Selective benzimidazole inhibitors of the antigen receptor-mediated NF-κB activation pathway

Article abstract of DOI:10.1016/j.bmc.2010.01.039

Dysregulated antigen receptor-mediated NF-κB activation can contribute to development of autoimmunity, chronic inflammation, and malignancy. A chemical biology screening strategy has identified a substituted benzimidazole that selectively inhibits antigen receptor-mediated NF-κB activation without blocking other NF-κB activation pathways. A library of analogs was synthesized and the structure-activity relationship and metabolic stability for the series is presented.

Full text of DOI:10.1016/j.bmc.2010.01.039

Bioorganic & Medicinal Chemistry 18 (2010) 1918–1924
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Selective benzimidazole inhibitors of the antigen receptor-mediated NF-
activation pathway
jB
Karl J. Okolotowicz a, , , Ranxin Shi b, , Xueying Zheng a, Mary MacDonald a, John C. Reed b, John R. Cashman a
*
a Human BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, CA 92121, USA
b Burnham Institute for Medical Research, 10901 N. Torrey Pines Rd., La Jolla, CA 92037, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Dysregulated antigen receptor-mediated NF-jB activation can contribute to development of autoimmu-
Received 30 October 2009
Revised 14 January 2010
Accepted 16 January 2010
Available online 25 January 2010
nity, chronic inflammation, and malignancy. A chemical biology screening strategy has identified a
substituted benzimidazole that selectively inhibits antigen receptor-mediated NF- B activation without
blocking other NF- B activation pathways. A library of analogs was synthesized and the structure–activ-
ity relationship and metabolic stability for the series is presented.
j
j
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Nf-jB
Benzimidazoles
Antigen receptor-mediated
Library synthesis
Medicinal chemistry optimization
1. Introduction
of a complex of IKK-alpha, IKK-beta, and the scaffold protein,
IKK-gamma. In all but the non-canonical NF-kB pathway, IKK acti-
Nuclear factor-kappa B (NF-
scription factors that play a critical role in many pathways includ-
ing host-defense, immune responses, inflammation, and cancer.
j
B) is a member of a family of tran-
vation results in phosphorylation of I
tein for ubiquitination and proteasome-dependent destruction,
thus releasing p65/p50 NF- B heterodimers from I B-alpha in
jB-alpha, targeting this pro-
j
j
Currently, nine pathways are known for the activation of NF-
jB
the cytosol, and allowing translocation into the nucleus where they
in mammals that include: (1) a canonical (classical) tumor necrosis
factor (TNF) pathway;1 (2) a non-canonical (alternative) pathway
activated by select TNF-family receptors;2 (3) the Toll-like receptor
(TLR) pathway that involves the TLR domain-containing adapters
and interleukin-1 receptor associated kinase (IRAK) family of pro-
tein kinases;3 (4) an exogenous RNA activated pathway that is of
importance for host-defenses against viruses;4 (5) a DNA damage
pathway involving the p53-induced protein with a death domain
(PIDD);5 (6) a pathway that involves the nucleotide-binding oligo-
merization domain (NOD)-family of proteins, cytosolic proteins
that oligomerize in response to microbial-derived molecules;6 (7)
an antigen receptor-activated pathway in B-cells and T-cells;7 (8)
inhibition of apoptosis (IAP2)-mucosa associated lymphoid tissue
lymphoma translocation gene 1 through tumor necrosis factor
(TNF)-receptor-associated factor 6 (TRAF6); and (9) X-linked inhi-
bition of Apoptosis Protein (XIAP) working via a transforming
growth factor b activated kinase 1 (TAB1) pathway.8 The core event
initiate transcription of various target genes.
The NF-jB pathway activated by antigen receptors is critical for
acquired (as opposed to innate) immunity and contributes to T-
and B-lymphocyte activation, proliferation, survival, and effector
functions. Dysregulated NF-
tribute to development of autoimmunity, chronic inflammation,
and lymphoid malignancy.7,9 The NF-
B activation pathway linked
jB activation in lymphocytes can con-
j
to antigen receptors involves a cascade of adapter and signal trans-
ducing proteins that minimally includes Carma1, Bcl-10, Paracas-
pase (MALT1), TRAF6, and Ubc13. In T- and B-cells, the NF-jB
pathway is initiated by protein kinase C (PKC)-theta and PKC-beta,
respectively, that induces phosphorylation of components of this
signaling pathway, leading ultimately to IKK activation through a
mechanism involving lysine 63-linked polyubiquitination of IKK-
gamma.10 Thus, the antigen receptor pathway for NF-
jB activation
is initiated and concluded by activation of protein kinases (i.e.,
PKCs and IKKs, respectively). With respect to drug discovery,
although IKKs represent logical drug targets, chemical inhibitors
that these NF-
jB activation pathways converge upon is the activa-
tion of Inhibitor of
jB Kinases (IKKs) that are typically comprised
of IKKs suppress all reported NF-jB activation pathways, and thus
lack the selectivity required to inhibit lymphocyte responses with-
out simultaneously interfering with innate immunity and creating
broad immunosuppression with considerable risk of infection.11
* Corresponding author. Tel.: +1 858 458 9305; fax: +1 858 458 9311.
