Synthesis and Antileishmanial Evaluation of Arylimidamide-Azole Hybrids Containing a Phenoxyalkyl Linker

Article abstract of DOI:10.1021/acsinfecdis.0c00855

Due to the limitations of existing medications, there is a critical need for new drugs to treat visceral leishmaniasis. Since arylimidamides and antifungal azoles both show oral activity in murine visceral leishmaniasis models, a molecular hybridization approach was employed where arylimidamide and azole groups were separated by phenoxyalkyl linkers in an attempt to capitalize on the favorable antileishmanial properties of both series. Among the target compounds synthesized, a greater antileishmanial potency against intracellular Leishmania donovani was observed as the linker length increased from two to eight carbons and when an imidazole ring was employed as the terminal group compared to a 1,2,4-triazole group. Compound 24c (N-(4-((8-(1H-imidazol-1-yl)octyl)oxy)-2-isopropoxyphenyl) picolinimidamide) displayed activity against L. donovani intracellular amastigotes with an IC50 value of 0.53 μM. When tested in a murine visceral leishmaniasis model, compound 24c at a dose of 75 mg/kg/day p.o. for five consecutive days resulted in a modest 33% decrease in liver parasitemia compared to the control group, indicating that further optimization of these molecules is needed. While potent hybrid compounds bearing an imidazole terminal group were also strong inhibitors of recombinant CYP51 from L. donovani, as assessed by a fluorescence-based assay, additional targets are likely to play an important role in the antileishmanial action of these compounds.

Full text of DOI:10.1021/acsinfecdis.0c00855

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Article
Synthesis and Antileishmanial Evaluation of Arylimidamide−Azole
Hybrids Containing a Phenoxyalkyl Linker
Ahmed Abdelhameed, Mei Feng, April C. Joice, Emilia M. Zywot, Yiru Jin, Chris La Rosa, Xiaoping Liao,
Heidi L. Meeds, Yena Kim, Junan Li, Craig A. McElroy, Michael Zhuo Wang, and Karl A. Werbovetz*
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ABSTRACT: Due to the limitations of existing medications, there is a critical need for new drugs to treat visceral leishmaniasis.
Since arylimidamides and antifungal azoles both show oral activity in murine visceral leishmaniasis models, a molecular hybridization
approach was employed where arylimidamide and azole groups were separated by phenoxyalkyl linkers in an attempt to capitalize on
the favorable antileishmanial properties of both series. Among the target compounds synthesized, a greater antileishmanial potency
against intracellular Leishmania donovani was observed as the linker length increased from two to eight carbons and when an
imidazole ring was employed as the terminal group compared to a 1,2,4-triazole group. Compound 24c (N-(4-((8-(1H-imidazol-1-
yl)octyl)oxy)-2-isopropoxyphenyl) picolinimidamide) displayed activity against L. donovani intracellular amastigotes with an IC50
value of 0.53 μM. When tested in a murine visceral leishmaniasis model, compound 24c at a dose of 75 mg/kg/day p.o. for five
consecutive days resulted in a modest 33% decrease in liver parasitemia compared to the control group, indicating that further
optimization of these molecules is needed. While potent hybrid compounds bearing an imidazole terminal group were also strong
inhibitors of recombinant CYP51 from L. donovani, as assessed by a fluorescence-based assay, additional targets are likely to play an
important role in the antileishmanial action of these compounds.
KEYWORDS: Leishmaniasis, azole antifungal, arylimidamide, CYP51, molecular hybridization
leishmaniasis in India than in East Africa,3 although higher
eishmaniasis continues to be an important public health
Lproblem. According to recent World Health Organization doses of amphotericin B are effective against East African
visceral leishmaniasis.4 Like the antimonials and amphotericin
estimates, approximately 30,000 and over one million new
B, paromomycin is given by injection (for 3 weeks in the case
of this aminoglycoside). As with amphotericin B, paromomycin
is more effective in treating visceral leishmaniasis on the Indian
subcontinent than in East Africa.5 Miltefosine has the
advantage of being the only oral drug of the four listed
above; however, it is relatively expensive and teratogenic, and
cases of visceral leishmaniasis and cutaneous leishmaniasis
occur each year, respectively, and over 1 billion people live in
areas where leishmaniasis occurs.1 The visceral form of
leishmaniasis is the most serious; without drug treatment,
symptomatic visceral leishmaniasis is usually fatal. The
treatment of visceral leishmaniasis generally involves the use
of pentavalent antimonials, amphotericin B, paromomycin, or
miltefosine or combinations of two of these drugs. Pentavalent
antimonials suffer from cardiotoxicity and the loss of efficacy
on the Indian subcontinent.2 Amphotericin B is expensive
when given as a liposomal formulation and nephrotoxic when
administered as the more economical deoxycholate formula-
tion; this drug is more effective for treating visceral
Special Issue: In Celebration of Jonathan Venner-
strom: A Pioneer in Neglected Tropical Diseases
Received: December 5, 2020
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© XXXX American Chemical Society
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A

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Figure 1. Structures of 1−4 and the AIA−azole scaffold explored here.
a
Scheme 1. Synthesis of 9a−l
a
Reagents and conditions: (a) dibromoalkane, K2CO3, CH3CN, reflux (62−93%); (b) imidazole or 1,2,4-triazole, K2CO3, CH3CN (49−86%); (c)
SnCl2·2H2O, EtOAc (81−99%); (d) naphthalen-2-ylmethyl pyridine-2-carbimidothioate·HBr, CH3CN/EtOH (1:3), rt (33−70%).
its efficacy has decreased for the treatment of visceral disease in
recent years.2 Given the weaknesses of the current anti-
leishmanial drugs, the need for new, effective oral treatments is
clear.
The potency of arylimidamides (AIAs) against Leishmania
has been known for some time. Initially termed reversed
amidines, AIAs displayed potent activity against Leishmania
donovani6,7 and Leishmania species responsible for cutaneous
disease.7,8 Such compounds contain pyridyl imidamide groups
at both ends of the molecule and are thus referred to as bis-
AIAs. Mono-AIAs (which contain only one pyridyl imidamide
group) were less potent than bis-AIAs, but several mono-AIAs
B
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a
Scheme 2. Synthesis of 9m,n
a
Reagents and conditions: (a) 1,8-dibromooctane, K2CO3, CH3CN, reflux (92%); (b) imidazole, K2CO3, CH3CN, reflux (86% 7h); (c) pyrrole,
KOH, DMF, reflux (35% 7m); (d) SnCl2·2H2O, EtOAc (85% 8h); (e) Zn, NH4Cl, MeOH (85% 8m); (f) naphthalen-2-ylmethyl
benzimidothioate·HBr, CH3CN/EtOH (1:3), rt (22% 9n); (g) naphthalen-2-ylmethyl pyridine-2-carbimidothioate·HBr, CH3CN/EtOH (1:3), rt
(17% 9m).
nevertheless showed submicromolar antileishmanial activity in
vitro.9 The most promising AIA identified to date, the bis-AIA
1 (DB766), also displayed good in vivo activity in rodent
models of visceral leishmaniasis when given orally, but >90%
inhibition of liver parasitemia was not observed at achievable
doses.10,11 Orally available azole-containing antifungal drugs
have also shown antileishmanial activity12,13 and have occa-
sionally been used in the clinic against leishmaniasis with
varying success.14−16 Although the target of the AIAs has not
been determined, azole antifungal drugs are thought to act on
sterol-14α-demethylase (CYP51), a critical enzyme in the
sterol biosynthesis pathway in both fungi and kinetoplastid
parasites such as Leishmania.17 Inhibition of this target is
believed to occur through coordination of the active site heme
group of the enzyme by an imidazole or triazole nitrogen lone
pair, while the remainder of the molecule makes important
contacts within the substrate binding channel of CYP51.17
We demonstrated that oral combinations of 1 with
posaconazole displayed moderately synergistic activity in a
murine model of visceral leishmaniasis as defined by the
combination index method, while combinations of 1 with
ketoconazole were less effective in this model.11 It was striking
that posaconazole and ketoconazole, which were over 2 orders
of magnitude less potent in our hands against intracellular
L. donovani in vitro compared to 1, displayed comparable, if
not superior, efficacy to 1 in the murine visceral leishmaniasis
model.11 In addition, L. donovani axenic amastigotes, which
became resistant to compound 1 due to prolonged pressure
with 1 in culture, exhibited hypersensitivity to posaconazole
and ketoconazole.18 On the basis of these observations as well
as the desire to take advantage of polypharmacology,19 the
synthesis of AIA−azole hybrid compounds displaying features
of mono-AIAs together with features of azole-containing
antifungal drugs was undertaken. Molecular hybridization is a
strategy in which two or more structural components
possessing distinct pharmacologic activities are incorporated
in one molecule to attack different targets.20 This strategy has
been used widely in infectious disease drug discovery in an
attempt to address resistance issues,21−24 to improve
pharmacokinetic properties,24 and even to combat different
infections using the same molecule.25 Structures of the
frontrunner bis-AIA 1, the mono-AIA 2 (DB2002), the azole
antifungals posaconazole (3) and ketoconazole (4), and the
scaffold of AIA−azole hybrid compounds are shown in Figure
1. The synthesis and evaluation of an initial series of such
hybrid compounds containing a phenoxyalkyl linker against
L. donovani are described here.
RESULTS
■
The synthesis of AIA−azole hybrids 9a−l is shown in Scheme
1. According to a previously published procedure,26 the
reaction of dibromo alkanes of different chain lengths with 5a−
c in dry acetonitrile under basic conditions provided 6a−i.
Similarly, the reaction of 6a−i with imidazole or 1,2,4-triazole
under the same basic conditions afforded compounds 7a−l.
Tin chloride-mediated reduction of the nitro group yielded
8a−l. Compounds 8a−l rapidly changed within less than an
hour to dark gummy materials, possibly due to auto-oxidation
upon exposure to air. This is reminiscent of our earlier
observation regarding the auto-oxidation of 1-benzyl-2,2,4-
trimethyl-1,2-dihydroquinolin-6-ols,27 which contain a similar
structural motif. Arylamines 8a−l were thus used without
further purification to yield target compounds 9a−l through
the reaction with the pyridyl thioimidate salt under the general
conditions reported previously.7 Since the resultant AIA−azole
hybrids were stable as free bases, permitting satisfactory
elemental analysis and testing in biological assays, conversion
to the salt form as mentioned in Reid et al.7 was not necessary.
The method for preparing AIA−azole hybrids possessing
additional terminal groups is shown in Scheme 2. Compound
9m contains a pyrrole group rather than an imidazole or
triazole substitution at the azole terminus while a phenyl group
is present in compound 9n instead of a pyridine at the
imidamide end. The route used to synthesize these molecules
is similar to the one shown in Scheme 1 except that the
installation of the pyrrole ring in target compound 9m required
the use of a stronger base (KOH) along with the more polar
DMF as a solvent to facilitate the reaction.28 In addition, the
reduction of nitroaromatic intermediate 7m to the correspond-
ing aniline 8m could be accomplished with either tin(II)
chloride dihydrate or Zn metal/ammonium chloride. Target
C
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a
Scheme 3. Synthesis of 17a−c
a
Reagents and conditions: (a) MOMCl, K2CO3, DMF, 30 °C (48%); (b) RI, K2CO3, CH3CN, sealed tube, 80 °C (91−97%); (c) HCl, MeOH/
CH2Cl2, rt (81−90%); (d) 1,8-dibromooctane, K2CO3, CH3CN, reflux (84−92%); (e) imidazole, K2CO3, CH3CN, reflux (73−90%); (f) SnCl2·
2H2O, EtOAc, reflux (98−99%); (g) naphthalen-2-ylmethyl pyridine-2-carbimidothioate·HBr, CH3CN/EtOH (1:3), rt (31−45%).
a
Scheme 4. Synthesis of 24a−c
a
Reagents and conditions: (a) RI, K2CO3, sealed tube, DMF, 80 °C (64−82%); (b) NaOH, DMSO/H2O, reflux (74−85%); (c) 1,8-
dibromooctane, K2CO3, CH3CN, reflux (76−94%); (d) imidazole, K2CO3, CH3CN, reflux (79−81%); (e) SnCl2·2H2O, EtOAc, reflux (95−
100%); (f) naphthalen-2-ylmethyl pyridine-2-carbimidothioate·HBr, CH3CN/EtOH (1:3), rt (34−45%).
a
Scheme 5. Synthesis of 28
a
Reagents and conditions: (a) 1,8-dibromooctane, DMF, 90 °C (60%); (b) imidazole, K2CO3, DMF, 80 °C (62%); (c) NH2NH2·H2O, EtOH,
reflux (67%); (d) naphthalen-2-ylmethyl pyridine-2-carbimidothioate·HBr, CH3CN/EtOH (1:3), rt (28%).
compound 9n was obtained using the phenyl thioimidate salt
rather than the pyridyl thioimidate salt.
imidamide moiety. Installation of the alkyl groups through
the reaction of alkyl iodides with 5-fluoro-2-nitrophenol (18)
followed by replacement of fluorine via nucleophilic aromatic
substitution using sodium hydroxide as reported by Lebold and
Kerr30 yielded nitrophenols 20a−c. Conversion of 20a−c to
the desired target compounds 24a−c followed the general
synthetic strategy described above.
The synthesis of the AIA−azole hybrid lacking a phenoxy
linker (28) is illustrated in Scheme 5. Compound 26 was
prepared through the reaction of potassium phthalimide (25)
with 1,8-dibromooctane. The reaction of 26 with imidazole
under basic conditions followed by subjecting the intermediate
to Gabriel synthesis conditions provided the primary amine 27
according to a previously published procedure.31 The reaction
of 27 with the pyridyl thioimidate salt under the conditions
described earlier yielded target compound 28 in low yield.
Structure−Activity and Structure−Toxicity Relation-
ships. When the hybrid target compounds were designed, the
effect of three factors was examined: (1) chain length, (2)
The synthesis of hybrid target compounds 17a−c containing
alkoxy substitutions meta to the imidamide group is shown in
Scheme 3. MOMCl protection of 4-nitrocatechol (10) was
carried out according to a previous method29 followed by the
reaction of methyl, ethyl, or isopropyl iodide with the
protected nitrophenol 11 under basic conditions, providing
compounds 12a−c. Deprotection under acidic conditions
followed by the reaction of the resulting phenols 13a−c with
dibromooctane under basic conditions in dry acetonitrile
provided the intermediate monobromo derivatives 14a−c,
which were transformed to 15a−c by treatment with imidazole
in the presence of potassium carbonate. Nitro reduction
followed by the reaction of the resulting arylamines 16a−c
with the pyridyl thioimidate salt as described above yielded
target compounds 17a−c.
Scheme 4 shows the preparation of AIA−azole target
compounds 24a−c bearing alkoxy groups ortho to the
D
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Table 1. In Vitro Antileishmanial Activity, Mammalian Cell Toxicity, and Inhibition of L. donovani CYP51 Activity for AIA−
Azole Hybrids
IC50 against
L. donovani
IC50 against J774
macrophages (μM)
IC50 against
HepG2 (μM)
IC50 against recombinant
a
a
a
a
compound
X
Y
Z
R1
R2
n
(μM)
L. donovani CYP51 (μM)
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
17a
17b
17c
24a
24b
24c
28
CH
N
CH
CH
N
N
N
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
2
2
3
4
4
5
6
8
8
8
8
10
8
8
8
8
8
8
>50
>50
>50
>50
>50
>50
>50
>40
11
6.8 0.7
8.6 1.2
36
4.9 0.9
11
0.48 0.02
16
9.0 1.3
7.8 0.3
9.3 0.5
6.8 0.1
6.8 0.2
not tested
not tested
0.025 0.001
not tested
not tested
not tested
not tested
not tested
>50
>100
>50
>20
>100
42
20
12
7.7 1.5
8.6 1.2
32
6.9 1.0
13
4.9 1.0
23
13
18
16
12
12
>100
21
35
22
6.4 0.1
89
4.8 0.1
1.3 0.1
0.36 0.03
0.18 0.01
0.15 0.01
1.9 0.1
1
2
0
N
N
N
N
N
N
N
N
N
N
N
CH
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
CH
N
N
N
N
N
N
>100
>25
18
>100
13
8.8 0.6
1.0 0.0
1.3 0.2
2.1 0.2
51 5%
1.4 0.2
1.7 0.3
1.2 0.1
2.2 0.3
1.9 0.3
0.97 0.07
1.5 0.4
0.73 0.15
0.53 0.05
>100
0.045 0.003
not tested
not tested
not tested
3
5
4
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
4
3
3
1
b
1
5
0.22 0.01
14
1
2
1
c
H
0.34 0.01
0.57 0.01
0.53 0.02
0.29 0.01
0.36 0.01
0.34 0.01
0.33 0.01
not tested
not tested
not tested
not tested
not tested
OMe
OEt
OiPr
H
H
H
1
2
0
1
2
2
2
OMe
OEt
OiPr
8
8
c
amphotericin B
podophyllotoxin
doxorubicin
quinacrine
ketoconazole
posaconazole
fluconazole
not tested
not tested
0.20 0.02
5.5 0.7
not tested
not tested
not tested
d
50 7%
47 2%
16 3%
0.048 0.004
0.059 0.001
0.96 0.03
e
d
a
b
c
Mean standard error of the mean (SEM; n = 3). Inhibition of parasitemia at 12.5 μM (mean SEM (n = 3)). Mean range (n = 2).
d
e
Inhibition of parasitemia at 50 μM (mean SEM (n = 3)). Inhibition of parasitemia at 7.5 μM (mean SEM (n = 3)).
identity of the terminal groups, and (3) substitution of the
phenoxy linker. Data from the evaluation of the potency of the
hybrid compounds against intracellular L. donovani and their
effect against two mammalian cell lines is shown in Table 1. An
increase in the length of the alkyl chain improved the
antileishmanial potency but also increased the activity against
both J774 macrophages and HepG2 cells. The best balance of
potency and toxicity in this series was achieved with an octyl
chain; compound 9h possessed an IC50 value of 1.0 μM against
L. donovani intracellular amastigotes and IC50 values of 11 and
12 μM against J774 macrophages and HepG2 cells,
respectively. An increase in the chain length to ten carbons
(compound 9l) did not improve the antileishmanial activity
but decreased the selectivity against both the macrophage and
hepatocarcinoma cell lines. Compounds containing imidazole
as the azole terminal group showed greater antileishmanial
potency than the corresponding triazoles. For the compounds
bearing four carbon alkyl linkers, imidazole 9d was at least 5-
fold more potent against intracellular L. donovani than triazole
9e. In the eight carbon linker series, the replacement of
imidazole with triazole led to a >10-fold decrease in potency
when 9h was compared with 9k (a precise IC50 value could not
be calculated for compound 9k in the intracellular L. donovani
assay because of toxicity to the host cells at higher
concentrations, but ∼50% inhibition of intracellular para-
sitemia was observed for 9k at a concentration of 12.5 μM as
indicated in Table 1). Little effect on antileishmanial potency
and mammalian cell toxicity was observed when the imidazole
ring was replaced with pyrrole (compare 9h to 9m). Regarding
the arylimidamide moiety, the replacement of the 2-pyridyl
group with a phenyl moiety (compound 9n) resulted in a 22-
fold increase in potency against J774 cells, a 2.4-fold increase in
HepG2 activity, and no increase in antileishmanial potency
compared to 9h. In terms of substitutions on the phenoxy
portion of the linker, the placement of chlorine atoms either
meta (9i) or ortho (9j) to the imidamide end did not increase
antileishmanial potency but may have decreased antileishma-
nial selectivity compared to 9h. Minor effects on antileishma-
nial potency and selectivity were observed upon substitution of
the phenoxy ring with alkoxy groups. The most favorable
E
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Figure 2. Gel electrophoresis (A), reduced CO-difference spectra (B), and ligand binding spectra (C and D) of the purified L. donovani CYP51.
