Design, synthesis and stepwise optimization of nitrile-based inhibitors of cathepsins B and L

Article abstract of DOI:10.1016/j.bmc.2020.115827

Human cathepsin B (CatB) is an important biological target in cancer therapy. In this work, we performed a knowledge-based design approach and the synthesis of a new set of 19 peptide-like nitrile-based cathepsin inhibitors. Reported compounds were assayed against a panel of human cysteine proteases: CatB, CatL, CatK, and CatS. Three compounds (7h, 7i, and 7j) displayed nanomolar inhibition of CatB and selectivity over CatK and CatL. The selectivity was achieved by using the combination of a para biphenyl ring at P3, halogenated phenylalanine in P2 and Thr-O-Bz group at P1. Likewise, compounds 7i and 7j showed selective CatB inhibition among the panel of enzymes studied. We have also described a successful example of bioisosteric replacement of the amide bond for a sulfonamide one [7e → 6b], where we observed an increase in affinity and selectivity for CatB while lowering the compound lipophilicity (ilogP). Our knowledge-based design approach and the respective structure–activity relationships provide insights into the specific ligand-target interactions for therapeutically relevant cathepsins.

Full text of DOI:10.1016/j.bmc.2020.115827

Journal Pre-proofs
Design, synthesis and stepwise optimization of nitrile-based inhibitors of
cathepsins B and L
Lorenzo Cianni, Fernanda Dos Reis Rocho, Vinícius Bonatto, Felipe Cardoso
Prado Martins, Jerônimo Lameira, Andrei Leitão, Carlos A. Montanari,
Anwar Shamim
PII:
S0968-0896(20)30657-X
https://doi.org/10.1016/j.bmc.2020.115827
BMC 115827
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
11 May 2020
21 October 2020
22 October 2020
Please cite this article as: L. Cianni, F. Dos Reis Rocho, V. Bonatto, F. Cardoso Prado Martins, J. Lameira, A.
Leitão, C.A. Montanari, A. Shamim, Design, synthesis and stepwise optimization of nitrile-based inhibitors of
cathepsins B and L, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115827
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Design, synthesis and stepwise optimization of nitrile-based inhibitors of
cathepsins B and L
Lorenzo Ciannia§, Fernanda Dos Reis Rochoa§, Vinícius Bonattoa, Felipe Cardoso Prado
Martinsa, Jerônimo Lameiraab, Andrei Leitãoa, Carlos A. Montanaria,*, Anwar Shamima*
§These authors contributed equally to the work
aMedicinal and Biological Chemistry Group, Institute of Chemistry of São Carlos, University
of São Paulo, Avenue Trabalhador Sancarlense, 400, 23566-590, São Carlos/SP, Brazil.
bOn leave from Drug Designing and Development Laboratory. Federal University of Pará.
Rua Augusto Correa S/N. Belém-PA.
*corresponding authors: Anwar Shamim, shamim.hej@gmail.com; Carlos A. Montanari,
carlos.montanari@usp.br
§These authors contributed equally to the work
1

Abstract
Human cathepsin B (CatB) is an important biological target in cancer therapy. In this
work, we performed a knowledge-based design approach and the synthesis of a new set
of 19 peptide-like nitrile-based cathepsin inhibitors. Reported compounds were assayed
against a panel of human cysteine proteases: CatB, CatL, CatK, and CatS. Three
compounds (7h, 7i, and 7j) displayed nanomolar inhibition of CatB and selectivity over
CatK and CatL. The selectivity was achieved by using the combination of a para
biphenyl ring at P3, halogenated phenylalanine in P2 and Thr-O-Bz group at P1.
Likewise, compounds 7i and 7j showed selective CatB inhibition among the panel of
enzymes studied. We have also described a successful example of bioisosteric
replacement of the amide bond for a sulfonamide one [7e6b], where we observed an
increase in affinity and selectivity for CatB while lowering the compound lipophilicity
(ilogP). Our knowledge-based design approach and the respective structure-activity
relationships provide insights into the specific ligand-target interactions for
therapeutically relevant cathepsins.
Keywords: human cysteine proteases, cathepsin B, cathepsin L, nitrile inhibitors
1. Introduction
Cysteine proteases (CPs) are found in every living organism1 and are known to be involved in
a wide range of diseases, such as cancer,2 infectious3 and neurodegenerative diseases.4 Human
cysteine cathepsins are a family of CPs that are widely studied for being essential targets for
cancer and immune system-related diseases.5,6
2

Human cathepsin B is an enzyme ubiquitously expressed in the lysosome while being
considered a crucial tumor-promoting factor.7,8 The enzyme is known to be involved in the
degradation of the extracellular matrix (ECM), which changes the tumor environment and
thus facilitates tumor migration, invasion and even metastasis.9–11 Therefore, CatB has been
validated as an essential drug target for various types of cancer.10,12,13
CatB has a pH-dependent activity that regulates its ability to exhibit exopeptidase or
endopeptidase activity. The two location-driven activities are believed to mediate tumour
progression.14,15 These two activities occur due to the presence of an occluding loop present in
the primed binding region of the enzyme. The occluding loop can change the form of the
enzyme to an open (neutral pH) or closed (acidic pH) conformation; the occluding-loop
orientation can also be influenced by other effects such as the substrate/inhibitor bimolecular
recognition process.7,10,16
Along with CatB, cathepsin L is overexpressed during tumour expansion, which leads to its
expulsion to outer cells.9 This could be an indicator of a possible role of both cathepsins
during cancer progression.5,9,17 Consequently, the inhibition of both CatB and CatL might be
advantageous when these cathepsins are upregulated in breast cancer.2 Hence, as shown by
Blatt and co-workers, the activity of different cathepsins inside the cells is too multifaceted,
and this could explain why potent and selective inhibitors of CatL did not advance to clinical
phases yet.18,19 Therefore, the current challenge is to develop a molecular scaffold based on a
structure design approach that can lead to a potent singular cathepsin inhibitor or a cross-class
cysteine protease inhibitor by just a single atom modification. The challenge can also be
expanded to activity-based probes (ABPs), which usually require a high degree of selectivity
to achieve the desired biological response with minor interference.18,20 As recently reported,18
the root mean square deviation (RMSD) values for the whole structure and active site regions
(amino acids near co-crystallized ligands) for these cysteine proteases highlight a substantial
3

structural similarity, excluding CatB. Indeed, the latter has three different main features: (i)
the presence of an occluding loop at the N-terminal of the active site; (ii) the S2 binding site is
broader and shallower than the other endopeptidases; (iii) the S2’ is a superficial subsite.
However, small differences in active site residues in the active site lead to observed strong
substrate specificities amongst CatL, CatK and CatS.18,21,22 The S1 subsite is conserved in all
cathepsins, while the surface of the only deep binding pocket (S2 subsite) is less conserved,
which has been used in the past to achieve a certain degree of selectivity. Specifically, CatB
has a larger S2 pocket than the other cathepsins and it is the only one containing polar amino
acid (Glu245) at the bottom of the pocket. CatK contains the smallest one, shaped by Leu209
and Tyr67, that uniquely recognizes a proline residue. Sequence and structure differences in
the area beyond the S2 subsite and on the prime side have been used recently to design nitrile
inhibitors with higher selectivity.21 The compounds described by Gütschow and collaborators
anchor the CatB occlusion loop to leverage the selectivity of the carboxylate moiety,
exhibiting Ki values of 41.3 nM, 27.3 nM, or 19.2 nM, depending on the substrate and pH of
the assay (Figure 1). Therefore, here we report the design and stepwise optimization of new
selective and potent dipeptidyl nitrile derivatives that act as low nanomolar inhibitors of CatB
and CatL by leveraging the S3 and S1’ subsites. The compounds were assayed against a panel
of cysteine proteases, which included CatB, CatL, CatK, and CatS. The effects of different
moieties in the P3 and P2 positions were evaluated through a structure-activity relationship,
seeking to identify selectivity between the different kinds of CPs.
2. Materials and Methods
2.1.
Synthesis
2.1.1. General consideration
4

