Structure-Activity Relationship Studies on Diversified Salicylamide Derivatives as Potent Inhibitors of Human Adenovirus Infection

Article abstract of DOI:10.1021/acs.jmedchem.9b01950

The effective treatment of adenovirus (HAdV) infections in immunocompromised patients still poses great challenges. Herein, we reported our continued efforts to optimize a series of salicylamide derivatives as potent inhibitors of HAdV infection. Of these, nine compounds (11, 13, 14, 17, 20, 58, 60, 62, and 70) showed significantly improved anti-HAdV activities with nanomolar to submicromolar IC₅₀ values and high selectivity indexes (SI > 100), indicating better safety windows, compared to those of the lead compound niclosamide. Our mechanistic assays suggest that compounds 13, 62, and 70 exert their activities in the HAdV entry pathway, while compounds 14 and 60 likely target the HAdV DNA replication, and 11, 17, 20, and 58 inhibit later steps after DNA replication. Given the broad anti-viral activity profile of niclosamide, these derivatives may also offer therapeutic potential for other viral infections.

Full text of DOI:10.1021/acs.jmedchem.9b01950

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Article
Structure-Activity Relationship Studies on Diversified Salicylamide
Derivatives as Potent Inhibitors of Human Adenovirus Infection
Jimin Xu, Judith Berastegui-Cabrera, Haiying Chen,
Jerónimo Pachón, Jia Zhou, and Javier Sanchez-Cespedes
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01950 • Publication Date (Web): 11 Feb 2020
Downloaded from pubs.acs.org on February 17, 2020
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Structure-Activity Relationship Studies on Diversified Salicylamide Derivatives as Potent
Inhibitors of Human Adenovirus Infection
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†
,‖
‡,‖
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‡,♯
†,
Jimin Xu , Judith Berastegui-Cabrera , Haiying Chen , Jerónimo Pachón-Díaz , Jia Zhou *
‡
,
Javier Sánchez-Céspedes *
†
Chemical Biology Program, Department of Pharmacology and Toxicology, University of Texas
Medical Branch, Galveston, Texas 77555, United States
‡
Unit of Infectious Diseases, Microbiology and Preventive Medicine, Institute of Biomedicine of
Seville (IBiS), University Hospital Virgen del Rocío/CSIC/University of Seville, E41013 Seville,
Spain
♯
Department of Medicine, University of Seville, E-41009 Seville, Spain
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ABSTRACT:
The effective treatment of adenovirus (HAdV) infections in immunocompromised patients still
poses great challenges. Herein, we reported our continued efforts to optimize a series of
salicylamide derivatives as potent inhibitors of HAdV infection. Of these, nine compounds (11,
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3, 14, 17, 20, 58, 60, 62 and 70) showed significantly improved anti-HAdV activities with
nanomolar to submicromolar IC50 values and high selectivity indexes (SI > 100), indicating
better safety windows, compared to the lead compound niclosamide. Our mechanistic assays
suggest that compounds 13, 62 and 70 exert their activities in the HAdV entry pathway, while
compounds 14 and 60 likely target the HAdV DNA replication, and 11, 17, 20 and 58 inhibit
later steps after DNA replication. Given the broad antiviral activity profile of niclosamide, these
derivatives may also offer therapeutic potential for other viral infections.
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INTRODUCTION
Human adenoviruses (HAdVs) are non-enveloped viruses with an icosahedral capsid which
contains a linear and double stranded DNA genome of approximately 36 kb in size that encodes
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about 35 genes. Currently, HAdVs consist of at least 70 different serotypes divided into seven
species (HAdV A-G) that belong to the Mastadenovirus genus of the family Adenoviridae; most
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-4
of these species spread globally with predominant types differing geographically. HAdVs can
cause a wide variety of clinical diseases including acute upper respiratory disease, conjunctivitis,
gastroenteritis, hepatitis, myocarditis and pneumonia. Primary infections occur in young children
with virus transmitted via multiple routes (respiratory, fecal-oral, direct conjunctival inoculation,
environmentally, etc.), and are typically self-limiting and rarely associated with severe clinical
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, 5
symptoms in immunocompetent individuals. However, HAdV infections can cause significant
morbidity and mortality in immunocompromised patients, such as solid-organ transplant (SOT)
or allogenic hematopoietic stem cell transplant (allo-HSCT) patients, AIDS patients and patients
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-9
with genetic immunodeficiencies. Pediatric allo-HSCT patients are particularly at risk of
HAdV infections with frequencies ranging from 5% to 47%; the reported mortality rates are up
to 26% for them with symptomatic infection and as high as 80% for them with disseminated
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-6, 10
disease.
Despite the significant clinical impact, there remains no specifically approved antiviral
therapy for HAdV infection to date. Several broad-spectrum antiviral drugs are used off-label to
treat HAdV infections in immunocompromised patients, being the most common the acyclic
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nucleoside phosphonates.
Cidofovir (Figure 1), an acyclic nucleoside phosphonate cytosine
analogue, is the most frequently used drug to treat HAdV infections. Cidofovir was approved to
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treat cytomegalovirus (CMV) retinitis and can inhibit viral DNA replication acting as a chain
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terminator. Cidofovir displays broad antiviral activities against all HAdV species, but it has
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Figure 1. Representative nucleoside and non-nucleoside inhibitors of adenovirus infection.
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low oral bioavailability, long plasma half-life, and significant nephrotoxicity.
Considering its proven efficacy, a lipid-linked derivative of cidofovir, named brincidofovir
(BCV, previously named CMX001, 3-hexadecyloxy-1-propanol-cidofovir), was developed with
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improved antiviral potency, safety, bioavailability and pharmacokinetics.
BCV was proven to
be effective in eliminating disseminated HAdV infection in the Syrian Hamster model which is
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permissive for HAdV-5 replication. More excitingly, the good results from the phase II and III
trials (NCT01231344 & NCT02087306) confirm the antiviral activity of BCV against
adenoviruses and support the continued development of BCV as the first therapeutic option for
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HAdV infection.
However, BCV administration caused gastrointestinal disturbances,
primarily diarrhea, in some patients and a new administration strategy is needed to avoid this
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adverse effect. USC-087 (3), an N-alkyl tyrosinamide phosphonate ester prodrug of HPMPA,
the adenine analog of cidofovir, was highly effective against multiple HAdV types in cell
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culture. Promisingly, USC-087 protected Syrian hamsters against lethal challenge with HAdV-
or -6 even when administered starting at 4 days post challenge. Besides the nucleoside
analogues reported in recent years and described above, a few non-nucleoside analogues have
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also been identified as anti-HAdV agents.
Benzoic acid analogue (4) was discovered by high-
31, 32
throughput screening (HTS) and subsequent optimization.
It inhibited HAdV-5 replication
with an EC of 0.58 μM and exhibited low cell toxicity. Our research team has been devoted to
50
anti-HAdV drug discovery for many years and reported a series of inhibitors of HAdV infection
including compounds 5-7. Piperazine derivative 5 significantly inhibited HAdV and CMV
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infections in different phases of their life cycle, with little or no cytotoxicity.
