Synthesis and biological evaluation of novel peptidomimetics as rhodesain inhibitors

Article abstract of DOI:10.3109/14756366.2015.1108972

Novel rhodesain inhibitors were developed by combining an enantiomerically pure 3-bromoisoxazoline warhead with a 1,4-benzodiazepine scaffold as specific recognition moiety. All compounds were proven to inhibit rhodesain with K _i values in the low-micromolar range. Their activity towards rhodesain was found to be coupled to an in vitro antitrypanosomal activity, with IC₅₀ values ranging from the mid-micromolar to a low-micromolar value for the most active rhodesain inhibitor (R,S,S)-3. All compounds showed a good selectivity against the target enzyme since all of them were proven to be poor inhibitors of human cathepsin L.

Full text of DOI:10.3109/14756366.2015.1108972

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and biological evaluation of novel
peptidomimetics as rhodesain inhibitors
Roberta Ettari, Santo Previti, Sandro Cosconati, Jochen Kesselring, Tanja
Schirmeister, Silvana Grasso & Maria Zappalà
To cite this article: Roberta Ettari, Santo Previti, Sandro Cosconati, Jochen Kesselring, Tanja
Schirmeister, Silvana Grasso & Maria Zappalà (2015): Synthesis and biological evaluation of
novel peptidomimetics as rhodesain inhibitors, Journal of Enzyme Inhibition and Medicinal
Chemistry, DOI: 10.3109/14756366.2015.1108972
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Published online: 16 Nov 2015.
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
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2015 Taylor & Francis. DOI: 10.3109/14756366.2015.1108972
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RESEARCH ARTICLE
Synthesis and biological evaluation of novel peptidomimetics as
rhodesain inhibitors
Roberta Ettari1, Santo Previti1, Sandro Cosconati2, Jochen Kesselring3, Tanja Schirmeister3, Silvana Grasso4, and
1
`
Maria Zappala
2
1Dipartimento Di Scienze Del Farmaco E Dei Prodotti per La Salute, University of Messina, Messina, Italy, DiSTABiF, Second University of Naples,
Naples, Italy, 3Institute of Pharmacy and Biochemistry, University of Mainz, Mainz, Germany, and 4Dipartimento Di Scienze Chimiche, University of
Messina, Messina, Italy
Abstract
Keywords
Novel rhodesain inhibitors were developed by combining an enantiomerically pure
3-bromoisoxazoline warhead with a 1,4-benzodiazepine scaffold as specific recognition
moiety. All compounds were proven to inhibit rhodesain with Ki values in the low-micromolar
range. Their activity towards rhodesain was found to be coupled to an in vitro antitrypanosomal
activity, with IC50 values ranging from the mid-micromolar to a low-micromolar value for the
most active rhodesain inhibitor (R,S,S)-3. All compounds showed a good selectivity against the
target enzyme since all of them were proven to be poor inhibitors of human cathepsin L.
Peptidomimetics, rhodesain, trypanosoma
History
Received 1 September 2015
Revised 5 October 2015
Accepted 6 October 2015
Published online 16 November 2015
Introduction
be administered3. For these reasons there is an urgent need to
develop new drugs endowed with trypanocidal activity and to
identify new potential molecular targets.
Human African Trypanosomiasis (HAT), also known as sleeping
sickness, is a neglected tropical disease transmitted by bite of a fly
(Glossina genus), afflicting millions of people in sub-Saharan
Africa, with an estimated number of cases of 30 000/year1.
HAT is caused by a unicellular protozoan in the class of
zooflagellates. The subspecies responsible for the human disease
are Trypanosoma brucei gambiese and T. b. rhodesiense2. The
first subspecies, widespread in west and central Africa, are able to
induce the chronic form of the disease, while T. b. rhodesiense,
prevalent in the south-eastern end of Africa causes the rapid onset
acute form of HAT.
The first hemolymphatic stage of the disease is characterized
by fever, itching, headache, joint pain, while during the late
neurological stage, the parasite crosses the blood–brain barrier
(BBB) reaching the central nervous system, causing the typical
symptoms of the disease such as sensory disturbances and
coordination, and finally sleep–wake cycle disorders. If left
untreated, sleeping sickness has a fatal outcome.
Because of the lack of an effective vaccine, chemotherapy is at
present the unique way to control the disease. There are four
currently available drugs to treat HAT: melarsoprol, eflornithine,
pentamidine and suramin. Only two which are able to cross the
BBB, and therefore are useful in the second stage of the disease,
are melarsoprol and eflornithine. However, the arsenical com-
pounds are highly toxic, while eflornithine, is ineffective towards
T. b. rhodesiense, is very expensive and requires hospitalization to
The cysteine proteases represent very promising targets for the
development of new drugs with antiparasitic activity. In this
context, rhodesain, a Clan CA papain-family cysteine protease
plays an important role in the Trypanosoma life cycle. Rhodesain
(TbCatL) is localized in the lysosome and is involved in the
degradation of parasite proteins and intracellularly transported
host proteins4. The protease is required to cross the BBB leading
to the lethal stage of the sleeping sickness; it is involved in the
turnover of variant surface glycoproteins (VSG) of T. brucei coat,
and in the degradation of host immunoglobulines, thus evading
the host immune response.