The expected molecular weight of the CYP51-32c construct is 53 kDa. The P450 content was determined to be 7.1 nmol/mg for the batch of
purified CYP51 used in this experiment. Various concentrations of 24c (C) and 9h (D) were titrated into CYP51 before recording binding spectra
and determining the dissociation constants (Kd).
Figure 3. Fluorescence-based inhibition assay for L. donovani CYP51 (A) and inhibition by 24c (B). The fluorogenic substrate BFC (50 μM) was
incubated with purified CYP51-32c (60 nM) in the presence of cofactor CuOOH (0.1 mM) or hydrogen peroxide (HOOH; 25 mM) at 37 °C.
Control incubations did not contain either the cofactor (but with CYP) or the CYP protein (but with CuOOH or HOOH). The IC50 value (mean
SEM of triplicate determinations) is shown for 24c.
substitutions of those tested appear to be ethoxy and
isopropoxy groups ortho to the imidamide (compounds 24b
and 24c, respectively). These changes resulted in slight
increases in antileishmanial potency, similar selectivity for
intracellular L. donovani vs J774 macrophages compared to 9h,
and slight improvements in selectivity for intracellular parasites
vs HepG2 cells compared to 9h. For example, compound 24c
displayed submicromolar potency against L. donovani with
selectivity indexes (IC50 vs mammalian cell line/IC50 vs
L. donovani) of 13 and 23 for intracellular parasites compared
to J774 macrophages and HepG2 cells, respectively. Notably,
the removal of the phenoxy linker (compound 28) resulted in
a total loss of activity against L. donovani and HepG2 cells. The
higher hydrophilicity of compound 28 and the presumed
higher pKa value of its imidamide moiety may contribute to its
lack of antileishmanial activity.
Binding to L. donovani CYP51. Purified CYP51 showed
good purity with a single major band in both SDS-PAGE and
Western blot analyses (Figure 2A) and exhibited a character-
istic absorbance at 450 nm (the active P450 form) upon
dithionite reduction and carbon monoxide (CO) binding
(Figure 2B). The P450 form of the protein was stabilized by
lanosterol as evidenced by a reduction in the inactive P420
form (Figure 2B),32 likely due to its binding at the active site.
F
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When titrated with 24c or 9h, CYP51 exhibited typical Type II
binding spectra (Figure 2C,D) with an absorbance maximum
at 431 nm and a minimum at 412−413 nm, indicating direct
binding to the active site of CYP51. A Type II binding with
CYP protein shows an absorption maximum above the 415−
418 nm Soret band range (red-shifted Soret band for low spin
Fe3+) and is indicative of inhibitory ligand binding as the ligand
replaces the water molecule to form the sixth coordination
with the heme iron, stabilizes low spin Fe3+, and prevents
substrate binding.33
were found to be strong inhibitors of recombinant human
CYP3A4, displaying IC50 values of 0.047 0.003 and 0.080
0.009 μM, respectively. For comparison, azole antifungal drugs
ketoconazole, posaconazole, and fluconazole exhibited IC50
values of 0.0070
0.0003, 0.15
0.01, and 18
2 μM,
respectively. Fluconazole is known to be a relatively weak
inhibitor of CYP3A4.37
Microsomal Stability. Selected hybrid compounds were
also evaluated for their in vitro microsomal stability (Table 2).
Inhibition of L. donovani CYP51. A fluorescence-based
inhibition assay was developed for the L. donovani CYP51
using 7-benzyloxy-4-trifluoromethylcoumarin (BFC) as sub-
strate, which was readily converted via oxidative O-
debenzylation to its fluorescent metabolite 7-hydroxy-4-
trifluoromethylcoumarin (HFC; excitation 410 nm, emission
538 nm) by the purified CYP51 in the presence of cumene
hydroperoxide (CuOOH) as a cofactor (Figure 3A). CuOOH
has previously been shown to enable CYP-catalyzed reactions
in the absence of CYP natural cofactors (NADPH:P450
oxidoreductase and NADPH).34
Table 2. In Vitro Metabolic Stability of Selected Hybrid
Compounds
human liver microsome t or mouse liver microsome t or
a1/2
a1/2
compound
percent remaining
percent remaining
9h
9l
100% after 60 min
57 min
70 min
68 min
17a
17c
24a
24b
24c
1
100% after 60 min
>80% after 60 min
>80% after 60 min
>80% after 60 min
52 min
>50% after 120 min
100% after 60 min
100% after 60 min
>80% after 60 min
100% after 60 min
67 min
>50% after 120 min
b
b
The azole antifungal drugs posaconazole, ketoconazole, and
fluconazole exhibited strong-to-moderate inhibition of the
L. donovani CYP51 with IC50 values (mean SEM of triplicate
determinations) of 0.059 0.001, 0.048 0.004, and 0.96
0.03 μM, respectively. Table 1 also shows the inhibition of
CYP51-mediated conversion of BFC to HFC by the hybrid
compounds. A representative inhibition curve is shown for 24c
(Figure 3B). Generally consistent with observations regarding
antiparasitic potency, compounds possessing longer linkers
display greater inhibition of CYP51. In the imidazole series, the
inhibitory activity increases as chain length increases from 3
carbons (9c) to 10 carbons (9l). Hybrids bearing an imidazole
ring at the “azole end” of the molecule have higher potency
than those containing the triazole ring. This is clearly
illustrated by comparing four carbon linkers containing
imidazole 9d and triazole 9e (CYP51 IC50 values of 6.4 and
89 μM, respectively) and eight carbon linkers containing
imidazole 9h and triazole 9k (CYP51 IC50 values of 0.36 and
1.9 μM, respectively). The importance of the imidazole ring in
conveying CYP51 inhibition is further demonstrated through
the comparison of the pyrrole terminal compound 9m and the
corresponding imidazole terminal compound 9h. Lacking
nitrogen at position 3 of the heterocycle, compound 9m
displays an IC50 value of 14 μM against CYP51 as opposed to
an IC50 value of 0.36 μM for the corresponding imidazole
compound 9h as mentioned earlier. This observation for 9m
represents a departure from the antiparasitic SAR described
above. Within the series of imidazole-containing phenoxyalkyl
hybrids bearing eight carbon linkers, the substitution on the
phenoxyalkyl ring and the opposite terminal group have
relatively little influence on CYP51 inhibition, as phenoxyalkyl
substituted compounds 17a−c and 24a−c along with phenyl
terminal compound 9n show CYP51 IC50 values similar to that
of 9h. It should be noted, however, that Cl substituted
compounds 9i and 9j are about 2-fold more potent as
inhibitors of L. donovani CYP51 compared to 9h.
c
c
d
d
2
100%, 111% after 60 min
44%, 61% after 60 min
a
Substrate concentration of 3 μM, measured by LC-MS detection
b
c
unless otherwise noted. Measured by UV detection. Measured at
substrate concentrations of 1 and 10 μM from Wang et al.10
d
Measured by UV detection at substrate concentrations of 1 and 10
μM, respectively.
These compounds were incubated with either human or mouse
liver microsomes at a substrate concentration of 3 μM; the
disappearance of the compound of interest was measured over
time by LC-MS or LC-UV detection. All compounds exhibited
t
1/2 > 50 min. Compounds 9l (bearing a ten carbon linker) and
24c (containing an eight carbon linker and an isopropoxy
substitution ortho to the imidamide) were less stable in the
presence of both human and mouse liver microsomes,
possessing t1/2 of 52−68 min under these conditions.
Compound 9h (containing an eight carbon linker and an
unsubstituted phenoxy ring) displayed t1/2 = 70 min in the
presence of mouse liver microsomes but was not degraded by
human liver microsomes over a period of 1 h. For the rest of
the hybrid compounds tested, 80% or more remained
unchanged in the presence of either human or mouse liver
microsomes after a 60 min incubation. Metabolic stability data
for AIAs 1 and 2 are provided for comparison.
In Vivo Evaluation of Compound 24c. Since 24c
displayed a t1/2 of >60 min in the presence of mouse liver
microsomes and was the most potent hybrid compound tested
in the phenoxyalkyl hybrid series, it was evaluated for
antileishmanial efficacy in vivo. When 24c was given by oral
gavage at a dose of 100 mg/kg/day for 5 days to a pair of
female 6−8 week old BALB/c mice, no weight loss, signs of
overt toxicity, or obvious effects on the GI tract, liver, spleen,
or kidneys were observed upon necropsy performed 1 day after
administration of the final dose. Oral doses of 24c at 75 mg/
kg/day × 5 and 37.5 mg/kg/day × 5 were then administered
to L. donovani-infected female BALB/c mice starting at 1 week
postinfection (these doses were chosen on the basis of
compound availability). Liver parasitemia was then determined
3 days after the final dose (Figure 4). In animals treated with
75 mg/kg 24c, a 33 7% (mean SEM) reduction in liver
parasitemia was observed, while the liver parasite burden was
Inhibition of Human CYP3A4. CYP3A enzymes are
critical in drug metabolism, being expressed in the liver,
intestine, and other tissues.35 Since several azole drugs are
potent inhibitors of CYP3A4, the inhibition of recombinant
human CYP3A4 by hybrid compounds 9h and 24c was
evaluated using BFC as a substrate.36 Compounds 9h and 24c
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In terms of their potency against intracellular L. donovani,
the hybrid compounds reported here are comparable to the
mono-AIAs.9 The most potent compounds reported thus far
from each group exhibit high nanomolar IC50 values against
these intracellular parasites and show toxicity to J774
macrophages at concentrations near 10 μM. There are caveats
to this comparison; the mono-AIAs appear to be slightly more
selective for Leishmania, and the intracellular L. donovani assays
reported in the two studies were performed by different
methods. Nonetheless, considering their comparable in vitro
biological activities given the available data, it is appropriate to
ask whether the hybrid target compounds reported here are
simply a subset of the mono-AIAs. Although the targets of the
AIAs remain unknown, the interactions of active compounds
from both series with L. donovani CYP51 are discussed below.
Azole antifungals disrupt sterol biosynthesis in Leishmania.47
Fluconazole inhibits CYP51, the 14α-demethylase involved in
sterol biosynthesis, from Leishmania as shown by Hargrove et
al.48 and as demonstrated in the present work. Imidazole-
containing compounds (R)-N-(1-(2,4-dichlorophenyl)-2-(1H-
imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)-
benzamide (VNI), (R)-N-(1-(3,4’-difluorobiphenyl-4-yl)-2-
(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)-
benzamide (VFV), and their analogs also inhibit Leishmania
CYP5144,49 and display in vitro and in vivo antileishmanial
effects.44 Molecules consisting of an imidazole group and an
aromatic substituent connected by a hydrocarbon chain were
identified by high throughput screening as strong binders to
T. cruzi CYP51 and also displayed submicromolar potency
against intracellular T. cruzi amastigotes.50 Most of the hybrid
compounds reported here that display single-digit micromolar
or greater potency against intracellular L. donovani also inhibit
Leishmania CYP51 (Table 1); imidazole containing hybrid
compounds are more active against both Leishmania CYP51
and intracellular parasites than the corresponding triazole
compounds. While these data show the ability of selected
hybrid compounds to bind to this parasite enzyme involved in
sterol biosynthesis, they do not clearly demonstrate that
CYP51 is the target for these compounds. Given that the bis-
AIA 1 and the related mono-AIA 2 are weak inhibitors of
L. donovani CYP51 at concentrations 5−10 times greater than
their IC50 against intracellular L. donovani (M. Z. Wang,
unpublished observations), CYP51 inhibition does not appear
to be required in order for molecules possessing an
arylimidamide group to display potent antileishmanial activity.
In addition, the pyrrole containing compound 9m is only
slightly less potent against intracellular L. donovani than the
corresponding imidazole 9h, yet it is 39-fold less active as a
CYP51 inhibitor. The protein CYP5122A1 also has a role in
ergosterol synthesis in Leishmania donovani, although its
precise function is currently unknown.46 Given the range of
inhibitors that have been identified for T. cruzi CYP51, it may
also be possible to target CYPs in Leishmania with a variety of
inhibitors. In an attempt to test the hypothesis mentioned
above that improved inhibitors of Leishmania sterol biosyn-
thesis may result in the development of new therapeutics, the
identification of new CYP450 inhibitors in Leishmania and the
characterization of their effects on this parasite are an area of
active investigation in our laboratories.
Figure 4. Efficacy of compound 24c in the murine model of visceral
leishmaniasis. Compound 24c, miltefosine, and vehicle were
administered by oral gavage for five consecutive days to groups of
four L. donovani-infected female BALB/c mice. Liver parasite burdens
are expressed as Leishman-Donovan units (LDU) with horizontal
bars indicating the mean for each group. #, p < 0.001 compared to the
PEG400 control group; *, p < 0.05 compared to the PEG400 control
group as assessed by the two-tailed Student’s t test.
reduced by 17 5% in those animals given 24c at 37.5 mg/kg.
Consistent with previous studies in this murine visceral
leishmaniasis model, oral treatment with miltefosine at 10
mg/kg/day × 5 led to a 95
2% reduction of liver
infection.7,11
DISCUSSION
■
Molecular hybridization is a powerful approach that has been
widely used in the search for promising antileishmanial and
antitrypanosomal candidates.38−41 The design and synthesis of
hybrid compounds displaying structural features of both
antifungal azoles and AIAs may be a promising strategy
toward the discovery of new antileishmanial leads because
members of both classes of compounds display in vivo
antileishmanial activity when given orally. Although they are
not FDA approved for antileishmanial treatment, antifungal
azoles are considered for off-label use against leishmaniasis,42
while AIAs have shown oral activity in rodent models of
visceral leishmaniasis.9,10,43 It is widely accepted that antifungal
azoles exert their activity against fungi by inhibiting CYP51,
the 14α-demethylase enzyme crucial for sterol biosynthesis;
this enzyme is also inhibited by azoles displaying antileishma-
nial activity.44 However, it has been speculated that the
development of new antileishmanial therapies targeting sterol
biosynthesis may require the discovery of more powerful
inhibitors, drug combinations, or molecular hybridization
approaches.17,45 While the precise mechanisms of action of
AIAs remain unknown, L. donovani half knockout parasites for
CYP5122A1, a protein involved in Leishmania ergosterol
biosynthesis,46 were less susceptible to 1 than wild type
parasites but were more susceptible to ketoconazole.18 Work
with these hybrid compounds may uncover additional insights
regarding Leishmania sterol metabolism that could aid
antileishmanial drug design.
Like ketoconazole and posaconazole, two hybrid compounds
(9h and 24c) are shown to be potent inhibitors of human
CYP3A4, which poses a risk for drug−drug interactions with
molecules that are metabolized by human CYP3A4. It was
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reported that human CYP51 is resistant to inhibition by azole
antifungals, including posaconazole, ketoconazole, and fluco-
nazole.51 As such, in this first report on AIA−azole hybrid
compounds, these agents were not evaluated for the inhibition
of human CYP51. Future studies should consider the
inhibition of a wider panel of human CYPs by the AIA−
azole hybrid compounds, especially those CYPs commonly
involved in drug metabolism, to prioritize hits for further
optimization and development.
The AIA−azole hybrid agents described here hold promise
as antileishmanials based on their strong activity against
intracellular parasites (Table 1) and the opportunity they
present to explore a wide range of chemical space in the design
of new derivatives to improve potency and drug-like properties.
