Synthesis and Evaluation of N-Phenyl-3-sulfamoyl-benzamide Derivatives as Capsid Assembly Modulators Inhibiting Hepatitis B Virus (HBV)

Article abstract of DOI:10.1021/acs.jmedchem.8b00654

Small molecule induced hepatitis B virus (HBV) capsid assembly modulation is considered an attractive approach for new antiviral therapies against HBV. Here we describe efforts toward the discovery of a HBV capsid assembly modulator in a hit-to-lead optimization, resulting in JNJ-632, a tool compound used to further profile the mode of action. Administration of JNJ-632 (54) in HBV genotype D infected chimeric mice resulted in a 2.77 log reduction of the HBV DNA viral load.

Full text of DOI:10.1021/acs.jmedchem.8b00654

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Article
Synthesis and Evaluation of N-phenyl-3-sulfamoyl-benzamide Derivatives
as Capsid Assembly Modulators inhibiting Hepatitis B Virus (HBV).
Koen Vandyck, Geert Rombouts, Bart Stoops, Abdellah Tahri, Ann Vos, Wim
Verschueren, Yiming Wu, Jingmei Yang, Fuliang Hou, Bing Huang, Karen Vergauwen,
Pascale Dehertogh, Jan-Martin Berke, and Pierre Jean Marie Bernard Raboisson
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00654 • Publication Date (Web): 15 Jun 2018
Downloaded from http://pubs.acs.org on June 16, 2018
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Synthesis and Evaluation of Nꢀphenylꢀ3ꢀsulfamoylꢀ
benzamide Derivatives as Capsid Assembly
Modulators inhibiting Hepatitis B Virus (HBV).
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†,*
†
†
†
†
Koen Vandyck, Geert Rombouts, Bart Stoops, Abdellah Tahri, Ann Vos, Wim
†
ǂ
ǂ
ǂ
ǂ
†
Verschueren, Yiming Wu, Jingmei Yang, Fuliang Hou, Bing Huang, Karen Vergauwen,
†
†
†
Pascale Dehertogh, Jan Martin Berke, Pierre Raboisson
†
Janssen Pharmaceutica NV, Janssen Pharmaceutical Companies of Johnson & Johnson,
Turnhoutseweg 30, 2340, Beerse, Belgium
ǂWuXi AppTec, 288 Fute Zhong Road, China (Shanghai) Pilot Free Trade Zone; Shanghai
2
00131, PR China
KEYWORDS
HBV, CAM, Capsid Assembly Modulators, JNJꢀ6379, JNJꢀ632
ABSTRACT
Small molecule induced Hepatitis B virus (HBV) capsid assembly modulation is considered an
attractive approach for new antiviral therapies against HBV. Here we describe efforts towards
the discovery of a HBV capsid assembly modulator in a hitꢀtoꢀlead optimization, resulting in
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JNJꢀ632, a tool compound used to further profile the mode of action. Administration of JNJꢀ632
(54) in HBV genotype D infected chimeric mice, resulted in a 2.77 log reduction of the HBV
DNA viral load.
0
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Introduction
Despite the availability of a safe and effective prophylactic HBV vaccine, it is estimated that
1
about 240 million people worldwide are chronically infected by hepatitis B virus. Chronic HBV
infection puts patients at high risk for developing cirrhosis and liver cancer, with estimation of
more than 686 000 people dying every year due to complications of hepatitis B. Besides
(Pegylated) interferonꢀalpha ((PEG)ꢀIFNα), multiple nucleos(t)ide analogs have been approved
for treatment of hepatitis B. This current treatment can slow the progression of cirrhosis, reduce
incidence of liver cancer and improve long term survival, but the majority of patients will not be
cured from HBV infection. Therefore, chronic HBV infection presents a major global health
concern.
HBV capsid assembly is a critical step in virus production and it is considered an attractive
target for new antiviral therapies against HBV. Capsid assembly modulators (CAMs),
compounds that modulate the kinetics/thermodynamics of HBV core protein aggregation, can
block encapsidation of pregenomic RNA (pgRNA) and will consequently inhibit the synthesis of
HBV DNA and infectious virion production. Recently, it has been shown that capsid assembly
2
modulators, for example JNJꢀ632 described in this paper, and JNJꢀ6379, currently in phase 2
3
, 4
clinical trial, are also able to block de novo HBV infection in vitro.
5
, 6
Prototypical examples of two distinct types of capsid assembly modulators are ATꢀ130
and
7
Bay 41ꢀ4109 (Fig 1). Both are inducing aggregation of the HBV core protein, but the first
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induces the formation of empty capsids (CAMꢀI class of compounds), lacking the pgRNAꢀ
polymerase, while the latter induces the formation of aberrant structures (CAMꢀII class of
compounds).
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Figure 1. Two prototypical capsid assembly modulators Bay 41ꢀ4109 (CAMꢀII) and ATꢀ130
(CAMꢀI)
Originating from our compound library, a series of Nꢀphenylꢀ3ꢀsulfamoylꢀbenzamide
8
derivatives (Table 1) was picked up, represented by compounds 1 to 5, that inhibit the hepatitis
B virus (HBV), evidenced by dose dependent reduction of HBV DNA in the HepG2.2.15 cell
line. In addition, compounds 1 to 4 induce HBV core protein aggregation, resulting in the
formation of particles of regular capsid size, but not larger aberrant structures, as indicated by
size exclusion chromatography (data not shown). A preliminary compound optimization was
initiated, exploring initial SAR while generating a suitable tool compound to assess the mode of
action in a relevant in vivo HBV mouse model, either by oral or subcutaneous administration, as
at the time of this work, in vivo data was only available for the heteroaryldihydropyrimidine
(HAP) CAMꢀII class of compounds (known to aggregate the core protein in aberrant structures).
8
ꢀ10
At this time, with the publication of both our own and other patent applications,
Campagna et
11
12
13
al. (Sulfamoylbenzamide; SBA) Klumpp et al. (Sulfamoyl Carboxamide (SOC)) and others
reported on Nꢀphenylꢀ3ꢀsulfamoylꢀbenzamide derivatives for HBV.
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1
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2
2
2
2
2
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4
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5
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Table 1. Initial N-phenyl-3-sulfamoyl-benzamide derivatives.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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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
*
#
$
SEC ratio
HLM/
EC50
CC50
Sol
1
2
3
4
ID
R
R
R
R
(ꢀM)
(ꢀM)
(ꢀM)
MLM (%)
Cpd/ctrl
1
F
H
Cl
0.66
17.0
1
98/100
0.01/11.5
2
3
H
H
Cl Cl
0.50
1.04
>25
>25
2
ꢀ
99/100
80/ND
0.2/13.7
0.7/9.9
Cl
H
H
H
4
5
H
H
0.60
2.23
27.2
16.4
91
ꢀ
94/98
0.3/15.2
ND
H
F
100/ND
*
#
HBV DNA HepG2.2.15; HepG2, 6 days; $ Kinetic Solubility pH 7.4 (see supporting information for
details)
HLM/MLM: Human/Mouse liver microsome stability, expressed as percentage metabolisation after 15
minutes of incubation (see supporting information for details).SEC: Size exclusion chromatography.
