Synthesis and antiproliferative activity of sulfonamide-based peptidomimetic calpain inhibitors

Article abstract of DOI:10.1016/j.bmc.2020.115433

The calpains are a conserved family of cysteine proteases that includes several isoforms of which μ–calpain and m-calpain are the most widely distributed in mammalian cells. Calpains have been implicated in normal physiological processes as well as cellular abnormalities such as neurodegenerative disorders, cataract, and cancer. Therefore, calpain inhibitors are of interest as potential therapeutic agents. We have synthesized four new sulfonamide-based peptidomimetic compounds 2–5 as inhibitors of μ-calpain that incorporate (E)-1-(phenyl)-2-phenyldiazene and (E)-1-(phenyl)-2-phenylethene functionalities as the N-terminal capping groups of the inhibitors. Compound 5 with K_i value of 9 nM versus μ-calpain was the most potent member of the group. The compounds were predicted to be more lipophilic compared to MDL28170 based on CLogP estimation. They displayed moderate to good antiproliferative activity versus melanoma cell lines (A-375 and B-16F1) and PC-3 prostate cancer cells in vitro. Additionally, one member of the group (compound 3) inhibited DU-145 cell invasion by 80% at 2 μM concentration in the Matrigel cell invasion assay.

Full text of DOI:10.1016/j.bmc.2020.115433

Bioorganic&MedicinalChemistry28(2020)115433
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antiproliferative activity of sulfonamide-based
peptidomimetic calpain inhibitors
Isaac O. Donkor⁎, Jin Xu, Jiuyu Liu, Keyuna Cameron
Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, 881 Madison Avenue, Memphis, TN 38163, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
The calpains are a conserved family of cysteine proteases that includes several isoforms of which µ–calpain and
m-calpain are the most widely distributed in mammalian cells. Calpains have been implicated in normal phy-
siological processes as well as cellular abnormalities such as neurodegenerative disorders, cataract, and cancer.
Therefore, calpain inhibitors are of interest as potential therapeutic agents. We have synthesized four new
sulfonamide-based peptidomimetic compounds 2–5 as inhibitors of μ-calpain that incorporate (E)-1-(phenyl)-2-
phenyldiazene and (E)-1-(phenyl)-2-phenylethene functionalities as the N-terminal capping groups of the in-
hibitors. Compound 5 with Ki value of 9 nM versus μ-calpain was the most potent member of the group. The
compounds were predicted to be more lipophilic compared to MDL28170 based on CLogP estimation. They
displayed moderate to good antiproliferative activity versus melanoma cell lines (A-375 and B-16F1) and PC-3
prostate cancer cells in vitro. Additionally, one member of the group (compound 3) inhibited DU-145 cell in-
vasion by 80% at 2 μM concentration in the Matrigel cell invasion assay.
Calpain
Calpain inhibitor
Antiproliferative
Antiinvasion
Peptidomimetic
Ketoamide
1. Introduction
synthesis commenced with treating commercially available (E)-4-phe-
nyldiazobenzenesulfonyl chloride 6 with either L-valine methyl ester
Calpain, a calcium-activated cysteine proteinase was identified in rat
brain over fifty years ago.1 Following its discovery a plethora of studies
have shown that calpain is a complex cysteine protease that consists of
several isoforms. Some of the isoforms are ubiquitously expressed in var-
ious tissues while others are localized in specific tissues.2 The cellular
actions of calpain are tightly controlled by the endogenous protein in-
hibitor, calpastatin. However, under pathological conditions over-
activation of calpain initiates a cascade of events that result in cellular
damage hence the enzyme has been implicated in a variety of disease
states including traumatic brain injury,3 stroke,4 cataract,5 and cancer.6,7
As a result of these studies calpain inhibitors have been investigated as
neuroprotectants, cardioprotectants, anticataract agents and anticancer
agents.8–11 This report describes the synthesis, calpain inhibition, and in
vitro antiproliferative activity of a series of peptidomimetic compounds
(1–5, Figure 1) and the anti-invasive potential of compound 3.
hydrochloride or
room temperatureL-tporgoilvineecmometphoyulnedsste7r ahnyddr8o,crhelsopreidcetivinelyp.yBriadsiincehayt-
drolysis of the ester group followed by coupling with L-leucinol and
oxidation with Dess-Martin periodinane gave compounds 1 and 2 in
51% and 50% yield, respectively. Compound 1 was transformed to
compound 3 by reacting its aldehyde group with KCN to give cyano-
hydrin 9, the cyano group of which was hydrolyzed in HCl/MeOH
mixture to give α-hydroxy-β-amino methyl ester 10. Treatment of 10
with MeOH/NH3 gave α-hydroxy-β-amino carboxamide 11, which was
oxidized with PySO3/DMSO mixture to give compound 3 in 71% yield.
Compound 1 was previously reported by Abell et al.12 as a mixture of
the E-, and Z-isomers in the ratio of 3.3:1 based on 1H NMR spectro-
scopy. The authors showed that irradiating 1 with ultraviolet light in-
creased the ratio of the (Z)-isomer, which was a less potent inhibitor of
calpain compared to the corresponding (E)-isomer. We have synthe-
sized compounds 4 and 5 as ethylene-bridged analogues to investigate
the significance of diazo group for calpain inhibition. The coupling
constant of the vinyl protons in the 1H NMR spectra of the ethylene-
bridged compounds was 16.5 Hz, which supports the (E)-geometry of
the compounds.
2. Results and discussion
2.1. Chemistry
Compounds 1–3 were synthesized as outlined in Scheme 1. The
The syntheses of compounds 4 and 5 are outlined in Scheme 2. The
⁎ Corresponding author at: University of Tennessee Health Science Center, 881 Madison Avenue, Room 562, Memphis, TN 38163, United States
E-mail address: idonkor@uthsc.edu (I.O. Donkor).
https://doi.org/10.1016/j.bmc.2020.115433
Received 26 November 2019; Received in revised form 1 March 2020; Accepted 11 March 2020
Availableonline13March2020
0968-0896/©2020ElsevierLtd.Allrightsreserved.

I.O. Donkor, et al.
Bioorganic&MedicinalChemistry28(2020)115433
Figure 1. Structures of sulfonamide-based peptidomimetics 1–5 and
MDL28170.
compounds were synthesized as described above for the synthesis of the
diazene derivatives 1–3. Briefly, the sodium salt of 4-styrene sulfonic
acid 12 was transformed to 4-styrylphenylsulfonic acid using the Heck
reaction, which is known to generate products with (E)-configuration.
