Design, synthesis, and in?vitro evaluation of BP-1-102 analogs with modified hydrophobic fragments for STAT3 inhibition

Article abstract of DOI:10.1080/14756366.2020.1871336

Twelve novel analogs of STAT3 inhibitor BP-1-102 were designed and synthesised with the aim to modify hydrophobic fragments of the molecules that are important for interaction with the STAT3 SH2 domain. The cytotoxic activity of the reference and novel co

Full text of DOI:10.1080/14756366.2020.1871336

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Design, synthesis, and in vitro evaluation of
BP-1-102 analogs with modified hydrophobic
fragments for STAT3 inhibition
Patrik Oleksak, Miroslav Psotka, Marketa Vancurova, Olena Sapega, Jana
Bieblova, Milan Reinis, David Rysanek, Romana Mikyskova, Katarina
Chalupova, David Malinak, Jana Svobodova, Rudolf Andrys, Helena
Rehulkova, Vojtech Skopek, Pham Ngoc Lam, Jiri Bartek, Zdenek Hodny &
Kamil Musilek
To cite this article: Patrik Oleksak, Miroslav Psotka, Marketa Vancurova, Olena Sapega, Jana
Bieblova, Milan Reinis, David Rysanek, Romana Mikyskova, Katarina Chalupova, David Malinak,
Jana Svobodova, Rudolf Andrys, Helena Rehulkova, Vojtech Skopek, Pham Ngoc Lam, Jiri Bartek,
Zdenek Hodny & Kamil Musilek (2021) Design, synthesis, and in vitro evaluation of BP-1-102
analogs with modified hydrophobic fragments for STAT3 inhibition, Journal of Enzyme Inhibition
and Medicinal Chemistry, 36:1, 410-424, DOI: 10.1080/14756366.2020.1871336
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2021, VOL. 36, NO. 1, 410–424
https://doi.org/10.1080/14756366.2020.1871336
RESEARCH PAPER
Design, synthesis, and in vitro evaluation of BP-1-102 analogs with modified
hydrophobic fragments for STAT3 inhibition
a
a
c
c
c
ꢀ
ꢀ
ꢀ
Patrik Oleksak , Miroslav Psotka
, Marketa Vancurovab , Olena Sapega , Jana Bieblova , Milan Reinis
,
David Rysanekb, Romana Mikyskovac, Katarina Chalupovaa , David Malinaka , Jana Svobodovaa
,
Rudolf Andrysa , Helena Rehulkovaa , Vojtech Skopeka, Pham Ngoc Lama, Jiri Bartekb,d, Zdenek Hodnyb
Kamil Musileka
and
b
aDepartment of Chemistry, Faculty of Science, University of Hradec Kralove, Hradec Kralove, Czech Republic; Department of Genome Integrity,
c
Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic; Laboratory of Immunological and Tumour Models,
d
Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic; Genome Integrity Unit, Danish Cancer Society
Research Center, Copenhagen, Denmark
ABSTRACT
ARTICLE HISTORY
Received 21 October 2020
Revised 21 December 2020
Accepted 28 December 2020
Twelve novel analogs of STAT3 inhibitor BP-1-102 were designed and synthesised with the aim to modify
hydrophobic fragments of the molecules that are important for interaction with the STAT3 SH2 domain.
The cytotoxic activity of the reference and novel compounds was evaluated using several human and two
mouse cancer cell lines. BP-1-102 and its two analogs emerged as effective cytotoxic agents and were fur-
ther tested in additional six human and two murine cancer cell lines, in all of which they manifested the
cytotoxic effect in a micromolar range. Reference compound S3I-201.1066 was found ineffective in all
tested cell lines, in contrast to formerly published data. The ability of selected BP-1-102 analogs to induce
apoptosis and inhibition of STAT3 receptor-mediated phosphorylation was confirmed. The structure–activ-
ity relationship confirmed a demand for two hydrophobic substituents, i.e. the pentafluorophenyl moiety
and another spatially bulky moiety, for effective cytotoxic activity and STAT3 inhibition.
KEYWORDS
STAT3 signalling pathway;
cancer; SH2 domain;
inhibitor; structure–activity
relationship
1. Introduction
cell cycle regulators MYC and CCND1 and anti-apoptotic BCL-2
family genes BCL2, BCL2L2 (BCL-W), BCL2L1 (BCL-XL), MCL1, and
BIRC5 (survivin; reviewed in refs.4,5).
Signal transducers and activators of transcription (STAT) signalling
pathways belong to signalling modules that are relatively simple
and straightforward: upon appropriate stimuli (binding of a poly-
peptide-based ligand), a plasma membrane receptor-associated
kinase phosphorylates, and thus activates (without use of a
second messenger) transcription factors of the STAT family to exe-
cute a specific biological function by regulating gene expression.
Human STAT transcription factors (STAT1, STAT2, STAT3, STAT4,
STAT5a, STAT5b, and STAT6) are involved in the response to auto-
crine or paracrine stimuli in a multitude of cellular functions such
as regulation of cell proliferation, apoptosis, differentiation, and
The signalling pathway executed by STAT3 (hence the STAT3
signalling pathway) attracts specific attention of pharmaceutical
research due to its involvement in human inflammatory and
malignant diseases. Under normal conditions, activation of the
STAT3 signalling pathway is transient due to a number of negative
regulators. However, STAT3 signalling was found to be constitu-
tively activated in several solid and haematological malignancies
(e.g. refs.6,7). In effect, STAT3 signalling contributes to all hallmarks
of cancer as defined by Hanahan and Weinberg8, including aber-
rant cell proliferation, regulation of cell death, tumour-promoting
inflammation, invasion and metastasis, immunosuppression,
genome instability, tumour metabolism, and treatment resist-
ance4,5,9,10. Constitutive STAT3 activation is associated with worse
prognosis, for instance, of patients with gliomas11,12. Due to this
functional role in malignant progression, the inhibition of activity
of STAT3 signalling is considered a potential target for cancer
stress response1–3
.
The STAT3 protein structure is characterised by four domains
involved in oligomerisation, binding to DNA, and transactivation
activity, and a Src homology 2 (SH2) domain mediating phosphor-
ylation-dependent dimerisation. The SH2 domain contains tyrosine
705 (Y705), which is phosphorylated in response to extracellular
signalling by kinases of the Jak and Src families. STAT3 proteins
with phosphorylated Y705 (pY705) form dimers via reciprocal
binding of SH2 domains and they are further translocated into the
nucleus to bind to specific DNA response elements. STAT3 are
treatment13,14
.
Current approaches to inhibiting the STAT3 signalling pathway
focus on several levels of the signalling module from inhibition of
both activators and repressors of hundreds of genes, including upstream activating kinase Jak1, inhibition of SH2 domain
CONTACT Kamil Musilek
kamil.musilek@uhk.cz
Department of Chemistry, Faculty of Science, University of Hradec Kralove, Rokitanskeho 62, Hradec Kralove
500 03, Czech Republic; Zdenek Hodny
142 20, Czech Republic
hodny@img.cas.cz Department of Genome Integrity, Institute of Molecular Genetics CAS, Videnska 1083, Prague 4 CZ
ꢀ
These authors contributed equally to this work.
Supplemental data for this article can be accessed here.
ß 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
411
Table 1. Prepared final products 1–14.
compartments. Therefore, it is possible to modify the hydrophobic
substituents by other moieties to plausibly decrease the hydro-
phobicity of the molecule and enhance the water/buffer solubility,
while at the same time evaluating the antitumor efficacy (Table 1).
First, the cyclohexylbenzyl moiety can be omitted or replaced by
less bulky hydrophobic fragments (e.g. t-butyl, methoxy, and tri-
fluoromethyl). Additionally, the pentafluorophenyl moiety can be
replaced by a tolyl fragment (similarly as in the effective STAT3
inhibitor S3I-201.106625, 2) or by mono-, di-, or trifluorophenyl
with very similar steric/spatial properties.
The physical–chemical properties (logP, logD7.4, and logS7.4) of the
designed molecules were predicted to depict differences from the par-
ent molecule 1 (Table 1). Using the proposed changes in hydrophobic
substituents, the logP values were usually decreased compared to
standards 1 or 2, reaching the optimal range for barrier penetrability
(ꢁ5)25 with the exception of molecules 5, 6, 10–12, and 14. In case of
logD, the values were decreased when compared to standards, again
with the exception of molecules 5, 6, 10–12, and 14, suggesting lower
lipid binding. Concerning the solubility prediction (logS7.4), the majority
of the proposed molecules, except for molecules 10 and 14, should be
at least slightly more soluble than the standard 1.
Compound
R1
R2
ClogP* ClogD7.4
*
ClogS7.4
*
Yield
BP-1-102 (1)
CyH
CyH
H
2,3,4,5,6-F-
4-CH3
6.91
5.76
4.92
4.94
5.46
6.41
4.76
4.89
5.08
5.35
5.37
5.28
5.07
5.45
3.77
2.62
1.78
1.8
−4.88
−2.63
−1.93
−3.74
−2.8
96 %
63 %
65 %
74 %
77 %
99 %
81 %
72 %
59 %
72 %
57 %
99 %
99 %
93 %
S3I-201.1066 (2)
3
4
4-CH3
H
2,3,4,5,6-F
4-CH3
5
t-Bu
t-Bu
OCH3
OCH3
CF3
CF3
CF3
CF3
CF3
CF3
2.32
3.27
1.62
1.75
1.94
2.21
2.23
2.14
1.93
2.31
6
2,3,4,5,6-F
2,4,6-F
2,3,4,5,6-F
-
−4.64
−2.64
−3.81
−2.67
−3.01
−2.76
−3.72
−3.81
−4.82
7
8
2.2. Synthesis of novel compounds
9
The synthetic strategy towards preparation of standards 1–2 and
designed p-aminosalicylic acid sulphonamides 3–14 was based on
the methodology reported by Page et al.26 The convergent syn-
thesis involved two main routes. Initial benzylation of p-aminosali-
cylic acid with BnBr and t-BuOK in DMF gives 59% yield of desired
dibenzylated derivative 15a, with tribenzylated 17c and mono-O-
benzylated 15b byproducts (Scheme 1). Dibenzylated amine 15a
was used for direct condensation with selected p-substituted ben-
zaldehydes 16a–e with subsequent reductive amination. These
conditions allowed preparation of a series of substituted second-
ary amines 17a–e, which represent the first coupling partners for
amide reaction. Substituted aldehyde 16a was prepared according
to formerly reported procedures27, other aldehydes 16b–e were
commercially purchased.
Sarcosine t-butyl ester hydrochloride was used as the starting
material for preparation of the carboxylic coupling partner
(Scheme 2). Sarcosine ester in reaction with selected benzenesul-
fonyl chlorides 18a–f provided corresponding sulphonamides
19a–f in good to excellent yields. The following acid hydrolysis
promoted with TFA in CH2Cl2 allowed preparation of desired sub-
stituted sulphonamide carboxylic acids 20a–f in excellent yields.
The substituted carboxylic acids 20a–f were the second partners
for the amide coupling reaction.
10
11
12
13
14
4-CH3
4-F
3,5-F
2,4,6-F
2,3,4,5,6-F
aClogP, ClogD7.4, and ClogS7.4 were calculated in ACDLabs PhysChemSuite 14.0.
phosphorylation and dimerisation, to inhibition of the transcrip-
tion-activating domain and DNA-binding activity15–22. However,
despite intensive efforts, currently there is no STAT3 inhibitor in
clinical practice. As summarised by Huang et al., the reasons for
that are multiple. The most unfavourable for development of
inhibitors targeting the SH2 domain is the fact that the pY705-
peptide binding site within the STAT3 SH2 domain spreads over
large, relatively flat surfaces and lacks well-defined deep binding
pockets. It makes the specific binding of the small molecular
inhibitors difficult. In addition, several direct STAT3 inhibitors are
also characterised by low aqueous solubility, low cell permeability
and poor oral bioavailability, which may hinder the drug develop-
ment and clinical studies23.
Among the small molecules, BP-1-102 was formerly found to
be a direct STAT3 inhibitor with reasonable in vivo tumour-inhibit-
ing activity and commercial availability. It was synthesised as an
analogue of S3I-201.1066 and it is proposed to bind specifically to
the STAT3 SH2 domain24.
In this study, novel analogs of commercially available STAT3
inhibitor BP-1-102 (1; Table 1) were designed, synthesised, and
their cytotoxic activity plus receptor-mediated STAT3 phosphoryl-
ation inhibitory ability were determined.
The microwave-assisted amide coupling28 between substituted
secondary amines 17a–e and substituted carboxylic acids 20a–f
was promoted with Ph3PCl2 and represents the key reaction
step29,30. Although yields of the coupling reactions were highly
variable (Table 1), provided amides 21a–n were the direct precur-
sors of the final products. Thus, final debenzylation catalysed with
5% Pd/C in CH3OH/THF (1:1) yielded target p-aminosalicylic acid
analogs 1–14 (Scheme 3). The structure of the final products was
characterised by NMR (Figures S1–S14) and HRMS analysis. The
purity was estimated using the HPLC method and was found to
be >95% for all final products.
