Discovery and Structural Optimization of 4-(Aminomethyl)benzamides as Potent Entry Inhibitors of Ebola and Marburg Virus Infections

Article abstract of DOI:10.1021/acs.jmedchem.0c00463

The recent Ebola epidemics in West Africa underscore the great need for effective and practical therapies for future Ebola virus outbreaks. We have discovered a new series of remarkably potent small molecule inhibitors of Ebola virus entry. These 4-(aminomethyl)benzamide-based inhibitors are also effective against Marburg virus. Synthetic routes to these compounds allowed for the preparation of a wide variety of structures, including a conformationally restrained subset of indolines (compounds 41-50). Compounds 20, 23, 32, 33, and 35 are superior inhibitors of Ebola (Mayinga) and Marburg (Angola) infectious viruses. Representative compounds (20, 32, and 35) have shown good metabolic stability in plasma and liver microsomes (rat and human), and 32 did not inhibit CYP3A4 nor CYP2C9. These 4-(aminomethyl)benzamides are suitable for further optimization as inhibitors of filovirus entry, with the potential to be developed as therapeutic agents for the treatment and control of Ebola virus infections.

Full text of DOI:10.1021/acs.jmedchem.0c00463

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Article
Discovery and Structural Optimization of 4-(Aminomethyl)benzamides
as Potent Entry Inhibitors of Ebola and Marburg Virus Infections
Irina N Gaisina, Norton P. Peet, Letitia Wong, Adam M. Schafer, Han Cheng,
Manu Anantpadma, Robert A. Davey, Gregory RJ Thatcher, and Lijun Rong
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00463 • Publication Date (Web): 03 Jun 2020
Downloaded from pubs.acs.org on June 4, 2020
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Discovery and Structural Optimization of 4-(Aminomethyl)benzamides as Potent Entry
Inhibitors of Ebola and Marburg Virus Infections
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Irina N. Gaisina,*,†,‡ Norton P. Peet,*,‡ Letitia Wong,‡ Adam M. Schafer,ǁ Han Cheng, ǁ Manu
Anantpadma,⊥,§ Robert A. Davey,⊥,§ Gregory R. J. Thatcher†, and Lijun Rong*,ǁ.
†
UICentre (Drug Discovery @ UIC) and Department of Pharmaceutical Sciences, College of
Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612,
United States
‡ Chicago BioSolutions Inc., 2242 W Harrison Street, Chicago, Illinois 60612, United States
ǁ
College of Medicine, Department of Microbiology and Immunology, University of Illinois at
Chicago, 909 S Wolcott Ave, Chicago, Illinois 60612, United States
⊥ Texas Biomedical Research Institute, 8715 W Military Drive, San Antonio, Texas 78227, United
States
§ Department of Microbiology, Boston University, 620 Albany Street, Boston, Massachusetts
02118, United States
ABSTRACT: The recent Ebola epidemics in West Africa underscore the great need for effective
and practical therapies for future Ebola virus outbreaks. We have discovered a new series of
remarkably potent small molecule inhibitors of Ebola virus entry. These 4-
(aminomethyl)benzamide-based inhibitors are also effective against Marburg virus. Synthetic
routes to these compounds allowed for the preparation of a wide variety of structures, including a
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conformationally restrained subset of indolines (compounds 41-50). Compounds 20, 23, 32, 33,
and 35 are superior inhibitors of Ebola (Mayinga) and Marburg (Angola) infectious viruses.
Representative compounds (20, 32 and 35) have shown good metabolic stability in plasma and
liver microsomes (rat and human), and 32 did not inhibit CYP3A4 nor CYP2C9. These 4-
(aminomethyl)benzamides are suitable for further optimization as inhibitors of filovirus entry, with
potential to be developed as therapeutic agents for the treatment and control of Ebola virus
infections.
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INTRODUCTION
Ebola virus (EBOV) and Marburg virus (MARV) are Category A emerging infectious agents
because of the possibility of an aerosol mode of transmission1, their high fatality rate, and the
unpredictable nature of the outbreaks.2-4 These viruses can cause a highly lethal hemorrhagic fever
with a 50-90% mortality rate in infected patients.2-3, 5 The 2014-2016 Ebola epidemic in West
Africa, which was the largest recorded outbreak, and the recent 2018 epidemic in the Democratic
Republic of Congo, which is the tenth outbreak since 1976 and the second biggest Ebola epidemic,
underscore the need for drug discovery and development efforts to produce effective treatments.
Several Ebola-specific vaccines have shown promising efficacy in animal or nonhuman primate
models; however, the production process for vaccines generally takes 6 to 36 months and is
considered impractical during a rapidly spreading EBOV outbreak. With the availability of
Merck’s investigational Ebola vaccine V920 (rVSV-ZEBOV),6 which is now FDA-approved as
Ervebo®, the number of cases has declined to date; however, the outbreak is not yet over. Thus,
there is still an urgent medical need to develop efficacious and broad-spectrum small molecule
therapeutic agents that are stable, cost-effective and easy to use, and most importantly, agents that
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can be readily available in an outbreak zone. Such agents could be used alone or in combination
with vaccines in future infections.
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Some representative antifilovirus compounds are shown in Figure 1. This set includes nucleosides
BCX4430 and C-c3-Ado;7-8 a rhodanine derivative LJ-001;9 compound 3.47 with a lipophilic
adamantyl group;10 polyaromatic amines FGI-103, FGI-104 and FGI-106;11-13 and our previously
published hits, including benzodiazepine-based “compound 7”14 and recently described coumarin-
based CBS112915. A group of known drugs with potetntial for repurposing as anti-Ebola agents is
the class of estrogen receptor modulators,16 as exemplified by toremefine17, which is displayed in
Figure 1. Toremefine has been shown to interact with and destabilize the Ebola virus
glycoprotein.18 Only a few compounds have advanced to clinical trials. A pyrazinecarboxamide
derivative T-705 (faviprivir)19-20 has shown no efficacy in patients with high levels of Ebola virus
in the blood. CMX001 (brincidofovir), a prodrug of the known antiviral medication cidofovir,
received an authorization from the U.S. FDA as an emergency investigational new drug, but was
subsequently withdrawn in clinical trials, due to the lack of convincing preclinical data. GS-5734
(remdesivir)21, a nucleotide analog was the only small molecule drug tested in the recent 2018
Kivu Ebola outbreak, but it did not demonstrate significant efficacy.
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NH2
HO
NH2
O
H
N
N
N
N
N
O
O
O
O
N
N
N
O
S
N
N
NH
H
HO
O
S
OH
OH
9
HO
HO
BCX4430
N
C-c3-Ado
NH
NH2
LJ-001
3.47
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N
HN
N
H
N
OH
O
N
O
H2N
N
HN
OH
NH
NH
FGI-103
FGI-104
FGI-106
Cl
N
CF2CHF2
O
N
N
N
H2N
OH
O
N
N
O
O
Cl
CBS1129
toremifene
Compound 7
NH2
O
O
O
O
N
O
H
N
N
N
O
N
(14)
F
N
N
P
O
P
O
NH2
OH
O
O
N
N
O
O
OH
OH
HO
GS-5734
HO
NH2
CMX001
T705
Figure 1. Small molecule antifilovirus compounds.
The EBOV and MARV genomes contain at least seven genes, including the gene that encodes
the viral envelope glycoprotein (GP).3 The GP consists of two subunits, GP1 and GP2. The GP1
subunit is responsible for receptor binding and host tropism, while the GP2 subunit mediates
viral/cell membrane fusion.3, 22-25 Blocking GP fusion prevents entry into the cell and downstream
replication processes. The structural studies of EBOV/MARV GPs, alone and in complex with
receptors/antibodies/inhibitors,26-31 provide insights into the elucidation of the filoviral entry
mechanism and development of antifiloviral therapeutics. Recent work on EBOV GP in complex
with toremifene18 suggests a novel binding mechanism. Toremifene was shown to bind to GP
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directly and block GP-mediated fusion. This finding has provided validation for the continued
development of the 4-(aminomethyl)benzamide antiviral agents reported herein.
