Highly Potent Non-Carboxylic Acid Autotaxin Inhibitors Reduce Melanoma Metastasis and Chemotherapeutic Resistance of Breast Cancer Stem Cells

Article abstract of DOI:10.1021/acs.jmedchem.6b01270

Autotaxin (ATX, aka. ENPP2) is the main source of the lipid mediator lysophosphatidic acid (LPA) in biological fluids. This study reports on inhibitors of ATX derived by lead optimization of the benzene-sulfonamide in silico hit compound 3. The new analogues provide a comprehensive structure-activity relationship of the benzene-sulfonamide scaffold that yielded a series of highly potent ATX inhibitors. The three most potent analogues (3a, IC₅₀ ～ 32 nM; 3b, IC₅₀ ～ 9 nM; and 14, IC₅₀ ～ 35 nM) inhibit ATX-dependent invasion of A2058 human melanoma cells in vitro. Two of the most potent compounds, 3b and 3f (IC₅₀ ～ 84 nM), lack inhibitory action on ENPP6 and ENPP7 but possess weak antagonist action specific to the LPA₁ G protein-coupled receptor. In particular, compound 3b potently reduced in vitro chemotherapeutic resistance of 4T1 breast cancer stem-like cells to paclitaxel and significantly reduced B16 melanoma metastasis in vivo.

Full text of DOI:10.1021/acs.jmedchem.6b01270

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Article
Highly Potent Non-Carboxylic Acid Autotaxin Inhibitors Reduce Melanoma
Metastasis and Chemotherapeutic Resistance of Breast Cancer Stem Cells
Souvik Banerjee, Derek D. Norman, Sue Chin Lee, Abby L Parrill, Truc
Chi T. Pham, Daniel L. Baker, Gabor G. Tigyi, and Duane D Miller
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01270 • Publication Date (Web): 23 Jan 2017
Downloaded from http://pubs.acs.org on January 24, 2017
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Highly Potent Non-Carboxylic Acid Autotaxin Inhibitors Reduce Melanoma
Metastasis and Chemotherapeutic Resistance of Breast Cancer Stem Cells
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Souvik Banerjee, Derek D. Norman, Sue Chin Lee, Abby L. Parrill, Truc Chi T.
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Pham , Daniel L. Baker, Gabor G. Tigyi , Duane D. Miller* .
1
. Department of Pharmaceutical Sciences, College of Pharmacy, University of
Tennessee Health Science Center, Memphis, TN 38163, USA
2. Department of Physiology, College of Medicine, University of Tennessee Health
Science Center, Memphis, TN 38163, USA
3
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. Department of Chemistry, The University of Memphis, Memphis TN 38152
. Computational Research on Materials Institute, The University of Memphis,
Memphis TN 38152
#
Senior coꢀauthors
Address correspondence to: Duane D. Miller, Ph.D.
Email. dmiller@uthsc.edu
Phone. (+1)ꢀ901ꢀ448ꢀ6026Fax. (+1)ꢀ901ꢀ448ꢀ3446
or
Gabor Tigyi :Ph.D., M.D.
Email. gtigyi@uthsc.edu
Phone. (+1)ꢀ901ꢀ448ꢀ2634
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ABSTRACT
Autotaxin (ATX, aka. ENPP2) is the main source of the lipid mediator lysophosphatidic
acid (LPA) in biological fluids. This study reports on inhibitors of ATX derived by lead
optimization of the benzeneꢀsulfonamide in silico hit compound 3. The new analogues
provide a comprehensive SAR of the benzeneꢀsulfonamide scaffold that yielded a
series of highly potent ATX inhibitors. The three most potent analogues (3a, IC ~ 32
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nM and 3b, IC ~ 9 nM as well as 14, IC ~ 35 nM) inhibit ATXꢀdependent invasion of
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A2058 human melanoma cells in vitro. Two of the most potent compounds, 3b and 3f
IC50 ~ 84 nM) lack inhibitory action on ENPP6 and ENPP7 but possess weak
(
antagonist action specific to the LPA GPCR. In particular, compound 3b potently
1
reduced in vitro chemotherapeutic resistance of 4T1 breast cancer stemꢀlike cells to
paclitaxel and reduced significantly B16 melanoma metastasis in vivo.
Key words: ATX, LPA, chemoresistance, cancer stem cell, metastasis, benzene
sulfonamide
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Introduction.
Autotaxin (ATX), a member of the ectonucleotide pyrophosphate and phosphatase
(
ENPP) family, is primarily known to catalyze the hydrolysis of lysophosphatidylcholine
LPC) resulting in the production of the growthꢀfactorꢀlike bioactive phospholipid
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(
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ꢀ3
lysophosphatidic acid (LPA). LPA activates a set of six GPCR LPA , ion channels,
1ꢀ6
the transcription factor PPARγ, which regulate a host of cellular responses, including
4, 5
cell proliferation and migration, vascular tone, and platelet aggregation. Many of
these responses are at the core of human diseases that include fibrotic diseases,
1, 4ꢀ6
neuropathic pain, rheumatoid arthritis, as well as cardiovascular diseases.
In recent
years, the ATXꢀLPA signaling pathway has been linked to several aspects of cancer cell
6ꢀ8
biology that include cancer growth, invasion, metastasis, and therapeutic resistance.
Recently, ATX has been found to play an important role in the maintenance and
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proliferation of ovarian cancer stem cells. Because of the explicit role of LPA in these
1, 3, 10ꢀ15
human pathologies development of drugꢀlike ATX inhibitors has begun.
These
synthetic efforts have resulted in a number of novel ATX inhibitors that can arbitrarily be
divided into two categories: Lipidꢀlike ATX inhibitors, that mimic either the LPC or LPA
1ꢀ3, 7, 11, 12, 14, 16ꢀ20
and; Nonꢀlipid ATX inhibitors
. Despite these efforts, limited success
has been achieved in preclinical and regulatory development of ATX inhibitor drug
1
candidates. In this context, the high partition coefficient (Log P > 5) renders most lipidꢀ
2, 7, 16
like inhibitors unsuitable for potential clinical development.
The elucidation of ATX
crystal structure with the inhibitor coꢀcrystalized in the active site, provided insight into
10
the potential active site surfaces. We and others pursued rational discovery that
yielded a new generation of small molecule drugꢀlike nonꢀlipid ATX inhibitors with
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pharmacological properties that meet Lippinski’s rule of five.
A subgroup of
recently developed nonꢀlipid ATX inhibitors, including (Z)ꢀ4ꢀ[(4ꢀ{[3ꢀ(4ꢀFluorobenzyl)ꢀ2,4ꢀ
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dioxoꢀ1,3ꢀthiazolanꢀ5ꢀyliden]methyl}phenoxy) methyl]phenyl boronic acid (HA 155) ,
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,5ꢀDichlorobenzylꢀ[4ꢀ[3ꢀoxoꢀ3ꢀ(2ꢀoxoꢀ2,3ꢀdihydrobenzoxazolꢀ6ꢀyl)propyl]]piperazineꢀ1ꢀ
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carboxylate (PF 8380) , compounds reported by pharmaceutical companies Novartis
and PharmAkea , have similar structural features and contain an acid or acidꢀlike
2+
moiety, to interact with one of the Zn ions at the catalytic site, a core spacer, and a
1ꢀ3, 12, 21ꢀ24
hydrophobic tail accommodated within the hydrophobic pocket of ATX.
However, to the best of our knowledge, none of the active site inhibitors have
successfully made it through clinical trials yet. Thus, recent approaches have focused
on developing small molecule nonꢀlipid ATX inhibitors without an acidic moiety (Figure
15
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1
), allosteric modulators of the enzyme , and DNA aptamers. Our group utilized highꢀ
throughput and in silico searching to identify a small molecule ATX inhibitor, 2,4ꢀ
dichloroꢀNꢀ(3ꢀfluorophenyl)ꢀ5ꢀ(morpholinosulfonyl)benzamide [GRI918013 (3), Figure
1
A], which exerts its inhibition by binding to the hydrophobic pocket remote from the
2, 16
catalytic site, and lacks an acidic moiety.