Refluxing the 4-styrylphenylsulfonic acid in SOCl2 gave sulfonyl
chloride 13, which was treated with L-valine methyl ester hydro-
chloride in the presence of N,N-diisopropylethylamine (DIPEA) to give
sulfonamide 14. Hydrolysis of the ester group of 14 followed by cou-
pling of the resulting acid with L-leucinol afforded 16, which was
transformed to aldehyde 4 (85% yield) and alpha-ketoamide 5 (53%
yield) as described for the synthesis of the corresponding diazene de-
rivatives 1 and 3. The geometries of the compounds were established
based on the coupling constants (J = 16.5 Hz) of the olefinic hydrogen
atoms.13
2.2. Biological activity
2.2.1. In vitro inhibition of calpain
Calpain inhibitors such as MDL28170 (Figure 1) were discovered
via N-terminal capping of dipeptide aldehyde inhibitors of the en-
zyme.14 The N-terminal capping group of calpain inhibitors occupies
the S3/S4 subsites of the enzyme. In a previous study we demonstrated
that the S3/S4 subsites of calpain prefer planar aromatic groups over
nonplanar saturated groups as the N-terminal capping functionality.15
Abell et al.12 reported compound 1 as a mixture of E- and Z-isomers (in
3.3:1 ratio) that potently inhibited m-calpain. Using molecular
Scheme 1. Synthesis of compounds 1–3. aReagents: (a) Pyridine,
OMe.HCI or L-Pro-OMe.HCI RT, 20 h; (b) 1.0 N NaOH, MeOH, 55 °C, 2 h;L(-cV)aLl--
leucinol, CDI, THF, CH2CI2, RT, 96 h; (d) Dess-Martin reagent, CH2CI2; (e)
NaHSO3/KCN; (f) Conc. HCI/MeOH; (g) 7.0 N NH3 in MeOH, 16 h; (h) PySO3,
DMSO.
2

I.O. Donkor, et al.
Bioorganic&MedicinalChemistry28(2020)115433
determine the significance of the diazene moiety itself for calpain in-
hibition. The compounds were tested as inhibitors of porcine ery-
throcyte μ-calpain and the results are displayed in Table 1. Except for
compound 2 (Ki = 273 nM), all members of the series were potent
inhibitors of μ-calpain and displayed Ki values between 9 nM and
18 nM. Compound 1 with L-valine as the P2 substituent was 15-fold
more potent than 2, which has a proline residue at the P2 position. This
is consistent with previous structure–activity relationship studies,
which demonstrated that the S2 subsite of calpain prefers small hy-
drophobic groups at the P2 position of inhibitors.16 The ethylene-
bridged analogues 4 and 5, with Ki values of 14 nM and 9 nM, re-
spectively, were also potent inhibitors of μ-calpain, suggesting that the
diazene group is not required for potent inhibition of calpain.
MDL28170 is a peptidyl aldehyde that is recognized as a benchmark
calpain inhibitor and has been used to investigate the cellular roles of
calpain.14,16 However, peptidyl aldehydes are known to form hydrates
in aqueous media and are prone to oxidation in vivo,17,18 which limit
the usefulness of the inhibitor as a pharmacological tool. Therefore, to
mitigate these properties of peptidyl aldehydes we synthesized and
evaluated the μ-calpain inhibitory activity of alpha-ketoamides 3 and 5
as oxidation stable analogues of aldehydes 1 and 4. As shown in
Table 1, alpha-ketoamides 3 and 5 were also potent inhibitors of μ-
calpain. The compounds are also more lipophilic (based on CLogP es-
timation) compared to MDL28170, which suggests that these new cal-
pain inhibitors may display greater cellular permeability compared to
MDL28170.
Collectively, these advantages (i.e., enhanced potential for cellular
stability and greater lipophilicity) could translate into greater in vivo
exposure of compounds 3 and 5 compared to MDL28170 but this will be
the subject matter of future pharmacokinetics studies.
2.2.2. Antiproliferation and antiinvasion activities
Calpain has been implicated in cellular functions such as cell mi-
gration, cell invasion, and tumorigenesis,6,7 hence we studied the
ability of the compounds to inhibit the growth of melanoma cells (A-
375 and B-16F1) and PC-3 prostate cancer cells in culture using the
sulforhodamine B assay and the MTT assay, respectively. The com-
pounds moderately inhibited proliferation of the cancer cell lines
(Table 1). The GI50 values for the antiproliferative activity of the
compounds versus the human melanoma cell line A-375 ranged from
4.1 μM to 22.2 μM and that for the mouse B-16F1 melanoma cells was
between 10.3 μM and 18.7 μM. Compound 2 was the least effective
calpain inhibitor of the series, however, it was the most effective an-
tiproliferative agent versus all of the cancer cell lines studied. It in-
hibited the growth of the cell lines with GI50 values of 4.1 μM (A-375
melanoma cells), 13.6 μM (B-16F1 melanoma cells), and 5.6 μM (PC-3
prostate cancer cells) suggesting that other factors besides calpain in-
hibition may also contribute to the antiproliferation activity of the
compound.
It has been demonstrated using in vitro and in vivo studies that an-
tisense suppression of calpain expression limits the invasion of DU-145
prostate carcinoma cells.19,20 Therefore, due to the important role that
calpain plays in cell motility21 and tumor invasion19,20 we studied the
effect of compound 3 on DU-145 prostate cancer cell invasion in vitro
using the Matrigel chamber assay. In this assay tumor invasion in the
presence of the inhibitor was expressed as the number of cells that in-
vaded through the Matrigel compared to untreated cells (control).
Compound 3 showed concentration-dependent inhibition of prostate
cancer cell invasiveness and inhibited DU-145 cell invasion by about
80% at 2 μM (Figure 2). Also, the compound (at 1 μM or 2 μM) did not
affect cell proliferation as determined by the MTT assay (Figure 3),
indicating that the decrease in cell transmigration was not secondary to
decreased cell proliferation.
Scheme 2. Synthesis of compounds 4 and 5. aReagents: (a) Bromobenzene, Pd
(OAc)2, TEA, (o-Tol)3P; (b) SOCI2, DMF; (c) L-Val-OMe.HCI, DIPEA, Pyridine;
(d) 4.0 N NaOH, MeOH; (e) L-Leucinol, TEA, Mukaiyama's reagent, DMF; (f)
PySO3, DMSO; (g) NaHSO3/KCN; (h) Conc. HCI/MeOH; (i) 7.0 N NH3 in MeOH.
modeling studies, it was demonstrated that the E-(diazo) derivative
extends deep into the S3 pocket of the enzyme and contributes to the
calpain inhibitory activity of the mixture of the diazo-dipeptide alde-
hydes. Here we disclose compounds 2 and 3 as derivatives of compound
1 to investigate if the (E)-1-(phenyl)-2-phenyldiazene group could serve
as an effective N-terminal capping functionality to achieve potent in-
hibition of calpain. Compounds 4 and 5 were synthesized as ethylene-
bridged analogues of the diazene derivatives 1 and 3, respectively, to
3

I.O. Donkor, et al.
Bioorganic&MedicinalChemistry28(2020)115433
Table 1
Calpain inhibition and antiproliferative action of compounds 1–5 on cancer cells.