2. Results and discussion
2.1. Molecular design and prediction of
physical–chemical properties
2.3. In vitro screening of cytotoxic effects
BP-1-102 (1) was found to be an effective inhibitor of STAT3 both Using BP-1-102 (1) and S3I-201.1066 (2) as reference compounds
in vitro and in vivo24, but with limited solubility in water/buffer with STAT3 inhibitory activity24,31, the cytotoxic effects of 12

412
P. OLEKSAK ET AL.
Scheme 1. Preparation of secondary amines 17a–e as the first coupling partners.
Scheme 2. Preparation of substituted carboxylic acids 20a–f as the second coupling partners.
Scheme 3. Amide coupling and deprotection leading to final products 1–14.
newly prepared BP-1-102 analogs (3–14) were compared using and 14 were cytotoxic starting from concentration 10 mM and
the human breast cancer MDA-MB-231 cell line with constitutive 20 mM, respectively (Figure 1(A)). Similar results were obtained
STAT3 signalling routinely used for testing of chemically induced with human immortalised retinal pigment epithelium RPE-1
STAT3 inhibition32,33. The cells were treated with the compounds (Figure 1(B)) and human glioblastoma U373 (Figure 1(C)) cell lines,
at concentrations 0, 5, 10, 20, and 50 mM for 24 h, and the cyto- which were exposed to BP-1-102 and to the novel analogs and
toxicity was estimated by the resazurin assay34. Apart from the analysed by the crystal violet assay. Again, compounds BP-1-102
compounds 3, 7–8, and 10–13 lacking any significant cytotoxicity, (1), 6 and 14 showed concentration-dependent cytotoxic activity.
the compounds 4 and 5 showed mild-to-negligible cytotoxicity in
To complement the resazurin assay, which estimates cell meta-
the tested concentration range. Note that the reference com- bolic activity as an indirect measure of the compound cytotoxicity,
pound S3I-201.1066 (2) did not show any observable cytotoxic cell death induction was confirmed by annexin V and propidium
effect in contrast to published data31. However, the compounds 6 iodide (PI) staining for apoptosis/necrosis and analysis by

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
413
Figure 1. The cytotoxic effect of newly synthesised compounds (concentration range 0–50 mM, 24 h) on breast cancer MDA-MB-231 (A), immortalised RPE-1 (B), and
glioblastoma U373 (C) cell lines was tested by the resazurin assay (A) or crystal violet staining (B, C). Data were normalised to control/untreated samples and plotted
as mean SD (n ꢂ 3).
fluorescence-activated flow cytometry (FACS) in MDA-MB-231 cells as 14.96 mM for BP-1-102 (1), 26.21 mM for 6, and 39.30 mM for 14
exposed to BP-1-102 (1), 6 and 14 at concentrations 0, 5, 10, 20, (Figure S15C). Thus, the compound 6 showed comparable inhibi-
and 50 mM for 24 h (Figure S15A–B).
To estimate IC50 of 6 and 14, MDA-MB-231 cells were treated
with 1 (0, 2, 5, 10, 15, 20, 25, 30, 40, and 50 mM), 6 (0, 5, 10, 20,
tory activity as the parent molecule BP-1-102 (1). Note that the
IC50 of BP-1-102 agreed with the previously estimated values24.
Further, the cytotoxicity and possible cell type-dependent dif-
25, 30, 40, 50, 70, and 100 mM), and 14 (0, 20, 40, 50, 60, 70, 80, ferences of the effective BP-1-102 analogs were tested in human
100, 150, and 200 mM) for 24 h. Cytotoxicity was estimated by the cancer cells of various origin. Cell lines derived from several
resazurin assay, and IC50 was calculated using a nonlinear regres- human solid malignancies, namely, human breast adenocarcinoma
sion module in GraphPad Prism (GraphPad Software, La Jolla, CA) MCF-7, cervix carcinoma HeLa, prostate carcinoma DU-145, and

414
P. OLEKSAK ET AL.
Figure 2. (A) The cytotoxic effect of BP-1-102 (1) and newly synthesised compounds 6 and 14 (concentration range 0–50 mM, 24 h) was tested on human prostate car-
cinoma DU-145, cervix carcinoma HeLa, breast adenocarcinoma MCF-7, and osteosarcoma U2-OS cell lines by the resazurin assay. (B) The cytotoxic effect of BP-1-102
(1) and newly synthesised compound 6 was tested on human glioblastoma U-87 and T98 cell lines using crystal violet staining. Data were normalised to control/
untreated samples and plotted as mean SD (n ¼ 3).
osteosarcoma U2-OS, were treated with the most potent com- decreased at lower concentrations, and the prostate carcinoma
pound 6 at concentrations 0, 5, 10, 20, and 50 mM for 24 h, and DU-145 cell line exhibited a weaker response to compound 14
(Figure 2(A)).
cytotoxicity was estimated by the resazurin assay. As shown in
Figure 2(A), all tested cell lines were sensitive to BP-1-102 (1) to a
comparable extent; nevertheless, HeLa and U2-OS cells were rela-
tively more sensitive to BP-1-102 (1) compared to cells with con-
stitutive STAT3 activity DU-145 and MCF-7. Compound 6 in the
DU-145, HeLa, MCF-7, and U2-OS cells manifested a similar or
slightly lower cytotoxic effect as the parent molecule BP-1-102 (1).
Furthermore, the cytotoxicity of compound 6 was compared to
BP-1-102 (1) in human glioblastoma cell lines U-87 and T98 in
concentration range 0–80 mM for 24 h using the crystal violet
assay. T98 cells, one of the most chemoresistant human glioblast-
oma cell lines, showed remarkable resistance to both BP-1-102 (1)
and 6 when compared to U-87 cells (Figure 2(B)). Altogether, the
novel analogue 6 manifested similar cytotoxic activity for human
cancer cells as parental compound BP-1-102.
To uncover the possible differences between mouse vs. human
cells, the whole set of compounds was screened in mouse lung
TC-1 and prostate TRAMP-C2 cancer cell lines using the MTT assay
(Figure S16). Similarly to human cells, compound 2 (S3I-201.1066)
did not demonstrate any cytotoxic effect in both murine cell lines,
whereas compounds 6 and 14 displayed cytotoxic activity com-
parable to BP-1-102 (1) (Figure 3) with IC50 summarised in Table
2. Additionally and in contrast to human cells, compound 8 mani-
fested a mild cytotoxic effect. Further, the cytotoxicity of the
selected compounds 1, 6, and 14 in murine cells was also con-
firmed by annexin V and PI staining for cell apoptosis/necrosis
and by FACS analysis (Figures S17–S18).
2.4. Inhibitory effect on STAT3 phosphorylation
The less potent compound 14 was tested in four human
cancer cell lines DU-145, HeLa, MCF-7, and U2-OS. Although the
The inhibitory effect of the selected compounds on receptor
cytotoxic effect on HeLa, MCF-7, and U2-OS was comparable to (JAK)-mediated phosphorylation of STAT3 were demonstrated in
BP-1-102 (1) and compound 6 at 50 mM, it was markedly MDA-MB-231 cells with constitutive STAT3 Y705 phosphorylation

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
415
Figure 3. The cytotoxic effect of BP-1-102 (1) and newly synthesised compounds 6 and 14 (concentration range 0–50 mM, 24h) was tested on mouse TC-1 lung and
TRAMP-C2 prostate cancer cell lines by the MTT assay. Data were normalised to control/untreated samples and plotted as mean SD (n ¼ 2).
Table 2. Antiproliferative activity of the tested compounds.
Compound
R1
R2
IC50 SD (mM)a
DU-145
IC50 SD (mM)b
TRAMP-C2
MDA-MB-231
MCF-7
RPE-1
TC-1
BP-1-102 (1)
S3I-201.1066 (2)
3
CyH
CyH
H
2,3,4,5,6- F
4-CH3
4-CH3
15.0 3.7
18.0 8.3
–
–
–
13.9 0.2
7.8 1.1
7.9 4.5
–
–
–
–
–
–
–
–
–
–
4
5
6
7
8
9
10
11
12
13
14
H
2,3,4,5,6-F
4-CH3
2,3,4,5,6-F
2,4,6-F
2,3,4,5,6-F
–
4-CH3
4-F
3,5-F
2,4,6-F
2,3,4,5,6-F
>50
–
26.2 5.6
>50
–
34.0 2.6
>50
–
19.6 3.6
>50
–
24.5 2.6
–
–
–
–
t-Bu
t-Bu
OCH3
OCH3
CF3
CF3
CF3
CF3
CF3
CF3
6.2 1.1
14.50 13.40
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
39.3 6.3
65.3 7.8
47.3 3.8
39.7 7.4
25.3 6.6
23.1 10.5
aThe inhibitory effect of the compounds on the proliferation of four human cell lines was determined by the resazurin assay: SD – standard deviation; data are the
mean SD from at least two independent experiments.
bThe inhibitory effect of the compounds on the proliferation of two mouse cell lines was determined by the MTT assay: SD – standard deviation; data are the
mean SD from at least two independent experiments.
(pY705). The cells were exposed to BP-1-102 (1), compounds 6
Additionally, replacement of the pentafluorophenyl moiety by
and 14 in a micromolar concentration range for 4 h, and then the mono-, di-, or trifluorophenyl led to complete loss of cytotoxic
activity irrespective of changes of the R1 substituent in com-
levels of STAT3 pY705 and total STAT3 were probed by immuno-
blotting with specific antibodies. As shown in Figure 4(A), both
the reference and two novel compounds inhibited constitutive
STAT3 phosphorylation. Similar effects of all three compounds
were also observed using mouse cell lines TC-1 and TRAMP-C2
(Figure 4(B); cytotoxic concentrations of individual drugs used in
these experiments were derived from annexin V/PI apoptosis ana-
lysis; see Figures S17–S18). It can be concluded that both novel
compounds 6 and 14 manifested inhibitory effects on receptor-
mediated phosphorylation of Y705 of STAT3 in both human and
mouse cell lines.
pounds 2–3, 5, 7, 9, and 10–13. Only one compound with the
intact pentafluorophenyl group (8) showed no cytotoxic activity in
human MDA-MB-231 and U373 cells, while mild cytotoxicity in
murine cells (Figure S16), indicating that the pentafluorophenyl
group highly contributes to the cytotoxic effect of the BP-1-102-
derived scaffold. Interestingly, our data obtained with compound
S3I-201.1066 (2) differ from the previously published studies. S3I-
201.1066 (2) was reported to affect viability of human breast can-
cer MDA-MB-231 and pancreatic cancer Panc-1 cell lines31.
Although our findings do not support that, as compound 2 did
not show any cytotoxic effect, they are still in line with the
importance of the pentafluorophenyl moiety for cytotoxic activity
and STAT3 inhibition.
2.5. Structure–activity relationship
Concerning prediction of the physical–chemical parameters,
the molecules (1, 6, 14) with increased logP, logD7.4 and
decreased logS7.4 were found to have better cytotoxic activity,
presuming these compounds to be well penetrable across the
membranes, but less soluble in the water/buffer environment.
Concerning the cytotoxic activity overall, hydrophobic substituents
with an optimal pentafluorophenyl moiety in the sulphonamidic
part of the molecule and a spatially bulky moiety in the benzyl
The structure–activity relationship was based on chemical modifi-
cation made in both hydrophobic regions and its correlation to
cytotoxic effects of the studied molecules. First, the cyclohexyl
moiety omission led to a decrease of cytotoxic activity (1>4), i.e.
increased IC50 values in the selected cell lines (Table 2; Figure 5).
The replacement of cyclohexyl by t-butyl or trifluoromethyl
resulted in higher cytotoxicity (decreased IC50) for all tested cell
lines, and spatially bulkier t-butyl was found twofold more potent
than trifluoromethyl, but still less effective than the original cyclo- part of the molecule are important prerequisites for the biological
hexyl moiety (1>6>14). effect of the compound.

416
P. OLEKSAK ET AL.
Figure 4. Human MDA-MB-231 (A) or mouse TC-1 and TRAMP-C2 (B) cells were treated with BP-1-102 (1) and two newly synthesised compounds 6 and 14 at the
given concentrations for 4 h. Whole cell lysates were subjected to SDS-PAGE/immunoblotting analysis and probed for pSTAT3 (Y705) and total STAT3. GAPDH was
used as a loading control.
Figure 5. Structure–activity relationship for the cytotoxicity of BP-1-102 and its analogs (1–14).
substituents, i.e. the pentafluorophenyl moiety and the second
spatially bulky moiety for effective cytotoxic activity and
STAT3 inhibition.