This chemical entity, even without an acidic
head group, shows the same pharmacological and biological effects as catalytic site
inhibitors do but inhibition is mediated via interference with the binding of the
hydrophobic chain of the lipid substrates, although it lacks sufficient potency and
2, 16
stability in vivo.
Recently, Galapagos Inc., has developed a number of potent
compounds that have a major structural similarity to 3, lacking the acidic group (Figure
3
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). One of the Galapagos compounds has been reported to successfully pass the first
inꢀhuman phase one clinical trial. Thus, the objectives of our synthetic campaign were:
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) To improve the potency of ATX inhibition with IC values in the low nanomolar range
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and 2) Obtain compounds that were effective in vitro and in vivo assays dependent on
ATX activity. Our lead optimization set four structural criteria for the novel compounds:
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) Lack of acidic moiety, 2) Lack of chiral center, 3) Molecular weight less than 500 D,
and 4) Partition coefficients (Log P) less than 3. Here we report on the structureꢀactivity
relationship of several modifications of the hit 3 that yielded two novel compounds with
potent in vitro and in vivo activity blocking B16 melanoma invasion, metastasis, and
reduced chemotherapeutic resistance of 4T1 breast cancer stemꢀlike cells to paclitaxel.
Results
1
1
. Chemical synthesis
.1. Modification of ring A. We designated the three rings in 3 as rings A, B, and C
(Figure 1A). Our previous screening experiments, mutagenesis, and molecular
modelling findings suggested that 3 binds into the hydrophobic pocket of ATX without
protruding into and blocking substrate access to the catalytic site as shown in Figure
1
B. First, we designed and synthesized a series of derivatives with varying substituents
on ring A as shown in Scheme 1.
Compound 3 was synthesized following the strategy described in Scheme 1 with 70%
yield. We first increased the number of fluorine substituents on ring A by synthesizing
the 3,4ꢀdiꢀfluoro analogue (3a), and the 3,4,5ꢀtriꢀfluoro analogue (3b) depicted in
Scheme 1 and tested them for ATX inhibitory activity (Table 1). The simple
manipulation of aromatic substituents resulted in fourꢀfold IC improvements in 3a (IC
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31 nM) and thirteenꢀfold in 3b (IC50 = 9 nM) relative to the IC = 120 nM parent
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compound. When 3b was docked in to the ATX crystal structure (PDB 3NKM) , we
found that three fluoro substituents promote a tighter binding of this compound over 3
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the angle of the halogenated ring A. 3 makes largely edgeꢀtoꢀface π interaction with
residue W260 whereas, 3b has a rotated aromatic ring that allows a hydrogen bond
between a fluorine substituent and aromatic NꢀH of W260 (Figure 3 A). We continued
this series by making pentaꢀfluoro analogue (3c) that was poorly active. Next we
synthesized and tested a relatively less electron deficient 3,4,5ꢀtrichloro analogue 3d,
and the electron donating 3,4,5ꢀtrimethoxy analogue 3e, both of which were predicted to
be sterically more congested than trifluoro analogue 3b in modeling studies. These two
analogues were poorly active, suggesting increased steric congestion by ring A, which
did not allow the molecule to be properly accommodated into the hydrophobic pocket.
Compound 3d was also docked into the ATX crystal structure and a dramatically
different orientation was observed in concert with our experimental findings (Figure 3B).
Top poses of 3b and 3d showed good volume overlap but 3d ran in the opposite
direction and central aromatic ring B was twisted completely out of conjugation with the
amide linker. This represents a very high energy conformation and unlikely to bind
because of the conformational energy penalty. Next, we tested the effects of combining
two different kinds of ring A substituents on the inhibitory activity of the molecule. We
synthesized 3,5ꢀdifluoroꢀ4ꢀchloro analogue (3f) and the 3,5ꢀdichloroꢀ4ꢀfluoro analogue
(3g) and tested them for the ATX inhibition. Both analogues were highly potent ATX
inhibitors with IC values of 83 nM and 40 nM, respectively. When docked to ATX, 3f
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b exposed to water in hydrophobic tunnel (Figure 3C). However, 3g (not shown)
adopted a completely different pose in which the halogenated aromatic A ring was in the
hydrophobic pocket not in the hydrophobic tunnel, whereas the polar ends of the
molecules overlapped, although running in opposite directions. Distances between the
chlorine atoms of 3g in the hydrophobic pocket predicted a waterꢀmediated hydrogen
bond with backbone atoms of L214 or A218 and a weak hydrogen bond with W276,
which can be the reason for the twoꢀfold higher potency of 3g over 3 f.
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In an attempt to introduce more polar substituents into ring A, we prepared the
3,5ꢀdifluoroꢀ4ꢀcyano analogue (3h) and the 3,5,ꢀdifluoro 4ꢀmethyl ester analogue (3i).
ATX activity measurements with these compounds showed that 3h although modestly
tolerated, 3i was much less potent than 3 with IC50 values of 190 nM and 863 nM,
respectively. Next, we hydrolyzed the ester bond in 3i to the corresponding carboxylic
acid and found that this new compound 4 was inactive.
1
.2. Modifications of the linker between rings A and B. Subsequent synthetic
modifications were aimed at the replacement of the amide bond of 3 to explore the
importance of amide linker in ATX inhibition and to improve biological stability to
amidases. To accomplish this, we first tried to synthesize the triꢀfluoro benzoxazole (6)
from intermediate 5 (Scheme 2). Although, intermediate 5 was obtained in good yield
(76%) following the same strategy as shown in scheme 1, the synthetic procedure
failed to turn intermediate 5 into desired oxazole 6. An alternative strategy was applied
to generate the oxazole 7 and the imidazole 8 using commercially available aminoꢀ
phenol and aminoꢀaniline precursors, respectively (Scheme 2). We also synthesized
the Nꢀmethyl analogue (9). The four compounds 5, 7, 8, and 9 obtained with the
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synthetic route shown in scheme 2 were tested for ATX inhibition (Table 1). Compound
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compound 3b. Compounds 7 and 8 closely mimic the amide framework, yet failed to
inhibit ATX. It is noteworthy that the A and B aromatic rings in compounds 7 and 8 were
predicted to be closer to planar than in the compounds with amide linkers. Additionally,
compound 9 showed only very weak potency inhibiting ATX. We next examined the role
of the carbonyl oxygen/carbonyl bond for the ATX inhibitory activity, because the SAR
of the analogues we synthesized to this point indicated that the orientation of ring A
relative to ring B around amide linkage is critical for the activity. To achieve this, we
reduced amide bonds of the two most active ATX inhibitors, 3a and 3b (Scheme 3).
The desired amines 10 and 11, were obtained in good yields and were subjected to
ATX inhibition analysis to find that the inhibitory activity of 3,4,5ꢀtrifluoro amine (11) was
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amine 10 was found inactive.
In the next phase of our SAR analysis, we prepared compounds with thioamide
linkers because of their higher stability against proteases compared to amides. We
selected the four most potent inhibitors and synthesized the corresponding thioamides
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utilizing the synthetic strategy shown in scheme 4. ATX inhibition assays showed that
compounds 12,13, 14, and 15 retained similar inhibitory activity to the amide analogues
27ꢀ30
(Table 1). We also replaced the amide linker with a sulfonamide (Scheme 5).
The
intermediate 18 was obtained in good yield and was converted into two different kinds of
sulfonamides. Compounds 19 and 20 have the sulfonamide nitrogen linked to the ring B
whereas, in compounds 22 and 23 the sulfonamide nitrogen is linked to ring A. All four
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compounds lost their ATX inhibitory activity as shown in Table 1. These results
underline the importance of linker geometry for ATX inhibition.
