Human toll-like receptor 8-selective agonistic activities in 1-alkyl-1 h -benzimidazol-2-amines

Article abstract of DOI:10.1021/jm500701q

Toll-like receptor (TLR)-8 agonists strongly induce the production of T helper 1-polarizing cytokines and may therefore serve as promising candidate vaccine adjuvants, especially for the very young and the elderly. Earlier structure-based ligand design led to the identification of 3-pentyl-quinoline-2-amine as a novel, human TLR8-specific agonist. Comprehensive structure-activity relationships in ring-contracted 1-alkyl-1H-benzimidazol-2-amines were undertaken, and the best-in-class compound, 4-methyl-1-pentyl-1H-benzo[d]imidazol-2-amine, was found to be a pure TLR8 agonist, evoking strong proinflammatory cytokine and Type II interferon responses in human PBMCs, with no attendant CD69 upregulation in natural lymphocytic subsets. The 1-alkyl-1H-benzimidazol-2-amines represent a novel, alternate chemotype with pure TLR8-agonistic activities and will likely prove useful not only in understanding TLR8 signaling but also perhaps as a candidate vaccine adjuvant.

Full text of DOI:10.1021/jm500701q

Subscriber access provided by UniSA Library
Article
Human Toll-like Receptor 8-Selective Agonistic
Activities in 1-Alkyl-1H-benzimidazol-2-amines
Mallesh Beesu, Subbalakshmi S. Malladi, Lauren M Fox,
Cassandra D. Jones, Anshuman Dixit, and Sunil A. David
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500701q • Publication Date (Web): 07 Aug 2014
Downloaded from http://pubs.acs.org on August 11, 2014
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 66
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Human Toll-like Receptor 8-Selective Agonistic
Activities in 1-Alkyl-1H-benzimidazol-2-amines
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
1
1
Mallesh Beesu, Subbalakshmi S. Malladi, Lauren M. Fox,
1
2
Cassandra D. Jones, Anshuman Dixit,
1
and Sunil A. David *
1
Department of Medicinal Chemistry,
University of Kansas, Lawrence, KS 66047, USA.
2
Department of Translational Research and Technology Development,
Institute of Life Sciences, Nalco Square, Bhubaneswar-751023, India.
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 66
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Running Title: TLR8 agonism in 1-alkyl-1H-benzimidazol-2-amines
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Keywords: TLR8, TLR8 agonists, Vaccine adjuvants, Innate immunity, 2ꢀ Aminobenzimidazoles.
2
ACS Paragon Plus Environment

Page 3 of 66
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Abstract
Tollꢀlike receptor (TLR)ꢀ8 agonists strongly induce the production of T helper 1ꢀpolarizing
cytokines, and may therefore serve as promising candidate vaccine adjuvants, especially for the
very young and the elderly. Earlier structureꢀbased ligand design led to the identification of 3ꢀ
pentylꢀquinolineꢀ2ꢀamine as a novel, human TLR8ꢀspecific agonist. Comprehensive structureꢀ
activity relationships in ringꢀcontracted 1ꢀalkylꢀ1Hꢀbenzimidazolꢀ2ꢀamines were undertaken, and
the bestꢀinꢀclass compound, 4ꢀmethylꢀ1ꢀpentylꢀ1Hꢀbenzo[d]imidazolꢀ2ꢀamine, was found to be a
pure TLR8 agonist, evoking strong proinflammatory cytokine and Type II interferon responses in
human PBMCs, with no attendant CD69 upregulation in natural lymphocytic subsets. The 1ꢀalkylꢀ
1Hꢀbenzimidazolꢀ2ꢀamines represent a novel, alternate chemotype with pure TLR8ꢀagonistic
activities, and will likely prove useful not only in understanding TLR8 signaling, but also perhaps
as a candidate vaccine adjuvant.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(118 words)
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Introduction
Page 4 of 66
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Although the role of vaccines in mitigating morbidity and mortality attributable to infectious
1
, 2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
diseases in modern times is indisputable, we are witnessing a reemergence of infectious diseases
previously declared eliminated in the United States. The Centers for Disease Control, for instance,
reports 154 cases of measles during the first four months of 2014, a number poised to surpass the
2
011 outbreak (the largest since 1996) — even as the recent World Health Organization (WHO)
3
report on antimicrobial resistance warns that “a postꢀantibiotic era — in which common infections
and minor injuries can kill — far from being an apocalyptic fantasy, is instead a very real possibility
for the 21st Century”. We can neither afford to rest on the numerous and extraordinary successes in
preventive medicine, nor cease to remind ourselves of the challenges that are yet to be overcome in
developing effective vaccines against ‘old foes’ such as the human immunodeficiency virus (HIV),
tuberculosis, or malaria.
The main thrust of immunization programs worldwide is generally on vaccinating infants
4
and young children, and it is pertinent to note in this context the WHO’s efforts toward developing
policy recommendations regarding immunization beyond childhood and into old age. The elderly
represent a particularly vulnerable subset of the population. The proportion of the world’s
5
population aged 60 and older is estimated to increase from 10% at present to about 21% by 2050.
In the United States, the annual mortality of vaccineꢀpreventable diseases in older adults is
5, 6
estimated to be about 50,000–70,000 (compared to 1000–3000 children), emphasizing the need
for effective immunization strategies adapted specifically for the epidemiological characteristics of
the spectrum of infectious diseases in the elderly. Efforts toward optimizing vaccines for the elderly
7
8, 9
have included increasing antigen dosage, as well as the development of better vaccine adjuvants.
4
ACS Paragon Plus Environment

Page 5 of 66
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Vaccine adjuvants are immune potentiators which initiate early innate immune responses
1
0ꢀ14
leading to the induction of robust and longꢀlasting adaptive immune responses.
Until the
15ꢀ17
relatively recent approval of 3ꢀOꢀdesacylꢀ4'ꢀmonophosphoryl lipid A (MPL®) by the FDA,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aluminum salts (primarily phosphate and hydroxide), discovered by Glenny and coworkers in
1
8
1
926, have been the only adjuvants in clinical use. Aluminum salts are weak adjuvants for
1
9, 20
antibody induction, promoting a T 2ꢀskewed, rather than a T 1 response,
and are virtually
H
H
ineffective at inducing cytotoxic T lymphocyte or mucosal IgA antibody responses.
Innate immune signals activated by candidate vaccine adjuvants include those originating
2
1ꢀ23
24
from Tollꢀlike receptors (TLRs),
as well as RIGꢀIꢀlike receptors and NODꢀlike receptors
25, 26
(
NLRs).
There are 10 functional TLRs encoded in the human genome, which are transꢀ
membrane proteins with an extracellular domain having leucineꢀrich repeats (LRR) and a cytosolic
2
2
domain called the Toll/ILꢀ1 receptor (TIR) domain. The ligands for these receptors are highly
conserved molecules such as lipopolysaccharides (LPS) (recognized by TLR4, also the target of
MPL®), lipopeptides (TLR2 in combination with TLR1 or TLR6), flagellin (TLR5), single
stranded RNA (TLR7 and TLR8), double stranded RNA (TLR3), CpG motifꢀcontaining DNA
2
2
(
recognized by TLR9), and profilin present on uropathogenic bacteria (TLR11). TLR1, ꢀ2, ꢀ4, ꢀ5,
and ꢀ6 recognize extracellular stimuli, while TLR3, ꢀ7, ꢀ8 and ꢀ9 function within the endolysosomal
compartment. The activation of TLRs by their cognate ligands leads to production of inflammatory
cytokines, and upꢀregulation of major histocompatibility complex (MHC) molecules and coꢀ
stimulatory signals in antigenꢀpresenting cells as well as activating natural killer (NK) cells (innate
immune response), which leads to the priming and amplification of Tꢀ, and Bꢀcell effector functions
2
1, 22, 27
(adaptive immune responses).
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 66
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
8ꢀ45
In the course of the last five years we have systematically explored
of a variety of TLR
agonists with a view to identifying safe and potent vaccine adjuvants. The chemotypes that we have
30, 37, 40, 41
29, 34, 35, 38, 42, 43, 46
28, 31, 32, 36, 46
explored thus far include agonists of TLR2,
TLR7,
TLR8,
3
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
nucleotide oligomerization domain 1 (NOD1), as well as CꢀC chemokine receptor type 1
33
(
CCR1).
Our interest in small molecule agonists of TLR8 as vaccine adjuvants was, in part, kindled
by the necessity of having to first identify chemotypes with pure TLR8 activity, given that the
4
3, 46, 47
31, 48ꢀ51
known agonists of TLR8 such as the imidazoquinolines,
thiazoloquinolines,
and the 2ꢀ
5
2
aminobenzazepines were found to exhibit mixed TLR7/TLR8ꢀagonism. Our discovery of the 2,3ꢀ
36
32
diaminoꢀfuro[2,3ꢀc]pyridines and 4ꢀaminoꢀfuro[2,3ꢀc]quinolines, leading to structureꢀbased
2
8
design of the 3ꢀalkylꢀquinolineꢀ2ꢀamines as pure TLR8 agonists with no detectable TLR7 activity
was also motivated by observations that the strongly Th1ꢀbiasing TLR8 agonists could be useful as
53, 54
candidate vaccine adjuvants for the newborn.
Maternal immunoglobulins acquired by passive
5
5
transplacental passage confer protection to the neonate for the first few weeks of life; thereafter,
the newborn is susceptible to a wide range of pathogens until early infancy. The very young do not
mount adequate adaptive immune responses and, consequently, even highly effective vaccines that
5
6, 57
confer excellent protection in adults may fail to elicit strong immune responses in them.
The
neonatal immunophenotype is characterized by decreased production of both type I and type II
interferons, as well as Th1ꢀbiasing cytokines such as TNFꢀα, ILꢀ12, ILꢀ18, ILꢀ23, the preferential
induction of memory B lymphocytes rather than immunoglobulinꢀsecreting plasma cells, as well as
5
8, 59
a pronounced Tꢀhelper type 2 (Th2) skewing of Tꢀcell responses.
TLR8 agonists induce the
2
8, 31, 32
production of ILꢀ12, ILꢀ18 and IFNꢀγ,
and may therefore be of value in developing vaccines
for the neonate.
6
ACS Paragon Plus Environment