Melting points were determined on a Büchi 510 oil bath apparatus and are uncorrected.
Infrared spectra were obtained from FT-IR Thermo Scientific Nicolet 380. Reagents, starting
materials and solvents were of commercial quality and were used without further purification
unless otherwise stated. All syntheses were started with enantiopure amino acids. TLC
analysis was carried out on Merck 60 F254 silica gel plates and visualized under UV light at
254 nm and 365 nm or by using a ninhydrin staining solution.
Purity was determined with an LC-MS instrument (AmaZon SL ESI-MS, Shimadzu LC) with
a cellulose-2 Phenomenex column (250 × 4.6 mm, 5 μm) or a Diacel column (IC-chiralpak,
250 × 4.6 mm, 5 μm). Isocratic elution with MeCN and water was applied as specified, stop
1
13
time 60 min, flow 0.5 mL/min. H and C NMR spectra were recorded on either 400 MHz
Agilent NMR spectrometer or 500 MHz Agilent NMR spectrometer. Chemical shifts (δ) are
reported in parts per million, and coupling constant (J) values are given in Hz, using
tetramethylsilane (TMS) as an internal reference. HRMS spectra were recorded on a Thermo
Scientific LTQ Velos Orbitrap in electrospray ionization (ESI) mode by direct injection.
2.1.2. General procedure for amide synthesis (GP1)
Method A: Isobutyl chloroformate (5.5 mmol, 1 equiv., 0.75 mL) was added dropwise to a
solution of Boc-(L) amino acid enantiomeric pure (5.0 mmol, 1 equiv.), DIPEA (13.00 mmol,
2.36 equiv., 2.28 mL) in dry DMF (20 mL), under argon atmosphere, at -30 °C and it was
stirred for 30 minutes. Then an aqueous 2 M NH4Cl solution (5.5 mmol, 1 equiv., 2.75 mL)
was added. The resulting solution was stirred at room temperature for 20 hours. The reaction
mixture was dried under reduced pressure and ethyl acetate (100 mL) was added, following a
washing step with a saturated NaHCO3 solution (3 × 50 mL) and brine (1 × 50 mL). The
organic phase was dried over Na2SO4 and evaporated under reduced pressure to give a crude
residue that was purified by flash column chromatography.
5

Method B: The primary free amine (1 mmol, 1 equiv.) was added to a solution of the
carboxylic acid (1.3 mmol, 1.3 equiv., see Supporting Information for the synthesis and
characterization of the carboxylic acids 11a and 11b and its precursor, the rest of the
carboxylic acids were purchased), HATU (1.3 mmol, 1 equiv., 0.49 g) and DIPEA (2.60
mmol, 2 equiv., 0.45 mL) in dry DMF (5 mL), under argon atmosphere. The resulting
solution was stirred at room temperature for 20 hours. The reaction mixture was diluted with
ethyl acetate (25 mL) and washed with a saturated NaHCO3 solution (3 × 20 mL) and brine (3
× 20 mL). The organic phase was dried over Na2SO4 and evaporated to give a crude residue
that was purified by flash column chromatography.
2.2.3 General procedure for dehydration of primary amide to nitrile (GP2)
Method A: The primary amide (1 mmol, 1 equiv.) was dissolved in dry THF (5 mL) and
DIPEA (2.6 mmol, 2.6 equiv., 0.45 mL) was added. Trifluoroacetic anhydride (1.3 mmol, 1
equiv., 0.18 mL) was added over 5 min, at 0 °C. The mixture was stirred and allowed to reach
room temperature within 2 hours. Then the reaction was quenched with H2O (20 mL), THF
removed in vacuum, and the product extracted into ethyl acetate (2 × 50 mL). The organic
phase was washed with a 1 M solution of KHSO4 (3 × 20 mL) then with a saturated NaHCO3
solution (3 × 20 mL) and brine (3 × 20 mL) and dried over Na2SO4. The solvent was
removed, and the crude product was purified by flash chromatography.
Method B: The primary amide (1 mmol, 1 equiv.) was dissolved in dry DMF (5 mL) at 0 °C.
Then cyanuric chloride (1.1 mmol, 1.1 equiv., 0.15 g) was added slowly to the solution under
the argon atmosphere. The resulted solution was stirred for 30 minutes and then 30 mL of a
saturated NaHCO3 solution was added and it was stirred at room temperature for 2 hours. The
product was extracted with ethyl acetate (2 × 50 mL) and then the combined organic phases
were washed with 1 M solution of KHSO4 (3 × 20 mL), brine (4 × 30 mL) and dried over
6

Na2SO4. The solvent was removed, and the crude product was purified by flash
chromatography.
2.2.4 General procedure for removal of Boc protecting group (GP2)
Method A: The Boc-protected compound was treated with formic acid (2 mL) at room
temperature. The resulting solution was stirred for 18 hours. The reaction mixture was
evaporated under reduced pressure to get a yellowish oil, which was then treated with an
aqueous solution of 1M NaOH until pH 9. The product was extracted with ethyl acetate (4 ×
20 mL) and then washed with brine (1 × 20 mL). The combined organic phase was
evaporated to obtain a colorless oil. The formation of the product was confirmed by TLC
(ethyl acetate). The product was used for the next step without any further purification.
Method B: To a solution of Boc-protected compound (1 mmol, 1 equiv.) in dry DCM (5 mL),
TFA (8 mmol, 8 equiv., 0.91 mL,) was added at 0 °C. The mixture was stirred and allowed to
reach room temperature within 2 hours. The progress of the reaction was monitored by TLC
(ethyl acetate). The reaction mixture was evaporated under reduced pressure to eliminate the
excess of TFA to get a yellowish solid. The product was used for the next step without any
further purification.
2.2.5 General Procedure for sulfonamides synthesis (GP4)
In a round bottom flask, the amine 5a (0.30 mmol, 1 equiv., 112 mg) and the appropriate
sulfonyl chloride (0.33 mmol, 1.1 equiv.) were added under argon atmosphere. Then dry
dichloromethane (5 mL) was added and the temperature was lowered to 0 °C. Then, DIPEA
(0.9 mmol, 3 equiv., 0.16 mL) was added slowly and the reaction mixture was stirred at room
temperature for 16 hours. After completion of the reaction (monitored by TLC), the mixture
was extracted with brine and dichloromethane. The organic phase was dried over Na2SO4,
7

filtered, and evaporated under low pressure. The crude product was purified via flash column
chromatography using an elution system of 30-40% ethyl acetate in hexane.
2.2.5 General Procedure for the formation of C-C bond via Suzuki Reaction
(GP5)
A stream of argon was passed through a suspension of aryl bromide 7d (0.3 mmol, 1 equiv.,
238 mg). The proper aryl/heteroaryl boronic acid (0.56 mmol. 1.3 equiv.), 2M Na2CO3 (1.52
mL) and DMF (5 mL) for 10 min. PdCl2dppf (5 mol%) was then added and the reaction was
warmed to 80 ºC and stirred under argon atmosphere. After 3 h, 15 mL of water was added
and the aqueous phase was extracted with ethyl acetate (3 × 15 mL). The combined organic
phases were dried with sodium sulfate and purified by flash column chromatography using a
gradient of ethyl acetate/n-hexane (3:7 to 1:0) to give a light yellow or white solid with a 50-
75% yield.
2.2.4 Characterization of the final compounds
(For the characterization of intermediates, please see Supporting Information)
N-((S)-1-(((1R,2R)-2-(benzyloxy)-1-cyanopropyl)amino)-3-(3-bromophenyl)-1-oxopropan-2-
yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (7b)
Compound 7b was synthesized according to procedure GP1, GP2, and GP3 as a light yellow
solid (28.0% overall yield); Mp: 117.1-118.0 °C; purity (HPLC): > 99%; 1H NMR (500 MHz,
CDCl3): δ 7.46 – 7.28 (m, 6H), 7.19 (dd, J = 12.5, 5.2 Hz, 2H), 6.65 (dd, J = 22.7, 8.2 Hz,
2H), 6.32 (s, 1H), 4.80 (ddd, J = 21.9, 11.4, 5.3 Hz, 2H), 4.58 (dd, J = 42.9, 11.7 Hz, 2H),
4.03 (s, 3H), 3.87 (dd, J = 6.3, 2.9 Hz, 1H), 3.13 (ddd, J = 21.5, 13.8, 7.1 Hz, 2H), 1.29 (s,
9H), 1.17 (d, J = 6.2 Hz, 3H); 13C NMR (126 MHz, CDCl3): δ 170.51, 160.44, 160.01,
137.97, 136.79, 134.17, 132.28, 130.64, 130.59, 128.58, 128.54, 128.22, 127.98, 127.91,
8

