Discovery of Sulfonamide-Derived Agonists of SOS1-Mediated Nucleotide Exchange on RAS Using Fragment-Based Methods

Article abstract of DOI:10.1021/acs.jmedchem.0c00511

The nucleotide exchange factor Son of Sevenless (SOS) catalyzes the activation of RAS by converting it from its inactive GDP-bound state to its active GTP-bound state. Recently, we have reported the discovery of small-molecule allosteric activators of SOS1 that can increase the amount of RAS-GTP in cells. The compounds can inhibit ERK phosphorylation at higher concentrations by engaging a feedback mechanism. To further study this process, we sought different chemical matter from an NMR-based fragment screen using selective methyl labeling. To aid this process, several Ile methyl groups located in different binding sites of the protein were assigned and used to categorize the NMR hits into different classes. Hit to lead optimization using an iterative structure-based design paradigm resulted in compounds with improvements in binding affinity. These improved molecules of a different chemical class increase SOS1cat-mediated nucleotide exchange on RAS and display cellular action consistent with our prior results.

Full text of DOI:10.1021/acs.jmedchem.0c00511

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Article
Discovery of Sulfonamide-Derived Agonists of SOS1-Mediated
Nucleotide Exchange on RAS Using Fragment-Based Methods
Dhruba Sarkar, Edward T Olejniczak, Jason Phan, Jesse A Coker, Jiqing Sai,
Allison L Arnold, Yugandhar Beesetty, Alex G. Waterson, and Stephen W. Fesik
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00511 • Publication Date (Web): 16 Jul 2020
Downloaded from pubs.acs.org on July 16, 2020
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Discovery of Sulfonamide-Derived Agonists of SOS1-Mediated Nucleotide Exchange on RAS Using
Fragment-Based Methods
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†
Dhruba Sarkar,† Edward T. Olejniczak,† Jason Phan,† Jesse A. Coker, Jiqing Sai,† Allison Arnold,†
Yugandhar Beesetty,† Alex G. Waterson,§,¥ and Stephen W. Fesik*,†,§,¥
†Department of Biochemistry, Department of Pharmacology, Vanderbilt University School of Medicine,
§
Nashville, Tennessee 37232-0146, USA
¥Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37232-0146, USA
KEYWORDS: RAS, Son of Sevenless (SOS), drug discovery, Cancer, fragment-based lead discovery.
ABSTRACT: The nucleotide exchange factor Son of Sevenless (SOS) catalyzes the activation of RAS by
converting it from its inactive GDP-bound state to its active GTP-bound state. Recently, we have reported
the discovery of small molecule allosteric activators of SOS1 that can increase the amount of RAS-GTP in
cells. The compounds can inhibit ERK phosphorylation at higher concentrations by engaging a feedback
mechanism. To further study this process, we sought different chemical matter from an NMR-based
fragment screen using selective methyl labeling. To aid this process, several Ile methyl groups located in
different binding sites of the protein were assigned and used to categorize the NMR hits into different
classes. Hit to lead optimization using an iterative structure-based design paradigm resulted in compounds
with improvements in binding affinity. These improved molecules of a different chemical class increase
SOS1cat-mediated nucleotide exchange on RAS and display cellular action consistent with our prior results.
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■ INTRODUCTION
The RAS family of proteins represent a target of extreme importance for cancer drug discovery.1-3
Indeed, more than 30 % of all human cancers have activating mutations in K-RAS, H-RAS, or N-RAS, and
many cancer types are driven by dysregulated signaling downstream of RAS.4-5 Despite this disease
association and recent advancements in the discovery of molecules that bind to, and affect RAS function,
no agents directly acting on RAS have yet been approved for patient use.6-7 The main reason for this is the
lack of a druggable pocket on RAS proteins to which small-molecules could bind with high affinity.8-9
However, in recent years, there has been a resurgence of research around RAS, driven by direct10-14 and
indirect15-17 approaches of targeting RAS, in both academia and the pharmaceutical industry.
The GTPase RAS is an important signaling molecule that cycles between an inactive GDP-bound
and an active GTP-bound state.7,18 The nucleotide-bound status of RAS is controlled by various regulatory
proteins: GTPase Associated Proteins (GAPs) that accelerate the hydrolysis of triphosphate nucleotides,
and Guanine Exchange Factors (GEFs) that facilitate the exchange of one nucleotide to the other.19-20 The
GEF Son of Sevenless (SOS) is the primary protein responsible for this exchange process on RAS, thereby
converting it to an active GTP-bound state that initiates downstream signaling pathways.21-23
We have previously reported on the discovery of small molecules that bind to the catalytic core of
the GEF SOS and activate nucleotide exchange on RAS, increasing the amount of GTP-bound RAS, yet
producing paradoxical inhibition of downstream RAS signaling in cells.17 This paradoxical activity results
from the engagement of a feedback loop in the signal transduction pathway.24-25 We have reported several
improved follow-up molecules, many of which were derived from hits discovered by a high-throughput
screen26 that were subsequently optimized using structure-based design.27-29 To obtain additional chemical
matter, we have conducted an NMR-based fragment screen on SOS1cat, the results of which are reported
herein. The same pocket was recently reported for fragment hits targeting HRAS–SOS1 using an X-ray
fragment screening approach and for fragment hits targeting KRASG12C–SOS1 using a saturation transfer
difference (STD)-NMR.15
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Fragment-based screening methods, by virtue of the smaller size of the screened compounds,
allow greater sampling of chemical space compared with traditional screening techniques, even when a
modestly sized compound library is used.30 However, due to the weaker binding affinities typically exhibited
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by fragment molecules, sensitive biophysical techniques are required to detect the binding event.31 HSQC
NMR represents an ideal method for this purpose. We have previously realized great success using
uniformly 15N labeled protein; however, the catalytic unit of SOS1 (SOS1cat), comprised of the CDC25 and
REM domains (residues 566-1046), is relatively large.32 Therefore, to execute an NMR-based fragment
screen on the 65 kD SOS1cat, we selectively methyl labeled the protein and used changes in the13C HMQC
spectra to track spectrum changes due to ligand binding.33-34 Using this approach, we identified 59 fragment
hits that bind to SOS1cat, representing multiple chemically distinct classes of compounds. Several of the
fragment hits provide novel optimization starting points.27-29 Using an iterative structure-based ligand
design, new molecules that bind to SOS1cat with low micromolar affinities were obtained. High resolution X-
ray structural studies of the compounds were used to understand the SAR and provide insight for the design
of improved molecules. As in our prior work, we verified that these novel fragment-derived sulfonamide-
based compounds can activate nucleotide exchange on RAS and affect downstream signaling.24-25 They
thus represent alternative starting points for additional compound optimization efforts
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■ RESULTS AND DISCUSSION
2D HMQC NMR-Based Fragment Screening
Since SOS1 is relatively large protein (Mw ~65 kD), we used selectively labeled IVLM methyl
SOS1cat (residues 566-1046). The protein was grown using selectively deuterated methyl precursors (i.e.
3,3 2H methyl 13C alpha ketobutyric acid to label the c1 methyl of Ile and 3-D1 2H, 13C-5 alpha ketoisovaleric
acid to label c1,2 Val and Leu c1,2 methyls) and 13C C methyl labeled Methionine. The 1H-13C HMQC
spectra obtained from this selectively IVLM methyl-labeled SOS1cat was well-resolved (Figure 1A) and
offers methyl reporter resonances throughout the protein (Figure S2, Supplementary information). To
assign the methyl signals and to identify Ile methyl groups located in the different potential SOS1cat binding
pockets, we used selective Ile to Ala mutations as well as 13C edited nOe experiments. For example,
comparison of a mutant I718A SOS1 spectra (Figure 1B, red overlay) compared to the wt-SOS1cat spectra
(Figure 1B, blue spectra) shows the disappearance of a single Ile peak. In this case, the peak can be
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assigned to the Ile 718 resonance by inspection. For spectra with multiple changes, we used 13C-edited
nOe studies to help assign the peaks by examination of nOe resonances to other assigned methyl groups.
For example, Met 878, assigned previously, is in close proximity to Ile 839 and has shown an nOe
enhancement to one of the Ile resonances that disappears in the mutant I839A spectra. To verify whether
this methyl labeling pattern was sufficient to detect binding of known ligands to SOS1cat, we added an
activator ligand (S1, Supplementary information) previously found to bind to SOS1cat with 0.14 M affinity
(Figure S2A, Supplementary information). This resulted in several spectral changes (Figure 1 C and D).
From the known Ile and Met assignments, we identified several methyl resonances (Ile 893, Ile 839, and
Met 878) that shift upon addition of the compound. The known structure of this activator ligand bound to
SOS1cat confirms that all of these residues are located in the activator site pocket (Figure S2B,
Supplementary information).
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Figure 1. 13C HMQC spectra of methyl labeled SOS1cat. (A) Full methyl spectra of SOS1cat. (B) Ile region for a Mutant
I718A SOS1cat protein. (C) Ile region and (D) Met region of SOS1cat after addition of the activator compound S1.
Hit Identification
The recombinant SOS1cat (residues 566-1046) selectively labeled with 13C at the methyl groups
was used to screen our in-house fragment library (>13,800 compounds) by recording 1H-13C HMQC spectra
(600 MHz, 298 K)33-34 of SOS1cat (60 µM). This process yielded 59 hits that shifted activator site methyl
resonances (e.g. Ile 856, Ile 839, Met 874), a calculated hit rate of 0.1%. Hits were ranked using NMR
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titration experiments (Figure 2), showing affinities of 330 μM to >2 mM, with 5 hits having a Kd of less than
1 mM. Notably, fragment F-2 is structurally similar to our earlier benzimidazole SOS1 agonist.29 Further,
we verified that these compounds bound to the activator site pocket by using a single concentration (400
μM) to test for displacement of a FITC-labeled compound from binding to SOS1cat in the published
fluorescence polarization anisotropy (FPA) assay.29 Several hits that competed with the labeled probe at
this site were selected for X-ray studies and chemical optimization.
