaction of the
chloromethyl compound 28a with 26a, affording, after Boc deprotection (conditions d,
Scheme 6) the thiazoline 30a. The unprotected alkylating agent 28b was then tested, and
proved effective, affording the thiazolines 30b and 30c. To our knowledge, the use of preꢀ
assembled aminoanilide linkers of types 22 and 28 that contain electrophilic centers
appropriate for subsequent reaction is a novel approach to aminoanilide HDAC inhibitors;
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this route has greater convergence and also avoids a lateꢀstage amination with the feebly
nucleophilic 1,2ꢀphenylenediamine derivative.
Scheme 6.a
0
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5
6
7
8
9
0
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H
R
OH
H
S
a
NH
2
R
N
S
H
25a R=CH
2
Ph
26a R=CH Ph
2
25b R=CH
2
(3-indolyl)
26b R=CH (3-indolyl)
2
25c R=Ph
26c R=Ph
Cl
Cl
NHR
b
c
H
N
Cl
27
O
O
2
2
8a R=Boc
8b R=H
d
e
H
S
H
d
S
R
N
R
N
S
NHBoc
S
NH
2
H
H
N
N
O
O
3
2
0a R=CH Ph
29
30b R=CH (3-indolyl)
2
30c R=Ph
a
Reagents and conditions: (a) CS , aq. KOH, reflux, 20 h; (b)
N
-Boc-phenylene-
, -10 1C to 20 1C, 2 h; (c) 28a, NaI, acetone, reflux,
h; then 26a, 20 1C, 28 h;(d) TFA, CH Cl , 20 1C, 2.5 h; (e) 26b/26c, K CO
acetone, reflux, 17 h;
2
3 2 2
1,2-diamine, Et N, CH Cl
2
2
2
2
3
,
Results and Discussion
To our knowledge, few SAR data or details of Mocetinosat analogues have been
reported, and no chiral heterocyclic analogs have been reported; indeed some planar analogs
showed less potency to the extent they were evaluated, namely against HDAC1 and a few
2
9,30
cell lines).
A 5ꢀ(2ꢀthienyl) substituent on the oꢀphenylenediamine ring enhanced potency
3
1
in some cases; regarding the zincꢀbinding group, replacement of the 2ꢀamino group by
3
2
hydroxy retained activity in some aminoanilides, but that specific analogue of Mocetinostat
has not been described. Since effects of 4ꢀsubstitution on the terminal aryl ring of
3
1
Mocetinostat were largely unknown, a range of 4ꢀsubstituted derivatives of Mocetinostat
were prepared and evaluated (Table 1). Of that series, 4ꢀmethoxy and 4ꢀhydroxy substituents
(entries 4 and 5) were most potent against HDAC2 and HDAC3ꢀNCoR1. For both isoforms,
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the 4ꢀmethoxy substituent conferred some sixꢀfold greater potency than the 4ꢀhydroxy
compound. An aminonaphthyl terminus (entry 1) conferred good selectivity for HDAC3ꢀ
NCoR1 over all other isoforms tested, including HDAC1 (>20 mM). In contrast, a 4ꢀphenyl
derivative (entry 2) showed no inhibition, suggesting that there is a strict limit on the length
of the 4ꢀsubstituent to retain HDAC3 inhibition. The greater inhibition of HDAC2 and
HDAC3ꢀNCoR1 for entries 4 and 5 in this series may reflect a stronger binding to zinc
arising from increased electron density provided by the mesomeric effect of the methoxy and
hydroxy groups, respectively. Since investigation of the series of compounds 8a-8i did not
disclose a substituted Nꢀ(2ꢀaminophenyl)ꢀbenzamide unit of improved potency, the binding
moiety present in Mocetinostat was used in further studies.
10
11
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19
20
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60
Table 1
There have been few reports of alternative ring systems to replace the pyrimidine ring
in Mocetinostat; only planar, fused rings have been substituted, leading to high molecular
2
9
weight compounds with nonꢀoptimal aqueous solubility, albeit with medicinal potential. Our
view was that fiveꢀmembered semiꢀsaturated ring systems could prove effective, also offering
the possibility of introducing a chiral centre, as well as variable substituents that would be
alternatives to the terminal 3ꢀpyridyl ring present in Mocetinostat. The imidazolinꢀ4ꢀone ring
indeed proved effective (Table 2, entries 1ꢀ6); these compounds showing appreciable isoform
selectivity for HDAC3ꢀNCoR1. Of particular note is entry 6; this suggests that the imidazolinꢀ
4
ꢀone ring is a good mimic of pyrimidine, and that the 5ꢀphenyl substituent occupies a closely
similar location in HDAC to that of the 3ꢀpyridyl group in Mocetinostat. Entry 6 showed at
least as great potency as did Mocetinosat against HDAC3ꢀNCoR1.
Table 2
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5
5
5
5
5
5
6
A thiazoline ring was next examined, in order to establish whether a single nitrogen
atom in the ring was compatible with HDAC inhibitory activity; that is the case, as shown by
entries 7ꢀ9 (Table 2). Once again, a phenyl substituent was the most potent in inhibiting
HDAC3ꢀNCoR1, with the 2ꢀarylmethylthio linkage in entry 9 being notable as the first Nꢀ(2ꢀ
aminophenyl)ꢀbenzamide HDAC inhibitor lacking an aminomethyl linker that exhibits an
appreciably low nanomolar IC50 value. Its aminomethyl analog (Table 3, entry 1) was an
even more potent inhibitor, and likely more potent than Mocetinostat against HDAC3ꢀ
NCoR1. For inhibition of HDAC1 and HDAC2, a (4R)ꢀconfiguration (entries 2, 5 and 7)
shows somewhat greater inhibition than the corresponding (4S)ꢀenantiomer (entries 1, 4 and
6). However for 24a/24b, the (4S)ꢀenantiomer (entry 1) is the more potent inhibitor of
HDAC3ꢀNCoR1. Although phenyl, 4ꢀhydroxyphenyl and benzyl monosubstitution was
consistent with good potency, bulkier 4,5ꢀdiphenyl substitution (entries 8 and 9) was found
to be deleterious to all HDAC inhibition investigated.
0
1
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2
3
4
5
6
7
8
9
0
Table 3
As expected, most thiazolines prepared (Tables 2 and 3) inhibited HDAC8 only weakly,
binding to the cap region being ineffective owing to the short tunnel that leads to the
3
3
catalytic site in HDAC8. As for compounds studied previously, inhibition of HDAC6 by
any of the novel benzamides was not detected. Molecular modeling of thiazoline 24a bound
to HDAC3 (modification of PDB code: 4A69) showed the coꢀordination to zinc to both the
carbonyl group (Fig. 2) of the benzamide linkage and to the aniline nitrogen atom. The
largely hydrophobic tunnel (including Leu266, Phe144, Phe200) accommodates the
benzamide ring, whereas contacts to the protein periphery (including Phe199) are made by
the terminal phenyl and thiazoline rings.
Figure 2
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Two benzamide inhibitors, including MSꢀ275, were previously shown to exhibit slow,
tightꢀbinding kinetics for HDAC1, HDAC2 and HDAC3, although the mechanism and rate
1
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of binding for HDAC3 differed from HDAC1 and HDAC2. This assay, in our hands,
identified a twoꢀstep binding mechanism for the binding of Mocetinostat to HDAC3ꢀNCoR1
and additionally revealed compound 24a as a slow tightꢀbinding inhibitor of HDAC3ꢀ
NCoR1 (see supporting information for graphs). These results contrasted with the
hydroxamic acid 31 which showed the expected fast, competitive inhibition of HDAC3ꢀ
NCoR1. The above slow tightꢀbinding behavior is therefore attributable to the Nꢀ(2ꢀ
aminophenyl)ꢀbenzamide moiety.
Thiazoline 24a possesses good physicochemical properties as exemplified by its
calculated lipophilicity value (Daylight clog P = 2.91), measured lipophilicity values (Chrom
35
35
36
log DpH7.4 = 4.24; Chrom log P = 4.65), and its calculated polar surface area (tpsa = 79.5
2
Å ). Inhibitor 24a has good measured solubility, with chemiluminescent nitrogen detection
37
solubility = 239 ꢁM, and is also predicted to have good cell permeability (artificial
38
membrane permeability = 510 nm/s). The cytochrome P450 inhibition profile of 24a
showed only weak inhibition of all tested isoforms except CYP2D6 (isoform IC values:
5
0
2C19 = 32 ꢁM; 2C9 = 16 ꢁM; 2D6 = 80 nM; 3A4 (Vivid® Red substrate) = 25 ꢁM; 3A4
(Vivid® Green substrate) = 16 ꢁM). Owing to the inhibition of CYP2D6, the risk of drugꢀ
drug interactions would need to be evaluated further during any development of 24a.