E-mail address: kokolotowicz@hbri.org (K.J. Okolotowicz).
Contributed equally to the manuscript.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.039

K. J. Okolotowicz et al. / Bioorg. Med. Chem. 18 (2010) 1918–1924
1919
A chemical biology strategy for identification of chemical com-
N
N
NH OH
pounds that selectively inhibit antigen receptor-mediated NF-jB
B
activation was devised.12 Two libraries comprising a total of
110,000 compounds13 were screened for inhibitors of antigen
O
A
receptor-mediated NF-
<3 M were further characterized by twelve additional counter-
screens that helped determine inhibition selectivity. From the ini-
tial screens, three hits with an IC50 <2 M were identified and con-
firmed as selective for the antigen receptor activation pathway by
the counter-screen assays. Of the three hits, benzimidazole (1)
(Fig. 1) was found to be a highly potent and selective inhibitor of
jB activation. Compounds with an IC50
l
OH
l
C
Figure 2. Three regions (i.e., A, B and C) of library diversification in ‘hit’ compound 1.
antigen receptor-mediated NF-
jB activation. The successful syn-
thesis of a diverse library of compounds based on benzimidazole
1 and their biological evaluation as inhibitors of NF-jB was accom-
plished and is reported herein.
S
H
N
HBr, Br2
R1
R1
R1
R1
NH2
NH2
S
O
+
S
K
+
EtOH / H2O
60 %
N
HOAc
32 %
H
2, R1 = H
3, R1 = CH3
4, R1 = H
5, R1 = CH3
2. Results and discussion
2.1. Chemistry
O
R1
N
K2CO3
DMF
R1
R1
R2
R3
Br
N
Br
Br
N
N
R1
Identification of regions in the ‘hit’ (i.e., compound 1) to diver-
sify by chemical synthesis was guided by biological evaluation of a
number of closely related compounds obtained from a commercial
supplier (ChemBridge Corporation, San Diego, CA). The results of
inhibition studies of antigen receptor-mediated NF-jB activation
from a small collection of commercial compounds suggested all
H
O
R4
75 - 88 %
8a-d
6, R1 = H
7, R1 = CH3
R4
R2
R3
9a-d, R1 = H
10a-d, R1 = CH3
n=1,2
X
n=1,2
HN
R1
R1
N
N
N
three regions (i.e., A, B, C) of the benzimidazole core were impor-
X
tant for NF-
jB inhibitory potency (Fig. 2).
R2 = H, t-butyl, OCH2
R3 = OH, H, OCH2
R4 = H, t-butyl
125 °C
50 - 70 %
O
Two synthetic approaches (Schemes 1 and 2), were designed
and utilized to vary either B or C portions of 1 during the last step
of the synthesis.
X = N, O, C
R4
R2
R3
The appropriate phenylenediamine (2 or 3) was treated with
potassium ethyl xanthate in EtOH/H2O to give 2-mercaptobenzimi-
dazole 4 or 5.14 2-Mercaptobenzimidazole, 4 or 5, was further trea-
ted with HBr/Br2 in acetic acid to afford the 2-bromobenzimidazole
6 or 7.15 Diversification of the C region was accomplished by addi-
tion of bromoacetophones 8a–d in the presence of potassium car-
bonate in DMF to provide compounds 9a–d and 10a–d. To further
diversify region B, compounds 9a–d or 10a–d were heated at
125 °C with the desired cyclic amine to give compounds 11a–d
and 12a–d as a library of tertiary amines. It was found that an
undesirable cyclic byproduct (i.e., 13) was isolated as the major
product when acyclic primary amines (e.g., 3-aminopropanol)
were used in the synthesis of 1 (Fig. 3).16
11a-d, R1 = H
12a-d, R1 = CH3
Scheme 1. Synthetic pathway for the variation of the B and C regions of 1 (Fig. 2).
OH
N
N
H2N
OH
125 °C
Br
NH
N
N
H
H
76 %
7
14
To help minimize the undesired cyclic byproduct, the synthesis
shown in Scheme 1 was further optimized as shown in Scheme 2.
Heating 3-aminopropanol with 7 at 125 °C gave 14. Compounds 14
and 15 were heated in n-butanol at 115 °C to give 1 as the major
product, with only ꢀ5% of 13 isolated. This optimized method gave
increased yields of the desired product over the method shown in
Scheme 1. For example, the method of Scheme 2 gave a ratio of
95:5 of 1/13, whereas the method of Scheme 1 only gave a ratio
OH
O
14
N
N
Br
NH
Butanol
115 °C
45 %
HO
O
15
OH
1
Scheme 2. Optimized synthesis of benzimidazole analog 1.