127.90, 122.92, 116.91, 103.20, 73.30, 71.64, 54.13, 44.94, 38.92, 37.95, 31.96, 30.47, 30.27,
16.13; IR (film, cm-1): 3312.05, 3222.44, 2245.42, 1672.05, 1641.04, 1540.66, 1514.05,
1455.35, 1381.41, 1286.98, 1097.39, 1040.29;
23 (Chloroform): -18.18.
[α]D
N-((S)-1-(((1R,2R)-2-(benzyloxy)-1-cyanopropyl)amino)-3-(3-chlorophenyl)-1-oxopropan-2-
yl)-4-bromobenzamide (7c)
Compound 7c was synthesized according to procedure GP1, GP2 and GP3 as a white solid
1
(27.7% overall yield); Mp: 134.1-134.5 °C; purity (HPLC): > 96%; H NMR (500 MHz,
DMSO-d6): δ 7.46 – 7.28 (m, 6H), 7.19 (dd, J = 12.5, 5.2 Hz, 2H), 6.65 (dd, J = 22.7, 8.2 Hz,
2H), 6.32 (s, 1H), 4.80 (ddd, J = 21.9, 11.4, 5.3 Hz, 2H), 4.58 (dd, J = 42.9, 11.7 Hz, 2H),
4.03 (s, 3H), 3.87 (dd, J = 6.3, 2.9 Hz, 1H), 3.13 (ddd, J = 21.5, 13.8, 7.1 Hz, 2H), 1.29 (s,
13
9H), 1.17 (d, J = 6.2 Hz, 3H); C NMR (126 MHz, DMSO- d6): δ 170.51, 160.44, 160.01,
137.97, 136.79, 134.17, 132.28, 130.64, 130.59, 128.58, 128.54, 128.22, 127.98, 127.91,
127.90, 122.92, 116.91, 103.20, 73.30, 71.64, 54.13, 44.94, 38.92, 37.95, 31.96, 30.47, 30.27,
16.13; IR (film, cm-1): 3271.27, 3145.90, 1737.86, 1666.50, 1633.71, 1591.27, 1330.88,
1298.09, 1274.95, 1240.23, 1190.08, 1143.79, 1111.00, 1072.42, 840.96, 775.38, 734.88;
[α]2D3
(Chloroform): -71.43.
(2S)‐N‐[(1R,2R)‐2‐(benzyloxy)‐1‐cyanopropyl]‐2‐[(3‐bromophenyl)formamido]‐3‐(3‐chlorop
henyl)propanamide (7d)
Compound 7d was synthesized according to procedure GP1, GP2 and GP3 as a yellow solid
1
(27.7% overall yield); Mp: 120.2-121.0 °C; purity (HPLC): > 99%; H NMR (500 MHz,
CDCl3): δ 7.86 (t, J = 1.8 Hz, 1H), 7.68 – 7.57 (m, 2H), 7.39 – 7.05 (m, 10H), 5.01 (q, J = 7.3
Hz, 1H), 4.88 (dd, J = 8.6, 3.1 Hz, 1H), 4.52 (dd, J = 38.7, 11.8 Hz, 2H), 3.79 (dd, J = 6.3, 3.2
13
Hz, 1H), 3.15 (d, J = 7.2 Hz, 2H), 1.13 (d, J = 6.3 Hz, 3H); C NMR (101 MHz, CDCl3): δ
9

171.14, 166.12, 137.74, 136.86, 135.08, 134.90, 134.60, 130.38, 130.19, 129.42, 128.52,
128.09, 127.78, 127.63, 127.47, 125.75, 122.81, 116.86, 73.23, 71.45, 54.79, 44.96, 38.21,
15.96; IR (film, cm-1): 3272.26, 2248.04, 1664.06, 1633.11, 1597.34, 1381.98, 1230.92,
1089.11, 1046.33, 739.49;
23 (Chloroform): -10.35.
[α]D
(2S)‐N‐[(1R,2R)‐2‐(benzyloxy)‐1‐cyanopropyl]‐2‐(3‐bromobenzenesulfonamido)‐3‐(3‐chloro
phenyl)propanamide (6a)
Compound 6a was synthesized according to procedure GP1, GP2, GP3 and GP4 as a light
1
yellow solid (26.9% overall yield); Mp: 44.2-45.0 °C; purity (HPLC) > 95%; H NMR (500
MHz, CDCl3): δ 7.77 (t, J = 1.8 Hz, 1H), 7.64 (dd, J = 8.0, 1.8 Hz, 1H), 7.58 (dd, J = 8.2, 1.3
Hz, 1H), 7.43 – 7.31 (m, 4H), 7.27 (t, J = 7.9 Hz, 1H), 7.20 – 7.09 (m, 2H), 6.97 (s, 1H), 6.89
(dd, J = 7.0, 1.6 Hz, 2H), 5.58 (dd, J = 15.9, 8.2 Hz, 1H), 4.81 (dd, J = 8.5, 3.1 Hz, 1H), 4.63
(dd, J = 45.0, 11.7 Hz, 2H), 3.99 (dd, J = 14.1, 8.3 Hz, 1H), 3.88 (qd, J = 6.2, 3.2 Hz, 1H),
3.00 (dd, J = 14.1, 5.8 Hz, 1H), 2.94 – 2.81 (m, 1H), 1.17 (d, J = 6.3 Hz, 3H); 13C NMR(126
MHz, CDCl3): δ 170.17, 140.80, 136.94, 136.90, 136.20, 134.64, 130.65, 130.09, 129.64,
129.13, 128.62, 128.23, 128.04, 127.86, 127.41, 125.41, 123.20, 116.86, 73.31, 71.64, 57.79,
45.19, 38.09, 16.08; IR (film, cm-1): 3285.53, 2249.50, 1670.83, 1598.44, 1454.97, 1382.49,
[α]2D3
1160.35, 1083.47, 1046.88, 997.78, 784.28;
(Chloroform): -70.31.
(S)-2-([1,1'-biphenyl]-3-ylsulfonamido)-N-((1R,2R)-2-(benzyloxy)-1-cyanopropyl)-3-(3-
chlorophenyl)propanamide (6b)
Compound 6b was synthesized according to procedure GP1, GP2, GP3 and GP4 as a white
1
solid (26.3 % overall yield); Mp 45.5-45.9 °C; purity (HPLC) > 99%; H NMR (400 MHz,
DMSO- d6): δ 8.90 (d, J = 8.4 Hz, 1H), 7.84 – 7.77 (m, 2H), 7.65 (d, J = 7.2 Hz, 2H), 7.57 –
7.26 (m, 9H), 7.23 (s, 1H), 7.20 – 7.05 (m, 3H), 4.88 (dd, J = 8.3, 3.6 Hz, 1H), 4.46 (q, 2H),
10

4.15 (dd, J = 12.4, 8.3 Hz, 1H), 3.45 (dd, J = 6.2, 3.6 Hz, 1H), 2.87 – 2.64 (m, 2H), 0.93 (d, J
13
= 6.2 Hz, 3H); C NMR (101 MHz, DMSO-d6): δ 171.04, 141.96, 141.08, 139.60, 139.09,
138.25, 133.03, 130.75, 130.05, 129.84, 129.53, 129.43, 128.70, 128.61, 128.46, 128.06,
127.28, 126.92, 125.44, 124.73, 117.95, 73.76, 70.79, 57.17, 44.70, 38.25, 15.61; IR (film,
cm-1) 3308.10, 2250.35, 1675.68, 1595.80, 1529.26, 1333.90, 1206.93, 1157.11, 1096.02,
765.10, 699.01;
23 (Chloroform): -198.04.
[α]D
(2S)‐N‐[(1R,2R)‐2‐(benzyloxy)‐1‐cyanopropyl]‐2‐{[1,1'‐biphenyl]‐4‐sulfonamido}‐3‐(3‐chlor
ophenyl)propanamide (6c)
Compound 6c was synthesized according to procedures GP1, GP2, GP3 and GP4 as a white
solid (26.8% overall yield); Mp: 160.1-161.0 °C; purity (HPLC): > 99%; 1H NMR (400 MHz,
CDCl3): δ 7.71 (d, J = 8.6 Hz, 2H), 7.63 – 7.56 (m, 4H), 7.53 – 7.29 (m, 7H), 7.16 – 7.05 (m,
2H), 6.98 (s, 1H), 6.89 (d, J = 7.2 Hz, 1H), 6.81 (d, J = 8.6 Hz, 1H), 5.26 (d, J = 7.7 Hz, 1H),
4.77 (dd, J = 8.6, 3.1 Hz, 1H), 4.59 (dd, J = 36.0, 11.7 Hz, 2H), 4.02 (dd, J = 14.0, 7.2 Hz,
1H), 3.85 (qd, J = 6.2, 3.1 Hz, 1H), 3.03 – 2.90 (m, 2H), 1.10 (d, J = 6.3 Hz, 3H); 13C NMR
(101 MHz, CDCl3): δ 170.15, 146.05, 138.93, 137.24, 136.94, 136.87, 134.73, 130.24,
129.32, 129.09, 128.67, 128.59, 128.21, 128.02, 127.81, 127.63, 127.46, 127.29, 116.81,
73.22, 71.62, 57.60, 45.08, 38.00, 16.03; IR (film, cm-1): 3308.18, 2249.65, 1675.63, 1595.84,
23
1521.76, 1396.34, 1332.94, 1208.13, 1158.91, 949.38, 763.30, 697.07;
22.95.
(Chloroform): -
[α]D
N-((S)-1-(((1R,2R)-2-(benzyloxy)-1-cyanopropyl)amino)-3-(3-chlorophenyl)-1-oxopropan-2-
yl)-[1,1'-biphenyl]-3-carboxamide (7e)
Compound 7e was synthesized according to procedure GP1, GP2 and GP3 as a white solid
1
(27.0% overall yield); Mp: 164.0-164.5 °C; purity (HPLC): > 99%; H NMR (400 MHz,
11