Niclosamide
(6) is an FDA-approved anthelminthic drug used in humans to treat tapeworm infections,
involving uncoupling of oxidative phosphorylation. Accumulated studies indicated that
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1-46
niclosamide can modulate multiple signaling pathways and biological processes,
and has
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7, 48
49
great antiviral potential against various viruses including Zika virus,
coronavirus, hepatitis
5
0
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52
53
C virus, Ebola Virus, HIV, Japanese encephalitis virus, human rhinoviruses or influenza
54
virus, etc. Our research team first found that niclosamide significantly inhibited HAdV
infection at low micromolar concentration (IC50 = 0.60 μM). However, it showed moderate
cytotoxicity (CC = 22.9 μM) and a relatively narrow safety window with a low selectivity
5
0
35
index (SI = 38.2). Mifepristone (7), a commercially available synthetic steroid drug, showed
potent in vitro anti-HAdV activity by interfering with HAdV genome accessibility into the
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nucleus. While significant progress has been made in anti-HAdV drug discovery, there is still
an urgent need to develop highly effective antiviral agents lacking major adverse effects. As part
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2, 55-57
of our ongoing antiviral drug discovery and development program,
herein we reported our
continued structure-activity relationship (SAR) optimization studies of niclosamide and its
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salicylamide derivatives, aiming to acquire an alternative for the treatment of HAdV infections
with increased activity and low toxicity.
RESULTS AND DISCUSSION
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Chemistry. The general synthesis of salicylanilide derivatives is summarized in Scheme 1.
Commercially available substituted 2-methoxybenzoic acids 8a-d were coupled with various
anilines 9a-i in the presence of PCl , followed by demethylation with BBr , to afford the
3
3
2
4
corresponding salicylanulides 10-20. R -R groups are defined in Table 1. Derivative 21 was
accessed under the same coupling conditions by condensation of 3-chlorobenzoic acid 8e with 3-
fluoro-5-(trifluoromethyl)aniline 9d.
a
Scheme 1. Synthesis of Salicylanilide Derivatives 10-21
a
Reagents and conditions: (a) PCl , toluene, 100 °C, 93%. (b) i. PCl , toluene, 100 °C; ii. BBr ,
3
3
3
CH Cl -78 °C to 0 °C, 33-95% (two steps).
2
2,
As described in Scheme 2, condensation of 5-chloro-2-methoxybenzoic acid 8a with
different substituted benzylamine 22a-p was followed by demethylation using BBr to give
3
salicylamide derivatives 30-45. Direct coupling of 5-chlorosalicylic acid 24 with substituted
1
4
benzylamine 25a-e afforded salicylamide derivatives 46-50. R -R groups are defined in Table
. Derivative 51 was synthesized by EDCI-mediated condensation of acid 8a and cumylamine 26
and subsequent demethylation with BBr Substitution of methyl 5-chloro-2-hydroxybenzoate 27
2
3
.
with 4-fluorophenethylamine 28 in methanol directly provided derivative 52.
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a
Scheme 2. Synthesis of Salicylamide Derivatives 30-52
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a
Reagents and conditions: (a) EDCI, DMAP, Et N, CH Cl , r.t. (b) BBr , CH Cl , -78 °C to 0 °C,
51-93% (two steps). (c) EDCI, DMAP, CH Cl , r.t., 39-64%. (d) EDCI, DMAP, CH Cl , 50 °C,
3
2
2
3
2
2
2
2
2
2
1
5%. (e) CH OH, r.t., 52%.
3
a
Scheme 3. Synthesis of 2-Phenylacetamide Derivatives 53-54
a
Reagents and conditions: (a) i. PCl , toluene, 100 °C; ii. BBr , CH Cl , -78 °C to 0 °C, 77-82%
3
3
2
2
(two steps).
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As shown in Scheme 3, 2-phenylacetamide derivatives 53-54 were prepared in a similar
manner to that described above for compounds 10-20. Condensation of (5-chloro-2-
methoxyphenyl)acetic acid 29 with anilines 9a or 9d was followed by demethylation to afford
compound 53 and 54, respectively.
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Compounds 56-72 were prepared according to the general synthesis outlined in Scheme
4
. Various anilines 9a-e were coupled with different Fmoc-protected amino acids in the presence
of PCl , followed by piperidine deprotection, to give the amino intermediates 55a-n. The
3
subsequent condensation of amino intermediates 55a-h, j-n with acid 8a was followed by
demethylation to afford salicylamide derivatives 56-63 and 65-69. The amino intermediate 55i
with tert-butoxycarbonyl moiety was condensed with 5-chlorosalicylic acid 24 to provide
compound 64. Derivatives 70-72 were accessed by the coupling reaction between amino 55d and
1
5
5
-chloro-2-(methylsulfonamido)benzoic acid 23. R -R groups are defined in Table 3.
a
Scheme 4. Synthesis of Salicylamide Derivatives 56-72
a
Reagents and conditions: (a) i. Fmoc-amino acid, PCl , toluene, 100 °C; ii. piperidine, CH CN,
3
3
r.t., 69-99% (two steps). (b) i. 8a, EDCI, DMAP, CH Cl , r.t.; ii. BBr , CH Cl , -78 °C to 0 °C,
2
2
3
2
2
2
9-83% (two steps). (c) 24, EDCI, DMAP, CH Cl , r.t., 18%. (d) 5-chloro-2-
2 2
(
methylsulfonamido)benzoic acid 23, HBTU, DIEA, CH Cl , r.t., 60-77%.
2 2
In Vitro Evaluation of Human Adenovirus Inhibition. All newly synthesized compounds were
first screened in plaque assay at the concentration of 10 μM, quantifying their abilities to inhibit
HAdV plaque formation. The active compounds screened out (inhibition > 90%) were further
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Table 1. Inhibition of HAdV in Plaque Assay, Cytotoxicity and Selectivity Index for
Compounds 10-21
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0
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a
Plaque Assay
Select
1
2
3
4
5
6
7
b
ivity
index
Compd
R
R
R
R
R
R
R
(%)
Inhibition
(10 μM)
CC (μM)
50
IC (μM)
50
c
(SI)
6
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
Cl Cl
H
NO
2
2
2
H
H
100 ± 0
100 ± 0
0.6 ± 0.05
22.9 ± 9.8
15.1 ± 0.5
10.9 ± 0.5
8.1 ± 3.2
27.1 ± 0.1
35.0 ± 3.7
8.0 ± 0.8
4.1 ± 1.8
38.2
15.1
10
Cl
Cl
Cl
Cl
H
F
Cl NO
1.00 ± 0.37
1
1
1
1
2
3
H
H
H
H
F
NO
H
H
98.7 ± 1.9 0.05 ± 0.01
100 ± 0 0.31 ± 0.10
98.0 ± 2.8 0.11 ± 0.01
218.2
26.2
F
NO
H
2
H
NO
246.6
437.5
29.6
2
14
Cl Cl
CF
F
3
H
100 ± 0
100 ± 0
100 ± 0
100 ± 0
0.08 ± 0.00
0.27 ± 0.02
0.06 ± 0.00
1
1
1
1
5
6
7
8
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
CF
H
H
H
H
H
H
CF
CF
CF
CF
CF
CF
68.3
3
3
F
0.18 ± 0.01 120.0 ± 33.6 666.7
3
F
98.7 ± 1.9 1.07 ± 0.09
16.3 ± 8.6
15.2
23.3
186.8
6.9
3
3
3
3
19
Me
H
F
98.0 ± 2.8 0.65 ± 0.43 15.2 ± 11.0
2
0
1
OH Cl
F
96.8 ± 1.9 0.11 ± 0.05
99.3 ± 0.9 5.61 ± 0.37
20.6 ± 1.0
38.6 ± 1.7
2
H
H
Cl
F
a
Percentage of control HAdV5-GFP inhibition at 10 μM and inhibitory concentration 50% (IC )
5
0
b
at low MOI in a plaque assay using the 293β5 cell line. Cytotoxic concentration 50% (CC ).
