Discovery of Novel Substituted N-(4-Amino-2-chlorophenyl)-5-chloro-2-hydroxybenzamide Analogues as Potent Human Adenovirus Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.0c01226

An effective therapy for human adenovirus (HAdV) infections in immunocompromised patients and healthy individuals with community-Acquired pneumonia remains an unmet medical need. We herein reported a series of novel substituted N-(4-Amino-2-chlorophenyl)-5-chloro-2-hydroxybenzamide analogues as potent HAdV inhibitors. Compounds 6, 15, 29, 40, 43, 46, 47, and 54 exhibited increased selectivity indexes (SI > 100) compared to the lead compound niclosamide, while maintaining sub-micromolar to low micromolar potency against HAdV. The preliminary mechanistic studies indicated that compounds 6 and 43 possibly target the HAdV DNA replication process, while compounds 46 and 47 suppress later steps of HAdV life cycle. Notably, among these derivatives, compound 15 showed improved anti-HAdV activity (IC50 = 0.27 μM), significantly decreased cytotoxicity (CC50 = 156.8 μM), and low in vivo toxicity (maximum tolerated dose = 150 mg/kg in hamster) as compared with niclosamide, supporting its further in vivo efficacy studies for the treatment of HAdV infections.

Full text of DOI:10.1021/acs.jmedchem.0c01226

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Article
Discovery of Novel Substituted N‑(4-Amino-2-chlorophenyl)-5-
chloro-2-hydroxybenzamide Analogues as Potent Human
Adenovirus Inhibitors
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∥
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Jimin Xu, Judith Berastegui-Cabrera, Na Ye, Marta Carretero-Ledesma, Jeronimo Pachon-Díaz,
́
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́
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Haiying Chen, Maria Eugenia Pachon-Ibanez, Javier Sanchez-Cespedes,* and Jia Zhou*
̃
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ABSTRACT: An effective therapy for human adenovirus (HAdV) infections in immunocompromised patients and healthy
individuals with community-acquired pneumonia remains an unmet medical need. We herein reported a series of novel substituted
N-(4-amino-2-chlorophenyl)-5-chloro-2-hydroxybenzamide analogues as potent HAdV inhibitors. Compounds 6, 15, 29, 40, 43, 46,
47, and 54 exhibited increased selectivity indexes (SI > 100) compared to the lead compound niclosamide, while maintaining sub-
micromolar to low micromolar potency against HAdV. The preliminary mechanistic studies indicated that compounds 6 and 43
possibly target the HAdV DNA replication process, while compounds 46 and 47 suppress later steps of HAdV life cycle. Notably,
among these derivatives, compound 15 showed improved anti-HAdV activity (IC50 = 0.27 μM), significantly decreased cytotoxicity
(CC50 = 156.8 μM), and low in vivo toxicity (maximum tolerated dose = 150 mg/kg in hamster) as compared with niclosamide,
supporting its further in vivo efficacy studies for the treatment of HAdV infections.
occasional cases and outbreaks of community-acquired
pneumonia (CAP) in healthy individuals.11−16 Despite the
great clinical significance, there are still no specific antiviral
drugs approved for the treatment of HAdV infections.17−22 At
present, the off-label use of intravenous cidofovir is the major
therapeutic option to treat HAdV diseases in many transplant
clinics. Cidofovir is an acyclic nucleotide phosphonate cytosine
analogue which was approved to treat cytomegalovirus (CMV)
retinitis in acquired immune deficiency syndrome (AIDS)
patients, acting as a chain terminator during DNA
replication.23 Cidofovir is a broad antiviral agent and effective
against all HAdV types in vitro. However, cidofovir has low oral
bioavailability, and its use is associated with significant
nephrotoxicity and myelotoxicity.24 To overcome these
drawbacks, brincidofovir (BCV, CMX001, and 3-hexadecy-
INTRODUCTION
■
Human adenoviruses (HAdVs) are nonenveloped, linear, and
double-stranded DNA viruses displaying a genome size of 34−
36 kb and encoding about 35 genes.1,2 Currently, HAdVs
comprise more than 100 different HAdV types classified into
seven species (HAdV A−G).3−5 HAdVs are endemic in the
human population, mainly associated with respiratory, ocular,
and gastrointestinal infections, particularly in infants and
young children.5 In general, species B, C, and E lead to
respiratory illness, whereas species D and F cause eye and
gastrointestinal infections, respectively.6−8 The infections are
usually self-limiting with mild clinical symptoms in healthy
individuals, resulting in lifelong immunity to this infected type.
However, HAdV infections can cause serious and often life-
threatening disease in immunocompromised patients, such as
solid-organ transplant (SOT) and allogenic hematopoietic
stem cell transplant (allo-HSCT) recipients receiving immu-
nosuppressive therapy.7,9 Pediatric allo-HSCT recipients are
the most severely affected group, where HAdV infections occur
in frequencies from 5−6 to 42−47% and mortality rates are up
to 80% with disseminated disease.7,8,10 In recent years, along
with the progress in molecular techniques of diagnosis, HAdV
was also identified as an important etiologic agent of both
Received: July 14, 2020
https://dx.doi.org/10.1021/acs.jmedchem.0c01226
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A

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Figure 1. Previous work and the discovery of lead compound 6 for the current work.
a
Scheme 1. Synthesis of Compounds 4−33 and 35−56
a
Reagents and conditions: (a) Zn, NH4Cl, H2O, MeOH, 0 °C to room temperature (RT), 16 h, quantitative. [b(i)] AcCl, Et3N, acetone, 50 °C, 2
h, 98% and (ii) LiOH, H2O, MeOH, RT, 1 h, 97%. (c) Aldehyde or ketone, NaBH3CN, AcOH, MeOH, 0 °C to RT, 12 h, 7−86%. (d) TsOH,
MeOH, RT, 12 h, 8−22% in two steps. (e) TFA, CH2Cl2, 0 °C to RT, 2 h, 78−79%. (f) HCl (4 M, aq), MeOH, RT, 12 h, 87%. [g(i)] 2-Methyl-1-
pentene or 1-methyl-1-cyclohexene or 3-methyl-3-buten-1-ol or 3-buten-1-ol, or 2-methyl-3-phenyl-1-propene, Fe(acac)3, PhSiH3, EtOH, 60 °C, 1
h and (ii) Zn, 2 M HCl (aq), 60 °C, 1 h, 21−58%.
loxy-1-propanol-cidofovir), a lipid conjugate of cidofovir, was
developed with decreased toxicity, increased oral bioavail-
ability, and enhanced anti-HAdV activity.25−27 Moreover, the
outcomes of the phase II and III trials (NCT01231344 and
NCT02087306) demonstrated the anti-HAdV activity of BCV
and its great potential to be developed as the first treatment
option for HAdV infections.28−31 Unfortunately, long-term
BCV treatment caused gastrointestinal adverse effects,
primarily diarrhea, in these trials.31 Therefore, it is still a
priority to develop highly effective antiviral agents against
HAdV infections without major adverse effects.
indicated that niclosamide is a multifunctional drug that has
great potential to treat other human conditions, such as cancer,
bacterial and viral infection, and metabolic diseases, more than
just helminthic diseases.35−45 Our research team first found
that niclosamide significantly inhibited HAdV infection with an
IC50 value of 0.6 μM. Nevertheless, it displayed moderate
cytotoxicity (CC50 = 22.9 μM), resulting in a relatively narrow
safety window with a low selectivity index (SI = 38.2).46 In our
previous work, we directed the chemical optimization mainly
involving substitutions with other electron-withdrawing groups
in the aniline moiety and introducing α-amino acid linkers
between two aromatic rings of niclosamide (Figure 1).47
Compounds 2 with the 3′-F-5′−CF3−aniline moiety and 3
with the L-valine linker were discovered with improved anti-
HAdV activities (IC50 = 0.18 μM and 0.45 μM, respectively)
Niclosamide (Figure 1) is an FDA-approved drug widely
used in humans to treat helminthic infections by inhibiting
oxidative phosphorylation and stimulating adenosine triphos-
phatase activity in the mitochondria.32−34 Recent studies have
B
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a
Scheme 2. Synthesis of Compounds 34, 57, and 63−68
a
Reagents and conditions: (a) R2NH2, K2CO3, DMF, 100 °C, 1 h, 87−96%. (b) Zn, NH4Cl, H2O, MeOH, 0 °C to RT, 16 h, 75−90%. [c(i)] 2-
Acetoxybenzoic acid, HBTU, DIEA, CH2Cl2, RT, 12 h and (ii) NaOH, H2O, MeOH, RT, 2 h, 35% in two steps. [d(i)] 2-Methoxy-5-
methylbenzoic acid, HBTU, DIEA, CH2Cl2, RT, 12 h, 88% and (ii) BBr3, DCM, −78 to 0 °C, 12 h, 96%. (e) Substituted benzoic acids, HBTU,
DIEA, CH2Cl2, RT, 12 h, 85−98%. [f(i)] 5-Chloro-2-methoxybenzoic acid, EDCI, DMAP, DCM, RT, 2 h, 89−93% and (ii) BBr3, DCM, −78 to 0
°C, 12 h, 58−77%.
a
Scheme 3. Synthesis of Compounds 69−75
a
Reagents and conditions: (a) Zn, NH4Cl, H2O, MeOH, 0 °C to RT, 16 h, 57−99%. (b) Cyclopentanone, NaBH3CN, AcOH, MeOH, 0 °C to RT,
12 h, 57−98%.
and better safety windows (SI = 666.7 and 444.4, respectively),
compared to niclosamide. Despite its long history of
application in medicinal chemistry and drug development,
the nitro group may possess potential toxicity issues and is
often classified as a structural alert or a toxicophore.48
Accordingly, we turned our attention to the chemical
optimization of the nitro group of niclosamide and attempted
to reduce it to an amino group. However, both amino
substitution (4) and acetamide substitution (5) led to a
dramatic loss of potency, not active against HAdV up to 10
μM. Intriguingly, compound 6 with cyclopentylamino
substitution regained the same level of potency (IC50 = 0.5
μM), with decreased cytotoxicity (CC50 = 92.9 μM) and
increased selectivity index (SI = 185.8), compared to the lead
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Table 1. Inhibition of HAdV in Plaque Assays, Cytotoxicity, and Selectivity Index for Compounds 4−21
a
Percentage of control HAdV5-GFP inhibition at 10 μM and inhibitory concentration 50% (IC50) at low MOI in a plaque assay using the 293β5
b
c
cell line. Cytotoxic concentration 50% (CC50). Selectivity index value was determined as the ratio of CC50 to IC50 in a plaque assay for each
compound. NT: not tested. The results represent means SD of triplicate samples from three independent experiments.
d
compound niclosamide. These preliminary results indicated
that it is a feasible strategy to reduce the nitro group of
niclosamide to an amino group and the N-alkylated amino
group is beneficial for potency against HAdV and the
improvement of aqueous solubility. As part of our ongoing
antiviral drug discovery and development program,39,49−53 we
herein reported our continued structure−activity relationship
(SAR) optimization efforts mainly focusing on the substituents
of the amino moiety, aiming to elucidate the chemical space for
the potential to acquire enhanced potency and pharmacoki-
netic properties such as better aqueous solubility and
decreased toxicity.
accessed from tetrahydropyran (THP) ethers 44 and 45,
respectively, via acidic depyranylation. Boc deprotection of 50,
52, and 54 under acidic conditions afforded derivatives 51, 53,
and 55, respectively. Direct hydroamination of niclosamide
with different olefins provided the corresponding amines 15,
46, and 47, while N,O-alkylated adducts 21 and 43 were
afforded as byproducts without undergoing further N−O bond
reduction under the same conditions.54
As shown in Scheme 2, substitution of 2-chloro-4-fluoro-1-
nitrobenzene with the corresponding amines gave the
intermediates 59a−c, which were then reduced to generate
amines 60a−c. With intermediates 60a−c in hand, the desired
compounds were readily synthesized by a series of con-
densations and deprotections. To this end, condensation of
60a with 2-acetoxybenzoic acid was followed by hydrolysis to
afford compound 63. Compound 64 was accessed under the
same coupling conditions, by condensation of 60a with 2-
methoxy-5-methylbenzoic acid, followed by the removal of the
methyl group. Direct coupling of 60a with different substituted
benzoic acids afforded derivatives 65−68. 5-Chloro-2-methox-
ybenzoic acid was condensed with 60b−c followed by
demethylation with BBr3 to provide compounds 34 and 57,
respectively.
RESULTS AND DISCUSSION
■
Chemistry. The general synthesis of new derivatives with
chemical optimizations on the amino group is outlined in
Scheme 1. Reduction of the nitro group of niclosamide 1 with
zinc dust provided amine 4, which was then converted into
acetamide 5 via acetylation and hydrolysis. Reductive
amination of amine 4 with various aldehydes or ketones
afforded a series of substituted N-(4-amino-2-chlorophenyl)-5-
chloro-2-hydroxybenzamide analogues 6−14, 16−20, 22−42,
44, 45, 50, 52, 54, and 56. Alcohol analogues 48 and 49 were
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Table 2. Inhibition of HAdV in Plaque Assays, Cytotoxicity, and Selectivity Index for Compounds 22−43
a
Percentage of control HAdV5-GFP inhibition at 10 μM and inhibitory concentration 50% (IC50) at low MOI in a plaque assay using the 293β5
b
c
cell line. Cytotoxic concentration 50% (CC50). Selectivity index value was determined as the ratio of CC50 to IC50 in a plaque assay for each
compound. The results represent means SD of triplicate samples from three independent experiments.
Compounds 69−75 were prepared according to the general
synthesis outlined in Scheme 3. Known nitro derivatives 61a−
g were reduced to afford anilines 62a−g, which were then
converted into compounds 69−75 via reductive amination
with cyclopentanone.47
In Vitro Evaluation of HAdV Inhibition. All newly
synthesized compounds were first screened in plaque assay at a
concentration of 10 μM, quantified as the percentage of HAdV
plaque formation inhibition, and the active compounds
(inhibition > 90%) were further evaluated to characterize
their antiviral activity (IC50) in plaque assay and their
cytotoxicity (CC50 values). Based on the potent anti-HAdV
activity of N-cyclopentyl-substituted derivative 6, we first
investigated the effect of different alkyl substitutions on the
amino moiety (Table 1). Excitingly, N-alkylated (7−16), N,N-
alkylated (17−20), and N,O-alkylated (21) derivatives were all
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Table 3. Inhibition of HAdV in Plaque Assays, Cytotoxicity, and Selectivity Index for Compounds 46−57
a
Percentage of control HAdV5-GFP inhibition at 10 μM and inhibitory concentration 50% (IC50) at low MOI in a plaque assay using the 293β5
b
c
cell line. Cytotoxic concentration 50% (CC50). Selectivity index value was determined as the ratio of CC50 to IC50 in a plaque assay for each
compound. NT: not tested. The results represent means SD of triplicate samples from three independent experiments.
d
active at a concentration of 10 μM, with full inhibition against
HAdV plaque formation. In contrast to lead compound 6, N-
ethyl derivative 7 and N-n-propyl derivative 8 showed
decreased potency (IC50 = 2.18 μM and 5.04 μM,
respectively), while compounds 9 with isopropyl and 10 with
isobutyl maintained the same level of potency (IC50 = 0.88 μM
and 0.89 μM, respectively). Long-length branched alkyl groups
(11 and 12) were also well tolerated, and compound 11 with
the octan-2-yl moiety showed very potent anti-HAdV activity
with an IC50 value of 0.32 μM. Nevertheless, these derivatives
also displayed increased cytotoxicity (CC50 = 11.0−60.0 μM)
with no improved selectivity index (SI = 4.5−73.4) compared
to lead compound 6. Introduction of cycloalkyl groups
produced compounds 13−16, and N-cyclopropylmethyl
substitution (13), N-cyclopentylmethyl substitution (14),
and N-cyclohexyl substitution (16) displayed slightly de-
creased activity (IC50 = 1.38 μM, 1.24 μM, and 1.34 μM,
respectively) and increased cytotoxicity with different degrees
(CC50 = 14.4 μM, 21.5 μM, and 68.3 μM, respectively) as
compared with N-cyclopentyl substitution (6). Remarkably,
compound 15 with the 1-methylcyclopentyl moiety possessed
improved potency against HAdV (IC50 = 0.27 μM); mean-
dialkylated derivatives 18−20. These results indicated that
branched alkyl groups are beneficial for potency, and N,N-
dialkyl substitution is superior to its corresponding N-alkyl
substitution.
Next, we attempted to introduce various aryl groups, and
compounds 22−43 were prepared and evaluated as shown in
Table 2. The substituents on the benzyl moiety (22−35) do
not seem to be critical for potency with IC50 values ranging
from 0.88 to 3.61 μM, and halogen (23−25), trifluoromethyl
(26, 27), methoxyl (28), hydroxyl (29), and dimethylamino
(30) were all well tolerated. Hydroxy derivatives 29 displayed
potent anti-HAdV activity (IC50 = 1.11 μM) and low
cytotoxicity (CC50 = 115.6 μM), resulting in a high selectivity
index (SI = 104.1). Intriguingly, disubstituted derivatives (31−
35) showed similar potency with low micromolar IC50 values
against HAdV and varied cytotoxicity. Among these com-
pounds, bis(trifluoromethyl)-substituted derivative 35 ex-
hibited a relatively high selectivity index (SI = 82.9) because
of its low cytotoxicity (CC50 = 92.8 μM). Compounds 36−39
with pyridine moieties were all active (IC50 = 1.66−4.78 μM)
along with relatively low cytotoxicity (CC50 = 65.3−125.7
μM). Notably, compound 40 with the pyridone moiety
showed sub-micromolar potency (IC50 = 0.78 μM), meanwhile
displaying low cytotoxicity (CC50 = 98.5 μM) and a high
selectivity index (SI = 126.3). Substitution with thiophene
(41) and indazole (42) moieties retained anti-HAdV activities
(IC50 = 2.58 μM and 4.65 μM, respectively). N,O-Dialkylated
derivative 43 displayed similar potency (IC50 = 1.27 μM) to its
analogue 21, with decreased cytotoxicity (CC50 = 164.7 μM)
and increased selectivity index (SI = 129.7).
while, it exhibited significantly decreased cytotoxicity (CC50
=
156.8 μM) with a very high selectivity index (SI = 580.7). N,N-
Dialkyl-substituted derivatives (17−20) displayed sub-micro-
molar to low micromolar potency against HAdV (IC50 = 0.62−
1.92 μM). When we increased the length of alkyl, compounds
17−19 showed an increasing trend in both potency and
cytotoxicity. Additionally, N,N-dialkylated derivatives 18−20
were more potent than their corresponding N-alkylated
derivatives 7, 8, and 13, respectively. Interestingly, N,O-
dialkylated derivative 21 retained similar potency (IC50 = 0.74
μM) and cytotoxicity (CC50 = 18.0 μM) to those of N,N-
To introduce aqueous functional groups, compounds 46−57
were designed, synthesized, and evaluated (Table 3).
Interestingly, branched hydroxyalkyl derivatives 46 and 47
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Table 4. Inhibition of HAdV in Plaque Assays, Cytotoxicity, and Selectivity Index for Compounds 63−75
a
Percentage of control HAdV5-GFP inhibition at 10 μM and inhibitory concentration 50% (IC50) at low MOI in a plaque assay using the 293β5
b
c
cell line. Cytotoxic concentration 50% (CC50). Selectivity index value was determined as the ratio of CC50 to IC50 in a plaque assay for each
compound. NT: not tested. The results represent means SD of triplicate samples from three independent experiments.
d
showed potent anti-HAdV activity (IC50 = 0.90 μM and 1.20
μM, respectively), while straight-chained hydroxyalkyl ana-
logues 48 and 49 were inactive at a concentration of 10 μM.
This series of derivatives also exhibited low cytotoxicity (CC50
= 105.5−200.0 μM), and consequently, the active molecules
46 and 47 possessed high selectivity index (SI = 188.3 and
166.7, respectively). The aminoalkyl groups (51, 53, and 55-
57) were unfavorable for potency, not active up to 10 μM,
while their N-Boc-protected derivatives 50, 52, and 54
maintained micromolar antiviral activities with IC50 values of
2.78 μM, 1.21 μM, and 0.93 μM, respectively. Of these,
compounds 52 and 54 showed low cytotoxicity (CC50 = 100.0
μM and 95.0 μM, respectively) and relatively high selectivity
index values (SI = 82.6 and 102.2, respectively).
Considering its potent antiviral activity, we made a quick
investigation on the effect of substituents on other positions of
this scaffold, while retaining the cyclopentylamino moiety of
lead compound 6. As listed in Table 4, removal of the 5-Cl
group on the salicylic ring (63) led to a significant loss of
potency with an inhibition of 77.5% at 10 μM, while replacing
Cl on the aniline ring (69) resulted in a slight loss of potency
(IC50 = 2.60 μM), while substitution of 2′-Cl with 2′-F (70) or
moving 2′-Cl to 3′-position (71) maintained the same level of
potency (IC50 = 1.04 μM and 0.84 μM, respectively) as
compared with lead compound 6. In contrast to compound 70,
moving the 4′-cyclopentylamino group to 5′-position produced
compound 71 with a similar potency (IC50 = 1.34 μM).