1.3. Modifications of the linker between ring B and C. Because these findings
identified the importance of the amideꢀbond linkage for the inhibitory activity, we
decided to further explore variations of the sulfonamide linkage between rings B and C
to assess the role of the sulfonyl group. First, we replaced the sulfonamide linkage with
a polar but smaller linkage keeping the morpholine ring intact. Additionally, we avoided
the introduction of chirality in linking ring B to C. We constructed the hydrazine moiety,
where the sulfonyl is replaced by a nitrogen atom as shown in Scheme 6. Compound
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5 was obtained through intermediate 24 using the BuchwaldꢀHartwig crossꢀcoupling
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reaction following the synthetic tools described by Cacchi et al. Compound 25
completely lost ATX inhibitory activity (Table 1). This result confirms that the sulfonyl
linkage provides a better contact with the polar surface of the ATX substrate binding
pocket and forms some electrostatic interactions to promote strong binding. We
hypothesized that introduction of a mesyl group (26) on the hydrazine in compound 25
could be as effective as compound 3b, in contacting the polar enzyme surface. Many
32ꢀ34
attempts to obtain 26b remained unsuccessful.
However, utilizing excess sodium
tertꢀbutoxide and 1:11 ratio of methane sulfonyl chloride, compound 26a was obtained.
Testing revealed that compound 26a was inactive, once again reinforcing the
importance of the linker geometry. We also replaced ring C with Nꢀmethylꢀpiperazine
shown in Scheme 7. The rationale behind this modification was that it would be more
water soluble than the morpholine analogues. We selected the two most potent
morpholine analogues 3b and 3g, and prepared their Nꢀmethyl piperazine analogues
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8a and 28b, following the synthetic strategy shown in Scheme 7. Analogues, 28a and
8b, were tested for ATX inhibition and found to inhibit the ATX with IC values of 66
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nM and 55 nM, respectively (Table 1). Finally, we selected the more potent analogue
8b, and converted it into the corresponding thioamide (29) which showed a potency of
IC = 100 nM.
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. Pharmacological characterization of the benzene sulfonamide ATX Inhibitors
ATX has dual nucleotide pyrophosphate phosphatase and lysophosholipase D activity
that can be selectively measured using the pNPꢀTMP and FSꢀ3 substrates,
2, 13, 16
respectively.
The inhibitory activities of the new compounds against the two
different substrates are listed in Tables 1. Our primary test was to assess the inhibitory
action of the new compounds on the lysophospholipase D activity of purified
recombinant human ATX. Compound 3b was found to be the most potent ATX inhibitor
with an IC50 of ~ 9 nM in this assay. The mechanism of action was competitive;
however, the traditional measure of a competitive inhibitor may not be appropriately
applied in this context. 3b inhibited hydrolysis of the lipidꢀlike substrate FSꢀ3 while
sparing effects on hydrolysis of the nucleotide substrate pNPꢀTMP. Molecular modeling
predicted that 3b does not bind at the active site in which case both substrates would be
affected. However, because the compound is targeted toward the hydrophobic pocket
of ATX, it shares space with the binding site for the hydrophobic tail of lipidꢀlike
substrates like FSꢀ3. As such, the mechanism of action presents itself as “competitive”
in the FSꢀ3 hydrolysis assay even though the binding site of the 3b exists outside the
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catalytic site of the enzyme. This particular behavior of ATX inhibitors has been noted
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before.
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3
. Selective dose-dependent inhibition of LPA GPCR by compounds 3b, 3f, and
1
3g
The product of ATX, LPA is ligand to at least six GPCRs and the benzene sulfonamide
compounds we have generated in this study could also possess some ligand activity at
the different LPAR subtypes. For this reason, we tested all new chemical entities with
useful ATX inhibitory potency (< 250 nM) for agonist and antagonist activity at
35
LPA1/2/3/4/5 using cells stably transfected with each individual GPCR . None of the
compounds tested exhibited agonist activity at any of these LPA receptor subtypes.
Additionally, of those screened, only 3b, 3f, and 3g exhibited antagonist activity, and
this inhibition was specific to the LPA receptor (Table 2, Figure 4). Compounds 3b
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(ATX IC = 9 nM, LPA1 IC ~ 14 ꢁM), 3f (ATX IC = 83 nM, LPA1 IC ~ 6 ꢁM), and
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3
g (ATX IC = 40 nM, LPA1 IC = 18 ꢁM), were dual inhibitors of both ATX and the
50 50
LPA receptor.
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. Effect of benzene sulfonamide ATX Inhibitors on other members of the ENPP
family
ATX constitutes one of three members of the ENPP family, comprised also of ENPP6
and ENPP7. Both ENPP6 and ENPP7 retain similar phosphodiesterase activity to ATX
but lack its hydrophobic pocket. To test the inhibitory effects of benzene sulfonamide
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ATX inhibitors versus purified recombinant ENPP6 and ENPP7, the nucleotide
substrate pꢀnitrophenylphosphorylcholine (pNPPC) was used. None of the compounds
tested exhibited inhibitory activity for ENPP6 or ENPP7 (data not shown) as the
benzene sulfonamide structures are targeted to the hydrophobic pocket of ATX, which
is not found in either of the other ENPP family members.
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5. Inhibition of ATX-mediated invasion of A2058 human melanoma in vitro
We utilized the Boyden chamber assay to assess the effect of benzene sulfonamide
analogues 3a, 3b, 3f and 14 on invasion of A2058 human melanoma cells in vitro. The
A2058 cells invade the matrigel layer in an LPAꢀdependent manner. Therefore, we used
exogenous LPC and recombinant ATX as a source of LPA. First, we screened the
compounds at 10µM concentration alongside (4ꢀpentadecylbenzyl)phosphonic acid [30
13
(
BMP22)] , a potent ATX inhibitor that we have previously identified and
13
characterized. We found that all four inhibitors were as effective as 30 in inhibiting
A2058 cell invasion across the matrigel layer in response to exogenous LPC and
recombinant ATX (figure 5A). Subsequently, we generated dose response curves for
compounds 3a, 3b, 3f, and 14 (figure 5B). All four inhibitors doseꢀdependently
decreased the A2058 cell invasion with similar potencies (Table 3).
6. In Vitro Stability Analysis
The lead 3, 3b, 3f, and 14 were analyzed for stability in the presence of cells in culture
using liquid chromatography mass spectrometry. All analogs tested showed significant
stability over the 40h of exposure to 4T1 cells in serum free culture medium. The lead, 3
maintained 79.7 ± 7.8% (16h) and 79.5 ± 12.6% (40h) of the 0h control signal. Whereas
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the polyꢀhalogenated amide and thioamide analogs appear to be a bit more stable in
this system over this limited time course (3b maintained 102.6 ± 2.6% (16h) and 105.3 ±
2
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.7% (40h), 3f 101.3 ± 3.7% (16h) and 108.2 ± 1.3%, and the thioamide 14 93.2 ±
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0.4% (16h) and 102.0 ± 14.5% (40h) of their corresponding 0h controls).
7
. Inhibition of B16 murine melanoma lung metastasis by benzene sulfonamide
ATX Inhibitors
To complement our in vitro invasion studies, we tested the effect of 3b and 14 in the
B16 lung metastasis in vivo model. We found that daily administration of compounds 3b
and 14 for a total of 10 days significantly reduced the number of metastatic lung nodules
formed in mice (Figure 6).
8
. Sensitization of therapy resistant 4T1 murine breast carcinoma cells and stem
cell-like cells (CSC) to paclitaxel by benzene sulfonamide ATX Inhibitors
To further characterize the three most potent compounds, we tested them on the
viability and growth of 4T1 murine breast cancer cells (Figure 7). Adherent cultures of
4
T1 cells were treated for 24h with a range of 0ꢀ10 µM compounds 3b, 3f, 14, or
reference inhibitor 30 in RPMI with 1% (v/v) charcoalꢀstripped FBS. After 48h treatment
with daily replenishment of inhibitor, cell number/viability was assessed (Figure 7A).