At present the main class of rhodesain inhibitors5 are peptides:
vinyl sulfones, peptidyl aldehydes, diazomethyl or halomethyl
ketones, peptidyl-a,b-epoxyesters, azadipeptide nitriles and pep-
tides
bearing
an
aziridine-2,3-dicarboxylate
warhead.
Furthermore, many other chemotypes have been identified as
non-peptide rhodesain inhibitors, such as, thiosemicarbazones,
isoquinolines, triazine nitriles, Michael acceptors6 or recently
reported non-covalent rhodesain inhibitors7.
In this research area, our research group has been actively
involved in the development of novel non-peptide8 or peptidomi-
metic cysteine protease inhibitors9,10, the latter characterized by
the presence of a 1,4-benzodiazepine scaffold, introduced into a
peptide backbone, in which the condensed aromatic ring could
mimic a Phe residue at P2 site, highly preferred by rhodesain,
while the hydroxylmethyl group at C3 of the scaffold was used to
tie different substituents, able to create additional interactions
with the S3 pocket of the target enzyme. In particular, we
identified a vinyl ester 1 (Figure 1) which behaves as Michael
Address for correspondence: Roberta Ettari, Dipartimento Di Scienze Del
Farmaco E Dei Prodotti per La Salute, University of Messina, Viale
Annunziata, Messina, Italy. E-mail: rettari@unime.it

2
R. Ettari et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
acceptor, leading to an irreversible alkylation of the target enantiomerically pure form starting from Garner ester 4, follow-
enzyme. Ester 1, which is characterized by the presence of an ing our previously described procedure (9a).
adamantyl nucleus directly linked to the methyl carbamoyl
Enantiomerically pure amines (R)-8 and (S)-8 were prepared
portion appended to C3 of the BDZ scaffold, showed an starting from enantiopure alcohols (R)-7 and (S)-7 (12a) in turn
impressive second-order rate constant of inhibition value obtained, with a 94% enantiomeric excess, following a previously
(k2nd ¼ 4 620 000 Mꢀ1 minꢀ1), coupled with a good antitrypano- reported methodology13.
somal activity (IC50 ¼ 4.8 mM)11.
We then coupled the carboxylic acid (R)-5 to the enantiopure
Starting from this lead compound we now decided to introduce amines (R)-8 and (S)-8 (Scheme 2), using carbodiimide EDCI and
at the C-terminal moiety the 3-bromo isoxazoline nucleus (i.e. HOBt as coupling reagent. The coupling products were then
compounds (R,R)-2 and (R,S)-2, Figure 2), successfully employed desilylated by treatment with TBAF to afford (R,R)-12 and (R,S)-
by our group12, to investigate the profile of these inhibitors. 12, which by subsequent reaction with the adamantyl isocyanate
Moreover, we have also designed peptidomimetics in which a gave the desired carbamates (R,R)-2 and (R,S)-2.
molecular rigidification has been introduced by incorporating the
3-bromo isoxazoline nucleus into a bicyclic system [i.e. (R,R,R)-3 (R,S,S)-3, the bicyclic amine ( )-1014 was first prepared via 1,3-
and (R,S,S)-3), Figure 2]. dipolar cycloaddition between the dipolarophile 9 and bromoni-
Conversely, for the synthesis of compounds (R,R,R)-3 and
Herein, we report the synthesis and the biological evaluation of trileoxide, generated in situ by dehydrohalogenation of the stable
compounds (R,R)-2, (R,S)-2, (R,R,R)-3 and (R,S,S)-3 against precursor dibromoformaldoxime (DBF). Enantiopure amines
rhodesain and T. brucei brucei, together with the selectivity (R,R)-11 and (S,S)-11 were obtained by resolving the racemic
towards the target enzyme, verified by testing the new compounds mixture of ( )-10 by chiral HPLC, both with a 99% enantiomeric
against human cathepsin L, like rhodesain also belonging to excess, followed by treatment with 30% TFA in CH2Cl2 (12a).
papain-family.
The condensation of the bicyclic amines (R,R)-11 and (S,S)-11
to the carboxylic acid (R)-5, under the same conditions estab-
lished for compounds (R,R)-2 and (R,S)-2, allowed us to recover
the two diastereoisomers (R,R,R)-3 and (R,S,S)-3 (Scheme 3).
Results and discussion
Chemistry
To synthesize compounds (R,R)-2 and (R,S)-2 we first accom- Biological activity
plished the synthesis of the key intermediate (R)-5 (Scheme 1) in
Compounds (R,R)-2, (R,S)-2, (R,R,R)-3 and (R,S,S)-3 (Table 1)
were tested for their inhibitory activity against rhodesain using
Cbz-Phe-Arg-AMC as fluorogenic substrate.
First a preliminary screening with an inhibitor concentration of
100 mM was performed and an equivalent volume of DMSO was
used as negative control. Compounds capable of inhibiting the
enzymatic activity by more than 70% were characterized in detail.