Page 7 of 66
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Our initial interest in TLR8 agonists as candidate adjuvants has been sustained also by
60ꢀ62
63ꢀ65
observations of impaired TLR signaling
contributing to immune senescence
in aging. In
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
particular, substantial decreases in TNFꢀα, ILꢀ6, and/or ILꢀ12p40 production have been
documented in myeloid dendritic cells isolated from older individuals in response to TLR8
6
0ꢀ62
engagement,
between the very young and the aged—perhaps an echo of Shakespeare’s characterization of the
Seventh Age’.
reflecting parallels in immune ontogeny of TLRꢀdriven cytokine responses
‘
A highꢀresolution (1.8 Å) structure of human TLR8 coꢀcrystallized with a pure TLR8ꢀ
agonistic lead compound (C2ꢀbutylꢀfuro[2,3ꢀc]quinoline) suggested that the furan ring was
dispensable, and led to the identification of 3ꢀpentylꢀquinolineꢀ2ꢀamine as a novel, structurally
28
simple, and highly potent human TLR8ꢀspecific agonist. We now report detailed structureꢀactivity
relationships in the ringꢀcontracted 1ꢀalkylꢀ1Hꢀbenzimidazolꢀ2ꢀamines. The bestꢀinꢀclass compound
of this novel chemotype, 4ꢀmethylꢀ1ꢀpentylꢀ1Hꢀbenzo[d]imidazolꢀ2ꢀamine, was found to retain a
pure TLR8 agonistic activity profile.
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 66
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Results and Discussion
With 3ꢀpentylꢀquinolineꢀ2ꢀamine as our point of departure, we initially targeted the ringꢀ
contracted 2ꢀaminoꢀ3ꢀalkylindole analogue 4, accessed via sequential Cꢀalkylation of commercially
available 2ꢀnitrophenylacetonitrile, reduction of the 2ꢀnitro group, and Brønsted acidꢀpromoted,
microwaveꢀassisted intramolecular cyclization (Scheme 1). The hydrochloride salt of 4 was isolated
and found to be inactive; its freeꢀbase, however, was exceedingly unstable, leading to the rapid
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
66
formation of the overꢀoxidized 3ꢀol derivative 5, presumably via autoxidation (Scheme 1), which
was also inactive in primary screens.
We therefore focused our attention on stable analogues possessing structural scaffolds
similar to the 2ꢀaminoindole 4, and began our investigations with 1ꢀalkylꢀ2ꢀaminobenzimidazole
analogues (Scheme 2). The 2ꢀaminobenzimidazole scaffold was conveniently accessed via the
reaction of oꢀphenylenediamine with cyanogen bromide (CNBr). The N1 position could be
selectively derivatized (Scheme 2), furnishing analogues that we were particularly interested in. In a
focused SAR assessment of these analogues, a clear dependence of substituent chain length at N1
was noted, consistent with our previous observations on other chemotypes, with the optimal
analogue being 8b (N1ꢀpentyl; EC50 = 3.23
ꢀM; Fig. 1). We specifically desired to examine
analogues with benzyl (8d) and 3ꢀaminomethylbenzyl (8f) substituents at N1, for these substituents
on the imidazoquinoline scaffold had yielded highꢀpotency pure TLR7 and mixed TLR8/7 agonists,
4
3
respectively. Somewhat to our surprise, we found that neither 8d nor 8f were active; substitution
of the N1ꢀpentyl group with butoxycarbonyl group (9), or acylation of the N2 amine (10) also
completely abrogated activity, suggestive of stringent structural requirements for activity, and
demonstrating that both the free amine at C2 and the N1 pentyl substituent are important for TLR8ꢀ
8
ACS Paragon Plus Environment

Page 9 of 66
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
agonistic activity.
We addressed modifications on the benzimidazole core, mindful of our previous
2
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
observations of benzologues of imidazo[4,5ꢀc]pyridines showing specific TLR7 activity. We
therefore synthesized all possible regioisomeric benzologues of 8b (Scheme 3). The naphtho[2,3ꢀ
d]imidazole analogue 13 was accessed readily via cyclization of the naphthaleneꢀ2,3ꢀdiamine with
CNBr. The symmetryꢀrelated regioisomers 18 and 23 could, in principle, have been obtained from
the rather expensive 1,2ꢀdiaminonaphthalene, followed by resolution of the regioisomers. But given
the nearꢀidentical R values (and retention times in analytical HPLC) of these two regioisomers, we
f
were fortunate (in hindsight) to have opted for an alternative route. We had originally envisaged a
sequence of chlorination of 1ꢀnitronaphthalenꢀ2ꢀol and 2ꢀnitronaphthalenꢀ1ꢀol precursors,
nucleophilic substitution with pentylamine, and subsequent formation of the aminoimidazole ring;
however, conversion of the nitronaphthols to corresponding chloronitronaphols proved
unexpectedly problematic. This was alleviated by conversion to the nitromethoxynaphthols 15 and
20, which underwent facile nucleophilic substitution reactions with pentylamine, affording the
desired regioisomers 18 and 23 in good yields (Scheme 3). Of the three different regioisomers, only
2
3 (EC = 3.16 ꢀM) showed nearꢀidentical activity to that of 8b (Table 1). These observations,
50
taken together, also suggested that substitutions could be tolerated at C4 and C5, but not at C6 and
C7, which were borne out as described below.
We next targeted all possible regioisomers of imidazopyridines (27a-d) for possible TLR7/8
2
9
activity, given that these analogues are congeneric to the imidazo[4,5ꢀc] pyridines (Scheme 4).
These analogues were synthesized via S Ar reactions of corresponding oꢀhalonitropyridines with
N
pentylamine, reduction of the nitro group, and final cyclization with CNBr (Scheme 4). None of
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 66
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
these analogues were active, and it remained for us to explore substitutions on the benzimidazole
core. In our final salvo, therefore, we systematically introduced methyl substituents at C4ꢀC7
positions (31aꢀ31d, Scheme 5) to ascertain steric effects of the benzimidazole ring, and their
consequences on biological activity. Only 31a, with a C4ꢀmethyl group showed significantly more
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
potent activity (EC = 1.13 ꢀM), relative to the parent compound 8b; the potency was comparable
5
0
3
2
to that of C2ꢀbutylꢀfuro[2,3ꢀc]quinoline, and marginally less than that of 3ꢀpentylꢀquinolineꢀ2ꢀ
2
8
amine (Fig. 1). We also examined different electronꢀdonating and ꢀwithdrawing substituents at C4
position. The methoxy analogue 31e (EC = 3.74 M) showed comparable activity to that of 8b,
ꢀ
5
0
but was less active than 31a (Table 1). Electronꢀwithdrawing substitutions (31fꢀ31i) resulted in
inactive compounds. Homologation of the methyl group at C4 to the ethylꢀsubstituted analogue 31j
(accomplished via Suzuki coupling of 3ꢀbromoꢀ2ꢀnitroꢀNꢀpentylaniline with ethylboronic acid, and
subsequent elaboration; Scheme 5) resulted in a slight reduction in TLR8ꢀagonistic potency
compared to 31a, alerting us to the possibility that longer (or bulkier) substituents would not be
favorable and, indeed, phenyl (31l) benzyl (31m), or benzyloxy (35) substituents at C4 abrogated
activity. The electronꢀdeficient 4ꢀnitro analogue 39 (Scheme 7) was inactive, and analogues with
electron–donating groups at C4 (31k, 36, 40; Schemes 5ꢀ7) displayed attenuated potency relative to
the 4ꢀmethyl compound 31a (Table 1).
32
We employed inducedꢀfit docking methods to compare the binding modes of the 2ꢀ
aminobenzimidazole analogues with known TLR8 ligands, utilizing highꢀresolution crystal
2
8, 67
structures of human TLR8.
The TLR8ꢀactive analogues such as 8b and 31a occupy the binding
pocket formed by both of the TLR8 protomers with the expected binding geometry involving strong
bidentate ionic H bonds between Asp543 of TLR8 and both the C2 amine as well as the N3 atom of
the benzimidazole compounds (Fig. 2). Stabilization derived from an H bond between Thr574
(
protomer B) and the C2ꢀNH as well as hydrophobic interactions of the N1ꢀalkyl group with the
2
1
0
ACS Paragon Plus Environment

