Structure-activity relationships in Toll-like receptor 7 agonistic 1H-imidazo[4,5-c]pyridines

Article abstract of DOI:10.1039/c3ob40816g

Engagement of TLR7 in plasmacytoid dendritic cells leads to the induction of IFN-α/β which plays essential functions in the control of adaptive immunity. We had previously examined structure-activity relationships (SAR) in TLR7/8-agonistic imidazoquinolines with a focus on substituents at the N ¹, C², N³ and N⁴ positions, and we now report SAR on 1H-imidazo[4,5-c]pyridines. 1-Benzyl-2-butyl-1H-imidazo[4,5-c] pyridin-4-amine was found to be a pure TLR7-agonist with negligible activity on TLR8. Increase in potency was observed in N⁶-substituted analogues, especially in those compounds with electron-rich substituents. Direct aryl-aryl connections at C6 abrogated activity, but TLR7 agonism was reinstated in 6-benzyl and 6-phenethyl analogues. Consistent with the pure TLR7-agonistic behavior, prominent IFN-α induction in human PBMCs was observed with minimal proinflammatory cytokine induction. A benzologue of imidazoquinoline was also synthesized which showed substantial improvements in potency over the parent imidazopyridine. Distinct differences in N⁶-substituted analogues were observed with respect to IFN-α induction in human PBMCs on the one hand, and CD69 upregulation in lymphocytic subsets, on the other.

Full text of DOI:10.1039/c3ob40816g

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PAPER
Structure-Activity Relationships in Toll-like Receptor 7 Agonistic 1H-
Imidazo[4,5-c]pyridines
Euna Yoo, Breanna M. Crall, Rajalakshmi Balakrishna, Subbalakshmi S. Malladi, Lauren M. Fox, Alec
R. Hermanson and Sunil A. David*
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Engagement of TLR7 in plasmacytoid dendritic cells leads to the induction of IFNꢀα/β which plays
essential functions in the control of adaptive immunity. We had previously examined structureꢀactivity
relationships (SAR) in TLR7/8ꢀagonistic imidazoquinolines with a focus on substituents at the N1, C2, N3
10 and N4 positions, and we now report SAR on 1Hꢀimidazo[4,5ꢀc]pyridines. 1ꢀBenzylꢀ2ꢀbutylꢀ1Hꢀ
imidazo[4,5ꢀc]pyridinꢀ4ꢀamine was found to be a pure TLR7ꢀagonist with negligible activity on TLR8.
Increase in potency was observed in N6ꢀsubstituted analogues, especially in those compounds with
electronꢀrich substituents. Direct arylꢀaryl connections at C6 abrogated activity, but TLR7 agonism was
reinstated in 6ꢀbenzyl and 6ꢀphenethyl analogues. Consistent with the pure TLR7ꢀagonistic behavior,
15 prominent IFNꢀα induction in human PBMCs was observed with minimal proinflammatory cytokine
induction. A benzologue of imidazoquinoline was also synthesized which showed substantial
improvements in potency over the parent imidazopyridine. Distinct differences in N6ꢀsubstituted
analogues were observed with respect to IFNꢀα induction in human PBMCs on the one hand, and CD69
upregulation in lymphocytic subsets, on the other.
20
stimuli, while TLR3, ꢀ7, ꢀ8 and ꢀ9 function within the
Introduction
endolysosomal compartment.13 The activation of TLRs by their
cognate ligands leads to production of inflammatory cytokines,
50 and upꢀregulation of major histocompatibility complex (MHC)
molecules and coꢀstimulatory 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 (adaptive immune responses).16ꢀ19
Host responses to pathogens are mediated via highly
coordinated mechanisms involving both the innate and adaptive
limbs of the immune system. The innate immune system utilizes
25 germlineꢀencoded pattern recognition receptors (PRRs) to detect
pathogenꢀassociated molecular patterns (PAMPs) that are distinct
and unique to the pathogen.1ꢀ3 PRRs encompass a broad range of
molecules4 that are secreted into the extracellular environment
(such as the collectins,5 ficolins,6 pentraxins,7 alarmins8), exist in
30 the cytosol (examples of which include the retinoic acid–
inducible gene I–like receptors,9 and the nucleotideꢀbinding
domain and leucineꢀrich repeat–containing receptors10), or are
present on membranes.
Important among the transmembrane PRRs include the Tollꢀ
35 like receptors11 (TLRs) which are either expressed on the plasma
membrane or in the endolysosomal compartments.1,12 At least 10
functional TLRs are encoded in the human genome, each with an
extracellular domain having leucineꢀrich repeats (LRR) and a
cytosolic domain called the Toll/ILꢀ1 receptor (TIR) domain.13
40 The ligands for these receptors are highly conserved microbial
molecules such as lipopolysaccharides (LPS) (recognized by
TLR4), lipopeptides (TLR2 in combination with TLR1 or TLR6),
flagellin (TLR5), single stranded RNA (TLR7 and TLR8), double
stranded RNA (TLR3), CpG motifꢀcontaining DNA (recognized
45 by TLR9), and profilin present on uropathogenic bacteria
(TLR11).14,15 TLR1, ꢀ2, ꢀ4, ꢀ5, and ꢀ6 recognize extracellular
55
The Type I interferon (IFN) family in humans include
approximately 20 IFNꢀα subtype genes in addition to individual
genes encoding IFNꢀβ, ꢀκ, ꢀε and ꢀω; these monomeric secreted
proteins bind to a single IFNꢀα/β receptor, which is constitutively
expressed in virtually all cell types.20 Occupancy of TLR721ꢀ23 or
60 TLR924,25 in professional antigenꢀpresenting cells (APCs),
particularly plasmacytoid dendritic cells (pDCs), leads to the
induction of IFNꢀα/β. Although the Type I IFNs are best known
historically for their antiviral activities,26 recent studies show that
they have many essential functions in the control of adaptive
65 immunity.2 First, Type I IFNs promote crossꢀpriming through
direct stimulation of DCs, leading to specific CD8+ lymphocytic
responses to soluble antigens.27 Second, Type I IFNs potently
enhance the primary antibody responses to soluble antigens,
inducing sustained and durable humoral responses with
70 appropriate isotype switching, as well as the induction of
immunological memory.28 B lymphocytes can differentiate into
two distinct types of functionally polarized effectors: Bꢀeffectorꢀ
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NH2
NH2
1ꢀcells (Beꢀ1), producing a Thꢀ1ꢀlike cytokine pattern, or Beꢀ2,
H
N
N
N
characterized by a Thꢀ2ꢀlike profile.29,30 It is of particular interest
that recent reports suggest that IFNꢀα may serve as an initial
trigger for Beꢀ1ꢀbiased differentiation pattern.31 Third, Type I
interferons secondarily induce Type II IFN (IFNꢀγ) secretion, also
driving Thꢀ1ꢀbiased adaptive immune responses.32 Type I IFNꢀ
inducing TLR ligands may therefore hold promise as vaccine
adjuvants.
N
N
O
OH
O
O
N
O
N
N
H
N
H
OH
N
O
5
SM360320
CL264
NH2
NH2
NH2
N
N
N
O
N
N
N
N
N
In an effort to identify optimal immunostimulatory
N
OH
10 chemotypes, we have screened representative members of
virtually the entire compendium of known TLR agonists in a
series of hierarchical assays including primary TLRꢀreporter
assays, secondary indices of immune activation such as IFNꢀ
α/β/γ and cytokine induction, activation of lymphocytic subsets
15 in whole human blood, and tertiary screens characterizing
transcriptomal activation patterns.33 In these assays, smallꢀ
molecule agonists of TLR7 were uniquely immunostimulatory;
they were potent inducers of Type I IFN and, unlike TLRꢀ4, ꢀ5,
or ꢀ8 agonists,33 did not evoke dominant proinflammatory
20 cytokine responses, suggesting that they may be effective, yet
safe vaccine adjuvants, a premise that we have been actively
exploring.34 Small molecule TLR7 agonists are also being
investigated as orally bioavailable, endogenous Type I IFN
inducers for the management of chronic viral diseases,35
25 especially hepatitis C and hepatitis B. Current therapeutic
regimens for the therapy of hepatitis C and hepatitis B include
parenteral IFNꢀα.36 Clinical trials of TLR7 agonists for
hematological malignancies are also currently underway.37
The currently known small molecule agonists of TLR7 occupy
30 a very small chemical space, and are represented by the 1Hꢀ
imidazo[4,5ꢀc]quinolines,38 8ꢀhydroxyꢀ39ꢀ41 or 8ꢀoxoadenines,42
and guanine nucleoside analogues.43,44 We had previously
reported structureꢀactivity relationships (SAR) in the
imidazoquinolines with a focus on substituents at the N1, C2, N3
35 and N4 positions,45,46 and we had observed that relatively minor
structural modifications at these positions yielded compounds
with widely differing immunomodulatory properties.34,47ꢀ49 It was
of interest, therefore, to extend our SAR studies to the quinoline
ring system. We asked if a partꢀstructure (imidazopyridine) or a
40 benzologue (benzoimidazoquinoline) would alter the biological
properties of the parent imidazoquinoline compound.
Examination of the structures of 3Mꢀ00350 and the 8ꢀhydroxyꢀ
and 8ꢀoxoadenines (Fig. 1) suggested that the quinoline system
may be dispensable, and activity would be retained in
45 imidazopyridines. Indeed, imidazopyridine derivatives with alkyl
groups at C6 and C7 positions,51 hydroxyalkyl,52 oxime and
hydroxylamineꢀbearing substituents53 at C2, and alkylsulfonamide
substituents at the N1 position54 have been reported in the patent
literature. Detailed activity profiles of these compounds,
50 however, are not available, perhaps owing to the fact that the
investigations of such compounds precede the discovery of the
TLRs.
Imiquimod
IMDQ
3M-003
60
Fig. 1
Structures of small molecule agonists of TLR7 represented by
the 8ꢀoxoadenine (SM360320, Ref. 42), 8ꢀhydroxyadenines (CL264, Ref.
39), 1Hꢀimidazo[4,5ꢀc]quinolines 3Mꢀ003 (Ref. 49), 1ꢀisobutylꢀ1Hꢀ
imidazo[4,5ꢀc]quinolinꢀ4ꢀamine (Imiquimod) and 1ꢀbenzylꢀ2ꢀbutylꢀ1Hꢀ
65 imidazo[4,5ꢀc]quinolinꢀ4ꢀamine (IMDQ, Ref. 45).
pyridinꢀ4ꢀamine, exhibited moderate TLR7ꢀagonistic activity.
N4ꢀacyl or ꢀalkyl substitutions abrogated activity. The majority of
C6 derivatives bearing aryl groups were also inactive, but
70 analogues with N6ꢀbenzyl substituents gained TLR7ꢀspecific
activity. Particular N6 substituents were found to augment TLR7ꢀ
specific agonistic potency without compromising specificity at
TLR7; consistent with their pure TLR7 activity (and undetectable
TLR8 agonism), these compounds potently induced IFNꢀα in
75 human peripheral blood mononuclear cells (PBMCs), upregulated
CD69 in lymphocytic subsets, and yet showed very weak
proinflammatory cytokineꢀinducing activities. Strong Type I IFN
inducers, especially in
conjunction with
proinflammatory profiles are expected to be potently adjuvantic
80 without inducing prominent local or systemic inflammation.
attenuated
Results and discussion
Our interest in exploring TLR7 agonists as vaccine adjuvants
has been greatly reinforced by our observations that pure TLR7
85 agonists, unlike other TLR ligands, are potently
immunostimulatory without prominently activating inflammatory
programs in human whole blood model systems.55 As mentioned
earlier, the structures of 3Mꢀ00350 and the 8ꢀhydroxyꢀ and 8ꢀ
oxoadenines (Fig. 1), as well as patent literature suggested that
90 the quinoline system may be dispensable, and activity would be
retained in imidazopyridines. Our previous SAR studies on the
imidazoquinolines had established that N1ꢀbenzyl and C2ꢀbutyl
substituents were optimal;46 our point of departure in examining
structureꢀactivity relationships in the imidazopyridines
95 consequently began with the evaluation of 1ꢀbenzylꢀ2ꢀbutylꢀ1Hꢀ
imidazo[4,5ꢀc]pyridinꢀ4ꢀamine (5), following the synthetic
strategy described earlier (Scheme 1).38,46 Compound 5, itself a
novel and unprecedented structure, was found to possess TLR7ꢀ
specific agonistic activity (EC50: 1.57 ꢀM, Fig. 2, Table 1), with
100 negligible TLR8 activity. The potency of the lead TLR7ꢀspecific
imidazoquinoline (1ꢀbenzylꢀ2ꢀbutylꢀ1Hꢀimidazo[4,5ꢀc]quinolinꢀ
4ꢀamine, structure in Fig. 1) was 0.06 ꢀM (Fig. 2).46
Incorporating substituents that we had previously determined
to be optimal in the imidazoquinolines (N1ꢀbenzyl and C2ꢀbutyl;
55 IMDQ, Fig. 1), we embarked on the syntheses and biological
evaluation of novel 1Hꢀimidazo[4,5ꢀc]pyridine analogues with
modifications at the N4ꢀ and
C6 positions. The parent
imidazopyridine compound, 1ꢀbenzylꢀ2ꢀbutylꢀ1Hꢀimidazo[4,5ꢀc]
2
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3
5
19b
19d
19f
19g
19k
19l
19m
19p
23a
IMDQ
Neg. Ctrl.
2
1
10-7
10-6
10-5
10-4
Compound Concentration (M)
40
O
N
Fig. 2
TLR7 agonistic activity of imidazopyridine compounds. Data
points represent means and standard deviations on quadruplicates.
NH2
R
NH
N
N
i
N
N
N
alkylated
intermediate
14.
The
6ꢀchloroꢀNꢀ(2,4,4ꢀ
trimethylpentanꢀ2ꢀyl)ꢀ1Hꢀimidazopyridinꢀ4ꢀamine intermediate
45 16 was obtained without difficulty, and we were able to
synthesize the N6ꢀsubstituted analogues 19aꢀo under conventional
BuchwaldꢀHartwig conditions (Scheme 4). TLRs signal via
ligandꢀinduced dimerization, but since that the crystal structure of
human TLR7 and of its ligand binding modes are as yet
50 unknown, we utilized intermediate 16 in constructing ‘dimeric’
imidazopyridines (using pꢀ and mꢀxylylenediamine, Scheme 5) to
ascertain if such preꢀorganized dimeric ligands could yield highꢀ
potency agonists. For the C6ꢀsubstituted compounds 23aꢀj
(Scheme 6), we observed mediocre yields in pilot Suzuki
55 coupling reactions with the advanced intermediate 16. We
therefore exploited the electronꢀwithdrawing resonance effect of
the 3ꢀnitro group in 14. As expected, the classical Suzuki reaction
on intermediate 14 using various aliphatic and aromatic boronic
acids/boronate esters resulted in the intermediates 20aꢀj (Scheme
60 6), which were further derivatized to obtain the desired C6
alkyl/aryl substituted imidazopyridines 23aꢀj.
5
6a
; R = CH3
6b; R = C3H7
Scheme 2. Reagents: (i) RCOCl, NEt3, CH2Cl2.
5
Acylation (6a, 6b, Scheme 2) of the C4ꢀNH2 resulted in
complete abrogation of activity (Table 1). C6ꢀmodified analogues
were synthesized via an alternate route. Nitration of 4ꢀaminoꢀ2ꢀ
chloropyridine resulted, as expected, in a mixture of the 3ꢀand 5ꢀ
nitro intermediates 7a and 7b, which were taken forward to
10 obtain the 4ꢀ and 6ꢀchloroimidazopyridines 10a and 10b (Scheme
3). Excellent chromatographic separation of these advanced
intermediates was possible. Pdꢀcatalyzed CꢀN crossꢀcoupling
reactions using nꢀbutylamine and benzylamine furnished the C4ꢀ
Nꢀalkylated analogues 11a and 11b, respectively (Scheme 3). A
15 4ꢀbutoxy analogue 11c was also obtained by ipsoꢀchloro
displacement with 1ꢀbutanol. Compounds 11a-c were, however,
inactive (Table 1). We had envisaged utilizing the 6ꢀchloro
imidazopyridine intermediate 10b for synthesizing C6ꢀ
functionalized analogues. However, this intermediate exhibited
20 unexpectedly low reactivity to displacement with nucleophiles or
to BuchwaldꢀHartwig coupling reactions. As outlined in Scheme
4, we utilized 4ꢀaminoꢀ2,6ꢀdichloropyridine as the starting
material and obtained 13 as a key intermediate which, upon
reaction with tertꢀoctylamine56 provided exclusively the N2ꢀ
25
The 6ꢀchloro imidazopyridine, 17 (Scheme 4) was inactive
(Table 1). BuchwaldꢀHartwigꢀderived N6ꢀsubstituted analogues
19aꢀq, however, showed a distinctive SAR. Compound 19a with
65 a free NH2 at C6, obtained by coupling the tertꢀoctylamine and
subsequent Nꢀdealkylation with HCl (Scheme 4, Table 1)
displayed TLR7ꢀspecific agonism with a potency comparable to
that of the parent C6ꢀunsubstituted compound 5.