Continuous assays were then performed (progress curve method,
at seven different concentrations) ranging from those that
minimally inhibited to those that fully inhibited the enzyme
(Figure 3), to determine the Ki values (Figure 4) are reported in
Table 1. All compounds were proven to inhibit rhodesain in the
low micromolar range, with the best binding affinity shown by the
inhibitor (R,S,S)-3 (Ki ¼ 1.26 mM).
O
N
H
N
N
OMe
O
N
H
O
O
O
1
Figure 1. Structure of the Michael acceptor 1.
In case of both couple of diastereoisomers 2 and 3, compounds
(S,S) configured on the warhead showed a slightly improved
O
N
O
N
H
N
(R)
H
N
N
(R)
O
N
O
Br
N
H
(S)
Br
N
H
(R)
O
O
N
O
O
O
O
N
(R,S)-2
(R,R)-2
Br
Br
O
O
N
N
(S)
(S)
(R)
H
H
(R)
(R)
N
N
N
O
N
O
N
N
N
N
O
O
(R)
O
O
O
O
(R,R,R)-3
(R,S,S)-3
Figure 2. Structure of target compounds.

DOI: 10.3109/14756366.2015.1108972
Peptidomimetics as rhodesain inhibitors
3
Scheme 1. Reagents and conditions: (a)
LiOH, MeOH, 0 ꢁC, rt, 6 h; (b) i-BuOCOCl,
NMM, CH2Cl2, 0 ꢁC, rt, 30 min, then 2-
aminobenzophenone, reflux, 20 min, rt, 12 h;
(c) HCl/MeOH, reflux, 5 h, then NaHCO3,
MeOH, rt, 12 h; (d) TBS-Cl, imidazole,
CH2Cl2, 0 ꢁC, rt, 12 h; (e) NaH,
BrCH2COOEt, 0 ꢁC, rt, 5 h; (f) LiOH, MeOH,
0 ꢁC, rt, 6 h; (g) lipase-PS, potassium phos-
phate buffer, pH ¼ 7, acetone, 650; (h) MsCl,
TEA, CH2Cl2, ꢀ10 ꢁC, rt, 16 h; (i) NaN3,
DMSO, 60 ꢁC, 4 h; (j) Ph3P, THF/H2O, rt,
24 h; (k) DBF, NaHCO3, EtOAc, rt, 48 h;
(l) preparative chiral HPLC; and (m) 30%
TFA, CH2Cl2, 0 ꢁC, rt, 4 h.
NBoc
a,b,c
O
d,e,f
N(R)
O
N(R)
NH
HO
N
(R) COOMe
4
OH
TBSO
Br
ref 9a
ref 9a
O
O
5
5
Br
h,i,j
OH
NH2
(R)
(R)
N
ref 12a
N
O
O
(R)-7
(R)-8
Br
OCOC3H7
ref 13)
g
N
O
(R)-6
g,f
Br
Br
OH
(S)
NH2
(S)
h,i,j
N
N
O
O
ref 12a
(S)-7
(S)-8
Br
N
(R)
HN
Br
O
(R)
k
l,m
ref 12a
(R,R)-11
BocN
N
N
Boc
ref 14
O
Br
(S)
-10
9
HN
N
O
(S)
(S,S)-11
O
N(R)
O
N(R)
H
(R)
N
N
O
c
a,b
HO
(R)
N
N
H
+
(R)-5 (R)-8
Br
N
Br
H
O
O
O
N
O
O
N
(R,R)-12
(R,R)-2
O
N(R)
O
N
N(R)
H
a,b
c
(R)-5 (S)-8
+
N
(S)
N
O
(S)
HO
N
Br
N
Br
H
H
O
O
O
N
O
O
N
(R,S)-12
(R,S)-2
Scheme 2. Reagents and conditions: (a) EDCI, HOBt, DIPEA, CH2Cl2, rt, 12 h; (b) TBAF, THF, rt; 6 h; and (c) 1-adamantylNCO, TEA, CH2Cl2,
rt, 72 h.
activity towards the target enzyme, e.g. (R,S,S)-3 versus (R,R,R)-3
with Ki values of 1.26 mM and 2.49 mM, respectively.
The antitrypanosomal activity of the rhodesain inhibitors
(R,R)-2, (R,S)-2, (R,R,R)-3 and (R,S,S)-3 was also evaluated on T.
Selectivity assays were also performed, testing inhibitors brucei brucei TC221 strains, with obtained IC50 values in the
against human cathepsin L: none of the synthesized compounds range 4.71–13.07 mM. Worthy of note, the most active rhodesain
2–3 passed the initial screening at 100 mM and thus demonstrating inhibitor (R,S,S)-3 showed the strongest antitrypanosomal activity
a good selectivity against the target enzyme.
