Synthesis of sulfamoylbenzamide derivatives as HBV capsid assembly effector

Article abstract of DOI:10.1016/j.ejmech.2017.06.062

The synthesis of novel series of sulfamoylbenzamides as HBV capsid assembly effector is reported. The structure was divided into five parts which were independently modified as part of our lead optimization. All synthesized compounds were evaluated for their anti-HBV activity and toxicity in human hepatocytes, lymphocytes and other cells. Additionally, we assessed their effect on HBV cccDNA formation in an HBeAg reporter cell-based assay. Among the 27 compounds reported, several analogs exhibited submicromolar activities and significant reduction of HBeAg secretion. Selected compounds were studied under negative-stain electron microscopy for their ability to disrupt the HBV capsid formation. Structures were modeled into a binding site recently identified in the HBV capsid protein for similar molecules to rationalize the structure-activity relationships for this family of compounds.

Full text of DOI:10.1016/j.ejmech.2017.06.062

Accepted Manuscript
Synthesis of sulfamoylbenzamide derivatives as HBV capsid assembly effector
Ozkan Sari, Sebastien Boucle, Bryan D. Cox, Tugba Ozturk, Olivia Ollinger Russell,
Leda Bassit, Franck Amblard, Raymond F. Schinazi
PII:
S0223-5234(17)30516-0
10.1016/j.ejmech.2017.06.062
EJMECH 9557
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 May 2017
Revised Date: 27 June 2017
Accepted Date: 28 June 2017
Please cite this article as: O. Sari, S. Boucle, B.D. Cox, T. Ozturk, O.O. Russell, L. Bassit, F. Amblard,
R.F. Schinazi, Synthesis of sulfamoylbenzamide derivatives as HBV capsid assembly effector, European
Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.06.062.
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KEYWORDS: sulfamoylbenzamide, antiviral agent, HBV, capsid assembly effector, cccDNA, HBeAg.