Page 11 of 66
Journal of Medicinal Chemistry
1
2
hydrophobic pocket lined by Phe346/Ile403/Gly376 within protomer A were also observed as
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
8
reported earlier. Favorable π–π interactions of the phenyl ring of the bezimidazoles and Phe405
were observed. We were, in particular, interested in understanding why only the C4ꢀmethyl
analogue 31a, and not congeners bearing methyl groups at C5, C6, or C7 (31bꢀ31d, respectively)
showed enhanced potency relative to 8b. An examination of 31a bound to TLR8 showed favorable
van der Waals interactions (3.7 Ǻ) between the C4ꢀmethyl and the side chain of Val520 (Fig. 2A),
which become unfavorable (5.2 Ǻ) in the C5ꢀmethyl analogue 31b, and lost entirely in 31c and 31d
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
not shown). The occupancy of the benzologues 13, 18 and 23 in the binding pocket is
compromised by unfavorable sterics, exemplified in the case of 13, forcing the binding of the
analogue in an inverted fashion with the consequent loss of the critical Hꢀbond interactions between
the C2 amine and Asp543 (Fig. 2B). As detailed above, the docking studies allowed a
rationalization of binding activities in terms of steric properties of the analogues, but proved less
useful in understanding electronic effects. For instance, favorable binding geometries and energies
were also observed for the inactive pyridyl analogues 27a-d, pointing to limitations inherent in
6
8
force field computations.
All analogues were counterꢀscreened in agonism screens using reporter cell lines specific for
human TLR2, TLR3, TLR4, TLR5, TLR7, TLR9, NOD1 and NOD2 (Figs. S1 and S2). No offꢀ
target effects were detected, confirming the specificity of the active analogues for human TLR8.
6
9
Certain benzimidazoles such as Noditinibꢀ1 (1ꢀ[(4ꢀmethylphenyl)sulfonyl]ꢀ1Hꢀbenzimidazolꢀ2ꢀ
amine) have been shown to inhibit NODꢀ1 signaling, and it was therefore of interest to also
characterize possible antagonistic activities. Weak antagonistic activities (IC : > 10 ꢀM) toward
5
0
NODꢀ1 and NODꢀ2 were observed for 10, 13, 18, 31l, 31m, and 35 (Fig. S3); these results
suggested that bulky substituents at C4 may yield NODꢀ1/NODꢀ2 antagonists, but were not a
1
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 66
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
priority for us in the context of discovering novel vaccine adjuvants.
The bestꢀinꢀclass of this novel TLR8ꢀagonistic chemotype, 31a was taken forward and
characterized further in cytokine/chemokine induction profiles in a panel of secondary screens
employing human peripheral blood mononuclear cells, as well as whole human blood. Consistent
with its specificity for TLR8, we observed induction of proinflammatory cytokines, as well as ILꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2p40 and IFNꢀγ (Fig. 2), and a complete absence of CD69 upregulation in natural killer (NK)
lymphocytes (Fig. 3). We had previously shown that CD69 upregulation in NK cells is ascribable
2
8, 32
purely to TLR7 activity,
and these results confirm absolute specificity of the lead 2ꢀ
aminobenzimidazole compounds for human TLR8. CD69 is a type II Cꢀlectin membrane receptor
7
0, 71
with immunoregulatory functions.
The absence of CD69 (in CD69 knockout mice) is correlated
with increased generation of Th1 lymphocytes and enhanced production of Th1ꢀbiasing
72, 73
cytokines,
consistent with our observation of induction of high levels of ILꢀ12 and IFNꢀγ in
human PBMCs (Fig. 3).
In summary, the 1ꢀalkylꢀ1Hꢀbenzimidazolꢀ2ꢀamines represent a novel chemotype with
human TLR8ꢀspecific agonistic activities, which will likely prove useful not only as tools to dissect
TLR7 visꢀàꢀvis TLR8 signaling, but also as candidate vaccine adjuvants with strong Th1 bias.
1
2
ACS Paragon Plus Environment

Page 13 of 66
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
Experimental Section
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Chemistry. All of the solvents and reagents used were obtained commercially and used as such
unless noted otherwise. Moistureꢀ or airꢀsensitive reactions were conducted under nitrogen
atmosphere in ovenꢀdried (120 ºC) glass apparatus. Solvents were removed under reduced pressure
using standard rotary evaporators. Flash column chromatography was carried out using RediSep Rf
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
‘
Gold’ high performance silica columns on CombiFlash R instruments unless otherwise mentioned,
f
while thinꢀlayer chromatography was carried out on silica gel CCM preꢀcoated aluminum sheets.
Purity for all final compounds was confirmed to be greater than 98% by LCꢀMS using a Zorbax
Eclipse Plus 4.6 mm x 150 mm, 5 ꢀm analytical reverse phase C column with H OꢀCH CN and
1
8
2
3
H OꢀMeOH gradients and an Agilent 6520 ESIꢀQTOF Accurate Mass spectrometer (mass accuracy
2
of 5 ppm) operating in the positive ion acquisition mode.
2
-(2-Nitrophenyl)hexanenitrile (2). To a solution of 2ꢀnitrophenylacetonitrile (162 mg, 1 mmol) in
anhydrous DMSO (5 mL) was added K CO (152 mg, 1.1 mmol) and the reaction mixture was
2
3
stirred for 10 min under nitrogen atmosphere. Butyl iodide (125 µL, 1.1 mmol) was added to the
reaction mixture and the stirring was continued for 3 h. Water was added to the reaction mixture
and it was extracted with EtOAc (3 x 20 mL). The combined organic layer was dried over Na SO ,
2
4
concentrated under reduced pressure, and the crude material was purified by silica gel column
chromatography (10% EtOAc/hexanes) to afford compound 2 as a pale yellow oil (174 mg, 80%).
1
R = 0.50 (10% EtOAc/hexanes). H NMR (500 MHz, CDCl ) δ 8.05 (dd, J = 8.2, 1.3 Hz, 1H), 7.79
f
3
(dd, J = 7.9, 1.4 Hz, 1H), 7.70 (td, J = 7.6, 1.3 Hz, 1H), 7.52 (ddd, J = 8.6, 7.5, 1.4 Hz, 1H), 4.70
(dd, J = 9.5, 4.9 Hz, 1H), 2.01 – 1.84 (m, 2H), 1.59 – 1.52 (m, 2H), 1.47 – 1.32 (m, 2H), 0.93 (t, J =
1
3
7
.3 Hz, 3H). C NMR (126 MHz, CDCl ) δ 147.64, 134.26, 131.59, 130.26, 129.39, 125.77,
3
1
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 66
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
1
20.22, 35.47, 33.98, 29.62, 22.08, 13.90. MS (ESIꢀTOF) for C H N O [M + H] calculated
12 14 2 2
2
19.1128, found 219.1095.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2-(2-Aminophenyl)hexanenitrile (3). To a solution of compound 2 (109 mg, 0.5 mmol) in
anhydrous EtOAc (10 mL) was added a catalytic amount of Pt/C (39 mg, 1 mol %), and the reaction
mixture was subjected to hydrogenation at 30 psi hydrogen pressure for 3 h. The reaction mixture
was filtered, and the filtrate concentrated under reduced pressure. The crude material was purified
using silica gel column chromatography (10% MeOH/CH Cl ) to obtain 3 as a pale yellow oil (70
2
2
1
mg, 74%). R = 0.40 (10% MeOH/CH Cl ). H NMR (500 MHz, CDCl ) δ 7.22 (dd, J = 7.7, 1.4 Hz,
f
2
2
3
1
H), 7.14 (td, J = 7.8, 1.5 Hz, 1H), 6.83 (td, J = 7.5, 1.2 Hz, 1H), 6.73 (dd, J = 8.0, 1.1 Hz, 1H),
.75 (dd, J = 9.0, 6.0 Hz, 1H), 3.69 (bs, 2H), 2.04 – 1.94 (m, 1H), 1.91 – 1.83 (m, 1H), 1.58 – 1.44
3
13
(
m, 2H), 1.42 – 1.33 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H). C NMR (126 MHz, CDCl ) δ 143.47,
3
1
29.22, 128.70, 120.76, 120.70, 119.85, 117.54, 33.47, 32.24, 29.62, 22.28, 13.96. MS (ESIꢀTOF)
+
for C H N [M + H] calculated 189.1386, found 189.1359.
1
2
16
2
2
-Amino-3-butyl-3H-indol-3-ol (5). To a solution of compound 3 (38 mg, 0.2 mmol) in anhydrous
dioxane (1 mL) was added 4N HCl/dioxane (0.1 mL). The reaction mixture was then heated under
microwave conditions (400 W, 100 ºC) in a sealed vial for 20 min. The reaction mixture was cooled
to room temperature and the solvent was removed under reduced pressure to obtain the compound
4
. To a solution of compound 4 in MeOH (1 mL) was added Et N (56 µL, 0.4 mmol). The reaction
3
mixture was then stirred for 3 h. The solvent was removed under reduced pressure and the crude
material was purified using silica gel column chromatography (20% MeOH/CH Cl ) to obtain the
2
2
1
compound 5 as white solid (23 mg, 56%). R = 0.30 (20% MeOH/CH Cl ). H NMR (500 MHz,
f
2
2
DMSO) δ 8.49 (bs, 2H), 7.20 (ddd, J = 11.4, 8.8, 4.2 Hz, 2H), 6.98 – 6.85 (m, 2H), 6.05 (s, 1H),
1
4
ACS Paragon Plus Environment