The aggregation of core protein, expressed as a ratio between the area under the curve for the observed
amount of core dimer and area under the curve for the aggregated core protein, as determined in size
exclusion chromatography after 24 hours of aggregation. Determined for both the compound at 20 ꢀM
(
cpd) and a noꢀcompoundꢀcontrol (ctrl), (see supporting information for details).
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ND not determined.
Results and discussion
The formation of a capsid containing preꢀgenomic RNA (pgRNA) and the viral polymerase, is
the first, cytoplasmic, step in the formation of an infectious HBV virion. Only within the viral
nucleocapsid, the pgRNA is reverse transcribed to produce relaxedꢀcircular DNA (rcDNA). As
CAMs accelerate the kinetics of capsid assembly, preventing encapsidation of the PolꢀpgRNA
complex, they block the reverse transcription of the pregenomic RNA and the formation of
rcDNA. It is hypothesized that CAMs should nucleate misassembly faster than normal HBV
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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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
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9
0
1
2
3
4
5
6
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9
0
1
2
3
4
5
6
7
8
9
0
1
4
virion assembly. Therefore, it is expected that, in case of CAMs, the kinetics of the compound
induced assembly will be indicative for its antiꢀHBV activity (HBV DNA). In this SARꢀ
exploration, in addition to measuring HBV DNA inhibition (EC ) in stable HepG2.117 (and
50
HepG2.2.15) cell lines, also the core protein assembly kinetics were monitored in the
15
fluorescence quenching assay (See Supporting Information).
However, it is expected that the intrinsic capability of a compound to accelerate assembly, in this
assay, might be clouded by a low kinetic solubility of a compound. To assess if a compound
targets the capsid assembly, even if it only induces a slow assembly, either due to intrinsic
capability or low kinetic solubility, the assembly was also monitored in a 24ꢀhours assay. Here,
the assembly domain (amino acids 1ꢀ149) of a recombinant HBV core protein (Cp149) was
incubated with the compounds for 24 hours and the compounds tendency to assemble the Cp149,
is expressed as a ratio between the area under the curve for the observed amount of Cp149 dimer
and area under the curve for the aggregated Cp149, as determined by size exclusion
chromatography. This is compared to the same ratio obtained without addition of compound, as
in this case, also a minor amount of empty assembled capsids is formed.
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5
5
6
As the eventual goal of this study was to assess the in vivo effect of this class of compounds in
a humanized mouse model and the initial hits suffered from low metabolic stability, both human
(HLM) and mouse liver microsome (MLM) stability were assessed. Stability in human
microsomes was considered relevant for the humanized mouse model, whereas mouse liver
microsome stability would allow generation of PK data in a relevant nonꢀhumanized mouse
strain, but also in the context of residual presence of mouse hepatocytes in the humanized mice.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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9
0
1
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9
0
1
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9
0
1
2
3
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5
6
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8
9
0
Initial SARꢀexploration included the synthesis of bioisosteric derivatives of the sulphamoyl
and Nꢀphenyl amide moieties as depicted in table 2 and table 3 respectively. Whereas the kinetic
solubility of a compound 1 and close analogues was low, possibly hampering further SAR
studies, compound 4 demonstrated higher solubility. As an example, as compound 1 and 2 where
clearly impacting capsid assembly as indicated by the SEC ratio (table 1), they however had
limited impact in a shorter timeframe as indicated by the quenching curves (supporting
information), likely explained by a low kinetic solubility of those two compounds. The higher
solubility of compound 4 however, together with its high potency, resulted in faster kinetics in
the quenching assay (supporting information). Compound 6, the pꢀFluoroꢀ analogue of
compound 4, showed slightly increased metabolic stability versus compound 4, and was used,
together with its cyclohexyl analogue compound 7, as a starting point for an initial SARꢀstudy.
Inversion of the sulfamoyl moiety, as for compound 8 and 9, retained some, although slightly
lower activity. Interestingly, this specifically improved HLM stability (compound 8 versus 6),
but not MLM stability.
Table 2. Variations and replacement of the eastern sulphamoyl part.
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SEC
Cpd/
ctrl
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
$
*
°
#
HLM/
MLM
Sol
EC50
EC50
CC50
5
ID
R
(ꢀM)
(ꢀM)
(ꢀM)
(
ꢀM)
(
%)
6
7
0.47
ND
0.6
32.7
>25
50/91
69/93
83
ND
0.58
43
92
86
19
53
0.2/12.0
8
9
1.2
1.2
>50
4/93
ND
0.3/6.8
ND
1.1
2.6
2.2
11.1
16.3
>25
>25
33.6
23.8
43.2
>100
57.7
46/91
98/100
86/99
56/93
55/87
10
11
12
13
7.5
2.0/6.6
>100
62.2
<0.4 6.4/7.2
7
7.2/7.7
*
#
HBV DNA HepG2.2.15; ° HBV DNA HepG2.117; HepG2, 4 days; $ Kinetic Solubility pH 7.4
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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
The methylated derivatives 10 and 11 showed reduced potency versus there NHꢀanalogues, 6 and
8
respectively. Replacing the sulfamoyl moiety by an amide, as in 12 and 13, rendered the
compound inactive, without any CAM property indicated by the SEC ratio.
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 3. Variation and replacement of the western Nꢀphenyl amide part
A
or
B
#
*
$
CC50
HLM/
MLM (%) (ꢀM)
Sol
SEC
Cpd/ctrl
7
2
EC50
(
EC50°
ID
R
R
ꢀM)
(ꢀM)
1.30
0.60
(ꢀM)
>50
>25
1
1
4
5
A
A
F
F
0.78
0.66
61/79
58
21
0.3/9.2
63/100
0.1/11.5
4
B
B
H
F
0.60
0.36
0.76
0.66
>25
>25
94/98
36/77
91
82
0.3/15.2
ND
16
1
1
7
8
A
A
F
F
>100
28.8
ꢀ
93.9
>25
74/ꢀ
ꢀ/ꢀ
29
ꢀ
8.4/11.0
ND
>25
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1
9
A
H
8.0
>25
>25
91/100
99/100
22
10
ND
20
21
22
23
24
25
A
B
A
A
A
A
H
H
F
F
F
F
9.6
89.6
1.7
>25
>25
>25
>25
19.2
>25
>25
>25
7.0/7.2
ND
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
100/100 >100
23.3
24.8
16.29
>25
75/99
57/99
68/75
85/100
3
10.2/9.9
ND
5.3
>100 9.8/11.8
>100 6.0/10.0
>100 9.7/10.0
29.3
2
6
A
F
15.6
>25
>25
94/100
12
ND
2
2
7
8
A
A
B
F
H
H
12.7
4.18
0.27
>25
ꢀ
13.0
36/56
86/ND
88/100
<0.2
ND
57
7.5/7.7
ND
^
>25
29
0.43
>25
>25
ND
3
0
B
H
0.093
0.36
45/86
46
ND
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1
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2
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2
2
2
2
2
2
2
3
3
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3
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3
3
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3
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
31
B
H
0.28
0.78
>25
79/100
28
ND
3
3
2
3
B
B
H
H
0.10
0.11
0.42
1.51
48.2
>23
66/97
33
11
0.02/9.3
ND
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
74/100
3
4
B
H
1.99
15.3
>25
67/99
12
ND
*
#
^
;
HBV DNA HepG2.2.15; ° HBV DNA HepG2.117; HepG2, 4 days; HepG2, 6 days ; $ Kinetic Solubility pH 7.4
Assessment of replacements of the western Nꢀphenyl amide part was performed relative to
compounds 4, 14, 15 and 16 (Table 3). First, when looking at the impact of R =F on the central
2
2
scaffold (R , Table 3) in 14 and 16 versus hydrogen substituted compound 7 and 6 respectively, a
2
limited impact was noticed. For isosteric replacements, both R : F and H (Table 3) were used.