3. Conclusions
Twelve novel analogs of STAT3 inhibitor BP-1-102 were designed
with the aim to modify hydrophobic fragments of the molecule,
and these compounds were successfully synthesised. The cytotoxic
activity of reference compounds BP-1-102 (1) or S3I-201.1066 (2)
and the novel compounds was screened using two human and
two mouse cancer cell lines, and two potent inhibitors (6, 14)
were selected. The selected compounds were further tested in
detail in other six human and two murine cancer cell lines, in all
of which they manifested the cytotoxic effect in a micromolar
range. Their ability to induce apoptosis and inhibition of STAT3
receptor-mediated phosphorylation was confirmed. Notably, com-
pound S3I-201.1066 (2) emerged as ineffective for the growth
inhibition of the used cell lines. Importantly, it was found that
glioblastoma cell line T98, one of the most chemoresistant glio-
blastoma cell lines, is also resistant to BP-1-102 (1) and its most
effective analogue in this study, compound 6. The structure–activ-
4. Experimental
4.1. General synthetic methods
All used commercial reagents and solvents were purchased in the
highest available purity from supplier Sigma-Aldrich (St. Louis,
MO). Prepared compounds were purified by column chromatog-
raphy on silica gel Kieselgel 60 (0.040–0.063 mm, 230–400 mesh,
Merck, Kenilworth, NJ). Thin layer chromatography was performed
on Merck silica gel 60 F254 analytical plates, using a mobile phase
corresponding to the mobile phase used for the glass column
chromatography. Structures on TLC were detected using either UV
light (254 nm) or spraying with the detection reagent (10% solu-
ity relationship confirmed the demand for two hydrophobic tion of phosphomolybdic acid in MeOH) with subsequent heating.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
417
Microwave-assisted reactions were accomplished in a focussed 128.7, 131.8, 136.0, 153.6, 163.8, 169.9; ESI-MS [M þ H]þ calculated
þ
Discover Microwave System (CEM Corporation, Stallings, NC), and for C14H14NO3 (m/z): 244.1, found 244.2.
the contents of the vessel were cooled down rapidly by a stream
of compressed air. Melting points of solid compounds were
4.2.2. General procedure for preparation of substituted secondary
amine (17a–e)
€
recorded in a Melting Point Apparatus-Buchi M-565 and were
uncorrected. Electrosprayionisation mass spectrometry (ESI-MS)
was evaluated in an Agilent 6470 Triple Quadrupole mass spec-
trometer (Agilent Technologies, Palo Alto, CA) in a positive or
negative mode. High resolution mass spectrometry (HRMS) was
determined by a Q Exactive Plus hybrid quadrupole-orbitrap spec-
trometer (ThermoFisher, Bremen, Germany). NMR spectra were
recorded in CD3OD-d4, CDCl3-d, or DMSO-d6 solution at ambient
temperature in a Varian S500 spectrometer (499.87 MHz for 1H
and 125.71 MHz for 13C). Chemical shifts were recorded as d values
in parts per million (ppm), were referred to solvent signal (CD3OD-
d4: 3.31 ppm for 1H and 49.00 ppm for 13C; CDCl3-d: 7.26 ppm for
1H and 77.16 ppm for 13C; DMSO-d6: 2.50 ppm for 1H and
39.52 ppm for 13C), and spin multiplicities are given as s (singlet),
d (doublet), dd (doublet of doublets), t (triplet), tt (triplet of trip-
lets), or m (multiplet). Coupling constants (J) are given in hertz
(Hz). The purity of all the final products was ꢂ92%, based on LC
with UV detection (k ¼ 254 nm).
To a stirred solution of corresponding aldehyde 16a–e (1.70 mmol)
in anhydrous MeOH (13.3 ml; 13.3 ml/mmol) under N2 atmosphere,
4 Å molecular sieves were added followed by acetic acid (0.10 ml;
1.80 mmol). The mixture was stirred for 1 h at RT, the dibenzylated
amine 15a (1 mmol) was added, and the mixture was heated to
50 ꢃC for 3.5 h. The reaction mixture was cooled to RT, NaBH3CN
(157 mg; 2.50 mmol) was added, and the mixture was stirred for
40 h. The solvent was removed under reduced pressure, the resi-
due was dissolved in CH2Cl2, filtered through a glass frit, and con-
centrated under reduced pressure. The organic phase was
transferred to
a separatory funnel, then saturated aqueous
NaHCO3 (8 ml) and H2O (8 ml) were added for extraction. The
aqueous layer was extracted with two portions of CH2Cl2 (both
8 ml). Combined organic layers were washed with H2O (12 ml) and
brine (12 ml). After drying with Na2SO4, filtration and evaporation,
the residue was purified by glass column chromatography on sil-
ica gel in mobile phase heptane:EA:Et3N (5:1:2%) to give corre-
sponding secondary amine 17a–e.
Benzyl 2-(benzyloxy)-4-((4-cyclohexylbenzyl)amino)benzoate (17a).
Isolated in 73% as a white crystalline solid. Mp: 135.5 1 ꢃC; dH
(500 MHz, CDCl3-d): 1.21–1.31 (1H, m, Cy), 1.35–1.46 (4H, m, Cy),
1.73–1.79 (1H, m, Cy), 1.81–1.91 (4H, m, Cy), 2.47–2.54 (1H, m, Cy),
4.27–4.31 (2H, m, CH2), 4.42 (1H, bs, NH), 5.08 (2H, s, CH2(Bn)),
5.31 (2H, s, CH2(Bn)), 6.17 (1H, d, J ¼ 2.0 Hz, Ar), 6.21 (1H, dd,
J ¼ 8.6 Hz, J ¼ 2.0 Hz, Ar), 7.19 (2H, d, J ¼ 8.1 Hz, Ar), 7.24 (2H, d,
J ¼ 8.1 Hz, Ar), 7.26–7.35 (6H, m, Ar), 7.38–7.41 (2H, m, Ar),
7.43–7.46 (2H, m, Ar), 7.84 (1H, d, J ¼ 8.6 Hz, Ar); dC (126 MHz,
CDCl3-d): 26.3, 27.0, 34.6, 44.4, 47.7, 66.0, 70.6, 97.5, 105.1, 108.6,
127.1, 127.4, 127.7, 127.7, 127.9, 128.2, 128.5, 128.6, 134.5, 135.7,
137.0, 137.0, 147.7, 153.1, 161.1, 165.9; ESI-MS [M þ H]þ calculated
4.2. Chemical synthesis and characterisation of the compounds
4.2.1. General procedure for benzylation (15a–b)
t-BuOK (2.418 g; 21.55 mmol) was added to p-aminosalicylic acid
(3000 g; 19.59 mmol) dissolved under N2 atmosphere in dry DMF
(103 ml; 5.26 ml/mmol) cooled to 0 ꢃC. After 15 min, BnBr (2.56 ml;
21.55 mmol) was added dropwise, and the mixture was stirred for
another 15 min at 0 ꢃC. The mixture was then allowed to warm to
RT and stirred for 5.5 h. Further, the reaction mixture was again
cooled down to 0 ꢃC and a second portion of t-BuOK (2.418 g;
21.55 mmol) was added. After 15 min, a second portion of BnBr
(2.56 ml; 21.55 mmol) was added dropwise to the reaction. The
mixture was stirred for another 15 min at 0 ꢃC then allowed to
warm to RT and stirred for 17 h. The reaction mixture was cooled
to 0 ꢃC, and then saturated aqueous solution of NaHCO3 (50 ml)
and H2O (75 ml) were added. The mixture was transferred to a
separatory funnel, and the organic product was extracted to ethyl
acetate (EA) (3 ꢄ 20 ml). Organic layers were combined, washed
with H2O (75 ml), then with saturated aqueous NaCl (40 ml), and
dried with Na2SO4 and filtered. The organic solvent was removed
under reduced pressure, and the residue was purified by column
chromatography on silica gel in the mobile phase petrolether
(PE):EA:Et3N (4:1:2%) to produce the desired product 15a with tri-
benzylated 17c and monobenzylated 15b as byproducts.
þ
for C34H36NO3 (m/z): 506.3, found 506.8.
Benzyl 2-(benzyloxy)-4-((4-tert-butylbenzyl)amino)benzoate (17b).
Isolated in 83% as a pale-yellow crystalline solid. Mp: 91.0 1 ꢃC;
dH (500 MHz, CDCl3-d): 1.33 (9H, s, t-Bu), 4.30 (2H, d, J ¼ 5.3 Hz,
CH2), 4.42 (1H, t, J ¼ 5.3 Hz, NH), 5.09 (2H, s, CH2(Bn)), 5.31 (2H, s,
CH2(Bn)), 6.17 (1H, d, J ¼ 2.2 Hz, Ar), 6.22 (1H, dd, J ¼ 8.6 Hz,
J ¼ 2.2 Hz, Ar), 7.24–7.34 (8H, m, Ar), 7.36–7.41 (4H, m, Ar),
7.43–7.46 (2H, m, Ar), 7.84 (1H, d, J ¼ 8.6 Hz, Ar); dC (126 MHz,
CDCl3-d): 31.5, 34.7, 47.6, 66.0, 70.6, 97.4, 105.1, 108.6, 125.8,
127.1, 127.4, 127.8, 128.2, 128.5, 128.6, 134.5, 135.4, 137.0, 137.0,
150.8, 153.1, 161.1, 165.9; ESI-MS [M þ H]þ calculated for
þ
C32H34NO3 (m/z): 480.2, found 480.4.
Benzyl 4-amino-2-(benzyloxy)benzoate (15a). Isolated in 59%
Benzyl 4-(benzylamino)-2-(benzyloxy)benzoate (17c). Isolated in
70% yield as a orange solid. Mp: 74.5 1 ꢃC; dH (500 MHz, DMSO-
d6): 4.35 (2H, d, J ¼ 6.0 Hz, CH2), 5.06 (2H, s, CH2(Bn)), 5.21 (2H, s,
CH2(Bn)), 6.26 (1H, dd, J ¼ 8.7 Hz, J ¼ 1.9 Hz, Ar), 6.38 (1H, d,
J ¼ 1.9 Hz, Ar), 7.07–7.11 (1H, m, Ar), 7.22–7.27 (1H, m, Ar),
7.27–7.39 (11H, m, Ar), 7.43–7.46 (2H, m, Ar), 7.63 (1H, d,
J ¼ 8.7 Hz, Ar); dC (126 MHz, DMSO-d6): 46.0, 64.9, 69.3, 96.5, 104.6,
105.9, 126.9, 127.1, 127.3, 127.5, 127.6, 127.7, 128.0, 128.2, 128.3,
128.4, 133.5, 137.0, 137.0, 153.9, 160.3, 164.8; ESI-MS [M þ H]þ cal-
yield as
a
pale-yellow crystalline solid. Mp: 86.0 1 ꢃC; dH
(500 MHz, DMSO-d6): 5.07 (2H, s, CH2(Bn)), 5.20 (2H, s, CH2(Bn)),
5.96 (2H, s, NH2), 6.19 (1H, dd, J ¼ 8.6 Hz, J ¼ 1.9 Hz, Ar), 6.32 (1H,
d, J ¼ 1.9 Hz, Ar), 7.28–7.39 (8H, m, Ar), 7.46–7.49 (2H, m, Ar), 7.60
(1H, d, J ¼ 8.6 Hz, Ar); dC (126 MHz, DMSO-d6): 64.8, 69.1, 97.6,
105.5, 105.9, 127.0, 127.4, 127.6, 127.7, 128.2, 128.3, 133.7, 137.0,
137.1, 154.7, 160.4, 164.9; ESI-MS [M þ H]þ calculated for
þ
C21H20NO3 (m/z): 334.1, found 334.2.
þ
4-Amino-2-benzyloxybenzoic acid (15b). Isolated in 15% yield as
a pale-orange crystalline solid. Mp: 140.0 1 ꢃC; dH (500 MHz,
DMSO-d6): 5.29 (2H, s, CH2(Bn)), 6.01 (1H, d, J ¼ 2.2 Hz, Ar), 6.13
(1H, dd, J ¼ 8.8 Hz, J ¼ 2.2 Hz, Ar), 6.16 (2H, bs, NH2), 7.31–7.36 (1H,
culated for C28H26NO3 (m/z): 424.2, found 424.3.
Benzyl 2-(benzyloxy)-4-((4-trifluoromethylbenzyl)amino)benzoate
(17d). Isolated in 52% yield as a yellow crystalline solid. Mp:
88.0 1 ꢃC; dH (500 MHz, CDCl3-d): 4.40 (2H, d, J ¼ 5.7 Hz, CH2), 4.59
m, Ar), 7.38–7.45 (4H, m, Ar), 7.49 (1H, d, J ¼ 8.8 Hz, Ar), 10.7 (1H, (1H, t, J ¼ 5.7 Hz, NH), 5.06 (2H, s, CH2(Bn)), 5.30 (2H, s, CH2(Bn)),
s, COOH); dC (126 MHz, DMSO-d6): 66.3, 100.8, 106.9, 128.2, 128.4, 6.12 (1H, d, J ¼ 2.0 Hz, Ar), 6.18 (1H, dd, J ¼ 8.6 Hz, J ¼ 2.0 Hz, Ar),

418
P. OLEKSAK ET AL.
7.27–7.34 (6H, m, Ar), 7.37–7.42 (6H, m, Ar), 7.58 (2H, d, J ¼ 8.2 Hz, DMSO-d6): 27.5, 35.4, 51.1, 81.4, 116.4 (d, J ¼ 22.7 Hz), 130.1 (d,
Ar), 7.82 (1H, d, J ¼ 8.6 Hz, Ar); dC (126 MHz, CDCl3-d): 47.3, 64.6, J ¼ 9.6 Hz), 134.6 (d, J ¼ 2.9 Hz), 164.4 (d, J ¼ 251.4 Hz), 167.4; ESI-
66.1, 70.6, 97.7, 105.1, 109.3, 125.6 (q, J ¼ 3.8 Hz), 125.8 (q, MS [M þ H]þ calculated for C13H19FNO4Sþ (m/z): 304.1,
J ¼ 3.7 Hz), 127.0, 127.4, 127.8, 127.9, 128.2, 128.6, 128.6, 129.9 (q, found 304.1.
tert-Butyl 2-(N-methyl-N-(p-toluenesulfonamido)acetate (19e).