OH
N
NH
N
of 2:98 of 1/13. The optimized method of Scheme 2 allowed for
synthesis of diversity of the C region using primary amine side-
chains on the benzimidazole ring system.
O
OH
2.2. SAR discussion
1
5,6-Dimethyl-2-bromobenzimidazole (7) and 2-bromobenzimi-
dazole (6) were chosen as the core moieties for library construction
because of the commercial availability of their phenylenediamine
Figure 1. An amino benzimidazole ‘hit’ from a screen of inhibition of antigen
receptor-mediated NF-jB activation.

1920
K. J. Okolotowicz et al. / Bioorg. Med. Chem. 18 (2010) 1918–1924
OH
N
N
N
N
N
OH
N
O
OH
OH
13
36
Figure 3. Cyclic benzimidazole byproduct from the synthesis of compound 1 using
Scheme 1.
Figure 4. N-Methyl analog of benzimidazole 1.
precursors (i.e., compounds 2 and 3). The benzimidazole cores
were alkylated with synthetically derived and/or commercially
available bromoacetophenones (6a–d) to further diversify the li-
brary. Substituted and unsubstituted cyclic amines (e.g., piperi-
dines, morpholines, and piperazines) were added in the last step
to give the final compounds for two reasons: (a) to diversify the li-
brary and (b) to preclude ring cyclization to 13. Preparation of this
focused library afforded 144 compounds each with a purity of
>90%. All 144 compounds were prepared and tested as inhibitors
The data of Table 2 show that compound 1 possessed a very
strict SAR. Placement of a methyl group at the phenolic oxygen
atom provided compound 37 that was a very potent NF-
jB inhib-
itor, (IC50 = 0.07 M). Incorporation of a methyl group into the ali-
l
phatic hydroxyl side chain oxygen atom afforded compound 38
that was somewhat less potent (i.e., the potency was decreased
approximately fivefold). Based on these results, analogs were syn-
thesized that were focused around modifying the aliphatic amino
side chain. It was hypothesized that the terminal end of the ali-
phatic substituent required an atom that possessed properties of
both a hydrogen donor and acceptor. Replacement of the terminal
nitrogen for a methyl group was predicted to decrease the inhibi-
tory nature of the molecule. However, the butyl analog (compound
against antigen receptor-mediated NF-
focused library contained only weakly potent compounds (IC50
>5 M). Table 1 shows a representative sample from the 144 com-
jB activation. However, this
l
pounds synthesized for the initial SAR study. We hypothesized that
the NH at the 2-position of the benzimidazole was a key interac-
tion for the receptor and tertiary amines at this position decreased
potency.
To test the hypothesis that a secondary amine was required for
optimal potency, the N-methyl analog of benzimidazole 1 was syn-
thesized (compound 36, Fig. 4). However, it was also found to be
44) possessed an IC50 = 0.1 lM. This result showed that the termi-
nal position was slightly more sensitive to the number of atoms
than the requirement for terminal atoms to act in hydrogen bond
donation or acceptor interactions.
Compounds 40–43 were synthesized to probe the effects of sub-
stituents on the phenyl ring. Compounds 40–43 were found to be
weakly potent (i.e., IC50 >5 lM) and similar to weakly potent mem-
weak inhibitors of antigen receptor-mediated NF-jB activation.
bers of the cyclic tertiary amine focus library. Consequently, sec-
ondary amines at the 2-position of benzimidazoles were utilized
exclusively in further studies.
A second focused library of secondary amine analogs of 1 pro-
vided considerable SAR information for the benzimidazole core.
Eleven additional analogs were synthesized to further probe the
SAR at the 2-amino and the acetophenone ring positions affording
only secondary alkylamines. Table 2 summarizes the biological re-
sults for these analogs.
This result suggested that bulky aliphatic groups may be necessary
for potent inhibition. Compound 46, was synthesized to determine
the influence of the presence of a ketone group on the potency of 1
(Fig. 5). The alcohol compound 46 was found to be weakly potent
with an IC50 >5 lM and the conclusion was that the ketone group
was necessary for greater potency.
To further explore the SAR of the terminal portion of the ali-
phatic side chain, an aminopropargyl group was appended by syn-
thesis. However, compound 45 was found to be weakly potent,
Table 1
(IC50 >5 lM) suggesting the amino side chain does not tolerate
groups that can form strong pi interactions. While several of the
NF-j
B inhibition by selected tertiary amine compounds
analogs were found to have similar potency as benzimidazole 1
at inhibiting the antigen receptor-mediated NF-jB activation path-
Structure
Compound R1
IC50
M)
(
l
way, none showed a greater inhibitory effect. Modification of cer-
tain regions of 1 (e.g., t-butyl, keto, NH), were found to have
significant impact on the inhibition of antigen receptor-mediated
16
17
18
19
Piperidine
Morpholine
Pyrrollidine
(S)-2(Methoxymethyl)-
pyrrollidine
(S)-1-(2-Pyrrolidinylmethyl)-
pyrrollidine
2,4-Dimethyl-3-
>5
>5
>5
>5
NF-jB activation while other groups, (e.g., 5,6-dimethyl substitu-
ent of the benzimidazole ring) did not have much effect. These re-
sults suggested that benzimidazole 1 possessed a strict SAR and
small modifications of the core significantly affected its inhibitory
potency.
