NMR conformational analysis in solution of a potent class of cysteine proteases inhibitors

Article abstract of DOI:10.1007/s11224-015-0597-5

Abstract Conformational analysis of a potent class of cysteine protease inhibitors is thoroughly studied by NMR, in both, polar and apolar solvents to get a better insight over the known biological activity and migration through biological media. These molecules are composed by a benzodiazepine (BDZ) scaffold connected to a bromo-isoxazoline (IOX) ring through an alkyl spacer (AS) with up to four-carbon atoms. Data, supported by theoretical calculations at DFT level, reveal that both BDZ and IOX keep a pretty rigid and asymmetric conformation, so that four diastereo-atropisomers (two mirror-image couples) are generated. The relative stiffness of these substrates, maintained also in different solvents, is confirmed by: (a) remarkable separation of diastereotopic protons; (b) specific through the space contacts (NOESY); and (c) very good fitting of the coupling constants evaluations. The prototypic compound with the longer AS shows two main conformations and a certain dynamic freedom around the AS torsional angles close to IOX; according to our data, the AS length is not fundamental for the functional BDZ and IOX fitting into the macromolecular complex; however, it does play a crucial role to cross the parasite cell membranes.

Full text of DOI:10.1007/s11224-015-0597-5

Struct Chem
DOI 10.1007/s11224-015-0597-5
ORIGINAL RESEARCH
NMR conformational analysis in solution of a potent class
of cysteine proteases inhibitors
Archimede Rotondo1
Roberta Ettari2 Silvana Grasso1 Maria Zappala
2
•
•
•
`
Received: 13 April 2015 / Accepted: 30 April 2015
Ó Springer Science+Business Media New York 2015
1
Abstract Conformational analysis of a potent class of
cysteine protease inhibitors is thoroughly studied by NMR,
in both, polar and apolar solvents to get a better insight
over the known biological activity and migration through
biological media. These molecules are composed by a
benzodiazepine (BDZ) scaffold connected to a bromo-
isoxazoline (IOX) ring through an alkyl spacer (AS) with
up to four-carbon atoms. Data, supported by theoretical
calculations at DFT level, reveal that both BDZ and IOX
keep a pretty rigid and asymmetric conformation, so that
four diastereo-atropisomers (two mirror-image couples) are
generated. The relative stiffness of these substrates, main-
tained also in different solvents, is confirmed by: (a) re-
markable separation of diastereotopic protons; (b) specific
‘‘through the space contacts’’ (NOESY); and (c) very good
fitting of the coupling constants evaluations. The prototypic
compound with the longer AS shows two main confor-
mations and a certain dynamic freedom around the AS
torsional angles close to IOX; according to our data, the AS
length is not fundamental for the functional BDZ and IOX
fitting into the macromolecular complex; however, it does
play a crucial role to cross the parasite cell membranes.
Keywords Proteases inhibitors Á H, 13C, 15N NMR Á
Solution chemical structure Á Conformational analysis
Introduction
Neglected tropical diseases (NTDs) are a group of tropical
infections, affecting more than 1.4 billion people [1], in
this context one of the most relevant disease is the Human
African Trypanosomiasis (HAT), also known as sleeping
sickness. HAT is caused by parasites of Trypanosoma
genus, two of which are able to transmit the disease to
humans: T. brucei gambiense, and T. brucei rhodesiense,
which cause the chronic and acute form of the disease,
respectively [2]. At present, there are only a few available
drugs for HAT treatment: suramine and pentamidine which
are active on the first (haemolymphatic) stage of the dis-
ease, while the second-stage active drugs melarsoprol and
eflornithine show many disadvantages that limit their use:
the arsenical derivative causes encephalopathy in 5–10 %
of treated patients, while eflornithine is active only against
T. brucei gambiense. In addition, the therapy is very ex-
pensive and difficult to administer, requiring hospitaliza-
tion [3]. Taking into consideration all these reasons, there
is an urgent need to identify promising targets and to de-
velop new drugs. To address this need, we focused our
attention on rhodesain, a clan CA, family C1 (papain
family) cysteine protease that plays essential roles in T. b.
rhodesiense life cycle [4]; specifically, it is required to
cross the blood–brain barrier (BBB), leading to the second
lethal stage of the disease; moreover, it plays a funda-
mental role in evading the host immune system.
Electronic supplementary material The online version of this
article (doi:10.1007/s11224-015-0597-5) contains supplementary
material, which is available to authorized users.
& Archimede Rotondo
arotondo@unime.it
1
Department of Chemical Sciences, University of Messina,
via D’Alcontres 31, 98166 Messina, Italy
In this context, our research group has been involved in
the last years into the development of cysteine protease
inhibitors for the treatment of NTDs [5–14]. In particular,
2
Department of Drug Sciences and Products for Health,
University of Messina, Viale Annunziata, 98168 Messina,
Italy
123