70
75
80
30
35
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Table 1. EC50 values of compounds in human TLR7ꢀspecific
reporter gene assay
Table 1. (contd.)
hTLR7ꢀagonistic
Activity (ꢀM)
Compound
Number
hTLR7ꢀagonistic
Activity (ꢀM)
Compound
Number
R1
H
R2
R1
R2
H
5
H
1.57
Inactivea
Inactive
Inactive
Inactive
Inactive
1.25
19n
Inactive
1.09
6a
6b
COCH3
H
19o
H
COC3H7
H
H
11a
11b
17
C4H9
19p
19q
H
H
0.26
0.37
CH2C6H5
H
H
H
H
H
H
H
H
Cl
19r
19s
H
H
1.08
19a
19b
19c
19d
19e
19f
NH2
NHC4H9
NHC7H15
0.34
Inactive
0.76
23a
23b
H
H
C4H9
C6H5
0.28
0.32
Inactive
NHC6H5
1.72
23c
23d
H
H
Inactive
Inactive
NHCH2C6H5
0.21
19g
H
0.075
23e
23f
H
H
H
H
Inactive
Inactive
0.28
19h
19i
H
H
0.61
1.04
23g
23h
19j
19k
19l
H
H
H
H
0.64
0.075
0.25
1.80
23i
23j
30
H
H
Inactive
0.57
19m
0.25
Benzologue (Scheme 7)
0.22
aInactive: no activity was detected up to a concentration of 500 ꢁg/mL.
5
4
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Cl
Cl
NH
NH
Cl
NO2
NO2
NH2
N
NH2
NH
NO2
NH
i
iii
ii
iv
N
N
N
N
Cl
NH
NH
Cl
Cl
Cl
Cl
NH2
NH
30
35
40
45
50
12
13
14
15
NH2
N
N
N
vii
vi
N
N
N
v
N
N
RHN
N
Cl
RHN
18a o
-
19a-o
16
R =
vii
e:
j:
a:
b:
c:
d:
i:
NH2
f:
g:
h:
H3CO
N
N
N
Cl
H3CO
F3C
n:
Cl
o:
k:
l:
m:
N
O
N
17
Scheme 4. Reagents: (i) a. H2SO4, HNO3; b. H2SO4; (ii) BnBr, NaH, THF; (iii) t-Octylamine, NEt3, CH2Cl2; (iv) Zn, HCOONH4, MeOH; (v) a. C4H9COCl,
NEt3, THF; b. NaOH, EtOH; (vi) RNH2, Pd2(dba)3, DavePhos, KOtBu, dioxane; (vii) HCl.
3.0
Modest gains in potency were obtained in analogues with short
5
30
Neg Ctrl
aliphatic substituents with N6ꢀbutyl (19b) and N6ꢀ
cyclohexylmethyl (19d), but potency diminished in the N6ꢀheptyl
analogue (19c). The N6ꢀphenylꢀsubstituted compound 19e was
marginally weaker than 5; however, the potency of the N6ꢀbenzyl
analogue 19f was ~7.6 times that of 5 (Table 1, Fig. 2), triggering
a detailed SAR investigation on various aryl substituents at N6.
10 Both steric and electronic effects appear to play a role in
governing TLR7ꢀagonistic potency, since the biphenylmethylꢀ
substituted compound 19o was active, whereas the
2.5
2.0
1.5
1.0
0.5
5
naphthylmethyl analogue 19n was quiescent; to
a first
approximation, electronꢀrich N6 substituents appear to be
15 preferred, with the methoxybenzyl derivatives (19g and 19h) and
the pyridinylmethyl compounds (19l and 19m) being marginally
more active than the trifluoromethylꢀ (19i) or chloroꢀ (19j)
substituted analogues. Compounds 19p and 19q were also active
in primary screens, with EC50 values of 0.26 and 0.37 ꢀM,
20 respectively (Table 1). In the C6ꢀalkyl or ꢀaryl analogues (Scheme
6), the SAR appeared more stringent. Whereas the C6ꢀbutyl
compound 23a was more active than 5, direct arylꢀaryl
connections at C6 (23bꢀf) abrogated activity, but TLR7 agonistic
properties were restored in the 6ꢀbenzyl (23g) and 6ꢀphenethyl
25 analogues (23j). Taken together with activity data of compounds
of the 19 series, we surmised that rotational constraints about the
10-7
10-6
10-5
10-4
Compound Concentration (M)
Fig. 3
compounds
Doseꢀresponse profiles of TLR7 agonistic activity of
and 30. Data points represent means and standard
55 deviations on quadruplicates.
5
C6ꢀaryl groups may hinder TLR7 occupancy. Unlike TLR2,
TLR3, TLR4,57 and TLR558 for which crystal structures are
available as complexes with their cognate ligands, a detailed
structural characterization of the mode of binding of TLR7
60 ligands is not yet available to guide structureꢀbased design of
small molecule agonists of TLR7, necessitating classical SAR
approaches to refine successive iterations of ligand design.
NH2
NH2
NH
NH2
N
N
N
N
N
N
N
N
i, ii
N
+
N
H
+
H
N
H
Cl
N
N
N
R
p; R = p-CH2NH2
R
N
16
19p; R = p-CH2NH2
19q
19r; p-dimer
NH2
q
19s
; R = m-CH2NH2
; R = m-CH2NH2
; m-dimer
Scheme 5. Reagents: (i) Pd2(dba)3, DavePhos, KOtBu, dioxane; (ii) HCl.
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800
IF N -
α
700
600
500
400
300
200
100
0
19b
19d
19f
19g
19k
19l
19m
19p
23a
C L075
5000
4000
3000
2000
1000
IL-8
0
5 00 0
IL -1
β
4 00 0
3 00 0
2 00 0
1 00 0
0
2500
TN F-
α
2000
1500
1000
500
0
10-7
10-6
10-5
Compound Concentration (M)
Fig. 4
Doseꢀresponse profiles of Type I interferon (IFNꢀα) and
proinflammatory cytokine (ILꢀ8, ILꢀ1β, and TNFꢀα) induction by
selected imidazopyridine (and reference) compounds. Representative data
20 from three independent experiments are presented.
5
The benzologue 30 was synthesized as shown in Scheme 7. It
showed substantial improvements in potency over the parent
imidazopyridine 5 (Fig. 3, Table 1), but the two most potent
compounds in the entire series as adjudged by primary screens
well as CL07561,62 (2ꢀpropylthiazolo[4,5ꢀc]quinolinꢀ4ꢀamine), a
predominantly TLR8ꢀactive agonist with an EC50 of 1.32 ꢀM in
hTLR8 assays.63 Given that the imidazopyridine compounds are
25 pure TLR7 agonists, we expected to find prominent IFNꢀα
induction,33 and this was indeed the case, with 19p, 19m and 19k
being the most potent (EC50: 0.3 ꢀM, 0.4 ꢀM and 0.7 ꢀM,
respectively; Fig. 4). CL075 was among the least potent in IFNꢀα
induction (EC50: 2.6 ꢀM; Fig. 4), and as expected for a TLR8
30 agonist, CL075 was dramatically more active in inducing
10 were the N6ꢀ(4ꢀmethoxybenzyl) and N6ꢀ(furanꢀ2ꢀylmethyl)
analogues (19g and 19k, respectively), both of which were
approximately twentyꢀfold more potent than 5 (Fig. 2, Table 1).
We chose the nine most active compounds (19b, 19d, 19fꢀg,
19kꢀm, 19p and 23a) for evaluation in secondary screens using
15 IFNꢀα and cytokine release in human PBMCs. We used, as
reference compounds, imiquimod, a known TLR7 agonist,59,60 as
6
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NK cells (CD3- CD56+)
We had previously shown that of all the various classes of
innate immune stimuli, TLR7 agonists were extraordinarily
20 immunostimulatory, stimulating virtually all subsets of
lymphocytes (assessed by quantifying CD69 expression), and yet
without inducing dominant proinflammatory cytokine
responses,33 and we wished to confirm the rankꢀorder potency
observed in IFNꢀα induction assays described above. We
25 observed considerable dissociation between Type I IFN induction
on the one hand (Fig. 4), and CD69 upregulation in lymphocytic
subsets on the other (Fig. 5). Whereas the subset of active
compounds induced IFNꢀα with similar potencies (EC50 values
between 0.3ꢀ2 ꢀM; Fig. 4), pronounced differences were
30 observed in CD69 expression in natural killer, cytokineꢀinduced
killer and B lymphocytic subsets with 19p being as active as the
reference TLR7 agonist IMDQ, and 19d showing virtually no
activity (Fig. 5). Possible mechanisms underlying the differential
activity in these two compounds are being investigated.
6000
4000
2000
0
19b
19d
19f
19g
19k
19l
19m
19p
23a
Controls:
IMDQ
Imiquimod
Neg. Ctrl.
10-6
CIK cells (CD3+ CD56+)
10-5
10-4
10-5
10-4
6000
5000
4000
3000
35
Conclusions
These findings raise the possibility of utilizing these
compounds in selectively targeting Type I IFN induction versus
lymphocytic activation, and are being explored in greater detail.
40 The potential advantages of strong Type I IFN inducers as
candidate vaccine adjuvants have been discussed earlier. Such
compounds, especially in conjunction with attenuated
proinflammatory cytokines, are expected to be potently
adjuvantic without inducing prominent local or systemic
45 inflammation. As mentioned earlier, the prominent Type I IFN
inducing abilities of the imidazopyridines may also find utility as
an alternative therapeutic strategy to address disease states
wherein systemic IFNꢀα is of proven benefit. A clear delineation
of structural features that confer TLR specificity not only charts a
50 rational course for the development of effective, yet safe vaccine
adjuvants, but also provides tools to understand innate immune
function in greater detail.
10-6
B cells (CD3- CD56-)
10000
8000
6000
4000
2000
0
10-6
10-5
Experimental
Compound Concentration (M)
CD69 upregulation in human natural killer (NK), cytokineꢀ
55 Materials and equipment. 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. The solvents were removed under reduced pressure
60 using standard rotary evaporators. Flash column chromatography
was carried out using RediSep Rf ‘Gold’ high performance silica
columns on CombiFlash Rf instrument unless otherwise
mentioned, while thinꢀlayer chromatography was carried out on
silica gel (200 ꢂm) CCM preꢀcoated aluminum sheets. Purity for
65 all final compounds was confirmed to be greater than 97% by
LCꢀMS using a Zorbax Eclipse Plus 4.6 mm x 150 mm, 5 ꢂm
analytical reverse phase C18 column with H2Oꢀisopropanol or
H2OꢀCH3CN gradients and an Agilent 6520 ESIꢀQTOF Accurate
Mass spectrometer (mass accuracy of 5 ppm) operating in the
70 positive ion (or negative ion, as appropriate) acquisition mode.
Fig. 5
induced killer (CIK) and nominal lymphoyctes by select
B
imidazopyridine (and reference) compounds.
5
proinflammatory cytokines such as TNFꢀα, ILꢀ1β, and ILꢀ8 (Fig.
4). We do not yet understand the basis for the slight discrepancy
between rankꢀorder potency in primary screens (19k ≈ 19g > 19f
> 19p; Fig. 2, Table 1) visꢀàꢀvis IFNꢀαꢀinducing potency in
human PBMCs (19p ≈ 19m > 19k; Fig. 4), and we surmise that
10 analogues with more basic C6 substituents may allow for higher
endolysosomal partitioning. The doseꢀresponse profiles show
characteristic biphasic responses (doseꢀdependent activation,
followed by apparent suppression) as we had previously observed
in several chemotypes. We verified that the apparent suppression
15 was not due to cytotoxicity using LDH release and mitochondrial
redoxꢀbased assays.
Synthesis of compound 1: N-Benzyl-3-nitropyridin-4-amine.
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To a solution of 4ꢀchloroꢀ3ꢀnitropyridine (1.0 g, 6.31 mmol) in 25
Synthesis of compound 4: 1-Benzyl-2-butyl-1H-imidazo[4,5-
mL of CH2Cl2 were added triethylamine (1.32 mL, 9.47 mmol) 60 c]pyridine 5-oxide. To a solution of compound 3 (210 mg, 0.79
and benzyl amine (0.83 mL, 7.57 mmol). The reaction mixture
was refluxed for 18 h. The solvent was then evaporated under
vacuum and H2O was added to the residue. The solution was
extracted with CH2Cl2 (3 × 20 mL), washed with water and dried
mmol) in 15 mL of was added mꢀchloroperoxybenzoic acid (443
mg, 1.98 mmol), and the solution was refluxed at 45ꢀ50 ºC for 1
h. The solvent was then removed and the residue was purified
using column chromatography (10% MeOH/CH2Cl2) to obtain
5
over sodium sulfate. The solvent was evaporated and the residue 65 the Nꢀoxide derivative (188 mg, 85%). 1H NMR (500 MHz,
was purified using silica gel column chromatography (5%
CDCl3) δ 8.71 (d, J = 1.3 Hz, 1H), 8.05 (dd, J = 7.0, 1.6 Hz, 1H),
7.36 – 7.30 (m, 3H), 7.01 (dd, J = 10.4, 4.3 Hz, 3H), 5.31 (s, 2H),
2.85 – 2.80 (m, 2H), 1.79 (dt, J = 15.4, 7.6 Hz, 2H), 1.44 – 1.34
(m, 2H), 0.90 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
MeOH/CH2Cl2) to obtain compound 1 as a yellow solid (1.4 g,
10 94%). 1H NMR (500 MHz, CDCl3) δ 9.22 (s, 1H), 8.53 (s, 1H),
8.25 (dd, J = 6.1, 0.6 Hz, 1H), 7.41 – 7.35 (m, 2H), 7.32 (dd, J =
7.2, 5.3 Hz, 3H), 6.69 (d, J = 6.2 Hz, 1H), 4.56 (d, J = 5.7 Hz, 70 160.72, 140.89, 134.42, 134.32, 133.94, 131.52, 129.48, 128.75,
2H). 13C NMR (126 MHz, CDCl3) δ 153.38, 149.06, 148.68,
135.96, 130.04, 129.21, 128.22, 127.10, 108.25, 46.85. MS (ESI)
15 calculated for C12H12N3O2, m/z 230.0924, found 230.0949 (M +
H)+.
126.24, 106.63, 47.75, 29.25, 27.49, 22.52, 13.78. MS (ESI)
calculated for C17H20N3O, m/z 282.1601, found 282.1612 (M +
H)+.
75 Synthesis of compound 5: 1-Benzyl-2-butyl-1H-imidazo[4,5-
c]pyridin-4-amine. To a solution of compound 4 (188 mg, 0.67
mol) in 15 mL of CH2Cl2 was added benzoyl isocyanate (197 mg,
1.34 mmol) and heated at 45 ºC for 2 h. The solvent was then
removed under vacuum, and the residue was dissolved in 15 mL
Synthesis of compound 2: N4-Benzylpyridine-3,4-diamine. To
a solution of compound 1 (1.0 g, 4.36 mmol) in 40 mL of MeOH
20 were added zinc dust (1.4 g, 21.8 mmol) and ammonium formate
(1.4 g, 21.8 mmol). The reaction mixture was stirred at room
temperature for 10 min and filtered through celite. Then the 80 of anhydrous MeOH, followed by the addition of excess sodium
solvent was evaporated and the residue was dissolved in water.
This was extracted with EtOAc (3 × 20 mL), washed with water
25 and dried over sodium sulfate. The solvent was evaporated under
methoxide. The reaction mixture was then heated at 80 ºC for 1 h.
The solvent was removed under vacuum and the residue was
purified using column chromatography (7% MeOH/CH2Cl2) to
obtain the compound 5 (56 mg, 30%). 1H NMR (500 MHz,
1
vacuum to obtain the compound 2 (0.8 g, 92%). H NMR (500
MHz, CDCl3) δ 7.81 (d, J = 5.4 Hz, 1H), 7.77 (s, 1H), 7.29 – 7.19 85 CDCl3) δ 7.78 (d, J = 5.8 Hz, 1H), 7.34 – 7.28 (m, 3H), 7.03 (d, J
(m, 5H), 6.36 (d, J = 5.4 Hz, 1H), 4.87 (s, 1H), 4.28 (d, J = 5.4
Hz, 2H), 3.28 (s, 2H). 13C NMR (126 MHz, CDCl3) δ 144.52,
30 143.51, 138.22, 137.53, 128.96, 128.79, 127.55, 127.48, 105.29,
47.22. MS (ESI) calculated for C12H14N3, m/z 200.1182, found
200.1242 (M + H)+.