J ¼ 32.3 Hz), 134.5, 136.9, 142.7, 152.6, 161.0, 165.8; ESI-MS
þ
[M þ H]þ calculated for C29H25F3NO3 (m/z): 492.2, found 492.3.
Isolated in 98% yield as a yellow oil. dH (500 MHz, CDCl3-d): 1.30
Benzyl 2-(benzyloxy)-4-((4-methoxybenzyl)amino)benzoate (17e). (9H, s, t-Bu), 2.31 (3H, s, CH3(tol)), 2.77 (3H, s, CH3N), 3.75 (2H, s,
Isolated in 75% as a pale-yellow crystalline solid. Mp: 112.7 1 ꢃC; CH2), 7.19–7.23 (2H, m, Ar), 7.56–7.61 (2H, m, Ar); dC (126 MHz,
dH (500 MHz, CDCl3-d): 3.81 (3H, s, CH3O), 4.25 (2H, d, J ¼ 5.2 Hz, CHCl3-d): 21.3, 27.7, 35.4, 51.4, 81.8, 127.2, 129.5, 135.3, 143.3,
CH2), 4.41 (1H, t, J ¼ 5.2 Hz, NH), 5.09 (2H, s, CH2(Bn)), 5.31 (2H, s, 167.3; ESI-MS [M þ H]þ calculated for C14H22NO4Sþ (m/z): 300.1,
CH2(Bn)), 6.16 (1H, d, J ¼ 2.1 Hz, Ar), 6.21 (1H, dd, J ¼ 8.6 Hz, found 300.2.
J ¼ 2.1 Hz, Ar), 6.86–6.89 (2H, m, Ar), 7.22–7.25 (2H, m, Ar),
tert-Butyl 2-(N-methylphenylsulfonamido)acetate (19f). Isolated in
7.26–7.35 (6H, m, Ar), 7.38–7.41 (2H, m, Ar), 7.42–7.45 (2H, m, Ar), 98% yield as a white crystalline solid. Mp: 60.9 1 ꢃC; dH (500 MHz,
7.84 (1H, d, J ¼ 8.6 Hz, Ar); dC (126 MHz, CDCl3-d): 47.3, 55.4, 66.0, DMSO-d6): 1.34 (9H, s, t-Bu), 2.81 (3H, s, CH3N), 3.90 (2H, s, CH2),
70.6, 97.4, 105.1, 108.6, 114.3, 127.1, 127.7, 127.9, 128.2, 128.5, 7.58–7.63 (2H, m, Ar), 7.66–7.70 (1H, m, Ar), 7.77–7.81 (2H, m, Ar);
128.6, 128.9, 130.3, 134.5, 137.0, 137.0, 153.1, 159.2, 161.1, 165.9; dC (126 MHz, DMSO-d6): 27.5, 35.4, 51.1, 81.3, 126.9, 129.3, 132.8,
ESI-MS [M þ H]þ calculated for C29H28NO4
(m/z): 454.2, 138.1, 167.4; ESI-MS [M þ H]þ calculated for C13H20NO4Sþ (m/z):
þ
found 454.3.
286.1, found 286.1.
4.2.3. General procedure for preparation of substituted t-butyl sul- 4.2.4. General procedure for preparation of substituted carboxylic
phonamide acetates (19a–f)
acid (20a–f)
The solution of sarcosine t-butyl ester hydrochloride (182 mg; To a solution of substituted t-butyl ester sulphonamide acetates
1.00 mmol) was dissolved in anhydrous CH3CN (3.59 ml; 3.59 ml/ 19a–f (1 mmol) in CH2Cl2 (6.3 ml; 6.3 ml/mmol), TFA (6.3 ml; 6.3 ml/
mmol) under N2 atmosphere and the mixture was cooled to 0 ꢃC. mmol) was added and the mixture was stirred at RT for 3.5 h.
Further, DIPEA (0.44 ml; 2.50 mmol) was added and the mixture Solvent and TFA were then evaporated under reduced pressure,
was stirred at 0 ꢃC for 10 min. Corresponding substituted sulph- to give pure desired substituted acids 20a–f.
2-(2,3,4,5,6-Pentafluoro-N-methylphenylsulfonamido)acetic
acid
onyl chloride 18a–f (1.50 mmol) was added dropwise and the mix-
ture was stirred for 30 min at 0 ꢃC, then it was allowed to warm to
RT and stirred for 30 min. The reaction was quenched with H2O
(5 ml) and the product was extracted to EA (5 ml). The aqueous
layer was extracted with two portions of EA (both 5 ml). Organic
layers were combined, washed with saturated aqueous NaHCO3
(8 ml), then with brine (8 ml). The organic layer was dried with
Na2SO4, filtered and evaporated. The crude product was purified
by glass column chromatography on silica gel in mobile phase
PE:EA (20:1) to produce desired t-butyl esters 19a–f.
(20a). Isolated in quantitative yield as a white crystalline solid. Mp:
192.0 1 ꢃC; dH (500 MHz, DMSO-d6): 2.97 (3H, s, CH3N), 4.12 (2H, s,
CH2), 13.08 (1H, bs, COOH); dC (126 MHz, DMSO-d6): 35.2, 50.5,
136.3–138.7 (m), 142.0–144.4 (m), 143.1–145.4 (m), 169.5; ESI-MS
[M–H]– calculated for C9H5F5NO4S– (m/z): 318.0, found 318.0.
2-(2,4,6-Trifluoro-N-methylphenylsulfonamido)acetic acid (20b).
Isolated in 94% as a white crystalline solid. Mp: 166.7 1 ꢃC; dH
(500 MHz, DMSO-d6): 2.94 (3H, s, CH3N), 4.07 (2H, s, CH2),
7.40–7.46 (2H, m, Ar), 12.93 (1H, bs, COOH); dC (126 MHz, DMSO-
d6): 35.1, 50.3, 102.5 (td, J ¼ 28.8 Hz, J ¼ 3.7 Hz), 113.9 (td,
tert-Butyl 2-(2,3,4,5,6-pentafluoro-N-methylphenylsulfonamido)ace-
tate (19a). Isolated in 88% as a white crystalline solid. Mp ¼ J ¼ 17.5 Hz, J ¼ 5.4 Hz), 159.9 (ddd, J ¼ 256.6 Hz, J ¼ 16.2 Hz,
86.3 1 ꢃC; dH (500 MHz, DMSO-d6): 1.38 (9H, s, t-Bu), 2.98 (3H, s, J ¼ 7.1 Hz), 164.5 (dt, J ¼ 254.1 Hz, J ¼ 16.6 Hz), 169.6; ESI-MS
CH3N), 4.13 (2H, s, CH2); dC (126 MHz, DMSO-d6): 27.5, 35.2, 51.1, [M–H]– calculated for C9H7F3NO4S– (m/z): 282.0, found 282.1.
2-(3,5-Difluoro-N-methylphenylsulfonamido)acetic acid (20c).
82.0, 136.3–138.6 (m), 142.1–144.5 (m), 143.0–145.3 (m), 167.2; ESI-
MS [M–H]– calculated for C13H13F5NO4S– (m/z): 374.0, found 374.2.
Isolated in 95% as a grey crystalline solid. Mp: 133.1 1 ꢃC; dH
tert-Butyl
2-(2,4,6-trifluoro-N-methylphenylsulfonamido)acetate (500 MHz, DMSO-d6): 2.84 (3H, s, CH3N), 3.99 (2H, s, CH2),
(19b). Isolated in 98% yield as a yellow oil. dH (500 MHz, DMSO- 7.53–7.58 (2H, m, Ar), 7.63 (1H, tt, J ¼ 9.2 Hz, J ¼ 2.3 Hz, Ar), 12.89
d6): 1.35 (9H, s, t-Bu), 2.95 (3H, s, CH3N), 4.08 (2H, s, CH2), (1H, bs, COOH); dC (126 MHz, DMSO-d6): 35.5, 50.5, 108.6 (t,
7.41–7.46 (2H, m, Ar); dC (126 MHz, DMSO-d6): 27.5, 35.0, 50.8, J ¼ 25.8 Hz), 110.8–111.0 (m), 141.6 (t, J ¼ 8.5 Hz), 162.3 (dd,
81.7, 102.5 (td, J ¼ 26.5 Hz, J ¼ 3.6 Hz), 113.9 (td, J ¼ 17.0 Hz, J ¼ 251.2 Hz, J ¼ 12.4 Hz), 169.8; ESI-MS [M–H]– calculated for
J ¼ 5.1 Hz), 159.9 (ddd, J ¼ 256.7 Hz, J ¼ 16.2 Hz, J ¼ 7.0 Hz), 164.5 C9H8F2NO4S– (m/z): 264.0, found 264.1.
(dt, J ¼ 254.1 Hz, J ¼ 16.4 Hz), 167.2; ESI-MS [M þ H]þ calculated for
C13H17F3NO4Sþ (m/z): 340.1, found 340.1.
2-(4-Fluoro-N-methylphenylsulfonamido)acetic
acid
(20d).
Isolated in 99% yield as a white crystalline solid. Mp: 161.2 1 ꢃC;
tert-Butyl
2-(3,5-difluoro-N-methylphenylsulfonamido)acetate dH (500 MHz, DMSO-d6): 2.79 (3H, s, CH3N), 3.91 (2H, s, CH2),
(19c). Isolated in 95% as a white crystalline solid. Mp: 79.6 1 ꢃC; 7.41–7.46 (2H, m, Ar), 7.84–7.89 (2H, m, Ar), 12.82 (1H, m, COOH);
dH (500 MHz, DMSO-d6): 1.35 (9H, s, t-Bu), 2.86 (3H, s, CH3N), 4.01 dC (126 MHz, DMSO-d6): 35.5, 50.5, 116.4 (d, J ¼ 22.6 Hz), 130.1 (d,
(2H, s, CH2), 7.52–7.58 (2H, m, Ar), 7.65 (1H, tt, J ¼ 9.2 Hz, J ¼ 9.6 Hz), 134.5 (d, J ¼ 2.9 Hz), 164.5 (d, J ¼ 251.3 Hz), 169.8; ESI-
J ¼ 2.3 Hz, Ar); dC (126 MHz, DMSO-d6): 27.5, 35.5, 51.0, 81.5, 108.6 MS [M–H]– calculated for C9H9FNO4S– (m/z): 246.0, found 246.1.
2-(N-Methyl-N-(p-toluenesulfonamido)acetic acid (20e). Isolated
(t, J ¼ 25.8 Hz), 110.7–111.0 (m), 141.6 (t, J ¼ 8.6 Hz), 162.3 (dd,
J ¼ 251.3 Hz, J ¼ 12.4 Hz), 167.3; ESI-MS [M þ H]þ calculated for
in 99% yield as a brown crystalline solid. Mp: 149.0 1 ꢃC; dH
C13H18F2NO4Sþ (m/z): 322.1, found 322.1.
(500 MHz, DMSO-d6): 2.38 (3H, s, CH3(tol)), 2.75 (3H, s, CH3N), 3.85
tert-Butyl 2-(4-fluoro-N-methylphenylsulfonamido)acetate (19d). (2H, s, CH2), 7.38–7.42 (2H, m, Ar), 7.66–7.69 (2H, m, Ar), 12.71 (1H,
Isolated in 95% as a white crystalline solid. Mp: 94.5 1 ꢃC; dH bs, COOH); dC (126 MHz, DMSO-d6): 21.0, 35.5, 50.6, 127.1, 129.8,
(500 MHz, DMSO-d6): 1.34 (9H, s, t-Bu), 2.80 (3H, s, CH3N), 3.91 (2H, 135.0, 143.3, 169.9; ESI-MS [M–H]– calculated for C10H12NO4S– (m/
s, CH2), 7.42–7.47 (2H, m, Ar), 7.84–7.89 (2H, m, Ar); dC (126 MHz, z): 242.1, found 242.1.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
419
2-(N-Methylphenylsulfonamido)acetic acid (20f). Isolated in 97% m, Ar), 7.84 (1H, d, J ¼ 8.1 Hz, Ar); dC (126 MHz, CDCl3-d): 31.5, 34.7,
yield as a grey crystalline solid. Mp: 181.1 1 ꢃC; dH (500 MHz, 36.1, 52.2, 52.9, 67.3, 70.9, 114.2, 120.1, 121.4, 125.7, 127.3, 128.3,
DMSO-d6): 2.78 (3H, s, CH3N), 3.89 (2H, s, CH2), 7.58–7.63 (2H, m, 128.4, 128.4, 128.7, 128.7, 128.8, 133.2, 133.5, 135.8, 135.8,
Ar), 7.65–7.69 (1H, m, Ar), 7.78–7.81 (2H, m, Ar), 12.47 (1H, bs, 136.7–139.1 (m), 143.8–146.1 (m), 144.5, 151.2, 158.9, 165.4, 166.0;
COOH); dC (126 MHz, DMSO-d6): 35.6, 50.6, 127.0, 129.4, 132.9, ESI-MS [M þ H]þ calculated for C41H38F5N2O6Sþ (m/z): 781.2,
138.0, 169.9; ESI-MS [M–H]– calculated for C9H10NO4S– (m/z): 228.0, found 781.1.