Only 30 inhibited cell growth at 3 µM, whereas all other compounds failed to cause a
significant reduction in cell number. However, unlike the other compounds, 14 was
toxic at 10µM, the highest concentration tested.
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LPA and ATX increase the resistance of cancer cells to chemoꢀ and radiation therapy.
To assess whether the new ATX inhibitors could overcome chemoresistance, the 4T1
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breast cancer cell line was exposed to increasing concentration of paclitaxel (PT) for
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8h with or without 24h preꢀtreatment with 3 µM of compound 3b or 3f. Both ATX
inhibitors caused a significant shift in the LD50 of PT doseꢀresponse relationship
indicating that ATX inhibitor treatment made them more sensitive to PT (Figure 7B).
The PT LD50 for reducing cell growth by 50% was 202 nM when applied alone. In
contrast, the 3b and 3f reduced the LD to PT to 127 and 66 nM, respectively.
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Next, we evaluated if these inhibitors were able to overcome chemoresistance to PT in
cancer stemꢀlike cells. To do this, we generated a paclitaxelꢀresistant 4T1 cell line (4T1ꢀ
TaxR) and grew them in an anchorageꢀindependent environment to induce sphere
formation. This culture method has been widely used to enrich for breast CSC. We
found that both 30 and compound 3b were able to further reduce cell viability by ~25 to
3
0% compared to PT alone, suggesting that the ATX inhibitors were able to resensitize
the CSC to PT treatment (Figure 7C).
Discussion and Conclusion
Here we report on the synthesis of ATX inhibitors that are benzeneꢀsulfonamide
analogues derived from hit compound 3 which, we identified via in silico screening of
the Genome Research Institute library at the University of Cincinnati Drug Discovery
2, 16
Center.
The parent compound 3, although a potent nonꢀlipid ATX inhibitor with an
IC value ~ 117 nM, lacked sufficient stability and activity in cellular and in vivo assays.
50
Thus, our study objectives were: 1) To improve the potency of ATX inhibition with IC50
values in the low nanomolar range and 2) Obtain compounds that were effective in in
vitro cellꢀbased assays and in vivo metastasis model dependent on ATX activity. We
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have conducted four different kinds of SAR explorations of the 3 scaffold. The structural
features explored included 1) modification of ring A substituents, 2) modification of the
amide bond AꢀB ring linker, 3) modification of sulfonyl BꢀC ring linker, and 4) the
modification of the ring C. The Aꢀring optimization has resulted in a number of
substantially more active ATX inhibitors, including inhibitor 3b (IC = 9 nM) and 5 (IC
50
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=
18 nM). When tested for agonist action at LPA1/2/3/4/5 GPCR none of the compound
were active up to 10µM, the highest concentration tested. However, three of the potent
2
ATX inhibitors, 3b, 3f, and 3g, were also effective in antagonizing the LPA ꢀelicited Ca
1
responses. The antagonist potency of these ATX inhibitors was in the low micromolar
range that is not negligible when assessing their cellular actions. Our synthetic
improvement to increase the LPA antagonist activity of derivatives of compounds 3b
1
and 3f without compromising their low nanomolar ATX inhibitory activity were
unsuccessful.
Unexpectedly, inhibition of ATX was highly sensitive to transformations of the amide
linkage as any alteration led to deterioration of potency. Thioamides, including
compound 14 (IC = 35 nM) were the most potent in this series, with threeꢀfold better
50
potency than the screening hit 3. This finding underlines the importance of linker
geometry for inhibition of ATX. The third optimization, was modification of the sulfonyl
bond and this did not yield any compound that was more potent than 3. Finally,
replacement of morpholine with piperazine, was tolerated resulting in three more active
compounds than the starting scaffold, the most potent 28b showing an IC of 55 nM. It
50
is noteworthy that the piperazine series with Log P values around 3.5 are water soluble.
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We selected the three most potent amide analogues (3a, 3b, and 3f) and the most
potent thioamide analogue (14) in cellular assays using the ATXꢀdependent invasion of
A2058 human melanoma cells in vitro. All four inhibitors showed good in vitro stability
and doseꢀdependently decreased A2058 cell invasion with overlapping potencies
(Table 3). In addition, two of these inhibitors, 3b and 14 were shown to be effective in
reducing the metastasis of B16F10 cells to the lungs of C57BL/6 mice. Compounds 30
and 3b effectively resensitize the resistant 4T1 cell line (4T1ꢀTaxR) to paclitaxel
treatment. Our results complement the findings of Venkatraman et al., in which
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activation of the LPA receptor promotes chemoresistance in breast cancer cells in part
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via the stabilization of Nrf2 and the subsequent increase in the transcription of various
36
genes involved in drug resistance. Thus compounds that have both inhibitory actions
against ATX and LPA , might have therapeutic utility as adjuvant for the treatment of
1
therapy resistant cancers.
In summary, this series of inhibitors meets the four objectives of our synthetic campaign
as they lack carboxylic acid functionality, contain no chiral center, their molecular weight
is less than 500 D, and each has IC50 values less than 85 nM for the ATX. During our
study, Galapagos Inc., has developed a number of potent compounds with structural
1
similarity to 3, (Figure 1A). One of the Galapagos compound has been reported to
1
successfully complete the first inꢀhuman phase one clinical trial giving us hope that our
inhibitors will yield similar outcome with higher potency than Galapagos compound.
Galapagos
Experimental.
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Chemistry.
General methods: All nonꢀaqueous reactions were performed in ovenꢀdried glassware
under an inert atmosphere of dry nitrogen. All the reagents and solvents were
purchased from Aldrich (St. Louis, MO), AlfaꢀAesar (Ward Hill, MA), CombiꢀBlocks (San
Diego, CA), Ark Pharm (Libertyville, IL) and used without further purification. Analytical
thinꢀlayer chromatography was performed on Silica Gel GHLF 10 x 20 cm Analtech TLC
Uniplates (Analtech, Newark, DE) and were visualized by fluorescence quenching under
UV light. Biotage SP1 Flash Chromatography Purification System (Charlotte, NC)
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(Biotage SNAP Cartridge, silica, 50g & 100g) was used to purify the compounds. 1H
NMR and 13C NMR spectra were recorded on a Varian Inovaꢀ500 spectrometer (500
MHz) (Agilent Technologies, Santa Clara, CA) or a Bruker Ascend 400 (400 MHz)
(Billerica, MA) spectrometer. Chemical shifts are reported in ppm on the δ scale and
referenced to the appropriate solvent peak. Mass spectra were collected on a Brucker
ESQUIRE electrospray/ion trap instrument in the positive and negative modes. High
resolution mass spectrometer (HRMS) data were acquired on a Waters Xevo G2ꢀS
QTOF (Milford, MA) system equipped with an Acquity Iꢀclass UPLC system. Purity of all
1
tested compounds was determined to be >95% as evident by H NMR and HPLC.
HPLC method used to determine purity is as follows: Compound purity was analyzed
using Agilent 1100 HPLC system (Santa Clara, CA) with a Zorbax SBꢀC18 column,
particle size 3.5 µm, 4.6 × 150 mm from Agilent. Mobile phases consist of water with
0
.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). Flow rate of 1 mL/min
was used. The gradient elution started at 50% B. It reached at 100% B from 0 to 9 min
and was maintained at it from 9 to 12 min, and was then decreased to 50% B from 12 to
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5 min and stopped. Compound purity was monitored with a DAD detector set at 254
nm.
O
Synthesis of 2,4-dichloro-5-(chlorosulfonyl)benzoic acid (1). To a cooled (0 C)
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chlorosulfonic acid (10.4 mL, 157 mmol) 2,4ꢀdichlorobenzoic acid (5 g, 26 mmol) was
O
added potion wise. The reaction mixture was subsequently heated to 140 C and
continued to stir for 16 hrs. The reaction mixture was cooled to room temperature and
poured into crushed ice. The formed white precipitates were collected by filtration and
37
dried under the vacuum. Yield = 80%. The characterizations comply with the literature.