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RESULTS AND DISCUSSION
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Identification of 4-(Aminomethyl)benzamides as Antifilovirus Agents. One of the challenges
of working with highly pathogenic viruses such as EBOV/MARV is that biosafety level 4 (BSL-
4) facilities are required to handle the infectious viruses. For the study of GP fusion of many
enveloped viruses, this obstacle can be circumvented by a surrogate system called viral
pseudotyping.32 This surrogate system has been widely utilized by virologists to study the entry
mechanisms of highly pathogenic viruses and to identify and develop antiviral therapeutics, in
our31 laboratories and other32-35 laboratories. It is generally accepted that pseudotyped assays for
filoviruses and other pathogenic viruses are valid surrogate assays. Thus, many of the antifilovirus
compounds displayed in Figure 1 have been identified using a viral pseudotyped assay followed
by validation with infectious virus. A recent publication from USAMRIID reports a “high degree
of correlation” between infectious BSL-4 neutralization assays and pseudotyped BSL-2
fluorescence assays.33 In this study, we used this system to rapidly identify potent EBOV and
MARV entry inhibitors. We have screened a ChemBridge Small Molecule Library of 10,000
compounds for filovirus entry inhibitors using a robust, cell-based high-throughput screening
(HTS) protocol.34 Several hit compounds, including a set of the substituted 4-
(aminomethyl)benzamides, have been shown to inhibit entry of both EBOV and MARV
pseudovirions35-36. To validate our initial hits, we tested them against wild type EBOV (1976
Mayinga variant) and MARV (Lake Victoria, 2005 Angola) strains in Vero cells.37 One of the 4-
(aminomethyl)benzamides, designated as CBS1118 (Figure 2), was found to have EC50 values <
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10 µM for both EBOV and MARV, suggesting that this compound has broad-spectrum
antifiloviral activity.
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Figure 2. Structural Optimization of 4-(Aminomethyl)-benzamides as Antifilovirus Agents.
Structure-Activity Relationship (SAR) Development. The structural modifications on the hit
compound CBS1118 were implemented to improve selectivity and potency toward EBOV or
MARV through the addition of appropriate substituents in the amide portion of the molecule, the
aromatic region or the tertiary amine region as outlined in Figure 2. Upon medicinal chemistry and
SAR optimization, we have assembled several sets of modified 4-(aminomethyl)benzamides.
Scheme 1. Synthesis of 4-(Aminomethyl)benzamides 5-35 and 4-(Aminomethyl)thiophenes 38
and 39 a.
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R1
R1
R1
d, or
d then e
O
O
O
OMe
3a-f
NH
+
R2
N
R2
N
a
b, c
X
X
OMe
Cl
2a-c
4a-f
1a-e
9
X
X
1
. R = H, R2 = H, X = CH2;
. R1 = H, X = 4-CH2Br;
. R1 = H, X = 3-CH2Br;
. R1 = F, X = 4-CH2Br;
. R1 = H, X = 4-CH(CH3)Br;
. R1 = H, X = 4-COCl
a
a
a
. X = CH2;
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1
b
b
. R = F, R2 = H, X = CH2;
b
c
. X = O;
. R = H, R2 = CH3, X = CH2;
(para-)
(meta-)
1
c
. X = N-CH3
c
1
. R = H, R2 = H, X = O;
d
d
1
. R = H, R2 = H, X = N-CH3
e
f
e
. R1 = H, R2 = H, X = CH2;
R1
R2
N
O
Me
O
O
O
N
HN
HN
HN
N
N
HN
11-24
O
R
R
30-31
OMe
25-27
5-9
X
30
31
: R =
O
: R1 = R2 = H
: R1 = F, R2 = H;
: R1 = H, R2 = Me
25
11
: R = 4-CO2H;
5
6
7
8
9
: X = CH2, 4-CO2Me;
: X = O, 4-CO2Me;
: X = N-CH3, 4-CO2Me;
: X = CH2, 3-CO2Me
: X = CH2, 4-CO2Me
O
26
27
12
13
14
15
16
17
18
19
20
21
22
23
24
: R = 4-SO2Me;
: R =
(para-)
(meta-)
O
: R = 4-SO2NHMe;
: R = 4-NHSO2Me;
: R = 4-CN;
O
R1
R2
: R = 4-CF3;
O
N
O
N
X
HN
: R = 3-CF3;
O
O
HN
: R = 3-Cl,4-CF3;
: R = 3-CF3,4-Cl;
: R = 3-Cl,4-SCF3;
: R = 4-cyclo-Pr;
: R = 4-i-Pr;
32,33
OMe
N
HN
(n)
10
32
33
: X= CH2;
: X = O
28,29
: R1 = R2 = H, n = 2
: R1 = R2 = F, n = 1
O
R3
N
28
29
R1
R2
: R = 2,4-di-t-Bu
N
HN
: R =
CN
34,35
X
: R1 = R2 = F, R3 = H, X = CH2;
: R1 = H, R2 = Me, R3 = CF3, X = O
34
35
O
H2N
O
NH
O
S
HN
S
R
R
Cl
d
N
a-c
N
S
38,39
OCH3
37
Br
36
38
39
: R = CO2Me;
: R = CF3
aReagents and conditions: a). NEt3, CH2Cl2; b). NaOH, MeOH-THF-H2O; c). SOCl2, reflux; d) NEt3, CH2Cl2;
e). LiOH-NaOH, THF-MeOH
Our general method for the synthesis of 4-(aminomethyl)benzamides is shown in Scheme 1.
Treatment of 4-(bromomethyl)benzoic acid esters 1a-e with cyclic amines such as piperidine (2a),
morpholine (2b) and 4-methylpiperazine (2c) cleanly produced the 4-(aminomethyl)benzoic acid
esters 3a-f. Basic hydrolysis of these esters gave the intermediate benzoic acids, which were
subsequently converted to the benzoyl chlorides 4a-f by heating at reflux in thionyl chloride. The
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benzoyl chlorides 4a-f were coupled with a variety of anilines by utilizing the traditional Schotten-
5
6
Baumann procedure to provide the 4-(aminomethyl)benzamides 5-35.
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Scheme 2. Synthesis of the indoline series of 4-(aminomethyl)benzamides 41-50 and heterocyclic
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analogs 52-55.
R2
R1
N
O
R1
N
O
N
NEt3, CH2Cl2
+
R3
R3
N
H
Cl
4a,c
40a-h
41-50
R2
3
2
3
1
1
2
a
b
. R = H; R = 5-CO2CH3
a
41
42
43
44
45
46
47
48
49
50
. R = H;
: R = R = H; R = 5-CO2CH3
2
3
1
1
2
3
. R = H; R = 5-CF3
b
. R = Me
: R = R = H; R = 5-CF3
2
3
1
2
3
c
d
. R = H; R = 6-CF3;
: R = R = H; R = 6-CF3;
2
2
1
2
2
. R = H; R = 6-Cl;
: R = R = H; R = 6-Cl;
2
3
1
2
3
e
. R = H; R = 6-F;
: R = R = H; R = 6-F;
2
3
1
2
3
f
. R = di-Me; R = H;
: R = H, R = di-Me; R = H;
2
3
1
2
3
g
. R = cyclo-Pr; R = H;
: R = H, R = cyclo-Pr; R = H;
2
3
1
2
3
h
. R = H; R = 6-CF3;
: R = Me, R = H; R = 6-CF3;
i. R2 = H; R3 = 5-t-Bu;
1
2
3
: R = Me, R = H; R = 5-t-Bu;
2
3
1
2
3
j
. R = t-Bu; R = H
: R = Me, R = t-Bu; R = H
O
O
H
R1
NEt3, CH2Cl2
N
+
R2
N
N R1
R2
N
Cl
52-54
4a
51a-c
H
H
N
1
2
1
2
a
: R = H; R =
N
52
53
54
: NR R =
N
CF3
N
CF3
1
2
1
2
b
c
: NR R =
: R R =
N
N
O
O
OCH3
CF3
OCH3
CF3
1
2
1
2
: R R =
: NR R =
N
N
O
NEt3, CH2Cl2
S
N
37 + 40a
N
CO2CH3
55
The thiophene analogs 38 and 39 were synthesized in a similar manner starting from
commercially available methyl 5-(bromomethyl)thiophene-2-carboxylate (36) through the
intermediate thiophene-2-carbonyl chloride (37) as shown in Scheme 1.
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To create conformationally restricted versions of our scaffold, indolines and other related
heterocycles were incorporated into the amide portion of the molecule. These compounds were
prepared as shown in Scheme 2. Benzoic acids 4a,c were coupled with substituted indolines 40a-
h, again using the Schotten-Baumann procedure, to provide compounds 41-50. Using an analogous
procedure, 4-(piperidin-1-ylmethyl)benzoyl chloride (4a) was treated with three different amines
51a-c to provide amides 52-54. The thiophene compound 55, bearing a 5-carboethoxy substituted
indoline, was synthesized as a constrained analog of compound 38. Conformational restraints can
produce compounds that bind with lower energy barriers by eliminating the entropic
preorganization component.