Increasing the length of the linker generated compounds 73−
75, and these modifications retained some activity with
micromolar IC50 values. Unfortunately, no compounds with a
high selectivity index were discovered during the modifications
at other sites of the scaffold because of either moderate to high
cytotoxicity or relatively low antiviral activity.
We next selected eight compounds with a high selectivity
index (SI > 100) and examined their effect on viral replication
in a virus burst assay by measuring the production of infectious
virus particles. In this assay, A549 cells were infected with
HAdV-5 wild-type virus, and the TCID50 of an infection in the
presence and absence of the anti-HAdV compounds was
calculated. As listed in Table 5, these selected derivatives
showed an overall virus yield reduction with varied folds,
ranging from 9.3-fold for compound 47 to 232-fold for
compound 54.
it with 5-methyl group (64) retained some activity (IC50
=
2.72 μM). Modifications of 2-phenolic hydroxyl yielded
compounds 65−68, and all these changes diminished the
anti-HAdV activities at different levels. Removal (65) or
methylation (66) of the 2-OH group resulted in a decrease in
activity with inhibition of 82.9% and 78.9% at 10 μM,
respectively, while substitution with 4-O-(1-methylpiperidinyl)
(67) led to a complete loss of potency. Unexpectedly,
replacing 2-OH with 2-methylsulfonamido (68) maintained
low micromolar anti-HAdV activity (IC50 = 1.72 μM). Next,
we turned our attention to the aniline moiety. Removal of 2′-
Impact on HAdV Entry. To gain a mechanistic under-
standing of their HAdV inhibition, we first evaluated these
selected derivatives in entry assay to determine their ability to
block the GFP gene expression when A549 cells are infected
with HAdV-GFP and incubated for 48 h (Figure S1). As
summarized in Table 6, compounds 15, 29, and 47 showed
significant inhibition against HAdV-GFP transgene expression
with percentages of 85.4%, 83.3%, and 88.6% at 50 μM,
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could quantify after the isolation of nuclei from the infected
cells. As shown in Figure 2A, at 45 min postinfection (p.i.),
cells treated with compounds 29 and 40 displayed significant
reductions in the amount of nuclear-associated HAdV
genomes versus those treated with dimethyl sulfoxide
(DMSO), while compounds 15 and 54 partially inhibited
the accessibility of HAdV DNA to the nucleus. Compounds 6,
43, 46, and 47 exhibited no significant differences in the
amount of nuclear-associated HAdV genome versus the control
group. These results suggested that compounds 29 and 40
acted in the early steps of the HAdV replication cycle, while
compounds 6, 43, 46, and 47 possibly suppressed later steps
after the HAdV genome was imported into the nucleus. As a
control for the purity of nuclear isolation, the DNA copy
number of the cellular housekeeping gene GAPDH in both the
nucleus and the cytoplasm was also measured. As shown in
Figure 2B, our results probe that we were specifically
evaluating the HAdV DNA that reached the cell nucleus,
suggesting that the compounds did not interfere with the steps
leading up to this late entry event.
Table 5. Virus Yield Reduction for Selected Compounds
with a High Selectivity Index
a
compound
virus yield reduction (fold)
1
6
82 35
17.1 3.1
52.6 6.2
17.4 14.2
26.6 5.0
21.5 0.0
15.7 6.8
9.3 2.2
15
29
40
43
46
47
54
232 85
a
Fold reduction in virus yield as the ratio of particles produced in the
presence of DMSO divided by the yield in the presence of each
selected compound at 10-fold IC50 concentration obtained in the
plaque assay for each compound. Virus yield reduction assay used the
A549 cell line, and the MOI of HAdV was 100 vp/cell. The results
represent means SD of triplicate samples from three independent
experiments.
Impact on HAdV Replication. The next step was to
evaluate the effect of these compounds on viral DNA
replication. Quantitative real-time PCR (qPCR) was used to
evaluate HAdV viral replication based on the efficiency of
DNA replication in the presence of the selected compounds.
To avoid the influence of newly generated viral particles from
subsequent rounds of infection occurring 32−36 h p.i., HAdV
DNA was extracted at 24 h p.i. and thus quantified in a single
round of infection a measurement of DNA replication
efficiency. As shown in Figure 3, compounds 43 and 54
significantly inhibited HAdV-5 DNA replication by more than
95%, while moderate inhibition was observed for compounds 6
and 15. Compounds 29, 40, 46, and 47 displayed low
inhibition against HAdV-5 DNA replication when compared to
the control treated with the same concentration of DMSO.
Taken together, compounds 6 and 43 caused a significant
decrease in HAdV DNA replication, while displaying no
obvious effect on HAdV entry, indicating that these two
compounds possibly target the HAdV DNA replication
process. Compounds 46 and 47 exhibited no obvious
inhibitory effect on either HAdV entry or HAdV DNA
replication, likely suppressing later steps of HAdV life cycle
such as the viral particle assembly, maturation, or release
processes. Compounds 15 and 54, which partially blocked the
accessibility of HAdV genomes to the nucleus, showed potent
inhibition against HAdV DNA replication. However, based on
our current data, it is still hard to tell whether these
compounds had direct impact on HAdV replication.
Interestingly, although compounds 29 and 40 significantly
suppressed the accessibility of HAdV DNA to the nucleus, they
showed a low decrease in HAdV DNA replication. Further
investigation is deemed necessary to clarify this phenomenon
and figure out their exact mechanisms of action.
Table 6. Inhibition of HAdV in Entry Assay for Selected
Compounds with a High Selectivity Index
(%) inhibition entry assay IC50 entry assay
a
b
compound
(50 μM)
(μM)
CC50 (μM)
1
6
100
0
1.22 0.44
NT
22.9 9.8
92.9 7.1
156.8 11.0
115.6 2.5
98.5 0.0
164.7 3.6
169.5 4.2
200.0 0.0
95.0 17.2
c
27.5 3.8
85.4 31.4
83.3 16.4
51.7 9.8
59.1 10.1
42.1 3.0
88.6 2.9
67.6 15.9
15
29
40
43
46
47
54
15.2 5.1
25.0 6.9
>50.00
34.1 6.9
NT
30.8 4.5
<3.13
a
Percentage of control HAdV5-GFP inhibition at 50 μM and
inhibitory concentration 50% (IC50) at high MOI in an entry assay
b
using the A549 cell line. Cell cytotoxic concentration 50% (CC50)
c
using the A549 cell line. NT: not tested.
respectively, and IC50 values ranging from 15.2 to 30.8 μM,
while derivatives 6, 40, and 46 displayed no obvious inhibitory
activity (IC50 > 50 μM). As for compounds 43 and 54, lower
percentage of inhibition (59.1% and 67.6%, respectively) at 50
μM was observed, with IC50 values of 34.1 μM and <3.13 μM,
respectively. However, these results could not provide us
conclusive indication of their potential mechanism of action, as
any interference at a step between HAdV attachment to its
cellular receptors and HAdV genome import into the cell
nucleus, or even a potential interference of these molecules
with the CMV promotor of the viral vector would eventually
lead to a decrease in the GFP expression. In addition, the use
of high MOIs imposes a limitation for this assay as it may
hamper the normal trafficking during HAdV entry, probably
resulting in a nonreliable test to evaluate viral entry.55
To further clarify whether these compounds were able to
block any of the steps in the entry pathway, we performed an
assay to quantitatively measure nuclear delivery of HAdV
genome in a 45 min assay. Once HAdV particles escape from
the endosome, the partially uncoated capsids are transported
to the nuclear membrane where the HAdV genome is
imported into the nucleus via the nuclear pore complex. We
hypothesized that if these molecules blocked any of these steps,
it would be reflected in the number of HAdV genomes that we
Time of Addition Assay. Next, we measured the ability of
these analogues to inhibit HAdV infection depending on their
time of addition as an alternative way to confirm our previous
results. All compounds exhibited a time-dependent decrease in
inhibitory activity, showing different patterns (Figure 4).
Compounds 6 and 29 showed an inhibition of HAdV infection
≥50% when they were added either at the beginning of the 60
min incubation at 4 °C (−60 min) or at 120 min p.i., just like
niclosamide. Compounds 40, 46, and 47 showed a sudden
decrease in inhibition from the beginning, showing less than
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Figure 2. (A) Percentage of the nuclear-associated HAdV genome of selected compounds after 45 min of incubation. (B) Control for the
specificity of nuclear DNA purification. Concentration of niclosamide was 5 μM and 10-fold IC50 concentration obtained in the plaque assay for
each compound. Bars represent means SD of triplicate samples. ***p ≤ 0.001 and ****p ≤ 0.0001.
compounds 15 and 43 showed a decrease in their antiviral
activity with inhibitions below 50% when they were added at 5
min p.i. In all these cases, these results would suggest a very
early target step during HAdV internalization after binding to
the cell. Compound 54 loses its anti-HAdV activity when
added between 10 and 20 min p.i., suggesting a target involved
in the HAdV escape from the endosome or the microtubular
transport of the partially uncoated particle to the nucleus of the
cell.
Impact on HCMV Replication. The selected compounds
were further tested to evaluate their potential broad antiviral
effects on human CMV (HCMV) quantifying the total DNA
72 h after the infection of HFF cells in a single round of
infection as a measure of DNA replication efficiency. The
addition of derivatives 1, 29, 43, 46, and 54 generated
significant reductions (p ≤ 0.001) in the quantification of total
HCMV DNA, showing a decrease from 84 to 98% compared
to the DMSO control (Figure 5). Compounds 40 and 47 also
decreased the number of HCMV DNA copies (p ≤ 0.001),
although the percentages of reduction were lower (82% and
Figure 3. Effect of selected compounds on HAdV DNA replication.
78%, respectively). Compounds 6 and 15 showed no
Concentration of these compounds for the assay was selected based
significant inhibition of HCMV DNA replication, indicating
on CC50: niclosamide and 6 were tested at 5 μM, 15 was tested at 2.7
a high antiviral specificity against HAdV.
μM, 29 was tested at 11.1 μM, 40 was tested at 7.8 μM, 43 was tested
Synergistic Activity of Selected Derivatives. Because
derivatives 2 and 3 were found to be targeting later steps after
HAdV DNA replication and compound 15 seems to be
addressing early stages during HAdV entry in the host cell, we
decided to evaluate the effect of their combination on their
anti-HAdV activity. We hypothesized that because these
at 12.7 μM, 46 was tested at 9.0 μM, 47 was tested at 12.0 μM, and
54 was tested at 9.3 μM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and
****p ≤ 0.0001.
50% inhibition after the addition at the moment of the
infection (0 min). In the same way, but a little later,
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Figure 4. Time-dependence curve assay. Effect of selected compounds on HAdV infection at different time points at concentrations 10-fold the
IC50 concentration obtained in the plaque assay of each compound. Results show three different patterns: (A) for compound 54, (B) for
compounds 1, 6, and 29, and (C) for compounds 15, 40, 43, 46, and 47. The line chart represents mean SD of duplicate samples.
hypothesis, we conducted a combination study based on the
Chou−Talalay method for drug combination using CalcuSyn
software56,57 and using those compounds with lower IC50 in
the plaque assay and higher SI, compound 15 from this work
and compounds 2 and 3 from our first library of derivatives.
The constant ratio for each combination was selected based on
the IC50 values for each drug. The data for all the combinations
showed good conformity with the mass action law principle (r
= 0.78−0.95) (Table 7). The combination of these three
compounds was classified as strong synergism for the
combination between compounds 2 and 3 at ED90 to slight
synergism for the combination of 15 and 3 at ED75. At ED50,
the combination of 2 and 3 (ratio 1:2.5) was classified as
synergistic (ED50 = 0.68) and the combination of 2 with 15
(ratio 1:1.5) showed slight synergism (ED50 = 0.88). However,
the combination of compounds 15 and 3 (ratio 1:1.7) resulted
in a clear antagonistic effect (ED50 = 1.81).
Maximum Tolerated Dose. To assist the selection of the
best candidates for further in vivo efficacy evaluation, the
maximum tolerated dose (MTD) values of compound 15,
which showed the best SI in this library of compounds,
together with derivatives 2 and 3, were tested in the
immunosuppressed Syrian hamster model. As shown in Figure
6, compound 15 presented the safest MTD at 150 mg/kg,
followed by derivatives 3 and 2 with MTD of 50 and 12.5 mg/
kg, respectively. Niclosamide showed high toxicity at doses ≥5
mg/kg, and finally, an MTD of 1 mg/kg that prevented the loss
of weight of the animals was determined. These results were
consistent with the trend in the cytotoxicity for each of the
molecules and showed a wide therapeutic window for the
further in vivo evaluation of their efficacy in the treatment of
HAdV infection, especially for derivatives 3 and 15.
Figure 5. Effect of selected compounds on HCMV DNA replication.
The concentration of these compounds in this assay was selected
based on their CC50: niclosamide and 6 were tested at 5 μM, 15 was
tested at 2.7 μM, 29 was tested at 11.1 μM, 40 was tested at 7.8 μM,
43 was tested at 12.7 μM, 46 was tested at 9.0 μM, 47 was tested at
12.0 μM, and 54 was tested at 9.3 μM. **p ≤ 0.01 and ***p ≤ 0.001.
compounds are targeting different processes during HAdV
replicative cycle, their combination would result in an
improved antiviral activity blocking HAdV infection at lower
concentrations because of their synergistic effect. To assess this
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a
Table 7. CalcuSyn Results for the Different Combinations of Compounds 2, 3, and 15
combinatory index (CI) values in plaque assay
combination (ratio)
derivative
IC50 single
IC50 combination
ED50
0.68
ED75
0.47
ED90
0.11
r
2 & 3 (1:2.5)
2
3
15
3
2
15
0.18 0.01
0.45 0.06
0.27 0.16
0.45 0.06
0.18 0.01
0.27 0.16
0.10 0.01
0.25 0.02
0.30 0.02
0.50 0.04
0.09 0.00
0.14 0.00
0.92
15 & 3 (1:1.7)
2 & 15 (1:1.5)
1.81
0.88
0.91
0.63
0.46
0.48
0.95
0.78
a
The combinatory index values are shown for the combinations at the IC50, IC75, and IC90 levels of inhibition. The r value for each combination is
also reported to indicate the correlation coefficient of the data to the mass-action law.
Figure 6. Percentage of body weight gains in three female hamsters treated with compounds 1, 2, 3, and 15. Dosing of the compounds started at
Day 0. Symbols represent the group mean, while the error bars signify the standard deviation.
selected for further in vivo characterization to evaluate first
their pharmacokinetics profiles and subsequently their efficacy
for the treatment of HAdV infection in the immunosuppressed
Syrian hamster model. The findings on such extensive in vivo
studies will be reported in due course.
CONCLUSIONS
■
In summary, we discovered a series of novel substituted N-(4-
amino-2-chlorophenyl)-5-chloro-2-hydroxybenzamide ana-
logues as potent HAdV inhibitors. Compounds 6 (NY0624),
15 (JMX0510-2), 29 (JMX0452), 40 (JMX0464), 43
(JMX0508), 46 (NY0611), 47 (NY0621), and 54
(JMX0461) exhibited significantly high selectivity indexes
(SI > 100), while maintaining sub-micromolar to low
micromolar potency against HAdV compared to niclosamide.
The preliminary mechanistic studies indicated that compounds
6 and 43 may target the transcription of the HAdV immediate
early and early genes (essential for subsequent DNA
replication) or alternatively the HAdV DNA replication by
interfering with key proteins involved in this process, while
compounds 46 and 47 suppress later steps of HAdV life cycle.
With respect to compounds 15, 29, 40, and 54, it is hard to
conclude what stage they act at, and further investigation is
imperative to clarify their exact mechanisms of action. Notably,
among all the derivatives tested in our two rounds of
optimization, compounds 2, 3, and 15 showed the highest
anti-HAdV activity (IC50 = 0.18, 0.45, and 0.27 μM,
EXPERIMENTAL SECTION
■
General Chemistry Information. All commercially available
starting materials and solvents were of reagent grade and used without
further purification. Reactions were performed under a nitrogen
atmosphere in dry glassware with magnetic stirring. Preparative
column chromatography was performed using silica gel 60, particle
size 0.063−0.200 mm (70−230 mesh, flash). Analytical thin layer
chromatography (TLC) was carried out employing silica gel 60 F254
plates (Merck, Darmstadt). Visualization of the developed chromato-
grams was performed with detection by ultraviolet (UV, 254 nm).
NMR spectra were recorded on a Brucker-300 (1H, 600 and 300
MHz; 13C, 150 and 75 MHz) spectrometer. 1H and 13C NMR spectra
were recorded with tetramethylsilane (TMS) as an internal reference.
Chemical shifts were expressed in ppm, and J values were given in Hz.
High-resolution mass spectra (HRMS) were obtained from Thermo
Fisher LTQ Orbitrap Elite mass spectrometer. Parameters include the
following: Nano ESI spray voltage was 1.8 kV; capillary temperature
was 275 °C, and the resolution was 60,000; ionization was achieved
by the positive mode. Melting points were measured on a Thermo
Scientific electrothermal digital melting point apparatus and
uncorrected. Purities of final compounds were established by
analytical high-performance liquid chromatography (HPLC), which
respectively), significantly decreased cytotoxicity (CC50
=
120.0, 200.0, and 156.8 μM, respectively), and very low in
vivo toxicity (MTD = 12.5, 50, and 150 mg/kg, respectively) as
compared with niclosamide (IC50 = 0.6 μM, CC50 = 22.9 μM,
and MTD = 1 mg/kg). Thus, compounds 2, 3, and 15 were
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was carried out on a Shimadzu HPLC system (model: CBM-20A LC-
20AD SPD-20A UV/VIS). HPLC analysis conditions: Waters
μBondapak C18 (300 × 3.9 mm); flow rate 0.5 mL/min; UV
detection at 270 and 254 nm; and linear gradient from 10%
acetonitrile in water to 100% acetonitrile in water in 20 min followed
by 30 min of the last-named solvent (0.1% TFA was added into both
acetonitrile and water). All biologically evaluated compounds are
>95% pure.
N-(4-Amino-2-chlorophenyl)-5-chloro-2-hydroxybenza-
mide (4). To a solution of niclosamide (500 mg, 1.53 mmol) in 20
mL of MeOH was added 4 mL of saturated NH4Cl (aq). Zinc dust
(994 mg, 15.3 mmol) was added into the solution at 0 °C. The
reaction was stirred at RT for 16 h. TLC indicated that the starting
material was gone. MeOH (100 mL) was added to the solution. The
zinc solid was filtered, and the filtrate was concentrated under
vacuum. Then, 15 mL of MeOH was added, and the resulting mixture
was stirred at RT for 20 min. Compound 4 (455 mg, 100%) was
filtered as a pale yellow solid. HPLC purity 99.8% (tR = 12.54 min).
1H NMR (300 MHz, DMSO-d6): δ 12.27 (s, 1H), 10.40 (s, 1H), 8.01
9−10 with NaHCO3 (aq) at 0 °C. The mixture was extracted with
DCM, dried (Na2SO4), and concentrated. The residue was purified by
column chromatography or recrystallization (MeOH/H2O) to give
the amino products.
5-Chloro-N-(2-chloro-4-(cyclopentylamino)phenyl)-2-hy-
droxybenzamide (6). Compound 6 (100 mg, 73%) was prepared as
a yellow solid according to general procedure B, starting from
compound 4 and cyclopentanone. HPLC purity 99.5% (tR = 17.54
1
min). H NMR (300 MHz, CDCl3): δ 11.97 (s, 1H), 8.07 (s, 1H),
7.89 (d, J = 9.0 Hz, 1H), 7.49 (d, J = 2.4 Hz, 1H), 7.38 (dd, J = 8.7,
2.4 Hz, 1H), 6.97 (d, J = 9.0 Hz, 1H), 6.65 (d, J = 2.7 Hz, 1H), 6.53
(dd, J = 8.7, 2.7 Hz, 1H), 3.82−3.69 (m, 2H), 2.11−1.95 (m, 2H),
1.80−1.60 (m, 4H), 1.53−1.37 (m, 2H). 13C NMR (75 MHz,
CDCl3): δ 166.9, 160.5, 146.7, 134.5, 126.7, 125.1, 124.9, 123.8,
122.5, 120.5, 115.8, 112.8, 112.5, 54.8, 33.6 (2C), 24.2 (2C). HRMS
(ESI): calcd for C18H19Cl2N2O2, 365.0824 (M + H)+; found,
365.0820.