Replacement of the FꢀPhenyl of 14, or phenyl of 15, by either a benzyl (17) or a cyclohexyl (18)
deteriorated the potency of the compound (as indicated by HBV DNA EC , SEC ratio and/or
50
quenching). In addition, inverting the amide of 7, resulting in 19, or the related compound 20,
diminished the activity significantly, without any indication of remaining CAM properties (table
3
, SEC ratio for 20). NꢀMethylation of the amide of 6, as in 21 also resulted in significantly
reduced activity (lack of quenching and significantly increased HBV DNA EC ), altogether
50
indicating the essential role of the ꢀNHꢀphenyl amide. Additionally, replacement of the ꢀNꢀ
phenyl amide by a phenylꢀimidazole, as in 22 (compared to 15), did not results in a CAM
compound as indicated by the high SEC ratio versus the control. When for this scaffold, the Nꢀ
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Page 11 of 54
Journal of Medicinal Chemistry
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3
4
5
6
7
8
9
phenyl was replaced by either a ꢀNꢀHeteroaryl (Table 3, 23, 24, 25) or a Nꢀnaphthyl (compounds
2
6, 27), no relevant CAM property could be picked up according to SEC and/or quenching.
An early assessment of the substitution pattern on the Nꢀphenyl, indicated that switching the pꢀ
Fluoro of 6 to the ortho position of the Nꢀphenyl, as in 29, retained activity. Reintroduction of a
pꢀFluoro (30) or a mꢀFluoro (31) both led to potent CAM’s. Similarly, replacing one of the mꢀ
Fluoro’s in 30 and 31 by a mꢀMethyl, as in 32 and 33 respectively maintained the high potency,
both on HBV reduction as on the SEC and quenching assays. Nevertheless, when replacing the
pꢀFluoro by a pꢀMethyl, as in 30 versus 34, or introducing an oꢀMethyl, as in 28 versus 4 or 15),
potency is significantly reduced (EC HBV DNA).
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
4
Table 4. SAR of the sulphonamide substitutent R .
*
°
#
$
HLM/
MLM
(%)
EC50 EC50
(
CC50
Sol
SEC
Cpd/ctrl
4
ID
R
ꢀM) (ꢀM) (ꢀM) (ꢀM)
3
5
6
4.50
2.7
70.4
9
65/99
0.4/7.2
3
3
3
26.5 11.1
>100
>100
>100
>100 53/84 6.3/10.8
7
8
7.10
6.3
86
19/52
0.9/7.7
0.6/9.2
8.49 11.0
>100 27/69
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Journal of Medicinal Chemistry
Page 12 of 54
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2
3
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
3
9
4.53 3.03
96.9
48
43
44/96
50/89
ND
4
0
1
5.02 2.99
0.74 0.96
>100
57.5
1.4/9.2
ND
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
4
>100 16/61
42
0.75 3.63
40.3
15
53/99
ND
4
3
4
0.75 8.63
0.34 0.89
39.6
46.0
92
59
18/82
21/88
ND
ND
4
4
5
1.24 2.28
52.5
1
15/31 0.9/10.8
4
4
6
7
0.83 0.90
2.10 3.05
57.7
84
51/99
ND
>100
>100 14/21
0.8/7.7
4
4
8
9
0.96 0.93
>100
>100
3/29
0.5/11.9
ND
1.18 2.03 >100
>100 21/24
>100 32/29
5
0
0.54 1.36 >100
ND
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Page 13 of 54
Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
5
5
5
1
2
3
6.77 4.68 >100
>100
0/23
2.9/10.9
4.2/9.6
ND
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
5.3
16.0 >100
94
10/13
0.93 1.67 >100
>100 14/48
*
#
$
HBV DNA HepG2.2.15; ° HBV DNA HepG2.117; HepG2, 4 days; Kinetic Solubility pH 7.4
In table 4, the alteration of the sulphone amide substitution was investigated. Introduction of
4
secondary amines on R (35ꢀ40) rendered compounds that were still showing CAM properties, in
the case of 35, 37ꢀ40 (SEC ratio and/or quenching), but nevertheless with reduced activity versus
6 and 7 on HBV DNA EC . Introduction of the basic piperazine as in 36, significantly impacted
50
the CAM properties according to quenching and SEC ratio, while maintaining some potency on
HBV DNA reduction. Interestingly, branched NHꢀalkyls like ꢀiPr (41), 1ꢀethylpropyl (42) or
secButyl, with a preference for the Rꢀisomer (44 versus 43), displayed good potency, and for
example in the case of 41 versus compounds 6 and 7 also improved metabolic stability on both
HLM and MLM was observed. Slightly decreased potency (HBV DNA), nevertheless improved
metabolic stability, was observed for the tBuꢀderivative 45. Introduction of a NHꢀcBu showed
similar stability (and slightly lower potency) versus compound 6, while the corresponding
oxetane (47) loses potency (HBV DNA), and gains in metabolic stability, both on MLM and
HLM. Introduction of an extra methyl on the oxetane (48), returned potency (HBV DNA) while
retaining metabolic stability (versus compound 47). The introduction of a heteroatom also
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Journal of Medicinal Chemistry
Page 14 of 54
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2
3
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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
improved metabolic stability for other ring sizes, as observed for tetrahydrofuryl (49 and 50)
versus cyclopentyl and tetrahydroꢀ2Hꢀpyranyl (53) versus cyclohexyl (7), in addition, an
improved kinetic solubility was observed. Although introduction of a nitrogen in the cyclohexyl
of 7 also increased metabolic stability (compound 51 and 52), a decreased potency was observed
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
(HBV DNA).
Using some of the optimal substituents from table 4, taking into account potency, solubility and
4
metabolic stability, R substituents A, B and C (Table 5) were selected for a further combination
with the optimal substituent patterns from table 3.
Table 5. SAR of NꢀPhenyl Substitution.