General procedure for the Synthesis of compounds 2 and 27. To a solution of 1 (3g,
0.3 mmol) in 20 mL CH Cl , trimethylamine was added (3.61 mL, 26 mmol). The
1
2
2
O
reaction mixture was cooled down to 0 C under the ice. The corresponding secondary
amine (11.4 mmol) was added dropꢀwise and the reaction mixture was continued to stir
over the night. The reaction mixture was concentrated and the crude was taken for next
step without further purification.
General procedure for the Synthesis of compounds 3 as well as 3a to 3i. To a
solution of compound 2 (0.46g, 1.4 mmol) in 20 mL of CH Cl , a volume of 1 mL SOCl
2
2
2
(13.6 mmol) and 2 drops of DMF were added under argon atmosphere. The reaction
was continued to stir at room temperature over the night. The reaction mixture was
concentrated and dissolved back again in 20 mL CH Cl . Pyridine (330 uL, 4.1 mmol)
2
2
and the corresponding aniline (1.2 mmol) were added under argon atmosphere. The
reaction mixture was allowed to stir over the night at room temperature. The reaction
mixture was extracted with 10% HCl, water, and brine solution. The organic layer was
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dried over MgSO , concentrated, and purified by column chromatography leading to the
4
pure product.
2,4-Dichloro-N-(3-fluorophenyl)-5-(morpholinosulfonyl)benzamide
compound 3 was prepared following general procedure for the synthesis of 3 as well as
aꢀ3i. The crude was purified in 1% MeOH/CH Cl yielding the pure product as white
(3).
The
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solid. Yield = 70%. H NMR (400 MHz, DMSOꢀd ) δ 10.94 (s, 1H), 8.18 (d, J = 3.8 Hz,
6
2
H), 7.77 – 7.67 (m, 1H), 7.53 – 7.37 (m, 2H), 7.12 – 6.95 (m, 1H), 3.69 (t, J = 4.70 Hz,
+
2
4
H), 3.28 (t, J = 5.0 Hz, 4H). HRMS [C H N O FSCl ]: calcd 433.0192, found
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2
4
433.0182.
2
,4-Dichloro-N-(3,4-difluorophenyl)-5-(morpholinosulfonyl)benzamide (3a).
compound 3a was prepared following general procedure for the synthesis of 3 as well
as 3aꢀ3i. The crude was purified in 1% MeOH/CH Cl yielding the pure product as white
The
2
2
O
1
solid. Yield = 72%. mp = 221.3 C. H NMR (400 MHz, DMSO) δ 10.90 (s, 1H), 8.13 (s,
2
H), 7.85 (ddd, J = 12.8, 7.4, 2.4 Hz, 1H), 7.45 (ddd, J = 27.0, 18.1, 9.0 Hz, 2H), 3.63 (t,
13
J = 4.46, 4H), 3.22 (t, J = 4.77, 4H). C NMR (101 MHz, DMSO) δ 162.71, 135.72,
1
4
35.29, 134.19, 133.20, 133.10, 131.49, 117.76, 117.58, 116.22, 109.00, 108.79, 65.72,
+
2
5.49. HRMS [C H N O F SCl ]: calcd 451.0098, found 451.0099.
17
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2
4
2
2
,4-Dichloro-5-(morpholinosulfonyl)-N-(3,4,5-trifluorophenyl)benzamide (3b). The
compound 3b was prepared following general procedure for the synthesis of 3 as well
as 3aꢀ3i. The crude was purified in 1% MeOH/CH Cl yielding the pure product as white
2
2
O
1
solid. Yield = 71.5%. mp = 237.8 C. H NMR (400 MHz, DMSO) δ 11.05 (s, 1H), 8.15
(d, J = 2.1 Hz, 2H), 7.58 (dd, J = 9.9, 6.5 Hz, 2H), 3.63 (t, J = 4.67, 4H), 3.22 (t, J =
13
4
.53, 4H). C NMR (101 MHz, DMSO) δ 162.99, 135.70, 134.94, 134.27, 133.42,
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33.17, 131.53, 104.39, 104.15, 65.72, 45.49. HRMS [C H N O F SCl ]: calcd
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69.0003, found 451.0012.
,4-Dichloro-5-(morpholinosulfonyl)-N-(perfluorophenyl)benzamide
(3c).
The
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compound 3c was prepared following general procedure for the synthesis of 3 as well
as 3aꢀ3i. The crude was purified in 1% MeOH/CH Cl yielding the pure product as white
2
2
O
1
solid. Yield = 65%. mp = 237.1 C. H NMR (400 MHz, DMSOꢀd ) δ 11.01 (s, 1H), 8.15
6
13
(
dd, J = 24.5, 19.0 Hz, 2H), 3.70 – 3.55 (m, 4H), 3.27 – 3.13 (m, 4H). C NMR (101
MHz, DMSOꢀd ) δ 166.95, 163.05, 136.00, 134.33, 133.82, 133.46, 131.49, 65.73,
6
+
2
4
5.43. HRMS [C H N O F SCl ]: calcd 504.9815, found 504.9838.
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2
4
5
2
,4-Dichloro-5-(morpholinosulfonyl)-N-(3,4,5-trichlorophenyl)benzamide (3d). The
compound 3d was prepared following general procedure for the synthesis of 3 as well
as 3aꢀ3i. The crude was purified in 1% MeOH/CH Cl yielding the pure product as white
2
2
O
1
solid. Yield = 75%. mp = 303.0 C. H NMR (400 MHz, DMSOꢀd ) δ 11.09 (s, 1H), 8.18
6
(
s, 1H), 8.15 (s, 1H), 7.94 (s, 2H), 3.68 – 3.57 (t, J = 4.32, 4H), 3.27 – 3.16 (t, J = 4.84,
13
4
H). C NMR (101 MHz, DMSOꢀd ) δ 163.10, 138.36, 135.74, 134.78, 134.30, 133.52,
6
+
5
133.20, 133.01, 131.59, 124.51, 119.89, 65.72, 45.48. HRMS [C H N O SCl ]: calcd
17
14
2
4
5
16.9117, found 516.9108.
,4-Dichloro-5-(morpholinosulfonyl)-N-(3,4,5-trimethoxyphenyl)benzamide
2
(3e).
The compound 3e was prepared following general procedure for the synthesis of 3 as
well as 3aꢀ3i. The crude was purified in 1% MeOH/CH Cl yielding the pure product as
2
2
O
1
white solid. Yield = 65%. mp = 281.2 C. H NMR (400 MHz, Chloroformꢀd) δ 8.34 (s,
H), 7.83 (s, 1H), 7.66 (s, 1H), 6.93 (s, 2H), 3.89 (s, 6H), 3.84 (s, 3H), 3.73 (dd, J = 5.9,
1
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+
3
5
.7 Hz, 4H), 3.39 – 3.18 (m, 4H). HRMS [C H N O SCl ]: calcd 505.0603, found
20 23 2 7 2
05.0606.
2,4-Dichloro-N-(4-chloro-3,5-difluorophenyl)-5-(morpholinosulfonyl)benzamide
3f). The compound 3f was prepared following general procedure for the synthesis of 3
as well as 3aꢀ3i. The crude was purified in 1% MeOH/CH Cl yielding the pure product
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
2
2
O
1
as white solid. Yield = 75%. mp = 275.7 C. H NMR (400 MHz, DMSOꢀd ) δ 11.17 (s,
6
1
4
H), 8.16 (d, J = 6.1 Hz, 2H), 7.60 (d, J = 9.1 Hz, 2H), 3.63 (t, J = 4.35, 4H), 3.22 (t, J =
13
.93, 4H). C NMR (101 MHz, CDCl ) δ 168.38, 140.96, 140.08, 139.55, 138.75,
3
+
3
138.43, 136.82, 109.16, 108.89, 70.97, 50.74. HRMS [C H N O F SCl ]: calcd
1
7
14
2
4
2
4
84.9708, found 484.9729.
