2-Phenyl-4-piperazinylbenzimidazoles: Orally active inhibitors of the gonadotropin releasing hormone (GnRH) receptor

Article abstract of DOI:10.1016/j.bmc.2008.05.024

Antagonism of the gonadotropin releasing hormone (GnRH) receptor has shown positive clinical results in numerous reproductive tissue disorders such as endometriosis, prostate cancer and others. Traditional therapy has been limited to peptide agonists and

Full text of DOI:10.1016/j.bmc.2008.05.024

Bioorganic & Medicinal Chemistry 16 (2008) 6617–6640
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2-Phenyl-4-piperazinylbenzimidazoles: Orally active inhibitors of the
gonadotropin releasing hormone (GnRH) receptor
b
b
c
a
a
Jeffrey C. Pelletier a, , Murty Chengalvala , Josh Cottom , Irene Feingold , Lloyd Garrick , Daniel Green ,
Diane Hauze a, Christine Huselton c, James Jetter a, Wenling Kao a, Gregory S. Kopf b, Joseph T. Lundquist IV a,
Charles Mann a, John Mehlmann a, John Rogers a, Linda Shanno b, Jay Wrobel a
*
a Department of Chemical & Screening Sciences, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA
b Department of Women’s Health & Musculoskeletal Biology, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA
c Department of Drug Safety and Metabolism, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Antagonism of the gonadotropin releasing hormone (GnRH) receptor has shown positive clinical results
in numerous reproductive tissue disorders such as endometriosis, prostate cancer and others. Traditional
therapy has been limited to peptide agonists and antagonists. Recently, small molecule GnRH antagonists
have emerged as potentially new treatments. This article describes the discovery of 2-phenyl-4-piperaz-
inylbenzimidazoles as small molecule GnRH antagonists with nanomolar potency in in vitro binding and
functional assays, excellent bioavailability (rat %F > 70) and demonstrated oral activity in a rat model
having shown significant serum leuteinizing hormone (LH) suppression.
Received 8 January 2008
Revised 4 May 2008
Accepted 7 May 2008
Available online 11 May 2008
Keywords:
Gonadotropin releasing hormone antagonist
Oral activity
Ó 2008 Elsevier Ltd. All rights reserved.
Small molecule
Pharmaceutical profiling
1. Introduction
delay for the onset of the antagonist effect and during this time pa-
tients experience exacerbation of symptoms (flare effect).
Gonadotropin releasing hormone (GnRH), or leuteinizing hor-
mone releasing hormone (LHRH), is a 10 amino acid peptide syn-
In recent years more extensive modifications to GnRH have gi-
ven rise to peptide antagonists such as abarelix and cetrorelix.4 Di-
rect blockade of the GnRHR leads to immediate and full
antagonism of the receptor with no flare effect. As expected, both
peptide superagonists and antagonists have poor pharmacokinetic
and bioavailability properties when taken orally. Hence, these
agents are only effective following parenteral administration. In
addition, many patients receive extended release forms of these
drugs that tend to lower hormones to castration levels. An orally
active small molecule GnRH antagonist could offer additional ben-
efits such as regimen compliance, the ability to titrate drug and
hormone levels as well as the flexibility to withdraw the drug rel-
atively quickly when adverse symptoms are seen.5
thesized and released in
a
pulsatile fashion from the
hypothalamus.1 The hormone stimulates a G-protein coupled
receptor (GPCR)—the GnRH receptor (GnRHR)—positively coupled
to phospholipase C on cell surfaces of the anterior pituitary gland.
Following stimulation of the GnRH receptor the gonadotropins, fol-
licle stimulating hormone (FSH) and leuteinizing hormone (LH) are
released into the general circulation where they act on the male
and female gonads to trigger gametogenesis as well as synthesis
and release of sex steroids.2 Early structural modifications to GnRH
led to agonists with enhanced activity over the native hormone. In
clinical settings these ‘superagonists’ have been used to treat var-
ious cancers of the reproductive organs as well as primary hirsut-
ism, endometriosis, and other hormone dependant conditions
through the suppression of sex steroids.3 The concept of hormone
suppression with an agonist/superagonist is unique. Constant
exposure of the GnRH receptor to a GnRH agonist leads to receptor
desensitization and eventual down-regulation causing an overall
antagonistic effect. During this process, however, there is a 2 week
Recently, numerous small molecule GnRH antagonists repre-
senting various pharmacophores bonded to several template
classes have been reported in the literature. Indoles,6 quino-
lones,7 furans,8 uracils,9 benzimidazoles,10 and thienopyridines11
are some of the GnRH receptor antagonists disclosed (Fig. 1).
Stoichiometric association and nanomolar affinity between
a
broad, structurally diverse class of small molecules and a single
receptor is unusual and a model has been proposed to explain
this promiscuity.12 Considering the breadth of chemical spatial
diversity and the acceptable ADME descriptors associated with
these series it would be expected to see good to excellent oral
* Corresponding author.
E-mail address: Pelletj@wyeth.com (J.C. Pelletier).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.024

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J. C. Pelletier et al. / Bioorg. Med. Chem. 16 (2008) 6617–6640
N
N
HN
H
N
N
O
N
N
H
O
N
Cl
N
O
H
H
Merck6
Merck7
O
O O
HN
N
O
O
N
O
N
N
N
NH
O
HN
HN
O
S
N
F
O
O
F
Pfizer8
Takeda11
O
F
F
O
N
NO2
N
N
O
S
H2N
O
N
N
N
H
H
MeO
F
Bayer10
Neurocrine9
Figure 1. Representative small molecule GnRH antagonists recently reported in the literature.
bioavailability from compounds in each class. In most cases,
however, oral bioavailability is poor to modest (F < 25%). In addi-
tion, small molecule association with the GnRH receptor can be
species selective. As a result, many compounds may require pre-
clinical evaluation in expensive monkey models. Reported here
are the lead discovery and in vitro structural optimization of a
series of 2-phenyl-4-piperazinylbenzimidazole antagonists of
the GnRH receptor. Optimization efforts resulted in the discovery
of a compound with excellent oral bioavailability and rat plasma
LH suppression properties.
sure that compounds had antagonist properties. This led to the
identification of two structurally related 4-piperazinylbenzimidaz-
oles, 1a and 1b (Fig. 2), that had modest binding and antagonist
properties on the human GnRH receptor as well as potent binding
properties to several central 5HT-1 receptor subtypes. In addition,
further screening indicated both compounds had affinity for the rat
GnRH receptor and both were antagonists as measured by LH re-
lease inhibition from primary rat pituitary cells. The compounds,
however, were weaker (25- to 50-fold) in this species. The possibil-
ity of in vivo evaluation in a lower species such as the rat was
attractive. The leads were suitable for analogue purposes, hence
an optimization effort was initiated to increase binding and func-
tional potency at the human and rat receptors as well as selectivity
over the 5HT-1 receptor subtypes.
Efforts to identify chemical leads were initiated through screen-
ing of a platform-based biogenic amine library rich in GPCR ligands
(ꢀ2200 compounds) using a previously reported hGnRH binding
assay13 for receptor affinity followed by a functional assay14 to en-
S
F3C
N
HN
NH
N
HN
HN
HN
F
N
N
N
F
N
N
F
F
O
O
F
F
1a
1b
hGnRH binding IC50 = 1.54 μM
hIP turnover inhibition IC50 = 9.2 μM
hGnRH binding IC50 = 0.80 μM
hIP turnover inhibition IC50 = 4.5 μM
rGnRH binding IC50 = 15.0 μM
rLH release inhibition IC50 = 83.0 μM
rGnRH binding IC50 = 21.0 μM
rLH release inhibition IC50 = 76.0 μM
h5-HT1A Ki = 19 nM
h5-HT1B Ki = 60 nM
h5-HT1D Ki = 0.55 nM
h5-HT1A Ki = 2.8 nM
h5-HT1B 67% inhibition @ 1000 nM
h5-HT1D = 67% inhibition @ 1000 nM
Figure 2. 4-piperazinylbenzimidazoles 1a and 1b, with GnRH antagonist properties, were identified from screening a GPCR privileged library.

J. C. Pelletier et al. / Bioorg. Med. Chem. 16 (2008) 6617–6640
6619
Preparation of compounds with modified ethoxy linkers con-
necting the piperazine with the pendant 2-trifluoromethylbenzim-
idazole or the 2-thiobenzimidazolone is shown in Schemes 1 and 2.
Treatment of 2,6-difluoronitrobenzene 32 with sodium azide then
piperazine gave the azido-piperazinyl nitrobenzene intermediate
34 which was protected and fully reduced to the phenylenedi-
amine 36. Treatment of this compound with hot trifluoroacetic
acid (TFA)15 gave the key deprotected intermediate benzimidazole
37. Reductive amination or N-alkylation with electrophiles, pre-
pared from literature procedures shown in the Scheme 1 inset,16–20
gave intermediates with the various linkers. Nitro groups on all
of these intermediates were reduced under the standard condi-
tions of hydrogenation or tin(II) chloride dihydrate (SnCl2ꢁ2H2O)
to provide the corresponding phenylenediamines, which were fur-
ther reacted with thiocarbonyldiimidazole (thioCDI) and/or hot
TFA to provide the desired products 3, 4, 5a, 5b, 6a, and 6b. In addi-
tion, the glycolamide 2 was prepared by converting the fluoro of 2-
azido-6-fluoronitrobenzene 33 to a phenol21 then alkylating the
phenol with tert-butyl bromoacetate to provide 57 (Scheme 2).
TFA deprotection of the acid followed by coupling with the piper-
azine 37 led to amide 59. The nitro and azide groups were reduced
by catalytic hydrogenation and conversion to the desired product 2
was achieved by reacting the phenylenediamine 60 with thioCDI.
Preparation of compound 7, with the ethoxy linker shifted one
carbon on the pendant thiobenzimidazolone, is shown in Scheme
3. The fluorine of 3,4-dinitrofluorobenzene 61 was displaced with
the sodium salt of glycolaldehyde diethyl acetal, and the nitro
groups were reduced to provide the phenylene diamine 63. Treat-
ment of this intermediate with thioCDI followed by hydrolysis gave
the thiobenzimidazolone glycolaldehyde 65, which was reductively
aminated with piperazine derivative 37 to provide 7. In addition,
the piperazine on the piperazinylbenzimidazole template was also
shifted one carbon. The preparation of this compound is shown in
Scheme 4. As in Scheme 3, the fluoro of 3,4-dinitrofluorobenzene
was displaced with monoprotected piperazine, and the product
was fully reduced to the phenylenediamine to provide intermediate
67. This underwent deprotection and trifluorobenzimidazole for-
mation upon treatment with hot TFA to give 68 which was reacted
with aldehyde 70, prepared in similar fashion to aldehyde 65 (see
Scheme 3) starting from 2-azido-6-fluoronitrobenzene, to provide
the nitroazide 71. Full reduction followed by thioCDI treatment as
above provided the desired product 9.
F3C
NO2
NO2
NH2
NBoc
NH
N
N3
R
F
F
i, ii, iii
iv
v
H2N
N
HN
N
33 R = F
32
37
36
34 R = 1-piperzine
35 R = 4-Boc-1-
piperazine
R1
xvi, xvii or xviii, xix or xx
F3C
HN
R
R2
N
N
xii, xiii, xiv or xv
N
40 R = CH2CH2CH2O, R1 = NH2, R2 = NO2
41 R = CH2CH2CH2, R1 = NH2, R2 = NO2
42 R = CH2, R1 = NH2, R2 = NO2
43 R = CH2CH2CH2O, R1, R2 = NH2
44 R = CH2CH2CH2, R1, R2 = NH2
45 R = CH2, R1, R2 = NH2
F3C
HN
N
R
N
N
R
38 R, R = NO2
39 R, R = NH2
5a R, R = NC(CF3)NH
5b R, R = NHCSNH
3 R = CH2CH2CH2O, R1, R2 = NHCSNH
4 R = CH2CH2CH2, R1, R2 = NC(CF3)NH
6a R = CH2, R1, R2 = NC(CF3)NH
6b R = CH2, R1, R2 = NHCSNH
NO2
NH2
NO2
NH2
NMe2
vii, viii
vi
OH
R
O2N
NO2
O2N
NO2
O
46
47
48
49 R = OH
50 R = Cl
OH
NO2
NO2
O
Cl
NH2
NO2
NH2
NH2
NH2
NO2
xi
ix, x
R
55
52 R = OH
53 R = Cl
54
51
Scheme 1. Preparation of analogues with a modified linker. Reagents and conditions: (i) NaN3, DMSO, 60 °C, 100%; (ii) piperazine, DMSO, 60 °C, 95%; (iii) (Boc)2O, 100%; (iv)
H2, 10% Pd/C, MeOH, 100%; (v) TFA, 70 °C, 89%; (vi) DMF–DMA, 150 °C (Ref. 16); (vii) SOCl2, PhH, Ref., then NaBH4, MeOH, 46% for two steps (Refs. 17,18); (viii) SOCl2, TEA,
CH2Cl2, 82%; (ix) 1.0 M BH3/THF, THF, 62%; (x) SOCl2, TEA, CH2Cl2, 57%; (xi) 1-bromo-3-chloropropane, K2CO3, DMF, 90 °C, 46%; (xii) 47, NaCNBH3, THF, AcOH, H2O, 44%; (xiii)
H2, 10% Pd/C, MeOH; (xiv) ThioCDI, THF, 70 °C, 23% for three steps to 5b; (xv) TFA, 70 °C, 61% for 5a; (xvi) 50 or 53 or 55, DIPEA, DMF, 80 °C, yield of 42 = 41%, 41 = 22%,
40 = 85%; (xvii) 40, H2, 10% Pd/C, MeOH, 100%; (xviii) 41 or 42, SnCl2ꢁ2H2O, NMP, yield of 44 = 63%, 45 = 50%; (xix) 43 or 45, ThioCDI, THF, 70 °C, yield of 3 = 40%, 6b = 32%; (xx)
44 or 45, TFA, 70 °C, yield of 4 = 42%, 6a = 24%.

6620
J. C. Pelletier et al. / Bioorg. Med. Chem. 16 (2008) 6617–6640
O
R1
F3C
HN
O
R2
NO2
NO2
N
N
iv, v
N3
OR
i, ii, iii
N3
F
N
59 R1 = NO2, R2 = N3
60 R1, R2 = NH2
33
56 R = H
57 R = CH2CO2t-Bu
58 R = CH2CO2H
2 R1, R2 = NHCSNH
Scheme 2. Preparation of glycolamide linker analogue 2. Reagents and conditions: (i) 2-(methanesulfonyl)ethanol, NaH, DMF, 57%; (ii) tert-butylbromoacetate, K2CO3,
acetone, 87%; (iii) TFA, CH2Cl2, 100%; (iv) 37, HATU, DIPEA, DMF, 56%; (v) H2, 10% Pd/C, MeOH then ThioCDI, THF, 70 °C, 18%.
OEt
R
R
O
O2N
O2N
F
i, ii
iii, iv
OEt
61
62 R = NO2
63 R = NH2
F3C
O
H
N
N
O
R
v
N
HN
N
S
NH
S
N
H
HN
7
64 R = CH(OEt)2
65 R = CHO
Scheme 3. Preparation of an analogue (7) with the ethoxy linker shifted one position on the thiobenzimidazolone. Reagents and conditions: (i) 2,2-diethoxyethanol, NaH,
THF, 0 °C, 39%; (ii) H2, 10% Pd/C, MeOH, 93%; (iii) ThioCDI, THF, 70 °C, 98%; (iv) 1 N HCl, THF, 70 °C; (v) 37, NaBH(OAc)3, NMP, 21%.
NBoc
NH
O2N
O2N
F
i, ii
iii
R
R
N
N
N
F3C
N
H
61
68
66 R = NO2
67 R = NH2
NO2
O
NO2
33
iv, v
vi
N3
R
N3
F
69 R = CH(OEt)2
70 R = CHO
O
O
N
N
vii, viii
N
N
N
R1
N
O2N
F3C
F3C
R2
N3
N
H
N
H
71
72 R1, R2 = NH2
9 R1, R2 = NHCSNH
Scheme 4. Preparation of an analogue (9) with the piperazine shifted to the 5-position of the benzimidazole. Reagents and conditions: (i) 1-(Boc)piperazine, NMM, NMP,
100%; (ii) H2, 10% Pd/C, MeOH, 97%; (iii) TFA, 70 °C, 66%; (iv) 2,2-diethoxyethanol, NaH, THF, 0 °C, 90%; (v) 1 N HCl, THF, 70 °C, 62%; (vi) 68, NaBH(OAc)3, DMF, 44%; (vii) H2,
10% Pd/C, MeOH; (viii) ThioCDI, THF, 70 °C, 36% for two steps.
N-Methyl derivatives 8a and 8b were synthesized as shown in
Scheme 5. Hence, the azide of intermediate 35, prepared as shown
in Scheme 1, was selectively reduced and trifluoroacetylated to
give 74, which was methylated under Mitsunobu conditions to
provide 75. Reduction of the nitro group on 75 followed by TFA re-
moval of the Boc protecting group gave 77. Reductive amination of
this piperazine derivative with aldehyde 70 (see Scheme 4) fol-
lowed by reduction and treatment with hot TFA or thioCDI gave
the desired products 8a and 8b, respectively.
Synthesis of molecules with additional modifications to the
template benzimidazole is shown in Scheme 6. The fluorine of
intermediate 33 was displaced with the sodium salt of 2-(1-piper-
azinyl)ethanol, and the resulting free amine was protected with a
Boc group. Full reduction of the azide and nitro groups provided

J. C. Pelletier et al. / Bioorg. Med. Chem. 16 (2008) 6617–6640
6621
F3C
N
R2 NO2
N
NBoc
NR
N
NO2
NBoc
iv, v
i, ii, iii
N
N
N3
N
R1
73 R1, R2 = H
35
76 R = Boc
77 R = H
74 R1 = H, R2 = COCF3
75 R1 = Me, R2 = COCF3
F3C
N
F3C
O
N
O
N
N
N
viii or ix
vi, vii
N
N
R1
N
R1
R2
R2
78 R1 = NO2, R2 = N3
79 R1, R2 = NH2
8a R1, R2 = NHC(CF3)N
8b R1, R2 = NHCSNH
Scheme 5. Preparation of N-methyl analogues 8a and 8b. Reagents and conditions: (i) SnCl2ꢁ2H2O, MeOH, 82%; (ii) TFAA, DIPEA, CH2Cl2, 99%; (iii) MeOH, TPP, DIAD, CH2Cl2,
89%; (iv) H2, 10% Pd/C, MeOH, 68%; (v)TFA, CH2Cl2, 71%; (vi) 70, NaBH(OAc)3, AcOH, NMP, 45%; (vii) H2, 5% Pd/C, MeOH, 90%; (viii) ThioCDI, THF, 70 °C, yield for 8b = 36%; (ix)
TFA, 70 °C, yield for 8a = 27%.
R1
R1
NO2
O
R2
O
R2
F
N3
iv (82 to 83)
N
i, ii, iii
N
N
R3
N
or v, vi (82 to 85)
R3
83 R1, R2 = NHC(CF3)N, R3 = H
84 R1, R2 = NHCSNH, R3 = Boc
85 R1, R2 = NHCSNH, R3 = H
80 R1 = NO2, R2 = N3, R3 = H
81 R1 = NO2, R2 = N3, R3 = Boc
82 R1, R2 = NH2, R3 = Boc
33
CF3
N
NH
O
R2
N
x or xi or xii
R1
R1
N
O
R2
R4
N
vii then
R3
N
10a R1, R2 = NHCONH
11 R1, R2 = NHCSNH
viii (86 to 88)
or ix (87 to 89)
20a R1, R2 = NHC(4-EtPh)N
21 R1, R2 = NHC(4-
22 R1, R2 = NHC(4-
i
t
-PrPh)N
-BuPh)N
86 R1, R2 = NHC(CF3)N, R3 = N3, R4 = NO2
87 R1, R2 = NHCSNH, R3 = N3, R4 = NO2
88 R1, R2 = NHC(CF3)N, R3, R4 = NH2
89 R1, R2 = NHCSNH, R3, R4 = NH2
x or xii or xiii or
xiv or xv or xvi
10b R1, R2 = NHCONH
S
12 R1, R2 = NHCOCONH
13 R1, R2 = NCHCHN
HN
NH
O
R2
N
14 R1, R2 = NHCHN
15 R1, R2 = NHC(CH3)N
16 R1, R2 = NHC(C2F6)N
17 R1, R2 = NHC(Ph)N
18 R1, R2 = NHC(3-MePh)N
19 R1, R2 = NHC(4-MePh)N
20b R1, R2 = NHC(4-EtPh)N
23 R1, R2 = NHC(2,4-diMePh)N
R1
N
Scheme 6. Preparation of additional analogues with template benzimidazole modifications. Reagents and conditions: (i) 2-(1-piperazinyl)ethanol, NaH, THF, ꢂ78 °C, 74%; (ii)
(Boc)2O, CH2Cl2, 100%; (iii) H2, 5% Pd/C, MeOH, 93%; (iv) TFA, 20–70 °C; (v) ThioCDI, THF, 47%; (vi) TFA, polymer bound dimethylsilane, 69%; (vii) 33, DIPEA, DMSO, yield of
86 = 63% for two steps, 87 = 64%; (viii) H2, 5% Pd/C, MeOH, 51% for 88; (ix) SnCl2ꢁ2H2O, NMP, 20–100 °C, 43% for 89; (x) CDI, THF, yield for 10a = 61%, 10b = 4%; (xi) ThioCDI,
THF, yield for 11 = 66%; (xii) RCO2H, HATU, NMP, then AcOH, 100 °C, yield for 18 = 13%, 19 = 36%, 20a = 10%, 20b = 15%, 21 = 38%, 22 = 34%, 23 = 10%; (xiii) 30% aq glyoxal,
MeOH, yield for 13 = 38%; (xiv) RCO2H, 90 °C, yield for 14 = 5%, 15 = 40%, 16 = 35%; (xv) PhCHO, i-PrOH, Ref., air, yield for 17 = 50%; (xvi) oxalyldiimidazole, THF, yield for
12 = 30%.
the phenylenediamine 82. Treatment of the intermediate with thi-
oCDI followed by TFA or with hot TFA alone gave the thiobenzim-
idazolone and trifluoromethylbenzimidazole derivatives 85 and
83, respectively. Reacting this compound with 33 gave the azidoni-
tro intermediates 86 and 87. Catalytic hydrogenation of 86 and tin
mediated reduction of 87 gave the phenylenediamines 88 and 89,
respectively. One or both of these intermediates were treated with
several reagents including CDI, thioCDI, oxalyldiimidazole, glycol
aldehyde, hot formic, acetic and perfluoropropionic acids, benzal-
dehyde and substituted benzoic acids to give the desired products
10–23.
Finally, compounds with the 2-(4-tert-butylphenyl)-1H-ben-
zimidazol-4-ylpiperazine core were prepared via reductive amina-
tion of appropriate aldehyde22 precursors and intermediate 91
(Scheme 7). Final products 25, 26, 30, and 31 were prepared as
shown as well as the intermediate nitrofluoro compound 92, which
was carried on through a series of straight-forward steps to pro-
vide the quinoxalinedione 29. Also shown in Scheme 7 is the prep-