The data shown in Table 2 also indicate that, when compared
to AIAs 1 and 2, the inclusion of the azole group in the hybrid
molecules does not have a clear negative effect on metabolic
stability. Lead compound 24c did not display signs of toxicity
when administered orally to BALB/c mice at doses up to 100
mg/kg, although the in vivo efficacy of this compound in a
murine model of visceral leishmaniasis was modest (Figure 4).
Work is in progress to improve the antileishmanial properties
of the AIA−azole hybrids to describe their interactions with
potential target molecules and to examine their influence on
sterol metabolism in Leishmania.
1-(2-Bromoethoxy)-4-nitrobenzene (6a).54 White powder,
2.2 g, yield 62% starting from 2.0 g of 4-nitrophenol (5a, 14.4
mmol).
1-(3-Bromopropoxy)-4-nitrobenzene (6b).54 White pow-
der, 3.0 g, yield 80% starting from 2.0 g of 4-nitrophenol (5a,
14.4 mmol).
1-(4-Bromobutoxy)-4-nitrobenzene (6c).54 White powder,
2.5 g, yield 63% starting from 2.0 g of 4-nitrophenol (5a, 14.4
mmol).
1-(5-Bromopentoxy)-4-nitrobenzene (6d).54 White pow-
der, 3.2 g, yield 77% starting from 2.0 g of 4-nitrophenol (5a,
14.4 mmol).
1-(6-Bromohexoxy)-4-nitrobenzene (6e).54 Yellow oil, 3.30
g, yield 76% starting from 2.0 g of 4-nitrophenol (5a, 14.4
mmol).
1-((8-Bromooctyl)oxy)-4-nitrobenzene (6f).54 White pow-
der, 1.10 g, yield 92% starting from 0.5 g 4-nitrophenol (5a,
3.60 mmol).
1-((8-Bromooctyl)oxy)-2-chloro-4-nitrobenzene (6g). Yel-
low oil, 0.98 g, yield 93% starting from 0.5 g of 2-chloro-4-
nitrophenol (5b, 2.88 mmol). 1H NMR (300 MHz, CDCl3) δ
1.31−1.56 (m, 8H), 1.80−1.94 (m, 4H), 3.40 (t, J = 6.8 Hz,
2H), 4.13 (t, J = 6.4 Hz, 2H), 6.96 (d, J = 9.1 Hz, 1H), 8.13
(dd, J = 9.1, 2.7 Hz, 1H), 8.28 (d, J = 2.7 Hz, 1H); 13C NMR
(75 MHz, CDCl3) δ 25.9, 28.2, 28.7, 28.9, 29.2, 32.9, 34.0,
70.0, 111.8, 123.6, 124.1, 126.2, 141.1, 159.9.
4-((8-Bromooctyl)oxy)-2-chloro-1-nitrobenzene (6h). Yel-
low oil, 0.97 g, yield 92% starting from 0.5 g of 3-chloro-4-
METHODS
■
General Chemistry Methods. Reactions were carried out
and monitored, and compounds were purified according to the
techniques described earlier52 unless noted otherwise.
Commercial solvents and reagents were purchased from
Sigma-Aldrich, VWR, Fisher Scientific, or Combi-Blocks and
were used without further purification. NMR spectra were
recorded on Bruker AV300 or DRX400 or Avance III HD
Ascend 700 MHz spectrometers at 300 K unless noted and
were calibrated using the residual undeuterated solvent peak
1
nitrophenol (5c, 2.88 mmol). H NMR (300 MHz, CDCl3) δ
1.30−1.52 (m, 8H), 1.75−1.92 (m, 4H), 3.41 (t, J = 6.7 Hz,
2H), 4.02 (t, J = 6.4 Hz, 2H), 6.85 (dd, J = 9.1, 2.6 Hz, 1H),
6.99 (d, J = 2.6 Hz, 1H), 7.99 (d, J = 9.1 Hz, 1H); 13C NMR
(75 MHz, CDCl3) δ 25.9, 28.2, 28.7, 29.0, 29.2, 32.9, 34.0,
69.3, 113.5, 117.4, 128.2, 129.8, 140.6, 162.7.
1-(10-Bromodecyoxy)-4-nitrobenzene (6i).54 White pow-
der, 1.0 g, yield 78%, starting from 0.5 g of 4-nitrophenol (5a,
3.60 mmol).
1
(CDCl3: δ 7.26 ppm for H NMR, 77.16 ppm for 13C NMR;
1
DMSO-d6: δ 2.50 ppm for H NMR, 39.52 ppm for 13C
NMR).53 Proton (1H) and carbon (13C) NMR data are
reported as outlined previously.52 High resolution mass spectra
(HRMS) and melting points were obtained as described
earlier.52
Synthesis of 4-Nitrophenoxy Alkyl Azoles (7a−l) and
Pyrrole 7m.
Synthesis of 4-Nitrophenoxy Alkyl Bromides (6a−i).
To a solution of 4-nitrophenols 5a−c (2.9−14.4 mmol) in dry
acetonitrile (20 mL) was added 2 equiv of K2CO3 (5.8−28.8
mmol) and 5 equiv of dibromoalkane (14.4−71.8 mmol). The
mixture was stirred at 80 °C overnight according to a
previously published procedure.26,54 After completion of the
reaction, the suspension was filtered and the filtrate was
concentrated under reduced pressure. The crude product was
subjected to silica gel chromatography employing ethyl
acetate/hexanes (1:10) as the eluent to afford the product in
62−93% yield.
Synthesis of 7a−l. To a solution of 6a−i (1.40−5.5 mmol) in
dry acetonitrile (20 mL) was added 2 equiv of K2CO3 (2.80−
11.00 mmol) and 2 equiv of imidazole or triazole (2.80−11.00
mmol), and the mixture was stirred at 80 °C overnight. After
the reaction was completed, the suspension was filtered and
the filtrate was concentrated under reduced pressure. The
crude product was subjected to silica gel chromatography
employing DCM/MeOH (20:1) as the eluent to afford the
product in 49−86% yield.
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1-(2-(4-Nitrophenoxy)ethyl)-1H-imidazole (7a). White
powder, 0.72 g, yield 76% starting from 1.00 g of 6a (4.06
mmol). H NMR (300 MHz, CDCl3) δ 4.28−4.33 (m, 2H),
MHz, CDCl3) δ 25.8, 26.6, 28.9, 29.1, 29.2, 31.1, 47.1, 69.2,
113.5, 117.3, 118.8, 128.1, 129.5, 129.8, 137.2, 140.6, 162.6.
1-(8-(4-Nitrophenoxy)octyl)-1H-1,2,4-triazole (7k). White
powder, 0.41 g, yield 70% starting from 0.6 g of 6f (1.81
1
4.37−4.43 (m, 2H), 6.93 (d, J = 9.3 Hz, 2H), 7.03 (t, 1.2 Hz,
1H), 7.08 (s, 1H), 7.60 (s, 1H), 8.20 (d, J = 9.3 Hz, 2H).
1-(2-(4-Nitrophenoxy)ethyl)-1H-1,2,4-triazole (7b). White
powder, 0.86 g, yield 75% starting from 1.20 g of 6a (4.87
1
mmol). H NMR (400 MHz, CDCl3) δ 1.24−1.49 (m, 8H),
1.75−1.84 (m, 2H), 1.85−1.94 (m, 2H), 4.03 (t, J = 6.4 Hz,
2H), 4.16 (t, J = 7.1 Hz, 2H), 6.92 (d, J = 9.2 Hz, 2H), 7.93 (s,
1H), 8.04 (s, 1H), 8.18 (d, J = 9.2 Hz, 2H); 13C NMR (100
MHz, CDCl3) δ 25.9, 26.5, 29.0, (overlapped 2Cs), 29.2, 29.9,
49.8, 68.9, 114.5, 126.0, 141.5, 142.9, 152.1, 164.3.
1
mmol). H NMR (300 MHz, CDCl3) δ 4.44 (t, J = 4.9 Hz,
2H), 4.62 (t, J = 5.0 Hz, 2H), 6.91 (d, J = 9.2 Hz, 2H), 7.96 (s,
1H), 8.14−8.23 (m, 3H); 13C NMR (75 MHz, CDCl3) δ 49.0,
66.4, 114.6, 126.1, 142.3, 144.1, 152.6, 162.8.
1-(3-(4-Nitrophenoxy)propyl)-1H-imidazole (7c).55 White
powder, 1.0 g, yield 78% starting from 1.35 g of 6b (5.19
mmol).
1-(10-(4-Nitrophenoxy)decyl)-1H-imidazole (7l). Off-white
powder, 0.32 g, yield 65%, starting from 0.5 g of 6i (1.40
1
mmol). H NMR (300 MHz, CDCl3) δ 1.22−1.50 (m, 12H),
1.71−1.86 (m, 4H), 3.91 (t, J = 7.1 Hz, 2H), 4.03 (t, J = 6.5
Hz, 2H), 6.88−6.95 (m, 3H), 7.04 (s, 1H), 7.45 (s, 1H), 8.18
(d, J = 9.2 Hz, 2H); 13C NMR (75 MHz, CDCl3) δ 26.0, 26.6,
29.1, 29.2, 29.35, 29.45, 29.49, 31.2, 47.1, 69.0, 114.5, 118.9,
126.0, 129.5, 137.2, 141.5, 164.3.
1-(4-(4-Nitrophenoxy)butyl)-1H-imidazole (7d).55 White
powder, 0.73 g, yield 51% starting from 1.5 g of 6c (5.47
mmol).
1-(4-(4-Nitrophenoxy)butyl)-1H-1,2,4-triazole (7e).55
White powder, 1.10 g, yield 76% starting from 1.5 g of 6c
(5.47 mmol).
Synthesis of 1-(8-(4-Nitrophenoxy)octyl)-1H-pyrrole (7m).
Compound 7m was synthesized according to a previously
published procedure.28 To a solution of pyrrole (0.21 g, 3.13
mmol) in DMF (5 mL) was added 1.14 equiv of potassium
hydroxide (0.20 g, 3.56 mmol), and the suspension was stirred
at rt for 15 min. 1.16 equiv of 6f (1.2 g, 3.56 mmol) was added,
and the mixture was stirred at 80 °C for 16 h. After the
reaction was complete, the mixture was quenched with water,
extracted with ethyl acetate, and dried over sodium sulfate. The
organic layer was evaporated under reduced pressure and
purified by column chromatography using hexanes/DCM
(5:1) as the eluent to yield the product as a yellow oil, 0.35
1-(5-(4-Nitrophenoxy)pentyl)-1H-imidazole (7f). White
powder, 0.96 g, yield 67% starting from 1.5 g of 6d (5.20
1
mmol). H NMR (300 MHz, CDCl3) δ 1.42−1.54 (m, 2H),
1.78−1.92 (m, 4H), 3.97 (t, J = 7.0 Hz, 2H), 4.02 (t, J = 6.1
Hz, 2H), 6.88−6.95 (m, 3H), 7.05 (s, 1H), 7.46 (s, 1H), 8.17
(d, J = 9.2 Hz, 2H); 13C NMR (75 MHz, CDCl3) δ 23.3, 28.6,
30.9, 46.9, 68.4, 114.5, 118.8, 126.0, 129.7, 137.2, 141.6, 164.0.
1-(6-(4-Nitrophenoxy)hexyl)-1H-imidazole (7g). White
powder, 1.10 g, yield 72% starting from 1.6 g of 6e (5.29
1
mmol). H NMR (300 MHz, CDCl3) δ 1.30−1.42 (m, 2H),
1
1.44−1.55 (m, 2H), 1.75−1.86 (m, 4H), 3.94 (t, J = 7.0 Hz,
2H), 4.01 (t, J = 6.3 Hz, 2H), 6.88−6.94 (m, 3H), 7.04 (s,
1H), 7.45 (s, 1H), 8.18 (d, J = 9.2 Hz, 2H); 13C NMR (75
MHz, CDCl3) δ 25.6, 26.4, 28.9, 31.1, 47.0, 68.6, 114.5, 118.8,
126.0, 129.6, 137.2, 141.6, 164.2.
g, 35%. H NMR (300 MHz, CDCl3) δ 1.25−1.52 (m, 8H),
1.72−1.87 (m, 4H), 3.87 (t, J = 7.1 Hz, 2H), 4.04 (t, J = 6.5
Hz, 2H), 6.14 (t, J = 2.1 Hz, 2H), 6.65 (t, J = 2.1 Hz, 2H), 6.94
(d, J = 9.3 Hz, 2H), 8.19 (d, J = 9.3 Hz, 2H); 13C NMR (75
MHz, CDCl3) δ 26.0, 26.8, 29.0, 29.2, 29.3, 31.6, 49.7, 68.9,
107.9, 114.5, 120.6, 126.0, 141.5, 164.3.
1-(8-(4-Nitrophenoxy)octyl)-1H-imidazole (7h). Off-white
powder, 0.41 g, yield 86% starting from 0.5 g of 6f (1.51
Synthesis of 4-Aminophenoxy Alkyl Azoles (8a−l)
and Pyrrole 8m.
1
mmol). H NMR (300 MHz, CDCl3) δ 1.25−1.50 (m, 8H),
1.72−1.85 (m, 4H), 3.93 (t, J = 7.1 Hz, 2H), 4.03 (t, J = 6.4
Hz, 2H), 6.88−6.96 (m, 3H), 7.05 (s, 1H), 7.45 (s, 1H), 8.19
(d, J = 9.3 Hz, 2H); 13C NMR (75 MHz, CDCl3) δ 25.9, 26.6,
29.0, 29.1, 29.2, 31.2, 47.1, 68.9, 114.5, 118.9, 126.0, 129.6,
137.2, 141.5, 164.3.
1-(8-(2-Chloro-4-nitrophenoxy)octyl)-1H-imidazole (7i).
Yellow oil, 0.41 g, yield 60%, starting from 0.71 g of 6f
1
(1.94 mmol). H NMR (400 MHz, CDCl3) δ 1.25−1.41 (m,
6H), 1.43−1.53 (m, 2H), 1.72−1.81 (m, 2H), 1.81−1.90 (m,
2H), 3.92 (t, J = 7.1 Hz, 2H), 4.11 (t, J = 6.4 Hz, 2H), 6.89 (s,
1H), 6.95 (d, J = 9.1 Hz, 1H), 7.04 (s, 1H), 7.44 (s, 1H), 8.12
(dd, J = 9.1, 2.7 Hz, 1H), 8.27 (d, J = 2.7 Hz, 1H); 13C NMR
(100 MHz, CDCl3) δ 25.9, 26.6, 28.8, 29.0, 29.1, 31.1, 47.1,
69.9, 111.8, 118.8, 123.5, 124.1, 126.1, 129.5, 137.2, 141.1,
159.8.
To a solution of 7a−m (0.60−1.70 mmol) in ethyl acetate (20
mL) was added 5 equiv of tin(II) chloride dihydrate (3.0−8.5
mmol), and the mixture was refluxed for 8−12 h. After the
reaction was complete, the suspension was made basic with 1
N NaOH solution (pH = 11) and extracted with ethyl acetate
(30 mL × 3). The organic layer was dried over sodium sulfate
and filtered. The filtrate was evaporated under reduced
pressure yielding the product in 81−99% yield, which was
taken directly to the next step without further purification. For
1-(8-(3-Chloro-4-nitrophenoxy)octyl)-1H-imidazole (7j).
Synthesis of 7j followed the general synthesis procedure
except for the use of 1.2 equiv of imidazole (115 mg, 1.69
mmol) and 1.2 equiv of potassium carbonate (231 mg, 1.66
mmol) with gentle reflux overnight. Yellow oil, 0.24 g, yield
1
49% starting from 0.51 g of 6f (1.39 mmol). H NMR (400
MHz, CDCl3) δ 1.22−1.48 (m, 8H), 1.71−1.83 (m, 4H), 3.92
(t, J = 7.1 Hz, 2H), 4.00 (t, J = 6.4 Hz, 2H), 6.83 (dd, J = 9.2,
2.6 Hz, 1H), 6.88 (s, 1H), 6.98 (d, J = 2.6 Hz, 1H), 7.03 (s,
1H), 7.44 (s, 1H), 7.97 (d, J = 9.2 Hz, 1H); 13C NMR (100
1
this reason, only H NMR spectra were obtained for 8a−l.
1
Since the compounds were crude, H NMR spectra for the
J
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Article
anilines reported in this manuscript often contain solvent peaks
such as DCM and ethyl acetate along with traces of starting
material in some cases (occasionally resulting in slightly higher
than expected integration for the alkyl protons, particularly for
the most upfield alkyl multiplet).
4-(2-(1H-Imidazol-1-yl)ethoxy)aniline (8a). Yellow pow-
der, 0.10 g, yield 82% starting from 0.14 g of 7a (0.60 mmol).
1H NMR (300 MHz, DMSO-d6) δ 4.08 (t, J = 5.2 Hz, 2H),
4.27 (t, J = 5.2 Hz, 2H), 4.62 (brs, ArNH2), 6.49 (d, J = 8.8 Hz,
2H), 6.63 (d, J = 8.8 Hz, 2H), 6.88 (s, 1H), 7.21 (s, 1H), 7.65
(s, 1H).
4-(2-(1H-1,2,4-Triazol-1-yl)ethoxy)aniline (8b). Yellow
powder, 0.28 g, yield 81% starting from 0.40 g of 7b (1.70
mmol). 1H NMR (300 MHz, CDCl3) δ 3.26−3.65 (brs,
ArNH2), 4.24 (t, J = 5.0 Hz, 2H), 4.50 (t, J = 5.0 Hz, 2H), 6.60
(d, J = 9.0 Hz, 2H), 6.67 (d, J = 9.3 Hz, 2H), 7.94 (s, 1H),
8.20 (s, 1H).