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Figure 2. Representative SOS1cat activator hits identified by 13C HMQC NMR-based fragment screening.
X-ray structures of fragments binding to SOS1cat and fragment merging
The hits identified by screening the fragment library were tested in co-crystallization trials with
SOS1cat to obtain structural information on their binding modes. All 16 successfully co-crystallized fragment
hits occupied the same binding site on SOS1cat initially identified as the binding site of compounds
discovered using an orthogonal HTS assay.28-29 The co-crystal structure of sulfonamide fragment F-7 bound
to SOS1cat (Figure 3A) shows a unique bidentate H-bonding interaction with the side chain amide of Asn879.
On the other hand, fragment F-4 (Figure 3B), containing a nitro group, picked up a similar H-bonding
interaction with the side chain amide of Asn 879. Interestingly, the phenyl ring of Phe 890 is flipped up
relative to its position in the co-crystal structures obtained with fragment F-4. A similar upward position of
Phe 890 was observed in the co-crystal structures of previously reported SOS1cat binding compounds.23,28-29
Moreover, the arene ring of F-7 and the imidazole ring of F-4 occupied the same hydrophobic area where
benzimidazole29 and quinazoline28 cores were previously found to bind. Based on the overlay of the crystal
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structures (Figure 3C) of fragments F-7 and F-4 bound to SOS1cat, we hypothesized that merging of these
two fragments may force the Phe 890 rotation and thus open the hydrophobic core of the binding pocket
under Phe 890 to enable additional optimization of F-7. Based on this hypothesis, as well as structural
information available from previous benzimidazole29 and quinazoline28 work, compound 1a was
synthesized. A measurable binding affinity of 1a (NMR Kd = 600 μM) was obtained from NMR titration
experiments (Figure S3, Supplementary information). Furthermore, the X-ray co-crystal structure of 1a
bound to SOS1cat (Figure 3D) confirmed that the aromatic ring of Phe 890 was flipped and the space
beneath was partially occupied by the phenyl ring from compound 1a.
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Figure 3. X-ray co-crystal structures of (A) fragment F-7 (magenta, PDB ID code 6V9M), (B) fragment F-4 (orange,
PDB ID code 6V94), (C) overlay of F-7 (magenta) with F-4 (orange), and (D) compound 1a (green, PDB ID code 6V9N)
bound to SOS1cat
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Aryl sulfonamide SAR at the 4-position
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On the basis of the co-crystal structure of 1a bound to SOS1cat (Figure 3D) and our earlier studies,
we initially focused on introducing a variety of lipophilic groups to the phenyl ring to occupy additional
hydrophobic space under Phe 890. The previously reported FPA binding assay29 was used to assess the
binding affinities of the analogues (Table 1). Introduction of a lipophilic substituent at the 4-position of the
phenol ring (1b) provided the first compound for which a binding affinity could be confidently obtained from
the competition FPA assay. Encouraged from this result, we investigated alternative substitution patterns.
However, 3,5-di-fluoro (1c) 3,5-di-methyl (1d), and 2,4-di-fluoro (1e) substituted analogues proved to be
inactive in the FPA assay. Alternatively, 3,4-di-fluoro (1f) and 3,4-di-chloro (1g) derivatives restored activity
and showed equal affinity. Since the aryl sulfonamide fragment (F-7) contained alkyl substituents on the
sulfonamide, we anticipated that introduction of lipophilic substituents on the N-H of the aryl sulfonamide
would boost affinity. However, alkyl substituted sulfonamides (1h-i) were not tolerated. Having explored the
biaryl sulfonamides connected by a phenolic ether, we subsequently examined a nitrogen connection. It
was found that smaller atoms, such as fluorine, at 3-, 4-, and 5-positions of the aniline ring were well
tolerated (1k-m) and provided the tightest binding analogue (1k) in this initial set of compounds. Conversely,
larger lipophilic groups, such as the 3-chloro group in compound 1n and the 3- and 5-methyl groups in
compound 1o, were not well tolerated. According to these SAR trends, compounds 1g and 1k were
identified as scaffolds for further optimization.
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Table 1. SAR of aryl sulfonamide at 4-position.
Compd
1a
X
O
R1
H
R2
H
LEb
ND
FPA Kd (M)a
>100
1b
1c
1d
1e
O
O
O
O
O
O
O
O
NH
NH
NH
NH
NH
H
H
H
H
H
H
n-Pr
n-Pr
H
H
H
H
H
4-F
86.4 ± 3.3
>100
0.32
ND
ND
3,5-di-F
3-Me, 5-Me
2,4-di-F
3,4-di-F
3-Cl, 4-F
3,4-di-F
3-Cl,4-F
4-F
3,4-diF
2,4-diF
3,5-diF
3-Cl, 4-F
>100
>100
85.3 ± 7.1
74.6 ± 4.9
>100
>100
>100
14.2 ± 2.3
69.2 ± 6.1
24.5 ± 4.0
89.4 ± 2.8
ND
1f
0.30
0.30
ND
ND
ND
0.35
0.34
0.34
0.29
1g
1h
1i
1j
1k
1l
1m
1n
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1o
NH
H
3-Me, 4-F, 5-Me
96.4 ± 5.4
0.28
5
6
7
aMeasurements are reported as the mean ± SD of two or more independent experiments, each conducted in duplicate.
bLigand efficiency index, LE =1.4 × pKd/HAC.
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Aryl sulfonamide SAR at the 3-position
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Analysis of the X-ray co-crystal structure of compound 1g revealed that the side chain carboxylate
group of Asp 887 lies in close proximity to the 3-position of the aryl sulfonamide (Figure 4A). Moreover, an
overlay of the bound structures of fragment hit F-2 and compound 1g showed that both ligands rest in the
same hydrophobic pocket and that the 2-position of the benzimidazole superimposes on the 3-position of
the aryl sulfonamide (Figure 4B). Notably, the 2-amino group of F-2 fragment achieved a water-mediated
interaction with the side chain carboxylate of Asp 887. We therefore adopted a fragment growing approach
for further optimization, anticipating that picking up an interaction with Asp 887 could be achieved from the
3-position of the aryl sulfonamide (Table 2). Engaging this residue provided a distinct affinity advantage in
our optimization work in other chemical series.29 Initial analogues evaluated the suitability of monodentate
cyclic amines (2a-b); among them, compound 2b showed five-fold better affinity compared with compound
2a. With these results in hand, we sought to introduce a monodentate linear amine (2c). While this moiety
did not show any binding affinity improvement, we were pleased to find that introduction of a linear amine
(2d) with an additional carbon in the linker showed single-digit micromolar affinity. Encouraged from this
result, we next investigated exocyclic monodentate (2e) and bidentate (2f) basic amine moieties. As might
be expected, compounds 2e-f showed comparable affinity to the linear amine (2d). In addition, we
examined the affinity of bidentate cyclic basic amine moiety (2g); this compound showed low micromolar
binding affinity. To improve affinity, we next examined substituted piperazines as well as additional ring
sizes and topologies, including 7-membered ring amines and various spirocyclic amines. However, this set
of compounds (2h-k) showed only weak affinity. Although spirocyclic compound 2l was tolerated with low
micromolar affinity, erosion of ligand efficiency made it less favorable for further optimization compared with
other analogs, such as 2d and 2g. Having explored extension of phenol-containing aryl sulfonamide
analogues, we turned our attention to extensions of aniline-containing aryl sulfonamide analogues toward
Asp 887. Surprisingly, compound 2m did not show any improvement in affinity relative to the parent
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compound 1k (Kd = 14.2 M). Moreover, the 3,4-difluorophenol containing compound 2n was roughly 2-
fold worse than the corresponding 3-fluoro,4-chlorophenol aryl sulfonamide analogue (2g). Based on the
binding affinity and ligand efficiency, we therefore selected 2g as a starting point for additional exploration.
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Figure 4. (A) X-ray co-crystal structures of 1g (cyan, PDB ID code 6V9J); (B) Overlay of 1g (cyan) with F-2 (orange,
PDB ID code 6V9F) bound to SOS1cat
.
Table 2. SAR of aryl sulfonamide at 3-position.
LEb
FPA Kd (M)a
Compd
2a
X
R
Y
O
3-Cl, 4-F
87.1 ± 6.2
0.19
2b
O
3-Cl, 4-F
13.3 ± 0.6
0.26
2c
2d
2e
O
O
O
3-Cl, 4-F
3-Cl, 4-F
3-Cl, 4-F
62.0 ± 4.4
4.51 ± 0.04
14.1 ± 1.3
0.20
0.34
0.28
2f
2g
2h
2i
O
O
O
O
3-Cl, 4-F
3-Cl, 4-F
3-Cl, 4-F
3-Cl, 4-F
8.10 ± 0.65
6.42 ± 0.95
>100
0.30
0.28
ND
>100
ND
2j
O
3-Cl, 4-F
73.4 ± 6.0
0.22
2k
2l
O
O
3-Cl, 4-F
3-Cl, 4-F
24.5 ± 4.3
0.23
0.24
3.12 ± 0.95
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2m
2n
NH
O
3-F, 4-F
3-F, 4-F
19.1 ± 1.1
10.1 ± 1.1
0.26
0.24
7
8
aMeasurements are reported as the mean ± SD of two or more independent experiments, each conducted in duplicate.
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bLigand efficiency index, LE =1.4 × pKd/HAC.
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SAR of the extension of 4-piperazine.