HLM/
MLM
(%)
*
$
EC50
EC °
50
CC50
Sol
4
8
9
10
11
ID
R
R
R
R
R
(
ꢀM) (ꢀM)
(ꢀM) (ꢀM)
5
5
5
5
4
5
6
7
A
A
A
A
A
A
A
B
B
Me
Me
Me
F
H
F
H
H
H
H
H
H
H
H
H
H
0.12
0.43
0.78
3.30
1.91
0.51
0.98
2.66
0.15
2.59
>50
>25
>25
>25
>50
>50
>25
47.5
28.5
82
26/51
H
F
0.29
2.22
3.63
0.38
0.81
2.33
0.07
0.31
>100 25/66
>100 17/69
>100 37/68
H
Me
H
H
H
H
H
H
H
58
F
>100
5/42
5
9
CN
OCH
Me
Et
H
>100 13/27
>100 20/35
60
3
H
6
6
1
2
H
41
15
26/80
31/88
H
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Page 15 of 54
Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
6
6
6
3
4
5
B
B
B
C
C
C
F
H
H
H
H
H
H
H
H
H
H
H
H
H
0.21
1.40
0.32
0.12
2.74
0.40
2.79
0.91
0.30
1.65
1.91
25.2
29.8
35.2
47.1
>25
>25
>100 18/60
iPr
cPr
Me
Me
Me
13
31/96
27/61
7/65
H
31
66
H
>100
>100
>100
6
6
7
8
Me
H
0/77
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
Me 3.06
1/78
*
#
$
HBV DNA HepG2.2.15; ° HBV DNA HepG2.117; HepG2, 4 days; Kinetic Solubility pH 7.4
Combination of substituents A, B or C with either the Nꢀ(4ꢀfluoroꢀ3ꢀmethylphenyl)amide (Table
: 54, 61 or 66 respectively) or Nꢀ(3,4ꢀdifluorophenyl)amide (Table 5: 58, 63 respectively) led
5
to compounds with improved potency and/or metabolic stability versus 6 (and very significant
9
effect on quenching, see supporting information). Further introduction of a Fluor on R (55) or
11
R
(56) versus compound 54 did not results in improved potency or significant stability (in
contrast the potency of compound 56 is significantly reduced). In addition, introduction of a
9
11
methyl on R (57 versus 58, or 67 versus 66) or R (68 versus 66), reduces potency (HBV DNA)
significantly. Variation of the NꢀPhenyl mꢀalkyl, as in compounds 62 (Et), 64 (iPr) and 65 (cPr),
8
reduced potency (HBV DNA) versus the methyl analogue 61. Replacing the mꢀMethyl (R ) in 54
by mꢀOMethyl (60) reduced potency (HBV DNA), while variation to mꢀcyano (59), resulted in
decreased potency, metabolic stability was further improved.
Among several suitable candidates (compounds 54, 58, 61, 63 and 66) that were further profiled
in vitro, 54 (JNJꢀ632) was selected based on it’s suitable in vitro profile, for further in vivo
characterization. In the highꢀcontent multiparameter cytotoxicity (HepG2), JNJꢀ632 showed
EC ’s in the 10ꢀ30 ꢀM range (considered weakly cytotoxic. Notably, in contrast, 63 was
20
1
6, 17
considered cytotoxic, with and EC ranging down to 1 ꢀM).
20
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Journal of Medicinal Chemistry
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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
While JNJꢀ632 was clean in kinase panel (1 ꢀM, n=240), a receptor, ion channel and transporter
assay profiling panel (n=80), resulted in one hit at 10 µM (5ꢀHT2B (71%)). The compound
didn’t show agonist activity of 5ꢀHT2B, but was an antagonist of 5ꢀHT2B with IC of 5.6 µM.
5
0
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
Activity on the serotonin receptor 2B was observed across several analogues in this series, with
2 and 63 as more potent examples (IC 5HT2B: 0.09 ꢀM and 0.23 ꢀM respectively versus 4.0
1
50
ꢀ
M for JNJꢀ632). JNJꢀ632 did not significantly bind to NaCH, CaCH or hERG with IC > 10
50
µM and an automated hERG patch clamp system indicated that 54 showed 31% of IK inhibition
r
at 3 µM (versus 14% of IK inhibition by the solvent). Moreover, JNJꢀ632 exhibits low CYP
r
inhibition with IC50 > 10 µM against six major CYP enzymes (CYP1A2, CYP2C8, CYP2C9,
CYP2C19, CYP2D6 and CYP3A4). 54 was found positive in GSH (glutathione) trapping assay,
(oxidative defluorination followed by GSH addition) in human liver microsomes. No cleavage of
the Nꢀphenylamide was observed in primary hepatocytes, in contrast to related analogues like
compound 66 (data not shown). Finally, JNJꢀ632 tested negative in Ames II and the in vitro
micronucleus test.
The permeability of JNJꢀ632 was measured in the LLCꢀPK1ꢀMDR1 cell line in A to B
direction in absence and presence of the Pꢀgp inhibitor GFꢀ120,918. The apparent permeability
ꢀ6
value Papp (AꢀB) (ꢀGF) was 7.3 x 10 cm/s and the apparent permeability value Papp (AꢀB)
ꢀ6
(
+GF) was 16.2 x 10 cm/s. The ratio Papp (AꢀB) (+GF)/Papp(AꢀB) (ꢀGF) indicated that the
compound was a weak Pꢀgp substrate. The unbound fraction of JNJꢀ632 was determined to be
2.6% and 8.1% in mouse and human plasma, respectively. Notably, when determining plasma
1
protein binding, for certain compounds, not for JNJꢀ632, a low recovery was noticed specifically
in the case of mouse plasma, not in human plasma, possibly indicating instability in the former
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Page 17 of 54
Journal of Medicinal Chemistry
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3
4
5
6
7
8
9
medium. Finally, JNJꢀ632 gave 51% turnover after 15 min incubation in mouse liver
microsomes and 26% turnover after 15 min incubation in human liver microsomes.
The single dose PK profile of JNJꢀ632 was evaluated in C57BL/6 mice following intravenous
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
(i.v.) and oral (p.o.) administration. JNJꢀ632 had a moderate plasma clearance of 34 mL/min/kg
and a moderate volume of distribution of 1.3 L/kg. The oral bioavailability was 40% following
oral administration of 10 mg/kg and 66% following oral administration of 50 mg/kg. To
circumvent the first pass metabolism, compound was also dosed subcutaneously at 50 mg/kg in
C57BL/6 mice and this resulted in a concentration in plasma after 24 h of dosing of 102 ng/mL
and concentration in liver after 24 h of dosing of 1297 ng/g (Table 6). Finally, the PK of 54 was
assessed in uninfected humanized mice (Phoenix Bio®) following subcutaneous administration
of 50 and 200 mg/kg as a methocel suspension. Compound JNJꢀ632 was dosed for 8 days and
PK was evaluated on days 1 and 8. Repeated subcutaneous dosing of a suspension resulted in
higher exposure at day 8 versus day 1 (Table 7).
Table 6. PK parameters C57BL/6 mice for 54
AUC
0ꢀlast
(ng.h
CL
Dose
C
max
(ng/
(
mL/
min/
kg)
V
ss
t
1/2
(h)
T
max
(h)
F
(%)
(
mg/
(L/ kg)
kg)
mL)
/
mL)
2
(i.v.)
10
.5
34
1.3
0.42
±0.06
ꢀ
ꢀ
1240
±303
ꢀ
±9.2 ±0.20
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
1.1
1940
±296
1990
±159
40
±3.3
(p.o)
0.5
±
0.67
50
(p.o)
2.4
9440
±1420
0.7
±0.3
16400
±2350 ±9.5
66
±
2.3
5
(s.c)
0
5.3
3367
±342
16877 68
±2959 ±12
1.0
±
0.1
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Journal of Medicinal Chemistry
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2
3
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
Table 7. Exposure of JNJꢀ632 in uninfected humanized mice after subcutaneous administration
of 50 and 200 mg/kg.