4-(3-(1H-Imidazol-1-yl)propoxy)aniline (8c). Brown oil,
0.26 g, yield 99% starting from 0.30 g of 7c (1.21 mmol).
1H NMR (300 MHz, CDCl3) δ 2.12−2.23 (m, 2H), 2.86−3.93
(brs, ArNH2), 3.83 (t, J = 5.7 Hz, 2H), 4.17 (t, J = 6.8 Hz, 2H),
6.63 (d, J = 8.8 Hz, 2H), 6.72 (d, J = 8.9 Hz, 2H), 6.91 (s,
1H), 7.05 (s, 1H), 7.48 (s, 1H).
1H), 6.73 (d, J = 2.8 Hz, 1H), 6.75 (d, J = 8.6 Hz, 1H), 6.89
(s, 1H), 7.04 (s, 1H), 7.45 (s, 1H).
4-((8-(1H-Imidazole-1-yl)octyl)oxy)-2-chloroaniline (8j).
Yellow oil, 0.23 g, yield 97%, starting from 0.26 g of 7j (0.74
1
mmol). H NMR (400 MHz, CDCl3) δ 1.23−1.45 (m, 8H),
1.66−1.81 (m, 4H), 3.61−3.80 (brs, ArNH2, partially under
other peaks), 3.84 (t, J = 6.5 Hz, 2H), 3.91 (t, J = 7.1 Hz, 2H),
6.62−6.71 (m, 2H), 6.83 (d, J = 2.4 Hz, 1H), 6.89 (s, 1H),
7.04 (s, 1H), 7.46 (s, 1H).
4-((8-(1H-1,2,4-Triazol-1-yl)octyl)oxy)aniline (8k). Buff
powder, 0.21 g, yield 93%, starting from 0.25 g of 7k (0.78
1
mmol). H NMR (400 MHz, CDCl3) δ 1.23−1.46 (m, 8H),
1.66−1.75 (m, 2H), 1.83−1.91 (m, 2H), 3.26−3.53 (brs,
ArNH2), 3.85 (t, J = 6.5 Hz, 2H), 4.14 (t, J = 7.1 Hz, 2H), 6.62
(brd, J = 8.8 Hz, 2H), 6.72 (brd, J = 8.8 Hz, 2H), 7.92 (s, 1H),
8.03 (s, 1H).
4-((10-(1H-Imidazole-1-yl)decyl)oxy)aniline (8l). Buff pow-
der, 0.20 g, yield 88%, starting from 0.25 g of 7l (0.72 mmol).
1H NMR (300 MHz, CDCl3) δ 1.23−1.47 (m, 12H), 1.67−
1.81 (m, 4H), 3.07−3.64 (brs, ArNH2), 3.83−3.95 (m, 4H),
6.63 (d, J = 9.0 Hz, 2H), 6.73 (d, J = 8.9 Hz, 2H), 6.89 (t, J =
1.2 Hz, 1H), 7.05 (s, 1H), 7.45 (s, 1H).
4-((8-(1H-Pyrrol-1-yl)octyl)oxy)aniline (8m). The general
procedure listed above was used to prepare 8m, but this
material was also prepared via a Zn-mediated reduction, which
is described here. To a solution of 7m (170 mg, 0.54 mmol) in
MeOH (10 mL) was added ammonium chloride (285 mg, 5.30
mmol) and Zn (350 mg, 5.40 mmol), and the mixture was
stirred at room temperature for 3 h. After the reaction was
complete, the reaction mixture was filtered through Celite. The
Celite was washed with MeOH (10 mL); then, the combined
MeOH filtrate was evaporated under reduced pressure to
obtain a crude solid. To the crude solid was added water (20
mL), followed by extraction with DCM (2 × 20 mL). The
organic layers were collected, dried over anhydrous sodium
sulfate, and evaporated under reduced pressure to obtain the
title product as yellow oil (130 mg, 85%). 1H NMR (300 MHz,
CDCl3) δ 1.23−1.47 (m, 8H), 1.68−1.80 (m, 4H), 3.83−3.89
(m, 4H), 6.13 (t, J = 2.1 Hz, 2H), 6.64 (t, J = 2.1 Hz, 2H),
6.67 (d, J = 8.8 Hz, 2H), 6.74 (d, J = 8.8 Hz, 2H).
4-(4-(1H-Imidazol-1-yl)butoxy)aniline (8d). Buff powder,
1
0.25 g, yield 95% starting from 0.3 g of 7d (1.14 mmol). H
NMR (300 MHz, DMSO-d6) δ 1.67−1.78 (m, 2H), 1.91−2.02
(m, 2H), 3.09−3.46 (brs, ArNH2), 3.88 (t, J = 6.0 Hz, 2H),
4.00 (t, J = 7.1 Hz, 2H), 6.62 (d, J = 8.9 Hz, 2H), 6.71 (d, J =
8.9 Hz, 2H), 6.92 (s, 1H), 7.05 (s, 1H), 7.47 (s, 1H).
4-(4-(1H-1,2,4-Triazol-1-yl)butoxy)aniline (8e). Buff pow-
der, 0.32 g, yield 91% starting from 0.40 g of 7e (1.52 mmol).
1H NMR (300 MHz, CDCl3) δ 1.68−1.79 (m, 2H), 2.02−2.13
(m, 2H), 3.16−3.69 (brs, ArNH2), 3.91 (t, J = 6.0 Hz, 2H),
4.24 (t, J = 7.1 Hz, 2H), 6.62 (d, J = 8.9 Hz, 2H), 6.71 (d, J =
8.9 Hz, 2H), 7.93 (s, 1H), 8.06 (s, 1H).
4-((5-(1H-Imidazole-1-yl)pentyl)oxy)aniline (8f). Brown
oil, 0.29 g, yield 99% starting from 0.33 g of 7f (1.19
1
mmol). H NMR (300 MHz, CDCl3) δ 1.40−1.52 (m, 2H),
1.70−1.89 (m, 4H), 3.08−3.41 (brs, ArNH2), 3.86 (t, J = 6.2
Hz, 2H), 3.94 (t, J = 7.1 Hz, 2H), 6.62 (d, J = 8.9 Hz, 2H),
6.71 (d, J = 8.9 Hz, 2H), 6.90 (s, 1H), 7.05 (s, 1H), 7.47 (s,
1H).
Synthesis of Hybrid Target Compounds 9a−n.
4-((6-(1H-Imidazole-1-yl)hexyl)oxy)aniline (8g). Brown oil,
1
0.30 g, yield 92% starting from 0.35 g of 7g (1.20 mmol). H
NMR (300 MHz, CDCl3) δ 1.29−1.41 (m, 2H), 1.42−1.54
(m, 2H), 1.67−1.86 (m, 4H), 2.80−3.35 (brs, ArNH2), 3.86 (t,
J = 6.3 Hz, 2H), 3.93 (t, J = 7.1 Hz, 2H), 6.63 (d, J = 8.9 Hz,
2H), 6.72 (d, J = 8.9 Hz, 2H), 6.89 (s, 1H), 7.05 (s, 1H), 7.46
(s, 1H).
4-((8-(1H-Imidazole-1-yl)octyl)oxy)aniline (8h). Orange
powder, 0.23 g, yield 85%, starting from 0.30 g of 7h (0.95
1
mmol). H NMR (400 MHz, CDCl3) δ 1.23−1.36 (m, 6H),
1.37−1.46 (m, 2H), 1.67−1.80 (m, 4H), 3.18−3.58 (brs,
ArNH2), 3.86 (t, J = 6.5 Hz, 2H), 3.90 (t, J = 7.1 Hz, 2H), 6.62
(brd, J = 8.7 Hz m, 2H), 6.72 (brd, J = 8.7 Hz, 2H), 6.89 (s,
1H), 7.04 (s, 1H), 7.44 (s, 1H).
The synthesis of AIA−azole hybrid target compounds followed
previously published procedures for the synthesis of
arylimidamides.6,7,56 Two equivalents of S-(2-naphthylmeth-
yl)-2-pyridyl thioimidate hydrobromide (0.76−1.38 mmol) or
S-(2-naphthylmethyl) benzimidothioate hydrobromide (1.04
mmol) were added to one equivalent of a cooled solution of
the arylamines 8a−m (0.38−0.69 mmol) in dry acetonitrile/
ethanol (3:10 mL) in an ice bath for 15 min; then, this solution
4-((8-(1H-Imidazole-1-yl)octyl)oxy)-3-chloroaniline (8i).
Yellow oil, 0.17 g, yield 93%, starting from 0.20 g of 7i (0.57
1
mmol). H NMR (300 MHz, CDCl3) δ 1.24−1.39 (m, 6H),
1.40−1.51 (m, 2H), 1.70−1.82 (m, 4H), 2.95−3.70 (brs,
ArNH2), 3.91 (t, J = 6.7 Hz, 4H), 6.52 (dd, J = 8.6, 2.8 Hz,
K
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Article
was warmed to room temperature. The reaction mixture was
stirred at room temperature for 24−48 h. After the
disappearance of the starting material, the organic solvent
was evaporated under reduced pressure to yield a crude oil
product. Dry ether (100 mL) was added to the crude material,
and the mixture was stirred at room temperature overnight.
The precipitate was filtered and washed with dry ether. The
solid was dissolved in ethanol (2 mL); then, the solution was
cooled to 0 °C in an ice bath, and 10% NaOH was added until
the pH reached approximately 11. The free base was extracted
with ethyl acetate (3 × 30 mL). The organic layer was washed
with distilled water, dried over anhydrous sodium sulfate,
filtered, and concentrated under reduced pressure yielding a
waxy or solid product, which was further purified by column
chromatography on triethylamine neutralized silica using
DCM/MeOH as an eluent (100:0.5 to 100:3). The product
was further crystallized using hexanes/ethyl acetate to yield the
final product in 33−70% yield.
imidamide NHs), 6.89 (d, J = 8.9 Hz, 2H), 6.92−6.98 (m, 3H),
7.07 (t, J = 0.9 Hz, 1H), 7.38 (ddd, J = 7.5, 4.8, 1.2 Hz, 1H),
7.49 (s, 1H), 7.80 (td, J = 7.7, 1.7 Hz, 1H), 8.40 (d, J = 7.9 Hz,
1H), 8.56 (ddd, J = 4.8, 1.7, 0.9 Hz, 1H); 13C NMR (100
MHz, CDCl3) δ 26.5, 28.2, 46.9, 67.5, 115.7, 118.9, 121.6,
122.7, 125.2, 129.7, 136.9, 137.2, 143.3, 148.0, 151.8, 153.1,
154.9; HRMS (ESI) m/z (M + H)+ calcd for C19H22N5O,
336.18189; found, 336.18155; Anal. Calcd for C19H21N5O: C,
68.04; H, 6.31; N, 20.88. Found: C, 68.20; H, 6.35; N, 20.77.
N-(4-(4-(1H-1,2,4-Triazol-1-yl)butoxy)phenyl) Picolinimi-
damide (9e). Buff powder; 141 mg; yield 70% (starting from
1
0.14 g of 8e, 0.60 mmol); mp 109−111 °C. H NMR (400
MHz, CDCl3) δ 1.75−1.83 (m, 2H), 2.08−2.16 (m, 2H), 3.99
(t, J = 6.0 Hz, 2H), 4.27 (t, J = 7.0 Hz, 2H), 5.32−6.31 (brs,
imidamide NHs), 6.89 (d, J = 8.9 Hz, 2H), 6.96 (brd, J = 8.7
Hz, 2H), 7.38 (ddd, J = 7.5, 4.8, 1.2 Hz, 1H), 7.80 (td, J = 7.8,
1.8 Hz, 1H), 7.95 (s, 1H), 8.08 (s, 1H), 8.40 (d, J = 7.9 Hz,
1H), 8.56 (ddd, J = 4.8, 1.7, 0.9 Hz, 1H); 13C NMR (100
MHz, CDCl3) δ 26.3, 27.1, 49.5, 67.5, 115.6, 121.62, 122.7,
125.2, 136.9, 143.1, 143.3, 148.0, 151.8, 152.1, 153.1, 154.9;
HRMS (ESI) m/z (M + H)+ calcd for C18H21N6O, 337.17714;
found, 337.17712; Anal. Calcd for C18H20N6O: C, 64.27; H,
5.99; N, 24.98. Found: C, 64.43; H, 6.00; N, 24.93.
N-(4-(2-(1H-Imidazol-1-yl)ethoxy)phenyl) Picolinimida-
mide (9a). Yellow powder; 63 mg; yield 42% (starting from
1
0.10 g of 8a, 0.49 mmol); mp 141−144 °C. H NMR (400
MHz, DMSO-d6) δ 4.21 (t, J = 5.2 Hz, 2H), 4.35 (t, J = 5.1
Hz, 2H), 6.26−6.59 (brs, imidamide NHs), 6.82−6.93 (m,
5H), 7.25 (t, J = 1.2 Hz, 1H), 7.53 (ddd, J = 7.5, 4.8, 1.2 Hz,
1H), 7.68 (s, 1H), 7.93 (td, J = 7.7, 1.8 Hz, 1H), 8.29 (d, J =
7.9 Hz, 1H), 8.61 (ddd, J = 4.8, 1.7, 0.9 Hz, 1H); 13C NMR
(100 MHz, DMSO-d6) δ 45.7, 67.4, 115.3, 119.7, 121.1, 122.4,
125.3, 128.3, 137.0, 137.6, 143.6, 148.0, 151.6, 151.8, 153.4;
HRMS (ESI) m/z (M + H)+ calcd for C17H18N5O, 308.15059;
found, 308.15057; Anal. Calcd for C17H17N5O: C, 66.43; H,
5.58; N, 22.79. Found: C, 66.15; H, 5.71; N, 22.49.
N-(4-((5-(1H-Imidazol-1-yl)pentyl)oxy)phenyl) Picolinimi-
damide (9f). Light brown powder; 70 mg; yield 33% (starting
1
from 0.15 g of 8f, 0.61 mmol); mp 128−131 °C. H NMR
(700 MHz, CDCl3) δ 1.47−1.53 (m, 2H), 1.78−1.83 (m, 2H),
1.84−1.89 (m, 2H), 3.94 (t, J = 6.2 Hz, 2H), 3.97 (t, J = 7.2
Hz, 2H), 5.37−6.48 (brs, imidamide NHs), 6.89 (d, J = 8.8 Hz,
2H), 6.91 (t, J = 1.1 Hz, 1H) 6.95 (brd, J = 7.5 Hz, 2H), 7.06
(s, 1H), 7.38 (ddd, J = 7.5, 4.8, 1.1 Hz, 1H), 7.47 (s, 1H), 7.80
(td, J = 7.7, 1.7 Hz, 1H), 8.40 (d, J = 8.0 Hz, 1H), 8.57 (ddd, J
= 4.8, 1.6, 0.8 Hz, 1H); 13C NMR (176 MHz, CDCl3) δ 23.4,
28.9, 31.0, 47.0, 67.8, 115.6, 118.9, 121.6, 122.6, 125.2, 129.6,
136.9, 137.2, 143.2, 148.0, 151.8, 153.1, 155.1; HRMS (ESI)
m/z (M + H)+ calcd for C20H24N5O, 350.19754; found,
350.19757; Anal. Calcd for C20H23N5O: C, 68.74; H, 6.63; N,
20.04. Found: C, 68.45; H, 6.60; N, 19.90.
N-(4-(2-(1H-1,2,4-Triazol-1-yl)ethoxy)phenyl) Picolinimi-
damide (9b). Buff powder; 107 mg; yield 59% (starting
1
from 0.12 g of 8b, 0.58 mmol); mp 115−118 °C. H NMR
(400 MHz, CDCl3) δ 4.33 (t, J = 5.0 Hz, 2H), 4.56 (t, J = 5.0
Hz, 2H), 5.40−6.21 (brs, imidamide NHs), 6.86 (brd, J = 8.8
Hz, 2H), 6.93 (brd, J = 8.6 Hz, 2H), 7.38 (ddd, J = 7.4, 4.9, 1.1
Hz, 1H), 7.80 (td, J = 7.8, 1.7 Hz, 1H), 7.96 (s, 1H), 8.23 (s,
1H), 8.38 (d, J = 8.0 Hz, 1H), 8.56 (br d, J = 4.4 Hz, 1H); 13C
NMR (100 MHz, CDCl3) δ 49.6, 66.2, 115.9, 121.6, 122.8,
125.3, 136.9, 144.2, 148.0, 151.7, 152.2, 153.1, 154.0; HRMS
(ESI) m/z (M + H)+ calcd for C16H17N6O, 309.14584; found,
309.14569; Anal. Calcd for C16H16N6O: C, 62.32; H, 5.23; N,
27.26. Found: C, 62.39; H, 5.28; N, 27.18.
N-(4-((6-(1H-Imidazol-1-yl)hexyl)oxy)phenyl) Picolinimi-
damide (9g). Light brown powder; 70 mg; yield 33% (starting
1
from 0.15 g of 8g, 0.57 mmol); mp 127−129 °C. H NMR
(400 MHz, CDCl3) δ 1.33−1.43 (m, 2H), 1.48−1.56 (m, 2H),
1.73−1.87 (m, 4H), 3.92−3.97 (m, 4H), 5.61−6.12 (brs,
imidamide NHs), 6.88−6.98 (m, 5H), 7.06 (s, 1H), 7.39 (ddd,
J = 7.4, 4.9, 1.0 Hz, 1H), 7.46 (s, 1H), 7.81 (td, J = 7.8, 1.7 Hz,
1H), 8.41 (d, J = 8.0 Hz, 1H), 8.57 (d, J = 4.6 Hz, 1H); 13C
NMR (100 MHz, CDCl3) δ 25.8, 26.5, 29.3, 31.2, 47.1, 68.0,
115.7, 118.9, 121.6, 122.6, 125.2, 129.6, 136.9, 137.2, 143.1,
148.0, 151.9, 153.1, 155.2; HRMS (ESI) m/z (M + H)+ calcd
for C21H26N5O, 364.21319; found, 364.21298; Anal. Calcd for
C21H25N5O: C, 69.40; H, 6.93; N, 19.27. Found: C, 69.43; H,
6.81; N, 19.13.