Page 15 of 66
Journal of Medicinal Chemistry
1
2
1
.91 (td, J = 12.6, 4.7 Hz, 1H), 1.80 (td, J = 12.6, 4.7 Hz, 1H), 1.21 – 1.08 (m, 2H), 0.98 – 0.80 (m,
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
2
H), 0.75 (t, J = 7.3 Hz, 3H). C NMR (126 MHz, DMSO) δ 176.36, 150.46, 135.76, 129.12,
+
1
22.59, 121.63, 113.90, 81.06, 37.84, 24.89, 22.18, 13.83. MS (ESIꢀTOF) for C H N O [M + H]
1
2
16
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
calculated 205.1335, found 205.1358.
2
-Aminobenzimidazole (7). To a solution of compound oꢀphenylenediamine (108 mg, 1 mmol) in
1:1 mixture of MeOH (5 mL) and water (5 mL) was added CNBr (318 mg, 3 mmol). The reaction
mixture was stirred for 3 h at 60 ºC. The reaction mixture was cooled to room temperature; the
MeOH was removed under reduced pressure, and the remaining mixture was basified with 1.0 M
aq. NaOH (to pH = 8.0) and extracted with EtOAc (3 × 30 mL). The combined organic layer was
dried over Na SO , concentrated under reduced pressure, and the crude material was purified by
2
4
silica gel column chromatography (20% MeOH/CH Cl ) to obtain the compound 7 as a white solid
2
2
1
(
109 mg, 82%). R = 0.20 (20% MeOH/CH Cl ). H NMR (500 MHz, DMSO) δ 10.68 (bs, 1H),
f 2 2
13
7
.12 – 7.01 (m, 2H), 6.83 (dd, J = 5.7, 3.2 Hz, 2H), 6.11 (s, 2H). C NMR (126 MHz, DMSO) δ
+
155.29, 138.79, 118.95, 111.52. MS (ESIꢀTOF) for C H N [M + H] calculated 134.0713, found
7
7
3
1
34.0705.
1-Butyl-1H-benz[d]imidazol-2-amine (8a). To a solution of 2ꢀaminobenzimidazole (7) (27 mg,
.2 mmol) in acetone (1 mL) were added KOH (22 mg, 0.4 mmol) and butyl iodide (23 µL, 0.2
0
mmol). The reaction mixture was stirred for 3 h at 60 ºC. The reaction mixture was cooled to room
temperature and the solvent was removed under reduced pressure. The reaction mixture was diluted
with water and extracted with EtOAc (3 x 10 mL). The combined organic layer was dried over
Na SO , concentrated under reduced pressure, and the crude material was purified by silica gel
2
4
column chromatography (20% MeOH/CH Cl ) to obtain the compound 8a as a white solid (28 mg,
2
2
1
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 66
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
7
1
4%). R = 0.45 (20% MeOH/CH Cl ). H NMR (500 MHz, CDCl ) δ 7.44 (dd, J = 7.7, 0.8 Hz,
f
2
2
3
H), 7.15 – 7.06 (m, 3H), 4.61 (bs, 2H), 3.92 (t, J = 7.2 Hz, 2H), 1.81 – 1.74 (m, 2H), 1.45 – 1.37
13
(
m, 2H), 0.96 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, CDCl ) δ 153.15, 141.86, 134.42, 121.59,
3
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
119.98, 116.73, 107.95, 42.80, 31.30, 20.41, 13.90. MS (ESIꢀTOF) for C H N [M + H]
11 15 3
calculated 190.1339, found 190.1393.
Compounds 8b-8e were synthesized similarly as compound 8a.
1
-Pentyl-1H-benzo[d]imidazol-2-amine (8b). 1ꢀIodopentane was used as reagent. White solid (25
1
mg, 61%). R = 0.50 (20% MeOH/CH Cl ). H NMR (500 MHz, DMSO) δ 7.11 (d, J = 1.0 Hz,
f
2
2
1
2
H), 7.09 (d, J = 1.0 Hz, 1H), 6.91 (td, J = 7.4, 1.1 Hz, 1H), 6.85 (td, J = 7.4, 1.1 Hz, 1H), 6.35 (s,
1
3
H), 3.94 (t, J = 7.2 Hz, 2H), 1.66 – 1.57 (m, 2H), 1.34 – 1.20 (m, 4H), 0.84 (t, J = 7.0 Hz, 3H). C
NMR (126 MHz, DMSO) δ 154.81, 142.80, 134.27, 120.09, 117.88, 114.65, 107.44, 41.34, 28.30,
+
2
8.18, 21.94, 13.95. MS (ESIꢀTOF) for C H N [M + H] calculated 204.1495, found 204.1544.
1
2
17
3
1
-Hexyl-1H-benzo[d]imidazol-2-amine (8c). 1ꢀIodohexane was used as reagent. White solid (30
1
mg, 69%). R = 0.52 (20% MeOH/CH Cl ). H NMR (500 MHz, CDCl ) δ 7.44 (d, J = 7.8 Hz, 1H),
f
2
2
3
7.16 – 7.04 (m, 3H), 4.66 (bs, 2H), 3.91 (t, J = 7.3 Hz, 2H), 1.82 – 1.75 (m, 2H), 1.39 – 1.25 (m,
13
6
H), 0.88 (t, J = 7.0 Hz, 3H). C NMR (126 MHz, CDCl ) δ 153.25, 142.00, 134.43, 121.53,
3
1
19.92, 116.72, 107.92, 43.02, 31.59, 29.20, 26.82, 22.64, 14.13. MS (ESIꢀTOF) for C H N [M +
13 19
3
+
H] calculated 218.1652, found 218.1706.
1-Benzyl-1H-benzo[d]imidazol-2-amine (8d). Benzyl bromide was used as reagent. White solid
1
(
28 mg, 63%). R = 0.44 (10% 20% MeOH/CH Cl ). H NMR (500 MHz, DMSO) δ 7.33 – 7.28 (m,
f
2
2
1
6
ACS Paragon Plus Environment

Page 17 of 66
Journal of Medicinal Chemistry
1
2
2
H), 7.24 (ddd, J = 6.4, 3.9, 1.3 Hz, 1H), 7.21 – 7.16 (m, 2H), 7.13 (d, J = 7.4 Hz, 1H), 7.04 (d, J =
.4 Hz, 1H), 6.91 (td, J = 7.6, 1.2 Hz, 1H), 6.81 (td, J = 7.6, 1.1 Hz, 1H), 6.52 (s, 2H), 5.25 (s, 2H).
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
13
C NMR (126 MHz, DMSO) δ 155.02, 142.92, 137.25, 134.17, 128.52, 127.27, 127.00, 120.47,
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
118.06, 114.77, 107.90, 44.66. MS (ESIꢀTOF) for C H N calculated [M + H] 224.1182, found
1
4
13
3
2
24.1183.
3-((2-Amino-1H-benzo[d]imidazol-1-yl)methyl)benzonitrile (8e). 3ꢀCyanobenzyl bromide was
1
used as reagent. White solid (38 mg, 76%). R = 0.40 (20% MeOH/CH Cl ). H NMR (500 MHz,
f
2
2
CDCl ) δ 7.61 (d, J = 7.7 Hz, 1H), 7.51 – 7.47 (m, 2H), 7.45 (d, J = 7.7 Hz, 1H), 7.39 – 7.35 (m,
3
1
2
1
H), 7.19 (td, J = 7.7, 1.2 Hz, 1H), 7.09 (td, J = 7.6, 1.1 Hz, 1H), 7.03 (d, J = 7.5 Hz, 1H), 5.19 (s,
13
H), 4.62 (bs, 2H). C NMR (126 MHz, CDCl ) δ 153.12, 141.95, 137.21, 134.26, 132.07, 130.90,
3
30.28, 130.09, 122.42, 120.72, 118.27, 117.25, 113.59, 107.81, 45.56. MS (ESIꢀTOF) for
+
C H N [M + H] calculated 249.1135, found 249.1136.
15
12
4
1-(3-(Aminomethyl)benzyl)-1H-benzo[d]imidazol-2-amine (8f). To a solution of compound 8e
25 mg, 0.1 mmol) in THF (2 mL) was added LiAlH (0.4 mL, 0.4 mmol, 1.0 M in THF) at 0 ºC
(
4
under nitrogen atmosphere. The reaction mixture was stirred for 1 h at 25 ºC and 5 h at 75 ºC. The
reaction mixture was cooled to room temperature and quenched carefully with iceꢀcold water. The
resulting mixture was basified with 10% NaOH (to pH = 8.0) and extracted with CH Cl (3 × 10
2
2
mL). The combined organic layer was dried over Na SO , concentrated under reduced pressure, and
2
4
the crude material was purified by neutralꢀalumina column chromatography (30% MeOH/CH Cl )
2
2
1
to obtain the compound 8f as a white solid (17 mg, 67%). R = 0.20 (40% MeOH/CH Cl ). H NMR
f
2
2
(400 MHz, DMSO) δ 7.25 – 7.20 (m, 3H), 7.12 (d, J = 7.7 Hz, 1H), 7.02 (d, J = 7.4 Hz, 1H), 7.00 –
.95 (m, 1H), 6.91 (td, J = 7.6, 1.1 Hz, 1H), 6.80 (td, J = 7.6, 1.0 Hz, 1H), 6.50 (s, 2H), 5.22 (s, 2H),
6
1
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 66
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
3
.67 (s, 2H). C NMR (126 MHz, DMSO) δ 155.03, 144.18, 142.95, 137.02, 134.23, 128.31,
26.09, 125.85, 124.92, 120.42, 118.03, 114.75, 107.92, 45.44, 44.83. MS (ESIꢀTOF) for C H N
4
1
1
5
16
+
[
M + H] calculated 253.1448, found 253.1470.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Butyl 2-amino-1H-benzo[d]imidazole-1-carboxylate (9). To a solution of 2ꢀaminobenzimidazole
7) (27 mg, 0.2 mmol) in anhydrous THF (2 mL) was added butyl chloroformate (27 µL, 0.2 mmol)
(
under nitrogen atmosphere. The reaction mixture was stirred for 3 h. The reaction mixture was
diluted with water and extracted with EtOAc (3 x 10 mL). The combined organic layer was dried
over Na SO , concentrated under reduced pressure, and the crude material was purified by silica gel
2
4
column chromatography (20 % MeOH/CH Cl ) to afford the compound 9 as a white solid (28 mg,
2
2
1
6
7
4
0%). R = 0.65 (10% MeOH/CH Cl ). H NMR (500 MHz, DMSO) δ 7.60 (d, J = 7.6 Hz, 1H),
f 2 2
.22 (bs, 2H), 7.18 (dd, J = 7.8, 0.7 Hz, 1H), 7.13 (td, J = 7.6, 1.2 Hz, 1H), 7.05 – 6.93 (m, 1H),
13
.45 (t, J = 6.6 Hz, 2H), 1.86 – 1.72 (m, 2H), 1.52 – 1.38 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H). C
NMR (126 MHz, DMSO) δ 153.58, 151.26, 142.89, 130.02, 124.11, 119.91, 115.54, 113.61, 67.42,
+
29.94, 18.68, 13.57. MS (ESIꢀTOF) for C H N O [M + H] calculated 234.1237, found 234.1260.
1
2
15
3
2
N-(1-Pentyl-1H-benzo[d]imidazol-2-yl)acetamide(10). To a stirred solution of compound 8b (41
mg, 0.2 mmol) in pyridine (2 mL) was added acetyl chloride (14 µL, 0.2 mmol). The reaction
mixture was stirred for 3 h. The solvent was removed under reduced pressure. The reaction mixture
was diluted with water and extracted with EtOAc (3 x 10 mL). The combined organic layer was
dried over Na SO , concentrated under reduced pressure, and the crude material was purified by
2
4
silica gel column chromatography (10% MeOH/CH Cl ) to afford the compound 10 as a white solid
2
2
1
(32 mg, 65%). R = 0.62 (10% MeOH/CH Cl ). H NMR (500 MHz, CDCl ) δ 12.19 (bs, 1H), 7.29
f
2
2
3
(
d, J = 8.1 Hz, 1H), 7.25 – 7.18 (m, 3H), 4.11 (t, J = 7.4 Hz, 2H), 2.24 (s, 3H), 1.87 – 1.73 (m, 2H),
1
8
ACS Paragon Plus Environment