QD
Dose
(mg/kg)
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
Cmax
Tmax AUC0ꢀ24
ng/mL) (h) (ng.h/mL) (ng.h/mL) (h)
AUC0ꢀ∞
t1/2
C
plasma 24h
,
C
(ng/g)
,
liver 24h
Dosing
day
(
(ng/mL)
2
±
4
190
246
380
4750*
±326
4780
±313
20700
±2850
12700
±561
1.1
±0.2
6.2
±1.1
5.6
±1.7
12.1
±3.2
Day 1
Day 8
Day 1
Day 8
1
50
0.8
±0.4
0.8
19200
±2180
12000
±1060
86900
±6520
167
±70.2
570
±168
±1280
2110
±941
7700
±0.4
200
119000
±4910
1820
±179
4880
±1060
1
±
2490
*
AUC(0ꢀ8h) was used instead of AUC(0ꢀ24h)
Antiviral activity of JNJ-632 in HBV-infected chimeric mice
The antiꢀHBV activity of JNJꢀ632 was tested in HBV infected primary human hepatocytes
(PHHs) derived from naive chimeric mice. qPCR analysis of the HBV DNA in the cell culture
supernatant showed antiꢀHBV activity with an EC of 200 nM for genotype D, which is similar
50
2
to the EC obtained in HepG2.2.15 cells (genotype D). The antiꢀHBV activity of JNJꢀ632 was
5
0
tested in an in vivo proof of mechanism study in genotype D HBVꢀinfected chimeric mice with
humanized liver. HBVꢀinfected chimeric mice were used extensively for HBV research in the
past due to authentic HBV infection of human hepatocytes and persistent formation of cccDNA
1
8
in vivo. All mice used in this study had a human hepatocyte replacement index of 70% or more
as assessed by the level of human albumin in the serum (data not shown).
JNJꢀ632 was dosed subcutaneously once a day over 28 days at a dose of 200 mg/kg, whereas
saline and ETV was dosed once a day orally and served as negative and positive control
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respectively. None of the compounds reduced human albumin levels in the serum over time (data
not shown), indicating that the treatment did not have cytotoxic effects on the human hepatocytes
in the liver of the mice. A mean ꢀ2.77 log reduction of HBV DNA in the serum was observed for
JNJꢀ632, demonstrating that the compound inhibited HBV replication in vivo (Figure 3). No
reduction of HBeꢀ and HBsAg levels was observed during this 28ꢀdays study (data not shown).
1
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0
1.0E+11
1.0E+10
1.0E+09
1.0E+08
Saline
JNJ-632
ETV
1.0E+07
.0E+06
1
1
4
7
10 13 16 19 22 25 28
day
Figure 3: Antiviral activity in HBV genotype D infected chimeric mice. Mean HBV DNA
change in serum after daily treatment with saline, ETV and JNJ-632 for 28 days.
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Table 8. Mean HBV DNA change of ETV and JNJ-632 versus the saline control in HBV
genotype D infected chimeric mice.
Mean HBV DNA
change (SD)*
0
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9
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Saline
ꢀ0.29 (0.14)
3.03 (0.19)
ETV 0.03mg/kg QD,
p.o.
ꢀ
JNJꢀ632 200mg/kg
QD, s.c.
ꢀ2.77 (0.10)
*
from baseline at day 29
HBV genotype D infected chimeric mice with a human hepatocyte replacement index of 70% or
more were treated once a day for 28 days with saline (oral), 0.03mg/kg ETV (oral) and 200
mg/kg JNJꢀ632 (subcutan; s.c.). Values in the table represent the mean HBV DNA change from
baseline at day 29 and the standard deviation (SD) from 4 (ETV) and 5 (saline and JNJꢀ632)
mice per group respectively.
Docking of JNJ-632
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0
Figure 2. Docked binding mode of JNJꢀ632.
JNJꢀ632 was docked to adopt a binding mode as depicted in figure 2. The MethylꢀFluorꢀphenyl
group binds in a hydrophobic pocket surrounded by residues Pro25, Asp29, Leu30, Thr33,
Trp102, Ile105, Ser106, Val124, Arg127 and Thr128. The amide group makes hydrogen bonds
with Trp102 and Thr128, while the sulfonamide group picks up a hydrogen bond with Leu140
and a water mediated hydrogen bond with Ser121. The tetrahydrofuryl group resides in a
solventꢀaccessible area. It is possible that this part of the inhibitor helps in stabilizing the
19
arginineꢀrich domain of the Cꢀterminus, which is not part of the crystal structure.
Chemistry
New 3ꢀsulfamoylꢀNꢀphenylbenzamide compounds were prepared according to the general
scheme 1 starting from either 3ꢀ(chlorosulfonyl)benzoic acid derivatives (Scheme 1a) or 3ꢀ
8
(
chlorosulfonyl)benzoyl chloride (scheme 1b). As depicted in scheme 1a, starting from 3ꢀ
(chlorosulfonyl)benzoic acid 69a, for compounds 7, 10, 29-34, 41-44, 49, 50, 55-57, 59-61 and
6
3 and from 5ꢀ(chlorosulfonyl)ꢀ2ꢀfluorobenzoic acid 69b for compounds 14 and 15, the
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5
6
chlorosulfone was condensed with the corresponding amine in the presence of a base, followed
by amide formation under influence of HATU, or by activation of the carboxylic acid to the
corresponding acid chloride followed by amide formation. Alternatively, for the synthesis of
0
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0
compounds 6, 35-40, 45-48, 51-54, 58, 66-68 applying the well described difference in reactivity
20,21
of carboxy acid chlorides and sulfonyl chlorides,
3ꢀ(chlorosulfonyl)benzoyl chloride is first
reacted with an aniline 72, resulting in the formation of an amide, followed by the reaction of
another amine 70, resulting in the formation of the sulfonamide. Synthesis of compounds 8, 9,
1
1ꢀ13, 17-27, 62, 64 and 65 is described in the supporting information.
Scheme 1. Synthesis new Nꢀphenylꢀ3ꢀsulfamoylꢀbenzamide derivatives.