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All synthesized compounds were tested in A549 cells infected by a HIV/MARV-GP or
HIV/EBOV-GP pseudotype virus containing a luciferase reporter gene and biological data are
summarized in Tables 1-3. Toremifene, a small molecule entry inhibitor previously shown to bind
and destabilize GP,18 was used as positive control in this assay. Our SAR studies described here
mainly focused on the potency of these compounds for EBOV infection inhibition. It started with
the replacement of the piperidine ring in ester 5 (HTS hit CBS1118) with morpholine and N-
methylpiperazine, to vary the amine substituent. Both of these modifications decreased potency;
compounds 6 and 7 showed only 57% and 29% inhibition at 12.5 µM, respectively. Regioisomer
8, with the methyl ester in the meta-position, was completely inactive. 3-
(Aminomethyl)benzamide 9 was much weaker than the original hit compound, 4-
(aminomethyl)benzamide 5 (only 13% inhibition vs 74%, respectively). Also, the introduction of
a carbonyl linker, such as in compound 10, was unfavorable (12.9% inhibition).
Next, we investigated replacements of the potentially metabolically unstable methyl ester with
different bioisosteres. Among the monosubstituted analogs 11-17, only compounds 16 and 17,
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bearing a lipophilic trifluoromethyl substituent in the para- and meta-positions, were more potent
than the hit compound 5 (EC50 = 3.87 and 2.97 µM, respectively, vs 9.86 µM for compound 5),
while the replacements with more polar or negatively charged groups provided only less potent
inhibitors. Introduction of an additional chlorine group in the aromatic ring was well-tolerated and
enhanced the potency of compounds 18 (2.34 µM) and 19 (1.52 µM) and its SCF3 analog 20 (1.27
µM) over the corresponding monosubstituted compounds 16 and 17 (Table 1).
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33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1. Antifiloviral Activity of 4-(Aminomethyl)benzamides 5-8 and 10-27.
Ebola pseudovirus
Marburg pseudovirus
A549 cells
A549 cells
N
Structure
a
a
%
EC50
(µM)
SIb
%
EC50
(µM)
inhibition
inhibition
O
O
O
O
O
O
N
HN
N
X
HN
HN
O
OMe
N
OMe
OMe
10
5-8
9
5
6
7
8
9
10
X = CH2; para-
X = O; para-
X = N-Me; para-
X = CH2; meta-
74.0
57.2
29.4
0
13.0
12.9
9.86 ± 2.14
15
90.7
68.8
30.3
0
5.90
0
0.53 ± 0.09
-
-
-
-
-
-
-
-
-
-
R1
R2
N
O
O
HN
N
HN
11-24
R
25-27
11
12
13
14
15
16
17
18
19
20
21
22
23
R = 4-CO2H
R = 4-SO2Me
R = 4-SO2NHMe
R = 4-NHSO2Me
R = 4-CN
3.12
0
0
3.91
0
89.9
82.2
99.7
99.6
99.9
74.3
93.1
99.5
-
-
-
-
10.3
17.4
3.30
6.81
12.4
79.3
57.5
98.5
98.2
99.8
66.2
67.5
85.0
-
-
-
-
-
-
R = 4-CF3
R = 3-CF3
3.87 ± 0.73
2.97 ± 0.20
2.34 ± 0.57
1.52 ± 0.28
1.27 ± 0.38
4.57 ± 1.50
2.87 ± 0.38
0.48 ± 0.17
9
15
9
7
5
22
10
76
5.70 ± 1.56
1.41 ± 0.32
2.05 ± 0.29
1.24 ± 0.24
1.61 ± 0.15
2.65 ± 1.64
2.10 ± 1.24
2.34 ± 0.64
R = 3-Cl, 4-CF3
R = 3-CF3, 4-Cl
R = 3-Cl, 4-SCF3
R = 4-cyclo-Pr
R = 4-i-Pr
R = 2,4-di-t-Bu
CN
24
96.8
0.73 ± 0.29
19
94.0
1.63 ± 0.42
R = 4-
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25
26
27
R1 = R2 = H
R1 = F, R2 = H
R1 = H, R2 = Me
toremifene
98.1
96.1
99.3
0.99 ± 0.18
0.38 ± 0.12
0.33 ± 0.08
0.09 ± 0.04
101
382
390
299
73.4
71.0
88.9
0.96 ± 0.32
1.33 ± 1.23
0.85 ± 0.10
a
Dose-response studies were conducted to determine EC50 values for those compounds that
showed more than 70% inhibition at 12.5 uM concentration; EC50 values were calculated by four-
parameter dose-response curve-fitting in GraphPad. Results are from three replicates. Percent
inhibition errors are estimated to be <10%; EC50 data are presented as mean ± standard deviation
(SD). b Selectivity index is the ratio of CC50/EC50, where CC50 is cytotoxicity assessed by utilizing
the “CellTiter 96 aqueous nonradioactive cell proliferation assay” (Promega, Madison, WI).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Among the alkyl substituted analogs, the potency correlated with the size of the alkyl group as
follows: 4-cyclopropyl (21, EC50 = 4.57 µM) < 4-isopropyl (22, 2.87 µM) < cyclohexyl (25, 0.99
µM) < 2-cyclopentylacetonitrile (24, 0.73 µM) < di-tert-butyl (23, 0.48 µM). We chose to maintain
the bulky cyclohexyl group to explore some further modifications targeting the benzyl ring, such
as introduction of fluorine, as in compound 26 (0.38 µM), and introduction of a methyl group in
the linker, as in compound 27 (0.33 µM). All three analogs 25-27 showed excellent activity against
EBOV pseudovirus and remarkable potency against pseudoviral MARV (Table 1), coupled with
improved selectivity. The selectivity index (SI) in Table 1-3 is a ratio between cytotoxicity (CC50)
and antiviral activity of the tested compounds. Additional modifications targeted the aniline
portion of the scaffold and comprised replacement of the aromatic ring with more flexible linkers,
such as alkyl or alkoxy chains between the cyclohexane moiety and the amide. Replacement with
a two-carbon linker, as in 28, led to a 2.3-fold drop in potency, while a shorter linker, as in 29, or
insertion of an oxygen, as in the ether 30 or acetal 31, caused greater reductions in potency in both
pseudoviral assays (Table 2).
Compounds 32 and 33, bearing an adamantyl group in the para-position of the aniline ring and
piperidine and morpholine rings attached to the benzylic portion of the molecule, respectively,
were both found to possess excellent potency, with EC50 < 100 nM. Since enhancement of potency
appeared to be correlated to the lipophilicity of the substituents on the phenyl ring, our objective
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was to strike a balance between potency and drug-likeness, including a drug-like LogP value. The
lipophilicity of our new lead compound 34 was decreased by replacement of the adamantyl group
with a 4,4-difluoropiperidinyl group: compound 34 (EC50 value < 1 µM) has a lower cLogP value
of 4.71 as compared to the adamantane analog 32 (cLogP = 5.97). The best compound in this series
was compound 35, with an EC50 value of 12 nM. This combination of a trifluoromethyl group in
the meta-position and 4-methylpiperidinyl in the para-position of the aniline moiety, with
morpholine as the cyclic amine, appeared to be the best combination of substituents with respect
to potency. Compound 35 displays low cytotoxicity with an excellent SI value.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. Antifiloviral Activity of 4-(Aminomethyl)benzamides 27-35 and 5-
(Aminomethyl)thiophenecarboxamides 38-39.