5-Chloro-N-(2-chloro-4-(ethylamino)phenyl)-2-hydroxyben-
zamide (7). Compound 7 (49 mg, 37%) was prepared as a yellow
solid according to general procedure B, starting from compound 4
(d, J = 2.7 Hz, 1H), 7.66 (d, J = 8.7 Hz, 1H), 7.47 (dd, J = 8.7, 2.7 Hz,
1H), 7.18 (br s, 1H), 7.04 (d, J = 8.7 Hz, 1H), 6.71 (d, J = 2.4 Hz,
1H), 6.55 (dd, J = 8.7, 2.4 Hz, 1H), 5.37 (s, 2H). 13C NMR (75 MHz,
DMSO-d6): δ 163.7, 156.5, 147.5, 133.1, 128.8, 126.6, 126.1, 123.0,
122.6, 119.1, 118.8, 113.4, 112.8. HRMS (ESI): calcd for
C13H11Cl2N2O2, 297.0198 (M + H)+; found, 297.0189.
1
and acetaldehyde. HPLC purity 98.6% (tR = 15.92 min). H NMR
(300 MHz, CDCl3 + CD3OD): δ 7.97 (s, 1H), 7.81 (d, J = 8.7 Hz,
1H), 7.29 (dd, J = 8.7, 2.4 Hz, 1H), 6.88 (d, J = 8.7 Hz, 1H), 6.65 (s,
1H), 6.54 (d, J = 9.0 Hz, 1H), 3.08 (q, J = 7.2 Hz, 2H), 1.21 (t, J = 7.2
Hz, 3H). 13C NMR (75 MHz, CDCl3 + CD3OD): δ 165.3, 156.8,
147.8, 133.6, 129.9, 127.8, 126.0, 125.1, 124.3, 119.6, 118.9, 113.0,
112.3, 38.8, 14.6. HRMS (ESI): calcd for C15H15Cl2N2O2, 325.0511
(M + H)+; found, 325.0504.
5-Chloro-N-(2-chloro-4-(propylamino)phenyl)-2-hydroxy-
benzamide (8). Compound 8 (80 mg, 68%) was prepared as a
yellow solid according to general procedure A, starting from
compound HJC0129 and propionaldehyde. HPLC purity 99.4% (tR
= 17.02 min). 1H NMR (300 MHz, CDCl3): δ 11.92 (s, 1H), 8.13 (s,
1H), 7.84 (d, J = 8.7 Hz, 1H), 7.49 (d, J = 2.1 Hz, 1H), 7.36 (dd, J =
9.0, 2.4 Hz, 1H), 6.95 (d, J = 8.7 Hz, 1H), 6.61 (d, J = 2.4 Hz, 1H),
6.50 (dd, J = 9.0, 2.4 Hz, 1H), 3.77 (s, 1H), 3.04 (t, J = 7.2 Hz, 2H),
1.70−1.54 (m, 2H), 0.99 (t, J = 7.2 Hz, 3H). 13C NMR (75 MHz,
CDCl3): δ 166.8, 160.2, 147.2, 134.4, 126.9, 125.3, 125.0, 123.8,
122.5, 120.3, 115.8, 112.2, 112.0, 45.8, 22.6, 11.6. HRMS (ESI): calcd
for C16H17Cl2N2O2, 339.0667 (M + H)+; found, 339.0663.
N-(4-Acetylamino-2-chlorophenyl)-5-chloro-2-hydroxyben-
zamide (5). To the mixture of compound 4 (200 mg, 0.67 mmol) in
10 mL of acetone was added Et3N (341 mg, 3.4 mmol). AcCl (159
mg, 2.0 mmol) was added at 0 °C. The resulting mixture was stirred at
50 °C for 2 h. The mixture was concentrated, and then, the residue
was washed with acetone (3 mL). The intermediate 2-((4-acetamido-
2-chlorophenyl)carbamoyl)-4-chlorophenyl acetate was filtered as a
1
pale yellow solid (250 mg, 98%). H NMR (600 MHz, CDCl3): δ
8.65 (s, 1H), 8.45 (d, 1H, J = 9.0 Hz), 7.98 (s, 1H), 7.96 (s, 1H), 7.50
(d, 1H, J = 8.4 Hz), 7.14−7.17 (m, 3 H), 2.37 (s, 3H), 2.17 (s, 3H).
13C NMR (150 MHz, DMSO-d6): δ 168.7, 168.6, 162.9, 146.9, 138.3,
131.4, 130.4, 129.9, 129.1, 129.0, 128.8, 128.2, 125.5, 119.3, 117.8,
24.0, 20.8.
To a solution of the intermediate 2-((4-acetamido-2-
chlorophenyl)carbamoyl)-4-chlorophenyl acetate (150 mg, 0.4
mmol) in 10 mL of MeOH and 2.5 mL of H2O was added LiOH
(66 mg, 1.6 mmol) at 0 °C. The resulting mixture was stirred at RT
for 1 h. The mixture was diluted with ethyl acetate (EtOAc, 100 mL)
and washed with water (20 mL) and 2 N HCl (5 mL). The organic
layer was separated and dried with anhydrous Na2SO4. The solution
was concentrated to give compound 5 (130 mg, 97%) as a yellow
5-Chloro-N-(2-chloro-4-(isopropylamino)phenyl)-2-hydrox-
ybenzamide (9). Compound 9 (70 mg, 53%) was prepared as a
yellow solid according to general procedure B, starting from
compound HJC0129 and acetone. HPLC purity 99.7% (tR = 16.14
1
min). H NMR (300 MHz, CDCl3): δ 11.91 (s, 1H), 8.15 (s, 1H),
7.84 (d, J = 8.7 Hz, 1H), 7.50 (d, J = 2.4 Hz, 1H), 7.36 (dd, J = 9.0,
2.4 Hz, 1H), 6.95 (d, J = 8.7 Hz, 1H), 6.60 (d, J = 2.7 Hz, 1H), 6.48
(dd, J = 9.0, 2.7 Hz, 1H), 3.73−3.43 (m, 2H), 1.20 (d, J = 6.3 Hz,
6H). 13C NMR (75 MHz, CDCl3): δ 166.8, 160.2, 146.2, 134.4,
126.9, 125.3, 125.1, 123.8, 122.4, 120.3, 115.9, 112.8, 112.4, 44.5, 22.9
(2C). HRMS (ESI): calcd for C16H17Cl2N2O2, 339.0667 (M + H)+;
found, 339.0664.
1
solid. HPLC purity 99.3% (tR = 15.97 min). H NMR (600 MHz,
DMSO-d6): δ 12.23 (s, 1H), 10.75 (s, 1H), 10.14 (s, 1H), 8.21 (d,
1H, J = 8.4 Hz), 7.99 (s, 1H), 7.96 (s, 1H), 7.50 (d, 1H, J = 9.0 Hz),
7.43 (d, 1H, J = 9.0 Hz), 7.06 (d, 1H, J = 9.0 Hz), 2.05 (s, 3H). 13C
NMR (150 MHz, DMSO-d6): δ 168.5, 162.9, 156.1, 136.6, 133.3,
130.0, 129.5, 123.9, 123.5, 123.1, 119.5, 119.2, 119.2, 118.0, 24.0.
HRMS (ESI): calcd for C15H13Cl2N2O3, 339.0303 (M + H)+; found,
339.0295.
General Procedure A for Reductive Amination Reaction.
Compound 4 (1.0 equiv) and aldehyde (1.5 equiv) or ketone (3.0
equiv) were suspended in DCE (5 mL/0.1 mmol) and treated with
AcOH (2.5 equiv). NaBH(OAc)3 (3 equiv) was added in portions at
0 °C, and the mixture was stirred at RT overnight. The pH of the
mixture was adjusted to 9−10 with NaHCO3 (aq) at 0 °C. The
aqueous phase was extracted with dichloromethane (DCM), dried
(Na2SO4), and concentrated. The residue was purified by column
chromatography or recrystallization (MeOH/H2O) to give the amino
products.
5-Chloro-N-(2-chloro-4-(isobutylamino)phenyl)-2-hydroxy-
benzamide (10). Compound 10 (52 mg, 42%) was prepared as a
yellow solid according to general procedure B, starting from
compound HJC0129 and isobutyraldehyde. HPLC purity 99.6% (tR
= 17.54 min). 1H NMR (300 MHz, CDCl3): δ 11.93 (s, 1H), 8.10 (s,
1H), 7.86 (d, J = 8.7 Hz, 1H), 7.49 (d, J = 2.4 Hz, 1H), 7.37 (dd, J =
8.7, 2.4 Hz, 1H), 6.96 (d, J = 8.7 Hz, 1H), 6.62 (d, J = 2.7 Hz, 1H),
6.52 (dd, J = 9.0, 2.7 Hz, 1H), 3.84 (s, 1H), 2.90 (d, J = 6.6 Hz, 2H),
1.96−1.80 (m, 1H), 0.99 (d, J = 6.6 Hz, 6H). 13C NMR (75 MHz,
CDCl3): δ 166.9, 160.3, 147.3, 134.5, 126.9, 125.2, 125.0, 123.8,
122.5, 120.4, 115.8, 112.2, 112.0, 51.8, 28.1, 20.5 (2C). HRMS (ESI):
calcd for C17H19Cl2N2O2, 353.0824 (M + H)+; found, 353.0821.
5-Chloro-N-(2-chloro-4-(octan-2-ylamino)phenyl)-2-hydrox-
ybenzamide (11). Compound 11 (80 mg, 74%) was prepared as a
yellow solid according to general procedure B, starting from
compound 4 and 2-octanone. HPLC purity 96.6% (tR = 22.46
General Procedure B for Reductive Amination Reaction.
Compound 4 (1.0 equiv) and aldehyde (1.5 equiv) or ketone (3.0
equiv) were dissolved in MeOH (7 mL/0.1 mmol), and then AcOH
(4.0 equiv) was added at 0 °C. The mixture was stirred at 0 °C for 30
min; then, NaBH3CN (3.0 equiv) was added. The resulting mixture
was stirred at RT overnight. The pH of the mixture was adjusted to
1
min). H NMR (300 MHz, CDCl3): δ 11.96 (s, 1H), 8.09 (s, 1H),
7.87 (d, J = 8.7 Hz, 1H), 7.49 (d, J = 2.4 Hz, 1H), 7.37 (dd, J = 8.7,
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2.4 Hz, 1H), 6.97 (d, J = 9.0 Hz, 1H), 6.62 (d, J = 2.7 Hz, 1H), 6.50
(dd, J = 9.0, 2.7 Hz, 1H), 3.90−3.25 (m, 2H), 1.67−1.21 (m, 10H),
1.18 (d, J = 6.3 Hz, 3H), 0.95−0.83 (m, 3H). 13C NMR (75 MHz,
CDCl3): δ 166.9, 160.4, 146.5, 134.5, 126.9, 125.2, 125.1, 123.8,
122.3, 120.5, 115.8, 112.6, 112.3, 48.8, 37.2, 31.9, 29.4, 26.2, 22.7,
20.8, 14.2. HRMS (ESI): calcd for C21H27Cl2N2O2, 409.1450 (M +
H)+; found, 409.1449.
5-Chloro-N-(2-chloro-4-(decan-2-ylamino)phenyl)-2-hy-
droxybenzamide (12). Compound 12 (570 mg, 77%) was prepared
as a yellow solid according to general procedure B, starting from
compound 4 and 2-decanone. HPLC purity 99.4% (tR = 20.76 min).
1H NMR (300 MHz, CDCl3): δ 11.90 (s, 1H), 8.21 (s, 1H), 7.82 (d, J
= 8.7 Hz, 1H), 7.51 (d, J = 2.1 Hz, 1H), 7.34 (dd, J = 8.7, 1.5 Hz,
1H), 6.94 (d, J = 8.7 Hz, 1H), 6.59 (d, J = 1.8 Hz, 1H), 6.47 (dd, J =
8.7, 1.5 Hz, 1H), 3.60 (s, 1H), 3.45−3.31 (m, 1H), 1.55−1.25 (m,
14H), 1.16 (d, J = 6.0 Hz, 3H), 0.94−0.84 (m, 3H). 13C NMR (75
MHz, CDCl3): δ 166.8, 160.0, 146.4, 134.3, 127.0, 125.4, 125.2,
123.8, 122.2, 120.2, 115.9, 112.6, 112.1, 48.7, 37.0, 31.9, 29.7, 29.6,
29.3, 26.1, 22.7, 20.6, 14.2. HRMS (ESI): calcd for C23H31Cl2N2O2,
437.1763 (M + H)+; found, 437.1753.
5-Chloro-N-(2-chloro-4-((cyclopropylmethyl)amino)-
phenyl)-2-hydroxybenzamide (13). Compound 13 (59 mg, 45%)
was prepared as a yellow solid according to general procedure A,
starting from compound 4 and cyclopropanecarboxaldehyde. HPLC
purity 95.1% (tR = 16.87 min). 1H NMR (300 MHz, CDCl3): δ 11.94
(s, 1H), 8.10 (s, 1H), 7.89 (d, J = 9.0 Hz, 1H), 7.49 (d, J = 2.1 Hz,
1H), 7.37 (dd, J = 8.7, 2.1 Hz, 1H), 6.96 (d, J = 9.0 Hz, 1H), 6.64 (d,
J = 2.7 Hz, 1H), 6.54 (dd, J = 8.7, 2.4 Hz, 1H), 3.93 (s, 1H), 2.93 (d, J
= 6.6 Hz, 2H), 1.14−0.95 (m, 1H), 0.61−0.53 (m, 2H), 0.29−0.21
(m, 2H). 13C NMR (75 MHz, CDCl3): δ 166.9, 160.4 147.1, 134.5,
126.7, 125.2, 124.9, 123.8, 122.8, 120.5, 115.8, 112.4, 112.1, 49.1,
10.8, 3.6 (2C). HRMS (ESI): calcd for C17H17Cl2N2O2, 351.0667 (M
+ H)+; found, 351.0663.
5-Chloro-N-(2-chloro-4-((cyclopentylmethyl)amino)-
phenyl)-2-hydroxybenzamide (14). Compound 14 (101 mg,
79%) was prepared as a yellow solid according to general procedure
A, starting from compound 4 and cyclopentanecarboxaldehyde.
HPLC purity 98.0% (tR = 20.39 min). 1H NMR (300 MHz,
CDCl3): δ 11.92 (s, 1H), 8.11 (s, 1H), 7.86 (d, J = 8.7 Hz, 1H), 7.49
(d, J = 2.4 Hz, 1H), 7.37 (dd, J = 9.0, 2.4 Hz, 1H), 6.96 (d, J = 9.0 Hz,
1H), 6.63 (d, J = 2.7 Hz, 1H), 6.52 (dd, J = 9.0, 2.7 Hz, 1H), 3.80 (s,
1H), 2.99 (d, J = 7.2 Hz, 2H), 2.20−2.08 (m, 1H), 1.89−1.76 (m,
2H), 1.71−1.45 (m, 4H), 1.32−1.18 (m, 2H). 13C NMR (75 MHz,
CDCl3): δ 166.9, 160.3, 147.3, 134.5, 126.8, 125.2, 125.0, 123.8,
122.6, 120.4, 115.8, 112.2, 112.0, 49.4, 39.4, 30.7 (2C), 25.4 (2C).
HRMS (ESI): calcd for C19H21Cl2N2O2, 379.0980 (M + H)+; found,
379.0977.
5-Chloro-N-(2-chloro-4-((1-methylcyclopentyl) amino)-
phenyl)-2-hydroxybenzamide (15). To a solution of niclosamide
(150 mg, 0.46 mmol) and Fe(acac)3 (49 mg, 0.14 mmol) in EtOH (4
mL) were added the donor olefin 1-methyl-1-cyclopentene (113 mg,
1.38 mmol) and PhSiH3 (99 mg, 0.92 mmol). The resulting mixture
was heated in an oil bath preheated to 60 °C with stirring for 1 h. The
reaction mixture was then cooled to room temperature, and Zn (595
mg, 9.2 mmol) and 2 N HCl (2 mL) were added to the reaction
mixture. After stirring at 60 °C for another 1 h, the reaction mixture
was cooled to room temperature and filtered through Celite. After the
filter cake was washed with EtOAc, the filtrate was neutralized with
sat. NaHCO3 (aq) and extracted with EtOAc three times. The
combined organic layers were washed with brine, dried over Na2SO4,
filtered, and concentrated under reduced pressure. The resulting
crude product was then purified on SiO2 to furnish compound 15 (52
mg, 30%) as an off-white solid. HPLC purity 96.8% (tR = 21.41 min).
1H NMR (300 MHz, CDCl3): δ 11.66 (br s, 1H), 8.12 (s, 1H), 7.84
(2C), 25.9, 24.4 (2C). HRMS (ESI): calcd for C19H21Cl2N2O2,
379.0980 (M + H)+; found, 379.0979.
5-Chloro-N-(2-chloro-4-(cyclohexylamino)phenyl)-2-hy-
droxybenzamide (16). Compound 16 (110 mg, 86%) was prepared
as an off-white solid according to general procedure B, starting from
compound 4 and cyclohexanone. HPLC purity 99.3% (tR = 17.47
1
min). H NMR (300 MHz, CDCl3): δ 11.94 (s, 1H), 8.08 (s, 1H),
7.85 (d, J = 8.7 Hz, 1H), 7.48 (s, 1H), 7.37 (d, J = 7.5 Hz, 1H), 6.96
(d, J = 9.0 Hz, 1H), 6.61 (s, 1H), 6.50 (d, J = 8.7 Hz, 2.1, 1H), 3.67
(s, 1H), 3.28−3.14 (m, 1H), 2.10−1.96 (m, 2H), 1.84−1.72 (m, 2H),
1.71−1.61 (m, 1H), 1.45−1.31 (m, 2H), 1.29−1.09 (m, 3H). 13C
NMR (75 MHz, CDCl3): δ 166.9, 160.4, 146.1, 134.5, 126.8, 125.2,
125.0, 123.8, 122.3, 120.4, 115.8, 112.6, 112.4, 51.9, 33.3 (2C), 25.9,
25.0 (2C). HRMS (ESI): calcd for C19H21Cl2N2O2, 379.0980 (M +
H)+; found, 379.0970.
5-Chloro-N-(2-chloro-4-(dimethylamino)phenyl)-2-hydroxy-
benzamide (17). Compound 17 (100 mg, 76%) was prepared as a
yellow solid according to general procedure B, starting from
compound 4 and paraformaldehyde. HPLC purity 96.1% (tR
=
18.82 min). 1H NMR (300 MHz, DMSO-d6): δ 12.21 (s, 1H), 10.49
(s, 1H), 8.02 (d, J = 2.7 Hz, 1H), 7.89 (d, J = 9.0 Hz, 1H), 7.48 (dd, J
= 9.0, 2.7 Hz, 1H), 7.03 (d, J = 9.0 Hz, 1H), 6.81 (d, J = 3.0 Hz, 1H),
6.73 (dd, J = 9.0, 3.0 Hz, 1H), 2.91 (s, 6H). 13C NMR (75 MHz,
DMSO-d6): δ 163.5, 156.3, 148.6, 133.1, 129.0, 126.5, 125.5, 123.3,
123.1, 119.1, 119.0, 112.0, 111.3, 40.0 (2C). HRMS (ESI): calcd for
C15H15Cl2N2O2, 325.0511 (M + H)+; found, 325.0509.
5-Chloro-N-(2-chloro-4-(diethylamino)phenyl)-2-hydroxy-
benzamide (18). Compound 18 was prepared by a procedure the
same as that used to prepare compound 7. The title compound (58
mg, 41%) was obtained as a yellow solid. HPLC purity 99.8% (tR =
1
16.50 min). H NMR (300 MHz, CDCl3): δ 11.99 (s, 1H), 8.10 (s,
1H), 7.88 (d, J = 9.0 Hz, 1H), 7.50 (s, 1H), 7.37 (d, J = 9.0 Hz, 1H),
6.96 (d, J = 9.0 Hz, 1H), 6.68 (d, J = 2.1 Hz, 1H), 6.59 (dd, J = 9.0,
2.1 Hz, 1H), 3.33 (q, J = 6.9 Hz, 4H), 1.17 (t, J = 6.9 Hz, 6H). 13C
NMR (75 MHz, CDCl3): δ 166.9, 160.4, 146.5, 134.4, 127.1, 125.2,
125.0, 123.7, 121.1, 120.4, 115.9, 111.7, 110.8, 44.6 (2C), 12.6 (2C).
HRMS (ESI): calcd for C17H19Cl2N2O2, 353.0824 (M + H)+; found,
353.0824.