,4-Dichloro-N-(3,5-dichloro-4-fluorophenyl)-5-(morpholinosulfonyl)benzamide
2
(
3g). The compound 3g was prepared following general procedure for the synthesis of 3
as well as 3aꢀ3i. The crude was purified in 1% MeOH/CH Cl yielding the pure product
2
2
O
1
as white solid. Yield = 76%. mp = 297.4 C. H NMR (400 MHz, DMSOꢀd ) δ 11.00 (s,
6
1
H), 8.16 (d, J = 9.9 Hz, 2H), 7.85 (d, J = 6.1 Hz, 2H), 3.63 (t, J = 4.8 Hz, 4H), 3.22 (t, J
13
=
5.22 Hz, 4H). C NMR (101 MHz, DMSOꢀd ) δ 162.96, 135.74, 134.89, 134.28,
6
133.45, 133.18, 131.56, 121.11, 120.93, 120.19, 65.72, 45.49. HRMS
+
4
[
C H N O FSCl ]: calcd 500.9412, found 500.9409.
17
14
2
4
2
,4-Dichloro-N-(4-cyano-3,5-difluorophenyl)-5-(morpholinosulfonyl)benzamide
(3h). The compound 3h was prepared following general procedure for the synthesis of 3
as well as 3aꢀ3i. The crude was purified in 1% MeOH/CH Cl yielding the pure product
2
2
O
1
as white solid. Yield = 72%. mp = 240.7 C. H NMR (400 MHz, DMSO) δ 11.51 (s, 1H),
8
.19 (d, J = 17.4 Hz, 2H), 7.63 (d, J = 10.1 Hz, 2H), 3.63 (t, J = 4.75 Hz, 4H), 3.22 (t, J =
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2
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2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
4
.68, 4H). C NMR (101 MHz, DMSOꢀd ) δ 163.67, 135.69, 134.51, 134.36, 133.73,
6
+
2
1
33.23, 131.69, 109.75, 103.18, 102.93, 65.72, 45.48. HRMS [C H N O F SCl ]:
18 14
3
4
2
calcd 476.0050, found 476.0048.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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4
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7
8
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0
1
2
3
4
5
6
7
8
9
0
Methyl
4-(2,4-dichloro-5-(morpholinosulfonyl)benzamido)-2,6-difluorobenzoate
(3i). The compound 3i was prepared following general procedure for the synthesis of 3
as well as 3aꢀ3i. The crude was purified in 1% MeOH/CH Cl yielding the pure product
2
2
O
1
as white solid. Yield = 69%. mp = 216.4.1 C. H NMR (400 MHz, DMSOꢀd ) δ 11.29 (s,
6
1H), 8.17 (d, J = 14.8 Hz, 2H), 7.50 (d, J = 10.6 Hz, 2H), 3.87 (s, 3H), 3.63 (t, J = 4.46
13
Hz, 4H), 3.23 (t, J = 4.69 Hz, 4H). C NMR (101 MHz, DMSOꢀd ) δ 163.37, 160.85,
6
1
4
43.00, 135.70, 134.69, 134.34, 133.61, 133.19, 131.63, 103.16, 102.90, 65.72, 52.70,
+
2
5.49. HRMS [C H N O F SCl ]: calcd 509.0152, found 509.0146.
19
17
2
6
2
4
-(2,4-Dichloro-5-(morpholinosulfonyl)benzamido)-2,6-difluorobenzoic acid (4). An
amount of 8.5 mg LiOH (0.35 mmol) was added to a solution of compound 3i (90 mg,
0.17 mmol) in 10 THF/ MeOH (2: 1) mixture. The reaction mixture was allowed to stir
over the night at room temperature. The reaction mixture was diluted with water,
acidified with 10% HCl (pH = 4), extracted with ethyl acetate. The organic layer was
dried over MgSO and purified through flash chromatography (2% MeOH/CH Cl )
4
2
2
O
1
leading to the pure product as white solid. Yield = 62%. mp = 256.2 C. H NMR (400
MHz, DMSOꢀd ) δ 13.63 (s, 1H), 10.73 (s, 1H), 8.12 (s, 1H), 7.57 (s, 1H), 7.52 (d, J =
6
13
10.4 Hz, 2H), 4.01 (s, 3H), 3.62 (t, J = 4.66 Hz, 4H), 3.14 (t, J = 4.51 Hz, 4H). C NMR
(
101 MHz, DMSOꢀd ) δ 163.12, 161.79, 159.94, 135.47, 132.99, 126.54, 123.22,
6
+
1
4
16.17, 103.04, 102.74, 65.67, 57.40, 45.50. HRMS [C H N O F SCl ]: calcd
19 18
2
7 2
91.0491, found 491.0500.
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N-(2-Bromo-3,4,5-trifluorophenyl)-2,4-dichloro-5-(morpholinosulfonyl)benzamide
(5). The compound 5 was prepared following general procedure for the synthesis of 3
as well as 3aꢀ3i. The crude was purified in 1% MeOH/CH Cl yielding the pure product
2
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
1
as white solid. Yield = 76%. mp = 196.1 C. H NMR (400 MHz, DMSOꢀd ) δ 10.64 (s,
6
1H), 8.21 (s, 1H), 8.12 (s, 1H), 7.86 (m, 1H), 3.64 (t, J = 4.7 Hz, 4H), 3.23 (t, J = 4.7 Hz,
13
4
H). C NMR (101 MHz, DMSOꢀd ) δ 163.37, 135.82, 134.80, 134.02, 133.29, 133.07,
6
+
2
1
31.77, 111.61, 65.71, 45.46. HRMS [C H N O F SCl ]: calcd 546.9109, found
19
17
2
6
2
546.9113.
2
-(2,4-Dichloro-5-(morpholinosulfonyl)phenyl)-7-fluorobenzo[d]oxazole (7). To a
solution of 0.28g of compound 2 (0.82 mmol) in 20 mL of CH2Cl2, was added a volume
of 0.6 mL SOCl (8.2 mmol) and catalytic amount of DMF (2 drops) under argon
2
atmosphere. The reaction was allowed to sir for 12 hrs. The reaction mixture was
concentrated and dissolved back in 20 mL CH2Cl2. A volume of 0.2 mL pyridine (2.5
mmol) as well as an amount of 0.105g (0.82 mmol) 2ꢀaminoꢀ6ꢀfluorophenol was added
to the solution under argon atmosphere. The reaction mixture was allowed to stir over
the night at room temperature. The reaction mixture was diluted with 20 mL CH2Cl2 and
extracted with 10% HCl. The organic layer was dried over MgSO4 and concentrated.
The crude was dissolved in 25 mL toluene. An amount of 156 mg pꢀTSA (0.82 mmol)
was added to the solution under argon atmosphere. The reaction mixture was refluxed
over 12 hrs. The reaction was cooled down to room temperature, poured into water and
extracted with ethyl acetate. The organic layer was separated then washed with water,
brine, dried (MgSO ), filtered and concentrated. The crude product was purified by
4
column chromatography (silica gel, 30% ethyl acetate/hexanes) to afford the desired
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
1
product as an offꢀwhite solid. Yield = 77%. mp = 167.4 C. H NMR (400 MHz,
Chloroformꢀd) δ 8.87 (s, 1H), 7.80 (s, 1H), 7.66 (dd, J = 8.0, 0.9 Hz, 1H), 7.37 (td, J =
8
.2, 4.6 Hz, 1H), 7.21 (ddd, J = 9.9, 8.3, 0.9 Hz, 1H), 3.90 – 3.62 (m, 4H), 3.49 – 3.20
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
(
m, 4H). C NMR (101 MHz, CDCl ) δ 158.89, 148.30, 145.78, 144.63, 138.44, 137.84,
3
135.44, 134.80, 125.61, 124.72, 116.62 (d, J = 4.3 Hz), 113.09, 112.93, 66.51, 45.90.