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J. C. Pelletier et al. / Bioorg. Med. Chem. 16 (2008) 6617–6640
aration of 24, 27 and 28 from 91 and the benzylchloride derivative
50.
IC50 = 3300 nM) but had no effect on 5-HT1a activity (Ki = 2.8 nM)
when compared to the lead compound 1b. Conversion of the eth-
oxy group to n-propyl resulted in a less active compound (Table
1, compound 4) as did the shorter alkyl modifications of 5a, 5b,
and 6a. When the thiobenzimidazolone heterocycle was coupled
with the piperazinylbenzimidazole via the methylene linker, how-
ever, a derivative of equal or slightly greater potency than the ori-
ginal lead 1b resulted (compound 6b). This represents an increase
of nearly 20-fold in potency over the similar trifluoromethylbenz-
imidazole analogue 6a and a clear separation of activities com-
pared to the nearly equipotent leads 1a and 1b. In addition,
human functional activity remained about equal to the lead com-
pound, while rat binding activity weakened slightly (Table 1). Fi-
nally, transposition of the ethoxy group by one carbon unit in the
aromatic region also resulted in a compound of less activity (7,
hGnRH IC50 = 4.5 lM).
Additional structure–activity relationship (SAR) optimization
focused on modification of the trifluoromethylbenzimidazole of
1a, 1b directly bonded to the piperazine (Table 2). Initial efforts
were directed at N-alkylation of the imidazole (8a and 8b), trans-
position of the linker-heterocycle location (9), and variation of
the bicyclic structure to other heterocycles (10a–13). All com-
pounds displayed relatively low activity as human GnRH binding
agents. Further tests on these molecules (rat binding, functional
activity) were not performed and structural focus remained on
the N-unsubstituted benzimidazole.
Human and rat binding assays were used as the primary drivers
of structure–activity relationship studies and were performed on
recombinant cells expressing either rat or human GnRH receptors
as described in the literature.13 Displacement of radioiodinated
(D-Trp6)-GnRH, a GnRH agonist, to these cells using test article
was used to determine percent inhibition and IC50’s. Human func-
tional data were measured through the inhibition of tritiated inos-
itols from cells overexpressing the human receptor treated with
the GnRH agonist, (D-Trp6)-GnRH, or agonist combined with vari-
ous concentrations of test article.14 Finally, rat functional activity
was estimated by measuring LH release from primary rat pituitary
cells following treatment with the GnRH agonist, (D-Trp6)-GnRH,
and test article at various concentrations (see Section 2 for details).
Initial optimization efforts focused on maximizing GnRH antag-
onism and selectivity through modification of the ethoxy unit link-
ing the piperazine ring with its pendant benzimidazole or
thiobenzimidazolone (Table 1). At this juncture the 2-trifluorom-
ethylbenzimidazole and 2-thiobenzimidazolone groups pendant
to the ethoxy linker were considered bioequivalent, therefore,
comparison of biological properties between structurally similar
compounds with either the pendant trifluoromethylbenzimidazole
or the thiobenzimidazolone was considered valid. Conversion to
the glycolamide 2 led to a sharp drop in activity, most likely indi-
cating the requirement for a basic nitrogen in position 4 of the
piperidine ring. Elongating to the propyloxy derivative 3 led to a
modest loss in GnRH activity (human GnRH binding
Additional optimizations on the proximal (template) benzimid-
azole centered on substitutions at the 2-position occupied by the
R
N
NR
NH2
NBoc
N
N
N
N
iii
H2N
N
i, ii
HN
HN
36
25 R = Ph
26 R = Naph
30 R = 5-uracil
90 R = Boc
91 R = H
31 R = 3-ethyl-5-uracil
92 R = 3-F-4-O2NPh
NH
O
R2
N
HN
N
O
N
vi
iv, v
R1
N
92
N
N
HN
HN
93 R1 = N3, R2 = NO2
94 R1, R2 = NH2
29
N
N
R1
R2
HN
NH
ix or x or xi
vii, viii
X
N
N
N
N
91
HN
HN
95 R1 = NH2, R2 = NO2
96 R1, R2 = NH2
24 X = CS
27 X = CO
28 X = COCO
Scheme 7. Preparation of additional benzylic analogues. Reagents and conditions: (i) 4-tert-butylPhCHO, IPA, air, 80 °C, 95%; (ii) TFA, CH2Cl2, 50%; (iii) RCHO, NaBH(OAc)3,
NMP, yield for 25 = 100%, 26 = 84%, 30 = 57%, 31 = 61%, 92 = 68%; (iv) NaN3, DMSO, 92%; (v) H2, 10% Pd/C, MeOH, 100%; (vi) oxalyldiimidazole, THF, 70 °C, 24%; (vii) 50, DIPEA,
NMP, 36%; (viii) SnCl2 ꢁ 2H2O, NMP, 70 °C, 20%; (ix) ThioCDI, THF, 60 °C, yield for 24 = 20%; (x) CDI, THF, 70 °C, yield for 27 = 28%; (xi) oxalyldiimidazole, THF, yield for 28 = 6%.

J. C. Pelletier et al. / Bioorg. Med. Chem. 16 (2008) 6617–6640
6623
Table 1
In vitro activity of ethoxy linker analogues of lead compounds 1a and 1b
F3C
HN
X
H
N
N
(a or b)
H
N
N
N
or
S
CF3
N
H
N
a
b
Compound
X
a or b
hGnRH binding
hGnRH IP inhibition
rGnRH binding
5-HT1a binding Ki
IC50 SD (nM)
IC50 SDa (lM)
IC50 SDb (lM)
IC50 SDa (lM)
1a
1b
2
3
4
5a
5b
6a
6b
–(CH2)2O–
–(CH2)2O–
–COCH2O–
–(CH2)3O–
–(CH2)3–
–(CH2)2–
–(CH2)2–
–CH2–
a
b
b
b
a
a
b
a
b
1.5 0.21
9.2 3.0
4.5 1.0
—
—
—
—
—
15 2.6
21 3.3
—
—
—
—
—
19 3.2
2.8 0.3
—
2.3
—
—
—
—
—
0.79 0.15
24% @ 10 lM
3.3 0.40
42% @ 10 lM
26% @ 10 lM
51% @ 10 lM
9.5 1.9
21 4.5
3.4 0.42
64 10
51 3.8
–CH2–
0.58 0.10
H
N
7
–(CH2)2O–
4.5 3.6
—
—
—
S
N
H
a
Binding to overexpressed human or rat GnRH receptors in competiton with 125I-(D-Trp6)-GnRH (Ref. 13).
b
Compound driven IP reduction in whole cells following stimulation with (D-Trp6)-GnRH (Ref. 14).
trifluoromethyl group in both lead compounds. A significant range
5-HT1a and alpha1 adrenergic receptors dropped to undetectable
levels indicating strong selectivity for GnRH over the catechol-
amine receptors (Scheme 8) and marked improvement over the
activity of the lead compounds.
of SAR were observed following substitution of the trifluoromethyl
group with hydrogen, lower alkyl, extended perfluoro lower alkyl,
and substituted phenyl groups (Table 3). Significant loss of activity
was observed with small, homologous replacement units such as
hydrogen (14), methyl (15), and perfluoroethyl (16). An investiga-
tion into phenyl ring replacement groups, however, revealed SAR
patterns of interest. Simply replacing the trifluoromethyl group
with phenyl resulted in a compound (17) with less activity
(IC50 = 11 lM) than the leads 1a and 1b. However, the possibility
of the phenyl group serving as additional template space was
attractive. As shown in Table 3 substitution with lower alkyl
groups in the 4-position on the phenyl ring led to compounds of
increasing activity with an increase in steric size. Activity peaked
with the 4-tert-butylphenyl compound (compare hGnRH binding
IC50: 19 = 1.3 lM, 22 = 0.050 lM), while the 3-methylphenyl was
about equipotent to the unsubstituted phenyl in the human bind-
ing assay. Substitution of the 2-position of the phenyl in the 2-phe-
nylbenzimidazoles led to inactive compounds (data not shown);
however, the 2,4-dimethylphenyl derivative 23 had binding activ-
ity nearly equivalent to the 4-methylphenylbenzimidazole 19.
Additional significant trends seen in Table 3 include rat binding
as well as human functional data. In nearly all examples where
hGnRH binding potency increased so too did rat binding and func-
tional human activity. Hence, the rat binding and human func-
tional activity of the lead compound 1b (rGnRH binding
IC50 = 21 lM, hIP IC50 = 4.5 lM) were improved with the 4-tert-
butylphenyl derivative 22 (rGnRH binding IC50 = 0.71 lM, hIP
IC50 = 1.2 lM).
At this juncture a combination of optimized SAR pharmaco-
phores from the studies mentioned above was investigated. This
practice frequently leads to molecules with greatly enhanced de-
sired activity. Hence, the favorable structural properties of com-
pounds 6b (Table 1) and 22 (Table 3) were blended into a single
molecule (Scheme 8). The resulting molecule 24 possessed GnRH
antagonism properties superior to all others previously tested. Hu-
man GnRH binding and functional IC50’s improved to 6.2 nM and
14 nM, respectively. Rat activity also improved as the binding
IC50 was measured at 110 nM, and LH release from rat primary
pituitary cells increased to 480 nM. Also notable was the lack of
binding activity of 24 at CNS GPCR’s. Binding activity at the
Although these improvements were noteworthy, we thought
that a greater potency and functional activity at the rat receptor
would be required for successful in vivo evaluation as serum LH
supressors. Hence, the thiobenzimidazolone portion of the mole-
cule was targeted for modification in an effort to address this is-
sue. The results are summarized in Table 4. Replacement of the
thiobenzimidazolone with the simple aromatic phenyl and naph-
thyl groups (25 and 26, respectively) resulted in compounds
with a substantial loss of hGnRH receptor binding. Human activ-
ity remained high with benzimidazolone 27 but rat binding and
functional activity diminished sixfold compared to 24. Some of
the rat activity returned with 5-substituted quinoxalinedione
28 but a dramatic increase in rat activity and human functional
activity was observed with the one carbon transposed, 6-substi-
tuted quinoxalinedione 29. Although the human and rat GnRH
antagonist properties of 29 were suitable for in vivo studies
the quinoxalinedione rendered the molecule highly insoluble
and a poor candidate for pharmacokinetic evaluation. The phenyl
group of 25 was clearly deleterious to GnRH activity and sug-
gested that the phenyl portion of the quinoxalinedione does
not add to the GnRH inhibitory activity of 29. Replacement of
the quinoxalinedione with a monocyclic heterocycle in this posi-
tion may retain activity similar to this bicyclic structure. Hence,
we evaluated the uracils 30 and 31 in the human and rat GnRH
assays. Both compounds showed excellent binding properties at
human and rat receptors. In addition they were potent antago-
nists in both functional assays. Hence, the uracils were attractive
candidates for in vivo evaluation based on their potent, non-
selective in vitro GnRH antagonist profile.
Prior to in vivo pharmacokinetic analysis uracils 30 and 31 were
evaluated for their absorption–distribution–metabolism–excretion
(ADME) properties. Table 5 summarizes the ADME molecular
descriptors generally thought to effect bioavailability and exposure
levels. All values are within recommended ranges as suggested
by established models.23,24 In addition, in vitro pharmaceutical
profiling properties of solubility, PAMPA membrane diffusion and
rat liver microsome half-life are also shown.25 Both compounds

6624
J. C. Pelletier et al. / Bioorg. Med. Chem. 16 (2008) 6617–6640
Table 2
tion. This would ensure an additional GnRH active component in
vivo. The data obtained from ADME molecular descriptors and in
vitro pharmaceutical property analysis supported in vivo pharma-
cokinetic evaluation of 30 and 31. Hence, rats were exposed to both
uracils via intravenous (iv) and oral (po) administration and their
pharmacokinetic parameters were established. The data are sum-
marized in Table 6. Uracil 30 had low bioavailability (%F ꢀ 5), while
the N-ethyl compound, 31, was highly bioavailable (%F = 74). These
data suggested in vivo, oral exposure for this series is absorption
dependant and supported the in vivo efficacy analysis of com-
pound 31.
In vitro activity results of analogues of the trifluoromethylbenzimidazole template
region of 1a, b
H
N
H
N
O
N
(a or b)
or
S
CF3
N
N
H
N
R
a
b
a
Compound
R
a or b
hGnRH binding IC50 (lM)
N
Intact animals have pulsatile patterns of serum LH making
accurate measurements of the gonadotropin difficult. Orchidec-
tomized rats, however, display elevated, stable levels of serum
LH due to the removal of the testosterone mediated feedback loop
that controls the central release of GnRH. Exposure of these ani-
mals to a GnRH antagonist can lower serum LH to normal levels.
Hence, castrated rats were treated orally with compound 31 and
their serum LH levels were monitored post-administration. As
shown in Figure 3, the N-ethyluracil derivative successfully low-
ered LH levels starting at 1 h post-administration and the effect
continued for at least 6 h. At the 24 h time point LH levels had re-
turned to levels equivalent to the control group (data not shown).
The in vivo experiment clearly indicated that antagonism of the
GnRH receptor is occurring in vivo with compound 31. It was also
subjected to limited off-target pharmacological analysis since ear-
lier generation leads were active at 5-HT and alpha1 adrenergic
receptors. Compound 31 was inactive in all assays tested with
the exception of the 5-HT2a subtype (Ki = 230 nM). Functional eval-
uation of 31 in 5-HT2a assay indicated compound 31 is a full antag-
onist and not expected to elicit detrimental side effects or toxicity
via this mechanism.
The goal of our program was to discover small molecule, orally
active antagonists of the GnRH receptor from two lead com-
pounds obtained from a GPCR directed library that focused on
GnRH receptor antagonist activity. Both compounds had micro-
molar binding potencies to the human and rat GnRH receptors
and they were shown to be antagonists in both species. Enhance-
ment of rat activity along with human activity was critically
important for in vivo evaluation. Compounds that are inactive
in rat are usually evaluated in monkeys making pre-clinical
discovery and development expensive and time consuming. A
compartmentalized structure activity optimization process led
to individual improvements to binding and efficacy properties.
Combining these improvements together into a single molecule
led to a nanomolar inhibitor of human GnRH and a modest
antagonist of rat GnRH (compound 24). Further refinements led
to enhanced rat activity in compound 31, which also had high
bioavailability when orally administered to rats at 20 mg/kg
(%F = 74). Suppression of serum LH levels leads to the suppression
of sex hormones, which in turn has therapeutic value for dis-
eases/conditions aggravated by these hormones. Proof of concept
can be achieved when serum LH levels are suppressed to a statis-
tically significant level. Uracil 31 dropped rat serum LH levels for
several hours following administration of a single oral dose
(30 mg/kg). LH levels returned to normal after 24 h. This data
confirms the discovery of uracil 31 as an orally active antagonist
of the GnRH receptor. Further studies on this and other com-
pounds will be reported in due course.
1a
a
b
a
b
b
a
b
a
b
b
1.5 0.21
CF3
N
H
N
1b
8a
8b
9
0.79 0.15
CF3
CF3
CF3
N
H
N
N
5% @ 10 lM
8% @ 10 lM
11% @ 10 lM
8% @ 10 lM
6% @ 10 lM
42% @ 10 lM
9% @ 10 lM
1% @ 10 lM
N
N
N
CF3
N
H
H
N
10a
10b
11
12
O
O
S
N
H
H
N
N
H
H
N
N
H
H
N
O
O
N
H
N
13
N
Binding to overexpressed human GnRH receptors in competiton with 125I-(D-
a
Trp6)-GnRH (Ref. 13).
displayed moderate solubility and permeability properties. The
unsubstituted uracil 30 was stable in the rat liver microsome assay
while the N-ethyluracil 31 was unstable in the same assay with a
half-life of 5 min. Generally speaking, compounds with a half-life
less than 15 min in the rat in vitro liver microsome assay are con-
sidered unstable and will not likely survive the first-pass effect in
the liver following oral absorption. These latter data suggested a
relatively higher in vivo dose may overcome any first-pass elimina-
tion effects brought on by high liver clearance. In addition, because
the structures differ only by an N-ethyl group the microsomal
instability of 31 may be due to N-dealkylation of the uracil which
could provide an in vivo source of compound 30 via biotransforma-
2. Experimental details
2.1. General methods
All experiments were conducted in well-ventillated fume
hoods. Anhydrous solvents were purchased from Aldrich Chemical

J. C. Pelletier et al. / Bioorg. Med. Chem. 16 (2008) 6617–6640
6625
Table 3
In vitro SAR following substitution of the trifluoromethyl group of the proximal benzimidazole region of 1a, b: GnRH binding and functional data
R
O
H
N
N
(a or b)
H
N
N
HN
N
or
S
CF3
N
H
N
a
b
Compound
R
a or b
hGnRH binding IC50 SDa (lM)
hGnRH IP inhibition IC50 SDb (lM)
rGnRH binding IC50 SDa (lM)
1a
CF3
CF3
H
CH3
C2F5
Ph
a
b
b
b
b
b
b
b
a
b
a
a
b
1.5 0.21
9.2 3.0
4.5 1.0
—
—
—
—
—
2.8 0.21
2.3 0.32
0.64 0.055
0.45 0.014
1.2 0.070
—
15 2.6
21 3.3
—
—
>100
—
—
—
5.2 0.40
3.9 0.25
0.99 0.044
0.71 0.048
—
1b
14
15
16
17
18
19
20a
20b
21
22
23
0.79 0.15
5% @ 10 lM
10% @ 10 lM
6.3 1.1
11
3-Methylphenyl
4-Methylphenyl
4-Ethylphenyl
4-Ethylphenyl
4-i-Propylphenyl
4-tert-Butylphenyl
2,4-Dimethylphenyl
47% @ 10 lM
1.3 0.12
0.22 0.05
0.12 0.01
0.12 0.002
0.050 0.0021
1.2 0.055
a
Binding to overexpressed human or rat GnRH receptors in competiton with 125I-(D-Trp6)-GnRH (Ref. 13).
Compound driven IP reduction in whole cells following stimulation with (D-Trp6)-GnRH (Ref. 14).
b
CF3
N
NH
O
N
N
HN
N
S
HN
NH
22
N
N
hGnRH binding IC50 = 50 nM
HN
N
24
S
HN
hGnRH binding IC50 = 6.2 nM
rGnRH binding IC50 = 110 nM
F3C
HN
NH
N
N
N
hIP inhibition IC50 = 14 nM
rLH release inhibition IC50 = 480 nM
5-HT1a binding = 5% @ 10.0 μM
6b
α
1 adrenergic binding = 7% @ 10.0 μM
hGnRH binding IC50 = 510 nM
Scheme 8. A combination of the features of 6b and 22 led to analogue 24 with multiple optimized features.
Co. (Milwaukee, WI) and used directly. Bulk solvents and chemicals
were purchased from EMD and used directly. 1H and 13C NMR were
recorded on Varian INOVA 400 MHz, Bruker AVANCE II 400 MHz
and Bruker AVANCE II 300 MHz instruments in the indicated sol-
vent at 20 °C. Chemical shifts (d) are expressed in ppm downfield
from tetramethylsilane (TMS). High resolution mass spectrometry
was recorded on an Agilent 6210 TOF instrument. Positive and neg-
ative electrospray mass spectrometries were recorded on Waters
ZQ or ZMD instruments. Elemental analysis was performed by Rob-
ertson Microlit (Madison, NJ) and agree with theoretical values
within 0.4%.
formic acid), 2 min, hold 1.5 min. Flow rate: 0.8 mL/min. Detec-
tion: 210–400 nm.
Method B: Column Xterra reverse phase 18, 3.5 l, 150 ꢃ 4.6 mm.
Mobile phase: 85/15–5/95 ammonium formate buffer (pH 3.5)/
acetonitrile and methanol (1:1) for 10 min, hold 4 min, 1.2 mL/
min. Detection: 210–370 nm.
Method
C: Column Xterra reverse phase C18, 3.5 l,
150 ꢃ 2.1 mm. Mobile phase: 78/22–5/95 phosphate buffer
(pH 2.1) acetonitrile and methanol (1:1) for 10 min, hold
4 min, 1.2 mL/min. Detection: 210–370 nm.
Method
D: Column: Xterra reverse phase 18, 3.5 l,
The following HPLC and LC/MS methods were used for the syn-
theses outlined in the examples:
150 ꢃ 4.6 mm. Mobile phase: 85/15–5/95 phosphate buffer
(pH 2.1)/acetonitrile and methanol (1:1) for 10 min, hold
4 min, 1.2 mL/min. Detection: 210–370 nm.
Method A: Column: Xterra MS C18, 5l, 50 ꢃ 2.1 mm. Mobile
Method E: Column: YMC CombiPrep ProC18 50 ꢃ 20 mm I.D.
phase: 90/10–5/95 water (0.1% formic acid)/acetonitrile (0.1%
S-5 lm, 12 nm. Mobile phase: 10/90 acetonitrile/water to

6626
J. C. Pelletier et al. / Bioorg. Med. Chem. 16 (2008) 6617–6640
Table 4
In vitro results for the analogues of compound 24
N
N
HN
N
Compound
Structure
hGnRH binding IC50 SDa (nM)
6.2 0.040
hIP IC50 SDb (nM)
rGnRH binding IC50 SDa (nM)
rLH release IC50 SDc (nM)
H
N
24
22 3.5
110
240
S
N
H
25
26
27
6% @ 10 lM
7% @ 10 lM
8.7 0.037
—
—
—
—
—
—
H
N
63 10
680
1500
O
N
H
H
O
O
N
28
5.7 2.4
24
72 12
480
N
H
H
N
O
O
29
30
4.9 0.20
5.5 2.3
1.7 0.65
9.4
40 17
25 9.0
41 11
59
N
H
O
O
HN
11 3.0
4.0 0.64
32 12
O
O
HN
HN
N
31
17.5 8.9
a
Binding to overexpressed human or rat GnRH receptors in competiton with 125I-(D-Trp6)-GnRH (Ref. 13).
Compound driven IP reduction in whole cells following stimulation with (D-Trp6)-GnRH (Ref. 14).
b
c
Compound driven reduction in LH release from primary rat pituitary cells stimulated with (D-Trp6)-GnRH (see Section 2).
100% acetonitrile over 10 min, ramp back to 10/90 acetonitrile/
water over 2 min. Detection: 210, 254 nm.
2.1.1. 1-Azido-3-fluoro-2-nitrobenzene (33)
A solution of 2,6-difluoronitrobenzene (4.8 g, 30 mmol) in
DMSO (35 mL) was treated with sodium azide (2.2 g, 33 mmol)
and stirred for 4 h. The mixture was diluted with ethyl acetate
(150 mL) and washed with water (2 ꢃ 150 mL), dried (MgSO4),
and evaporated to provide the title product as a semi-crystalline
solid (5.4 g, 100%). 1H NMR (300 MHz, CDCl3) d 7.51 (td, 1H,
J = 8.2 Hz, J = 6.0 Hz), 7.10 (d, 1H, J = 8.2 Hz), 7.03 (dd, 1H,
J = 8.2 Hz, J = 8.2 Hz).
Method F: Column: YMC CombiPrep ProC18 50 ꢃ 20 mm I.D. S-
5 lm, 12 nm. Mobile phase: 10/90 acetonitrile/water (0.1%
TFA in both solvents) to 100% acetonitrile (0.1% TFA) over
10 min, ramp back to 10/90 acetonitrile/water (0.1% TFA in both
solvents) over 2 min. Detection: 210, 254 nm.
Method G: (LC/MS slow gradient) Column: Waters Xterra MS
C18, 5 lm, 50 ꢃ 2 mm. Mobile phase: 90/10 to 5/95 water
(0.1% formic acid)/acetonitrile (0.1% formic acid),10 min, hold
2 min. Flow rate: 0.8 mL/min. Detection 210–400 nm.
2.1.2. 3-Azido-2-nitrophenol (56)
Method H: (LC/MS very fast gradient) Column: Phenomenex C18
monolith, 3 ꢃ 100 mm. Mobile phase: 95/5–5/95 water (0.1%
formic acid)/acetonitrile (0.1% formic acid), 1.5 min, hold
0.5 min. Flow rate: 1.5 mL/min. Detection 210–400 nm.
Method I: (LC/MS long gradient) Column: Phenomenex C18
monolith, 3 ꢃ 100 mm. Mobile phase: 95/5 to 5/95 water
(0.1% formic acid)/acetonitrile (0.1% formic acid), 4.5 min, hold
0.4 min. Flow rate: 1.5 mL/min. Detection 210–400 nm.
A solution of 1-Azido-3-fluoro-2-nitrobenzene (33, 1.0 g,
5.5 mmol) and 2-(methylsulfonyl)ethanol (1.0 g, 8.2 mmol) in
anhydrous dimethylformamide (10 mL) was cooled to 5 °C and
treated with sodium hydride (60% in mineral oil, 0.63 g,
16.5 mmol). The reaction mixture was stirred and allowed to warm
to room temperature over 1 h. The mixture was treated with 1 N
HCl (100 mL) and extracted with ethyl acetate (3ꢃ 50 mL). The
combined organic extracts were washed with water (3ꢃ 100 mL)