Inspection of the X-ray co-crystal structure (Figure 5) of 2d bound to SOS1cat confirmed the water-
mediated interaction between the ethyl amine moiety and Asp887, and also revealed regions that are
solvent-exposed, occupied by molecules of water. We hypothesized that these regions would allow
considerable tolerance for functional groups that may offer the ability to both tune the overall molecular
properties of the analogues and also improve the binding affinity. To exploit these opportunities, we
explored a wide range of chemical diversity at the piperazine nitrogen (Table 3). Expectedly, N-methyl
piperazine analogue 3a showed comparable binding affinity to the parent compound 2g. However,
introduction of additional lipophilic substituents did not improve affinity (3b-d) compared to the parent
compound 2g. Moreover, cyclic analogues (3e-f) were not tolerated, and exhibited diminished affinity.
Linear amine 3g also exhibited 2-fold less affinity than the parent compound and a longer chain (3h) was
not tolerated. Furthermore, compounds bearing cyclic ether (3i) or alcohol (3j) moieties exhibited
comparable ligand efficiency (LE) and AlogP values, but marginal improvement in the FPA assay relative
to the parent compound 2g (Kd = 6.42 M, LE = 0.28, and AlogP = 2.46).
Figure 5. X-ray co-crystal structure of 2d (salmon, PDB ID code 6V9L) bound to SOS1cat
.
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Table 3. SAR of the extended amines.
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FPA Kd (M)a
Compd
3a
R
LEb
AlogP
6.51 ± 1.65
0.27
3.2
3b
3c
3d
80.3 ± 5.5
23.0 ± 4.9
30.5 ± 2.2
0.20
0.22
0.20
3.72
3.48
3.94
3e
3f
>100
ND
3.09
2.44
79.8 ± 5.2
0.19
3g
3h
16.5 ± 0.8
63.4 ± 4.1
0.23
0.19
2.17
2.23
3i
5.69 ± 1.76
0.24
2.71
3j
3.08 ± 0.52
28.4 ± 4.0
0.27
0.21
2.45
2.86
3k
aMeasurements are reported as the mean ± SD of two or more independent experiments, each conducted in duplicate.
bLigand efficiency index, LE =1.4 × pKd/HAC.
Nucleotide Exchange Assay
We evaluated the ability of selected compounds to affect SOS1cat-mediated nucleotide exchange
on RAS (Table 4). To modulate RAS−GTP signaling and achieve potency in cancer cells, the compounds
need to elicit a marked increase in the maximum rate of nucleotide exchange. Compound efficacy was
expressed in terms of the maximal percent activation (defined as the maximum exchange rate elicited by
an individual compound at 100 μM relative to the DMSO vehicle control), normalized to the effect of the
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published compound S3 (Supplementary information).17 Compound potency was defined as the half
maximal effective concentration (EC50), calculated from the concentration−response curve (0−100 μM
compound) generated from the nucleotide exchange assay. A general agreement between nucleotide
exchange EC50 and FPA Kd was observed, and moderate to good efficacy was observed for selected
compounds relative to the control compound (S3). Compound 2d displayed the most potent exchange
activation yet observed in this series; its 1.32 µM EC50 is comparable to many of the molecules identified
from the medicinal chemistry campaigns that began with molecules from our earlier HTS.26
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Table 4. Nucleotide Exchange Results.
Rel. Act. (%)b
69.9 ± 7.1
67.0 ± 13.4
68.1 ± 2.5
89.3 ± 7.4
53.0 ± 1.4
91.6 ± 9.1
84.6 ± 9.2
FPA Kd (M)a
4.51 ± 0.04
14.1 ± 1.3
6.42 ± 0.46
3.12 ± 0.95
16.5 ± 0.8
EC50 (M)a
1.32 ± 0.15
4.32 ± 2.39
8.30 ± 0.92
3.31 ± 0.86
16.3 ± 0.4
Compd
2d
2e
2g
2l
3g
3i
3j
5.69 ± 1.76
3.08 ± 0.52
7.88 ± 2.72
4.76 ± 0.98
aMeasurements are reported as the mean ± SD of two or more independent experiments, each conducted in duplicate.
bActivation values calculated as the percentage activation for each compound at 100 μM relative to the activation of
control compound (S3) at 100 μM.
Cellular Assessment
To verify that the sulfonamide series compounds discovered from the NMR screen can elicit the
same signaling changes in cells as our prior series discovered from an HTS, we investigated the effect of
compounds 2d, 2g, and 3g in cancer cells, using western blotting for p-ERK (Figure 6). The levels of p-
ERK1/2T202/Y204 and the total ERK1/2 protein levels were measured in response to compound treatment.
Previously discovered SOS1 activator indole compound S3 (Supplementary information) was also tested
and served as a control compound.17 Importantly, all three of these compounds (2d, 2g, and 3g) elicited
the expected biphasic modulation of ERK1/2 phosphorylation at higher concentrations. Notably, the rank
order of ERK1/2 phosphorylation (2d > 2g > 3g) is consistent with the rank order in binding affinity (FPA
Kd), nucleotide exchange EC50, and in vitro efficacy (relative activation). Although the cellular activity of the
sulfonamides 2d, 2g, and 3g is weaker than the previously reported SOS1 activator indole compounds
(S3), the trend of modulation of ERK1/2 phosphorylation data is consistent and exemplifies the
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characteristic activity of our compounds that bind to this pocket on SOS1. Together, these results suggest
that the sulfonamide series compounds likely act via the same biological mechanism as previously
described, wherein compound-induced increases in the levels of RAS−GTP produce biphasic modulation
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of ERK1/2 phosphorylation via negative feedback on SOS1 by pERK1/2T202/Y204 24,27
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Figure 6. pERK1/2T202/Y204 levels from HeLa cells that were treated for 30 min with up to 200 μM of compound 2d, 2g,
3g, or S3. EGF (50 ng/mL for 5 min) was used as a positive control for pathway activation. Data are representative of
two independent experiments.
Compound Synthesis
To access various O-linked aryl sulfonamide derivatives, commercially available 4-
fluorobenzenesulfonyl chlorides 4 were treated with ammonium hydroxide or alkyl amines. Subsequent
displacement of the 4-fluoro atom with an array of phenols via an SNAr-type reaction furnished compounds
1a-i and 5-6 (Scheme 1). From there, alkylation of the 3-bromosulfonamides (5-6) via a Suzuki-Miyaura
cross-coupling reaction with alkyltrifluoroboronates and Boc removal (if required) furnished analogue 2a-e,
2g, 2n, and 3a. Subsequently, a reductive amination or N-alkylation of compounds 2g, followed by acid-
mediated removal of the N-Boc protecting group (if required) gave compounds 3d-h.
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Scheme 1. Synthesis of O-linked aryl sulfonamide derivatives.a
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aReaction conditions: (a) NH4OH, H2O, CH2Cl2, rt; or n-propylamine, Et3N, CH2Cl2, rt; (b) phenol derivatives, Cs2CO3,
DMSO, 120 °C; (c) potassium alkyl trifluoroborate, Pd(OAc)2, XPhos, Cs2CO3, THF/ H2O, 80 °C; or potassium alkyl
trifluoroborate, PdCl2(dppf).CH2Cl2, Cs2CO3, toluene/ H2O, 80 °C; (d) TFA, CH2Cl2, rt when Boc removal necessary;
(e) alkyl trifluoroborate, Pd(OAc)2, XPhos, Cs2CO3, THF/ H2O, 80 °C; (f) TFA, CH2Cl2, rt; (g) alkyl ketone or aldehyde
derivatives, NaBH(OAc)3, CH2Cl2, rt; or bromoalkylamines, K2CO3, NaI, MeCN, reflux; (h) TFA, CH2Cl2, rt when Boc
removal necessary.
To synthesize various N-linked aryl sulfonamide derivatives, commercially available 4-
fluorobenzenesulfonyl chlorides 7 were treated with ammonia, followed by Boc protection of the
sulfonamide nitrogen atom to afford intermediate 8 (Scheme 2). Subsequently, a Buchwald-Hartwig cross-
coupling reaction of intermediates 8 with anilines and Boc removal afforded compounds 1j-o and 9. Finally,
alkylation of the 3-bromosulfonamide (9) via a Suzuki-Miyaura cross-coupling reaction with
alkyltrifluoroboronates and Boc-removal (when required) furnished analogue 2m.
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Scheme 2. Synthesis of N-linked aryl sulfonamide derivatives.a
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aReaction conditions: (a) NH3, MeOH, rt; (b) Boc2O, Et3N, 4-DMAP, CH2Cl2, rt; (c) anilines, Pd(OAc)2, BINAP, Cs2CO3,
dioxane, 100 °C; (d) TFA, CH2Cl2, rt; (e) potassium alkyl trifluoroborate, Pd(OAc)2, XPhos, Cs2CO3, THF/ H2O, 100 °C;
(f) TFA, CH2Cl2, rt.
To access various O-linked aryl sulfonamide derivatives in an alternative route, commercially available 5-
(chlorosulfonyl)-2-fluorobenzoic acid 10 was treated with ammonium hydroxide and esterified with ethyl
iodide. Then an SNAr-type reaction with 3-chloro-4-fluorophenol was used to produce the intermediate 11
(Scheme 3). Subsequently, saponification of ester 11, followed by coupling with amines afforded
intermediate 12. Lastly, reduction of 12 with borane and Boc removal (when required) furnished analogues
2f, 2h-l, 3b-c, and 3i-k.
Scheme 3. Synthesis of O-linked aryl sulfonamides using alternative route.a
aReaction conditions: (a) NH4OH, H2O, EtOAc, rt; (b) methyl iodide, K2CO3, DMF, rt; (c) phenols, Cs2CO3, DMSO,
120 °C; (d) LiOH, H2O, MeOH, rt; (e) alkyl amines, HATU, N-methylmorpholine, CH2Cl2, DMF, rt; (f) BH3-THF, THF,
reflux; (g) TFA, CH2Cl2, rt when Boc removal necessary.