5
0
c
Selectivity index value was determined as the ratio of CC to IC in a plaque assay for each
50
50
compound. The results represent means ± SD of triplicate samples from three independent
experiments.
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6
evaluated to characterize their antiviral activity (IC ) in plaque assay and their cytotoxicity
5
0
(
CC values). Starting from niclosamide, we initially made a simple exploration on the effect
50
generated in its antiviral activity by the replacement of the substituents on its two phenyl rings.
As shown in Table 1, moving the 2´-Cl group on the aniline ring to the 3´-position (10)
maintained the same level of potency (IC = 1.00 μM) and cytotoxicity (CC = 15.1 μM), in
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
50
comparison with the parent compound (6). Interestingly, substitution of 2´-Cl with 2´-F (11)
resulted in a 11-fold increase in potency (IC = 0.05 μM) and an improved selectivity index (SI
5
0
=
218.2), while it also displayed increased cytotoxicity (CC50 = 10.9 μM). In contrast to
compound 11, moving the 4´-NO group to 5´-position (12) led to a slight loss of activity (IC =
2
50
0.31 μM). Nevertheless, it still showed the same level of potency compared to niclosamide.
Removal of the 2´-Cl group of niclosamide yielded compound 13 with enhanced potency (IC =
5
0
0
.11 μM) and selectivity index (SI = 246.6). Excitingly, compound 14 with 2´-Cl-4´-CF -aniline
3
moiety showed significantly increased potency (IC50 = 0.08 μM) and decreased cytotoxicity
CC = 35.0 μM), resulting in a high selectivity index (SI = 437.5). Compounds 15 with 3´,4´-
(
50
difluoro substitution and 16 with 3´,5´-bis(trifluoromethyl) substitution exhibited potent anti-
HAdV activities with IC values of 0.27 μM and 0.06 μM, respectively. However, these two
50
compounds also showed increased cytotoxicity (CC = 8.0 μM and 4.1 μM, respectively), and
50
consequently no obvious improvement on their selectivity index was observed (SI = 29.6 and
8.3, respectively). Remarkably, compound 17 with 3´-F-5´-CF -aniline moiety possessed
6
3
improved potency against HAdV (IC = 0.18 μM), meanwhile it showed significantly decreased
5
0
cytotoxicity (CC = 120.0 μM), with a high selectivity index (SI = 666.7). These explorations
5
0
described above indicated that the introduction of fluoro and trifluoromethyl groups at the proper
positions on the aniline ring was beneficial for antiviral potency, consistent with the previous
1
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58
results. Considering its potent activity and low cytotoxicity, we next explored the effect of the
substitution on the salicylic ring while retaining the 3´-F-5´-CF -aniline moiety of compound 17.
3
Removal of the 5-Cl group (18) or replacement of the 5-Cl group with 5-methyl group (19) led to
a slight loss of potency (IC = 1.07 μM and 0.65 μM, respectively) and increased cytotoxicity
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
(
CC = 16.3 μM and 15.2 μM, respectively), compared to compound 17. Moving the 5-Cl group
50
to 4-position (20) maintained the same level of potency (IC50 = 0.11 μM), with increased
cytotoxicity (CC = 20.6 μM) and decreased selectivity index (SI = 186.8). However, removal
5
0
of the 2-OH group resulted in a significant loss of potency (IC50 = 5.6 μM). These results
suggested the 2-phenolic hydroxyl group at the salicylic part was essential for salicylanilide
derivatives to maintain their anti-HAdV activities.
Table 2. Inhibition of HAdV in Plaque Assay, Cytotoxicity and Selectivity Index for
Compounds 30-54
a
Plaque Assay
Selecti
1
2
3
4
5
6
b
vity
index
Compd
R
R
R
R
R
R
(%)
Inhibition
(10 μM)
CC (μM)
50
IC (μM)
50
c
(SI)
3
3
3
0
1
2
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
F
H
H
H
H
F
NO
2
H
H
100 ± 0
1.40 ± 0.11
51.4 ± 3.7
5.5
12.1
17.9
NT
F
97.1 ± 4.1 2.56 ± 0.06 30.9 ± 10.2
Cl
F
H
99.2 ± 0.4 1.73 ± 0.51 30.9 ± 13.6
d
33
34
H
74.7 ± 6.9
NT
NT
NT
NT
H
F
H
37.2 ± 23.4
NT
3
5
H
F
H
CF
3
98.1 ± 1.4 2.40 ± 0.05
36.3 ± 7.9
15.1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
6
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
F
F
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
CF
3
3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
99.0 ± 1.5 1.40 ± 0.02
98.8 ± 0.8 1.32 ± 0.04
97.4 ± 3.7 3.57 ± 1.43
22.3 ± 7.2
45.6 ± 7.3
27.1 ± 1.1
NT
15.9
34.6
7.6
37
38
H
H
CF
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
F
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
72.0 ± 1.5
72.0 ± 3.0
0.0 ± 0.0
NT
NT
NT
NT
NT
NT
9.2
3
4
4
4
4
4
4
4
9
0
1
2
3
4
5
6
H
NT
H
F
NT
H
CF
99.6 ± 0.2 2.59 ± 0.42
23.9 ± 9.2
3
H
NO
99.5 ± 0.7 6.56 ± 0.47 175.0 ± 43.9 26.7
2
H
CH
H
96.6 ± 4.0 8.12 ± 2.16
87.7 ± 4.7
NT
10.8
NT
3
H
12.7 ± 2.9
100 ± 0
NT
NT
(R)-Me
(S)-Me
(R)-Me
(R)-Me
(R)-Me
H
84.6±20.5
199.3 ± 8.7
31.1±3.2
>200
NT
47
48
H
100 ± 0
2.54 ± 0.1
NT
78.5
NT
Cl
F
100 ± 0
4
5
9
0
100 ± 0
3.02 ± 0.07
NT
66.2
NT
OMe
33.5 ± 14.8
NT
5
5
5
5
1
2
3
4
100 ± 0
3.46 ± 0.88
>200
57.8
98.7 ± 1.8 3.94 ± 1.38 154.1 ± 13.1 39.1
91.0 ± 3.6 4.92 ± 0.01
0.0 ± 0.0 NT
23.7 ± 1.5
NT
4.8
NT
a
Percentage of control HAdV5-GFP inhibition at 10 μM and inhibitory concentration 50% (IC )
at low MOI in a plaque assay using the 293β5 cell line. Cytotoxic concentration 50% (CC ).
Selectivity index value was determined as the ratio of CC to IC in a plaque assay for each
compound. NT: not tested. The results represent means ± SD of triplicate samples from three
independent experiments.
5
0
b
5
0
c
50
50
d
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Next, we turned our attention to the amide linker region. In order to improve the
flexibility of salicylanilide derivatives, we attempted to insert one or two additional carbon atoms
between two aromatic rings, and compounds 30-54 were designed, synthesized, and evaluated
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Table 2). We first replaced the aniline moiety with various substituted benzylamines to yield a
series of N-benzylsalicylamide derivatives (30-51), and most of these compounds showed
potency at the concentration of 10 μM, with IC values ranging from 1 to 10 μM. In general,
50
disubstituted benzylamine derivatives (30-32 and 35-37) were more potent than monosubstituted
benzylamine derivatives (38-40 and 42-44). Unexpectedly, difluoro substitution (33 and 34)
reduced the potency, especially 3,4-difluoro substituted derivative 34 with no inhibition against
HAdV at 10 μM, and inconsistent with the trend observed before. Methylation of amide (41)
resulted in a complete loss of potency (0% at 10 μM) in contrast to 40. Compound 45 with
unsubstituted benzylamine moiety displayed no inhibitory activity against HAdV up to 10 μM.