N-(4-(3-(1H-Imidazol-1-yl)propoxy)phenyl) Picolinimida-
mide (9c). Yellow powder; 85 mg; yield 38% (starting from
1
0.15 g of 8c, 0.69 mmol); mp 115−118 °C. H NMR (700
MHz, CDCl3) δ 2.20−2.25 (m, 2H), 3.91 (t, J = 5.7 Hz, 2H),
4.21 (t, J = 6.8 Hz, 2H), 5.35−6.49 (brs, imidamide NHs), 6.90
(d, J = 8.8 Hz, 2H), 6.94 (t, J = 1.2 Hz, 1H), 6.97 (brd, J = 8.6
Hz, 2H), 7.07 (s, 1H), 7.39 (ddd, J = 7.5, 4.8, 1.1 Hz, 1H),
7.49 (s, 1H), 7.81 (td, J = 7.7, 1.7 Hz, 1H), 8.41 (d, J = 7.9 Hz,
1H), 8.57 (ddd, J = 4.8, 1.7, 0.9 Hz, 1H); 13C NMR (176
MHz, CDCl3) δ 31.0, 43.6, 64.1, 115.7, 119.1, 121.6, 122.8,
125.3, 129.8, 136.9, 137.6, 143.5, 148.0, 151.7, 153.2, 154.7;
HRMS (ESI) m/z (M + H)+ calcd for C18H20N5O, 322.16624;
found, 322.16592; Anal. Calcd for C18H19N5O: C, 67.27; H,
5.96; N, 21.79. Found: C, 67.42; H, 5.98; N, 21.78.
N-(4-((8-(1H-Imidazol-1-yl)octyl)oxy)phenyl) Picolinimi-
damide (9h). Light brown powder; 85 mg; yield 44% (starting
from 0.14 g of 8h, 0.49 mmol); mp 89−92 °C. 1H NMR (400
MHz, CDCl3) δ 1.28−1.40 (m, 6H), 1.41−1.50 (m, 2H),
1.73−1.82 (m, 4H), 3.90−3.97 (m, 4H), 5.47−6.30 (brs,
imidamide NHs), 6.89−6.93 (m, 3H), 6.96 (d, J = 8.9 Hz, 2H),
7.05 (t, J = 1.0 Hz, 1H), 7.39 (ddd, J = 7.5, 4.8, 1.2 Hz, 1H),
7.45 (s, 1H), 7.81 (td, J = 7.8, 1.8 Hz, 1H), 8.42 (d, J = 8.0 Hz,
1H), 8.57 (ddd, J = 4.9, 1.7, 0.9 Hz, 1H); 13C NMR (100
MHz, CDCl3) δ 26.1, 26.6, 29.1, 29.3, 29.4, 31.2, 47.2, 68.3,
115.7, 118.9, 121.7, 122.7, 125.2, 129.6, 136.9, 137.2, 142.7,
N-(4-(4-(1H-Imidazol-1-yl)butoxy)phenyl) Picolinimida-
mide (9d). Buff powder; 132 mg; yield 65% (starting from
1
0.14 g of 8d, 0.60 mmol); mp 103−105 °C. H NMR (400
MHz, CDCl3) δ 1.74−1.82 (m, 2H), 1.96−2.05 (m, 2H), 3.97
(t, J = 6.0 Hz, 2H), 4.03 (t, J = 7.0 Hz, 2H), 5.43−6.30 (brs,
L
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148.0, 151.8, 153.2, 155.4; HRMS (ESI) m/z (M + H)+ calcd
for C23H30N5O, 392.24449; found, 392.24451; Anal. Calcd for
C23H29N5O: C, 70.56; H, 7.47; N, 17.89. Found: C, 70.27; H,
7.29; N, 17.70.
47.2, 68.4, 115.7, 118.9, 121.7, 122.7, 125.2, 129.5, 136.9,
137.2, 142.5, 148.0, 151.8, 153.2, 155.4; HRMS (ESI) m/z (M
+ H)+ calcd for C25H34N5O, 420.27579; found, 420.27637;
Anal. Calcd for C25H33N5O: C, 71.57; H, 7.93; N, 16.69.
Found: C, 71.27; H, 7.66; N, 16.47.
N-(4-((8-(1H-Imidazol-1-yl)octyl)oxy)-3-chlorophenyl) Pi-
colinimidamide (9i). Yellow powder; 99 mg; yield 54%
N-(4-((8-(1H-Pyrrol-1-yl)octyl)oxy)phenyl) Picolinimida-
mide (9m). The synthesis of 9m follows the general synthesis
of 9a−l with the modification of using 1.5 equiv of S-(2-
naphthylmethyl)-2-pyridyl thioimidate hydrobromide and,
after completion of the reaction, no diethyl ether treatment
was performed. The compound was purified by column
chromatography using hexanes/DCM (1:1.5) on triethylamine
neutralized silica. After the column chromatography purifica-
tion step, the powder was triturated with diethyl ether/hexanes
(3 × 10 mL) followed by filtration. The filtrate was further
crystallized using hexanes/ethyl acetate to yield the final
product. Light brown powder, 58 mg, yield 17% (starting from
1
(starting from 0.14 g of 8i, 0.43 mmol); mp 102−104 °C. H
NMR (400 MHz, CDCl3) δ 1.25−1.41 (m, 6H), 1.45−1.55
(m, 2H), 1.74−1.86 (m, 4H), 3.92 (t, J = 7.1 Hz, 2H), 4.01 (t,
J = 6.4 Hz, 2H), 5.42−6.39 (brs, imidamide NHs), 6.86 (dd, J =
8.6, 2.4 Hz, 1H), 6.90 (t, J = 1.2 Hz, 1H), 6.93 (d, J = 8.6 Hz,
1H), 7.05 (s, 1H), 7.08 (d, J = 2.4 Hz, 1H), 7.39 (ddd, J = 7.5,
4.8, 1.2 Hz, 1H), 7.45 (s, 1H), 7.81 (td, J = 7.7, 1.7 Hz, 1H),
8.37 (d, J = 8.0 Hz, 1H), 8.57 (ddd, J = 4.8, 1.7, 0.9 Hz, 1H);
13C NMR (100 MHz, CDCl3) δ 26.0, 26.6, 29.1, 29.2, 29.3,
31.2, 47.2, 69.7, 115.0, 118.9, 120.8, 121.7, 123.6, 123.9, 125.4,
129.6, 137.0, 137.2, 143.6, 148.0, 150.7, 151.5, 153.3; HRMS
(ESI) m/z (M + H)+ and ((M + 2) + H)+ calcd for
C23H29N5ClO, 426.20551 and 428.20261; found, 426.20490
and 428.20236; Anal. Calcd for C23H28N5ClO: C, 64.85; H,
6.63; N, 16.44. Found: C, 64.81; H, 6.67; N, 16.32.
1
0.25 g of 8m, 0.87 mmol); mp 93−95 °C. H NMR (400
MHz, CDCl3) δ 1.26−1.40 (m, 6H), 1.42−1.51 (m, 2H),
1.73−1.82 (m, 4H), 3.87 (t, J = 7.2 Hz, 2H), 3.94 (t, J = 6.5
Hz, 2H), 5.52−6.09 (brs, imidamide NHs), 6.14 (t, J = 2.1 Hz,
2H), 6.65 (t, J = 2.1 Hz, 2H), 6.89−6.99 (m, 4H), 7.39 (ddd, J
= 7.5, 4.8, 1.1 Hz, 1H), 7.81 (td, J = 7.8, 1.7 Hz, 1H), 8.41 (d, J
= 8.0 Hz, 1H), 8.57 (d, J = 4.8 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ 26.2, 26.9, 29.3, 29.4, 29.5, 31.7, 49.8, 68.4, 107.9,
115.7, 120.6, 121.6, 122.6, 125.2, 136.9, 143.0, 148.0, 151.9,
153.0, 155.4; HRMS (ESI) m/z (M + H)+ calcd for
C24H31N4O, 391.24924; found, 391.24988; Anal. Calcd for
C24H30N4O: C, 73.81; H, 7.74; N, 14.35. Found: C, 74.03; H,
7.89; N, 14.25.
N-(4-((8-(1H-Imidazol-1-yl)octyl)oxy)-2-chlorophenyl) Pi-
colinimidamide (9j). White powder; 71 mg; yield 39%
1
(starting from 0.14 g of 8j, 0.43 mmol); mp 74−76 °C. H
NMR (400 MHz, CDCl3) δ 1.24−1.40 (m, 6H), 1.40−1.50
(m, 2H), 1.72−1.83 (m, 4H), 3.89−3.95 (m, 4H), 5.40−6.38
(brs, imidamide NHs), 6.82 (dd, J = 8.7, 2.7 Hz, 1H), 6.90 (s,
1H), 6.95 (d, J = 8.7 Hz, 1H), 7.00 (d, J = 2.7 Hz, 1H), 7.05
(s, 1H), 7.40 (ddd, J = 7.4, 4.8, 1.0 Hz, 1H), 7.46 (s, 1H), 7.82
(td, J = 7.7, 1.6 Hz, 1H), 8.46 (d, J = 8.0 Hz, 1H), 8.58 (d, J =
4.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 26.0, 26.6, 29.1,
29.27, 29.30, 31.2, 47.2, 68.6, 114.7, 116.2, 118.9, 122.0, 123.4,
125.4, 126.7, 129.6, 137.0, 137.2, 139.7, 148.0, 151.3, 153.4,
155.6; HRMS (ESI) m/z (M + H)+ and ((M + 2) + H)+ calcd
for C23H29N5ClO, 426.20551 and 428.20261; found,
426.20663 and 428.20416; Anal. Calcd for C23H28N5ClO: C,
64.85; H, 6.63; N, 16.44. Found: C, 64.68; H, 6.58; N, 16.38.
N-(4-((8-(1H-1,2,4-Triazol-1-yl)octyl)oxy)phenyl) Picolini-
midamide (9k). Light brown powder; 85 mg; yield 39%
N-(4-((8-(1H-Imidazol-1-yl)octyl)oxy)phenyl)-
benzimidamide (9n). Buff powder; 45 mg; yield 22% (starting
1
from 0.15 g of 8h, 0.52 mmol); mp 107−109 °C. H NMR
(700 MHz, CDCl3) δ 1.28−1.38 (m, 6H), 1.42−1.48 (m, 2H),
1.74−1.80 (m, 4H), 3.90−3.95 (m, 4H), 4.68−5.18 (brs,
imidamide NHs), 6.87−6.94 (m, 5H), 7.04 (s, 1H), 7.41−7.48
(m, 4H), 7.85 (brs, 2H); 13C NMR (176 MHz, CDCl3) δ 26.1,
26.6, 29.1, 29.3, 29.4, 31.2, 47.1, 68.3, 115.6, 118.9, 122.7,
126.9, 128.7, 129.5, 130.6, 136.1, 137.2, 142.6, 155.3; HRMS
(ESI) m/z (M + H)+ calcd for C24H31N4O, 391.24924; found,
391.24905; Anal. Calcd for C24H30N4O: C, 73.81; H, 7.74; N,
14.35. Found: C, 73.53; H, 7.80; N, 14.26.
1
(starting from 0.16 g of 8k, 0.55 mmol); mp 98−100 °C. H
NMR (700 MHz, CDCl3) δ 1.28−1.39 (m, 6H), 1.42−1.48
(m, 2H), 1.74−1.79 (m, 2H), 1.87−1.92 (m, 2H), 3.94 (t, J =
6.4 Hz, 2H), 4.16 (t, J = 7.1 Hz, 2H), 5.29−6.44 (brs,
imidamide NHs), 6.91 (d, J = 8.8 Hz, 2H), 6.96 (brd, J = 8.6
Hz, 2H), 7.38 (ddd, J = 7.4, 4.9, 1.0 Hz, 1H), 7.80 (td, J = 7.7,
1.7 Hz, 1H), 7.93 (s, 1H), 8.04 (s, 1H), 8.41 (d, J = 7.9 Hz,
1H), 8.56 (d, J = 4.7 Hz, 1H); 13C NMR (176 MHz, CDCl3) δ
26.1, 26.5, 29.1, 29.3, 29.4, 29.9, 49.8, 68.3, 115.7, 121.7, 122.7,
125.2, 136.9, 142.9, 148.0, 151.7, 152.0, 153.2, 155.4; HRMS
(ESI) m/z (M + H)+ calcd for C22H29N6O, 393.23974; found,
393.23932; Anal. Calcd for C22H28N6O: C, 67.32; H, 7.19; N,
21.41. Found: C, 67.18; H, 7.16; N, 21.47.
2-(Methoxymethoxy)-5-nitrophenol (11).
Compound 11 was synthesized following a previously
published procedure.29 To a solution of 4-nitrocatechol (10,
1.0 g, 6.44 mmol) in dry DMF (10 mL) was added 1.2 equiv of
potassium carbonate (1.06 g, 7.67 mmol) and 1.1 equiv of
MOMCl (0.57 g, 7.08 mmol). The reaction mixture was
heated to 40 °C for 2 h. After completion of the reaction, the
solvent was removed under reduced pressure; water was added,
and the product was extracted with ethyl acetate (3 × 30 mL).
The combined organic layer was washed with brine, dried over
sodium sulfate, and evaporated under reduced pressure. The
crude product was purified by column chromatography using
hexanes/ethyl acetate (4:1) as eluent, yielding the product as a
buff powder, 0.61 g, 48%.
N-(4-((10-(1H-Imidazol-1-yl)decyl)oxy)phenyl) Picolinimi-
damide (9l). Light brown powder; 61 mg; yield 38% (starting
1
from 0.12 g of 8l, 0.38 mmol); mp 104−106 °C. H NMR
(400 MHz, CDCl3) δ 1.24−1.39 (m, 10H), 1.41−1.50 (m,
2H), 1.73−1.81 (m, 4H), 3.89−3.97 (m, 4H total, over-
lapped), 3.91 (t, J = 7.1 Hz, overlapped), 3.94 (t, J = 6.5 Hz,
overlapped), 5.38−6.29 (brs, imidamide NHs), 6.89−6.98 (m,
5H), 7.05 (s, 1H), 7.38 (ddd, J = 7.4, 4.8, 1.1 Hz, 1H), 7.45 (s,
1H), 7.80 (td, J = 7.7, 1.7 Hz, 1H), 8.41 (d, J = 8.0 Hz, 1H),
8.56 (ddd, J = 4.8, 1.7, 0.9 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ 26.2, 26.7, 29.2, 29.46, 29.47, 29.50, 29.55, 31.2,
M
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Synthesis of 2-Alkoxy-1-(methoxymethoxy)-4-nitro-
benzenes (12a−c).
CDCl3) δ 1.42 (d, J = 6.1 Hz, 6H), 4.71 (sep, J = 6.1 Hz, 1H),
6.31 (brs, 1H), 6.97 (d, J = 8.8 Hz, 1H), 7.75 (d, J = 2.5 Hz,
1H), 7.84 (dd, J = 8.8, 2.5 Hz, 1H); 13C NMR (75 MHz,
CDCl3) δ 22.0, 72.7, 108.4, 114.1, 118.4, 141.2, 144.3, 152.6.
Synthesis of 1-((8-Bromooctyl)oxy)-2-alkoxy-4-nitro-
benzenes (14a−c).
To a solution of 11 (1.30−1.50 mmol) in dry acetonitrile (5
mL) was added 2 equiv of potassium carbonate (2.60−3.00
mmol) and 3 equiv of alkyl iodide (3.90−4.50 mmol). The
mixture was heated in a sealed tube at 80 °C for 4−5 h. After
the reaction was complete, the solution was cooled and the
solvent was removed under reduced pressure. Ice was added to
precipitate the pure product, which was filtered as a white
powder in 91−97% yield.
2-Methoxy-1-(methoxymethoxy)-4-nitrobenzene (12a).
0.29 g, yield 97%, starting from 0.28 g of 11 (1.40 mmol).
1H NMR (300 MHz, CDCl3) δ 3.51 (s, 3H), 3.96 (s, 3H),
5.32 (s, 2H), 7.21 (d, J = 8.9 Hz, 1H), 7.77 (d, J = 2.6 Hz,
1H), 7.86 (dd, J = 8.9, 2.6 Hz, 1H); 13C NMR (75 MHz,
CDCl3) δ 56.5, 56.8, 95.3, 107.1, 114.4, 117.6, 142.5, 149.6,
152.2.
2-Ethoxy-1-(methoxymethoxy)-4-nitrobenzene (12b).
0.28 g, yield 95%, starting from 0.26 g of 11 (1.30 mmol).
1H NMR (300 MHz, CDCl3) δ 1.49 (t, J = 7.0 Hz, 3H), 3.51
(s, 3H), 4.18 (q, J = 7.0 Hz, 2H), 5.31 (s, 2H), 7.20 (d, J = 9.0
Hz, 1H), 7.75 (d, J = 2.6 Hz, 1H), 7.84 (dd, J = 9.0, 2.6 Hz,
1H); 13C NMR (75 MHz, CDCl3) δ 14.7, 56.7, 65.1, 95.4,
108.2, 115.0, 117.4, 142.6, 149.0, 152.4.