Page 19 of 66
Journal of Medicinal Chemistry
1
2
1
3
1
1
.45 – 1.30 (m, 4H), 0.90 (t, J = 7.0 Hz, 3H). C NMR (126 MHz, CDCl ) δ 183.60, 153.38,
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
29.66, 128.46, 123.01, 122.89, 111.34, 109.47, 42.07, 29.85, 28.92, 28.12, 22.42, 14.08. MS (ESIꢀ
+
TOF) for C H N O [M + H] calculated 246.1601, found 246.1637.
1
4
19
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound 12 was synthesized similarly as compound 7.
1H-Naphtho[2,3-d]imidazol-2-amine (12). Naphthaleneꢀ2,3ꢀdiamine was used as reagent. Offꢀ
1
white solid (135 mg, 74%). R = 0.35 (20% MeOH/CH Cl ). H NMR (500 MHz, DMSO) δ 10.78
f
2
2
(
bs, 1H), 7.77 (dd, J = 6.2, 3.3 Hz, 2H), 7.47 (s, 2H), 7.20 (dd, J = 6.3, 3.3 Hz, 2H), 6.60 (s, 2H).
1
3
C NMR (126 MHz, DMSO) δ 158.41, 129.03, 126.87, 126.81, 121.99, 106.25. MS (ESIꢀTOF) for
+
C H N [M + H] calculated 184.0869, found 184.0877.
11
9
3
Compound 13 was synthesized similarly as compound 8a.
1-Pentyl-1H-naphtho[2,3-d]imidazol-2-amine (13). Compound 12 was used as reagent. Offꢀwhite
1
solid (40 mg, 79%). R = 0.45 (10% MeOH/CH Cl ). H NMR (500 MHz, DMSO) δ 7.85 – 7.77
f
2
2
(
m, 2H), 7.53 (s, 1H), 7.52 (s, 1H), 7.27 – 7.21 (m, 2H), 6.97 (s, 2H), 4.05 (t, J = 7.3 Hz, 2H), 1.75
1
3
– 1.64 (m, 2H), 1.36 – 1.27 (m, 4H), 0.85 (t, J = 6.9 Hz, 3H). C NMR (126 MHz, DMSO) δ
1
2
57.56, 135.95, 129.84, 128.17, 126.97, 126.91, 122.45, 122.13, 109.25, 102.56, 41.58, 28.32,
+
7.85, 21.99, 13.98. MS (ESIꢀTOF) for C H N [M + H] calculated 254.1652, found 254.1714.
16 19
3
1
-Methoxy-2-nitronaphthalene (15). To a solution of compound 2ꢀNitroꢀ1ꢀnaphthol (189 mg, 1
mmol) in acetone (5 mL) were added KOH (168 mg, 3 mmol) and MeI (124 µL, 2 mmol). The
reaction mixture was refluxed for 12 h. The reaction mixture was cooled to room temperature and
1
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 66
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
solvent was removed under reduced pressure. Water was then added to the reaction mixture and
extracted with EtOAc (3 x 30 mL). The combined organic layer was dried over Na SO ,
2
4
concentrated under reduced pressure, and the crude material was purified by silica gel column
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
chromatography (10% EtOAc/hexanes) to obtain the compound 15 as a pale yellow solid (178 mg,
1
8
8%). R = 0.50 (10% EtOAc/hexanes). H NMR (500 MHz, CDCl ) δ 8.32 (ddd, J = 3.0, 1.6, 0.7
f
3
1
3
Hz, 1H), 7.90 (d, J = 8.9 Hz, 2H), 7.71 – 7.60 (m, 3H), 4.15 (s, 3H). C NMR (126 MHz, CDCl ) δ
3
151.85, 139.19, 136.61, 129.61, 128.70, 128.34, 127.74, 124.49, 124.30, 121.16, 63.88.
2
-Nitro-N-pentylnaphthalen-1-amine (16). To a solution of compound 15 (102 mg, 0.5 mmol) in
DMF (3 mL), was added amyl amine (87 µL, 0.75 mmol). The reaction mixture was stirred for 10 h
at 60 ºC. The reaction mixture was diluted with water and extracted with EtOAc (3 x 20 mL). The
combined organic layer was dried over Na SO , concentrated under reduced pressure, and the crude
2
4
material was purified by silica gel column chromatography (10% EtOAc/hexanes) to obtain the
1
compound 16 as a red solid (115 mg, 89%). R = 0.62 (10% EtOAc/hexanes). H NMR (500 MHz,
f
CDCl ) δ 9.01 (bs, 1H), 8.31 (d, J = 8.5 Hz, 1H), 8.08 (d, J = 9.3 Hz, 1H), 7.77 – 7.70 (m, 1H), 7.60
3
(ddd, J = 8.1, 6.9, 1.1 Hz, 1H), 7.46 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.10 (d, J = 9.3 Hz, 1H), 3.77
1
3
(
td, J = 7.0, 5.3 Hz, 2H), 1.79 – 1.67 (m, 2H), 1.46 – 1.28 (m, 4H), 0.90 (t, J = 7.1 Hz, 3H). C
NMR (126 MHz, CDCl ) δ 149.00, 138.03, 130.98, 130.18, 128.60, 128.10, 125.92, 125.25, 121.89,
3
+
1
18.44, 51.31, 31.63, 29.09, 22.46, 14.07. MS (ESIꢀTOF) for C H N O [M + H] calculated
1
5
18
2
2
2
59.1441, found 259.1386.
1
-Pentyl-1H-naphtho[1,2-d]imidazol-2-amine (18). To a solution of compound 16 (52 mg, 0.2
mmol) in anhydrous EtOAc (5 mL) was added a catalytic amount of 5% Pt on carbon (16 mg, 2 mol
). The reaction mixture was subjected to hydrogenation at 30 psi H pressure for 3 h. The reaction
%
2
2
0
ACS Paragon Plus Environment