Benzyl 2-(benzyloxy)-4-(N-(4-tert-butylbenzyl)-2-(N-methyl-N-(p-tol-
uenesulfonyl))acetamido)benzoate (21d). Isolated in 31% yield as a
yellow oil. dH (500 MHz, CDCl3-d): 1.29 (9H, s, t-Bu), 2.40 (3H, s,
CH3(tol)), 2.82 (3H, s, CH3N), 3.66 (2H, s, CH2), 4.76 (2H, s, CH2),
4.96 (2H, s, CH2(Bn)), 5.35 (2H, s, CH2(Bn)), 6.52–6.57 (1H, m, Ar),
6.69 (1H, dd, J ¼ 8.2 Hz, J ¼ 1.5 Hz, Ar), 7.04 (2H, d, J ¼ 8.2 Hz, Ar),
7.24–7.27 (2H, m, Ar), 7.27–7.35 (10H, m, Ar), 7.38–7.41 (2H, m, Ar),
7.61 (2H, d, J ¼ 8.2 Hz, Ar), 7.84 (1H, d, J ¼ 8.2 Hz, Ar); dC (126 MHz,
CDCl3-d): 21.7, 31.5, 34.7, 36.1, 51.5, 52.9, 67.2, 70.9, 114.4, 120.2,
120.8, 125.6, 125.8, 127.2, 127.6, 128.2, 128.4, 128.4, 128.7, 128.7,
129.6, 133.3, 133.7, 135.5, 135.9, 135.9, 143.4, 145.3, 150.9, 158.9,
165.5, 166.9; ESI-MS [M þ H]þ calculated for C42H45N2O6Sþ (m/z):
705.3, found 705.4.
Benzyl 2-(benzyloxy)-4-(N-(benzyl)-2-(2,3,4,5,6-pentafluoro-N-meth-
ylphenylsulfonamido)acetamido)benzoate (21e). Isolated in 32%
yield as a yellow oil. dH (500 MHz, CDCl3-d): 3.05 (3H, s, CH3N),
3.87 (2H, s, CH2), 4.71 (2H, s, CH2), 4.98 (2H, s, CH2(Bn)), 5.35 (2H,
s, CH2(Bn)), 6.47–6.51 (1H, m, Ar), 6.62 (1H, dd, J ¼ 8.2 Hz,
J ¼ 1.4 Hz, Ar), 7.00–7.04 (2H, m, Ar), 7.25–7.29 (3H, m, Ar),
7.29–7.36 (8H, m, Ar), 7.37–7.40 (2H, m, Ar), 7.82 (1H, d, J ¼ 8.2 Hz,
Ar); dC (126 MHz, CDCl3-d): 36.1, 52.2, 53.2, 67.3, 70.9, 114.1, 120.1,
121.5, 127.2, 128.1, 128.3, 128.4, 128.7, 128.8, 128.8, 128.9, 133.5,
135.8, 135.8, 136.2, 136.6–139.0 (m), 143.7–146.1 (m), 144.3, 159.0,
165.4, 166.2; ESI-MS [M þ H]þ calculated for C37H30F5N2O6Sþ (m/z):
725.2, found 725.3.
Benzyl 2-(benzyloxy)-4-(N-(benzyl)-2-(N-methyl-N-(p-toluenesulfo-
nyl))acetamido)benzoate (21f). Isolated in 19% yield as a yellow oil.
dH (500 MHz, CDCl3-d): 2.40 (3H, s, CH3(tol)), 2.82 (3H, s, CH3N),
3.67 (2H, s, CH2), 4.79 (2H, s, CH2), 4.99 (2H, s, CH2(Bn)), 5.35 (2H,
s, CH2(Bn)), 6.55–6.58 (1H, m, Ar), 6.65 (1H, dd, J ¼ 8.2 Hz,
J ¼ 1.2 Hz, Ar), 7.07–7.12 (2H, m, Ar), 7.24–7.28 (5H, m, Ar),
7.28–7.36 (8H, m, Ar), 7.38–7.41 (2H, m, Ar), 7.60–7.62 (2H, m, Ar),
7.82 (1H, d, J ¼ 8.2 Hz, Ar); dC (126 MHz, CDCl3-d): 21.7, 36.1, 51.5,
53.2, 67.2, 70.9, 114.3, 120.2, 127.2, 127.6, 127.9, 128.2, 128.4,
128.4, 128.7, 128.8, 129.0, 129.6, 133.3, 135.5, 135.9, 136.0, 136.7,
143.5, 145.1, 158.9, 165.5, 167.0; ESI-MS [M þ H]þ calculated for
C38H37N2O6Sþ (m/z): 649.2, found 649.3.
found 228.1.
4.2.5. General procedure for the key amide coupling reac-
tion (21a–n)
Selected acid 20a–f (0.8 mmol) and Ph3PCl2 (666 mg; 2.00 mmol)
were dissolved under N2 atmosphere in anhydrous CHCl3 (3.57 ml;
7.13 ml/mmol) in a 35 ml microwave pressure vessel. The reaction
mixture was stirred for 1 h at RT, then the selected secondary
amine 17a–e (0.5 mmol) was quantitatively transferred to the reac-
tion in anhydrous CHCl3 (3.57 ml; 7.13 ml/mmol). The pressure vial
was transferred to a microwave reactor and heated to 65 ꢃC for
1.5 h. The mixture was transferred to a separatory funnel, then
saturated aqueous NaHCO3 (4 ml) and H2O (4 ml) were added.
After extraction and separation, the aqueous phase was extracted
with two portions of CHCl3 (both 5 ml). Organic phases were com-
bined, washed with saturated aqueous NaHCO3 (7 ml) and brine
(7 ml), dried with Na2SO4, filtered and evaporated. The residue
was purified by column glass chromatography on silica gel in
mobile phase heptane:EA (3:1) to produce desired debenzylated
amides 21a–n.
Benzyl 2-(benzyloxy)-4-(N-(4-cyclohexylbenzyl)-2-(2,3,4,5,6-penta-
fluoro-N-methylphenylsulfonamido)acetamido)benzoate
(21a).
Isolated in 58% yield as a white crystalline solid. Mp: 56.5 1 ꢃC;
dH (500 MHz, CDCl3-d): 1.19–1.29 (1H, m, Cy), 1.32–1.43 (4H, m,
Cy), 1.71–1.77 (1H, m, Cy), 1.78–1.87 (4H, m, Cy), 2.43–2.50 (1H, m,
Cy), 3.05 (3H, s, CH3N), 3.85 (2H, s, CH2), 4.67 (2H, s, CH2), 4.94 (2H,
s, CH2(Bn)), 5.35 (2H, s, CH2(Bn)), 6.42–6.45 (1H, m, Ar), 6.65 (1H,
dd, J ¼ 8.2 Hz, J ¼ 1.6 Hz, Ar), 6.94 (2H, d, J ¼ 8.0 Hz, Ar), 7.11 (2H,
d, J ¼ 8.0 Hz, Ar), 7.28–7.36 (8H, m, Ar), 7.37–7.40 (2H, m, Ar), 7.83
(1H, d, J ¼ 8.2 Hz, Ar); dC (126 MHz, CDCl3-d): 26.2, 27.0, 34.6, 36.1,
44.4, 52.2, 53.0, 67.2, 70.9, 114.2, 120.1, 121.4, 127.2, 127.3, 128.3,
128.4, 128.4, 128.7, 128.8, 129.0, 133.5, 133.6, 135.8, 135.8,
136.6–139.1 (m), 143.8–146.1 (m), 144.4, 148.1, 158.9, 165.4, 166.0;
ESI-MS [M þ H]þ calculated for C43H40F5N2O6Sþ (m/z): 807.2,
found 807.4.
Benzyl 2-(benzyloxy)-4-(N-(4-cyclohexylbenzyl)-2-(N-methyl-N-(p-
toluenesulfonyl))acetamido)benzoate (21b). Isolated in 28% as a yel-
low oil. dH (500 MHz, CDCl3-d): 1.23–1.26 (1H, m, Cy), 1.33–1.38
(4H, m, Cy), 1.70–1.74 (1H, m, Cy), 1.78–1.84 (4H, m, Cy), 2.39 (3H,
s, CH3(tol.)), 2.42–2.48 (1H, m, Cy), 2.80 (3H, s, CH3N), 3.64 (2H, s,
CH2), 4.74 (2H, s, CH2), 4.93 (2H, s, CH2(Bn)), 5.34 (2H, s, CH2(Bn)),
6.48–6.51 (1H, m, Ar), 6.66 (1H, dd, J ¼ 8.2 Hz, J ¼ 1.2 Hz, Ar), 7.00
(2H, d, J ¼ 8.0 Hz, Ar), 7.09 (2H, d, J ¼ 8.0 Hz, Ar), 7.23–7.25 (2H, m,
Ar), 7.27–7.34 (8H, m, Ar), 7.37–7.39 (2H, m, Ar), 7.60 (2H, d,
Benzyl
2-(benzyloxy)-4-(N-(4-trifluoromethylbenzyl)-2-(2,3,4,5,6-
pentafluoro-N-methylphenylsulfonamido)acetamido)benzoate (21g).
Isolated in 66% yield as a yellow oil. dH (500 MHz, CDCl3-d): 3.05
(3H, s, CH3N), 3.90 (2H, s, CH2), 4.74 (2H, s, CH2), 5.05 (2H, s,
CH2(Bn)), 5.36 (2H, s, CH2(Bn)), 6.54 (1H, d, J ¼ 1.7 Hz, Ar), 6.63 (1H,
dd, J ¼ 8.2 Hz, J ¼ 1.7 Hz, Ar), 7.15 (2H, d, J ¼ 8.0 Hz, Ar), 7.31–7.36
(8H, m, Ar), 7.37–7.41 (2H, m, Ar), 7.52 (2H, d, J ¼ 8.0 Hz, Ar), 7.84
(1H, d, J ¼ 8.0 Hz, Ar); dC (126 MHz, CDCl3-d): 36.1, 52.1, 52.9, 67.3,
J ¼ 8.2 Hz, Ar), 7.82 (1H, d, J ¼ 8.2 Hz, Ar); dC (126 MHz, CDCl3-d): 71.0, 113.8, 119.9, 121.8, 125.8 (q, J ¼ 3.8 Hz), 127.1, 128.4, 128.5,
21.7, 26.2, 27.0, 34.6, 36.1, 44.4, 51.5, 53.0, 67.2, 70.9, 127.1, 127.3, 128.5, 128.7, 128.9, 129.1, 130.4 (q, J ¼ 32.5 Hz), 133.7, 135.6, 135.7,
127.7, 128.2, 128.4, 128.4, 128.7, 128.8, 129.1, 129.6, 133.3, 134.1, 136.7–139.1 (m), 140.1, 143.7–146.0 (m), 144.1, 159.1, 165.3, 166.5;
135.9, 136.0, 143.5, 147.9, 158.9, 165.6, 166.9; ESI-MS [M þ H]þ cal- ESI-MS [M þ H]þ calculated for C38H29F8N2O6Sþ (m/z): 793.2,
culated for C44H47N2O6Sþ (m/z): 731.3, found 731.5.
found 793.3.
Benzyl
2-(benzyloxy)-4-(N-(4-tert-butylbenzyl)-2-(2,3,4,5,6-penta-
Benzyl
2-(benzyloxy)-4-(N-(4-trifluoromethylbenzyl)-2-(2,4,6-tri-
fluoro-N-methylphenylsulfonamido)acetamido)benzoate
(21c). fluoro-N-methylphenylsulfonamido)acetamido)benzoate
(21h).