DMSO-d6): δ 9.06 (d, J = 8.3 Hz, 1H), 8.88 (d, J = 8.3 Hz, 1H), 8.07 (s, 1H), 7.85 – 7.70 (m,
4H), 7.53 (dt, J = 11.4, 7.7 Hz, 4H), 7.46 – 7.22 (m, 8H), 5.11 (dd, J = 8.3, 4.1 Hz, 1H), 4.82
(ddd, J = 10.6, 8.4, 4.5 Hz, 1H), 4.63 (q, J = 11.9 Hz, 2H), 3.88 (dt, J = 10.4, 5.2 Hz, 1H),
3.08 (ddd, J = 24.2, 13.6, 7.6 Hz, 2H), 1.22 (d, J = 6.2 Hz, 3H); 13C NMR (101 MHz, DMSO-
d6): δ 172.11, 166.81, 141.10, 140.49, 139.87, 138.39, 134.90, 133.14, 130.33, 130.02,
129.62, 129.44, 128.68, 128.45, 128.27, 128.17, 128.04, 127.24, 127.07, 126.83, 126.04,
118.43, 73.92, 70.91, 54.90, 45.24, 36.87, 16.06; IR (film, cm-1): 3266.89, 2246.62, 1665.46,
[α]2D3
1630.82, 1382.80, 1307.76, 1266.37, 1095.56, 1028.25, 1008.30, 887.87, 695.84;
(Chloroform): -82.35.
N-((S)-1-(((1R,2R)-2-(benzyloxy)-1-cyanopropyl)amino)-3-(3-chlorophenyl)-1-oxopropan-2-
yl)-3-(pyridin-3-yl)benzamide (7f)
Compound 7f was synthesized according to procedure GP1, GP2 and GP3 as a white solid
1
(26.9% overall yield); Mp: 44.0-44.8 °C; purity (HPLC): > 97%; H NMR (500 MHz.
DMSO-d6): δ 9.05 (d, J = 8.3 Hz, 1H), 8.96 (s, 1H), 8.87 (d, J = 8.3 Hz, 1H), 8.61 (d, J = 4.0
Hz, 1H), 8.16 – 8.10 (m, 2H), 7.91 – 7.87 (m, 1H), 7.84 – 7.79 (m, 1H), 7.61 – 7.51 (m, 3H),
7.40 – 7.21 (m, J = 10.3, 8.0, 4.0, 3.2 Hz, 7H), 5.09 (dd, J = 8.3, 4.2 Hz, 1H), 4.82 (ddd, J =
10.8, 8.4, 4.4 Hz, 1H), 4.62 (dd, J = 26.8, 11.9 Hz, 2H), 3.91 – 3.84 (m, 1H), 3.07 (ddd, J =
24.3, 13.5, 7.6 Hz, 2H), 1.21 (d, J = 6.2 Hz, 3H); 13C NMR (126 MHz, DMSO-d6): δ 172.05,
166.54, 149.32, 148.23, 141.03, 138.40, 137.46, 135.04, 134.68, 133.15, 130.35, 130.25,
129.70, 129.62, 128.69, 128.44, 128.17, 128.05, 127.79, 126.85, 126.14, 124.37, 118.40,
73.92, 70.93, 54.88, 45.26, 36.96, 16.09; IR (film, cm-1): 3275.42, 1740.47, 1656.12, 1582.04,
1333.29, 1246.56, 1092.29, 799.16,;
23 (Chloroform): -73.77.
[α]D
12

N-((S)-1-(((1R,2R)-2-(benzyloxy)-1-cyanopropyl)amino)-3-(3-bromophenyl)-1-oxopropan-2-
yl)-3-(pyridin-3-yl)benzamide (7g)
Compound 7g was synthesized according to procedure GP1, GP2 and GP3 as a light yellow
1
solid (26.8% overall yield); Mp: 49.7-50.5 °C; purity (HPLC): > 99%; H NMR (400 MHz,
DMSO-d6): δ 9.06 (d, J = 8.3 Hz, 1H), 8.95 (d, J = 1.8 Hz, 1H), 8.87 (d, J = 8.4 Hz, 1H), 8.60
(dd, J = 4.8, 1.6 Hz, 1H), 8.12 (dd, J = 5.8, 3.5 Hz, 2H), 7.88 (d, J = 7.7 Hz, 1H), 7.80 (d, J =
7.9 Hz, 1H), 7.66 (s, 1H), 7.61 – 7.48 (m, 2H), 7.43 – 7.17 (m, 7H), 5.09 (dd, J = 8.3, 4.1 Hz,
1H), 4.81 (ddd, J = 10.9, 8.4, 4.3 Hz, 1H), 4.60 (q, J = 11.9 Hz, 2H), 3.86 (dd, J = 6.2, 4.2 Hz,
1H), 3.13 – 2.95 (m, 2H), 1.19 (d, J = 6.2 Hz, 3H); 13C NMR (101 MHz, DMSO-d6): δ
172.06, 166.54, 149.30, 148.22, 141.33, 138.38, 137.45, 135.33, 135.01, 134.68, 132.48,
130.64, 130.24, 129.72, 129.69, 128.83, 128.68, 128.17, 128.04, 127.79, 126.13, 124.36,
121.83, 118.42, 73.91, 70.91, 54.91, 45.25, 36.92, 16.06; IR (film, cm-1): 3273.56, 1740.47,
1653.22, 1582.04, 1381.01, 1243.52, 1092.21, 797.10;
23 (Chloroform): -89.22.
[α]D
N-((S)-1-(((1R,2R)-2-(benzyloxy)-1-cyanopropyl)amino)-3-(3-chlorophenyl)-1-oxopropan-2-
yl)-3-(pyrimidin-5-yl)benzamide (8)
Compound 8 was synthesized according to procedure GP1, GP2, GP3 and GP5 as a white
solid (27.9% overall yield); Mp: 112.2-112.8 °C; purity (HPLC): > 99%; 1H NMR (400 MHz,
CDCl3): δ 9.19 (s, 1H), 8.92 (s, 2H), 7.97 (t, J = 1.6 Hz, 1H), 7.73 (dddd, J = 7.7, 2.8, 2.2, 1.1
Hz, 2H), 7.53 (t, J = 7.8 Hz, 1H), 7.35 (d, J = 7.9 Hz, 1H), 7.29 – 7.08 (m, 8H), 5.09 (q, J =
7.3 Hz, 1H), 4.93 (dd, J = 8.6, 3.1 Hz, 1H), 4.49 (dd, J = 30.1, 11.8 Hz, 2H), 3.81 (dd, J = 6.3,
3.2 Hz, 1H), 3.17 (d, J = 7.2 Hz, 2H), 1.14 (d, J = 6.3 Hz, 3H); 13C NMR (101 MHz, CDCl3):
δ 171.20, 166.78, 157.79, 154.92, 137.82, 136.86, 134.87, 134.50, 134.32, 133.21, 130.42,
130.17, 129.75, 129.44, 128.45, 128.02, 127.70, 127.58, 127.51, 127.37, 126.12, 117.02,
73.41, 71.43, 54.81, 44.99, 38.32, 15.96; IR (film, cm-1): 3271.43, 1735.19, 1646.40, 1574.65,
13