5-Chloro-N-(2-chloro-4-(dipropylamino)phenyl)-2-hydroxy-
benzamide (19). Compound 19 was prepared by a procedure the
same as that used to prepare compound 8. The title compound (10
mg, 7%) was obtained as a yellow solid. HPLC purity 95.7% (tR
=
1
18.11 min). H NMR (300 MHz, CDCl3): δ 12.02 (s, 1H), 8.05 (s,
1H), 7.89 (d, J = 9.0 Hz, 1H), 7.49 (d, J = 2.4 Hz, 1H), 7.38 (dd, J =
9.0, 2.4 Hz, 1H), 6.97 (d, J = 8.7 Hz, 1H), 6.65 (d, J = 2.7 Hz, 1H),
6.57 (dd, J = 9.0, 2.7 Hz, 1H), 3.23 (d, J = 7.5 Hz, 4H), 1.65−1.56
(m, 4H), 0.94 (t, J = 7.5 Hz, 6H). 13C NMR (75 MHz, CDCl3): δ
166.8, 160.4, 147.0, 134.4, 127.0, 125.2, 124.9, 123.7, 121.0, 120.5,
115.9, 111.6, 110.8, 53.0 (2C), 20.5 (2C), 11.5 (2C). HRMS (ESI):
calcd for C19H23Cl2N2O2, 381.1137 (M + H)+; found, 381.1133.
N-(4-(Bis(cyclopropylmethyl)amino)-2-chlorophenyl)-5-
chloro-2-hydroxybenzamide (20). Compound 20 was prepared
by a procedure the same as that used to prepare compound 13. The
title compound (53 mg, 43%) was obtained as a yellow solid. HPLC
purity 97.7% (tR = 17.75 min). 1H NMR (300 MHz, CDCl3): δ 11.97
(s, 1H), 8.15 (s, 1H), 7.91 (d, J = 9.3 Hz, 1H), 7.50 (d, J = 2.4 Hz,
1H), 7.36 (dd, J = 8.7, 2.4 Hz, 1H), 6.95 (d, J = 8.7 Hz, 1H), 6.82 (d,
J = 3.0 Hz, 1H), 6.73 (dd, J = 9.3, 3.0 Hz, 1H), 3.25 (d, J = 6.0 Hz,
4H), 1.11−0.98 (m, 2H), 0.58−0.50 (m, 4H), 0.26−0.19 (m, 4H).
13C NMR (75 MHz, CDCl3): δ 166.8, 160.3, 147.6, 134.4, 126.8,
125.2, 124.7, 123.7, 121.5, 120.4, 115.9, 112.5, 111.6, 55.1 (2C), 9.4
(2C), 4.0 (4C). HRMS (ESI): calcd for C21H23Cl2N2O2, 405.1137
(M + H)+; found, 405.1129.
5-Chloro-N-(2-chloro-4-((1-methylcyclopentyl) ((1-
methylcyclopentyl)oxy)amino) phenyl)-2-hydroxybenzamide
(21). Compound 21 was prepared by a procedure the same as that
used to prepare compound 15. The title compound (46 mg, 21%) was
obtained as a pale yellow solid. HPLC purity 95.5% (tR = 25.71 min).
1H NMR (300 MHz, CDCl3): δ 11.78 (s, 1H), 8.38 (s, 1H), 8.20 (d, J
(d, J = 9.0 Hz, 1H), 7.50 (d, J = 2.4 Hz, 1H), 7.37 (dd, J = 9.0, 2.4 Hz,
1H), 6.96 (d, J = 9.0 Hz, 1H), 6.70 (d, J = 2.7 Hz, 1H), 6.57 (dd, J =
9.0, 2.7 Hz, 1H), 1.98−1.84 (m, 2H), 1.82−1.65 (m, 6H), 1.40 (s,
3H). 13C NMR (75 MHz, CDCl3): δ 166.9, 160.3, 145.6, 134.5,
126.4, 125.2, 124.7, 123.8, 122.4, 120.4, 115.8, 114.5, 114.0, 61.7, 40.7
= 9.0 Hz, 1H), 7.51 (d, J = 2.4 Hz, 1H), 7.46 (d, J = 2.1 Hz, 1H), 7.40
M
https://dx.doi.org/10.1021/acs.jmedchem.0c01226
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(dd, J = 8.7, 2.4 Hz, 1H), 7.28−7.22 (m, 1H), 6.99 (d, J = 9.0 Hz,
1H), 2.10−1.84 (m, 3H), 1.82−1.41 (m, 10H), 1.40−1.19 (m, 3H),
1.17 (s, 3H), 1.05 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 166.9,
160.5, 151.0, 134.9, 129.8, 125.9, 125.2, 124.8, 124.0, 123.2, 121.2,
120.6, 115.8, 90.1, 71.9, 38.0, 37.7 (2C), 36.8, 25.8, 24.2, 24.0, 23.2,
22.9, 20.6. HRMS (ESI): calcd for C25H31Cl2N2O3, 477.1712 (M +
H)+; found, 477.1711.
5-Chloro-N-(2-chloro-4-((3-(trifluoromethyl)benzyl)amino)-
phenyl)-2-hydroxybenzamide (27). Compound 27 (131 mg,
85%) was prepared as a white solid according to general procedure B,
starting from compound 4 and 3-(trifluoromethyl)benzaldehyde.
1
HPLC purity 95.6% (tR = 20.07 min). H NMR (300 MHz, DMSO-
d6): δ 12.20 (s, 1H), 10.40 (s, 1H), 7.99 (d, J = 2.4 Hz, 1H), 7.76−
7.53 (m, 5H), 7.47 (dd, J = 8.7, 2.7 Hz, 1H), 7.02 (d, J = 9.0 Hz, 1H),
6.73 (d, J = 2.4 Hz, 1H), 6.67 (t, J = 6.0 Hz, 1H), 6.61 (dd, J = 8.7,
2.4 Hz, 1H), 4.40 (d, J = 6.0 Hz, 2H). 13C NMR (75 MHz, CDCl3): δ
163.7, 156.4, 146.9, 141.4, 133.2, 131.3, 129.4, 129.1 (q, J = 31.2 Hz),
128.9, 126.8, 126.1, 124.3 (q, J = 270.8 Hz), 123.1, 123.6 (m, 2C),
123.0, 119.1, 118.8, 111.9, 111.5, 45.7. HRMS (ESI): calcd for
C21H16Cl2F3N2O2, 455.0541 (M + H)+; found, 455.0534.
5-Chloro-N-(2-chloro-4-((4-methoxybenzyl)amino)phenyl)-
2-hydroxybenzamide (28). Compound 28 (55 mg, 39%) was
prepared as a yellow solid according to general procedure B, starting
from compound 4 and 4-methoxybenzaldehyde. HPLC purity 99.1%
(tR = 19.07 min). 1H NMR (300 MHz, CDCl3): δ 11.93 (s, 1H), 8.07
(s, 1H), 7.91 (d, J = 8.7 Hz, 1H), 7.48 (d, J = 2.1 Hz, 1H), 7.38 (dd, J
= 9.0, 2.1 Hz, 1H), 7.27 (d, J = 7.8 Hz, 2H), 6.97 (d, J = 9.0 Hz, 1H),
6.89 (d, J = 8.4 Hz, 2H), 6.68 (d, J = 2.4 Hz, 1H), 6.57 (dd, J = 8.7,
2.4 Hz, 1H), 4.24 (s, 2H), 4.09 (s, 1H), 3.81 (s, 3H). 13C NMR (75
MHz, CDCl3): δ 166.9, 160.5, 159.2, 146.8, 134.6, 130.5, 128.9 (2C),
126.7, 125.1, 124.9, 123.8, 123.1, 120.5, 115.8, 114.3 (2C), 112.7,
112.3, 55.5, 47.9. HRMS (ESI): calcd for C21H19Cl2N2O3, 417.0773
(M + H)+; found, 417.0771.
5-Chloro-N-(2-chloro-4-((4-hydroxybenzyl)amino)phenyl)-
2-hydroxybenzamide (29). Compound 29 (70 mg, 51%) was
prepared as a yellow solid according to general procedure B, starting
from compound 4 and 4-hydroxybenzaldehyde. HPLC purity 96.8%
(tR = 17.31 min). 1H NMR (300 MHz, CD3OD + CDCl3): δ 7.98 (d,
J = 2.4 Hz, 1H), 7.77 (d, J = 9.0 Hz, 1H), 7.29 (dd, J = 8.7, 2.7 Hz,
1H), 7.15 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.7 Hz, 1H), 6.79−6.72
(m, 2H), 6.67 (d, J = 2.7 Hz, 1H), 6.55 (dd, J = 8.7, 2.4 Hz, 1H), 4.16
(s, 2H). 13C NMR (75 MHz, CD3OD + CDCl3): δ 165.4, 156.9,
156.7, 147.9, 133.8, 130.7, 130.1, 129.3 (2C), 128.0, 126.2, 125.3,
124.4, 119.8, 119.1, 115.9 (2C), 113.3, 112.5, 48.0. HRMS (ESI):
calcd for C20H17Cl2N2O3, 403.0616 (M + H)+; found, 403.0610.
5-Chloro-N-(2-chloro-4-((4-(dimethylamino)benzyl)amino)-
phenyl)-2-hydroxybenzamide (30). Compound 30 (60 mg, 41%)
was prepared as a yellow solid according to general procedure B,
starting from compound 4 and 4-dimethylaminobenzaldehyde. HPLC
purity 98.9% (tR = 14.97 min). 1H NMR (300 MHz, CDCl3): δ 11.96
(s, 1H), 8.07 (s, 1H), 7.92 (d, J = 9.0 Hz, 1H), 7.49 (d, J = 2.1 Hz,
1H), 7.38 (dd, J = 8.7, 2.1 Hz, 1H), 7.22 (d, J = 8.7 Hz, 2H), 6.98 (d,
J = 9.0 Hz, 1H), 6.76−6.69 (m, 3H), 6.59 (dd, J = 9.0, 2.4 Hz, 1H),
4.19 (s, 2H), 4.03 (s, 1H), 2.95 (s, 6H). 13C NMR (75 MHz, CDCl3):
δ 166.9, 160.5, 150.4, 147.0, 134.6, 128.8 (2C), 126.6, 126.1, 125.1,
124.8, 123.8, 122.9, 120.5, 115.8, 112.9 (2C), 112.6, 112.2, 48.0, 40.8
(2C). HRMS (ESI): calcd for C22H22Cl2N3O2, 430.1089 (M + H)+;
found, 430.1082.
N-(4-(Benzylamino)-2-chlorophenyl)-5-chloro-2-hydroxy-
benzamide (22). Compound 22 (110 mg, 84%) was prepared as a
yellow solid according to general procedure A, starting from
compound 4 and benzaldehyde. HPLC purity 95.2% (tR = 19.59
min). 1H NMR (300 MHz, CD3OD + CDCl3): δ 7.93 (d, J = 2.7 Hz,
1H), 7.81 (d, J = 8.7 Hz, 1H), 7.34−7.19 (m, 6H), 6.87 (d, J = 8.7
Hz, 1H), 6.66 (d, J = 2.4 Hz, 1H), 6.54 (dd, J = 9.0, 2.7 Hz, 1H), 4.27
(s, 2H). 13C NMR (75 MHz, CD3OD + CDCl3): δ 165.2, 156.8,
147.1, 139.2, 133.5, 129.3, 128.9 (2C), 127.5 (2C), 127.5, 127.5,
125.8, 124.8, 124.3, 119.1, 118.9, 113.0, 112.2, 48.2. HRMS (ESI):
calcd for C20H17Cl2N2O2, 387.0667 (M + H)+; found, 387.0659.
5-Chloro-N-(2-chloro-4-((4-fluorobenzyl)amino)phenyl)-2-
hydroxybenzamide (23). Compound 23 (82 mg, 39%) was
prepared as a white solid according to general procedure B, starting
from compound 4 and 4-fluorobenzaldehyde. HPLC purity 99.5% (tR
= 19.67 min). 1H NMR (300 MHz, CD3OD + CDCl3): δ 7.85 (d, J =
2.7 Hz, 1H), 7.80 (d, J = 9.0 Hz, 1H), 7.30−7.22 (m, 3H), 7.01−6.93
(m, 2H), 6.86 (d, J = 8.7 Hz, 1H), 6.62 (d, J = 2.7 Hz, 1H), 6.52 (dd,
J = 8.7, 2.7 Hz, 1H), 4.22 (s, 2H). 13C NMR (75 MHz, CD3OD +
CDCl3): δ 165.2, 162.0 (d, J = 243.5 Hz), 157.1, 146.5, 134.5 (d, J =
3.1 Hz), 133.4, 128.8 (d, J = 8.0 Hz, 2C), 128.4, 127.2, 125.5, 124.4,
124.0, 118.8, 118.2, 115.4 (d, J = 243.5 Hz, 2C), 112.7, 112.0, 47.2.
HRMS (ESI): calcd for C20H16Cl2FN2O2, 405.0573 (M + H)+; found,
405.0570.
5-Chloro-N-(2-chloro-4-((4-chlorobenzyl)amino)phenyl)-2-
hydroxybenzamide (24). Compound 24 (126 mg, 88%) was
prepared as a pale yellow solid according to general procedure B,
starting from compound 4 and 4-chlorobenzaldehyde. HPLC purity
97.3% (tR = 20.01 min). 1H NMR (300 MHz, DMSO-d6): δ 12.20 (s,
1H), 10.39 (s, 1H), 7.99 (d, J = 2.7 Hz, 1H), 7.70 (d, J = 8.7 Hz, 1H),
7.47 (dd, J = 9.0, 2.7 Hz, 1H), 7.42−7.34 (m, 4H), 7.01 (d, J = 8.7
Hz, 1H), 6.68 (d, J = 2.7 Hz, 1H), 6.65−6.54 (m, 2H), 4.29 (d, J =
6.0 Hz, 2H). 13C NMR (75 MHz, DMSO-d6): δ 163.7, 156.4, 147.0,
138.8, 133.2, 131.2, 129.0 (2C), 128.9, 128.3 (2C), 126.8, 126.1,
123.0, 123.0, 119.1, 118.8, 111.8, 111.5, 45.5. HRMS (ESI): calcd for
C20H16Cl3N2O2, 421.0277 (M + H)+; found, 421.0270.
N-(4-((4-Bromobenzyl)amino)-2-chlorophenyl)-5-chloro-2-
hydroxybenzamide (25). Compound 25 (135 mg, 86%) was
prepared as a pale yellow solid according to general procedure B,
starting from compound 4 and 4-bromobenzaldehyde. HPLC purity
97.0% (tR = 20.18 min). 1H NMR (300 MHz, DMSO-d6): δ 12.20 (s,
1H), 10.39 (s, 1H), 7.99 (d, J = 2.7 Hz, 1H), 7.70 (d, J = 8.7 Hz, 1H),
7.56−7.50 (m, 2H), 7.47 (dd, J = 8.7, 2.7 Hz, 1H), 7.32 (d, J = 8.4
Hz, 2H), 7.01 (d, J = 8.7 Hz, 1H), 6.68 (d, J = 2.4 Hz, 1H), 6.65−
6.54 (m, 2H), 4.27 (d, J = 6.0 Hz, 2H). 13C NMR (75 MHz, DMSO-
d6): δ 163.7, 156.4, 147.0, 139.2, 133.2, 131.2 (2C), 129.4 (2C),
128.9, 126.8, 126.0, 123.0, 123.0, 119.7, 119.1, 118.8, 111.8, 111.5,
45.6. HRMS (ESI): calcd for C20H16BrCl2N2O2, 464.9772 (M + H)+;
found, 464.9764.
5-Chloro-N-(2-chloro-4-((3-chloro-5-fluorobenzyl)amino)-
phenyl)-2-hydroxybenzamide (31). Compound 31 (125 mg,
84%) was prepared as a pale solid according to general procedure B,
starting from compound 4 and 3-chloro-5-fluorobenzaldehyde. HPLC
1
purity 97.7% (tR = 20.29 min). H NMR (300 MHz, DMSO-d6): δ
12.21 (s, 1H), 10.41 (s, 1H), 7.99 (d, J = 2.7 Hz, 1H), 7.73 (d, J = 8.7
Hz, 1H), 7.47 (dd, J = 8.7, 2.7 Hz, 1H), 7.33−7.23 (m, 2H), 7.19 (d, J
= 9.3 Hz, 1H), 7.02 (d, J = 8.7 Hz, 1H), 6.71 (d, J = 2.7 Hz, 1H), 6.66
(t, J = 6.3 Hz, 1H), 6.60 (dd, J = 9.0, 2.4 Hz, 1H), 4.34 (d, J = 6.3 Hz,
2H). 13C NMR (75 MHz, DMSO-d6): δ 163.7, 162.2 (d, J = 245.9
Hz), 156.4, 146.7, 145.0 (d, J = 7.6 Hz), 133.8 (d, J = 10.9 Hz), 133.2,
128.9, 126.8, 126.1, 123.3, 123.2 (d, J = 2.8 Hz), 123.0, 119.1, 118.8,
114.3 (d, J = 25.1 Hz), 112.9 (d, J = 21.5 Hz), 112.0, 111.5, 45.3.
HRMS (ESI): calcd for C20H15Cl3FN2O2, 439.0183 (M + H)+; found,
439.0175.
5-Chloro-N-(2-chloro-4-((4-(trifluoromethyl)benzyl)amino)-
phenyl)-2-hydroxybenzamide (26). Compound 26 (132 mg,
86%) was prepared as a pale yellow solid according to general
procedure B, starting from compound 4 and 4-(trifluoromethyl)-
1
benzaldehyde. HPLC purity 95.0% (tR = 20.12 min). H NMR (300
MHz, DMSO-d6): δ 12.20 (s, 1H), 10.39 (s, 1H), 7.99 (d, J = 2.7 Hz,
1H), 7.71 (dd, J = 8.6, 3.2 Hz, 3H), 7.57 (d, J = 8.0 Hz, 2H), 7.47
(dd, J = 8.8, 2.8 Hz, 1H), 7.01 (d, J = 8.8 Hz, 1H), 6.76−6.66 (m,
2H), 6.59 (dd, J = 8.8, 2.6 Hz, 1H), 4.41 (d, J = 6.0 Hz, 2H). 13C
NMR (75 MHz, DMSO-d6): δ 163.7, 156.4, 146.9, 144.9, 133.2,
128.9, 127.8 (2C), 127.5 (q, J = 31.5 Hz), 126.8, 126.1, 125.2 (q, J =
3.8 Hz), 124.3 (q, J = 31.5 Hz, 2C), 123.1, 123.0, 119.1, 118.8, 111.9,
111.4, 45.8. HRMS (ESI): calcd for C21H16Cl2F3N2O2, 455.0541 (M
+ H)+; found, 455.0533.
5-Chloro-N-(2-chloro-4-((3,5-dichlorobenzyl)amino)-
phenyl)-2-hydroxybenzamide (32). Compound 32 (130 mg,
84%) was prepared as a white solid according to general procedure B,
starting from compound 4 and 3,5-dichlorobenzaldehyde. HPLC
N
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1
purity 97.9% (tR = 20.95 min). H NMR (300 MHz, DMSO-d6): δ
purified by preparative TLC to afford compound 34 (68 mg, 58%) as
a pale yellow solid. HPLC purity 98.4% (tR = 20.33 min). H NMR
1
12.20 (s, 1H), 10.41 (s, 1H), 7.99 (d, J = 2.7 Hz, 1H), 7.73 (d, J = 8.7
Hz, 1H), 7.50−7.44 (m, 2H), 7.41 (d, J = 1.8 Hz, 2H), 7.02 (d, J =
8.7 Hz, 1H), 6.72 (d, J = 2.4 Hz, 1H), 6.66 (t, J = 6.3 Hz, 1H), 6.60
(dd, J = 9.0, 2.7 Hz, 1H), 4.33 (d, J = 6.3 Hz, 2H). 13C NMR (75
MHz, DMSO-d6): δ 163.7, 156.4, 146.6, 144.6, 134.0 (2C), 133.2,
128.9, 126.8, 126.4, 126.1, 125.8 (2C), 123.3, 123.0, 119.1, 118.8,
112.0, 111.5, 45.2. HRMS (ESI): calcd for C20H15Cl4N2O2 454.9888
(M + H)+, found 454.9880.
5-Chloro-N-(2-chloro-4-((3-chloro-5-(trifluoromethyl)-
benzyl)amino)phenyl)-2-hydroxybenzamide (33). Compound
33 (96 mg, 58%) was prepared as a white solid according to general
procedure B, starting from compound 4 and 3-chloro-5-
(trifluoromethyl)benzaldehyde. HPLC purity 96.9% (tR = 20.91
(300 MHz, CDCl3 + CD3OD): δ 7.97 (d, J = 2.4 Hz, 1H), 7.82 (d, J
= 8.7 Hz, 1H), 7.41 (s, 1H), 7.31−7.24 (m, 2H), 7.19 (d, J = 8.4 Hz,
1H), 6.88 (d, J = 8.7 Hz, 1H), 6.63 (d, J = 2.7 Hz, 1H), 6.52 (dd, J =
8.7, 2.7 Hz, 1H), 4.36 (s, 2H). 13C NMR (75 MHz, CDCl3
+
CD3OD): δ 165.2, 163.3 (d, J = 247.0 Hz), 156.6, 146.8, 144.6 (d, J =
6.8 Hz), 133.6, 133.0 (qd, J = 32.9, 8.1 Hz), 129.9, 127.7, 126.0,
125.1, 124.9, 123.8 (qd, J = 270.0, 3.0 Hz), 119.8 (quint, J = 3.6 Hz),
119.5, 118.9, 117.8 (d, J = 21.8 Hz), 113.2, 112.2, 111.7 (dq, J = 24.7,
3.8 Hz), 47.3 (d, J = 1.1 Hz). HRMS (ESI): calcd for
C21H15Cl2F4N2O2, 473.0447 (M + H)+; found, 473.0439.