Cell growth inhibition data for four cell lines are given in Table 4. Despite good in
vitro inhibitory potency against HDAC2 and HDAC3, the imidazolinꢀ4ꢀones 19a and 19c
showed poor inhibition of the cancer cell lines. However, thiazolines 24a, 24d and 30b were
more potent, having low micromolar IC50 values for the four cell lines tested, their pIC50 values
approaching those of Mocetinostat and Vorinostat.
Table 4
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6
Changes in histone acetylation status were assayed by analyzing histone H3 acetylated
on lysine 9 (H3K9Ac) following addition of Nꢀ(2ꢀaminophenyl)ꢀbenzamides. To determine
whether Mocetinostat, 19c or 24a increase histone H3 acetylation levels, the human cell lines
HeLa and Jurkat were cultured in the presence of each compound (10 ꢁM or 1 ꢁM), or an
equivalent volume of the diluent (DMSO) added as the control. The cells were cultured for
24 h and H3K9Ac was analyzed by Western blotting. The data show that 10 ꢁM or 1 ꢁM
Mocetinostat and 24a both cause an increase in histone H3K9Ac with respect to histone H3
expression in Jurkat and in HeLa cells. Imidazolinꢀ4ꢀone 19c (10 ꢁM or 1 ꢁM) also caused
an increase in H3K9Ac in Jurkat cells but not in HeLa cells (Fig. 3). These data are
consistent with an inhibition of endogenous cellular HDAC activity which would cause net
acetylation of histone H3K9.
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Previous studies showed that Mocetinostat causes an increase in H3K9Ac in HeLa cells
3
9
and leads to cell death by apoptosis. Consequently, in the present study flow cytometry
analyses of DNA and protein content of cells cultured with the compounds were carried out
to determine whether 19c and 24a also cause apoptosis. These data (Figs. 4 and 5) show that
Mocetinostat (10 ꢁM or 1 ꢁM) and 24a increase the percentage of Jurkat cells with sub
G1
DNA content, consistent with an induction of apoptosis. However, HeLa cells cultured with
2
either 19c or 24a at 10 ꢁM appeared to accumulate in G /M, whereas there was an increase
in the percentage of Jurkat cells in S
phase, indicating a difference in underlying
mechanisms in these two cell types. In contrast, culturing Jurkat cells with 19c did not result
1
in the appearance of cells with sub
G DNA content and did not significantly alter the
percentage of cells in each cell cycle phase. The Poly
ADP Ribose Polymerase (PARP)
protein is degraded during apoptosis by activation of Caspase 3 as was here observed in
Jurkat cells (Fig. 3). However, no apparent cleavage of PARP occurred in HeLa cells,
3
9
consistent with data previously reported on apoptosis induced by Mocetinostat.
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Summary
For the first time, chiral cyclic capping groups have been shown to be effective for Nꢀ(2ꢀ
aminophenyl)ꢀbenzamide HDAC inhibitors. Inhibition of HDAC1, HDAC2 and HDAC3
(Class I) was generally potent, and in a few of the examples studied, this novel class of
inhibitors exceeded (low nanomolar IC50) potency against HDAC3ꢀNCoR1, compared with
Mocetinostat, one of the most potent benzamide HDAC inhibitors previously reported. The
present study shows the importance of future work to address the central question of whether
improved isoform selectivity and potency over existing Nꢀ(2ꢀaminophenyl)ꢀbenzamide
inhibitors can be achieved by new, enantiopure compounds of the appropriate absolute
configuration and of the classes here disclosed. This study has shown the efficacy of chiral
heterocyclic capping groups, in particular substituted imidazolinꢀ4ꢀones and thiazolines,
especially the latter, which conferred lowꢀnanomolar potency for the inhibition of HDAC3ꢀ
NCoR1 and may be of use as an alternative to the 2ꢀ[3ꢀ(3’ꢀpyridyl)]pyrimidinyl group present
in the clinical benzamide inhibitor MGCD0103. Both Mocetinostat and thiazoline 24a (each at
1 ꢁM) increased histone H3K9 acetylation in Jurkat and HeLa cell lines and caused cell death
of Jurkat cells consistent with induction of apoptosis. Additionally, convergent routes were
established to HDAC inhibitors containing a variety of chiral heterocyclic capping groups by
use of a preassembled Nꢀ(2ꢀaminophenyl)ꢀbenzamide unit, new methodology that should apply
to heterocyclic systems other than those described here.
10
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19
20
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23
24
25
26
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36
37
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Further work is needed to establish the detailed advantages of this novel class of HDAC
inhibitors over existing Nꢀ(2ꢀaminophenyl)ꢀbenzamides, but these may include benefits from
the absence of the commonly required 3ꢀpyridyl terminal capping group and its metabolism to
the Nꢀoxide, and of particular interest, a new means by which HDAC isoform selectivity could
be increased. This study demonstrates that, in terms of HDAC inhibition, other ring systems
can replace either of or both the pyridine and pyrimidine rings present in Mocetinostat, and
hence supports design of new, chiral, Class Iꢀselective HDAC inhibitors.
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Experimental Section
Chemistry. All chemicals were used as supplied, except 1,2ꢀphenylenediamine which was
recrystallized from ethanol. Solvents used were reagent grade, and anhydrous solvents were
obtained from Anhydrous Engineering (USA) solvent systems after passing through an
alumina column. Compound homogeneity was monitored by ascending thinꢀlayer
chromatography, performed on Merck 0.2 mm aluminumꢀbacked silica gel 60 F254 plates and
visualized using an alkaline potassium permanganate dip or by ultraviolet light. Flash
column chromatography was performed using Merck 0.040 to 0.063 mm, 230 to 400 mesh
silica gel. Evaporation refers to the removal of solvent under reduced pressure. Melting
points were determined using an Electrothermal digital melting point apparatus. Infrared (IR)
spectra were recorded on a PerkinꢀElmer spectrum 100 FTꢀIR spectrometer as neat powders
0
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0
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4
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7
8
9
0
1
or as thin films. H NMR spectra were recorded at 300 MHz on a Bruker AMX300
spectrometer, at 400 MHz on a Bruker AMX400, at 500 MHz on a Bruker Avance 500
spectrometer, or at 600 MHz on a Bruker Avance 600 spectrometer in the stated solvent;
chemical shifts are reported in δ (ppm) relative to the internal reference tetramethylsilane.
Mass spectra were obtained on a Fisons VG70ꢀSE mass spectrometer or Thermo Finnigan
MAT900xp instrument. Purity of tested compounds was assessed to be at least 95% by
HPLC analysis unless indicated otherwise. (Dionex) on an Supelco Discovery® BIO
widepore C18 column (250 mm × 4.6 mm, 10 ꢁm particle size) using gradient elution of
acetonitrile in water, 2 to 98% for 20 min at a flow rate of 1 mL/min with detection at 254
nm wavelength. For all samples 0.1% TFA was added to both eluents.
The following compounds were prepared according to the literature: methyl 4ꢀaminoꢀ3ꢀ
4
0
41
23
nitrobenzoate (2a); 2ꢀnitroꢀ4ꢀphenylaniline (6); methyl 4ꢀ(guanidinomethyl)benzoate;
2
4
(E)ꢀ3ꢀ(dimethylamino)ꢀ1ꢀ(pyridinꢀ3ꢀyl)propꢀ2ꢀenꢀ1ꢀone;
methyl
4ꢀ((4ꢀ(pyridinꢀ3ꢀ
2
4
yl)pyrimidinꢀ2ꢀylamino)methyl)benzoate;
4ꢀ((4ꢀ(pyridinꢀ3ꢀyl)pyrimidinꢀ2ꢀ
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2
4
ylamino)methyl)benzoic acid (7); Nꢀ(2ꢀaminophenyl)ꢀ4ꢀ({[4ꢀ(pyridinꢀ3ꢀyl)pyrimidinꢀ2ꢀ
2
4
yl]amino}methyl)benzamide
(Mocetinostat).