J. C. Pelletier et al. / Bioorg. Med. Chem. 16 (2008) 6617–6640
6627
Table 5
in acetone (10 mL) was purged with nitrogen, treated with 3-azi-
do-2-nitrophenol (56, 150 mg, 0.83 mmol) and stirred rapidly at
60 °C for an hour. The solvent was evaporated, and the residue
was dissolved in water (25 mL) and methylene chloride (25 mL).
The aqueous layer was separated, extracted with methylene chlo-
ride (25 mL) and the combined organic extracts were dried
(MgSO4), and evaporated. The crude material was purified by silica
gel chromatography (Isco instrument, 40 g normal phase column,
eluted with a gradient of 15–35% ethyl acetate in hexanes) to leave
the pure product 57 as a light yellow solid (213 mg, 87%). 1H NMR
(300 MHz, CDCl3) d = 7.39 (dd, 1H, J = 8.3 Hz, J = 8.3 Hz), 6.90 (d,
1H, J = 8.3 Hz), 6.67 (d, 1H, J = 8.3 Hz), 4.59 (s, 2H), 1.46 (s, 2H).
ADME associated molecular descriptors and in vitro pharmaceutical profiling values
for 30 and 31a
Compound
30
31
Molecular descriptors
Molecular weight
clogP
458
3.88
3
5
4
486
4.26
2
5
5
H-bond donors
H-bond acceptors
Rotatable bonds
Total polar surface area (A2)
90
81
In vitro pharma. profiling
Solubility @ pH 7.4 (lg/mL)
PAMPA (10ꢂ6 cm/s)
25
0.60
30
13
2.4
5
2.1.4. 2-(3-Azido-2-nitrophenoxy)acetic acid (58)
Rat liver microsome t1/2 (min)
A solution of tert-butyl 2-(3-azido-2-nitrophenoxy)acetate (57,
0.18 g, 0.61 mmol) in trifluoroacetic acid (0.5 mL) and methylene
chloride (0.5 mL) was stirred for 30 min then evaporated under re-
duced pressure to leave the acid product as a solid (149 mg, 100%).
1H NMR (300 MHz, CDCl3) d 7.44 (dd, 1H, J = 8.5 Hz, J = 8.4 Hz), 7.00
(d, 1H, J = 8.4 Hz), 6.87 (d, 1H, J = 8.5 Hz), 4.74 (s, 2H). LC/MS
(Method A), tR = 1.17 min (purity = 100%), [MꢂH]ꢂ = 237.
a
For a description of the assays see Ref. 25.
Table 6
Pharmacokinetic parameters for compounds 30 and 31
Compound
30
PO (PEG 400)
31
PO (PEG 400)
Method (vehicle)
IV (DMSO)
IV (DMSO)
2.1.5. 1-(3-Azido-2-nitrophenyl)piperazine (34)
Dose (mg/kg)
Plasma t1/2 (h)
Tmax (h)
Cmax (ng/mL)
AUC0–1 (ng-h/mL)
Bioavailability (%F)
Clp (mL/min/kg)
Vss (L/kg)
10
5.8
1.8
19
200
5
1.0
3.5
—
—
355
—
20
1.0
1.1
—
—
190
—
To a solution of 1-azido-3-fluoro-2-nitrobenzene (33, 0.20 g,
1.1 mmol) in dimethylsulfoxide (2 mL) was added piperazine
(0.19 g, 2.2 mmol). The mixture was stirred for 2 h, diluted with
ethyl acetate (30 mL), washed with water (2ꢃ 30 mL), dried
(MgSO4), and evaporated under reduced pressure to leave the
product as a yellow solid (0.26 g, 95%). 1H NMR (300 MHz, CDCl3)
d 7.41 (dd, 1H, J = 8.2 Hz, J = 8.0 Hz), 6.98 (d, 1H, J = 8.2 Hz), 6.96
(d, 1H, J = 8.0 Hz), 2.95 (br s, 8H). LC/MS (Method A), tR = 0.75 min
(purity = 100%), [M+H]+ = 249.
3.1
0.67
480
2717
74
—
—
47
3.1
—
—
95
4.8
and brine (100 mL). The organic layer was dried (MgSO4) and evap-
orated under reduced pressure. The crude product was purified by
silica chromatography (Isco instrument, 80 g normal phase col-
umn, eluted with a gradient of 15–50% ethyl acetate in hexanes)
to leave the product as a yellow solid (0.56 g, 57%). 1H NMR
(300 MHz, CDCl3) d 10.50 (br s, 1H), 7.48 (dd, 1H, J = 7.8 Hz,
J = 7.8 Hz), 6.93 (d, 1H, J = 7.8 Hz), 6.86 (d, 1H, J = 7.8 Hz).
2.1.6. tert-Butyl 4-(3-azido-2-nitrophenyl)piperazine-1-
carboxylate (35)
To
a solution of 1-(3-azido-2-nitrophenyl)piperazine (34,
0.23 g, 0.93 mmol) in methylene chloride (5 mL) was added di-
tert-butyl dicarbonate (0.26 g, 1.2 mmol). The solution stirred an
hour then was treated with aminomethyl polystyrene resin
(2.4 mmol/g, 0.39 g, 0.93 mmol). After an additional hour of stir-
ring the mixture was filtered, the residue was washed with meth-
ylene chloride (3ꢃ 10 mL) and the combined organic washes were
evaporated under reduced pressure to afford the product as a yel-
low gum (0.32 g, 100%). 1H NMR (300 MHz, CDCl3) d = 7.45 (dd, 1H,
J = 8.2 Hz, J = 8.2 Hz), 7.02 (d, 1H, J = 8.2 Hz), 6.98 (d, 1H, J = 8.2 Hz),
3.50 (m, 4H), 2.93 (m, 4H), 1.48 (s, 9H). LC/MS (Method A),
tR = 1.93 min (purity = 90.9%), [M+H]+ = 349.
2.1.3. tert-Butyl 2-(3-azido-2-nitrophenoxy)acetate (57)
A
mixture of tert-butylbromoacetate (0.49 g, 2.5 mmol,
0.37 mL) and well-ground potassium carbonate (1.1 g, 8.3 mmol)
300
Vehicle
30mpk cmpd 31
250
2.1.7. tert-Butyl 4-(2,3-diaminophenyl)piperazine-1-
carboxylate (36)
200
150
100
50
tert-Butyl 4-(3-azido-2-nitrophenyl)piperazine-1-carboxylate
(35, 0.30 g, 0.86 mmol) was hydrogenated over 10% Pd/C (40 mg)
at one atmosphere hydrogen pressure in methanol (10 mL) for
4 h. The catalyst was filtered, washed (methanol, 2ꢃ 10 mL), and
the filtrate was evaporated under reduced pressure to afford the
product 36 as
a
dark brown gum (0.25 g, 100%). 1H NMR
(300 MHz, CDCl3) d = 6.69 (dd, 1H, J = 7.7 Hz, J = 7.8 Hz), 6.59 (d,
1H, J = 7.8 Hz), 6.55 (d, 1H, J = 7.7 Hz), 3.79 (br s, 2H), 3.60 (m,
4H), 3.40 (br s, 2H), 2.82 (br s, 4H), 1.49 (s, 9H). LC/MS (Method
A), tR = 1.06 min (purity = 93.8%), [M+H]+ = 293.
0
0 h
1 h
2 h
4 h
6 h
2.1.8. 4-(Piperazin-1-yl)-2-(trifluoromethyl)-1H-
benzo[d]imidazole (37)
Time
A solution of tert-butyl 4-(2,3-diaminophenyl)piperazine-1-
carboxylate (36, 0.23 g, 0.79 mmol) in trifluoroacetic acid
(5 mL) was heated to 70 °C for 2 h then evaporated to dryness
Figure 3. Serum LH levels in orchidectomized rats following oral administration of
31 (levels at t = 0 adjusted to 100). Vehicle and test article groups consisted of nine
animals each. Vehicle = PEG 400.

6628
J. C. Pelletier et al. / Bioorg. Med. Chem. 16 (2008) 6617–6640
under reduced pressure. The residue was dissolved in ethyl ace-
tate (25 mL) and washed with saturated aqueous sodium bicar-
bonate solution (2ꢃ 25 mL). The organic layer was dried and
evaporated to leave the product as a brown gum (0.19 g, 89%).
1H NMR (300 MHz, DMSO-d6) d = 7.26 (dd, 1H, J = 8.0 Hz,
J = 7.7 Hz), 7.19 (d, 1H, J = 8.0 Hz), 6.72 (d, 1H, J = 7.7 Hz), 3.62
(br s, 4H), 3.26 (m, 4H). LC/MS (Method A), tR = 0.32 min (pur-
ity = 84.5%), [M+H]+ = 271.
(0.2 mL) and water (0.2 mL) and stirred for 90 min. Sodium
cyanoborohydride (47 mg, 0.74 mmol) was added and stirring
continued an additional 90 min. The reaction mixture was diluted
with ethyl acetate (20 mL) and washed with saturated aqueous
sodium bicarbonate solution (15 mL), water (15 mL), and brine
(15 mL). The organic layer was dried (MgSO4), and evaporated
under reduced pressure to leave the crude product which was
purified by reversed phase HPLC (Method 2). The product
fractions were combined, diluted with ethyl acetate (50 mL),
washed with saturated aqueous sodium bicarbonate solution
(50 mL), dried (MgSO4) and evaporated to leave the product 38
as a yellow gum (75 mg, 44%). A 64 mg sample of 38 (0.14 mmol)
was hydrogenated over 10% Pd/C (10 mg) in methanol (2 mL) at 1
atmosphere hydrogen pressure for 2 h. The catalyst was
filtered, washed with methanol (2ꢃ 5 mL), and the combined
filtrates were evaporated under reduced pressure to leave the
phenylenediamine product 39 as a brown gum (50 mg, 88%). A
22 mg sample of 39 (54 lmol) was dissolved in tetrahydrofuran
(0.50 mL) in an 8-mL scintillation vial and treated with 1,10-thi-
ocarbonyldiimidazole (19 mg, 0.11 mmol). The reaction mixture
was purged with nitrogen, capped and heated to 70 °C for 3 h.
Water (0.1 mL) was added and the product was purified by
reversed phase HPLC (Method 2), and the product fractions
were lyophilized. The product 5b was obtained as a yellow
powder (14 mg, 58%). 1H NMR (500 MHz, DMSO-d6) d = 13.78
(br s, 1H), 12.80 (br s, 1H), 12.47 (br s, 1H), 7.24 (dd, 1H,
J = 8.0 Hz, J = 8.2 Hz), 7.09 (d, 1H, J = 7.4 Hz), 7.05 (dd, 1H,
J = 7.4 Hz, J = 7.7 Hz), 7.01 (dd, 1H, J = 7.7 Hz, J = 1.1 Hz), 6.98
(dd, 1H, J = 7.7 Hz, J = 1.1 Hz), 6.64 (d, 1H, J = 6.9 Hz), 3.55 (br s,
4H), 2.99 (t, 2H, J = 7.4 Hz), 2.73 (br s, 4H), 2.63 (t, 3H,
J = 7.4 Hz). Anal. Calcd for C21H22F3N6S: C, 56.49; H, 4.74; N,
18.82. Found: C, 56.35; H, 5.02; N, 18.70. ESMS [MꢂH]ꢂ = 445,
[M+H]+ = 447.
2.1.9. 2-(3-Azido-2-nitrophenoxy)-1-(4-(2-(trifluoromethyl)-
1H-benzo[d]imidazol-4-yl)piperazin-1-yl)ethanone (59)
A sample of 2-(3-azido-2-nitrophenoxy)acetic acid (58, 75 mg,
0.32 mmol) was dissolved in anhydrous dimethylformamide
(2 mL) and treated with 4-(piperazin-1-yl)-2-(trifluoromethyl)-
1H-benzo[d]imidazole (37, 94 mg, 0.35 mmol) and diisopropyl-
ethylamine (45 mg, 0.35 mmol, 63 lL). The mixture was purged
with a nitrogen atmosphere and cooled in an ice bath. HATU
(0.13 g, 0.35 mmol) was added and the mixture stirred for 2 h.
The reaction mixture was treated with water (0.5 mL), and the
product was purified directly without workup by reversed phase
HPLC (Method E) to leave the product as a foamy solid (88 mg,
56%). 1H NMR (300 MHz, CD3OD) d = 7.44 (dd, 1H, J = 8.5,
J = 8.3), 7.25 (dd, 1H, J = 8.2 Hz, J = 7.7 Hz), 7.18 (d, 1H,
J = 7.7 Hz), 6.99 (d, 1H, J = 8.5 Hz), 6.94 (d, 1H, J = 8.3 Hz), 6.72
(d, 1H, J = 7.6 Hz), 4.83 (s, 2H), 3.79 (m, 2H), 3.72 (m, 2H), 3.38
(m, 2H), 3.32 (m, 2H). LC/MS (Method B), tR = 1.33 min (pur-
ity = 99.9%), [M+H]+ = 491.
2.1.10. 2-(2-Thioxo-2,3-dihydro-1H-benzo[d]imidazol-4-yloxy)-
1-(4-(2-(trifluoromethyl)-1H-benzo[d]imidazol-4-yl)piperazin-
1-yl)ethanone (60 and 2)
2-(3-Azido-2-nitrophenoxy)-1-(4-(2-(trifluoromethyl)-1H-ben-
zo[d]imidazol-4-yl)piperazin-1-yl)ethanone (59, 80 mg, 0.16 mmol)
was hydrogenated over 10% palladium on carbon (25 mg) in meth-
anol (2 mL) for 5 h. The catalyst was filtered over diatomaceous
earth, washed with methanol (2ꢃ 5 mL), and the combined
filtrates were evaporated under reduced pressure to leave the
phenylenediamine 60, which was used without further purification
or characterization. The residue was dissolved in anhydrous
tetrahydrofuran (3 mL) and treated with 1,10-thiocarbonyldiimi-
dazole (43 mg, 0.24 mmol). The reaction mixture stirred for 18 h
and then it was diluted with water (25 mL) and extracted with
ethyl acetate (3ꢃ 25 mL). The combined organic extracts were
dried (MgSO4) and evaporated. The crude product was purified
by silica gel chromatography (Isco instrument, 12 g normal phase
column, eluted with a gradient of 50–100% ethyl acetate in
hexanes) to leave the pure product 2 as a gum (14 mg, 18%). 1H
NMR (300 MHz, CD3COCD3) d = 7.25–7.10 (m, 3H), 7.03 (dd, 1H,
J = 8.1 Hz, J = 8.0 Hz), 6.84 (d, 1H, J = 8.1 Hz), 6.81 (d, 1H,
J = 8.3 Hz), 6.67 (br s, 1H), 5.01 (s, 1H), 3.60 (m, 4H), 2.77 (dd,
2H, J = 7.5 Hz, J = 8.3 Hz), 2.55 (dd, 2H, J = 6.0 Hz, J = 7.0 Hz). HPLC
(Method D), tR (purity) = 8.67 min (79.6%). HR ESMS
[M+H]+ = 477.1313, calcd for C21H19F3N6O2S: 477.1315.
2.1.12. 2-(Trifluoromethyl)-4-(4-(2-(2-(trifluoromethyl)-1H-
benzo[d]imidazol-4-yl)ethyl)piperazin-1-yl)-1H-benzo[d]-
imidazole (5a)
A
sample of the phenylenediamine (39, 22 mg, 54 lmol)
was dissolved in trifluoroacetic acid (1 mL) and heated to
70 °C for 3 h. The solvent was evaporated and the residue
was purified by reversed phase HPLC (Method 2). The product
fractions were lyophilized to leave 5a as a free base (16 mg,
61%). 1H NMR (500 MHz, DMSO-d6) d = 13.80 (br s, 2H), 7.47
(br s, 1H), 7.30 (dd, 1H, J = 7.4 Hz, J = 7.7 Hz), 7.25 (dd, 1H,
J = 6.0 Hz, J = 6.9 Hz), 7.24 (d, 1H, J = 8.0 Hz), 7.11 (br s, 1H),
6.65 (br s, 1H), 3.50 (br s, 4H), 3.20 (t, 2H, J = 7.4 Hz), 2.77
(m, 6H). Anal. Calcd for
C22H20F6N6: C, 54.77; H, 4.18; N,
17.42. Found: C, 54.60; H, 4.30; N, 17.05. ESMS [MꢂH]ꢂ = 481,
[M+H]+ = 483.
2.1.13. 2-(3-Chloropropoxy)-6-nitroaniline (55)
A solution of 2-amino-3-nitrophenol (54, 2.0 g,12.98 mmol) in
dimethylformamide (26 mL) was treated with potassium carbon-
ate (1.97 g, 14.27 mmol) followed by 1-bromo-3-chloropropane
(1.92 mL, 19.46 mmol), and the reaction mixture was heated to
90 °C in an oil bath and stirred overnight. The reaction mixture
was cooled to room temperature, diluted with ethyl acetate
(300 mL), washed with water (3ꢃ 75 mL), 1 N sodium hydroxide
(75 mL), brine (100 mL), dried (MgSO4), and concentrated to dry-
ness. The crude product was purified by flash chromatography on
silica gel eluted with 20% ethyl acetate in hexanes to yield 1.38 g
of 2-(3-chloropropoxy)-6-nitroaniline, 55, as an orange solid
(46%). 1H NMR (DMSO) d = 7.59 (d, 1H, J = 8.8 Hz), 7.11 (m, 3H),
6.59 (t, 1H, J = 8.4 Hz), 4.15 (t, 2H, J = 5.7 Hz), 3.92 (t, 2H,
J = 6.5 Hz), 2.24 (t, 2H, J = 6.1 Hz). LC/MS (Method A), tR = 1.60 min,
[M+H]+ = 231/233 [Cl pattern].
2.1.11. 4-(2-(4-(2-(Trifluoromethyl)-1H-benzo[d]imidazol-4-
yl)piperazin-1-yl)ethyl)-1H-benzo[d]imidazole-2(3H)-thione
(47, 38, 39 and 5b)
A
solution of 2,3-dinitrotoluene (46, 7.3 g, 40 mmol) in
dimethylformamide (20 mL) was treated with dimethylformam-
ide dimethylacetal (9.9 g, 83 mmol, 11 mL). The mixture was
stirred at 150 °C for 2.5 h. The solvents were removed by vacuum
distillation, and the crude product was crystallized from acetoni-
trile. The solid was filtered and air-dried to leave 47 as a dark pur-
ple solid. A 0.20 g sample of this solid and 4-(piperazin-1-yl)-2-
(trifluoromethyl)-1H-benzo[d]imidazole (37, 0.10 g, 0.37 mmol)
was dissolved in a mixture of tetrahydrofuran (2 mL), acetic acid