N-(4-((3,5-Bis(trifluoromethyl)benzyl)amino)-2-chlorophen-
yl)-5-chloro-2-hydroxybenzamide (35). Compound 35 (119 mg,
67%) was prepared as an off-white solid according to general
procedure B, starting from compound 4 and 3,5-bis(trifluoromethyl)-
1
min). H NMR (300 MHz, DMSO-d6): δ 12.19 (s, 1H), 10.41 (s,
1H), 7.99 (d, J = 2.7 Hz, 1H), 7.78−7.69 (m, 4H), 7.47 (dd, J = 8.7,
2.7 Hz, 1H), 7.02 (d, J = 8.7 Hz, 1H), 6.75 (d, J = 2.4 Hz, 1H), 6.69
(t, J = 6.3 Hz, 1H), 6.62 (dd, J = 9.0, 2.4 Hz, 1H), 4.42 (d, J = 6.0 Hz,
2H). 13C NMR (75 MHz, DMSO-d6): δ 163.7, 156.4, 146.6, 144.3,
134.0, 133.2, 131.1, 130.8 (q, J = 32.2 Hz), 128.9, 126.8, 126.1, 123.4,
123.4 (q, J = 271.1 Hz), 123.6 (q, J = 3.8 Hz), 123.1, 122.6 (q, J = 3.7
Hz), 119.1, 118.8, 112.0, 111.5, 45.2. HRMS (ESI): calcd for
C21H15Cl3F3N2O2, 489.0151 (M + H)+; found, 489.0146.
5-Chloro-N-(2-chloro-4-((3-fluoro-5-(trifluoromethyl)-
benzyl)amino)phenyl)-2-hydroxybenzamide (34). To a solution
of 2-chloro-4-fluoronitrobenzene (300 mg, 1.71 mmol) and 3-fluoro-
5-(trifluoromethyl)benzylamine (396 mg, 2.05 mmol) in 5 mL of
DMF was added K2CO3 (473 mg, 3.42 mmol). The resulting mixture
was stirred at 100 °C for 1 h. Then, the mixture was cooled to RT and
poured into 50 mL of H2O. The mixture was extracted with EtOAc (2
× 80 mL), washed with H2O (3 × 20 mL) and brine (30 mL), dried
(Na2SO4), and concentrated. The residue was purified by column
chromatography (Hex/EtOAc = 6/1 to 3/1) to give 3-chloro-N-(3-
fluoro-5-(trifluoromethyl)benzyl)-4-nitroaniline 59b as a yellow solid
(450 mg, 87%). 1H NMR (300 MHz, CDCl3): δ 7.97 (d, J = 9.0 Hz,
1H), 7.39 (s, 1H), 7.32−7.17 (m, 2H), 6.64 (d, J = 2.7 Hz, 1H), 6.48
(dd, J = 9.3, 2.7 Hz, 1H), 5.05 (t, J = 5.4 Hz, 1H), 4.50 (d, J = 6.0 Hz,
2H).
1
benzaldehyde. HPLC purity 98.2% (tR = 21.01 min). H NMR (300
MHz, CD3OD + CDCl3): δ 7.98 (d, J = 2.4 Hz, 1H), 7.88 (s, 2H),
7.82 (d, J = 8.7 Hz, 1H), 7.76 (s, 1H), 7.30 (dd, J = 8.7, 2.4 Hz, 1H),
6.89 (d, J = 8.7 Hz, 1H), 6.68 (d, J = 2.1 Hz, 1H), 6.55 (dd, J = 8.7,
2.4 Hz, 1H), 4.45 (s, 2H). 13C NMR (75 MHz, CD3OD + CDCl3): δ
165.5, 156.9, 147.3, 144.0, 133.9, 132.5 (q, J = 132.5 Hz, 2C), 130.3,
128.2, 128.1 (q, J = 2.7 Hz, 2C), 126.5, 125.4, 125.3, 124.3 (q, J =
270.5 Hz, 2C), 121.5 (septet, J = 3.8 Hz), 119.9, 119.2, 113.6, 112.3,
47.4. HRMS (ESI): calcd for C22H15Cl2F6N2O2, 523.0415 (M + H)+;
found, 523.0413.
5-Chloro-N-(2-chloro-4-((pyridin-4-ylmethyl)amino)-
phenyl)-2-hydroxybenzamide (36). Compound 36 (54 mg, 41%)
was prepared as a yellow solid according to general procedure B,
starting from compound 4 and 4-pyridinecarboxaldehyde. HPLC
1
purity 98.3% (tR = 16.26 min). H NMR (300 MHz, DMSO-d6): δ
12.16 (s, 1H), 10.40 (s, 1H), 8.51 (d, J = 4.2 Hz, 2H), 7.99 (d, J = 2.7
Hz, 1H), 7.72 (d, J = 9.0 Hz, 1H), 7.46 (dd, J = 8.7, 2.7 Hz, 1H), 7.35
(d, J = 5.7 Hz, 2H), 7.01 (d, J = 8.7 Hz, 1H), 6.72−6.62 (m, 2H),
6.58 (dd, J = 8.7, 2.4 Hz, 1H), 4.35 (d, J = 6.3 Hz, 2H). 13C NMR (75
MHz, DMSO-d6): δ 163.7, 156.4, 149.5 (2C), 149.0, 146.9, 133.2,
128.9, 126.8, 126.1, 123.2, 123.0, 122.2 (2C), 119.01, 118.8, 111.9,
111.4, 45.2. HRMS (ESI): calcd for C19H16Cl2N3O2, 388.0620 (M +
H)+; found, 388.0615.
To a solution of compound 59b (450 mg, 1.29 mmol) in 10 mL of
MeOH was added 5 mL of saturated NH4Cl (aq). Zinc dust (838 mg,
12.9 mmol) was added into the solution at 0 °C. The reaction was
stirred at RT for 16 h. TLC indicated that the starting material was
gone. EtOAc (150 mL) was added to the solution. The zinc solid was
filtered, and the filtrate was washed with 30 mL of brine, dried
(Na2SO4), and concentrated under vacuum. The residue was purified
by column chromatography to afford 3-chloro-N1-(3-fluoro-5-
(trifluoromethyl)benzyl)benzene-1,4-diamine 60b (310 mg, 75%) as
5-Chloro-N-(2-chloro-4-(((2-chloropyridin-3-yl)methyl)-
amino)phenyl)-2-hydroxybenzamide (37). Compound 37 (90
mg, 63%) was prepared as a yellow solid according to general
procedure B, starting from compound 4 and 2-chloro-3-formylpyr-
1
idine. HPLC purity 96.0% (tR = 18.73 min). H NMR (300 MHz,
DMSO-d6): δ 12.20 (s, 1H), 10.42 (s, 1H), 8.32 (dd, J = 4.8, 1.8 Hz,
1H), 7.99 (d, J = 2.7 Hz, 1H), 7.84−7.72 (m, 2H), 7.47 (dd, J = 8.7,
2.7 Hz, 1H), 7.42 (dd, J = 7.5, 4.8 Hz, 1H), 7.02 (d, J = 8.7 Hz, 1H),
6.72 (d, J = 2.4 Hz, 1H), 6.65 (t, J = 6.0 Hz, 1H), 6.59 (dd, J = 9.0,
2.4 Hz, 1H), 4.36 (d, J = 5.7 Hz, 2H). 13C NMR (75 MHz, DMSO-
d6): δ 163.7, 156.4, 149.4, 148.1, 146.6, 137.8, 133.2, 133.2, 128.9,
126.9, 126.1, 123.5, 123.3, 123.0, 119.1, 118.8, 111.9, 111.3, 43.6.
HRMS (ESI): calcd for C19H15Cl3N3O2, 422.0230 (M + H)+; found,
422.0228.
5-Chloro-N-(2-chloro-4-(((4-chloropyridin-3-yl)methyl)-
amino)phenyl)-2-hydroxybenzamide (38). Compound 38 (110
mg, 77%) was prepared as a yellow solid according to general
procedure B, starting from compound 4 and 4-chloropyridine-3-
carboxaldehyde. HPLC purity 99.1% (tR = 18.96 min). 1H NMR (300
MHz, DMSO-d6): δ 12.20 (s, 1H), 10.42 (s, 1H), 8.57 (s, 1H), 8.46
(d, J = 5.1 Hz, 1H), 8.00 (d, J = 2.7 Hz, 1H), 7.76 (d, J = 9.0 Hz, 1H),
7.57 (d, J = 5.43 Hz, 1H), 7.47 (dd, J = 8.7, 2.7 Hz, 1H), 7.02 (d, J =
8.7 Hz, 1H), 6.77 (d, J = 2.4 Hz, 1H), 6.64 (dd, J = 9.0, 2.4 Hz, 1H),
6.55 (t, J = 6.0 Hz, 1H), 4.41 (d, J = 5.7 Hz, 2H). 13C NMR (75
MHz, DMSO-d6): δ 163.7, 156.4, 150.3, 149.6, 146.6, 142.6, 133.2,
132.2, 128.9, 126.9, 126.1, 124.4, 123.4, 123.0, 119.1, 118.8, 111.9,
111.3, 42.2. HRMS (ESI): calcd for C19H15Cl3N3O2, 422.0230 (M +
H)+; found, 422.0223.
1
a yellow solid. H NMR (300 MHz, CDCl3): δ 7.42 (s, 1H), 7.30−
7.19 (m, 2H), 6.65 (d, J = 8.4 Hz, 1H), 6.57 (d, J = 2.7 Hz, 1H), 6.41
(dd, J = 8.7, 2.7 Hz, 1H), 4.30 (s, 2H), 3.96−3.46 (m, 3H).
To a solution of 3-chloro-N1-(3-fluoro-5-(trifluoromethyl)benzyl)-
benzene-1,4-diamine (84 mg, 0.26 mmol), 5-chloro-2-methoxyben-
zoic acid (59 mg, 0.32 mmol), and 4-(dimethylamino)pyridine
(DMAP, 7 mg, 0.05 mmol) in 15 mL of DCM was added 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI,
101 mg, 0.53 mmol) at 0 °C. The resulting mixture was stirred at
RT overnight and then concentrated. The residue was purified by
column chromatography (Hex/EtOAc = 3/1) to afford 5-chloro-N-
(2-chloro-4-((3-fluoro-5-(trifluoromethyl)benzyl)amino)phenyl)-2-
1
methoxybenzamide (120 mg, 93%) as a yellow solid. H NMR (300
MHz, CDCl3): δ 10.23 (s, 1H), 8.33 (d, J = 8.7 Hz, 1H), 8.20 (d, J =
2.7 Hz, 1H), 7.42−7.34 (m, 2H), 7.26−7.17 (m, 2H), 6.92 (d, J = 8.7
Hz, 1H), 6.61 (d, J = 2.7 Hz, 1H), 6.51 (dd, J = 9.0, 2.7 Hz, 1H), 4.43
(br s, 1H), 4.34 (s, 2H), 4.01 (s, 3H).
To
a solution of 5-chloro-N-(2-chloro-4-((3-fluoro-5-
(trifluoromethyl)benzyl)amino)phenyl)-2-methoxybenzamide (120
mg, 0.25 mmol) in DCM (15 mL) was added BBr3 (0.74 mL, 0.74
mmol, 1 M in DCM) dropwise at 0 °C. The mixture was stirred at 0
°C for 2 h. The mixture was diluted with DCM, washed with H2O
and brine, dried (Na2SO4), and concentrated. The residue was
N-(4-(((6-Bromopyridin-2-yl)methyl)amino)-2-chlorophen-
yl)-5-chloro-2-hydroxybenzamide (39). Compound 39 (90 mg,
57%) was prepared as a yellow solid according to general procedure B,
O
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starting from compound HJC0129 and 6-bromo-2-pyridinecarbox-
aldehyde. HPLC purity 96.7% (tR = 19.06 min). 1H NMR (300 MHz,
DMSO-d6): δ 12.19 (s, 1H), 10.40 (s, 1H), 7.99 (d, J = 2.7 Hz, 1H),
7.77−7.68 (m, 2H), 7.52 (d, J = 7.8 Hz, 1H), 7.47 (dd, J = 9.0, 2.7
Hz, 1H), 7.39 (d, J = 7.5 Hz, 1H), 7.02 (d, J = 8.7 Hz, 1H), 6.75−
6.67 (m, 2H), 6.59 (dd, J = 8.7, 2.4 Hz, 1H), 4.38 (d, J = 6.0 Hz, 2H).
13C NMR (75 MHz, DMSO-d6): δ 163.7, 161.4, 156.4, 146.8, 140.8,
140.1, 133.2, 128.9, 126.9, 126.3, 126.1, 123.3, 123.1, 120.5, 119.1,
118.8, 111.9, 111.3, 47.7. HRMS (ESI): calcd for C19H15BrCl2N3O2,
465.9725 (M + H)+; found, 465.9724.
1H), 7.00−6.89 (m, 2H), 6.75 (dd, J = 8.7, 1.8 Hz, 1H), 5.85 (br s,
2H), 3.88 (t, J = 6.0 Hz, 2H), 1.89 (t, J = 6.0 Hz, 2H), 1.31 (s, 6H).
13C NMR (75 MHz, CDCl3): δ 166.6, 159.6, 143.7, 134.4, 126.1,
125.9, 125.9, 124.3, 124.1, 120.2, 119.1, 118.3, 116.3, 59.9, 55.2, 42.7,
28.1 (2C). HRMS (ESI): calcd for C18H21Cl2N2O3, 383.0929 (M +
H)+; found, 383.0920.
5-Chloro-N-(2-chloro-4-((4-hydroxybutan-2-yl)amino)-
phenyl)-2-hydroxybenzamide (47). Compound 47 was prepared
by a procedure similar to that used to prepare compound 15, starting
from niclosamide and 3-buten-1-ol. The title compound (20 mg,
27%) was obtained as a yellow solid. HPLC purity 95.1% (tR = 15.65
5-Chloro-N-(2-chloro-4-(((6-hydroxypyridin-3-yl)methyl)-
amino)phenyl)-2-hydroxybenzamide (40). Compound 40 (120
mg, 88%) was prepared as a yellow solid according to general
procedure B, starting from compound 4 and 6-hydroxynicotinalde-
1
min). H NMR (300 MHz, CDCl3): δ 8.28 (s, 1H), 7.85 (d, J = 8.7
Hz, 1H), 7.54 (d, J = 2.4 Hz, 1H), 7.37 (dd, J = 9.0, 2.4 Hz, 1H), 6.96
(d, J = 8.7 Hz, 1H), 6.68 (d, J = 2.7 Hz, 1H), 6.55 (dd, J = 9.0, 2.7 Hz,
1H), 3.87−3.73 (m, 2H), 3.70−3.61 (m, 1H), 1.80−1.72 (m, 2H),
1.21 (d, J = 6.3 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 166.9, 160.2,
146.2, 134.5, 127.0, 125.5, 125.2, 123.9, 123.1, 120.4, 116.0, 113.4,
112.9, 60.6, 47.5, 39.3, 21.0. HRMS (ESI): calcd for C17H19Cl2N2O3,
369.0773 (M + H)+; found, 369.0767.
1
hyde. HPLC purity 98.6% (tR = 16.79 min). H NMR (300 MHz,
DMSO-d6): δ 12.20 (s, 1H), 11.43 (s, 1H), 10.41 (s, 1H), 8.00 (d, J =
2.7 Hz, 1H), 7.74 (d, J = 8.7 Hz, 1H), 7.46 (td, J = 9.3, 2.4 Hz, 2H),
7.33 (s, 1H), 7.02 (d, J = 8.7 Hz, 1H), 6.74 (d, J = 2.1 Hz, 1H), 6.62
(dd, J = 8.7, 2.1 Hz, 1H), 6.37−6.29 (m, 2H), 4.00 (d, J = 4.8 Hz,
2H). 13C NMR (75 MHz, DMSO-d6): δ 163.7, 162.0, 156.4, 146.9,
141.5, 133.2, 128.9, 126.8, 126.0, 123.0, 119.9, 119.1, 118.8, 115.7,
111.9, 111.5, 42.8. HRMS (ESI): calcd for C19H16Cl2N3O3, 404.0569
(M + H)+; found, 404.0567.
N-(4-(((5-Bromothiophen-2-yl)methyl)amino)-2-chloro-
phenyl)-5-chloro-2-hydroxybenzamide (41). Compound 41 (95
mg, 60%) was prepared as a yellow solid according to general
procedure B, starting from compound 4 and 5-bromothiophene-2-
carboxaldehyde. HPLC purity 99.5% (tR = 20.16 min). 1H NMR (300
MHz, DMSO-d6): δ 12.20 (s, 1H), 10.42 (s, 1H), 8.00 (d, J = 2.7 Hz,
1H), 7.75 (d, J = 8.7 Hz, 1H), 7.47 (dd, J = 8.7, 2.7 Hz, 1H), 7.07 (d,
J = 3.6 Hz, 1H), 7.02 (d, J = 8.7 Hz, 1H), 6.92 (d, J = 3.9 Hz, 1H),
6.77 (d, J = 2.4 Hz, 1H), 6.68−6.57 (m, 2H), 4.44 (d, J = 5.7 Hz,
2H). 13C NMR (75 MHz, DMSO-d6): δ 163.6, 156.4, 146.5, 146.2,
133.2, 130.0, 128.9, 126.7, 125.9, 125.8, 123.5, 123.1, 119.1, 118.9,
112.2, 111.8, 109.3, 42.0. HRMS (ESI): calcd for C18H14Cl2N2O2S,
470.9336 (M + H)+; found, 470.9334.
5-Chloro-N-(2-chloro-4-((3-hydroxypropyl)amino)phenyl)-
2-hydroxybenzamide (48). To a solution of 3-((tetrahydro-2H-
pyran-2-yl)oxy)propanal (260 mg, 1.64 mmol) and compound 4 (250
mg, 1.10 mmol) in 10 mL of MeOH was added AcOH (197 mg, 3.30
mmol) at 0 °C. The mixture was stirred at 0 °C for 30 min; then,
NaBH3CN (226 mg, 3.30 mmol) was added. The resulting mixture
was stirred at RT overnight. The pH of the mixture was adjusted to
9−10 with NaHCO3 (aq) at 0 °C. The mixture was extracted with
DCM (2 × 70 mL), dried (Na2SO4), and concentrated. The residue
was purified by column chromatography to give the product as a
mixture. The mixture was dissolved in 20 mL of MeOH, and p-TsOH
(30 mg, 0.17 mmol) was added. The resulting mixture was stirred at
RT for 12 h and then concentrated. The residue was purified by
column chromatography (DCM/MeOH = 20/1) to afford compound
48 (86 mg, 22% in two steps) as a yellow solid. HPLC purity 95.5%
1
(tR = 16.81 min). H NMR (300 MHz, CD3OD): δ 8.00 (d, J = 2.1
Hz, 1H), 7.76 (d, J = 8.7 Hz, 1H), 7.37 (dd, J = 8.7, 2.4 Hz, 1H), 6.95
(d, J = 8.7 Hz, 1H), 6.71 (d, J = 2.1 Hz, 1H), 6.59 (dd, J = 8.7, 2.1 Hz,
1H), 3.68 (t, J = 6.3 Hz, 2H), 3.17 (t, J = 6.9 Hz, 2H), 1.90−1.76 (m,
2H). 13C NMR (75 MHz, CD3OD): δ 166.2, 157.7, 149.3, 134.3,
130.4, 128.8, 127.1, 125.7, 124.5, 120.3, 119.7, 113.3, 112.6, 60.8,
41.6, 33.0. HRMS (ESI): calcd for C16H17Cl2N2O3, 355.0616 (M +
H)+; found, 355.0608.
N-(4-(((1H-Indazol-6-yl)methyl)amino)-2-chlorophenyl)-5-
chloro-2-hydroxybenzamide (42). Compound 42 (122 mg, 84%)
was prepared as a yellow solid according to general procedure B,
starting from compound 4 and 1H-indazole-6-carboxaldehyde. HPLC
1
purity 97.2% (tR = 18.05 min). H NMR (300 MHz, DMSO-d6): δ
12.95 (s, 1H), 12.20 (s, 1H), 10.39 (s, 1H), 8.05−7.95 (m, 2H), 7.71
(dd, J = 8.7, 2.7 Hz, 2H), 7.52−7.41 (m, 2H), 7.12 (d, J = 8.1 Hz,
1H), 7.01 (d, J = 8.7 Hz, 1H), 6.75−6.58 (m, 3H), 4.43 (d, J = 5.7
Hz, 2H). 13C NMR (75 MHz, DMSO-d6): δ 163.7, 156.4, 147.3,
140.2, 137.8, 133.3, 133.2, 128.9, 126.8, 126.1, 123.0, 122.8, 121.9,
120.4, 120.2, 119.1, 118.8, 111.8, 111.4, 107.7, 46.4. HRMS (ESI):
calcd for C21H17Cl2N4O2, 427.0729 (M + H)+; found, 427.0720.