(5S)ꢀ1ꢀacetylꢀ5ꢀbenzylꢀ2ꢀthiohydantoin
25
25
(10a);
(5S)ꢀ1ꢀacetylꢀ5ꢀ(1Hꢀindolꢀ3ꢀylmethyl)ꢀ2ꢀthiohydantoin
(10b);
Nꢀ(tert-
4
2
43
10
11
12
13
14
15
16
17
18
19
20
21
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23
24
25
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butoxycarbonyl)ꢀ
L
ꢀphenylalanine (14a), Nꢀ(tert-butoxycarbonyl)ꢀ
L
ꢀtryptophan (14b); N-
4
4
(tertꢀbutoxycarbonyl)ꢀ
D
ꢀphenylglycine (14c);
(5S)ꢀ1ꢀ(tert-butoxycarbonyl)ꢀ5ꢀbenzylꢀ2ꢀ
2
6
45
thiohydantoin (15a); 4ꢀ[(trifluoroacetamido)methyl]benzoic acid (20); tertꢀbutyl (2ꢀ
4
6
aminophenyl)carbamate);
tertꢀbutyl
Nꢀ(2ꢀ{4ꢀ
4
7
[(trifluoroacetamido)methyl]benzamido}phenyl)carbamate (21a);
tertꢀbutyl Nꢀ{2ꢀ[4ꢀ
4
7
48
(aminomethyl)benzamido]phenyl}carbamate (21b);
Dꢀphenylalaninol;
Lꢀphenylalaninol
49
48
31
(25a); (2R)ꢀ2ꢀaminoꢀ2ꢀphenylethanꢀ1ꢀol; (2S)ꢀ2ꢀaminoꢀ2ꢀphenylethanꢀ1ꢀol (25c); (4S)ꢀ
5
0
32
4ꢀbenzylꢀ1,3ꢀthiazolidineꢀ2ꢀthione (26a). (4S)ꢀ4ꢀphenylꢀ1,3ꢀthiazolidineꢀ2ꢀthione (26c).
N-(2-Amino-4-bromophenyl)-4-({[4-(pyridin-3-yl)pyrimidin-2-
yl]amino}methyl)benzamide (8c). To a solution of iron(II) sulfate heptahydrate (1.87 g,
6.74 mmol) in water (4 mL) and 30% aqueous ammonia (4 mL) heated to 60 °C was added a
suspension
of
Nꢀ(4ꢀbromoꢀ2ꢀnitrophenyl)ꢀ4ꢀ({[4ꢀ(pyridinꢀ3ꢀyl)pyrimidinꢀ2ꢀ
yl]amino}methyl)benzamide (0.28 g, 0.56 mmol) in ethanol (4 mL). The mixture was stirred
at 60 °C for 4 h then cooled to 20 °C, filtered through celite and the celite washed with 1:9
methanol:dichloromethane (100 mL) and dichloromethane 100 mL). The combined filtrates
4
were washed with water (100 mL) and then with brine (100 mL), dried over MgSO , filtered
and evaporated to give a pale yellow solid (0.24 g) that was purified by column
chromatography on silica gel (5% methanol in chloroform) to give 8c as a white solid (0.164
ꢀ1
1
g, 61%), mp 206ꢀ208 °C; νmax (cm ) 3247, 3055, 1633, 1608; H NMR (500 MHz, DMSO-
6
d ) δ
4.64 (2H, d, J=6.2 Hz), 5.19 (2H, s), 6.69 (1H, dd, J=8.4, 2.0 Hz), 6.93 (1H, d, J=2.0
Hz), 7.09 (1H, d, J=8.4 Hz), 7.24 (1H, d, J=5.2 Hz), 7.44 ꢀ 7.55 (3H, m), 7.90 (2H, d, J=8.2
Hz), 7.97 (1H, t, J=6.2 Hz), 8.40 (2H, d, J=5.2 Hz), 8.67 (1H, d, J=3.6 Hz), 9.23 (1H, br. s),
13
9
6
.53 (1H, s); C NMR (150 MHz, DMSO-d ) δ 44.0, 106.0, 117.7, 118.3, 118.8, 122.5,
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4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
123.9, 126.9, 127.9, 128.6, 132.4, 132.8, 134.2, 144.3, 145.1, 148.1, 151.3, 159.1, 159.6,
+
+
162.4, 165.4; m/z (ES , %) 475 (53), 307 (100); HRMS C23
H
20BrN
6
O calcd. for [M+H]
475.0882, found: 475.0866.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N-(2-Amino-4-methoxyphenyl)-4-({[4-(pyridin-3-yl)pyrimidin-2-
yl]amino}methyl)benzamide (8d). To a solution of acid 5 (0.306 g, 1.0 mmol), 4ꢀmethoxyꢀ
1,2ꢀphenylenediamine (0.531 g, 1.1 mmol) and (benzotriazolꢀ1ꢀ
yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) (0.531 g, 1.2 mmol) in
DMF (10 mL) was added triethylamine (0.28 mL, 2.0 mmol). The mixture was stirred under
a blanket of nitrogen at 20 °C for 27 h then diluted with ethyl acetate (50 mL), washed with
brine (3 x 50 mL) and then with saturated aqueous sodium hydrogen carbonate (25 mL). The
combined aqueous layers were extracted with ethyl acetate (25 mL) and the combined
organic layers were washed with brine (2 x 25 mL), dried over Na SO and evaporated.
2
4
Column chromatography on silica gel (ethyl acetate) gave 8d as a cream solid (0.21 g, 49%),
ꢀ1
1
mp 176ꢀ178 °C; νmax (cm ) 3435, 3249, 1628; H NMR (400 MHz, DMSO-d ) δ 3.67 (3H,
6
s), 4.65 (2H, d, J=5.9 Hz), 4.91 (2H, br. s), 6.18 (1H, dd, J=8.5, 2.5 Hz), 6.35 (1H, d, J=2.5
Hz), 6.99 (1H, d, J=8.5 Hz), 7.27 (1H, d, J=5.0 Hz), 7.41ꢀ7.60 (3H, m), 7.92 (2H, d, J=8.0
Hz), 8.02 (1H, t, J=5.9 Hz), 8.42 (2H, d, J=5.3 Hz), 8.69 (1H, d, J=3.3 Hz), 9.25 (1H, br. s),
1
3
9.49 (1H, br. s); C NMR (150 MHz, DMSO-d
6
) δ 44.0, 54.9, 100.7, 101.9, 106.1, 116.6,
123.9, 126.9, 127.7, 127.9, 132.4, 133.0, 134.2, 144.1, 144.6, 148.1, 151.3, 158.2, 159.2,
ꢀ
ꢀ
1
59.6, 162.4, 165.3; m/z (ES , %) 425 (100); HRMS calcd. for C24
21 6 2
H N O [MꢀH] 425.1731,
found: 425.1732.
(±)-1-Acetyl-5-benzyl-2-(methylsulfanyl)-4,5-dihydro-1H-imidazol-4-one (11a). To a
stirred mixture of the thiohydantoin 10a (0.70 g, 2.82 mmol) and anhydrous potassium
carbonate (0.468 g, 3.38 mmol) in acetonitrile (35 mL) under nitrogen at 20 °C was added
iodomethane (0.263 mL, 4.23 mmol), dropwise. The mixture was stirred vigorously at 20 °C
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under nitrogen for 3.5 h, then filtered and the filtrate evaporated. The resulting cream solid
was purified by column chromatography (1:9 to 2:1 ethyl acetate:hexane) to give 11a as a
ꢀ
1
1
white solid (0.203 g, 27%), mp 142ꢀ143 °C; νmax (cm ) 1725, 1698; H NMR (500 MHz,
DMSOꢀd ) δ 2.24 (3H, s), 2.39 (3H, s), 3.17 (1H, dd, J=14.3, 2.7 Hz), 3.35 (1H, dd, J=14.3,
6.0 Hz), 4.96 (1H, dd, J=6.0, 2.8 Hz), 6.97 ꢀ 7.06 (2H, m), 7.22 (3H, s); C NMR (125
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
1
3
MHz, DMSOꢀd
6
) δ 15.7, 23.6, 36.1, 64.1, 127.1, 128.2, 129.4, 134.0, 168.5, 184.5, 185.4;
+
+
m/z (EI , %) 262 (12); HRMS calcd. for C H N O S M 262.0771, found: 262.0765.
1
3
14
2
2
(±)-1-Acetyl-5-(1H-indol-3-ylmethyl)-2-(methylsulfanyl)-4,5-dihydro-1H-imidazol-
4-one (11b). The thiohydantoin 10b (0.907 g, 3.38 mmol) and anhydrous potassium
carbonate (0.56 g, 4.05 mmol) in acetonitrile (40 mL) under nitrogen at 20 °C was added
iodomethane (0.32 mL, 5.06 mmol), dropwise. The mixture was stirred vigorously at 20 °C
under nitrogen for 2.5 h, then filtered and the filtrate evaporated. Purification of the residue
by column chromatography (1:1 then 2:1 ethyl acetate:hexane) gave 11b as a cream solid
ꢀ
1
1
(0.61 g, 60%), mp 187ꢀ190 °C; νmax (cm ) 3234, 1725, 1698; H NMR (500 MHz, DMSOꢀ
d
6
) δ 2.22 (3H, s), 2.39 (3H, s), 3.35 (1H, dd, J=15.4, 2.9 Hz), 3.47 (1H, dd, J=15.4, 5.8 Hz),
4.94 (1H, dd, J=5.8, 3.0 Hz), 6.93 (1H, t, J=7.5 Hz), 6.96 (1H, d, J=2.4 Hz), 7.03 (1H, t,
13
J=7.5 Hz), 7.29 (1H, d, J=8.0 Hz), 7.40 (1H, d, J=8.0 Hz), 10.90 (1H, br. s); C NMR (125
MHz, DMSOꢀd
6
) δ 15.6, 23.6, 26.4, 64.1, 106.4, 111.3, 118.3, 118.5, 121.0, 124.1, 127.2,
+
135.7, 168.5, 185.1, 185.3; m/z (EI , %) 301 (10), 130 (100); HRMS calcd. for C15
H
15
N
3
O
2
S
+
M 301.0879, found: 301.0878.