= 6.4 Hz, 2H), 6.59 (d, J = 5.8 Hz, 1H), 5.27 (s, 2H), 5.15 (s, 2H),
2.83 – 2.75 (m, 2H), 1.72 (ddd, J = 13.0, 9.0, 7.7 Hz, 2H), 1.44 –
1.34 (m, 2H), 0.90 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz,
CDCl3) δ 154.06, 151.00, 140.61, 140.41, 135.76, 129.21,
90 128.23, 126.33, 97.74, 47.52, 30.10, 27.42, 22.68, 13.89. HRMS
(ESI) calculated for C17H21N4, m/z 281.1761, found 281.1795 (M
+ H)+.
Synthesis of compound 3: 1-Benzyl-2-butyl-1H-imidazo[4,5-
35 c]pyridine. To a solution of compound 2 (400 mg, 2.00 mmol) in
20 mL of anhydrous THF were added triethylamine (0.29 mL,
Synthesis of compound 6a: N-(1-Benzyl-2-butyl-1H-
2.10 mmol) and valeryl chloride (0.27 mL, 2.20 mmol). The 95 imidazo[4,5-c]pyridin-4-yl)acetamide. To
a
solution of
reaction mixture was refluxed for 2 h. The solvent was then
removed under vacuum, and the residue was dissolved in EtOAc
40 and washed with water. The EtOAc fraction was dried using
sodium sulfate and evaporated under vacuum to obtain the
compound 5 (30 mg, 0.11 mmol) in 2 mL of CH2Cl2 were added
triethylamine (17 ꢀL, 0.12 mmol) and acetyl chloride (8 ꢀL, 0.11
mmol). The reaction mixture was stirred at room temperature for
3
h
and purified using column chromatography (5%
intermediate amide compound. This was dissolved in 20 mL of 100 MeOH/CH2Cl2) to obtain the compound 6a as white solid (6 mg,
EtOH and NaOH (160 mg, 4.00 mmol) in 2 mL of H2O was
added. The reaction mixture was refluxed for 4 h. The solvent
45 was then removed under vacuum, and the residue was dissolved
in EtOAc and washed with water. The organic layer was dried
16%). 1H NMR (500 MHz, CDCl3) δ 11.60 (s, 1H), 8.20 (s, 1H),
7.37 (dd, J = 7.7, 5.5 Hz, 3H), 7.20 (d, J = 5.6 Hz, 1H), 7.05 (dd,
J = 6.3, 2.5 Hz, 2H), 5.46 (s, 2H), 3.03 – 2.94 (m, 2H), 2.55 (s,
3H), 1.85 (dt, J = 15.0, 7.6 Hz, 2H), 1.43 (dq, J = 14.6, 7.3 Hz,
using sodium sulfate and evaporated and purified using column 105 2H), 0.92 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
chromatography (5% MeOH/CH2Cl2) to obtain the compound 3
(210 mg, 40%). 1H NMR (500 MHz, CDCl3) δ 9.04 (d, J = 0.6
50 Hz, 1H), 8.34 (d, J = 5.6 Hz, 1H), 7.34 – 7.28 (m, 3H), 7.14 (dd,
J = 5.6, 1.0 Hz, 1H), 7.01 (dd, J = 7.7, 1.8 Hz, 2H), 5.33 (s, 2H),
141.48, 129.64, 129.40, 129.17, 126.30, 126.25, 48.47, 29.06,
24.87, 22.44, 13.60. HRMS (ESI) calculated for C19H23N4O, m/z
323.1866, found 323.1911 (M + H)+.
2.86 – 2.81 (m, 2H), 1.82 (dt, J = 15.5, 7.7 Hz, 2H), 1.46 – 1.36 110 Compounds 6b was synthesized similarly as compound 6a.
(m, 2H), 0.91 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
157.30, 142.14, 142.00, 140.48, 140.01, 135.26, 129.28, 128.37,
55 126.27, 105.12, 47.25, 29.44, 27.38, 22.61, 13.85. MS (ESI)
calculated for C17H20N3, m/z 266.1652, found 266.1715 (M +
H)+.
N-(1-Benzyl-2-butyl-1H-imidazo[4,5-c]pyridin-4-yl)
butyramide (6b). Butyryl chloride was used as a reagent. (4 mg,
1
14 %). H NMR (500 MHz, CDCl3) δ 11.31 (s, 1H), 8.30 (d, J =
115 4.8 Hz, 1H), 7.38 – 7.34 (m, 3H), 7.27 (s, 1H), 7.04 (dd, J = 6.4,
2.6 Hz, 2H), 5.50 (s, 2H), 2.94 (t, J = 7.5 Hz, 2H), 2.78 (t, J = 7.4
8
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Hz, 2H), 1.80 (dp, J = 22.1, 7.5 Hz, 4H), 1.41 (dt, J = 14.7, 7.4
1H), 7.01 (dd, J = 7.4, 2.0 Hz, 2H), 5.34 (s, 2H), 2.91 – 2.86 (m,
Hz, 2H), 1.01 (t, J = 7.4 Hz, 3H), 0.91 (t, J = 7.4 Hz, 3H). 13C 60 2H), 1.78 (dt, J = 15.7, 7.7 Hz, 2H), 1.45 – 1.36 (m, 2H), 0.90 (t,
NMR (126 MHz, CDCl3) δ 174.32, 141.35, 133.50, 129.69,
129.16, 126.46, 103.38, 48.58, 39.27, 29.16, 27.45, 22.55, 18.38,
13.75. HRMS (ESI) calculated for C21H27N4O, m/z 351.2179,
found 351.2240 (M + H)+.
J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 158.03, 141.88,
141.75, 141.12, 137.03, 134.84, 129.39, 128.58, 126.25, 105.18,
47.90, 30.04, 27.63, 22.73, 13.82. MS (ESI) calculated for
C17H19ClN3, m/z 300.1262, found 300.1159 (M + H)+.
5
65
Synthesis of compound 10a: 1-Benzyl-2-butyl-4-chloro-1H-
imidazo[4,5-c]pyridine. 4ꢀAminoꢀ2ꢀchloropyridine (2.0 g, 15.6
10 mmol) was taken in 20 mL of conc. H2SO4 in an iceꢀbath to
which was added 10 mL of conc. HNO3 slowly. The reaction
Synthesis of compound 11a: 1-Benzyl-N,2-dibutyl-1H-
imidazo[4,5-c]pyridin-4-amine. To a solution of compound 10
(50 mg, 0.17 mmol) in 1 mL of dioxane were added potassium
tertꢀbutoxide (57 mg, 0.51 mmol), catalytic amount of 2ꢀ
mixture was gradually brought to room temperature and stirred 70 dicyclohexylphosphinoꢀ2′ꢀ(N,Nꢀdimethylamino)biphenyl
for 1 h. The reaction was quenched by pouring the reaction
mixture on ice. Ammonium hydroxide solution was slowly added
15 until a pH of 3.0 was reached. A white solid was obtained which
was filtered, washed with water, and dried. This (Nꢀ
(DavePhos)
and
tris(dibenzylideneacetone)dipalladium(0)
(Pd2(dba)3) and butyl amine (83 ꢀL, 0.83 mmol). The reaction
mixture was then heated under microwave conditions (500 W,
100 ºC) in a sealed vial for 1 h. It was cooled to room temperature
nitro)aminopyridine intermediate was taken up in 10 mL of conc. 75 and filtered through celite and washed with MeOH. The solvent
H2SO4 and the reaction solution was heated at 90 ºC for 30 min. It
was cooled to room temperature and poured into ice. It was
20 slowly neutralized with ammonium hydroxide solution until a pH
of 7 and the formed yellow solid was filtered, washed with water
was removed and the crude residue was purified using column
chromatography (5% MeOH/CH2Cl2) to obtain the compound
11a (21 mg, 36%). 1H NMR (500 MHz, CDCl3) δ 7.84 (d, J = 5.9
Hz, 1H), 7.33 – 7.27 (m, 3H), 7.03 (dd, J = 4.5, 3.6 Hz, 2H), 6.48
and dried to obtain compound 7 as a mixture of 2ꢀchloroꢀ3ꢀ 80 (d, J = 5.9 Hz, 1H), 5.41 (s, 1H), 5.25 (s, 2H), 3.60 (dt, J = 12.9,
nitropyridinꢀ4ꢀamine
and
2ꢀchloroꢀ5ꢀnitropyridinꢀ4ꢀamine
6.5 Hz, 2H), 2.80 – 2.74 (m, 2H), 1.73 – 1.67 (m, 4H), 1.49 (dt, J
= 15.0, 7.4 Hz, 3H), 1.38 (dd, J = 15.0, 7.5 Hz, 2H), 0.96 (t, J =
7.4 Hz, 3H), 0.89 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz,
CDCl3) δ 153.15, 151.39, 135.95, 129.24, 129.15, 128.15,
intermediates. Sodium hydride (275 mg, 6.90 mmol) was
25 carefully added to 20 mL of THF under N2 and compound 7 (1.0
g, 5.76 mmol) was slowly added to the solution at 0 ºC. The
reaction mixture was stirred for 1 h, followed by the addition of 85 127.25, 126.34, 126.18, 96.16, 47.43, 41.09, 32.22, 30.27, 27.41,
benzyl bromide (0.75 mL, 6.34 mmol). The reaction mixture was
stirred at room temperature for 2 h and poured into ice water.
30 Then it was extracted with EtOAc (3 × 20 mL), washed with
water, dried over sodium sulfate. The solvent was removed and
the crude residue was purified using column chromatography
(20% EtOAc/hexane) to obtain the compound 8 as a mixture of
regioisomeric Nꢀbenzylꢀ2ꢀchloroꢀ3ꢀnitropyridinꢀ4ꢀamine and Nꢀ
35 benzylꢀ2ꢀchloroꢀ5ꢀnitropyridinꢀ4ꢀamine intermediates. To this
regioisomeric mixture (1.0 g, 4.2 mmol) in 20 mL of MeOH were
22.69, 20.47, 14.11, 13.89. HRMS (ESI) calculated for C21H29N4,
m/z 337.2387, found 337.2451 (M + H)+.
Compounds 11b was synthesized similarly as compound 11a.
N,1-Dibenzyl-2-butyl-1H-imidazo[4,5-c]pyridin-4-amine
90
1
(11b). Benzyl amine was used as a reagent. (33 mg, 52%). H
NMR (500 MHz, CDCl3) δ 7.86 (d, J = 5.9 Hz, 1H), 7.45 (dd, J =
7.9, 0.9 Hz, 2H), 7.35 – 7.27 (m, 6H), 7.03 (d, J = 6.4 Hz, 2H),
added zinc dust (1.4 g, 21.0 mmol) and ammonium formate (1.4 95 6.54 (d, J = 5.9 Hz, 1H), 5.76 (s, 1H), 5.26 (s, 2H), 4.83 (d, J =
g, 21.0 mmol). The reaction mixture was stirred at room
temperature for 10 min and filtered through celite. Then the
40 solvent was evaporated and the residue was dissolved in water.
This was extracted with EtOAc (3 × 20 mL), washed with water
5.6 Hz, 2H), 2.79 – 2.74 (m, 2H), 1.72 – 1.67 (m, 2H), 1.37 (dq, J
= 14.8, 7.4 Hz, 2H), 0.88 (t, J = 7.4 Hz, 3H). 13C NMR (126
MHz, CDCl3) δ 153.39, 150.92, 140.51, 139.81, 139.78, 135.88,
129.18, 128.62, 128.23, 128.19, 127.24, 126.36, 126.24, 96.73,
and dried over sodium sulfate. The filtrate evaporated under 100 47.46, 45.40, 30.14, 27.38, 22.67, 13.88. HRMS (ESI) calculated
vacuum, and chromatographed (20% EtOAc/hexane) to obtain
the required N4ꢀbenzylꢀ2ꢀchloropyridineꢀ3,4ꢀdiamine compound
45 9a. Also obtained was N4ꢀbenzylꢀ6ꢀchloropyridineꢀ3,4ꢀdiamine
as a sideꢀproduct. To a solution of compound 9a (495 mg, 2.12
for C24H27N4, m/z 371.2230, found 371.2303 (M + H)+.
Synthesis of compound 11c: 1-Benzyl-4-butoxy-2-butyl-1H-
imidazo[4,5-c]pyridine. To a suspension of sodium hydride (48
mmol) in 20 mL of anhydrous THF were added triethylamine 105 mg, 2.00 mmol) in 2 mL of anhydrous THF was added 1ꢀbutanol
(0.31 mL, 2.23 mmol) and valeryl chloride (0.28 mL, 2.33
mmol). The reaction mixture was refluxed for 1 h. The solvent
50 was then removed under vacuum, and the residue was dissolved
in 20 mL of EtOH and NaOH (170 mg, 4.24 mmol) in 2 mL of
(0.18 mL, 2.00 mmol). It was stirred at room temperature for 1 h,
followed by the addition of compound 10 (100 mg, 0.33 mmol).
The reaction mixture was heated at 60 ºC for 18 h and then
solvent was evaporated under vacuum. The residue was extracted
H2O was added. The reaction mixture was refluxed for 2 h. The 110 with EtOAc (3 × 10 mL), washed with water and dried over
solvent was then removed under vacuum, and the residue was
dissolved in EtOAc and washed with water. The EtOAc fraction
55 was dried using sodium sulfate and evaporated and purified using
sodium sulfate. The solvent was removed and the crude residue
was purified using column chromatography (5% MeOH/CH2Cl2)
to obtain the compound 11c (61 mg, 55%). H NMR (500 MHz,
1
column chromatography (5% MeOH/CH2Cl2) to obtain the
compound 10a (203 mg, 32%). H NMR (500 MHz, CDCl3) δ 115 J = 7.7, 1.7 Hz, 2H), 6.77 (d, J = 5.8 Hz, 1H), 5.30 (s, 2H), 4.52
CDCl3) δ 7.85 (d, J = 5.8 Hz, 1H), 7.33 – 7.28 (m, 3H), 7.01 (dd,
1
8.10 (d, J = 5.6 Hz, 1H), 7.35 – 7.30 (m, 3H), 7.08 (d, J = 5.6 Hz,
(t, J = 7.0 Hz, 2H), 2.87 – 2.82 (m, 2H), 1.90 (dd, J = 15.0, 7.1
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Hz, 2H), 1.75 (dt, J = 15.7, 7.7 Hz, 2H), 1.58 – 1.48 (m, 2H),
amine. To a solution of compound 14 (260 mg, 0.66 mmol) in 20
1.38 (dq, J = 14.7, 7.4 Hz, 2H), 0.97 (t, J = 7.4 Hz, 3H), 0.88 (t, J 60 mL of MeOH were added zinc dust (434 mg, 6.60 mmol) and
= 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 156.04, 155.18,
142.02, 138.95, 135.59, 129.21, 128.27, 127.41, 126.27, 100.47,
66.25, 47.61, 31.34, 30.21, 27.44, 22.75, 19.45, 14.09, 13.85.
HRMS (ESI) calculated for C21H28N3O, m/z 338.2227, found
338.2307 (M + H)+.
ammonium formate (416 mg, 6.60 mmol). The reaction mixture
was stirred at room temperature for 10 min and filtered through
celite. Then the solvent was evaporated and the residue was
dissolved in water. This was extracted with EtOAc (3 × 20 mL),
65 washed with water and dried over sodium sulfate. The solvent
was removed under vacuum to obtain compound 15, brown oil
(184 mg, 77%). To a solution of compound 15 (184 mg, 0.50
mmol) in 10 mL of anhydrous THF were added triethylamine (74
ꢀL, 0.52 mmol) and valeryl chloride (62 ꢀL, 0.50 mmol). The
5
Synthesis of compound 12: 2,6-Dichloro-3-nitropyridin-4-
10 amine. 4ꢀAminoꢀ2,6ꢀdichloropyridine (2.0 g, 12.27 mmol) was
added to 20 mL of conc. H2SO4. The mixture was cooled to 0 ºC
and 10 mL of conc. HNO3 was dropwise at 0 ºC. The reaction 70 reaction mixture was refluxed for 1 h. The solvent was then
mixture was stirred at room temperature for 1 h and then poured
into crushed ice. The white solid was filtered, washed with water
15 and dried. This intermediate was dissolved in 10 mL of conc.
H2SO4 and the reaction solution was heated at 90 ºC for 30 min. It
removed under vacuum, and the residue was dissolved in 10 mL
of EtOH and NaOH (40 mg, 1.00 mmol) in 1 mL of H2O was
added. The reaction mixture was refluxed for 5 h. The solvent
was then removed under vacuum, and the residue was dissolved
was cooled to room temperature and poured into ice. It was 75 in EtOAc and washed with water. The EtOAc fraction was dried
slowly neutralized with ammonium hydroxide solution until a pH
of 9 and the formed yellow solid was filtered, washed with water
using sodium sulfate and evaporated and purified using column
chromatography (20% EtOAc/hexane) to obtain the compound 16
1
1
20 and dried to obtain compound 12 as light yellow solid. H NMR
as yellow solid (120 mg, 56%). H NMR (500 MHz, CDCl3) δ
(500 MHz, MeOD) δ 6.84 (s, 1H). 13C NMR (126 MHz, MeOD)
δ 152.53, 151.18, 144.57, 111.12.