5-Chloro-N-(2-chloro-4-((2-methyl-1-phenylpropan-2-yl)
((2-methyl-1-phenylpropan-2-yl)oxy)amino)phenyl)-2-hy-
droxybenzamide (43). Compound 43 was prepared by a procedure
similar to that used to prepare compound 15, starting from
niclosamide and 2-methyl-3-phenyl-1-propene. The title compound
(115 mg, 43%) was obtained as a yellow solid. HPLC purity 97.1% (tR
= 21.59 min). 1H NMR (300 MHz, CDCl3): δ 8.38 (s, 1H), 7.91 (d, J
= 9.0 Hz, 1H), 7.55 (d, J = 2.4 Hz, 1H), 7.36−7.16 (m, 10H), 7.08
(dd, J = 7.5, 1.5 Hz, 2H), 6.93 (d, J = 8.7 Hz, 1H), 6.79 (d, J = 2.7 Hz,
1H), 6.65 (dd, J = 9.0, 2.7 Hz, 1H), 2.93 (s, 2H), 2.74 (s, 2H), 1.30
(s, 6H), 1.21 (s, 6H). 13C NMR (75 MHz, CDCl3): δ 166.6, 159.9,
145.3, 137.8, 137.7, 134.3, 130.6 (2C), 130.5 (2C), 128.3 (2C), 128.1
(2C), 126.6, 126.5, 126.3, 125.7, 124.7, 123.9, 123.5, 120.2, 116.1,
116.0, 115.4, 71.0, 54.4, 49.8, 46.2, 29.2 (2C), 28.4 (2C). HRMS
(ESI): calcd for C33H35Cl2N2O3 577.2025 (M + H)+, found 577.2017.
5-Chloro-N-(2-chloro-4-((4-Hydroxy-2-methylbutan-2-yl)-
amino)phenyl)-2-hydroxybenzamide (46). Compound 46 was
prepared by a procedure similar to that used to prepare compound 15,
starting from niclosamide and 3-methyl-3-buten-1-ol. The title
compound (44 mg, 58%) was obtained as a yellow solid. HPLC
purity 95.6% (tR = 15.89 min). 1H NMR (300 MHz, CDCl3): δ 8.66
(s, 1H), 7.93 (d, J = 8.7 Hz, 1H), 7.61 (s, 1H), 7.35 (d, J = 8.4 Hz,
N-(4-(Bis(3-hydroxypropyl)amino)-2-chlorophenyl)-5-
chloro-2-hydroxybenzamide (49). Compound 49 was prepared
by a procedure the same as that used to prepare compound 48. The
title compound (34 mg, 8% in two steps) was obtained as a yellow
1
solid. HPLC purity 95.8% (tR = 16.79 min). H NMR (300 MHz,
CD3OD + CDCl3): δ 8.00 (d, J = 2.7 Hz, 1H), 7.85 (d, J = 9.0 Hz,
1H), 7.37 (dd, J = 9.0, 2.7 Hz, 1H), 6.95 (d, J = 8.7 Hz, 1H), 6.83 (d,
J = 3.0 Hz, 1H), 6.72 (dd, J = 9.0, 2.7 Hz, 1H), 3.63 (t, J = 6.0 Hz,
4H), 3.43 (t, J = 7.2 Hz, 4H), 1.87−1.73 (m, 4H). 13C NMR (75
MHz, CD3OD): δ 165.9, 157.6, 148.1, 134.3, 130.5, 128.8, 126.9,
125.7, 124.0, 120.4, 119.7, 113.2, 112.1, 60.5 (2C), 48.1 (2C), 31.0
(2C). HRMS (ESI): calcd for C19H23Cl2N2O4, 413.1035 (M + H)+;
found, 413.1028.
tert-Butyl (2-((3-chloro-4-(5-chloro-2-hydroxybenzamido)-
phenyl)amino)ethyl)carbamate (50). Compound 50 (100 mg,
67%) was prepared as a yellow solid according to general procedure B,
starting from compound 4 and N-Boc-2-aminoacetaldehyde. HPLC
purity 98.2% (tR = 17.72 min). 1H NMR (300 MHz, CDCl3): δ 11.85
(s, 1H), 8.32 (s, 1H), 7.83 (d, J = 8.7 Hz, 1H), 7.55 (d, J = 2.1 Hz,
1H), 7.36 (dd, J = 8.7, 2.4 Hz, 1H), 6.95 (d, J = 8.7 Hz, 1H), 6.61 (d,
J = 2.4 Hz, 1H), 6.51 (dd, J = 8.7, 2.7 Hz, 1H), 4.92 (s, 1H), 4.35 (br
s, 1H), 3.39−3.29 (m, 2H), 3.23−3.15 (m, 2H), 1.45 (s, 9H). 13C
NMR (75 MHz, CDCl3): δ 166.8, 160.0, 156.8, 146.8, 134.4, 127.1,
125.6, 125.3, 123.9, 123.1, 120.3, 116.0, 112.4, 112.0, 80.1, 44.7, 40.1,
28.5 (3C). HRMS (ESI): calcd for C20H24Cl2N3O4, 440.1144 (M +
H)+; found, 440.1146.
N-(4-((2-Aminoethyl)amino)-2-chlorophenyl)-5-chloro-2-
hydroxybenzamide Hydrochloride (51). To a solution of
P
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compound 50 (40 mg, 0.091 mmol) in 5 mL of DCM was added 1
mL of TFA at 0 °C. The resulting mixture was stirred at 0 °C for 2 h
and concentrated. Then, the residue was dissolved in 5 mL of MeOH,
and 1 mL of ammonium hydroxide was added. The mixture was
stirred at RT for 10 min and then concentrated. The residue was
purified through column chromatography on reverse-phase C-18 silica
gel (eluent, H2O (1% HCl)/CH3CN = 2/1 to 1/1) to afford
compound 51 (28 mg, 78%) as a yellow solid. HPLC purity 99.4% (tR
3.08 (m, 3H), 2.96−2.71 (m, 4H), 1.94−1.70 (m, 3H), 1.37−1.16
(m, 2H). 13C NMR (75 MHz, DMSO-d6): δ 167.1, 164.8, 145.5,
131.5, 128.4, 125.9, 124.2, 124.1, 122.2, 119.4, 114.2, 111.4, 111.1,
48.2, 43.4 (2C), 33.2, 27.3 (2C). HRMS (ESI): calcd for
C19H22Cl2N3O2, 394.1089 (M + H)+; found, 394.1085.
5-Chloro-N-(2-chloro-4-(((1-methylpiperidin-4-yl)methyl)-
amino)phenyl)-2-hydroxybenzamide (56). Compound 56 (85
mg, 61%) was prepared as a white solid according to general
procedure B, starting from compound 4 and N-methyl-piperidine-4-
carboxaldehyde. HPLC purity 96.0% (tR = 16.02 min). 1H NMR (300
MHz, DMSO-d6): δ 7.96 (d, J = 8.4 Hz, 1H), 7.88 (s, 1H), 7.35−7.21
(m, 1H), 6.90 (d, J = 8.7 Hz, 1H), 6.67 (s, 1H), 6.56 (d, J = 9.0 Hz,
1H), 5.87 (s, 1H), 3.04−2.82 (m, 4H), 2.35 (s, 3H), 2.27−2.12 (m,
2H), 1.85−1.70 (m, 2H), 1.57 (s, 1H), 1.34−1.14 (m, 2H). 13C NMR
(75 MHz, DMSO-d6): δ 163.8, 160.8, 146.7, 132.4, 128.8, 125.4,
124.9, 123.9, 120.3, 119.5, 119.4, 111.4, 110.9, 54.4 (2C), 48.3, 44.9,
33.6, 28.9 (2C). HRMS (ESI): calcd for C20H24Cl2N3O2, 408.1246
(M + H)+; found, 408.1236.
5-Chloro-N-(2-chloro-4-((2-morpholinoethyl)amino)-
phenyl)-2-hydroxybenzamide (57). To a solution of 2-chloro-4-
fluoronitrobenzene (400 mg, 2.28 mmol) and 2-morpholinoethan-
amine (355 mg, 2.73 mmol) in 10 mL of DMF was added K2CO3
(630 mg, 4.56 mmol). The resulting mixture was stirred at 100 °C for
1 h. Then, the mixture was cooled to RT and poured into 50 mL of
H2O. The yellow precipitate was isolated by filtration and dried to
afford 3-chloro-N-(2-morpholinoethyl)-4-nitroaniline 59c (570 mg,
87%). 1H NMR (300 MHz, CDCl3): δ 7.95 (d, J = 9.0 Hz, 1H), 6.57
(d, J = 2.7 Hz, 1H), 6.44 (dd, J = 9.0, 2.7 Hz, 1H), 5.25 (t, J = 4.5 Hz,
1H), 3.71 (t, J = 4.5 Hz, 4H), 3.26−3.13 (m, 2H), 2.68−2.58 (m,
2H), 2.52−2.40 (m, 4H). 13C NMR (75 MHz, CDCl3): δ 152.4,
136.0, 130.8, 129.0, 113.7, 110.3, 66.9 (2C), 56.3, 53.2 (2C), 39.1.
To a solution of compound 59c (570 mg, 1.99 mmol) in 20 mL of
MeOH was added 10 mL of saturated NH4Cl (aq). Zinc dust (648
mg, 9.97 mmol) was added to the solution at 0 °C. The reaction was
stirred at RT for 16 h. TLC indicated that the starting material was
gone. EtOAc (200 mL) was added to the solution. The zinc solid was
filtered, and the filtrate was washed with 30 mL of brine, dried
(Na2SO4), and concentrated under vacuum. The residue was purified
by column chromatography to afford 3-chloro-N1-(2-
morpholinoethyl)benzene-1,4-diamine 60c (450 mg, 88%) as a
yellow solid.
To a solution of compound 60c (88 mg, 0.34 mmol), 5-chloro-2-
methoxybenzoic acid (71 mg, 0.38 mmol), and DMAP (10 mg, 0.08
mmol) in 10 mL of DCM was added EDCI (130 mg, 0.68 mmol) at 0
°C. The resulting mixture was stirred at RT overnight and then
concentrated. The residue was purified by column chromatography
(DCM/MeOH = 100/1 to 30/1) to afford the intermediate 5-chloro-
N-(2-chloro-4-((2-morpholinoethyl)amino)phenyl)-2-methoxybenza-
mide (130 mg, 89%) as a yellow solid. 1H NMR (300 MHz CDCl3):
δ 10.24 (s, 1H), 8.36 (d, J = 8.7 Hz, 1H), 8.26 (d, J = 2.7 Hz, 1H),
7.41 (dd, J = 8.7, 2.7 Hz, 1H), 6.96 (d, J = 8.7 Hz, 1H), 6.67 (d, J =
2.7 Hz, 1H), 6.58 (dd, J = 9.0, 2.7 Hz, 1H), 4.34 (br s, 1H), 4.05 (s,
3H), 3.72 (t, J = 4.5 Hz, 4H), 3.14 (t, J = 6.0 Hz, 2H), 2.62 (t, J = 6.0
Hz, 2H), 2.47 (t, J = 4.5 Hz, 4H). 13C NMR (75 MHz, CDCl3): δ
161.4, 155.9, 145.8, 132.7, 132.2, 127.0, 126.0, 124.5, 123.6, 123.4,
113.1, 112.7, 112.4, 67.1 (2C), 57.1, 56.7, 53.5 (2C), 40.1.
To a solution of the intermediate 5-chloro-N-(2-chloro-4-((2-
morpholinoethyl)amino)phenyl)-2-methoxybenzamide (130 mg, 0.38
mmol) in DCM (15 mL) was added BBr3 (1.53 mL, 1.53 mmol, 1 M
in DCM) dropwise at 0 °C. The mixture was stirred at RT for 2 h.
The mixture was diluted with DCM, washed with H2O and brine,
dried (Na2SO4), and concentrated. The residue was purified by
preparative TLC developed by 6% MeOH in DCM to afford
compound 57 (97 mg, 77%) as a yellow solid. HPLC purity 98.8% (tR
= 15.54 min). 1H NMR (300 MHz, CDCl3): δ 8.30 (s, 1H), 7.86 (d, J
= 8.7 Hz, 1H), 7.54 (d, J = 2.4 Hz, 1H), 7.36 (dd, J = 9.0, 2.4 Hz,
1H), 6.95 (d, J = 9.0 Hz, 1H), 6.66 (d, J = 2.4 Hz, 1H), 6.55 (dd, J =
8.7, 2.4 Hz, 1H), 3.73 (t, J = 4.5 Hz, 4H), 3.15 (t, J = 6.0 Hz, 2H),
2.64 (t, J = 6.0 Hz, 2H), 2.49 (t, J = 4.5 Hz, 4H). 13C NMR (75 MHz,
CDCl3): δ 166.8, 160.0, 147.0, 134.4, 127.0, 125.6, 125.2, 123.8,
1
= 14.84 min). H NMR (300 MHz, CD3OD): δ 7.96 (d, J = 2.4 Hz,
1H), 7.81 (d, J = 9.0 Hz, 1H), 7.42 (dd, J = 8.7, 2.7 Hz, 1H), 7.02 (d,
J = 8.7 Hz, 1H), 6.87 (d, J = 2.4 Hz, 1H), 6.73 (dd, J = 8.7, 2.4 Hz,
1H), 3.47 (t, J = 6.0 Hz, 2H), 3.19 (t, J = 6.0 Hz, 2H). 13C NMR (75
MHz, CD3OD): δ 165.9, 156.9, 147.6, 134.6, 130.5, 128.7, 127.0,
125.8, 125.6, 120.2, 119.7, 114.2, 113.2, 41.8, 39.6. HRMS (ESI):
calcd for C15H16Cl2N3O2, 340.0620 (M − Cl)+; found, 340.0610.
tert-Butyl 4-((3-chloro-4-(5-chloro-2-hydroxybenzamido)-
phenyl)amino)piperidine-1-carboxylate (52). Compound 52
(305 mg, 72%) was prepared as an off-white solid according to
general procedure B, starting from compound 4 and 1-tert-
butoxycarbonylpiperidin-4-one. HPLC purity 99.5% (tR = 18.48
min). 1H NMR (300 MHz, DMSO-d6): δ 11.53 (br s, 1H), 7.99−7.85
(m, 2H), 7.32 (dd, J = 8.7, 2.7 Hz, 1H), 6.94 (d, J = 8.7 Hz, 1H), 6.72
(d, J = 2.1 Hz, 1H), 6.59 (dd, J = 9.0, 2.1 Hz, 1H), 5.71 (d, J = 8.1 Hz,
1H), 3.92−3.80 (m, 2H), 3.48−3.36 (m, 1H), 3.00−2.84 (m, 2H),
1.98−1.78 (m, 2H), 1.40 (s, 9H), 1.29−1.14 (m, 2H). 13C NMR (75
MHz, DMSO-d6): δ 163.7, 159.7, 153.9, 145.5, 132.5, 128.8, 125.6,
125.1, 123.8, 120.4, 120.0, 119.3, 112.0, 111.5, 78.5, 48.6, 42.2 (2C),
31.5 (2C), 28.1 (3C). HRMS (ESI): calcd for C23H28Cl2N3O4,
480.1457 (M + H)+; found, 480.1450.
5-Chloro-N-(2-chloro-4-(piperidin-4-ylamino)phenyl)-2-hy-
droxybenzamide Hydrochloride (53). To a solution of compound
52 (245 mg, 0.51 mmol) in 5 mL of MeOH was added 4 M HCl (5
mL) at 0 °C. The resulting mixture was stirred at RT for 48 h. Then,
most of MeOH was evaporated, and compound 53 (185 mg, 87%)
was isolated as a pale yellow solid by filtration. HPLC purity 99.8% (tR
1
= 15.40 min). H NMR (300 MHz, DMSO-d6): δ 12.22 (s, 1H),
10.42 (s, 1H), 9.10−8.80 (m, 2H), 8.01 (d, J = 2.4 Hz, 1H), 7.80 (d, J
= 9.0 Hz, 1H), 7.46 (dd, J = 8.7, 2.4 Hz, 1H), 7.09 (d, J = 9.0 Hz,
1H), 6.86 (d, J = 2.4 Hz, 1H), 6.71 (dd, J = 8.7, 2.1 Hz, 1H), 3.64−
3.50 (m, 1H), 3.36−3.22 (m, 2H), 3.08−2.90 (m, 2H), 2.13−1.97
(m, 2H), 1.74−1.56 (m, 2H). 13C NMR (75 MHz, DMSO-d6): δ
163.5, 156.3, 144.6, 133.1, 129.0, 126.5, 125.7, 124.1, 123.1, 119.1,
119.0, 113.1, 112.6, 47.1, 41.9 (2C), 27.9 (2C). HRMS (ESI): calcd
for C18H20Cl2N3O2, 380.0933 (M − Cl)+; found, 380.0922.
tert-Butyl 4-(((3-chloro-4-(5-chloro-2-hydroxybenzamido)-
phenyl)amino)methyl)piperidine-1-carboxylate (54). Com-
pound 54 (120 mg, 72%) was prepared as a yellow solid according
to general procedure B, starting from compound 4 and N-Boc-4-
1
piperidinecarboxaldehyde. HPLC purity 97.0% (tR = 20.32 min). H
NMR (300 MHz, DMSO-d6): δ 12.22 (s, 1H), 10.39 (s, 1H), 8.01 (d,
J = 2.7 Hz, 1H), 7.70 (d, J = 8.7 Hz, 1H), 7.47 (dd, J = 8.7, 2.7 Hz,
1H), 7.02 (d, J = 9.0 Hz, 1H), 6.70 (d, J = 2.4 Hz, 1H), 6.58 (dd, J =
8.7, 2.4 Hz, 1H), 6.00 (t, J = 5.7 Hz, 1H), 4.02−3.88 (m, 2H), 2.91
(d, J = 5.4 Hz, 2H), 2.79−2.58 (m, 2H), 1.79−1.63 (m, 3H), 1.39 (s,
9H), 1.14−0.95 (m, 2H). 13C NMR (75 MHz, DMSO-d6): δ 163.8,
156.5, 153.8, 147.7, 133.1, 128.8, 127.0, 126.2, 123.0, 122.3, 119.1,
118.8, 111.3, 110.9, 78.4, 48.3 (2C), 43.2, 35.0 (2C), 29.6, 28.1 (3C).
HRMS (ESI): calcd for C24H30Cl2N3O4, 494.1613 (M + H)+; found,
494.1602.
5-Chloro-N-(2-chloro-4-((piperidin-4-ylmethyl)amino)-
phenyl)-2-hydroxybenzamide (55). To a solution of compound
54 (60 mg, 0.12 mmol) in 5 mL of DCM was added 1 mL of TFA at
0 °C. The resulting mixture was stirred at 0 °C for 2 h and
concentrated. Then, the residue was dissolved in 5 mL of MeOH, and
1 mL of ammonium hydroxide was added. The mixture was stirred at
RT for 10 min. The yellow solid was isolated by filtration to afford
compound 55 (38 mg, 79%). HPLC purity 96.3% (tR = 15.61 min).
1H NMR (300 MHz, DMSO-d6): δ 13.83 (s, 1H), 8.12 (d, J = 9.0 Hz,
1H), 7.69 (d, J = 3.0 Hz, 1H), 7.00 (dd, J = 8.7, 3.0 Hz, 1H), 6.63 (d,
J = 1.5 Hz, 1H), 6.57−6.45 (m, 2H), 5.74 (t, J = 5.1 Hz, 1H), 3.35−
Q
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122.9, 120.3, 116.0, 112.6, 112.1, 66.9 (2C), 56.9, 53.3 (2C), 39.8.
HRMS (ESI): calcd for C19H22Cl2N3O3, 410.1038 (M + H)+; found,
410.1028.
162.8, 155.3, 145.1, 133.5, 132.5, 130.7, 125.8, 124.3, 123.4, 121.3,
112.8, 112.5, 111.4, 56.2, 54.8, 33.4 (2C), 24.1 (2C), 20.4.
To
a solution of the intermediate N-(2-chloro-4-
N-(2-Chloro-4-(cyclopentylamino)phenyl)-2-hydroxybenza-
mide (63). To a solution of 2-chloro-4-fluoronitrobenzene (760 mg,
4.3 mmol) and cyclopentylamine (442 mg, 5.2 mmol) in 10 mL of
DMF was added K2CO3 (1.2 g, 8.6 mmol). The resulting mixture was
stirred at 100 °C for 1 h. Then, the mixture was cooled to RT and
poured into 50 mL of H2O. The mixture was extracted with EtOAc (2
× 120 mL), washed with H2O (3 × 30 mL) and brine (30 mL), dried
(Na2SO4), and concentrated. The residue was purified by column
chromatography (Hex/EtOAc = 6/1 to 3/1) to afford 3-chloro-N-
(cyclopentylamino)phenyl)-2-methoxy-5-methylbenzamide (190 mg,
0.51 mmol) in DCM (20 mL) was added BBr3 (2.5 mL, 2.5 mmol, 1
M in DCM) dropwise at 0 °C. The mixture was stirred at RT for 2 h.