Ebola pseudovirus
Marburg pseudovirus
A549 cells
A549 cells
N
Structure
a
a
%
EC50
(µM)
SIb
%
EC50
(µM)
inhibition
inhibition
R1
R2
Me
N
O
O
HN
R
N
HN
(n)
29
28
(n = 2),
(n = 1)
30 31
,
28
29
n = 2, R1 = R2 = H
90.9
42.9
2.32 ± 0.47
-
25
27.3
23.0
7.74 ± 2.33
-
n = 1, R1 = R2 = F
O
30
31
52.4
56.0
-
-
42.5
37.2
-
R =
O
-
O
R =
O
O
R3
O
R1
R2
N
N
HN
N
X
HN
N
R
HN
S
X
38,39
34,35
32,33
32
33
X = CH2
X = O
99.4
99.0
0.035 ± 0.02
0.018 ± 0.01
231
5437
99.0
72.5
0.38 ± 0.09
2.79 ± 4.78
R1 = R2 = F, R3 =
H, X = CH2
R1 = H, R2 = Me,
R3 = CF3, X = O
CO2Me
34
35
99.7
99.3
0.82 ± 0.47
69
88.8
92.3
1.88 ± 0.34
0.18 ± 0.19
0.012 ± 0.01
2088
38
39
33.8
80.2
-
27.2
70.0
-
CF3
toremifene
5.60 ± 0.67
0.09 ± 0.04
9
299
3.65 ± 0.76
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a
Dose-response studies were conducted to determine EC50 values for those compounds that showed
more than 70% inhibition at 12.5 uM concentration; EC50 values were calculated by four-parameter dose-
response curve-fitting in GraphPad. Results are from three replicates. Percent inhibition errors are
estimated to be <10%; EC50 data are presented as mean ± SD. b Selectivity index is the ratio of CC50/EC50,
where CC50 is cytotoxicity assessed by utilizing the “CellTiter 96 aqueous nonradioactive cell
proliferation assay” (Promega, Madison, WI).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
On the other hand, replacement of the phenyl ring with thiophene was detrimental to activity
and compounds 38 and 39 showed only insignificant inhibition at the test concentration. Perhaps
this loss of activity with the thiophenes reflects an unfavorable change in the trajectory of the two
aromatic substituents on the thiophene ring compared with the phenyl ring.
Table 3 shows the indoline series compounds, which are amide-constrained versions of the
benzamide 5. Indoline 41, having a methyl ester at the 5-position, was less potent than the
unconstrained version (41.7% vs 74% inhibition, respectively). While introduction of a
trifluoromethyl group at the 6-position afforded compound 43 with augmented potency (EC50 =
1.68 µM), its 5-regioisomer 42 showed only minor improvement over the ester 42 (56.4%
inhibition at 12.5 µM vs 41.7%, respectively). Among the two halogenated analogs 44 and 45, the
6-chloro substituted compound 44 showed enhanced potency (EC50 = 2.22 µM). The introduction
of gem-dimethyl groups in the 3-position of the indoline ring yielded compound 46 with moderate
potency; its cyclopropyl analog 47 was less active. The bulky tert-butyl group at the 3-position
improved activity, producing analog 50 with an EC50 value of 0.16 µM. Interestingly, compound
48, bearing a methyl group at the benzyl methylene position, was similar in potency to its
desmethylated analog 43.
The final iteration of optimization (Table 3) was to replace the anilide aromatic ring with
various heterocycles. Pyridine in 52 represents a simple bioisosteric replacement of carbon with
nitrogen in compound 16, which led to a drastic decrease in potency (5.4% vs 89.9% inhibition).
Tetrahydroquinoline 53 was less potent than its indoline analog 41 and so was indole 54.
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5
6
Replacement of the core phenyl ring with thiophene as in compound 55 had a lesser (negative)
impact on the potency than in the aniline series.
7
8
9
Table 3. Antifiloviral Activity of 4-(Aminomethyl)benzamides 41-50, 52-54 and 5-
(Aminomethyl)thiophenecarboxamide 55.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Ebola pseudovirus
A549 cells
Marburg pseudovirus
A549 cells
N
Structure
%
%
inhi
bition
a
a
EC50
EC50
inhi
(µM)
bition
SIb
(µM)
R1
N
O
N
R3
41-50
R2
41
R1 = R2 = H, R3 = 5-CO2Me
R1 = R2 = H, R3 = 5-CF3
R1 = R2 = H, R3 = 6-CF3
R1 = R2 = H, R3 = 6-Cl
41.7
56.4
83.7
81.5
20.1
87.3
74.5
95.6
99.8
99.9
-
-
51.2
49.6
59.2
42.4
13.5
21.4
34.4
76.9
98.1
92.8
-
-
42
43
44
45
46
47
48
49
50
1.68 ± 0.43
2.22 ± 0.39
-
2.22 ± 1.02
-
1.56 ± 0.69
2.05 ± 0.33
0.16 ± 0.07
38
14
1.46 ± 0.41
2.31 ± 0.37
-
2.90 ± 0.87
-
1.73 ± 0.88
3.09 ± 0.53
3.18 ± 1.33
R1 = R2 = H, R3 = 6-F
R1 = H, R2 = di-Me, R3 = H
R1 = H, R2 = cyclo-Pr, R3 = H
R1 = Me, R2 = H, R3 = 6-CF3
R1 = Me, R2 = H, R3 = 5-t-Bu
R1 = Me, R2 = t-Bu, R3 = H
33
21
9
224
O
O
N
N
S
N
R
52-54
55
CO2CH3
HN
N
CF3
52
53
5.4
-
-
9.4
0
-
-
N
R =
R =
CO2CH3
CF3
25.1
N
54
55
0
-
-
0
0
-
-
R =
38.9
toremifene
0.09 ± 0.04
299
a Dose-response studies were conducted to determine EC50 values for those compounds that showed
more than 75% inhibition at 12.5 uM concentration; EC50 values were calculated by four-parameter
dose-response curve-fitting in GraphPad. Results are from three replicates. Percent inhibition errors
b
are estimated to be <10%; EC50 data are presented as mean ± SD. Selectivity index is the ratio of
CC50/EC50, where CC50 is cytotoxicity assessed by utilizing the “CellTiter 96 aqueous nonradioactive
cell proliferation assay” (Promega, Madison, WI).
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The HTS hit, compound 5, surprisingly, was more potent toward Marburg virus than Ebola
virus, as shown in Table 1. The variety of structural modifications described herein, especially the
incorporation of bulky alkyl or cycloalkyl substituents, gave compounds either almost equipotent
against both viruses, or more potent against Ebola virus. It has been our experience that structural
changes which enhance activity for Ebola virus generally do the same for Marburg virus, even
though the potencies may differ.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4. Antiviral Activity of Selected 4-(Aminomethyl)benzamides in the Wild Type EBOV
and MARV Infection Inhibition Assays.
EBOV (HeLa)
MARV (HeLa)
N
EC50 (µM)a
2.83b (>25)
1.12 ± 0.21
0.59 ± 0.16
1.04 ± 0.50
1.80 ± 0.51
0.11 ± 0.03
0.83 ± 0.19
0.31 ± 0.07
3.22 ± 0.39
2.08 ± 0.14
0.87 ± 0.21
0.67 ± 0.02
SId
35b
14
227
128
54
202
134
58
39
19
137
51
EC50 (µM) a
5
NDc
20
23
26
27
32
33
35
43
49
50
2.40 ± 0.17
2.00 ± 0.44
16.55 ± 0.96
5.56 ± 0.75
1.23 ± 0.12
0.52 ± 0.18
0.82 ± 0.16
8.17 ±0.29
3.38 ± 0.64
6.32 ± 0.09
3.25 ± 0.42
toremifene
a
EC50 values were calculated by four-parameter dose-response curve-
fitting in GraphPad. Results are from three replicates. EC50 data are
presented as mean ± SD. bTested in Vero cells. cND denotes dose response-
dependent inhibition not detected within the range of compound
concentrations tested. d Selectivity index is the ratio of CC50/EC50, where
CC50 is cytotoxicity assessed by utilizing the “CellTiter 96 aqueous
nonradioactive cell proliferation assay” (Promega, Madison, WI).
All new compounds with promising activity in the pseudovirus assay were evaluated in wild
type EBOV and MARV virus assays in HeLa cells. As shown in Table 4, several compounds have
greatly improved potency against EBOV, combined with low cytotoxicity. For example,
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compounds 32 and 35 display EC50 values of 0.11 µM and 0.31 µM, respectively, with excellent
SI values. In addition, they both retained good potency against MARV (EC50 = 1.23 µM for 32
and 0.82 µM for 35). These results imply that the newly developed 4-(aminomethyl)benzamides
are broad-spectrum antifiloviral agents that target the GP-mediated entry process. In general, there
was a good agreement with the EC50 values for a compound between the HIV-based pseudoviral
entry assay and the infectious EBOV virus assays, as shown in Tables 1-4. However, discrepancies
were observed for some compounds, which is not surprising since these are different bioassays
and the discrepancies demonstrate the need for validating antiviral activity for the lead compounds
with infectious EBOV and MARV. Remarkably, the HTS hit 5 (CBS1118), which displayed an
EC50 value of 2.83 µM against the Ebola strain in Vero E6 cells, was found to be inactive when
tested in the HeLa cell line. This inconsistency could be attributed to a nonspecific esterase activity
in HeLa cells resulting in metabolism of the parent ester to the inactive carboxylic acid 11. In
addition to the higher potency and selectivity displayed by our new hit and lead compounds (Table
4), we have attempted to remove metabolic liabilities.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Mechanism of action (MOA) studies of the 4-(aminomethyl)benzamides with filovirus
GPs. The recently published co-crystal structure of GP with toremifene shows that this ligand
binds in a cavity between the attachment (GP1) and fusion (GP2) subunits at the entrance to a large
tunnel that links with equivalent tunnels from the other monomers of the trimer at the three-fold
axis (Figure 3a,b).18 The key interactions within the binding site are formed between the aromatic
rings of toremifene and Y517 and T519 on GP2, as well as multiple hydrophobic residues on the
GP2 internal fusion loop such as L554 and M548. Additional interactions include hydrogen
bonding with R64 and the side chain of L186 on the GP1 (Figure 3b).