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■ CONCLUSION
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We have utilized a fragment screening approach, based on selectively labeled SOS1cat and 13C
HMQC spectra, to discover small-molecules that bind to SOS1cat. X-ray co-crystal structures of multiple
fragment hits confirmed that they bind to the same hydrophobic pocket of SOS1cat as previously reported
compounds, but in a distinctive manner. Beginning with a weakly binding sulfonamide fragment (F-7), we
used a structure-guided fragment merging approach to attach a phenol group to the 4-position, and a
subsequent fragment-growing approach to tether a basic amine. These strategies improved the affinity of
the compounds by > 500-fold. Compounds in the series exhibit low micromolar activation of SOS1cat-
catalyzed nucleotide exchange on RAS and also display biphasic modulations in ERK1/2 phosphorylation
at higher concentrations. These observations are in agreement with our previously proposed mechanism
in which compound-mediated agonism of SOS1 can modulate RAS signaling via negative feedback on
SOS1 by active pERK1/2T202/Y204. It is well precedented that sulfonamide-based compounds can show a
range of biological activities, including antiviral, antibacterial, antiallergic, antimalarial, and antitumor
properties.35-37 Indeed, more than 72 sulfonamide-containing small molecule drugs have been approved to
date by the FDA.38 In preliminary cellular experiments, the sulfonamide-based compounds described here
display on-target SOS1 agonist activity identical to multiple unrelated chemical series. Thus, these
compounds, which are derived from a fragment-based approach, provide an excellent starting point within
a different chemical series for the discovery and design of future modulators of RAS signaling via binding
to SOS1.
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■ EXPERIMENTAL
General Procedures. All chemical reagents and reaction solvents were purchased from commercial
suppliers and used as received. Proton nuclear magnetic resonance (1H NMR) spectra were recorded at
400 MHz on a Bruker spectrometer. For 1H NMR spectra, chemical shifts are reported in parts per million
(ppm) and are reported relative to residual non-deuterated solvent signals. Coupling constants are reported
in hertz (Hz). The following abbreviations (or a combination, thereof) are used to describe splitting patterns:
s, singlet; d, doublet; t, triplet; q, quartet; quint, quintet; septate, sept; m, multiplet; comp, overlapping
multiplets of non-magnetically equivalent protons; br, broad. All final compounds were of 95% purity or
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higher, unless otherwise noted, as measured by analytical reversed-phase HPLC. Analytical HPLC was
performed on an Agilent 1200 series system with UV detection at 214 and 254 nm, along with evaporative
light scattering detection (ELSD). Low-resolution mass spectra were obtained on an Agilent 6140 mass
spectrometer with electrospray ionization (ESI). LC-MS experiments were performed with the following
parameters: Phenomenex Kinetex 2.6 μm XB-C18 100 Å, LC column 50 x 2.1 mm; 2 min gradient, 5%–
95% MeCN in H2O, and 0.1% TFA or 0.1% formic acid. Analytical thin layer chromatography (TLC) was
performed on Kieselgel 60 F254 glass plates precoated with a 0.25 mm thickness of silica gel. TLC plates
were visualized with UV light and iodine. Silica gel chromatography was performed using a Teledyne ISCO
Combiflash® Rf system, eluting with varying concentrations of EtOAc in hexanes or MeOH in CH2Cl2.
Preparative reversed-phase HPLC was performed on a Gilson instrument equipped with a Phenomenex
Kinetex C18 column, using varying concentrations of MeCN in H2O, and 0.1% TFA. Starting materials 4, 7,
10, and phenol derivatives, aniline derivatives, potassium alkyl trifluoroborates, alkyl ketones, and alkyl
aldehydes were purchased from commercial suppliers and used as received.
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General Procedure A: Synthesis of 1a–i and 5–6. Step 1: A stirred solution 4 (10 mmol) in
dichloromethane (10 ml) at room temperature, the ammonium hydroxide (30% aqueous solution) (10 ml)
was added dropwise. The reaction mixture was stirred at room temperature for 1-2 h. The product had
formed as a white solid precipitate. The solid was collected by filtration, and washed with cold water (3 x
10 mL). The obtained crude was used for next step without further purification. Or, to a solution of n-
propylamine (0.6 mmol) in CH2Cl2 (6 ml) was added Et3N (0.8 mmol). After 10 minutes of stirring, 4-
fluorobenzene sulfonyl chloride (0.5 mmol) was added and the mixture was allowed to stir for overnight at
room temperature. The reaction mixture was quenched with water and the organic layer was washed with
HCl (1N), dried over magnesium sulfate and then concentrated in vacuo. The obtained crude was used for
next step without further purification. Step 2: To a solution of aryl phenol (0.6 mmol) and arylsulfonamide
(0.18 g, 0.5 mmol) in dimethylsulfoxide (3 mL) was added Cesium carbonate (1.2 mmol) and the reaction
mixture was stirred at 120 °C for 8 h and allowed to cool to ambient temperature. The mixture was diluted
with ethyl acetate (80 mL) and washed with water (2x10 mL) and brine (10 mL), dried over anhydrous
sodium sulfate, filtered and concentrated in vacuo. The crude solid was purified by silica gel
chromatography.
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4-phenoxybenzenesulfonamide
(1a).
The
title
compound
was
prepared
from
4-
5
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7
8
fluorobenzenesulfonamide and phenol in similar fashion to general procedure A in 31% yield over two steps.
1H NMR (400 MHz, CDCl3): δ 7.92-7.88 (m, 2H), 7.46-7.42 (m, 2H), 7.27-7.23 (m, 1H), 7.10-7.05 (m, 4H),
4.82 (s, 2H). MS (ESI) m/z = 267.1 [M+NH4]+. Purity (AUC), ≥95%.
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4-(4-fluorophenoxy)benzenesulfonamide (1b). The title compound was prepared from 4-
fluorobenzenesulfonamide and 4-fluorophenol in similar fashion to general procedure A in 41% yield over
two steps. 1H NMR (400 MHz, CDCl3): δ 7.82-7.79 (m, 2H), 7.05-6.92 (comp, 6H), 4.82 (s, 2H). MS (ESI)
m/z = 285.1 [M+NH4]+. Purity (AUC), ≥95%.
4-(3,5-difluorophenoxy)benzenesulfonamide (1c). The title compound was prepared from 4-
fluorobenzenesulfonamide and 3,5-difluorophenol in similar fashion to general procedure A in 35% yield
over two steps. 1H NMR (400 MHz, CDCl3): δ 7.90-7.86 (m, 2H), 7.08-7.05 (m, 2H), 6.61-6.55 (comp, 1H),
6.53-6.48 (comp, 2H), 4.76 (s, 2H). MS (ESI) m/z = 303.1 [M+NH4]+. Purity (AUC), ≥95%.
4-(3,5-dimethylphenoxy)benzenesulfonamide (1d). The title compound was prepared from 4-
fluorobenzenesulfonamide and 3,5-dimethylphenol in similar fashion to general procedure A in 30% yield
over two steps. 1H NMR (400 MHz, CDCl3): δ 7.80-7.78 (m, 2H), 6.97-7.95 (m, 2H), 6.78 (s, 1H), 6.61 (s,
2H), 4.70 (br, 2H), 2.25 (s, 6H). MS (ESI) m/z = 295.1 [M+NH4]+. Purity (AUC), ≥95%.
4-(2,4-difluorophenoxy)benzenesulfonamide (1e). The title compound was prepared from 4-
fluorobenzenesulfonamide and 2,4-difluorophenol in similar fashion to general procedure A in 35% yield
over two steps. 1H NMR (400 MHz, CDCl3): δ 7.83-7.79 (m, 2H), 7.11-7.06 (comp, 1H), 6.95-6.84 (comp,
4H), 4.77 (s, 2H). MS (ESI) m/z = 303.1 [M+NH4]+. Purity (AUC), ≥95%.
4-(3,4-difluorophenoxy)benzenesulfonamide (1f). The title compound was prepared from 4-
fluorobenzenesulfonamide and 3,4-difluorophenol in similar fashion to general procedure A in 38% yield
over two steps. 1H NMR (400 MHz, CDCl3): δ 7.86-7.7.82 (m, 2H), δ 7.16-7.09 (comp, 1H), 7.00-6.96 (m,
2H), 6.87-6.82 (comp, 1H), 6.76-6.72 (comp, 1H), 4.85 (s, 2H). MS (ESI) m/z = 303.1 [M+NH4]+. Purity
(AUC), ≥95%.
4-(3-chloro-4-fluorophenoxy)benzenesulfonamide (1g). The title compound was prepared from 4-
fluorobenzenesulfonamide and 4-chloro-3-fluorophenol in similar fashion to general procedure A in 31%
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yield over two steps. 1H NMR (400 MHz, CDCl3): δ 7.93-7.90 (m, 2H), 7.20-7.12 (comp, 2H), 7.06-7.03 (m,
2H), 6.97-6.94 (comp, 1H), 4.85 (s, 2H). MS (ESI) m/z = 319.1 [M+NH4]+. Purity (AUC), ≥95%.
4-(3,4-difluorophenoxy)-N-propylbenzenesulfonamide (1h). The title compound was prepared from 4-
fluoro-N-propylbenzenesulfonamide and 3,4-difluorophenol in similar fashion to general procedure A in
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38% yield over two steps. H NMR (400 MHz, CDCl3): δ 7.79-7.76 (m, 2H), 7.16-7.09 (comp, 1H), 6.99-
6.95 (m, 2H), 6.87-7.82 (comp, 1H), 6.76-6.72 (comp, 1H), 4.41 (s, 1H), 2.86 (t, J = 7.3 Hz, 2H), 1.44 (sept,
J = 7.3 Hz, 2H), 0.82 (t, J = 7.3 Hz, 3H). MS (ESI) m/z = 328.2 [M+H]+. Purity (AUC), ≥95%.