20
21
>5
>5
N
R1
ethylpyrrollidine
N
22
23
24
25
26
27
28
29
30
3-Hydroxypyrrollidine
2-(Hydroxymethyl)piperidine
4-Benzylpiperidine
3-Methylpiperidine
4-Hydroxypiperidine
2-Methylpiperidine
3,5-Dimethylpiperidine
4-Methylpiperidine
N,N-Dimethyl-1-
piperidin-2-ylmethanamine
N-Methylpiperazine
N-Ethylpiperazine
N-Benzylpiperazine
N-Hydroxyethylpiperazine
N-(2-Methoxyphenyl)-
piperazine
>5
>5
>5
>5
>5
>5
>5
>5
>5
O
2.3. Metabolic stability study
OH
Inspection of the molecule suggested that several sites in com-
pound 1 could be prone to metabolic attack and possibly decrease
the potency of the parent molecule during planned in vivo evalua-
tion. Metabolic stability studies were thus undertaken for the most
potent compounds to determine the utility of 1 and close analogs
for studies in vivo. In vitro metabolic stability studies in the pres-
ence of both human liver microsomes (HLM) and S9 and a reduced
nicotinamide adenine dinucleotide phosphate (NADPH) generating
system were done with the oxalate salt of compounds 1, 37, and
38. Table 3 summarizes the results from these studies.
31
32
33
34
35
>5
>5
>5
>5
>5

K. J. Okolotowicz et al. / Bioorg. Med. Chem. 18 (2010) 1918–1924
1921
M)
Table 2
Inhibition of antigen receptor-mediated NF-
j
B by compounds 1 and 37–45
R1
Structure
Compound
R2
R4
R3
IC50 (l
1
37
3-Hydroxypropylamino
3-Hydroxypropylamino
t-Butyl
t-Butyl
t-Butyl
t-Butyl
OH
OMe
0.07
0.07
N
38
39
40
41
42
43
44
45
3-Methoxypropylamino
3-Methoxypropylamino
3-Hydroxypropylamino
3-Methoxypropylamino
3-Hydroxypropylamino
3-Methoxypropylamino
n-Butylamino
t-Butyl
t-Butyl
Methyl
Methyl
H
t-Butyl
t-Butyl
Methyl
Methyl
H
OH
OMe
OH
OH
OH
OH
OH
OH
0.25
0.25
>5
>5
>5
>5
0.1
>8
R1
N
O
R4
H
H
R2
R3
t-Butyl
t-Butyl
t-Butyl
t-Butyl
Propargylamino
3. Conclusions
N
N
NH
OH
In summary, a library of >160 compounds was synthesized as
inhibitors of the antigen receptor-mediated NF- B activation path-
HO
j
way. The results showed that several compounds exhibited the
same range of potency as compound 1. A concise SAR was deter-
mined from the library of compounds that was synthesized. The
SAR was found to be very steep and small changes made in 1 affor-
ded significant changes in the potency of inhibition. Finally, meta-
bolic stability studies were done on select compounds. The results
showed compounds 1, 37, and 38 were metabolically stable, and
only very modestly inhibited their own metabolism. It is likely that
compounds such as 1, 37, and 38 possess sufficient pharmacologi-
cal and pharmaceutical properties (i.e., potency, solubility, and
metabolic stability) to test as inhibitors of inflammation in appro-
priate animal models.
OH
46
Figure 5. Chemical structure of the ketone reduction product of 1.
Metabolic stability studies showed compounds 1, 37, and 38
were quite stable to hepatic metabolism compared with testoster-
one that was run as a positive control. One possible reason for this
result was that 1, 37, or 38 inhibited its own metabolism. To ex-
plore this point, inhibition of cytochrome 3A4-mediated micro-
somal hydroxylation of testosterone (known to be hydroxylated
by cytochrome P-450, the major cytochrome P-450 in adult human
liver microsomes) by 1, 37, or 38 was studied. Table 4 summarizes
the results from the study of the inhibition of testosterone hydrox-
ylase by 1, 37, and 38.
Results of Table 4 show that 1, 37, and 38 possess only modest
inhibitory activity of testosterone hydroxylase functional activity.
From these results and the knowledge that imidazoles are potent
inhibitors of cytochrome P-450’17,18 any significant inhibition of
cytochrome P-450 3A4 by compounds 1, 37, and 38 could account
for the metabolic stability observed. That potent inhibition of
cytochrome P-450 3A4 was not observed suggested that inhibi-
tion of its own metabolism was not occurring for 1, 37, and 38.