Compounds 14a−c were prepared from 13a−c (0.92−1.11
mmol) following the general synthesis of compounds 6a−i.
1-((8-Bromooctyl)oxy)-2-methoxy-4-nitrobenzene (14a).
Yellowish white powder, 0.33 g, yield 91% starting from 0.17
1
g of 13a (1.00 mmol). H NMR (300 MHz, CDCl3) δ 1.30−
1.54 (m, 8H), 1.80−1.93 (m, 4H), 3.40 (t, J = 6.8 Hz, 2H),
3.94 (s, 3H), 4.10 (t, J = 6.7 Hz, 2H), 6.88 (d, J = 9.0 Hz, 1H),
7.73 (d, J = 2.6 Hz, 1H), 7.88 (dd, J = 8.9, 2.6 Hz, 1H); 13C
NMR (75 MHz, CDCl3) δ 25.9, 28.2, 28.7, 28.9, 29.2, 32.8,
34.0, 56.4, 69.6, 106.8, 110.9, 117.9, 141.4, 149.2, 154.3.
1-((8-Bromooctyl)oxy)-2-ethoxy-4-nitrobenzene (14b).
Yellowish white powder, 0.32 g, yield 92% starting from 0.17
1
g of 13b (0.92 mmol). H NMR (300 MHz, CDCl3) δ 1.31−
1.52 (m, 11H total, overlapped), 1.48 (t, J = 7.0 Hz,
overlapped) 1.80−1.92 (m, 4H), 3.40 (t, J = 6.8 Hz, 2H),
4.09 (t, J = 6.7 Hz, 2H, overlapped), 4.15 (q, J = 7.0 Hz, 2H,
overlapped), 6.88 (d, J = 8.9 Hz, 1H), 7.73 (d, J = 2.6 Hz, 1H),
7.87 (dd, J = 8.9, 2.6 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ
14.7, 25.9, 28.2, 28.8, 29.0, 29.2, 32.9, 34.0, 65.1, 69.6, 108.2,
111.2, 117.8, 141.4, 148.6, 154.7.
2-Isopropoxy-1-(methoxymethoxy)-4-nitrobenzene (12c).
1
0.33 g, yield 91%, starting from 0.30 g of 11 (1.50 mmol). H
NMR (300 MHz, CDCl3) δ 1.40 (d, J = 6.1 Hz, 6H), 3.51 (s,
3H), 4.62 (sep, J = 6.1 Hz, 1H), 5.28 (s, 2H), 7.18 (d, J = 9.0
Hz, 1H), 7.77 (d, J = 2.6 Hz, 1H), 7.83 (dd, J = 9.0, 2.6 Hz,
1H); 13C NMR (75 MHz, CDCl3) δ 22.0, 56.7, 72.4, 95.3,
110.7, 115.5, 117.5, 142.6, 148.1, 153.4.
1-((8-Bromooctyl)oxy)-2-isopropoxy-4-nitrobenzene
(14c). White powder, 0.36 g, yield 84% starting from 0.22 g of
13c (1.11 mmol). H NMR (300 MHz, CDCl3) δ 1.31−1.54
1
(m, 14H total, overlapped), 1.38 (d, J = 6.1 Hz, overlapped),
1.80−1.92 (m, 4H), 3.41 (t, J = 6.8 Hz, 2H), 4.07 (t, J = 6.5
Hz, 2H), 4.57 (sep, J = 6.0 Hz, 1H), 6.88 (d, J = 9.0 Hz, 1H),
7.76 (d, J = 2.6 Hz, 1H), 7.87 (dd, J = 9.0, 2.6 Hz, 1H); 13C
NMR (75 MHz, CDCl3) δ 22.1, 25.9, 28.2, 28.8, 29.0, 29.2,
32.9, 34.0, 69.5, 72.8, 111.7, 111.7, 118.3, 141.3, 147.5, 156.0.
Synthesis of 1-(8(2-Alkoxy-4-nitrophenoxy)octyl)-1H-
imidazoles (15a−c).
Synthesis of 2-Alkoxy-4-nitrobenzenes (13a−c).
To a cooled solution of 12a−c (1.11−1.31 mmol) in
methanol/DCM (6:3) at 0 °C was added 4 N HCl (1 mL),
and the mixture was stirred for 30 min. The mixture was then
warmed to rt and allowed to stir for another 2 h. After the
reaction was complete, ethyl acetate (30 mL) was added and
the product was extracted (2 × 30 mL) with ethyl acetate. The
combined organic layer was washed with water and brine,
dried over sodium sulfate, and evaporated under reduced
pressure. The crude product was purified by column
chromatography affording the product in 82−89% yield.
2-Methoxy-4-nitrophenol (13a).57 Chromatography sol-
vent: hexanes/DCM (1:1), yellow powder, 0.18 g, yield 81%
starting from 0.28 g of 12a (1.31 mmol).
Compounds 15a−c were synthesized from 14a−c (0.83−0.98
mmol) following the general synthesis of compounds 7a−l.
1-(8(2-Methoxy-4-nitrophenoxy)octyl)-1H-imidazole
(15a). Yellowish white powder, 0.23 g, yield 74% starting from
1
0.32 g of 14a (0.89 mmol). H NMR (300 MHz, CDCl3) δ
1.23−1.50 (m, 8H), 1.71−1.90 (m, 4H), 3.88−3.94 (m, 5H
total, overlapped), 3.94 (s, overlapped), 4.08 (t, J = 6.7 Hz,
2H), 6.85−6.89 (m, 2H), 7.04 (s, 1H), 7.44 (s, 1H), 7.72 (d, J
= 2.6 Hz, 1H), 7.87 (dd, J = 8.9, 2.6 Hz, 1H); 13C NMR (75
MHz, CDCl3) δ 25.9, 26.6, 28.9, 29.1, 29.2, 31.1, 47.1, 56.4,
69.5, 106.8, 110.9, 117.9, 118.8, 129.5, 137.2, 141.4, 149.2,
154.3.
2-Ethoxy-4-nitrophenol (13b).58 Chromatography solvent:
hexanes/DCM (2:1 to 2:3), yellow powder, 0.18 g, yield 89%
starting from 0.25 g of 12b (1.11 mmol).
2-Isopropoxy-4-nitrophenol (13c). Chromatography sol-
vent: hexanes/DCM (2:1), yellow waxy oil, 0.23 g, yield 90%
starting from 0.31 g of 12c (1.29 mmol). 1H NMR (300 MHz,
1-(8(2-Ethoxy-4-nitrophenoxy)octyl)-1H-imidazole (15b).
Yellowish white powder, 0.22 g, yield 73% starting from 0.31 g
N
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1
of 14b (0.83 mmol). H NMR (300 MHz, CDCl3) δ 1.23−
yield 45% starting from 220 mg of 16a (0.69 mmol); mp 117−
1
1.51 (m, 11H total, overlapped), 1.47 (t, J = 7.0 Hz,
overlapped), 1.72−1.90 (m, 4H), 3.92 (t, J = 7.1 Hz, 2H),
4.07 (t, J = 6.7 Hz, 2H), 4.14 (q, J = 7.0 Hz, 2H), 6.84−6.90
(m, 2H), 7.04 (s, 1H), 7.45 (s, 1H), 7.72 (d, J = 2.6 Hz, 1H),
7.86 (dd, J = 8.9, 2.6 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ
14.7, 25.9, 26.6, 28.9, 29.1, 29.2, 31.2, 47.1, 65.1, 69.5, 108.2,
111.2, 117.8, 118.9, 129.5, 137.2, 141.4, 148.6, 154.6.
1-(8(2-Isopropoxy-4-nitrophenoxy)octyl)-1H-imidazole
(15c). Yellow oil, 0.33 g, yield 90% starting from 0.38 g of 14c
(0.98 mmol). H NMR (300 MHz, CDCl3) δ 1.24−1.50 (m,
14H total, overlapped), 1.36 (d, J = 6.1 Hz, overlapped) 1.71−
1.88 (m, 4H), 3.91 (t, J = 7.1 Hz, 2H), 4.05 (t, J = 6.6 Hz,
2H), 4.55 (sep, J = 6.1 Hz, 1H), 6.84−6.89 (m, 2H), 7.03 (s,
1H), 7.43 (s, 1H), 7.74 (d, J = 2.7 Hz, 1H), 7.85 (dd, J = 9.0,
2.7 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 22.1, 25.9, 26.6,
28.9, 29.1, 29.2, 31.1, 47.1, 69.4, 72.7, 111.6, 111.7, 118.2,
118.8, 129.5, 137.1, 141.3, 147.5, 155.9.
119 °C. H NMR (400 MHz, CDCl3) δ 1.28−1.39 (m, 6H),
1.41−1.51 (m, 2H), 1.74−1.87 (m, 4H), 3.84 (s, 3H), 3.92 (t,
J = 7.2 Hz, 2H), 3.99 (t, J = 6.8 Hz, 2H), 5.50−6.34 (brs,
imidamide NHs), 6.55 (dd, J = 8.4, 2.3 Hz, 1H), 6.62 (d, J = 2.3
Hz, 1H), 6.87−6.91 (m, 2H), 7.05 (s, 1H), 7.39 (ddd, J = 7.5,
4.8, 1.2 Hz, 1H), 7.45 (s, 1H), 7.81 (td, J = 7.7, 1.7 Hz, 1H),
8.41 (d, J = 8.0 Hz, 1H), 8.57 (ddd, J = 4.8, 1.7, 0.9 Hz, 1H);
13C NMR (100 MHz, CDCl3) δ 26.0, 26.6, 29.1, 29.3, 29.4,
31.2, 47.2, 56.0, 69.6, 106.5, 112.8, 114.6, 118.9, 121.6, 125.3,
129.6, 136.9, 137.2, 143.5, 144.6, 148.0, 150.6, 151.7, 153.2;
HRMS (ESI) m/z (M + H)+ calcd for C24H32N5O2,
422.25505; found, 422.25616; Anal. Calcd for C24H31N5O2:
C, 68.38; H, 7.41; N, 16.61. Found: C, 68.18; H, 7.46; N,
16.38.
1
N-(4-((8-(1H-Imidazol-1-yl)octyl)oxy)3-ethoxyphenyl) Pi-
colinimidamide (17b). Yellowish white powder, 65 mg, yield
33% starting from 150 mg of 16b (0.45 mmol); mp 100−102
1
Synthesis of 4-((8-(1H-Imidazol-1-yl)octyl)oxy)-3-al-
koxyanilines (16a−c).
°C. H NMR (400 MHz, CDCl3) δ 1.25−1.51 (m, 11H total,
overlapped), 1.42 (t, J = 7.0 Hz, overlapped), 1.74−1.84 (m,
4H), 3.92 (t, J = 7.1 Hz, 2H), 3.98 (t, J = 6.7 Hz, 2H), 4.06 (q,
J = 7.0 Hz, 2H), 5.49−6.23 (brs, imidamide NHs), 6.54 (d, J =
8.3 Hz, 1H), 6.61 (s, 1H), 6.87−6.91 (m, 2H), 7.05 (s, 1H),
7.38 (ddd, J = 7.5, 4.8, 1.1 Hz, 1H), 7.45 (s, 1H), 7.80 (td, J =
7.8, 1.7 Hz, 1H), 8.40 (d, J = 7.9 Hz, 1H), 8.56 (d, J = 4.9 Hz,
1H); 13C NMR (100 MHz, CDCl3) δ 15.0, 26.1, 26.6, 29.1,
29.3, 29.5, 31.2, 47.2, 64.5, 70.0, 108.0, 113.0, 115.7, 118.9,
121.6, 125.2, 129.6, 136.9, 137.2, 143.9, 145.0, 148.0, 150.1,
151.8, 153.1; HRMS (ESI) m/z (M + H)+ calcd for
C25H34N5O2, 436.27070; found, 436.27029; Anal. Calcd for
C25H33N5O2: C, 68.94; H, 7.64; N, 16.08. Found: C, 69.09; H,
7.54; N, 16.03.
Compounds 16a−c were synthesized from 15a−c (0.49−0.86
mmol) following the general synthesis of compounds 8a−m.
4-((8-(1H-Imidazol-1-yl)octyl)oxy)-3-methoxyaniline
(16a). Orange oil, 0.27 g, yield 99% starting from 0.30 g of 15a
1
(0.86 mmol). H NMR (300 MHz, CDCl3) δ 1.23−1.46 (m,
8H), 1.70−1.81 (m, 4H), 3.00−3.39 (brs, ArNH2), 3.79 (s,
3H), 3.86−3.93 (m, 4H), 6.19 (dd, J = 8.4, 2.6 Hz, 1H), 6.29
(d, J = 2.6 Hz, 1H), 6.70 (d, J = 8.4 Hz, 1H), 6.88 (t, J = 1.1
Hz, 1H), 7.04 (s, 1H), 7.44 (s, 1H).
N-(4-((8-(1H-Imidazol-1-yl)octyl)oxy)3-isopropoxyphenyl)
Picolinimidamide (17c). Yellowish white powder, 90 mg, yield
31% starting from 220 mg of 16c (0.63 mmol); mp 88−91 °C.
1H NMR (400 MHz, CDCl3) δ 1.28−1.39 (m, 12H), 1.42−
1.52 (m, 2H), 1.74−1.82 (m, 4H), 3.92 (t, J = 7.2 Hz, 2H),
3.97 (t, J = 6.6 Hz, 2H), 4.48 (sep, J = 6.1 Hz, 1H), 5.50−6.30
(brs, imidamide NHs), 6.57 (d, J = 8.4 Hz, 1H), 6.64 (s, 1H),
6.88−6.91 (m, 2H), 7.05 (s, 1H), 7.38 (ddd, J = 7.5, 4.9, 1.2
Hz, 1H), 7.45 (s, 1H), 7.80 (td, J = 7.8, 1.7 Hz, 1H), 8.40 (d, J
= 8.0 Hz, 1H), 8.57 (ddd, J = 4.8, 1.6, 0.9 Hz, 1H); 13C NMR
(100 MHz, CDCl3) δ 22.4, 26.1, 26.7, 29.2, 29.3, 29.6, 31.2,
47.2, 70.0, 72.1, 111.7, 114.1, 116.3, 118.9, 121.6, 125.2, 129.6,
136.9, 137.2, 143.9, 146.4, 148.0, 149.1, 151.8, 153.1; HRMS
(ESI) m/z (M + H)+ calcd for C26H36N5O2, 450.28635; found,
450.28671; Anal. Calcd for C26H35N5O2: C, 69.46; H, 7.85; N,
15.58. Found: C, 69.19; H, 7.77; N, 15.28.
4-((8-(1H-Imidazol-1-yl)octyl)oxy)-3-ethoxyaniline (16b).
Brown oil, 0.16 g, yield 98% starting from 0.18 g of 15b
(0.49 mmol). H NMR (300 MHz, CDCl3) δ 1.24−1.48 (m,
11H total, overlapped), 1.40 (t, J = 7.0 Hz, overlapped), 1.68−
1.82 (m, 4H), 1.92−2.43 (brs, ArNH2), 3.86−3.95 (m, 4H),
4.01 (q, J = 7.0 Hz, 2H), 6.20 (dd, J = 8.4, 2.6 Hz, 1H), 6.30
(d, J = 2.7 Hz, 1H), 6.72 (d, J = 8.4 Hz, 1H), 6.89 (brs, 1H),
7.04 (s, 1H), 7.45 (s, 1H).
1-((8-(1H-Imidazol-1-yl)octyl)oxy)-3-isopropoxyaniline
(16c). Brown oil, 0.27 g, yield 98% starting from 0.30 g of 15c
(0.79 mmol). H NMR (300 MHz, CDCl3) δ 1.22−1.47 (m,
14H total, overlapped), 1.30 (d, J = 6.1 Hz, overlapped), 1.66−
1.80 (m, 4H), 3.08−3.43 (brs, ArNH2), 3.84−3.93 (m, 4H),
4.42 (sep, J = 6.1 Hz, 1H), 6.22 (dd, J = 8.4, 2.7 Hz, 1H), 6.31
(d, J = 2.7 Hz, 1H), 6.71 (d, J = 8.4 Hz, 1H), 6.88 (t, J = 1.1
Hz, 1H), 7.03 (s, 1H), 7.44 (s, 1H).
1
1
Synthesis of 4-Fluoro-2-alkoxy-1-nitrobenzenes
(19a−c).
Synthesis of N-(4-((8-(1H-Imidazol-1-yl)octyl)oxy)3-
alkoxyphenyl) Picolinimidamides (17a−c).
To a solution of 18 (0.50−0.55 g, 3.18−3.50 mmol) in dry
DMF (5 mL) was added 1.5 equiv of potassium carbonate
(4.77−5.25 mmol) and 3 equiv of alkyl iodide (9.54−10.50
mmol), and the mixture was heated in a sealed tube at 80 °C
for 4−5 h. After the reaction was complete, the solution was
cooled down and the solvent was removed under reduced
pressure. The crude product was purified by column
Compounds 17a−c were synthesized from 16a−c (0.45−0.69
mmol) following the general synthesis of compounds 9a−m.
N-(4-((8-(1H-Imidazol-1-yl)octyl)oxy)3-methoxyphenyl)
Picolinimidamide (17a). Yellowish white powder, 130 mg,
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chromatography to afford the products 24a−c in 64−82%
yield.
4-((8-Bromooctyl)oxy)-2-ethoxy-1-nitrobenzene (21b).
Yellow powder, 0.51 g, yield 76% starting from 0.33 g of
4-Fluoro-2-methoxy-1-nitrobenzene (19a).59 Chromatog-
raphy solvent: hexanes/DCM (3:1), yellow powder, 0.43 g,
yield 79% starting from 0.50 g of 18 (3.18 mmol).