(IC50 ¼ 4.71 mM) when compared to the other three derivatives,

4
R. Ettari et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
Br
O
N(R)
Br
N
(R)
H
N
O
N(R)
(R)
(R)
N
N
N
O
a,b
N
HO
c
+
(R)-5 (R,R)-11
N
O
(R)
O
O
O
O
(R,R,R)-3
(R,R,R)-13
Br
N
Br
N
O
N(R)
O
N
(S)
(S)
N(R)
H
N
a,b
(S)
(S)
N
O
c
(R)-5
+ (S,S)-11
HO
N
N
O
O
O
O
O
(R,S,S)-13
(R,S,S)-3
Scheme 3. Reagents and conditions: (a) EDCI, HOBt, DIPEA, CH2Cl2, rt, 12 h; (b) TBAF, THF, rt; 6 h; and (c) 1-adamantylNCO, TEA, CH2Cl2,
rt, 72 h.
These data clearly indicate that the 3-bromo isoxazoline
moiety, coupled with the 1,4-BDZ scaffold, as recognition moiety,
Table 1. Activity against rhodesain, hCatL and T. B. Brucei.
has been successfully employed. The presence of the adamantly
nucleus, directly linked to the methyl carbamoyl portion appended
Rhodesain (TbCatL)
Ki (mM) or %
inhibition
hCatL Ki (mM)
or % inhibition
at 100 mM
to C3 of the BDZ core, is probably fundamental for the
physicochemical properties of the inhibitor, making it suitable
to cross the parasite membrane. This hypothesis is supported by
the interesting PK properties calculated for the most active
derivative.
In view of these consideration, compound (R,S,S)-3 could be
considered as a lead compound for further development in the
design of new inhibitors which can be used as antitrypanosomal
agents.
T. b. brucei
IC50 (mM)
Compound
at 100 mM
(R,R)-2
(R,S)-2
(R,R,R)-3
(R,S,S)-3
2.36 0.15
1.91 0.04
2.49 0.43
1.26 0.04
24%
44%
16%
33%
11.43 0.67
13.07 0.97
12.27 0.14
4.71 1.23
thus giving an evidence of a clear correlation between inhibitory
properties and antiparasitic activity.
Experimental
Given the satisfactory antitrypanosomal properties of (R,S,S)-3
we decided to profile its physicochemical and pharmacokinetic
(PK) features as a rough assessment of its drug-like character. For
Chemistry
General: All reagents and solvents were obtained from commercial
suppliers and were used without further purification. Elemental
analyses were carried out on a C. Erba Model 1106 (Elemental
Analyzer for C, H and N) instrument, and the obtained results are
within 0.4% of the theoretical values. Merck silica gel 60 F254
plates were used for analytical TLC; flash column chromatography
was performed on Merck silica gel (200–400mesh) using a MP-LC
¨
such a purpose, we employed the Qikprop program (Schrodinger,
LLC, New York, NY). The results are summarized in Table 2.
These predictions would indicate that all the predicted parameters
fall into the ranges calculated for the 95% on the marketed drugs.
Of note, (R,S,S)-3 is predicted to have acceptable gut/blood
barrier permeability (see QPPCaco values in Table 2), high
potential of passing the blood/brain barrier (see QPlogBB and
QPPMDCK values in Table 2) and more than 80% human oral
absorption. All these data underscore the potentiality of this
compound to be considered as the ideal candidate lead compound
for the discovery of potent antitrypanosomal agents endowed with
promising PK parameters.
BUCHI system. H and 13C and NMR spectra were recorded on
1
Bruker Avance 300 MHz NMR spectrometer equipped with a BBI
probe and operating at frequencies of 300.13 and 75.47 MHz. We
used the residual signal of the deuterated solvent as an internal
standard. Splitting patterns are described as singlet (s), doublet (d),
doublet of doublet (dd), triplet (t), quartet (q), multiplet (m), or
broad singlet (br s). 1H and 13C NMR chemical shifts (ꢀ) are
expressed in ppm, and coupling constants (J) are given in Hz. MS
analyses were performed on a Varian 320-MS triple-quadrupole
mass spectrometer equipped with an electron-spray ionization (ESI)
source. Polarimetric analyses were carried out on a Perkin-Elmer
Polarimeter 341.
Conclusions
In conclusion, starting from the lead compound 1, we designed
two couples of diastereoisomers: a first one (R,R)-2/(R,S)-2
containing a 3-bromo isoxazoline nucleus as novel warhead, and a
second one in which the 3-bromo isoxazoline moiety was
incorporated into a bicyclic system [i.e. (R,R,R)-3/(R,S,S)-3].
All compounds were proven to inhibit rhodesain with Ki values in Synthesis of compounds (R,R)-2 and (R,S)-2
the low micromolar range, coupled with a good target selectivity,
N-(((R)-3-bromo-4,5-dihydroisoxazol-5-yl)methyl)-2-((R,Z)-2,3-
dihydro-3-(hydroxymethyl)-2-oxo-5-phenylbenzo[e][1,4]diazepin-
1-yl)acetamide [(R,R)-12]
evaluated by testing compounds against the human cathepsin L.
The most active rhodesain inhibitor (R,S,S)-3 (Ki ¼ 1.26 mM)
showed a good match of enzyme inhibitory properties and
antitrypanosomal activity, confirmed by an IC50 value of 4.71 mM, Step 1. To a solution of (R)-[3-(tert-butyl-dimethyl-silanylox-
comparable to that of the potent Michael acceptor
(IC50 ¼ 4.8 mM).