7.34 – 7.28 (m, 3H), 7.02 (d, J = 6.5 Hz, 2H), 6.42 (s, 1H), 5.38
80 (s, 1H), 5.16 (s, 2H), 2.76 – 2.69 (m, 2H), 2.02 (s, 2H), 1.71 –
1.65 (m, 4H), 1.60 (s, 6H), 1.37 (dd, J = 15.0, 7.5 Hz, 2H), 1.01
(s, 9H), 0.89 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
153.37, 149.45, 141.67, 140.77, 135.64, 129.21, 128.22, 126.31,
125.50, 93.95, 55.74, 51.42, 47.42, 31.91, 31.73, 30.12, 29.96,
Synthesis of compound 13: N-Benzyl-2,6-dichloro-3-nitro
25 pyridin-4-amine. Sodium hydride (138 mg, 5.77 mmol) was
carefully suspended in 10 mL of dry THF under N2. The grey
suspension was cooled to 0 ºC and compound 12 (1.0 g, 4.81 85 27.37, 22.66, 13.87. MS (ESI) calculated for C25H36ClN4, m/z
mmol) was slowly added to the suspension at 0 ºC. The reaction
mixture was stirred for 1 h, followed by the addition of benzyl
30 bromide (0.5 mL, 5.29 mmol). The reaction mixture was stirred at
room temperature for 2 h and poured into ice water. Then it was
427.2623, found 427.2635 (M + H)+.
Synthesis of compound 17: 1-Benzyl-2-butyl-6-chloro-1H-
imidazo[4,5-c]pyridin-4-amine. Compound 16 (34 mg, 0.082
extracted with EtOAc (3 × 20 mL), washed with water, dried over 90 mmol) was dissolved in 1 mL of HCl (4M in dioxane) and stirred
sodium sulfate. The solvent was removed and the crude residue
was purified using column chromatography (20% EtOAc/hexane)
35 to obtain the compound 13 as yellow solid. 1H NMR (500 MHz,
CDCl3) δ 7.44 – 7.34 (m, 3H), 7.32 – 7.27 (m, 2H), 6.99 (s, 1H),
at room temperature for 30 min. Then the solvent was removed
under vacuum to obtain compound 17 (11 mg, 42%). H NMR
(500 MHz, CDCl3) δ 7.36 – 7.30 (m, 3H), 7.01 (d, J = 6.4 Hz,
1
2H), 6.58 (s, 1H), 5.23 (s, 2H), 5.21 (s, 2H), 2.80 – 2.73 (m, 2H),
6.67 (s, 1H), 4.47 (d, J = 5.4 Hz, 2H). 13C NMR (126 MHz, 95 1.72 (dt, J = 15.5, 7.7 Hz, 2H), 1.39 (dq, J = 14.8, 7.4 Hz, 2H),
CDCl3) δ 152.07, 149.97, 145.68, 135.01, 129.48, 128.71,
127.32, 106.62, 47.74.
0.90 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 154.83,
149.94, 142.31, 141.73, 135.26, 129.32, 128.57, 128.41, 126.26,
96.29, 47.59, 29.92, 27.36, 22.63, 13.87. HRMS (ESI) calculated
for C17H20ClN4, m/z 315.1371, found 315.1422 (M + H)+.
40
Synthesis of compound 14: N4-Benzyl-6-chloro-3-nitro-N2-
(2,4,4-trimethylpentan-2-yl)pyridine-2,4-diamine.
To
a
100
solution of compound 13 (300 mg, 1.02 mmol) in 20 mL of
CH2Cl2 were added triethylamine (0.21 mL, 1.53 mmol) and tertꢀ
45 octylamine (0.5 mL, 3.06 mmol). The reaction mixture was
refluxed for 48 h and the solvent was removed under vacuum.
Synthesis of compound 19a: 1-Benzyl-2-butyl-1H-imidazo
[4,5-c]pyridine-4,6-diamine. To a solution of compound 16 (70
mg, 0.16 mmol) in 1 mL of dioxane were added potassium tertꢀ
butoxide (92 mg, 0.82 mmol), catalytic amount of DavePhos and
The crude residue was purified using column chromatography 105 Pd2(dba)3 and tertꢀoctylamine (83 ꢀL, 0.83 mmol). The reaction
(20% EtOAc/hexane) to obtain the compound 14 as yellow solid
(360 mg, 91%). H NMR (500 MHz, CDCl3) δ 9.57 (s, 1H), 9.51
mixture was then heated under microwave conditions (500 W,
100 ºC) in a sealed vial for 1 h. It was cooled to room temperature
and filtered through celite and washed with MeOH. The solvent
was removed and the crude residue was purified using column
1
50 (s, 1H), 7.39 (ddd, J = 7.5, 4.4, 1.3 Hz, 2H), 7.36 – 7.30 (m, 3H),
5.92 (s, 1H), 4.44 (d, J = 5.4 Hz, 2H), 1.95 (s, 2H), 1.56 (s, 6H),
0.98 (s, 9H). 13C NMR (126 MHz, CDCl3) δ 155.05, 154.25, 110 chromatography (20% EtOAc/hexane) to obtain the compound
153.44, 136.10, 129.24, 128.24, 127.43, 115.80, 94.21, 57.32,
51.54, 47.64, 31.94, 31.66, 29.84. MS (ESI) calculated for
55 C20H28ClN4O2, m/z 391.1895, found 391.1901 (M + H)+.
18a, brown solid (41 mg, 49%). Compound 18a (33 mg, 0.063
mmol) was dissolved in 1 mL of HCl (4M in dioxane) and stirred
at room temperature for 30 min. Then the solvent was removed
under vacuum to obtain compound 19a as brown solid (11 mg,
Synthesis of compound 16: 1-Benzyl-2-butyl-6-chloro-N- 115 58%). 1H NMR (500 MHz, DMSO) δ 7.37 – 7.31 (m, 2H), 7.30 –
(2,4,4-trimethylpentan-2-yl)-1H-imidazo[4,5-c]pyridin-4-
7.25 (m, 1H), 7.09 – 7.04 (m, 2H), 6.41 (s, 2H), 5.65 (s, 1H), 5.24
10 | Journal Name, [year], [vol], 00–00
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DOI: 10.1039/C3OB40816G
(s, 2H), 2.73 – 2.67 (m, 2H), 1.59 (dt, J = 15.3, 7.5 Hz, 2H), 1.31
154.37, 150.36, 149.19, 142.41, 136.65, 128.97, 127.76, 126.44,
(dq, J = 14.7, 7.4 Hz, 2H), 0.83 (t, J = 7.4 Hz, 3H). 13C NMR 60 120.35, 73.62, 55.26, 51.67, 50.18, 47.02, 37.88, 31.91, 31.80,
(126 MHz, DMSO) δ 151.81, 147.80, 144.08, 136.91, 128.81,
128.77, 127.47, 126.29, 117.96, 76.19, 46.11, 29.27, 26.17,
21.80, 13.68. HRMS (ESI) calculated for C17H22N5, m/z
296.1870, found 296.1906 (M + H)+.
31.48, 30.51, 30.31, 27.46, 26.80, 26.17, 22.72, 13.91. MS (ESI)
calculated for C32H50N5, m/z 504.4061, found 504.41 (M + H)+.
5
1-Benzyl-2-butyl-N6-(cyclohexylmethyl)-1H-imidazo[4,5-c]
65 pyridine-4,6-diamine (19d). Light brown (28 mg, 80%). 1H
NMR (500 MHz, DMSO) δ 7.35 (t, J = 7.3 Hz, 2H), 7.31 – 7.27
(m, 1H), 7.10 (d, J = 7.2 Hz, 2H), 6.34 (d, J = 3.1 Hz, 1H), 5.85
(s, 1H), 5.33 (s, 2H), 2.92 (t, J = 6.1 Hz, 2H), 2.71 – 2.66 (m,
2H), 1.75 – 1.64 (m, 4H), 1.57 (dt, J = 15.3, 7.6 Hz, 3H), 1.49
Compounds 19bꢀ19o were synthesized similarly as compound
19a.
10
1-Benzyl-N6,2-dibutyl-1H-imidazo[4,5-c]pyridine-4,6-diamine
(19b). Butyl amine was used as a reagent. Brown solid (23 mg, 70 (dtd, J = 11.2, 7.4, 3.7 Hz, 1H), 1.30 (dt, J = 14.8, 7.4 Hz, 2H),
1
79%). H NMR (500 MHz, CDCl3) δ 7.31 (ddd, J = 8.6, 6.4, 3.4
Hz, 3H), 7.05 (d, J = 6.7 Hz, 2H), 5.46 (s, 1H), 5.26 (s, 1H), 5.16
1.21 – 1.09 (m, 4H), 0.91 (dd, J = 22.3, 10.5 Hz, 2H), 0.81 (t, J =
7.4 Hz, 3H). 13C NMR (126 MHz, DMSO) δ 153.32, 146.28,
145.05, 136.69, 128.79, 127.61, 126.53, 116.94, 74.21, 48.49,
46.22, 36.76, 30.47, 29.02, 26.21, 26.05, 25.43, 21.75, 13.65.
15 (s, 2H), 3.03 (t, J = 7.1 Hz, 2H), 2.72 – 2.66 (m, 2H), 1.68 (dd, J
= 15.5, 7.6 Hz, 2H), 1.55 (dd, J = 14.8, 7.2 Hz, 2H), 1.43 – 1.33
(m, 4H), 0.90 (dt, J = 16.4, 7.4 Hz, 6H). 13C NMR (126 MHz, 75 HRMS (ESI) calculated for C24H34N5, m/z 392.2809, found
CDCl3) δ 152.42, 148.50, 144.42, 135.99, 129.14, 128.06,
126.33, 74.80, 47.20, 43.09, 31.41, 30.03, 27.37, 22.66, 20.40,
20 14.02, 13.89. HRMS (ESI) calculated for C21H30N5, m/z
352.2496, found 352.2515 (M + H)+.
392.2806 (M + H)+.
1-Benzyl-2-butyl-N6-phenyl-N4-(2,4,4-trimethylpentan-2-yl)-
1H-imidazo[4,5-c]pyridine-4,6-diamine (18e). Aniline was used
1
80 as a reagent. Yellow solid (51 mg, 55%). H NMR (500 MHz,
1-Benzyl-2-butyl-N6-heptyl-N4-(2,4,4-trimethylpentan-2-yl)-
1H-imidazo[4,5-c]pyridine-4,6-diamine (18c). Heptylamine
25 was used as a reagent. Brown oil (43 mg, 65%). H NMR (500
MHz, CDCl3) δ 7.32 – 7.26 (m, 3H), 7.05 (d, J = 6.7 Hz, 2H),
CDCl3) δ 7.31 (dt, J = 16.5, 5.5 Hz, 3H), 7.22 (t, J = 7.8 Hz, 2H),
7.16 (d, J = 7.6 Hz, 2H), 7.05 (d, J = 6.7 Hz, 2H), 6.90 (t, J = 6.9
Hz, 1H), 6.16 (s, 1H), 6.02 (s, 1H), 5.23 (s, 1H), 5.12 (s, 2H),
2.74 – 2.67 (m, 2H), 2.03 (s, 2H), 1.66 (dd, J = 15.8, 8.2 Hz, 4H),
1
5.39 (s, 1H), 5.13 (s, 1H), 5.11 (s, 2H), 3.13 (t, J = 7.2 Hz, 2H), 85 1.60 (s, 6H), 1.38 (dd, J = 15.0, 7.5 Hz, 2H), 1.02 (s, 9H), 0.89 (t,
2.68 – 2.64 (m, 2H), 2.04 (s, 2H), 1.65 – 1.55 (m, 11H), 1.41 –
1.22 (m, 12H), 1.01 (s, 9H), 0.89 – 0.84 (m, 6H). 13C NMR (126
30 MHz, CDCl3) δ 154.28, 150.38, 149.22, 142.39, 136.63, 128.97,
128.05, 127.76, 126.40, 120.44, 95.52, 73.57, 55.27, 51.72,
J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 151.49, 149.42,
149.11, 142.46, 141.51, 136.32, 129.16, 129.06, 127.93, 126.53,
121.98, 120.91, 118.83, 55.42, 51.80, 47.26, 31.93, 31.80, 30.44,
30.19, 27.46, 22.71, 13.91. MS (ESI) calculated for C31H42N5,
47.00, 43.63, 31.97, 31.94, 31.90, 31.79, 30.47, 30.29, 29.97, 90 m/z 484.3435, found 484.3459 (M + H)+.
29.32, 27.44, 27.39, 22.77, 22.71, 14.25, 13.90. MS (ESI)
calculated for C32H52N5, m/z 506.4217, found 506.4209 (M +
35 H)+.
1-Benzyl-2-butyl-N6-phenyl-1H-imidazo[4,5-c]pyridine-4,6-
1
diamine (19e). Light yellow solid (28 mg, 76%). H NMR (500
MHz, DMSO) δ 9.10 (s, 1H), 8.08 (s, 1H), 7.39 – 7.35 (m, 2H),
95 7.33 – 7.28 (m, 3H), 7.11 (t, J = 7.3 Hz, 4H), 7.01 (t, J = 7.3 Hz,
1H), 6.41 (s, 1H), 5.44 (s, 2H), 2.82 (t, J = 7.5 Hz, 2H), 1.63 (dt,
J = 15.3, 7.6 Hz, 2H), 1.33 (dd, J = 14.9, 7.4 Hz, 2H), 0.84 (t, J =
7.4 Hz, 3H). 13C NMR (126 MHz, DMSO) δ 146.34, 140.41,
136.06, 129.52, 128.92, 127.84, 126.69, 122.44, 118.87, 46.68,
1-Benzyl-2-butyl-N6-heptyl-1H-imidazo[4,5-c]pyridine-4,6-
diamine (19c). Light brown solid (24 mg, 80%). H NMR (500
MHz, DMSO) δ 8.03 (s, 2H), 7.38 – 7.33 (m, 2H), 7.32 – 7.28
40 (m, 1H), 7.12 (d, J = 7.2 Hz, 2H), 6.84 (s, 1H), 5.98 (s, 1H), 5.39
(s, 2H), 3.08 (t, J = 7.0 Hz, 2H), 2.73 – 2.68 (m, 2H), 1.59 (dt, J =
1
15.3, 8.2 Hz, 2H), 1.50 (dd, J = 14.3, 7.2 Hz, 2H), 1.33 – 1.21 (m, 100 28.89, 26.15, 21.72, 13.63. HRMS (ESI) calculated for C23H26N5,
12H), 0.85 (t, J = 7.0 Hz, 3H), 0.81 (t, J = 7.4 Hz, 3H). 13C NMR
(126 MHz, DMSO) δ 154.62, 145.66, 145.15, 136.32, 128.96,
45 128.86, 127.73, 126.64, 126.55, 74.21, 46.34, 42.24, 31.25,
28.78, 28.42, 28.09, 26.34, 26.18, 22.07, 21.71, 13.98, 13.62.
m/z 372.2183, found 372.2219 (M + H)+.
N6,1-Dibenzyl-2-butyl-N4-(2,4,4-trimethylpentan-2-yl)-1H-
imidazo[4,5-c]pyridine-4,6-diamine (18f). Benzyl amine was
HRMS (ESI) calculated for C24H36N5, m/z 394.2965, found 105 used as a reagent. Light brown solid (30 mg, 46%). 1H NMR (500
394.3001 (M + H)+.
MHz, CDCl3) δ 7.35 (d, J = 7.4 Hz, 2H), 7.26 (dddd, J = 9.4, 7.2,
6.1, 1.6 Hz, 6H), 7.03 (d, J = 6.8 Hz, 2H), 5.44 (s, 1H), 5.17 (s,
1H), 5.07 (s, 2H), 4.42 (s, 2H), 2.70 – 2.64 (m, 2H), 1.67 – 1.60
(m, 2H), 1.55 (s, 6H), 1.35 (dq, J = 14.7, 7.4 Hz, 2H), 1.02 – 0.94
50 1-Benzyl-2-butyl-N6-(cyclohexylmethyl)-N4-(2,4,4-
trimethylpentan-2-yl)-1H-imidazo[4,5-c]pyridine-4,6-diamine
(18d). NꢀCyclohexylmethylamine was used as a reagent. Yellow 110 (m, 9H), 0.87 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
1
solid (50 mg, 62%). H NMR (500 MHz, CDCl3) δ 7.32 – 7.27
(m, 3H), 7.06 (d, J = 6.7 Hz, 2H), 5.37 (s, 1H), 5.10 (s, 2H), 2.99
55 (d, J = 6.7 Hz, 2H), 2.69 – 2.64 (m, 2H), 2.04 (s, 2H), 1.79 (d, J =
13.2 Hz, 2H), 1.73 – 1.60 (m, 9H), 1.58 (s, 6H), 1.35 (dd, J =
153.83, 150.58, 149.20, 142.27, 140.52, 136.49, 128.98, 128.56,
127.80, 127.68, 126.97, 126.46, 126.39, 74.17, 55.29, 51.62,
47.59, 47.07, 31.87, 31.76, 30.45, 30.28, 30.20, 27.42, 22.70,
13.90. MS (ESI) calculated for C32H44N5, m/z 498.3591, found
15.0, 7.5 Hz, 2H), 1.27 – 1.13 (m, 4H), 1.01 (s, 9H), 0.98 – 0.91 115 498.3599 (M + H)+.