4-Fluoro-2-ethoxy-1-nitrobenzene (19b).59 Chromatogra-
phy solvent: hexanes/DCM (4:1), yellow powder, 0.53 g, yield
82% starting from 0.55 g of 18 (3.50 mmol).
1
20b (1.80 mmol). H NMR (300 MHz, CDCl3) δ 1.32−1.52
(m, 11H total, overlapped), 1.48 (t, J = 7.0 Hz, overlapped)
1.75−1.91 (m, 4H), 3.41 (t, J = 6.8 Hz, 2H), 4.00 (t, J = 6.5
Hz, 2H), 4.15 (q, J = 7.0 Hz, 2H), 6.46 (dd, J = 9.0, 2.4 Hz,
1H), 6.50 (d, J = 2.3 Hz, 1H), 7.95 (d, J = 9.0 Hz, 1H); 13C
NMR (100 MHz, CDCl3) δ 14.7, 26.0, 28.2, 28.8, 29.1, 29.2,
32.9, 34.1, 65.5, 68.8, 101.0, 105.3, 128.4, 133.2, 155.2, 164.3.
4-((8-Bromooctyl)oxy)-2-isopropoxy-1-nitrobenzene
(21c). White powder, 0.48 g, yield 94% starting from 0.26 g of
4-Fluoro-2-isopropoxy-1-nitrobenzene (19c).59 Chroma-
tography solvent: hexanes/DCM (4:1), yellow oil, 0.42 g,
yield 64% starting from 0.52 g of 18 (3.30 mmol).
Synthesis of 3-Alkoxy-4-nitrophenols (20a−c).
1
20c (1.32 mmol). H NMR (400 MHz, CDCl3) δ 1.30−1.50
(m, 14 H total, overlapped), 1.39 (d, J = 6.1 Hz, overlapped)
1.74−1.89 (m, 4H), 3.40 (t, J = 6.8 Hz, 2H), 3.99 (t, J = 6.5
Hz, 2H), 4.62 (sep, J = 6.1 Hz, 1H), 6.45 (dd, J = 9.1, 2.5 Hz,
1H), 6.50 (d, J = 2.5 Hz, 1H), 7.89 (d, J = 9.1 Hz, 1H); 13C
NMR (100 MHz, CDCl3) δ 22.0, 25.9, 28.1, 28.7, 29.1, 29.2,
32.8, 34.0, 68.7, 72.8, 102.5, 105.4, 128.2, 134.2, 154.1, 164.0.
Synthesis of 1-(8(3-Alkoxy-4-nitrophenoxy)octyl)-1H-
imidazoles (22a−c).
Compounds 20a−c were prepared according to a previously
published procedure.30 To a solution of 19a−c (1.30−1.80
mmol) in DMSO (5 mL) was added NaOH (0.5 g) dissolved
in distilled water (5 mL), and the mixture was stirred
vigorously at 80 °C for 20 h. After completion of the reaction,
the solution was cooled and the pH was rendered acidic with 6
N HCl. The product was extracted with ethyl acetate (3 × 30
mL), washed with water and brine, and dried over sodium
sulfate. The combined organic layer was evaporated under
reduced pressure and then purified by column chromatography
using hexanes/ethyl acetate (2:1) as eluent to afford the pure
product in 74−85% yield.
Compounds 22a−c were synthesized from 21a−c (0.85−1.38
mmol) following the general synthesis of compounds 7a−l.
1-(8-(3-Methoxy-4-nitrophenoxy)octyl)-1H-imidazole
(22a). Yellow powder, 0.28 g, yield 81% starting from 0.36 g of
3-Methoxy-4-nitrophenol (20a).60 Yellow powder, 0.32 g,
yield 83%, starting from 0.39 g of 19a (2.27 mmol). 1H NMR
(300 MHz, DMSO-d6) δ 3.86 (s, 3H), 6.47 (dd, J = 9.1, 2.4
Hz, 1H), 6.60 (d, J = 2.4 Hz, 1H), 7.88 (dd, J = 9.1 Hz, 1H),
10.87 (brs); 13C NMR (75 MHz, DMSO-d6) δ 56.4, 100.4,
107.5, 128.3, 130.9, 155.7, 164.0.
1
21a (1.00 mmol). H NMR (300 MHz, CDCl3) δ 1.24−1.50
(m, 8H), 1.72−1.84 (m, 4H), 3.89−3.95 (m, 5H total,
overlapped), 3.93 (s, overlapped), 4.00 (t, J = 6.5 Hz, 2H),
6.47 (dd, J = 9.1, 2.5 Hz, 1H), 6.51 (d, J = 2.4 Hz, 1H), 6.89
(s, 1H), 7.05 (s, 1H), 7.45 (s, 1H), 7.98 (d, J = 9.0 Hz, 1H);
13C NMR (75 MHz, CDCl3) δ 26.0, 26.6, 29.09, 29.11, 29.3,
31.2, 47.1, 56.6, 68.8, 100.2, 105.3, 118.9, 128.6, 129.6, 132.9,
137.2, 155.9, 164.5.
3-Ethoxy-4-nitrophenol (20b). Yellow powder, 0.36 g, yield
74%, starting from 0.49 g of 19b (2.64 mmol). 1H NMR (300
MHz, DMSO-d6) δ 1.35 (t, J = 6.9 Hz, 3H), 4.13 (q, J = 6.9
Hz, 2H), 6.46 (dd, J = 9.0, 2.3 Hz, 1H), 6.58 (d, J = 2.3 Hz,
1H), 7.86 (d, J = 9.0 Hz, 1H), 10.83 (brs); 13C NMR (75
MHz, DMSO-d6) δ 14.3, 64.8, 101.0, 107.5, 128.1, 131.1,
154.8, 163.8.
1-(8-(3-Ethoxy-4-nitrophenoxy)octyl)-1H-imidazole (22b).
Yellow powder, 0.25 g, yield 81% starting from 0.32 g of 21b
3-Isopropoxy-4-nitrophenol (20c).61 Yellow powder, 0.30
g, yield 85%, starting from 0.36 g of 19c (1.80 mmol).
Synthesis of 4-((8-Bromooctyl)oxy)-2-alkoxy-1-nitro-
benzenes (21a−c).
1
(0.85 mmol). H NMR (300 MHz, CDCl3) δ 1.25−1.50 (m,
11H total, overlapped), 1.47 (t, J = 7.0 Hz, overlapped), 1.72−
1.83 (m, 4H), 3.92 (t, J = 7.1 Hz, 2H), 3.99 (t, J = 6.5 Hz,
2H), 4.14 (q, J = 7.0 Hz, 2H), 6.45 (dd, J = 9.0, 2.5 Hz, 1H),
6.49 (d, J = 2.4 Hz, 1H), 6.89 (s, 1H), 7.04 (s, 1H), 7.45 (s,
1H), 7.94 (d, J = 9.0 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ
14.6, 25.9, 26.6, 29.08, 29.10, 29.2, 31.1, 47.1, 65.5, 68.7, 101.0,
105.3, 118.9, 128.4, 129.6, 133.3, 137.2, 155.1, 164.3.
1-(8-(3-Isopropoxy-4-nitrophenoxy)octyl)-1H-imidazole
Compounds 21a−c were synthesized from 20a−c (1.32−2.06
mmol) following the general synthesis of compounds 6a−i.
4-((8-Bromooctyl)oxy)-2-methoxy-1-nitrobenzene (21a).
Yellow powder, 0.67 g, yield 90% starting from 0.35 g of 20a
(22c). Yellow oil, 0.35 g, yield 79% starting from 0.46 g of 21c
1
(1.18 mmol). H NMR (300 MHz, CDCl3) δ 1.25−1.48 (m,
14H total, overlapped), 1.40 (d, J = 6.1 Hz, overlapped), 1.73−
1.81 (m, 4H), 3.92 (t, J = 7.1 Hz, 2H), 3.98 (t, J = 6.5 Hz,
2H), 4.61 (sep, J = 6.1 Hz, 1H), 6.45 (dd, J = 9.1, 2.5 Hz, 1H),
6.49 (d, J = 2.4 Hz, 1H), 6.89 (s, 1H), 7.04 (s, 1H), 7.45 (s,
1H), 7.90 (d, J = 9.1 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ
22.0, 26.0, 26.6, 29.10, 29.11, 29.3, 31.2, 47.1, 68.7, 72.8, 102.5,
105.4, 118.9, 128.2, 129.6, 134.3, 137.2, 154.1, 164.0.
1
(2.06 mmol). H NMR (300 MHz, CDCl3) δ 1.33−1.53 (m,
8H), 1.75−1.91 (m, 4H), 3.41 (t, J = 6.8 Hz, 2H), 3.94 (s,
3H), 4.02 (t, J = 6.5 Hz, 2H), 6.48 (dd, J = 9.0, 2.5 Hz, 1H),
6.52 (d, J = 2.4 Hz, 1H), 7.98 (d, J = 9.0 Hz, 1H); 13C NMR
(75 MHz, CDCl3) δ 26.0, 28.2, 28.8, 29.1, 29.2, 32.9, 34.0,
56.6, 68.9, 100.2, 105.3, 128.6, 132.9, 155.9, 164.5.
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Synthesis of 4-((8-(1H-imidazol-1-yl)octyl)oxy)-3-al-
koxyanilines (23a−c).
C24H31N5O2: C, 68.38; H, 7.41; N, 16.61. Found: C, 68.47; H,
7.54; N, 16.61.
N-(4-((8-(1H-Imidazol-1-yl)octyl)oxy)-2-ethoxyphenyl) Pi-
colinimidamide (24b). Buff powder, 100 mg, yield 42%
1
starting from 180 mg of 23b (0.54 mmol); mp 86−89 °C. H
NMR (700 MHz, CDCl3) δ 1.28−1.38 (m, 9H total,
overlapped), 1.34 (t, J = 7.0 Hz), 1.43−1.48 (m, 2H), 1.74−
1.81 (m, 4H), 3.91−3.95 (m, 4H), 4.03 (q, J = 7.0 Hz, 2H),
5.51−6.41 (brs, imidamide NHs), 6.51 (dd, J = 8.5, 2.5 Hz,
1H), 6.56 (d, J = 2.5 Hz, 1H), 6.90 (s, 1H), 6.98 (brs, 1H),
7.05 (s, 1H), 7.39 (ddd, J = 0.9, 4.8, 7.4 Hz, 1H), 7.45 (s, 1H),
7.81 (td, J = 7.8, 1.6 Hz, 1H), 8.47 (d, J = 7.8 Hz, 1H), 8.57 (d,
J = 4.6 Hz, 1H); 13C NMR (176 MHz, CDCl3) δ 15.0, 26.1,
26.6, 29.1, 29.3, 29.5, 31.2, 47.1, 64.5, 68.3, 102.2, 106.1, 118.9,
122.0, 123.5, 125.2, 129.6, 137.0, 137.2, 148.0, 151.2, 151.5,
153.5, 156.5; HRMS (ESI) m/z (M + H)+ calcd for
C25H34N5O2, 436.27070; found, 436.27139; Anal. Calcd for
C25H33N5O2: C, 68.94; H, 7.64; N, 16.08. Found: C, 68.66; H,
7.65; N, 15.86.
Compounds 23a−c were synthesized from 22a−c (0.66−0.86
mmol) following the general synthesis of compounds 8a−m.
4-((8-(1H-Imidazol-1-yl)octyl)oxy)-2-methoxyaniline
(23a). Yellow oil, 0.26 g, yield 95% starting from 0.30 g of 22a
1
(0.86 mmol). H NMR (300 MHz, CDCl3) δ 1.23−1.48 (m,
8H), 1.67−1.82 (m, 4H), 2.88−3.30 (brs, ArNH2), 3.83 (s,
3H), 3.87 (t, J = 6.4 Hz, 2H), 3.91 (t, J = 7.1 Hz, 2H), 6.33
(dd, J = 8.4, 2.6 Hz, 1H), 6.44 (d, J = 2.6 Hz, 1H), 6.62 (d, J =
8.4 Hz, 1H), 6.89 (t, J = 1.1 Hz, 1H), 7.05 (s, 1H), 7.45 (s,
1H).
N-(4-((8-(1H-Imidazol-1-yl)octyl)oxy)-2-isopropoxyphen-
yl) Picolinimidamide (24c). Yellow powder, 92 mg, yield 34%
4-((8-(1H-Imidazol-1-yl)octyl)oxy)-2-ethoxyaniline (23b).
Brown oil, 0.21 g, yield 95% starting from 0.24 g of 22b
1
starting from 210 mg of 23c (0.60 mmol); mp 82−85 °C. H
1
(0.66 mmol). H NMR (300 MHz, CDCl3) δ 1.26−1.47 (m,
NMR (700 MHz, CDCl3) δ 1.25 (brd, J = 5.9 Hz, 6H), 1.28−
1.38 (m, 6H), 1.42−1.48 (m, 2H), 1.74−1.80 (m, 4H), 3.92 (t
(apparent), J = 6.9 Hz, 4H), 4.44 (sep, J = 5.9 Hz, 1H), 5.38−
6.16 (brs, imidamide NHs), 6.55 (dd, J = 8.5, 2.5 Hz, 1H), 6.57
(d, J = 2.5 Hz, 1H), 6.80−6.98 (brs, 2H total, overlapped),
6.90 (s), 7.05 (s, 1H), 7.36−7.39 (m, 1H), 7.46 (s, 1H), 7.78−
7.82 (m, 1H), 8.43 (brs, 1H), 8.57 (d, J = 3.9 Hz, 1H); 13C
NMR (176 MHz, CDCl3) δ 22.4, 26.1, 26.6, 29.2, 29.3, 29.5,
31.2, 47.2, 68.3, 72.1, 105.9, 107.7, 118.9, 121.8, 123.7, 125.0,
129.6, 133.8, 136.8, 137.2, 147.9, 149.8, 152.0, 152.6, 156.1;
HRMS (ESI) m/z (M + H)+ calcd for C26H36N5O2,
450.28635; found, 450.28778; Anal. Calcd for C26H35N5O2:
C, 69.46; H, 7.85; N, 15.58. Found: C, 69.40; H, 7.90; N,
15.37.
overlapped, 11H total), 1.42 (t, J = 7.0 Hz),1.67−1.83 (m,
4H), 2.34−3.02 (brs, ArNH2), 3.86 (t, J = 6.6 Hz, 2H), 3.92 (t,
J = 7.1 Hz, 2H), 4.03 (q, J = 7.0 Hz, 2H), 6.32 (dd, J = 8.4, 2.6
Hz, 1H), 6.43 (d, J = 2.6 Hz, 1H), 6.63 (d, J = 8.4 Hz, 1H),
6.89 (s, 1H), 7.05 (s, 1H), 7.46 (s, 1H).
4-((8-(1H-Imidazol-1-yl)octyl)oxy)-2-isopropoxyaniline
(23c). Brown oil, 0.24 g, yield 100% starting from 0.26 g of 22c
1
(0.69 mmol). H NMR (300 MHz, CDCl3) δ 1.25−1.47 (m,
overlapped, 14H total), 1.34 (d, J = 6.0 Hz), 1.67−1.82 (m,
4H), 3.02−3.36 (brs, ArNH2), 3.85 (t, J = 6.5 Hz, 2H), 3.91 (t,
J = 7.1 Hz, 2H), 4.48 (sep, J = 6.1 Hz, 1H), 6.32 (dd, J = 8.5,
2.6 Hz, 1H), 6.44 (d, J = 2.6 Hz, 1H), 6.62 (d, J = 8.5 Hz, 1H),
6.89 (s, 1H), 7.04 (s, 1H), 7.45 (s, 1H).
Synthesis of N-(4-((8-(1H-Imidazol-1-yl)octyl)oxy)2-
alkoxyphenyl) Picolinimidamides (24a−c).
2-(8-Bromooctyl)isoindoline-1,3-dione (26).
To a solution of 1,8-dibromooctane (8.8 g, 32.4 mmol) in dry
DMF (30 mL) was added phthalimide potassium salt (2.0 g,
10.8 mmol), and the mixture was stirred at 90 °C for 24 h. The
reaction mixture was extracted with CH2Cl2 (2 × 30 mL) and
washed with 0.1 N NaOH (50 mL). The combined organic
layer was dried over anhydrous Na2SO4, filtered, and
evaporated under reduced pressure. The crude product was
purified by column chromatography using hexanes/ethyl
acetate (15:1) as eluent to afford the pure product as a
white powder, 2.2 g, yield 60%. 1H NMR (400 MHz, CDCl3) δ
1.25−1.46 (m, 8H), 1.62−1.72 (m, 2H), 1.79−1.87 (m, 2H),
3.38 (t, J = 6.9 Hz, 2H), 3.67 (t, J = 7.3 Hz, 2H), 7.67−7.73
(m, 2H), 7.81−7.86 (m, 2H); 13C NMR (100 MHz, CDCl3) δ
26.8, 28.2, 28.6, 28.7, 29.1, 32.9, 34.1, 38.1, 123.3, 132.3, 134.0,
168.6.
Compounds 24a−c were synthesized from 23a−c (0.54−0.63
mmol) following the general synthesis of compounds 9a−m.