Introduction of one or two methyl groups at the benzylic site produced compounds 46-51, and all
these compounds except compound 50 with (R)-4-methoxy-α-methylbenzylamine moiety fully
inhibited HAdV plaque formation at the concentration of 10 μM. Interestingly, compounds 47
with (S)-α-methylbenzylamine moiety, 49 with (R)-4-fluoro-α-methylbenzylamine moiety and
5
1 with α, α -dimethyl-4-fluorobenzylamine showed similar potency (IC = 2.54 μM, 3.02 μM
50
and 3.46 μM, respectively) and very low cytotoxicity (CC = 199.3 μM, >200 μM and >200
5
0
μM, respectively), along with similar selectivity index as well (SI = 78.5, 57.8 and 39.1,
respectively). Insertion of two carbon atoms between the nitrogen atom of the amide and the
aromatic ring (52) also retained the potency with an IC50 of 3.94 μM, while it showed low
cytotoxicity (CC50 = 154.1 μM) and a similar selectivity index (SI = 39.1) compared to
niclosamide. Inserting one carbon between the amide carbonyl and the aromatic ring yielded two
1
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
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5
5
5
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5
5
5
5
6
derivatives 53 and 54 with entirely different inhibitory activities against HAdV. Intriguingly,
compound 53 with 3´-F-5´-CF -aniline moiety exhibited potency with an IC of 4.92 μM, while
3
50
54 with 2´-Cl-4´-NO -aniline moiety showed no inhibitory activity against HAdV at 10 μM. Due
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
to the weak potency, no more optimization efforts on this series of compounds were further
pursued.
Table 3. Inhibition of HAdV in Plaque Assay, Cytotoxicity and Selectivity Index for
Compounds 56-72
a
Plaque Assay
(%)
Inhibition
10 μM)
100 ± 0
Select
1
2
3
4
5
6
7
b
ivity
Compd
R
R
R
R
R
R
R
CC (μM)
50
index
IC (μM)
5
0
c
(SI)
(
1
1
29.2 ±
4.7
00.1 ±
13.3
56
OH
H
H
Cl
H
NO
2
H
2.43 ± 0.59
53.2
26.0
3
5
5
5
6
7
8
9
0
OH
OH
OH
OH
OH
OH
OH
Me
iso-Pr
Bn
H
H
H
Cl
Cl
Cl
H
H
H
H
H
H
H
NO
NO
NO
NO
NO
NO
NO
2
2
2
2
2
2
2
H
H
H
H
H
H
H
97.4 ± 2.7 3.85 ± 0.04
100 ± 0
100 ± 0
0.45 ± 0.06 200.0 ± 1.9 444.4
d
NT
15.2 ± 2.1
NT
H
Bn Cl
100 ± 0
0.30 ± 0.00 77.9 ± 10.2 259.7
NT NT NT
61
62
63
iso-Bu
sec-Bu
H
H
H
Cl
Cl
Cl
17.4 ± 12.3
99.0 ± 0.3 0.16± 0.00 20.2 ± 6.9 127.6
34.8 ± 16.9
NT
NT
NT
6
4
OH
H
Cl
H
NO
2
H
97.3 ± 0.3 0.67± 0.11 22.9 ± 5.2
99.5 ± 0.7 0.40 ± 0.09 30.9 ± 0.3
34.2
6
6
5
6
OH
OH
iso-Pr
iso-Pr
H
H
H
CF
3
H
CF
3
77.4
NT
Cl
H
CF
H
24.8 ± 12.3
NT
NT
3
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OH
OH
OH
iso-Pr
iso-Pr
Bn
H
H
H
H
H
H
F
H
H
F
CF
3
3
95.8 ± 5.9 0.66 ± 0.03 43.0 ± 14.4 65.2
98.3 ± 2.4 2.96 ± 0.50 95.0 ± 18.7 32.1
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
68
69
F
CF
H
CF
99.0 ± 1.4 0.40 ± 0.02 17.9 ± 1.1
44.9
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
174.5 ±
7
7
7
0
1
2
iso-Pr
Bn
H
H
H
Cl
Cl
Cl
H
H
H
NO
NO
NO
2
2
2
H
H
H
99.1 ± 1.2 0.68 ± 0.08
256.0
1
2.1
88.9 ± 15.7
NT
NT
NT
141.1 ±
13.5
H
99.6 ± 5.5 9.65 ± 0.01
14.6
a
Percentage of control HAdV5-GFP inhibition at 10 μM and inhibitory concentration 50% (IC )
at low MOI in a plaque assay using the 293β5 cell line. Cytotoxic concentration 50% (CC ).
Selectivity index value was determined as the ratio of CC to IC in a plaque assay for each
compound. NT: not tested. The results represent means ± SD of triplicate samples from three
independent experiments.
5
0
b
5
0
c
50
50
d
Amino acid linkers were widely used to tune the flexibility and improve the
pharmacokinetic profiles for those compounds with two aromatic rings linked by a simple amide
5
9
bond. To explore the effect of introducing various α-amino acid linkers, compounds 56-72
were prepared and evaluated as shown in Table 3. Introducing different substitution on the linker
moiety yielded compounds 56-64 while retaining the salicylic and aniline moieties of
niclosamide. Use of an unsubstituted glycine linker (56) reduced the potency (IC = 2.43 μM) as
5
0
well as cytotoxicity (CC = 129.2 μM) compared to niclosamide. The linker with (S)-methyl
5
0
group (57) was similar in potency (IC = 3.85 μM) and cytotoxicity (CC = 100.1 μM) to
5
0
50
compound 56. Encouragingly, compound 58 with (S)-isopropyl substitution displayed potent
activity (IC = 0.45 μM) similar to niclosamide, with decreased cytotoxicity (CC = 200 μM)
5
0
50
and significantly improved selectivity index (SI = 444.4). Compound 60 with (R)-benzyl
substitution also showed improved potency (IC = 0.30 μM) and diminished cytotoxicity (CC
50
50
=
77.9 μM), with a high SI value of 259.7. However, its enantiomer 59 with (S)-benzyl
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
substitution exhibited increased cytotoxicity (CC = 15.2 μM). Linkers with (S)-isobutyl (61)
5
0
and (S)-2-(methylthio)ethyl (63) displayed no significant inhibitory activity at the concentration
of 10 μM, while compounds 62 with (S)-sec-butyl and 64 with (S)-3-(tert-butoxy)-3-oxopropyl
maintained the same level of activity (IC50 = 0.16 μM and 0.67 μM, respectively) and
cytotoxicity (CC = 20.2 μM and 22.9 μM, respectively) compared to niclosamide, with SI
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
values of 127.6 and 34.2, respectively. Considering the potent activity and low cytotoxicity of
compound 58, we next investigated the effect of the substitution on the aniline ring, using L-
valine as a linker. Compounds 65 with 3´,5´-bis(trifluoromethyl) substitution and 67 with 3´-
fluoro-5´-trifluoromethyl substitution retained similar potency (IC = 0.40 μM and 0.66 μM,
5
0
respectively) to compound 58, with increased cytotoxicity (CC50 = 30.9 μM and 43.0 μM,
respectively). Interestingly, 2´-chloro-4´-trifluoromethyl substitution (66) led to a complete loss
of potency at 10 μM, while compounds 68 with 3´,4´-difluoro substitution retained some activity
(IC = 2.96 μM). Compared to compound 65, use of L-phenylalanine linker (69) maintained the
5
0
same level of potency (IC = 0.40 μM), with a slight increase in cytotoxicity (CC = 17.9 μM).