Page 21 of 66
Journal of Medicinal Chemistry
1
2
mixture was filtered, and the filtrate was concentrated under the reduced pressure to obtain
compound 17. To a solution of compound 17 in a 1:1 mixture of MeOH (1 mL) and water (1 mL)
was added CNBr (64 mg, 0.6 mmol). The reaction mixture was stirred for 3 h at 60 ºC. The reaction
mixture was cooled to room temperature, and MeOH removed under reduced pressure. The
remaining mixture was basified with 1.0 M aq. NaOH (to pH = 8.0) and extracted with EtOAc (3 ×
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0 mL). The combined organic layer was dried over Na SO , concentrated under reduced pressure,
2 4
and the crude material was purified by silica gel column chromatography (10% MeOH/CH Cl ) to
2
2
1
obtain the compound 18 as a purple solid (35 mg, 69%). R = 0.45 (10% MeOH/CH Cl ). H NMR
f
2
2
(
500 MHz, CDCl ) δ 8.08 (d, J = 8.5 Hz, 1H), 7.94 (d, J = 7.5 Hz, 1H), 7.69 – 7.59 (m, 2H), 7.52
3
(ddd, J = 8.4, 6.9, 1.3 Hz, 1H), 7.38 (ddd, J = 8.0, 6.9, 1.0 Hz, 1H), 4.76 (bs, 2H), 4.35 (t, J = 7.6
13
Hz, 2H), 2.05 – 1.92 (m, 2H), 1.53 – 1.43 (m, 2H), 1.43 – 1.36 (m, 2H), 0.93 (t, J = 7.2 Hz, 3H). C
NMR (126 MHz, CDCl ) δ 152.28, 138.22, 130.26, 129.91, 126.14, 125.95, 123.19, 122.84, 121.52,
3
+
1
2
18.84, 118.07, 45.55, 29.62, 29.19, 22.57, 14.12. MS (ESIꢀTOF) for C H N [M + H] calculated
1
6
19
3
54.1652, found 254.1715.
Compound 20 was synthesized similarly as compound 15.
2-Methoxy-1-nitronaphthalene (20). 1ꢀNitroꢀ2ꢀnaphthol was used as reagent. Green solid (164
1
mg, 81%). R = 0.50 (10% EtOAc/hexanes). H NMR (500 MHz, CDCl ) δ 7.97 (d, J = 9.1 Hz,
f
3
1
1
1
H), 7.85 (d, J = 8.3 Hz, 1H), 7.68 (dd, J = 8.6, 0.8 Hz, 1H), 7.63 – 7.57 (m, 1H), 7.49 – 7.42 (m,
1
3
H), 7.35 (d, J = 9.2 Hz, 1H), 4.04 (s, 3H). C NMR (126 MHz, CDCl ) δ 148.76, 132.33, 129.30,
3
28.35, 128.18, 125.83, 125.31, 120.59, 113.19, 57.21.
Compound 21 was synthesized similarly as compound 16.
2
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 66
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
-Nitro-N-pentylnaphthalen-2-amine (21). Compound 20 was used as reagent. Orange solid (102
1
mg, 79%). R = 0.60 (10% EtOAc/hexanes). H NMR (500 MHz, CDCl ) δ 8.94 (bs, 1H), 8.79 (d, J
f
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
= 8.8 Hz, 1H), 7.79 (d, J = 9.4 Hz, 1H), 7.67 (dd, J = 7.9, 1.3 Hz, 1H), 7.60 (ddd, J = 8.6, 7.0, 1.5
Hz, 1H), 7.33 (ddd, J = 7.9, 7.1, 1.0 Hz, 1H), 7.07 (d, J = 9.3 Hz, 1H), 3.44 (td, J = 7.1, 5.4 Hz,
1
3
2
H), 1.83 – 1.73 (m, 2H), 1.51 – 1.35 (m, 4H), 0.94 (t, J = 7.2 Hz, 3H). C NMR (126 MHz,
CDCl ) δ 146.99, 137.46, 130.37, 129.11, 128.89, 126.47, 125.56, 124.03, 123.44, 114.23, 43.71,
3
+
2
2
9.26, 29.14, 22.52, 14.11. MS (ESIꢀTOF) for C H N O [M + H] calculated 259.1441, found
15
18
2
2
59.1384.
Compound 23 was synthesised similarly as compound 18.
3
-Pentyl-3H-naphtho[1,2-d]imidazol-2-amine (23). Compound 21 was used as reagent. Purple
1
solid (37 mg, 73%). R = 0.45 (10% MeOH/CH Cl ). H NMR (500 MHz, DMSO) δ 8.18 (d, J =
f
2
2
8.3 Hz, 1H), 7.85 (d, J = 8.2 Hz, 1H), 7.48 (d, J = 8.6 Hz, 1H), 7.44 – 7.40 (m, 2H), 7.31 (ddd, J =
8
.1, 6.8, 1.3 Hz, 1H), 6.42 (s, 2H), 4.08 (t, J = 7.2 Hz, 2H), 1.75 – 1.63 (m, 2H), 1.35 – 1.16 (m,
1
3
4
H), 0.83 (t, J = 6.9 Hz, 3H). C NMR (126 MHz, DMSO) δ 153.77, 136.96, 129.24, 129.19,
128.18, 124.60, 124.58, 122.75, 121.13, 117.88, 109.93, 41.60, 28.56, 28.29, 21.95, 13.95. MS
+
(ESIꢀTOF) for C H N [M + H] calculated 254.1652, found 254.1713.
1
6
19
3
2
-Nitro-N-pentylpyridin-3-amine (25a). To a solution of compound 3ꢀfluoroꢀ2ꢀnitropyridine (142
mg, 1 mmol) in DMSO (2 mL), were added amyl amine (116 µL, 1 mmol) and DIPEA (174 µL, 1
mmol). The reaction mixture was stirred for 6 h at 60 ºC. After the completion of the reaction
(monitored by TLC), the reaction mixture was diluted with water and extracted with EtOAc (3 × 20
2
2
ACS Paragon Plus Environment

Page 23 of 66
Journal of Medicinal Chemistry
1
2
mL). The combined organic layer was dried over Na SO and concentrated under reduced pressure,
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
4
and the crude material was purified by flash chromatography (10% EtOAc/hexanes) to afford the
1
compound 25a as a yellow oil (184 mg, 88%). R = 0.30 (10% EtOAc/hexanes). H NMR (500
f
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MHz, CDCl ) δ 7.88 (dd, J = 1.43, 3.96 Hz, 1H), 7.75 (s, 1H), 7.43 (ddd, J = 0.62, 3.96, 8.59 Hz,
3
1
H), 7.33 (dd, J = 1.29, 8.62 Hz, 1H), 3.29 (td, J = 5.29, 7.12 Hz, 2H), 1.79 – 1.68 (m, 2H), 1.48 –
13
1
.31 (m, 4H), 0.93 (t, J = 7.12 Hz, 3H). C NMR (126 MHz, CDCl ) δ 141.21, 140.82, 135.10,
3
+
130.58, 123.44, 42.92, 29.26, 28.67, 22.48, 14.09. MS (ESIꢀTOF) for C H N O [M + H]
10
15
3
2
calculated 210.1237, found 210.1214.
Compounds 25b-25d were synthesized similarly as compound 25a.
3
-Nitro-N-pentylpyridin-4-amine (25b). 4ꢀChloroꢀ3ꢀnitropyridine was used as reagent. Yellow oil
1
(
179 mg, 86%). R = 0.20 (10% EtOAc/hexanes). H NMR (500 MHz, CDCl ) δ 9.20 (s, 1H), 8.28
f
3
(dd, J = 6.2, 0.7 Hz, 1H), 8.16 (bs, 1H), 6.70 (d, J = 6.2 Hz, 1H), 3.32 (td, J = 7.2, 5.4 Hz, 2H), 1.79
1
3
– 1.69 (m, 2H), 1.47 – 1.32 (m, 4H), 0.94 (t, J = 7.1 Hz, 3H). C NMR (126 MHz, CDCl ) δ
3
1
53.22, 149.30, 148.82, 129.79, 107.87, 42.92, 29.18, 28.50, 22.44, 14.06. MS (ESIꢀTOF) for
+
C H N O [M + H] calculated 210.1237, found 210.1307.
10
15
3
2
4
-Nitro-N-pentylpyridin-3-amine (25c). 3ꢀFluoroꢀ4ꢀnitropyridine was used as reagent. Yellow
1
solid (184 mg, 88%). R = 0.20 (10% EtOAc/hexanes). H NMR (500 MHz, CDCl ) δ 8.15 (d, J =
f
3
8
.9 Hz, 1H), 7.86 (d, J = 2.9 Hz, 1H), 6.91 (dd, J = 9.0, 2.9 Hz, 1H), 3.23 (t, J = 7.2 Hz, 2H), 1.73 –
13
1
.64 (m, 2H), 1.45 – 1.34 (m, 4H), 0.93 (t, J = 7.1 Hz, 3H). C NMR (126 MHz, CDCl ) δ 148.64,
3
147.60, 133.24, 120.64, 117.58, 43.46, 29.18, 28.77, 22.50, 14.09. MS (ESIꢀTOF) for C H N O
1
0
15
3
2
+
[
M + H] calculated 210.1237, found 210.1197.
2
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 66
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
-Nitro-N-pentylpyridin-2-amine (25d). 2ꢀChloroꢀ3ꢀnitropyridine was used as reagent. Yellow oil
1
(188 mg, 90%). R = 0.22 (10% EtOAc/hexanes). H NMR (500 MHz, CDCl ) δ 8.43 – 8.38 (m,
f
3
2
H), 8.25 (bs, 1H), 6.62 (dd, J = 8.0, 4.8 Hz, 1H), 3.65 – 3.58 (m, 2H), 1.74 – 1.64 (m, 2H), 1.46 –
13
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1.33 (m, 4H), 0.92 (t, J = 7.1 Hz, 3H). C NMR (126 MHz, CDCl ) δ 156.02, 152.91, 135.45,
3
+
1
28.01, 111.55, 41.46, 29.34, 29.20, 22.56, 14.15. MS (ESIꢀTOF) for C H N O [M + H]
10 15 3 2
calculated 210.1237, found 210.1220.
Compounds 27a-27d were synthesized similarly as compound 18.
1
-Pentyl-1H-imidazo[4,5-b]pyridin-2-amine (27a). Compound 25a was used as reagent. White
1
solid (29 mg, 71%). R = 0.42 (10% MeOH/CH Cl ). H NMR (500 MHz, DMSO) δ 7.94 (dd, J =
f
2
2
5
.0, 1.3 Hz, 1H), 7.42 (dd, J = 7.6, 1.4 Hz, 1H), 6.86 (bs, 2H), 6.81 (dd, J = 7.6, 5.0 Hz, 1H), 3.96
1
3
(t, J = 7.2 Hz, 2H), 1.65 – 1.56 (m, 2H), 1.32 – 1.17 (m, 4H), 0.83 (t, J = 7.1 Hz, 3H). C NMR
(126 MHz, DMSO) δ 156.80, 156.50, 140.62, 126.91, 113.48, 113.42, 41.32, 28.22, 28.09, 21.91,
+
13.93. MS (ESIꢀTOF) for C H N [M + H] calculated 205.1448, found 205.1491.
1
1
16
4
1
-Pentyl-1H-imidazo[4,5-c]pyridin-2-amine (27b). Compound 25b was used as reagent. White
1
solid (29 mg, 71%). R = 0.20 (10% MeOH/CH Cl ). H NMR (500 MHz, DMSO) δ 8.33 (s, 1H),
f
2
2
8
1
1
.01 (d, J = 5.21 Hz, 1H), 7.19 (d, J = 5.17 Hz, 1H), 6.73 (s, 2H), 3.98 (t, J = 7.21 Hz, 2H), 1.67 –
1
3
.55 (m, 2H), 1.34 – 1.14 (m, 4H), 0.83 (t, J = 7.12 Hz, 3H). C NMR (126 MHz, DMSO) δ
55.64, 140.08, 139.51, 138.55, 135.79, 103.52, 41.57, 28.19, 28.08, 21.88, 13.92. MS (ESIꢀTOF)
+
for C H N [M + H] calculated 205.1448,found 205.1477.
1
1
16
4
2
4
ACS Paragon Plus Environment