(m, 2H), 0.86 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 11

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DOI: 10.1039/C3OB40816G
N6,1-Dibenzyl-2-butyl-1H-imidazo[4,5-c]pyridine-4,6-diamine
(19f). White solid (22 mg, 85%). H NMR (500 MHz, DMSO) δ 60 (126 MHz, DMSO) δ 150.57, 149.14, 143.25, 140.18, 137.18,
(dd, J = 14.9, 7.4 Hz, 2H), 0.82 (t, J = 7.4 Hz, 3H). 13C NMR
1
8.07 (s, 2H), 7.38 – 7.35 (m, 2H), 7.34 – 7.29 (m, 5H), 7.29 –
7.25 (m, 1H), 7.07 (d, J = 6.3 Hz, 2H), 6.06 (s, 1H), 5.33 (s, 2H),
4.38 (s, 2H), 2.75 – 2.69 (m, 2H), 1.58 (dt, J = 15.3, 7.6 Hz, 2H),
1.30 (dt, J = 14.8, 7.4 Hz, 2H), 0.81 (t, J = 7.4 Hz, 3H). 13C NMR
(126 MHz, DMSO) δ 154.68, 145.29, 137.99, 136.12, 128.85,
128.45, 127.78, 127.51, 127.23, 126.74, 75.08, 46.43, 45.67,
28.76, 26.16, 21.70, 13.62. HRMS (ESI) calculated for C24H28N5,
130.77, 129.08, 128.63, 127.96, 127.37, 126.56, 118.72, 75.34,
46.06, 44.71, 40.11, 40.02, 39.94, 29.40, 26.20, 21.82, 13.69.
HRMS (ESI) calculated for C24H27ClN5, m/z 420.1950, found
420.1976 (M + H)+.
5
65
1-Benzyl-2-butyl-N6-(furan-2-ylmethyl)-N4-(2,4,4-
trimethylpentan-2-yl)-1H-imidazo[4,5-c]pyridine-4,6-diamine
(18k). Furfuryl amine was used as a reagent. Yellow oil (53 mg,
54%). 1H NMR (500 MHz, CDCl3) δ 7.30 (ddd, J = 10.9, 4.6, 1.1
70 Hz, 4H), 7.04 (d, J = 6.6 Hz, 2H), 6.28 (dd, J = 3.2, 1.9 Hz, 1H),
6.15 (dd, J = 3.2, 0.6 Hz, 1H), 5.50 (s, 1H), 5.10 (s, 2H), 4.43 (s,
2H), 2.72 – 2.64 (m, 2H), 2.03 (s, 2H), 1.68 – 1.59 (m, 4H), 1.58
(s, 6H), 1.35 (dt, J = 14.7, 7.4 Hz, 2H), 1.00 (s, 9H), 0.87 (t, J =
7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 153.92, 153.28,
10 m/z 386.2339, found 386.2389 (M + H)+.
1-Benzyl-2-butyl-N6-(4-methoxybenzyl)-1H-imidazo[4,5-c]
pyridine-4,6-diamine (19g). 4ꢀMethoxybenzyl amine was used
1
as a reagent. Light yellow solid (22 mg, 69%). H NMR (500
15 MHz, DMSO) δ 8.07 (s, 2H), 7.34 – 7.28 (m, 5H), 7.08 (d, J =
6.4 Hz, 2H), 6.87 (d, J = 8.7 Hz, 2H), 6.04 (s, 1H), 5.34 (s, 2H),
4.29 (s, 2H), 3.72 (s, 3H), 2.75 – 2.70 (m, 2H), 1.58 (dt, J = 15.3, 75 150.77, 149.15, 142.19, 141.69, 136.47, 129.00, 127.83, 126.42,
7.6 Hz, 2H), 1.29 (dd, J = 14.9, 7.4 Hz, 2H), 0.81 (t, J = 7.4 Hz,
3H). 13C NMR (126 MHz, DMSO) δ 158.48, 154.67, 145.28,
20 145.27, 136.13, 129.73, 128.92, 128.84, 127.78, 126.74, 113.82,
75.11, 55.08, 46.43, 45.18, 28.76, 26.16, 21.71, 13.62. HRMS
110.36, 106.53, 74.85, 55.32, 51.68, 47.09, 40.78, 31.89, 31.77,
30.47, 30.31, 27.43, 22.71, 13.90. MS (ESI) calculated for
C30H42N5O, m/z 488.3384, found 488.3446 (M + H)+.
(ESI) calculated for C25H30N5O, m/z 416.2445, found 416.2490 80 1-Benzyl-2-butyl-N6-(furan-2-ylmethyl)-1H-imidazo[4,5-c]
(M + H)+.
pyridine-4,6-diamine (19k). Brown solid (21 mg, 55%). 1H
NMR (500 MHz, DMSO) δ 7.55 (s, 1H), 7.34 (t, J = 7.3 Hz, 2H),
7.31 – 7.27 (m, 1H), 7.10 (d, J = 7.3 Hz, 2H), 6.36 (dd, J = 3.0,
1.9 Hz, 1H), 6.29 (d, J = 2.6 Hz, 1H), 5.99 (s, 1H), 5.31 (s, 2H),
25 1-Benzyl-2-butyl-N6-(3-methoxybenzyl)-1H-imidazo[4,5-c]
pyridine-4,6-diamine (19h). 3ꢀMethoxybenzyl amine was used
1
as a reagent. Brown solid (27 mg, 69%). H NMR (500 MHz, 85 4.35 (d, J = 5.8 Hz, 2H), 2.73 – 2.67 (m, 2H), 1.58 (dt, J = 15.2,
DMSO) δ 7.32 – 7.27 (m, 3H), 7.23 (t, J = 7.9 Hz, 1H), 7.06 (d, J
= 6.1 Hz, 2H), 6.94 (dd, J = 12.3, 4.8 Hz, 2H), 6.83 (dd, J = 8.1,
30 2.2 Hz, 1H), 6.03 (s, 1H), 5.31 (s, 2H), 4.33 (d, J = 5.8 Hz, 2H),
3.71 (s, 3H), 2.70 (t, J = 7.6 Hz, 2H), 1.57 (dt, J = 15.3, 7.6 Hz,
7.6 Hz, 2H), 1.30 (dd, J = 14.9, 7.4 Hz, 2H), 0.82 (t, J = 7.4 Hz,
3H). 13C NMR (126 MHz, DMSO) δ 142.17, 136.66, 128.79,
127.62, 126.61, 110.39, 107.43, 75.34, 60.19, 46.26, 38.71,
29.07, 26.21, 21.76, 13.65. HRMS (ESI) calculated for
90 C22H26N5O, m/z 376.2132, found 376.2184 (M + H)+.
2H), 1.29 (dd, J = 14.9, 7.4 Hz, 2H), 0.81 (t, J = 7.4 Hz, 3H). 13
C
NMR (126 MHz, DMSO) δ 159.37, 136.32, 129.52, 128.80,
127.72, 126.70, 119.55, 113.13, 112.50, 75.05, 55.00, 46.33,
35 45.63, 40.11, 40.02, 39.94, 28.89, 26.21, 21.72, 13.63. HRMS
(ESI) calculated for C25H30N5O, m/z 416.2445, found 416.2506
(M + H)+.
1-Benzyl-2-butyl-N6-(pyridin-4-ylmethyl)-N4-(2,4,4-trimethyl
pentan-2-yl)-1H-imidazo[4,5-c]pyridine-4,6-diamine (18l). 4ꢀ
Picolylamine was used as a reagent. Yellow solid (25 mg, 31%).
95 1H NMR (500 MHz, CDCl3) δ 8.48 (dd, J = 4.5, 1.6 Hz, 2H),
7.29 – 7.25 (m, 3H), 7.24 (dd, J = 4.5, 1.5 Hz, 2H), 7.01 (d, J =
6.1 Hz, 2H), 5.40 (s, 1H), 5.06 (s, 2H), 4.49 (s, 2H), 2.70 – 2.64
(m, 2H), 1.92 (s, 2H), 1.63 (dt, J = 15.5, 7.7 Hz, 2H), 1.48 (s,
6H), 1.36 (dt, J = 14.9, 7.4 Hz, 2H), 0.95 (s, 9H), 0.87 (t, J = 7.4
1-Benzyl-2-butyl-N6-(4-(trifluoromethyl)benzyl)-1H-imidazo
40 [4,5-c]pyridine-4,6-diamine (19i). 4ꢀ(Trifluoromethyl)benzyl
1
amine was used as a reagent. Brown solid (17 mg, 77%). H
NMR (500 MHz, DMSO) δ 7.60 (d, J = 8.2 Hz, 2H), 7.51 (d, J = 100 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 149.83, 136.31, 129.01,
8.1 Hz, 2H), 7.29 – 7.21 (m, 3H), 7.02 (d, J = 6.7 Hz, 2H), 6.23
(s, 1H), 5.70 (s, 2H), 5.57 (s, 1H), 5.15 (s, 2H), 4.41 (d, J = 6.3
45 Hz, 2H), 2.69 – 2.63 (m, 2H), 1.56 (dt, J = 15.3, 7.5 Hz, 2H),
1.29 (dd, J = 14.9, 7.4 Hz, 2H), 0.82 (t, J = 7.4 Hz, 3H). 13C
NMR (126 MHz, DMSO) δ 153.45, 150.32, 149.41, 146.48, 105
143.12, 137.24, 128.58, 127.82, 127.31, 126.51, 124.88, 124.85,
118.91, 75.32, 46.04, 44.94, 29.43, 26.19, 21.82, 13.69. HRMS
50 (ESI) calculated for C25H27F3N5, m/z 454.2213, found 454.2284
(M + H)+.
127.89, 126.40, 122.20, 74.67, 55.25, 51.56, 47.13, 46.09, 31.81,
31.75, 31.71, 30.44, 30.23, 27.41, 22.69, 13.89. MS (ESI)
calculated for C31H43N6, m/z 499.3544, found 499.3475 (M +
H)+.
1-Benzyl-2-butyl-N6-(pyridin-4-ylmethyl)-1H-imidazo[4,5-c]
1
pyridine-4,6-diamine (19l). Brown solid (7 mg, 47%). H NMR
(500 MHz, DMSO) δ 8.43 (dd, J = 4.5, 1.5 Hz, 2H), 7.27 (ddd, J
= 9.7, 6.8, 4.4 Hz, 5H), 7.03 (d, J = 6.7 Hz, 2H), 6.50 – 6.28 (m,
110 2H), 5.65 (s, 1H), 5.18 (s, 2H), 4.37 (d, J = 6.3 Hz, 2H), 2.69 –
2.65 (m, 2H), 1.57 (dt, J = 15.3, 7.5 Hz, 2H), 1.30 (dd, J = 14.9,
7.4 Hz, 2H), 0.81 (d, J = 7.4 Hz, 3H). 13C NMR (126 MHz,
DMSO) δ 149.28, 137.09, 130.51, 128.65, 127.43, 126.53,
122.35, 113.97, 75.37, 46.09, 44.38, 29.34, 26.19, 21.80, 13.68.
1-Benzyl-2-butyl-N6-(4-chlorobenzyl)-1H-imidazo[4,5-c]
pyridine-4,6-diamine (19j). 4ꢀChlorobenzylamine was used as a
55 reagent. Yellow solid (15 mg, 63%). 1H NMR (500 MHz,
DMSO) δ 7.33 – 7.25 (m, 8H), 7.02 (d, J = 6.6 Hz, 2H), 6.18 (s,
1H), 5.83 (s, 2H), 5.59 (s, 1H), 5.16 (s, 2H), 4.30 (d, J = 6.0 Hz, 115 HRMS (ESI) calculated for C23H27N6, m/z 387.2292, found
2H), 2.68 – 2.64 (m, 2H), 1.56 (dt, J = 15.3, 7.5 Hz, 2H), 1.29
387.2299 (M + H)+.
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DOI: 10.1039/C3OB40816G
1
19p as light yellow solid (5 mg, 63%). H NMR (500 MHz,
60 DMSO) δ 8.37 (s, 2H), 8.06 (s, 2H), 7.42 (q, J = 8.5 Hz, 4H),
7.36 – 7.30 (m, 3H), 7.08 (d, J = 6.8 Hz, 2H), 6.08 (s, 1H), 5.34
(s, 2H), 4.40 (s, 2H), 3.98 (q, J = 5.8 Hz, 2H), 2.75 – 2.67 (m,
2H), 1.57 (dt, J = 15.3, 7.6 Hz, 2H), 1.28 (dd, J = 14.9, 7.4 Hz,
2H), 0.80 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, DMSO) δ
1-Benzyl-2-butyl-N6-(pyridin-3-ylmethyl)-1H-imidazo[4,5-
c]pyridine-4,6-diamine (19m). 3ꢀPicolylamine was used as a
1
reagent. Light brown solid (33 mg, 63%). H NMR (500 MHz,
5
DMSO) δ 8.92 (s, 1H), 8.80 (d, J = 5.3 Hz, 1H), 8.46 (d, J = 8.0
Hz, 1H), 8.12 (s, 2H), 7.94 (dd, J = 7.9, 5.7 Hz, 1H), 7.69 (s, 1H),
7.33 – 7.25 (m, 3H), 7.04 (d, J = 6.9 Hz, 2H), 6.03 (s, 1H), 5.32 65 154.71, 145.41, 145.34, 138.43, 136.19, 132.98, 129.02, 128.89,
(s, 2H), 4.64 (s, 2H), 2.72 (t, J = 7.6 Hz, 2H), 1.56 (dt, J = 15.2,
7.6 Hz, 2H), 1.28 (dd, J = 14.9, 7.4 Hz, 2H), 0.80 (t, J = 7.4 Hz,
127.79, 127.75, 126.65, 46.40, 45.31, 41.89, 28.78, 26.18, 21.70,
13.62. HRMS (ESI) calculated for C25H31N6, m/z 415.2605,
found 415.2606 (M + H)+.
10 3H). 13C NMR (126 MHz, DMSO) δ 154.65, 145.80, 145.01,
143.55, 141.29, 141.12, 138.17, 136.10, 128.83, 127.76, 126.68,
126.52, 75.49, 46.45, 42.51, 28.77, 26.12, 21.68, 13.60. HRMS 70 N6,N6'-(1,4-phenylenebis(methylene))bis(1-benzyl-2-butyl-1H-
(ESI) calculated for C23H27N6, m/z 387.2292, found 387.2296 (M
+ H)+.
imidazo[4,5-c]pyridine-4,6-diamine) (19r). Compound 18r (35
mg, 0.038 mmol) was dissolved in 1 mL of HCl (4M in dioxane)
and stirred at room temperature for 30 min. Then the solvent was
removed under vacuum to obtain compound 19r as light yellow
15
1-Benzyl-2-butyl-N6-(naphthalen-1-ylmethyl)-1H-imidazo
[4,5-c]pyridine-4,6-diamine (19n). 1ꢀNaphthylmethylamine was 75 solid (12 mg, 45%).1H NMR (500 MHz, MeOD) δ 7.27 (s, 4H),
used as a reagent. Light brown solid (28 mg, 70%). 1H NMR (500
7.21 – 7.17 (m, 6H), 7.02 – 6.98 (m, 4H), 5.20 (s, 4H), 4.33 (s,
4H), 2.78 – 2.73 (m, 4H), 1.67 (dt, J = 15.3, 7.6 Hz, 4H), 1.37
(dd, J = 15.0, 7.5 Hz, 4H), 0.89 (t, J = 7.4 Hz, 6H). 13C NMR
(126 MHz, MeOD) δ 155.94, 150.96, 148.16, 146.47, 138.77,
MHz, DMSO) δ 12.12 (s, 1H), 8.10 (d, J = 8.0 Hz, 1H), 8.04 (s,
20 2H), 7.99 – 7.96 (m, 1H), 7.88 (d, J = 8.2 Hz, 1H), 7.61 – 7.53
(m, 3H), 7.45 (dd, J = 8.1, 7.1 Hz, 1H), 7.33 – 7.28 (m, 3H), 7.08
(d, J = 6.4 Hz, 2H), 6.22 (s, 1H), 5.35 (s, 2H), 4.83 (s, 2H), 2.74 – 80 137.12, 130.00, 129.03, 128.70, 127.70, 118.63, 48.01, 47.26,
2.68 (m, 2H), 1.58 (dt, J = 15.3, 7.6 Hz, 2H), 1.29 (dd, J = 14.9,
7.4 Hz, 2H), 0.81 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz,
25 DMSO) δ 154.69, 145.51, 145.31, 136.23, 133.44, 132.90,
130.92, 128.83, 128.63, 127.99, 127.76, 126.67, 126.39, 125.97,
30.28, 27.76, 23.29, 14.05. HRMS (ESI) calculated for
C42H49N10, m/z 693.4136, found 693.4331 (M + H)+.