1
ymethyl)-2-oxo-5-phenyl-2,3-dihydro-benzo[e][1,4]diazepin-1-
yl]-acetic acid (R)-5 (9a) (500 mg, 1.14 mmol) in CH2Cl2

DOI: 10.3109/14756366.2015.1108972
Peptidomimetics as rhodesain inhibitors
5
eluting with EtOAc to give the deprotected title compound (R,R)-
12 (504 mg, 98%) Rf (EtOAc/light petroleum, 8:2) ¼ 0.18. 1H
NMR (300 MHz, CDCl3) ꢀ: 2.93 (bs, 1H), 3.03 (dd, J ¼ 7.3,
17.2 Hz, 1H), 3.33 (dd J ¼ 10.2, 17.2 Hz, 1H), 3.40–3.56 (m, 2H),
3.88 (t, J ¼ 7.4 Hz, 1H), 4.07 (d, J ¼ 16.0 Hz, 1H), 4.19–4.29 (m,
1H), 4.36–4.45 (m, 1H), 4.73 (d, J ¼ 16.0 Hz, 1H), 4.76–4.84 (m,
1H), 6.64 (bs, 1H), 7.18–7.68 (m, 9H).
14000
12000
10000
8000
6000
4000
2000
0
N-(((S)-3-bromo-4,5-dihydroisoxazol-5-yl)methyl)-2-((R,Z)-2,3-
dihydro-3-(hydroxymethyl)-2-oxo-5-phenylbenzo[e][1,4]diazepin-
1-yl)acetamide [(R,S)-12]
Compound (R,S)-12 was prepared as described for its diastereo-
isomer (R,R)-12, starting from (R)-5 (9a) (500 mg, 1.14 mmol)
and amine (S)-8 (12a) (203 mg, 1.14 mmol), followed by
deprotection with TBAF (1.6 mL of 1 M solution in THF,
1.6 mmol). (Overall yield 90%, 497 mg); Rf (EtOAc/light petrol-
0
200
400
time
600
Figure 3. Progress curves of substrate hydrolysis in the presence of the
inhibitor (R,S,S)-3. F ¼ fluorescence units. Inhibitor concentrations (from
top to bottom): 0, 10, 20, 40, 50, 60, 80 and 100 mM.
1
eum, 8:2) ¼ 0.18; H NMR (300 MHz, CDCl3) ꢀ: 3.01 (bs, 1H),
3.08 (dd, J ¼ 7.2, 17.2 Hz, 1H), 3.35 (dd J ¼ 10.3, 17.2 Hz,
1H), 3.42–3.51 (m, 1H), 3.60–3.78 (m, 1H), 3.87 (t, J ¼ 7.4 Hz,
1H), 4.09 (d, J ¼ 16.0 Hz, 1H), 4.18–4.26 (m, 1H), 4.34–4.43
(m, 1H), 4.72 (d, J ¼ 16.0 Hz, 1H), 4.74–4.82 (m, 1H), 6.60 (bs,
1H), 7.20–7.67 (m, 9H).
20
18
16
14
((R-1-(2-(((R)-3-bromo-4,5-dihydroisoxazol-5-yl)methylamino)-2-
oxoethyl)-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-
3-yl)methyl adamantan-1-ylcarbamate [(R,R)-2]
b
12
10
8
To a solution of compound (R,R)-12 (504 mg, 1.04 mmol), in dry
CH2Cl2 (20 mL), under nitrogen atmosphere, adamantyl isocyan-
ate (369 mg, 2.08 mmol) and TEA (210 mg, 289 mL, 2.08 mmol)
were added and the resulting mixture was stirred for 72 h at room
temperature. After this time the mixture was washed with water
(2 ꢂ 100 mL), dried over Na2SO4 and the solvent was removed
under reduced pressure. The crude material was purified by flash
column chromatography eluting with EtOAc/light petroleum 8:2
to give the title compound (R,R)-2. (262 mg, 38% yield). Rf
(EtOAc/light petroleum, 8:2) ¼ 0.61; Anal. Calcd. for
C33H36BrN5O5: C 59.26, H 5.28, N 10.80; found: C 59.01, H
6
4
0
20
40
60
80
100
I
20
5.39, N 10.44; ½ꢁꢃD ¼ ꢀ35.0 (c ¼ 0.25, CHCl3); MS [M + 1]+:
Figure 4. The slopes of the progress curve (b) from Figure 2 were plotted
against the inhibitor concentrations and fitted to the 4 parameter
IC50 equation. Ki was obtained from the Cheng–Prusoff equation
Ki ¼ IC50/(1 + [S]Kmꢀ1).