Isolated in 68% as a white crystalline solid. Mp: 188.3 1 ꢃC; dH Isolated in 18% yield as a yellow oil. dH (500 MHz, CDCl3-d): 3.05
(500 MHz, CDCl3-d): 1.30 (9H, s, t-Bu), 3.05 (3H, s, CH3N), 3.86 (2H, (3H, s, CH3N), 3.87 (2H, s, CH2), 4.77 (2H, s, CH2), 5.05 (2H, s,
s, CH2), 4.68 (2H, s, CH2), 4.95 (2H, s, CH2(Bn)), 5.35 (2H, s, CH2(Bn)), 5.35 (2H, s, CH2(Bn)), 6.58 (1H, d, J ¼ 1.5 Hz, Ar), 6.65 (1H,
CH2(Bn)), 6.47 (1H, s, Ar), 6.66 (1H, dd, J ¼ 8.2 Hz, J ¼ 1.5 Hz, Ar), dd, J ¼ 8.2 Hz, J ¼ 1.5 Hz, Ar), 6.67–6.72 (2H, m, Ar), 7.18 (2H, d,
6.96 (2H, d, J ¼ 8.1 Hz, Ar), 7.28–7.36 (10H, m, Ar), 7.37–7.40 (2H, J ¼ 8.1 Hz, Ar), 7.30–7.36 (8H, m, Ar), 7.38–7.41 (2H, m, Ar), 7.51

420
P. OLEKSAK ET AL.
(2H, d, J ¼ 8.1 Hz, Ar), 7.81 (1H, d, J ¼ 8.2 Hz, Ar); dC (126 MHz,
Benzyl 2-(benzyloxy)-4-(N-(4-methoxybenzyl)-2-(2,4,6-trifluoro-N-
CDCl3-d): 36.1, 51.7, 52.9, 67.3, 71.0, 101.7–102.2 (m), 113.9, 119.9, methylphenylsulfonamido)acetamido)benzoate (21m). Isolated in
121.6, 124.1 (q, J ¼ 271.9 Hz), 125.7 (q, J ¼ 3.7 Hz), 127.1, 128.3, 51% as a yellow oil. dH (500 MHz, CDCl3-d): 3.05 (3H, s, CH3N), 3.78
128.5, 128.7, 128.8, 129.2, 130.2 (q, J ¼ 32.5 Hz), 133.6, 135.8, 135.8,
(3H, s, CH3O), 3.83 (2H, s, CH2), 4.66 (2H, s, CH2), 5.02 (2H, s,
140.5, 144.6, 159.1, 160.8 (ddd, J ¼ 259.0 Hz, J ¼ 15.4 Hz, J ¼ 7.1 Hz),
CH2(Bn)), 5.35 (2H, s, CH2(Bn)), 6.53–6.55 (1H, m, Ar), 6.62 (1H, dd,
164.9 (dt, J ¼ 31.7 Hz, J ¼ 15.6 Hz), 165.4, 166.9; ESI-MS [M þ H]þ
J ¼ 8.2 Hz, J ¼ 1.7 Hz, Ar), 6.69–6.75 (2H, m, Ar), 6.75–6.79 (2H, m,
Ar), 6.93–6.97 (2H, m, Ar), 7.28–7.37 (8H, m, Ar), 7.38–7.40 (2H, m,
Ar), 7.82 (1H, d, J ¼ 8.2 Hz, Ar); dC (126 MHz, CDCl3-d): 36.1, 51.8,
52.6, 55.4, 67.2, 71.0, 101.7–102.2 (m), 114.0, 120.3, 127.2, 128.2,
128.4, 128.4, 128.6, 128.7, 128.8, 130.4, 133.4, 135.9, 135.9, 144.8,
159.0, 159.4, 165.5, 166.4; ESI-MS [M þ H]þ calculated for
C38H34F3N2O7Sþ (m/z): 719.2, found 719.3.
calculated for C38H31F6N2O6Sþ (m/z): 757.2, found 757.1.
Benzyl 2-(benzyloxy)-4-(N-(4-trifluoromethylbenzyl)-2-(3,5-difluoro-
N-methylphenylsulfonamido)acetamido)benzoate (21i). Isolated in
21% yield as a yellow oil. dH (500 MHz, CDCl3-d): 2.89 (3H, s,
CH3N), 3.72 (2H, s, CH2), 4.79 (2H, s, CH2), 5.05 (2H, s, CH2(Bn)),
5.36 (2H, s, CH2(Bn)), 6.53–6.55 (1H, m, Ar), 6.63 (1H, dd, J ¼ 8.2 Hz,
J ¼ 1.7 Hz, Ar), 7.02 (1H, tt, J ¼ 8.5 Hz, J ¼ 2.3 Hz, Ar), 7.19 (2H, d,
J ¼ 8.1 Hz, Ar), 7.28–7.36 (10H, m, Ar), 7.38–7.41 (2H, m, Ar), 7.53
(2H, d, J ¼ 8.1 Hz, Ar), 7.84 (1H, d, J ¼ 8.2 Hz, Ar); dC (126 MHz,
CDCl3-d): 36.3, 51.6, 52.9, 67.3, 71.0, 108.4 (t, J ¼ 25.1 Hz),
111.0–111.3 (m), 113.9, 119.9, 121.6, 124.1 (q, J ¼ 272.0 Hz), 125.8
(q, J ¼ 3.6 Hz), 127.1, 128.3, 128.4, 128.7, 128.9, 129.2, 130.3 (q,
J ¼ 32.5 Hz), 133.6, 135.7, 135.8, 140.5, 142.3 (t, J ¼ 8.4 Hz), 144.5,
159.1, 162.8 (dd, J ¼ 254.0 Hz, J ¼ 11.6 Hz), 165.3, 166.9, 171.3; ESI-
MS [M þ H]þ calculated for C38H32F5N2O6Sþ (m/z): 739.2,
found 739.2.
Benzyl 2-(benzyloxy)-4-(N-(4-trifluoromethylbenzyl)-2-(4-fluoro-N-
methylphenylsulfonamido)acetamido)benzoate (21j). Isolated in 21%
yield as a yellow oil. dH (500 MHz, CDCl3-d): 2.83 (3H, s, CH3N),
3.70 (2H, s, CH2), 4.81 (2H, s, CH2), 5.05 (2H, s, CH2(Bn)), 5.36 (2H,
s, CH2(Bn)), 6.58–6.60 (1H, m, Ar), 6.65 (1H, d, J ¼ 8.1 Hz, J ¼ 1.7 Hz,
Ar), 7.11–7.15 (2H, m, Ar), 7.21 (2H, d, J ¼ 8.2 Hz, Ar), 7.28–7.36
(10H, m, Ar), 7.38–7.42 (2H, m, Ar), 7.52 (2H, d, J ¼ 8.2 Hz, Ar), 7.84
(1H, d, J ¼ 8.1 Hz, Ar); dC (126 MHz, CDCl3-d): 36.2, 51.5, 53.0, 67.3,
71.0, 114.0, 116.2 (d, J ¼ 22.5 Hz), 125.7 (q, J ¼ 3.6 Hz), 127.1, 127.4,
128.3, 128.5, 128.6, 128.7, 128.8, 129.2, 130.3 (d, J ¼ 9.3 Hz), 133.5,
135.8, 140.6, 142.1, 144.8, 165.2 (d, J ¼ 254.7 Hz), 165.4, 165.9,
167.3; ESI-MS [M þ H]þ calculated for C38H33F4N2O6Sþ (m/z): 721.2,
found 721.2.
Benzyl 2-(benzyloxy)-4-(N-(4-trifluoromethylbenzyl)-2-(N-methyl-N-
(p-toluenesulfonyl))acetamido)benzoate (21k). Isolated in 50% yield
as a yellow oil. dH (500 MHz, CDCl3-d): 2.40 (3H, s, CH3(tol)), 2.82
(3H, s, CH3N), 3.67 (2H, s, CH2), 4.83 (2H, s, CH2), 5.06 (2H, s,
CH2(Bn)), 5.36 (2H, s, CH2(Bn)), 6.61–6.62 (1H, m, Ar), 6.65 (1H, dd,
J ¼ 8.2 Hz, J ¼ 1.8 Hz, Ar), 7.22 (2H, d, J ¼ 8.1 Hz, Ar), 7.25 (2H, d,
J ¼ 8.3 Hz, Ar), 7.28–7.36 (8H, m, Ar), 7.38–7.42 (2H, m, Ar), 7.52
(2H, d, J ¼ 8.1 Hz, Ar), 7.60 (2H, d, J ¼ 8.3 Hz, Ar), 7.84 (1H, d,
J ¼ 8.2 Hz, Ar); dC (126 MHz, CDCl3-d): 21.6, 36.3, 51.5, 52.9, 67.2,
70.9, 114.1, 119.9, 124.1 (q, J ¼ 271.4 Hz), 125.6 (q, J ¼ 3.6 Hz),
127.1, 127.6, 128.2, 128.4, 128.7, 128.8, 129.2, 129.7, 130.1 (q,
J ¼ 32.6 Hz), 133.4, 135.8, 140.7, 143.6, 144.9, 159.0, 165.4, 167.4;
ESI-MS [M þ H]þ calculated for C39H36F3N2O6Sþ (m/z): 717.2,
found 717.3.
Benzyl
2-(benzyloxy)-4-(N-(4-methoxybenzyl)-2-(2,3,4,5,6-penta-
(21n).
fluoro-N-methylphenylsulfonamido)acetamido)benzoate
Isolated in 42% yield as a yellow oil. dH (500 MHz, CDCl3-d): 3.05
(3H, s, CH3N), 3.78 (3H, s, CH3O), 3.85 (2H, s, CH2), 4.64 (2H, s,
CH2), 5.02 (2H, s, CH2(Bn)), 5.35 (2H, s, CH2(Bn)), 6.48–6.51 (1H, m,
Ar), 6.59 (1H, dd, J ¼ 8.1 Hz, J ¼ 1.2 Hz, Ar), 6.78 (2H, d, J ¼ 8.5 Hz,
Ar), 6.93 (2H, d, J ¼ 8.5 Hz, Ar), 7.29–7.37 (8H, m, Ar), 7.38–7.41 (2H,
m, Ar), 7.82 (1H, d, J ¼ 8.1 Hz, Ar); dC (126 MHz, CDCl3-d): 36.2, 52.2,
52.6, 55.4, 67.3, 71.0, 114.1, 114.2, 120.2, 127.2, 128.3, 128.4, 128.4,
128.7, 128.8, 130.3, 133.5, 135.8, 144.3, 159.0, 159.5, 165.4, 166.0;
ESI-MS [M þ H]þ calculated for C38H32F5N2O7Sþ (m/z): 755.2,
found 755.3.
4.2.6. General procedure for preparation of final products (1–14)
To a stirring mixture of 5% Pd/C (200 mg; 10 mg/mmol) in dry
MeOH (16 ml; 16 ml/mmol) in a two-neck round bottom flask con-
nected to the Schlenk apparatus, dibenzylated amide 21a–n
(1 mmol) in dry THF (16 ml; 16 ml/mmol) was quantitatively trans-
ferred under N2 atmosphere. The mixture was evacuated, and
then H2 was introduced into the flask. The mixture was stirred for
3 h at RT, then filtered through a column of celite on a glass frit
and washed with MeOH. The solvent was evaporated under
reduced pressure, and the residue was purified by glass column
chromatography
on silica
gel in
a
mobile phase
CH2Cl2:MeOH:AcOH (150:1:1) to give final p-aminosalicylic
acids 1–14.
4-(N-(4-Cyclohexylbenzyl)-2-(2,3,4,5,6-pentafluoro-N-methylphenyl-
sulfonamido)acetamido)-2-hydroxybenzoic acid (1). Isolated in 96%
yield as a white solid. Mp: 89.0 1 ꢃC; dH (500 MHz, CD3OD-d4):
1.23–1.32 (1H, m, Cy), 1.36–1.47 (4H, m, Cy), 1.71–1.77 (1H, m, Cy),
1.78–1.86 (4H, m, Cy), 2.44–2.50 (1H, m, Cy), 3.07 (3H, s, CH3N),
4.10 (2H, s, CH2), 4.77 (2H, s, CH2), 6.62 (1H, dd, J ¼ 8.4 Hz,
J ¼ 2.0 Hz, Ar), 6.69 (1H, d, J ¼ 2.0 H, Ar), 7.02 (2H, d, J ¼ 8.0 Hz, Ar),
7.12 (2H, d, J ¼ 8.1 Hz, Ar), 7.87 (1H, d, J ¼ 8.4 Hz, Ar); dC (126 MHz,
CD3OD-d4): 27.2, 28.0, 35.6, 36.4, 45.7, 52.9, 53.8, 114.1–114.5 (m),
117.9, 119.9, 128.0, 129.5, 133.1, 135.1, 138.0–140.5 (m),
143.9–146.3 (m), 145.0–147.3 (m), 147.7, 148.9, 164.0, 168.1, 172.6;
Benzyl
2-(benzyloxy)-4-(N-(4-trifluoromethylbenzyl)-2-(N-methyl-
phenylsulfonyl))acetamido)benzoate (21l). Isolated in 38% yield as a HRMS (HESIþ): [M þ H]þ: calculated for C29H28F5N2O6Sþ (m/z):
pale-yellow oil. dH (500 MHz, CDCl3-d): 2.84 (3H, s, CH3N), 3.68 (2H, 627.1588, found 627.1585.
4-(N-(4-Cyclohexylbenzyl)-2-(N-methyl-N-(p-toluenesulfonyl))aceta-
mido)-2-hydroxybenzoic acid (2). Isolated in 63% yield as a pale-
pink crystalline solid. Mp: 125 1 ꢃC; dH (500 MHz, CD3OD-d4):
1.27–1.31 (1H, m, Cy), 1.37–1.43 (4H, m, Cy), 1.71–1.75 (1H, m, Cy),
1.78–1.84 (4H, m, Cy), 2.37 (3H, s, CH3(tol.)), 2.43–2.48 (1H, m, Cy),
2.82 (3H, s, CH3N), 3.74 (2H, s, CH2), 4.80 (2H, s, CH2), 6.53 (1H, d,
J ¼ 6.4 Hz, Ar), 6.60 (1H, s, Ar), 7.06–7.12 (4H, m, Ar), 7.29 (2H, d,
s, CH2), 4.82 (2H, s, CH2), 5.06 (2H, s, CH2(Bn)), 5.36 (2H, s,
CH2(Bn)), 6.59–6.61 (1H, m, Ar), 6.63–6.66 (1H, m, Ar), 7.22 (2H, d,
J ¼ 8.0 Hz, Ar), 7.27–7.36 (8H, m, Ar), 7.38–7.42 (2H, m, Ar),
7.44–7.48 (2H, m, Ar), 7.51 (2H, d, J ¼ 8.0 Hz, Ar), 7.53–7.57 (1H, m,
Ar), 7.71–7.73 (2H, m, Ar), 7.84 (1H, dd, J ¼ 8.2 Hz, J ¼ 1.0 Hz, Ar); dC
(126 MHz, CDCl3-d): 35.3, 50.5, 51.9, 66.3, 70.0, 113.1, 119.0, 120.4,
123.1 (q, J ¼ 272.0 Hz), 124.7 (q, J ¼ 3.7 Hz), 126.1, 126.5, 127.2,
127.4, 127.5, 127.7, 127.8, 128.1, 128.2, 129.2 (q, J ¼ 32.5 Hz), 131.8, J ¼ 8.0 Hz, Ar), 7.55 (2H, d, J ¼ 8.1 Hz, Ar), 7.86 (1H, d, J ¼ 6.6 Hz,
132.5, 134.8, 137.5, 139.7, 143.9, 158.0, 164.4, 166.3; ESI-MS Ar); dC (126 MHz, CD3OD-d4): 21.5, 27.2, 28.0, 35.6, 36.9, 45.6, 52.5,
[M þ H]þ calculated for C38H34F3N2O6Sþ (m/z): 703.2, found 703.3.