1540.33, 1385.01, 1345.17, 1307.35, 1241.95, 1189.54, 1095.47, 1040.54, 773.40, 695.09;
[α]2D3
(Chloroform): -178.57.
(2S)‐N‐[(1R,2R)‐2‐(benzyloxy)‐1‐cyanopropyl]‐2‐({[1,1'‐biphenyl]‐4‐yl}formamido)‐3‐(3‐chl
orophenyl)propanamide (7h)
Compound 7h was synthesized according to procedure GP1, GP2 and GP3 as a white solid
1
(27.1% overall yield); Mp: 185.0-185.9 °C; purity (HPLC): > 99%; H NMR (500 MHz,
CDCl3): δ 7.81 (d, J = 8.5 Hz, 2H), 7.60 (dd, J = 12.5, 7.8 Hz, 3H), 7.53 (d, J = 8.1 Hz, 1H),
7.47 (t, J = 7.4 Hz, 2H), 7.41 (t, J = 7.3 Hz, 1H), 7.34 – 7.18 (m, 7H), 7.13 (t, J = 7.8 Hz, 2H),
5.12 (q, J = 7.3 Hz, 1H), 4.93 (dd, J = 8.7, 3.3 Hz, 1H), 4.51 (dd, J = 33.3, 11.9 Hz, 2H), 3.78
13
(qd, J = 6.2, 3.3 Hz, 1H), 3.20 (d, J = 7.2 Hz, 2H), 1.13 (d, J = 6.3 Hz, 3H); C NMR (126
MHz, CDCl3): δ 171.43, 167.36, 144.96, 139.72, 138.05, 137.04, 134.55, 131.49, 130.11,
129.53, 128.94, 128.47, 128.16, 127.98, 127.82, 127.70, 127.53, 127.26, 127.19, 116.96,
73.41, 71.40, 54.71, 44.96, 38.33, 15.94; IR (film, cm-1): 3267.49, 2248.42, 1666.26, 1630.32,
[α]2D3
1610.41, 1538.45, 1382.84, 1309.56, 1231.92, 1207.33, 1094.36, 746.01, 696.34;
(Chloroform): -18.00.
N-((S)-1-(((1R,2R)-2-(benzyloxy)-1-cyanopropyl)amino)-3-(3-bromophenyl)-1-oxopropan-2-
yl)-[1,1'-biphenyl]-4-carboxamide (7i)
Compound 7i was synthesized according to procedure GP1, GP2 and GP3 as a white solid
1
(8.0% overall yield); Mp: 185.0-186.0 °C; purity (HPLC): > 99%; H NMR (400 MHz,
CDCl3): δ 7.80 (d, J = 8.4 Hz, 2H), 7.59 (dd, J = 9.7, 7.7 Hz, 5H), 7.49 – 7.38 (m, 4H), 7.36 –
7.26 (m, 4H), 7.19 – 7.10 (m, 3H), 5.15 – 5.05 (m, 1H), 4.96 – 4.87 (m, 1H), 4.59 – 4.41 (m,
13
2H), 3.76 (qd, J = 6.2, 3.2 Hz, 1H), 3.19 (d, J = 7.1 Hz, 2H), 1.12 (d, J = 6.2 Hz, 3H); C
NMR (101 MHz, CDCl3): δ 171.22, 167.31, 144.96, 139.74, 138.30, 137.00, 132.42, 131.57,
130.48, 130.44, 128.95, 128.50, 128.16, 128.03, 128.01, 127.77 (d, J = 1.1 Hz), 127.30,
14

127.20, 122.83, 116.97, 73.39, 71.44, 54.64, 44.94, 38.20, 29.70, 15.98; IR (film, cm-1):
23 (Chloroform): -7.6.
3259.71, 1670.44, 1633.72, 1537.34, 746.41, 694.40;
[α]D
N-((S)-1-(((1R,2R)-2-(benzyloxy)-1-cyanopropyl)amino)-3-(4-bromophenyl)-1-oxopropan-2-
yl)-[1,1'-biphenyl]-4-carboxamide (7j)
Compound 7j was synthesized according to procedure GP1, GP2 and GP3 as a white solid
1
(26.6% overall yield); Mp: 199.9-200.6 °C; purity (HPLC): > 96%; H NMR (500 MHz,
CDCl3): δ 7.79 (d, J = 8.4 Hz, 2H), 7.66 – 7.58 (m, 4H), 7.50 – 7.38 (m, 5H), 7.35 – 7.23 (m,
J = 15.9, 7.6 Hz, 6H), 7.13 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 7.7 Hz, 1H), 5.03 (q, J = 7.1 Hz,
1H), 4.88 (dd, J = 8.6, 3.2 Hz, 1H), 4.54 (dd, J = 38.8, 11.9 Hz, 2H), 3.81 (qd, J = 6.2, 3.2 Hz,
1H), 3.19 (d, J = 6.9 Hz, 2H), 1.14 (d, J = 6.3 Hz, 3H); 13C NMR (126 MHz, CDCl3): δ
170.94, 167.10, 145.00, 139.74, 136.90, 134.82, 132.05, 131.69, 131.07, 128.95, 128.56,
128.17, 128.13, 127.85, 127.60, 127.37, 127.20, 121.41, 116.99, 73.23, 71.52, 54.47, 44.97,
37.87, 16.11; IR (film, cm-1): 3258.17, 2941.22, 2851.65, 1671.42, 1631.90, 1535.98, 993.21,
745.67, 693.55;
23 (Chloroform): -117.46.
[α]D
N-((S)-1-(((1R,2R)-2-(benzyloxy)-1-cyanopropyl)amino)-3-(3-bromophenyl)-1-oxopropan-2-
yl)-4-(pyridin-3-yl)benzamide (7k)
Compound 7k was synthesized according to procedure GP1, GP2 and GP3 as a white solid
1
(27.2% overall yield); Mp: 45.7-46.4 °C; purity (HPLC): > 99%; H NMR (500 MHz,
CDCl3): δ 8.85 (d, J = 1.9 Hz, 1H), 8.65 (dd, J = 4.8, 1.4 Hz, 1H), 7.89 (ddd, J = 7.9, 2.3, 1.7
Hz, 2H), 7.84 (d, J = 8.5 Hz, 2H), 7.63 (d, J = 8.5 Hz, 1H), 7.44 (s, 1H), 7.42 – 7.25 (m, 6H),
7.22 – 7.16 (m, 3H), 6.99 (d, J = 7.7 Hz, 1H), 4.99 (dd, J = 14.4, 7.5 Hz, 1H), 4.89 (dd, J =
8.6, 3.2 Hz, 1H), 4.55 (dd, J = 39.8, 11.8 Hz, 2H), 3.84 (dd, J = 6.3, 3.2 Hz, 1H), 3.28 – 3.11
13
(m, 2H), 1.17 (d, J = 6.3 Hz, 3H); C NMR (101 MHz, CDCl3): δ 171.55, 166.92, 148.96,
15

148.02, 140.91, 138.97, 137.25, 135.43, 134.46, 133.05, 132.42, 130.12, 130.01, 128.38,
128.16, 128.03, 127.87, 127.76, 127.04, 123.70, 122.41, 117.16, 73.82, 71.44, 54.60, 44.97,
37.77, 15.92; IR (film, cm-1): 3275.71, 2979.32, 1743.67, 1653.22, 1331.23, 904.27, 797.17,
743.30;
23 (Chloroform): -91.37.
[α]D
N-((S)-1-(((1R,2R)-2-(benzyloxy)-1-cyanopropyl)amino)-3-(3-chlorophenyl)-1-oxopropan-2-
yl)-4-(pyrimidin-5-yl)benzamide (7l)
Compound 7l was synthesized according to procedure GP1, GP2 and GP3 as a white solid
1
(28.0% overall yield); Mp: 134.0-134.7 °C; purity (HPLC): > 99%; H NMR (500 MHz,
CDCl3): δ 9.25 (s, 1H), 8.95 (s, 2H), 7.88 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 8.5 Hz, 2H), 7.38 –
7.18 (m, 8H), 7.13 (d, J = 7.4 Hz, 1H), 7.09 (d, J = 7.8 Hz, 1H), 5.06 (q, J = 7.3 Hz, 1H), 4.91
(dd, J = 8.7, 3.1 Hz, 1H), 4.53 (dd, J = 37.0, 11.8 Hz, 2H), 3.83 (qd, J = 6.2, 3.1 Hz, 1H), 3.19
13
(dd, J = 7.0, 2.6 Hz, 2H), 1.16 (d, J = 6.3 Hz, 3H); C NMR (101 MHz, CDCl3): δ 170.94,
166.59, 158.08, 154.93, 137.89, 137.75, 136.89, 134.62, 133.41, 133.12, 130.21, 129.44,
128.50, 128.29, 128.08, 127.75, 127.64, 127.52, 127.23, 116.96, 73.40, 71.50, 54.67, 44.95,
38.36, 16.00; IR (film, cm-1): 3271.49, 2931.10, 1736.69, 1648.60, 1346.57, 1307.35,
1046.54, 773.48, 693.99, 650.12;
23 (Chloroform): -80.64.
[α]D
N-((S)-1-(((1R,2R)-2-(benzyloxy)-1-cyanopropyl)amino)-3-(3-bromophenyl)-1-oxopropan-2-
yl)-4-(pyrimidin-5-yl)benzamide (7m)
Compound 7m was synthesized according to procedure GP1, GP2 and GP3 as a white solid
1
(28.0% overall yield); Mp: 99.2-99.8 °C; purity (HPLC): > 99%; H NMR (400 MHz,
DMSO-d6): δ 9.20 (s. 1H), 9.19 (s, 2H). 9.05 (d, J = 8.3 Hz, 1H), 8.83 (d, J = 8.3 Hz, 1H),
7.93 (q, J = 8.7 Hz, 4H), 7.65 (t, J = 1.7 Hz, 1H), 7.45 – 7.17 (m, 8H), 5.09 (dd, J = 8.3, 4.1
Hz, 1H), 4.79 (ddd, J = 10.4, 8.4, 4.6 Hz, 1H), 4.61 (q, J = 11.9 Hz, 2H), 3.87 (dd, J = 6.2, 4.1
16