4-(3-chloro-4-fluorophenoxy)-N-propylbenzenesulfonamide (1i). The title compound was prepared
from 4-fluoro-N-propylbenzenesulfonamide and 3-chloro-4-fluorophenol in similar fashion to general
procedure A in 38% yield over two steps. 1H NMR (400 MHz, CDCl3): δ 7.85-7.84 (m, 2H), 7.23-7.15 (comp,
2H), 7.08-7.04 (m, 2H), 7.00-6.96 (comp, 1H), 4.42 (s, 1H), 2.96 (q, J = 7.3 Hz, 2H), 1.53 (sept, J = 7.3 Hz,
2H), 0.91 (t, J = 7.3 Hz, 3H). MS (ESI) m/z = 344.1 [M+H]+. Purity (AUC), ≥95%.
3-bromo-4-(3-chloro-4-fluorophenoxy)benzenesulfonamide (5). The title compound was prepared from
3-bromo-4-fluorobenzenesulfonamide and 3-chloro-4-fluorophenol in similar fashion to general procedure
A in 42% yield over two steps.
3-bromo-4-(3,4-difluorophenoxy)benzenesulfonamide (6). The title compound was prepared from 3-
bromo-4-fluorobenzenesulfonamide and 3,4-difluorophenol in similar fashion to general procedure A in 35%
yield over two steps.
General Procedure B: Synthesis of 2a–c, 2g, 2n and 3a. Step 1: A mixture of an potassium alkyl
trifluoroborate (0.75 mmol), Pd(OAc)2 (0.05 mmol), XPhos (0.15 mmol), Cs2CO3 (1.5 mmol), and 5 or 6
(0.5 mmol) under argon was charged with dry THF/ H2O (4:1, 2 mL) and heated to a temperature of 80 °C.
After 12 h, the reaction mixture was cooled to rt, diluted with sat. aqueous ammonium chloride, and
extracted with CH2Cl2 (5 mL). The organic phases were combined, dried (Na2SO4), and concentrated under
reduced pressure. The crude residue was purified by silica gel chromatography/ reversed-phase
preparative HPLC. Step 2: The compounds obtained from step 1 (0.2 mmol) was dissolved in CH2Cl2 (0.06
M) and stirred at rt. TFA (0.02 M) was added and the mixture was stirred for 2 h before being concentrated
under reduced pressure. The residue was purified by reversed-phase preparative HPLC and the pure
fractions were concentrated under a stream of warm air to afford the target compound as a TFA salt.
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4-(3-chloro-4-fluorophenoxy)-3-(piperidin-1-ylmethyl)benzenesulfonamide (2a as TFA salt). The title
compound was prepared as the corresponding TFA salt from 3-bromo-4-(3-chloro-4-
fluorophenoxy)benzenesulfonamide (5) and potassium 1-trifluoroboratomethylpiperidine in similar fashion
to general procedure B in 51% yield. 1H NMR (400 MHz, CD3OD): δ 8.15 (d, J = 2.2 Hz, 1H), 7.98 (dd, J =
2.2 Hz, J = 8.5 Hz, 1H), 7.45-7.38 (comp, 2H), 7.22-7.18 (m, 1H), 7.01 (d, J = 8.5 Hz, 1H), 4.52 (s, 2H),
3.50 (br, 2H), 3.15 (br, 2H), 1.96-1.59 (m, 6H). MS (ESI) m/z = 399.1 [M+H]+. Purity (AUC), ≥95%.
4-(3-chloro-4-fluorophenoxy)-3-(pyrrolidin-1-ylmethyl)benzenesulfonamide (2b as TFA salt). The title
compound was prepared as the corresponding TFA salt from 3-bromo-4-(3-chloro-4-
fluorophenoxy)benzenesulfonamide (5) and potassium 1-trifluoroboratomethylpyrrolidine in similar fashion
to general procedure B in 48% yield. 1H NMR (400 MHz, CD3OD): δ 8.15 (d, J = 2.2 Hz, 1H), 7.97 (dd, J =
2.2 Hz, J = 8.5 Hz, 1H), 7.46-7.37 (comp, 2H), 7.23-7.19 (comp, 1H), 7.00 (d, J = 8.5 Hz, 1H), 4.62 (s, 2H),
3.66 (br, 2H), 3.34 (br, 2H), 2.23 (br, 2H), 2.19 (br, 2H). MS (ESI) m/z = 385.1 [M+H]+. Purity (AUC), ≥95%.
4-(3-chloro-4-fluorophenoxy)-3-((dimethylamino)methyl)benzenesulfonamide (2c as TFA salt). The
title compound was prepared as the corresponding TFA salt from 3-bromo-4-(3-chloro-4-
fluorophenoxy)benzenesulfonamide (5) and potassium N,N-dimethylaminomethyltrifluoroborate in similar
fashion to general procedure B in 41% yield. 1H NMR (400 MHz, CD3OD): δ 8.13 (d, J = 2.2 Hz, 1H), 7.99
(dd, J = 2.2 Hz, J = 8.5 Hz, 1H), 7.46-7.38 (comp, 2H), 7.24-7.20 (comp, 1H), 7.00 (d, J = 8.5 Hz, 1H), 4.57
(s, 2H), 3.53 (s, 6H). MS (ESI) m/z = 359.1 [M+H]+. Purity (AUC), ≥95%.
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4-(3-chloro-4-fluorophenoxy)-3-(piperidin-1-ylmethyl)benzenesulfonamide (2g as TFA salt). The title
compound was prepared as the corresponding TFA salt from 3-bromo-4-(3-chloro-4-
fluorophenoxy)benzenesulfonamide (5) and potassium 1-trifluoroboratomethyl-4-(N-Boc) piperazine in
similar fashion to general procedure B in 26% yield over two steps. 1H NMR (400 MHz, CD3OD): δ 8.06 (d,
J = 2.3 Hz, 1H), 7.86 (dd, J = 2.3 Hz, J = 8.7 Hz, 1H), 7.34-7.30 (comp, 1H), 7.23-7.21 (comp, 1H), 7.05-
7.01 (m, 2H), 3.26-3.23 (m, 4H), 2.85-2.83 (br, 4H), 3.15 (br, 2H). MS (ESI) m/z = 399.9 [M+H]+. Purity
(AUC), ≥95%.
4-(3-chloro-4-fluorophenoxy)-3-((4-methylpiperazin-1-yl)methyl)benzenesulfonamide (3a as TFA
salt). The title compound was prepared as the corresponding TFA salt from 3-bromo-4-(3-chloro-4-
fluorophenoxy)benzenesulfonamide (5) and potassium 1-trifluoroboratomethyl-4-(N-Boc) piperazine in
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similar fashion to general procedure B in 32% yield. H NMR (400 MHz, CD3OD): δ 8.06 (d, J = 2.4 Hz,
5
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7
8
1H), 7.85 (dd, J = 2.4 Hz, J = 8.7 Hz, 1H), 7.35-7.30 (comp, 1H), 7.23-7.20 (comp, 1H), 7.05-7.00 (m, 2H),
3.79 (s, 2H), 3.50 (br, 2H), 3.15 (br, 2H), 3.12 (br, 2H), 2.90 (s, 3H), 2.52 (br, 2H). MS (ESI) m/z = 414.1
[M+H]+. Purity (AUC), ≥95%.
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4-(3,4-difluorophenoxy)-3-(piperazin-1-ylmethyl)benzenesulfonamide (2n as TFA salt). The title
compound
was
prepared
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the
corresponding
TFA
salt
from
3-bromo-4-(3,4-
difluorophenoxy)benzenesulfonamide (6) and potassium 1-methyl-4-trifluoroboratomethylpiperidine in
similar fashion to general procedure B in 32% yield over two steps. 1H NMR (400 MHz, CD3OD): δ 8.07 (d,
J = 2.4 Hz, 1H), 7.85 (dd, J = 2.4 Hz, J = 8.6 Hz, 1H), 7.37-7.30 (comp, 1H), 7.07-7.02 (m, 2H), 6.87-6.84
(comp, 1H), 3.78 (s, 2H), 3.24-3.21 m, 4H), 2.79-2.77 (m, 4H). MS (ESI) m/z = 384.1 [M+H]+. Purity (AUC),
≥95%.
General Procedure C: Synthesis of 2d–e. Step 1: A mixture of an potassium alkyl trifluoroborate (0.75
mmol), Pd(OAc)2 (0.05 mmol), XPhos (0.15 mmol), Cs2CO3 (1.5 mmol), and 5 (0.5 mmol) under argon was
charged with dry Toluene/ H2O (8:1, 2 mL) and heated to a temperature of 80 °C. After 12 h, the reaction
mixture was cooled to rt, diluted with sat. aqueous ammonium chloride, and extracted with CH2Cl2 (5 mL).
The organic phases were combined, dried (Na2SO4), and concentrated under reduced pressure. The crude
residue was purified by silica gel chromatography. Step 2: The compounds obtained from step 1 (0.2 mmol)
was dissolved in CH2Cl2 (0.06 M) and stirred at rt. TFA (0.02 M) was added and the mixture was stirred for
2 h before being concentrated under reduced pressure. The residue was purified by reversed-phase
preparative HPLC and the pure fractions were concentrated under a stream of warm air to afford the target
compound as a TFA salt.
3-(2-aminoethyl)-4-(3-chloro-4-fluorophenoxy)benzenesulfonamide (2d as TFA salt). The title
compound was prepared as the corresponding TFA salt from 3-bromo-4-(3-chloro-4-
fluorophenoxy)benzenesulfonamide (5) and potassium t-butyl N-[2-(trifluoroboranuidyl)ethyl] carbamate in
similar fashion to general procedure C in 21% yield over two steps. 1H NMR (400 MHz, CD3OD): δ 7.93 (d,
J = 2.3 Hz, 1H), 7.83 (dd, J = 2.2 Hz, J = 8.4 Hz, 1H), 7.37-7.30 (comp, 2H), 7.12-7.08 (comp, 1H), 6.96-
6.94 (d, J = 8.4 Hz, 1H), 3.28 (t, J = 6.3 Hz, 2H), 3.16 (t, J = 6.3 Hz, 2H). MS (ESI) m/z = 345.1 [M+H]+.