Page 25 of 66
Journal of Medicinal Chemistry
1
2
3
-Pentyl-3H-imidazo[4,5-c]pyridin-2-amine (27c). Compound 25c was used as reagent. White
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
solid (30 mg, 74%). R = 0.40 (10% MeOH/CH Cl ). H NMR (500 MHz, DMSO) δ 7.84 (d, J =
f
2
2
2
.6 Hz, 1H), 7.32 (dd, J = 8.9, 3.0 Hz, 1H), 6.48 (dd, J = 8.9, 0.5 Hz, 1H), 5.98 (s, 2H), 3.47 (t, J =
13
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7.1 Hz, 2H), 1.64 – 1.56 (m, 2H), 1.38 – 1.26 (m, 4H), 0.87 (t, J = 7.1 Hz, 3H). C NMR (126
MHz, DMSO) δ 157.42, 138.53, 129.16, 126.49, 114.87, 108.44, 50.58, 27.89, 26.70, 21.71, 13.86.
+
MS (ESIꢀTOF) for C H N [M + H] calculated 205.1448, found 205.1495.
1
1
16
4
3
-Pentyl-3H-imidazo[4,5-b]pyridin-2-amine (27d). Compound 25d was used as reagent. White
1
solid (34 mg, 83%). R = 0.45 (10% MeOH/CH Cl ). H NMR (500 MHz, DMSO) δ 7.83 (dd, J =
f
2
2
5
.0, 1.4 Hz, 1H), 7.37 (dd, J = 7.7, 1.4 Hz, 1H), 6.93 (dd, J = 7.7, 5.0 Hz, 1H), 6.75 (s, 2H), 4.00 (t,
13
J = 7.3 Hz, 2H), 1.70 – 1.60 (m, 2H), 1.34 – 1.19 (m, 4H), 0.83 (t, J = 7.1 Hz, 3H). C NMR (126
MHz, DMSO) δ 155.48, 147.91, 137.37, 135.70, 119.96, 116.61, 40.06, 28.26, 28.00, 21.85, 13.92.
+
MS (ESIꢀTOF) for C H N [M + H] calculated 205.1448, found 205.1470.
1
1
16
4
Compounds 29a-29i were synthesized similarly as compound 25a.
3
-Methyl-2-nitro-N-pentylaniline (29a). 1ꢀFluoroꢀ3ꢀmethylꢀ2ꢀnitrobenzene was used as reagent.
1
Red oil (200 mg, 90%). R = 0.70 (10% EtOAc/hexanes). H NMR (500 MHz, CDCl ) δ 7.25 – 7.14
f
3
(
m, 1H), 6.65 (d, J = 8.5 Hz, 1H), 6.60 (bs, 1H), 6.50 (d, J = 7.4 Hz, 1H), 3.19 (td, J = 7.1, 5.1 Hz,
1
3
2
H), 2.47 (s, 3H), 1.73 – 1.60 (m, 2H), 1.46 – 1.31 (m, 4H), 0.92 (t, J = 7.1 Hz, 3H). C NMR (126
MHz, CDCl ) δ 144.34, 135.92, 135.59, 133.41, 119.09, 111.31, 43.55, 29.36, 28.86, 22.55, 21.71,
3
+
1
4.13. MS (ESIꢀTOF) for C H N O [M + H] calculated 223.1441, found 223.1400.
1
2
18
2
2
2
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 66
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
-Methyl-2-nitro-N-pentylaniline (29b). 1ꢀFluoroꢀ4ꢀmethylꢀ2ꢀnitrobenzene was used as reagent.
1
Red oil (180 mg, 81%). R = 0.65 (10% EtOAc/hexanes). H NMR (500 MHz, CDCl ) δ 8.02 – 7.91
f
3
(m, 2H), 7.27 – 7.24 (m, 1H), 6.76 (d, J = 8.8 Hz, 1H), 3.28 (td, J = 7.1, 5.3 Hz, 2H), 2.26 (s, 3H),
1
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1.78 – 1.66 (m, 2H), 1.47 – 1.34 (m, 4H), 0.93 (t, J = 7.1 Hz, 3H). C NMR (126 MHz, CDCl ) δ
3
1
44.12, 137.93, 131.39, 126.20, 124.67, 113.93, 43.22, 29.37, 28.86, 22.54, 20.11, 14.13. MS (ESIꢀ
+
TOF) for C H N O [M + H] calculated 223.1441, found 223.1410.
1
2
18
2
2
5
-Methyl-2-nitro-N-pentylaniline (29c). 2ꢀChloroꢀ4ꢀmethylꢀ1ꢀnitrobenzene was used as reagent.
1
Orange solid (180 mg, 81%). R = 0.68 (10% EtOAc/hexanes). H NMR (400 MHz, CDCl ) δ 8.11
f
3
(bs, 1H), 8.08 (d, J = 8.8 Hz, 1H), 6.63 (s, 1H), 6.46 (dd, J = 8.8, 1.6 Hz, 1H), 3.30 (td, J = 7.1, 5.3
13
Hz, 2H), 2.36 (s, 3H), 1.83 – 1.66 (m, 2H), 1.52 – 1.35 (m, 4H), 0.96 (t, J = 7.1 Hz, 3H). C NMR
(126 MHz, CDCl ) δ 147.85, 145.86, 129.96, 126.99, 116.96, 113.47, 43.12, 29.37, 28.79, 22.52,
3
+
2
2.35, 14.13. MS (ESIꢀTOF) for C H N O [M + H] calculated 223.1441, found 223.1400.
1
2
18
2
2
2-Methyl-6-nitro-N-pentylaniline (29d). 2ꢀChloroꢀ1ꢀmethylꢀ3ꢀnitrobenzene was used as reagent.
1
Red oil (175 mg, 79%). R = 0.70 (10% EtOAc/hexanes). H NMR (500 MHz, CDCl ) δ 7.97 – 7.86
f
3
(m, 1H), 7.28 (ddd, J = 7.3, 1.6, 0.8 Hz, 1H), 6.96 (bs, 1H), 6.74 (dd, J = 8.4, 7.3 Hz, 1H), 3.24 (t, J
1
3
= 7.2 Hz, 2H), 2.39 (s, 3H), 1.62 – 1.53 (m, 2H), 1.34 ꢀ 1.31 (m, 4H), 0.89 (t, J = 7.1 Hz, 3H). C
NMR (126 MHz, CDCl ) δ 146.08, 138.52, 137.93, 130.14, 124.44, 118.36, 48.02, 30.93, 29.13,
3
+
2
2.53, 20.93, 14.11. MS (ESIꢀTOF) for C H N O [M + H] calculated 223.1441, found 223.1445.
12 18
2
2
3
-Methoxy-2-nitro-N-pentylaniline (29e). 1ꢀFluoroꢀ3ꢀmethoxyꢀ2ꢀnitrobenzene was used as
1
reagent. Red oil (200 mg, 84%). R = 0.62 (10% EtOAc/hexanes). H NMR (500 MHz, CDCl ) δ
f
3
7
.22 (dd, J = 12.5, 4.3 Hz, 1H), 6.36 (d, J = 8.6 Hz, 1H), 6.24 (dd, J = 8.2, 0.8 Hz, 1H), 6.19 (bs,
2
6
ACS Paragon Plus Environment