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32
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34
35
36
37
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. Toremifene bound to Ebola virus glycoprotein and modeling of binding poses for 32
and 49. (a) Crystal structure of Ebola glycoprotein trimer in complex with toremifene (PDB 5JQ7).
(b) Enlarged image of the binding site, where toremifene appears to bind in a cleft between GP1
(blue) and GP2 (red). Residues involved in the protein-ligand interactions are labeled. Overlays of
49 (c) and 32 (d) with toremifene in the Ebola GP structure. Binding poses indicate that all ligands
interact with T519; however, only toremifene and 49 interact directly with Y517.
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To investigate the potential binding mode of our new compounds, selected 4-
(aminomethyl)bezamides were docked into the toremifene hydrophobic binding site of EBOV GP
(PDB 5JQ7) as exemplified by 49 and 32 (Figure 3c,d). Docking of indoline 49 (Figure 3c) shows
that flexibility of this ligand allows the range of amino acid interactions observed for toremifene,
including π-stacking of the indoline heterocycle with Y517 (Figure 3b). While ligand 32 does not
appear to be within an acceptable proximity to hydrogen bond with Y517, the carbonyl group of
32 is able to hydrogen bond with T519. The adamantyl ring of 32 is involved in multiple
interactions with L554, M548 and other residues in this hydrophobic pocket. Since the adamantyl
moiety makes compound 32 more rigid, its binding mode is different from compounds where the
amide portion is smaller. Although this ligand is more shifted toward the side chain of R64, it is
well-accommodated in the pocket and it overlaps with toremifene. Thus, the modeling results
suggest that our 4-(aminomethyl)benzamides can bind to GP in the toremifene site. A recent report
on the structural studies of the binding mode of another set of anti-EBOV small molecule agents
supports this statement.38
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. The effects of toremifene and the new 4-(aminomethyl)benzamides 49 and 32 on the
infectivity of Y517S and T519V mutants of HIV/EBOV-GP pseudovirus in 293T cells. Functional
activities of representative compounds (a) indoline 49; and (b) adamantyl substituted 4-
(aminomethyl)benzamide 32. Toremifene was used as a reference compound (EC50 = 62.8 nM).
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To provide support for the conclusions of the docking studies, including the binding of 32 and
49 to the toremifene binding site, two GP mutants were used: Y517S and T519V. In the Y517S
mutant, concentration-response was right-shifted for toremifene and to a lesser extent 49 (Figure
4a), but not shifted for 32 (Figure 4b). In the T519V mutant, response was right-shifted for
toremifene and, to a lesser extent, for 32 (Figure 4b). These results are in agreement with the
docking studies and provide further support for the proposed MOA that our new compounds inhibit
viral entry by binding directly to the EBOV-GP in a similar fashion to toremifene.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 5. ADME Properties of the Selected 4-(Aminomethyl)benzamides.
Metabolic stabilitya
Plasma Liver microsome
Human
CYP450 inhibitionb
N
CYP3A4
(IC50, µM)
NTc
CYP2C9
(IC50, µM)
Rat
78
88
82
86
82
96
86
89
0
Human
54
Rat
0
5
63
100
85
87
88
100
86
100
85
NT
20
23
26
27
32
33
35
43
49
50
90
17
28
31
75
34
64
31
94
99
23
24
71
57
75
0
15.1
>100
NT
4.49
18.6
NT
NT
NT
>100
>100d
>100d
NT
>100
3.14d
4.98d
NT
94
88
62
2
46
2
27
0
24.0
NT
7.94
NT
a
b
% of compound remaining after 60 min related to t0. 10 µM quinidine and 30 µM
sulfaphenazole were used as the positive inhibitor controls in the CYP3A4 inhibition assay and
the CYP2C9 inhibition assay, respectively. Results are from three replicates: errors are
estimated to be <10%. cNT, not tested. d tested at Reaction Biology Corp.
Compound metabolic stability and CYP inhibition data are shown in Table 5 for selected
compounds. Indolines 43 and 50 were found to be surprisingly unstable in rat plasma, and both rat
and human liver microsomes, while compounds 20, 23, 32, 33 and 35 possessed acceptable
stability and were tested for inhibition of the two most important Phase 1 metabolism human
CYPs, CYP3A4 and CYP2C9. Although CYP inhibition is observed at concentrations higher than
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those required for antiviral activity, the observed weak inhibition of CYP2C9 will need to be
considered in further lead optimization studies with this group of potential drug candidates.
CONCLUSIONS
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We have described the synthesis of directed libraries from phenyl-substituted, indoline and
heterocyclic derivatives of the 4-(aminomethyl)benzamide scaffold. Using the EBOV/MARV
pseudotype and filoviral assays for SAR development, we defined inhibitors with high potency
(EC50 < 1 µM) and low cytotoxicity (SI > 100). 4-(Aminomethyl)benzamides 32 and 35 are two
of the superior compounds that we have identified, with broad-spectrum activity (EC50 values of
0.11 and 0.31 µM; 1.25 and 0.82 µM, respectively) against both Ebola and Marburg infectious
viruses. These compounds have favorable ADME properties, suggesting that these inhibitors can
be optimized and developed as potential antifiloviral drugs. Several other compounds show
promise, including compounds in other series, such as indoline 50. Development of small, orally
active molecules as therapeutic agents for prophylaxis or treatment of filovirus infections is
critically important. To date, we have witnessed the administration of two Ebola antibodies39-40
and one small molecule drug with an inappropriate mechanism of action41 to Ebola virus patients
in Western Africa. Positive results with the Merck vaccine42 have inspired a second vaccine from
J&J, which is presently being tested in Uganda.43 And, interestingly, these vaccines have
additionally inspired the development of a human antibody cocktail.44 Notwithstanding the partial
success of vaccination, it is generally recognized that vaccines will not be ideal for controlling
epidemics, and this realization underscores the continuing need for the development of small
molecule therapeutics with appropriate mechanisms.
EXPERIMENTAL SECTION
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General. EC50 values obtained from HIV/EBOV-GP or HIV/MARV-GP pseudotype and
EBOV (1976 Mayinga variant) or MARV (Lake Victoria, 2005 Angola) assays are reported as the
average of two or more replicates. All solvents and reagents were purchased from commercial
suppliers and used without further purification.
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1H and 13C NMR spectra were recorded on Bruker DPX-400 or AVANCE-400 spectrometers,
at 400 MHz and 100 MHz, respectively. NMR chemical shifts were reported in δ (ppm) using
residual solvent peaks as standards (CDCl3: 7.26 ppm (1H), 77.23 ppm (13C); CD3OD: 3.31 ppm
(1H), 49.15 ppm (13C); DMSO-d6: 2.50 ppm (1H), 39.52 ppm (13C)). Mass spectra were measured
in the ESI mode at an ionization potential of 70 eV with an LC-MSD on Hewlett-Packard
spectrometer. All key compounds (5−35, 38, 39, 41-50, 52−55) possess a purity of at least 95% as
assessed by analytical reversed phase HPLC using an ACE 3AQ C8 column (150 × 4.6 mm,
particle size 3 μM) with detection at 254 and 280 nm on a Shimadzu SPD-20A VP detector; flow
rate =1.0 mL/min; gradient of 10−95% acetonitrile in water (both containing 0.1 vol % of FA) in
20 min.
Cell-based protocol to identify entry inhibitors using a pseudotype virus.34 Low-passage
A549 cells were infected by a HIV/MARV-GP or HIV/EBOV-GP pseudotype virus containing a
luciferase reporter gene in the presence and absence of compounds at 12.5 µM.45 Plates were
incubated for 48 h and the infection was then quantified by the luciferase activity of the infected
A549 cells using the Neolite Reporter Gene Assay System (Perkin Elmer; Boston, MA).