4-({[(±)-1-Acetyl-5-benzyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl]amino}methyl)benzoic
acid (12a) To a solution of the imidazolinꢀ4ꢀone 11a (179 mg, 0.682 mmol) in ethanol (8
mL) was added 4ꢀ(methylamino)benzoic acid (103 mg, 0.682 mmol). The mixture was
stirred at reflux under nitrogen for 18 h. The solution was cooled to 20 °C, then cooled in a
refrigerator, filtered and the solid dried under vacuum to give 12a as a cream solid (122 mg,
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2
2
2
2
2
2
2
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3
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4
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5
5
5
5
5
5
5
6
ꢀ
1
1
49%), mp 232ꢀ234 °C; νmax (cm ) 3263, 2938, 1688; H NMR (500 MHz, DMSOꢀd ) δ 2.42
6
(3H, s), 3.14 (1H, dd, J=14.2, 1.9 Hz), 3.30 (1H, dd, J=14.2, 5.7 Hz), 4.37 (1H, dd, J=15.8,
6.0 Hz), 4.52 (1H, dd, J=15.8, 6.6 Hz), 4.77 (1H, dd, J=5.7, 1.9 Hz), 6.91 (2H, d, J=8.2 Hz),
1
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6.99 ꢀ 7.08 (2H, m), 7.18 ꢀ 7.32 (3H, m), 7.77 (2H, d, J=8.2 Hz), 9.28 (1H, t, J=6.3 Hz); C
NMR (125 MHz, DMSOꢀd
6
) δ 24.2, 35.7, 45.4, 63.1, 126.6, 126.8, 128.1, 129.2, 129.6,
+
134.4, 142.9, 167.2, 167.6, 171.2, 183.4; m/z (ES, %) 366 ([M+H] 100); HRMS calcd. for
+
C H N O [M+H] 366.1448, found: 366.1448.
2
0
20
3
4
4-({[(±)-1-Acetyl-5-(1H-indol-3-ylmethyl)-4-oxo-4,5-dihydro-1H-imidazol-2-
yl]amino}methyl)benzoic acid (12b). The imidazolinꢀ4ꢀone 11b (0.60 g, 1.99 mmol) was
reacted with 4ꢀ(methylamino)benzoic acid according to the above procedure for 12a.
Filtration gave 12b as a tan solid (0.295 g, 37%); the mother liquor was evaporated and the
residue recrystallized from ethanol to give further 12b as a tan solid (0.13 g, 16%), overall
ꢀ
1
1
yield 53%, mp 163ꢀ166 °C; νmax (cm ) 3315, 1728, 1674, 1663; H NMR (500 MHz,
DMSOꢀd ) δ 2.46 (3H, s), 3.31 (1H, dd, J=15.1, 2.2 Hz), 3.45 (1H, dd, J=15.1, 5.4 Hz), 4.32
6
(1H, dd, J=15.8, 5.7 Hz), 4.49 (1H, dd, J=15.8, 6.9 Hz), 4.75 (1H, dd, J=5.4, 2.2 Hz), 6.74
(2H, d, J=8.0 Hz), 6.92 (1H, t, J=7.3 Hz), 6.99 (1H, d, J=2.0 Hz), 7.04 (1H, t, J=7.4 Hz),
7.36 (1H, d, J=8.2 Hz), 7.41 (1H, d, J=7.9 Hz), 7.74 (2H, d, J=8.2 Hz), 9.20 (1H, t, J=6.3
1
3
Hz), 11.00 (1H, br. s); C NMR (125 MHz, DMSOꢀd
6
) δ 24.2, 26.2, 45.3, 63.3, 106.6,
111.3, 118.3, 118.5, 120.8, 124.3, 126.2, 127.5, 129.3, 129.3, 135.9, 142.9, 167.2, 167.8,
ꢀ
ꢀ
ꢀ
1
71.2, 184.1; m/z (ES , %) 403 ([MꢀH] , 95), HRMS calcd. for C22
19 4 4
H N O [MꢀH] 403.1412,
found: 403.1412.
4
-({[(±)-1-Acetyl-5-benzyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl]amino}methyl)-N-
(
2-aminophenyl)benzamide (13a). To a solution of acid 12a (114 mg, 0.312 mmol) and
(
benzotriazolꢀ1ꢀyloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) (165
mg, 0.374 mmol) in anhydrous DMF (1.4 mL) was added triethylamine (174 ꢁl, 1.25 mmol)
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and the mixture stirred at 20 °C under nitrogen for 15 min. Phenyleneꢀ1,2ꢀdiamine (40 mg,
0.374 mmol) was then added and the solution stirred at 20 °C under nitrogen for 3.5 h. The
mixture was diluted with ethyl acetate (50 mL) then washed with water (25 mL), saturated
aqueous sodium hydrogen carbonate (25 mL), and lastly with brine (25 mL). The organic
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
layer was dried over MgSO
4
, filtered and evaporated to give an orange solid. Purification by
column chromatography (4% methanol in dichloromethane) gave 13a as a white solid (21
ꢀ1
1
mg, 15%), mp 235ꢀ236 °C; νmax (cm ) 3283, 1720, 1671; H NMR (600 MHz, DMSOꢀd ) δ
6
2.44 (3H, s), 3.16 (1H, dd, J=14.0, 2.3 Hz), 3.31 (1H, dd, J=14.0, 5.6 Hz), 4.40 (1H, dd,
J=15.6, 5.6 Hz), 4.54 (1H, dd, J=15.6, 6.6 Hz), 4.80 (1H, dd, J=5.6, 2.3 Hz), 4.91 (2H, br. s),
6.61 (1H, t, J=7.3 Hz), 6.79 (1H, d, J=7.2 Hz), 6.94 ꢀ 7.00 (3H, m), 7.07 (2H, d, J=7.2 Hz),
7.17 (1H, d, J=7.5 Hz), 7.28 (2H, t, J=7.5 Hz), 7.32 (1H, t, J=7.2 Hz), 7.84 (2H, d, J=7.5
1
3
Hz), 9.30 (1H, t, J=6.4 Hz), 9.64 (1H, s); C NMR (150 MHz, DMSOꢀd ) δ 24.3, 35.8, 45.4,
6
63.1, 116.1, 116.3, 123.3, 126.5, 126.6, 126.8, 127.0, 127.7, 128.3, 129.6, 133.2, 134.4,
+
141.4, 143.2, 165.1, 167.6, 171.3, 183.5; m/z (ES, %) 456 (M , 80); HRMS calcd. for
+
C H N O M 456.2030, found: 456.2017.
2
6
26
5
3
4-({[(±)-1-Acetyl-5-(1H-indol-3-ylmethyl)-4-oxo-4,5-dihydro-1H-imidazol-2-
yl]amino}methyl)-N-(2-aminophenyl)benzamide (13b). Acid 12b (240 mg, 0.593 mmol)
was reacted with phenyleneꢀ1,2ꢀdiamine according to the above procedure for 13a. Column
chromatography of the residue (6% methanol in dichloromethane) afforded a solid that was
recrystallized from methanolꢀdichloromethane to give 13b as a cream crystalline solid (182
ꢀ
1
1
mg, 62%), mp 168ꢀ170 °C; νmax (cm ) 3267, 1720, 1676; H NMR (500 MHz, DMSOꢀd
.45 (3H, s), 3.31 (1H, dd, J=15.1, 2.4 Hz), 3.46 (1H, dd, J=15.1, 5.4 Hz), 4.36 (1H, dd,
J=15.6, 6.0 Hz), 4.49 (1H, dd, J=15.6, 6.8 Hz), 4.76 (1H, dd, J=5.4, 2.4 Hz), 4.90 (2H, br. s),
.61 (1H, t, J=8.0 Hz), 6.79 (1H, dd, J=8.0, 1.1 Hz), 6.84 (2H, d, J=8.0 Hz), 6.92 ꢀ 7.00 (3H,
6
) δ
2
6
m), 7.05 (1H, t, J=8.0 Hz), 7.19 (1H, d, J=7.7 Hz), 7.38 (1H, d, J=8.0 Hz), 7.43 (1H, d,
J=8.0 Hz), 7.79 (2H, d, J=8.0 Hz), 9.22 (1H, t, J=6.4 Hz), 9.55 (1H, s), 10.96 (1H, br. d,
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2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
J=1.6 Hz); C NMR (125 MHz, DMSOꢀd
6
) δ 24.2, 26.2, 45.3, 63.3, 106.6, 111.4, 116.1,
116.3, 118.3, 118.5, 120.9, 123.4, 124.2, 126.2, 126.5, 126.6, 127.5, 127.7, 133.2, 135.8,
+
+
141.3, 143.1, 165.2, 167.7, 171.2, 184.1; m/z (ES , %) 495 ([M+H] , 100); HRMS calcd. for
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
C
28
H
27
N
6
O
3
[M+H] 495.2139, found: 495.2129.