ACCEPTED MANUSCRIPT
Synthesis of sulfamoylbenzamide derivatives as HBV capsid assembly effector
Ozkan Sari, Sebastien Boucle, Bryan D. Cox, Tugba Ozturk, Olivia Ollinger Russell, Leda Bassit, Franck Amblard, and Raymond
F. Schinazi*
Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of
Medicine, Atlanta, GA 30322, USA.
ABSTRACT: The synthesis of novel series of sulfamoylbenzamides as HBV capsid assembly effector is reported. The structure
was divided into five parts which were independently modified as part of our lead optimization. All synthesized compounds were
evaluated for their anti-HBV activity and toxicity in human hepatocytes, lymphocytes and other cells. Additionally, we assessed
their effect on HBV cccDNA formation in an HBeAg reporter cell-based assay. Among the 27 compounds reported, several analogs
exhibited submicromolar activities and significant reduction of HBeAg secretion. Selected compounds were studied under negative-
stain electron microscopy for their ability to disrupt the HBV capsid formation. Structures were modeled into a binding site recently
identified in the HBV capsid protein for similar molecules to rationalize the structure-activity relationships for this family of
compounds.
1. Introduction
Hepatitis B virus (HBV) remains a major public health concern with over 2 billion people estimated to be infected worldwide.
However, while most healthy adults are capable of recovering from the infection, up to 50% of children will develop chronic
infections if the infection occurs before the age of 6 [1]. If left untreated, these subjects, adults and children, will experience liver
diseases such as cirrhosis and hepatocellular carcinoma. A constant suppression of the viral replication is then required with
available treatments involving polymerase inhibitors such as lamivudine (3TC), entecavir (ETV) and tenofovir-diisoproxyl
fumarate (TDF) or tenofovir alafenamide (TAF) [2]. Although these drugs are very effective at controlling virus infection, long-
term treatments may cause drug resistance or severe side effects and more importantly, none of these drugs can cure HBV
infections [3, 4].
Fig 1. Capsid assembly effectors.
Nucleocapsids have an important role in the HBV viral replication cycle since they play a role during genome packaging, reverse
transcription, intracellular trafficking and maintenance of chronic infection [5]. Early discoveries have shown that interactions of
the core proteins with small heterocyclic molecules can induce faulty assembly which leads to the formation of dysfunctional
nucleocapsids [6, 7]. By acting on the encapsidation process of viral material, capsid assembly effectors (CAEs) are expected to
significantly decrease the formation of cccDNA partly responsible of the viral persistence [8]. Disrupting HBV capsids allows
exposure of cccDNA to degrading enzymes. In recent years, several classes of CAE targeting the HBV nucleocapsid
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formation/disruption have emerged. Among them, heteroarylpyrimidines (GLS-4, phase II) [9], phenylpropenamides (AT-130)[10]
and sulfamoylbenzamide derivatives (DVR-23 [11], NVR 3-778 [12], phase IIa, structure not yet disclosed) exhibited promising
antiviral effects and could potentially lead to a curative treatment (Figure 1). As part of our HBV research program, we turned our
attention towards the sulfamoyl scaffold of DVR-23 and designed new series of derivatives. Herein, we wish to report their detailed
synthesis along with their antiviral evaluation and characterization.
2. Results and discussion
2.1. Chemical synthesis
To simplify the structure-activity relationship (SAR) study, the sulfamoylbenzamide scaffold was divided in five parts labeled A, B,
C, D and E (Figure 2).
Fig 2. Structure of the sulfamoylbenzamide scaffold.
Part A: Various groups selected from alkyls, cycloalkyls, alkylaryls or sulfonyls were introduced at this position.
Sulfamoylbenzamide derivatives 4a-s were prepared from 2-fluorobenzoic acid 1 by reaction with chlorosulfonic acid leading to
sulfonyl chloride 2 in 77% yield (Scheme 1) [13]. Subsequent reaction with thionyl chloride followed by treatment with 3,4-
difluoroaniline provided the amide 3 in 65% yield. This key intermediate was then reacted with various primary and secondary
aliphatic and benzyl amines, amino acids and sulfonamides to afford a small library of structurally diverse sulfamoyl analogs
(Compounds 4a-s).
Scheme 1. Reagents and conditions: (i) ClSO3H, 80 °C, 4 h, 77%; (ii) SOCl2, 80 °C, 16 h then 3,4-difluoroaniline, toluene, 110 °C,
2 h, 65%; (iii) amine, Et3N, CH2Cl2, 0 °C to rt (or 40 °C), 2 h, 26-94%.
Part B: Inversion of the sulfonamide portion was also investigated. The synthesis of derivatives 9-11 was achieved in four steps
from commercially available 5-amino-2-fluorobenzoic acid 5 by first Boc-protection of the amino group to afford derivative 6 in
74% yield (Scheme 2). The resulting intermediate was then activated using HATU and reacted with 3,4-difluoroaniline to afford
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compound 7 in 90% yield. Finally, deprotection of the Boc group using TFA followed by reaction with different cycloalkylsulfonyl
chlorides (cycloproyl, cyclopentyl and cyclohexyl) provided the desired analogs 9-11 in moderate yields (23-47%).
Scheme 2. Reagents and conditions: (i) Boc2O, NaHCO3, 1,4-dioxane/H2O, 0 °C to rt, 16 h, 74%; (ii) 3,4-difluoroaniline, HATU,
DIPEA, DMF, rt, 1 h, 90%; (iii) TFA, DCM, 0 °C to rt, 2 h, 60%; (iv) cycloalkylsulfonyl chloride, Et3N, CH2Cl2, DMAP, 40 °C, 3
h, 23-47%.
Part C: Inversion of the amide group was also evaluated through the synthesis of compound 16. The synthesis was initiated by a
Friedel-Craft sulfonylation of 2-fluoroacetanilide 12 by treatment with chlorosulfonic acid [14]. As expected, the reaction produced
a mixture of regioisomers separable by column chromatography. Structure of both isomers was established by careful analysis of
1
coupling constant observed in H NMR. The regioisomer 13 was then reacted with cyclopentylamine to afford intermediate 14 in
88% yield. Finally, deacetylation under acidic conditions followed by reaction with 3,4-difluorobenzoyl chloride gave the targeted
analog 16 (Scheme 3).
Scheme 3. Reagents and conditions: (i) ClSO3H, 80 °C, 5 h, 62%; (ii) cyclopentylamine, Et3N, CH2Cl2, rt, 2 h, 88%; (iii) HCl 6N,
100 °C, 1 h, 74%; (iv) 3,4-difluorobenzoyl chloride, Et3N, CH2Cl2, 0 °C to rt, 2 h, 36%.
Part D: The original aniline moiety of our scaffold was replaced with various substituted benzyl amines using the chemistry
described in scheme 4. Cyclopropyl-substituted benzylamine 18 was prepared by treatment of 3,4-difluorobenzonitrile 17 with
ethylmagnesium bromide and titanium isopropoxide [15], while α-dimethyl substituted benzylamine 20 was prepared from 17 by
treatment with methyl lithium in presence of cerium chloride [16]. Coupling of acid 2 with either 18 or 20 and subsequent reaction
with cyclopentylamine afforded derivatives 19 and 21, respectively.
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Scheme 4. Reagents and conditions: (i) EtMgBr, Ti(Oi-Pr)4, BF3.Et2O, Et2O, -78 °C, 1 h, 56%; (ii) MeLi, CeCl3, THF, -25 °C, 1 h,
51%; (iii) 2, SOCl2, 80 °C, 16 h then 18 or 20, toluene, 110 °C, 2 h; (iv) cyclopentylamine, Et3N, CH2Cl2, rt, 2 h, 34% for 19 and
25% for 21 over two steps.
Part E: Finally, a new series of more rigid bicyclic derivatives was synthesized (Scheme 5). Commercially available
phenylethylamine 22 and 3-phenyl-1-propylamine 23 were acylated using methyl chloroformate and subsequently treated with
trifluoromethanesulfonic acid to form derivatives 24 and 25, respectively. These intermediates were then regioselectively
sulfonylated by treatment with chlorosulfonic acid to afford the corresponding 3-sulfonyl chloride derivatives 26 and 27 in high
yields. 26 and 27 were then reacted with cyclopentylamine and N-methylcyclopentylamine to afford compounds 28-30 in yields
ranging from 64% to 94%. These intermediates were then N-alkylated with 3,4-difluorobromobenzene via a copper iodide
catalyzed Ullmann reaction. Overall, these reactions were sluggish, requiring 12-15 hr of heating at high temperature (150 °C) and
a stoichiometric amount (and sometimes an excess) of copper iodide to reach full conversion. Interestingly, the coupling was found
to proceed faster with N-methyl substrates 30 and 31 under microwave irradiation after 90 minutes while no improvement was
observed for the unsubstituted substrates 28 and 29. It is noteworthy that additional arylation of the sulfonamide moiety was also
observed when starting from substrates 28 and 29.
Scheme 5. Reagents and conditions: (i) (a) methyl chloroformate, Et3N, DMF, 0 °C to rt, 1 h; b) CF3SO3H, 70 °C, 24 h, 82% for 24
and 84% for 25 over two steps; (ii) ClSO3H, 60 °C, 16 h, 83 % for 26 and 79% for 27; (iii) cyclopentylamine or N-
methylcyclopentylamine, Et3N, CH2Cl2, rt, 2 h, 81% for 28, 97% for 29, 69% for 30, 94% for 31; (iv) 3,4-difluorobromobenzene,
CuI, K2CO3, DMF, 150 °C, 16 h (or microwave, 150 °C, 90 min), 56% for 32, 58% for 33, 28% for 34 and 35% for 35.
2.2. Biological evaluation
The in vitro anti-HBV activity and safety profile of all new compounds were assessed by RT-PCR in HepAD38 cells as previously
described by Stuyver et al. [17] The concentration of compound that inhibited HBV DNA replication by 50% (EC50) was
determined by linear regression. All data were given relative to the untreated control. In addition, cytotoxicity was determined by
using the CellTiter 96 non-radioactive cell proliferation colorimetric assay (Promega) in peripheral blood mononuclear (PBM) cells
and in human T lymphoblast (CEM), African green monkey kidney (Vero), and human hepatocellular carcinoma (HepG2) cell
lines. Toxicity levels were measured as the concentration of test compound that inhibited cell growth by 50% (CC50). Several
derivatives exhibited potent activities with EC50 comprised between 0.8 and 9.2 µM while only displaying moderate toxicities in
primary human lymphocytes, Vero and CEM cells (compounds 4a-s, 9-11, 16, 19, 21, 32-35, Table 1). Overall, cycloalkyls gave
the best results among the first 19 synthesized analogs while benzyl, amino acid or disulfonamide substituted compounds were
found to be less potent or devoid of antiviral activity (Part A). The potency of cyclopentyl sulfonamide 4a, previously reported by
Campagna et al. [11] (DVR-56), was confirmed in our assay with an EC50 of 0.8 ± 0.2 µM (reported EC50 in HepDES19 cells: 0.14
± 0.09 µM). The difluoro azetidine substituted derivative 4f exhibited a comparable anti-HBV activity (EC50 = 1.2 ± 0.6 µM) but
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displayed higher toxicity in CEM and Vero cells. It is noteworthy that inversion of the sulfonamide group on part B (compounds 9-
11) mostly preserved the potency of the parent compound with the cyclopentyl-substituted analog 10 being the most active
compound of this series (EC50 = 2.1 ± 1.3 µM). Regarding modifications on both part C and D, the inversion of the amide group
(compound 16) and replacement of the 3,4-difluoroarylamide group with its α-substituted benzyl counterparts (compound 19 and
21) led to complete loss of activity. Similarly, the bicyclic analogs 32-35 did not exhibit any activity suggesting the relative
importance of preserving the NH-aryl amide moiety or the flexibility of the molecule (Part E).
Table 1. Anti-HBV activity and cytotoxicity
Anti-HBV activity (µM)
Cytotoxicity CC50 (µM)
Compound
EC50
EC90
PBM
CEM
Vero
HepG2
37.1 ± 0.04
48.6 ± 0.01
> 100
7.8 ± 0.6
0.8 ± 0.2
7.7 ± 1.6
4.5 ± 0.3
6.7 ± 0.6
9.2 ± 1.3
1.2 ± 0.6
> 10
15.6 ± 0.01
25.8 ± 0.03
> 100
14.0 ± 0.01
14.5 ± 0.07
10.3 ± 0.09
4.8 ± 0.78
≤ 1
32.7 ± 0.11
18.3 ± 0.13
> 100
4a
4b
4c
4d
4e
4f
≥ 10 (87 ± 0.2)
9.4 ± 0.1
> 10 (83 ± 1.7)
> 10 (59 ± 14)
6.3 ± 2.9
N/A
> 100
21.3 ± 0.06
9.0 ± 0.02
14.7 ± 0.04
39.6 ± 0.13
79.2 ± 0.03
30.7 ± 0.02
39.4 ± 0.26
68.0 ± 0.03
13.1 ± 0.06
> 100
> 100
> 100
> 100
69.1 ± 0.07
> 100
3.7 ± 0.04
37.8 ± 0.16
13.6 ± 0.12
22.9 ± 0.05
3.8 ± 0.07
3.6 ± 0.16
31.1 ± 0.16
31.6 ± 0.08
10.8 ± 0.05
6.4 ± 0.04
> 100
27.0 ± 0.07
>100
4g
4h
4i
8.9 ± 0.05
10 ± 0.01
9.6 ± 0.1
9.3 ± 0.03
N/A
3.2 ± 0.4
4.6 ± 1.2
6.8 ± 0.9
7.7 ± 2.5
> 10
30.1 ± 0.06
> 100
62.3 ± 0.13
> 100
> 100
88.2 ± 0.08
> 100
4j
> 100
4k
4l
> 100
45.5 ± 0.11
76.8 ± 0.01
> 100
N/A
> 10
> 100
4m
4n
4o
4p
4q
4r
4s
N/A
> 10
58.1 ± 0.04
13.0 ± 0.05
> 100
39.1 ± 0.09
5.8 ± 0.04
> 100
> 10 (82 ± 3.2)
N/A
2.7 ± 1.1
> 10
26.4 ± 0.03
> 100
N/A
> 10
> 100
> 100
> 100
> 100
N/A
> 10
> 100
> 100
> 100
> 100
N/A
> 10
> 100
> 100
> 100
> 100
> 10 (76 ± 1.6)
9.5 ± 0.8
≥ 10 (86 ± 2.1)
N/A
6.9 ± 0.1
2.1 ± 1.3
3.7 ± 0.1
≥ 10
39.2 ± 0.04
> 100
11.8 ± 0.12
54.2 ± 0.11
74.5 ± 0.07
13.6 ± 0.01
> 100
30.7 ± 0.04
29.1 ± 0.08
6.4 ± 0.06
87.5 ± 0.01
12.7 ± 0.03
84.3 ± 0.04
> 100
61.8 ± 0.03
> 100
9
10
11
16
19
21
32
33
34
35
3TC
> 100
80.4 ± 0.11
>100
81.0 ± 0.01
> 100
N/A
> 10
> 100
N/A
> 10
> 100
64.0 ± 0.04
> 100
> 100
N/A
> 10
> 100
> 100
N/A
> 10
> 100
50.6 ± 0.10
36.0 ± 0.07
> 100
> 100
> 100
N/A
> 10
71.2 ± 0.03
> 100
> 100
> 100
N/A
> 10
> 100
> 100
0.3 ± 0.02
0.04 ± 0.03
> 100
> 100
> 100
> 10
Data are the mean of replicates of two or three ± standard deviation (SD) from 2 independent experiments with representative
results. N/A: not applied. Results in parenthesis: % of inhibition ± SD at 10 µM.
To determine whether our active derivatives have an effect on the levels of cccDNA formation, secretion of HBe antigen was
measured (by ELISA, Biochain) as a cccDNA-dependent marker using the HepAD38 cells and tested them in parallel with GLS4,
and 3TC or entecavir [18, 19]. Potent polymerase-based inhibitors, such as 3TC or entecavir, are expected to have negligible on
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cccDNA formation/amplification both in vivo and in vitro. Although they suppress HBV DNA levels in serum, they do not inhibit
de novo formation of viral cccDNA in infected hepatocytes [18-21]. Thus, as expected, GLS4 reduced secretion of HBeAg whereas
10 µM 3TC or ETV had no effect (Table 2). All five selected derivatives reduced secreted HBeAg, with EC50s varying from 1.0 to
9.7 µM, and with drug-induced inhibition of intra/extra viral replication. Remarkably, HBeAg secretion was reduced by 90% at 3-4
µM with compounds 4a and 4f versus 2 µM with GLS-4. The novel sulfamoyl derivatives could be negatively acting on the
stabilization maturation-associated nucleocapsid, consequently interrupting the release of relaxed capsid into the nucleus, and the
subsequent cccDNA formation.
Table 2. Sulfamoyl derivatives reduce HBV cccDNA formation in an HBeAg reporter cell-based assay.
HBeAg Secretion
EC50 (µM)
1.3 ± 0.4
HBV DNA - Extracellular
HBV DNA - Intracellular
Compound
EC90 (µM)
4.6 ± 2.0
EC50 (µM)
0.8 ± 0.2
1.2 ± 0.6
6.9 ± 0.1
2.1 ± 1.3
EC90 (µM)
EC50 (µM)
0.6 ± 0.002
0.9 ± 0.05
EC90 (µM)
7.8 ± 0.6
6.3 ± 2.9
4.3 ± 0.07
9.7 ± 0.04
8.1 ± 0.001
3.6 ± 0.14
7.9 ± 0.17
0.7 ± 0.05
4a
4f
1.0 ± 0.05
5.5 ± 0.02
6.0 ± 2.9
3.2 ± 0.01
> 10 (61 ± 1.7)
> 10 (66 ± 5.9)
> 10 (76 ± 1.6) 0.8 ± 0.01
9.5 ± 0.8 0.3 ± 0.04
≥ 10 (86 ± 2.1) < 0.1 (63 ± 2.3)
0.3 ± 0.02 < 0.01 (74 ± 4.4)
0.0007 ± 0.00001 0.1 ± 0.03
9
10
11
3TC
9.7 ± 2.5
> 10 (67 ± 15.3) 3.7 ± 0.1
0.04 ± 0.03
> 10 (<1 ± 0.03) N/A
N/A
≤ 0.0001 (53 ± 0.5) 0.009 ± 0.002
< 0.01 (82 ± 3.4) 0.05 ± 0.0001
> 10 (<1 ± 0.2)
0.7 ± 0.2
ETV
1.9 ± 0.04
0.2 ± 0.1
1.0 ± 0.1
GLS-4
Data are the mean of replicates of two or three ± standard deviation (SD) from 2 independent experiments with representative
results. N/A: not applied. Results in parenthesis: % of inhibition ± SD at 10 µM.
2.3. Electron microscopy
Recently published results show that a compound chemically similar to 4f inhibits replication by disrupting the HBV capsid protein
[22]. To determine if our compounds act by this mechanism, HBV capsid formation was monitored by negative-stain electron
microscopy. In the absence of drug, the HBV Cp149 dimers assemble upon addition of NaCl into regular hollow spheres with a
diameter of approximately 40 nm (Figure 3A) [23]. Three compounds including 4a, 4f and the inactive compound 21 were tested at
20 µM in a single-blind series of experiments. HBV Cp149 dimers were incubated with the agents for 1 hr, and assembly was
induced with the addition of NaCl overnight. One sample yielded properly formed HBV Cp149 capsids of similar shape, size and
yield as the drug-lacking images (Figure 3B and 3C). The other two samples exhibited morphology dissimilar to the wild-type
capsids correctly identifying 4a and 4f as the active agents. Compound 4a induces Cp149 to form greater numbers of disfigured
circular assemblies (Figure 3D). Pre-incubation with compound 4f yielded a large number of incomplete, but typically spherical
capsids aggregated into clusters (Figure 3E and 3F). These results support that sulfamoyl agents like 4f inhibit HBV replication by
binding to the capsid protein and affecting assembly.
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Fig 3. Perturbation of HBV Cp149 capsid formation by sulfamoyl compounds at 20 µM determined by negative-stain TEM. A)
HBV Cp149 capsids formed in the absence of drug, B) in the presence of 21, C) properly formed capsid D) in the presence of 4a,
E) in the presence of 4f and F) incomplete and aggregated capsid.
2.4. Molecular modeling
A recent crystal structure revealed the binding site for sulfamoyl-based HBV capsid effectors at the Cp149 chain B-C dimer-dimer
interface (Figure 4A) [22]. Chain B formed a “concave” that contributes most protein-ligand interactions while chain C acted as a
“lid”. The compound 4f was modeled into this pocket at the interface of chain B and chain C using Glide Docking. To refine the
docked model and identify transient interactions, the entire HBV Cp149 hexamer was simulated for 10 ns in explicit solvent using
Desmond MD (Figure 4A). The simulation rapidly equilibrated in ~2.5 ns, and protein RMSF agreed with the experimentally
observed thermal factors (see Supporting Information). The resulting protein-ligand contacts from the molecular dynamics
simulation provided insight into the structure-activity relationships observed for this series (Figure 4B, 4C). In most frames, the
amide oxygen accepted a hydrogen bond from Trp102 NH while also engaging in a minor water-mediated bridge with the Phe23
backbone (observed in 5% of frames). The amide nitrogen acted as a hydrogen bond donor to the Thr128 side chain alcohol on the
other interfacial partner. These amide noncovalent bonds bridged the two proteins comprising the binding pocket. Compounds 32-
35 are cyclized at the amide N thereby lacking the hydrogen-bond donating functionality, and these agents do not inhibit HBV
replication supporting the necessity of the hydrogen bonding interactions. The difluorobenzyl ring positioned into a hydrophobic
pocket formed by Pro25, Leu30, Trp102, and Val124 from the interfacial partner. The central monofluorobenzyl ring falls into a
hydrophobic pocket lined by Phe110, Leu140, and Thr128 from chain C. The sulfonamide group formed transient water bridges
with Ser141. The Ser141 water bridge must be weak and dynamic since the “flipped” sulfonamide compounds 9-11 retain modest
anti-HBV activity. The difluoroazetidine group positions in a narrow, hydrophobic solvent-exposed tunnel. Since this group does
not form specific interactions with the HBV capsid protein, several small, hydrophobic substitutions are tolerated (compounds 4a,
4b, 4c, 4d, 4e, 4h, 4i, 4j, 4k, and 4o). Bulkier substitutions on the sulfonamide (compounds 4l, 4m, and 4n) are predicted to
sterically clash with the HBV protein resulting in inactivity. This model and subsequent dynamical observations help explain the
structure-activity relationships for this series and will be useful in optimizing future sulfamoyl agents as HBV inhibitors.
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Fig 4. Model of sulfonamide compound 4f bound to HBV capsid protein from dynamic simulation. The model was generated by
docking compound 4f into HBV Cp149 Y132A hexamer crystal structure (PDBID 5T2P) followed by 10ns dynamic simulation. A)
Binding locus for compound 4f in HBV capsid hexamer. B) The binding pocket lies at the interface of two HBV Cp149 proteins
(chain B in red and chain C in green). Residues that interact with compound 4f from the simulation are shown. C) Simulation
interaction diagram of compound 4f bound to HBV Cp149 Y132A hexamer from the productive 7.5 ns of a 10 ns simulation. Green
residues are hydrophobic, hydrogen bond interactions are indicated by purple arrows, polar residues are shown in cyan, and water
bridges are shown in grey. The values in purple indicate the percentage of frames from the production phase in which these
interactions were observed.
3. Conclusion
Among the 27 sulfamoylbenzamides prepared, several analogs exhibited potent anti-HBV activities in the low micromolar range.
Antiviral activity was correlated with reduction in cccDNA level by measuring HBeAg secretion. These sulfamoyl derivatives were
found to inhibit HBV replication by disrupting the capsid assembly leading to misshaped unviable assemblies. Results from this
work validate this novel class of small molecule as CAE and the model used to rationalize our SAR could lead to the discovery of
more potent and effective analogs. Ultimately these sulfamoyl derivatives, when combined with other modalities (e.g., nucleoside
analogs), could lead to a novel therapeutic strategy with reduced treatment duration and a functional cure for HBV infection.
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4. Experimental section
4.1. Chemistry
Commercially available chemicals were of reagent grade and used as received. Nuclear magnetic resonance (NMR) spectra (1H, 13
and 19F) were recorded on a Bruker Ascend™ 400 MHz Fourier transform spectrometer at rt, with tetramethylsilane (TMS) as an
internal standard. Chemical shifts ( ) are reported in parts per million (ppm) and signals are quoted as s (singlet), d (doublet), t
C
δ
(triplet), q (quartet), m (multiplet), bs (broad signal), dd (doublet of doublets) or ddd (doublet of doublets of doublets). 13C NMR
data is reported as observed, that is, some carbon signals overlap with solvent signals. High-resolution mass spectra (HRMS) were
recorded on a ThermoFisher Q exactive Plus high-resolution mass spectrometer with electrospray ionization (ESI). Thin-layer
chromatography (TLC) was performed on 0.25 mm silica gel. Purifications were performed on silica gel column chromatography
(60 Å, 63-200 or 40-75
µm). Reactions under microwave irradiation were performed on a CEM Discovery SP Microwave
Synthesizer using 5 mL sealed tubes.
4.1.1. 5-Chlorosulfonyl-2-fluorobenzoic acid (2)
To chlorosulfonic acid (23.8 mL, 0.35 mol, 10 equiv.) cooled to 0 °C was added portion wise 2-fluorobenzoic acid 1 (5.0 g, 35
mmol). After addition, the yellow solution was allowed to warm to room temperature and heated at 75 °C for 16 h. The reaction
mixture was cooled to room temperature and carefully added dropwise into crushed ice. The white precipitate was filtered, washed
with water and dried in vacuo to afford compound 2 as a white solid (6.4 g, 77%). Spectral data was consistent with that previously
reported. CAS: 37098-75-2.
4.1.2. 3-((3,4-Difluorophenyl)carbamoyl)-4-fluorobenzenesulfonyl chloride (3)
A solution of 2 (3 g, 12.6 mmol) in SOCl2 (20 mL) was heated at 80 °C for 16 h. The mixture was concentrated under reduced
pressure and co-evaporated with toluene. The crude residue was dissolved in toluene (25 mL) and 3,4-difluoroaniline (1.24 mL,
12.6 mmol) was added. The mixture was heated at 110 °C for 2 h and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography using hexanes/EtOAc (8:2) to afford 3 (2.87 g, 65% over two steps). 1H NMR (400
MHz, Acetone-d6
1H), 7.54 – 7.45 (m, 1H), 7.33 (dt,
(dd, = 244.5, 13.3 Hz), 146.8 (dd,
130.1 (d, = 4.9 Hz), 125.9 (d, = 16.7 Hz), 118.9 (d,
= 22.1 Hz). 19F NMR (377 MHz, Acetone-d6
)
δ
9.99 (s, 1H), 8.54 (dd,
J
= 6.1, 2.6 Hz, 1H), 8.43 – 8.32 (m, 1H), 7.98 – 7.87 (m, 1H), 7.73 (dd,
J
= 9.7, 8.9 Hz,
= 262.3 Hz), 160.2, 149.6
= 8.9, 3.2 Hz), 132.2 (d, = 11.2 Hz),
= 6.1, 3.5 Hz), 109.6 (d,
J
= 10.5, 9.0 Hz, 1H). 13C NMR (101 MHz, Acetone-d6
)
δ
163.4 (d,
J
J
J
= 243.6, 12.8 Hz), 140.2 (d,
J
= 3.2 Hz), 135.3 (dd,
J
J
J
J
J
= 25.0 Hz), 117.3 (d,
J
= 18.3 Hz), 116.5 (dd,
J
J
)
δ
-103.6, -139.3 – -139.4 (m), -145.6 – -145.7 (m).
4.1.3. General procedure to compounds 4a-n
To a solution of sulfonyl chloride derivative 3 (100 mg, 0.286 mmol) in CH2Cl2 (3 mL) were added the appropriate amine (0.286
mmol) and Et3N (0.315 mmol) at 0 °C. The mixture was stirred 2 h at room temperature, diluted with CH2Cl2 and washed with
water. The organic layer was dried over MgSO4 and concentrated under reduced pressure. The residue was purified by silica gel
column chromatography using hexanes/EtOAc to afford the desired sulfonamide derivatives.
4.1.3.1. 5-(
Yield 81%. 1H NMR (400 MHz, Acetone-d6
1H), 7.58 – 7.47 (m, 2H), 7.43 – 7.28 (m, 1H), 6.72 (d,
2H), 1.55 – 1.37 (m, 4H). 13C NMR (101 MHz, Acetone-d6
N
-Cyclopentylsulfamoyl)-
N
-(3,4-difluorophenyl)-2-fluorobenzamide (4a)
9.89 (s, 1H), 8.29 (dd, = 6.6, 2.4 Hz, 1H), 8.12 – 8.05 (m, 1H), 8.01 – 7.92 (m,
= 7.3 Hz, 1H), 3.72 – 3.57 (m, 1H), 1.83 – 1.71 (m, 2H), 1.68 – 1.58 (m,
)
δ
J
J
)
δ
161.4 (d,
J
= 255.9 Hz), 161.2, 149.6 (dd,
J
= 244.2, 13.3 Hz), 146.6
9