GI50 (µM) for inhibition of cancer cell growthc
Compound
Ki (nM)a
CLogPb
A-375
B-16F1
P-C3
1
18
273
16
14
9
2
5.54
4.57
4.01
5.73
4.20
3.64
ND
12.9
4.1
1.5
0.5
2.1
2.1
1.3
13.7 + 5
27.3
5.6
2
2
20
13.6
18.7
10.3
17.2
ND
1.7
9
3
2
1
22.2
9.4
3.0
2.0
2.8
24.1
23.0
6.02
ND
3
2
1
4
5
1
16
MDL28170
Taxol
10
NDd
2
ND
0.01
0.02
0.03
a
Ki values were determined by Dixon plots using the average of triplicate assays and plotting 1/v versus I (inhibitor concentration) to give intersecting lines with
correlation coefficient ≥ 0.95;
b
c
CLog P values are the calculated partition coefficients of the inhibitors obtained with ChemDraw Ultra ver. 9.0;
GI50 (half maximal growth inhibition) values of the compounds versus the melanoma cell lines A-375 and B-16F1- were determined using the sulforhodamine B
colorimetric cytotoxicity assay while those for the PC3 cell line were determined using the MTT assay. All experiments were performed in triplicate and the results are
reported as
standard error of the mean.
d
ND = Not determined.
3. Conclusion
In summary, four new sulfonamide-based peptidomimetic inhibitors
of porcine erythrocyte μ-calpain were synthesized. All of the com-
pounds were estimated to be more lipophilic than MDL28170.
Compound 2 moderately inhibited μ-calpain but the other three mem-
bers of the series (i.e., compounds 3, 4, and 5) were potent inhibitors of
the enzyme, suggesting that the (E)-1-(phenyl)-2-phenyldiazene func-
tionality is a good N-terminal capping group for potent inhibition of the
enzyme. The study showed that the diazene linker is not necessary for
μ-calpain inhibition because the ethylene bridged derivatives were as
potent (4) or more potent (5) than the diazene bridged analogue 3. In
concert with literature evidence that calpain plays an important role in
tumorigenesis and/or tumor invasion,6,7,19,20 the inhibitors demon-
strated antiproliferative activity as evidenced by their ability to inhibit
the growth of melanoma and prostate cancer cells as well as limit the
migration of the DU-143 prostate cancer cells in vitro.
Figure 2. Concentration (μM) dependent inhibition of DU-145 cell invasion by
compound 3. Data shown are the average +/- S.E. of triplicate experiments.
4. Experimental section
4.1. Chemistry
All reagents and solvents were purchased from Sigma Aldrich,
Fisher Scientific, TCI, or Calbiochem, and were used without further
purification. Thin layer chromatography (TLC) was performed on silica
gel plates purchased from Analtech, Inc. Fisher silica gel S732-25
(100–400 mesh) was used for column chromatography. Melting points
were determined on a Fisher-Johns melting point apparatus. Molecular
masses were determined with electron spray ionization mass spectra
(ESI-MS) on a Bruker/Hewlett Packard Esquire LC/MS instrument.
Proton nuclear magnetic resonance (1H NMR) spectra were recorded on
Bruker ARX 300 MHz and Varian Inova-500 MHz instruments.
Deuterated solvents were purchased from Cambridge Isotope
Laboratories, Inc. The chemical shifts (δ) are reported in parts per
million (ppm) relative to TMS, and coupling constants (J) are reported
in hertz (Hz). Splitting patterns are indicated as follows: s, singlet; d,
doublet; t, triplet; m, multiplet. IR spectra (neat) were recorded on a
Perkin Elmer Spectrum 100 FT-IR spectrophotometer, and the re-
presentative absorption bands are reported. Elemental analyses (C, H,
N) were performed by Atlantic Microlab Inc., Norcross, GA and are
within
0.4% of the theoretical values.
4.1.1. General procedure 1. Dess-Martin oxidation
Figure 3. DU-145 cell proliferation in the presence of 1 (μM) and 2 (μM) of
compound 3. Data are shown as the average of +/- S.E. of triplicate experi-
ments.
Dess-Martin reagent (17.2 mmol) was slowly added to an ice-cooled
solution of the appropriate alcohol (15.6 mmol) in anhydrous CH2Cl2
(80 mL). After 10 min, the ice bath was removed and the milky reaction
4

I.O. Donkor, et al.
Bioorganic&MedicinalChemistry28(2020)115433
mixture was stirred at RT for 2–4 h (checked by TLC). A solution of
Na2S2O3 (160 mmol) in saturated NaHCO3 was added and the mixture
was stirred for an additional 10 min at RT. After separation, the aqu-
eous layer was extracted with CH2Cl2 (3 × 30 mL) and the combined
organic layer was washed successively with NaHCO3 solution, water,
and brine followed by drying (MgSO4), concentration, and purification
either by flash chromatography or crystallization.
(dd, J = 5.4 Hz and 5.1 Hz, 1H, CH2OH), 3.21–3.29 (m, 1H, NCH2CH2),
2.10–2.23 (m, 1H, NCH2CH2), 1.66–1.86 (m, 6H, NCH2CH2CH2 and
CH2CH(CH3)2), 1.41–1.54 (m, 1H, CH(CH3)2), 0.98–1.03 (dd CH(CH3)2,
6H).
Dess-Martin oxidation (general procedure 1) of the orange solid
(557 mg, 1.21 mmol) followed by column chromatographic purification
(EtOAc/hexane, 2:1) of the resulting product gave 2 as a yellow solid in
50% yield. m.p. 79–82 °C. IR (cm−1) 1655 (C]O), 1735 (CHO), 3366
(amide). 300 MHz 1H NMR (CDCl3) δ 9.63 (s, 1H, CHO), 7.98–8.13 (m,
6H, Ar), 7.57–7.60 (m, 3H, Ar), 7.26 (s, 1H, CONH), 4.43–4.50 (m, 1H,
CHCH2CH(CH3)2), 4.22–4.25 (m, 1H, NCHCO), 3.61–3.68 (m, 1H,
NCH2CH2), 3.25–3.34 (m, 1H, NCH2CH2), 2.24–2.30 (m, 1H, CH
(CH3)2), 1.66–1.86 (m, 6H, NCH2CH2CH2 and CH2CH(CH3)2), 1.01 (dd
CH(CH3)2, 6H). MS (ESI) 454 [M−H]-. Anal. Calcd. for:
4.1.2. General procedure 2. Pyridine-sulfur trioxide oxidation
A solution of pyridine-sulfur trioxide complex (7.18 mmol) in DMSO
(15 mL) was added dropwise to an ice-cooled solution of the alcohol
(0.798 mmol) and DIPEA (7.36 mmol) in CH2Cl2/DMSO (30 mL) and
the mixture was stirred for 1 h at 0 °C. The reaction mixture was diluted
with EtOAc (300 mL), washed with aqueous 1 N HCl (2x), saturated
aqueous NaHCO3 (2x), and brine. The organic phase was recovered and
dried over MgSO4 followed by concentration and purification either by
flash chromatography or crystallization.
C
23H28N4O4S·0·.5H2O, C, 59.34; H, 6.28; N, 12.03; S, 6.89. Found: C,
59.28; H, 6.16; N, 11.96; S, 6.69.