However, it is possible that some of the metabolic stability of
compounds 1, 37, and 38 was due at least in part to inhibition
of other cytochrome P-450s that are more prominent in the
metabolism of 1, 37, and 38. Based on the results presented in Ta-
ble 4, sufficient metabolic stability is present for 1, 37, and 38 for
these compounds to be taken forward in animal studies for effi-
cacy testing.
4. Experimental
4.1. Chemistry
1H NMR (300 MHz) spectra were determined on a Varian
Mercury 300 instrument in the indicated solvent. 13C NMR (125
MHz) spectra were determined on a Bruker AMX-500 II in the indi-
cated solvent. Mass spectra were recorded with a Hitachi M8000.
The active compounds were checked for purity using LC/MS at
two wavelengths (220 and 254 nm), (Platform: Agilent: equipped
with an autosampler, detector Diode Array Detector (monitored
at 220 and 254 nm), a MS detector (Perkin Elmer API 150EX); HPLC
column: Sunfire C18, 3.5
lm, 2.1 ꢁ 50 mm; HPLC gradient: 800
lL/
min, from 10% acetonitrile in water to 90% acetonitrile in water in
for 5.5 min. Both acetonitrile and water have 0.025% TFA). tR is
retention time (TIC% purity). All compounds were checked by TLC
on silica gel G plates with fluorescent indicator at 254 nm. Devel-
oped plates were visualized by UV light. Solvents were of reagent
grade and, when necessary, were purified and dried by standard
methods. Concentration of the reaction solutions involved the
use of rotary evaporator under reduced pressure. Compound 1
was purchased from Chembridge (San Diego).
Table 3
Metabolic stability of oxalate salts for compounds 1, 37 and 38
4.1.1. 2-Mercapto-5,6-benzimidazole (5)
Cofactor system
Enzyme
Half life (h)
A mixture of 10 g (73 mmol) of 4,5-dimethylphenylenediamine,
16 g (111 mmol) of potassium ethyl xanthate, 100 mL ethanol, and
14 mL of water were added to a 500 mL Erlenmeyer flask and
heated to reflux. After 3 h, 3.4 g of charcoal (Norit A) was added
and refluxed for an additional 10 min. The Norit was filtered and
the filtrate was heated to 60–70 °C. To the warm solution was
added 100 mL of warm water and 8 mL of acetic acid in 16 mL of
water with efficient stirring. Upon the addition of the acetic acid
solution, the mixture became a foamy solid. The mixture was
placed in a 4 °C refrigerator for 3 h. The solid was filtered and dried
1
33
34
Testosterone
0.8
+NADPH
+UDPG
+PAPS
HLMa
S9a
HLM
S9
HLM
S9
5.5
15.2
2.9
4.4
3.1
4.6
Not determined
Stableb
Stable
Stable
a
HLM, human liver microsomes, S-9 is the supernatant from a 10,000g centri-
fugation of a human liver homogenate.
b
Stable indicates no detectable loss of compound in 1 h as judged by HPLC.

1922
K. J. Okolotowicz et al. / Bioorg. Med. Chem. 18 (2010) 1918–1924
Table 4
isolated with an Rf = 0.25 gave 37.3 mg (76%) of a light brown res-
idue. 1H NMR (DMSO-d6, 300 MHz) d 1.62–1.70 (m, 2H), 2.18 (s,
6H), 3.25–3.33 (m, 2H), 3.43–3.47 (m, 2H), 4.86 (br s, 1H), 6.33
(br s, 1H), 6.87 (s, 2H), 10.46 (br s, 1H). MS: calculated
C12H17N3O m/z = 219.14 found m/z = 220.56 [M+H].
Inhibition of testosterone hydroxylase in the presence of HLMa and an NADPH
generating system
Compound
Calculated IC50 (lM)
1
37
38
85
67
97
4.1.6. General procedure for the synthesis of the target benzimi-
dazoles
a
HLM, human liver microsomes.
To a 1-dram vial, 1 equiv of the appropriate aklylaminobenzim-
idazole and 1.2 equiv of the appropriate bromoacetophenone 5
were added to 0.5 mL butanol. The reaction was heated to 115 °C
until the reaction was complete based on TLC analysis. Butanol
was removed and the crude mixture was extracted with saturated
sodium bicarbonate/EtOAc. The organic layer was dried over so-
dium sulfate and concentrated to dryness. The crude mixture
was purified via preparative TLC using 5% MeOH/CH2Cl2. Yields
range from 30% to 50%.
over P2O5 to give 8.1 g (62%) of a tan solid. The compound was used
without further purification. 1H NMR (300 MHz, DMSO-d6) d 2.21
(s, 6H), 6.91 (s, 2H), 12.31 (br s, 2H). MS: calculated C9H10N2S m/
z = 178.06 found m/z = 179.25 [M+H].