Compounds 19q and 19s were synthesized similarly as
125.49, 125.43, 123.63, 74.91, 46.39, 43.99, 28.80, 26.20, 21.71, 85 compounds 19p and 19r.
13.62. HRMS (ESI) calculated for C28H30N5, m/z 436.2496,
found 436.2549 (M + H)+.
N6-(3-(aminomethyl)benzyl)-1-benzyl-2-butyl-1H-imidazo
30
[4,5-c]pyridine-4,6-diamine (19q). mꢀxylylenediamine was used
as a reagent. Light yellow solid. H NMR (500 MHz, MeOD) δ
1
N6-([1,1'-Biphenyl]-4-ylmethyl)-1-benzyl-2-butyl-1H-imidazo
[4,5-c]pyridine-4,6-diamine (19o). [1,1'ꢀbiphenyl]ꢀ4ꢀyl methan 90 7.49 (s, 1H), 7.41 (dd, J = 3.9, 1.5 Hz, 2H), 7.39 – 7.35 (m, 1H),
amine was used as a reagent. Light brown solid (21 mg, 51%). 1H
NMR (500 MHz, DMSO) δ 12.36 (s, 1H), 8.04 (s, 2H), 7.63 (dd,
35 J = 12.4, 7.8 Hz, 4H), 7.50 – 7.42 (m, 4H), 7.40 – 7.33 (m, 1H),
7.28 (t, J = 7.3 Hz, 2H), 7.23 (t, J = 7.2 Hz, 1H), 7.07 (d, J = 7.3
7.32 – 7.28 (m, 3H), 7.05 – 7.02 (m, 2H), 5.31 (s, 2H), 4.44 (s,
2H), 4.07 (s, 2H), 2.79 – 2.75 (m, 2H), 1.70 (dt, J = 15.3, 7.6 Hz,
2H), 1.38 (dq, J = 14.8, 7.4 Hz, 2H), 0.89 (t, J = 7.4 Hz, 3H). 13
C
NMR (126 MHz, MeOD) δ 157.36, 147.23, 146.79, 140.00,
Hz, 2H), 6.07 (s, 1H), 5.33 (s, 2H), 4.42 (s, 2H), 2.71 (t, J = 7.6 95 136.86, 134.96, 130.63, 130.12, 129.26, 129.19, 129.14, 129.00,
Hz, 2H), 1.57 (dt, J = 15.2, 7.7 Hz, 2H), 1.29 (dq, J = 14.6, 7.3
Hz, 2H), 0.81 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, DMSO) δ
40 154.65, 145.40, 145.35, 139.80, 139.07, 137.24, 136.17, 128.95,
128.80, 128.06, 127.67, 127.43, 126.71, 126.69, 126.57, 75.11,
127.62, 48.10, 47.15, 44.18, 30.00, 27.75, 23.24, 14.03. HRMS
(ESI) calculated for C25H31N6, m/z 415.2605, found 415.2610 (M
+ H)+.
46.38, 45.31, 28.81, 26.18, 21.69, 13.61. HRMS (ESI) calculated 100 N6,N6'-(1,3-phenylenebis(methylene))bis(1-benzyl-2-butyl-1H-
for C30H32N5, m/z 462.2652, found 462.2698 (M + H)+.
imidazo[4,5-c]pyridine-4,6-diamine) (19s). Light yellow solid.
1H NMR (500 MHz, DMSO) δ 12.38 (s, 2H), 8.04 (s, 4H), 7.38
(s, 1H), 7.30 – 7.22 (m, 9H), 7.04 (d, J = 6.8 Hz, 4H), 6.01 (s,
2H), 5.28 (s, 4H), 4.34 (s, 4H), 2.73 – 2.68 (m, 4H), 1.56 (dd, J =
45 Synthesis of compound 19p and 19r: N6-(4-(aminomethyl)
benzyl)-1-benzyl-2-butyl-1H-imidazo[4,5-c]pyridine-4,6-
diamine. To a solution of compound 16 (70 mg, 0.16 mmol) in 1 105 15.3, 7.7 Hz, 4H), 1.29 (dd, J = 14.9, 7.4 Hz, 4H), 0.81 (t, J = 7.4
mL of dioxane were added potassium tertꢀbutoxide (90 mg, 0.80
mmol), catalytic amount of DavePhos and Pd2(dba)3 and pꢀ
50 xylylenediamine (109 mg, 0.80 mmol). The reaction mixture was
then heated under microwave conditions (500 W, 100 ºC) in a
sealed vial for 1 h. It was cooled to room temperature and filtered 110
through celite and washed with MeOH. The solvent was removed
and the crude residue was purified using column chromatography
55 (20% EtOAc/hexane) to obtain the compound 18p and 18r.
Compound 18p (33 mg, 0.063 mmol) was dissolved in 1 mL of
Hz, 6H). 13C NMR (126 MHz, DMSO) δ 154.62, 145.36, 145.31,
138.22, 136.15, 128.80, 128.61, 127.72, 126.66, 126.56, 126.38,
46.36, 45.73, 28.80, 26.19, 21.71, 13.62. HRMS (ESI) calculated
for C42H49N10, m/z 693.4136, found 693.4131 (M + H)+.
Synthesis of compound 23a: To a solution of compound 14 (120
mg, 0.31 mmol) in 1 mL of dioxane were added cesium carbonate
(303 mg, 0.93 mmol) in H2O (0.5 mL), [1,1′ꢀ
bis(diphenylphosphino)ferrocene]dichloropalladium(II)
HCl (4M in dioxane) and stirred at room temperature for 30 min. 115 (Pd(dppf)Cl2) (15 mg, 0.019 mmol) and nꢀbutylboronic acid (98
Then the solvent was removed under vacuum to obtain compound
ꢀL, 0.46 mmol) under N2. The reaction mixture was then heated
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DOI: 10.1039/C3OB40816G
at 90 ºC in a sealed vial for 18 h. It was cooled to room
Hz, 2H), 7.45 – 7.42 (m, 3H), 7.40 – 7.38 (m, 3H), 7.34 – 7.31
temperature and filtered through celite and washed with MeOH. 60 (m, 1H), 7.26 (s, 1H), 6.39 (s, 1H), 4.58 (d, J = 5.4 Hz, 2H), 2.09
The solvent was removed and the crude residue was purified
using column chromatography (15% EtOAc/hexane) to obtain the
compound 20a (97mg, 76%). To a solution of compound 20a (94
mg, 0.23 mmol) in 10 mL of MeOH were added zinc dust (149
(s, 2H), 1.64 (s, 6H), 0.99 (s, 9H). 13C NMR (126 MHz, CDCl3) δ
159.01, 154.74, 153.13, 138.88, 136.87, 130.20, 129.19, 128.66,
128.06, 127.50, 127.44, 92.16, 56.72, 51.69, 47.62, 32.02, 31.74,
30.15. MS (ESI) calculated for C26H33N4O2, m/z 433.2598, found
5
mg, 2.30 mmol) and ammonium formate (145 mg, 2.30 mmol). 65 433.2615 (M + H)+.
The reaction mixture was stirred at room temperature for 10 min
and filtered through celite. Then the solvent was evaporated and
1-Benzyl-2-butyl-6-phenyl-N-(2,4,4-trimethylpentan-2-yl)-1H-
10 the residue was dissolved in water. This was extracted with
EtOAc (3 × 20 mL), washed with water and dried over sodium
imidazo[4,5-c]pyridin-4-amine (22b). Yellow solid (31 mg,
47%). H NMR (500 MHz, CDCl3) δ 8.02 (dd, J = 8.3, 1.2 Hz,
1
sulfate. The solvent was removed under vacuum to obtain 70 2H), 7.39 (t, J = 7.7 Hz, 2H), 7.34 – 7.27 (m, 4H), 7.07 (d, J = 6.7
compound 21a (45 mg, 51%). To a solution of compound 21a (42
mg, 0.11 mmol) in 7 mL of anhydrous THF were added
15 triethylamine (16 ꢀL, 0.12 mmol) and valeryl chloride (13 ꢀL,
0.11 mmol). The reaction mixture was refluxed for 1 h. The
Hz, 2H), 6.94 (s, 1H), 5.34 (s, 1H), 5.28 (s, 2H), 2.79 – 2.73 (m,
2H), 2.18 (s, 2H), 1.73 – 1.66 (m, 8H), 1.39 (dd, J = 15.0, 7.5 Hz,
2H), 1.03 (s, 9H), 0.89 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz,
CDCl3) δ 153.22, 150.01, 148.43, 141.28, 140.32, 136.16,
solvent was then removed under vacuum, and the residue was 75 129.61, 129.42, 129.15, 128.41, 128.07, 127.67, 127.54, 126.72,
dissolved in 5 mL of EtOH and NaOH (10 mg, 0.22 mmol) in 1
mL of H2O was added. The reaction mixture was refluxed for 18
20 h. The solvent was then removed under vacuum, and the residue
was dissolved in EtOAc and washed with water. The EtOAc
126.36, 126.28, 126.22, 91.77, 55.45, 51.63, 47.28, 31.98, 31.81,
30.33, 30.25, 27.50, 22.72, 13.90. MS (ESI) calculated for
C31H41N4, m/z 469.3326, found 469.3350 (M + H)+.
fraction was dried using sodium sulfate and evaporated and 80 1-Benzyl-2-butyl-6-phenyl-1H-imidazo[4,5-c]pyridin-4-amine
1
purified using column chromatography (20% EtOAc/hexane) to
obtain the compound 22a (25 mg, 51%). Compound 22a (22 mg,
25 0.049 mmol) was dissolved in 1 mL of HCl (4M in dioxane) and
stirred at room temperature for 30 min. Then the solvent was
removed under vacuum to obtain compound 23a (11 mg, 69%).
(23b). White solid (13 mg, 73%). H NMR (500 MHz, CDCl3) δ
7.91 (d, J = 7.2 Hz, 2H), 7.54 – 7.44 (m, 3H), 7.42 – 7.34 (m,
3H), 7.05 (dd, J = 6.4, 1.1 Hz, 2H), 6.83 (s, 1H), 5.37 (s, 2H),
2.88 – 2.78 (m, 2H), 1.78 (dd, J = 15.2, 7.8 Hz, 2H), 1.42 (dq, J =
85 14.7, 7.4 Hz, 2H), 0.93 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz,
CDCl3) δ 157.54, 149.18, 142.11, 141.93, 134.08, 132.07,
130.82, 129.68, 129.66, 129.01, 127.38, 126.25, 95.25, 48.13,
29.31, 27.40, 22.52, 13.84. HRMS (ESI) calculated for C23H25N4,
m/z 357.2074, found 357.2155 (M + H)+.
N4-Benzyl-6-butyl-3-nitro-N2-(2,4,4-trimethylpentan-2-
30 yl)pyridine-2,4-diamine (20a). Yellow oil (97 mg, 76%). 1H
NMR (500 MHz, CDCl3) δ 9.51 (t, J = 4.4 Hz, 1H), 9.40 (s, 1H),
7.37 (ddd, J = 7.1, 4.4, 1.6 Hz, 2H), 7.34 – 7.30 (m, 3H), 5.74 (s,
1H), 4.45 (d, J = 5.4 Hz, 2H), 2.44 (t, J = 7.5 Hz, 2H), 2.01 (s,
2H), 1.67 – 1.61 (m, 2H), 1.30 (dd, J = 15.0, 7.4 Hz, 2H), 0.96 (s,
35 9H), 0.89 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
166.49, 154.69, 152.58, 136.94, 129.07, 127.92, 127.42, 115.70,
90
4-(4-(Benzylamino)-5-nitro-6-((2,4,4-trimethylpentan-2-yl)
amino)pyridin-2-yl)benzonitrile (20c). 4ꢀCyanophenylboronic
acid was used as a reagent. Yellow solid (30 mg, 55%). 1H NMR
(500 MHz, CDCl3) δ 9.72 (s, 1H), 9.51 (s, 1H), 7.97 (d, J = 8.6
94.41, 56.59, 51.17, 47.44, 38.87, 31.95, 31.64, 30.61, 30.19, 95 Hz, 2H), 7.72 (d, J = 8.6 Hz, 2H), 7.44 – 7.31 (m, 5H), 6.38 (s,
22.43, 14.11. MS (ESI) calculated for C24H37N4O2, m/z 413.2911,
found 413.3267 (M + H)+.
1H), 4.59 (d, J = 5.4 Hz, 2H), 2.06 (s, 2H), 1.63 (s, 6H), 0.99 (s,
9H). 13C NMR (126 MHz, CDCl3) δ 156.75, 154.69, 153.26,
143.14, 136.53, 132.50, 129.28, 128.23, 127.93, 127.35, 118.79,
113.38, 93.05, 56.87, 51.72, 47.68, 32.01, 31.72, 30.10. MS (ESI)
40
1-Benzyl-2,6-dibutyl-1H-imidazo[4,5-c]pyridin-4-amine (23a).
1
White solid (11 mg, 69%). H NMR (500 MHz, CDCl3) δ 7.37 – 100 calculated for C27H32N5O2, m/z 458.2551, found 458.2571 (M +
7.31 (m, 3H), 7.10 (d, J = 6.9 Hz, 2H), 6.81 (s, 1H), 5.47 (s, 2H),
2.85 (t, 2H), 2.72 (t, 2H), 1.74 (dd, J = 15.3, 7.6 Hz, 2H), 1.67
H)+.
45 (ddd, J = 15.3, 10.4, 7.0 Hz, 2H), 1.38 (ddq, J = 14.8, 10.2, 7.4
Hz, 4H), 0.94 (t, J = 7.4 Hz, 3H), 0.90 (t, J = 7.4 Hz, 3H). 13C
4-(1-Benzyl-2-butyl-4-((2,4,4-trimethylpentan-2-yl)amino)-
1H-imidazo[4,5-c]pyridin-6-yl)benzamide (22c). Cyano group
NMR (126 MHz, CDCl3) δ 157.75, 150.13, 143.74, 137.10, 105 was converted into amide in the basic condition. Yellow solid (22
1
130.16, 129.22, 127.59, 124.62, 97.63, 35.61, 32.84, 30.27,
27.85, 23.31, 23.22, 14.12, 14.04. HRMS (ESI) calculated for
50 C21H29N4, m/z 337.2387, found 337.2476 (M + H)+.
mg, 41%). H NMR (500 MHz, CDCl3) δ 8.10 (d, J = 8.5 Hz,
2H), 7.84 (d, J = 8.5 Hz, 2H), 7.34 – 7.28 (m, 3H), 7.08 – 7.04
(m, 2H), 6.99 (s, 1H), 6.14 (s, 1H), 5.79 (s, 1H), 5.41 (s, 1H),
5.29 (s, 2H), 2.81 – 2.74 (m, 2H), 2.17 (s, 2H), 1.72 – 1.66 (m,
Compounds 23bꢀ23j were synthesized similarly as compound 110 8H), 1.43 – 1.33 (m, 2H), 1.03 (s, 9H), 0.89 (t, J = 7.4 Hz, 3H).
23a.
13C NMR (126 MHz, CDCl3) δ 169.46, 153.74, 150.08, 147.00,
144.87, 140.11, 135.97, 131.83, 129.20, 128.15, 127.62, 126.76,
126.74, 126.31, 92.63, 55.50, 51.55, 47.31, 31.95, 31.79, 30.25,
30.17, 27.46, 22.69, 13.89. MS (ESI) calculated for C32H42N5O,
55 N4-Benzyl-3-nitro-6-phenyl-N2-(2,4,4-trimethylpentan-2-
yl)pyridine-2,4-diamine (20b). Phenylboronic acid was used as
a reagent. Yellow solid (28 mg, 57%). 1H NMR (500 MHz, 115 m/z 512.3384, found 512.3408 (M + H)+.