J. C. Pelletier et al. / Bioorg. Med. Chem. 16 (2008) 6617–6640
6629
2.1.14. 2-Nitro-6-(3-(4-(2-(trifluoromethyl)-1H-benzo[d]-
imidazol-4-yl)piperazin-1-yl)propoxy)aniline (40)
2.72 (t, 2H, J = 7.6 Hz), 1.90 (m, 2H). LC/MS (Method A),
tR = 0.91 min (purity = 100%); ESMS [M+H]+ = 197.
A
solution of 4-(piperazin-1-yl)-2-(trifluoromethyl)-1H-
benzo[d]imidazole (37, 0.10 g, 0.26 mmol) in DMSO (3 mL) was
added triethylamine (0.08 mL, 0.57 mmol) followed by 2-(3-chlo-
ropropoxy)-6-nitroaniline (55, 0.09 g, 0.39 mmol), and the reaction
was heated to 90 °C in an oil bath and stirred for 20 h. The reaction
mixture was cooled to room temperature, diluted with ethyl ace-
tate (100 mL) washed with water (3ꢃ 50 mL) and brine (50 mL),
and dried (MgSO4). The solvents were concentrated to dryness
and the crude product was purified by flash chromatography on
silica gel with 5% methanol in methylene chloride as eluant to yield
50 mg of 40 as an orange-yellow solid. (41% yield). 1H NMR (CDCl3)
d = 7.72 (d, 1H, J = 8.9 Hz), 7.27 (m, 2H), 7.08 (br s, 1H), 6.91 (d, 1H,
J = 7.6 Hz), 6.70 (br s, 1H), 6.60 (m, 3H), 4.11 (dd, 2H, J = 11.9 Hz,
J = 6.0 Hz), 3.58 (br s, 4H), 2.71 (br s, 4H), 2.62 (t, 2H, J = 7.1 Hz),
2.08 (m, 2H). LC/MS (Method A), tR = 0.94 min [M+H]+ = 465,
[MꢂH]ꢂ = 463.
2.1.18. 2-(3-Chloropropyl)-6-nitroaniline (53)
A solution of 3-(2-amino-3-nitrophenyl)propan-1-ol (52, 0.58 g,
3.0 mmol) and triethylamine (0.38 g, 3.8 mmol, 0.54 mL) in meth-
ylene chloride (40 mL) was stirred and cooled in an ice bath. Thio-
nyl chloride (0.45 g, 3.8 mmol, 0.27 mL) was added dropwise to the
reaction mixture. After the addition was completed the mixture
was allowed to warm to room temperature and stirred for 18 h.
Ice (50 g) and 1 N HCl were added to the reaction mixture. After
the ice melted the organic layer was separated and washed with
saturated aqueous sodium bicarbonate solution (50 mL) and brine
(50 mL). The organic layer was dried (Na2SO4) and evaporated in
vacuo. The residue was filtered through a pad of silica gel eluted
with 50% ethyl acetate in hexanes to leave the product as a yel-
low-orange powder (0.37 g, 57%). 1H NMR (300 MHz, CDCl3)
d = 8.06 (dd, 1H, J = 8.7 Hz, J = 1.3 Hz), 7.30 (d, 1H, J = 8.6 Hz), 6.67
(dd, 1H, J = 8.7 Hz, J = 8.6 Hz), 6.35 (br s, 2H), 3.63 (t, 2H,
J = 6.0 Hz), 2.78 (t, 2H, J = 7.3 Hz), 2.11 (m, 2H). LC/MS (Method
A), tR = 1.64 min (purity = 90.0%), ESMS [M+H]+ = 215, 217 (Cl
pattern).
2.1.15. 3-(3-(4-(2-(Trifluoromethyl)-1H-benzo[d]imidazol-4-
yl)piperazin-1-yl)propoxy)benzene-1,2-diamine (43)
2-Nitro-6-(3-(4-(2-(trifluoromethyl)-1H-benzo[d]imidazol-4-
yl)piperazin-1-yl)propoxy)aniline (40, 50 mg, 0.108 mmol) was
hydrogenated at 1 atmosphere H2 pressure in methanol (2 mL)
over 10% Pd/C (11 mg) for 18 h. The reaction mixture was filtered
and washed with methanol (2ꢃ 5 mL) and the filtrate was concen-
trated to dryness in vacuo to leave the product 43 as an off-white
solid (49 mg, 100%). 1H NMR (DMSO) d = 14.0 (br s, 1H), 7.31 (t, 1H,
J = 7.8 Hz), 7.19 (d, 1H, J = 8.1 Hz), 6.74 (d, 1H, J = 7.8 Hz), 6.40 (m,
1H), 6.26 (m, 2H), 3.99 (m, 2H), 3.58 (br s, 4H), 2.71 (br s, 4H), 2.62
(m, 2H), 2.08 (m, 2H). LC/MS (Method A), tR = 0.48 min,
[M+H]+ = 435, [MꢂH]ꢂ = 433.
2.1.19. 2-Nitro-6-(3-(4-(2-(trifluoromethyl)-1H-benzo[d
]imidazol-4-yl)piperazin-1-yl)propyl)aniline (41)
A
solution of 2-(3-chloropropyl)-6-nitroaniline (53, 0.29 g,
1.3 mmol), 4-(piperazin-1-yl)-2-(trifluoromethyl)-1H-benzo[d]-
imidazole (37, 0.30 g, 1.1 mmol), and diisopropylethylamine
(0.14 g, 1.1 mmol, 0.20 mL) in anhydrous dimethylformamide
(20 mL) was stirred at 80 °C for 18 h. The mixture was cooled to
room temperature, diluted with ethyl acetate (50 mL) and washed
with water (3ꢃ 50 mL) and brine (50 mL). The organic layer was
dried (Na2SO4) and evaporated. The residue was purified by silica
gel chromatography eluted with a gradient of methanol in methy-
lene chloride (2–5–10%) to leave the product as an orange powder
(0.11 g, 22%). 1H NMR (300 MHz, CDCl3) d = 9.70 (br s, 1H), 8.04
(dd, 1H, J = 8.7 Hz, J = 1.4 Hz), 7.42 (br s, 1H), 7.30 (dd, 1H,
J = 9.4 Hz, J = 1.3 Hz), 7.09 (d, 1H, J = 8.1 Hz), 6.72 (d, 1H,
J = 7.8 Hz), 6.62 (dd, 1H, J = 8.7 Hz, J = 8.1 Hz), 3.64 (br s, 4H), 2.77
(m, 4H), 2.72 (t, 2H, J = 6.5 Hz), 2.42 (t, 2H, J = 6.0 Hz), 1.90 (m,
2H). ESMS [M+H]+ = 449.
2.1.16. 4-(3-(4-(2-(Trifluoromethyl)-1H-benzo[d]imidazol-4-
yl)piperazin-1-yl)propoxy)-1H-benzo[d]imidazole-2(3H)-thione
(3)
A solution of 3-(3-(4-(2-(trifluoromethyl)-1H-benzo[d]imida-
zol-4-yl)piperazin-1-yl)propoxy)benzene-1,2-diamine (43, 50 mg,
0.115 mmol) in THF (5 mL) was treated with thiocarbonyldiimi-
dazole (31 mg, 0.17 mmol), and the mixture was stirred and
heated to 70 °C for 18 h. The crude product was purified by direct
injection reversed phase HPLC (Method E) to yield 3 as a light
yellow solid (22 mg, 40% yield). 1H NMR (DMSO) d = 14.0 (s, 1H),
12.61 (d, 2H, J = 21 Hz), 7.32 (t, 1H, J = 7.9 Hz), 7.21 (m, 1H), 7.08
(t, 1H, J = 8.1 Hz), 6.78 (m, 3H), 4.45 (d, 2H, J = 10.7 Hz), 4.20 (t,
2H, J = 5.3 Hz), 3.77–3.54 (m, 4H), 3.37 (q, 2H, J = 10.4 Hz), 3.19
(m, 2H), 2.21 (m, 2H). HPLC (Method C), tR = 12.9 min (pur-
ity = 93.6%). HR ESMS [M+H]+ = 477.1689, calcd for C22H23F3N6OS:
471.1686.
2.1.20. 2-(Trifluoromethyl)-4-(3-(4-(2-(trifluoromethyl)-1H-
benzo[d]imidazol-4-yl)piperazin-1-yl)propyl)-1H-benzo[d]-
imidazole (44 and 4)
A
solution
of
2-nitro-6-(3-(4-(2-(trifluoromethyl)-1H-
benzo[d]imidazol-4-yl)piperazin-1-yl)propyl)aniline (41, 0.34 g,
0.76 mmol) in N-methylpyrrolidinone (10 mL) was treated with ti-
n(II) chloride dihydrate (0.86 g, 4.6 mmol) and heated to 65 °C for
3.5 h. Another portion of tin(II) chloride dihydrate (0.43 g) was
added and heating continued for 2 h. The mixture was cooled to
room temperature and stirred 18 h then diluted with methylene
chloride (50 mL) and extracted with 1 N HCl (3ꢃ 50 mL). The com-
bined acidic layers were neutralized with 25% sodium hydroxide
solution and extracted with ethyl acetate (3ꢃ 50 mL). The com-
bined organic layers were washed with water (5ꢃ 50 mL) and
brine (50 mL) and dried (Na2SO4) and evaporated to leave 44 as a
brown powder (0.20 g, 63%). A portion of this powder (45 mg,
0.11 mmol) was dissolved in trifluoroacetic acid (4 mL), capped
tightly and heated to 70 °C for 2 h. The solvent was evaporated
and the residue was purified by reversed phase HPLC (Method 2).
The product fractions were lyophilized to leave the product 4 as
a bistrifluoroacetate salt (28 mg, 42%). 1H NMR (300 MHz, DMSO-
d6) d = 14.01 (br s, 1H), 9.58 (br s, 1H), 7.53 (d, 1H, J = 6.1 Hz),
7.38 (br s, 1H), 7.30 (dd, 1H, J = 8.0 Hz, J = 8.0 Hz), 7.25 (br s, 1H),
7.19 (d, 1H, J = 8.0 Hz), 6.73 (d, 1H, J = 7.7 Hz), 4.40 (d, 2H,
2.1.17. 3-(2-Amino-3-nitrophenyl)propan-1-ol (52)
A solution of 2-allyl-6-nitroaniline (51, 1.94 g, 10.9 mmol) in
THF (10 mL) under a nitrogen atmosphere was cooled in ice, stirred
and treated with a solution of borane in THF (1.0 M, 10.9 mL,
10.9 mmol). After 2.5 h the reaction mixture was treated with so-
dium perborate tetrahydrate (1.67 g, 10.9 mmol) and water
(20 mL). The mixture was allowed to warm to room temperature
and stirred for 18 h. The tetrahydrofuran was evaporated, the
aqueous residue was treated with ethyl acetate (50 mL) and brine
(30 mL). The aqueous layer was further extracted with ethyl ace-
tate (50 mL) and the combined layers were dried (Na2SO4) and
evaporated. The residue was purified by silica gel chromatography
using a gradient of ethyl acetate in hexanes (40–60–75–100%) to
leave 52 (1.3 g, 62%). 1H NMR (300 MHz, CDCl3) d = 8.04 (dd, 1H,
J = 8.7 Hz, J = 1.3 Hz), 7.28 (dd, 1H, J = 8.7 Hz, J = 1.3 Hz), 6.64 (dd,
1H, J = 8.7 Hz, J = 8.7 Hz), 6.55 (br s, 1H), 3.74 (t, 2H, J = 5.8 Hz),

6630
J. C. Pelletier et al. / Bioorg. Med. Chem. 16 (2008) 6617–6640
J = 12.6 Hz), 3.69 (d, 2H, J = 11.5 Hz), 3.29 (br s, 4H), 3.12 (t, 2H,
J = 12.6 Hz), 3.05 (br s, 4H). Mass. Spec. (ESI) m/z [M+H]+ = 497.
Anal. Calcd for C27H24F12N6O4: C, 44.76; H, 3.34; N, 11.60. Found:
C, 44.86; H, 3.71; N, 11.12.
imidazole (37, 0.11 g, 0.42 mmol), and diisopropylethylamine
(81 mg, 0.63 mmol, 0.11 mL) in anhydrous dimethylformamide
(10 mL) was stirred at 80 °C for 18 h. The mixture was cooled
to room temperature, diluted with water (50 mL) and ethyl ace-
tate (50 mL). The layers were separated, and the aqueous layer
was extracted with ethyl acetate (50 mL). The combined organic
layers were washed with water (4ꢃ 50 mL) and brine (50 mL),
dried (Na2SO4) and evaporated under reduced pressure. The
product was purified by flash chromatography on silica gel
eluted with a gradient of 2–3% methanol in methylene chloride
to leave 42 as a foamy, orange solid (0.15 g, 85%). 1H NMR
(300 MHz, CDCl3) d = 11.22 (br s, 1H), 8.09 (dd, 1H, J = 8.7 Hz,
J = 1.0 Hz), 7.69 (br s, 2H), 7.25 (dd, 1H, J = 8.0 Hz, J = 8.0 Hz),
7.18 (d, 1H, J = 6.8 Hz), 7.09 (d, 1H, J = 8.1 Hz), 6.70 (d, 1H,
J = 7.8 Hz), 6.58 (dd, 1H, J = 8. Hz, J = 8.6 Hz), 3.56 (s, 2H), 3.52
(br s, 4H), 2.54 (br s, 4H). LC/MS (Method A), tR = 1.04 min. ESMS
[M+H]+ = 421.
2.1.21. 2-Amino-3-nitrobenzoic acid (48)
To a suspension of 3-nitrophthallic anhydride (5.0 g, 26 mmol) in
benzene (anhydrous, 70 mL) was added trimethylsilyl azide, and the
resulting suspension was heated in an 80 °C bath for 2.5 h. The
solution was cooled to rt and concentrated under reduced pressure
to one-fourth its original volume. The concentrated solution was
heated in a 100 °C bath overnight. The yellow solution was cooled
and concentrated to afford a yellow solid. To the crude material
was added EtOH (20 mL), and the solution was concentrated to
dryness to provide 8-nitro isatoic anhydride (5.4 g, 99%). 1H NMR
300 MHz (300 MHz, DMSO-d6): d = 11.2 (br s, 1H), 8.51 (dd, 1H,
J = 8.5, 1.5 Hz), 8.35 (dd, 1H, J = 7.7, 1.5 Hz), 7.42 (t, 1H, J = 8.0 Hz).
A suspensionof 8-nitro isatoic anhydride (3.0 g, 14.4 mmol) in concd
HCl (100 mL) was heated in a 95 °C bath for 3 h. The solution was
cooled to rt. The precipitate which formed was collected to provide
the product 48 as a yellow powder (1.50 g, 57%). LC/MS (Method
A), tR = 1.05 min (purity = 95%), MS (ESI-NEG) [MꢂH]ꢂ = 181.
2.1.25. 3-((4-(2-(Trifluoromethyl)-1H-benzo[d]imidazol-4-
yl)piperazin-1-yl)methyl)benzene-1,2-diamine (45)
To
a
solution of 2-nitro-6-((4-(2-(trifluoromethyl)-1H-
benzo[d]imidazol-4-yl)piperazin-1-yl)methyl)aniline (42, 0.15 g,
0.36 mmol) in N-methlpyrrolidinone (10 mL) was added tin(II)
chloride dihydrate (0.41 g, 2.1 mmol) and the mixture was
heated to 70 °C for 1.5 h. Another portion of tin(II) chloride
dehydrate was added (0.41 g, 2.1 mmol), and heating continued
another 1.5 h. The mixture was cooled to room temperature, di-
luted with 1 N HCl (50 mL) and methylene chloride (50 mL). The
layers were separated, the organic layer was extracted with 1 N
HCl (50 mL), and the combined aqueous layers were neutralized
to pH 7 with 25% aqueous sodium hydroxide, extracted with
ethyl acetate (3ꢃ 25 mL) and the combined organic layers were
dried (Na2SO4) and evaporated to a yellow oil (70 mg, 50%). 1H
NMR (300 MHz, MeOH-d4) d = 7.26 (dd, 1H, J = 7.9 Hz,
J = 7.9 Hz), 7.17 (d, 1H, J = 8.0 Hz), 6.74 (d, 1H, J = 8.8 Hz), 6.70
(dd, 1H, J = 7.4 Hz, J = 3.0 Hz), 6.56 (d, 1H, J = 6.4 Hz), 6.55 (d,
1H, J = 3.0 Hz), 3.58 (s, 2H), 3.40 (br s, 4H), 2.67 (m, 4H). LC/
2.1.22. (2-Amino-3-nitrophenyl)methanol (49)
To a suspension of 2-amino-3-nitro benzoic acid (48, 2.12 g,
10 mmol) in benzene (75 mL) was added thionyl chloride
(1.8 mL, 25 mmol) dropwise. The suspension was heated to reflux
overnight. The mixture was cooled to room temperature and con-
centrated under reduced pressure to afford a golden solid. The
crude material was dissolved in THF (40 mL) and cooled in an ice
bath. Sodium borohydride (0.83 g, 22 mmol) was added in por-
tions, and the reaction was allowed to come to room temperature
with stirring. The mixture was again cooled in ice, and H2O (15 mL)
was slowly added to the reaction. Once gas evolution had subsided,
the solution was concentrated under reduced pressure. The residue
was dissolved in EtOAc (200 mL) and washed with saturated aque-
ous NaHCO3 (2ꢃ 50 mL) and brine (50 mL). The organic layer was
dried (Na2SO4), filtered, and concentrated under reduced pressure.
The reside was adsorbed onto silica gel and purified by column
chromatography, eluting with a gradient of 25% EtOAc/hexane to
50% EtOAc/hexane to afford the alcohol 49 (0.89 g, 46%) as an or-
ange solid. 1H NMR 300 MHz (300 MHz, DMSO-d6): d = 7.92 (dd,
1H, J = 8.7, 1.4 Hz), 7.49 (d, 1H, J = 6.8 Hz), 7.12 (br s, 2H), 6.66
(dd, 1H, J = 8.7, J = 7.1 Hz), 5.45 (t, 1H, J = 5.4 Hz), 4.52 (d, 2H,
J = 5.4 Hz). MS (ESI-POS): [M+H]+ = 169.
MS
(Method
A),
tR = 0.71 min
(purity = 94.0%);
ESMS
[M+H]+ = 391.
2.1.26. 4-((4-(2-(Trifluoromethyl)-1H-benzo[d]imidazol-4-
yl)piperazin-1-yl)methyl)-1H-benzo[d]imidazole-2(3H)-thione
(6b)
A solution of 3-((4-(2-(trifluoromethyl)-1H-benzo[d]imidazol-
4-yl)piperazin-1-yl)methyl)benzene-1,2-diamine
(45,
50 mg,
0.13 mmol) and 1,10-thiocarbonyldiimidazole (30 mg, 0.22 mmol)
in anhydrous THF (10 mL) under a nitrogen atmosphere was stir-
red and heated to 70 °C for 2 h. The mixture was cooled to room
temperature, water (0.2 mL) was added, and the mixture was
evaporated. The crude product was purified by reversed phase
HPLC (Method 2) and the product fractions were lyophilized to
leave 6b as a bistrifluoroacetate salt (18 mg, 32%). 1H NMR
(300 MHz, DMSO-d6) d = 14.00 (br s, 1H), 12.88 (br s, 1H),
12.80 (br s, 1H), 9.80 (br s, 1H), 7.18–7.35 (m, 5H), 6.75 (d,
1H, J = 7.3 Hz), 4.56 (s, 2H), 4.30–4.45 (m, 2H), 3.32–3.40 (m,
4H), 3.08–3.20 (m, 2H). Mass. Spec. (ESI) m/z [M+H]+ = 433. Anal.
Calcd for C24H21F9N6O4S: C, 43.64; H, 3.20; N, 12.72. Found: C,
43.75; H, 3.18; N, 13.00.
2.1.23. 2-Chloromethyl-6-nitroaniline (50)
To a solution of (2-amino-3-nitrophenyl)methanol (49, 5.83 g,
35 mmol) in CH2Cl2 (250 mL) cooled in an ice bath was added tri-
ethyl amine (6.3 mL, 45 mmol), followed by dropwise addition of
thionyl chloride (3.16 mL, 43 mmol). The solution was stirred at
room temperature overnight. The ice bath was replaced, and ice
was added to quench the reaction. The reaction mixture was
washed with 0.1 N HCl (30 mL), H2O (2ꢃ 30 mL), and brine
(30 mL). The organic layer was dried (Na2SO4), filtered, and concen-
trated under reduced pressure. The crude material was adsorbed
onto silica gel and purified by column chromatography, eluting
with a gradient of 20% EtOAc/hexane to 30% EtOAc/hexane to afford
the chloromethyl compound 50 (5.3 g, 82%) as a yellow powder. 1H
NMR (300 MHz, DMSO-d6): d = 7.89 (dd, 1H, J = 8 Hz, J = 2 Hz), 7.44
(d, 1H, J = 7 Hz), 7.12 (br s, 2H), 6.69–6.66 (m, 1H), 4.61 (s, 2H).
2.1.27. 2-(Trifluoromethyl)-4-(4-((2-(trifluoromethyl)-1H-
benzo[d]imidazol-4-yl)methyl)piperazin-1-yl)-1H-
benzo[d]imidazole (6a)
2.1.24. 2-Nitro-6-((4-(2-(trifluoromethyl)-1H-
A solution of 3-((4-(2-(trifluoromethyl)-1H-benzo[d]imidazol-
benzo[d]imidazol-4-yl)piperazin-1-yl)methyl)aniline (42)
4-yl)piperazin-1-yl)methyl)benzene-1,2-diamine
(45,
80 mg,
A
solution of 2-chloromethyl-6-nitroaniline (50, 79 mg,
0.20 mmol) in trifluoroacetic acid (4 mL) was capped tightly and
0.42 mmol), 4-(piperazin-1-yl)-2-(trifluoromethyl)-1H-benzo[d]-
heated to 70 °C for 2 h. The solvent was evaporated and the residue