13
Hz, 1H), 3.11 – 2.97 (m, 2H), 1.20 (d, J = 6.2 Hz, 3H); C NMR (101 MHz, DMSO- d6): δ
172.07, 166.27, 158.13, 155.35, 141.38, 138.38, 137.02, 134.25, 132.72, 132.41, 130.62,
129.71, 128.85, 128.76, 128.70, 128.18, 128.06, 127.20, 121.86, 118.44, 73.91, 70.91, 55.00,
45.25, 36.77, 16.03; IR (film, cm-1): 3276.99, 1651.87, 1548.14, 1327.41, 734.68, 697.75;
[α]2D3
(Chloroform): -98.47.
N-((S)-1-(((1R,2R)-2-(benzyloxy)-1-cyanopropyl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-
yl)-[1,1'-biphenyl]-4-carboxamide (7n)
Compound 7n was synthesized according to procedure GP1, GP2 and GP3 as a white solid
1
(14.0% overall yield); Mp: 166.0-167.0 °C; purity (HPLC): > 99%; H NMR (400 MHz,
CDCl3): δ 7.94 – 7.76 (m, 4H), 7.65 – 7.58 (m, 4H), 7.50 – 7.44 (m, 2H), 7.42 – 7.32 (m, 2H),
7.23 – 7.18 (m, 1H), 7.18 – 7.11 (m, 2H), 7.07 (d, J = 2.4 Hz, 1H), 6.61 (d, J = 8.7 Hz, 1H),
5.05 (ddd, J = 8.9, 7.2, 5.0 Hz, 1H), 4.81 (dd, J = 8.7, 3.0 Hz, 1H), 4.54 – 4.38 (m, 2H), 3.78
(qd, J = 6.2, 3.0 Hz, 1H), 3.51 (ddd, J = 14.3, 5.1, 0.9 Hz, 1H), 3.21 (dd, J = 14.4, 8.9 Hz,
1H), 1.08 (d, J = 6.3 Hz, 2H); 13C NMR (101 MHz, CDCl3): δ 171.73, 166.88, 144.70,
139.88, 137.15, 136.32, 132.14, 128.93, 128.55, 128.15, 128.08, 128.06, 127.66, 127.26,
127.21, 126.90, 123.83, 122.56, 120.06, 119.01, 117.25, 111.43, 110.08, 73.44, 71.55, 53.90,
45.00, 28.89, 15.94; IR (film, cm-1): 3406.28, 3267.41, 2358.94, 1633.70, 1539.2, 1097.5,
744.52, 698.23;
23 (Chloroform): -11.44.
[α]D
(S)-2-(2-([1,1'-biphenyl]-4-yl)acetamido)-N-((1R,2R)-2-(benzyloxy)-1-cyanopropyl)-3-(3-
bromophenyl)propanamide (7o)
Compound 7o was synthesized according to procedure GP1, GP2 and GP3 as a white solid
1
(6.0% overall yield); Mp: 198.0-199.5 °C; purity (HPLC): > 95%; H NMR (500 MHz,
DMSO-d6): δ 8.96 (d, J = 8.3 Hz, 1H), 8.50 (d, J = 8.4 Hz, 1H), 7.61 (dd, J = 8.3, 1.3 Hz,
17

2H), 7.54 – 7.43 (m, 5H), 7.41 – 7.32 (m, 6H), 7.30 – 7.26 (m, 2H), 7.21 (t, J = 7.8 Hz, 1H),
7.15 (d, J = 8.3 Hz, 2H), 5.07 (dd, J = 8.3, 4.1 Hz, 1H), 4.68 – 4.53 (m, 3H), 3.82 (qd, J = 6.2,
4.0 Hz, 1H), 3.50 – 3.31 (m, 2H), 2.97 (dd, J = 13.6, 4.3 Hz, 1H), 2.80 (dd, J = 13.6, 10.3 Hz,
13
1H), 1.15 (d, J = 6.2 Hz, 3H); C NMR (101 MHz, DMSO-d6): δ 171.99, 170.49, 140.84,
140.47, 138.64, 138.40, 135.90, 132.41, 130.63, 129.89, 129.75, 129.38, 128.89, 128.72,
128.17, 128.07, 127.72, 126.96, 126.86, 121.89, 118.34, 73.88, 70.88, 53.99, 45.13, 42.07,
37.43, 15.99; IR (film, cm-1): 3265.34, 2972.30, 2931.80, 2358.94, 2339.65, 1670.35,
1647.21, 1541, 740.70, 696.30.
2.3
Enzymatic Inhibition Studies
The cathepsins L (P07711), K (P43235), S (P25774), and B (P07858) were purchased from
Enzo Life Science®. The protocols used for the enzymatic inhibition studies performed for
CatL, CatK, and CatS are reported elsewhere.22
2.3.3
Determination of the inhibition constant (Ki) for CatB
Human cathepsin B was assayed on a Biotek Synergy HT plate reader with a fluorescence
emission at 460 nm (excitation at 355 nm) at a temperature of 37 °C. The fluorogenic
substrate Z-Phe-Arg-AMC was used in a final concentration of 71 µM (0.71 × KM) in the
assay buffer (100 mM sodium phosphate buffer at pH 6.0, 100 mM of NaCl and 5 mM
EDTA). CatB solution was prepared with a final concentration of 0.7 nM in the assay buffer
containing 1 mM DTT and 0.014% of Triton X-100. The activation was performed at a
temperature of 37 °C for 30 minutes. Inhibitor solutions were prepared in DMSO (final
concentration of 5% v/v), with initial concentrations varying from 50 to 0.1 µM. After 2
minutes of incubation of the enzyme with the inhibitor, the substrate solution is added, and
18

the fluorescence emission is recorded for 5 minutes. The experiment was performed in
triplicate for each inhibitor.
Initial velocities of the substrate hydrolysis under the first-order reaction were calculated by
Gen5TM Biotek software. The apparent inhibition constant Ki’ was determined by non-linear
regression using the equation Vs = Vo/(1+[I]/Ki’), where Vs is the steady-state rate, Vo is the
rate in the absence of inhibitor, and [I] is the inhibitor concentration. The actual inhibition
constant Ki was calculated by the correction of Ki’ according to Ki = Ki’/(1+[S]/KM), where [S]
is the substrate concentration and KM is the Michaelis-Menten constant.
Visual inspection and a pre-reading of plate wells were performed to check for possible
precipitation and background fluorescence, respectively. None of the substances displayed a
significant fluorescence signal in the emission wavelength used in the assay. Analysis and
manipulation of the data were performed with Origin Pro 8.5.
2.4
Molecular Dynamics Simulations
Herein, the crystal structure from protein data bank (PDB code: 2XU1)23 was used as a
starting point to obtain the coordinates of CatL enzyme, while initial coordinates to draw the
ligands were taken from a crystal structure of a dipeptidyl nitrile in complex with cruzain.24
The initial coordinates for protein and inhibitor were taken from protein data bank
(PDB code: 1GMY)25 for the molecular dynamics (MD) simulations of CatB. The protonation
states of the residues were evaluated through the PROPKA program.26,27 For the compounds
involved in a possible halogen bond interaction (6a and 7d) between the halogen at the P3
position of the ligand with the enzyme, we first calculated the electrostatic potential of the
inhibitor through Gaussian 09 program28, with the 6-31G(d) basis set29 at the Hartree-Fock
level of theory. Then, partial charges were obtained using a restrained electrostatic potential
(RESP) approach.30(p) We include an extra-point (EP) of charge bounded with Br atom to
19