Struct Chem
Table 1 Activity of the
peptidomimetics 1–3 towards
rhodesain, FP-2, T.b.brucei and
P. falciparum
Comp
Rhodesain
Falcipain-2
T. b. brucei
P. falciparum
(
(
(
)-1
)-2
)-3
2.85
3.33
2.44
4.86
4.49
10.6
[40
[40
12.22
1.17
1.59
0.71
Br
N
N
N
O
n
O
(
(
(
)-1 n=2
)-2 n=3
)-3 n=4
we focused our attention on the design of peptidomimetics
whose use, with respect to peptides, is particularly advan-
tageous in terms of potency and selectivity, as widely
demonstrated in these years by our group within the de-
velopment of protease inhibitors [15–19]. More recently,
we developed novel peptidomimetics with a 3-bromo-
isoxazoline group (IOX) as innovative warhead, able to
react with the active site cysteine of rhodesain, thus leading
to a reversible inhibition of the target enzyme [20, 21].
Promising inhibitors were obtained by coupling a 1,4-
benzodiazepine (BDZ) scaffold, a recognition motif widely
employed by our group, with IOX, connected by means of
an aliphatic chain (AC) of different length (2–4 carbon
atoms). When tested against rhodesain compounds 1–3
showed Ki values in the range 2.44–3.33 lM, with the
inhibitor 3, with the longest AC, endowed with an antit-
rypanosomal activity of 12.22 lM with respect to the poor
activity of the homologous molecules 1 and 2. At the same
time, compounds 1–3 were tested on falcipain-2 (FP-2), a
cysteine protease of P. falciparum, belonging like rhode-
sain to papain family. The results of this investigation
clearly pointed out that, while compounds 1 and 2 showed
a better binding affinity with respect to compound 3 (see
Table 1); however, also in this case, the isoxazoline
derivative 3 showed the best antiplasmodial activity
(IC50 = 0.71 lM), highlighting its strong ability to cross
the parasite cell membrane. Starting from these consid-
erations, we decided to perform an NMR structural char-
acterization and conformational analysis of the three
inhibitors 1–3, in order to better correlate their biologically
active conformation to their antiparasitic activity.
4,5-dihydro-1,2-oxazol-5-yl)butyl]-5-phenyl-1,3-dihydro-
2H-1,4-benzodiazepin-2-one, is chosen as the most repre-
sentative as it issues the best biological response, and we
will mainly focus on this structure with the systematic la-
belling indicated in Scheme 1.
1,4-Benzodiazepine, such as diazepam, is among the
most important scaffolds in medicinal chemistry, being
considered ‘‘privileged structures’’ [22]. Despite the ab-
sence of a stereogenic centre, the seven-membered ring
assumes two chiral boat conformations being 3-CH2 proS
over the benzo-fused ring, or far away from it. Tradition-
ally, these two conformational enantiomers were indicated
with the P and M notation (helicoidal descriptors of en-
docyclic dihedral angles), respectively (Fig. 1) [23, 24]. It
is also known that racemization rate is tuned by the sub-
stituent on the N1 position which at least might prevent the
process allowing asymmetric resolution even for pre-
parative purposes [25, 26] and M to P interconversion
needs very high temperatures to fall within NMR time-
scales (spectral line broadenings) [22, 26].
Conformational analysis
Conformational analysis of biologically active compounds
is by itself a fundamental step within the whole knowledge
about the interaction mechanisms occurring in the
Results and discussion
Structural considerations concerning the investigated
compounds are a key step towards the understanding of
their promising antiparasitic activity. Compounds 1–3 are
composed by the 5-substituted IOX warhead connected to
the BDZ scaffold through an AS ranging from two to four
C atoms. Spectral data concerning the six samples of 1–3,
dissolved either in CDCl3 or CD3OD, reveal that there are
not noteworthy spectral differences among these; therefore,
BDZBuIOX (3), with the systematic name 1-[4-(3-bromo-
Scheme 1 Molecular representation of 3 with the labelling scheme
used in the text
123