J. C. Pelletier et al. / Bioorg. Med. Chem. 16 (2008) 6617–6640
6631
was purified by reversed phase HPLC (Method 2), and the product
fractions were lyophilized to leave the product 6a as a powder
(33 mg, 24% based on the bistrifluoroacetate salt). 1H NMR
(300 MHz, DMSO-d6) d = 14.38 (br s, 1H), 13.99 (br s, 1H), 10.00
(br s, 1H), 7.79 (br s, 1H), 7.60 (d, 1H, J = 7.3 Hz), 7.53 (br s, 1H),
7.27 (dd, 1H, J = 7.9 Hz, J = 7.9 Hz), 7.17 (d, 1H, J = 6.1 Hz), 6.70
(br s, 1H), 4.81 (s, 2H), 4.38 (br s, 2H), 3.45 (br s, 2H), 3.15 (br s,
4H). Mass. Spec. (ESI) m/z [MꢂH]ꢂ = 467. Anal. Calcd for
evaporated under reduced pressure to leave the product as an
amorphous solid. Used as is with no characterization.
2.1.32. 5-(2-(4-(2-(Trifluoromethyl)-1H-benzo[d]imidazol-4-
yl)piperazin-1-yl)ethoxy)-1H-benzo[d]imidazole-2(3H)-thione
(7)
A
solution of 4-(piperazin-1-yl)-2-(trifluoromethyl)-1H-
benzo[d]imidazole (37, 46 mg, 0.17 mmol) and 2-(2-thioxo-2,3-
dihydro-1H-benzo[d]imidazol-5-yloxy)acetaldehyde (65, 29 mg,
0.14 mmol) in N-methylpyrrolidinone (1.3 mL) was stirred and trea-
ted with sodium triacetoxyborohydride (40 mg, 0.19 mmol) for an
hour. The reaction mixture was diluted with water (0.3 mL) and
purified by reversed phase HPLC (Method F) to leave the product 7
as an amorphous solid bistrifluoroacetate salt (20 mg, 21%). 1H
NMR (300 MHz, DMSO-d6) d = 14.00 (br s, 1H), 12.55 (br s, 1H),
12.44 (br s, 1H), 9.90 (br s, 1H), 7.31 (dd, 1H, J = 8.0 Hz, J = 7.8 Hz),
7.20 (d, 1H, J = 8.0 Hz), 7.09 (d, 1H, J = 8.4 Hz), 6.85 (d, 1H, obscured),
6.83 (s, 1H), 6.77 (d, 1H, J = 7.8 Hz), 4.40 (m, 2H), 3.65 (m, 10H). LC/
MS (Method A), tR = 0.74 min (purity = 100%), [M+H]+ = 463. Anal.
Calcd for C25H23F9N6O5S: C, 43.48; H, 3.36; N, 12.17. Found: C,
43.91; H, 3.81; N, 12.51.
C25H20F12N6O4: C, 43.11; H, 2.89; N, 12.07. Found: C, 43.28; H,
3.38; N, 12.27.
2.1.28. 4-(2,2-Diethoxyethoxy)-1,2-dinitrobenzene (62)
To a solution of 2,2-diethoxyethanol (0.25 g, 1.9 mmol) in THF
(5 mL) under nitrogen atmosphere was added a 60% dispersion of
sodium hydride in mineral oil (76 mg, 1.9 mmol). The mixture stir-
red for 20 min and was then added dropwise via syringe to an ice
cooled solution of 4-fluoro-1,2-dinitrobenzene (61, 0.35 g,
1.9 mmol) in THF (5 mL) under a nitrogen atmosphere. The reac-
tion mixture stirred an hour and was diluted with water (50 mL)
and ethyl acetate (50 mL). The organic layer was separated, dried
(MgSO4) and evaporated. The crude product was purified by chro-
matography on silica gel eluted with 25% ethyl acetate in hexanes.
The purified product 62 was isolated as an oil (0.22 g, 39%). 1H
NMR (300 MHz, CDCl3) d = 8.02 (d, 1H, J = 9.0 Hz), 7.29 (d, 1H,
J = 2.5 Hz), 7.18 (dd, 1H, J = 9.0 Hz, J = 2.5 Hz), 4.85 (t, 1H,
J = 5.0 Hz), 4.16 (d, 2H, J = 5.0 Hz), 3.79 (dq, 2H, J = 9.0 Hz,
J = 7.1 Hz), 3.63 (dq, 2H, J = 9.0 Hz, J = 7.1 Hz), 1.27 (t, 6H,
J = 7.1 Hz).
2.1.33. tert-Butyl 4-(3,4-dinitrophenyl)piperazine-1-
carboxylate (66)
A mixture of 4-fluoro-1,2-dinitrobenzene (61, 0.50 g, 2.7 mmol),
tert-butyl piperazine-1-carboxylate (0.65 g, 3.5 mmol), and N-
methylmorpholine on polystyrene (3.4 mmol/g, 1.6 g, 5.4 mmol)
in N-methylpyrrolidinone (10 mL) was shaken for 2 h. Isocyanate
on polystyrene resin (1.2 mmol/g, 1.2 g, 1.4 mmol) and additional
solvent (10 mL) were added and the mixture was shaken at 60 °C
for 2 h. After cooling to room temperature the mixture was filtered,
the residue washed (ethyl acetate, 3ꢃ 20 mL) and the combined or-
ganic layers were washed with water (5ꢃ 100 mL), dried (MgSO4)
and evaporated to leave the product as a yellow solid (0.95 g,
100%). 1H NMR (300 MHz, CDCl3) d = 8.01 (d, 1H, J = 9.2 Hz),
6.92–6.85 (m, 2H), 3.63 (m, 4H), 3.48 (m, 4H), 1.50 (s, 9H). LC/MS
(Method A), tR = 1.73 min (purity = 88%), [M+H]+ = 353.
2.1.29. 4-(2,2-Diethoxyethoxy)benzene-1,2-diamine (63)
4-(2,2-Diethoxyethoxy)-1,2-dinitrobenzene
(62,
0.20 g,
0.67 mmol) was hydrogenated over 10% Pd/C (40 mg) at one atmo-
sphere hydrogen pressure in methanol (3 mL) for 18 h. The mixture
was filtered, washed (methanol, 2ꢃ 2 mL) and the filtrate was
evaporated under reduced pressure to provide the product 63 as
a dark gum (0.15 g, 93%). 1H NMR (300 MHz, CDCl3) d = 6.61 (d,
1H, J = 8.3 Hz), 6.35 (d, 1H, J = 2.6 Hz), 6.27 (dd, 1H, J = 8.3 Hz,
J = 2.6 Hz), 4.80 (t, 1H, J = 5.2 Hz), 3.92 (d, 2H, J = 5.2 Hz), 3.75
(dq, 2H, J = 9.0 Hz, J = 7.0 Hz), 3.62 (dq, 2H, J = 9.0 Hz, J = 7.0 Hz),
1.24 (t, 6H, J = 7.0 Hz). LC/MS (Method A), tR = 0.21 min (pur-
ity = 99%), [M+H]+ = 241.
2.1.34. tert-Butyl 4-(3,4-diaminophenyl)piperazine-1-
carboxylate (67)
tert-Butyl 4-(3,4-dinitrophenyl)piperazine-1-carboxylate (66,
0.92 g, 2.6 mmol) was hydrogenated over 10% Pd/C (0.10 g) at 1
atmosphere hydrogen pressure in methanol (20 mL) for 18 h. The
catalyst was filtered, washed (methanol, 2ꢃ 10 mL), and the filtrate
was evaporated to leave the product as a dark oil (0.74 g, 97%). 1H
NMR (300 MHz, CDCl3) d = 6.65 (d, 1H, J = 8.3 Hz), 6.37 (d, 1H,
J = 2.3 Hz), 6.32 (dd, 1H, J = 8.3 Hz, J = 2.3 Hz), 3.56 (m, 4H), 3.40
(br s, 4H), 2.96 (m, 4H), 1.49 (s, 9H). LC/MS (Method A),
tR = 0.77 min (purity = 91%), [M+H]+ = 293.
2.1.30. 5-(2,2-Diethoxyethoxy)-1H-benzo[d]imidazole-2(3H)-
thione (64)
A solution of 4-(2,2-diethoxyethoxy)benzene-1,2-diamine (63,
0.13 g, 0.54 mmol) and 1,10-dithiocarbonyldiimidazole (0.19 g,
1.1 mmol) in THF (2 mL) was stirred and heated to 70 °C for
2 h. After cooling to room temperature the mixture was diluted
with ethyl acetate (20 mL) and washed with 1 N HCl (10 mL)
and water (10 mL). The organic layer was dried (MgSO4) and
evaporated under reduced pressure to leave the product 64 as a
yellow solid (0.15 g, 98%). 1H NMR (300 MHz, DMSO-d6)
d = 12.47 (s, 1H), 12.40 (s, 1H), 7.02 (d, 1H, J = 8.6 Hz), 6.75 (dd,
1H, J = 9.7 Hz, J = 2.2 Hz), 6.70 (d, 1H, J = 2.2 Hz), 4.79 (t, 1H,
J = 5.1 Hz), 3.93 (d, 2H, J = 5.1 Hz), 3.66 (dq, 2H, J = 9.4 Hz,
J = 7.2 Hz), 3.57 (dq, 2H, J = 9.4 Hz, J = 7.2 Hz), 1.14 (t, 6H,
J = 7.2 Hz). LC/MS (Method A), tR = 1.14 min (purity = 93.3%),
[M+H]+ = 283.
2.1.35. 5-(Piperazin-1-yl)-2-(trifluoromethyl)-1H-
benzo[d]imidazole (68)
A solution of tert-butyl 4-(3,4-diaminophenyl)piperazine-1-car-
boxylate (67, 0.70 g, 2.4 mmol) in TFA (15 mL) was stirred and
heated to 70 °C for 2 h. The solvent was evaporated and the residue
was dissolved in ethyl acetate (50 mL), washed with saturated
aqueous sodium bicarbonate solution (50 mL), dried (MgSO4) and
evaporated to leave the product as a tan amorphous solid (0.43 g,
66%). 1H NMR (300 MHz, CDCl3) d = 8.50 (br s, 2H), 7.62 (d, 1H,
J = 8.9 Hz), 7.17 (dd, 1H, J = 8.9 Hz, J = 1.6 Hz), 7.09 (d, 1H,
J = 1.6 Hz), 3.30 (m, 4H), 3.21 (m, 4H). LC/MS (Method A),
tR = 0.33 min (purity = 88%), [M+H]+ = 271.
2.1.31. 2-(2-Thioxo-2,3-dihydro-1H-benzo[d]imidazol-5-
yloxy)acetaldehyde (65)
A solution of 5-(2,2-diethoxyethoxy)-1H-benzo[d]imidazole-
2(3H)-thione (64, 0.13 g, 0.46 mmol) in 1 N HCl (1 mL) and
terahydrofuran (2 mL) was stirred at 70 °C for 2 h. The reaction
mixture was cooled to room temperature, diluted with ethyl
acetate (20 mL), washed with water (10 mL), dried (MgSO4) and
2.1.36. 1-Azido-3-(2,2-diethoxyethoxy)-2-nitrobenzene (69)
A solution of 2,2-diethoxyethanol (0.12 g, 0.90 mmol) in THF
(1 mL) under nitrogen atmosphere was treated with sodium

6632
J. C. Pelletier et al. / Bioorg. Med. Chem. 16 (2008) 6617–6640
hydride (36 mg, 0.90 mmol, 60% dispersion in mineral oil) and
stirred 20 min. The mixture was added dropwise via syringe to a
stirring, ice cooled solution of 1-azido-3-fluoro-2-nitrobenzene
(33, 0.11 g, 0.60 mmol) in tetrahydrofuran (1 mL) under a nitrogen
atmosphere. After 2 h the reaction mixture was diluted with
water (15 mL) and ethyl acetate (15 mL), the layers were sepa-
rated, and the aqueous layer was extracted with ethyl acetate
(15 mL). The combined organic layers were dried (MgSO4) and
evaporated to leave the product as a yellow foamy solid (0.16 g,
90%) which was used for subsequent reactions without further
purification. 1H NMR (300 MHz, CDCl3) d = 7.40 (dd, 1H, J = 8.2,
J = 8.4 Hz), 6.47 (d, 1H, J = 8.2), 6.33 (d, 1H, J = 8.4 Hz), 4.75 (t, 1H,
J = 4.8 Hz), 4.10 (d, 2H, J = 5.2 Hz), 3.75 (m, 2H), 3.59 (m, 2H),
1.22 (t, 6H, J = 6.4 Hz). HPLC (Method 1), tR = 1.74 min
(purity = 84%).
2.1.40. tert-Butyl 4-(3-amino-2-nitrophenyl)piperazine-1-
carboxylate (73)
A solution of tert-butyl 4-(3-azido-2-nitro-phenyl)-piperazine-
1-carboxylate (35, 300 mg, 0.861 mmol) in methanol (15 mL) at
0 °C under nitrogen atmosphere was treated with tin(II) chloride
(171 mg, 0.904 mmol) and the resulting yellow mixture was stirred
for 1 h then allowed to warm to room temperature. After 2 h at 20 °C
the dark orange mixture was concentrated in vacuo. The resulting
dark red powder was partitioned between water (30 mL) and ethyl
acetate (30 mL). Aqueous sodium hydroxide 15% (2 mL) was added
and the aqueous layer was extracted with ethyl acetate (2ꢃ
30 mL). Brine (20 mL) was added and the aqueous layer was further
extracted with ethyl acetate (2ꢃ 15 mL). The combined organic lay-
ers were dried (MgSO4) and concentrated in vacuo. The product was
purified by chromatography on silica gel eluted with 0.5% MeOH in
CH2Cl2 to afford 73 as a red powder (226 mg, 82% yield). 1H NMR
(300 MHz, DMSO-d6) d = 7.10 (t, 1H, J = 7.88 Hz) 6.56 (dd, 1H,
J = 7.88, J = 0.83 Hz), 6.39 (dd, 1H, J = 7.88 Hz, J = 0.83 Hz), 5.91 (br
s, 2H), 3.79 (m, 4H), 3.34 (m, 4H), 1.41 (s, 9H). MS (ESI) m/z
[M+H]+ = 323. Anal. Calcd for C15H22N4O4: C, 55.89; H, 6.88; N,
17.38. Found: C, 55.47; H, 6.66; N, 16.99.
2.1.37. 2-(3-Azido-2-nitrophenoxy)acetaldehyde (70)
A solution of 1-azido-3-(2,2-diethoxyethoxy)-2-nitrobenzene
(69, 0.16 g, 0.54 mmol) was stirred in 1 N HCl (1 mL) and tetra-
hydrofuran (1 mL) at 70 °C for 3 h. The reaction mixture was
cooled to room temperature, diluted with ethyl acetate
(20 mL), washed with water (2ꢃ 15 mL), dried (MgSO4), and
evaporated. The crude product was chromatographed on silica
gel eluted with 50% ethyl acetate in hexanes to afford the prod-
uct as a light yellow amorphous solid (74 mg, 62%). 1H NMR
(300 MHz, CDCl3) d = 9.81 (s, 1H), 7.45 (dd, 1H, J = 8.6 Hz,
J = 8.1 Hz), 6.97 (d, 1H, J = 8.1 Hz), 6.68 (d, 1H, J = 8.6 Hz), 4.66
(s, 2H).
2.1.41. tert-Butyl 4-{2-nitro-3-[(trifluoroacetyl)amino]phenyl}-
piperazine-1-carboxylate (74)
To a solution of 4-(3-nitro-2-nitro-phenyl)-piperazine-1-carbox-
ylic acid tert-butyl ester (73, 200 mg, 0.620 mmol) in methylene
chloride (10 mL) at 0 °C under a nitrogen atmosphere was added
di-isopropylethylamine (137 lL, 0.744 mmol) followed by trifluoro-
acetic anhydride (92 lL, 0.651 mmol). The resulting red solutionwas
stirred at 0 °C for 1 h. The reaction was terminated with the addition
of saturated aqueous sodium bicarbonate solution (10 mL). The or-
ganic layer was separated and the aqueous layer was diluted with
brine (10 mL) and extracted with methylene chloride (2ꢃ 10 mL).
The combined organic extracts were dried (MgSO4) and concen-
trated in vacuo. The product was purified by chromatography on sil-
ica gel eluted with 0.5% MeOH in CH2Cl2 to afford 74 as a red powder
(220 mg, 99% yield). 1H NMR (300 MHz, DMSO-d6) d = 11.57 (br s,
1H), 7.28 (dd, 1H, J = 7.5 Hz, J = 0.8 Hz), 7.10 (t, 1H, J = 7.9 Hz), 6.56
(dd, 1H, J = 7.6 Hz, J = 0.8 Hz), 3.37 (m, 4H), 2.91 (m, 4H), 1.41 (s,
9H). MS (ESI) m/z [M+H]+ = 417. Anal. Calcd for C17H21F3N4O5: C,
48.81; H, 5.06; N, 13.39. Found: C, 48.61; H, 4.82; N, 12.95.
2.1.38. 5-(4-(2-(3-Azido-2-nitrophenoxy)ethyl)piperazin-1-yl)-
2-(trifluoromethyl)-1H-benzo[d]imidazole (71)
A
solution of 2-(3-azido-2-nitrophenoxy)acetaldehyde (70,
60 mg, 0.27 mmol) and 5-(piperazin-1-yl)-2-(trifluoromethyl)-
1H-benzo[d]imidazole (37, 73 mg, 0.27 mmol) in DMF (2 mL) was
stirred and treated with sodium triacetoxyborohydride (63 mg,
0.30 mmol). The reaction mixture was stirred for 2 h, diluted with
ethyl acetate (20 mL), washed with water (3ꢃ 20 mL), dried
(MgSO4), and evaporated to leave the product as a thick gum
(57 mg, 44%). 1H NMR (300 MHz, CDCl3) d = 10.60 (br s, 1H), 7.71
(d, 1H, J = 8.9 Hz), 7.40 (dd, 1H, J = 8.3 Hz, J = 8.5 Hz), 7.08 (d, 1H,
J = 8.3 Hz), 6.88 (s, 1H), 6.87 (d, 1H, J = 8.2 Hz), 9.80 (d, 1H,
J = 8.5 Hz), 4.25 (t, 2H, J = 5.4 Hz), 3.20 (m, 4H), 2.88 (t, 2H,
J = 5.4 Hz), 2.67 (m, 4H). LC/MS (Method A), tR = 1.05 min (pur-
ity = 91%), [MꢂH]ꢂ = 475.
2.1.42. tert-Butyl 4-{3-[methyl(trifluoroacetyl)amino]-2-
nitrophenyl}piperazine-1-carboxylate (75)
To a solution of 4-[2-nitro-3-(2,2,2,-trifluoro-acetylamino)-phe-
nyl]-piperazine-1-carboxylic acid tert-butyl ester (74, 220 mg,
0.526 mmol) in tetrahydrofuran (10 mL) at 0 °C under a nitrogen
atmosphere was added triphenylphosphine (268 mg, 1.03 mmol),
methanol (48 lL, 1.18 mmol), and diisopropylazodicarboxylate
(207 lL, 1.03 mmol). The resulting yellow solution was allowed
to warm to room temperature and stirred 15 h. Concentration in
vacuo followed by chromatography on silica gel eluted with 0.5%
MeOH in CH2Cl2 afforded 75 as an orange gum (201 mg, 89% yield).
1H NMR (300 MHz, DMSO-d6) d = 7.63 (t, 1H, J = 7.4 Hz), 7.59 (dd,
1H, J = 7.4, J = 0.7 Hz), 7.43 (dd, 1H, J = 7.2, J = 0.7 Hz), 3.33 (m,
4H), 3.22 (s, 3H), 2.91 (m, 4H), 1.41 (s, 9H). ESMS m/z
[M+H]+ = 433. Anal. Calcd for C18H23F3N4O5: C, 50.00; H, 5.36; N,
12.96. Found: C, 49.88; H, 5.40; N, 12.46.
2.1.39. 4-(2-(4-(2-(Trifluoromethyl)-1H-benzo[d]imidazol-5-
yl)piperazin-1-yl)ethoxy)-1H-benzo[d]imidazole-2(3H)-thione
(72 and 9)
5-(4-(2-(3-Azido-2-nitrophenoxy)ethyl)piperazin-1-yl)-2-(tri-
fluoromethyl)-1H-benzo[d]imidazole (71, 50 mg, 0.11 mmol)
was hydrogenated over 10% Pd/C (10 mg, 1 atmosphere hydrogen
pressure) in methanol (1.5 mL) for 18 h. The catalyst was filtered,
washed (methanol, 2ꢃ 2 mL), and the filtrate was evaporated un-
der reduced pressure to leave phenylenediamine 72. The residue
was dissolved in anhydrous tetrahydrofuran (1 mL), treated with
1,10-thiocarbonyldiimidazole and shaken at 70 °C under a nitro-
gen atmosphere for 1.5 h. The mixture was diluted with water
(250 lL) and purified by reversed phase HPLC (Method F) to leave
the product as a bistrifluoroacetate salt (27 mg, 36%). 1H NMR
(300 MHz, CD3OD) d = 7.63 (d, 1H, J = 8.9 Hz), 7.27 (dd, 1H,
J = 9.0 Hz, J = 2.1 Hz), 7.22 (d, 1H, J = 2.1 Hz), 7.17 (dd, 1H,
J = 8.1 Hz, J = 8.1 Hz), 6.92 (d, 1H, J = 8.1 Hz), 6.88 (d, 1H,
J = 8.1 Hz), 4.60 (t, 2H, J = 4.1 Hz), 3.79 (t, 2H, J = 4.1 Hz), 3.70
(br m, 8H). ESMS [MꢂH]ꢂ = 461, [M+H]+ = 463. Anal. Calcd for
2.1.43. tert-Butyl 4-[1-methyl-2-(trifluoromethyl)-1H-
benzimidazol-4-yl]piperazine-1-carboxylate (76)
tert-Butyl 4-{3-[methyl(trifluoroacetyl)amino]-2-nitrophenyl}-
piperazine-1-carboxylate (75, 2.92 g, 6.75 mmol) was hydroge-
nated over 5% Pd/C (300 mg) at 20 °C and one atmosphere hydro-
gen pressure in methanol (75 mL). The catalyst was filtered
through Celite, and the filtrate was concentrated in vacuo. The
C25H23F9N6O5S: C, 43.48; H, 3.36; N, 12.17. Found: C, 43.10; H,
3.60; N, 11.96.

J. C. Pelletier et al. / Bioorg. Med. Chem. 16 (2008) 6617–6640
6633
product was purified by chromatography on silica gel eluted with
40–70% ethyl acetate in hexanes to afford 76 as a white powder
(1.78 g, 68% yield). 1H NMR (300 MHz, DMSO-d6) d = 7.30 (t, 1H,
J = 8.7 Hz), 7.23 (d, 1H, J = 8.7 Hz), 6.29 (d, 1H, J = 8.7 Hz), 3.93 (s,
3H), 3.52 (m, 4H), 3.46 (m, 4H), 1.48 (s, 9H). MS (ESI) m/z
[M+H]+ = 385. Anal. Calcd for C18H23F3N4O2: C, 56.24; H, 6.03; N,
14.58. Found: C, 55.87; H, 5.74; N, 14.34.
J = 12.37 Hz), 3.71 (m, 2H), 3.48 (t, 2H, J = 11.54 Hz), 3.23 (t, 2H,
J = 11.54 Hz). MS (ESI) m/z [M+H]+ = 476. Anal. Calcd for
C26H25F9N6O5S: C, 44.32; H, 3.58; N, 11.93. Found: C, 44.60; H,
4.03; N, 11.80.
2.1.47. 1-Methyl-2-(trifluoromethyl)-4-[4-(2-{[2-(trifluoro-
methyl)-1H-benzimidazol-4-yl]oxy}ethyl)piperazin-1-yl]-1H-
benzimidazole (8a)
2.1.44. 1-Methyl-4-piperazin-1-yl-2-(trifluoromethyl)-1H-
benzimidazole (77)
A solution of 3-(2-{4-[1-methyl-2-(trifluoromethyl)-1H-ben-
zimidazol-4-yl]piperazin-1-yl}ethoxy)benzene-1,2-diamine (79,
51 mg, 0.117 mmol) in TFA (2 mL) was capped and heated to
70°C for 2.5 h. The solvent was evaporated, and the product was
purified by reverse phase HPLC (Method F) to afford 8a as a brown
powder (19.6 mg, 27% yield) as the bistrifluoroacetate. 1H NMR
(300 MHz, DMSO-d6) d = 3.21 (t, 2H, J = 11.54 Hz), 3.49 (t, 2H,
J = 11.54 Hz), 3.75 (m, 2H), 3.84 (d, 2H, J = 12.37 Hz), 3.95 (s, 3H),
4.44 (d, 2H, J = 12.37 Hz), 4.68 (m, 2H), 6.79 (d, 1H, J = 7.69 Hz),
6.99 (br s, 1H), 7.32 (m, 4H), 9.99 (br s, 1H), 14.05 (br s, 1H). MS
(ESI) m/z [M+H]+ = 512. Anal. Calcd for C27H24F12N6O5: C, 43.79;
H, 3.27; N, 11.35. Found: C, 43.88; H, 3.50; N, 11.51.
A solution of tert-butyl 4-[1-methyl-2-(trifluoromethyl)-1H-
benzimidazol-4-yl]piperazine-1-carboxylate
(76,
1.75
g,
4.55 mmol) in methylene chloride (25 mL) and TFA (10 mL) was
stirred 1 h. Methylene chloride (30 mL) was added, and the mix-
ture was washed with 30% aq NaOH (100 mL). The aqueous layer
was extracted with methylene chloride (30 mL), and the combined
organic layers were dried (MgSO4) and filtered through a silica pad
eluted with 10% methanol in methylene chloride. The filtrate was
concentrated in vacuo to a brown gum (920 mg, 71% yield). 1H
NMR (DMSO-d6): d = 7.28 (t, 1H, J = 6.9 Hz), 7.18 (d, 1H,
J = 6.9 Hz), 6.65 (d, 1H, J = 6.9 Hz), 3.91 (br s, 1H), 3.43 (m, 4H),
3.36 (s, 3H), 2.91 (m, 4H). HPLC (Method A) tR = 0.23 min (pur-
ity = 95.6%), m/z [M+H]+ = 285.
2.1.48. 1-(2-(3-Azido-2-nitrophenoxy)ethyl)piperazine (80)
A solution of 2-(piperazin-1-yl)ethanol (5.7 g, 43 mmol) in tet-
rahydrofuran (40 mL) under a nitrogen atmosphere was cooled in
an ice bath and treated with sodium hydride (60% mineral oil dis-
persion, 1.7 g, 43 mmol) in portions over 5 min. After 1 h the mix-
ture was cooled to ꢂ78 °C and a solution of 1-azido-3-fluoro-2-
nitrobenzene (33, 6.0 g, 33 mmol) in tetrahydrofuran (40 mL) was
added dropwise over 15 min. The mixture warmed to 20 °C over
2 h, 1 N HCl (50 mL) was added cautiously followed by ethyl ace-
tate (200 mL) and water (200 mL). The aqueous layer was sepa-
rated, washed with ethyl acetate (100 mL), neutralized with solid
sodium carbonate and the product was extracted with chloroform
(3ꢃ 100 mL). The combined chloroform layers were dried (MgSO4)
and evaporated under reduced pressure to leave the product as a
gum (1.4 g, 15%). The original ethyl acetate layers were combined
and extracted with 1 N HCl (2ꢃ 100 mL). The combined acidic lay-
ers were neutralized with solid sodium carbonate and extracted
with chloroform (3ꢃ 100 mL). The combined chloroform layers
were dried (MgSO4) and evaporated to leave additional product
(5.7 g, 59%) for a total yield of 7.1 g (74%). 1H NMR (300 MHz,
CDCl3) d = 7.40 (dd, 1H, J = 8.4 Hz, J = 8.3 Hz), 6.85 (d, 1H,
J = 8.3 Hz), 6.80 (d, 1H, J = 8.4 Hz), 4.20 (t, 2H, J = 5.7 Hz), 2.88 (m,
4H), 2.78 (t, 2H, J = 5.7 Hz), 2.51 (m, 4H).
2.1.45. 3-(2-{4-[1-Methyl-2-(trifluoromethyl)-1H-benzimi-
dazol-4-yl]piperazin-1-yl}ethoxy)benzene-1,2-diamine
(78 and 79)
To a solution of 1-methyl-4-piperazin-1-yl-2-(trifluoromethyl)-
1H-benzimidazole (77, 500 mg, 1.76 mmol) and 2-(3-azido-2-nitro-
phenoxy)-acetaldehyde (70, 469 mg, 2.11 mmol), in N-methyl-2-
pyrrolidone (10 mL), at room temperature under nitrogen were
added sodium triacetoxyborohydride (746 mg, 3.52 mmol) and ace-
tic acid (10 lL). The resulting mixture was stirred for 90 min,
quenched with water (1 mL) and partitioned between ethyl acetate
(50 mL) and saturated aq sodium bicarbonate solution (50 mL). The
organic layer was dried (MgSO4)and concentrated in vacuo. The
product was purified by chromatography on silica gel eluted with
0.5% methanol in methylene chloride to afford 78 as an orange
gum (390 mg, 45% yield) whichwas usedimmediately withoutchar-
acterization. 4-{4-[2-(Azido-2-nitro-phenoxy)-ethyl]piperazin-1-
yl}-1-methyl-2-trifluoromethyl-1H-benzimidazole (78, 390 mg,
0.795 mmol) was hydrogenated over 5% Pd/C in methanol (8 mL)
at room temperature and one atmosphere of hydrogen pressure.
The catalyst was filtered, washed (methanol), and the filtrate was
evaporated in vacuo to leave 79 as a light brown powder (310 mg,
90% yield). 1H NMR (300 MHz, DMSO-d6) d = 7.30 (t, 1H, J = 7.4 Hz),
7.21 (d, 1H, J = 7.4 Hz), 6.67 (d, 1H, J = 7.4 Hz), 6.35 (d, 1H,
J = 7.7 Hz), 6.22 (td, 2H, J = 7.7 Hz, J = 1.4 Hz), 4.43 (br s, 2H), 4.11
(br s, 2H), 4.03 (t, 2H, J = 5.7 Hz), 3.92 (s, 3H), 3.54 (m, 4H), 2.74 (t,
2H, J = 5.7 Hz), 2.72 (m, 4H). HPLC (Method A), tR = 0.22 min, m/z
[M+H]+ = 435.
2.1.49. tert-Butyl 4-(2-(3-azido-2-nitrophen-
oxy)ethyl)piperazine-1-carboxylate (81)
To a solution of 1-(2-(3-azido-2-nitrophenoxy)ethyl)piperazine
(80, 7.1 g, 24 mmol) in methylene chloride (100 mL) was added di-
tert-butyldicarbonate (7.4 g, 34 mmol), and the mixture stirred for
30 min. Aminomethylpolystyrene resin was added (3.2 mmol/g,
7.5 g, 24 mmol) and the mixture stirred an additional hour. The resin
was filtered, washed with methylene chloride (3ꢃ 100 mL), and the
combined filtrates were evaporated under reduced pressure to leave
the product 81 as a brown gum (9.4 g, 100%). 1H NMR (300 MHz,
CDCl3) d = 7.41 (dd, 1H, J = 8.4 Hz, J = 8.3 Hz), 6.87 (d, 1H,
J = 8.3 Hz), 6.81 (d, 1H, J = 8.4 Hz), 4.20 (t, 2H, J = 5.6 Hz), 3.41 (m,
4H), 2.78 (t, 2H, J = 5.6 Hz), 2.47 (m, 4H), 1.47 (s, 9H). LC/MS (Method
A), tR = 1.11 min (purity = 90.0%), [M+H]+ ꢂ56 (tert-butyl) = 337.
2.1.46. 4-(2-{4-[1-Methyl-2-(trifluoromethyl)-1H-benzimi-
dazol-4-yl]piperazin-1-yl}ethoxy)-1,3-dihydro-2H-benzimi-
dazole-2-thione (8b)
To a solution of 3-(2-{4-[1-methyl-2-(trifluoromethyl)-1H-ben-
zimidazol-4-yl]piperazin-1-yl}ethoxy)benzene-1,2-diamine (79,
50 mg, 0.12 mmol) in tetrahydrofuran (5 mL) at room temperature
under nitrogen was added 1,10-thiocarbonyldiimidazole (61 mg,
0.345 mmol). The resulting mixture was capped and heated to
70 °C for 2.5 h. The solvent was evaporated, and the product was
purified by reverse phase HPLC (Method F) to afford a yellow pow-
der (38 mg, 56% yield) as the bistrifluoroacetate. 1H NMR
(300 MHz, DMSO-d6) d = 12.68 (s, 1H), 12.64 (s, 1H), 9.60 (br s,
1H), 7.34 (m, 2H), 7.08 (t, 1H, J = 8.2 Hz), 6.82 (m, 3H), 4.50 (m,
2H), 4.44 (d, 2H, J = 12.37 Hz), 3.96 (s, 3H), 3.83 (d, 2H,
2.1.50. tert-Butyl 4-(2-(2,3-diaminophenoxy)ethyl)piperazine-
1-carboxylate (82)
A
solution
of
tert-butyl
4-(2-(3-azido-2-nitrophen-
oxy)ethyl)piperazine-1-carboxylate (81, 4.46 g, 11.4 mmol) in
methanol (120 mL) was hydrogenated at one atmosphere hydro-
gen pressure over 5% palladium on carbon (0.63 g) for 4 h. The cat-