+
2
HRMS [C H N O FSCl ]: calcd 431.0035, found 431.0045.
17
14
2
4
4
-((2,4-Dichloro-5-(5,6-difluoro-1H-benzo[d]imidazol-2-yl)phenyl)sulfonyl)-
morpholine (8). To a solution of 0.28g of compound 2 (0.82 mmol) in 20 mL of CH Cl ,
2
2
was added a volume of 0.6 mL SOCl (8.2 mmol) and catalytic amount of DMF (2 drops)
2
under argon atmosphere. The reaction was allowed to sir for 12 hrs. The reaction
mixture was concentrated and dissolved back in 20 mL CH Cl . A volume of 0.2 mL
2
2
pyridine (2.5 mmol) as well as an amount of 0.118g (0.82 mmol) 4, 5ꢀdifluorobenzeneꢀ
,2ꢀdiamine was added to the solution under argon atmosphere. The reaction mixture
1
was allowed to stir over the night at room temperature. The mixture was diluted with
dichloromethane (30ml) and washed with saturated aqueous sodium bicarbonate
solution (80ml). The organic phase was dried over MgSO and the solvent removed to
4
provide the amide as a mixture of isomers. The crude amide was combined with
phosphorus oxychloride (10ml) under an argon atmosphere, heated to reflux for 100
minutes, then allowed to cool down to room temperature. The mixture was poured into
iceꢀwater (100ml) and neutralized with 2N sodium hydroxide solution. Saturated
aqueous sodium bicarbonate solution (60ml) was added and the aqueous phase was
extracted with ethyl acetate (3 x 70ml). Water (150ml) was added to the combined
organic phases and the pH was adjusted to 3 with 2N aqueous hydrochloric acid. The
2
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organic layer was washed with brine (100ml), dried over MgSO and the solvent
4
removed to give a brown solid. The crude was purified by flash chromatography (35%
EtOAc/hexanes) to afford the desired product as light brown solid. Yield = 71%. mp =
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
1
1
75.2 C. H NMR (400 MHz, DMSOꢀd ) δ 8.51 (s, 1H), 8.19 (s, 1H), 7.83 – 7.71 (m,
6
13
2H), 3.64 (t, J = 4.8 Hz, 5H), 3.24 (dd, J = 5.9, 3.6 Hz, 4H). C NMR (101 MHz, DMSOꢀ
d6) δ 148.55, 148.41, 146.18, 136.45, 134.30, 134.20, 133.91, 132.88, 128.32, 65.71,
+
2
5
4.87, 45.48. HRMS [C H N O F SCl ]: calcd 448.0101, found 448.0106.
17
14
3
3
2
2,4-Dichloro-N-(3-fluorophenyl)-N-methyl-5-(morpholinosulfonyl)benzamide
The compound 9 was prepared following general procedure for the synthesis of 3 as
well as 3aꢀ3i. The crude was purified in 1% MeOH/CH Cl yielding the pure product as
(9).
2
2
O
1
white solid. Yield = 75%. mp = 128.7 C. H NMR (400 MHz, DMSOꢀd ) δ 8.16 – 7.72
6
(
m, 2H), 7.47 – 7.24 (m, 2H), 7.22 – 6.99 (m, 2H), 3.57 (t, J = 4.6 Hz, 4H), 3.40 (s, 3H),
13
2
1
4
.95 (bt, J = 5.2 Hz, 4H). C NMR (101 MHz, DMSOꢀd ) δ 164.16, 160.61, 144.17,
6
35.50, 135.09, 133.32, 133.03, 132.68, 132.05, 131.84, 130.86, 123.31, 114.57, 65.53,
+
2
5.47, 36.60. HRMS [C H N O FSCl ]: calcd 447.0348, found 447.0369.
18
18
2
4
N-(2,4-Dichloro-5-(morpholinosulfonyl)benzyl)-3,4-difluoroaniline (10). To
a
solution of compound 3a (0.15g, 0.33 mmol) in anhydrous THF (20 mL), a volume of 47
µL of BH .DMS (0.5 mmol) was added under argon atmosphere. The reaction mixture
3
was allowed to reflux over 72 hrs and reaction progress was monitored by TLC. The
reaction was quenched with 30 mL of water, extracted with ethyl acetate. The organic
layer was extracted with brine and dried over MgSO . The crude was purified through
4
flash chromatography (silica gel, 40% EtOAc/hexanes) providing access to the pure
O
1
product as white solid. Yield = 75%. mp = 154.8 C. H NMR (400 MHz, CDCl ) δ 7.89
3
2
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
5
6
(
s, 1H), 7.53 (s, 1H), 6.96 – 6.79 (m, 1H), 6.33 – 6.10 (m, 2H), 4.35 (d, J = 6.0 Hz, 2H),
13
4
.23 (t, J = 5.9 Hz, 1H), 3.58 (t, J = 4.44 Hz, 4H), 3.03 (t, J = 4.54 Hz, 4H). C NMR
(101 MHz, DMSOꢀd ) δ 145.36, 137.86, 136.88, 133.19, 132.53, 131.01, 130.11,
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
17.66, 117.48, 108.10, 100.73, 100.53, 65.50, 45.45, 43.83. HRMS
+
2
[C H N O3F SCl ]: calcd 437.0305, found 437.0307.
17
17
2
2
N-(2,4-Dichloro-5-(morpholinosulfonyl)benzyl)-3,4,5-trifluoroaniline (11). To
a
solution of compound 3b (0.15g, 0.32 mmol) in anhydrous THF (20 mL), a volume of 46
µL of BH .DMS (0.48 mmol) was added under argon atmosphere. The reaction mixture
3
was allowed to reflux over 24 hrs and reaction progress was monitored by TLC. The
reaction was quenched with 30 mL of water, extracted with ethyl acetate. The organic
layer was extracted with brine and dried over MgSO . The crude was purified through
4
flash chromatography (silica gel, 40% EtOAc/hexanes) providing access to the pure
O
1
product as white solid. Yield = 72%. mp = 149.4 C. H NMR (400 MHz, CDCl ) δ 7.89
3
(s, 1H), 7.54 (s, 1H), 6.16 – 5.94 (m, 2H), 4.31 (dd, J = 13.5, 5.0 Hz, 3H), 3.61 (t, J =
13
4
.84 Hz, 4H), 3.08 (t, J = 5.0 Hz, 4H). C NMR (101 MHz, DMSO) δ 152.26, 149.91,
144.61, 137.99, 136.42, 133.27, 132.60, 131.01, 130.33, 96.08, 95.84, 65.54, 45.47,
+
2
4
3.66. HRMS [C H N O3F SCl ]: calcd 455.0211, found 455.0206.
17
16
2
3
General procedure for the synthesis of compounds from 12-15. To a solution of 0.2
g (1 equivalent) 3, 3a, 3b, or 3g in toluene (20 mL), was Lawesson’s reagent (0.9
26
equivalent) under argon atmosphere as demonstrated in the literature. The reaction
was allowed to reflux over 12 hrs. The reaction was cooled down to room temperature.
The solvent was evaporated and the crude was purified through flash chromatography
(silica gel, 30%ꢀ35% EtOAc/hexanes) yielding the pure product as the light yellow solid.