4.1.2. 2-Bromo-5,6-benzimidazole (7)
To a cooled solution of 40 mL of acetic acid and 4.2 mL
(37 mmol) of 48% aqueous HBr was added 5 g (28 mmol) of com-
pound 5. To this slurry was slowly added 5.2 mL (101 mmol) of
bromine dropwise over 10 min. The reaction turned orange and be-
came unstirrable and after half of the bromine was added, manual
or mechanical agitation was needed to break up solids. After the
addition of the bromine, 80 mL of acetic acid was added and the
mixture was stirred at room temperature. After 4.5 h, the mixture
was diluted with 90 mL of water and cooled to 0 °C. The pH of the
mixture was adjusted to pH 4 by the addition of solid NaOH. Upon
basification a solid precipitated out of solution. The solid was fil-
tered and dried overnight to give 2 g (32%) of product as a light or-
ange solid. 1H NMR (300 MHz, DMSO-d6) d 2.25 (s, 6H), 7.20 (s, 2H),
13.0 (br s, 1H). 13C NMR (CDCl3, 125 MHz) d 138.0, 130.8, 125.2,
114.5, 19.8. MS: calculated C9H9BrN2 m/z = 223.99, 225.99 found
m/z = 225.24, 227.25 [M+H].
4.1.6.1. 2-(2-(3-Hydroxypropylamino)-5,6-dimethyl-1H-benzo[d]-
imidazol-1-yl)-1-(3,5-di-tert-butyl-4-hydroxyphenyl)ethanone
(1). Rf = 0.3. 1H NMR (CDCl3, 300 MHz) d 0.87–1.00 (m, 2H), 1.49
(s, 18H), 2.22 (s, 3H), 2.26 (s, 3H), 3.52 (t, J = 5.6, 2H), 3.61 (t,
J = 5.5, 2H), 5.60 (s, 2H), 6.87 (s, 1H), 7.05 (s, 1H), 7.97 (s, 2H).
LC/MS tR = 2.39 (99%). MS: calculated C28H39N3O3 m/z = 465.30,
found m/z = 466.82 [M+H].
4.1.6.2. 1-(3,5-Di-tert-butyl-4-methoxyphenyl)-2-(2-(3-hydroxy-
propylamino)-5,6-dimethyl-1H-benzo[d]imidazol-1-yl)ethanone
(37). Rf = 0.4. 1H NMR (CDCl3, 300 MHz) d 1.43 (s, 18H), 1.70–1.77
(m, 2H), 2.27 (s, 3H), 2.28 (s, 3H), 3.56–3.60 (m, 2H), 3.61–3.65 (m,
2H), 3.72 (s, 3H), 5.23 (s, 2H), 6.78 (s, 1H), 7.20 (s, 1H), 7.94 (s, 2H).
13C NMR (CDCl3, 125 MHz) d 192.9, 165.7, 155.6, 145.3, 139.1,
132.7, 130.4, 129.3, 128.8, 127.5, 117.2, 108.1, 64.8, 58.7, 48.6,
39.8, 36.3, 33.8, 32.1, 20.4, 20.2. LC/MS tR = 2.55 (96%). MS: calcu-
lated C29H41N3O3 m/z = 479.31, found m/z = 480.53 [M+H].
4.1.3. General procedure for the alkylation of bromobenzimid-
azole (8a–d)
To a flask was added the appropriate bromobenzimidazole
(0.6 mmol), brominated acetophenone (0.66 mmol), and K2CO3
(1.3 mmol) in 1.2 mL DMF. The solution was stirred at room tem-
perature overnight. The reaction mixture was poured into 10% cit-
ric acid and extracted 3 times with EtOAc. The organic layers were
combined, dried over Na2SO4 and concentrated in vacuo. The crude
material was purified via column chromatography with the appro-
priate solvent system, generally 4:1 hexane/EtOAc.
4.1.6.3. 2-(2-(3-Methoxypropylamino)-5,6-dimethyl-1H-benzo-
[d]imidazol-1-yl)-1-(3,5-di-tert-butyl-4-hydroxyphenyl)ethanone
(38). Rf = 0.4. 1H NMR (CDCl3, 300 MHz) d 1.49 (s, 18H), 1.89–1.97
(m, 2H), 2.28 (s, 3H), 2.29 (s, 3H), 3.25 (s, 3H), 3.52 (t, J = 5.6, 2H),
3.60 (t, J = 5.5, 2H), 5.16 (s, 2H), 6.79 (s, 1H), 7.27 (s, 1H), 7.91 (s,
2H). 13C NMR (CDCl3, 125 MHz) d 192.7, 165.5, 154.9, 145.16,
140.22, 132.94, 129.9, 129.4, 128.4, 127.4, 117.4, 108.0, 72.2,
64.8, 58.7, 48.7, 42.5, 36.3, 32.09, 29.2, 20.4, 20.3. LC/MS tR = 2.65
(99%). MS: calculated C29H41N3O3 m/z = 479.31, found m/
z = 480.23 [M+H].