6634
J. C. Pelletier et al. / Bioorg. Med. Chem. 16 (2008) 6617–6640
alyst was filtered through diatomaceous earth, washed with meth-
anol (2ꢃ 20 mL), and the combined filtrates were evaporated un-
der reduced pressure to leave the product as a dark brown foamy
solid (3.55 g, 93%). 1H NMR (300 MHz, CDCl3) d = 6.64 (dd, 1H,
J = 8.1 Hz, J = 8.1 Hz), 6.41 (d, 1H, J = 8.1 Hz), 6.40 (d, 1H,
J = 8.1 Hz), 4.12 (t, 2H, J = 5.8 Hz), 3.53 (br s, 2H), 3.49 (br s, 2H),
3.45 (m, 4H), 2.81 (t, 2H, J = 5.8 Hz), 2.52 (m, 4H), 1.46 (s, 9H).
LC/MS (Method A), tR = 0.57 min (purity = 99%), [M+H]+ = 337.
2.1.54. 4-(4-(2-(2-(Trifluoromethyl)-1H-benzo[d]imidazol-4-
yloxy)ethyl)piperazin-1-yl)-1H-benzo[d]imidazole-2(3H)-
thione (11)
A solution of 3-(4-(2-(2-(trifluoromethyl)-1H-benzo[d]imida-
zol-4-yloxy)ethyl)piperazin-1-yl)benzene-1,2-diamine (88, 40 mg,
0.095 mmol) in tetrahydrofuran (1 mL) was treated with 1,10-thi-
ocarbonyldiimidazole (25 mg, 0.14 mmol) and stirred for 18 h.
The mixture was diluted with water (0.2 mL) and purified by re-
versed phase HPLC (Method 2) to leave the bistrifluoroacetate
product 11 as a gum (43 mg, 66%). 1H NMR (300 MHz, CD3OD)
d = 7.42 (dd, 1H, J = 8.1 Hz, J = 8.1 Hz), 7.31 (d, 1H, J = 8.2 Hz), 7.04
(dd, 1H, J = 8.0 Hz, J = 8.0 Hz), 6.97 (d, 1H, J = 8.0 Hz), 6.86 (d, 1H,
J = 8.0 Hz), 6.82 (d, 1H, J = 8.2 Hz), 4.64 (t, 2H, J = 4.9 Hz), 3.86 (m,
4H), 3.53 (m, 4H), 3.23 (t, 2H, J = 4.9 Hz). 13C NMR (300 MHz,
CD3OD) d = 157.00, 150.50, 141.41, 140.88, 137.05, 135.79,
133.05, 131.85, 127.65, 124.01, 123.29, 122.00, 118.43, 112.01,
107.85, 106.93, 106.10, 62.37, 56.70, 53.43, 53.40. HPLC (Method
C), tR = 6.68 min (purity = 99.9%). HRMS [M+H]+ = 463.1522, calcd
for C21H21F3N6OS: 463.1522.
2.1.51. 4-(2-(4-(3-Azido-2-nitrophenyl)piperazin-1-yl)ethoxy)-
2-(trifluoromethyl)-1H-benzo[d]imidazole (83 and 86)
A solution of tert-butyl 4-(2-(2,3-diaminophenoxy)ethyl)piper-
azine-1-carboxylate (82, 260 mg, 0.77 mmol) in TFA (3 mL) was
stirred for 30 min at room temperature then 2 h at 75 °C. The
TFA was evaporated and the residue was dissolved in water
(10 mL), and lyophilized to leave product 83 as a dark brown
gum. The residue was dissolved in dimethylsulfoxide (4 mL), trea-
ted with diisopropylethylamine (0.60 g, 4.6 mmol, 0.83 mL) and 1-
azido-3-fluoro-2-nitrobenzene (37, 0.21 g, 1.2 mmol) and stirred at
60 °C for 20 h. The reaction mixture was cooled to 20 °C, diluted
with ethyl acetate (50 mL) and washed with 1 M sodium carbonate
(50 mL), water (2ꢃ 50 mL), and brine (50 mL). The organic layer
was dried (MgSO4) and evaporated under reduced pressure. The
residue was chromatographed on silica gel (Isco instrument, 40 g
column; 50% ethyl acetate in hexanes to 100% ethyl acetate) to
leave the product 86 as a yellow, foamy solid (232 mg, 63%, two
steps). 1H NMR (300 MHz, CD3OD) d = 7.49 (dd, 1H, J = 8.3 Hz,
J = 8.2 Hz), 7.26 (d, 1H, J = 7.2 Hz), 7.25 (dd, 1H, J = 8.2 Hz,
J = 7.2 Hz), 7.14 (d, 1H, J = 8.2 Hz), 7.14 (d, 1H, J = 8.2 Hz), 6.87 (d,
1H, J = 8.3 Hz), 4.34 (t, 2H, J = 5.5 Hz), 3.00 (m, 4H), 2.94 (t, 2H,
J = 5.5 Hz), 2.70 (m, 4H). LC/MS (Method A), tR = 1.40 min (pur-
ity = 87.2%), [M+H]+ = 476.
2.1.55. tert-Butyl 4-(2-(2-thioxo-2,3-dihydro-1H-benzo[d]-
imidazol-4-yloxy)ethyl)piperazine-1-carboxylate (84)
A solution of tert-butyl 4-(2-(2,3-diaminophenoxy)ethyl)piper-
azine-1-carboxylate (82, 8.0 g, 24 mmol) was dissolved in anhy-
drous tetrahydrofuran (100 mL), purged with
a
nitrogen
atmosphere, treated with 1,10-thiocarbonyldiimidazole (7.6 g,
43 mmol) and stirred 18 h. Water (15 mL) was added and the reac-
tion mixture stirred an additional 24 h. The solvent was evapo-
rated, the residue was dissolved in ethyl acetate (300 mL),
washed with water (3ꢃ 100 mL) and brine (100 mL). The organic
layer was dried and evaporated under reduced pressure to leave
the crude product 84, which was purified by flash chromatography
on silica gel eluted with a gradient of 50% ethyl acetate in hexanes
to 100% ethyl acetate. The desired product was obtained as a color-
less gum (4.3 g, 47%). 1H NMR (300 MHz, DMSO-d6) d = 12.70 (br s,
1H), 12.48 (br s, 1H), 7.03 (dd, 1H, J = 8.1 Hz, J = 8.1 Hz), 6.77 (d, 1H,
J = 8.1 Hz), 6.74 (d, 1H, J = 8.1 Hz), 4.20 (t, 2H, J = 5.6 Hz), 3.31 (m,
4H), 2.56 (t, 2H, J = 5.6 Hz), 2.46 (m, 4H), 1.38 (s, 9H). LC/MS (Meth-
od A), tR = 0.64 min (purity = 95.3%), [M+H]+ = 378.
2.1.52. 3-(4-(2-(2-(Trifluoromethyl)-1H-benzo[d]imidazol-4-
yloxy)ethyl)piperazin-1-yl)benzene-1,2-diamine (88)
A
solution of 4-(2-(4-(3-azido-2-nitrophenyl)piperazin-1-
yl)ethoxy)-2-(trifluoromethyl)-1H-benzo[d]imidazole (86, 0.21 g,
0.44 mmol) in methanol (4 mL) was hydrogenated at 1 atmosphere
hydrogen pressure over 5% palladium on carbon (40 mg) for 6 h.
The mixture was filtered, the catalyst was washed with methanol
(2ꢃ 5 mL), and the combined filtrates were evaporated under re-
duced pressure to leave the product 88 as a tan foamy solid
(94 mg, 51%). 1H NMR (300 MHz, CD3OD) d = 7.20–7.30 (m, 2H),
6.88 (d, 1H, J = 7.0 Hz), 6.46–6.57 (m, 3H), 4.38 (t, 2H, J = 5.5 Hz),
2.98 (t, 2H, J = 5.5 Hz), 2.88 (m, 4H), 2.82 (m, 4H). LC/MS (Method
A), tR = 1.07 min (purity = 95.3%), [M+H]+ = 421.
2.1.56. 4-(2-(4-(3-Azido-2-nitrophenyl)piperazin-1-yl)ethoxy)-
1H-benzo[d]imidazole-2(3H)-thione (85 and 87)
A
mixture of tert-butyl 4-(2-(2-thioxo-2,3-dihydro-1H-
benzo[d]imidazol-4-yloxy)ethyl)piperazine-1-carboxylate (84,
4.3 g, 11 mmol) and dimethylsilane on polystyrene resin
(1.7 mmol/g, 13 g, 22 mmol) in methylene chloride (80 mL) was
treated with trifluoroacetic acid (20 mL) and stirred for an hour.
The reaction mixture was filtered, the polymer residue was washed
with methylene chloride (2ꢃ 50 mL), and the combined filtrates
were evaporated under reduced pressure. The crude product was
dissolved in water (100 mL), saturated with sodium carbonate,
and extracted with n-butanol (3ꢃ 100 mL). The combined organic
layers were dried (Na2SO4) and evaporated under reduced pressure
to leave the product 85 as a sticky, light tan gum (2.1 g, 69%). A
portion of this intermediate (85, 1.8 g, 6.5 mmol) was dissolved
in DMSO (18 mL), treated with 1-azido-3-fluoro-2-nitrobenzene
(37, 1.8 g, 9.7 mmol) and stirred at 60 °C for 24 h. The reaction mix-
ture was cooled to room temperature, diluted with ethyl acetate
(300 mL), washed with 1 M sodium carbonate (100 mL), and the
aqueous layer was further extracted with ethyl acetate (50 mL).
The combined ethyl acetate layers were washed with water (3ꢃ
100 mL) and brine (100 mL), dried (MgSO4), and evaporated under
reduced pressure. The residue was purified by flash chromatogra-
phy on silica gel eluted with a gradient of 75% ethyl acetate in hex-
anes to 100% ethyl acetate. The product 87 was obtained as a
yellow foamy solid (1.9 g, 64%). 1H NMR (300 MHz, CDCl3)
2.1.53. 4-(4-(2-(2-(Trifluoromethyl)-1H-benzo[d]imidazol-4-
yloxy)ethyl)piperazin-1-yl)-1H-benzo[d]imidazol-2(3H)-one
(10a)
A solution of 3-(4-(2-(2-(trifluoromethyl)-1H-benzo[d]imida-
zol-4-yloxy)ethyl)piperazin-1-yl)benzene-1,2-diamine (88, 40 mg,
0.095 mmol) in tetrahydrofuran (1 mL) was treated with 1,10-car-
bonyldiimidazole (23 mg, 0.14 mmol) and stirred for 18 h. The
mixture was diluted with water (0.2 mL) and purified by reversed
phase HPLC (Method 2) to leave the bistrifluoroacetate product 10a
as a gum (39 mg, 61%). 1H NMR (300 MHz, CD3OD) d = 7.42 (dd, 1H,
J = 8.1 Hz, J = 8.2 Hz), 7.31 (d, 1H, J = 8.1 Hz), 7.17 (dd, 1H,
J = 8.0 Hz, J = 8.0 Hz), 6.99 (d, 1H, J = 8.0 Hz), 6.97 (d, 1H,
J = 8.1 Hz), 6.92 (d, 1H, J = 8.2 Hz), 4.65 (t, 2H, J = 4.9 Hz), 3.87 (m,
4H), 3.57 (m, 4H), 3.27 (t, 2H, J = 4.9 Hz). 13C NMR (300 MHz,
CD3OD) d = 169.30, 150.40, 141.00 (q), 136.99, 136.27, 134.89,
132.99, 127.57, 127.17, 124.83, 118.50, 112.99, 107.86, 107.09,
106.05, 62.29, 56.61, 53.20, 53.19. HPLC (Method C), tR = 7.01 min
(purity = 99.9%). HRMS [M+H]+ = 447.1744, calcd for C21H21F3N6O2:
447.1751.

J. C. Pelletier et al. / Bioorg. Med. Chem. 16 (2008) 6617–6640
6635
d = 12.70 (br s, 1H), 10.42 (br s, 1H), 7.48 (dd, 1H, J = 8.1 Hz,
2.1.60. 4-(2-(4-(Quinoxalin-5-yl)piperazin-1-yl)ethoxy)-1H-
J = 8.1 Hz), 7.37 (dd, 1H, J = 8.3 Hz, J = 1.1 Hz), 7.06 (dd, 1H,
J = 8.0 Hz, J = 8.1 Hz), 7.02 (dd, 1H, J = 8.3 Hz, J = 1.1 Hz), 6.92 (d,
1H, J = 7.9 Hz), 6.80 (d, 1H, J = 7.9 Hz), 4.22 (t, 2H, J = 5.4 Hz), 3.35
(m, 4H), 2.81 (m, 6H). LC/MS (Method A), tR = 1.03 min (pur-
ity = 92.3%), [M+H]+ = 441.
benzo[d]imidazole-2(3H)-thione (13)
A solution of 4-(2-(4-(2,3-diaminophenyl)piperazin-1-yl)eth-
oxy)-1H-benzo[d]imidazole-2(3H)-thione (89, 10 mg, 26 lmol) in
methanol (1 mL) was treated with 40% aqueous glyoxal (8 lL,
52 lmol) and stirred for 2 h. Water (0.4 mL) was added and the
reaction mixture was purified by reversed phase HPLC (Method
2) to leave the monotrifluoracetate product 13 as an amorphous
solid (5.2 mg, 38%). 1H NMR (300 MHz, CD3OD) d = 8.86 (d, 1H,
J = 2.0 Hz), 8.84 (d, 1H, J = 2.0 Hz), 7.75 (d, 1H, J = 4.0 Hz), 7.74 (d,
1H, J = 4.0 Hz), 7.34 (dd, 1H, J = 4.0 Hz, J = 4.0 Hz), 7.13 (dd, 1H,
J = 8.0 Hz, J = 8.2 Hz), 6.88 (d, 1H, J = 8.0 Hz), 6.85 (d, 1H,
J = 8.2 Hz), 4.58 (t, 2H, J = 4.7 Hz), 4.20 (m, 2H), 3.86 (m, 2H), 3.81
(t, 2H, J = 4.7 Hz), 3.68 (m, 2H), 3.31 (m, 2H). HPLC (Method C),
tR = 5.21 min (purity = 98.5%). HR ESMS [M+H]+ = 407.1653, calcd
for C21H23N6OS: 407.1649.
2.1.57. 4-(2-(4-(2,3-Diaminophenyl)piperazin-1-yl)ethoxy)-1H-
benzo[d]imidazole-2(3H)-thione (89)
A
solution of 4-(2-(4-(3-azido-2-nitrophenyl)piperazin-1-
yl)ethoxy)-1H-benzo[d]imidazole-2(3H)-thione (87, 1.8 g,
4.1 mmol) in N-methylpyrrolidinone (40 mL) was treated with ti-
n(II) chloride dihydrate (9.2 g, 41 mmol) and stirred 5 min at
20 °C then 1.5 h at 100 °C. The reaction mixture was cooled to
20 °C, diluted with 1 N HCl (30 mL) and filtered. The filtrate was
neutralized with solid sodium carbonate, diluted with ethyl ace-
tate (200 mL), stirred for 15 min, and filtered. The filtrate layers
were separated, and the organic layer was washed with water
(5ꢃ 100 mL) and brine (100 mL). The organic layer was dried
(MgSO4) and evaporated under reduced pressure to leave the prod-
uct as a light yellow amorphous solid (0.68 g, 43%). 1H NMR
(300 MHz, CDCl3) d = 12.40 (br s, 1H), 10.65 (br s, 1H), 7.05 (dd,
1H, J = 8.1 Hz, J = 8.1 Hz), 6.85 (d, 1H, J = 8.0 Hz), 6.81 (dd, 1H,
J = 7.9, Hz, J = 1.1 Hz), 6.73 (d, 1H, J = 7.9 Hz), 6.70 (dd, 1H,
J = 7.9 Hz, J = 7.9 Hz), 6.55 (dd, 1H, J = 7.9 Hz, J = 1.1 Hz), 4.38 (t,
2H, J = 4.8 Hz), 3.60 (br s, 4H), 3.15 (t, 2H, J = 4.8 Hz), 2.89 (m,
4H). LC/MS (Method A), tR = 0.22 min (purity = 92.5%),
[M+H]+ = 385.
2.1.61. 4-(2-(4-(1H-Benzo[d]imidazol-4-yl)piperazin-1-
yl)ethoxy)-1H-benzo[d]imidazole-2(3H)-thione (14)
A solution of 4-(2-(4-(2,3-diaminophenyl)piperazin-1-yl)eth-
oxy)-1H-benzo[d]imidazole-2(3H)-thione (89, 41 mg, 0.12 mmol)
in 90% formic acid (1 mL) was heated to 90 °C for 2 h. The formic
acid was evaporated, and the product was purified by reversed
phase HPLC (Method F). The pure product 14 was obtained as
a
bistrifluoroacetate salt (4.0 mg, 5.4%). 1H NMR (300 MHz,
DMSO-d6) d = 12.62 (br s, 2H), 9.80 (br s, 1H), 9.15 (br s, 1H),
7.42 (s, 1H), 7.43 (d, 1H, J = 6.6 Hz), 7.12 (dd, 1H, J = 6.7 Hz,
J = 8.1 Hz), 7.00 (d, 1H, J = 5.7 Hz), 6.88 (d, 1H, J = 8.1 Hz), 6.85
(d, 1H, J = 7.8 Hz), 4.68 (br t, 2H, J = 5.6 Hz), 4.55 (br s, 4H),
3.74 (br s, 4H), 3.21 (br t, 2H, J = 5.7 Hz). HPLC (Method C),
tR = 6.63 min (purity = 91.0%). HR ESMS [M+H]+ = 395.1650, calcd
for C20H23N6OS: 395.1656.
2.1.58. 4-(4-(2-(2-Thioxo-2,3-dihydro-1H-benzo[d]imidazol-4-
yloxy)ethyl)piperazin-1-yl)-1H-benzo[d]imidazol-2(3H)-one
(10b)
A solution of 4-(2-(4-(2,3-diaminophenyl)piperazin-1-yl)eth-
oxy)-1H-benzo[d]imidazole-2(3H)-thione (89, 57 mg, 0.16 mmol)
and carbonyldiimidazole (22 mg, 0.12 mmol) in tetrahydrofuran
(1 mL) was stirred for 18 h. The reaction mixture was diluted
with water (0.2 mL) and purified by reversed phase HPLC (Meth-
od 2). The product 10b was obtained as a bistrifluoroacetate salt
(2.4 mg, 3.7%). 1H NMR (300 MHz, DMSO-d6) d = 12.62 (br s, 1H),
12.59 (br s, 1H), 10.80 (br s, 1H), 10.68 (br s, 1H), 9.55 (s, 1H),
7.12 (dd, 1H, J = 8.1 Hz), 6.92 (dd, 1H, J = 8.0 Hz, J = 8.0 Hz),
6.85 (d, 1H, J = 7.3 Hz), 6.82 (d, 1H, J = 7.4 Hz), 6.79 (d, 1H,
J = 7.3 Hz), 6.76 (d, 1H, J = 8.0), 4.50 (br t, 2H, J = 5.7 Hz), 3.72
(br s, 4H), 3.48 (partially obscured bs, 4H), 3.03 (br t, 2H,
J = 5.7 Hz). HPLC (Method C), tR = 7.34 min (purity = 97.8%). ESMS
[MꢂH]ꢂ = 409. HRMS [M+H]+ = 411.1605, calcd for C20H23N6O2S:
411.1611.
2.1.62. 4-(2-(4-(2-Methyl-1H-benzo[d]imidazol-4-yl)piperazin-
1-yl)ethoxy)-1H-benzo[d]imidazole-2(3H)-thione (15)
Prepared from 89 and acetic acid as above for compound 14 on
50 mg scale. Purification by HPLC (Method F). Yield = 58%. 1H NMR
(300 MHz, DMSO-d6) d = 12.66 (br s, 1H), 12.61 (br s, 1H), 9.65 (br
s, 1H), 7.34 (dd, 1H, J = 8.0 Hz, J = 7.9 Hz), 7.22 (d, 1H, J = 8.1 Hz),
7.10 (dd, 1H, J = 8.1, Hz, J = 8.0 Hz), 6.88 (d, 1H, J = 8.1 Hz), 6.81
(d, 1H, J = 8.0 Hz), 6.80 (d, 1H, J = 7.9 Hz), 4.50 (br t, 2H), 3.84
(br t, 2H), 3.68 (br s, 4H), 3.40 (obscured m, 4H), 2.81 (s, 3H).
HPLC (Method C), tR = 9.8 min (purity = 91.0%). HR ESMS
[M+H]+ = 408.1728, calcd for C21H24N6OS: 408.1732.
2.1.63. 4-(2-(4-(2-(Perfluoroethyl)-1H-benzo[d]imidazol-4-
yl)piperazin-1-yl)ethoxy)-1H-benzo[d]imidazole-2(3H)-thione
(16)
2.1.59. 5-(4-(2-(2-Thioxo-2,3-dihydro-1H-benzo[d]imidazol-4-
yloxy)ethyl)piperazin-1-yl)quinoxaline-2,3(1H,4H)-dione (12)
A solution of 4-(2-(4-(2,3-diaminophenyl)piperazin-1-yl)eth-
oxy)-1H-benzo[d]imidazole-2(3H)-thione (89, 30 mg, 0.078 mmol)
and oxalyldiimidazole (22 mg, 0.12 mmol) in tetrahydrofuran
(1 mL) was stirred for 18 h. The reaction mixture was diluted with
water (0.2 mL) and purified by reversed phase HPLC (Method 2).
The product was obtained as a bistrifluoroacetate salt (13 mg,
30%). 1H NMR (300 MHz, DMSO-d6) d = 12.63 (br s, 2H), 12.01 (br
s, 1H), 10.96 (br s, 1H), 9.94 (br s, 1H), 7.12 (d, 1H, J = 8.0 Hz),
7.09 (m, 2H), 7.00 (d, 1H, J = 8.9 Hz), 6.86 (d, 1H, J = 7.8 Hz), 6.84
(d, 1H, J = 7.8 Hz), 4.53 (br t, 2H, J = 4.7 Hz), 3.74 (br t, 2H,
J = 4.7 Hz), 3.68 (m, 4H), 3.12 (m, 4H). 13C NMR (300 MHz,
DMSO-d6) d = 167.71, 158.00 (q), 155.85, 155.05, 141.86, 138.11,
133.53, 126.29, 123.39,123.21, 121.93, 120.84, 115.59, 112.20,
105.71, 103.44, 62.72, 54.99, 51.72, 48.71. HPLC (Method C),
tR = 4.50 min (purity = 96.2%). HRMS [M+H]+ = 438.1471, calcd for
Prepared as above for compound 14 from 89 (15 mg, 39 lmol)
and perfluoropropionic acid (0.30 mL). Purified by reversed phase
HPLC (Method F) to leave the product 16 bistrifluoroacetate salt
as a foamy solid (10 mg, 35%). 1H NMR (300 MHz, DMSO-d6)
d = 14.10 (br s, 1H), 12.68 (br s, 1H), 12.65 (br s, 1H), 9.65 (br s,
1H), 7.33 (dd, 1H, J = 8.0 Hz, J = 7.9 Hz), 7.21 (d, 1H, J = 8.1 Hz),
7.10 (dd, 1H, J = 8.1, Hz, J = 8.0 Hz), 6.85 (d, 1H, J = 8.1 Hz), 6.83
(d, 1H, J = 8.0 Hz), 6.80 (d, 1H, J = 7.9 Hz), 4.52 (broad t, 2H), 3.85
(br t, 2H), 3.70 (br s, 4H), 3.90 (obscured m, 4H). HPLC (Method
3), tR = 10.2 min (purity = 82.6%). ESMS [M+H]+ = 513. HR ESMS
[M+H]+ = 513.1506, calcd for C22H22F5N6OS: 513.1498.
2.1.64. 4-(2-(4-(2-Phenyl-1H-benzo[d]imidazol-4-yl)piperazin-
1-yl)ethoxy)-1H-benzo[d]imidazole-2(3H)-thione (17)
A solution of 4-(2-(4-(2,3-diaminophenyl)piperazin-1-yl)eth-
oxy)-1H-benzo[d]imidazole-2(3H)-thione (89, 40 mg, 0.10 mmol)
and benzaldehyde (22 mg, 0.20 mmol) in isopropyl alcohol was
kept open to the atmosphere, stirred rapidly, and heated to 70 °C
C21H23N6O3S: 438.1474.