50
50
Substitution of 2-phenolic hydroxyl at the salicylic part with 2-methylsulfonamido yielded
compounds 70-72 with different amino acid linkers. Of these, compound 70 with a L-valine
linker displayed similar potency (IC = 0.68 μM) and lower cytotoxicity (CC = 174.5 μM) to
5
0
50
compound 58, with a high selectivity index (SI = 256.0). For the comparison using the same in
vitro assay protocols, the antiviral activity and safety of these niclosamide derivatives
demonstrated to be significantly better than those of cidofovir (IC = 24.06 ± 5.9 µM; CC =
5
0
50
5
0.06 ± 9.8 µM; SI = 7.5), the drug of current choice for the treatment of HAdV infections.
Those compounds with high selectivity index (SI > 100) were selected for further
evaluation in entry assay, using human A549 epithelial cells infected with HAdV-GFP in
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presence of the candidate compound and incubated for 48 h. As shown in Table 4, most of the
selected molecules inhibited expression of the HAdV-GFP transgene in a significant way (>
90%) like niclosamide (6). Compounds 58 and 60 showed lower percentage of inhibition, 81.8%
and 68.6%, respectively, but they were selected based on their low IC in the plaque assay (0.45
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
μM and 0.30 μM, respectively). As for their IC in entry assay, we observed that derivatives
5
0
whose percentage of inhibition was higher than 90% also showed a low IC50 value, ranging
between 0.2 μM and 2.8 μM. Compounds 58 and 60 were the only ones whose IC values were
5
0
higher than this range (11.20 μM and 9.70 μM, respectively). These results provided us with
preliminary information regarding their potential mechanisms of inhibition since the effect of
these compounds in a step between HAdV attachment to its cellular receptors and HAdV DNA
import into the cell nucleus would result in decreased GFP expression.
Table 4. Inhibition of HAdV in Entry Assay for Selected Compounds with High Selectivity
Index
(
%) Inhibition Entry
Assay (50 μM)
Compound
a
IC Entry Assay (μM)
CC (μM)
5
0
50
6
100.0 ± 0.0
92.4 ± 0.2
100.0 ± 0.0
100.0 ± 0.0
100.0 ± 0.0
100.0 ± 0.0
81.8 ± 8.6
68.6 ± 10.7
98.2 ± 0.5
99.1 ± 1.2
1.22 ± 0.44
1.57 ± 0.97
2.71 ± 0.07
1.67 ± 0.25
2.23 ± 0.58
0.26 ± 0.09
11.20 ± 3.60
9.70 ± 3.80
2.50 ± 0.22
1.38 ± 0.10
22.9 ± 9.8
10.9 ± 0.5
27.1 ± 0.1
35.0 ± 3.7
120.0 ± 30.6
20.6 ± 1.0
200.0 ± 1.9
77.9 ± 10.2
20.2 ± 6.9
174.5 ± 12.1
1
1
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1
1
2
5
4
7
0
8
60
6
7
2
0
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a
Percentage of control HAdV5-GFP inhibition at 50 μM and inhibitory concentration 50% (IC )
5
0
b
at high MOI in an entry assay using the A549 cell line. Cell cytotoxic concentration 50% (CC )
using A549 cell line.
5
0
The next step was to evaluate the effect of these selected compounds on virus replication
using a virus burst assay which measures the production of virus particles. In this assay, A549
cells were infected with the HAdV-5 wild-type virus, and the TCID values of an infection in
0
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8
9
0
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8
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1
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
the presence and absence of the selected compounds were calculated. As summarized in Table 5,
the overall reductions in virus yield varied from as low as 1.8-fold for compound 11 to as high as
9
89-fold for compound 17.
Table 5. Virus Yield Reduction for Selected Compounds with High Selectivity Index
Virus Yield Reduction
Compound
a
(fold)
6
82±35
1.8±0.3
1
1
1
1
3
4
137±71
175±33
17
989±361
385±273
213 ± 78
528 ± 100
10.0 ± 3.8
1.0 ± 0.4
2
5
6
6
0
8
0
2
70
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 the concentration based on CC (11
5
0
was tested at 2 μM, 6, 13, 20 and 62 at 5 μM, 14 at 10 μM, 60 at 20 μM and 17, 58 and 70 at 50
μM). Virus Yield Reduction assay used A549 cell line and the MOI of HAdV was 100 vp/cell.
The results represent means ± SD of triplicate samples from three independent experiments.
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To gain further mechanistic understanding for inhibition we then measured the time
dependence of these analogues addition on their ability to inhibit HAdV infection as an
alternative step toward identifying the specific stage of HAdV replicative cycle that was
inhibited by these compounds. HAdV has been shown to be internalized within 5 min after
binding, to escape the endosome after 15 min, and to attach to the nuclear pore complex after 35-
1
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5
6
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8
9
0
60
4
5 min. We observed that all compounds exhibited a time-dependent decrease in inhibitory
activity although with different patterns (Figure 2). Compounds 13, 20, 62 and 70 inhibited
infection in more than a 50% when they were added either at the beginning of the 60 min
incubation at 4 °C (-60 min) or at 120 min post-infection (p.i.). Compounds 11 and 14 showed
abrupt decreases in their antiviral activity at early time points. Compound 14 lost 20% of
inhibition when added between 5 and 10 min p.i. while compound 11 lost 34% of inhibition
when added between 20 and 40 p.i. In contrast, compound 60 showed a constant decrease from
the beginning of the 60 min incubation at 4 °C losing the 50% inhibition when added after 20
min p.i. and compounds 17 and 58 showed a little bit more but also constant decrease in
inhibition from the beginning of the 60 min incubation at 4 °C showing less than 50% inhibition
after the addition at the moment of the infection (0 min) (Figure 2). Collectively, the results
indicated that these analogues were inhibiting an early step in virus entry occurring after cell
attachment and also that the observed inhibition of HAdV infection were due to an effect on
virus entry and not to an indirect effect on GFP expression.
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6 0
6 2
7 0
4 0
2
0
0
-
6 0
0
5
1 0
2 0
4 0
6 0
1 2 0
T im e (m in )
Figure 2. Effect of selected compounds on HAdV infection at different time points. The
concentrations selected were depending on their CC : 10 μM for derivatives 14, 17, 58, 60 and
0; 5 μM for derivatives 11, 13, 20 and 62; and 1 μM for 6. Line chart represent meansꢀ±ꢀSD of
50
7
duplicate samples.
Impact on HAdV Entry. The HAdV cell entry pathway is a coordinated multi-stage process in
which following attachment and internalization of the HAdV particle, the exposure of protein VI
provokes endosome lysis and subsequent endosomal escape of virions into cytoplasm. Then, the
partially uncoated HAdV capsid is translocated along microtubules towards the nuclear pore
complex where further disassembly occurs and the HAdV genome is finally delivered into the
cell nucleus. If a compound blocks any step of the HAdV entry, this inhibitory effect will be
reflected in the number of HAdV genomes that reach the host nucleus after a synchronized
infection. To further validate whether these compounds could inhibit any of the steps in the entry
pathway, we performed an assay to quantitatively measure the HAdV genome accessibility to the
nucleus. As shown in Figure 3, cells treated with compounds 13, 62 and 70 showed significant
(
p≤0.0001) reductions in the amount of HAdV genomes isolated from the nucleus versus those
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treated with DMSO at 45 min post infection, consistent with the corresponding IC50 values
obtained from the entry assay. Compounds 11, 14, 20 and 60 partially inhibited the accessibility
of HAdV genomes to the nucleus while compounds 17 and 58 showed no significant differences
in the amount of nuclear-associated HAdV genomes versus the control group. These results
indicated that compounds 13, 62 and 70 exerted their main antiviral activity in the early steps of
the HAdV replication cycle while compounds 17 and 58 inhibited later steps after the HAdV
genome is imported into the nucleus.