The mixture was diluted with DCM, washed with H2O and brine,
dried (Na2SO4), and concentrated. The residue was purified by
preparative TLC to afford compound 64 (170 mg, 96%) as a yellow
1
solid. HPLC purity 96.4% (tR = 19.65 min). H NMR (300 MHz,
CDCl3): δ 11.83 (s, 1H), 8.14 (s, 1H), 7.94 (d, J = 8.7 Hz, 1H), 7.29
(s, 1H), 7.28−7.22 (m, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.65 (d, J = 2.4
Hz, 1H), 6.54 (dd, J = 9.0, 2.7 Hz, 1H), 3.82−3.66 (m, 2H), 2.34 (s,
3H), 2.10−1.96 (m, 2H), 1.80−1.59 (m, 4H), 1.53−1.40 (m, 2H).
13C NMR (75 MHz, CD3OD + CDCl3): δ 167.1, 156.5, 147.3, 134.9,
129.6, 129.3, 127.9, 126.1, 124.2, 117.4, 117.2, 113.4, 112.7, 55.1, 33.5
(2C), 24.4 (2C), 20.6. HRMS (ESI): calcd for C19H22ClN2O2,
345.1370 (M + H)+; found, 345.1364.
1
cyclopentyl-4-nitroaniline 59a (1.01 g, 96%) as yellow oil. H NMR
(300 MHz, CDCl3): δ 7.97 (d, J = 9.3 Hz, 1H), 6.57 (d, J = 2.7 Hz,
1H), 6.42 (dd, J = 9.13, 2.7 Hz, 1H), 4.59 (d, J = 4.8 Hz, 1H), 3.87−
3.75 (m, 1H), 2.15−1.97 (m, 2H), 1.82−1.57 (m, 4H), 1.58−1.41
(m, 2H). 13C NMR (75 MHz, CDCl3): δ 152.2, 135.8, 130.9, 129.1,
113.9, 110.6, 54.5, 33.4 (2C), 24.0 (2C).
To a solution of compound 59a (1.01 g, 4.2 mmol) in 20 mL of
MeOH was added 10 mL of saturated NH4Cl (aq). Zinc dust (1.36 g,
21.0 mmol) was added into the solution at 0 °C. The reaction was
stirred at RT for 16 h. TLC indicated that the starting material was
gone. EtOAc (200 mL) was added to the solution. The zinc solid was
filtered, and the filtrate was washed with 30 mL of brine, dried
(Na2SO4), and concentrated under vacuum. The residue was purified
by column chromatography to afford 3-chloro-N1-cyclopentylben-
3-Chloro-N-(2-chloro-4-(cyclopentylamino)phenyl)-
benzamide (65). To a solution of compound 60a (97 mg, 0.43
mmol) and 3-chlorobenzoic acid (67 mg, 0.43 mmol) in 20 mL of
DCM were added DIEA (111 mg, 0.86 mmol) and HBTU (326 mg,
0.86 mmol) successively at 0 °C. The resulting mixture was stirred at
RT overnight. The mixture was concentrated and purified by column
chromatography (Hex/EtOAc = 6/1 to 3/1) to give compound 65
(145 mg, 96%) as a purple solid. HPLC purity 99.2% (tR = 19.58
1
1
zene-1,4-diamine 60a (800 mg, 90%) as brown oil. H NMR (300
min). H NMR (300 MHz, CDCl3): δ 8.09−7.99 (m, 2H), 7.87 (t, J
MHz, chloroform-d): δ 6.64 (d, J = 8.7 Hz, 1H), 6.58 (d, J = 2.7 Hz,
1H), 6.41 (dd, J = 8.7, 2.7 Hz, 1H), 3.75−3.46 (m, 3H), 3.30 (s, 1H),
2.07−1.89 (m, 2H), 1.79−1.51 (m, 4H), 1.51−1.33 (m, 2H). 13C
NMR (75 MHz, CDCl3): δ 141.8, 134.0, 120.8, 117.7, 114.3, 114.2,
55.6, 33.6 (C), 24.2 (2C).
= 1.5 Hz, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.53−7.47 (m, 1H), 7.40 (t, J
= 7.8 Hz, 1H), 6.62 (d, J = 2.7 Hz, 1H), 6.51 (dd, J = 9.0, 2.4 Hz,
1H), 4.02 (s, 1H), 3.79−3.66 (m, 1H), 2.09−1.93 (m, 2H), 1.78−
1.54 (m, 4H), 1.52−1.35 (m, 2H). 13C NMR (75 MHz, CDCl3): δ
163.9, 146.1, 136.8, 135.1, 131.9, 130.1, 127.6, 125.7, 125.0, 124.1,
123.9, 112.7, 112.6, 54.8, 33.5 (2C), 24.1 (2C). HRMS (ESI): calcd
for C18H19Cl2N2O, 349.0874 (M + H)+; found, 349.0874.
To a solution of 2-acetoxybenzoic acid (163 mg, 0.90 mmol) and
N,N-diisopropylethylamine (DIEA, 85 mg, 0.66 mmol) in 20 mL of
DCM was added 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluro-
nium hexafluorophosphate (HBTU, 686 mg, 1.80 mmol) at 0 °C. The
resulting mixture was stirred at 0 °C for 30 min; then, compound 60a
(153 mg, 0.73 mmol) was added. The mixture was stirred at RT for 2
h and then concentrated. The residue was purified by column
chromatography (Hex/EtOAc = 6/1 to 3/1) to afford the
intermediate. The intermediate was dissolved in 10 mL of MeOH,
and NaOH (145 mg, 3.62 mmol) in 8 mL of H2O was added at 0 °C.
The resulting mixture was stirred at RT for 1 h. Then, the pH of the
mixture was adjusted to 6−7 with 2 M HCl (aq). The mixture was
extracted with EA (2 × 80 mL), dried (Na2SO4), and concentrated.
The residue was purified by column chromatography (Hex/EtOAc =
8/1 to 5/1) to afford compound 63 (85 mg, 35% in two steps) as a
5-Chloro-N-(2-chloro-4-(cyclopentylamino)phenyl)-2-me-
thoxybenzamide (66). Compound 66 was prepared by a procedure
similar to that used to prepare compound 65, starting from compound
60a and 5-chloro-2-methoxybenzoic acid. The title compound (80
mg, 89%) was obtained as a yellow solid. HPLC purity 98.1% (tR =
21.77 min). 1H NMR (300 MHz, CDCl3): δ 10.21 (s, 1H), 8.33 (d, J
= 9.0 Hz, 1H), 8.25 (d, J = 2.7 Hz, 1H), 7.38 (dd, J = 8.7, 2.7 Hz,
1H), 6.93 (d, J = 8.7 Hz, 1H), 6.62 (d, J = 2.4 Hz, 1H), 6.52 (dd, J =
9.0, 2.7 Hz, 1H), 4.02 (s, 3H), 3.79−3.62 (m, 2H), 2.10−1.93 (m,
2H), 1.78−1.54 (m, 4H), 1.50−1.37 (m, 2H). 13C NMR (75 MHz,
CDCl3): δ 161.3, 155.8, 145.4, 132.6, 132.1, 126.9, 125.5, 124.5,
123.5, 123.3, 113.0, 112.8, 112.6, 56.7, 54.9, 33.6 (2C), 24.1 (2C).
HRMS (ESI): calcd for C19H21Cl2N2O2, 379.0980 (M + H)+; found,
379.0970.
1
gray solid. HPLC purity 99.7% (tR = 19.01 min). H NMR (300
MHz, CDCl3): δ 12.07 (s, 1H), 8.20 (s, 1H), 7.94 (d, J = 9.0 Hz,
1H), 7.54 (dd, J = 8.1, 1.5 Hz, 1H), 7.48−7.39 (m, 1H), 7.05−6.99
(m, 1H), 6.96−6.89 (m, 1H), 6.63 (d, J = 2.4 Hz, 1H), 6.53 (dd, J =
9.0, 2.7 Hz, 1H), 3.86−3.66 (m, 2H), 2.10−1.95 (m, 2H), 1.80−1.55
(m, 4H), 1.52−1.38 (m, 2H). 13C NMR (75 MHz, CDCl3): δ 167.9,
161.8, 146.4, 134.6, 126.4, 125.5, 124.7, 122.9, 119.1, 118.9, 114.8,
112.7, 112.5, 54.8, 33.5 (2C), 24.1 (2C). HRMS (ESI): calcd for
C18H20ClN2O2, 331.1213 (M + H)+; found, 331.1204.
N-(2-Chloro-4-(cyclopentylamino)phenyl)-2-Hydroxy-5-
methylbenzamide (64). To a solution of compound 60a (127 mg,
0.60 mmol) and 2-methoxy-5-methylbenzoic acid (100 mg, 0.60
mmol) in 20 mL of DCM was added DIEA (156 mg, 1.20 mmol) and
HBTU (388 mg, 1.20 mmol) successively at 0 °C. The resulting
mixture was stirred at RT overnight and then concentrated. The
residue was purified by column chromatography (Hex/EtOAc = 5/1)
to afford the intermediate N-(2-chloro-4-(cyclopentylamino)phenyl)-
2-methoxy-5-methylbenzamide (190 mg, 88%) as yellow oil. 1H NMR
(300 MHz, CDCl3): δ 10.32 (s, 1H), 8.38 (d, J = 8.7 Hz, 1H), 8.09
(d, J = 2.1 Hz, 1H), 7.23 (dd, J = 8.1, 2.1 Hz, 1H), 6.87 (d, J = 8.4 Hz,
1H), 6.63 (d, J = 2.7 Hz, 1H), 6.53 (dd, J = 9.0, 2.7 Hz, 1H), 3.98 (s,
3H), 3.78−3.67 (m, 2H), 2.33 (s, 3H), 2.06−1.93 (m, 2H), 1.77−
1.53 (m, 4H), 1.50−1.37 (m, 2H). 13C NMR (75 MHz, CDCl3): δ
5-Chloro-N-(2-chloro-4-(cyclopentylamino)phenyl)-2-((1-
methylpiperidin-4-yl)oxy)benzamide (67). To a solution of
compound 60a (70 mg, 0.33 mmol) and 5-chloro-2-((1-methylpiper-
idin-4-yl)oxy)benzoic acid (90 mg, 0.33 mmol) in 20 mL of DCM
were added DIEA (85 mg, 0.66 mmol) and HBTU (250 mg, 0.66
mmol) successively at 0 °C. The resulting mixture was stirred at RT
overnight and then concentrated. The residue was purified by column
chromatography (DCM/MeOH = 10:1 to DCM/MeOH (7 M NH3
in MeOH) = 10:1) to afford compound 67 (150 mg, 98%) as a yellow
1
solid. HPLC purity 99.4% (tR = 15.54 min). H NMR (300 MHz,
CDCl3): δ 9.74 (s, 1H), 8.20 (d, J = 2.7 Hz, 1H), 8.06 (d, J = 9.0 Hz,
1H), 7.33 (d, J = 8.7, 2.7 Hz, 1H), 6.94 (d, J = 9.0 Hz, 1H), 6.59 (d, J
= 2.7 Hz, 1H), 6.50 (dd, J = 9.0, 2.7 Hz, 1H), 4.50−4.37 (m, 1H),
3.82−3.60 (m, 2H), 2.79−2.66 (m, 2H), 2.23 (s, 3H), 2.21−1.88 (m,
8H), 1.76−1.52 (m, 4H), 1.50−1.34 (m, 2H). 13C NMR (75 MHz,
CDCl3): δ 161.9, 153.9, 145.8, 132.4, 132.2, 126.8, 125.6, 124.8,
124.6, 124.5, 115.7, 112.7, 112.4, 76.1, 54.7, 53.2 (2C), 46.0, 33.46
(2C), 31.2 (2C), 24.06 (2C). HRMS (ESI): calcd for
C24H30Cl2N3O2, 462.1715 (M + H)+; found, 462.1708.
5-Chloro-N-(2-chloro-4-(cyclopentylamino)phenyl)-2-
(methylsulfonamido)benzamide (68). Compound 68 was pre-
pared by a procedure similar to that used to prepare compound 65,
R
https://dx.doi.org/10.1021/acs.jmedchem.0c01226
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Article
starting from compound 60a and 5-chloro-2-(methylsulfonamido)-
115.7, 113.1 (d, J = 11.6 Hz), 109.0 (d, J = 2.5 Hz), 99.6 (d, J = 23.3
Hz), 54.8, 33.5 (2C), 24.1 (2C). HRMS (ESI): calcd for
C18H19ClFN2O2, 349.1119 (M + H)+; found, 349.1111.
benzoic acid. The title compound (90 mg, 85%) was obtained as a
1
pale yellow solid. HPLC purity 99.4% (tR = 19.42 min). H NMR
(300 MHz, CDCl3): δ 10.25 (s, 1H), 7.98 (s, 1H), 7.88 (d, J = 8.7
Hz, 1H), 7.76 (d, J = 8.7 Hz, 1H), 7.64 (d, J = 2.4 Hz, 1H), 7.50 (dd,
J = 8.7, 2.4 Hz, 1H), 6.65 (d, J = 2.7 Hz, 1H), 6.54 (dd, J = 8.7, 2.7
Hz, 1H), 3.84−3.68 (m, 2H), 3.03 (s, 3H), 2.1−1.96 (m, 2H), 1.80−
1.58 (m, 4H), 1.53−1.39 (m, 2H). 13C NMR (75 MHz, CDCl3): δ
165.2, 147.0, 138.0, 133.1, 129.1, 127.1, 127.1, 125.1, 122.5, 122.3,
121.8, 112.6, 112.3, 54.7, 40.1, 33.5 (2C), 24.1 (2C). HRMS (ESI):
calcd for C19H22Cl2N3O3S, 442.0759 (M + H)+; found, 442.0751.
The amino intermediates 62a−g were prepared by a procedure
similar to that used to prepare compound 4, starting from the known
nitro derivatives 61a−g.
N-(4-Aminophenyl)-5-chloro-2-hydroxybenzamide 62a. 165
mg, 57%. Yellow solid. 1H NMR (300 MHz, CDCl3): δ 12.06 (s, 1H),
7.71 (s, 1H), 7.46 (d, J = 2.1 Hz, 1H), 7.37 (dd, J = 9.0, 2.4 Hz, 1H),
7.31 (d, J = 8.7 Hz, 2H), 6.97 (d, J = 9.0 Hz, 1H), 6.71 (d, J = 8.7 Hz,
2H), 3.71 (s, 2H).
N-(4-Amino-2-fluorophenyl)-5-chloro-2-hydroxybenzamide
62b. 67 mg, 82%. Yellow solid. 1H NMR (300 MHz, CDCl3): δ 11.90
(s, 1H), 7.85−7.74 (m, 2H), 7.48 (d, J = 2.4 Hz, 1H), 7.39 (dd, J =
9.0, 2.4 Hz, 1H), 6.98 (d, J = 8.7 Hz, 1H), 6.54−6.45 (m, 2H), 3.80
(s, 2H).
N-(4-Amino-3-chlorophenyl)-5-chloro-2-hydroxybenza-
mide 62c. 63 mg, 78%. Yellow solid. 1H NMR (300 MHz, CDCl3): δ
11.91 (s, 1H), 7.69 (s, 1H), 7.56 (d, J = 2.4 Hz, 1H), 7.45 (d, J = 2.4
Hz, 1H), 7.38 (dd, J = 9.0, 2.4 Hz, 1H), 7.20 (dd, J = 8.7, 2.4 Hz,
1H), 6.98 (d, J = 9.0 Hz, 1H), 6.79 (d, J = 8.7 Hz, 1H), 4.08 (s, 2H).
N-(5-Amino-2-fluorophenyl)-5-chloro-2-hydroxybenzamide
62d. 66 mg, 81%. Yellow solid. 1H NMR (300 MHz, CDCl3): δ 11.72
(s, 1H), 8.03 (s, 1H), 7.70 (dd, J = 6.6, 2.7 Hz, 1H), 7.48 (d, J = 2.4
Hz, 1H), 7.41 (dd, J = 8.7, 2.4 Hz, 1H), 7.03−6.91 (m, 2H), 6.46−
6.39 (m, 1H), 3.66 (s, 2H).
5-Chloro-N-(3-chloro-4-(cyclopentylamino)phenyl)-2-hy-
droxybenzamide (71). 78 mg, 91%. Yellow solid. HPLC purity
1
95.0% (tR = 21.36 min). H NMR (300 MHz, CDCl3): δ 11.98 (s,
1H), 7.76 (s, 1H), 7.51 (d, J = 2.1 Hz, 1H), 7.46 (d, J = 2.1 Hz, 1H),
7.37 (dd, J = 8.7, 2.1 Hz, 1H), 7.22 (dd, J = 8.7, 2.1 Hz, 1H), 6.96 (d,
J = 9.0 Hz, 1H), 6.67 (d, J = 9.0 Hz, 1H), 4.28 (s, 1H), 3.80 (s, 1H),
2.11−1.98 (m, 2H), 1.84−1.72 (m, 2H), 1.72−1.62 (m, 2H), 1.58−
1.46 (m, 2H). 13C NMR (75 MHz, CDCl3): δ 167.3, 160.4, 142.3,
134.4, 125.2, 125.2, 123.7, 123.7, 122.2, 120.5, 118.9, 115.6, 111.7,
54.7, 33.6 (2C), 24.2 (2C). HRMS (ESI): calcd for C18H19Cl2N2O2,
365.0824 (M + H)+; found, 365.0815.
5-Chloro-N-(5-(cyclopentylamino)-2-fluorophenyl)-2-hy-
droxybenzamide (72). 68 mg, 83%. Yellow solid. HPLC purity
1
97.7% (tR = 20.13 min). H NMR (300 MHz, CDCl3): δ 8.11 (s,
1H), 7.56 (dd, J = 6.6, 2.7 Hz, 1H), 7.49 (d, J = 2.4 Hz, 1H), 7.38
(dd, J = 9.0, 2.4 Hz, 1H), 7.00−6.89 (m, 2H), 6.35−6.27 (m, 1H),
3.80−3.66 (m, 1H), 2.10−1.96 (m, 2H), 1.81−1.57 (m, 4H), 1.52−
1.38 (m, 2H). 13C NMR (75 MHz, CDCl3): δ 166.9 (d, J = 20.9 Hz),
160.2, 146.0 (d, J = 231.8 Hz), 145.0 (d, J = 1.7 Hz), 134.7, 125.4,
125.4 (d, J = 10.1 Hz), 123.9, 120.4, 115.7 (d, J = 24.4 Hz), 115.2,
109.2 (d, J = 6.8 Hz), 106.9, 55.2, 33.6 (2C), 24.2 (2C). HRMS
(ESI): calcd for C18H19ClFN2O2, 349.1119 (M + H)+; found,
349.1108.
5-Chloro-N-(2-chloro-4-(cyclopentylamino)benzyl)-2-hy-
droxybenzamide (73). 74 mg, 67%. Pale yellow solid. HPLC purity
1
99.2% (tR = 20.56 min). H NMR (300 MHz, CDCl3): δ 12.27 (s,
1H), 7.36−7.23 (m, 2H), 7.17 (d, J = 8.4 Hz, 1H), 6.91 (d, J = 9.3
Hz, 1H), 6.64−6.54 (m, 2H), 6.45 (dd, J = 8.4, 2.4 Hz, 1H), 4.56 (d, J
= 5.4 Hz, 2H), 3.85−3.68 (m, 2H), 2.08−1.95 (m, 2H), 1.78−1.56
(m, 4H), 1.51−1.37 (m, 2H). 13C NMR (75 MHz, CDCl3): δ 168.6,
160.2, 149.1, 134.9, 134.1, 131.8, 125.2, 123.4, 121.9, 120.1, 115.4,
113.3, 112.0, 54.6, 41.7, 33.5 (2C), 24.1 (2C). HRMS (ESI): calcd for
C19H21Cl2N2O2, 379.0980 (M + H)+; found, 379.0971.
N-(4-Amino-2-chlorobenzyl)-5-chloro-2-hydroxybenzamide
62e. 90 mg, 76%. Yellow solid. 1H NMR (300 MHz, CDCl3): δ 12.20
(br s, 1H), 7.33−7.27 (m, 2H), 7.16 (d, J = 8.1 Hz, 1H), 6.94−6.86
(m, 1H), 6.74−6.64 (m, 2H), 6.51 (dd, J = 8.1, 2.4 Hz, 1H), 4.56 (d,
J = 5.7 Hz, 2H), 3.79 (s, 2H).