Purity (AUC), ≥95%.
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3-(azetidin-3-ylmethyl)-4-(3-chloro-4-fluorophenoxy)benzenesulfonamide (2e as TFA salt). The title
compound was prepared as the corresponding TFA salt from 3-bromo-4-(3-chloro-4-
fluorophenoxy)benzenesulfonamide (5) and potassium (1-Boc-azetidin-3-yl)methylfluoroborate in similar
fashion to general procedure C in 24% yield over two steps. 1H NMR (400 MHz, CD3OD): δ 7.85 (d, J = 2.4
Hz, 1H), 7.79 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.37-7.32 (comp, 1H), 7.29-7.26 (comp, 1H), 7.09-7.05
(comp, 1H), 6.93 (d, J = 8.8 Hz, 1H), 4.18-4.13 (m, 2H), 3.99-3.95 (m, 2H), 3.40-3.34 (m, 1H), 3.15 (d, J =
7.8 Hz, 2H). MS (ESI) m/z = 371.1 [M+H]+. Purity (AUC), ≥95%.
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General Procedure D: Synthesis of 3d–f. Step 1: A mixture of 2g (0.2 mmol), and alkyl aldehydes or
ketones (0.4 mmol) in CH2Cl2 (3 mL) was stirred for 1h, then NaBH(OAc)3 (0.5 mmol) was added. The
resulting mixture was stirred for 2h, then was poured into 2 N aqueous KOH solution (15 mL) and extracted
with EtOAc. The organic phases were combined, dried (Na2SO4), and concentrated under reduced
pressure. The residue was purified by reversed-phase preparative HPLC and the pure fractions were
concentrated under a stream of warm air to afford the target compound as a TFA salt. Step 2: The
compound obtained from step 1 (0.1 mmol) was dissolved in CH2Cl2 (0.06 M) and stirred at rt. TFA (0.02
M) was added and the mixture was stirred for 2 h before being concentrated under reduced pressure. The
residue was purified by reversed-phase preparative HPLC and the pure fractions were concentrated under
a stream of warm air to afford the target compound as a TFA salt.
4-(3-chloro-4-fluorophenoxy)-3-((4-cyclopentylpiperazin-1-yl)methyl)benzenesulfonamide (3d as
TFA salt). The title compound was prepared as the corresponding TFA salt from 4-(3-chloro-4-
fluorophenoxy)-3-(piperazin-1-ylmethyl)benzenesulfonamide (2g) and cyclopentanone in similar fashion to
general procedure D in 26% yield. 1H NMR (400 MHz, CD3OD): δ 8.06 (d, J = 2.4 Hz, 1H), 7.85 (dd, J = 2.4
Hz, J = 8.6 Hz, 1H), 7.35-7.30 (comp, 1H), 7.23-7.20 (comp, 1H), 7.05-7.00 (m, 2H), 3.81 (s, 2H), 3.56-3.52
(m, 3H), 3.16-3.12 (m, 4H), 2.60-2.57 (m, 2H), 2.18-2.15 (m, 2H), 1.85-1.84 (m, 2H), 1.74-1.71 (m, 4H). MS
(ESI) m/z = 468.1 [M+H]+. Purity (AUC), ≥95%.
4-(3-chloro-4-fluorophenoxy)-3-((4-(pyrrolidin-2-ylmethyl)piperazin-1-yl)methyl)
benzenesulfonamide (3e as TFA salt). The title compound was prepared as the corresponding TFA salt
from 4-(3-chloro-4-fluorophenoxy)-3-(piperazin-1-ylmethyl)benzenesulfonamide (2g) and tert-butyl 2-
1
formylpyrrolidine-1-carboxylate in similar fashion to general procedure D in 31% yield over two steps. H
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NMR (400 MHz, CD3OD): δ 8.16 (d, J = 2.3 Hz, 1H), 7.98 (dd, J = 2.3 Hz, J = 8.8 Hz, 1H), 7.44-7.38 (comp,
2H), 7.22-7.18 (comp, 1H), 7.01 (d, J = 8.8 Hz, 1H), 4.58 (s, 2H), 3.86-3.80 (m, 2H), 3.57-3.52 (m, 2H),
3.39-3.35 (m, 2H), 3.31-3.27 (m, 2H), 2.74-2.68 (m, 5H), 2.22-2.05 (m, 4H). (ESI) m/z = 483.2 [M+H]+.
Purity (AUC), ≥95%.
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3-((4-(azetidin-3-yl)piperazin-1-yl)methyl)-4-(3-chloro-4-fluorophenoxy)benzenesulfonamide (3f as
TFA salt). The title compound was prepared as the corresponding TFA salt from 4-(3-chloro-4-
fluorophenoxy)-3-(piperazin-1-ylmethyl)benzenesulfonamide (2g) and tert-butyl 3-oxoazetidine-1-
1
carboxylate in similar fashion to general procedure D in 15% yield over two steps. H NMR (400 MHz,
CD3OD): δ 8.16 (d, J = 2.3 Hz, 1H), 7.98 (dd, J = 2.3 Hz, J = 8.7 Hz, 1H), 7.44-7.37 (comp, 2H), 7.22-7.18
(comp, 1H), 7.05-7.00 (d, J = 8.7 Hz, 1H), 4.56 (s, 2H), 4.17-4.12 (m, 2H), 4.05-4.00 (m, 2H), 3.56-3.37 (m,
5H), 2.85-2.71 (m, 2H), 2.50-2.39 (m, 2H). MS (ESI) m/z = 455.2 [M+H]+. Purity (AUC), ≥95%.
General Procedure E: Synthesis of 3g–h. Step 1: A mixture of 2g (0.2 mmol), alkyl bromide (75 mg, 0.4
mmol), KI (0.4 mmol), and K2CO3 (0.4 mmol), in MeCN (3 mL) was stirred and heated to 80 °C for 15 h.
The reaction mixture was cooled to rt, diluted with sat. aqueous ammonium chloride, and extracted with
CH2Cl2 (5 mL). The organic phases were combined, dried (Na2SO4), and concentrated under reduced
pressure. The crude residue was purified by silica gel chromatography. Step 2: The compound obtained
from step 1 (0.1 mmol) was dissolved in CH2Cl2 (0.06 M) and stirred at rt. TFA (0.02 M) was added and the
mixture was stirred for 2 h before being concentrated under reduced pressure. The residue was purified by
reversed-phase preparative HPLC and the pure fractions were concentrated under a stream of warm air to
afford the target compound as a TFA salt.
3-((4-(2-aminoethyl)piperazin-1-yl)methyl)-4-(3-chloro-4-fluorophenoxy)benzenesulfonamide (3g as
TFA salt). The title compound was prepared as the corresponding TFA salt from 4-(3-chloro-4-
fluorophenoxy)-3-(piperazin-1-ylmethyl)benzenesulfonamide (2g) and tert-butyl (2-bromoethyl)carbamate
in similar fashion to general procedure E in 41% yield over two steps. 1H NMR (400 MHz, CD3OD): δ 8.05
(d, J = 2.3 Hz, 1H), 7.86 (dd, J = 2.3 Hz, J = 8.8 Hz, 1H), 7.33-7.26 (comp, 2H), 7.10-7.06 (comp, 1H), 6.99
(d, J = 8.8 Hz, 1H), 4.45 (s, 2H), 3.49-3.14 (m, 8H), 2.99 (t, J = 5.6 Hz, 2H), 2.63 (t, J = 5.6 Hz, 2H). MS
(ESI) m/z = 443.1 [M+H]+. Purity (AUC), ≥95%.
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3-((4-(3-aminopropyl)piperazin-1-yl)methyl)-4-(3-chloro-4-fluorophenoxy)benzenesulfonamide (3h
as TFA salt). The title compound was prepared as the corresponding TFA salt from 4-(3-chloro-4-
fluorophenoxy)-3-(piperazin-1-ylmethyl)benzenesulfonamide (2g) and tert-butyl (3-bromopropyl)carbamate
in similar fashion to general procedure E in 38% yield over two steps. 1H NMR (400 MHz, CD3OD): δ 8.10
(d, J = 2.3 Hz, 1H), 7.91 (dd, J = 2.3 Hz, J = 8.8 Hz, 1H), 7.37-7.7.32 (comp, 2H), 7.22-7.20 (comp, 1H),
7.02 (d, J = 8.8 Hz, 1H), 4.08 (s, 2H), 3.23 (t, J = 5.6 Hz, 2H), 3.15-3.00 (m, 8H), 2.82-2.79 (m, 2H), 2.07-
1.98 (m, 2H). MS (ESI) m/z = 457.0 [M+H]+. Purity (AUC), ≥95%.
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General Procedure F: Synthesis of 8. Step 1: A stirred solution 7 (8 mmol) in methanol (10 ml) at room
temperature, the methanol/ammonia solution (10 ml, excess) was added dropwise. The reaction mixture
was stirred at room temperature for 10 h. The reaction mixture was concentrated under reduced vacuum
to yield intermediate arylsulfonamide as a white solid. This material was used without further purification.
Step 2: A mixture of aryllsulfonamide (6 mmol) and di-tert-butyl dicarbonate (6.9 mmol) in CH2Cl2 (40 mL)
under nitrogen was treated with triethylamine (7.2 mmol) and DMAP (0.6 mmol) and stirred at room
temperature for 12 h. The crude residue was purified by silica gel chromatography.