CDCl3) δ 9.68 (s, 1H), 9.54 (s, 1H), 7.92 (ddd, J = 4.4, 2.5, 1.4
14 | Journal Name, [year], [vol], 00–00
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Page 15 of 19
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C3OB40816G
4-(4-Amino-1-benzyl-2-butyl-1H-imidazo[4,5-c]pyridin-6-
0.88 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 152.80,
yl)benzamide (23c). White solid (11 mg, 79%). H NMR (500 60 150.15, 143.24, 142.70, 140.71, 140.01, 136.16, 129.15, 128.83,
1
MHz, MeOD) δ 8.09 – 8.05 (m, 2H), 7.89 – 7.85 (m, 2H), 7.51 (s,
1H), 7.41 – 7.32 (m, 3H), 7.17 (d, J = 7.0 Hz, 2H), 5.62 (s, 2H),
3.76 – 3.72 (m, 1H), 3.68 – 3.64 (m, 1H), 3.58 (dd, J = 7.0, 2.7
Hz, 1H), 2.94 – 2.89 (m, 2H), 1.78 (dt, J = 15.3, 7.6 Hz, 2H),
1.42 (dd, J = 15.0, 7.5 Hz, 2H), 0.92 (t, J = 7.4 Hz, 3H). 13C
NMR (126 MHz, MeOD) δ 171.03, 159.91, 149.79, 143.90,
141.45, 137.23, 136.75, 136.73, 130.29, 129.74, 129.43, 128.43,
128.06, 126.33, 125.88, 108.87, 91.20, 55.44, 51.45, 47.25,
31.93, 31.79, 30.29, 30.16, 27.45, 22.71, 13.90. MS (ESI)
calculated for C29H39N4O, m/z 459.3118, found 459.3168 (M +
H)+.
5
65
1-Benzyl-2-butyl-6-(furan-3-yl)-1H-imidazo[4,5-c]pyridin-4-
1
amine (23e). Light yellow solid (12 mg, 64%). H NMR (500
10 127.76, 125.80, 98.94, 73.57, 72.45, 62.18, 43.73, 30.01, 27.99,
23.28, 14.04. HRMS (ESI) calculated for C24H26N5O, m/z
400.2132, found 400.2177 (M + H)+.
MHz, MeOD) δ 8.23 – 8.19 (m, 1H), 7.73 – 7.70 (m, 1H), 7.42 (s,
1H), 7.40 – 7.32 (m, 3H), 7.15 (d, J = 6.9 Hz, 2H), 6.97 (dd, J =
70 2.0, 0.9 Hz, 1H), 5.58 (s, 2H), 2.90 – 2.85 (m, 2H), 1.75 (dt, J =
15.3, 7.6 Hz, 2H), 1.40 (dq, J = 14.8, 7.4 Hz, 2H), 0.91 (t, J = 7.4
Hz, 3H). 13C NMR (126 MHz, MeOD) δ 159.50, 149.42, 146.46,
144.24, 142.92, 136.72, 135.38, 130.27, 129.41, 127.69, 124.89,
121.45, 109.21, 96.82, 48.27, 29.95, 27.91, 23.26, 14.03. HRMS
15 N4-Benzyl-5-nitro-N6-(2,4,4-trimethylpentan-2-yl)-[2,3'-
bipyridine]-4,6-diamine (20d). 4ꢀPyridinylboronic acid was
1
used as a reagent. Orange solid (135 mg, 82%). H NMR (500 75 (ESI) calculated for C21H23N4O, m/z 347.1866, found 347.1921
MHz, CDCl3) δ 9.74 (t, J = 5.1 Hz, 1H), 9.54 (s, 1H), 9.10 (dd, J
= 2.2, 0.6 Hz, 1H), 8.65 (dd, J = 4.8, 1.6 Hz, 1H), 8.21 – 8.16 (m,
20 1H), 7.42 – 7.36 (m, 5H), 7.35 – 7.31 (m, 1H), 6.38 (s, 1H), 4.59
(d, J = 5.5 Hz, 2H), 2.07 (s, 2H), 1.63 (s, 6H), 0.99 (s, 9H). 13C
(M + H)+.
N4-Benzyl-3-nitro-6-(thiophen-3-yl)-N2-(2,4,4-
trimethylpentan-2-yl)pyridine-2,4-diamine
(20f).
3ꢀ
NMR (126 MHz, CDCl3) δ 156.52, 154.78, 153.22, 150.86, 80 Thienylboronic acid was used as a reagent. Yellow solid (107 mg,
1
148.93, 136.60, 134.75, 134.36, 129.26, 128.17, 127.35, 123.54,
116.37, 92.38, 56.86, 51.65, 47.65, 32.01, 31.72, 30.11. MS (ESI)
25 calculated for C25H32N5O2, m/z 434.2551, found 434.2563 (M +
H)+.
76%). H NMR (500 MHz, CDCl3) δ 9.69 (s, 1H), 9.52 (s, 1H),
7.89 (dd, J = 3.0, 1.2 Hz, 1H), 7.49 (dd, J = 5.1, 1.2 Hz, 1H), 7.42
– 7.30 (m, 6H), 6.26 (s, 1H), 4.56 (d, J = 5.4 Hz, 2H), 2.08 (s,
2H), 1.63 (s, 6H), 0.99 (s, 9H). 13C NMR (126 MHz, CDCl3) δ
85 154.88, 154.61, 153.25, 142.21, 136.90, 129.19, 128.05, 127.38,
126.59, 126.56, 126.28, 91.99, 56.68, 51.59, 47.61, 32.02, 31.72,
30.16. MS (ESI) calculated for C24H31N4O2S, m/z 439.2162,
found 439.2183 (M + H)+.
1-Benzyl-2-butyl-6-(pyridin-3-yl)-1H-imidazo[4,5-c]pyridin-4-
1
amine (23d). Light yellow solid (10 mg, 72%). H NMR (500
30 MHz, MeOD) δ 9.28 (d, J = 1.9 Hz, 1H), 8.94 (dd, J = 5.5, 1.2
Hz, 1H), 8.83 (d, J = 8.2 Hz, 1H), 8.11 (ddd, J = 8.2, 5.6, 0.5 Hz,
1H), 7.72 (s, 1H), 7.40 – 7.32 (m, 3H), 7.18 (d, J = 6.9 Hz, 2H),
5.65 (s, 2H), 2.98 – 2.91 (m, 2H), 1.78 (dd, J = 15.3, 7.7 Hz, 2H),
1.42 (dd, J = 15.0, 7.5 Hz, 2H), 0.92 (t, J = 7.4 Hz, 3H). 13C
35 NMR (126 MHz, MeOD) δ 150.19, 143.97, 143.57, 143.11,
136.48, 130.41, 130.32, 129.52, 128.03, 127.74, 127.73, 100.47,
90
1-Benzyl-2-butyl-6-(thiophen-3-yl)-N-(2,4,4-trimethylpentan-
2-yl)-1H-imidazo[4,5-c]pyridin-4-amine (22f). Brown solid (25
mg, 35%). H NMR (500 MHz, CDCl3) δ 7.79 (dd, J = 3.1, 1.2
1
Hz, 1H), 7.58 (dd, J = 5.0, 1.2 Hz, 1H), 7.33 – 7.27 (m, 4H), 7.06
48.55, 29.93, 27.90, 23.26, 14.02. HRMS (ESI) calculated for 95 (d, J = 6.6 Hz, 2H), 6.79 (s, 1H), 5.35 (s, 1H), 5.25 (s, 2H), 2.78 –
C22H24N5, m/z 358.2026, found 358.2067 (M + H)+.
2.70 (m, 2H), 2.17 (s, 2H), 1.70 (dd, J = 8.7, 7.0 Hz, 3H), 1.67 (s,
6H), 1.38 (dq, J = 14.7, 7.4 Hz, 2H), 1.26 (dd, J = 8.7, 5.6 Hz,
1H), 1.02 (s, 9H), 0.89 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz,
CDCl3) δ 153.04, 150.02, 144.89, 144.32, 140.11, 136.13,
40 N4-Benzyl-6-(furan-3-yl)-3-nitro-N2-(2,4,4-trimethylpentan-2-
yl)pyridine-2,4-diamine (20e). 3ꢀFurylboronic acid was used as
a reagent. Yellow solid (95 mg, 70%). 1H NMR (500 MHz, 100 129.14, 128.07, 126.45, 126.34, 125.94, 125.41, 121.55, 91.54,
CDCl3) δ 9.68 (t, J = 4.8 Hz, 1H), 9.49 (s, 1H), 7.95 (dd, J = 1.5,
0.8 Hz, 1H), 7.45 (t, J = 1.7 Hz, 1H), 7.41 – 7.34 (m, 4H), 7.34 –
45 7.30 (m, 1H), 6.69 (dd, J = 1.9, 0.8 Hz, 1H), 6.07 (s, 1H), 4.53 (d,
J = 5.4 Hz, 2H), 2.05 (s, 2H), 1.60 (s, 6H), 0.98 (s, 9H). 13C NMR
55.43, 51.49, 50.96, 47.26, 31.96, 31.80, 30.26, 30.21, 27.46,
22.71, 13.89. MS (ESI) calculated for C29H39N4S, m/z 475.2890,
found 475.2922 (M + H)+.
(126 MHz, CDCl3) δ 154.88, 153.66, 153.09, 143.99, 143.68, 105 1-Benzyl-2-butyl-6-(thiophen-3-yl)-1H-imidazo[4,5-c]pyridin-
1
136.85, 129.19, 128.06, 127.39, 127.36, 108.83, 91.66, 56.68,
51.51, 47.61, 31.99, 31.71, 30.13. MS (ESI) calculated for
50 C24H31N4O3, m/z 423.2391, found 423.2401 (M + H)+.
4-amine (23f). White solid (9 mg, 53%). H NMR (500 MHz,
MeOD) δ 8.04 (dd, J = 2.9, 1.4 Hz, 1H), 7.66 (dd, J = 5.1, 2.9 Hz,
1H), 7.58 (dd, J = 5.1, 1.4 Hz, 1H), 7.48 (s, 1H), 7.41 – 7.32 (m,
3H), 7.17 (d, J = 6.9 Hz, 2H), 5.59 (s, 2H), 2.94 – 2.86 (m, 2H),
110 1.76 (dt, J = 15.3, 7.6 Hz, 2H), 1.41 (dq, J = 14.8, 7.4 Hz, 2H),
0.91 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, MeOD) δ 159.63,
149.38, 144.24, 137.90, 136.73, 135.24, 130.28, 129.42, 129.31,
127.74, 126.73, 126.03, 124.88, 97.21, 49.07, 29.96, 27.94,
23.26, 14.03. HRMS (ESI) calculated for C21H23N4S, m/z
1-Benzyl-2-butyl-6-(furan-3-yl)-N-(2,4,4-trimethylpentan-2-
yl)-1H-imidazo[4,5-c]pyridin-4-amine (22e). Brown oil (30 mg,
1
41%). H NMR (500 MHz, CDCl3) δ 7.94 (dd, J = 1.6, 0.7 Hz,
55 1H), 7.41 (t, J = 1.7 Hz, 1H), 7.34 – 7.27 (m, 3H), 7.05 (d, J = 6.6
Hz, 2H), 6.76 (dd, J = 1.8, 0.8 Hz, 1H), 6.61 (s, 1H), 5.30 (s, 1H),
5.24 (s, 2H), 2.76 – 2.71 (m, 2H), 2.13 (s, 2H), 1.70 – 1.65 (m, 115 363.1638, found 363.1686 (M + H)+.
2H), 1.64 (s, 6H), 1.36 (dt, J = 14.7, 7.4 Hz, 2H), 1.02 (s, 9H),
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 15

Organic & Biomolecular Chemistry
Page 16 of 19
View Article Online
DOI: 10.1039/C3OB40816G
N4,6-Dibenzyl-3-nitro-N2-(2,4,4-trimethylpentan-2-
yl)pyridine-2,4-diamine (20g). Benzylboronic acid pinacol ester 60 136.53, 134.52, 130.68, 130.25, 129.85, 129.42, 127.74, 124.41,
(126 MHz, MeOD) δ 159.11, 149.35, 144.27, 143.93, 138.37,
1
was used as a reagent. Orange oil (135 mg, 91%). H NMR (500
MHz, CDCl3) δ 9.54 (t, J = 5.0 Hz, 1H), 9.37 (s, 1H), 7.36 (ddd, J
= 7.4, 4.5, 1.4 Hz, 2H), 7.31 (dt, J = 9.8, 4.4 Hz, 1H), 7.27 (ddd, J
= 6.7, 3.3, 1.4 Hz, 5H), 7.22 – 7.18 (m, 3H), 5.75 (s, 1H), 4.40 (d,
99.41, 39.40, 30.01, 27.84, 23.26, 21.08, 14.03. HRMS (ESI)
calculated for C25H29N4, m/z 385.2387, found 385.2451 (M +
H)+.
5
J = 5.4 Hz, 2H), 3.75 (s, 2H), 1.89 (s, 2H), 1.49 (s, 6H), 0.90 (s, 65 N4-Benzyl-3-nitro-6-(4-(trifluoromethoxy)benzyl)-N2-(2,4,4-
9H). 13C NMR (126 MHz, CDCl3) δ 164.70, 154.68, 152.84,
138.77, 136.80, 129.45, 129.07, 128.54, 127.91, 127.46, 126.50,
trimethylpentan-2-yl)pyridine-2,4-diamine
(20i).
4ꢀ
(Trifluoromethoxy)benzylboronic acid pinacol ester was used as
1
10 124.96, 115.72, 94.56, 56.63, 51.05, 47.44, 45.61, 31.87, 31.60,
30.09, 24.86. MS (ESI) calculated for C27H35N4O2, m/z 447.2755,
found 447.2737 (M + H)+.
a reagent. Yellow solid (115 mg, 70%). H NMR (500 MHz,
CDCl3) δ 9.57 (t, J = 5.0 Hz, 1H), 9.37 (s, 1H), 7.38 – 7.29 (m,
70 3H), 7.28 (t, J = 1.8 Hz, 2H), 7.21 – 7.17 (m, 2H), 7.10 (d, J = 7.9
Hz, 2H), 5.74 (s, 1H), 4.43 (d, J = 5.5 Hz, 2H), 1.83 (s, 2H), 1.45
(s, 6H), 0.88 (s, 9H). 13C NMR (126 MHz, CDCl3) δ 163.87,
154.70, 152.91, 137.54, 136.71, 130.77, 129.10, 127.95, 127.33,
121.09, 115.75, 94.57, 56.63, 50.96, 47.44, 44.69, 31.82, 31.55,
1,6-Dibenzyl-2-butyl-N-(2,4,4-trimethylpentan-2-yl)-1H-
15 imidazo[4,5-c]pyridin-4-amine (22g). Light brown solid (55
mg, 60%). H NMR (500 MHz, CDCl3) δ 7.33 – 7.27 (m, 5H),
1
7.24 (t, J = 7.6 Hz, 2H), 7.14 (t, J = 7.3 Hz, 1H), 7.01 (d, J = 6.4 75 30.02. MS (ESI) calculated for C28H34F3N4O3, m/z 531.2578,
Hz, 2H), 6.31 (s, 1H), 5.22 (s, 1H), 5.16 (s, 2H), 3.96 (s, 2H),
2.74 – 2.67 (m, 2H), 2.02 (s, 2H), 1.66 – 1.61 (m, 2H), 1.56 (s,
20 6H), 1.35 (dd, J = 15.0, 7.5 Hz, 2H), 0.93 (s, 9H), 0.86 (t, J = 7.4
Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 152.37, 151.90, 150.06,
found 531.2738 (M + H)+.
1-Benzyl-2-butyl-6-(4-(trifluoromethoxy)benzyl)-1H-imidazo
[4,5-c]pyridin-4-amine (23i). White solid (21 mg, 47%). 1H
141.57, 140.10, 136.28, 129.31, 129.06, 128.21, 127.95, 126.38, 80 NMR (500 MHz, CDCl3) δ 7.37 – 7.29 (m, 5H), 7.21 (d, J = 8.0
125.78, 124.94, 93.59, 55.35, 50.98, 47.19, 45.39, 31.85, 31.68,
30.41, 30.27, 27.42, 22.67, 13.87. MS (ESI) calculated for
25 C32H43N4, m/z 483.3482, found 483.3526 (M + H)+.
Hz, 2H), 7.08 (d, J = 6.5 Hz, 2H), 6.79 (s, 1H), 5.45 (s, 2H), 4.09
(s, 2H), 2.87 (dd, J = 9.3, 6.1 Hz, 2H), 1.74 (dt, J = 15.3, 7.6 Hz,
2H), 1.41 (dt, J = 15.0, 7.4 Hz, 2H), 0.91 (t, J = 7.4 Hz, 3H). 13C
NMR (126 MHz, MeOD) δ 157.96, 150.50, 149.43, 149.42,
1,6-Dibenzyl-2-butyl-1H-imidazo[4,5-c]pyridin-4-amine (23g). 85 143.48, 138.54, 136.96, 131.59, 130.16, 129.25, 127.66, 124.97,
1
White solid (23 mg, 58%). H NMR (500 MHz, DMSO) δ 13.63
(s, 1H), 8.27 (s, 2H), 7.37 – 7.29 (m, 7H), 7.27 – 7.23 (m, 1H),
30 7.15 (s, 1H), 7.11 (d, J = 6.9 Hz, 2H), 5.52 (s, 2H), 4.08 (s, 2H),
2.86 – 2.79 (m, 2H), 1.61 (dt, J = 15.3, 7.6 Hz, 2H), 1.31 (dt, J =
122.37, 99.02, 40.76, 30.29, 27.86, 23.31, 14.05. HRMS (ESI)
calculated for C25H26F3N4O, m/z 455.2053, found 455.2131 (M +
H)+.