(5S)-1-(tert-Butoxycarbonyl)-5-(1H-indol-3-ylmethyl)-2-thiohydantoin (15b). To a stirred
solution of N-(tert-butoxycarbonyl)ꢀLꢀtryptophan (14b) (2.11 g, 6.93 mmol) in acetonitrile
(155 mL) was added ethoxycarbonyl isothiocyanate (0.98 mL, 8.3 mmol) and pyridine (0.67
mL, 8.3 mmol). The mixture was stirred at 20 °C for 17 h, evaporated and the residue
purified by column chromatography (1:2 ethyl acetate:hexane) to give 15b as a white solid
ꢀ1
1
(1.98 g, 82%), mp 85ꢀ87 °C; νmax (cm ) 3354, 1736; H NMR (400 MHz, CDCl ) δ 1.64
3
(9H, s), 3.53 (1H, dd, J=15.0, 2.8 Hz), 3.63 (1H, dd, J=15.1, 5.3 Hz), 4.81 (1H, dd, J=5.3,
2.9 Hz), 6.93 (1H, d, J=2.0 Hz), 7.10 (1H, t, J=7.2 Hz), 7.17 (1H, t, J=7.2 Hz), 7.31 (1H, d,
1
3
J=8.0 Hz), 7.56 (1H, d, J=8.0 Hz), 8.12 (1H, br. s), 8.35 (1H, br. s); C NMR (125 MHz,
CDCl ) δ 26.1, 28.2, 64.4, 85.5, 107.1, 111.3, 118.7, 120.0, 122.4, 123.7, 127.4, 135.9,
3
+
+
19 3 3
148.8, 171.5, 178.6; m/z (EI , %) 345 (M , 12), 130 (97); HRMS calcd. for C17H N O S
+
[M+H] 345.1142, found: 345.1144.
(5S)-1-(tert-Butoxycarbonyl)-5-phenyl-2-thiohydantoin (15c). To a stirred solution of Nꢀ
Boc ꢀphenylglycine, (14c) (2.50 g, 9.95 mmol) in acetonitrile (200 mL) was added
D
ethoxycarbonyl isothiocyanate (1.4 mL, 12.0 mmol) and pyridine (0.96 mL, 12.0 mmol). The
mixture was stirred at 20 °C for 17 h, evaporated and the residue purified by column
chromatography (1:3 ethyl acetate:hexane) to give a sticky mass that was recrystallized from
ꢀ
1
chloroform to give 15c as a white solid (1.71 g, 59%), mp 172ꢀ173 °C; νmax (cm ) 3111,
1
1
740; H NMR (500 MHz, DMSO-d
6
)
δ
1.17 (9H, s), 5.62 (1H, s), 7.23 (2H, d, J=6.9 Hz),
1
3
7
.34 ꢀ 7.47 (3H, m), 12.63 (1H, br. s); C NMR (125 MHz, DMSO-d
6
)
δ
27.2, 66.6, 83.3,
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+
+
126.4, 128.7, 129.0, 134.9, 147.5, 171.3, 180.9 (HNC=O); m/z (EI , %) 292 (M , 3%), 192
+
(100); HRMS calcd. for C14
H
16
N
2
O
3
S M 292.0876, found: 292.0870.
10
11
12
13
14
15
16
17
18
19
20
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(±)-1-(tert-Butoxycarbonyl)-5-benzyl-2-(methylsulfanyl)-4,5-dihydro-1H-imidazol-4-one
(16a). To a vigorously stirred mixture of the thiohydantoin 15a (0.90 g, 2.9 mmol) and
anhydrous potassium carbonate (0.481 g, 3.48 mmol) in acetonitrile (35 mL) under nitrogen
at 20 °C was added iodomethane (0.27 mL, 4.35 mmol), dropwise. The mixture was stirred
at 20 °C under nitrogen for 2.5 h, then filtered and the filtrate evaporated. Purification by
column chromatography (1:2 ethyl acetate:hexane) gave 16a as a white solid (0.586 g, 63%),
ꢀ
1
1
mp 105ꢀ107 °C; νmax (cm ) 2979, 2933, 1722; H NMR (500 MHz, CDCl
3
) δ 1.61 (9H, s),
2.37 (3H, s), 3.32 (1H, dd, J=13.9, 2.5 Hz), 3.42 (1H, dd, J=13.9, 6.0 Hz), 4.52 (1H, dd,
1
3
J=6.0, 2.5 Hz), 7.02 ꢀ 7.10 (2H, m), 7.18 ꢀ 7.25 (3H, m); C NMR (125 MHz, CDCl
3
) δ
16.2, 28.2, 36.1, 64.5, 85.7, 127.4, 128.6, 129.5, 133.8, 148.4, 184.7, 186.0; m/z (ES, %) 321
+
+
21 2 3
([M+H] , 100); HRMS calcd. for C16H N O S [M+H] 321.1267, found: 321.1273.
(±)-1-(tert-Butoxycarbonyl)-5-(1H-indol-3-ylmethyl)-2-(methylsulfanyl)-4,5-dihydro-
1H-imidazol-4-one (16b). To a vigorously stirred mixture of the thiohydantoin 15b (1.85 g,
5.36 mmol) and anhydrous potassium carbonate (0.89 g, 6.4 mmol) in acetonitrile (65 mL)
under nitrogen at 20 °C was added iodomethane (0.50 mL, 8.04 mmol), dropwise. The
mixture was stirred vigorously at 20 °C under nitrogen for 2.5 h, then filtered and the filtrate
evaporated. Purification of the residue by column chromatography (2:3 then 1:1 ethyl
ꢀ
1
acetate:hexane) gave 16b as a cream solid (1.35 g, 70%), mp 96ꢀ98 °C; νmax (cm ) 3312,
1
1
720; H NMR (500 MHz, CDCl
3
)
δ
1.60 (9H, s), 2.33 (3H, s), 3.49 (1H, dd, J=15.1, 3.0
Hz), 3.64 (1H, dd, J=15.0, 5.5 Hz), 4.60 (1H, dd, J=5.5, 3.0 Hz), 6.95 (1H, d, J=2.4 Hz),
.07 (1H, t, J=7.5 Hz), 7.14 (1H, t, J=7.6 Hz), 7.30 (1H, d, J=8.0 Hz), 7.53 (1H, d, J=8.0
7
1
3
Hz), 8.26 (1H, br. s); C NMR (125 MHz, CDCl
3
) δ 16.2, 26.0, 28.2, 64.8, 85.8, 107.9,
1
11.2, 118.5, 119.6, 122.0, 123.5, 127.8, 135.9, 148.5, 185.4, 186.1; m/z (ES, %) 382
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2
2
2
2
2
2
3
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3
3
3
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3
3
3
3
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
+
([M+Na] , 90); HRMS calcd. for C18
H
21
N
3
NaO
3
S [M+Na] 382.1196, found: 382.1188.
(±)-1-(tert-Butoxycarbonyl)-5-phenyl-2-(methylsulfanyl)-4,5-dihydro-1H-imidazol-4-
one (16c). To a vigorously stirred mixture of the imidazolꢀ4ꢀone 15c (0.25 g, 0.855 mmol)
and anhydrous potassium carbonate (0.141 g, 1.03 mmol) in acetonitrile (10 mL) at 20 °C
was added iodomethane (80 ꢂL, 1.28 mmol) dropwise. The mixture was stirred vigorously at
20°C under nitrogen for 2.5 h, then evaporated. Ethyl acetate (20 mL) was added to the
residue and the mixture was stirred and filtered. The filtrate was evaporated to give 16c as a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢀ
1
1
white solid (0.26 g, 99%), mp 145ꢀ147 °C; νmax (cm ) 1737, 1713; H NMR (500 MHz,
CDCl 1.26 (9H, s), 2.67 (3H, s), 5.18 (1H, s), 7.15 ꢀ 7.23 (2H, m), 7.30 ꢀ 7.39 (3H, m);
3
) δ
1
3
C NMR (125 MHz, CDCl3)
δ 16.4, 27.7, 67.6, 85.4, 126.4, 128.7, 129.0, 134.5, 148.4,
+
+
+
183.0, 186.6; m/z (EI , %) 306 (M , 3), 205 (100); HRMS calcd. for C15
H
18
N
2
O
3
S [M+H]
306.1033, found: 306.1036.