Compounds that showed ≥70% inhibition at 12.5 µM concentration of EBOV or MARV
pseudovirions35, 46 were further evaluated to determine their EC50 values using four-parameter
logistic regression analysis in GraphPad (Graphpad Software; San Diego, CA).
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Infectivity assay using infectious EBOV or MARV virus. Wild type Ebola virus or Marburg
virus was used for testing the efficacy of compounds. All viral infections were done in a BSL-4
facility at the Texas Biomedical Research Institute and in the NEIDL, Boston University. Briefly,
4,000 HeLa cells per well were grown overnight in 384-well tissue culture plates; the volume of
DMEM (Fisher scientific, Cat#MT10017CV) culture media supplemented with 10% fetal bovine
serum (Gemini Bio-Products, Cat#100106) was 25 µL. On the day of assay, test drugs were diluted
to 1 mM concentration in complete media. A 25 µL volume of this mixture was added to the cells
already containing 25 µL of media to achieve a concentration of 500 µM. All treatments were done
in triplicate. A 25 µL volume of media was removed from the first wells and added to the next
well. This type of serial dilution was performed 12 times and treated cells were then incubated at
37 °C in a humidified CO2 incubator for 1 hour. Final concentrations of 250, 125, 62.5, 31.25,
15.62, 7.81, 3.9, 1.9, 0.97, 0.48, 0.24 and 0.12 µM were achieved upon addition of 25 µL of
infection mix containing EBOV or MARV virus. Bafilomycin, at a final concentration of 10 nM,
was used as a positive control drug. Infections were designed to achieve a MOI of 0.05 to 0.15.
Infected cells were incubated for 24 hours. At 24 hours post-infection, cells were fixed by
immersing the plates in formalin for 24 hours at 4 °C. Fixed plates were decontaminated and
removed from the BSL-4 facility. Formalin from fixed plates was decanted and plates were washed
with PBS. EBOV (or MARV)-infected cells were stained for nuclei using the Hoechst stain at
1:50,000 dilution and virus specific antibody (IBT Bioservices) followed by a fluorescently labeled
secondary antibody (Alexa488). The plates were imaged. Nuclei (blue) and infected cells (green)
were counted using CellProfiler software (Broad Institute) Version 2.1.1. Total number of nuclei
(blue) was used as a proxy for cell numbers and a loss of cell number was assumed to reflect
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cytotoxicity. Concentrations where total cell numbers were 20% less than the control were rejected
from the analysis.
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Chemistry. General Method A. N-Alkylation of cyclic amines. To a solution of methyl
bromide (1 eq) in anhydrous dichloromethane (3 mL/mmol), cyclic amine (1.0 eq) and
triethylamine (5 eq) were added and the reaction mixture was stirred at room temperature under
argon for 8-24 hours to reaction completion as monitored by TLC. The solvents were evaporated,
and the solid was treated with a mixture of dichloromethane and sodium bicarbonate (aq). The
organic phase was collected, washed with water and brine, and dried over sodium sulfate. Solvent
was removed under vacuum to provide crude product, which was further purified by flash
chromatography (CombiFlash) by elution with methanol in dichloromethane (5-30%) to yield the
desired product.
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General Method B. Preparation of acyl chlorides. A solution of methyl ester in
tetrahydrofuran (3 mL/mmol), methanol (1 mL/mmol) and water (0.5 mL/mmol) was treated with
sodium hydroxide (2 eq). The reaction mixture was stirred overnight at room temperature. The
solvents were removed under vacuum and the residue was treated with thionyl chloride (2
mL/mmol). The reaction mixture was heated at reflux for 1 h and concentrated under vacuum. The
product was used in next step without purification.
General Method C. Amide formation from acyl chloride. To a solution of acyl chloride (1
eq) in anhydrous dichloromethane (3 mL/mmol), was added amine (0.8 eq), followed by
triethylamine (5 eq), and the reaction mixture was stirred at room temperature under argon for 8-
24 hours until reaction was complete as monitored by TLC. The reaction mixture was diluted with
dichloromethane and washed with sodium bicarbonate (aq), and brine, and dried over sodium
sulfate. Solvent was removed under vacuum to provide crude product, which was further purified
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by flash chromatography (CombiFlash) by elution with methanol in dichloromethane (5-30%) to
yield the desired product.
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Methyl 4-(4-(piperidin-1-ylmethyl)benzamido)benzoate (5). Yield: 165 mg, 59%. HPLC
purity: 99%. 1H NMR (400 MHz, DMSO-d6) δ 1.39 (m, 2H), 1.49 (m, 4H), 2.33 (m, 4H), 3.50 (s,
2H), 3.84 (s, 3H), 7.44 (d, J = 8.1 Hz, 2H), 7.90 (d, J = 8.1 Hz, 2H), 7.95 (m, 4H), 10.51 (s, 1H).
13C NMR (100 MHz, DMSO-d6) δ 24.4, 26.0, 52.3, 54.3, 62.8, 119.9, 124.6, 128.1, 129.0, 130.5,
133.5, 143.5, 144.1, 166.2, 166.3. HRMS (ESI) calculated for C21H25N2O3 ([M+H]+): 353.1865,
found: 353.1860.
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Methyl 4-(4-(morpholinomethyl)benzamido)benzoate (6). Yield: 150 mg, 66%. HPLC
purity: 99%. 1H NMR (400 MHz, CDCl3) δ 2.50-2.51 (m, 4H), 3.60 (s, 2H), 3.73 (t, J = 4.5 Hz,
4H), 7.47 (d, J = 8.1 Hz, 2H), 7.83 (d, J = 8.8 Hz, 2H), 7.92 (d, J = 8.1 Hz, 2H), 8.01 (d, J = 8.8
Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 55.5, 57.2, 66.6, 70.5, 123.7, 129.1, 131.5, 133.2, 134.2,
137.5, 145.4, 147.0, 171.0, 171.2. HRMS (ESI) calculated for C20H23N2O4 ([M+H]+): 355.1658,
found: 355.1651.
Methyl 4-(4-((4-methylpiperazin-1-yl)methyl)benzamido)benzoate (7). Yield: 140 mg, 38%.
HPLC purity: 99%. 1H NMR (400 MHz, MeOD) δ 2.31 (s, 3H), 2.47-2.54 (m, 8H), 3.63 (s, 2H),
3.91 (s, 3H), 7.50 (d, J = 8.2 Hz, 2H), 7.87 (dd, J = 1.9, 7.1 Hz, 2H), 7.91 (d, J = 8.2 Hz, 2H), 8.02
(dd, J = 1.9, 7.1 Hz, 2H). 13C NMR (100 MHz, MeOD) δ 44.4, 51.0, 52.0, 54.2, 61.7, 119.6, 125.2,
127.3, 129.1, 130.0, 133.5, 141.9, 143.2, 166.7, 167.2. HRMS (ESI) calculated for C21H26N3O3
([M+H]+): 368.1974, found: 368.1977.
Methyl 3-(4-(piperidin-1-ylmethyl)benzamido)benzoate (8). Yield: 115 mg, 83%. HPLC
purity: 99%. 1H NMR (400 MHz, CDCl3) δ 1.46-1.47 (m, 2H), 1.57-1.63 (m, 4H), 2.40 (m, 4H),
3.54 (s, 2H), 3.93 (s, 3H), 7.45-7.49 (m, 3H), 7.83-7.85 (m, 3H), 7.95 (s, 1H), 8.07 (dd, J = 1.1,
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8.1 Hz, 1H), 8.15 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 24.2, 25.8, 52.2, 54.5, 63.2, 121.0, 124.7,
125.4, 127.0, 129.1, 129.3, 130.8, 133.1, 128.3, 143.2, 165.8, 166.7. HRMS (ESI) calculated for
C21H25N2O3 ([M+H]+): 353.1865, found: 353.1865.