ACCEPTED MANUSCRIPT
(dd,
= 15.7 Hz), 117.4 (d,
(377 MHz, Acetone-d6
C18H18F3N2O3S: 399.0990, found: 399.0985.
J
= 243.4, 12.7 Hz), 138.8 (d,
= 6.1 Hz), 117.2, 116.3 (dd,
-110.5 – -110.6 (m), -139.5 – -139.6 (m), -146.0 – -146.2 (m). HRMS (ESI): m/z [M+H]+ calcd for
J
= 3.5 Hz), 135.5 (dd,
J
= 9.0, 3.2 Hz), 131.9 (d,
J = 10.0 Hz), 129.7 (d, J = 3.8 Hz), 124.5 (d, J
J
J
= 6.0, 3.5 Hz), 109.4 (d,
J
= 22.1 Hz), 78.3, 55.1, 54.1, 32.9, 23.0. 19F NMR
)
δ
4.1.3.2. 5-(
Yield 84%. 1H NMR (400 MHz, Acetone-d6
1H), 7.61 – 7.49 (m, 2H), 7.35 (dt, = 10.5, 9.0 Hz, 1H), 4.38 (p,
4H). 13C NMR (101 MHz, Acetone-d6
161.6 (d, = 256.4 Hz), 161.1 (d,
= 243.3, 12.7 Hz), 136.4 (d, = 3.7 Hz), 135.5 (dd, = 9.0, 3.2 Hz), 132.1 (d,
Hz), 117.5 (d, = 24.4 Hz), 117.3 (d, = 18.5 Hz), 116.3 (dd,
NMR (377 MHz, Acetone-d6
C19H19F3N2O3S: 413.1147, found: 413.1142.
N
-Cyclopentyl-
N
-methylsulfamoyl)-
9.88 (s, 1H), 8.24 (dd,
= 8.1 Hz, 1H), 2.77 (s, 3H), 1.71 – 1.54 (m, 4H), 1.52 – 1.35 (m,
= 1.7 Hz), 149.6 (dd, = 244.2, 13.3 Hz), 146.6 (dd,
= 10.0 Hz), 129.9 (d, = 3.8 Hz), 124.7 (d, = 15.8
= 22.2 Hz), 58.3, 28.2, 27.6, 23.8. 19F
N
-(3,4-difluorophenyl)-2-fluorobenzamide (4b)
)
δ
J
= 6.5, 2.5 Hz, 1H), 8.10 – 8.02 (m, 1H), 8.02 – 7.94 (m,
J
J
)
δ
J
J
J
J
J
J
J
J
J
J
J
J
= 6.1, 3.6 Hz), 109.4 (d, J
) δ
-110.1 – -110.2 (m), -139.4 – -139.6 (m), -146.0 – -146.1 (m). HRMS (ESI): m/z [M+H]+ calcd for
4.1.3.3.
Yield 86%. 1H NMR (400 MHz, DMSO-d6
1H), 7.68 (t,
= 9.1 Hz, 1H), 7.52 – 7.41 (m, 2H), 3.18 – 3.09 (m, 4H), 2.18 – 1.99 (m, 4H). 13C NMR (101 MHz, DMSO-d6
161.8 (d, = 257.5 Hz), 161.7, 149.4 (dd,
10.5 Hz), 130.0 (d, = 4.3 Hz), 126.1 (d,
109.4 (d, = 21.7 Hz), 43.8 (t, = 5.7 Hz), 33.2 (t,
23.0 Hz), -143.5 (d,
= 23.0 Hz). HRMS (ESI): m/z [M+H]+ calcd for C18H15F5N2O3S: 435.0802, found: 435.0796.
N
-(3,4-Difluorophenyl)-5-((4,4-difluoropiperidin-1-yl)sulfonyl)-2-fluorobenzamide (4c)
)
δ
10.88 (s, 1H), 8.07 (dd, = 6.2, 2.4 Hz, 1H), 8.06 – 7.97 (m, 1H), 7.92 – 7.83 (m,
J
J
)
δ
J
J
= 243.7, 13.2 Hz), 146.3 (dd,
J
= 242.4, 12.7 Hz), 135.9 (d,
J
= 8.9 Hz), 132.6 (d,
J
=
J
J
= 17.0 Hz), 124.5, 122.1, 119.7, 118.5 (d,
J
= 23.5 Hz), 118.1 (d, J = 17.8 Hz), 116.8,
J
J
J ) δ -97.5, -107.0, -136.9 (d, J =
= 23.6 Hz). 19F NMR (377 MHz, DMSO-d6
J
4.1.3.4.
Yield 79%. 1H NMR (400 MHz, DMSO-d6
– 7.41 (m, 2H), 3.68 (t, = 12.9 Hz, 2H), 3.42 (t,
= 257.7 Hz), 161.7, 149.4 (dd, = 243.8, 13.2 Hz), 146.3 (dd,
= 3.3 Hz), 130.4, 130.3 (d, = 4.3 Hz), 128.0, 125.9 (d,
= 21.6 Hz), 54.1 (t,
N
-(3,4-Difluorophenyl)-5-((3,3-difluoropyrrolidin-1-yl)sulfonyl)-2-fluorobenzamide (4d)
10.87 (s, 1H), 8.22 – 8.05 (m, 2H), 7.98 – 7.83 (m, 1H), 7.68 (t,
= 7.4 Hz, 2H), 2.43 – 2.25 (m, 2H). 13C NMR (101 MHz, DMSO-d6
= 242.6, 12.6 Hz), 135.9 (dd, = 9.0, 3.0 Hz), 132.8 (d,
= 16.5 Hz), 125.5, 118.4 (d, = 23.5 Hz), 118.1 (d,
= 23.9 Hz). 19F NMR (377 MHz, Acetone-
)
δ
J
= 9.2 Hz, 1H), 7.52
162.0 (d,
= 10.1
J
J
) δ
J
J
J
J
J
Hz), 132.4 (d,
J
J
J
J
J =
18.0 Hz), 117.0 – 116.4 (m), 109.5 (d,
d6
C17H13F5N2O3S: 421.0645, found: 421.0639.
J
J
= 32.0 Hz), 46.1, 33.9 (t, J
)
δ
-101.5 – -101.6 (m), -109.0 – -109.1 (m), -139.6 – -139.7 (m), -146.1 – -146.2 (m). HRMS (ESI): m/z [M+H]+ calcd for
4.1.3.5.
Yield 82%. 1H NMR (400 MHz, DMSO-d6
1H), 7.70 (t,
= 9.2 Hz, 1H), 7.56 – 7.42 (m, 2H), 4.19 – 3.92 (m, 4H). 13C NMR (101 MHz, DMSO-d6
161.6, 149.4 (dd,
130.8 (d, = 4.4 Hz), 126.0 (d,
(m), 115.8 (t, = 24.9 Hz), 109.4 (d,
N
-(3,4-Difluorophenyl)-2-fluoro-5-((3,3,4,4-tetrafluoropyrrolidin-1-yl)sulfonyl)benzamide (4e)
10.88 (s, 1H), 8.22 (dd, = 6.3, 2.5 Hz, 1H), 8.21 – 8.12 (m, 1H), 7.97 – 7.83 (m,
162.3 (d, = 258.4 Hz),
= 8.9, 2.9 Hz), 133.0 (d, = 10.3 Hz), 132.1,
= 17.7 Hz), 117.2 – 116.3
-107.97 – -108.05 (m),
)
δ
J
J
)
δ
J
J
= 243.8, 13.3 Hz), 146.3 (dd,
= 16.6 Hz), 121.0 (t,
= 21.6 Hz), 51.1 (t,
J
= 242.6, 12.6 Hz), 136.0 (dd,
J
J
J
J
J
= 24.8 Hz), 118.6 (d,
J
= 23.3 Hz), 118.1 (d, J
J
J
J
= 30.7 Hz). 19F NMR (377 MHz, Acetone-d6
)
δ
-124.1 – -124.2 (m), -139.5 – -139.7 (m), -146.1 – -146.2 (m). HRMS (ESI): m/z [M+H]+ calcd for C17H11F7N2O3S: 457.0457,
found: 457.0456.
10

ACCEPTED MANUSCRIPT
4.1.3.6. 5-((3,3-Difluoroazetidin-1-yl)sulfonyl)-
Yield 79%. 1H NMR (400 MHz, DMSO-d6
10.92 (s, 1H), 8.21 (dd,
1H), 7.73 (t, = 9.2 Hz, 1H), 7.52 – 7.42 (m, 2H), 4.36 (t,
= 12.7 Hz, 4H). 13C NMR (101 MHz, DMSO-d6
Hz), 161.6, 149.4 (dd, = 243.8, 13.2 Hz), 146.3 (dd, = 242.7, 12.6 Hz), 135.9 (dd,
(d, = 4.4 Hz), 130.4 (d,
6.0, 3.4 Hz), 114.8, 112.1, 109.5 (d,
N
-(3,4-difluorophenyl)-2-fluorobenzamide (4f)
= 6.3, 2.5 Hz, 1H), 8.20 – 8.11 (m, 1H), 7.93 – 7.86 (m,
162.4 (d, = 258.7
= 10.2 Hz), 131.3
= 17.8 Hz), 117.5, 116.9 (dd,
-98.3, -105.8, -
)
δ
J
J
J
)
δ
J
J
J
J
= 9.1, 2.9 Hz), 133.6 (d, J
J
J
= 3.2 Hz), 126.1 (d,
J
= 16.5 Hz), 118.6 (d,
J
= 23.7 Hz), 118.1 (d,
J
J =
J
= 21.5 Hz), 62.4 (t,
J
= 27.6 Hz), 46.1. 19F NMR (377 MHz, DMSO-d6
)
δ
136.9 (d,
J
= 22.8 Hz), -143.5 (d, J
= 23.1 Hz). HRMS (ESI): m/z [M+H]+ calcd for C16H11F5N2O3S: 407.0489, found: 407.0484.
4.1.3.7.
N-(3,4-Difluorophenyl)-2-fluoro-5-((2-oxopyrrolidin-1-yl)sulfonyl)benzamide (4g)
Note: 2-pyrolidinone (24 mg, 0.286 mmol) was treated with sodium hydride (12 mg, 0.286 mmol, 60% dispersion in mineral oil) in
THF (3 mL) prior to the addition of 3. Yield 37%. 1H NMR (400 MHz, Acetone-d6
9.93 (s, 1H), 8.41 (dd, = 6.5, 2.5 Hz, 1H),
8.30 – 8.20 (m, 1H), 8.03 – 7.92 (m, 1H), 7.57 (dd, = 10.0, 8.8 Hz, 1H), 7.55 – 7.49 (m, 1H), 7.36 (dt, = 10.5, 9.0 Hz, 1H), 4.01
(t, = 7.0 Hz, 2H), 2.46 (dd,
= 8.4, 7.6 Hz, 2H), 2.20 – 2.11 (m, 2H). 13C NMR (101 MHz, Acetone-d6 13C NMR (101 MHz,
Acetone-d6 173.5, 162.5 (d, = 258.3 Hz), 160.9, 149.6 (dd, = 244.3, 13.3 Hz), 146.6 (dd, = 243.4, 12.9 Hz), 135.5 (dd,
8.9, 3.2 Hz), 135.2, 133.3 (d, = 10.6 Hz), 130.8 (d, = 4.4 Hz), 124.7 (d, = 16.2 Hz), 117.5 (d, = 5.5 Hz), 117.2, 116.6 – 115.9
(m), 109.4 (d, -107.9, -139.5 – -139.7 (m), -146.0 – -146.2 (m).
= 22.2 Hz), 47.4, 31.6, 18.1. 19F NMR (377 MHz, Acetone-d6
HRMS (ESI): m/z [M+H]+ calcd for C17H13F3N2O4S: 399.0626, found: 399.0619.
)
δ
J
J
J
J
J
) δ
)
δ
J
J
J
J =
J
J
J
J
J
) δ
4.1.3.8. 5-(
Yield 77%. 1H NMR (400 MHz, DMSO-d6
Hz, 1H), 7.48 – 7.42 (m, 2H), 2.21 – 2.08 (m, 1H), 0.58 – 0.47 (m, 2H), 0.45 – 0.36 (m, 2H). 13C NMR (101 MHz, DMSO-d6
N
-Cyclopropylsulfamoyl)-
N
-(3,4-difluorophenyl)-2-fluorobenzamide (4h)
)
δ
10.86 (s, 1H), 8.09 (s, 2H), 8.07 – 7.98 (m, 1H), 7.91 – 7.84 (m, 1H), 7.65 (t, J = 9.2
)
δ
162.0, 161.2 (d,
6.1 Hz), 132.0 (d,
109.5 (d,
= 21.7 Hz), 24.6, 5.6. 19F NMR (377 MHz, DMSO-d6
(ESI): m/z [M+H]+ calcd for C16H13F3N2O3S: 371.0677, found: 371.0672.
J
= 256.1 Hz), 149.4 (dd,
J
= 243.7, 13.2 Hz), 146.3 (dd,
= 16.7 Hz), 118.2, 118.0 (d,
-108.2, -136.9 (d,
J
= 242.7, 12.9 Hz), 137.3 (d,
J
= 3.2 Hz), 135.9 (d,
J
=
J
= 9.9 Hz), 129.4 (d, = 4.0 Hz), 125.6 (d,
J
J
J
= 4.6 Hz), 116.8 (dd, J = 5.7, 3.4 Hz),
J
)
δ
J
= 23.0 Hz), -143.5 (d,
J
= 23.0 Hz). HRMS
4.1.3.9.
Yield 26%. 1H NMR (400 MHz, Acetone-d6
1H), 7.58 – 7.50 (m, 2H), 7.36 (dt, = 10.5, 9.0 Hz, 1H), 3.68 – 3.60 (m, 1H), 3.57 – 3.49 (m, 1H), 3.30 – 3.20 (m, 1H), 1.96 – 1.79
(m, 3H), 1.72 – 1.53 (m, 5H), 1.44 – 1.34 (m, 2H). 13C NMR (101 MHz, Acetone-d6
161.7 (d, = 256.4 Hz), 161.2, 149.6 (dd,
= 244.1, 13.2 Hz), 146.6 (dd, = 243.1, 12.6 Hz), 135.6 (dd, = 9.0, 3.0 Hz), 135.4 (d, = 3.4 Hz), 132.2 (d, = 10.0 Hz), 129.9 (d,
= 3.9 Hz), 124.6 (d, = 15.7 Hz), 117.5 (d, = 24.2 Hz), 117.5 – 117.1 (m), 116.5 – 116.1 (m), 109.3 (d, = 22.2 Hz), 59.6, 47.3,
37.7, 29.7, 27.3, 25.9, 22.9, 21.0. 19F NMR (377 MHz, Acetone-d6
-110.3 – -110.4 (m), -139.6 – -139.7 (m), -146.2 – -146.3 (m).
HRMS (ESI): m/z [M+H]+ calcd for C21H21F3N2O3S: 439.1303, found: 439.1297.
N-(3,4-Difluorophenyl)-2-fluoro-5-((octahydro-1H-indol-1-yl)sulfonyl)benzamide (4i)
)
δ
9.89 (s, 1H), 8.26 (dd, = 6.6, 2.4 Hz, 1H), 8.14 – 8.06 (m, 1H), 8.02 – 7.94 (m,
J
J
)
δ
J
J
J
J
J
J
J
J
J
J
)
δ
4.1.3.10.
Yield 69%. 1H NMR (400 MHz, DMSO-d6
7.62 (m, 1H), 7.52 – 7.44 (m, 2H), 7.44 – 7.35 (m, 1H), 7.30 – 7.23 (m, 2H), 7.18 – 7.04 (m, 3H), 1.53 (s, 6H). 13C NMR (101 MHz,
DMSO-d6 161.9, 160.7 (d, = 255.4 Hz), 149.4 (dd, = 243.6, 13.2 Hz), 146.3 (dd, = 242.5, 12.6 Hz), 145.5, 140.2 (d, = 3.3
Hz), 136.0 (dd, = 8.8, 2.9 Hz), 131.3 (d, = 9.9 Hz), 129.0 (d, = 3.9 Hz), 128.1, 126.9, 126.1, 124.7 (d, = 16.3 Hz), 118.0 (d,
= 17.8 Hz), 117.4 (d, = 23.7 Hz), 116.9 (dd, = 6.2, 3.3 Hz), 109.5 (d,
= 21.6 Hz), 58.1, 30.2. 19F NMR (377 MHz, Acetone-d6
N-(3,4-Difluorophenyl)-2-fluoro-5-(
N-(2-phenylpropan-2-yl)sulfamoyl)benzamide (4j)
)
δ
10.69 (s, 1H), 8.25 (s, 1H), 8.05 – 7.82 (m, 1H), 7.75 (dd,
J = 6.5, 2.4 Hz, 1H), 7.74 –
)
δ
J
J
J
J
J
J
J
J
J
J
J
J
)
11