4-({[(±)-1-(tert-Butoxycarbonyl)-5-benzyl-4-oxo-4,5-dihydro-1H-imidazol-2-
yl]amino}methyl)benzoic acid (17a). To a stirred solution of the imidazolꢀ4ꢀone 16a (0.550
g, 1.72 mmol) in ethanol (15 mL) was added 4ꢀ(methylamino)benzoic acid (0.260 g, 1.72
mmol). The mixture was stirred at reflux under nitrogen for 18 h. The solvent was
evaporated and the residue shaken with ethyl acetate (50 mL). The organic layer was washed
with water (50 mL) then with brine (50 mL), dried over MgSO
4
, filtered and evaporated.
Purification of the residue by column chromatography (ethyl acetate) gave 17a as a cream
ꢀ
1
1
solid (0.238 g, 33%), mp 137ꢀ140 °C; νmax (cm ) 3386, 2975, 1702; H NMR (500 MHz,
CDCl 1.62 (9H, br. s), 3.22 ꢀ 3.40 (2H, m), 4.36 (1H, m), 4.49 (1H, br. s), 4.77 (1H, dd,
J=14.8, 6.6 Hz), 6.96 (2H, d, J=7.3 Hz), 7.10 (2H, d, J=6.3 Hz), 7.15 ꢀ 7.34 (3H, m), 7.94
3
) δ
1
3
(
2H, d, J=7.3 Hz), 8.43 (1H, br. s), 8.95 (1H, br. s); C NMR (125 MHz, CDCl
3
) δ 28.3,
3
1
5.8, 46.4, 63.5, 85.7, 127.2, 127.4, 128.6, 128.9, 129.7, 130.6, 134.1, 142.2, 167.8, 170.5,
ꢀ
ꢀ
24 3 5
74.1, 184.3; m/z (ES, %) 422 ([MꢀH] , 100), HRMS calcd. for C23H N O [MꢀH]
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422.1721, found: 422.1707.
4-({[(±)-1-(tert-Butoxycarbonyl)-5-(1H-indol-3-ylmethyl)-4-oxo-4,5-dihydro-1H-
imidazol-2-yl]amino}methyl)benzoic acid (17b). To a solution of the imidazolꢀ4ꢀone 16b
(1.30 g, 3.62 mmol) in ethanol (30 mL) was added 4ꢀ(methylamino)benzoic acid (0.547 g,
3.62 mmol). The mixture was stirred at reflux under nitrogen for 4 h. The solvent was
evaporated and the resulting yellow solid purified by column chromatography (6% methanol
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ꢀ
1
in dichloromethane) to give 17b as a cream solid (1.0 g, 60%), mp 187ꢀ190 °C; νmax (cm )
1
3271, 1746, 1697, 1693; H NMR (500 MHz, DMSOꢀd
6)
δ
1.58 (9H, s), 3.23 (1H, dd,
J=14.7, 2.3 Hz), 3.44 (1H, dd, J=14.7, 5.3 Hz), 4.31 (1H, dd, J=15.8, 5.8 Hz), 4.45 ꢀ 4.52
(2H, m), 6.76 (2H, d, J=8.2 Hz), 6.88 (1H, d, J=2.2 Hz), 6.92 (1H, t, J=7.5 Hz), 7.04 (1H, t,
J=7.5 Hz), 7.36 (1H, d, J=8.0 Hz), 7.44 (1H, d, J=8.0 Hz), 7.74 (2H, d, J=8.2 Hz), 8.72 (1H,
1
3
br. t, J=5.7, 5.7 Hz), 10.97 (1H, br. d, J=2.2 Hz); C NMR (125 MHz, DMSOꢀd
6
) δ 25.2,
27.7, 45.3, 63.2, 84.0, 107.0, 111.3, 118.2, 118.4, 120.8, 123.8, 126.2, 127.6, 129.2, 135.8,
+
+
150.2, 167.2, 184.0; m/z (ES, %) 463 ([M+H] , 10), HRMS calcd. for C H N O [M+H]
2
5
27
4
5
463.1976, found: 463.1993.
4-(([(±)-1-(tert-Butoxycarbonyl)-5-phenyl-4-oxo-4,5-dihydro-1H-imidazol-2-
yl]amino)methyl)benzoic acid (17c). To a solution of the imidazolꢀ4ꢀone 16c (0.25 g, 0.817
mmol) in ethanol (5 mL) was added 4ꢀ(aminomethyl)benzoic acid (0.124 g, 0.817 mmol).
The mixture was heated under reflux for 4 h with stirring. After cooling to 20 °C, the mixture
was placed in the refrigerator (1 h), and then filtered. The filtrate was evaporated and the
residue was purified by column chromatography (4:1 ethyl acetate:hexane) to give 17c as a
ꢀ
1
1
white solid (0.131 g, 39%), mp 166ꢀ168 °C; νmax (cm ) 3315, 1702; H NMR (500 MHz,
methanol-d
4
)
δ
δ
1.24 (9H, s), 4.76 ꢀ 4.84 (2H, m), 5.28 (1H, s), 7.24 (2H, d, J=7.3 Hz), 7.32 ꢀ
1
3
7.43 (3H, m), 7.52 (2H, d, J=8.2 Hz), 8.03 (2H, d, J=8.5 Hz); C NMR (125 MHz,
methanol-d
4
)
27.9, 47.5, 68.1, 86.1, 127.8, 128.5, 129.5, 129.9, 131.2, 131.4, 137.2, 143.9,
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5
5
5
5
5
5
5
5
6
+
151.7, 169.4, 169.5, 185.7; m/z (ES, %) 410 ([M+H] , 8); HRMS calcd. for C22
H
24
N
3
O
5
+
[M+H] 410.1710, found: 410.1704.
0
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9
0
1-tert-Butyloxycarbonyl (±)-2-[({4-[(2-aminophenyl)carbamoyl]phenyl}methyl)amino]-
5-benzyl-4-oxo-4,5-dihydro-1H-imidazole (18a). To a solution of acid 17a (230 mg, 0.543
mmol) and (benzotriazolꢀ1ꢀyloxy)tris(dimethylamino)phosphonium hexafluorophosphate
(BOP) (0.287 g, 0.65 mmol) in anhydrous DMF (2.5 mL) was added triethylamine (0.30 ml,
2.16 mmol) and the mixture stirred at 20 °C under nitrogen for 30 min. Phenyleneꢀ1,2ꢀ
diamine (70 mg, 0.65 mmol) was then added and the solution stirred at 20 °C under nitrogen
for 5.5 h. The mixture was diluted with water (50 mL) then extracted with ethyl acetate (100
mL, then 50 mL). The combined organic layers were washed with saturated aqueous sodium
hydrogen carbonate (50 mL), and lastly with brine (50 mL). The organic layer was dried over
MgSO , filtered and evaporated to give an orange solid. Purification by column
4
chromatography (2% methanol in dichloromethane) gave 18a as a cream solid (154 mg,
ꢀ1
1
55%), mp 121ꢀ124 °C; νmax (cm ) 3304, 1699; H NMR (400 MHz, CDCl ) δ 1.65 (9H, s),
3
3.29 ꢀ 3.39 (2H, m), 3.92 (2H, br. s), 4.40 (1H, dd, J=15.4, 5.4 Hz), 4.44ꢀ 4.50 (1H, m), 4.73
(1H, dd, J=15.4, 7.2 Hz), 6.81 ꢀ 6.89 (2H, m), 7.02 (2H, d, J=8.0 Hz), 7.07 ꢀ 7.14 (3H, m),
1
3
7.17 ꢀ 7.40 (4H, m), 7.81 (2H, d, J=8.0 Hz), 8.15 (1H, br. s), 8.41 (1H, br. s); C NMR (125
MHz, CDCl ) δ 28.3, 35.8, 46.2, 63.4, 85.6, 118.4, 119.8, 124.7, 125.4, 127.3, 127.3, 127.6,
127.9, 128.6, 129.7, 133.6, 134.2, 140.6, 140.8, 151.1, 165.5, 167.8, 184.1; m/z (ES, %) 514
3
+
+
+
(
[M+H] , 10), 536 ([M+Na] , 19); HRMS calcd. for C29
31 5 4
H N NaO [M+Na] 536.2268,
found: 536.2277.
1
-tert-Butyloxycarbonyl (±)-2-[({4-[(2-aminophenyl)carbamoyl]phenyl}methyl)amino]-
-(1H-indol-3-ylmethyl)-4-oxo-4,5-dihydro-1H-imidazole (18b). To a solution of acid 17b
5
(
0.97 g, 2.10 mmol) and (benzotriazolꢀ1ꢀyloxy)tris(dimethylamino)phosphonium
hexafluorophosphate (BOP) (1.12 g, 2.52 mmol) in anhydrous DMF (10 mL) was added
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triethylamine (1.17 ml, 8.4 mmol) and the mixture stirred at 20 °C under nitrogen for 15 min.