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Methyl 4-(3-(piperidin-1-ylmethyl)benzamido)benzoate (9). Yield: 122 mg, 54%. HPLC
purity: 99%. 1H NMR (400 MHz, CDCl3) δ 1.46 (m, 2H), 1.59 (m, 4H), 2.40 (m, 4H), 3.52 (d, J =
5.4 Hz, 2H), 3.92 (s, 3H), 7.40-7.46 (m, 1H), 7.50-7.51 (m, 1H), 7.72 (d, J = 8.7 Hz, 3H), 7.86 (s,
1H), 8.03-8.07 (m, 2H), 8.20-8.30 (m, 1H). 13C NMR (100 MHz, CDCl3) δ 24.2, 25.8, 52.0, 54.5,
63.3, 119.16, 119.19, 125.6, 125.9, 127.5, 128.6, 130.8, 132.9, 134.4, 139.5, 142.2, 142.3, 165.9,
166.6. HRMS (ESI) calculated for C21H25N2O3 ([M+H]+): 353.1865, found: 353.1863.
Methyl 4-(4-(piperidine-1-carbonyl)benzamido)benzoate (10). Yield: 88 mg, 22%. HPLC
1
purity: 97%. H NMR (400 MHz, CDCl3) δ 1.49 (m, 2H), 1.69 (m, 4H), 3.26 (m, 2H), 3.74 (m,
2H), 3.92 (s, 3H), 7.20 (d, J = 8.2 Hz, 2H), 7.79 (d, J = 8.2 Hz, 2H), 7.93 (d, J = 8.8 Hz, 2H), 8.04
(d, J = 8.8 Hz, 2H), 9.52 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 24.3, 24.4, 25.5, 26.4, 43.2, 48.7,
51.9, 119.4, 125.4, 126.4, 127.8, 130.6, 135.7, 138.8, 142.9, 165.7, 166.7, 169.5. HRMS (ESI)
calculated for C21H23N2O4 ([M+H]+): 367.1658, found: 367.1650.
4-(4-(Piperidin-1-ylmethyl)benzamido)benzoic acid (11). Yield: 8.0 mg, 27%. HPLC purity:
98%. 1H NMR (400 MHz, MeOD) δ 1.47-1.48 (m, 2H), 1.59-1.61 (m, 4H), 2.44 (m, 4H), 3.53 (s,
1H), 3.56 (s, 1H), 7.45 (d, J = 8.2 Hz, 2H), 7.66 (d, J = 9.7 Hz, 2H), 7.92-7.97 (m, 4H). 13C NMR
(100 MHz, MeOD) δ 23.7, 25.1, 54.0, 62.8, 120.0, 127.3, 129.2, 129.5, 132.9, 134.6, 141.0, 142.5,
167.5, 173.9. HRMS (ESI) calculated for C20H23N2O3 ([M+H]+): 339.1709, found: 339.1712.
N-(4-(Methylsulfonyl)phenyl)-4-(piperidin-1-ylmethyl)benzamide (12). Yield: 76 mg, 56%.
HPLC purity: 98%. 1H NMR (400 MHz, DMSO-d6) δ 1.39 (m, 2H), 1.49 (m, 4H), 2.33 (m, 4H),
3.18 (s, 3H), 3.31 (s, 2H), 7.45 (d, J = 8.1 Hz, 2H), 7.91 (m, 4H), 8.03 (d, J = 8.7 Hz, 2H), 10.60
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(s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 24.3, 26.0, 44.2, 54.3, 62.8, 120.3, 128.2, 128.4, 129.0,
133.3, 135.4, 143.6, 144.2, 166.4. HRMS (ESI) calculated for C20H25N2O3S ([M+H]+): 373.1586,
found: 373.1582.
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N-(4-(N-Methylsulfamoyl)phenyl)-4-(piperidin-1-ylmethyl)benzamide (13). Yield: 70 mg,
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46%. HPLC purity: 99%. H NMR (400 MHz, CDCl3) δ 1.42 (m, 2H), 1.55-1.57 (m, 4H), 2.39
(m, 4H), 2.55 (s, 3H), 3.51 (s, 2H), 7.38 (d, J = 8.1 Hz, 2H), 7.59 (d, J = 8.8 Hz, 2H), 7.82-7.85
(m, 4H). 13C NMR (100 MHz, CDCl3) δ 23.8, 25.2. 28.7, 54.3, 63.2, 120.2, 127.4, 128.1, 129.7,
133.3, 133.4, 142.5, 166.9. HRMS (ESI) calculated for C20H26N3O3S ([M+H]+): 388.1695, found:
388.1692.
N-(4-(Methylsulfonamido)phenyl)-4-(piperidin-1-ylmethyl)benzamide (14). Yield: 79 mg,
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55%. HPLC purity: 99%. H NMR (400 MHz, MeOD) δ 1.43-1.45 (m, 2H), 1.55-1.60 (m, 4H),
2.40 (m, 4H), 2.93 (s, 3H), 3.53 (s, 3H), 7.20-7.24 (m, 2H), 7.39-7.41 (m, 2H), 7.64 (dd, J = 2.0,
7.9 Hz, 2H), 7.84 (d, J = 8.2 Hz, 2H). 13C NMR (100 MHz, MeOD) δ 23.8, 25.2, 38.4, 54.2, 63.2,
121.7, 121.9, 127.3, 129.7, 133.64, 133.67, 135.5, 166.8. HRMS (ESI) calculated for C20H26N3O3S
([M+H]+): 388.1695, found: 388.1690.
N-(4-Cyanophenyl)-4-(piperidin-1-ylmethyl)benzamide (15). Yield: 110 mg, 54%. HPLC
purity: 97.8%. 1H NMR (400 MHz, CDCl3) δ 1.48 (m, 2H), 1.64 (m, 4H), 2.46 (m, 4H), 3.60 (s,
2H), 7.49 (d, J = 8.1 Hz, 2H), 7.65 (d, J = 8.6 Hz, 2H), 7.87 (m, 4H), 8.31 (bs, 1H). 13C NMR (100
MHz, CDCl3) δ 23.9, 25.4, 45.8, 54.4, 62.9, 107.2, 118.8, 119.9, 127.2, 129.7, 133.0, 133.2, 142.1,
165.8. HRMS (ESI) calculated for C20H22N3O ([M+H]+): 320.1763, found: 320.1761.
4-(Piperidin-1-ylmethyl)-N-(4-(trifluoromethyl)phenyl)benzamide (16). Yield: 91 mg, 68%.
HPLC purity: 99%. 1H NMR (400 MHz, MeOD) δ 1.49 (m, 2H), 1.61-1.64 (m, 4H), 2.47 (m, 4H),
3.60 (s, 2H), 7.49 (d, J = 8.0 Hz, 2H), 7.63 (d, J = 8.3 Hz, 2H), 7.92-7.94 (m, 4H). 13C NMR (100
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MHz, MeOD) δ 27.7, 29.1, 67.0, 124.2, 129.71, 129.75, 129.9, 131.4, 133.7, 137.4, 145.1, 145.7,
171.1. HRMS (ESI) calculated for C20H22F3N2O ([M+H]+): 363.1684, found: 363.1688.
4-(Piperidin-1-ylmethyl)-N-(3-(trifluoromethyl)phenyl)benzamide (17). Yield: 48 mg, 72%.
HPLC purity: 99%. 1H NMR (400 MHz, CDCl3) δ 1.40-1.47 (m, 2H), 1.57-1.62 (m, 4H), 2.39 (m,
4H), 3.52 (s, 2H), 7.38-7.47 (m, 4H), 7.80 (d, J = 8.1 Hz, 2H), 7.87 (d, J = 8.1 Hz, 1H), 7.97 (s,
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1H), 8.36 (s, 1H). C NMR (100 MHz, CDCl3) δ 24.2, 25.8, 54.5, 63.2, 117.0, 117.04, 120.9,
1225, 123.3, 125.2, 127.0, 129.45, 129.55, 131.2, 131.5, 132.9, 138.6, 143.2, 166.0. HRMS (ESI)
calculated for C20H22F3N2O ([M+H]+): 363.1684, found: 363.1680.
N-(3-Chloro-4-(trifluoromethyl)phenyl)-4-(piperidin-1-ylmethyl)benzamide (18). Yield: 49
mg, 58%. HPLC purity: 98.8%. 1H NMR (400 MHz, CDCl3) δ 1.40-1.44 (m, 2H), 1.51-1.55 (m,
4H), 2.36 (m, 4H), 3.47 (s, 2H), 7.35 (d, J = 7.8 Hz, 2H), 7.56 (d, J = 8.6 Hz, 1H), 7.66 (d, J = 8.6
Hz, 1H), 7.79 (d, J = 7.8 Hz, 2H), 7.95 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 23.8, 25.2, 54.3,
63.2, 117.6, 121.5, 122.3, 123.0, 123.4, 124.2, 127.4, 127.9, 127.95, 129.7, 132.6, 133.1, 141.7,
142.6, 166.8. HRMS (ESI) calculated for C20H21ClF3N2O ([M+H]+): 397.1295, found: 397.1293.