ACCEPTED MANUSCRIPT
δ
-111.4 – -111.5 (m), -139.7 – -139.8 (m), -146.3 – -146.4 (m). HRMS (ESI): m/z [M+H]+ calcd for C22H19F3N2O3S: 449.1147,
found: 449.1142.
4.1.3.11.
Yield 75%. 1H NMR (400 MHz, Acetone-d6
1H), 7.59 – 7.47 (m, 1H), 7.45 – 7.27 (m, 2H), 7.24 – 7.09 (m, 6H), 4.67 – 4.51 (m, 1H), 1.41 (d,
MHz, Acetone-d6 161.2 (d, = 255.8 Hz), 161.0 (d, = 9.6 Hz), 149.6 (dd, = 244.1, 13.3 Hz), 146.6 (dd,
142.7, 138.7 (t, = 3.9 Hz), 136.0 – 135.0 (m), 131.7 (d, = 10.2 Hz), 129.8 (d, = 4.0 Hz), 128.2, 127.1, 126.2, 123.9 (dd,
15.5, 4.6 Hz), 117.3 (d, = 18.1 Hz), 117.0 (d, = 24.5 Hz), 116.6 – 116.0 (m), 109.4 (dd, = 22.2, 8.6 Hz), 53.8 (d, = 9.6 Hz),
23.1 (d, -109.4 – -109.5 (m), -138.3 – -138.4 (m), -144.8 – -145.0 (m). HRMS
= 3.6 Hz). 19F NMR (377 MHz, Acetone-d6
(ESI): m/z [M+H]+ calcd for C21H17F3N2O3S: 435.0990, found: 435.0986.
N
-(3,4-Difluorophenyl)-2-fluoro-5-(
N
-(1-phenylethyl)sulfamoyl)benzamide (4k)
9.73 (s, 1H), 8.06 (dd, = 6.7, 2.5 Hz, 1H), 8.02 – 7.93 (m, 1H), 7.89 – 7.77 (m,
= 7.0 Hz, 3H). 13C NMR (101
= 243.3, 12.8 Hz),
)
δ
J
J
)
δ
J
J
J
J
J
J
J
J =
J
J
J
J
J
) δ
4.1.3.12. Isopropyl ((3-((3,4-difluorophenyl)carbamoyl)-4-fluorophenyl)sulfonyl)-L-alaninate (4l)
Yield 94%. 1H NMR (400 MHz, Chloroform-
8.61 (d, = 10.4 Hz, 1H), 8.48 (dd, = 6.9, 2.5 Hz, 1H), 8.16 – 7.93 (m, 1H),
7.82 – 7.69 (m, 1H), 7.32 – 7.26 (m, 1H), 7.25 – 7.20 (m, 1H), 7.18 – 7.04 (m, 1H), 5.89 (d, = 7.4 Hz, 1H), 4.99 – 4.71 (m, 1H),
4.00 (t, = 7.4 Hz, 1H), 1.38 (d, = 7.2 Hz, 3H), 1.14 (d, = 6.3 Hz, 3H), 1.11 (d,
= 6.3 Hz, 3H). 13C NMR (101 MHz,
Chloroform- 171.6, 162.1 (d, = 256.4 Hz), 160.1 (d, = 2.9 Hz), 150.0 (dd, = 247.3, 13.2 Hz), 147.4 (dd, = 246.6, 12.8
Hz), 137.5 (d, = 3.0 Hz), 133.8 (dd, = 8.8, 3.3 Hz), 132.5 (d, = 10.6 Hz), 131.1 (d, = 3.4 Hz), 122.7 (d, = 13.4 Hz), 117.4 (d,
= 6.1, 3.6 Hz), 110.4 (d,
= 21.9 Hz), 69.9, 51.8, 21.4, 21.4, 19.6. 19F NMR (377 MHz,
= 21.2 Hz), -141.4 (d,
= 21.7 Hz). HRMS (ESI): m/z [M+H]+ calcd for C19H19F3N2O5S:
d
)
δ
J
J
J
J
J
J
J
d
)
δ
J
J
J
J
J
J
J
J
J
J
= 41.9 Hz), 117.4, 116.3 (dd,
J
J
Chloroform-
d
)
δ
-106.5, -135.4 (d,
J
J
445.1045, found: 445.1038.
4.1.3.13. Methyl ((3-((3,4-difluorophenyl)carbamoyl)-4-fluorophenyl)sulfonyl)-L-phenylalaninate (4m)
Yield 91%. 1H NMR (400 MHz, Chloroform-
8.44 (d, = 10.8 Hz, 1H), 8.23 (dd, = 6.9, 2.5 Hz, 1H), 7.78 – 7.51 (m, 2H),
7.16 – 7.00 (m, 6H), 6.99 – 6.93 (m, 2H), 5.81 (d, = 9.0 Hz, 1H), 4.26 – 4.10 (m, 1H), 3.49 (s, 3H), 2.98 (dd, = 13.8, 5.5 Hz, 1H),
2.86 (dd, 171.5, 162.0 (d, = 256.2 Hz), 160.0 (d, = 3.0 Hz), 150.0
= 13.8, 7.5 Hz, 1H). 13C NMR (101 MHz, Chloroform-
(dd, = 247.4, 13.3 Hz), 147.4 (dd, = 2.9 Hz), 135.1, 133.8 (dd, = 8.8, 3.3 Hz), 132.3 (d, = 10.7
Hz), 131.0 (d, = 13.3 Hz), 117.5 (d, = 7.5 Hz), 117.2, 116.3 (dd, = 6.0, 3.6 Hz),
110.4 (d, -106.5, -135.3 (d, = 21.8 Hz), -141.3 (d, = 21.6
d
)
δ
J
J
J
J
J
d
)
δ
J
J
J
J
= 246.6, 12.7 Hz), 137.2 (d,
J
J
J
J
= 3.4 Hz), 129.3, 128.6, 127.2, 122.4 (d,
J
J
J
J
= 21.9 Hz), 57.2, 52.7, 39.0. 19F NMR (377 MHz, Chloroform-
d
)
δ
J
J
Hz). HRMS (ESI): m/z [M+H]+ calcd for C23H19F3N2O5S: 493.1045, found: 493.1038.
4.1.3.14. Ethyl ((3-((3,4-difluorophenyl)carbamoyl)-4-fluorophenyl)sulfonyl)-L-leucinate (4n)
Yield 89%. 1H NMR (400 MHz, Chloroform-
7.77 – 7.59 (m, 1H), 7.25 – 7.12 (m, 2H), 7.10 – 6.97 (m, 1H), 5.64 (d,
2H), 1.76 – 1.60 (m, 1H), 1.43 (t, = 7.2 Hz, 2H), 1.03 (t, = 7.1 Hz, 3H), 0.81 (t,
Chloroform- 171.2, 161.1 (d, = 256.6 Hz), 159.0 (d, = 2.9 Hz), 149.0 (dd, = 247.4, 13.3 Hz), 146.4 (dd,
Hz), 136.2 (d, = 3.2 Hz), 132.8 (dd, = 8.8, 3.2 Hz), 131.5 (d, = 10.6 Hz), 130.2 (d, = 3.3 Hz), 121.6 (d, = 13.4 Hz), 116.7 –
116.3 (m), 116.2 (d, = 5.9, 3.6 Hz), 109.3 (d,
= 21.9 Hz), 60.8, 53.6, 41.0, 23.3, 21.6, 20.3, 12.9. 19
= 21.7 Hz), -141.4 (d,
= 21.4 Hz). HRMS (ESI): m/z [M+H]+ calcd for
d
)
δ
8.51 (d,
J
= 10.3 Hz, 1H), 8.37 (dd,
= 9.9 Hz, 1H), 3.95 – 3.88 (m, 1H), 3.84 (q,
= 6.6 Hz, 6H). 13C NMR (101 MHz,
= 246.6, 12.8
J
= 6.8, 2.5 Hz, 1H), 7.93 – 7.81 (m, 1H),
J
J
= 7.1 Hz,
J
J
J
d
)
δ
J
J
J
J
J
J
J
J
J
J
= 5.1 Hz), 115.2 (dd,
J
J
F
NMR (377 MHz, Chloroform-
d)
δ
-106.4, -135.4 (d,
J
J
C21H23F3N2O5S: 473.1358, found: 473.1351.
12

ACCEPTED MANUSCRIPT
4.1.3.15. 5-(
N-(Cyclopentyloxy)sulfamoyl)-
N-(3,4-difluorophenyl)-2-fluorobenzamide (4o)
To a solution of 3 (100 mg, 0.3 mmol) in acetonitrile (5 mL) were added
O
-cyclopentylhydroxylamine hydrochloride (0.04 mg, 0.3
mmol) and Et3N (0.2 mL, 1.5 mmol). The mixture was stirred 4 h at 65 ºC. After completion (checked by LC-MS), the reaction
mixture was absorbed on silica and purified by flash chromatography using hexanes/EtOAc (7:3) to afford 4o (48 mg, 39%) as a
white solid. 1H NMR (400 MHz, Acetone-d6
)
δ
9.88 (s, 1H), 9.36 (s, 1H), 8.29 (dd,
= 10.5, 9.0 Hz, 1H), 4.70 – 4.55 (m, 1H), 1.87 – 1.76 (m, 2H), 1.76 – 1.66 (m, 2H),
162.2 (d, = 257.3 Hz), 161.0 (d, = 1.7 Hz), 149.7 (dd, = 244.3, 13.2
= 9.0, 3.2 Hz), 134.5 (d, = 3.4 Hz), 133.6 (d, = 10.4 Hz), 131.2 (d, = 4.2 Hz),
= 6.1, 3.5 Hz), 109.4 (d, = 22.1 Hz), 88.1, 30.9,
J = 6.5, 2.5 Hz, 1H), 8.19 – 8.06 (m, 1H), 8.05
– 7.91 (m, 1H), 7.64 – 7.48 (m, 2H), 7.37 (dt,
J
1.65 – 1.47 (m, 4H). 13C NMR (101 MHz, Acetone-d6
Hz), 146.7 (dd, = 243.4, 12.7 Hz), 135.5 (dd,
124.6 (d, = 16.1 Hz), 117.3 (d, = 18.0 Hz), 117.3 (d,
23.2. 19F NMR (377 MHz, Acetone-d6
calcd for C18H18F3N2O4S: 415.0939, found: 415.0936.
)
δ
J
J
J
J
J
J
J
J
J
J
J
= 24.6 Hz), 116.3 (dd,
J
J
)
δ
-108.6 – -108.7 (m), -139.5 – -139.7 (m), -146.0 – -146.1 (m). HRMS (ESI): m/z [M+H]+
4.1.3.16.
Title compound 4p was obtained from 3 using the same procedure as for compound 4o. Yield 42%, triethylamine salt. H NMR
(400 MHz, Acetone-d6 9.89 (s, 1H), 8.78 (s, 1H), 8.24 (dd, = 6.8, 2.4 Hz, 1H), 8.09 – 8.03 (m, 1H), 8.03 – 7.94 (m, 1H), 7.57 –
7.48 (m, 1H), 7.38 – 7.28 (m, 2H), 3.32 (q, = 7.3 Hz, 4H), 2.89 (s, 3H), 1.34 (t,
= 7.3 Hz, 6H). 13C NMR (101 MHz, Acetone-d6
162.0 (d, = 1.7 Hz), 160.5 (d, = 253.4 Hz), 149.6 (dd, = 243.8, 13.2 Hz), 146.5 (dd, = 242.9, 12.8 Hz), 142.8 (d, = 3.5
Hz), 135.8 (dd, = 9.0, 3.1 Hz), 131.9 (d, = 9.6 Hz), 129.2 (d, = 3.5 Hz), 123.4 (d, = 15.3 Hz), 117.2 (d, = 18.1 Hz), 116.3
(dd, = 6.1, 3.5 Hz), 116.0 (d, = 23.8 Hz), 109.3 (d, -113.6 – -
= 22.2 Hz), 46.2, 42.3, 8.1. 19F NMR (377 MHz, Acetone-d6
N-(3,4-Difluorophenyl)-2-fluoro-5-(N-(methylsulfonyl)sulfamoyl)benzamide (4p)
1
)
δ
J
J
J
)
δ
J
J
J
J
J
J
J
J
J
J
J
J
J
) δ
113.7 (m), -139.7 – -139.8 (m), -146.4 – -146.5 (m). HRMS (ESI): m/z [M+H]+ calcd for C14H12F3N2O5S2: 409.0140, found:
409.0133.
4.1.3.17. 5-(
Title compound 4q was obtained from 3 using the same procedure as for compound 4o. Yield 59% yield, triethylamine salt. H
NMR (400 MHz, Acetone-d6 9.86 (s, 1H), 8.93 (s, 1H), 8.26 (dd, = 6.8, 2.4 Hz, 1H), 8.13 – 8.04 (m, 1H), 8.03 – 7.94 (m, 1H),
7.62 – 7.47 (m, 1H), 7.39 – 7.28 (m, 2H), 3.32 (q, = 7.3 Hz, 4H), 2.79 – 2.64 (m, 1H), 1.35 (t, = 7.3 Hz, 6H), 0.97 – 0.76 (m,
4H). 13C NMR (101 MHz, Acetone-d6
161.9, 160.5 (d, = 253.3 Hz), 149.6 (dd, = 244.0, 13.2 Hz), 146.5 (dd, = 242.5, 13.2
Hz), 143.2 (d, = 3.3 Hz), 135.8 (dd, = 9.0, 3.0 Hz), 131.9 (d, = 9.6 Hz), 129.3 (d, = 3.4 Hz), 123.4 (d, = 15.3 Hz), 117.2 (d,
= 18.1 Hz), 116.4 – 116.1 (m), 116.0 (d, = 23.9 Hz), 109.3 (d,
= 22.1 Hz), 46.1, 32.2, 8.1, 4.7. 19F NMR (377 MHz, Acetone-d6
-113.7 – -113.8 (m), -139.7 – -139.8 (m), -146.5 – -146.6 (m). HRMS (ESI): m/z [M+H]+ calcd for C16H14F3N2O5S2: 435.0296,
N-(Cyclopropylsulfonyl)sulfamoyl)-N-(3,4-difluorophenyl)-2-fluorobenzamide (4q)
1
)
δ
J
J
J
)
δ
J
J
J
J
J
J
J
J
J
J
J
)
δ
found: 435.0289.
4.1.3.18.
Title compound 4r was obtained from 3 using the same procedure as for compound 4o. Yield 48%, triethylamine salt. H NMR
(400 MHz, Acetone-d6 9.90 (s, 1H), 8.27 (dd, = 6.8, 2.4 Hz, 1H), 8.13 – 8.03 (m, 1H), 8.04 – 7.97 (m, 1H), 7.63 – 7.51 (m, 1H),
7.43 – 7.27 (m, 2H), 3.36 (q, = 7.3 Hz, 4H), 2.63 (s, 6H), 1.37 (t, 162.0,
= 7.3 Hz, 6H). 13C NMR (101 MHz, Acetone-d6
160.4 (d, = 253.2 Hz), 149.6 (dd, = 243.8, 13.2 Hz), 146.5 (dd, = 242.9, 12.7 Hz), 142.9 (d, = 3.5 Hz), 135.8 (dd, = 9.1, 3.1
Hz), 131.8 (d, = 9.6 Hz), 129.2 (d, = 3.4 Hz), 123.3 (d, = 15.2 Hz), 117.2 (d, = 17.8 Hz), 116.3 (dd,
= 23.9 Hz), 109.3 (d,
= 22.0 Hz), 46.4, 38.1, 8.1. 19F NMR (377 MHz, Acetone-d6
146.4 – -146.6 (m). HRMS (ESI): m/z [M+H]+ calcd for C15H15F3N3O5S2: 438.0405, found: 438.0399.
N-(3,4-Difluorophenyl)-5-(N-(N,N-dimethylsulfamoyl)sulfamoyl)-2-fluorobenzamide (4r)
1
)
δ
J
J
J
) δ
J
J
J
J
J
J
J
J
J
J
= 6.1, 3.5 Hz), 116.0 (d,
J
J
) δ -113.8 – -113.9 (m), -139.7 – -139.8 (m), -
13