4.1.5. 5-Methyl-3-(3-methyl-2-(4-(phenyldiazenyl)phenylsulfonamido)-
butanamido)-2-oxohexanamide (3)
4.1.3. 3-Methyl-N-(4-methyl-1-oxopentan-2-yl)-2-(4–phenyldiazenyl)
phenyl-sulfonamido)butanamide (1)
Oxidation of 11 (350 mg, 0.696 mmol) with pyridine-sulfur trioxide
complex as described under general procedure 2 followed by column
chromatographic purification (acetone/hexane 1:1) gave 3 as an orange
solid in 71% yield. m.p. 134–136 °C. IR (cm−1) 3464, 3360 and 3281
(NH), 1736 (C]O), 1693 (C]O). 300 MHz 1H NMR (acetone‑d6) δ
7.97–8.09 (m, 6H, Ar), 7.61–7.68 (m, 3H, Ar), 7.46 (d, J = 6.9 Hz, 2H,
CONH2), 6.99 (s, 1H, SO2NH), 6.56 (d, J = 9.3 Hz, 1H, CONH),
5.03–5.10 (m, 1H, CHCH2CH(CH3)2), 3.84–3.89 (m, 1H, NHCHCO),
1.83–1.88 (m, 1H, NCHCH(CH3)2), 1.22–1.30 (m, 2H, CH2CH(CH3)2),
1.12–1.16 (m, 1H, CH2CH(CH3)2), 1.02 (d, J = 6.9 Hz, 3H, CH(CH3)2),
0.91 (d, J = 6.9 Hz, 3H, CH(CH3)2), 0.71–1.02 (dd, J = 6.3 Hz and
9.3 Hz, 6H, CH2CH(CH3)2). MS (ESI) 500.6 [M−H]-. Anal. Calcd. for:
1,1′-Carbonyldiimidazole (CDI) (494 mg, 3.05 mmol) was added to
a stirred ice-cooled solution of acid 7 (1.0 g, 2.77 mmol) in a mixture of
THF (15 mL) and CH2Cl2 (20 mL). Stirring was continued at RT for
30 min. and L-leucinol (531 µL, 4.16 mmol) was added and the mixture
was stirred at RT for 96 h. The solvent was removed and the residue was
recrystallized from acetone/CH2Cl2 mixture to give N-(1-hydroxy-4-
methylpentan-2-yl)-3-methyl-2-(4-(phenyldiazenyl)-phenyl-sulfona-
mido)butanamide as a yellow solid in 51.0% yield. m.p.237–238 °C. IR
(cm−1) 1639 (C]O), 3284 (amide), 3505 (amide). 300 MHz 1H NMR
(acetone‑d6) δ 7.98–8.08 (m, 6H, Ar), 7.62–7.68 (m, 3H, Ar), 6.98 (d,
J = 8.1 Hz, 1H, SO2NH), 6.52 (d, J = 8.7 Hz, 1H, CONH), 3.69–3.81
(m, 3H, NHCHCONHCHCH2OH), 3.36–3.42 (m, 2H, CH2OH),
2.00–2.06 (m, 1H, NHCHCH(CH)3), 1.10–1.19 (m, 3H, CH2CH(CH3)2),
1.00 (d, J = 6.9 Hz, 3H, CHCH3), 0.89 (d, J = 6.9 Hz, 3H, CHCH3),
0.69 (d, J = 6.0 Hz, 3H, CH2CHCH3), 0.64 (d, J = 6.0 Hz, 3H,
CH2CHCH3). Addition of D2O to the NMR tube resulted in reduction of
the number of protons for the signals between δ 3.69–3.81 from 3
protons to 2 protons, which suggested that the OH signal of the product
is buried under the multiplex in this region of the spectrum. MS (ESI)
459.0 [M−H]-. Dess-Martin oxidation (general procedure 1) of the
yellow solid (300 mg, 0.65 mmol) followed by flash chromatographic
purification (acetone /CH2Cl2/hexane, 2:1:4) gave 1 in 51% yield. m.p.
146–149 °C. IR (cm−1) 1715 (CHO), 1668 (C]O). 300 MHz 1H NMR
(CDCl3) δ 9.50 and 9.42 (s, 1H, CHO), 7.94–8.10 (m, 6H, Ar), 7.54–7.63
(m, 3H, Ar), 6.30 and 6.01 (d, J = 7.2 Hz and 7.8 Hz, 1H, SO2NH), 5.45
and 5.26 (d, J = 8.7 Hz and 8.1 Hz, 1H, CONH), 4.40–4.48 (m, 1H,
CHCHO), 3.63–3.70 (m, 1H, NHCHCH(CH3)2), 2.11–2.20 (m, 1H,
NHCHCH(CH3)2), 1.19–1.70 (m, 3H, CH2CH(CH3)2), 0.79–1.01 (m,
12H, CH(CH3)2 and CH2CH(CH3)2). MS (ESI) 457.0 [M−H]-. Anal.
Calcd. for: C23H30N4O4S, C, 60.24; H, 6.59; N, 12.22; S, 6.99. Found: C,
60.05; H, 6.66; N, 12.17; S, 6.83.
C
24H31N5O5S·0·.5H2O, C, 56.45; H, 6.32; N, 13.72; S, 6.28. Found: C,
56.30; H, 6.09; N, 13.59; S, 6.16.
4.1.6. 3-Methyl-N-(4-methyl-1-oxopentan-2-yl)-2-(4-
styrylphenylsulfonamido) butanamide (4)
Oxidation of 16 (2.58 g, 5.63 mmol) with pyridine-sulfur trioxide
complex (general procedure 2) followed by recrystallization of the
crude product from EtOAc/hexane to give 4 as white solid in 85% yield.
m.p. 174–175 °C. IR (cm−1) 1637 (C]O), 1728 (CHO). 500 MHz 1H
NMR (CDCl3) δ 9.51 (s, 1H, CHO), 7.85 (d, J = 8.5 Hz, 2H, Ar), 7.63 (d,
J = 8.5 Hz, 2H, Ar), 7.56 (d, J = 8.0 Hz, 2H, Ar), 7.04–7.43 (t,
J = 7.5 Hz, 2H, Ar), 7.36 (t, J = 7.5 Hz, 1H, Ar), 7.25 (d, J = 16.5 Hz,
1H, CH = CH), 7.12 (d, J = 16.5 Hz, 1H, CH = CH), 6.12 (d,
J = 7.5 Hz, 1H, SO2NH), 5.34 (d, J = 8.5 Hz, 1H, CONH), 4.43 (m, 1H,
CHCH2CH(CH3)2), 3.62 (dd, J = 5.0 Hz and 8.0 Hz, 1H, NHCHCO),
2.15 (m, 1H, NCHCH(CH3)2), 1.56 (m, 1H, CH2CH(CH3)2), 1.41–1.49
(m, 1H, CH2CH(CH3)2), 1.26–1.32 (m, 1H, CH2CH(CH3)2), 0.95–0.97
(d, J = 6.5 Hz 3H, NHCHCHCH3), 0.90 (d, J = 2.5 Hz 3H, CH2CHCH3),
0.89 (d, J = 2.5 Hz, 3H, CH2CHCH3), 0.86 (d, J = 6.5 Hz, 3H,
NHCHCHCH3). MS (ESI) 455 [M−H]-. Anal. Calcd. for: C25H32N2O4S,
C, 65.76; H, 7.06; N, 6.14; S, 7.02. Found: C, 65.51; H, 7.10; N, 6.13; S,
7.24.