1
1
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0
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.
.
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0
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8
1
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±
±
±
±
±
±
±
±
±
±
4
4
2
1
±
2
1
0
8
3
6
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4
8
.
5
6
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9
.
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6
.
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.
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5
.
0
.
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1
2
0
9
7
5
6
5
0
4
5
4
6
1
*
***
*
***
*
**
*
*
*
*
1
5 0
*
***
*
**
1 0 0
5
0
0
6
1
3
4
7
0
8
0
2
0
7
O
1
1
1
1
2
5
6
6
S
M
D
Figure 3. Percentage of nuclear-associated HAdV genome of selected compounds.
Concentration of these compounds for the assay was selected based on CC : 11 was tested at 2
50
μM, 6, 13, 20 and 62 at 5 μM, 14 at 10 μM, 60 at 20 μM and 17, 58 and 70 at 50 μM. Bars
represent meansꢀ±ꢀSD of triplicate samples. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤
0
.0001.
Impact on HAdV Replication. To evaluate HAdV DNA replication efficiency we next carried
out quantitative real-time PCR (qPCR) in the presence of these selected compounds in a 24 h
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6
assay to avoid the influence of subsequent rounds of infection occurring 32-36 h post infection.
We used qPCR to quantify the newly synthesized HAdV DNA copies in a single round of
infection as a measure of DNA replication efficiency. As shown in Figure 4, compounds 13, 62
and 70 significantly inhibited HAdV-5 DNA replication by more than 90% in the same way as
that of niclosamide, while compounds 11, 17, 20 and 58 showed low inhibition on DNA
replication when compared to a control treated with the same concentration of DMSO.
Compounds 14 and 60 which did not show a significant inhibition in the nuclear-associate
genomes assay exhibited a significant (p≤0.0001) inhibitory activity, indicating that they may be
targeting the HAdV DNA replication process.
0
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.
1
0
.
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.
.
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3
.
6
.
.
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4
9
1
0
±
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5
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0
±
±
±
0
±
±
±
±
±
±
±
0
5
1
3
1
7
9
8
3
.
5
8
7
6
0
0
.
.
5
.
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.
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.
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.
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8
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0
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1
6
*
***
*
***
*
***
*
**
*
*
1 5 0
*
***
*
***
*
****
1
0 0
5
0
0
6
1
3
4
1
7
0
8
0
2
0
O
1
1
1
2
5
6
6
7
S
M
D
Figure 4. Effect of selected compounds on HAdV DNA replication. Concentration of these
compounds for the assay was selected based on CC : compound 11 was tested at 2 μM,
50
compounds 6, 13, 20 and 62 at 5 μM, 14 at 10 μM, 60 at 20 μM and 17, 58 and 70 at 50 μM.
Bars represent meansꢀ±ꢀSD of triplicate samples. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤
0
.0001.
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In summary, compounds 13, 62 and 70 caused a significant decrease in HAdV DNA
replication, and these activities were possibly attributed to their potent inhibitory effects during
the HAdV entry pathway. Compounds 17 and 58 did not show obvious inhibition against either
during HAdV entry or HAdV DNA replication, indicating that their mechanism of HAdV
inhibition may be related to later steps of the HAdV infection cycle, such as the viral protein
maturation, viral particles assembly, or release processes. Compounds 14 and 60 showed
moderate potency against HAdV-5 DNA replication, meanwhile partially blocking the
accessibility of HAdV genomes to the nucleus. Thus, it is hard to conclude whether these two
compounds had direct impacts on HAdV replication by interfering with a protein involved in this
process or impacting transcription of the immediate early genes, which is essential for
subsequent DNA replication. Despite their partial inhibition on the accessibility of HAdV
genomes to the nucleus, compounds 11 and 20 did not display significant effects on HAdV-5
DNA replication. Based on the current data, it is still unable to determine at what stage
compounds 11 and 20 were acting, and more studies are needed to elucidate their exact
mechanisms of action.
1
1
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1
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0
CONCLUSION
In conclusion, a series of salicylamide derivatives was optimized to identify potent inhibitors of
HAdV infection. Of these, nine compounds (11, 13, 14, 17, 20, 58, 60, 62 and 70) showed
improved anti-HAdV activities with nanomolar to submicromolar IC50 values and high
selectivity indexes (SI > 100), indicating better safety windows, compared to niclosamide.
Moreover, our preliminary mechanistic assays demonstrate that compounds 13, 62 and 70 exert
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their activities in the HAdV entry pathway, while compounds 11, 17, 20 and 58 possibly inhibit
later steps after DNA replication. With respect to compounds 14 and 60, they seem to be
targeting the HAdV DNA replication process although more studies are imperative to elucidate
their specific mechanisms of action. Given the broad antiviral activities of niclosamide, and once
their antiviral properties are validated in vivo, these derivatives may also offer great therapeutic
potential to be developed for other viral infections. The next step in the development of these
new antiviral drugs will be the further extensive evaluation of their efficacy and safety in the
animal model of viral infections as well as exact modes of actions to advance them into potential
clinical applications.
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EXPERIMENTAL SECTION
General Chemistry Information. All commercially available starting materials and
solvents were 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 TLC was carried out employing silica gel 60 F254 plates (Merck, Darmstadt).
Visualization of the developed chromatograms was performed with detection by UV (254 nm).
1
13
NMR spectra were recorded on a Brucker-300 ( H, 600 and 300 MHz; C, 150 and 75 MHz)
1
13
spectrometer. H and C NMR spectra were recorded with 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 positive mode. Melting
points were measured on a Thermo Scientific Electrothermal Digital Melting Point Apparatus
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and uncorrected. Purities of final compounds were established by analytical HPLC, which was
carried out on a Shimadzu HPLC system (model: CBM-20A LC-20AD SPD-20A UV/VIS).
HPLC analysis conditions: Waters Bondapak C18 (300 × 3.9 mm); flow rate 0.5 mL/min; UV
detection at 270 and 254 nm; 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.
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5-Chloro-N-(3-chloro-4-nitrophenyl)-2-hydroxybenzamide (10). To a solution of 5-
chloro-2-methoxybenzoic acid (281 mg, 1.5 mmol) and 3-chloro-4-nitroaniline (200 mg, 1.2
mmol) in 20 mL of toluene was added PCl (286 mg, 2.1 mmol) at r.t. The resulting mixture was
3
stirred at 100 °C for 6 h and concentrated. Then MeOH (20 mL) and H O (5 mL) was added
2
successively, and the mixture was stirred at r.t. for 20 min. The amide intermediate was isolated
by filtration as a pale yellow solid. The amide intermediate was dissolved in 40 mL of DCM, and
BBr (5.0 mL, 5.0 mmol, 1 M in DCM) was added dropwise at 0 °C. The mixture was stirred at 0
3
°
C for 2 h until the reaction was completed monitored by TLC. Then the mixture was diluted
with DCM, washed with H O, and concentrated. The residue was purified by recrystallization
2
(
MeOH/H O) to afford compound 10 as a pale solid (310 mg, 95% in two steps). HPLC purity
2
1
99.1% (t = 19.10 min). H NMR (300 MHz, DMSO-d ) δ 11.39 (s, 1H), 10.82 (s, 1H), 8.20 –
R
6
8
1
.14 (m, 2H), 7.86 (dd, J = 9.0, 2.4 Hz, 1H), 7.80 (d, J = 2.7 Hz, 1H), 7.48 (dd, J = 9.0, 2.7 Hz,
13
H), 7.04 (d, J = 8.7 Hz, 1H). C NMR (75 MHz, DMSO-d ) δ 165.1, 155.8, 143.3, 142.0,
6
133.1, 128.8, 127.4, 126.6, 122.8, 121.6, 120.8, 118.9 (2C). HRMS (ESI) calcd for
+
C H Cl N O , 326.9939 (M + H) ; found, 326.9931.