ACCEPTED MANUSCRIPT
4.1.3.19.
N-(3,4-Difluorophenyl)-5-(
N-((3,4-difluorophenyl)sulfonyl)sulfamoyl)-2-fluorobenzamide (4s)
1
Title compound 4s was obtained from 3 using the same procedure as for compound 4o. Yield 69%, triethylamine salt. H NMR
(400 MHz, Acetone-d6 9.81 (s, 1H), 8.13 (dd, = 6.8, 2.4 Hz, 1H), 8.05 – 7.91 (m, 2H), 7.74 – 7.64 (m, 1H), 7.64 – 7.59 (m, 1H),
7.56 – 7.49 (m, 1H), 7.40 – 7.27 (m, 3H), 3.38 (q,
161.68 (d, = 1.7 Hz), 160.58 (d, = 253.9 Hz), 151.40 (dd,
249.4, 13.4 Hz), 147.9 – 145.1 (m), 142.8 (d, = 4.1 Hz), 142.2 (d,
129.3 (d, = 3.5 Hz), 124.0 (dd, = 7.3, 3.8 Hz), 123.3 (d,
= 22.0 Hz), 46.6, 8.2. 19F NMR (377 MHz, Acetone-d6
-113.0, -137.6 – -137.7 (m), -139.6 – -139.8 (m), -140.1 – -140.2 (m), -
)
δ
J
J
= 7.3 Hz, 6H), 1.35 (t,
= 250.6, 12.7 Hz), 149.62 (dd,
= 3.3 Hz), 135.7 (dd,
J
= 7.3 Hz, 9H). 13C NMR (101 MHz, Acetone-d6
)
δ
J
J
J
J
= 244.0, 13.3 Hz), 149.12 (dd,
J
=
J
J
J
= 9.1, 3.1 Hz), 131.8 (d, J = 9.6 Hz),
J
J
J = 15.3 Hz), 117.3, 117.1, 117.0, 116.5 – 116.2 (m), 116.1, 109.4 (d, J
)
δ
146.3 – -146.4 (m). HRMS (ESI): m/z [M+H]+ calcd for C19H12F5N2O5S2: 507.0108, found: 507.0104.
4.1.4. 5-((tert-Butoxycarbonyl)amino)-2-fluorobenzoic acid (6)
To a solution of 5-amino-2-fluorobenzoic acid 5 (2 g, 12.9 mmol) in a mixture of 1,4-dioxane/H2O (14 mL, 1:1) were added Boc2O
(4.22 g, 19.3 mmol) and NaHCO3 (2.16 g, 25.8 mmol) at 0 °C. The mixture was stirred for 16 h at room temperature and the
volatiles were removed under reduced pressure. The aqueous layer was extracted with EtOAc (3 x 10 mL). The combined organic
layers were washed with 1M HCl, water, brine and dried over MgSO4. Concentration under reduced pressure afforded 6 (2.45 g,
74%) as a white solid. 1H NMR (400 MHz, DMSO-d6
= 10.5, 9.0 Hz, 1H), 1.47 (s, 9H). 13C NMR (101 MHz, DMSO-d6
(d, = 2.9 Hz), 124.3 (d, = 8.1 Hz), 121.1, 119.5 (d, = 11.1 Hz), 117.5 (d,
DMSO-d6 -119.1 – -121.9 (m).
)
δ
9.56 (s, 1H), 8.05 (dd,
J
= 6.6, 2.9 Hz, 1H), 7.74 – 7.54 (m, 1H), 7.21 (dd,
= 3.3 Hz), 156.7 (d, = 251.9 Hz), 153.2, 136.1
= 23.5 Hz), 79.9, 28.5. 19F NMR (377 MHz,
J
)
δ
165.4 (d,
J
J
J
J
J
J
)
δ
4.1.5. tert-Butyl (3-((3,4-difluorophenyl)carbamoyl)-4-fluorophenyl)carbamate (7)
To a solution of 6 (3 g, 11.7 mmol) in DMF (20 mL) were added 3,4-difluoroaniline (1.4 mL, 14.1 mmol), DIPEA (6.1 mL, 35.2
mmol) and HATU (6.7 g, 17.6 mmol). The mixture was stirred 1 h at room temperature, diluted with EtOAc and washed with 1N
HCl, water and brine. The organic layer was dried over MgSO4 and concentrated under reduced pressure. The residue was purified
1
by silica gel column chromatography using hexanes/EtOAc (8:2) to afford 7 (3.87 g, 90%) as a brown solid. H NMR (400 MHz,
DMSO-d6
7.51 – 7.36 (m, 2H), 7.28 (t,
149.4 (dd, = 243.5, 13.3 Hz), 146.1 (dd,
119.1, 118.0 (d, = 18.0 Hz), 117.6 – 116.30 (m), 116.6, 109.2 (d,
)
δ
10.63 (s, 1H), 9.60 (s, 1H), 7.97 – 7.84 (m, 1H), 7.78 (dd,
= 9.4 Hz, 1H), 1.48 (s, 9H). 13C NMR (101 MHz, DMSO-d6
= 242.3, 12.7 Hz), 136.9 – 135.7 (m), 136.3 – 136.1 (m), 124.7 (d,
= 21.6 Hz), 79.9, 28.5. 19F NMR (377 MHz, DMSO-d6
) δ -
J
= 6.3, 2.8 Hz, 1H), 7.57 (ddd,
J
= 9.0, 4.5, 2.8 Hz, 1H),
= 244.1 Hz), 153.3,
= 16.2 Hz), 122.4,
J
)
δ
163.3, 154.3 (d,
J
J
J
J
J
J
124.1 – -124.2 (m), -138.3 – -138.5 (m), -145.3 – -145.4 (m). HRMS (ESI): m/z [M+H]+ calcd for C18H18F3N2O3: 367.1270, found:
367.1263.
4.1.6. 5-Amino-
N-(3,4-difluorophenyl)-2-fluorobenzamide (8)
To a solution of 7 (250 mg, 0.68 mmol) in CH2Cl2 (7 mL) was added TFA (522
µL, 6.8 mmol) at 0 °C. The mixture was stirred 1 h
at 0 °C and then 1 h at room temperature. The solution was diluted with CH2Cl2 and neutralized by addition of solid NaHCO3. The
mixture was washed with water. The organic layer was separated, dried over MgSO4 and concentrated under reduced pressure. The
1
residue was purified by silica gel column chromatography with hexanes/EtOAc (7:3) to afford 8 (109 mg, 60%). H NMR (400
MHz, Chloroform-
= 11.9, 8.7 Hz, 1H), 6.86 – 6.75 (m, 1H), 3.77 (s, 2H). 13C NMR (101 MHz, Chloroform-
236.5 Hz), 150.1 (dd, = 247.2, 13.2 Hz), 147.2 (dd, = 245.9, 12.8 Hz), 143.5 (d, = 1.9 Hz), 134.2 (dd,
(d, = 12.1 Hz), 120.0 (d, = 9.0 Hz), 117.0, 116.7, 116.0 (dd, = 5.9, 3.6 Hz), 110.3 (d,
d
)
δ
8.51 (d,
J
= 17.6 Hz, 1H), 7.92 – 7.76 (m, 1H), 7.43 (dd,
J
= 6.5, 3.1 Hz, 1H), 7.25 – 7.10 (m, 2H), 7.00 (dd,
161.5 (d, = 3.8 Hz), 153.8 (d,
= 8.7, 3.2 Hz), 120.8
J
d
)
δ
J
J =
J
J
J
J
J
J
J
J
= 22.0 Hz). 19F NMR (377 MHz,
14

ACCEPTED MANUSCRIPT
Chloroform-
d
)
δ
-127.9 – -128.0 (m), -136.9 – -137.0 (m), -143.5 – -143.6 (m). HRMS (ESI): m/z [M+H]+ calcd for C13H10F3N2O:
267.0745, found: 267.0738.
4.1.7. 5-(Cyclopropanesulfonamido)-N-(3,4-difluorophenyl)-2-fluorobenzamide (9)
To a solution of 8 (180 mg, 0.68 mmol) in CH2Cl2 (6 mL) were added cyclopropylsulfonyl chloride (69
µL, 0.68 mmol), Et3N (104
µ
L, 0.75 mmol) and DMAP (4 mg, 0.03 mmol). The mixture was heated at 40 °C during 3 h and cooled down to room temperature.
The solution was diluted with CH2Cl2 and washed with 1M HCl and brine. The organic layer was dried over MgSO4 and
concentrated under reduced pressure. The residue was purified by silica gel column chromatography using hexanes/EtOAc (7:3) to
afford 9 (119 mg, 47%). 1H NMR (400 MHz, Acetone-d6
Hz, 1H), 7.63 – 7.56 (m, 1H), 7.56 – 7.48 (m, 1H), 7.41 – 7.27 (m, 2H), 2.72 – 2.56 (m, 1H), 1.07 – 0.96 (m, 4H). 13C NMR (101
MHz, Acetone-d6 161.9 (d, = 2.2 Hz), 156.6 (d, = 246.4 Hz), 149.6 (dd, = 244.0, 13.2 Hz), 146.5 (dd, = 243.1, 12.8 Hz),
135.7 (dd, = 9.0, 3.1 Hz), 135.0 (d, = 2.9 Hz), 126.2 (d, = 8.8 Hz), 124.1 (d, = 15.6 Hz), 123.3 (d, = 2.9 Hz), 117.3, 117.1,
116.9, 116.3 (dd, -121.3 – -121.4 (m), -139.6 – -
= 22.2 Hz), 4.8. 19F NMR (377 MHz, Acetone-d6
) δ 9.67 (s, 1H), 8.79 (s, 1H), 8.03 – 7.94 (m, 1H), 7.82 (dd, J = 6.2, 2.9
)
δ
J
J
J
J
J
J
J
J
J
J
= 6.1, 3.6 Hz), 109.4 (d,
J
) δ
139.7 (m), -146.3 – -146.4 (m). HRMS (ESI): m/z [M+H]+ calcd for C16H14F3N2O3S: 371.0677, found: 371.0669.
4.1.8. 5-(Cyclopentanesulfonamido)-
To a solution of 8 (180 mg, 0.68 mmol) in CH2Cl2 (6 mL) were added cyclopentylsulfonyl chloride (86
L, 0.75 mmol) and DMAP (4 mg, 0.03 mmol). The mixture was heated at 40 °C for 3 h and cooled down to room temperature.
N-(3,4-difluorophenyl)-2-fluorobenzamide (10)
µL, 0.68 mmol), Et3N (104
µ
The solution was diluted with CH2Cl2 and washed with 1M HCl and brine. The organic layer was dried over MgSO4 and
concentrated under reduced pressure. The residue was purified by silica gel column chromatography using hexanes/EtOAc (7:3) to
afford 10 (62 mg, 23%). 1H NMR (400 MHz, Acetone-d6
Hz, 1H), 7.61 – 7.55 (m, 1H), 7.55 – 7.50 (m, 1H), 7.40 – 7.24 (m, 2H), 3.68 – 3.58 (m, 1H), 2.04 – 1.93 (m, 4H), 1.81 – 1.68 (m,
2H), 1.67 – 1.55 (m, 2H). 13C NMR (101 MHz, Acetone-d6
161.9, 156.3 (d, = 246.1 Hz), 149.6 (dd, = 244.0, 13.2 Hz), 146.5
= 2.9 Hz), 125.1 (d, = 8.7 Hz), 124.2 (d, = 15.6 Hz), 122.1 (d,
= 24.7 Hz), 116.2 (dd, = 6.2, 3.4 Hz), 109.3 (d, = 22.2 Hz), 60.5, 27.6, 25.5.
) δ 9.67 (s, 1H), 8.82 (s, 1H), 8.13 – 7.92 (m, 1H), 7.79 (dd, J = 6.2, 2.9
)
δ
J
J
(dd,
J
= 242.9, 12.8 Hz), 135.7 (dd,
J
= 9.1, 3.1 Hz), 135.3 (d,
J
J
J
J
= 2.6 Hz), 117.2 (dd,
J
= 18.2, 0.9 Hz), 117.1 (d,
J
J
J
) δ
19F NMR (377 MHz, Acetone-d6 -122.0 – -122.1 (m), -139.7 – -139.8 (m), -146.4 – -146.6 (m). HRMS (ESI): m/z [M+H]+ calcd
for C18H18F3N2O3S: 399.0990, found: 399.0985.
4.1.9. 5-(Cyclohexanesulfonamido)-
To a solution of 8 (180 mg, 0.68 mmol) in CH2Cl2 (6 mL) were added cyclohexyllsulfonyl chloride (98
L, 0.75 mmol) and DMAP (4 mg, 0.03 mmol). The mixture was heated at 40 °C during 3 h and cooled down to room temperature.
N-(3,4-difluorophenyl)-2-fluorobenzamide (11)
µL, 0.68 mmol), Et3N (104
µ
The solution was diluted with CH2Cl2 and washed with 1M HCl and brine. The organic layer was dried over MgSO4 and
concentrated under reduced pressure. The residue was purified over silica gel column chromatography using hexanes/EtOAc (7:3)
to afford 11 (98 mg, 35%). 1H NMR (400 MHz, Acetone-d6
(dd, = 6.1, 2.9 Hz, 1H), 7.58 (ddd, = 8.9, 4.3, 2.9 Hz, 1H), 7.56 – 7.51 (m, 1H), 7.40 – 7.25 (m, 2H), 3.17 – 3.03 (m, 1H), 2.19 –
2.12 (m, 2H), 1.89 – 1.79 (m, 2H), 1.70 – 1.62 (m, 1H), 1.60 – 1.44 (m, 2H), 1.37 – 1.23 (m, 3H). 13C NMR (101 MHz, Acetone-d6
) δ 9.68 (s, 1H), 8.84 (s, 1H), 7.99 (ddd, J = 13.0, 7.4, 2.6 Hz, 1H), 7.79
J
J
)
δ
161.9, 156.1 (d,
(d, = 2.8 Hz), 124.5 (d,
116.2 (dd, = 5.9, 3.6 Hz), 109.3 (d,
139.7 – -139.9 (m), -146.5 – -146.6 (m). HRMS (ESI): m/z [M+H]+ calcd for C19H20F3N2O3S: 413.1147, found: 413.1139.
J
= 245.6 Hz), 149.6 (dd,
J
= 243.9, 13.2 Hz), 146.5 (dd,
J
= 242.9, 12.7 Hz), 135.7 (dd,
J
= 9.1, 3.2 Hz), 135.4
J
J
= 8.6 Hz), 124.2 (d,
J
= 15.7 Hz), 121.6 (d, = 2.6 Hz), 117.2 (d,
J
J
= 18.3 Hz), 117.1 (d, J = 24.6 Hz),
J
J ) δ -122.4 – -122.5 (m), -
= 22.1 Hz), 60.0, 26.3, 24.9, 24.7. 19F NMR (377 MHz, Acetone-d6
15