1
662.1; H NMR (300 MHz, CDCl3) ꢀ: 1.56–1.74 (m, 8H), 1.86–
1.96 (m, 4H), 2.02–2.08 (m, 3H), 2.97 (dd, J ¼ 8.0 and 17.3 Hz,
1H), 3.20 (dd, J ¼ 10.5 and 17.3 Hz, 1H), 3.47–3.55 (m, 2H), 3.9
(t, J ¼ 6.3 Hz, 1H), 4.22 (d, J ¼ 16.0 Hz, 1H), 4.70 (d, J ¼ 16.0 Hz,
1H), 4.71–4.80 (m, 1H), 4.81–4.88 (m, 2H), 4.95 (bs,1H), 6.66
(bs, 1H), 7.26–7.64 (m, 9H); 13C NMR (75 MHz, CDCl3):
ꢀ ¼ 29.93, 36.57, 41.96, 42.01, 44.14, 52.68, 62.18, 65.53, 80.35,
121.53, 124.89, 128.60, 129.33, 129.46, 130.01, 130.86, 132.47,
134.48, 138.01, 138.41, 153.47, 166.78, 169.04, 170.16.
(20 mL), HOBt (185 mg, 1.37 mmol) and EDCI (263 mg,
1.37 mmol) at 0 ꢁC were added. After 50 the ice bath was removed
and ((R)-3-bromo-4,5-dihydroisoxazol-5-yl)methanamine (R)-8
(12a) (203 mg, 1.14 mmol) and DIPEA (177 mg, 234 mL,
1.37 mmol) were added to the resulting mixture and the latter
was stirred at room temperature for 12 h. The organic layer was
separated, dried (Na2SO4), filtered and concentrated under
reduced pressure. The residue was purified by flash chromatog-
raphy eluting with light petroleum/EtOAc (8/2) to give N-(((R)-3-
bromo-4,5-dihydroisoxazol-5-yl)methyl)-2-((R,Z)-2,3-dihydro-3-
(tert-butyl-dimethyl-silanyloxymethyl)-2-oxo-5-phenylben-
zo[e][1,4]diazepin-1-yl)acetamide (636 mg, 93%).
Step 2. To a solution of N-(((R)-3-bromo-4,5-dihydroisoxazol-
5-yl)methyl)-2-((R,Z)-2,3-dihydro-3-(tert-butyl-dimethyl-silany-
loxymethyl)-2-oxo-5-phenylbenzo[e][1,4]diazepin-1-yl)acetamide
(636 mg, 1.06 mmol) in dry THF (20 mL) TBAF (1.6 mL of 1 M
solution in THF, 1.60 mmol) was added dropwise. The mixture
was stirred at room temperature until disappearance of the starting
material (TLC monitoring) and then it was diluted with EtOAc
(40 mL) and washed with water (2 ꢂ 100 mL). The organic layer
was separated, dried (Na2SO4), filtered, concentrated under
reduced pressure and purified by flash column chromatography
((R-1-(2-(((S)-3-bromo-4,5-dihydroisoxazol-5-yl)methylamino)-2-
oxoethyl)-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-
3-yl)methyl adamantan-1-ylcarbamate [(R,S)-2]
Compound (R,S)-2 was synthesized as described for its diastereo-
isomer (R,R)-2 starting from compound (R,S)-12 (497 mg,
1.02 mmol), adamantyl isocyanate (361 mg, 2.04 mmol) and
TEA (206 mg, 284 mL, 2.04 mmol). (243 mg, 36%); Rf (EtOAc/
light petroleum, 8:2) ¼ 0.61; Anal. Calcd. for C33H36BrN5O5: C
59.26, H 5.28, N 10.80; found: C 59.08, H 5.51, N 10.51;
20
1
½ꢁꢃD ¼ -15.0 (c ¼ 0.25, CHCl3); MS [M + 1]+: 662.2. H NMR
(300 MHz, CDCl3) ꢀ: 1.55–1.79 (m, 9H), 1.85–1.98 (m, 4H),
2.01–2.09 (m, 2H), 3.02 (dd, J ¼ 7.4 and 17.6 Hz, 1H), 3.26 (dd,
J ¼ 11.0 and 17.6 Hz, 1H), 3.43–3.54 (m, 1H), 3.62–3.80 (m, 1H),
3.88–3.96 (m, 1H), 4.10 (d, J ¼ 15.4 Hz, 1H), 4.72–4.91 (m, 4H),
5.16 (bs, 1H), 6.89 (bs, 1H), 7.32–7.64 (m, 9H); 13C NMR
(75 MHz, CDCl3): ꢀ ¼ 29.90, 36.55, 41.98, 42.03, 44.10, 52.63,

6
R. Ettari et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
Table 2. Calculated physicochemical and pharmacokinetic properties of compound (R,S,S)-3.
Range covered
by the 95%
of drugs
(R,S,S)-3
Description
#stars
3
0–5
Number of property or descriptor values that fall outside the 95%
range of similar values for known drugs.
#rotor
#rtvFG
5
0
0–15
0–2
Number of non-trivial, non-hindered rotatable bonds.
Number of reactive functional groups. The presence of these groups
can lead to false positives in HTS assays and to decomposition,
reactivity or toxicity problems in vivo.
mol_MW
dipole
SASA
674.593
7.74
990.263
1
130.0–725.0
1.0–12.5
300.0–1000.0
0.0–6.0
Molecular weight of the molecule.