Reagents and conditions: i) 69a, 70, CH Cl , NEt , room temperature, overnight or 69a, 70,
2
2
3
NaOH, THF, 20ꢀ25°C, 2ꢀ3 hours, 71a-d: 33ꢀ93% yield; 71b: EtOAc, iPrNH , 25°C, 3 hours, 79
2
%
yield, 71e: 69b, cyclohexamine, room temperature, EtOAc, 10 minutes, 86 % yield ii) HATU,
CH Cl DIPEA, 0°C to room temperature, 2 h to overnight, 9ꢀ66 % yield; or 13ꢀ39 % yield over
2
2,
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two steps from 69a (7, 10, 42, 44); 14 and 15: 71e, 72, HATU, CH Cl , DIPEA or NEt , 2ꢀ3
2
2
3
hours, 28ꢀ52 % yield; 55, 56, 59 and 60: 71a, 72, DMF/CH Cl , (COCl) , 2 hours, 0 to 20°C, 2
2
2
2
hours then NEt , CH Cl , 0°C to 20°C, 2 hours, 14ꢀ61 % yield; 57: 71a, 3,4ꢀdifluoroꢀ2ꢀmethyl
3
2
2
1
1
1
1
1
1
1
1
1
1
2
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2
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6
0
1
2
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9
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8
9
0
1
2
3
4
5
6
7
8
9
0
aniline, DIPEA, Pybrop, DMF, 18°C, 23 % yield. iiia) 72, CH Cl NEt , 0°C then 20°C, 1 h or
2
2,
3
toluene, reflux, 30 min iiib) 70, NEt 0°C then 20°C, 1h; 10ꢀ50 % yield over two steps, or (54)
3
7
0, toluene/CH Cl , DIPEA, 4 h room temperature, 39 % two steps.
2 2
Conclusions
In summary, the authors described a hitꢀtoꢀlead optimization, resulting in the discovery of JNJꢀ
32 (54), a Nꢀphenylꢀ3ꢀsulfamoylꢀbenzamide derivative and HBV capsid assembly modulator
6
that can inhibit HBV in vitro (HepG2.117, HepG2.2.15 and HBV infected primary human
hepatocytes) and in vivo (HBV infected humanized mice). Further optimization towards JNJꢀ
3
6
379, a capsid assembly modulator currently in phase 2 clinical trials, will be published in due
course.
Experimental section
Chemistry
Reagents and solvents were purchased from commercial sources and used without purification.
Purity of commercial, tested compounds was assessed by at least one LCꢀMS method and that of
all synthetized and tested compounds by two independent LCꢀMS methods. Compounds
exhibited greater than 95% purity, except for compound 26 which was tested at 94ꢀ95 % purity.
Purities and LC methods are listed in the supporting information.
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NMR spectra were recorded on Bruker DPXꢀ400, Bruker Avance Iꢀ400, Bruker Avance IIIꢀ400
and Bruker Avance IIIꢀHꢀ 600 spectrometers, operating at the frequencies specified. High
resolution mass spectrometry was performed on a Waters Acquity IClass UPLCꢀDAD and Xevo
G2ꢀS QTOF. The samples were run on a Waters BEH C18 (1.7 ꢁm, 2.1 mm × 50 mm, at 50 °C)
column using reversedꢀphase chromatography with a gradient from 95% A to 5% A in 4.6 min,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
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9
0
1
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9
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9
0
held for 0.4 min (A, 95% 6.5 mM CH COONH /5% CH CN; B, CH CN).
3
4
3
3
3
-(cyclopentylsulfamoyl)-N-(4-fluorophenyl)benzamide (6). Synthesis according to the
representative procedure of scheme 1b as described for compound 53. 21% yield. Method B; Rt:
+
+
4
.27 min. m/z: 363.1 (M+H) ESIꢀHRMS (TOF) m/z: [M + H] 363.1184 (calcd for
1
C H FN O S: 362.1100). H NMR (600 MHz, DMSOꢀd ) δ 10.50ꢀ10.62 (m, 1H), 8.36 (t,
18
19
2
3
6
J=1.70 Hz, 1H), 8.19 (ddd, J=1.00, 1.70, 7.80 Hz, 1H), 8.00 (ddd, J=1.00, 1.70, 7.80 Hz, 1H),
.74ꢀ7.82 (m, 4H), 7.19ꢀ7.25 (m, 2H), 3.41ꢀ3.48 (m, 1H), 1.49ꢀ1.64 (m, 4H), 1.33ꢀ1.43 (m, 1H),
7
1
3
1.24ꢀ1.32 (m, 2H). C NMR (151 MHz, DMSOꢀd ) δ 164.2, 158.5, 142.1, 135.6, 135.2, 131.1,
6
1
29.5, 129.3, 125.9, 122.4, 115.3, 54.5, 32.4, 22.8.
3
-(cyclohexylsulfamoyl)-N-(4-fluorophenyl)benzamide (7). To
a
solution of 3ꢀ
(chlorosulfonyl)benzoic acid (1 g, 4.53 mmol) in CH Cl (20 mL) at 5°C, cyclohexanamine
2 2
(0.899 g, 9.06 mmol) and triethylamine (1.38 g, 13.60 mmol) were successively added drop
wise. The solution was stirred at room temperature overnight. The mixture was washed with 1N
HCl (50 mL). The organic phase was dried over MgSO and concentrated resulting in 3ꢀ(Nꢀ
4
cyclohexylsulfamoyl)benzoic acid as a white solid (1.2 g, 93%), which was used in the next step
without purification. To a solution of 3ꢀ(Nꢀcyclohexylsulfamoyl)benzoic acid (1.2 g, 4.24 mmol)
in DMF (15 mL) at 5°C, 4ꢀfluoroaniline (0.52 g, 4.66 mmol) and DIPEA (1.64 g, 12.71 mmol)
were successively added.. The mixture was stirred for 20 minutes and then HATU (1.93 g, 5.08
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mmol) was added. The solution was stirred at room temperature overnight. To the reaction
mixture aqueous NaHCO (50 mL) was added followed by EtOAc (50 mL). The organic layer
3
was washed with HCl (5%; 50 mL) and brine. The organic layer was dried with MgSO and
4
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
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2
3
4
5
6
7
8
9
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2
3
4
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7
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9
0
concentrated, resulting in a residue. The obtained residue was purified by a silica gel
chromatography column (Petroleum ether:EtOAc=2:1) resulting in compound 7 as a white solid
+
(
850 mg, 25 % yield over two steps). Method B; Rt: 4.50 min. m/z: 377.2 (M+H) ESIꢀHRMS
1
(
TOF) m/z: [M + H]+ 377.1339 (calcd for C H FN O S: 376.1257). H NMR (400 MHz,
1
9
21
2
3
DMSOꢀd ) δ 10.55 (s, 1H), 8.36ꢀ8.39 (m, 1H), 8.16ꢀ8.22 (m, J=8.10 Hz, 1H), 7.99ꢀ8.05 (m,
6
J=8.10 Hz, 1H), 7.74ꢀ7.83 (m, 4H), 7.19ꢀ7.25 (m, 2H), 2.94ꢀ3.04 (m, 1H), 1.51ꢀ1.63 (m, 4H),
1
3
1.40ꢀ1.51 (m, 1H), 1.00ꢀ1.23 (m, 5H). C NMR (101 MHz, DMSOꢀd ) δ 164.2, 158.4, 142.8,
6
1
35.5, 135.2, 131.0, 129.4, 129.0, 125.6, 122.4, 115.2, 52.1, 33.2, 24.8, 24.3
3
-[cyclopentyl(methyl)sulfamoyl]-N-(4-fluorophenyl)benzamide (10).