ACCEPTED MANUSCRIPT
4.1.10. 3-Acetamido-4-fluorobenzenesulfonyl chloride (13)
To chlorosulfonic acid (20 mL) at 0 °C was added portion wise 2-fluoroacetanilide (5 g, 32.6 mmol). The solution was heated at
80 °C for 5 h, cooled down to room temperature and poured into crushed ice. The white precipitate was filtered, washed with cold
water and dried in vacuo
.
1H NMR revealed the presence of 2 regioisomers which were separated by silica gel column
9.74 (s,
= 10.9, 8.5 Hz, 1H), 2.08 (s, 3H). 13C NMR
chromatography using hexanes/EtOAc (8:2) to afford the desired isomer 13 (5.1 g, 62%). 1H NMR (400 MHz, DMSO-d6
)
δ
1H), 8.11 (dd,
J
= 7.7, 2.2 Hz, 1H), 7.35 (ddd,
J
= 8.5, 4.9, 2.2 Hz, 1H), 7.17 (dd,
J
(101 MHz, DMSO-d6
)
δ
169.1, 153.9 (d, = 246.5 Hz), 144.6, 125.8 (d,
J
J
= 12.2 Hz), 122.9 (d, J = 8.4 Hz), 122.2, 115.1 (d, J =
20.4 Hz), 23.9. 19F NMR (377 MHz, DMSO-d6 -125.6 – -125.7 (m). HRMS (ESI): m/z [M+H]+ calcd for C8H8ClFNO3S:
) δ
251.9897, found: 251.9889.
4.1.11.
To a solution of compound 13 (1 g, 3.97 mmol) in CH2Cl2 (10 mL) was added cyclopentylamine (196
(610 L, 4.37 mmol). The mixture was stirred 2 h at room temperature, diluted with CH2Cl2 and washed with water. The organic
layer was dried over MgSO4 and concentrated under reduced pressure. The residue was purified by silica gel column
N-(5-(N-Cyclopentylsulfamoyl)-2-fluorophenyl)acetamide (14)
µ
L, 3.97 mmol) and Et3N
µ
chromatography using hexanes/EtOAc (8:2) to afford 14 (1.05 g, 88%). 1H NMR (400 MHz, Chloroform-
Hz, 1H), 7.79 (d, = 3.1 Hz, 1H), 7.68 – 7.59 (m, 1H), 7.19 (dd, = 10.3, 8.6 Hz, 1H), 5.17 (d, = 7.3 Hz, 1H), 3.61 (h,
1H), 2.26 (s, 3H), 1.89 – 1.73 (m, 2H), 1.70 – 1.56 (m, 2H), 1.56 – 1.33 (m, 4H). 13C NMR (101 MHz, Chloroform-
154.3 (d, = 251.5 Hz), 137.5 (d, = 3.3 Hz), 127.1 (d, = 11.3 Hz), 123.6 (d, = 8.8 Hz), 120.8, 115.4 (d, = 21.0 Hz), 55.3,
-124.6. HRMS (ESI): m/z [M+H]+ calcd for C13H18FN2O3S: 301.1022,
d
)
δ
8.81 (dd,
J
= 7.3, 2.3
J = 6.8 Hz,
J
J
J
d) δ 168.8,
J
J
J
J
J
33.4, 24.5, 23.2. 19F NMR (377 MHz, Chloroform-
d) δ
found: 301.1017.
4.1.12. 3-Amino-N-cyclopentyl-4-fluorobenzenesulfonamide (15)
A solution of compound 14 (1 g, 3.33 mmol) in HCl 6N (5 mL) was stirred at 100 °C for 1 h and neutralized with NaOH 1M. The
mixture was extracted with CH2Cl2 (3 x 10 mL). The organic layer was dried over MgSO4 and concentrated in vacuo. The residue
1
was purified by silica gel column chromatography using hexanes/EtOAc (1:1) to afford 15 (639 mg, 74%). H NMR (400 MHz,
DMSO-d6
2H), 3.43 – 3.32 (m, 1H), 1.65 – 1.49 (m, 4H), 1.43 – 1.26 (m, 4H). 13C NMR (101 MHz, DMSO-d6
138.2 (d, = 2.8 Hz), 137.5 (d, = 13.9 Hz), 115.8 (d, = 19.9 Hz), 114.6 (d,
= 13.6 Hz), 114.6, 54.9, 32.9, 23.3. 19F NMR (377
MHz, DMSO-d6
-131.2 – -131.4 (m). HRMS (ESI): m/z [M+H]+ calcd for C11H16FN2O2S: 259.0917, found: 259.0910.
)
δ
7.48 (d,
J
= 7.0 Hz, 1H), 7.22 (dd,
J
= 8.4, 2.4 Hz, 1H), 7.16 (dd,
J
= 11.3, 8.4 Hz, 1H), 6.97 – 6.91 (m, 1H), 5.62 (s,
)
δ
152.5 (d, = 243.2 Hz),
J
J
J
J
J
)
δ
4.1.13.
To a solution of 15 (250 mg, 0.968 mmol) in CH2Cl2 (5 mL) were added 3,4-difluorobenzoyl chloride (122
Et3N (148 L, 1.06 mmol) at 0 °C. The mixture was stirred 2 h at room temperature, diluted with CH2Cl2 and washed with water.
The organic layer was dried over MgSO4 and concentrated under reduced pressure. The residue was purified by silica gel column
chromatography using hexanes/EtOAc (8:2) to afford 16 (138 mg, 36%). 1H NMR (400 MHz, Acetone-d6
9.60 (s, 1H), 8.63 (dd,
= 7.2, 2.4 Hz, 1H), 8.06 – 7.99 (m, 1H), 7.97 – 7.91 (m, 1H), 7.77 – 7.69 (m, 1H), 7.62 – 7.49 (m, 1H), 7.45 (dd, = 10.4, 8.6 Hz,
= 7.2 Hz, 1H), 3.73 – 3.56 (m, 1H), 1.87 – 1.73 (m, 2H), 1.70 – 1.57 (m, 2H), 1.56 – 1.38 (m, 4H). 13C NMR (101
163.7, 156.2 (d, = 252.4 Hz), 152.5 (dd, = 252.4, 12.7 Hz), 149.9 (dd, = 247.7, 13.1 Hz), 138.4 (d, = 3.6
Hz), 132.1 – 131.1 (m), 126.8 (d, = 12.5 Hz), 125.2 (dd, = 7.5, 3.7 Hz), 124.8 (d, = 8.9 Hz), 123.5 (d, = 2.8 Hz), 117.6 (d,
18.0 Hz), 117.3 (dd, = 18.8, 1.3 Hz), 116.1 (d, -120.3 – -120.4
= 21.4 Hz), 55.1, 32.8, 23.0. 19F NMR (377 MHz, Acetone-d6
(m), -134.5 – -134.6 (m), -138.8 – -138.9 (m). HRMS (ESI): m/z [M+H]+ calcd for C18H18F3N2O3S: 399.0990, found: 399.0985.
N-(5-(N-Cyclopentylsulfamoyl)-2-fluorophenyl)-3,4-difluorobenzamide (16)
µL, 0.97 mmol) and
µ
)
δ
J
J
2H), 6.67 (d,
J
MHz, Acetone-d6
)
δ
J
J
J
J
J
J
J
J
J =
J
J
) δ
16

ACCEPTED MANUSCRIPT
4.1.14. 1-(3,4-Difluorophenyl)cyclopropan-1-amine (18)
To a solution of 3,4-difluorobenzonitrile (278 mg, 2 mmol) in dry Et2O (10 mL) at -78 °C was added dropwise Ti(OiPr)4 (0.64 mL,
2.2 mmol) followed by EtMgBr (1.5 mL, 2.2 mmol, 3M in Et2O). After 10 minutes, BF3.Et2O (0.5 mL, 4 mmol) was added and the
solution was stirred for 1 h at -78 °C. The reaction was quenched by addition of 1N HCl (2 mL) and diluted with Et2O (10 mL). The
organic layer was separated, washed with water, brine and dried over MgSO4. The volatiles were evaporated under reduced
pressure and the residue was purified by silica gel column chromatogrpahy using hexanes/EtOAc (1:1) to afford 18 (189 mg, 56%).
1H NMR (400 MHz, Chloroform-
2H). 13C NMR (101 MHz, Chloroform-
Hz), 121.2 (dd, = 6.2, 3.4 Hz), 116.9 (d,
d) δ 7.16 – 7.04 (m, 2H), 7.03 – 6.98 (m, 1H), 1.91 (s, 2H), 1.16 – 1.05 (m, 2H), 1.01 – 0.90 (m,
d
)
δ
150.1 (dd,
J
= 247.5, 12.8 Hz), 148.6 (dd,
J = 246.2, 12.8 Hz), 144.2 (dd, J = 5.0, 3.5
J
J
= 17.0 Hz), 114.7 (d,
J
= 17.7 Hz), 36.2, 18.2. 19F NMR (377 MHz, Chloroform-
d) δ -
139.3 – -139.4 (m), -143.3 – -143.4 (m). HRMS (ESI): m/z [M+H]+ calcd for C9H10F2N: 170.0781, found: 170.0776.
4.1.15. 5-(N-Cyclopentylsulfamoyl)-N-(1-(3,4-difluorophenyl)cyclopropyl)-2-fluorobenzamide (19)
A solution of 2 (240 mg, 1 mmol) in SOCl2 (2.5 mL) was heated at 80 °C for 16 h. The mixture was concentrated under reduced
pressure and co-evaporated with toluene. The crude mixture was dissolved in toluene (2.5 mL) and a solution of 18 (170 mg, 1
mmol) in toluene (2 mL) was added via cannula. The mixture was heated at 110 °C for 2 h and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography using hexanes/EtOAc (8:2) to afford the desired sulfonyl chloride
derivative. To a solution of this sulfonyl chloride (314 mg) in CH2Cl2 (5 mL) were added cyclopentylamine (83
and Et3N (129 L, 0.929 mmol). The mixture was stirred 2 h at room temperature, diluted with CH2Cl2 and washed with water. The
organic layer was dried over MgSO4 and concentrated under reduced pressure. The residue was purified by silica gel column
chromatography using hexanes/EtOAc (8:2) to afford 19 (149 mg, 34% over two steps). 1H NMR (400 MHz, Acetone-d6
8.49 (s,
1H), 8.25 (dd, = 6.6, 2.5 Hz, 1H), 8.07 – 7.99 (m, 1H), 7.47 (dd, = 10.4, 8.7 Hz, 1H), 7.41 – 7.32 (m, 1H), 7.31 – 7.12 (m, 2H),
6.68 (d,
= 7.2 Hz, 1H), 3.68 – 3.48 (m, 1H), 1.80 – 1.69 (m, 2H), 1.67 – 1.56 (m, 2H), 1.53 – 1.31 (m, 8H). 13C NMR (101 MHz,
Acetone-d6 162.7 (d, = 2.3 Hz), 161.7 (d, = 254.9 Hz), 149.8 (dd, = 244.8, 12.9 Hz), 148.5 (dd, = 244.4, 12.6 Hz), 140.9
(dd, = 5.5, 3.6 Hz), 138.8 (d, = 3.5 Hz), 131.5 (d, = 10.2 Hz), 130.0 (d, = 4.3 Hz), 124.2 (d, = 15.9 Hz), 122.2 (dd, = 6.3,
3.4 Hz), 117.1 (d, = 25.0 Hz), 116.8 (d,
= 18.3 Hz), 55.1, 34.7, 32.8, 22.9, 17.6. 19F NMR (377 MHz,
Acetone-d6
µL, 0.844 mmol)
µ
)
δ
J
J
J
)
δ
J
J
J
J
J
J
J
J
J
J
J
J
= 17.2 Hz), 115.0 (d, J
)
δ
-110.5 – -110.6 (m), -141.9 – -142.0 (m), -145.2 – -145.3 (m). HRMS (ESI): m/z [M+H]+ calcd for C21H22F3N2O3S:
439.1303, found: 439.1300.
4.1.16. 2-(3,4-Difluorophenyl)propan-2-amine (20)
A solution of anhydrous CeCl3 (2.84g, 11.5 mmol) in THF (18 mL) was stirred at 45 °C for 3 h and cooled down to room
temperature. Then, 3,4-difluorobenzonitrile (800 mg, 5.75 mmol) was added and the mixture was cooled down to -25 °C before
addition of MeLi (9.6 mL, 14.4 mmol, 1.5 M in Et2O). The solution was stirred 1 h at -25 °C and quenched with NaOH 30% (4 mL).
The mixture was stirred for 16 h at room temperature and the cerium salts were filtered and washed with THF. The filtrate was
dried over MgSO4 and concentrated under reduced pressure. The residue was dissolved in THF and HCl (4N in dioxane) was added
and the solution was concentrated in vacuo. The resulting salts were filtered, washed with hexanes and then treated with aqueous
ammonium hydroxide (5 mL). The solution was then extracted with CH2Cl2, dried over MgSO4 and concentrated in vacuo. The
residue was purified by silica gel column chromatography using CH2Cl2/MeOH (98:2) to afford 20 (502 mg, 51%). 1H NMR (400
MHz, Chloroform-
Chloroform- 150.51 (dd,
(d, = 16.8 Hz), 114.26 (d,
143.4 – -143.5 (m). HRMS (ESI): m/z [M+H]+ calcd for C9H12F2N: 172.0938, found: 172.0932.
d
)
δ
7.41 – 7.32 (m, 1H), 7.27 – 7.19 (m, 1H), 7.11 (dt,
= 121.6, 12.6 Hz), 148.83 – 147.43 (m), 147.48 (d,
= 17.8 Hz), 52.12 (d,
= 1.4 Hz), 33.01. 19F NMR (377 MHz, Chloroform-
J
= 10.2, 8.4 Hz, 1H), 1.49 (s, 6H). 13C NMR (101 MHz,
d
)
δ
J
J
= 16.9 Hz), 120.69 (dd,
J
= 6.1, 3.5 Hz), 116.62
-139.4 – -139.5 (m), -
J
J
J
d)
δ
17