Page 27 of 66
Journal of Medicinal Chemistry
1
2
1
H), 3.87 (s, 3H), 3.17 (td, J = 7.1, 5.2 Hz, 2H), 1.70 – 1.61 (m, 2H), 1.42 – 1.32 (m, 4H), 0.92 (t, J
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
=
7.1 Hz, 3H). C NMR (126 MHz, CDCl ) δ 155.44, 144.62, 133.74, 127.24, 105.23, 99.34,
3
+
5
6.57, 43.60, 29.31, 28.89, 22.54, 14.12. MS (ESIꢀTOF) for C H N O [M + H] calculated
1
2
18
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
239.1390, found 239.1362.
3
-Fluoro-2-nitro-N-pentylaniline (29f). 1, 3ꢀDifluoroꢀ2ꢀnitrobenzene was used as reagent. Red oil
1
(169 mg, 75%). R = 0.62 (5% EtOAc/hexanes). H NMR (500 MHz, CDCl ) δ 7.31 – 7.27 (m, 1H),
f
3
7
.25 (bs, 1H), 6.57 (d, J = 8.8 Hz, 1H), 6.40 (ddd, J = 11.5, 8.1, 1.2 Hz, 1H), 3.23 (td, J = 7.1, 5.2
1
3
Hz, 2H), 1.76 – 1.65 (m, 2H), 1.47 – 1.33 (m, 4H), 0.93 (t, J = 7.1 Hz, 3H). C NMR (126 MHz,
CDCl ) δ 158.08 (d, J = 261.9 Hz), 145.85, 134.70 (d, J = 12.0 Hz), 108.71 (d, J = 3.7 Hz), 103.08,
3
+
1
02.90, 43.58, 29.14, 28.57, 22.36, 13.96. MS (ESIꢀTOF) for C H FN O [M + H] calculated
1
1
15
2
2
2
27.1190, found 227.1135.
3
-Chloro-2-nitro-N-pentylaniline (29g). 1ꢀChloroꢀ3ꢀfluoroꢀ2ꢀnitrobenzene was used as reagent.
1
Orange solid (200 mg, 83%). R = 0.65 (5% EtOAc/hexanes). H NMR (500 MHz, CDCl ) δ 7.21
f
3
(
ddd, J = 8.5, 7.9, 0.5 Hz, 1H), 6.71 (ddd, J = 18.3, 8.3, 1.0 Hz, 2H), 5.83 (bs, 1H), 3.17 (td, J = 7.1,
13
5
.2 Hz, 2H), 1.73 – 1.58 (m, 2H), 1.54 – 1.22 (m, 4H), 0.92 (t, J = 7.1 Hz, 3H). C NMR (126
MHz, CDCl ) δ 144.10, 134.65, 132.91, 128.81, 118.17, 111.83, 43.67, 29.25, 28.78, 22.51, 14.10.
3
+
MS (ESIꢀTOF): for C H ClN O [M + H] calculated 243.0895, found 243.0841.
1
1
15
2
2
2
-Nitro-N-pentyl-3-(trifluoromethyl)aniline (29h). 1ꢀChloroꢀ2ꢀnitroꢀ3ꢀ(trifluoromethyl) benzene
1
was used as reagent. Orange solid (179 mg, 65%). R = 0.70 (5% EtOAc/hexanes). H NMR (500
f
MHz, CDCl ) δ 7.41 (dd, J = 8.5, 7.8 Hz, 1H), 7.01 (d, J = 8.1 Hz, 2H), 5.94 (bs, 1H), 3.20 (td, J =
3
13
7
.1, 5.2 Hz, 2H), 1.74 – 1.59 (m, 2H), 1.44 – 1.31 (m, 4H), 0.93 (t, J = 7.1 Hz, 3H). C NMR (126
2
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 66
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
MHz, CDCl ) δ 142.94, 133.02, 132.67, 125.97 (q, J = 32.78 Hz), 122.69 (q, J = 273.57 Hz),
3
1
17.43, 114.82 (q, J = 5.71 Hz), 43.67, 29.22, 28.76, 22.50, 14.09. MS (ESIꢀTOF) for
+
C H F N O [M + H] calculated 277.1158, found 277.1105.
12
15
3
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
-Bromo-2-nitro-N-pentylaniline (29i). 1ꢀBromoꢀ3ꢀfluoroꢀ2ꢀnitrobenzene was used as reagent.
1
Orange solid (250 mg, 87%). R = 0.70 (5% EtOAc/hexanes). H NMR (500 MHz, CDCl ) δ 7.13
f
3
(ddd, J = 8.5, 7.9, 0.5 Hz, 1H), 6.92 (dd, J = 7.8, 1.1 Hz, 1H), 6.73 (dd, J = 8.6, 0.8 Hz, 1H), 5.74
bs, 1H), 3.17 (td, J = 7.1, 5.2 Hz, 2H), 1.74 – 1.59 (m, 2H), 1.46 – 1.27 (m, 4H), 0.92 (t, J = 7.1
(
1
3
Hz, 3H). C NMR (126 MHz, CDCl ) δ 143.98, 136.28, 133.15, 121.52, 116.46, 112.50, 43.65,
3
+
2
2
9.24, 28.78, 22.51, 14.10. MS (ESIꢀTOF) for C H BrN O [M + H] calculated 287.0390, found
1
1
15
2
2
87.0330.
3
-Ethyl-2-nitro-N-pentylaniline (29j). To a stirred solution of compound 29i (144 mg, 0.5 mmol)
in 1, 4ꢀdioxane (3 mL) were added ethylboronic acid (56 mg, 0.75 mmol), Pd(dppf)Cl (36 mg, 0.05
2
mmol) and K CO (207 mg, 1.5 mmol). The resulting reaction mixture was stirred at 90 ºC under
2
3
nitrogen atmosphere for 12 h. The reaction mixture was diluted with water and extracted with
EtOAc (3 x 20 mL). The combined organic layer was dried over Na SO and concentrated under
2
4
reduced pressure, crude material was purified by flash chromatography (10% EtOAc/hexanes) to
1
obtain the compound 29j as a red oil (72 mg, 61%). R = 0.70 (10% EtOAc/hexanes). H NMR (500
f
MHz, CDCl ) δ 7.23 (d, J = 8.0 Hz, 1H), 6.67 – 6.61 (m, 1H), 6.57 – 6.52 (m, 1H), 6.15 (bs, 1H),
3
3
1
.17 (td, J = 7.1, 5.1 Hz, 2H), 2.76 (q, J = 7.5 Hz, 2H), 1.74 – 1.60 (m, 2H), 1.52 – 1.34 (m, 4H),
13
.24 (t, J = 7.5 Hz, 3H), 0.92 (t, J = 7.1 Hz, 3H). C NMR (126 MHz, CDCl ) δ 143.50, 141.04,
3
136.04, 133.22, 117.65, 111.07, 43.60, 29.36, 28.90, 27.09, 22.56, 15.25, 14.13. MS (ESIꢀTOF) for
+
C H N O [M + H] calculated 237.1598, found 237.1581.
13
20
2
2
2
8
ACS Paragon Plus Environment

Page 29 of 66
Journal of Medicinal Chemistry
1
2
3
4
5
N,N-Dimethyl-2-nitro-N'-pentyl-benzene-1,3-diamine (29k). To a solution of compound 29g
242 mg, 1 mmol) in DMF (3 mL), was added dimethyl amine (2 mL, 4 mmol, 2.0 M in MeOH).
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The reaction mixture was stirred for 12 h at 75 ºC. The reaction mixture was cooled to room
temperature, diluted with water and extracted with EtOAc (3 x 30 mL). The combined organic layer
was dried over Na SO , concentrated under reduced pressure, and the crude material was purified
2
4
by silica gel column chromatography (10% EtOAc/hexanes) to obtain the compound 29k as a red
1
solid (200 mg, 80%). R = 0.3 (10% EtOAc/hexanes). H NMR (500 MHz, CDCl ) δ 7.19 – 7.11 (m,
f
3
1
2
1
H), 6.60 (bs, 1H), 6.22 – 6.11 (m, 2H), 3.16 (td, J = 7.1, 5.1 Hz, 2H), 2.85 (s, 6H), 1.72 – 1.62 (m,
1
3
H), 1.44 – 1.31 (m, 4H), 0.92 (t, J = 7.1 Hz, 3H). C NMR (126 MHz, CDCl ) δ 149.09, 145.16,
3
33.87, 126.83, 103.86, 101.81, 43.69, 42.37, 29.38, 28.94, 22.56, 14.14. MS (ESIꢀTOF) for
+
C H N O [M + H] calculated 252.1707, found 252.1734.
13 21
3
2
Compounds 31a-31k were synthesized similarly as compound 18.
4
-Methyl-1-pentyl-1H-benzo[d]imidazol-2-amine (31a). Compound 29a was used as reagent.
1
White solid (33 mg, 76%). R = 0.50 (10% MeOH/CH Cl ). H NMR (500 MHz, DMSO) δ 6.93
f
2
2
(dd, J = 7.3, 1.2 Hz, 1H), 6.82 – 6.61 (m, 2H), 6.34 (s, 2H), 3.92 (t, J = 7.2 Hz, 2H), 2.34 (s, 3H),
13
1
.70 – 1.46 (m, 2H), 1.40 – 1.10 (m, 4H), 0.83 (t, J = 7.1 Hz, 3H). C NMR (126 MHz, DMSO) δ
1
54.21, 141.56, 133.59, 123.77, 120.83, 117.83, 105.20, 41.44, 28.30, 28.21, 21.95, 16.41, 13.95.
+
MS (ESIꢀTOF) for C H N [M + H] calculated 218.1652, found 218.1657.
1
3
19
3
5-Methyl-1-pentyl-1H-benzo[d]imidazol-2-amine (31b). Compound 29b was used as reagent.
1
White solid (35 mg, 81%). R = 0.47 (10% MeOH/CH Cl ). H NMR (500 MHz, DMSO) δ 6.97 (d,
f
2
2
2
9
ACS Paragon Plus Environment