Struct Chem
Fig. 1 Structural asymmetric
conformations of BDZ labelled
with helicoidal descriptors
P and M. In the case of 1–3, the
quite big R substituent increases
the M to P interconversion
barrier
chemistry of life [27]. Moreover, many chemicals travel-
ling inside biological systems pass through different envi-
ronments sometimes undergoing conformational changes
[28]. Being compound 3 very soluble in chloroform, it was
straightforward to perform the first NMR analysis in this
solvent (Fig. 2b). From the 1H NMR spectrum, it was
immediately clear that, beyond the presence of the chiral
centre, all diastereotopic CH2 methylene signals look
definitely split. We have also observed that compound 3 is
soluble in CD3OD (4–6 mg/mL, temperature-dependent)
so that we could run a complete NMR characterization also
in this ‘‘polar-like’’ medium (Fig. 2a). Unlike other asses-
sed cases [28], the overall spectral pattern does not change
keeping apart the proton diastereotopic resonances (Fig. 2):
Fig. 2 1H NMR profile of the aliphatic region for compound 3: a in
the CD3OD polar solvent; b in the less polar CDCl3 environment. The
assignment referred to the P–S isomer (see Table 2) is also reported,
whereas peaks with * label are impurities and the ** resonance is the
residual hydrogenated solvent signal
123

Struct Chem
123

Struct Chem
this suggests there is not a dramatic effect of the solvent on
the conformational features of 1–3. Usually, this effect is
remarkable in the case of rather rigid conformations; con-
regardless the length of the spacer. This is a clear evidence
of a strong shielding anisotropic effect on the second
methylene group imposed by a specific arrangement. Albeit
pretty close, these two resonances give different Nuclear
Overhauser Effect (NOE) being the b-CH2proS, specifically
close to the a-CH2proR and the b-CH2proR closer to the a-
1
sistently, after the complete and total assignment of the H
and 13C signals, it was possible, in both solvents, to ob-
serve specific scalar (dihedral angles evaluation) and
dipolar couplings (‘‘through the space’’ information)
matching conformations with rather limited degrees of
freedom. Measured scalar and dipolar coupling occurring
between geminal protons are obvious and taken as a ref-
erence and will not be further mentioned or drown in order
to simplify the discussion.
3
CH2proS. These evidences also fit the great J coupling
constants (&7 Hz) between a-CH2proS and b-CH2proS,
and between a-CH2proR and b-CH2proR against the small
values (&1 Hz) for the crossing over homologous ones ([b-
CH2proS/a-CH2proR] and [b-CH2proR/a-CH2proS]); ac-
cording to the Karplus law, indeed [29, 30], coupling con-
stants strongly support the typical staggered-trans
conformation around the aC-bC dihedral angle already en-
visaged by the nuclear contacts. Nonetheless, NOE in any
solvent, show close contacts between the BDZ-11-CH and
both of the two a-CH2proS and b-CH2proS (far away from
each other). As the simultaneous presence of these space
contacts is physically impossible (Fig. 3), provided that the
dihedral angle around aC-bC is rather locked, experimental
data unambiguously show the presence of two rotamers
around the N-aC dihedral angle in fast equilibrium within
the NMR time scale. These two configurations lead to
pseudo-axial or pseudo-equatorial orientation of the AS
respect to the average mean plane of the BDZ (Fig. 4). DFT
optimizations assessed that these conformations, as well as
the different diastereoisomers, are energetically close
(Table 3); therefore, it is reasonable to think they equally
1
Another important feature of the H profiles is the pro-
nounced line broadening involving especially resonances
close to the 5-IOX chiral centre; a careful analysis of the
13C traces together with HSQC also reveal the 1:1 splitting
of the related parent 13C atoms. This accounts for the very
rigid conformation of the asymmetric M and P BDZ joined
to the 5-IOX asymmetric centre; it generates an atropo-
diastereomeric mixture (say M–S and P–S together with
their respective mirror images P–R and M–R), whose very
slight differences are reasonably perceived far away from
the BDZ fragment and close to the 5-IOX centre. From
now on, the structural considerations will be discussed for
the P–S form to be eventually extended to the other iso-
mers. The stiffness of BDZ is further confirmed by the
NOE contacts, between the BDZ-8-CH and BDZ-11-CH
with the BDZ-3-CH2 at around 3.55 ppm (assigned as proS
in the case of Fig. 3).
Further NMR considerations
Looking at the assignments (Table 2), it is immediately
clear that all of the AS-CH2 in the b position present two
different signals at very low frequencies (0.64–0.95 ppm)
Fig. 4 3D models of P–S isomer of compound 3: double-headed
harrows evidence the most non-geminal through the space vicinities:
grey/coloured connections are reasonable just for a pseudo-equatorial
or just for b pseudo-axial rotamer. Dashed lines indicate weak signals
due to: (a) long-range contacts, (b) averaged contacts due to
conformational freedom
Fig. 3 Ball and stick 3D model of 3 P–S diastereo-atropisomer with
axial orientation of the AS; P and S labels, as well as proR and proS,
are indicated on the specific positions according to the conventional
use
123