2 - ( 5 - C h lo r o - 2 - hy d r o x y p h e n y l ) -N- (2 - c h l o r o - 4 -
(cyclopentylamino)phenyl)acetamide (74). 72 mg, 84%. Gray
1
solid. HPLC purity 99.3% (tR = 17.47 min). H NMR (300 MHz,
N-(4-Amino-2-chlorophenyl)-2-(5-chloro-2-hydroxyphenyl)-
CD3OD + CDCl3): δ 7.55 (d, J = 8.7 Hz, 1H), 7.18 (s, 1H), 7.06 (d, J
= 8.4 Hz, 1H), 6.79 (d, J = 8.7 Hz, 1H), 6.57 (s, 1H), 6.47 (d, J = 8.7
Hz, 1H), 3.74−3.54 (m, 3H), 2.01−1.86 (m, 2H), 1.74−1.51 (m,
4H), 1.49−1.33 (m, 2H). 13C NMR (75 MHz, CD3OD + CDCl3): δ
171.5, 154.5, 147.6, 131.1, 129.0, 127.6, 126.0, 125.0, 124.1, 124.1,
117.2, 113.4, 112.8, 55.2, 39.6, 33.6 (2C), 24.5 (2C). HRMS (ESI):
calcd for C19H21Cl2N2O2, 379.0980 (M + H)+; found, 379.0970.
(S)-5-Chloro-N-(1-((2-chloro-4-(cyclopentylamino)phenyl)-
amino)-3-methyl-1-oxobutan-2-yl)-2-hydroxybenzamide (75).
1
acetamide 62f. 180 mg, 99%. Yellow solid. H NMR (300 MHz,
CD3OD): δ 7.40 (dd, J = 8.7, 2.7 Hz, 1H), 7.26−7.19 (m, 1H), 7.13−
7.03 (m, 1H), 6.81 (dd, J = 8.7, 2.7 Hz, 1H), 6.76−6.69 (m, 1H),
6.61−6.53 (m, 1H), 3.70−3.63 (m, 2H).
(S)-N-(1-((4-Amino-2-chlorophenyl)amino)-3-methyl-1-oxo-
butan-2-yl)-5-chloro-2-hydroxybenzamide 62g. 127 mg, 80%.
1
Yellow solid. H NMR (300 MHz, CD3OD): δ 7.94 (d, J = 2.4 Hz,
1H), 7.36 (dd, J = 9.0, 2.4 Hz, 1H), 7.21 (d, J = 8.7 Hz, 1H), 6.93 (d,
J = 8.7 Hz, 1H), 6.76 (d, J = 2.4 Hz, 1H), 6.60 (dd, J = 8.7, 2.4 Hz,
1H), 4.64 (d, J = 6.6 Hz, 1H), 2.39−2.25 (m, 1H), 1.15−1.05 (m,
6H).
1
48 mg, 98%. Yellow solid. HPLC purity 99.4% (tR = 17.19 min). H
NMR (300 MHz, CDCl3): δ 11.91 (s, 1H), 7.84−7.72 (m, 2H),
7.66−7.48 (m, 2H), 7.32−7.24 (m, 1H), 6.87 (d, J = 9.0 Hz, 1H),
6.58 (d, J = 2.1 Hz, 1H), 6.45 (dd, J = 8.7, 2.1 Hz, 1H), 4.57 (t, J =
7.5 Hz, 1H), 3.95−3.58 (m, 2H), 2.40−2.25 (m, 1H), 2.07−1.93 (m,
2H), 1.77−1.56 (m, 4H), 1.49−1.36 (m, 2H), 1.10 (d, J = 6.6 Hz,
6H). 13C NMR (75 MHz, CDCl3): δ 169.4, 168.7, 159.6, 146.5,
134.3, 126.3, 126.2, 124.8, 123.8, 123.1, 119.9, 115.5, 112.8, 112.4,
59.7, 54.8, 33.5 (2C), 31.6, 24.1 (2C), 19.4, 18.7. HRMS (ESI): calcd
for C23H28Cl2N3O3, 464.1508 (M + H)+; found, 464.1499.
Cell Lines and Virus Strain. Human A549 (ATCC CCL-185),
HEK-293 (ATCC CRL-1573), and HFF (ATCC SCRC-1041) cell
lines were obtained from the American Type Culture Collection
(ATCC, Manassas, VA). The 293β5 stable cell line overexpressing the
human β5 integrin subunit was kindly provided by Nemerow.58 The
cell lines were propagated in Dulbecco’s modified Eagle medium
(DMEM, Life Technologies/Thermo Fisher) supplemented with 10%
fetal bovine serum (FBS, Omega Scientific, Tarzana, CA), 10 mM
HEPES, 4 mM L-glutamine, 100 units/ml penicillin, 100 μg/mL
streptomycin, and 0.1 mM nonessential amino acids (complete
DMEM). Wild-type HAdV-5, and HCMV (AD169) were obtained
from the ATCC. The HAdV-GFP used in this study is a replication-
Compounds 69−75 were prepared according to general procedure
B, starting from the amino intermediates 62a−g and cyclopentanone.
5-Chloro-N-(4-(cyclopentylamino)phenyl)-2-hydroxybenza-
mide (69). 65 mg, 57%. Yellow solid. HPLC purity 99.2% (tR = 20.29
1
min). H NMR (300 MHz, CDCl3): δ 12.12 (s, 1H), 7.84 (s, 1H),
7.47 (d, J = 2.1 Hz, 1H), 7.34 (dd, J = 9.0, 2.1 Hz, 1H), 7.26 (d, J =
8.7 Hz, 2H), 6.94 (d, J = 8.7 Hz, 1H), 6.57 (d, J = 8.7 Hz, 2H), 3.84−
3.50 (m, 2H), 2.10−1.91 (m, 2H), 1.80−1.56 (m, 4H), 1.54−1.37
(m, 2H). 13C NMR (75 MHz, CDCl3): δ 167.3, 160.3, 146.4, 134.2,
125.6, 125.3, 123.9 (2C), 123.5, 120.3, 115.9, 113.4 (2C), 54.9, 33.6
(2C), 24.2 (2C). HRMS (ESI): calcd for C18H20ClN2O2, 331.1213
(M + H)+; found, 331.1203.
5-Chloro-N-(4-(cyclopentylamino)-2-fluorophenyl)-2-hy-
droxybenzamide (70). 80 mg, 95%. Yellow solid. HPLC purity
1
95.7% (tR = 19.53 min). H NMR (300 MHz, CDCl3): δ 7.87 (s,
1H), 7.67 (t, J = 8.7 Hz, 1H), 7.48 (d, J = 1.8 Hz, 1H), 7.35 (dd, J =
8.7, 1.8 Hz, 1H), 6.95 (d, J = 9.0 Hz, 1H), 6.41−6.29 (m, 2H), 3.77−
3.65 (m, 1H), 2.09−1.93 (m, 2H), 1.81−1.54 (m, 4H), 1.52−1.38
(m, 2H). 13C NMR (75 MHz, CDCl3): δ 167.1, 160.2, 155.7 (d, J =
241.6 Hz), 147.5 (d, J = 10.5 Hz), 134.4, 125.4 (2C), 123.7, 120.3,
S
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defective virus containing a CMV promoter-driven enhanced green
fluorescent protein (eGFP) reporter gene cassette in place of the E1/
E3 regions.59 HAdV was propagated in 293β5 cells and isolated from
the cellular lysate by cesium chloride density centrifugation. Virus
concentration, in mg/mL, was calculated with the Bio-Rad protein
assay (Bio-Rad Laboratories) and converted to virus particles/mL
(vp/mL) using 4 × 1012 vp/mg.
Plaque Assay. Compounds were tested using low MOI infections
(0.06 vp/cell) at concentrations of 10 μM and in a dose−response
assay ranging from 10 to 0.375 μM in a plaque assay. Briefly, 293β5
cells were seeded in six-well plates at a density of 4 × 105 cells per well
in duplicate for each condition. When cells reached 80−90%
confluency, they were infected with HAdV5-GFP (0.06 vp/cell) and
rocked for 2 h at 37 °C. After incubation, the inoculum was removed,
and the cells were washed once with PBS. The cells were then
carefully overlaid with 4 mL/well of equal parts of 1.6% (water/vol)
Difco Agar Noble (Becton, Dickinson & Co., Sparks, MD) and 2×
EMEM (Minimum Essential Medium Eagle, BioWhittaker) supple-
mented with 2× penicillin/streptomycin, 2× L-glutamine, and 10%
FBS. The mixture also contained the drugs in concentrations ranging
from 10 to 0.375 μM. Following incubation for 7 days at 37 °C, plates
were scanned with a Typhoon FLA 9000 imager (GE Healthcare Life
Sciences) and plaques were quantified with ImageJ.60
Entry Assay. The anti-HAdV activity was measured in an entry
assay using human A549 epithelial cells (3 × 105 cells/well in corning
black wall, clear bottom 96-well plates) infected with HAdV5-GFP
(2000 vp/cell) in the presence 50 μM of the candidates and in a
dose−response assay. A standard infection curve was generated in
parallel by infecting cells in the absence of compounds using serial
two-fold dilutions of the virus. All reactions were carried out in
triplicate. Cells, virus, and drugs were incubated for 48 h at 37 °C and
5% CO2. Infection, as measured by HAdV5-mediated GFP
expression, was analyzed using a Typhoon 9410 imager (GE
Healthcare Life Sciences) and quantified with ImageQuantTL (GE
Healthcare Life Sciences).
Cytotoxicity Assay. The cytotoxicity of the compounds was
analyzed using the commercial kit AlamarBlue (Invitrogen, Ref.
DAL1025). A549 cells at a density of 5 × 103 cells per well in 96-well
plates were seeded. Decreasing concentrations of each derivative
(200, 150, 100, 80, 60, 40, 30, 20, 10, 5, 2.5, and 0 μM) were diluted
in 100 μL of DMEM. Cells were then incubated at 37 °C for 48 h
following the kit protocol. The cytotoxic concentration 50 (CC50)
value was obtained using the statistical package GraphPad Prism. This
assay was performed in duplicate.
isolation from infected cells. 1 × 106 A549 cells in six-well plates were
infected with wild-type HAdV5 at MOI 2000 vp/cell in the presence
of 10-fold IC50 concentration obtained in the plaque assay of the
compounds or the same volume of DMSO for positive control. Forty-
five minutes after infection, A549 cells were trypsinized and collected
and then washed twice with PBS. Then, cytoplasmic and nuclear
fractions were separated using a hypotonic buffer solution and NP-40
detergent. The cell pellet was resuspended in 500 μL of 1× hypotonic
buffer (20 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) and
incubated for 15 min at 4 °C. Then, 25 μL of NP-40 was added, and
the samples were vortexed. The homogenates were centrifuged for 10
min at 835g and 4 °C. Following the removal of the cytoplasmic
fraction (supernatant), HAdV DNA was isolated from the nuclear
fraction (pellet) and from the cytoplasmic fraction using the E.Z.N.A.
tissue DNA kit (Omega Biotek, Norcross, GA).
HAdV DNA Quantification by Real-Time PCR. A549 cells (1.5
× 105 cells/well in a 24-well plate) were incubated for 24 h in 500 μL
of complete DMEM, and they were infected with wild-type HAdV5
(100 vp/cell) when more than 90% of confluency was observed.
Infected cells were incubated for 24 h at 37 °C in 500 μL of complete
DMEM containing 10-fold IC50 concentration obtained in the plaque
assay of the compounds or the same volume of DMSO (positive
control). All reactions were carried out in duplicate. After 24 h of
incubation at 37 °C, DNA was purified from the cell lysate with the
E.Z.N.A. tissue DNA kit (Omega Biotek, Norcross, GA) following the
manufacturer’s instructions. TaqMan primers and probes for a
common region of the HAdV5 were designed with GenScript Real-
Time PCR (TaqMan) Primer Design software (GenScript).
Oligonucleotides sequences were AQ1: 5′-GCC ACG GTG GGG
TTT CTA AAC TT-3′; AQ2: 5′-GCC CCA GTG GTC TTA CAT
GCA CAT-3′; and Probe: 6-FAM-5′-TGC ACC AGA CCC GGG
CTC AGG TAC TCC GA-3′-TAMRA. Real-time PCR mixtures
consisted of 9.5 μL of the purified DNA, AQ1, and AQ2 at a
concentration of 200 nM each and the probe at a concentration of 50
nM in a total volume of 25 μL. The PCR cycling protocol was 95 °C
for 3 min followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s.
The human glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
gene was used as an internal control. Oligonucleotide sequences for
GAPDH and conditions were those previously reported by Henke-
Gendo et al.61
For quantification, gene fragments from hexon and GAPDH were
cloned into the pGEM-T Easy vector (Promega) and known
concentrations of template were used to generate a standard curve
in parallel for each experiment. All assays were performed in the
thermal cycler LightCycler 96 system (Roche).
HCMV DNA Quantification by Real-Time PCR. To test the
anti-HCMV activity of these new analogs, HFF cells (ATCC SCRC-
1041) were seeded in a 24-well plate (1 × 106 cells/plate), infected
with HCMV (MOI of 0.05 vp/cell), and incubated in complete
DMEM in the presence of 10-fold IC50 concentration obtained in the
plaque assay of the compounds or the same volume of DMSO in
triplicate. Then, cells were incubated for 72 h at 37 °C and 5% CO2,
and HCMV DNA was purified from the cell lysate using the QIAamp
DNA Mini kit (Qiagen, Valencia, CA) following the manufacturer’s
instructions. Real-time PCR primers, mixtures, and protocols were the
same as previously reported.21
Virus Yield Reduction. A549 cells (1.5 × 105 cells/well in a 24-
well plate) were incubated for 24 h in 500 μL of complete DMEM,
and they were infected with wild-type HAdV5 (100 vp/cell) when
more than 90% of confluency was observed. Infected cells were
incubated for 48 h at 37 °C in 500 μL of complete DMEM containing
10-fold IC50 concentration obtained in the plaque assay of the
compounds or the same volume of DMSO (positive control). After 48
h, cells were harvested and subjected to three rounds of freeze−thaw.
Serial dilutions of clarified lysates were titrated on A549 cells (3 × 104
cells/well), and TCID50 values were calculated using an end-point
dilution method (Reed and Muench, 1938).
Time of Addition Assay. The anti-HAdV effect of derivatives at
different points of time was measured in a time-curve assay using
293β5 cells (3 × 105 cells/well in corning black wall, clear bottom 96-
well plates) infected with HAdV5-GFP (2000 vp/cell) in the presence
of 10-fold IC50 concentration obtained in the plaque assay for each
compound or the same volume of DMSO (positive control). Parallel
samples of HAdV-5 were incubated with or without the selected
derivatives on ice for 1 h. Virus was then added to 293β5 cells and
incubated at 37 °C. The derivatives were added at the indicated time
points before or during this incubation. After a total of 2 h at 37 °C,
cells were incubated for an additional 48 h at 37 °C and 5% CO2
before being analyzed for GFP expression using the Typhoon 9410
imager (GE Healthcare Life Sciences) as mentioned above.
Maximum Tolerated Dose. Experiments were approved by the
Committee on the Ethics of Animal Experiments of the Virgen
́
Macarena y Virgen del Rocio University Hospitals, Seville, Spain
(JSC-PIP-2018-01), and from the Ministry of Agricultura, Pesca y
Desarrollo Rural (17/09/2018/143). The Reed and Munch method62
was used to determine in vivo toxicity. Briefly, four female (RjHan/
AURA) golden Syrian hamsters per group (weight 80−100 g) were
immunosuppressed with a dose of 140 mg/kg of cyclophosphamide 7
days before beginning with the treatments and then twice weekly at a
dose of 100 mg/kg. Before dosing, compounds were diluted in 10%
DMSO, 10% Cremophor EL, and 80% sodium chloride physiological
solution. Hamsters were inoculated with 1 mL volume of the
appropriate suspension for the selected doses for compounds 2, 3, and
15 and niclosamide along 14 days. At day 14, animals were sacrificed
Nuclear-Associated HAdV Genomes. The nuclear delivery of
HAdV genomes was assessed by real-time PCR following nuclear
T
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Article
with a lethal dose of thiopental. All inoculations were performed
intraperitoneally in unanesthetized hamsters.
Notes
The authors declare no competing financial interest.
Statistical Analyses. One-way ANOVA tests (Dunnet method)
were carried out using the GraphPad Prism 6. We considered a
statistical significance with a p value under 0.05. This statistical
significance was pointed out with asterisk in graphs, and the numbers
of them indicate the level of significance (*p ≤ 0.05, **p ≤ 0.01, ***p
≤ 0.001, and ****p ≤ 0.0001).
ACKNOWLEDGMENTS
■
This work was supported by UTMB Technology Commerci-
alization Program, John Sealy Memorial Endowment Fund,
and Institute for Translational Sciences (ITS) at UTMB, Plan
Nacional de I + D + i 2013−2016 and Instituto de Salud Carlos
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01226.
■
́
́
III, Subdireccion General de Redes y Centros de Investigacion
sı
́
Cooperativa, Ministerio de Economia, Industria y Competitividad,
Spanish Network for Research in Infectious Diseases (REIPI
RD16/0016/0009), co-financed by European Development
Regional Fund “A way to achieve Europe”, Operative program
Intelligent Growth 20142020, the Instituto de Salud Carlos III,
Proyectos de Investigacion en Salud (PI17/01055; PI18/
01191) and Proyectos de Desarrollo Tecnologico en Salud
(DTS17/00130), the Spanish Adenovirus Network (Adeno-
Net, BIO2015/68990-REDT), and the program “Nicolas
Monardes” (C-0059-2018), Servicio Andaluz de Salud, Junta
de Andalucia. J.Z. is also partially supported by John D. Stobo,
1H and 13C NMR spectra of all new compounds (PDF)
Molecular formulation strings and some data (CSV)
́
́
AUTHOR INFORMATION
Corresponding Authors
■
́
́
́
Javier Sanchez-Cespedes − Unit of Infectious Diseases,
Microbiology and Preventive Medicine, Institute of Biomedicine
́
́
of Seville (IBiS), University Hospital Virgen del Rocio/CSIC/
M.D., Distinguished Chair Endowment Fund at UTMB.
M.E.P.I. is a postdoctoral researcher belonging to the program
“Nicolas Monardes” (C1-0038-2019), Servicio Andaluz de
University of Seville, E-41013 Seville, Spain; Phone: +34 955
923 100; Email: jsanchez-ibis@us.es; Fax: +34 955 923 101
Jia Zhou − Chemical Biology Program, Department of
Pharmacology and Toxicology, University of Texas Medical
Branch, Galveston, Texas 77555, United States; orcid.org/
0000-0002-2811-1090; Phone: (409) 772-9748;
́
́
Salud, Junta de Andalucia, Spain.
ABBREVIATIONS
■
HAdVs, human adenoviruses; SAR, structure−activity relation-
ship; allo-HSCT, allogenic hematopoietic stem cell transplant;
SOT, solid-organ transplant; AIDS, acquired immune
deficiency syndrome; BCV, brincidofovir; CAP, community-
acquired pneumonia; CMV, cytomegalovirus; HCMV, human
cytomegalovirus; IC50, half maximal inhibitory concentration;
CC50, cytotoxicity concentration 50%; ED50, median effective
dose; TCID50, median tissue culture infective dose; MTD,
maximum tolerated dose; p.i., postinfection; qPCR, quantita-
tive real-time PCR; TLC, thin layer chromatography; UV,
ultraviolet; TMS, tetramethylsilane; THP, tetrahydropyran;
HRMS, high-resolution mass spectrometry; HPLC, high-
performance liquid chromatography; HBTU, 2-(1H-benzotria-
zole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate;
DIEA, N,N-diisopropylethylamine; DCM, dichloromethane;
DMAP, 4-(dimethylamino)pyridine; DMSO, dimethyl sulf-
oxide; EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride; EtOAc, ethyl acetate
Email: jizhou@utmb.edu; Fax: (409) 772-9818
Authors
Jimin Xu − Chemical Biology Program, Department of
Pharmacology and Toxicology, University of Texas Medical
Branch, Galveston, Texas 77555, United States
Judith Berastegui-Cabrera − Unit of Infectious Diseases,
Microbiology and Preventive Medicine, Institute of Biomedicine
́
of Seville (IBiS), University Hospital Virgen del Rocio/CSIC/
University of Seville, E-41013 Seville, Spain
Na Ye − Chemical Biology Program, Department of
Pharmacology and Toxicology, University of Texas Medical
Branch, Galveston, Texas 77555, United States; orcid.org/
0000-0003-3612-1771
Marta Carretero-Ledesma − Unit of Infectious Diseases,
Microbiology and Preventive Medicine, Institute of Biomedicine
́
of Seville (IBiS), University Hospital Virgen del Rocio/CSIC/
University of Seville, E-41013 Seville, Spain
́
́
Jeronimo Pachon-Díaz − Unit of Infectious Diseases,
Microbiology and Preventive Medicine, Institute of Biomedicine
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