represent the σ-hole for the ligands in this step. The AM1-BCC charges31 was employed to
assign partial charges for the remaining ligands (7b, 7f, 7g, and 7k). Note that both AM1-
BCC and HF/6-31G(d) potentials are recommended for computing the atomic charges for
ligands in AMBER simulations.32 After the charge calculations, all compounds were
parameterized with the General Amber Force Field (GAFF)3321
Next, each system (covalent and noncovalent bound ligand to the protein) was solvated with
TIP3P34 water molecules in a truncated octahedron box and the counterions (Na+ for both
settings) were added to neutralize each ligand-protein complex system. The AMBER 18 suite
of programs32, together with the Amber ff14SB force field35 was used to perform MD
simulations. The bonds containing hydrogen atoms were restrained through the SHAKE
algorithm.36 Initially, the hydrogen atoms, water molecules, and ions were minimized using
10,000 cycles of steepest descent and conjugate gradient algorithms. Then, gradually the
systems were heated until they reach 300K through several heating steps. Finally, a 100 ns
production run was carried out using the NPT ensemble for each system (covalent and
noncovalent complexes).
3
Results and Discussion
3.2 Molecular Design, Synthesis and Structure-Activity Relationships (SARs)
The approach of reversible covalent inhibition of cathepsins by using a nitrile warhead has
been applied successfully throughout the years, where peptidomimetic nitriles form a
covalent-reversible thioimidate adduct.18,37
In our previous structure-activity relationships (SARs) on cathepsins, we have found that
compound 7a is a moderate reversible inhibitor of CatB (pKi of 6.3) and high-affinity CatL
inhibitor (pKi of 8.1).22 Compound 7a bears in P1 an unusual Thr-O-Bn moiety, which is a
privileged building block for CatB inhibition due to the recognition of aliphatic and aromatic
20

interactions in the S1/S1´ area, Figure 1. Our previous modelling study suggested that Thr-O-
Bn in the P1 position is accommodated in the S1´ pocket through hydrophobic interactions
without interfering in the general mode of binding.22 The S1´ pocket, which it is well
conserved in other human cathepsins (CatL, CatK, and CatS), is characterized by the amino
acids Val176, Leu181, Met 196, His199, Trp221 shaping a large hydrophobic pocket.18
Indeed, substrate mapping studies elucidate that the S1’ subsite of CatB prefers hydrophobic
residues with either aromatic or aliphatic side chains.38
Therefore, by applying a structure-based design approach, we have expanded our previous
inhibitor series to enhance the affinity and selectivity towards CatB. Considering that CatB
has a larger P2 and P3 pocket than CatL, we have explored different amino acids for the P2
and different benzoic acids or sulfonic acids for the P3 moiety, while the same P1 substituent
was kept unchanged (Figure 1). We selected the different building blocks by considering the
most advanced selective dipeptidyl nitrile inhibitors of CatB (Figure 1).7
21

Figure 1. Structure representation of known CatB inhibitor and prototypical scaffold applied in this
work. *pKi is pH-dependent.7
The synthesis of compounds 7i, 7n, and 7o was performed similarly, as described in our
recent work.22 For the synthesis of 7a-7h and 7j-7m, a different route was followed involving
the dehydration of amide to nitrile, in the beginning, followed by deprotection and coupling
reactions. Both synthetic routes are summarized in Scheme 1. The synthetic routes were
developed to optimize the set of substituents to be placed in P1, P2 and P3 that were defined
after the design studies. Due to the diversity of building blocks, it was necessary to evaluate
different coupling and dehydrating reagents, aiming at the best yield and preventing
racemization. The synthesis of compound 8 was performed by the Suzuki cross-coupling
reaction as a final step. For the synthesis of compounds 6a-6c, the formation of the
sulfonamide bond was performed by using the corresponding sulfonyl chloride (Scheme 1).
22

P2
O
S
P2
P2
H
H
N
BocHN
CN
H
(S)
N
CN
CN
CN
(S)
(S)
N
CN
c, d
(S)
c
e
(S)
(S)
BocHN
N
(S)
H2N
P1
3a
R
O
H
O
P1
O
P1
O
P1
4a-c
6a-c
5a-c
d
b
O
O
O
P2
O
P2
H
N
H
N
a
BocHN
(S)
BocHN
(S)
CN
(S)
OH
(S)
NH2
(S)
(S)
N
N
H
f
R
R
R1
H
P
P1
1 1
O
P1
O
7a-o
P1
8
2
c
g
R = aryl, heteroaryl
1 = 5-pyrimidinyl
P2
O
R
H
O
O
P2
O
N
(S)
H
N
d
(S)
H N
2
BocHN
NH2
c, d
(S)
(S)
NH2
3b
(S)
N
NH2
O
P1
H
P1
4d-e
O
P1
5d-f
Scheme 1. General synthetic scheme for the synthesis of dipeptidyl nitriles. Reagents and conditions:
a) Isobutyl chloroformate, DIPEA, DMF, argon, -30 °C for 0.5 h, NH4Cl, r.t., 20 h; b) DIPEA, THF
dry, TFAA added at 0 °C, 2 h, r.t.; c) HCO2H, r.t., 5-18 h; d) The appropriate sulfonyl chloride, N-
Boc amino acid or carboxylic acid, HATU, DIPEA, DMF, r.t., 12-20 h; e) The appropriate sulfonyl
chloride, DCM, DIPEA (added at 0 °C), r.t., 16 h; f) 5-pyrimidinyl boronic acid, PdCl2dppf, Na2CO3,
DMF, 80 °C, 3-7 h; g) Cyanuric chloride, DMF, NaHCO3, from 0 °C to r.t., 2 h.
The pKi values were determined for four human cysteine cathepsins (CatB, CatK, CatL, CatS)
and are reported in Table 1.
Table 1. Inhibitions of human cathepsins. The pKi values were calculated using the equation pKi =
log10(Ki/M).
pKi valuesa or percentage of inhibition (%)b
N°
6a
Structure
CatB
CatL
CatS
CatK
Cl
O
H
O
7.4
9.6
7.1
6.2
S
N
CN
(S)
N
(R)
H
(R)
O
OBn
Br
23

Cl
O
< 5.0
(50%)
H
N
(R)
O
6b
7.6
6.7
6.2
S
CN
N (S)
H
(R)
O
OBn
Ph
Cl
O
H
N
O
6c
7a
6.7
6.3
6.8
8.1
6.3
6.5
5.0
6.0
S
CN
N (S)
(R)
H
(R)
O
OBn
Ph
N
O
Cl
H
N
CN
N
N
(S)
(R)
H
(R)
OBn
O
O
O
Br
H
N
(R)
CN
N
7b
7c
7d
6.8
7.9
7.2
9.3
8.1
9.2
6.8
6.9
7.8
5.6
5.3
5.4
N
H
(S)
N
(R)
OBn
O
Cl
H
N
CN
N
(S)
(R)
H
(R)
O
Br
OBn
O
Cl
CN
H
N
N
(S)
(S)
(S)
(R)
H
(R)
OBn
O
Br
O
O
Cl
H
N
(R)
< 5.0
(> 90%)
CN
7e
7f
6.9
8.3
7.5
7.3
9.0
7.7
7.3
7.9
7.7
N
H
(R)
OBn
O
O
Ph
Cl
H
N
(R)
< 5.0
(58%)
CN
N
H
(R)
OBn
3-Pyridyl
O
Br
H
N
(R)
< 5.0
(> 90%)
CN
7g
N
H
(S)
(R)
OBn
O
3-Pyridyl
24