13
9
2
2
4
5-Chloro-N-(2-fluoro-4-nitrophenyl)-2-hydroxybenzamide (11). Compound 11 (318
mg, 80% in two steps) was prepared by a procedure similar to that used to prepare compound 10.
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The title compound was obtained as a grey solid. HPLC purity 99.3% (t = 18.54 min). H NMR
R
(
300 MHz, DMSO-d ) δ 12.34 (s, 1H), 11.1 (d, J = 2.1 Hz, 1H), 8.66 (t, J = 8.4 Hz, 1H), 8.28 –
6
8.13 (m, 2H), 7.92 (d, J = 2.7 Hz, 1H), 7.51 (dd, J = 8.7, 2.7 Hz, 1H), 7.06 (d, J = 8.7 Hz, 1H).
1
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
C NMR (75 MHz, DMSO-d ) δ 162.7, 155.2, 151.1 (d, J = 245.6 Hz), 142.4 (d, J = 8.8 Hz),
6
1
1
3
33.9, 133.0 (d, J = 10.3 Hz), 129.8, 123.7, 121.1 (d, J = 2.9 Hz), 120.8 (d, J = 0.8 Hz), 119.5,
+
19.2, 111.3 (d, J = 24.5 Hz). HRMS (ESI) calcd for C H ClFN O , 311.0235 (M + H) ; found,
13
9
2
4
11.0226.
5
-Chloro-N-(2-fluoro-5-nitrophenyl)-2-hydroxybenzamide (12). Compound 12 (370
mg, 93% in two steps) was prepared by a procedure similar to that used to prepare compound 10.
1
The title compound was obtained as a pale solid. HPLC purity 99.7% (t = 18.27 min). H NMR
R
(
300 MHz, DMSO-d ) δ 12.25 (s, 1H), 10.94 (s, 1H), 9.24 (dd, J = 6.6, 3.0 Hz, 1H), 8.11 – 8.03
6
(
m, 1H), 7.93 (d, J = 3.0 Hz, 1H), 7.62 (t, J = 9.6 Hz, 1H), 7.50 (dd, J = 8.7, 2.7 Hz, 1H), 7.05 (d,
1
3
J = 8.7 Hz, 1H). C NMR (75 MHz, DMSO-d ) δ 162.9, 155.9 (d, J = 252.6 Hz), 155.3, 143.8
6
(d, J = 2.5 Hz), 133.7, 129.7, 127.2 (d, J = 12.4 Hz), 123.6, 120.4 (d, J = 9.7 Hz), 119.4, 119.1,
1
16.9 (d, J = 3.5 Hz), 116.3 (d, J = 22.1 Hz). HRMS (ESI) calcd for C H ClFN O , 311.0235
13 9 2 4
+
(
M + H) ; found, 311.0229.
5
-Chloro-2-hydroxy-N-(4-nitrophenyl)benzamide (13). Compound 13 (400 mg, 63%
in two steps) was prepared by a procedure similar to that used to prepare compound 10. The title
1
compound was obtained as a yellow solid. HPLC purity 99.4% (t = 18.19 min). H NMR (300
R
MHz, DMSO-d ) δ 11.45 (s, 1H), 10.82 (s, 1H), 8.31 – 8.24 (m, 2H), 8.03 – 7.95 (m, 2H), 7.83
6
1
3
(d, J = 2.7 Hz, 1H), 7.48 (dd, J = 8.7, 2.7 Hz, 1H), 7.04 (d, J = 9.0 Hz, 1H). C NMR (75 MHz,
DMSO-d ) δ 165.0, 155.8, 144.5, 142.7, 133.0, 128.8, 124.9 (2C), 122.8, 120.9, 120.0 (2C),
6
+
1
18.9. HRMS (ESI) calcd for C H ClN O , 293.0329 (M + H) ; found, 293.0318.
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3
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Journal of Medicinal Chemistry
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-Chloro-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-hydroxybenzamide
(14).
Compound 14 (120 mg, 42% in two steps) was prepared by a procedure similar to that used to
prepare compound 10. The title compound was obtained as a white solid. HPLC purity 98.7% (tR
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
=
21.02 min). H NMR (300 MHz, CDCl ) δ 11.42 (s, 1H), 8.59 (d, J = 8.4 Hz, 2H), 7.74 (d, J =
3
1
1
.8 Hz, 1H), 7.62 (dd, J = 8.7, 1.8 Hz, 1H), 7.53 (d, J = 2.4 Hz, 1H), 7.45 (dd, J = 9.0, 2.4 Hz,
1
3
H), 7.02 (d, J = 9.0 Hz, 1H). C NMR (75 MHz, CDCl ) δ 167.3, 160.6, 136.7, 135.5, 127.8 (q,
3
J = 33.6 Hz), 126.6 (q, J = 3.9 Hz), 125.3 (q, J = 3.8 Hz), 125.3, 124.4, 123.9, 123.3 (q, J =
+
2
70.5 Hz), 122.0, 120.9, 115.4. HRMS (ESI) calcd for C H Cl F NO , 349.9962 (M + H) ;
1
4
9
2
3
2
found, 349.9950.
5
-Chloro-N-(3,4-difluorophenyl)-2-hydroxybenzamide (15). Compound 15 (120 mg,
5
0% in two steps) was prepared by a procedure similar to that used to prepare compound 10. The
1
title compound was obtained as a grey solid. HPLC purity 99.9% (t = 19.46 min). H NMR (300
R
MHz, DMSO-d ) δ 11.60 (s, 1H), 10.50 (s, 1H), 7.94 – 7.82 (m, 2H), 7.51 – 7.37 (m, 3H), 7.02
6
1
3
(d, J = 8.7 Hz, 1H). C NMR (75 MHz, DMSO-d ) δ 164.9, 156.4, 148.9 (dd, J = 242.0, 13.1
6
Hz), 145.9 (dd, J = 241.2, 12.7 Hz), 135.1 (dd, J = 9.0, 3.0 Hz), 133.0, 128.4, 122.7, 119.9,
1
19.0, 117.4 (d, J = 17.3Hz), 117.1 (dd, J = 6.0, 3.4 Hz), 109.8 (d, J = 21.4 Hz). HRMS (ESI)
+
calcd for C H ClF NO , 284.0290 (M + H) ; found, 284.0282.