Computed dipole moment of the molecule.
Total solvent accessible surface area (SASA) in square angstroms.
Estimated number of hydrogen bonds that would be donated by
the solute to water molecules in an aqueous solution. Values are
averages taken over a number of configurations, so they can be
non-integer.
donorHB
accptHB
12.2
2.0–20.0
Estimated number of hydrogen bonds that would be accepted by the
solute from water molecules in an aqueous solution. Values are
averages taken over a number of configurations, so they can be
non-integer.
QPlogPo/w
QPPCaco
4.554
247.687
ꢀ2.0–6.5
Predicted octanol/water partition coefficient.
Predicted apparent Caco-2 cell permeability in nm/s. Caco-2 cells are
a model for the gut blood barrier.
Predicted brain/blood partition coefficient for orally delivered drugs.
Predicted apparent MDCK cell permeability in nm/s. MDCK cells are
considered to be a good mimic for the blood–brain barrier.
Number of likely metabolic reactions.
525 poor,
4500 great
ꢀ3.0 to 1.2
525 poor,
4500 great
1–8
QPlogBB
QPPMDCK
ꢀ1.454
407.382
#metab
4
83.5
Percent Human
Oral Absorption
Rule Of Five
480% is high,
525% is poor
Maximum is 4
Predicted human oral absorption on 0–100% scale.
1
Number of violations of Lipinski’s rule of five. The rules are:
mol_MW5500, QPlogPo/w55, donorHB ꢄ 5, accptHB ꢄ 10.
Compunds that satisfy these rules are considered drug-like.
62.20, 65.50, 80.32, 121.50, 124.90, 128.62, 129.30, 129.49, ((R,4Z)-1-(2-((3aR,6aR)-3-bromo-3a,4,6,6a-tetrahydropyr-
130.05, 130.83, 132.48, 134.50, 138.03, 138.40, 153.50, 166.80, rolo[3,4-d]isoxazol-5-yl)-2-oxoethyl)-2,3-dihydro-2-oxo-5-
169.05, 170.12.
phenyl-1H-benzo[e][1,4]diazepin-3-yl)methyl adamantylcarba-
mate [(R,R,R)-3]
Synthesis of compounds (R,R,R)-3 and (R,S,S)-3
The title compound has been synthesized following the procedure
reported for compound (R,R)-2, starting from (R,R,R)-13 (516 mg,
1.04 mmol), adamantly isocyanate (369 mg; 2.08 mmol) and TEA
(210 mg, 289 mL, 2.08 mmol). (224 mg, 32%); Rf (EtOAc/light
petroleum, 8:2) ¼ 0.46; Anal. calcd for C34H36BrN5O5: C 60.54,
(c ¼ 0.36, CHCl3); MS [M + 1]+: 674.1; 1H NMR (300 MHz,
CDCl3) ꢀ: 1.56–1.67 (m, 8H), 1.88–1.93 (m, 4H), 2.00–2.07 (m,
3H), 3.59–3.66 (m, 1H), 3.80–4.03 (m, 5H), 4.13 (d, J ¼ 16.1 Hz,
1H), 4.28 (d, J ¼ 16.1 Hz, 1H), 4.72–4.83 (m, 2H), 5.12 (bs, 1H),
5.31–5.40 (m, 1H), 7.12–7.66 (m, 9H); 13C NMR (75 MHz,
CDCl3): ꢀ ¼ 29.55, 36.44, 42.54, 48.61, 50.81, 51.18, 53.97,
62.38, 63.29, 84.83, 122.20, 124.60, 128.31, 128.91, 129.52,
130.22, 130.67, 132.12, 138.34, 138.83, 139.66, 156.25, 166.77,
169.15, 169.42.
(R,4Z)-1-(2-((3aR,6aR)-3-bromo-3a,4,6,6a-tetrahydropyr-
rolo[3,4-d]isoxazol-5-yl)-2-oxoethyl)-3-(hydroxymethyl)-5-
phenyl-1H-benzo[e][1,4]diazepin-2(3H)-one [(R,R,R)-13]
Compound (R,R,R)-13 was prepared as described for compound
(R,R)-12, starting from acid (R)-5 (9a) (500 mg, 1.14 mmol) and
amine (R,R)-11(12a) (218 mg, 1.14 mmol); followed by deprotec-
tion with TBAF (1.6 mL of 1M solution in THF, 1.6 mmol).
(Overall yield 91%, 516 mg); Rf (EtOAc/light petroleum,
20
H 5.38, N 10.38; found C 60.71, H 5.67, N 10.22; ½ꢁꢃD ¼ ꢀ58.8
1
8:2) ¼ 0.11. H NMR (300 MHz, CDCl3) ꢀ: 3.55–3.63 (m, 1H),
3.82–4.05 (m, 5H), 4.12–4.19 (d, J ¼ 16.1 Hz, 1H), 4.26–4.35 (m,
2H), 4.70–4.77 (d, J ¼ 16.1, 1H), 5.34–5.41 (m, 1H), 7.17–7.65
(m, 9H).