53.8, 117.2, 119.1, 127.9, 128.5, 129.6, 130.7, 133.1, 135.4, 136.3,

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145.0, 146.4, 148.7, 163.6, 169.2; ESI-MS [M þ H]þ calculated for 114.3, 114.9, 118.1, 120.1, 129.7, 131.0, 133.1, 138.1–140.4 (m),
C30H35N2O6Sþ (m/z): 551.2, found 551.3.
4-(N-(Benzyl)-2-(N-methyl-N-(p-toluenesulfonyl))acetamido)-2-
hydroxybenzoic acid (3). Isolated in 65% yield as a pale-yellow crys-
talline solid. Mp: 131.0 1 ꢃC; dH (500 MHz, DMSO-d6): 2.36 (3H, s,
143.9–145.3 (m), 147.6, 160.8, 164.0, 168.0, 172.7; HRMS (HESIþ):
[M þ H]þ: calculated for C24H20F5N2O7Sþ (m/z): 575.0867,
found: 575.0914.
4-(N-(4-Trifluoromethylbenzyl)-2-(N-methylphenylsulfonyl)aceta-
CH3(tol)), 2.80 (3H, s, CH3N), 3.82 (2H, s, CH2), 4.77 (2H, s, CH2), mido)-2-hydroxybenzoic acid (9). Isolated in 59% yield as a pale-
6.44–6.46 (1H, m, Ar), 6.51 (1H, d, J ¼ 1.9 Hz, Ar), 7.15 (2H, d, brown solid. Mp: 81.5 1 ꢃC; dH (500 MHz, CD3OD-d4): 2.87 (3H, s,
J ¼ 7.1 Hz, Ar), 7.21–7.25 (1H, m, Ar), 7.26–7.31 (2H, m, Ar), 7.35 CH3N), 3.85 (2H, s, CH2), 4.96 (2H, s, CH2), 6.72 (1H, dd, J ¼ 8.4 Hz,
(2H, d, J ¼ 8.2 Hz, Ar), 7.54 (2H, d, J ¼ 8.3 Hz, Ar), 7.67 (1H, d, J ¼ 2.1 Hz, Ar), 6.80 (1H, d, J ¼ 2.0 Hz, Ar), 7.41 (2H, d, J ¼ 8.1 Hz,
J ¼ 8.1 Hz, Ar); dC (126 MHz, d6-DMSO-d6): 21.0, 35.9, 50.6, 52.0, Ar), 7.49–7.53 (2H, m, Ar), 7.58–7.62 (3H, m, Ar), 7.70–7.72 (2H, m,
115.3, 115.8, 118.9, 126.9, 127.1, 127.8, 128.3, 129.6, 130.9, 135.3, Ar), 7.89 (1H, d, J ¼ 8.4 Hz, Ar); dC (126 MHz, CD3OD-d4): 36.9, 52.6,
137.3, 143.1, 143.9, 163.5, 166.7, 170.9; HRMS (HESIþ): [M þ H]þ: 53.6, 114.3, 117.8, 119.8, 125.6 (q, J ¼ 271.6 Hz), 126.5 (q,
calculated for C24H25N2O6Sþ (m/z): 469.1433, found 469.1430.
J ¼ 3.8 Hz), 128.4, 130.0, 130.2, 130.8 (q, J ¼ 32.4 Hz), 133.2, 134.0,
139.4, 142.6, 148.0, 164.0, 169.4, 172.6; HRMS (HESIþ): [M þ H]þ:
4-(N-(Benzyl)-2-(2,3,4,5,6-pentafluoro-N-methylphenylsulfonami-
do)acetamido)-2-hydroxybenzoic acid (4). Isolated in 74% yield as a calculated for C24H22F3N2O6Sþ (m/z): 523.1151, found 523.1153.
pale-pink crystalline solid. Mp: 176.0 1 ꢃC, dH (500 MHz, DMSO-
4-(N-(4-Trifluoromethylbenzyl)-2-(N-methyl-N-(p-toluenesulfonyl))a-
d6): 3.02 (3H, s, CH3N), 4.09 (2H, s, CH2), 4.75 (2H, s, CH2), cetamido)-2-hydroxybenzoic acid (10). Isolated in 72% yield as a
6.40–6.42 (1H, m, Ar), 6.47 (1H, d, J ¼ 1.8 Hz, Ar), 7.12 (2H, d, white solid. Mp: 176.5 1 ꢃC; dH (500 MHz, CD3OD-d4): 2.39 (3H, s,
J ¼ 7.0 Hz, Ar), 7.22–7.30 (3H, m, Ar), 7.69 (1H, d, J ¼ 8.1 Hz, Ar); dC CH3(tol.)), 2.84 (3H, s, CH3N), 3.82 (2H, s, CH2), 4.96 (2H, s, CH2),
(126 MHz, d6-DMSO-d6): 35.6, 51.2, 52.1, 115.4, 115.6, 119.3–119.5 6.71 (1H, dd, J ¼ 8.4 Hz, J ¼ 2.1 Hz, Ar), 6.80 (1H, d, J ¼ 2.1 Hz, Ar),
(m), 127.2, 127.9, 128.2, 131.1, 136.2–138.6 (m), 136.9, 143.0–145.3 7.31 (2H, d, J ¼ 8.1 Hz, Ar), 7.41 (2H, d, J ¼ 8.1 Hz, Ar), 7.56–7.61
(m), 163.6, 166.1, 170.8; HRMS (HESIþ): [M þ H]þ: calculated for (4H, m, Ar), 7.88 (1H, d, J ¼ 8.4 Hz, Ar); dC (126 MHz, CD3OD-d4):
C23H18F5N2O6Sþ (m/z): 545.0806, found 545.0807.
21.4, 36.9, 52.6, 53.6, 114.2, 117.8, 119.8, 125.6 (q, J ¼ 271.3 Hz),
126.4 (q, J ¼ 3.6 Hz), 128.5, 130.0, 130.7, 130.8 (q, J ¼ 32.3 Hz),
4-(N-(4-tert-Butylbenzyl)-2-(N-methyl-N-(p-toluenesulfonyl))aceta-
mido)-2-hydroxybenzoic acid (5). Isolated in 77% yield as a pale- 133.2, 136.3, 142.6, 145.1, 148.1, 164.0, 169.5, 172.6; HRMS (HESIþ):
brown solid. Mp: 136.0 1 ꢃC; dH (500 MHz, CD3OD-d4): 1.29 (9H, s, [M þ H]þ: calculated for C25H24F3N2O6Sþ (m/z): 537.1307,
t-Bu), 2.39 (3H, s, CH3(tol)), 2.83 (3H, s, CH3N), 3.76 (2H, s, CH2), found 537.1307.
4.82 (2H, s, CH2), 6.56–6.60 (1H, m, Ar), 6.64 (1H, s, Ar), 7.11 (2H, d,
4-(N-(4-Trifluoromethylbenzyl)-2-(4-fluoro-N-methylphenylsulfona-
J ¼ 8.3 Hz, Ar), 7.29–7.34 (4H, m, Ar), 7.56 (2H, d, J ¼ 8.2 Hz, Ar), mido)acetamido)-2-hydroxybenzoic acid (11). Isolated in 57% as a
7.87 (1H, d, J ¼ 8.2 Hz, Ar); dC (126 MHz, CD3OD-d4): 21.5, 31.8, white solid. Mp: 218.0 1 ꢃC; dH (500 MHz, CD3OD-d4): 2.88 (3H, s,
35.4, 36.9, 52.6, 53.8, 117.4, 119.2, 126.4, 128.5, 129.4, 130.7, 133.1, CH3N), 3.89 (2H, s, CH2), 4.95 (2H, s, CH2), 6.72 (1H, dd, J ¼ 8.4 Hz,
135.1, 136.3, 145.1, 146.8, 151.7, 163.7, 169.3; HRMS (HESIþ): J ¼ 2.0 Hz, Ar), 6.80 (1H, d, J ¼ 2.0 Hz, Ar), 7.25 (2H, tt, J ¼ 8.8 Hz,
[M þ H]þ: calculated for C28H33N2O6Sþ (m/z): 525.2059, J ¼ 2.0 Hz, Ar), 7.40 (2H, d, J ¼ 8.1 Hz, Ar), 7.60 (2H, d, J ¼ 8.2 Hz,
found 525.2060.
Ar), 7.77–7.81 (2H, m, Ar), 7.90 (1H, d, J ¼ 8.4 Hz, Ar); dC (126 MHz,
CD3OD-d4): 36.7, 52.5, 53.6, 114.4, 117.2 (d, J ¼ 22.9 Hz), 117.8,
4-(N-(4-tert-Butylbenzyl)-2-(2,3,4,5,6-pentafluoro-N-methylphenyl-
sulfonamido)acetamido)-2-hydroxybenzoic acid (6). Isolated in 99% 119.8, 125.6 (q, J ¼ 271.0 Hz), 126.5 (q, J ¼ 3.7 Hz), 130.1, 130.8 (q,
yield as a white solid. Mp: 87.1 1 ꢃC; dH (500 MHz, CDCl3-d): 1.30 J ¼ 32.0 Hz), 131.4 (d, J ¼ 9.5 Hz), 133.2, 135.9 (d, J ¼ 3.1 Hz), 142.6,
(9H, s, t-Bu), 3.11 (3H, s, CH3N), 4.08 (2H, s, CH2), 4.76 (2H, s, CH2), 147.9, 164.1, 166.6 (d, J ¼ 253.0 Hz), 169.3, 172.7; HRMS (HESIþ):
6.58–6.62 (1H, m, Ar) 6.73 (1H, d, J ¼ 2.0 Hz, Ar), 7.02 (2H, d, [M þ H]þ: calculated for C24H21F4N2O6Sþ (m/z): 541.1056,
J ¼ 8.2 Hz, Ar), 7.30 (2H, d, J ¼ 8.2 Hz, Ar), 7.91 (1H, d, J ¼ 8.4 Hz, found 541.1057.
Ar), 10.60 (1H, s, COOH); dC (126 MHz, CDCl3-d): 31.4, 34.7, 36.1,
4-(N-(4-Trifluoromethylbenzyl)-2-(3,5-difluoro-N-methylphenylsulfo-
52.4, 53.2, 109.9, 111.9, 117.2–117.5 (m), 119.4, 125.8, 128.2, 132.8, namido)acetamido)-2-hydroxybenzoic acid (12). Isolated in quantita-
136.6–139.1 (m), 143.8–146.1 (m), 147.7, 151.2, 163.3, 166.2, 172.7; tive yield as a white solid. Mp: 82.1 1 ꢃC; dH (500 MHz, CD3OD-
HRMS (HESIþ): [M þ H]þ: calculated for C27H26F5N2O6Sþ (m/z): d4): 2.94 (3H, s, CH3N), 3.95 (2H, s, CH2), 4.94 (2H, s, CH2), 6.72 (1H,
601.1432, found 601.1431.
dd, J ¼ 8.4 Hz, J ¼ 2.0 Hz, Ar), 6.81 (1H, d, J ¼ 2.0 Hz, Ar), 7.26 (1H,
tt, J ¼ 9.0 Hz, J ¼ 2.2 Hz, Ar), 7.36–7.41 (4H, m, Ar), 7.60 (2H, d,
4-(N-(4-Methoxybenzyl)-2-(2,4,6-trifluoro-N-methylphenylsulfona-
mido)acetamido)-2-hydroxybenzoic acid (7). Isolated in 81% as a J ¼ 8.1 Hz, Ar), 7.90 (1H, d, J ¼ 8.4 Hz, Ar); dC (126 MHz, CD3OD-d4):
white crystalline solid. Mp: 85.5 1 ꢃC; dH (500 MHz, CD3OD-d4): 36.8, 52.5, 53.5, 109.2 (t, J ¼ 25.7 Hz), 111.8–112.2 (m), 114.4, 117.8,
3.05 (3H, s, CH3N), 3.76 (3H, s, CH3O), 4.03 (2H, s, CH2), 4.75 (2H, s, 119.8, 125.6 (q, J ¼ 271.0 Hz), 126.5 (q, J ¼ 3.8 Hz), 130.0, 130.9 (q,
CH2), 6.61 (1H, dd, J ¼ 8.4 Hz, J ¼ 2.0 Hz, Ar), 6.67 (1H, d, J ¼ 2.0 Hz, J ¼ 32.3 Hz), 133.3, 142.5, 143.4 (t, J ¼ 8.4 Hz), 147.8, 164.1, 164.3
Ar), 6.80–6.84 (2H, m, Ar), 6.99–7.06 (4H, m, Ar), 7.86 (1H, d, (dd, J ¼ 252.4 Hz, J ¼ 12.0 Hz), 169.0, 172.6; HRMS (HESIþ):
J ¼ 8.0 Hz, Ar); dC (126 MHz, CD3OD-d4): 36.3, 52.6, 53.3, 55.7, 103.0 [M þ H]þ: calculated for C24H20F5N2O6Sþ (m/z): 559.0962,
(td, J ¼ 29.0 Hz, J ¼ 4.0 Hz), 114.2, 114.9, 118.1, 120.1, 129.8, 131.0, found 559.0963.