4.1.4. General procedure for the synthesis of the benzimidazole
library (9a–d, 10a–d)
To a 1-dram vial was added the appropriate alkylated bromo-
benzimidazole 8a–d (0.01 mmol) and the cyclic amines (0.1 mmol)
and heated to 125 °C. After 4 h, the reactions were cooled to room
temperature and the products were isolated. The reaction mixture
was dissolved in 0.5 mL EtOAc and washed with 2 M HCl (0.5 mL).
The acid was neutralized with saturated sodium bicarbonate ex-
tracted twice with EtOAc (0.5 mL). The organic layers were com-
bined, dried over sodium sulfate and concentrated to dryness.
The isolated material was used without further purification.
4.1.6.4. 2-(2-(3-Methoxypropylamino)-5,6-dimethyl-1H-benzo[d]-
imidazol-1-yl)-1-(3,5-di-tert-butyl-4-methoxyphenyl)ethanone
(39). Rf = 0.6. 1H NMR (CDCl3, 300 MHz) d 1.44 (s, 18H), 1.89–1.97
(m, 2H), 2.28 (s, 3H), 2.29 (s, 3H), 3.25 (s, 3H), 3.52 (t, J = 5.6, 2H),
3.60 (t, J = 5.5, 2H), 3.72 (s, 3H), 5.18 (s, 2H), 6.79 (s, 1H), 7.27 (s,
1H), 7.95 (s, 2H). 13C NMR (CDCl3, 125 MHz) d 192.2, 159.7,
155.0, 140.4, 136.7, 133.0, 129.8, 128.2, 126.6, 126.3, 117.5,
107.9, 72.0, 58.9, 48.4, 42.3, 34.7, 30.3, 29.3, 20.4, 20.3. LC/MS
tR = 2.63 (99%). MS: calculated C30H43N3O3 m/z = 493.33, found m/
z = 494.13 [M+H].
4.1.5. 3-(5,6-Dimethyl-1H-benzo[d]imidazol-2-ylamino)propan-
1-ol (14)
4.1.6.5. 1-(3,5-Di-tert-butyl-4-hydroxyphenyl)-2-(2-(butylami-
no)-5,6-dimethyl-1H-benzo[d]imidazol-1-yl)ethanone (44).
Rf = 0.8. 1H NMR (CDCl3, 300 MHz) d 0.90–0.95 (m, 3H), 1.47 (s,
18H), 1.57–1.68 (m, 4H), 2.29 (s, 6H), 3.44–3.50 (m, 2H), 5.14 (s,
2H), 6.82 (s, 1H), 7.28 (s, 1H), 7.92 (s, 2H). 13C NMR (CDCl3,
125 MHz) d 192.9, 159.9, 155.0, 136.7, 132.8, 131.1, 130.4, 129.9,
128.3, 126.6, 117.5, 108.0, 48.4, 43.7, 34.7, 32.1, 30.3, 20.2, 20.4,
50 mg (0.22 mmol) of 7 and 100 lL (1.3 mmol) 3-aminopropa-
nol were added to a 1-dram vial and heated to 125 °C until the
reaction was complete based on TLC analysis. The reaction was
cooled to room temperature, extracted with saturated sodium
bicarbonate/EtOAc. The organic layer was dried over sodium sul-
fate and concentrated to give a brown residue. The crude product
was purified via preparative TLC using 10% MeOH/CH2Cl2. A band

K. J. Okolotowicz et al. / Bioorg. Med. Chem. 18 (2010) 1918–1924
1923
20.3, 14.1. LC/MS tR = 2.57 (98%). MS: calculated C29H41N3O2 m/
z = 463.32, found m/z = 464.35 [M+H].
PAC(used asan antioxidant), and 4 IU/mLglucose-6-phosphatedehy-
drogenase in potassium phosphate buffer (pH 7.4, 50 mM).
4.2. Biology
4.2.2.2. Glucuronidation with UDPG. Substrate (100
lM), 1.6 mg/
mL HLM (or S9), 200 M UDPG, 40 g/mL alamethicin, and 72 l
l
l
g/
Phorbol myristic acetate (PMA), ionomycin, the NADPH gener-
ating system, uridine diphosphoglucose (UDPG), alamethicin, and
adenosine 30-phosphate 50-phosphosulfate lithium salt hydrate
(PAPS) were from Sigma–Aldrich (St. Louis, MO). Lecithin was ob-
tained from Calbiochem (La Jolla, CA). Human liver microsomes
(HLM) and human liver S9 were purchased from B. D. Gentest (Wo-
burn, MA). RP-HPLC analysis of synthetic compounds was done
using a Hitachi HPLC system (D-7000 interface, L-7100 pump, L-
7200 autosampler, and the L-7400 UV detector) and a HS Supelco
mL lecithin was incubated in potassium phosphate buffer (pH 7.4,
50 mM).