ACCEPTED MANUSCRIPT
4.1.17. 3-(
N-Cyclopentylsulfamoyl)-
N-(2-(3,4-difluorophenyl)propan-2-yl)benzamide (21)
A solution of 2 (507 mg, 2.12 mmol) in SOCl2 (5 mL) was heated at 80 °C for 16 h. The mixture was concentrated under reduced
pressure and co-evaporated with toluene. The crude product was dissolved in toluene (3 mL) and a solution of 20 (360 mg, 2.12
mmol) in toluene (2 mL) was added via cannula. The mixture was heated at 110 °C again for 2 h and concentrated under reduced
pressure. The crude residue was purified by silica gel column chromatography using hexanes/EtOAc (8:2) to afford the sulfonyl
chloride intermediate. To a solution of this sulfonyl chloride (338 mg) in CH2Cl2 (5 mL) were added cyclopentylamine (89
µL,
0.904 mmol) and Et3N (139 L, 0.995 mmol). The mixture was stirred 2 h at room temperature, diluted with CH2Cl2 and washed
µ
with water. The organic layer was dried over MgSO4 and concentrated under reduced pressure. The residue was purified by silica
gel column chromatography using hexanes/EtOAc (8:2) to afford 21 (230 mg, 25% over two steps). 1H NMR (400 MHz, Acetone-
d6
6.66 (d,
(101 MHz, Acetone-d6
Hz), 146.0 – 144.6 (m), 138.7 (d,
6.4, 3.4 Hz), 117.1, 116.9, 116.7 (d,
Acetone-d6
-111.0 – -111.1 (m), -141.9 – -142.0 (m), -145.4 – -145.5 (m). HRMS (ESI): m/z [M+H]+ calcd for C21H23F3N2O3S:
)
δ
8.15 (dd,
= 7.2 Hz, 1H), 3.73 – 3.34 (m, 1H), 1.80 (s, 6H), 1.78 – 1.69 (m, 2H), 1.68 – 1.57 (m, 2H), 1.54 – 1.33 (m, 4H). 13C NMR
161.6 (d, = 253.8 Hz), 161.4 (d, = 2.0 Hz), 149.7 (dd, = 244.4, 12.7 Hz), 148.5 (dd, = 244.4, 12.7
= 3.3 Hz), 131.2 (d, = 10.0 Hz), 129.7 (d, = 4.3 Hz), 125.0 (d, = 16.3 Hz), 121.6 (dd,
= 17.0 Hz), 114.4 (d, = 18.3 Hz), 55.8 (d,
= 1.3 Hz), 55.1, 32.8, 22.9. 19F NMR (377 MHz,
J = 6.7, 2.5 Hz, 1H), 8.03 – 7.98 (m, 1H), 7.97 (s, 1H), 7.54 – 7.42 (m, 2H), 7.40 – 7.33 (m, 1H), 7.33 – 7.21 (m, 1H),
J
)
δ
J
J
J
J
J
J
J
J
J =
J
J
J
)
δ
441.1460, found: 441.1453.
4.1.18. 3,4-Dihydroisoquinolin-1(2H)-one (24)
To a solution of phenethylamine (3 mL, 24 mmol) and Et3N (3.6 mL, 26.4 mmol) in DMF (50 mL) was added methyl
chloroformate (2.0 mL, 26.4 mmol) at 0 °C. The reaction mixture was stirred 1 h at room temperature and diluted with EtOAc (100
mL). The solution was washed three times with water (3 x 30 mL). The organic layer was dried over MgSO4 and concentrated in
vacuo. The residue was purified by silica gel column chromatography using hexanes/EtOAc (1:1 to 0:1) to afford the carbamate
intermediate (3.72 g, 86%) as a colorless liquid. CAS: 26011-68-7. The obtained carbamate (3 g, 16.7 mmol) was dissolved in
trifluoromethanesulfonic acid (30 mL) at 0 °C and the mixture was stirred for 24 h at 70 °C. The mixture was then poured into
crushed ice and extracted with CH2Cl2. The organic layer was dried over MgSO4 and concentrated in vacuo. The residue was
purified by silica gel column chromatography using hexanes/EtOAc (1:1) to afford 24 (2.34 g, 95%) as a yellow oil. CAS: 1196-38-
9.
4.1.19. 2,3,4,5-Tetrahydro-1H-benzo[c]azepin-1-one (25)
To a solution of phenylpropylamine (3 mL, 21.1 mmol) and Et3N (3.2 mL, 23.2 mmol) in DMF (50 mL) was added methyl
chloroformate (1.8 mL, 23.2 mmol) at 0 °C. The reaction mixture was stirred 1 h at room temperature and diluted with EtOAc (100
mL). The solution was washed three times with water (3 x 30 mL). The organic layer was dried over MgSO4 and concentrated in
vacuo. The resulting liquid was purified by silica gel column chromatography using hexanes/EtOAc (1:1 to 0:1) to afford the
carbamate intermediate (3.80 g, 93%) as a colorless liquid. CAS: 111944-09-3. This carbamate (3.65 g, 18.9 mmol) was dissolved
in trifluoromethanesulfonic acid (30 mL) at 0 °C and the mixture was stirred for 24 h at 70 °C. The mixture was poured into
crushed ice and extracted with CH2Cl2. The organic layer was dried over MgSO4 and concentrated in vacuo. The residue was
purified by silica gel column chromatography using hexanes/EtOAc (1:1) to afford 25 (2.74 g, 90%) as a yellow oil. CAS: 6729-50-
6.
4.1.20.
N-Cyclopentyl-1-oxo-1,2,3,4-tetrahydroisoquinoline-7-sulfonamide (28)
To chlorosulfonic acid (25 mL) cooled to 0 °C was added portion wise compound 24 (2 g, 13.6 mmol). After complete addition, the
yellow solution was allowed to warm up to room temperature, then heated at 60 °C for 16 h. The reaction mixture was then cooled
down to room temperature and poured dropwise into crushed ice. The light yellow precipitate was filtered, washed with water and
18

ACCEPTED MANUSCRIPT
cold Et2O and dried in vacuo to afford the desired sulfonyl chloride intermediate 26. To a solution of compound 26 (1 g, 4.07 mmol)
in CH2Cl2 (10 mL) were added cyclopentylamine (400
at room temperature and quenched with 1M HCl. The precipitate was filtered and washed with water and cold Et2O. The solid was
dried in vacuo to afford 28 (971 mg, 81%) as a light yellow solid. 1H NMR (400 MHz, DMSO-d6
8.27 (d, = 2.1 Hz, 1H), 8.19
(s, 1H), 7.87 (dd, = 7.9, 2.1 Hz, 1H), 7.75 (d, = 6.9 Hz, 1H), 7.53 (d, = 8.0 Hz, 1H), 3.46 – 3.39 (m, 2H), 3.06 – 2.96 (m, 2H),
1.64 – 1.49 (m, 4H), 1.44 – 1.23 (m, 4H). 13C NMR (101 MHz, DMSO-d6
163.7, 144.0, 140.7, 130.4, 129.7, 129.1, 125.7, 54.9,
39.2, 32.9, 28.0, 23.2. HRMS (ESI): m/z [M+H]+ calcd for C14H19N2O3S: 295.1116, found: 295.1110.
µL, 4.07 mmol) and Et3N (624 µL, 4.48 mmol). The mixture was stirred 2 h
)
δ
J
J
J
J
)
δ
4.1.21.
N-Cyclopentyl-1-oxo-2,3,4,5-tetrahydro-1H-benzo[c]azepine-8-sulfonamide (29)
To chlorosulfonic acid (10 mL) cooled to 0 °C was added portion wise compound 25 (955 mg, 5.92 mmol). After complete addition,
the yellow solution was allowed to warm up to room temperature, then heated at 60 °C for 16 h. The reaction mixture was cooled
down to room temperature and poured dropwise into crushed ice. The light yellow precipitate was filtered, washed with water and
cold Et2O and dried in vacuo to afford the desired sulfonyl chloride intermediate 27. To a solution of compound 27 (250 mg g,
0.963 mmol) in CH2Cl2 (5 mL) were added cyclopentylamine (144 µL, 0.963 mmol) and Et3N (148 µL, 1.06 mmol). The mixture
was stirred 2 h at room temperature, diluted with CH2Cl2 and washed with water. The organic layer was dried over MgSO4 and
concentrated under reduced pressure. The residue was purified by silica gel column chromatography using hexanes/EtOAc (7:3 to
8:2) to afford the desired sulfonamide derivative 29 (183 mg, 97%) as a light yellow solid. 1H NMR (400 MHz, DMSO-d6
= 5.9 Hz, 1H), 7.91 (d, = 2.1 Hz, 1H), 7.82 (dd, = 7.9, 2.1 Hz, 1H), 7.70 (d, = 7.1 Hz, 1H), 7.49 (d, = 7.9 Hz, 1H), 3.44 –
3.37 (m, 1H), 2.91 (q, = 6.3 Hz, 2H), 2.82 (t, = 7.1 Hz, 2H), 1.93 (p,
= 6.7 Hz, 2H), 1.65 – 1.22 (m, 8H). 13C NMR (101 MHz,
DMSO-d6
170.9, 142.7, 140.7, 137.1, 130.1, 128.9, 126.7, 54.9, 38.8, 32.9, 30.11, 30.04, 23.3. HRMS (ESI): m/z [M+H]+ calcd
for C15H21N2O3S: 309.1273, found: 309.1266.
) δ 8.27 (t,
J
J
J
J
J
J
J
J
)
δ
4.1.22.
To a solution of compound 26 (1 g, 4.07 mmol) in CH2Cl2 (10 mL) were added
and Et3N (624 L, 4.48 mmol). The mixture was stirred for 2 h at room temperature, diluted with CH2Cl2 and washed with water.
N-Cyclopentyl-
N-methyl-1-oxo-1,2,3,4-tetrahydroisoquinoline-7-sulfonamide (30)
N-methylcyclopentylamine (480
µL, 4.07 mmol)
µ
The organic layer was dried over MgSO4 and concentrated under reduced pressure. The residue was purified by silica gel column
chromatography using hexanes/EtOAc (7:3 to 8:2) to afford the desired sulfonamide derivative 30 (1.13 g, 90%) as a light yellow
solid. 1H NMR (400 MHz, DMSO-d6
1H), 4.25 (p, = 8.1 Hz, 1H), 3.48 – 3.38 (m, 2H), 3.02 (t,
1.35 – 1.25 (m, 2H). 13C NMR (101 MHz, DMSO-d6
163.6, 144.6, 137.7, 130.5, 130.2, 129.4, 126.0, 58.3, 39.2, 28.9, 28.0, 27.8,
24.0. HRMS (ESI): m/z [M+H]+ calcd for C15H21N2O3S: 309.1273, found: 309.1267.
)
δ
8.22 (s, 1H), 8.17 (d,
= 6.6 Hz, 2H), 2.64 (s, 3H), 1.55 – 1.45 (m, 4H), 1.45 – 1.35 (m, 2H),
J = 2.1 Hz, 1H), 7.87 (dd, J = 8.0, 2.1 Hz, 1H), 7.56 (d, J = 8.0 Hz,
J
J
)
δ
4.1.23.
To a solution of compound 27 (250 mg, 0.963 mmol) in CH2Cl2 (5 mL) were added
mmol) and Et3N (148 L, 1.06 mmol). The mixture was stirred for 2 h at room temperature, diluted with CH2Cl2 and washed with
N-Cyclopentyl-
N-methyl-1-oxo-2,3,4,5-tetrahydro-1H-benzo[c]azepine-8-sulfonamide (31)
N-methylcyclopentylamine (144
µL, 0.963
µ
water. The organic layer was dried over MgSO4 and concentrated under reduced pressure. The residue was purified by silica gel
column chromatography using hexanes/EtOAc (7:3 to 8:2) to afford the desired sulfonamide derivative 31 (292 mg, 94%) as a light
yellow solid. 1H NMR (400 MHz, DMSO-d6
1H), 2.92 (q, = 6.3 Hz, 2H), 2.83 (t, = 7.1 Hz, 2H), 2.64 (s, 3H), 1.93 (t,
2H), 1.35 – 1.23 (m, 2H). 13C NMR (101 MHz, DMSO-d6
170.7, 143.3, 137.6, 137.4, 130.4, 129.4, 127.0, 58.3, 38.8, 30.09,
30.04, 28.9, 27.7, 24.1. HRMS (ESI): m/z [M+H]+ calcd for C16H23N2O3S: 323.1429, found: 323.1423.
)
δ
8.30 (t,
J
= 5.9 Hz, 1H), 7.82 (s, 2H), 7.52 (d,
J = 8.7 Hz, 1H), 4.24 (p, J = 8.1 Hz,
J
J
J
= 6.8 Hz, 2H), 1.56 – 1.45 (m, 4H), 1.44 – 1.36 (m,
)
δ
19