14.9, 7.4 Hz, 2H), 0.81 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, 90 N4-Benzyl-3-nitro-6-phenethyl-N2-(2,4,4-trimethylpentan-2-
DMSO) δ 156.72, 148.03, 142.12, 141.72, 137.39, 135.93,
128.96, 128.72, 128.70, 127.92, 126.98, 126.63, 123.29, 97.60,
35 46.84, 40.11, 38.20, 28.89, 26.28, 21.69, 13.61. HRMS (ESI)
calculated for C24H27N4, m/z 371.2230, found 371.2285 (M +
H)+.
yl)pyridine-2,4-diamine (20j). 2ꢀPhenylethylboronic acid was
used as a reagent. Yellow oil (85 mg, 59%). 1H NMR (500 MHz,
CDCl3) δ 9.52 (t, J = 4.9 Hz, 1H), 9.43 (s, 1H), 7.36 (t, J = 7.2
Hz, 2H), 7.33 – 7.28 (m, 4H), 7.27 – 7.25 (m, 3H), 7.19 (d, J =
95 7.3 Hz, 1H), 7.17 (d, J = 7.0 Hz, 2H), 5.69 (s, 1H), 4.39 (d, J =
5.4 Hz, 2H), 2.99 (dd, J = 9.1, 6.8 Hz, 2H), 2.75 (dd, J = 9.1, 6.8
Hz, 2H), 2.04 (s, 2H), 1.58 (s, 6H), 0.98 (s, 9H). 13C NMR (126
MHz, CDCl3) δ 165.06, 154.74, 152.65, 141.77, 136.85, 129.09,
128.52, 128.49, 127.95, 127.39, 126.10, 115.78, 94.52, 56.68,
N4-Benzyl-6-(4-methylbenzyl)-3-nitro-N2-(2,4,4-
40 trimethylpentan-2-yl)pyridine-2,4-diamine (20h). 4ꢀMethyl
benzylboronic acid pinacol ester was used as a reagent. Yellow
oil (130 mg, 91%). 1H NMR (500 MHz, CDCl3) δ 9.52 (t, J = 5.0 100 51.35, 47.41, 40.87, 34.61, 31.97, 31.69, 30.20. MS (ESI)
Hz, 1H), 9.37 (s, 1H), 7.37 – 7.30 (m, 3H), 7.28 – 7.24 (m, 3H),
7.10 – 7.03 (m, 7H), 5.74 (s, 1H), 4.39 (d, J = 5.4 Hz, 2H), 3.70
45 (s, 2H), 2.31 (s, 3H), 1.90 (s, 2H), 1.51 (s, 6H), 0.90 (s, 9H). 13C
NMR (126 MHz, CDCl3) δ 165.00, 154.68, 152.83, 136.82,
calculated for C28H37N4O2, m/z 461.2911, found 461.3096 (M +
H)+.
1-Benzyl-2-butyl-6-phenethyl-1H-imidazo[4,5-c]pyridin-4-
1
135.98, 135.70, 135.50, 134.26, 129.28, 129.23, 129.12, 129.05, 105 amine (23j). Yellow solid (11 mg, 42%). H NMR (500 MHz,
128.98, 127.89, 127.47, 115.70, 94.47, 83.50, 56.63, 51.03,
47.44, 45.22, 31.87, 31.57, 30.12, 24.87, 21.19, 21.11. MS (ESI)
50 calculated for C28H37N4O2, m/z 461.2911, found 461.3003 (M +
H)+.
CDCl3) δ 7.36 – 7.30 (m, 3H), 7.23 – 7.18 (m, 2H), 7.13 (ddd, J =
5.7, 3.7, 1.5 Hz, 3H), 7.02 (d, J = 6.5 Hz, 2H), 6.68 (s, 1H), 5.38
(s, 2H), 2.99 (s, 4H), 2.85 – 2.79 (m, 2H), 1.69 (ddd, J = 13.2,
8.5, 6.7 Hz, 2H), 1.37 (dq, J = 14.8, 7.4 Hz, 2H), 0.89 (t, J = 7.4
110 Hz, 3H). 13C NMR (126 MHz, MeOD) δ 157.35, 150.48, 148.64,
143.39, 141.89, 137.14, 130.14, 129.53, 129.43, 129.16, 127.59,
127.22, 124.68, 97.93, 48.17, 38.42, 36.84, 30.28, 27.83, 23.31,
14.03. HRMS (ESI) calculated for C25H29N4, m/z 385.2387,
found 385.2446 (M + H)+.
1-Benzyl-2-butyl-6-(4-methylbenzyl)-1H-imidazo[4,5-c]
pyridin-4-amine (23h). White solid (19 mg, 66%). 1H NMR
55 (500 MHz, MeOD) δ 7.38 – 7.32 (m, 3H), 7.16 (d, J = 7.9 Hz,
2H), 7.13 – 7.08 (m, 4H), 6.89 (s, 1H), 5.49 (s, 2H), 4.08 (s, 2H),
2.92 – 2.87 (m, 2H), 2.32 (s, 3H), 1.76 (dt, J = 15.3, 7.6 Hz, 2H), 115
1.40 (dt, J = 14.8, 7.4 Hz, 2H), 0.91 (t, J = 7.4 Hz, 3H). 13C NMR
Synthesis of compound 24: 3-Nitrobenzo[g]quinolin-4-ol.
16 | Journal Name, [year], [vol], 00–00
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Organic & Biomolecular Chemistry
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DOI: 10.1039/C3OB40816G
Nitromethane (0.96 mL, 18 mmol) was added dropwise to a
7.47 (m, 4H), 7.42 (ddd, J = 11.0, 5.4, 3.0 Hz, 1H), 5.28 (d, J =
solution of NaOH (2.2 g, 54 mmol) in water (5 mL), at 0 °C. The 60 5.9 Hz, 2H). 13C NMR (126 MHz, CDCl3) δ 152.50, 147.73,
mixture was then warmed to 40 ºC and nitromethane (0.96 mL,
18 mmol) was again added slowly at 40ꢀ45 ºC. The temperature
was maintained until a clear solution was obtained. The reaction
mixture was then heated to 55 ºC for 2ꢀ5 minutes, cooled to 30
ºC, poured onto crushed ice and acidified with conc. HCl (5 mL).
The resultant solution of methazoic acid was added immediately
to a filtered solution of 3ꢀaminoꢀ2ꢀnaphtholic acid (3 g, 16 mmol)
145.88, 136.85, 135.48, 130.53, 129.64, 129.12, 129.03, 128.98,
128.83, 128.11, 128.04, 127.29, 126.86, 124.21, 118.09, 53.19.
MS (ESI) calculated for C20H16N3O2, m/z 330.1237, found
330.1304 (M + H)+.
5
65
Synthesis of compound 30: 1-Benzyl-2-butyl-1H-benzo[g]
imidazo[4,5-c]quinolin-4-amine. To a solution of compound 26
(300 mg, 0.91 mmol) in 20 mL of MeOH were added zinc dust
(594 mg, 9.10 mmol) and ammonium formate (574 mg, 9.10
10 and conc. HCl (1 mL) in water (20 mL). The reaction mixture
was allowed to stand at room temperature for 12 h. After
filtration, the residue obtained was washed with water, and dried 70 mmol). The reaction mixture was stirred at room temperature for
(1.1 g, 90%). A solution of intermediate (2 g, 7.75 mmol) in
acetic anhydride (10 mL) was placed in a 2ꢀneck flask fitted with
15 a reflux condenser. It was stirred and heated to 105 ºC until a
clear solution was obtained. Heating was then discontinued and
30 min and filtered through celite. Then the solvent was
evaporated and the residue was dissolved in water. This was
extracted with EtOAc (3 × 20 mL), washed with water and dried
over sodium sulfate. The solvent was removed under vacuum to
potassium acetate (0.77 g, 7.90 mmol) was added. The mixture 75 obtain compound 27 (100 mg, 37%). To a solution of compound
was then refluxed for 15 min with vigorous stirring, until a solid
started to precipitate. The reaction mixture was then slowly
20 cooled to room temperature. The residue was filtered, washed
with glacial acetic acid until the washings were colorless, then
27 (98 mg, 0.33 mmol) in 10 mL of anhydrous THF were added
triethylamine (48 ꢀL, 0.35 mmol) and valeryl chloride (40 ꢀL,
0.33 mmol). The reaction mixture was refluxed for 2 h. The
solvent was then removed under vacuum, and the residue was
suspended in water, filtered, washed with water and dried at 110 80 dissolved in 10 mL of EtOH and NaOH (26 mg, 0.66 mmol) in 1
ºC to get compound 24 (0.93 g, 50%). 1H NMR (500 MHz,
DMSO) δ 13.12 (s, 1H), 9.28 (s, 1H), 8.95 (s, 1H), 8.27 – 8.21
25 (m, 2H), 8.11 (d, J = 8.4 Hz, 1H), 7.69 (t, J = 7.1 Hz, 1H), 7.61 (t,
J = 7.1 Hz, 1H). 13C NMR (126 MHz, DMSO) δ 168.61, 144.40,
mL of H2O was added. The reaction mixture was refluxed for 2 h.
The solvent was then removed under vacuum, and the residue
was dissolved in EtOAc and washed with water. The EtOAc
fraction was dried using sodium sulfate and evaporated and
135.05, 134.63, 130.14, 129.54, 128.94, 128.71, 127.46, 127.41, 85 purified using column chromatography (10% MeOH/CH2Cl2) to
126.65, 126.48, 116.78. MS (ESI) calculated for C13H8N2O3, m/z
240.05, found 263.04 (M + Na)+.
obtain the compound 28 (76 mg, 63%). To a solution of
compound 28 (76 mg, 0.21 mmol) in a solvent mixture of
MeOH:CH2Cl2:CHCl3 (1:10:10) was added 3ꢀchloroperoxy
benzoic acid (443 mg, 1.98 mmol), and the solution was refluxed
30
Synthesis of compound 25: 4-Chloro-3-nitrobenzo[g]
quinoline. A suspension of compound 24 (2.0 g, 8.30 mmol) in 90 at 45ꢀ50 ºC for 1 h. The solvent was then removed and the
phosphorus(V) oxychloride was placed in a pressure vessel and it
was heated at 150 ºC. After a clear solution was obtained, the
35 reaction mixture was kept at 150 ºC for 1 h. Then it was slowly
cooled to room temperature and the solvent was evaporated under
residue was purified using column chromatography (10%
MeOH/CH2Cl2) to obtain the Nꢀoxide derivative 29 (64 mg,
80%). To a solution of compound 29 (64 mg, 0.17 mol) in 10 mL
of CH2Cl2 was added benzoyl isocyanate (37 mg, 0.25 mmol) and
vacuum. The residue was poured over crushed ice while stirring 95 heated at 45 ºC for 18 h. The solvent was then removed under
and the formed solid was filtered, washed with water and dried to
obtain compound 25 (1.95 g, 91%). 1H NMR (500 MHz, DMSO)
40 δ 13.34 (d, J = 6.9 Hz, 1H), 9.26 (d, J = 7.3 Hz, 1H), 8.95 (s, 1H),
8.25 (d, J = 9.4 Hz, 2H), 8.11 (d, J = 8.4 Hz, 1H), 7.72 – 7.67 (m,
vacuum, and the residue was dissolved in 15 mL of anhydrous
MeOH, followed by the addition of excess sodium methoxide.
The reaction mixture was then heated at 80 ºC for 2 h. The
solvent was removed under vacuum and the residue was purified
1H), 7.61 (ddd, J = 8.1, 6.8, 1.1 Hz, 1H). 13C NMR (126 MHz, 100 using column chromatography (10% MeOH/CH2Cl2) to obtain
DMSO) δ 168.64, 144.23, 134.96, 134.64, 130.16, 129.56,
128.98, 128.70, 127.48, 127.43, 126.69, 126.48, 116.74.
the compound 30 (20 mg, 30%).1H NMR (500 MHz, DMSO) δ
8.40 (s, 1H), 8.05 (s, 1H), 7.88 (d, J = 8.3 Hz, 1H), 7.77 (d, J =
8.3 Hz, 1H), 7.38 (d, J = 7.2 Hz, 1H), 7.33 (dd, J = 12.9, 5.2 Hz,
3H), 7.24 (d, J = 7.4 Hz, 1H), 7.16 (d, J = 7.4 Hz, 2H), 6.91 (s,
45
Synthesis of compound 26: N-Benzyl-3-nitrobenzo[g]quinolin-
4-amine. To a solution of compound 25 (1.0 g, 3.90 mol) in 20 105 2H), 6.02 (s, 2H), 3.00 – 2.92 (m, 2H), 1.74 (dt, J = 15.4, 7.6 Hz,
mL of CH2Cl2 was added triethylamine (0.81 mL, 5.80 mmol)
and benzylamine (0.50 mL, 4.60 mmol). The reaction mixture
2H), 1.40 (dd, J = 14.9, 7.4 Hz, 2H), 0.88 (t, J = 7.4 Hz, 3H). 13
C
NMR (126 MHz, DMSO) δ 136.73, 131.80, 128.91, 127.99,
127.81, 127.46, 126.82, 125.81, 123.63, 118.72, 48.20, 29.71,
26.27, 21.85, 13.69. HRMS (ESI) calculated for C25H25N4, m/z
50 was refluxed for 2 h. The solvent was then evaporated under
vacuum and H2O was added to the residue. The solution was
extracted with CH2Cl2 (3 × 20 mL), washed with water and dried 110 381.2074, found 381.2089 (M + H)+.
over sodium sulfate. The solvent was evaporated and the residue
was purified using silica gel column chromatography (5%
55 MeOH/CH2Cl2) to obtain compound 26 as a yellow solid (1.1 g,
88%). 1H NMR (500 MHz, CDCl3) δ 10.57 (s, 1H), 9.41 (s, 1H),
Human TLR-7/-8 Reporter Gene assays (NF-κB induction).
The induction of NFꢀκB was quantified using HEKꢀBlueꢀ7
(hTLR7ꢀspecific) and HEKꢀBlueꢀ8 (hTLR8ꢀspecific) cells as
8.87 (s, 1H), 8.50 (s, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 115 previously described by us.33,45,46 HEK293 cells stably coꢀ
8.4 Hz, 1H), 7.65 – 7.60 (m, 1H), 7.55 – 7.51 (m, 1H), 7.50 –
transfected with human TLR7 or human TLR8, MD2, and
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 17

Organic & Biomolecular Chemistry
Page 18 of 19
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DOI: 10.1039/C3OB40816G
secreted alkaline phosphatase (sAP), were maintained in HEKꢀ
nominal T lymphocytes (CD3+CD56ꢀ) were quantified using
Blue™ Selection medium containing zeocin and normocin. 60 FlowJo v 7.0 software (Treestar, Ashland, OR).
Stable expression of secreted alkaline phosphatase (sAP) under
control of NFꢀκB/APꢀ1 promoters is inducible by appropriate
5
TLR agonists, and extracellular sAP in the supernatant is
Acknowledgments
proportional to NFꢀκB induction. HEKꢀBlue cells were incubated
This work was supported by NIH/NIAID contract
at a density of ~105 cells/ml in a volume of 80 ꢂl/well, in 384ꢀ 65 HSN272200900033C.
well, flatꢀbottomed, cell cultureꢀtreated microtiter plates until
confluency was achieved, and subsequently stimulated with
10 graded concentrations of stimuli. sAP was assayed
spectrophotometrically using an alkaline phosphataseꢀspecific
chromogen (present in HEKꢀdetection medium as supplied by the
vendor) at 620 nm.
Notes and references
Department of Medicinal Chemistry, University of Kansas,
Multidisciplinary Research Building, Room 320D, 2030 Becker Drive,
70 Lawrence KS 66047. Tel: 785-864-1610; Fax: 785-864-1961
E-mail: sdavid@ku.edu
15 Immunoassays for Interferon (IFN)-α, and cytokines. Fresh
human peripheral blood mononuclear cells (hPBMC) were
isolated from human blood obtained by venipuncture with
informed consent and as per institutional guidelines on Ficollꢀ
Hypaque gradients as described elsewhere.64 Aliquots of PBMCs
20 (105 cells in 100 ꢀL/well) were stimulated for 12 h with graded
concentrations of test compounds. Supernatants were isolated by
centrifugation, and were assayed in triplicates using either highꢀ
sensitivity multiꢀsubtype IFNꢀα ELISA kits (PBL Interferon
Source, Piscataway, NJ and R&D Systems, Inc., Minneapolis,
25 MN), or analyteꢀspecific multiplexed cytokine/chemokine bead
array assays as reported by us previously.65
† Electronic Supplementary Information (ESI) available: Characterization
of intermediates and final compounds (1H, 13C, mass spectra).
75
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