4.1.4. N-(4-Methyl-1-oxopentan-2-yl)-1-(4-((phenyldiazenyl)
phenylsulfonyl)-pyrrolidine-2-carboxamide (2)
CDI (474 mg, 2.93 mmol) was added to an ice-cooled solution of
acid 8 (955 g, 2.66 mmol) in a mixture of THF (20 mL) and CH2Cl2
(15 mL) and stirred for 30 min. at RT. L-Leucinol was added and the
mixture was stirred at RT for 70 h, after which the solvent was removed
and the residue was recrystallized from CH2Cl2/hexane to give N-(1-
hydroxy-4-methylpentan-2-yl)-1-(4-((phenyldiazenyl)phenylsulfonyl)
pyrrolidine-2-carboxamide as an orange solid in 60.0% yield. m.p.
152–154 °C. IR (cm−1) 1636 (C]O), 3376 (amide). 300 MHz 1H
NMR (CDCl3) δ 7.97–8.10 (m, 6H, Ar), 7.55–7.59 (m, 3H, Ar), 6.75
(d, J = 9.0 Hz, 1H, CONH), 4.08–4.18 (m, 1H, CH2OH), 4.02–4.06 (dd,
J = 3.3 Hz and 3.6 Hz, 1H, NCHCO), 3.79–3.84 (dd, J = 3.6 Hz and
3.3 Hz, 1H, CH2OH), 3.67–3.73 (m, 1H, CHCH2CH(CH3)2), 3.53–3.58
4.1.7. 5-Methyl-3-methyl-2-(4-styrylphenylsulfonamido)butanamido)-2-
oxohexanamide (5)
Oxidation of 19 (400 mg, 0.798 mmol) with pyridine-sulfur trioxide
complex (general procedure 2) followed by flash chromatographic
purification (acetone/hexane, 1:1) of the crude product gave 5 as a
white solid in 52% yield. m.p. 209–211 °C. IR (cm−1) 1637 (C]O),
1693 (C]O), 1740 (C]O) 3124, 3276, 3359, 3463 (amide). 500 MHz
1H NMR (CDCl3) δ 7.82 (d, J = 8.5 Hz, 2H, Ar), 7.61 (d, J = 8.0 Hz,
2H, Ar), 7.54 (d, J = 8.0 Hz, 2H, Ar), 7.41 (t, J = 7.2 Hz, 2H, Ar), 7.32
(t, J = 7.2 Hz , 1H, Ar), 7.24 (d, J = 16.5 Hz, 1H, CH = CH), 7.10 (d,
J = 16 Hz, 1H, CH = CH), 6.71 (s, 1H, CONH2), 6.21 (d, J = 7.5.0 Hz,
1H, SO2NH), 5.43 (s, 1H, CONH2), 5.33 (d, J = 8.0 Hz, 1H, CONH),
5

I.O. Donkor, et al.
Bioorganic&MedicinalChemistry28(2020)115433
5.13 (m, 1H, CHCH2CH(CH3)2), 3.58 (dd, 1H, NHCHCO), 2.06–2.11 (m,
1H, NCHCH(CH3)2), 1.31–1.35 (m, 2H, CH2CH(CH3)2), 1.26 (m, 1H,
CH2CH(CH3)2), 0.92 (d, J = 7.0 Hz, 3H, NHCHCHCH3), 0.84–0.87 (m,
9H, CH(CH3)2). MS (ESI) 498.0 [M−H]-. Anal. Calcd. for:
1.85 mmol) in MeOH (30 mL) and stirred at RT for 30 h. EtOAc (25 mL)
was added followed by 3.2% KCN solution and stirring was continued
for another 5 h. The EtOAc layer was separated and the aqueous layer
was extracted twice with EtOAc. The combined organic layer was wa-
shed with water, brine, and dried (MgSO4). The solvent was removed in
vacuo to give an orange solid in 90% yield as a mixture of diaster-
eomers. m.p 93–98 °C. IR (cm−1) 3322 (OH), 1659 (C]O). 300 MHz 1H
NMR (CDCl3) δ 7.96–8.04 (m, 6H, Ar), 7.56–7.63 (m, 3H, Ar),
6.40–6.53 (m, 1H, SO2NH), 5.43–5.52 (m, 1H, CONH), 4.50–4.57 (m,
1H, OHCHCN), 4.08–4.24 (m, 1H, OHCHCN), 3.55–3.66 (m, 1H,
NHCHCONH), 2.12–2.24 (m, 1H, NHCHCH2CH(CH3)2), 1.40–1.58 (m,
4H, NHCHCH(CH3)2, and CH2CH(CH3)2), 0.74–1.00 (m, 12H, CH3
C
26H33N3O5S·0·.2H2O, C, 62.06; H, 6.69; N, 8.35; S, 6.37. Found: C,
62.01; H, 6.59; N, 8.33; S, 6.27.
4.1.8. 3-Methyl-2-(4-(phenyldiazenyl)phenylsulfonamido)butanoic
acid
(7)
(E)-4-(Phenyldiazenyl)benzene-1-sulfonyl chloride
6
(3 g,
10.69 mmol) was added to a stirred ice-cooled solution of L-Val-
OMe·HCl (2.15 g, 12.8 mmol) in anhydrous pyridine (15 mL) and the
resulting crimson solution was stirred at RT for 44 h. Following this the
mixture was poured into 2.0 N HCl (100 mL) and extracted with EtOAc
(2 × 60 mL). The extract was washed successively with 1.0 N HCl,
saturated NaHCO3, brine, dried (MgSO4), and concentrated. The re-
sidue was purified by column chromatography (EtOAc/hexane, 1:2) to
give methyl 3-methyl-2-(4-(phenyldiazenyl)phenylsulfonamido)bu-
protons). MS (ESI) 508.1 [M + H]+
.
4.1.11. Methyl-2-hydroxy-5-methyl-3-(3-methyl-2-(4-phenyldiazenyl)
phenyl sulfonamido) butanamido)hexanoate (10)
Concentrated HCl (15 mL) was added to a solution of 9 (810 mg,
1.67 mmol) in MeOH (20 mL) and the mixture was stirred at 62 °C for
24 h followed by concentration in vacuo and purification by column
chromatography (EtOAc/hexane, 1:1) to give an orange solid in 69%
yield. m.p 163–168 °C. IR (cm−1) 3522, 3355 and 3270 (NH and OH),
1737 and 1648 (C]O). 300 MHz 1H NMR (CDCl3) δ 7.95–8.02 (m, 6H,
Ar), 7.55–7.57 (m, 3H, Ar), 5.96 (d, J = 9.0 Hz, 1H, SO2NH), 5.57 (d,
J = 7.8 Hz, 1H, CONH), 4.23–4.32 (m, 2H, OHCHCOOCH3 and
NHCHCH2), 3.80 (s, 3H, OCH3), 3.59 (dd, J = 4.8 Hz and 7.8 Hz, 1H,
NHCHCO), 3.10 (br s, 1H, OHCHCOOCH3), 2.07–2.15 (m, 1H,
NHCHCH(CH3)2), 1.04–1.41 (m, 3H, CH2CH(CH3)2), 0.68–0.98 (m,
12H, CH3 protons). MS (ESI) 517.0 [M−H]-.