To a solution of 3ꢀ(chlorosulfonyl)benzoic acid (1.10 g, 4.97 mmol) in THF (60 mL), sodium
hydroxide was added (aq., 2 mL, 5N) while cooled in an ice bath, followed by adding N-methylꢀ
cyclopentanamine (0.50 g, 4.97 mmol). After stirring at 25°C for 3 hours, the reaction mixture
was diluted with H O (50mL) and extracted with EtOAc (50 mL). The aqueous layer was
2
adjusted to pH=3 by HCl (2N) and extracted with EtOAc (3 x 50 mL). The combined organic
layer was washed with brine, dried over anhydrous MgSO and concentrated in vacuo resulting
4
in 3ꢀ(NꢀcyclopentylꢀNꢀmethylsulfamoyl)benzoic acid (0.8 g). To a solution of 3ꢀ(Nꢀcyclopentylꢀ
Nꢀmethylsulfamoyl)benzoic acid (0.80 g, 2.82 mmol), 4ꢀfluoroaniline (0.31 g, 2.82 mmol), and
HATU (1.61 g, 4.24 mmol) in CH Cl (10 mL), cooled in an icebath, DIPEA (1.09 g, 8.47mmol)
2
2
was added under N atmosphere. The resulting mixture was diluted with CH Cl (15 mL) and
2
2
2
washed with saturated aqueous NaHCO (15 mL) and brine (10 mL), dried over anhydrous
3
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MgSO and the solvent was removed in vacuo. The obtained residue was purified by preparative
4
high performance liquid chromatography over RPꢀ18 (eluent: CH CN in H O from 30% to 80%,
3
2
v/v; 0.05% TFA as addition). The pure fractions were collected and the volatiles were removed
in vacuo. The aqueous layer was adjusted to Ph=7 with Amberlite IRAꢀ900 ion exchange resin
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
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3
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2
3
4
5
6
7
8
9
0
(OH form), filtrated and lyophilized to dryness resulting in compound 10 (0.73 g, 39 % two
+
+
steps). Method B; Rt: 4.43 min. m/z: 377.2 (M+H) ESIꢀHRMS (TOF) m/z: [M + H] 377.1337
1
(
calcd for C H FN O S: 376.1257). H NMR (400 MHz, DMSOꢀd ) δ 10.55 (s, 1H), 8.31ꢀ8.35
1
9
21
2
3
6
(m, 1H), 8.22ꢀ8.27 (m, J=7.70 Hz, 1H), 7.97ꢀ8.02 (m, J=8.10 Hz, 1H), 7.74ꢀ7.82 (m, 3H), 7.18ꢀ
1
3
7
.25 (m, 2H), 4.29 (quin, J=7.97 Hz, 1H), 2.68 (s, 3H), 1.26ꢀ1.54 (m, 8H); C NMR (101 MHz,
DMSOꢀd ) δ 163.9, 158.5, 139.1, 135.6, 135.1, 131.7, 129.8, 129.7, 125.9, 122.5, 115.2, 57.8,
6
2
8.5, 27.3, 23.6
5
-(cyclohexylsulfamoyl)-2-fluoro-N-(4-fluorophenyl)benzamide (14).
5
ꢀ(Nꢀcyclohexylsulfamoyl)ꢀ2ꢀfluorobenzoic acid (2.0 g, 6.637 mmol), 4ꢀfluoroaniline (0.740 g,
6.660 mmol) and HATU (3.78 g, 9.941 mmol) were dissolved in dichloromethane (40 ml) at
0°C. Triethylamine (1.34 g, 13.3 mmol) was added to the reaction mixture. The reaction
2
mixture was stirred for 2 hours at 20°C. The mixture was was washed with saturated aqueous
K CO (20 ml), brine and dried over Na SO . The solvent was removed under vacuum. The
2
3
2
4
crude product was purified by preparative high performance liquid chromatography (column:
Phenomenex Synergi C18 250*50mm*10um, mobile phase: CH CN in water (TFA 0.1%) from
3
2
0% to 65%, flow rate: 60 ml/min). The pure fractions were collected and washed with aqueous
Na CO , extracted with DCM (40 mL). The organic layer was concentrated in vacuum to give
2
3
+
compound 14 (1080 mg, 52 %). Method C; Rt: 4.21 min. m/z: 395.1 (M+H) ESIꢀHRMS (TOF)
+
1
m/z: [M + H] 395.1244 (calcd for C H F N O S: 394.1163). H NMR (400 MHz, DMSOꢀd )
19 20
2
2
3
6
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δ ppm 10.68 (1 H, br. s), 8.08 (1 H, dd, J=6.0, 2.5 Hz), 8.01 (1 H, ddd, J= 8.5, 4.5, 2.5 Hz), 7.83
(1 H, br. s), 7.70 ꢀ 7.77 (2 H, m), 7.60 (1 H, app. t, J= 9.0 Hz), 7.18 ꢀ 7.27 (2H, m), 2.90 ꢀ 3.07 (1
1
3
H, m), 1.53 ꢀ 1.67 (4 H, m), 1.40 ꢀ 1.53 (1 H, m), 0.96 ꢀ 1.25 (5 H, m). C NMR (101 MHz,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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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
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7
8
9
0
1
2
3
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7
8
9
0
DMSOꢀd ) δ 161.2, 160.5, 158.5, 138.9, 134.9, 130.7, 128.4, 125.3, 121.7, 117.4, 115.4, 52.1,
6
3
3.2, 24.8, 24.3.
5-(cyclohexylsulfamoyl)-2-fluoro-N-phenyl-benzamide (15).
To an ice cooled mixture of 5ꢀ(Nꢀcyclohexylsulfamoyl)ꢀ2ꢀfluorobenzoic acid (602 mg, 2 mmol,
1
.0 eq.), aniline (223 mg, 2.4mmol, 1.2 eq.), HATU (1.14 g, 3.06 mmol, 1.5 eq.) in CH Cl (15
2 2
mL) was added DIPEA (0.774 g, 6 mmol, 3.0 eq.) under N atmosphere. The resulting mixture
2
was stirred at room temperature for 3 h. The reaction mixture was diluted with CH Cl (30 mL)
2
2
and washed with saturated aq.NaHCO solution (10 mL), brine (10 mL) and dried over Na SO .
3
2
4
The organic layer was concentrated under vacuum. The crude product was purified by
preparative high performance liquid chromatography preparative highꢀperformance liquid
chromatography (column: C18, eluent: CH3CN/H2O from 35/65 to 60/40, 0.1% CF COOH).