N-(4-Chloro-3-(trifluoromethyl)phenyl)-4-(piperidin-1-ylmethyl)benzamide (19). Yield: 52
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mg, 62%. HPLC purity: 99%. H NMR (400 MHz, CDCl3) δ 1.46-1.49 (m, 2H), 1.58-1.63 (m,
4H), 2.41 (m, 4H), 3.51 (s, 2H), 7.45 (d, J = 8.1 Hz, 2H), 7.49 (d, J = 8.7 Hz, 1H), 7.81 (d, J = 8.1
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Hz, 2H), 7.89 (dd, J = 2.7, 8.7 Hz, 2H), 7.99 (d, J = 2.7 Hz, 1H), 8.11-8.20 (m, 1H). C NMR
(100 MHz, CDCl3) δ 24.1, 25.8, 54.5, 63.1, 119.3, 119.35, 121.2, 123.9, 124.3, 126.9, 127.1, 128.5,
128.8, 129.4, 131.9, 132.6, 136.9, 143.4, 166.1. HRMS (ESI) calculated for C20H21ClF3N2O
([M+H]+): 397.1295, found: 397.1299.
N-(3-Chloro-4-((trifluoromethyl)thio)phenyl)-4-(piperidin-1-ylmethyl)benzamide
(20).
Yield: 45 mg, 25%. HPLC purity: 99.9%. 1H NMR (400 MHz, CDCl3) δ 1.40-1.48 (m, 2H), 1.58-
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1.63 (m, 4H), 2.40 (m, 4H), 3.55 (s, 2H), 7.49 (d, J = 8.1 Hz, 2H), 7.60 (dd, J = 2.0, 8.5 Hz, 1H),
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7.75 (d, J = 8.5 Hz, 1H),7.81 (d, J = 8.1 Hz, 2H),. 7.90 (b.s, 1H), 8.03 (d, J = 2.0 Hz, 1H). C
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NMR (100 MHz, CDCl3) δ 24.2, 26.0, 54.6, 63.3, 118.1, 118.7, 121.4, 127.0, 129.4, 132.4, 139.4,
141.2, 141.8, 144.2, 166.1. HRMS (ESI) calculated for C20H21ClF3N2OS ([M+H]+): 429.1015,
found: 429.1017.
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N-(4-Cyclopropylphenyl)-4-(piperidin-1-ylmethyl)benzamide (21). Yield: 34 mg, 45%.
HPLC purity: 99.7%. 1H NMR (400 MHz, CDCl3) δ 0.61-0.65 (m, 2H), 0.90-0.94 (m, 2H), 1.42-
1.43 (m, 2H), 1.53-1.59 (m, 4H), 1.82-1.89 (m, 1H), 2.37 (m, 4H), 3.49 (s, 2H), 7.01 (d, J = 6.5
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Hz, 2H), 7.36 (d, J = 8.3 Hz, 2H), 7.50 (d, J = 8.3 Hz, 2H), 7.78 (d, J = 6.5 Hz, 2H). C NMR
(100 MHz, CDCl3) δ 8.9, 14.9, 24.0, 25.4, 54.4, 63.3, 120.5, 120.6, 126.1, 127.1, 129.6, 133.8,
135.5, 140.2, 141.5, 166.1. HRMS (ESI) calculated for C22H27N2O ([M+H]+): 335.2123, found:
335.2126.
N-(4-Isopropylphenyl)-4-(piperidin-1-ylmethyl)benzamide (22). Yield: 46 mg, 32%. HPLC
purity: 97.9%. 1H NMR (400 MHz, CDCl3) δ 1.27 (d, J = 6.8 Hz, 6H), 1.49 (m, 2H), 1.59 (m, 4H),
2.40 (m, 4H), 2.93 (quintet, J = 6.8 Hz, 1H), 3.54 (s, 2H), 7.23 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 7.8
Hz, 2H), 7.57 (d, J = 8.2 Hz, 1H), 7.82 (d, J = 7.8 Hz, 2H), 7.95 (b.s, 1H). 13C NMR (100 MHz,
CDCl3) δ 24.0, 24.3, 26.0, 33.6, 54.5, 63.4, 120.4, 126.9, 129.3, 133.6, 135.7, 143.1, 145.2, 165.6.
HRMS (ESI) calculated for C22H29N2O ([M+H]+): 337.2280, found: 337.2280.
N-(2,4-Di-tert-butylphenyl)-4-(piperidin-1-ylmethyl)benzamide (23). Yield: 24 mg, 28%.
HPLC purity: 97.7%. 1H NMR (400 MHz, MeOD) δ 1.36 (s, 9H), 1.44 (s, 9H), 1.51 (m, 2H), 1.64
(m, 4H), 2.49 (m, 4H), 3.62 (s, 2H), 7.10 (d, J = 8.2 Hz, 1H), 7.32 (dd, J = 2.2, 8.2, Hz, 1H), 7.51
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(d, J = 8.2, Hz, 1H), 7.56 (d, J = 2.2 Hz, 1H), 7.95 (d, J = 7.9 Hz, 2H). C NMR (100 MHz,
MeOD) δ 23.6, 25.1, 30.1, 10.4, 34.2, 34.9, 54.0, 62.7, 123. 4, 123.7, 127.2, 129.6, 130.8, 132.6,
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133.5, 141.2, 146.3, 150.1, 168.3. HRMS (ESI) calculated for C27H39N2O ([M+H]+): 407.3062,
found: 407.3066.
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N-(4-(Cyano(cyclopentyl)methyl)phenyl)-4-(piperidin-1-ylmethyl)benzamide (24). Yield:
22 mg, 26%. HPLC purity: 98%. 1H NMR (400 MHz, CDCl3) δ 1.46-1.47 (m, 2H), 2.59-1.64 (m,
4H), 1.94-2.10 (m, 6H), 2.42-2.51 (m, 6H), 3.56 (s, 2H), 7.43 (d, J = 8.6 Hz, 2H), 7.47 (d, J = 8.1
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Hz, 2H), 7.70 (d, J = 8.6 Hz, 2H), 7.84 (d, J = 8.1 Hz, 2H), 8.09 (s, 1H). C NMR (100 MHz,
CDCl3) δ 24.1, 25.7, 40.4, 47.3, 54.5, 63.2, 120.5, 124.4, 126.7, 127.0, 129.5, 133.3, 135.6, 137.6,
142.8, 165.7. HRMS (ESI) calculated for C26H32N3O ([M+H]+): 402.2545, found: 402.2551.
N-(4-Cyclohexylphenyl)-4-(piperidin-1-ylmethyl)benzamide (25). Yield: 14 mg, 17%. HPLC
purity: 98%. 1H NMR (400 MHz, CDCl3) δ 1.20-1.40 (m, 7H), 1.55 (m, 4H), 1.68-1.80 (m, 5H),
2.37-2.43 (m, 4H), 3.50 (m, 3H), 7.14 (d, J = 6.5 Hz, 2H), 7.36 (d, J = 6.3 Hz, 2H), 7.50 (d, J =
6.5 Hz, 2H), 7.78 (d, J = 6.3 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 23.8, 25.2, 26.0, 26.7, 34.4,
43.9, 54.3, 63.2, 120.6, 127.1, 129.7, 133.9, 135.7, 144.4, 166.3. HRMS (ESI) calculated for
C25H33N2O ([M+H]+): 377.2593, found: 377.2589.
N-(4-Cyclohexylphenyl)-3-fluoro-4-(piperidin-1-ylmethyl)benzamide (26). Yield: 43 mg,
55%. HPLC purity: 99.6%. 1H NMR (400 MHz, MeOD) δ 1.20-1.44 (m, 7H), 1.51-1.62 (m, 4H),
1.71 (d, J = 7.3 Hz, 1H), 1.83 (m, 4H), 2.47 (m, 5H), 3.62 (s, 2H), 7.17 (d, J = 8.4 Hz, 2H), 7.45
(dd, J = 7.5, 10.4 Hz, 1H), 7.54 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 10.4 Hz, 1H), 7.66 (d, J = 7.5 Hz,
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1H). C NMR (100 MHz, MeOD) δ 23.7, 25.2, 26.0, 26.7, 34.4, 44.0, 53.9, 55.3, 114.4, 114.6,
121.0, 122.7, 127.0, 127.2, 132.1, 135.6, 136.2, 136.3, 144.7, 160.0, 162.4, 165.4. HRMS (ESI)
calculated for C25H32FN2O ([M+H]+): 395.2499, found: 395.2501.
N-(4-Cyclohexylphenyl)-4-(1-(piperidin-1-yl)ethyl)benzamide (27). Yield: 22 mg, 28%.
HPLC purity: 98.6%. 1H NMR (400 MHz, MeOD) δ 1.25-1.35 (m, 11H), 1.38-1.40 (m, 4H), 1.72
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