ACCEPTED MANUSCRIPT
4.1.24.
N-Cyclopentyl-2-(3,4-difluorophenyl)-1-oxo-1,2,3,4-tetrahydroisoquinoline-7-sulfonamide (32)
To a solution of compound 28 (50 mg, 0.170 mmol) in DMF (0.5 mL) were added 3,4-difluorobromobenzene (23 µL, 0.170 mmol),
CuI (32 mg, 0.170 mmol) and K2CO3 (47 mg, 0.340 mmol). The mixture was heated at 150 °C for 16 h and then diluted with
EtOAc and water and filtered over a pad of Celite. The filtrate was washed with a saturated solution of Na2CO3, water, dried over
MgSO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using
hexanes/EtOAc (9:1 to 7:3) to afford 32 (39 mg, 56%). 1H NMR (400 MHz, DMSO-d6
2.1 Hz, 1H), 7.80 (d, = 6.9 Hz, 1H), 7.67 – 7.58 (m, 2H), 7.57 – 7.47 (m, 1H), 7.38 – 7.28 (m, 1H), 5.76 (s, 1H), 4.04 – 3.95 (m,
2H), 3.24 (t, 162.6, 149.9 (dd,
= 6.4 Hz, 2H), 1.68 – 1.50 (m, 4H), 1.42 – 1.27 (m, 4H). 13C NMR (101 MHz, DMSO-d6
137.6, 13.0 Hz), 147.4 (dd, = 137.0, 12.9 Hz), 143.8, 141.0, 139.9 (dd, = 8.3, 3.4 Hz), 130.2 (d, = 21.4 Hz), 129.1, 126.4,
123.0 (dd, = 6.5, 3.3 Hz), 117.7 (d, = 17.8 Hz), 115.8 (d,
= 18.8 Hz), 55.4, 54.9, 49.0, 32.9, 28.0, 23.3. 19F NMR (377 MHz,
DMSO-d6
-139.0, -142.5. HRMS (ESI): m/z [M+H]+ calcd for C20H21F2N2O3S: 407.1241, found: 407.1235.
) δ 8.36 (d, J = 2.1 Hz, 1H), 7.95 (dd, J = 7.9,
J
J
)
δ
J =
J
J
J
J
J
J
)
δ
4.1.25.
N-Cyclopentyl-2-(3,4-difluorophenyl)-1-oxo-2,3,4,5-tetrahydro-1H-benzo[c]azepine-8-sulfonamide (33)
To a solution of compound 29 (50 mg, 0.170 mmol) in DMF (0.5 mL) were added 3,4-difluorobromobenzene (23
µ
L, 0.170 mmol),
CuI (32 mg, 0.170 mmol) and K2CO3 (47 mg, 0.340 mmol). The mixture was heated at 150 °C for 16 h and then diluted with
EtOAc and water and filtered over a pad of Celite. The filtrate was washed with a saturated solution of Na2CO3, water, dried over
MgSO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using
hexanes/EtOAc (9:1 to 7:3) to afford 33 (63 mg, 58%). 1H NMR (400 MHz, Chloroform-
7.9, 2.1 Hz, 1H), 7.37 (d, = 7.9 Hz, 1H), 7.32 – 7.19 (m, 2H), 7.17 – 7.08 (m, 1H), 5.21 (d,
3.05 (t, = 7.1 Hz, 2H), 2.19 (p, = 6.8 Hz, 2H), 1.88 – 1.71 (m, 2H), 1.66 – 1.53 (m, 2H), 1.51 – 1.41 (m, 2H), 1.40 – 1.27 (m,
2H). 13C NMR (101 MHz, Chloroform-
169.5, 150.1 (dd, = 249.8, 13.4 Hz), 149.0 (dd, = 248.9, 12.5 Hz), 141.8, 140.5,
138.6 (dd, = 7.7, 3.7 Hz), 136.6, 129.8, 129.3, 127.8, 122.3 (dd, = 6.4, 3.6 Hz), 117.6 (d, = 18.2 Hz), 116.0 (d, = 18.5 Hz),
-136.6 – -136.7 (m), -140.2 – -140.3 (m). HRMS (ESI): m/z
d
)
δ
8.25 (d,
J = 2.0 Hz, 1H), 7.95 (dd, J =
J
J
= 7.3 Hz, 1H), 3.67 – 3.54 (m, 3H),
J
J
d)
δ
J
J
J
J
J
J
55.3, 49.6, 33.3, 30.0, 29.2, 23.2. 19F NMR (377 MHz, Chloroform-
[M+H]+ calcd for C21H23F2N2O3S: 421.1397, found: 421.1392.
d) δ
4.1.26.
N-Cyclopentyl-2-(3,4-difluorophenyl)-N-methyl-1-oxo-1,2,3,4-tetrahydroisoquinoline-7-sulfonamide (34)
To a solution of compound 30 (300 mg, 0.972 mmol) in DMF (2 mL) were added 3,4-difluorobromobenzene (220
µ
L, 1.95 mmol),
CuI (371 mg, 1.95 mmol) and K2CO3 (269 mg, 1.95 mmol). The mixture was heated at 150 °C for 90 minutes under microwave
irradiation. The mixture was then diluted with EtOAc and filtered over a pad of Celite. The filtrate was washed with a saturated
solution of Na2CO3, water, dried over MgSO4 and concentrated under reduced pressure. The residue was purified by silica gel
1
column chromatography using hexanes/EtOAc (9:1) to afford 34 (116 mg, 28%). H NMR (400 MHz, Acetone-d6
)
δ
8.43 (d,
= 8.1
= 6.4 Hz, 2H), 2.76 (s, 3H), 1.73 – 1.34 (m, 8H). 13C NMR (101 MHz, Acetone-d6
= 245.7, 13.5 Hz), 148.0 (dd, = 245.1, 12.7 Hz), 143.6, 139.9 (dd, = 8.2, 3.5 Hz), 138.7, 130.4, 128.6,
= 6.4, 3.5 Hz), 116.9 (d, = 18.4 Hz), 115.2 (d,
= 19.2 Hz), 58.3, 48.8, 28.1, 28.0, 27.6, 23.7. 19F NMR (377
-140.4 – -140.5 (m), -144.2 – -144.3 (m). HRMS (ESI): m/z [M+H]+ calcd for C21H23F2N2O3S: 421.1397,
J =
2.0 Hz, 1H), 7.97 (dd,
Hz, 1H), 4.14 (dd, = 6.9, 6.0 Hz, 2H), 3.35 (t,
162.2, 149.5 (dd,
J = 8.0, 2.1 Hz, 1H), 7.63 (dd, J = 8.0, 0.7 Hz, 1H), 7.59 – 7.51 (m, 1H), 7.46 – 7.29 (m, 2H), 4.39 (p, J
J
J
)
δ
J
J
J
126.8, 122.0 (dd,
MHz, Acetone-d6
found: 421.1391.
J
J
J
)
δ
4.1.27.
N
-Cyclopentyl-2-(3,4-difluorophenyl)-
N
-methyl-1-oxo-2,3,4,5-tetrahydro-1H-benzo[c]azepine-8-sulfonamide (35)
L, 1.86 mmol),
To a solution of compound 31 (300 mg, 0.930 mmol) in DMF (2 mL) were added 3,4-difluorobromobenzene (210
µ
CuI (354 mg, 1.86 mmol) and K2CO3 (258 mg, 1.86 mmol). The mixture was heated at 150 °C for 90 minutes under microwave
irradiation. The mixture was then diluted with EtOAc and filtered over a pad of Celite. The filtrate was washed with a saturated
20

ACCEPTED MANUSCRIPT
solution of Na2CO3, water, dried over MgSO4 and concentrated under reduced pressure. The residue was purified by silica gel
1
column chromatography using hexanes/EtOAc (9:1) to afford 35 (141 mg, 35%). H NMR (400 MHz, Acetone-d6
)
δ
8.03 (d,
= 8.2 Hz, 1H), 3.71 (t, = 6.4 Hz,
= 6.8 Hz, 2H), 1.72 – 1.31 (m, 8H). 13C NMR (101 MHz, Acetone-d6
168.8,
= 245.3, 12.7 Hz), 142.3, 139.8 (dd, = 8.2, 3.5 Hz), 138.4, 137.2, 127.4, 122.8 (dd,
= 18.0 Hz), 116.0 (d, -138.7 –
= 18.9 Hz), 58.3, 49.2, 28.1, 27.6, 23.8. 19F NMR (377 MHz, Acetone-d6
J =
2.1 Hz, 1H), 7.90 (dd,
2H), 3.11 (t, = 7.1 Hz, 2H), 2.74 (s, 3H), 2.23 (p,
149.6 (dd, = 246.1, 13.3 Hz), 148.3 (dd,
6.4, 3.5 Hz), 117.2 (d,
J
= 7.9, 2.0 Hz, 1H), 7.64 – 7.50 (m, 2H), 7.47 – 7.28 (m, 2H), 4.37 (p,
J
J
J
J
) δ
J
J
J
J =
J
J
) δ
-138.8 (m), -142.4 – -142.6 (m). HRMS (ESI): m/z [M+H]+ calcd for C22H25F2N2O3S: 435.1554, found: 435.1546.
4.2. Biological evaluation
Cell culture – HepAD38 cells were seeded at 50,000 cells/well in collagen-coated 96-well plates with DMEM/F12 medium
(Thermo Scientific) supplemented with 10% heat-inactivated fetal bovine serum. Cells were treated with 0.3 µg/ml tetracycline as
needed. Test compounds and controls were added to cells to a final concentration of 10 µM or in a dose-dependent manner ranging
from 0.001 to 10 µM. Cells were cultured in the absence of tetracycline for 7 days to induce DNA synthesis and cccDNA formation,
at day 7, test compounds plus tetracycline were added back to the cultures to inhibit transcription of viral RNA from integrated viral
genome. Medium and test compounds were replenished every 5 days in culture. Supernatants were harvested at day 14, clarified by
centrifugation at 5,000 rpm for 5 min, and stored at -70 °C until use. ELISA - The levels of HBeAg secreted in the culture medium
were measured by using HBeAg ELISA kit (BioChain Institute Inc. Hayward, CA) according to the manufacturer’s protocol. The
effective concentration of compound that reduced levels of secreted HBeAg by 50% (EC50) was determined by linear regression.
4.3. Electron microscopy
Expression and isolation of HBV Cp149 dimeric protein was carried out following previous literature [23]. The truncated HBV
protein (residues 1-149) was cloned into a pET-29b vector at the BAMHI and XhoI sites with a c-terminal stop codon (no tag).
This vector was transformed into BL21 e. coli grown on LB media with AMP100 restriction. The e. coli were grown in LB broth at
37 ᵒC to an OD600 = 0.8, and expression of HBV Cp149 was induced with the addition of 1mM IPTG at 16 °C overnight. The cell
pellet was solubilized 3 g/10 ml lysis buffer [50 mM Tris, 5 mM DTT, 1 mM EDTA, 0.1 mg/ml RNase/DNase, pH = 7.4] and lysed
by sonication. Cell debris was pelleted by centrifugation at 26 k x g for 1 hr. Sucrose was added to the supernatant at a final
concentration of 0.15M, and the debris was pelleted by centrifugation at 100 k x g for 1 hr. The remaining solubilized protein,
including HBV Cp149, was pelleted by ammonium sulfate (40% saturation, 26 k x g for 1 hr). The ammonium sulfate precipitant
was resolubilized in Capsid Buffer (50 mM Tris, 500 mM NaCl, 2 mM DTT, pH = 7.4). High molecular weight assemblies
(include HBV Cp149 capsids) were separated from low molecular weight protein using size exclusion chromatography. The capsid
fractions were dialized in Dimer Buffer (100 mM carbonate, 2 mM DTT, pH = 9.5), and capsids were completely dissociated into
the composite HBV Cp149 dimers by addition of 4 M urea. The HBV Cp149 dimers were isolated from remaining high molecular
weight proteins using size exclusion chromatography. The yield of HBV Dimers was ~8-10 mg/L at > 95% purity as determined by
SDS-PAGE.
HBV Capsids were prepared for electron microscopy (EM) imaging by adding capsid buffer at a 3:2 ratio (final concentration of
NaCl was 300 mM) to a sample of HBV Cp149 dimers (in dimer buffer) yielding a final concentration of HBV Cp149 monomer of
10 µM. The sample was maintained at 4 °C overnight. To evaluate the effects of compounds on capsid assembly, HBV Cp149
dimers were incubated with the agents for 1 hr prior to the addition of Capsid buffer. HBV Cp149 capsid assemblies were foxed
onto a charged carbon grid and stained by uranyl acetate contrast agent for 15 minutes. EM images were collected using a JEOL
21

ACCEPTED MANUSCRIPT
JEM-1400 electron microscope operating at 120 kV at 25,000 – 35,000 x magnification (Emory University Robert P. Apkarian
electron microscopy core facility).
4.4. Molecular modeling
Molecular modeling was carried out using Schrödinger Suite version 2016-3 using an EXXACT MD GPU server [24]. Unless
otherwise stated, the default parameters for all modules were used. The HBV Cp149 Y132A crystal structure (PDBID 5T2P) was
prepared using Protein Prep in Maestro that assigned bond orders, added hydrogens and disulfide bonds. Waters within 5 Å of
ligands were retained in the model, and hydrogen bonding was optimized using Epik for a pH = 7 [25]. Hydrogen positions were
minimized using OPLS3 forcefield in implicit solvation model [26]. A docking grid was created using Glide centered on the
cocrystallized ligand (K89) located at the interface of chains B and C [27, 28]. The grid box was 10 x 10 x 10 Å and no groups were
permitted to rotate. Re-docking of the co-crystallized ligand K89 using flexible Glide docking at the standard precision yielded a
pose similar to that observed in the crystal structure (RMSD = 0.37 Å) supporting the accuracy of the methods. Compound 4f was
prepared and docked into a grid using Glide dock with standard precision and permitted flexibility. Desmond molecular dynamics
was used to simulation the capsid complexed to compound 4f [29-31]. To avoid artifacts from misplaced amino acids not present in
the structure, the proteins were pruned to 1 – 141/142/143aa. All co-crystallized small molecules were removed leaving a system of
only six HBV capsid proteins with compound 4f between chains B and C. This system was placed in a periodic SPC water box of
buffered by solvent at 10 Å in all directions. Salt was added to a final concentration of 150 mM with an additional 30 Na+ ions to
neutralize the system. The final number of atoms for the simulation was 115,834. The molecular dynamics simulation was carried
out in the default eight steps of progressive relaxation by Desmond molecular dynamics. The final simulation stage lasted 10 ns at
300K and 1.01325 bar pressure under NPT conditions with frames recorded every 10ps. The simulation analysis was performed
using the Simulation Interactions Diagram tool.
Corresponding Author
*E-mail address: rschina@emory.edu (R.F. Schinazi).
ACKNOWLEDGMENT
This work was supported in part by NIH Grant 1-R01-AI-132833, and 5P30-AI-50409 (CFAR). The authors declare no competing
financial interest. We gratefully acknowledge Elizabeth Wright, Ph.D. and Hong Yi (Emory University Robert P. Apkarian electron
microscopy core) for their technical assistance.
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•
•
•
•
•
The synthesis and characterization of new sulfamoyl derivatives is described
All 27 compounds were evaluated for their anti-HBV activity and cytotoxicity
Several compounds reduced cccDNA level in an HBeAg reporter cell-based assay
Disruption of HBV capsid formation was investigated using electron microscopy
Molecular modelling into the capsid protein was used to rationalize the SAR