General Procedure G: Synthesis of 1j-o and 9. Step 1: A mixture of a aniline (0.5 mmol), Pd(OAc)2 (0.05
mmol), BINAP (0.1 mmol), Cs2CO3 (1.5 mmol), and 8 (0.5 mmol) under argon was charged with degassed
doixane (2 mL) and heated to a temperature of 100 °C. After 12 h, the reaction mixture was cooled to rt,
diluted with sat. aqueous ammonium chloride, and extracted with CH2Cl2 (10 mL). The organic phases were
combined, dried (Na2SO4), and concentrated under reduced pressure. The crude residue was purified by
silica gel chromatography. Step 2: The compound obtained from step 1 (0.2 mmol) was dissolved in CH2Cl2
(0.06 M) and stirred at rt. TFA (0.02 M) was added and the mixture was stirred for 2 h before being
concentrated under reduced pressure. The residue was purified by silica gel chromatography.
4-((4-fluorophenyl)amino)benzenesulfonamide (1j). The title compound was prepared from tert-butyl
((4-bromophenyl)sulfonyl)carbamate (obtained from general procedure F) and 4-fluoroaniline in similar
fashion to general procedure G in 42% yield over two steps. 1H NMR (400 MHz, CDCl3): δ 7.70-7.67 (m,
2H), 7.10-7.07 (comp, 2H), 7.02-6.98 (comp, 2H), 6.85-6.82 (m, 2H), 5.86 (s, 1H), 4.59 (s, 2H). MS (ESI)
m/z = 267.1 [M+H]+. Purity (AUC), ≥95%.
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4-((3,4-difluorophenyl)amino)benzenesulfonamide (1k). The title compound was prepared from tert-
butyl ((4-bromophenyl)sulfonyl)carbamate (obtained from general procedure F) and 3,4-difluoroaniline in
similar fashion to general procedure G in 45% yield over two steps. 1H NMR (400 MHz, CD3OD): δ 7.75 (d,
J = 8.5 Hz, 2H), 7.24-7.17 (comp, 1H), 7.11-7.04 (m, 3H), 6.98-6.95 (comp, 1H). MS (ESI) m/z = 285.1
[M+H]+. Purity (AUC), ≥95%.
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4-((2,4-difluorophenyl)amino)benzenesulfonamide (1l). The title compound was prepared from tert-butyl
((4-bromophenyl)sulfonyl)carbamate (obtained from general procedure F) in similar fashion to general
1
procedure G in 38% yield over two steps. H NMR (400 MHz, CD3OD): δ 7.71 (d, J = 8.5 Hz, 2H), 7.41-
7.35 (comp, 1H), 7.10-7.04 (comp, 1H), 7.00-6.96 (comp, 1H), 6.90 (d, J = 8.5 Hz, 2H). MS (ESI) m/z =
285.1 [M+H]+. Purity (AUC), ≥95%.
4-((3,5-difluorophenyl)amino)benzenesulfonamide (1m). The title compound was prepared from tert-
butyl ((4-bromophenyl)sulfonyl)carbamate (obtained from general procedure F) and 3,5-difluoroaniline in
similar fashion to general procedure G in 31% yield over two steps. 1H NMR (400 MHz, CDCl3): δ 7.72 (m,
2H), 7.09-7.04 (comp, 1H), 6.97-6.93 (m, 3H), 6.82-6.80 (m, 1H), 5.89 (s, 1H), 4.62 (s, 2H). MS (ESI) m/z
= 285.1 [M+H]+. Purity (AUC), ≥95%.
4-((3-chloro-4-fluorophenyl)amino)benzenesulfonamide (1n). The title compound was prepared from
tert-butyl ((4-bromophenyl)sulfonyl)carbamate (obtained from general procedure F) and 4-chloro-3-
1
fluoroaniline in similar fashion to general procedure G in 38% yield over two steps. H NMR (400 MHz,
CD3OD): δ 7.64 (d, J = 8.6 Hz, 2H), 7.15-7.13 (comp, 1H), 7.10-7.01 (comp, 2H), 6.96 (d, J = 8.6 Hz, 2H).
MS (ESI) m/z = 302.2 [M+H]+. Purity (AUC), ≥95%.
4-((4-fluoro-3,5-dimethylphenyl)amino)benzenesulfonamide (1o). The title compound was prepared
from tert-butyl ((4-bromophenyl)sulfonyl)carbamate (obtained from general procedure F) and 4-fluoro-3,5-
dimethylaniline in similar fashion to general procedure G in 49% yield over two steps. 1H NMR (400 MHz,
CDCl3): δ 7.67 (d, J = 8.8 Hz, 2H), 6.81 (d, J = 8.8 Hz, 2H), 6.77-6.75 (comp, 2H), 5.76 (s, 1H), 4.56 (s,
2H), 2.19 (s, 6H). MS (ESI) m/z = 295.2 [M+H]+. Purity (AUC), ≥95%.
3-chloro-4-((3,4-difluorophenyl)amino)benzenesulfonamide (9). The title compound was prepared from
tert-butyl ((3-chloro-4-fluorophenyl)sulfonyl)carbamate (obtained from general procedure F) and 3,4-
difluoroaniline in similar fashion to general procedure G in 49% yield over two steps.
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Synthesis of 3-chloro-4-((3,4-difluorophenyl)amino)benzenesulfonamide (2m as TFA salt). Step 1: A
mixture of an potassium alkyl trifluoroborate (0.3 mmol), Pd(OAc)2 (0.02 mmol), XPhos (0.03 mmol),
Cs2CO3 (0.6 mmol), and 9 (0.2 mmol) under argon was charged with dry THF/ H2O (4:1, 1 mL) and heated
to a temperature of 100 °C. After 12 h, the reaction mixture was cooled to rt, diluted with sat. aqueous
ammonium chloride, and extracted with CH2Cl2 (3 mL). The organic phases were combined, dried
(Na2SO4), and concentrated under reduced pressure. The crude residue was purified by silica gel
chromatography. Step 2: The compounds obtained from step 1 (0.1 mmol) was dissolved in CH2Cl2 (0.06
M) and stirred at rt. TFA (0.02 M) was added and the mixture was stirred for 2 h before being concentrated
under reduced pressure. The residue was purified by reversed-phase preparative HPLC and the pure
fractions were concentrated under a stream of warm air to afford the target compound as a TFA salt with
30% yield over two steps. 1H NMR (400 MHz, CD3OD): δ 7.62-7.60 (m, 2H), 7.15-7.09 (comp, 2H), 7.04-
6.99 (m, 1H), 6.89-6.85 (comp, 1H), 3.63 (s, 2H), 3.22-3.19 (m, 4H), 2.63 (br, 4H). MS (ESI) m/z = 383.2
[M+H]+. Purity (AUC), ≥95%.
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Synthesis of methyl 2-fluoro-5-sulfamoylbenzoate (11). Step 1: To a solution of 5-(chlorosulfonyl)-2-
fluorobenzoic acid (16.76 mmol) in CH2Cl2 (20 mL), the ammonium hydroxide (30% aqueous solution) (20
mL) was added dropwise at 0 °C and the mixture was stirred overnight at room temperature. After
consumption of the starting material, the reaction mixture was concentrated to half volume with a rotary
evaporator and acidified with 6 M HCl to ~pH 5. The resulting solution was left at room temperature until
the product precipitated. The precipitate was filtered, washed with water, and dried under vacuum to give
2-fluoro-5-sulfamoylbenzoic acid (yield 52%) as a white solid. Step 2: 2-fluoro-5-sulfamoylbenzoic acid
(8.67 mmol) in anhydrous DMF (15 mL) were added to iodomethane (9 mmol) and potassium carbonate (5
mmol). The reaction mixture was stirred overnight at room temperature. The reaction was quenched with
water, extracted with ethyl acetate (50 mL), dried (Na2SO4), and concentrated under reduced pressure. The
crude residue was purified by silica gel chromatography to afford methyl 2-fluoro-5-sulfamoylbenzoate
(yield 56%) as a white solid.
Synthesis of ethyl 2-(3-chloro-4-fluorophenoxy)-5-sulfamoylbenzoate (12). Step 1: To a solution of 3-
chloro-4-fluorophenol (5.82 mmol), and 11 (4.85 mmol) in dimethylsulfoxide (20 mL) was added Cesium
carbonate (14.55 mmol) and the reaction mixture was stirred at 120 °C for 12 h and allowed to cool to
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ambient temperature. The mixture was diluted with ethyl acetate (30 mL) and washed with water (2x10 mL)
and brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude solid
was purified by silica gel chromatography to afford methyl 2-(3-chloro-4-fluorophenoxy)-5-
sulfamoylbenzoate (yield 61%) as a white solid. Step 2: To a solution of 2 methyl 2-(3-chloro-4-
fluorophenoxy)-5-sulfamoylbenzoate (2.94 mmol) in 10% H2O in methanol was added to lithium hydroxide
(81.6 mmol). The reaction mixture was stirred for 4 h at room temperature. The reaction was quenched with
water, extracted with ethyl acetate (20 mL), dried (Na2SO4), and concentrated under reduced pressure. The
crude residue was purified by silica gel chromatography to afford 2-(3-chloro-4-fluorophenoxy)-5-
sulfamoylbenzoic acid (yield 78 %) as a white solid. 1H NMR (400 MHz, CD3OD): δ 8.35 (d, J = 2.5 Hz, 1H),
7.92 (dd, J = 2.5 Hz, J = 8.7 Hz, 1H), 7.21-7.17 (comp, 1H), 7.11-7.09 (comp, 1H), 7.01 (d, J = 8.7 Hz, 1H),
6.93-6.89 (comp, 1H), 4.10 (s, 2H) 3.38-3.33 (m, 4H), 3.23-3.21 (m, 2H), 3.08-3.05 (m, 2H), 2.12-2.10 (m,
2H). MS (ESI) m/z = 346.1 [M+H]+.