tanoate as an orange solid in 92.5% yield. m.p. 150–152 °C. IR (cm−1
)
1736 (ester), 3282 (amide). 300 MHz 1H NMR (CDCl3) δ 7.94–8.03 (m,
6H, Ar), 7.53–7.60 (m, 3H, Ar), 5.18 (d, J = 9.9 Hz, 1H, SO2NH), 3.83
(dd, J = 10.2 Hz and 5.1 Hz, 1H, NHCHCO), 3.47 (s, 3H, OCH3),
2.04–2.11 (m, 1H, CH3CHCH3), 0.99 (d, J = 6.9 Hz, 3H, CH3), 0.90 (d,
J = 6.9 Hz, 3H, CH3). MS (ESI) 374.0 [M−H]-. NaOH (1.0 N, 100 mL)
was added to a solution of the orange solid (3.3 g, 8.8 mmol) in MeOH
(50 mL) and the mixture was stirred at 55 °C for 4 h after which the pH
was adjusted to 2 with 2.0 N HCl. The solvent was removed, and the
crude product was recrystallized from CH2Cl2/ethyl acetate to give 7 as
yellow solid in 75.5% yield. m.p.220–222 °C. IR (cm−1) 1694 (C]O),
2876 (br, COOH), 3294 (amide). 300 MHz 1H NMR (CDCl3) δ 7.95–8.00
(m, 6H, Ar), 7.54–7.59 (m, 3H, Ar), 5.17 (d, J = 10.2 Hz, 1H, SO2NH),
3.87 (dd, J = 4.5 Hz and 9.9 Hz, 1H, NHCHCO), 2.07–2.18 (m, 1H,
CH3CHCH3), 0.99 (d, J = 6.6 Hz, 3H, CH3), 0.88 (d, J = 6.6 Hz, 3H,
CH3). MS (ESI) 360.0 [M−H]-.
4.1.12. 2-Hydroxy-5-methyl-3-(3-methyl-2-(4-(phenyldiazenyl)
phenylsulfonamido) butanamido) hexanamide (11)
Compound 10 (600 mg, 1.15 mmol) was dissolved in 7.0 N me-
thanolic ammonia (30 mL) and stirred at RT for 48 h following which
the solvent was removed and the product was purified by column
chromatography (EtOAc/hexane, 2:1) to give an orange solid in 68%
yield. m.p 243–248 °C. IR (cm−1) 3454, 3357 and 3322 (NH and OH),
1668 and 1644 (C]O). 300 MHz 1H NMR (DMSO‑d6) δ 7.58–8.06 (m,
9H, Ar), 7.40 (d, 2H, NH2), 7.03–7.14 (m, 2H, SO2NH and CONH),
5.48–5.55 (dd, J = 5.7 Hz and 5.7 Hz, 1H, OHCHCONH2), 3.93–3.96
(m, 1H, OHCHCONH2), 3.64–3.74 (m, 2H, NHCHCH2CH(CH3)2 and
NHCHCH(CH3)2), 1.85–1.92 (m, 1H, NHCHCH(CH3)2), 1.23–1.26 (m,
1H, CH2CH(CH3)2), 0.70–1.11 (m, 8H, CH2CH(CH3)2), 0.43–0.63 (m,
6H, CH(CH3)2). MS (ESI) 501.9 [M−H]-.
4.1.9. 1-(4-(Phenyldiazenyl)phenylsulfonyl)pyrrolidine-2-carboxylic acid
(8)
(E)-4-(Phenyldiazenyl)benzene-1-sulfonyl chloride
6
(5 g,
17.8 mmol) was added to an ice-cooled solution of D-Pro-OMe·HCl
(3.54 g, 21.3 mmol) in anhydrous pyridine (25 mL) and stirred for 20 h
at RT after which the mixture was poured into 2.0 N HCl (50 mL) so-
lution and extracted with EtOAc (2 × 40 mL). The EtOAc extract was
washed successively with 2.0 N HCl, saturated NaHCO3, brine, dried
(MgSO4), and concentrated. The residue was purified by column chro-
matography (acetone/hexane, 1:2) to give methyl 1-(4-(phenyldia-
zenyl)phenyl-sulfonyl)pyrrolidine-2-carboxylate as a yellow solid in
87.3% yield. m.p. 126–128 °C. IR(cm−1) 1743 (C]O). 300 MHz 1H
NMR (CDCl3) δ 8.04 (s, 4H, Ar), 7.95–7.98 (m, 2H, Ar), 7.53–7.60 (m,
3H, Ar), 4.40 (dd, J = 5.6 Hz and 6.9 Hz, 1H, NCHCOOCH3), 3.73 (s,
3H, OCH3), 3.52–3.58 (m, 1H, NCH2CH2), 3.39–3.45 (m, 1H,
NCH2CH2), 1.98–2.12 (m, 3H, NCH2CH2CH2), 1.84–1.87 (m, 1H,
NCH2CH2CH2). The yellow solid (1.5 g, 4 mmol) was dissolved in
MeOH (50 mL) and 1.0 N NaOH (90 mL) was added and the mixture
was stirred at RT for 16 h followed by 2 h of refluxing. The mixture was
adjusted to pH 2 with 2.0 N HCl and extracted with EtOAc. The extract
was washed with brine, dried (MgSO4), concentrated and purified by
recrystallization from CH2Cl2/hexane to give 8 as orange solid in 87.3%
yield. m.p. 174–177 °C. IR (cm−1) 1730 (C]O), 2883 (br, COOH).
300 MHz 1H NMR (CDCl3) δ 8.05 (s, 4H, Ar), 7.96–7.99 (m, 2H, Ar),
7.55–7.57 (m, 3H, Ar), 4.38 (br, 1H, NCHCOOH), 3.58 (m, 1H,
NCH2CH2), 3.35–3.38 (m, 1H, NCH2CH2), 1.83–2.18 (m, 4H,
NCH2CH2CH2). MS (ESI) 358.0 [M−H]-.
4.1.13. Methyl 3-methyl-2-(4-styrylphenylsulfonamido)butanoate (14)
Bromobenzene (12.2 mL, 116 mmol), Pd(OAc)2 (387 mg,
1.74 mmol), TEA (17 mL), and tris(2-methylphenyl)phosphine (1.06 g,
3.5 mmol) were added to a solution of p-styrene sulfonic acid sodium
salt (20 g, 97 mmol) in DMF (200 mL)/ H2O (4 mL) mixture. The
mixture was stirred at 100 °C for 2 h. After cooling to RT, a gray solid
(21.9 g, 80%) separated out and was recovered by filtration, dried, and
used in the next step without further purification. The solid was dis-
solved in (87 mL)/anhydrous DMF (4.5 mL) mixture and refluxed at
90 °C for 3 h. The solvent was removed in vacuo and the residue was
dissolved in EtOAc (150 mL) and washed successively with 2.0 N HCl,
saturated NaHCO3, brine, and dried (MgSO4). The EtOAc layer was
recovered and concentrated under vacuum to give a gray solid (20.19 g,
93.3%), which was used without further purification. The solid (5 g,
17.95 mmol) was mixed with
hydrochloride (3.6 g,
21.47 mmol) and DIPEA (4 mL) inL-pVyarli-dOinMee(25 mL) and the mixture
was stirred for 4 days at RT, poured into 2.0 N HCl and extracted with
EtOAc. The extract was washed twice with aqueous 1 N HCl, saturated
aqueous NaHCO3, brine, and dried (MgSO4). The solvent was con-
centrated in vacuo and the residue was purified by column chromato-
graphy (acetone/hexane, 1:1) to give a white solid in 45% yield. m.p.