6636
J. C. Pelletier et al. / Bioorg. Med. Chem. 16 (2008) 6617–6640
for 3 h. The mixture was cooled to room temperature, diluted with
DMSO (0.5 mL) and water (0.5 mL), and purified by reversed phase
HPLC (Method 2). The product 17 was obtained as a bistrifluoro-
acetate salt (35 mg, 50%). 1H NMR (300 MHz, DMSO-d6) d = 12.67
(br s, 1H), 12.64 (br s, 1H), 10.08 (br s, 1H), 8.21 (d, 1H,
J = 8.1 Hz), 8.19 (d, 1H, J = 8.1 Hz), 7.59 (m, 3H), 7.25 (m, 2H),
7.12 (dd, 1H, J = 8.1 Hz, J = 8.1 Hz), 6.87 (d, 1H, J = 8.1 Hz), 6.85 (d,
1H, J = 8.1 Hz), 6.79 (d, 1H, J = 8.1 Hz), 4.56 (t, 3H, J = 4.7 Hz), 4.40
(m, 2H), 3.87 (m, 2H), 3.74 (t, 2H, J = 4.7 Hz), 3.57 (m, 2H), 3.28
(m, 2H). 13C NMR (300 MHz, DMSO-d6) d = 167.71, 158.40 (q),
148.87, 141.90, 141.89, 141.88, 140.24, 135.96, 133.51, 130.32,
128.93, 128.62, 126.82, 124.00, 123.19, 108.41, 105.79, 103.42,
62.90, 54.91, 51.70, 46.47. HPLC (Method C), tR = 7.28 min (pur-
ity = 92.2%). HR ESMS [M+H]+ = 470.1974, calcd for C26H27N6OS:
471.1969.
(br s, 1H), 12.66 (br s, 1H), 9.71 (br s, 1H), 7.95 (d, 1H, J = 8.0 Hz),
7.35 (d, 1H, J = 8.0 Hz), 7.30 (s, 1H), 7.23 (m, 2H), 7.15 (t, 1H,
J = 8.3 Hz), 6.89 (m, 2H), 6.73 (m, 1H), 4.59 (br s, 4H), 3.89 (m,
2H), 3.78 (m, 2H), 3.60 (m, 2H), 3.31 (t, 2H, J = 15 Hz), 2.38 (s,
3H), 2.29 (s, 3H). HRMS [M+H]+ = 499.2270, calcd for C28H31N6OS:
499.2273. HPLC (Method D), tR = 8.6 min (purity = 99.1%).
2.1.68. 2-(4-tert-Butylphenyl)-4-(4-(2-(2-(trifluoromethyl)-1H-
benzo[d]imidazol-4-yloxy)ethyl)piperazin-1-yl)-1H-
benzo[d]imidazole (22)
To an 8-mL screw cap vial were added N-methyl pyrrolidinone
(3 mL/vial) and 1 mL of a 0.14 M solution of O-(7-azabenzotriazol-
1-yl)-N,N,N0,N0-tetramethyl-uroniumhexafluoro phosphate (HATU)
in NMP. To this was added 50.0 mg (0.119 mmol) of 3-(4-(2-(2-(tri-
fluoromethyl)-1H-benzo[d]imidazol-4-yloxy)ethyl)piperazin-
1-yl)benzene-1,2-di-amine (88) followed by 4-tert-butylbenzoic
acid (25 mg, 0.143 mmol), and the vial was capped tightly. After
shaking overnight on an orbital shaker the vial was treated with
0.5 mL of glacial acetic acid, recapped and shaken at 110 °C for
2 h. It was then allowed to cool to room temperature and
shaken overnight. Sulfonic acid resin (Argonaut, 340 mg,
1.4 mmol/g) was then added and the vial was shaken for 6 h fol-
lowed by filtration using a polypropylene filter tube (15 mL). The
resin was washed with methanol (2ꢃ 3 mL) followed by dichloro-
methane (2ꢃ 3 mL). The resin was shaken with 1.0 mL of 9:1 meth-
anol/triethylamine for 3 min, and the mixture was filtered into a
test tube (13 ꢃ 100 mm) and the solvent removed by vacuum.
The crude product was then purified using automated RP-HPLC
(Method F) and the fractions evaporated in a 8-mL scintillation vial
to yield 23 mg (34% yield) of 2-(4-tert-butyl-phenyl)-4-(4-(2-(2-
(trifluoromethyl)-1H-benzo[d]imidazol-4-yloxy)ethyl) piperazin-
1-yl)-1H-benzo[d]-imidazole (22). 1H NMR (300 MHz, DMSO-d6)
d = 10.2 (br s, 1H), 8.09 (d, 2H, J = 8.4 Hz), 7.60 (d, 2H, J = 8.4 Hz),
7.35 (m, 2H), 7.20 (m, 2H), 7.02 (d, 1H, J = 8.1 Hz), 6.72 (d, 1H,
J = 8.1 Hz), 4.71 (br s, 2H), 4.42 (br s, 2H), 3.86 (br s, 2H), 3.79 (br
s, 2H), 3.57 (br s, 2H), 3.23 (br s, 2H), 1.35 (s, 9H). 13C NMR
(300 MHz, DMSO-d6) d = 158.39, 153.02, 148.87, 140.17, 139.20,
135.97, 126.49, 126.08, 125.66, 125.25, 123.67, 122.02, 120.71,
118.12, 117.12, 114.21, 110.3, 106.39, 105.76, 63.45, 54.81, 51.71,
46.37, 34.56, 30.85. HRMS [M+H]+ = 563.2739, calcd for
C31H34F3N6O: 563.2741. HPLC (Method D), tR = 10.5 min
(purity = 100%).
2.1.65. 4-(2-(4-(2-m-Tolyl-1H-benzo[d]imidazol-4-yl)piperazin-
1-yl)ethoxy)-1H-benzo[d]imidazole-2(3H)-thione (18)
To an 8-mL screw cap vial were added N-methyl pyrrolidinone
(4 mL/vial) and 279 lL of a 0.2 M solution of O-(7-azabenzotriazol-
1-yl)-N,N,N0,N0-tetramethyl-uronium hexafluorophosphate (HATU)
in NMP. To this was added 17.8 mg (0.046 mmol) of 4-{2-[4-(2,3-
diaminophenyl)-piperazin-1-yl]-ethoxy}-1,3-dihydro-benzimid-
azole-2-thione (89) followed by m-toluic acid (7.5 mg,
0.056 mmol) and the vial was capped tightly. After shaking over-
night on an orbital shaker the vial was treated with 0.5 mL of gla-
cial acetic acid, recapped and shaken at 110 °C for 2 h. It was then
allowed to cool to room temperature and was shaken overnight.
Sulfonic acid resin (Argonaut, 132 mg, 1.4 mmol/g) was then added
and the vial was shaken for 6 h followed by filtration using poly-
propylene filter tubes (15 mL). The resin was washed with metha-
nol (3ꢃ 3 mL) followed by dichloromethane (2ꢃ 3 mL). The filter
tube containing the washed resin was treated with 1.5 mL of 9:1
methanol/triethylamine. After loosely shaking for 3 min the
reaction was filtered into a test tube (13 ꢃ 100 mm) and the sol-
vent removed by vacuum. The crude product was then purified
using automated RP-HPLC (Method E) and the product fractions
were evaporated in a 8 mL scintillation vial to yield 3 mg (13%
yield) of 4-(2-(4-(2-m-tolyl-1H-benzo[d]imidazol-4-yl)piperazin-
1-yl)ethoxy)-1H-benzo[d]imidazole-2(3H)-thione (18). 1H NMR
(300 MHz, DMSO-d6) d = 12.68 (s, 1H), 12.66 (s, 1H), 9.72 (br s,
1H), 8.13 (s, 1H), 7.95 (d, 1H, J = 7.8 Hz), 7.70 (d, 1H, J = 7.8 Hz),
7.35 (t, 1H, J = 7.8 Hz), 7.23 (m, 2H), 7.15 (t, 1H, J = 8.3 Hz), 6.89
(m, 2H), 6.73 (m, 1H), 4.59 (br s, 4H), 3.89 (m, 2H), 3.78 (m, 2H),
3.60 (m, 2H), 3.31 (t, 2H, J = 15 Hz), 2.35 (s, 3H). HRMS
[M+H]+ = 485.2112, calcd for C27H29N6OS: 485.2116. HPLC (Meth-
od D): tR = 9.8 min (purity = 96.4%).
2.1.69. 2-(4-Isopropylphenyl)-4-(4-(2-(2-(trifluoromethyl)-1H-
benzo[d]imidazol-4-yloxy)ethyl)piperazin-1-yl)-1H-
benzo[d]imidazole (21)
Prepared as above for compound 22 88 and 4-i-propylbenzoic
acid (38% yield). 1H NMR (300 MHz, DMSO-d6) d = 10.2 (br s, 1H),
8.11 (d, 2H, J = 8.3 Hz), 7.48 (d, 2H, J = 8.3 Hz), 7.39 (m, 2H), 7.22
(m, 2H), 7.04 (d,1H, J = 7.9 Hz), 6.74 (d, 1H, J = 7.9 Hz), 4.74 (br s,
2H), 4.48 (br s,2H), 3.91 (br s, 2H), 3.82 (br s, 2H), 3.60 (br s, 2H),
3.25 (br s, 2H), 3.04 (m, 1H), 1.30 (d, 6H, J = 10.6 Hz). HRMS
[M+H]+ = 549.2593, calcd for C30H32F3N6O: 549.2582. HPLC (Meth-
od D), tR = 8.1 min (purity = 100%).
By the same method and scale were prepared:
2.1.66. 4-(2-(4-(2-(4-Ethylphenyl)-1H-benzo[d]imidazol-4-
yl)piperazin-1-yl)ethoxy)-1H-benzo[d]imidazole-2(3H)-thione
(20b)
Prepared as above for compound 18 from 89 and 4-ethylbenzoic
acid (15% yield). 1H NMR (300 MHz, DMSO-d6): d = 12.68 (s, 1H),
12.66 (s, 1H), 9.71 (br s, 1H), 8.11 (d, 2H, J = 8.3 Hz), 7.44 (d, 2H,
J = 8.3 Hz), 7.23 (m, 2H), 7.15 (t, 1H, J = 8.3 Hz), 6.89 (m, 2H), 6.73
(m, 1H), 4.59 (br s, 4H), 3.89 (m, 2H), 3.78 (m, 2H), 3.60 (m, 2H),
3.31 (t, 2H, J = 15 Hz), 2.72 (m, 2H), 1.26 (t, 3H, J = 9 Hz). HRMS
[M+H]+ = 499.2272, calcd for C28H31N6OS: 499.2275. A-HPLC
(Method D), tR = 8.6 min (purity = 90.2%).
2.1.70. 2-(4-Ethylphenyl)-4-(4-(2-(2-(trifluoromethyl)-1H-
benzo[d]imidazol-4-yloxy)ethyl)piperazin-1-yl)-1H-
benzo[d]imidazole (20a)
Prepared as above for compound 18 from 88 and 4-ethylbenzoic
acid (10% yield). 1H NMR (300 MHz, DMSO-d6) d = 10.2 (br s, 1H),
8.11 (d, 2H, J = 8.3 Hz), 7.45 (d, 2H, J = 8.3 Hz), 7.39 (m, 2H), 7.23
(m, 2H), 7.04 (d, 1H, J = 7.8 Hz), 6.75 (d, 1H, J = 7.8 Hz), 4.75 (m,
2H), 4.48 (br s, 2H), 3.91 (br s, 2H), 3.82 (m, 2H), 3.60 (br s, 2H),
3.25 (br s, 2H), 2.73 (q, 2H, J = 9.0 Hz), 1.28 (t, 3H, J = 9.0 Hz). HRMS
[M+H]+ = 535.2430, calcd for C29H30F3N6O: 535.2426. HPLC (Meth-
od D), tR = 8.1 min (purity = 100%).
2.1.67. 4-(2-(4-(2-(2,4-Dimethylphenyl)-1H-benzo[d]imidazol-
4-yl)piperazin-1-yl)ethoxy)-1H-benzo[d]imidazole-2(3H)-
thione (23)
Prepared as above for compound 18 from 89 and 2,4-dimethyl-
benzoic acid (10% yield). 1H NMR (300 MHz, DMSO-d6) d = 12.68