3
The desired fraction was collected and basified by saturated NaHCO (aq.). The mixture was
3
concentrated and extracted with CH Cl (30 mL* 3). The organic layer was separated, dried over
2
2
Na SO and the solvent was removed under vacuum. The product was dried under vacuum to
2
4
+
result in compound 15 (210 mg, 28%). Method C; Rt: 4.17 min. m/z: 377.1 (M+H) ESIꢀHRMS
+
1
(TOF) m/z: [M + H] 377.1334 (calcd for C H FN O S: 376.1257). H NMR (400 MHz,
1
9
21
2
3
DMSOꢀd ) δ 10.59 (s, 1H), 8.06ꢀ8.11 (m, 1H), 7.97ꢀ8.05 (m, 1H), 7.81 (br s, 1H), 7.68ꢀ7.76 (m,
6
2
H), 7.59 (t, J=9.15 Hz, 1H), 7.38 (t, J=7.53 Hz, 2H), 7.09ꢀ7.19 (m, 1H), 2.99 (br s, 1H), 1.60
13
(
br d, J=7.73 Hz, 4H), 1.46 (br d, J=11.80 Hz, 1H), 0.96ꢀ1.28 (m, 6H); C NMR (101 MHz,
DMSOꢀd ) δ 161.3, 160.4, 138.8, 138.5, 130.6, 128.8, 128.4, 125.5, 124.1, 119.8, 117.4, 52.1,
6
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3.2, 24.8, 24.3;
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-(cyclopentylsulfamoyl)-N-(3-fluorophenyl)benzamide (29).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
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66% yield from 3ꢀ(Nꢀcyclopentylsulfamoyl)benzoic acid according to the representative
procedure of scheme 1a as described for compound 32. Method B; Rt: 5.45 min. m/z: 363.2
+
+
1
(M+H) ESIꢀHRMS (TOF) m/z: [M + H] 363.1178 (calcd for C H FN O S: 362.1100). H
18 19 2 3
NMR (400 MHz, DMSOꢀd ) δ ppm 1.23 ꢀ 1.43 (m, 4 H) 1.47 ꢀ 1.64 (m, 4 H) 3.38 ꢀ 3.51 (m, 1
6
H) 6.97 (td, J=8.3, 2.5 Hz, 1 H) 7.41 (td, J=8.3, 7.0 Hz, 1 H) 7.54 ꢀ 7.59 (m, 1 H) 7.71 ꢀ 7.85 (m,
3
H) 8.02 (ddd, J=7.8, 1.5, 1.0 Hz, 1 H) 8.19 (ddd, J=7.8, 1.5, 1.0 Hz, 1 H) 8.35 (t, J=1.5 Hz, 1
1
3
H) 10.68 (br s, 1 H). C NMR (101 MHz, DMSOꢀd ) δ 164.5, 162.0, 142.1, 140.6, 135.4, 131.2,
6
130.3, 129.5 (2xC), 125.9, 116.2, 110.4, 107.2, 54.5, 32.4, 22.8
3
-(cyclopentylsulfamoyl)-N-(3,4-difluorophenyl)benzamide (30).
4
7 % yield from 3ꢀ(Nꢀcyclopentylsulfamoyl)benzoic acid according to the representative
procedure of scheme 1a as described for compound 32. Method B; Rt: 4.34 min. m/z: 381.2
+
+
1
(M+H) ESIꢀHRMS (TOF) m/z: [M + H] 381.1085 (calcd for C H F N O S: 380.1006). H
18 18 2 2 3
NMR (400 MHz, DMSOꢀd ) δ ppm 1.20 ꢀ 1.44 (m, 4 H), 1.44 ꢀ 1.68 (m, 4 H), 3.44 (sxt, J=6.8
6
Hz, 1 H), 7.45 (dt, J=10.6, 9.0 Hz, 1 H), 7.51 ꢀ 7.60 (m, 1 H), 7.77 (t, J=7.8 Hz, 1 H), 7.80 (d,
J=7.2 Hz, 1 H), 7.93 (ddd, J=13.2, 7.5, 2.5 Hz, 1 H), 8.02 (d, J=7.8 Hz, 1 H), 8.19 (d, J=7.7 Hz,
1
3
1
H), 8.35 (t, J=1.7 Hz, 1 H), 10.70 (s, 1 H). C NMR (101 MHz, DMSOꢀd ) δ 164.4, 148.8,
6
145.7, 142.1, 135.8, 135.2, 131.1, 129.5 (2xC), 125.8, 117.3, 116.8, 109.5, 54.5, 32.4, 22.8
3
-(cyclopentylsulfamoyl)-N-(3,5-difluorophenyl)benzamide (31).
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% yield from 3ꢀ(Nꢀcyclopentylsulfamoyl)benzoic acid according to the representative
procedure of scheme 1a as described for compound 32. Method B; Rt: 4.43 min. m/z: 381.2
+
+
1
(M+H) ESIꢀHRMS (TOF) m/z: [M + H] 381.1087 (calcd for C H F N O S: 380.1006). H
1
8
18
2
2
3
1
1
1
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0
NMR (400 MHz, DMSOꢀd ) δ 10.86 (br s, 1H), 8.37ꢀ8.40 (m, 1H), 8.19ꢀ8.24 (m, J=7.70 Hz,
6
1
H), 8.00ꢀ8.06 (m, J=8.50 Hz, 1H), 7.71ꢀ7.88 (m, 2H), 7.54ꢀ7.62 (m, 2H), 6.99 (tt, J=2.42, 9.28
1
3
Hz, 1H), 3.38ꢀ3.50 (m, 1H), 1.48ꢀ1.65 (m, 4H), 1.23ꢀ1.43 (m, 4H); C NMR (101 MHz, DMSOꢀ
d6) δ 164.7, 162.3, 142.2, 141.4, 135.0, 131.3, 129.7, 129.5, 125.9, 103.2, 99.0, 54.5, 32.4, 22.8
3
-(cyclopentylsulfamoyl)-N-(4-fluoro-3-methyl-phenyl)benzamide (32)
Representative procedure route scheme 1a: To an icedꢀcooled mixture of cyclopentanamine (1.93
g, 22.66 mmol) and a solution of NaOH (1.81 g, 45.32 mmol) in H O (25 mL) and THF (25 mL)
2
was added 3ꢀ(chloroꢀsulfonyl)benzoic acid (5.0 g, 22.66 mmol) in portions. The reaction
mixture was stirred at 20°C for 2 hours. The resulting mixture was diluted with H O (20 mL) and
2
extracted with ethyl acetate (30 mL). The aqueous layer was separated and adjusted pH =2 by 4
N HCl and extracted with dichloromethane (3 x 30 mL). The combined organic layers were
washed with brine (15 mL), dried over anhydrous Na SO and concentrated under reduced
2
4
1
pressure to afford 3ꢀ(Nꢀcyclopentylsulfamoyl)benzoic acid (71d, 4.5 g, 73 %). H NMR (400
MHz, DMSOꢀd ) δ 8.30 ꢀ 8.34 (m, 1H), 8.10 ꢀ 8.20 (m, 1H), 8.00 ꢀ 8.05 (m, 1H), 7.80 (d, J = 7.2
6
Hz, 1H), 7.65 ꢀ 7.75 (m, 1H), 1.40 ꢀ 1.60 (m, 4H), 1.30 ꢀ 1.38 (m, 2H), 1.20 ꢀ 1.28 (m, 2H), one
CH signal under residual water peak. To an ice cooled mixture of 3ꢀ(Nꢀ
cyclopentylsulfamoyl)benzoic acid (250 mg, 0.928 mmol), 4ꢀfluoroꢀ3ꢀmethylaniline (116.2 mg,
0
.928 mmol) and HATU (388.2 mg, 1.021 mmol) in CH Cl (15 mL) DIPEA (359.8 mg, 2.784
2 2
mmol) was added under a N atmosphere. The resulting mixture was stirred at 20°C for 16 hours.
2
The solvent was removed in vacuo. The mixture was washed with saturated aqueous critic acid
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