N-(4-((8-(1H-Imidazol-1-yl)octyl)oxy)-2-methoxyphenyl)
Picolinimidamide (24a). Yellowish white powder, 120 mg,
yield 45% starting from 200 mg of 23a (0.63 mmol); mp 109−
1
112 °C. H NMR (400 MHz, CDCl3) δ 1.26−1.40 (m, 6H),
1.41−1.51 (m, 2H), 1.73−1.83 (m, 4H), 3.80 (s, 3H), 3.90−
3.97 (m, 4H), 5.53−6.18 (brs, imidamide NHs), 6.50 (dd, J =
8.5, 2.6 Hz, 1H), 6.56 (d, J = 2.6 Hz, 1H), 6.88−6.99 (brs, 2H
total, overlapped), 6.90 (t, J = 1.2 Hz), 7.05 (t, J = 1.0 Hz,
1H), 7.37 (ddd, J = 1.2, 4.8, 7.5 Hz, 1H), 7.46 (s, 1H), 7.80
(td, J = 7.7, 1.7 Hz, 1H), 8.47 (d, J = 8.0 Hz, 1H). 8.56 (ddd, J
= 0.9, 1.7, 4.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 26.1,
26.6, 29.2, 29.4, 29.5, 31.2, 47.2, 56.0, 68.3, 100.7, 105.6, 118.9,
122.0, 123.0, 125.2, 129.6, 136.9, 137.2, 147.9, 151.7, 152.0,
153.6, 156.5; HRMS (ESI) m/z (M + H)+ calcd for
C24H32N5O2, 422.25505; found, 422.25452; Anal. Calcd for
8-(1H-Imidazol-1-yl)octan-1-amine (27).
Q
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Compound 27 was prepared over two steps with a slight
modification to a previously published procedure.31 To a
solution of 26 (2.0 g, 5.91 mmol) in dry DMF (20 mL) was
added 1.5 equiv of K2CO3 (1.22 g, 8.86 mmol) and 2 equiv of
imidazole (0.80 g, 11.82 mmol), and the mixture was stirred at
80 °C for 12 h. After the reaction was completed, the
suspension was filtered and the filtrate was concentrated under
reduced pressure. The crude product was subjected to silica gel
chromatography using DCM/MeOH (10:0.3) as the eluent to
afford the pure product as a white powder, 1.20 g, yield 62%.
The 2-(8-(1H-imidazol-1-yl)octyl)isoindoline-1,3-dione prod-
uct (1.1. g, 3.38 mmol) was heated to reflux with 6 equiv of
80% NH2NH2·H2O (1.2 mL, 20.28 mmol) in absolute ethanol
(40 mL) under a N2 atmosphere for 8 h. The mixture was
cooled and treated with 4 N HCl (12 mL) and heated to reflux
for a further 6 h. The suspension was filtered, and the filtrate
was concentrated under reduced pressure. The solution was
rendered alkaline with 2 N NaOH; then, the product was
extracted with DCM (100 mL). The organic layer was dried
over anhydrous sodium sulfate and evaporated under reduced
pressure to afford the product as yellow oil, which was taken to
the next step without further purification, 0.44 g, yield 67%. 1H
NMR (300 MHz, CDCl3) δ 1.19−1.34 (m, 8H), 1.35−1.48
(m, 2H), 1.67−1.87 (m, 4H), 2.66 (t, J = 7.0 Hz, 2H), 3.89 (t,
J = 7.1 Hz, 2H), 6.87 (s, 1H), 7.02 (s, 1H), 7.44 (s, 1H); 13C
NMR (75 MHz, CDCl3) δ 26.6, 26.8, 29.1, 29.3, 31.1, 33.6,
42.2, 47.1, 118.8, 129.4, 137.2
length polymorphism (RFLP) analysis of the ribosomal
internal transcribed spacer region obtained by PCR from
promastigote genomic DNA.62 The evaluation of the activity of
hybrid compounds against intracellular L. donovani was
performed as outlined by Abdelhameed et al.52
J774 Macrophage Toxicity Assay. J774 murine macro-
phages were confirmed to be of mouse origin by species-
specific PCR evaluation and to be free of Mycoplasma
contamination by IDEXX BioResearch (Columbia, MO).
The assay to determine the toxicity of hybrid compounds on
J774 murine macrophages was conducted as described by Zhu
et al.63
HepG2 Toxicity Assay. HepG2 cells were authenticated as
an exact match to ATCC HB-8065 (HepG2) by the American
Type Culture Collection (ATCC, Manassas, VA) using Short
Tandem Repeat analysis.64 The HepG2 assay was performed
as described previously65 with minor modifications. HepG2
cells were maintained in a complete MEM Alpha medium
(MEM Alpha (Life Technologies) containing 10% heat-
inactivated FBS [Sigma-Aldrich], 50 U/mL penicillin, and 50
μg/mL streptomycin (Life Technologies)). Cells were trypsi-
nized with TrypLE Express (Life Technologies) and were
plated at a density of 5 × 103 cells/well in a 100 μL volume in
96-well plates. Cells were allowed to adhere overnight at 37 °C
in a 5% CO2 atmosphere. The medium was then replaced with
medium containing either compound or control drug, and test
plates were allowed to incubate for approximately 72 h at 37
°C in a 5% CO2 atmosphere. Following this incubation, the
medium was removed and replaced with 100 μL/well of fresh
medium and 25 μL/well of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT; 5 mg/mL in sterile PBS).
After an additional 2 to 3 h of incubation, 100 μL of SDS lysis
buffer (100 mg/mL SDS in 50% aqueous DMF) was added to
lyse the cells. After an additional 3−5 h of incubation, the
absorbance in each well was read at 570 nm with the aid of a
SpectraMax M5 microplate reader (Molecular Devices,
Sunnyvale, CA).
L. donovani CYP51 Binding Spectra and Dissociation
Constant Determination. To examine the binding of hybrid
compounds to L. donovani CYP51 (XP_003859085.1), a
plasmid containing an N-terminal truncated construct
(CYP51-32c; removal of 31 N-terminal amino acids to
increase solubility) and a C-terminal histidine tag (6xHis; to
facilitate affinity purification) was selected for the heterologous
expression in Escherichia coli and protein purification as
described previously for L. infantum CYP5148 with modifica-
tions. Emulgen 911, instead of Triton X-100, was used as
detergent to solubilize CYP51 from E. coli cell homogenate.
Purified CYP51 was analyzed by SDS-PAGE and Western blot
(anti-His tag) analyses for purity and by carbon monoxide
(CO)-difference spectra upon reduction by sodium dithionite
as previously described.32 To determine dissociation constants
(Kd) of CYP−ligand complexes, titration of L. donovani CYP51
with selected AA hybrid compounds was performed as
described previously66 with modifications. Titration of
CYP51 (0.5 μM) was carried out in 30 mM potassium
phosphate buffer, pH 7.4, containing 0.1 mM EDTA and 20%
glycerol. The difference spectra were obtained by recording the
absorbance in the sample cuvette versus the absorbance in the
reference cuvette. For compounds without absorbance
interference in the 350 to 500 nm range, both reference and
sample cuvettes contained the same amount of the protein.
Compounds were added to the sample cuvette from stock
N-(8-(1H-Imidazol-1-yl)octyl)picolinimidamide (28).
The synthesis of 28 follows the general synthesis and workup
procedure for 9a−l with the modification of using 2.2 equiv of
S-(2-naphthylmethyl)-2-pyridyl thioimidate hydrobromide.
The compound was further purified by column chromatog-
raphy using DCM/methanol/triethylamine (10:1.5:0.5) fol-
lowed by trituration from hexanes/ether (3 × 10 mL). White
powder, 65 mg, yield 28% (starting from 0.15 g of 26, 0.77
1
mmol); mp 65−67 °C. H NMR (400 MHz, DMSO-d6) δ
1.16−1.40 (m, 8H), 1.53−1.62 (m, 2H), 1.64−1.73 (m, 2H),
3.15 (t, J = 7.0 Hz, 2H), 3.93 (t, J = 7.1 Hz, 2H), 6.86 (s, 1H),
7.14 (t, J = 1.1 Hz, 1H), 7.45 (ddd, J = 7.4, 4.8, 1.1 Hz, 1H),
7.59 (s, 1H), 7.86 (td, J = 7.7, 1.7 Hz, 1H), 8.12 (d, J = 8.0 Hz,
1H), 8.55 (ddd, J = 4.8, 1.6, 0.9 Hz, 1H); 13C NMR (100
MHz, DMSO-d6) δ 25.9, 27.0, 28.5, 28.8, 30.0, 30.6, 45.4, 45.9,
119.2, 120.5, 124.7, 128.3, 136.8, 137.2, 147.8, 151.9, 154.1;
HRMS (ESI) m/z (M + H)+ calcd for C17H26N5, 300.21827;
found, 300.21811; Anal. Calcd for C17H25N5: C, 68.19; H,
8.42; N, 23.39. Found: C, 68.30; H, 8.33; N, 23.35.
Biological Assays. IC50 Determinations (General). For in
vitro cell-based susceptibility assays, validity criteria concerning
Z′ scores and R2 values for dose−response curves were as
defined in Abdelhameed et al.52 For colorimetric assays
employing J774 macrophages and HepG2 cells, an additional
validity criterion was that the mean absorbance of the positive
control wells was ≥1.0. Absolute IC50 values were determined
for all assays as described earlier.11
L. donovani-Infected Macrophage Assay. The species
identity of the LV82 strain L. donovani parasites used in this
work was verified by HaeIII-mediated restriction fragment
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solutions in DMSO, and the corresponding volume of DMSO
was added to the reference cuvette. For compounds with
absorbance interference in the 350 to 500 nm range, only the
sample cuvette contained the protein solution. Compounds
were then added to both sample and reference cuvettes from a
conducted according to procedures approved by the Institu-
tional Animal Care and Use Committee at The Ohio State
University.
In Vivo Toxicity. The toxicity of compound 24c to 6−8
week old female BALB/c mice was assessed as outlined by
Joice et al.11 Compound 24c was dissolved in 50% PEG400/
50% water and was administered by oral gavage. No particles
or cloudiness were noted when the required solution of 24c
(10 mg/mL) was prepared.
In Vivo Efficacy. The efficacy of compound 24c and
miltefosine in a murine visceral leishmaniasis model were
determined according to the methods described by Joice et
al.11 In the present study, the uninfected vehicle control group
received PEG-400; compound 24c was dissolved in 50%
PEG400/50% water, and miltefosine was dissolved in water.
Administration of the test agents and vehicle was by oral
gavage.
stock solution in DMSO. The dissociation constants were
Bmax × [S]
Kd + [S]
calculated by fitting the equation y =
to the Δ(Amax −
A
min) versus substrate concentration curves.
L. donovani CYP51 Inhibition Assay. A fluorescence-based
inhibition assay was developed for the L. donovani CYP51
using 7-benzyloxy-4-trifluoromethylcoumarin (BFC; Corning
Inc.) as substrate and its fluorescent metabolite 7-hydroxy-4-
trifluoromethylcoumarin (HFC; excitation 410 nm, emission
538 nm). Cumene hydroperoxide (CuOOH) was used as a
cofactor to enable the CYP-catalyzed reactions in place of CYP
natural cofactors (NADPH/P450 oxidoreductase and
NADPH).34 Inhibition assays were performed in 96-well
plates in a 200 μL volume. A stock solution of fluorogenic
substrate BFC (5 mM) was prepared in 1:1 [v/v] DMSO/
DMA (dimethylacetamide). The hybrid compounds (1−10
mM) were dissolved in DMSO. The reaction mixture
contained CYP51 (60 nM P450), BFC (50 μM), and various
concentrations (0.01−100 μM) of hybrid compounds in a
phosphate buffer (100 mM, pH 7.4) containing MgCl2 (3.3
mM). DMSO was used as negative control. The reaction was
initiated with the addition of 100 μM CuOOH (Alfa Aesar)
and monitored at 37 °C for emission at 538 nm under
excitation at 410 nm on a Tecan Infinite M200 Pro microplate
reader. For each compound, the percent inhibition at different
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsinfecdis.0c00855.
1H and 13C NMR spectra for all target compounds
(PDF)
AUTHOR INFORMATION
■
Corresponding Author
concentrations was calculated as (1 − RFUcompound
/
Karl A. Werbovetz − Division of Medicinal Chemistry and
Pharmacognosy, College of Pharmacy, The Ohio State
University, Columbus, Ohio 43210, United States;
orcid.org/0000-0003-3583-7241; Email: werbovetz.1@
osu.edu
RFUnegative control) × 100, where RFU is the relative fluorescence
unit. The following two-parameter logistic equation was fitted
to the percent inhibition versus the logarithm of the compound
concentration curves to obtain the IC50 values.
100
y =
Hill slope
x
Authors
1 +
(IC )
50
Ahmed Abdelhameed − Division of Medicinal Chemistry and
Pharmacognosy, College of Pharmacy, The Ohio State
University, Columbus, Ohio 43210, United States;
Pharmaceutical Chemistry Department, Faculty of Pharmacy,
Helwan University, Cairo 11795, Egypt
Mei Feng − Department of Pharmaceutical Chemistry, School
of Pharmacy, University of Kansas, Lawrence, Kansas 66047,
United States
April C. Joice − Division of Medicinal Chemistry and
Pharmacognosy, College of Pharmacy, The Ohio State
University, Columbus, Ohio 43210, United States
Emilia M. Zywot − Division of Medicinal Chemistry and
Pharmacognosy, College of Pharmacy, The Ohio State
University, Columbus, Ohio 43210, United States
Yiru Jin − Department of Pharmaceutical Chemistry, School of
Pharmacy, University of Kansas, Lawrence, Kansas 66047,
United States
Chris La Rosa − Division of Medicinal Chemistry and
Pharmacognosy, College of Pharmacy, The Ohio State
University, Columbus, Ohio 43210, United States
Xiaoping Liao − Division of Medicinal Chemistry and
Pharmacognosy, College of Pharmacy, The Ohio State
University, Columbus, Ohio 43210, United States
Heidi L. Meeds − Division of Medicinal Chemistry and
Pharmacognosy, College of Pharmacy, The Ohio State
University, Columbus, Ohio 43210, United States
Microsomal Stability Determinations. The metabolic
stability of selected hybrid compounds (3 μM) was evaluated
using human and mouse liver microsomes (0.5 mg/mL)
supplemented with NADPH (1 mM). Microsomal incubations
were carried out as described previously10,11 with modifica-
tions. Reactions were allowed to proceed for up to 60 min at
37 °C; then, aliquots (10 μL each) were removed and
quenched with ice-cold acetonitrile (200 uL) containing an
internal standard (2 nM). After centrifugation (2000g), the
supernatants (100 μL) were dried down with nitrogen gas and
then reconstituted with 15% methanol (150 μL) and analyzed
by UPLC-MS/MS to quantify the amount of hybrid
compound remaining. For LC-UV detection, reaction aliquots
(200 μL) were removed and quenched with ice-cold
acetonitrile (100 μL) containing an internal standard (5
μM). After centrifugation (2000g), the supernatants (250 μL)
were dried down with nitrogen gas and then reconstituted with
15% methanol (100 μL) and quantified. Control incubations
were run as described above in the absence of NADPH.
Microsomal half-life (t1/2) values were obtained by fitting the
one-phase exponential decay equation (C = C0 × e−kt; t1/2
0.693/k) to percent substrate remaining versus time curves.
=
In Vivo Experiments. Experiments assessing the in vivo
toxicity and antileishmanial efficacy of compound 24c were
S
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Yena Kim − Division of Medicinal Chemistry and
Pharmacognosy, College of Pharmacy, The Ohio State
University, Columbus, Ohio 43210, United States
Junan Li − College of Pharmacy, The Ohio State University,
Columbus, Ohio 43210, United States
Craig A. McElroy − Division of Medicinal Chemistry and
Pharmacognosy, College of Pharmacy, The Ohio State
University, Columbus, Ohio 43210, United States
Michael Zhuo Wang − Department of Pharmaceutical
Chemistry, School of Pharmacy, University of Kansas,
Lawrence, Kansas 66047, United States; orcid.org/0000-
0003-2494-4172
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acsinfecdis.0c00855
Author Contributions
A.A., M.Z.W., and K.A.W. conceived and designed the
experiments. A.A., A.C.J., E.M.Z., X.L., H.L.M., Y.K., M.F.,
and Y.J. performed the experiments. A.A., A.C.J., E.M.Z.,
C.L.R., X.L., H.L.M., Y.K., J.L., C.A.M., M.F., Y.J., M.Z.W., and
K.A.W. analyzed the results. A.A., M.Z.W., and K.A.W. wrote
and revised the manuscript. All authors approved the final
version of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Office of the Assistant
Secretary of Defense for Health Affairs under the Peer
Reviewed Medical Research Program through award no.
W81XWH-14-2-0017 (to K.A.W.), by the National Institutes
of Health through R01 AI139198 (to M.Z.W.), and by the
Neglected Disease Drug Discovery Fund of The Ohio State
University College of Pharmacy. Opinions, interpretations,
conclusions, and recommendations are those of the authors
and are not necessarily endorsed by the Department of
Defense or the U.S. Army. We thank Dr. Chunhua Yuan for
assistance in collecting NMR spectra. We also thank Dr. Pankaj
Sharma for assistance with chemical characterization and Dr.
Liva Rakotondraibe for assistance in interpreting NMR spectra.
(12) de Macedo-Silva, S., Urbina, J., de Souza, W., and Rodrigues, J.
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DEDICATION
■
This manuscript is dedicated to Dr. Jonathan Vennerstrom in
honor of his significant contributions to the field of antimalarial
drug discovery and development.
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ABBREVIATIONS
■
AIA, arylimidamide; BFC, 7-benzyloxy-4-trifluoromethylcou-
marin; CuOOH, cumene hydroperoxide; HFC, 7-hydroxy-4-
(trifluoromethyl)coumarin; LDU, Leishman-Donovan units;
SEM, standard error of the mean
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as drug targets for fungi and protozoan parasites: past, present and
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