4-(N-(4-Trifluoromethylbenzyl)-2-(2,4,6-trifluoro-N-methylphenylsul-
133.0, 147.8, 160.7, 162.0 (ddd, J ¼ 257.7 Hz, J ¼ 15.7 Hz, J ¼ 7.0 Hz),
164.0, 165.3–167.6 (m), 168.3, 172.7; HRMS (HESIþ): [M þ H]þ: cal- fonamido)acetamido)-2-hydroxybenzoic acid (13). Isolated in 99%
culated for C24H22F3N2O7Sþ (m/z): 539.1055, found 539.1083.
yield as a white solid. Mp: 85.5 1 ꢃC; dH (500 MHz, CD3OD-d4):
4-(N-(4-Methoxybenzyl)-2-(2,3,4,5,6-pentafluoro-N-methylphenyl-
3.05 (3H, s, CH3N), 4.09 (2H, s, CH2), 4.91 (2H, s, CH2), 6.69 (1H, dd,
sulfonamido)acetamido)-2-hydroxybenzoic acid (8). Isolated in 72% J ¼ 8.3 Hz, J ¼ 2.0 Hz, Ar), 6.78 (1H, d, J ¼ 2.0 Hz, Ar), 7.00 (2H, t,
yield as a white crystalline solid. Mp: 89.0 1 ꢃC; dH (500 MHz, J ¼ 9.3 Hz, Ar), 7.37 (2H, d, J ¼ 8.1 Hz, Ar), 7.60 (2H, d, J ¼ 8.2 Hz,
CD3OD-d4): 3.07 (3H, s, CH3N), 3.76 (3H, s, CH3O), 4.08 (2H, s, CH2), Ar), 7.88 (1H, d, J ¼ 8.4 Hz, Ar); dC (126 MHz, CD3OD-d4): 36.3, 52.6,
4.74 (2H, s, CH2), 6.60 (1H, dd, J ¼ 8.3 Hz, J ¼ 1.1 Hz, Ar), 6.67 (1H, s, 53.5, 102.7–103.2 (m), 114.5, 115.6 (td, J ¼ 17.2 Hz, J ¼ 5.3 Hz),
Ar), 6.81 (2H, d, J ¼ 8.2 Hz, Ar), 7.03 (2H, d, J ¼ 8.2 Hz, Ar), 7.87 (1H, 117.8, 119.8, 125.6 (q, J ¼ 271.1 Hz), 126.4 (q, J ¼ 3.5 Hz), 130.1,
d, J ¼ 8.3 Hz, Ar); dC (126 MHz, CD3OD-d4): 36.4, 52.9, 53.3, 55.6, 130.8 (q, J ¼ 32.2 Hz), 133.3, 142.5, 147.7, 162.0 (ddd, J ¼ 257.5 Hz,

422
P. OLEKSAK ET AL.
J ¼ 15.6 Hz, J ¼ 6.9 Hz), 164.1, 166.5 (dt, J ¼ 255.3 Hz, J ¼ 16.1 Hz), tested compounds (dissolved in DMSO as 50 mM stock) in a con-
168.8, 172.6; HRMS (HESIþ): [M þ H]þ: calculated for
centration range as indicated. After 24 h, 200 ll of culture medium
C24H19F6N2O6Sþ (m/z): 577.0868, found 577.0869.
of each well was exchanged for 100 ll of resazurin (stock 30 mg/
4-(N-(4-Trifluoromethylbenzyl)-2-(2,3,4,5,6-pentafluoro-N-methyl-
phenylsulfonamido)acetamido)-2-hydroxybenzoic acid (14). Isolated
in 93% yield as a white solid. Mp: 154.5 1 ꢃC; dH (500 MHz,
CD3OD-d4): 3.08 (3H, s, CH3N), 4.15 (2H, s, CH2), 4.91 (2H, s, CH2),
6.69 (1H, dd, J ¼ 8.3 Hz, J ¼ 2.0 Hz, Ar), 6.78 (1H, d, J ¼ 2.0 Hz, Ar),
7.36 (2H, d, J ¼ 8.1 Hz, Ar), 7.59 (2H, d, J ¼ 8.2 Hz, Ar), 7.91 (1H, d,
J ¼ 8.4 Hz, Ar); dC (126 MHz, CD3OD-d4): 36.4, 52.9, 53.6, 114.6,
117.8, 119.8, 126.4–126.5 (m), 130.0, 130.9 (q, J ¼ 32.0 Hz), 133.4,
138.0–140.6 (m), 142.4, 143.8–146.3 (m), 144.9–147.3 (m), 147.5,
164.2, 168.5, 172.6; HRMS (HESIþ): [M þ H]þ: calculated for
C24H17F8N2O6Sþ (m/z): 613.0680, found 613.0680.
ml; Sigma, St. Louis, MO) diluted 10 times in the culture medium,
and the cells were incubated at 37 ꢃC for 1–3 h. Reading of fluor-
escence was done using an Envision reader (PerkinElmer,
Waltham, MA). Absolute values of fluorescence were related to
the values of control/untreated cells. To estimate the cytotoxic
concentration (half maximal inhibitory concentration, IC50), log-
scale data were processed in GraphPad Prism software (La Jolla,
CA) using nonlinear fit curve.
4.6. Crystal violet assay
To determine cell viability by crystal violet assay39, cells were
seeded in 96-well plates at densities of 3000 (RPE-1), 5000 (U373
and T98-G), and 8000 (U-87 MG) cells per well. The next day the
cells were treated with the tested compounds in triplicate in a
concentration range as indicated. After 24 h, the cells in each well
4.3. Antibodies
The following primary antibodies were used: mouse monoclonal
STAT3 (Santa Cruz Biotechnology, Inc., Dallas, TX, cat. no. sc-8019;
dilution 1:1000), mouse monoclonal pSTAT3 (Y705; Cell Signaling
Technology, Danvers, MA, cat. no. 9138; dilution 1:500), mouse were washed twice with 150 ml PBS and then stained in 30 ml
monoclonal GAPDH (Santa Cruz, cat. no. sc-47724; dilution
1:10,000), rabbit monoclonal anti-mouse STAT3 (Cell Signaling
Technology, Danvers, MA, cat. no. 4904; dilution 1:2000), rabbit
monoclonal anti-mouse pSTAT3 (Y705; Cell Signaling Technology,
Danvers, MA, cat. no. 9145S; dilution 1:2000), and rabbit monoclo-
nal anti-mouse GAPDH (Cell Signaling Technology, Danvers, MA,
cat. no. 2118S; dilution 1:10,000). The following secondary anti-
bodies were used: goat anti-mouse HRP-conjugated (Bio-Rad,
Hercules, CA, cat. no. 170–6516; dilution 1:10,000) and goat IgG-
HRP anti-rabbit (Cell Signaling Technology, Danvers, MA, cat. no.
7074S; dilution 1:2000).
0.5% w/v crystal violet in 20% methanol for 15 min. Plates were
washed three times with double distilled H2O and left to dry over-
night. Crystal violet was solubilised with 75 ml 0.2% Triton X-100
(Sigma, St. Louis, MO) in PBS for 15 min. Absorbance of crystal vio-
let was measured at 595 nm using a microplate reader (Multiskan
EX, Thermo Electron Corporation, Waltham, MA). Absorbance of
the treated samples was expressed as a percentage of absorbance
of untreated cells.
4.7. MTT assay and IC50 cytotoxicity estimation
Cells were seeded at the density of 2000 cells per well (96-well F
microplates; Nunc, Roskilde, Denmark). The following day, the
tested compounds were added. In the next 24 h, MTT (3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added. After
6 h, DMF (dimethylformamide) was added, and after additional
24 h, absorbance was measured at 560 nm by a microplate reader.
Absorbance of the treated samples was expressed as a percentage
of absorbance of untreated cells. To estimate the cytotoxic con-
4.4. Cell cultures
All cell lines used in the study were obtained from the American
Type Culture Collection (ATCC, Manassas, VA). Human breast
adenocarcinoma MDA-MB-231, prostate carcinoma DU-145, cervix
carcinoma HeLa, osteosarcoma U2-OS, glioblastoma U373, U-
87 MG and T98-G, and telomerase-immortalised retinal epithelium
RPE-1-hTERT cell lines were cultured in high-glucose (4.5 g/l)
DMEM. Media were supplemented with 10% foetal bovine serum centration (half maximal inhibitory concentration, IC50), log-scale
(Gibco/Thermo Fisher Scientific, Waltham, MA), 100 U/ml penicillin,
and 100 lg/ml streptomycin sulphate (Gibco/Thermo Fisher
Scientific, Waltham, MA). Cells were kept at 37 ꢃC under 5% CO2
atmosphere and 95% humidity.
data were processed in GraphPad Prism software (La Jolla, CA)
using a nonlinear fit curve.
The mouse TC-1 cell line was obtained in vitro by co-transfec-
tion of murine lung C57BL/6 cells with HPV16 E6/E7 and activated
human H-Ras (G12V) oncogenes. TC-1 cells were cultured in RPMI-
1640 medium supplemented with 10% foetal calf serum, L-glutam-
ine, and antibiotics35. Prostate carcinoma cell line TRAMP-C236 was
maintained in DMEM medium (Sigma-Aldrich, St. Louis, MO) sup-
plemented with 5% FCS, Nu-Serum IV (5%; BD Biosciences,
4.8. Detection of apoptosis by annexin V and propidium
iodide staining
Cells were harvested by centrifugation, resuspended in cold PBS
and again centrifuged. Cell pellets were then resuspended in
Annexin V Binding Buffer (ApoFlowEx FITC Kit, ED7044, Exbio,
Vestec, Czech Republic), annexin V-APC and PI were added to the
Bedford, MA), 5 mg/ml human insulin (Sigma-Aldrich, St. Louis, cell pellet and mixed. Cells were incubated in the dark at room
MO), dehydroisoandrosterone (DHEA, 10 nM; Sigma-Aldrich, St.
temperature for 15 min. After the incubation, cells were centri-
fuged, resuspended in Annexin V Binding Buffer and analysed by
flow cytometry. Five thousand cells per sample were measured by
analysing annexin V and PI using a BD FACSVerseTM flow cytome-
ter (BD Biosciences, Franklin Lakes, NJ). Data were analysed using
FlowJo 10 software (FlowJo LLC, Ashland, OR), and the percentage
of viable (annexin V-negative/PI-negative) cells relative to control
Louis, MO), and antibiotics37.
4.5. Resazurin assay and IC50 cytotoxicity estimation
The resazurin reduction assay34 was performed as described previ-
ously38. Briefly, cells were seeded onto a 96-well plate (2000 cells
per well) in hexaplicate and the following day treated with the was plotted.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
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4.9. SDS-PAGE and immunoblotting
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ꢀ
ꢀꢀ
determine statistical significance: p < 0.05 ( ), p < 0.01 ( ), and
p < 0.001 ( ).
ꢀꢀꢀ
Disclosure statement
14. Deng J, Grande F, Neamati N. Small molecule inhibitors of
Stat3 signaling pathway. Curr Cancer Drug Targets 2007;7:
91–107.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
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Funding
This work was supported by the Czech Science Foundation [No.
18-14259S], Institutional Grant [Project No. RVO 68378050], and
University of Hradec Kralove [Faculty of Science, No. VT2019-2021].
ORCID
Miroslav Psotka
Milan Reinis
Katarina Chalupova
David Malinak
Jana Svobodova
Rudolf Andrys
Helena Rehulkova
Zdenek Hodny
Kamil Musilek
http://orcid.org/0000-0002-7857-7411
http://orcid.org/0000-0001-7083-2898
http://orcid.org/0000-0003-3838-9149
http://orcid.org/0000-0002-0665-0667
http://orcid.org/0000-0001-7754-4231
http://orcid.org/0000-0003-1108-3924
http://orcid.org/0000-0003-1220-6631
http://orcid.org/0000-0002-6454-6796
http://orcid.org/0000-0002-7504-4062
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new small-molecule inhibitor of the STAT3 signaling path-
way. ACS Med Chem Lett 2010;1:454–9.
21. Horiguchi A, Asano T, Kuroda K, et al. STAT3 inhibitor
WP1066 as a novel therapeutic agent for renal cell carcin-
oma. Br J Cancer 2010;102:1592–9.
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