1
3
9
2
2
N-(3,5-Bis(trifluoromethyl)phenyl)-5-chloro-2-hydroxybenzamide (16). Compound
6 (105 mg, 55% in two steps) was prepared by a procedure similar to that used to prepare
1
compound 10. The title compound was obtained as a light yellow solid. HPLC purity 99.5% (t =
R
1
21.53 min). H NMR (300 MHz, CD OD) δ 8.26 (s, 2H), 7.89 (d, J = 2.7 Hz, 1H), 7.62 (s, 1H),
3
13
7
1
.29 (dd, J = 9.0, 2.7 Hz, 1H), 6.87 (d, J = 8.7 Hz, 1H). C NMR (75 MHz, CD OD) δ 167.4,
3
58.7, 141.2, 135.0, 133.2 (q, J = 33.2 Hz, 2C), 129.8, 125.5, 124.6 (q, J = 270.3 Hz, 2C), 121.5
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5
5
5
5
5
5
5
6
(
q, J = 3.2 Hz, 2C), 120.0, 119.1, 118.2 (hept, J = 3.8 Hz). HRMS (ESI) calcd for
+
C H ClF NO , 384.0226 (M + H) ; found, 384.0219.
15
9
6
2
5-Chloro-N-(3-fluoro-5-(trifluoromethyl)phenyl)-2-hydroxybenzamide
(17).
0
1
2
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7
8
9
0
1
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3
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9
0
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0
Compound 17 (100 mg, 33% in two steps) was prepared by a procedure similar to that used to
prepare compound 10. The title compound was obtained as a white solid. HPLC purity 96.7% (tR
1
=
20.86 min). H NMR (300 MHz, CDCl ) δ 11.42 (s, 1H), 8.04 (s, 1H), 7.79 (dt, J = 10.2, 2.1
3
Hz, 1H), 7.58 (s, 1H), 7.52 (d, J = 2.4 Hz, 1H), 7.43 (dd, J = 8.7, 2.4 Hz, 1H), 7.18 (d, J = 7.8
13
Hz, 1H), 7.02 (d, J = 8.7 Hz, 1H). C NMR (75 MHz, DMSO-d ) δ 165.2, 162.0 (d, J = 242.8
6
Hz), 156.2, 141.0 (d, J = 11.3 Hz), 133.1, 131.0 (qd, J = 32.4, 9.8 Hz), 128.6, 123.3 (q, J = 270.6
Hz), 122.7, 120.4, 119.0, 112.9 (quint, J = 3.4 Hz), 110.9 (d, J = 26.1 Hz), 107.8 (dq, J = 28.7,
+
3
.8 Hz). HRMS (ESI) calcd for C H ClF NO , 334.0258 (M + H) ; found, 334.0259.
1
4
9
4
2
N-(3-Fluoro-5-(trifluoromethyl)phenyl)-2-hydroxybenzamide (18). Compound 18
165 mg, 82% in two steps) was prepared by a procedure similar to that used to prepare
(
compound 10. The title compound was obtained as a pale yellow solid. HPLC purity 99.3% (t =
R
1
1
8.99 min). H NMR (300 MHz, CD OD+ CDCl ) δ 7.96 – 7.89 (m, 1H), 7.84 – 7.70 (m, 2H),
3
3
13
7
.40 – 7.31 (m, 1H), 7.05 (d, J = 8.4 Hz, 1H), 6.96 – 6.87 (m, 2H). C NMR (75 MHz, CD OD+
3
CDCl ) δ 168.2, 163.4 (d, J = 244.5 Hz), 159.5, 141.4 (d, J = 11.1 Hz), 135.0, 133.1 (qd, J =
3
33.0, 9.3 Hz), 129.7, 124.0 (qd, J = 270.5, 3.5 Hz), 120.3, 118.0, 113.8 (m), 117.2, 111.8 (d, J =
+
2
6.0 Hz), 108.5 (dq, J = 24.9, 3.8 Hz). HRMS (ESI) calcd for C H F NO , 300.0648 (M + H) ;
1
4
10
4
2
found, 300.0640.
N-(3-Fluoro-5-(trifluoromethyl)phenyl)-2-hydroxy-5-methylbenzamide
(19).
Compound 19 (210 mg, 92% in two steps) was prepared by a procedure similar to that used to
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Page 29 of 68
Journal of Medicinal Chemistry
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prepare compound 10. The title compound was obtained as a white solid. HPLC purity 98.4% (tR
1
=
19.72 min). H NMR (300 MHz, CDCl ) δ 11.28 (s, 1H), 8.09 (s, 1H), 7.82 (dt, J = 10.2, 2.1
3
Hz, 1H), 7.56 (s, 1H), 7.33 – 7.27 (m, 2H), 7.15 (d, J = 7.8 Hz, 1H), 6.98 – 6.93 (m, 1H), 2.35 (s,
1
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
3
3
3
4
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4
4
4
4
4
4
4
4
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5
5
5
5
5
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5
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0
1
2
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4
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6
7
8
9
0
1
2
3
4
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
1
1
H). C NMR (75 MHz, CD OD+ CDCl ) δ 168.1, 163.3 (d, J = 244.4 Hz), 157.1, 141.3 (d, J =
3 3
1.0 Hz), 135.7, 133.0 (qd, J = 33.2, 9.3 Hz), 129.6, 129.6, 124.0 (qd, J = 270.4, 3.5 Hz), 117.7,
16.7, 113.7 (quint, J = 3.7 Hz), 111.7 (d, J = 26.5 Hz), 108.3 (dq, J = 25.0, 3.8 Hz), 20.5.
+
HRMS (ESI) calcd for C H F NO , 314.0804 (M + H) ; found, 314.0797.
1
5
12
4
2
4-Chloro-N-(3-fluoro-5-(trifluoromethyl)phenyl)-2-hydroxybenzamide
(20).
Compound 20 (320 mg, 89% in two steps) was prepared by a procedure similar to that used to
prepare compound 10. The title compound was obtained as a yellow solid. HPLC purity 99.9%
1
(
t = 20.02 min). H NMR (300 MHz, DMSO-d ) δ 11.70 (s, 1H), 10.67 (s, 1H), 7.99 (s, 1H),
R
6
7
2
.93 (dt, J = 11.1, 1.8 Hz, 1H), 7.88 – 7.81 (m, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.07 – 7.01 (m,
1
3
H). C NMR (75 MHz, DMSO-d ) δ 165.6, 162.0 (d, J = 242.7 Hz), 158.1, 141.0 (d, J = 11.3
6
Hz), 137.5, 131.1, 130.0 (qd, J = 25.0, 9.9 Hz),123.3 (qd, J = 270.9, 3.5 Hz), 119.4, 118.0, 116.7,
1
12.8 (m), 110.8 (d, J = 26.0 Hz), 107.7 (dq, J = 25.0, 3.6 Hz). HRMS (ESI) calcd for
+
C H ClF NO , 334.0258 (M + H) ; found, 334.0251.
14
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4
2
3-Chloro-N-(3-fluoro-5-(trifluoromethyl)phenyl)benzamide (21). To a solution of 3-
chlorobenzoic acid (100 mg, 0.64 mmol) and 3-fluoro-5-(trifluoromethyl)aniline (137 mg, 0.77
mmol) in toluene (15 mL) was added PCl (132 mg, 0.96 mmol). The resulting mixture was
3
stirred at 100 °C for 24 h, and then concentrated. The residue was purified by column
chromatography (Hex/EtOAc = 5/1 to 3/1) to afford compound 21 as a white solid (190 mg,
1
9
7
3%). HPLC purity 99.9% (t = 19.73 min). H NMR (300 MHz, CDCl ) δ 8.83 (s, 1H), 7.80 –
R
3
.70 (m, 2H), 7.66 (dt, J = 7.8, 1.2 Hz, 1H), 7.59 (s, 1H), 7.50 – 7.40 (m, 1H), 7.31 (t, J = 7.8 Hz,
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