O
O
Cl
< 5.0
(61%)
H
N
(R)
7h
7i
7.5
7.3
6.8
7.1
6.3
5.8
CN
N
H
(S)
(S)
(R)
OBn
O
O
Ph
Ph
Br
< 5.0
(55%)
< 5.0
(> 90%)
H
N
(R)
CN
N
H
(R)
OBn
Br
O
< 5.0
(> 90%)
< 5.0
(86%)
< 5.0
(51%)
H
N
(R)
7j
CN
N
H
(S)
(R)
OBn
O
Ph
O
Br
CN
< 5.0
(84%)
H
7k
7l
7.9
7.9
7.7
7.8
7.4
7.2
N
N
(S)
(R)
H
(R)
OBn
O
3-Pyridyl
O
Cl
H
N
CN
N
(S)
5.2
5.3
(R)
H
(R)
OBn
O
5-pyrimidinyl
O
Br
H
N
CN
N
H
7m
7n
(S)
7.9
7.3
7.7
6.1
7.5
6.8
(R)
(R)
OBn
O
5-pyrimidinyl
NH
< 5.0
(70%)
O
H
N
CN
(R)
N
H
(S)
(R)
O
Ph
Ph
OBn
O
Br
< 5.0
(> 90%)
< 5.0
(59%)
< 5.0
(> 90%)
H
N
7o
8
6.3
7.8
CN
N
(S)
(R)
H
(R)
OBn
O
O
Br
CN
(R)
OBn
H
N
(R)
< 5.0
(82%)
7.2
7.7
N
(S)
H
O
5-pyrimidinyl
a The standard deviation was lower than 15% for all reported pKi values. All experiments were performed with seven
inhibitor concentrations and in triplicate. For the most potent compounds (pKi > 7.0), two other independent experiments
were performed.
b Percentage of inhibition at 10 µM inhibitor concentration.
25

One common approach to the SAR analysis is to examine pKi values associated with
particular structural transformations for molecular pairs that can be specified concisely using
the square bracket notation previously described.24,39 For example, replacement of the
chlorine atom in the meta position of phenylalanine in P2 of compound 7a to the
corresponding 3-bromo-phenylalanine (7b), noted as [7a→7b], led to an increase of 0.5 log
unit in affinity for CatB and over one log unit for CatL. This positive effect for both enzymes
was already described previously.7,37 Secondly, the replacement of N-methyl-tert-butyl-
pyrazole for 4-bromo-benzoic acid [7a→7c] strongly increases the affinity toward CatB of
almost one log unit (10-fold) while maintaining the same affinity for CatL. This replacement
shows how CatB has a preference for aromatic and hydrophobic moieties at the P3 position.
Compound 7c can be considered one of the best dipeptidyl nitrile inhibitors of CatB reported
up to now in literature, but without showing the desired selectivity over CatL. Interestingly,
the displacement of the bromine from para to meta [7c→7d] is detrimental for the affinity of
CatB (loss of 0.7 log units) and beneficial for CatL (gain of 1.1 log units). This effect for
CatL can be rationalized by considering the formation of a halogen bond with the Gly61
present on the S3 subsite, as recently reported.40 For CatB, our in-silico study suggests that no
halogen-bond is formed during the recognition process (see below), even if further structural
studies are needed to elucidate this trend. Besides, compound 7d is an excellent inhibitor of
CatL (pKi of 9.2) with good selectivity over CatB, CatS, and CatK.
Bioisosteric replacement of the amide bond in the P3/P2 position is a known strategy for
cysteine inhibitors to gain metabolic stability while maintaining an affinity for the desired
biological target.18,41 Therefore, sulfonamides were used in this work as bioisosteres of the
amide bond, despite being a non-conventional replacement owing to the presence of two
oxygens bound to the sulfur atom, causing the loss of planarity. Sulfonamides are known to
have conformational preferences in which the nitrogen of the sulfonamide bond lone pair
26

bisects the O=S=O angle in the Newman projection.42 Moreover, it has been already reported
that the structural replacement of the peptide bond for a sulfonamide maintained the affinity
for cruzain, the protozoan related CatL.43 In our case, the sulfonamide [7d→6a] slightly
improved the affinity towards both CatB (0.2 log units) and CatL (0.4 log units). It is
important to emphasize that compound 6a is a single-digit nanomolar inhibitor of CatL with
over two log units of selectivity over the other cathepsins. However, the real challenge is to
create a potent inhibitor of CatB and selective for CatL. Visual structure inspection and a
knowledge-based design approach10 suggest, due to the difference in the size of the S3 subsite
in CatB and CatL, that the insertion of large hydrophobic moieties in P3 for our archetype
(7a) would maintain the affinity for CatB while lowering it for CatL. Accordingly, structure
replacement of the 4-bromo-benzoyl moiety in P3 for a para bi-phenyl [7c→7h] produced the
desired effect with a decrease of over 3.1 log units (over 1000-fold) for CatL and just 0.4 for
CatB and a total selectivity of over 2.5 log units (about 500-fold) in favour of CatB (Figure
2). However, compound 7h was not so selective for cathepsin B over cathepsin S (∆pKi =
0.4).
27

Figure 2: SAR analysis considering matched molecular pairs for a set of structural modifications.
Grey areas depict structural modification in P3, sand-colour area depicts modification in the amide
bond P3/P2. Blue arrows are related to an increase in affinity for CatL and a decrease for CatB. Green
arrows show an increase in affinity for CatB and a decrease for CatL. Red arrows mean that a decrease
in affinity was observed for both cathepsins.
Fascinatingly, when the same replacement is performed for the meta substituent [7d→7e], we
observed a reduced affinity for both enzymes, without any improvement in selectivity.
Moreover, for compounds 7e and 7h, the amide bond in P3 was replaced by the sulfonamide
[7e→6b] and [7h→6c] in which we experienced a divergent effect. Indeed, compound 6b,
which bears a meta-biphenyl sulfonyl amide in P3, shows an increment in the affinity of 0.7
log units for CatB and a loss of 0.6 log units for CatL concerning the dipeptidyl nitrile pair 7e
(Figure 2). On the other hand, compound 6c, which bears a para-biphenyl sulfonyl amide in
P3, had an affinity reduction by 0.8 log units for CatB, with an increment of at least 1.8 log
units (almost 1000-fold) for CatL in comparison to 7h. The reverse behaviour observed for
these pairs can be an effect of the loss of planarity of the sulfonamide bond as for the amide
one, which allows the biphenyl moiety to have a different orientation in the S3 region.42
Moreover, the substitution of 3-chloro-phenylalanine to tryptophan in P2 [7h→7n] provided
an excellent affinity (pKi of 7.3) with a moderate CatB selectivity over CatL (12-fold).
Surprisingly, the insertion of methylene as a spacer in P3 [7i→7o] to increase the degree of
freedom led to a complete loss in affinity for CatB and CatL. More importantly, 7o, with a
pKi of 6.3 and a good selectivity over the other cathepsins (SI > 50), could be considered a
prototype inhibitor of CatS. Further studies should be implemented to understand the effect of
the spacer in the bimolecular recognition process.
Compound 7h is one of the best inhibitors of CatB (considering affinity and selectivity over
CatL), and it was further modified by replacing the chlorine atom in P2 for bromine in meta
28

[7h→7i] and para [7h→7j] position. The first replacement did not influence the affinity and
selectivity, while for the second replacement, we observed a slight decrease in affinity for
CatB (0.7 log units). At the same time, compounds 7i and 7j displayed no affinity for all the
other cathepsins studied (except CatS for 7i), making these compounds necessary prototypes
as selective CatB inhibitors for further optimization.
Compounds 7h, 7i, 7j, and 6b reached and even overpassed the affinity and selectivity level
for CatB described by Gütschow and colleagues.7 The enzymatic kinetic assay with cathepsin
B was also performed at pH 4.5 for compounds 7f, 7h, and 7i, to observe how the pH-
dependency of enzyme opening and closing movements of the occluding loop could affect the
ligand recognition (see Table S1 for more details). For compounds 7h and 7i, which do not
contain basic moieties, the affinity remains in the same order of magnitude or even increase of
over two-fold (7i). For compound 7f, we observed a 1000-fold decrease probably due to the
basic character of the pyridine ring, which reduces at acidic pH the hydrophobic interactions
with the P3 region.
Compounds described so far have high lipophilicity that was expected due to the basis of the
lead-likeness.44 Lead optimization that results in increased lipophilicity can decrease water
solubility. It is known that these parameters may be regarded as the primary physicochemical
risk factors in drug design.45 Hence, we tried to modify the P3 by inserting a pyridine or
pyrimidine moiety to reduce the lipophilicity. Interestingly, these compounds (7k, 7l, 7m, and
8) provided a strong affinity for both CatL and CatB (Figure 3).
29