J. C. Pelletier et al. / Bioorg. Med. Chem. 16 (2008) 6617–6640
6637
2.1.71. 2-p-Tolyl-4-(4-(2-(2-(trifluoromethyl)-1H-
2.1.75. 2-({4-[2-(4-tert-Butylphenyl)-1H-benzimidazol-4-
benzo[d]imidazol-7-yloxy)-ethyl)piperazin-1-yl)-1H-
benzo[d]Imidazole (19)
yl]piperazin-1-yl}methyl)-6-nitroaniline (96)
2-{4-[2-(4-tert-Butylphenyl)-1H-benzoimidazol-4-yl]pipera-
zin-1-ylmethyl}-6-nitrophenylamine (290 mg, 0.60 mmol) and ti-
n(II) chloride (681 mg, 3.59 mmol) were stirred in anhydrous
NMP (40 mL) at 70 °C under nitrogen for 18 h. The reaction was
quenched with 1 N HCl (100 mL) and extracted with methylene
chloride (2ꢃ 50 mL). The organic extracts were combined and ex-
tracted with 1 N HCl (2ꢃ 50 mL). The aqueous extracts were com-
bined and neutralized with 2 N NaOH solution. This aqueous phase
was extracted with ethyl acetate (4ꢃ 100 mL). The organic extracts
were combined and washed with distilled water (10ꢃ 50 mL). The
ethyl acetate extracts were combined, dried (Na2SO4), and the sol-
vent removed to give a brown oil. This material was dissolved in
aqueous DMSO and purified by RP-HPLC. The product fractions
were collected and the acetonitrile removed in vacuo. The aqueous
residue lyophilized to give a white solid (55 mg, 20% yield). 1H
NMR (300 MHz, DMSO-d6) d = 8.08 (d, 2H, J = 8.2 Hz), 7.57 (d, 2H,
J = 8.5 Hz), 7.15 (m, 2H), 6.90 (m, 2H), 6.65 (m, 2H), 4.35 (s, 2H),
3.50 (br s, 8H), 1.35 (s, 9H). HPLC (Method D), tR = 4.9 min (pur-
ity = 99.0%), [M+H]ꢂ = 453, [M+H]+ = 455.
Prepared as above for compound 18 from 88 and p-toluic acid
(36% yield). 1H NMR (300 MHz, DMSO-d6) d = 10.2 (br s, 1H), 8.04
(d, 2H, J = 8.1 Hz), 7.42 (d, 2H, J = 8.1 Hz), 7.39 (m, 2H), 7.23 (m,
2H), 7.04 (d, 1H, J = 7.8 Hz), 6.75 (d, 1H, J = 7.8 Hz), 4.75 (m, 2H),
4.48 (br s, 2H), 3.91 (br s, 2H), 3.82 (m, 2H), 3.60 (br s, 2H), 3.25
(br s, 2H), 2.34 (s, 3H). HRMS [M+H]+ = 521.2268, calcd for
C28H28F3N6O: 521.2271. HPLC (Method D), tR = 9.4 min
(purity = 91%).
2.1.72. tert-Butyl 4-(2-(4-tert-butylphenyl)-1H-
benzo[d]imidazol-4-yl)piperazine-1-carboxylate (90)
A mixture of tert-butyl 4-(2,3-diaminophenyl)piperazine-1-car-
boxylate (36, 1.8 g, 5.9 mmol), 4-tert-butylbenzaldehyde (1.1 g,
7.1 mmol, 1.2 mL), and 10% palladium on carbon (0.60 g) in isopro-
panol (40 mL) was stirred rapidly and heated to 80 °C for 2 h open
to air. After cooling to room temperature the catalyst was filtered
with the aid of diatomaceous earth, washed with isopropanol,
and the combined filtrates were evaporated under reduced pres-
sure. The crude product was chromatographed on silica gel eluted
with 75% hexanes in ethyl acetate to leave the product as a foamy
solid (2.5 g, 95%). 1H NMR (300 MHz, DMSO-d6) d = 12.72 (br s, 1H),
8.06 (d, 2H, J = 8.3 Hz), 7.56 (d, 2H, J = 8.3 Hz), 7.05 (m, 2H), 6.53 (d,
1H, J = 5.1 Hz), 3.56 (brs, 4H), 3.51 (brs, 4H), 1.44 (s, 9H), 1.33 (s,
2.1.76. 4-{4-[2-(4-tert-Butyl-phenyl)-1H-benzoimidazol-4-yl]-
piperazin-1-ylmethyl}-1,3-dihydro-benzoimidazole-2-thione
(24)
2-Amino-3-({4-[2-(4-tert-butylphenyl)-1H-benzimidazol-4-yl]-
piperazin-1-yl}methyl)phenylamine (96, 140 mg, 0.31 mmol) and
1,10-thiocarbonyldiimidazole (67 mg, 0.34 mmol) were stirred in
anhydrous THF (10 mL) at 60 °C for 2 h. The solvent was removed,
in vacuo, to give a reddish brown oil (220 mg). This material was
adsorbed onto silica and purified by column chromatography, elut-
ing with a 1:1 solution of hexane in ethyl acetate to give an off-
white solid (30 mg, 20% yield). 1H NMR (300 MHz, DMSO-d6)
d = 8.05 (d, 2H, J = 8.4 Hz), 7.55 (d, 2H, J = 8.5 Hz), 7.05 (m, 5H),
6.48 (m, 1H), 3.75 (s, 2H), 3.55 (br s, 4H), 2.65 (br s, 4H), 1.30 (s,
9H). HPLC (Method D) tR = 9.0 min (purity = 90.7%). HRMS
[M+H]+ = 497.2484, calcd for C29H33N6S: 497.2482.
9H).
LC/MS
(Method
I),
tR = 2.83 min
(purity = 100%),
[M+H]+ = 435.
2.1.73. 2-(4-tert-Butylphenyl)-4-(piperazin-1-yl)-1H-
benzo[d]imidazole (91)
A
solution of tert-butyl 4-(2-(4-tert-butylphenyl)-1H-
benzo[d]imidazol-4-yl)piperazine-1-carboxylate (90, 2.4 g,
5.3 mmol) in trifluoroacetic acid (20 mL) and dichloromethane
(20 mL) was stirred for 2 h. The solvents were evaporated, and
the crude product was purified by reversed phase HPLC (Method
E). The product containing fractions were combined, neutralized
with 1 M sodium carbonate solution, and extracted with ethyl ace-
tate (2ꢃ 50 mL). The extracts were combined, dried (MgSO4), and
evaporated under reduced pressure to provide the product as a
white, foamy solid (1.0 g, 54%). 1H NMR (300 MHz, DMSO-d6)
d = 12.68 (br s, 1H), 8.05 (d, 2H, J = 8.1 Hz), 7.56 (d, 2H,
J = 8.1 Hz), 7.05 (m, 2H), 6.49 (d, 1H, J = 7.1 Hz), 3.48 (brs, 4H),
2.97 (brs, 4H), 1.33 (s, 9H). LC/MS (Method I), tR = 1.40 min (pur-
ity = 95.0%), [M+H]+ = 335.
2.1.77. 4-{4-[2-(4-tert-Butyl-phenyl)-1H-benzoimidazol-4-yl]-
piperazin-1-ylmethyl}-1,3-dihydro-benzoimidazol-2-one (27)
2-Amino-3-({4-[2-(4-tert-butylphenyl)-1H-benzimidazol-4-yl]-
piperazin-1-yl}methyl)phenylamine (96, 150 mg, 0.33 mmol) and
1,10-carbonyldiimidazole (59 mg, 0.36 mmol) were stirred in anhy-
drous THF at 60 °C for 2 h. The solvent was removed in vacuo, dis-
solved in aqueous DMSO and purified by RP-HPLC (Method F). The
product fractions were combined and lyophilized to give a white
solid (45 mg, 28% yield). 1H NMR (300 MHz, DMSO-d6) d = 11.10
(s, 1H), 10.90 (s, 1H), 8.08 (d, 2H, J = 8.5 Hz), 7.60 (d, 2H,
J = 8.5 Hz), 7.10 (m, 3H), 7.05 (m, 2H), 6.65 (m, 1H), 4.45 (s, 2H),
3.55 (br s, 8H), 1.33 (s, 9H). HPLC tR = 8.9 min (purity = 88.0%).
HRMS [M+H]+ = 481.2708 calcd for C29H33N6O: 481.2710.
2.1.74. 2-{4-[2-(4-tert-Butyl-phenyl)-1H-benzoimidazol-4-
yl]piperazin-1-ylmethyl}-6-nitro-phenylamine (95)
To a solution of 2-(4-tert-butyl-phenyl)-4-piperazin-1-yl-1H-
benzoimidazole (91, 1.30 g, 3.89 mmol) and 2-chloromethyl-6-ni-
tro-phenylamine (50, 660 mg, 3.53 mmol) in anhydrous NMP
(40 mL) was added diisopropylethylamine (1.0 mL, 4.24 mmol)
and the solution stirred at room temperature under nitrogen for
18 h. The solution was diluted with ethyl acetate (150 mL) and
washed with water (10ꢃ 50 mL). The organic extracts were com-
bined and washed with brine (2ꢃ 100 mL), dried (Na2SO4), filtered,
and the solvent removed to give a tan solid with some amber col-
ored oil (1.84 g). This material was adsorbed onto silica gel and
purified by column chromatography eluted with a solution of
20% ethyl acetate in hexane to afford the nitro amino product
(610 mg, 36% yield) as a yellow solid. 1H NMR (300 MHz, CDCl3)
d = 8.25 (d, 1H, J = 7.5 Hz), 8.00 (d, 2H, J = 8.2 Hz), 7.50 (d, 1H,
J = 6.9 Hz), 7.35 (d, 2H, J = 8.4 Hz), 7.00 (m, 1H), 6.95 (m, 1H),
6.75 (m, 1H), 6.65 (d, 1H, J = 6.7 Hz), 4.15 (s, 2H), 3.33 (br s, 4H),
3.15 (br s, 4H), 1.25 (s, 9H). HPLC (Method D) tR = 12.5 min (pur-
ity = 100%). MS, [M+H]ꢂ = 483, [M+H]+ = 485.
2.1.78. 5-({4-[2-(4-tert-Butylphenyl)-1H-benzimidazol-4-
yl]piperazin-1-yl}methyl)-1,4-dihydroquinoxaline-2,3-dione
(28)
2-Amino-3-({4-[2-(4-tert-butylphenyl)-1H-benzimidazol-4-yl]-
piperazin-1-yl}methyl)phenylamine (96, 100 mg, 0.22 mmol) and
oxalyldiimidazole (63 mg, 0.33 mmol) were stirred at room tem-
perature in anhydrous tetrahydrofuran (10 mL) for 2 h. The reac-
tion was quenched with water (5 mL) and stirred for 1 h. The
solvent was removed in vacuo and the aqueous residue dissolved
in dimethylsulfoxide and purified by RP-HPLC (Method F). The
product fractions were combined and lyophilized to afford the
product as a white powder (7 mg, 6%). 1H NMR (300 MHz,
DMSO-d6) d = 8.07 (d, 2H, J = 8.4 Hz), 7.58 (d, 2H, 8.4 Hz), 7.27
(m, 3H), 7.14 (m, 2H), 6.75 (m, 1H), 4.64 (m, 4H), 3.16 (s, 2H),

6638
J. C. Pelletier et al. / Bioorg. Med. Chem. 16 (2008) 6617–6640
1.34 (s, 9H). HPLC (Method D) tR = 9.1 min (purity = 92.3%). MS
[MꢂH]ꢂ = 507, [M+H]+ = 509.
(2ꢃ 50 mL) and brine (50 mL). The organic layer was dried (MgSO4)
and concentrated to leave 190 mg (92% yield) of 4-[4-(3-azido-4-
nitrobenzyl)-piperazin-1-yl]-2-(4-tert-butylphenyl)-1H-benzoimi-
dazole as a light yellow solid. 1H NMR (300 MHz, DMSO-d6)
d = 12.69 (s, 1H), 8.04 (d, 2H, J = 8.7 Hz), 8.01 (d, 1H, J = 8.3 Hz),
7.57 (m, 1H), 7.54 (d, 2H, J = 8.6 Hz), 7.39 (dd, 1H, J = 8.5, 1.2 Hz),
7.03 (m, 2H), 6.50 (dd, 1H, J = 5.5, 1.4 Hz), 3.70 (s, 2H), 3.60 (br s,
4H), 2.68 (br s, 4H), 1.33 (s, 9H). LC/MS (Method A), tR = 1.41 min
(purity = 89.8%), [M+H]+ = 511, [MꢂH]ꢂ = 509.
2.1.79. 4-(4-Benzylpiperazin-1-yl)-2-(4-tert-butylphenyl)-1H-
benzimidazole (25)
2-(4-tert-Butylphenyl)-4-piperazin-1-yl-1H-benzimidazole (91,
40 mg, 0.12 mmol), benzaldehyde (15.8 lL, 0.16 mmol), and so-
dium triacetoxyborohydride (51 mg, 0.24 mmol) were stirred in
dichloroethane (2 mL) at room temperature for 18 h. The product
was concentrated, in vacuo, and dissolved in dimethylsulfoxide/
water and purified by RP-HPLC (method). The product fractions
were combined and the solvent removed to afford the product as
a clear oil as the di-TFA salt (51 mg, 100% yield). 1H NMR
(500 MHz, DMSO-d6) d = 1.36 (s, 9H), 3.02–3.21 (m, 2H), 3.40 (d,
J = 2.44 Hz, 2H), 3.47–3.59 (m, 2H), 4.49 (s, 4H), 6.69 (d,
J = 7.32 Hz, 1H), 7.20 (s, 1H), 7.18 (d, J = 7.32 Hz, 1H), 7.58–7.66
(m, 5H), 8.10 (d, J = 7.50 Hz, 2H), 9.97 (s, 1H). HPLC (Method D),
tR = 9.7 min (purity = 100%). HRMS, [M+H]+ = 425.2702, [M+H]+
calcd for C28H33N4: 425.2700.
2.1.83. 4-{4-[2-(4-tert-Butyl-phenyl)-1H-benzoimidazol-4-yl]-
piperazin-1-ylmethyl}-benzene-1,2-diamine (94)
In a round bottom flask under nitrogen were combined 4-[4-(3-
azido-4-nitro-benzyl)-piperazin-1-yl]-2-(4-tert-butyl-phenyl)-1H-
benzoimidazole (93, 0.19 g, 0.37 mmol), methanol (20 mL), and 5%
platinum on carbon (0.145 g). A hydrogen balloon was attached,
the flask evacuated, and a hydrogen atmosphere established. After
stirring for 4 h, the balloon was removed, the flask purged with
nitrogen, and the reaction mixture filtered thru Celite with a large
excess of methanol. The solution was concentrated to dryness on
a rotary evaporator to yield 170 mg (100% yield) of 4-{4-[2-(4-
tert-butyl-phenyl)-1H-benzoimidazol-4-yl]-piperazin-1-ylmethyl}-
benzene-1,2-diamine as a brown oil. LC/MS (Method A), tR =
0.90 min (purity = 100%), [M+H]+ = 455, [MꢂH]ꢂ = 453.
2.1.80. 2-(4-tert-Butylphenyl)-4-[4-(2-
naphthylmethyl)piperazin-1-yl]-1H-benzimidazole (26)
2-(4-tert-Butylphenyl)-4-piperazin-1-yl-1H-benzimidazole (91,
40 mg, 0.12 mmol), 2-napthaldehyde (24 mg, 0.16 mmol), and so-
dium triacetoxyborohydride (51 mg, 0.24 mmol) were stirred in
dichloroethane (2 mL) at room temperature for 18 h. The product
was concentrated, in vacuo, and dissolved in dimethylsulfoxide/
water and purified by RP-HPLC (Method F). The product fractions
were combined, and the solvent was removed to afford the prod-
uct as a clear oil as the di-TFA salt (48 mg, 84% yield). 1H NMR
(500 MHz, DMSO-d6) d = 1.36 (s, 9H), 3.01–3.25 (m, 4H), 3.61 (br
s, 4H), 4.43 (br s, 1H), 4.99 (br s, 2H), 6.68 (d, J = 7.32 Hz, 1H),
2.1.84. 6-{4-[2-(4-tert-Butyl-phenyl)-1H-benzoimidazol-4-yl]-
piperazin-1-ylmethyl}-1,4-dihydro-quinoxaline-2,3-dione (29)
In a round bottom flask under nitrogen were combined 4-{4-
[2-(4-tert-butyl-phenyl)-1H-benzoimidazol-4-yl]-piperazin-1-yl-
methyl}-benzene-1,2-diamine (94, 0.17 g, 0.37 mmol), THF
(20 mL), and 1,10-oxalyldiimidazole (0.284 g, 1.5 mmol) and the
solution stirred overnight. The solution was diluted with ethyl ace-
tate (100 mL) and washed with water (100 mL). A precipitate
forms and was collected by filtration. The filtrate was concentrated
to dryness on a rotary evaporator and combined with the precipi-
tate. Purification by RP-HPLC (Method E) yielded 46 mg (24% yield)
of 6-{4-[2-(4-tert-butyl-phenyl)-1H-benzoimidazol-4-yl]-pipera-
zin-1-ylmethyl}-1,4-dihydro-quinoxaline-2,3-dione as an off-white
solid. 1H NMR (300 MHz, DMSO-d6) d = 12.19 (s, 1H), 12.09 (s, 1H),
9.82 (br s, 1H), 8.07 (d, 2H, J = 8.6 Hz), 7.58 (d, 2H, J = 8.6 Hz), 7.27
(m, 2H), 7.22 (d, 1H, J = 8.6 Hz), 7.13 (m, 2H), 6.64 (m, 1H), 4.48 (br
s, 2H), 4.44 (s, 2H), 3.50 (br s, 2H), 3.38 (br s, 2H), 3.09 (m, 2H),
1.34 (s, 9H). HPLC (Method D), tR = 5.84 min (purity = 79%). HRMS
[M+H]+ = 509.2659, calcd for C30H33N6O2: 509.2659.
7.19 (d, J = 4.39 Hz, 2H), 7.49
- 7.77 (m, 6H), 7.88 (d,
J = 6.84 Hz, 1H), 8.09 (d, J = 8.30 Hz, 2H), 8.49 (d, J = 8.79 Hz,
2H), 9.73–9.97 (m, 1H). HPLC (Method D), tR = 11.0 min (pur-
ity = 92.0%). HRMS, [M+H]+ = 475.2860, calcd for C32H35N4
[M+H]+: 475.2856.
2.1.81. 2-(4-tert-Butylphenyl)-4-(4-(3-fluoro-4-
nitrobenzyl)piperazin-1-yl)-1H-benzoimidazole (92)
In
a round bottom flask were combined the 3-fluoro-4-
nitrobenzaldehyde (0.52 g, 3.07 mmol) and NMP (20 mL). To
the stirring solution were added the 2-(4-tert-butyl-phenyl)-4-
(piperazin-1-yl)-1H-benzo[d]imidazole (91, 0.86 g, 2.56 mmol)
and sodium triacetoxyborohydride (1.36 g, 6.4 mmol), and the
resulting mixture stirred overnight. The reaction mixture was
diluted with ethyl acetate (200 mL) and washed with saturated
sodium bicarbonate solution, water, and brine. The organic
layer was dried (MgSO4) and concentrated. The crude product
was purified by column chromatography with 40% ethyl ace-
tate in hexane as eluant to leave 850 mg (68% yield) of 2-(4-
tert-butylphenyl)-4-(4-(3-fluoro-4-nitrobenzyl)piperazin-1-yl)-
1H-benzimidazole as a light yellow solid. 1H NMR (300 MHz,
DMSO-d6) d = 12.69 (s, 1H), 8.16 (t, 1H, J = 8.2 Hz), 8.04 (d, 2H,
J = 8.5 Hz), 7.59 (m, 1H), 7.55 (d, 2H, J = 8.5 Hz), 7.47 (d, 1H,
J = 8.6 Hz), 7.03 (m, 2H), 6.50 (dd, 1H, J = 7.0 Hz, 1.8 Hz), 3.71 (s,
2H), 3.60 (br s, 4H), 2.66 (br s, 4H), 1.33 (s, 9H). LC/MS (Method A),
tR = 1.24 min (purity = 100%), [M+H]+ = 488, [MꢂH]ꢂ = 486.
2.1.85. 5-((4-(2-(4-tert-Butylphenyl)-1H-benzo[d]imidazol-4-
yl)piperazin-1-yl)methyl)pyrimidine-2,4(1H,3H)-dione (30)
2-(4-tert-Butylphenyl)-4-piperazin-1-yl-1H-benzimidazole (91,
75 mg, 0.22 mmol), uracil-5-carboxaldehyde (41 mg, 0.29 mmol),
and sodium triacetoxyborohydride (95 mg, 0.45 mmol) were stirred
in NMP (2 mL) at room temperature for 18 h. The mixture was trea-
ted with ethyl acetate (50 mL), washed with water (7ꢃ 30 mL) and
brine (40 mL), dried (MgSO4), and evaporated. The crude product
was purified by RP-HPLC (Method F). The product fractions were
combined, treated with saturated sodium bicarbonate solution
(50 mL), and extracted with ethyl acetate (3ꢃ 30 mL). The combined
extracts were washed with brine (50 mL), dried (MgSO4), and evap-
orated to leave the product as a white solid (15 mg, 15%). 1H NMR
(400 MHz, MeOH-d4) d = 7.98 (d, 2H, J = 8.5 Hz), 7.51 (d, 2H,
J = 8.5 Hz), 7.43 (s, 1H), 7.10 (m, 2H), 6.67 (m, 1H), 3.40 (m, 4H),
3.35 (s, 2H), 2.77 (m,. 4H), 1.33 (s, 9H). 13C NMR (300 MHz, DMSO-
d6) d = 164.33, 152.03, 151.23, 148.08, 142.79, 140.32, 136.08,
135.26, 127.56, 125.96, 125.57, 123.09, 107.88, 106.19, 103.29,
52.70, 52.36, 49.08, 34.48, 30.95. HPLC (Method D), tR = 8.5 min
(purity = 99.9%). HRMS, [M+Na]+ = 481.2327, calcd for C26H30N6O2:
481.2323.
2.1.82. 4-[4-(3-Azido-4-nitro-benzyl)-piperazin-1-yl]-2-(4-tert-
butyl-phenyl)-1H-benzo-imidazole (93)
In a round bottom flask were combined 2-(4-tert-butylphenyl)-
4-[4-(3-fluoro-4-nitrobenzyl)piperazin-1-yl]-1H-benzoimidazole
(0.197 g, 0.40 mmol), dimethyl sulfoxide (3 mL), and sodium azide
(0.029 g, 0.44 mmol), and the mixture stirred overnight. The mix-
ture was diluted with ethyl acetate (100 mL), washed with water

J. C. Pelletier et al. / Bioorg. Med. Chem. 16 (2008) 6617–6640
6639
2.1.86. 5-((4-(2-(4-tert-Butylphenyl)-1H-benzo[d]imidazol-4-
yl)piperazin-1-yl)methyl)-1-ethylpyrimidine-2,4(1H,3H)-dione
(31)
tion and replaced with an HBSS enzyme solution consisting of
3.5 mg/mL collagenase type (Worthington Biochemicals),
1
1.2 mg/mL hyaluronidase type III (Calbiochem), and 3% BSA (Sig-
ma). Tissue was incubated at 37 for 1 h and then dissociated by
gentle trituration through a fire polished Pasteur pipet. Cells were
strained through a cell strainer (Costar) to remove clumps and pel-
leted by centrifugation. Dissociated cells were resuspended in
DMEM media (low glucose—Invitrogen) supplemented with
25 mM Hepes, 1ꢃ glutamax (Invitrogen), 1ꢃ antibiotic/antimycotic
(Invitrogen), and 10% fetal bovine serum (growth media) and pla-
ted into flat bottom 96-well tissue culture plates at 30,000 cells/
well. Cultures were incubated at 37 °C with 5% CO2 for 72 h.
Treatments prepared in challenge media, which is growth med-
ia where the FBS is replaced with 0.1% BSA. Treatment consisted of
a dose response of an antagonist of choice in the presence of an
ED50 concentration of the GnRH analogue Dtrp6-LHRH. Cultures
were washed 2ꢃ with challenge media to remove serum and then
incubated with 100 lL/well of treatment for 3 h. Media was re-
moved and saved for LH RIA analysis. Toxicity of compound was
assessed directly on the cells using the cell titer-glo assay
(Promega).
2-(4-tert-Butylphenyl)-4-piperazin-1-yl-1H-benzimidazole (91,
75 mg, 0.22 mmol), 1-ethyluracil-5-carboxaldehyde (49 mg,
0.29 mmol) and sodium triacetoxyborohydride (95 mg, 0.45 mmol)
were stirred in NMP (2 mL) at room temperature for 18 h. The mix-
ture was treated with ethyl acetate (50 mL), washed with water
(7ꢃ 30 mL) and brine (40 mL), dried (MgSO4), and evaporated.
The crude product was purified by RP-HPLC (Method F). The prod-
uct fractions were combined, treated with saturated sodium bicar-
bonate solution (50 mL), and extracted with ethyl acetate (3ꢃ
30 mL). The combined extracts were washed with brine (50 mL),
dried (MgSO4), and evaporated to leave the product as a white solid
(50 mg, 46%). 1H NMR (400 MHz, MeOH-d4) d = 7.97 (d, 2H,
J = 8.6 Hz), 7.59 (s, 1H), 7.51 (d, 2H, J = 8.6 Hz), 7.10 (m, 2H), 6.65
(m, 1H), 3.77 (q, 2H, J = 7.2 Hz), 3.38 (m, 4H), 3.34 (s, 2H), 2.75
(m,. 4H), 1.31 (s, 9H), 1.25 (t, 3H, J = 7.2 Hz). 13C NMR (300 MHz,
DMSO-d6) d = 163.81, 152.03, 150.72, 158.08, 143.80, 142.79,
139.09, 135.27, 127.57, 125.97, 125.55, 123.08, 108.66, 106.18,
103.30, 52.82, 52.37, 49.03, 42.64, 34.47, 30.94, 14.16. HPLC (Meth-
od D), tR = 8.8 min (purity = 90.5%). HRMS, [M+Na]+ = 481.2638,
calcd for C28H34N6O2: 481.2323.
2.5. Efficacy studies in rats
2.2. Radio-ligand binding assay
Male Sprague–Dawley rats ten days post-castration were
instrumented with indwelling catheters in jugular vein to facilitate
frequent blood sampling, Basal blood samples were drawn from all
the animals and either vehicle (2% Tween 80/0.04 N HCl) or small
molecule GnRH antagonist was administered orally. Blood samples
were drawn at regular intervals and plasma LH levels measured
using a commercial ELISA kit (Endocrine Technologies, CA).
Radio-ligand binding assays were done according to the proce-
dure described by Millar et al. (Methods Neurosci. 1995, 25, 145–
162). Briefly, the recombinant cells expressing human or rat GnRH
receptors were harvested in binding buffer, (25 mM Tris–Cl, pH 7.4,
0.1% sodium azide, 0.1% BSA), homogenized with a polytron and
centrifuged at 12,000g for 15 min. The membrane pellets were
washed, resuspended in binding buffer and the protein concentra-
tion was determined (Pierce, Rockford, IL). Typically, 50 lg of the
membrane protein was incubated with approximately
50,000 cpm of 125I-(D-trp6)-LHRH for 2 h at 4 °C in a total volume
of 200 lL. Competition studies were performed by the addition of
increasing concentrations of unlabeled competitor. The samples
were filtered through glass-fiber filters (Skatron Corp. Sterling,
VA) presoaked in wash buffer (50 mM Tris, pH 7.4, 10 mM MgCl2,
0.5 mM EDTA) containing 1% BSA. The filters were washed twice
with the wash buffer, and the retained radioactivity was counted
on a gamma counter (Packard Instruments). Assays were always
performed in triplicates. Non-specific binding was assessed in the
presence of 1 lM unlabeled (D-trp6)-LHRH.
2.6. Statistical analysis
Data are analyzed using Excel/SAS analysis system (SAS Insti-
tute Inc., Cary, NC).
Acknowledgments
The authors thank Magid Abou-Gharbia, Ron Magolda, Richard
Lyttle and Len Freedman for support of this work. We also thank
our colleagues in analytical chemistry and pharmaceutical profil-
ing for compound characterization and Terry Andree for additional
biological evaluation.
References and notes
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