Phenyleneꢀ1,2ꢀdiamine (0.272 g, 2.52 mmol) was then added and the solution stirred at 20
°C under nitrogen for 5 h. The mixture was diluted with ethyl acetate (240 mL), washed with
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
water (90 mL), then with brine (50 mL), dried over MgSO , filtered and evaporated to give
an orange solid. Purification by column chromatography (6% methanol in chloroform) gave
ꢀ
1
1
18b as a cream solid (0.398 g, 34%), mp 185ꢀ186 °C; νmax (cm ) 3288, 1732, 1699; H
NMR (500 MHz, CDCl ) δ 1.66 (9H, s), 3.30 (1H, dd, J=14.8, 2.4 Hz), 3.49 (1H, dd, J=14.8,
3
3.7 Hz), 3.98 (2H, br. s), 4.12 (1H, dd, J=15.8, 5.2 Hz), 4.43 (1H, m), 4.58 (1H, dd, J=15.8,
7.7 Hz), 6.52 (2H, d, J=8.0 Hz), 6.67 (1H, d, J=1.7 Hz), 6.75 ꢀ 6.82 (2H, m), 6.99 (1H, t,
J=7.5 Hz), 7.03 ꢀ 7.12 (2H, m), 7.34 (1H, d, J=7.7 Hz), 7.41 (1H, d, J=8.4 Hz), 7.44 (1H, d,
1
3
J=8.2 Hz), 7.66 (2H, d, J=7.7 Hz), 8.31 (1H, br. s), 8.71 (1H, br. s), 9.11 (1H, br. s); C
NMR (125 MHz, CDCl ) δ 25.7, 28.3, 45.9, 64.2, 85.7, 107.1, 111.8, 117.8, 118.1, 119.3,
119.4, 121.7, 124.2, 124.6, 125.8, 126.5, 127.2, 127.9, 128.0, 133.2, 136.0, 140.3, 141.2,
3
+
+
151.0, 166.0, 168.0, 185.4; m/z (ES, %) 553 ([M+H] , 15), 575 ([M+Na] , 8); HRMS calcd.
+
for C H N NaO , [M+Na] 575.2377, found: 575.2389.
3
1
32
6
4
1-tert-Butyloxycarbonyl (±)-2-[({4-[(2-aminophenyl)carbamoyl]phenyl}methyl)amino]-
5-phenyl-4-oxo-4,5-dihydro-1H-imidazole (18c). To a solution of acid 17c (0.115 g, 0.281
mmol), (benzotriazolꢀ1ꢀyloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP)
(0.143 g, 0.323 mmol) and phenyleneꢀ1,2ꢀdiamine (0.061 g, 0.562 mmol) in DMF (3 mL)
was added triethylamine (0.075 mL, 0.562 mmol). The mixture was stirred at 20 °C for 4 h.
Some DMF was removed in vacuo and the residual oil was diluted with ethyl acetate (50
mL) and the organic layer washed with saturated aqueous lithium chloride (20 mL), then
with brine (2 x 20 mL). Evaporation gave a residue that was purified by column
chromatography on silica gel (ethyl acetate) to give 18c as a cream solid (0.14 g, 98%), mp
ꢀ
1
1
1
78ꢀ180 °C; νmax (cm ) 3318, 1736, 1700, 1596; H NMR (500 MHz, CDCl
3
) δ 1.22 (9H, s),
4.02 (2H, br. s), 4.71 (1H, d, J=15.1 Hz), 4.76 (1H, d, J=15.1 Hz), 5.10 (1H, s), 6.78 (2H, d,
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2
2
2
2
2
2
2
2
3
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3
4
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4
4
4
4
4
4
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5
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6
J=7.3 Hz), 7.03 (1H, t, J=7.6 Hz), 7.17 (2H, d, J=7.3 Hz), 7.24 (1H, d, J=7.6 Hz), 7.27 ꢀ 7.39
1
3
(5H, m), 7.82 (2H, d, J=7.3 Hz), 8.44 (1H, br. s), 8.79 (1H, br. s); C NMR (125 MHz,
CDCl 27.7, 46.8, 66.7, 85.5, 118.5, 119.9, 124.6, 125.7, 126.5, 127.3, 128.0, 128.2,
128.6, 128.9, 133.8, 135.2, 140.4, 140.5, 150.8, 165.8, 168.0, 183.1; m/z (ES, %) 522
3
) δ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
29 5 4
([M+Na] , 45); HRMS calcd. for C28H N NaO [M+Na] 522.2112, found: 522.2103.
(±)-N-(2-Aminophenyl)-4-({5-benzyl-4-oxo-4,5-dihydro-1H-imidazol-2-
yl]amino}methyl)benzamide (19a). To a solution of the imidazole 18a (100 mg, 0.195
mmol) in dichloromethane (1 mL) was added trifluoroacetic acid (1 mL). The solution was
stirred at 20 °C for 3 h, then evaporated and toluene (3 mL) added to the residue. Evaporation
of the solvent afforded a pink solid that was partitioned between saturated aqueous sodium
hydrogen carbonate (50 mL) and ethyl acetate (50 mL). The organic layer was washed with
brine, dried over MgSO , filtered and evaporated to give a solid that was purified by column
4
chromatography (10% methanol in dichloromethane) to give 19a as a pale yellow solid (58
ꢀ
1
1
mg, 72%), mp 157ꢀ160 °C; νmax (cm ) 3197, 1699, H NMR (2:1 mixture of tautomers) (600
MHz, DMSOꢀd ) δ 2.76 (1H, dd, J=13.6, 6.4 Hz), 2.97 (1H, m, minor tautomer), 3.03 (1H,
6
dd, J=13.4, 3.2 Hz, major tautomer), 4.06 (1H, app. t, J=5.1 Hz), 4.28 ꢀ 4.52 (2H, m), 4.91
(2H, br. s), 6.60 (1H, t, J=7.3 Hz), 6.79 (1H, d, J=7.9 Hz), 6.97 (1H, t, J=7.5 Hz), 7.10 ꢀ 7.31
(8H, m), 7.68 (2H, br. s, major tautomer) 7.89 (2H, d, J=7.2 Hz, major tautomer), 7.95 (2H,
1
3
br. d, J=7.2 Hz, minor tautomer), 8.28 (2H, br. s, minor tautomer), 9.67 (1H, br. s);
NMR (150 MHz, DMSOꢀd ) δ 37.2, 43.7, 44.7, 61.2, 116.1, 116.3, 123.3, 126.3, 126.5,
26.7, 126.7, 126.9, 127.8, 128.0, 128.1, 129.5, 129.6, 133.2, 133.3, 136.9, 137.3, 141.8,
C
6
1
1
+
42.8, 143.2, 165.1, 170.8, 171.5, 187.6, 188.1(tautomers); m/z (CI, %) 414 ([M+H] , 15);
+
HRMS calcd. for C24
H
24
N
5
O
2
[M+H] 414.1925, found: 414.1935.
(±)-N-(2-Aminophenyl)-4-({[5-(1H-indol-3-ylmethyl)-4-oxo-4,5-dihydro-1H-imidazol-
2-yl]amino}methyl)benzamide (19b). To a solution of the imidazole 18b (300 mg, 0.543
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mmol) in dichloromethane (3 mL) was added trifluoroacetic acid (1.5 mL). The solution was
stirred at 20 °C for 3 h, then evaporated and toluene (3 mL) added to the residue. Evaporation
of the solvent afforded a solid that was partitioned between saturated aqueous sodium
hydrogen carbonate (100 mL) and ethyl acetate (150 mL). The organic layer was washed
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
with brine, dried over MgSO
4
, filtered and evaporated to give 19b as a pale yellow solid
(0.24 g, 98%), mp 205ꢀ207 °C. Recrystallization of 50 mg from dichloromethaneꢀmethanol
ꢀ
1
1
gave 19b (30 mg) as a cream solid, mp 214ꢀ215 °C; νmax (cm ) 3216, 1689, 1662; H NMR
(2:1 mixture of tautomers) (400 MHz, DMSOꢀd ) δ 2.86 (1H, dd, J=14.8, 7.0 Hz), 3.16 (1H,
6
br. dd, J=14.8, 5.3 Hz), 4.06 (1H, m), 4.34 ꢀ 4.50 (2H, m), 4.91 (2H, br. s), 6.61 (1H, t, J=7.4
Hz), 6.79 (1H, d, J=7.5 Hz), 6.90 ꢀ 7.39 (8H, m), 7.57 (6H, m), 9.62 (1H, br. s), 10.87 (1H,
1
3
br. s); C NMR (125 MHz, DMSOꢀd ) δ 27.4, 43.7, 44.7, 48.6, 54.9, 61.1, 76.6, 106.8,
6
109.9, 111.2, 116.1, 116.3, 118.2, 118.6, 120.8, 123.3, 126.5, 126.6, 127.5, 127.8, 133.2,
+
136.0, 142.8, 143.1, 165.1, 171.7, 178.1, 184.9, 188.6 (tautomers); m/z (ES , %) 453
+
+
25 6 2
([M+H] , 100); HRMS calcd. for C26H N O [M+H] 453.2034, found: 453.2023.