(R,4Z)-1-(2-((3aS,6aS)-3-bromo-3a,4,6,6a-tetrahydropyrrolo[3,4-
d]isoxazol-5-yl)-2-oxoethyl)-3-(hydroxymethyl)-5-phenyl-1H-ben-
zo[e][1,4]diazepin-2(3H)-one [(R,S,S)-13]
((R,4Z)-1-(2-((3aS,6aS)-3-bromo-3a,4,6,6a-tetrahydropyr-
rolo[3,4-d]isoxazol-5-yl)-2-oxoethyl)-2,3-dihydro-2-oxo-5-
phenyl-1H-benzo[e][1,4]diazepin-3-yl)methyl
adamantylcarbamate [(R,S,S)-3]
Compound (R,S,S)-13 was synthesized as described for compound
(R,R)-12, starting from acid (R)-5 (9a) (500 mg, 1.14 mmol) and
amine (S,S)-11 (12a) (218 mg, 1.14 mmol), followed by subse-
quent deprotection with TBAF (1.6 mL of 1M solution in THF,
1.6 mmol). (Overall yield 89%, 505 mg); Rf (EtOAc/light petrol-
The title compound has been synthesized following the procedure
reported for compound (R,R)-2, starting from (R,S,S)-13 (505 mg,
1.01 mmol), adamantly isocyanate (358 mg; 2.02 mmol) and TEA
(204 mg, 281 mL, 2.02 mmol). (221 mg, 31%); Rf (EtOAc/light
petroleum, 8:2) ¼ 0.46; Anal. calcd for C34H36BrN5O5: C 60.54,
1
eum, 8:2) ¼ 0.11. H NMR (300 MHz, CDCl3) ꢀ: 3.38–3.48 (m,
1H), 3.69–3.83 (m, 2H), 3.84–3.98 (m, 3H), 4.11–4.18 (d,
J ¼ 15.2 Hz, 1H), 4.22–4.34 (m, 2H), 4.74–4.81 (d, J ¼ 15.2 Hz,
1H), 5.28–5.36 (m, 1H), 7.20–7.66 (m, 9H).
20
H 5.38, N 10.38; found C 60.83, H 5.54, N 10.13; ½ꢁꢃD ¼ ꢀ15.5
(c ¼ 0.18, CHCl3);MS [M + 1]+: 674.2; 1H NMR (300 MHz,

DOI: 10.3109/14756366.2015.1108972
Peptidomimetics as rhodesain inhibitors
7
CDCl3) ꢀ: 1.59–1.68 (m, 8H), 1.93–1.97 (m, 4H), 2.04–2.10 (m, running on a Dell Precision with Fedora Core. Minimizations
3H), 3.61–3.69 (m, 1H), 3.85–4.08 (m, 5H), 4.15 (d, J ¼ 15.8 Hz, were performed using the OPLS 2005 force field, an
1H), 4.31 (d, J ¼ 15.8 Hz, 1H), 4.75–4.88 (m, 2H), 5.18 (bs, 1H), enhanced version of Jorgensen’s OPLS force field, 4 with 5000
5.32–5.46 (m, 1H), 7.16–7.67 (m, 9H); 13C NMR (75 MHz, steps of steepest descent followed by conjugate-gradient energy
˚
CDCl3): ꢀ ¼ 29.53, 36.42, 42.55, 48.60, 50.79, 51.20, 53.96, calculations with
a
convergence of 0.0005 kJAꢀ1 molꢀ1
.
62.35, 63.28, 84.80, 122.18, 124.58, 128.29, 128.92, 129.50, Subsequently, the constructed (R,S,S)-3 was subjected to
130.20, 130.69, 132.10, 138.37, 138.80, 139.64, 156.27, 166.78, QikProp software calculations (QikProp, version 3.4 (2011);
¨
169.16, 169.40.
Schrodinger, LLC, New York, NY). In addition to predicting
molecular properties, QikProp provides ranges for comparing
each compound’s property with those of 95% of known drugs.
This software was also used because it allows for flagging reactive
functional groups that may cause false positives in biological
Biology
Enzyme assays
The preliminary screening was performed with 100 mM inhibitor assays.
concentration using an equivalent amount of DMSO as negative
control. The enzyme was recombinantly expressed as previously
Declaration of interest
described4. Product release from substrate hydrolysis (Cbz-Phe-
Arg-AMC, 10 mM) was determined continuously over a period of
10 min. Compounds showing at least 70% inhibition at 100 mM
were subjected to detailed assays. These were performed in a
50 mM sodium acetate buffer, pH 5.5 containing 10 mM DTT
with Cbz-Phe-Arg-AMC (10 mM) as substrate15. The Km value
used to calculate Ki values from IC50 values was determined as
0.9 mM (rhodesain)16. Inhibitor solutions were prepared from
stocks in DMSO. Each assay was performed twice in 96-well-
plates in a total volume of 200 mL. Fluorescence of the product
AMC of the substrate hydrolyses was measured using an Infinite
The authors report no declarations of interest.
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`
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