4.2.2.3. Sulfation with PAPS. Substrate (100
lM) was incubated
with 1.6 mg/mL HLM (or S9) and 100
(50 mM, pH 7.4).
lM PAPS in Tris–HCl buffer
4.2.2.4. Testosterone oxidation. Testosterone oxidation was
studied to investigate the inhibitory effect of 1 and analogs. The
column (4.6 mm ꢁ 250 mm, 5
l
m). All of the chemicals or reagents
incubation mixture contained 50
human liver microsomes, 400
M NADP+, 400
phate, 250 M DETAPAC, 4 IU/mL glucose-6-phosphate dehydroge-
l
M
testosterone, 1.6 mg/mL
were obtained in the highest purity available commercially.
l
lM glucose-6-phos-
l
4.2.1. Cell engineering
nase, and 1 mM MgCl2 in potassium phosphate buffer (pH 7.4,
50 mM). All incubations were initiated by the addition of substrate,
and were run at 37 °C in a shaking incubator. The assay was termi-
nated at 10 and 20 min by the addition of 1 mL of cold ethyl ace-
tate. The terminated incubations were centrifuged for 5 min at
1800 rpm to separate the organic and aqueous layers. The organic
layer was decanted into a new tube and concentrated under an ar-
HEK293 cells were co-transfected with pUC13-4xNF-jB-Luc
and p-TK-puromycin-resistance plasmids. Stable clones were se-
lected by culture in Dulbecco’s Modified Eagle’s Media (Invitrogen)
supplemented with 10% heat-inactivated fetal bovine serum (FBS)
(Hyclone), 1% v/v penicillin–streptomycin mix (Invitrogen) con-
taining 1 lg/mL puromycin. Individual clones were tested for
responsiveness to PMA/ionomycin-induced NF-kB reporter gene
gon stream until dry. The residue was reconstituted with 100 lL of
activity, and a clone was selected.
methanol. When test compounds were added to the incubation to
PMA/Iono induced NF-
NF- B-Luc stable cells HEK293-NF-
medium) were seeded in 96-well white plates (Greiner Bio-one)
overnight. Compounds were diluted in culture medium and 5
was added to the cells to reach a desired final concentration. 2 h la-
ter, 5 L PMA/Iono (200 ng/mL) was added (final concentra-
tion = 10 ng/mL) and cells were treated for another 16 h. 50
j
B luciferase assay was done in HEK293-
determine the IC50 of 1 or analogs, the buffer volume was reduced
to maintain the over-all final volume at 100 lL. Samples were ana-
lyzed using RP-HPLC; mobile phase was 32% (acetonitrile + 0.1%
TFA) and 68% (H2O + 0.1% TFA), and the flow rate was 1.2 mL/
min. Testosterone had a retention time of 13.4 min and the 6-OH
testosterone eluted at 4.4 min. A gradient mobile phase was se-
lected for the inhibition study to elute 1 and analogs.
j
j
B-Luc cells (104 cells in 90
lL
lL
l
lL
BriteLite solution (Perkin–Elmer) was added to each well and lumi-
nescence was read using a luminometor (LJL Biosystems). The
luminescence of cells treated by PMA/Ion was set to 100% and
the data of each compound were processed and presented as the
percentage of control. IC50 values were calculated using GraphPad
Prism 5.0.
Acknowledgments
We thank the National Institutes of Health (Grant # 1 X01
MH077633-01) and the CLL Global Research Foundation (John
Reed, PI) for the financial support for this project. We also thank
our summer interns, Michelle Wolfe and Logan Fink for their help
with the synthesis of the analogs tested.
4.2.2. Metabolic stability studies
The in vitro oxidation, glucuronidation, and sulfation of 1 and
analogs were studied with human liver microsomes or human liver
S9 in the presence of 3 different cofactors: an NADPH generating
system, UDPG and PAPS.19–21 All incubations at different time
points were measured in duplicate. All incubations were initiated
by the addition of HLM or S9, and were conducted at 37 °C in a
shaking incubator. Aliquots of the incubation were stopped at 5,
15, 30, 45, and 60 min by the addition of two volumes of cold ace-
tonitrile. For the 0 min time point, samples were aliquoted into
acetonitrile before adding the human liver microsomes or S9. Sam-
ples were analyzed using RP-HPLC with UV detection. Flow rate:
1.2 mL/min, UV wavelength was selected at 235 nm. Two solvent
phases were used: Solvent A was 85% acetonitrile (ACN) with
15% H2O and 0.1% trifluoroacetic acid (TFA), and Solvent B was
0.1% TFA in H2O. The mobile phase was 90% Solvent A with 10% Sol-
vent B for 37 with human liver S9 and NADPH incubation; reten-
tion time (tR) was 11.2 min. The mobile phase was 100% Solvent
A for all other incubation samples, and the tR for 1, 37, and 38 were
7.9, 10.5, and 10.3 min, respectively. The percent of 1 and analogs
oxidized, glucuronidated, or sulfated were calculated based on the
disappearance of the parent compound.
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l
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l
M NADP+, 400
l
M DETA-

1924
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