(±)-N-(2-Aminophenyl)-4-(4-oxo-5-phenyl-4,5-dihydro-1H-imidazol-2-
yl]amino)methyl)benzamide (19c). To a solution of the imidazole 18c (0.135 g, 0.27 mmol)
in dichloromethane (1.5 mL) was added trifluoroacetic acid (0.75 mL). The solution was
stirred at 20 °C for 3 h, then evaporated and toluene (3 mL) added to the residue. The solvent
was evaporated and the process repeated three more times. The residue was then dissolved in
methanol (10 mL), and stirred with saturated aqueous sodium hydrogen carbonate (10 mL)
for 30 min. The methanol was then evaporated and then water (10 mL) was added. The
suspension was sonicated for 5 min. The precipitate was filtered, washed with water then
with diethyl ether, and dried under vacuum to give 19c as a cream solid (75 mg, 70%), mp
ꢀ
1
1
2
21ꢀ223 °C; νmax (cm ) 3263, 3062, 1706, 1654; H NMR (500 MHz, DMSO-d
mixture of NH tautomers) 4.47 ꢀ 4.63 (2H, m), 4.85 (1H, s), 4.89 (2H, br. s), 6.59 (1H, t,
J=7.1 Hz), 6.77 (1H, d, J=7.9 Hz), 6.96 (1H, t, J=7.1 Hz), 7.12 ꢀ 7.20 (2H, m), 7.20 ꢀ 7.30
6
) (3:2
δ
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2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
4
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4
4
4
4
4
4
4
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5
5
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5
5
6
(2H, m), 7.30 ꢀ 7.39 (2H, m), 7.41 ꢀ 7.49 (2H, m), 7.92 ꢀ 8.02 (2H, m), 8.12 ꢀ 8.24 (1H, m,
1
3
major tautomer), 8.72 (1H, br. s, minor tautomer), 9.63 (1H, br. s); C NMR (150 MHz,
DMSOꢀd 43.4, 43.8, 45.0, 45.1, 63.8, 116.2, 116.5, 123.2, 123.3, 125.6, 125.8, 126.5,
6
) δ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
126.6, 126.7, 127.0, 127.1, 127.2, 127.4, 127.5, 127.5, 127.7, 128.0, 128.2, 128.3, 128.4,
128.6, 133.3, 133.4, 137.7, 141.6, 142.7, 143.0, 143.1, 165.2, 171.7, 186.7 (tautomers); m/z
+
+
(ES, %) 400 ([M+H] , 100); HRMS calcd. for C23
H
22
N
5
O
2
[M+H] 400.1768, found:
400.1755. Purity by HPLC was determined to be 92%.
tert-Butyl
N-{2-[4-isothiocyanatomethyl)benzamido]phenyl}carbamate
(22).
A
5
1
previously described protocol for preparing isothiocyanates was used. To a stirred solution of
amine 21b (0.341 g, 1.0 mmol) in dichloromethane (10 mL) was added carbon disulfide (1 mL,
10.4 mmol, CAUTION: FLAMMABLE). The mixture was stirred at 20 °C for 1 min, then
triethylamine (0.14 mL, 1.0 mmol) was added and stirring at 20 °C continued for a further 10
min. The mixture was cooled in an iceꢀbath and pꢀtoluenesulfonyl chloride (0.191 g, 1.0 mmol)
was added. The mixture was stirred at 0 °C for 10 min, then hydrochloric acid (10 mL, 1M)
added. The mixture was extracted with dichloromethane (2 x 10 mL) and the combined organic
layers washed with brine (20 mL), dried over Na SO and evaporated. Purification of the residue
2
4
by column chromatography on silica gel (1:3 ethyl acetate:hexane) gave 22 as a white solid
ꢀ
1
1
(0.26 g, 68%), mp 152ꢀ153 °C
;
ν
max (cm ) 3277, 2086, 1693, 1656; H NMR (400 MHz,
CDCl
3
)
δ
1.54 (9H, s), 4.81 (2H, s), 6.87 (1H, s), 7.12 ꢀ 7.26 (3H, m), 7.43 (2H, d, J=8.3 Hz),
1
3
7
.81 (1H, d, J=7.8 Hz), 8.01 (2H, d, J=8.3 Hz), 9.35 (1H, br. s); C NMR (125 MHz,
CDCl
3
)
δ
28.4, 48.4, 81.6, 124.6, 125.9, 126.1, 126.1, 127.0, 128.2, 129.9, 130.9, 133.6,
+
1
34.5, 138.2, 154.8, 164.9; m/z (ES, %) 406 ([M+Na] , 93), 284 (20); HRMS calcd. for
+
C
20
H
21
N
3
NaO
3
S [M+Na] 406.1196, found: 406.1198.
Synthesis of thioureas: general procedure A. To a solution of the amino alcohol (1 equiv.)
in acetonitrile (4 mL/mmol) was added isothiocyanate 22 (1 equiv.) and the resulting
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suspension was stirred at 20 °C for 16 h, then evaporated, and either purified or used
directly, as described below.
10
11
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15
16
17
18
19
20
21
22
23
24
25
26
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28
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42
43
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45
46
47
48
49
50
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53
54
55
56
57
58
59
60
tert-Butyl
N-(2-{4-[({[(1S)-2-hydroxy-1-phenylethyl]carbamothioyl}amino)methyl]
benzamido} phenyl)carbamate (23a). To a solution of (S)-2ꢀphenylglycinol (0.165 g, 1.2
mmol) in acetonitrile (4 mL) was added 22 (0.384 g, 1.0 mmol). The resulting suspension
was stirred at 20 °C for 16.5 h, then evaporated. The residue was purified by column
chromatography on silica gel (3:2 then 2:1 ethyl acetate:hexane) to give 23a as a white solid
ꢀ1
1
(0.464 g, 89%), mp 108ꢀ111 °C; νmax (cm ) 3285, 1691, 1646; H NMR (600 MHz, CDCl )
3
δ
1.46 (9H, s), 1.84 (1H, s), 3.45 (1H, br. s), 3.61 ꢀ 3.69 (1H, m), 3.77 (1H, m), 4.53 ꢀ 4.81
(2H, m), 5.19 (1H, br. s), 7.08 (2H, br. s), 7.11 ꢀ 7.18 (3H, m), 7.20 (2H, d, J=7.2 Hz), 7.24 ꢀ
1
3
7.32 (5H, m), 7.59 (1H, d, J=6.8 Hz), 7.67 (2H, d, J=7.5 Hz), 9.33 (1H, br. s); C NMR
(150 MHz, CDCl3) 28.4, 48.4, 60.2, 66.5, 81.7, 124.9, 125.9, 126.0, 126.5, 126.9, 127.4,
127.8, 128.3, 129.1, 130.4, 130.7, 132.7, 138.2, 142.3, 154.9, 166.2, 182.7; m/z (ES, %) 521
δ
+
ꢀ
([M+H] , 20), 543 (50); HRMS calcd. for C H N O S [MꢀH] 519.2071, found: 519.2094.
2
8
31
4
4
tert-Butyl
N-(2-{4-[({[(1R)-2-hydroxy-1-
phenylethyl]carbamothioyl}amino)methyl]benzamido} phenyl)carbamate (23b). (R)-2ꢀ
phenylglycinol (0.112 g, 0.82 mmol) was reacted according to general procedure A and the
product purified by column chromatography on silica gel (3:2 then 2:1 ethyl acetate:hexane)
ꢀ1
1
to give 23b as a white solid (0.37 g, 96%), mp 107ꢀ109 °C; νmax (cm ) 3278, 1692, 1648; H
NMR (400 MHz, CDCl
3
) δ 1.49 (9H, s), 1.88 (1H, br. s), 3.49 (1H, br. s), 3.66 (1H, m), 3.79
(1H, d, J=8.8 Hz), 4.69 (2H, d, J=2.8 Hz), 5.20 (1H, br. s), 6.97 ꢀ 7.39 (11H, m), 7.59 (1H, d,
1
3
J=6.8 Hz), 7.69 (2H, d, J=8.0 Hz), 9.35 (1H, br. s); C NMR (150 MHz, CDCl
3
) δ 28.4,
4
1
8.4, 60.2, 66.5, 81.7, 124.9, 125.9, 126.0, 126.5, 126.9, 127.4, 127.8, 128.3, 129.1, 130.4,
+
30.7, 132.7, 138.2, 142.3, 154.9, 166.2, 182.7; m/z (ES, %) 521 ([M+H] , 80), 421 (100);
+
HRMS calcd. for C28
H
33
N
4
O
4
S [M+H] 521.2217, found: 521.2195.
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