173–175 °C. IR (cm−1) 1735 (ester), 3278 (NH). 300 MHz 1H NMR
4.1.10. N-(1-Cyano-1-hydroxy-4-methylpentan-2-yl)-3-methyl-2-(4-
phenyl-diazenyl) phenylsulfonamido)butanamide (9)
NaHSO3 (3.2%, 11 mL) was added to a solution of 1 (847 mg,
6

I.O. Donkor, et al.
Bioorganic&MedicinalChemistry28(2020)115433
(DMSO‑d6) δ 8.26 (d, J = 9.3 Hz, 1H, SO2NH), 7.71–7.80 (q, 4H, Ar),
7.74–7.66 (d, 2H, CH = CH), 7.30–7.47 (m, 5H, Ar), 3.56 (dd,
J = 7.2 Hz and 9.0 Hz, 1H, NHCHCOOCH3), 3.36 (s, 3H, OCH3),
1.87–1.94 (m, 1H, NHCHCH(CH3)2), 0.82 (dd, J = 6.9 Hz and 9.0 Hz,
6H, CH(CH3)2). MS (ESI) 372.0 [M−H]-.
OCH3), 1.79–1.83 (m, 1H, NHCHCH(CH3)2), 1.01–1.27 (m, 3H, CH2CH
(CH3)2), 0.55–0.91 (m, 12H, CH3 protons). MS (ESI) 374.0 [M−H]-.
4.1.18. 2-Hydroxy-5-methyl-3-(3-methyl-2-(4-styrylphenylsulfonamido)-
butanamido) hexanamide (19)
A solution of 18 (750 mg, 1.4 mmol) in 7.0 N methanolic ammonia
(30 mL) was stirred at RT for 96 h and the mixture was concentrated to
4.1.14. 3-Methyl-2-(4-styrylphenylsulfonamido)butanoic acid (15)
NaOH (4.0 N, 40 mL) was added to a solution of 14 (6.85 g,
18.4 mmol) in MeOH (40 mL) and acetone (40 mL) mixture and stirred
for 16 h at 48 °C followed by adjustment of the pH to 2 with 2.0 N HCl
and extraction of the solid that separated out with EtOAc. The extract
was washed with brine, dried (MgSO4), filtered, and concentrated. The
residue was purified by recrystallization from acetone/hexane to give a
yellow solid in 80% yield. m.p. 193–195 °C. IR (cm−1) 1747 (C]O),
3313 (w, COOH). 1H NMR (DMSO‑d6) δ 12.58 (s, 1H, COOH), 8.01 (d,
J = 9.7 Hz, 1H, SO2NH), 7.75 (br. s, 4H, Ar), 7.63–7.66 (d, 2H,
CH = CH), 7.29–7.46 (m, 5H, Ar), 3.54 (dd, J = 6.0 Hz and 9.0 Hz, 1H,
NHCHCOOH), 1.90–1.97 (m, 1H, NHCHCH(CH3)2), 0.81 (dd,
J = 6.9 Hz and 9.6 Hz, 6H, CH(CH3)2). MS (ESI) 358.0 [M−H]-.
give
a residue, which was purified by column chromatography
(acetone/hexane, 2:1) to obtain 19 as a white solid in 64% yield. m.p.
234–242 °C. IR (cm−1) 1660, 1643 (C]O), 3326 (OH). 1H NMR
(DMSO‑d6) δ 7.04–7.77 (m, 15H, Ar, CONH, SO2NH, CH = CH, and
CONH2. Addition of D2O decreased the protons in this region from 15 to
11 (i.e., 9 Ar protons plus 2 ethylene protons) , 5.49–5.54(m, 1H, OH),
3.96–4.03 (m, 1H, OHCHCONH2), 3.58–3.71 (m, 2H, NHCHCH2 and
NHCHCO), 1.84–1.93 (m, 1H, NHCHCH(CH3)2), 1.24–1.34 (m, 1H,
CH2CH(CH3)2), 1.03–1.15 (m, 2H, CH2CH(CH3)2), 0.53–0.87 (m, 12H,
CH3 protons). MS (ESI) 500.0 [M−H]-.
4.2. Biological studies
4.1.15. N-(1-Hydroxy-4-methylpentan-2-yl)-3-methyl-2-(4-
4.2.1. Calpain inhibition assay
styrylphenylsulfonamido)-butanamide (16)
The Ki values for inhibition of μ-calpain activity was monitored in a
reaction mixture containing 50 mM Tris HCl (pH 7.4), 50 mM NaCl,
10 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA, 0.2 mM or 1.0 mM
Suc-Leu-Tyr-AMC (Calbiochem), 1 μg porcine erythrocyte μ-calpain
(Calbiochem), varying concentrations of inhibitor dissolved in DMSO
(2% total concentration) and 5 mM CaCl2 in a final volume of 250 μL in
a polystyrene microtiter plate as previously reported.22 The Ki values
were estimated from the semi-reciprocal plots of the initial velocity
versus the concentration of the inhibitor according to the method of
Dixon.23 The correlation coefficients for the Dixon plots were above
0.95. No other attempt was made to correct for slow binding or auto-
lysis. The reported Ki values are the average of triplicate determina-
tions.
werLe-Laedudceidnotlo(a2.s5o1lumtioLn, 1o9f .125m(5m.3olg),a1n4d.7T6EmAm(4ol.5) 3inmanL,hy3d2r.o5ums DmMolF)
(150 mL) at 0 °C followed by the addition of Mukaiyama’s reagent
(4.53 g, 17.7 mmol). After stirring for 4 days at RT, the solution was
poured into H2O (1.5 L) to give the crude product, which was purified
by recrystallization from EtOAc to obtain a white solid in 47% yield.
m.p. 196–197 °C. IR (cm−1) 1638 (C]O), 3282 (sulfamide), 3205
(amide), 3503 (br, OH). 300 MHz 1H NMR (DMSO‑d6) δ 7.62–7.79 (m,
7H, Ar), 7.54 (d, 1H, SO2NH), 7.28–7.43 (m, 5H, CONH, CH = CH, and
Ar), 4.55 (br s, 1H, OH), 3.55 (m, 1H, CH2OH), 3.49 (m, 1H, CH2OH),
3.19 (m, 1H, NHCHCH2), 3.05 (m, 1H, NHCHCH(CH3)2, 1.80–1.86 (m,
1H, NHCHCH(CH3)2), 1.10–1.18 (m, 2H, CH2CH(CH3)2), 0.99–1.04 (m,
1H, CH2CH(CH3)2), 0.63–0.86 (m, 12H, CH3 protons). MS (ESI) 457.0
[M−H]-.