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General Procedure H: Synthesis of 2f, 2h–l, 3b-c, and 3i-k. Step 1: To a mixture of 2-(3-chloro-4-
fluorophenoxy)-5-sulfamoylbenzoic acid (0.2 mmol) and HATU (0.3 mmol) in 3:1 anhydrous DCM: DMF (2
ml) was added to the corresponding alkyl amines (0.25 mmol) and 4-methylmorpholine (0.75 mmol). The
reaction mixture was stirred for 18 h at room temperature. The reaction was diluted with water, extracted
with ethyl acetate (3 mL), dried (Na2SO4), and concentrated under reduced pressure. The crude residue
was purified by silica gel chromatography. Step 2: The compound obtained from step 1 (0.1 mmol) was
dissolved in THF (0.05 M), was treated with 1 M BH3.THF complex (0.4 mmol) at room temperature, and
was refluxed at 100°C for 12 hours. The mixture was then cooled to room temperature, was treated with 6
M HCl solution (1 ml), and was refluxed at 100°C for 30 minutes. The mixture was cooled to room
temperature, diluted with water and basified by addition of K2CO3 to pH 9. The mixture was extracted with
ethyl acetate (2 mL), dried (Na2SO4), and concentrated under reduced pressure. The residue was purified
by reversed-phase preparative HPLC and the pure fractions were concentrated under a stream of warm air
to afford the target compound as a TFA salt. Step 3: The compound obtained from step 2 (0.05 mmol) was
dissolved in CH2Cl2 (0.06 M) and stirred at rt. TFA (0.02 M) was added and the mixture was stirred for 2 h
before being concentrated under reduced pressure. The residue was purified by reversed-phase
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preparative HPLC and the pure fractions were concentrated under a stream of warm air to afford the target
compound as a TFA salt.
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3-((3-aminoazetidin-1-yl)methyl)-4-(3-chloro-4-fluorophenoxy)benzenesulfonamide (2f as TFA salt).
The title compound was prepared as the corresponding TFA salt from 2-(3-chloro-4-fluorophenoxy)-5-
sulfamoylbenzoic acid and tert-butyl azetidin-3-ylcarbamate in similar fashion to general procedure H in
19% yield over three steps.1H NMR (400 MHz, CD3OD): δ 8.16 (d, J = 2.3 Hz, 1H), 7.97 (dd, J = 2.3 Hz, J
= 8.8 Hz, 1H), 7.44-7.37 (comp, 2H), 7.21-7.17 (comp, 1H), 7.00 (d, J = 8.8 Hz, 1H), 4.54 (s, 2H), 3.49-3.43
(m, 3H), 3.35-3.20 (m, 2H). MS (ESI) m/z = 386.1 [M+H]+. Purity (AUC), ≥95%.
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(S)-4-(3-chloro-4-fluorophenoxy)-3-((2-methylpiperazin-1-yl)methyl)benzenesulfonamide (2h as TFA
salt). The title compound was prepared as the corresponding TFA salt from 2-(3-chloro-4-fluorophenoxy)-
5-sulfamoylbenzoic acid and tert-butyl (S)-3-methylpiperazine-1-carboxylate in similar fashion to general
procedure H in 21% yield over three steps.1H NMR (400 MHz, CD3OD): δ 7.97 (d, J = 2.3 Hz, 1H), 7.73
(dd, J = 2.3 Hz, J = 8.7 Hz, 1H), 7.23-7.19 (comp, 1H), 7.12-7.10 (comp, 1H), 4.21-4.17 (m, 1H), 3.53-3.49
(m, 1H), 3.05-2.98 (m, 4H), 2.85-2.76 (m, 4H), 1.19-1.18 (m, 3H). MS (ESI) m/z = 414.0 [M+H]+. Purity
(AUC), ≥95%.
3-((1,4-diazepan-1-yl)methyl)-4-(3-chloro-4-fluorophenoxy)benzenesulfonamide (2i as TFA salt). The
title compound was prepared as the corresponding TFA salt from 2-(3-chloro-4-fluorophenoxy)-5-
sulfamoylbenzoic acid and tert-butyl 1,4-diazepane-1-carboxylate in similar fashion to general procedure H
in 31% yield over three steps. 1H NMR (400 MHz, CD3OD): δ 8.13 (d, J = 2.3 Hz, 1H), 7.88 (dd, J = 2.3 Hz,
J = 8.7 Hz, 1H), 7.36-7.32 (comp, 1H), 7.27-7.26 (comp, 1H), 7.09-7.06 (comp, 1H), 7.07 (d, J = 8.7 Hz,
1H), 4.10 (s, 2H) 3.38-3.33 (m, 4H), 3.23-3.21 (m, 2H), 3.08-3.05 (m, 2H), 2.12-2.10 (m, 2H). MS (ESI) m/z
= 414.0 [M+H]+. Purity (AUC), ≥95%.
3-((2,7-diazaspiro[3.5]nonan-7-yl)methyl)-4-(3-chloro-4-fluorophenoxy)benzenesulfonamide (2j as
TFA salt). The title compound was prepared as the corresponding TFA salt from 2-(3-chloro-4-
fluorophenoxy)-5-sulfamoylbenzoic acid and tert-butyl 2,7-diazaspiro[3.5]nonane-2-carboxylate in similar
fashion to general procedure H in 21% yield over three steps. 1H NMR (400 MHz, CD3OD): δ 8.14 (d, J =
2.4 Hz, 1H), 7.97 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.43-7.41 (comp, 1H), 7.38-7.36 (comp, 1H), 7.20-7.16
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(comp, 1H), 7.01 (d, J = 8.8 Hz, 1H), 4.45 (s, 2H) 3.99-3.36 (m, 4H), 3.22-3.18 (m, 4H), 2.23-2.18 (m, 4H).
MS (ESI) m/z = 439.9 [M+H]+. Purity (AUC), ≥95%.
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4-(3-chloro-4-fluorophenoxy)-3-((hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)methyl)
benzenesulfonamide (2k as TFA salt). The title compound was prepared as the corresponding TFA salt
from 2-(3-chloro-4-fluorophenoxy)-5-sulfamoylbenzoic acid and tert-butyl hexahydropyrrolo[3,4-c]pyrrole-
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2(1H)-carboxylate in similar fashion to general procedure H in 25% yield over three steps. H NMR (400
MHz, CD3OD): δ 8.13 (d, J = 2.3 Hz, 1H), 7.92 (dd, J = 2.3 Hz, J = 8.7 Hz, 1H), 7.39-7.35 (m, 2H), 7.14-
7.12 (comp, 1H), 7.00 (d, J = 8.8 Hz, 1H), 4.34 (s, 2H) 3.54-3.51 (m, 2H), 3.36-3.3.33 (m, 2H), 2.29-2.18
(m, 6H). MS (ESI) m/z = 426.0 [M+H]+. Purity (AUC), ≥95%.
3-((2,8-diazaspiro[4.5]decan-2-yl)methyl)-4-(3-chloro-4-fluorophenoxy)benzenesulfonami (2l as TFA
salt). The title compound was prepared as the corresponding TFA salt from 2-(3-chloro-4-fluorophenoxy)-
5-sulfamoylbenzoic acid and tert-butyl 1,7-diazaspiro[3.5]nonane-7-carboxylate in similar fashion to general
procedure H in 23% yield over three steps. 1H NMR (400 MHz, CD3OD): δ 8.06 (d, J = 2.3 Hz, 1H), 7.5 (dd,
J = 2.3 Hz, J = 8.8 Hz, 1H), 7.34-7.30 (comp, 1H), 7.22-7.19 (comp, 1H), 7.04-7.02 (m, 2H), 3.81 (s, 2H),
3.40-3.37 (m, 3H), 3.16-3.10 (m, 4H), 2.53 (t, J = 11.2 Hz, 2H), 2.31 (t, J = 11.2 Hz, 2H), 1.32-1.30 (m,
3H). MS (ESI) m/z = 453.9 [M+H]+. Purity (AUC), ≥95%.
4-(3-chloro-4-fluorophenoxy)-3-((4-isopropylpiperazin-1-yl)methyl)benzenesulfonamide (3b as TFA
salt). The title compound was prepared as the corresponding TFA salt from 2-(3-chloro-4-fluorophenoxy)-
5-sulfamoylbenzoic acid and 1-isopropylpiperazine in similar fashion to general procedure H in 38% yield
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over two steps. H NMR (400 MHz, CD3OD): δ 8.05 (d, J = 2.3 Hz, 1H), 7.86 (dd, J = 2.3 Hz, J = 8.7 Hz,
1H), 7.34-7.30 (comp, 1H), 7.21-7.19 (comp, 1H), 7.05-7.01 (m, 2H), 3.81 (s, 2H), 3.55-3.44 (m, 5H), 3.15-
3.13 (m, 4H), 1.37 (d, J = 6.7 Hz, 6H). MS (ESI) m/z = 441.9 [M+H]+. Purity (AUC), ≥95%.
4-(3-chloro-4-fluorophenoxy)-3-((4-cyclopropylpiperazin-1-yl)methyl)benzenesulfonamide (3c as
TFA salt). The title compound was prepared as the corresponding TFA salt from 2-(3-chloro-4-
fluorophenoxy)-5-sulfamoylbenzoic acid and 1-cyclopropylpiperazine in similar fashion to general
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procedure H in 31% yield over two steps. H NMR (400 MHz, CD3OD): δ 8.10 (d, J = 2.4 Hz, 1H), 7.90 (dd,
J = 2.4 Hz, J = 8.6 Hz, 1H), 7.37-7.29 (comp, 2H), 7.12-7.09 (comp, 1H), 7.01 (d, J = 8.6 Hz, 1H), 4.10 (s,
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