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,4-Dichloro-N-(3-fluorophenyl)-5-(morpholinosulfonyl)benzothioamide (12). The
compound 12 was obtained following the general procedure for the preparation of
compounds 12ꢀ15. The crude was purified through flash column (silica gel, 30%
EtOAc/hexanes) leading to the pure product as the light yellow solid. Yield = 68%. mp =
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
1
181.0 C. H NMR (400 MHz, DMSOꢀd ) δ 12.45 (s, 1H), 8.11 (s, 1H), 8.06 (dt, J = 11.4,
6
2
.3 Hz, 1H), 8.00 (s, 1H), 7.71 – 7.64 (m, 1H), 7.53 (td, J = 8.2, 6.7 Hz, 1H), 7.19 (td, J
13
=
8.5, 1.8 Hz, 1H), 3.65 (t, J = 4.64 Hz, 4H), 3.21 (t, J = 4.72 Hz, 4H). C NMR (101
MHz, DMSOꢀd ) δ 141.97, 133.92, 133.71, 132.78, 131.68, 131.18, 130.68, 130.59,
6
+
2
1
18.73, 109.48, 109.23, 65.68, 45.51. HRMS [C H N O3FS Cl ]: calcd 448.9963,
17
16
2
2
found 448.9956.
2
,4-Dichloro-N-(3,4-difluorophenyl)-5-(morpholinosulfonyl)benzothioamide
(13).
The compound 13 was obtained following the general procedure for the preparation of
compounds 12ꢀ15. The crude was purified through flash column (silica gel, 32%
1
EtOAc/hexanes) leading to the pure product as the light yellow solid. Yield = 70%. H
NMR (400 MHz, CDCl ) δ 9.55 (s, 1H), 8.19 – 7.86 (m, 2H), 7.71ꢀ7.40 (m, 2H), 7.31 –
3
13
7.15 (m, 1H), 3.71 (bs, 4H), 3.23 (bs, 4H). C NMR (101 MHz, CDCl ) δ 192.37, 151.23
3
(
d, J = 13.4 Hz), 150.07 (d, J = 12.8 Hz), 148.76 (d, J = 13.4 Hz), 147.58 (d, J = 12.7
Hz), 141.70, 134.58, 134.44, 133.99, 133.39, 132.76, 132.39, 119.40 (d, J = 2.4 Hz),
+
2
117.69, 117.50, 112.99, 112.78, 66.31, 45.87 HRMS [C H N O F S Cl ]: calcd
.
17 15
2
3
2
2
4
66.9869, found 466.9869.
,4-Dichloro-5-(morpholinosulfonyl)-N-(3,4,5-trifluorophenyl)benzothioamide (14).
2
The compound 14 was obtained following the general procedure for the preparation of
compounds 12ꢀ15. The crude was purified through flash column (silica gel, 35%
2
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1
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1
1
1
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1
1
1
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2
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2
2
2
2
3
3
3
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3
3
3
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5
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5
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EtOAc/hexanes) leading to the pure product as the light yellow solid. Yield = 71%. mp =
O
1
1
52.1 C. H NMR (400 MHz, CDCl ) δ 9.41 (s, 1H), 8.05 (s, 1H), 7.68 (dd, J = 8.7, 6.2
3
13
Hz, 2H), 7.59 (s, 1H), 3.73 (t, J = 5.23 Hz, 4H), 3.25 (t, J = 5.23 Hz, 4H). C NMR (101
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MHz, CDCl ) δ 191.63, 151.41, 140.50, 133.31, 133.21, 132.36, 132.08, 131.50,
3
+
2
106.82, 106.57, 65.33, 44.87. HRMS [C H N O F S Cl ]: calcd 484.9775, found
17
14
2
3
3
2
4
84.9778.
,4-Dichloro-N-(3,5-dichloro-4-fluorophenyl)-5-(morpholinosulfonyl)-
2
benzothioamide (15). The compound 15 was obtained following the general procedure
for the preparation of compounds 12ꢀ15. The crude was purified through flash column
(silica gel, 35% EtOAc/hexanes) leading to the pure product as the light yellow solid.
O
1
Yield = 72%. mp = 175.2 C. H NMR (400 MHz, DMSOꢀd ) δ 12.51 (s, 1H), 8.17 (d, J =
6
6
4
4
.0 Hz, 2H), 8.11 (s, 1H), 8.05 (s, 1H), 3.65 (bt, J = 4.41 Hz, 4H), 3.21 (bt, J = 4.31 Hz,
13
H). C NMR (101 MHz, DMSOꢀd ) δ 192.75, 152.43, 149.96, 141.53, 135.81 (d, J =
6
.4 Hz), 134.01, 133.78, 132.83, 131.92, 131.20, 123.89, 121.07, 120.88, 65.68, 45.51.
+
4
HRMS [C H N O FS Cl ]: calcd 516.9184, found 516.9227.
17
14
2
3
2
2,4-Dichloro-5-nitrobenzene-1-sulfonyl chloride (16). Intermediate 16 was prepared
following the experimental procedure for the preparation of intermediate 1 starting with
5
g of 2,4ꢀdichloroꢀ1ꢀnitrobenzene (26 mmol). An amount of 5.7g (19.6 mmol, 75%) pure
1
product was obtained as off white solid upon drying under the vacuum. H NMR (400
MHz, DMSOꢀd ) δ 8.44 (s, 1H), 7.91 (s, 1H).
6
4
-((2,4-Dichloro-5-nitrophenyl)sulfonyl)morpholine (17). Intermediate 17 was
prepared following the general procedure for the preparation of intermediate 2 and 27
staring with 3.5g of intermediate 16 (12 mmol). An amount of 3.2g (9.3 mmol, 78%) pure
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product was obtained as the off white solid. H NMR (400 MHz, CDCl ) δ 8.61 (s, 1H),
3
7
.79 (s, 1H), 3.75 (t, J = 4.71 Hz, 4H), 3.35 (t, J = 4.31 Hz, 4H).
2
,4-Dichloro-5-(morpholinosulfonyl)aniline (18). The nitro intermediate 17 (3g, 8.8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
mmol) was dissolved in a mixture of EtOH and H O (40mL, 10:1). Powdered iron (2.25g,
2
35.2 mmol) and five drops of HCl (12 M) were added. The mixture was refluxed 5 hrs.
The mixture was cooled to room temperature and the solvent was evaporated. HCl (1N,
1
00 mL) was added and the mixture was extracted with EtOAc (100 mL). The organic
phase was extracted with brine, dried over MgSO4 and concentrated providing the pure
1
product as the light red solid (2.6g, 8.3 mmol, 95%). H NMR (400 MHz, DMSOꢀd ) δ
6
7
4
.58 (s, 1H), 7.41 (s, 1H), 6.04 (bs, 2H), 3.54 (t, J = 5.0 Hz, 4H), 3.13 (t, J = 4.86 Hz,
H).
Synthesis of compound 19 and 20. To a solution of 0.2g 18 (0.64 mmol) in 15 mL
CH Cl , was added Et N (267 µL, 1.9 mmol), 3ꢀfluorobenzenesulfonyl chloride (0.149g,
2
2
3
0
.77 mmol), and catalytic amount of DMAP under argon atmosphere. The reaction was
allowed to stir at room temperature over 96 hrs and monitored by TLC. Reaction led to
formation of two new spots as determined by TLC. The reaction mixture was diluted
with 30 mL CH Cl . Organic layer was extracted with 10% HCl, H O, brine, and
2
2
2
evaporated under reduced pressure. The crude with two spots were isolated by flash
chromatography (silica gel, 30%~45% EtOAc/hexanes) providing pure compound 19
1
(
yield = 33%) and 20 (yield = 60%) as white solids. Compound 19. H NMR (400 MHz,
CDCl ) δ 8.24 (s, 1H), 7.72 – 7.43 (m, 4H), 7.33 (tdd, J = 8.3, 2.5, 0.9 Hz, 1H), 3.74 (t, J
3
1
3
=
1
5.10 Hz, 4H), 3.28 (t, J = 5.04 Hz, 4H). C NMR (101 MHz, CDCl ) δ 163.71, 161.19,
3
40.34 (d, J = 6.8 Hz), 135.81, 132.61, 132.43, 131.42 (d, J = 7.8 Hz), 129.09, 128.41,
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