4.2.2. Cell lines and cell cultures
The melanoma cell lines (human A375 cells and mouse B16F1 cells)
and the prostate cancer cell lines (PC3 and DU-145) were acquired from
ATCC and cultured as follows: The melanoma cells were cultured in
DMEM supplemented with 5% (v/v) fetal bovine serum (FBS), 1% (v/v)
antibiotic/antimycotic solution, and 0.05% bovine insulin (25 mM, pH
8.2). The prostate cancer cell lines were cultured in RPMI-1640 medium
(PC3 cells) or EMEM (DU145 cells) supplemented with 10% FBS, 1%
penicillin/streptomycin and 2% L-glutamin. All of the cell cultures were
maintained at 37 °C in a humidified atmosphere of 5% CO2 in air.
4.1.16. N-(1-Cyano-1-hydroxy-4-methylpentan-2-yl)-3-methyl-2-(4-
styrylphenylsulfonamido) butanamide (17)
NaHSO3 (3.2%, 14 mL) was added to a solution of 4 (2 g,
4.38 mmol) in MeOH (30 mL) and stirred at RT for 40 h followed by the
addition of 3.2% KCN solution (10 mL) and EtOAc (50 mL). After
stirring for another 5 h, the mixture was extracted with EtOAc
(3 × 50 mL). The extract was concentrated, and the residue was pur-
ified by recrystallization from acetone/hexane to give diasteromeric
mixture of 17 as a white solid in 90% yield. m.p. 125–128 °C. IR (cm−1
)
1736 (ester), 3282 (amide). 1H NMR (CDCl3)
δ 7.86–7.88 (d,
J = 8.4 Hz, 2H, Ar), 7.64–7.67 (m, 2H Ar), 7.54–7.57 (d, J = 8.1 Hz,
2H, Ar), 7.32–7.44 (m, 3H, Ar), 7.24–7.28 (d, J = 13.8 Hz, 1H,
CH = CH), 7.09–7.15 (d, J = 16.2 Hz, 1H, CH = CH), 6.53–6.69 (m,
1H, CONH), 5.33–5.40 (m, 1H, SO2NH), 4.51–4.59 (m, 2H, OHCHCN
and NHCHCH2), 4.10–4.27 (m, 1H, NHCHCO), 3.50–3.61 (m, 1H,
NHCHCH(CH3)2), 2.15–2.25 (m, 1H, CH2CH(CH3)2), 1.43–1.62 (m, 3H,
OH and CH2CH(CH3)2), 0.81–0.98 (m, 12H, CH3 protons). MS (ESI)
374.0 [M−H]-.
4.2.3. Sulforhodamine B colorimetric cytotoxicity assay
During the initial screen, compounds dissolved in DMSO were
added to plates to give a 20 μM solution followed by sulforhodamine B
(SRB). To determine IC50 values, human melanoma cell line A375 and
mouse melanoma cell line B16F1 were trypsinized and harvested by
centrifugation for 3 min. Cells were then resuspended in 5% FBS, 1%
penicillin/streptomycin and 0.05% insulin in DMEM and counted using
a hemocytometer. Cells were seeded into U-bottom 96-well microtiter
plate at 5000 cells/well. After 12 h, media was changed, and the test
compound was added at 8 different concentrations. Cells were in-
cubated with each compound for 48 h. Taxol was used as positive
control and wells containing media and cells only served as negative
controls. Following the incubation period cell death was quantitated by
sulforhodamine B assay according to the manufacturer's protocol
(Sigma-Aldrich). Cells were fixed with 10% TCA, washed five times
with water, and incubated with 0.4% sulforhodamine B in 1% acetic
acid solution for 30 min at RT. A solution of Tris Base (10 mM) was
added to release the dye from the cells for 1 h and the absorbance was
measured at 490 nm using an EL800 microplate reader (Biotek).
4.1.17. 3-Methyl-2-hydroxy-5-methyl-3-(3-methyl-2-(4-
styrylphenylsulfonamido)-butanamido) hexanoate (18)
Compound 17 (932 mg, 19.4 mmol) was dissolved in MeOH (40 mL)
containing conc. HCl (11 mL) and the mixture was stirred for 24 h at
54 °C. The solvent was removed in vacuo and the residue was purified
by column chromatography (acetone/hexane, 1:1) to give 18 as a white
solid in 75.2% yield. m.p. 180–183 °C. IR (cm−1) 1736 (ester), 3282
(amide). 1H NMR (DMSO‑d6) δ 7.28–7.73 (m, 13H, Ar, CONH, SO2NH,
and CH = CH), 5.35–5.58 (m, 1H, OH), 3.83–3.97 (m, 2H, OHCHCO-
OCH3 and NHCHCH2), 3.58–3.68 (m, 1H, NHCHCO), 3.54 (s, 3H,
7

I.O. Donkor, et al.
Bioorganic&MedicinalChemistry28(2020)115433
4.2.4. MTT cytotoxicity assay
processes. J Nat Sci. 2016;2(9):e218.
3. Wang Y, Liu Y, Lopez D, et al. Protection against TBI-induced neuronal death with
post-treatment with a selective calpain-2 inhibitor in mice. J Neurotrauma.
2018;35:105–117. https://doi.org/10.1089/neu.2017.5024.
Cells were distributed into 96-well plates at a density of 2,000 to
15,000 cells/well, and exposed to a range of drug concentrations (1 to
100 μM) for 72 h at 37 °C in a 5% CO2 atmosphere. Wells to which no
drug was added were used as negative controls. At the end of treatment,
an aliquot (25 μL) of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
2H-tetrazolium bromide) dye (5 mg/mL) was added to each well for the
final two to four hours of incubation. The plates were then centrifuged
at 300 g for 15 min. The supernatant medium in each well was aspi-
rated, and the formazan product was solubilized with 100 μL DMSO.
The absorbance (A) values of wells were determined at 595 nm using a
MRX microplate reader (DYNEX Technologies, VA). Percentage cell
survival was plotted against the drug concentration and the IC50, the
concentration of drug required to reduce living cell number by 50% as
compared to non-drug treated wells, was determined by nonlinear re-
gression analysis using WinNonlin (Pharsight Corporation, Mountain
View, CA).
4. Curcio M, Salazar IL, Mele M, Canzoniero LM, Duarte CB. Calpains and neuronal
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4.2.5. Matrigel cell invasion assay
DU-145 cells (20,000 cells) in serum-free medium containing 1%
BSA was seeded on the inner surface of the upper chamber and in-
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10% FBS as chemoattractant. For pharmacological inhibition, the
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serum-free medium in the absence or presence of compound 3 at three
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Acknowledgement
17. Shaw E. Cysteinyl proteinases and their selective inactivation. Adv. Enzymol.
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This work was supported in part by a Seed Grant from the College of
Pharmacy, University of Tennessee Health Science Center.
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Appendix A. Supplementary data
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