Struct Chem
In conclusion provided, there are two specific rotameric
conformations around the N-aC bond for 1–3, the most
conformational freedom is localized on the segment AS-
cC-dC-5C which is specifically present in the compound 3.
Albeit it is usually obvious the free rotation around C–C
bonds of alkyl chains, this is not the case because of the
great steric hindrance between two big and very rigid
heterocyclic moieties (BDZ and IOX). According to the
differences into the activities of compounds 1–3 shown in
Table 1, regardless the chemical affinity with the proteases,
it is probable that the key dynamic role of the 3 butylic AS
is played by crossing the biological barriers made by the
pathogenic organisms. In this crucial step, BDZ and IOX
might be dynamically kept in an advantageous position
from each other, afterwards BDZ and IOX can easily ac-
commodate into their biologically active molecular com-
plex. Another important role to be mentioned is certainly
played by both the BDZ and IOX stereogenic centres
which probably lead to one over the four isomers par-
ticularly active into the macromolecular complex.
Table 3 Calculated energies of configurational and conformational
isomers of 3, at DFT level, with LSDA method using Gaussian 6-3-1
basis sets
Isomeric form
DE (kJ/mol) Dipolar moment (D)
3 S-P-pseudo-equatorial AS
3 R-M-pseudo-equatorial AS
3 S-P-pseudo-axial AS
0
1.74
1.74
8.58
8.58
7.96
7.96
3.97
3.97
0
?5.05
?5.05
?3.68
?3.68
?2.35
?2.35
3 R-M-pseudo-axial AS
3 S-M-pseudo-equatorial AS
3 R-P-pseudo-equatorial AS
3 S-M-pseudo-axial AS
3 R-P-pseudo-axial AS
Couples of mirror images display obviously the same properties
share almost the entire molecular population. In summary,
the pseudo-axial arrangement, with a-CH2proS very close to
the aromatic BDZ-11-CH, accounts for the great anisotropic
shielding effect sensed by the b-methylene group as it is
permanently facing both the aromatic BDZ rings (Fig. 4b);
on the other hand, pseudo-equatorial orientation matches the
intense NOE cross-peaks between b-CH2proS and the BDZ-
11-CH and the weaker NOE between a-CH2proR and the
BDZ-11-CH (Fig. 4a).
Experimental section
NMR
The staggered-trans conformation seems to be assumed
also by the next alkyl connection as demonstrated by the
definite NOE 1,3 contacts (a-CH2proS/c-CH2proR and a-
CH2proR/c-CH2proS; see Fig. 4); however, starting from
the b-position the relatively weak but detectable 1,2 and
1,3 methylene vicinities witness that dihedral angle around
cC-dC is endowed with a certain degree of freedom.
Finally, as expected by the model, the IOX ring is almost
planar. Coupling constants between the two IOX-4-CH2 and
the stereogenic IOX-5-CH are pretty big and very similar to
each other (9 and 10 Hz); according to the Karplus law,
dihedral angles are either close to 0° or around 180°,
therefore, albeit the two-carbon atoms are sp3; nonetheless,
the planar conformation clearly driven by the conjugation of
the heterocyclic segment prevents possible envelope-like
folding. For the given configuration S, the IOX-4-CH2 proS
can be assigned considering the strong dipolar coupling to
the IOX-5-CH (dihedral angle close to 0°) together with the
lack of other detectable vicinities. On the other hand, ex-
pectedly, the geminal IOX-4-CH2 proR does not show NOE
with IOX-5-CH (dihedral angle not far from 180°); but does
present space contacts towards AS-d and AS-c methylene
protons. Again the clear contacts of both spatially separated
5-IOX-CH and 4-IOX-CH2proR towards d-CH2 protons;
and, with a lesser intensity, towards the next and opposite c-
CH2 (Fig. 4), warrantees free rotation around the (AS-dC)-
(IOX-5-C) bond, with the most probable staggered-trans
arrangement drawn in Fig. 4.
1H, 13C{1H} and 15N{1H} NMR spectra of 1–3 were recorded
on Bruker Avance 300 MHz NMR spectrometer equipped
with a BBI probeandoperating at frequencies of 300.13, 75.47,
30.42 MHz; many experiments were double checked on a
Varian 500 MHz spectrometer equipped with a ONE_NMR
probe and operating at 499.74, 125.73, 50.65 MHz, respec-
tively. Compounds 1–3 were dissolved in 500 lL of CDCl3
(10–20 mg) and in 500 lL of CD3OD (saturated solution,
about 10 mg/mL). The complete and unambiguous assign-
ment is confirmed by homo-nuclear 2D-COSY and NOSY
[31] and heteronuclear [32] 13C{1H}-HSQC, 13C{1H}-HMBC
and 15N{1H}-HMBC experiments. Extended NMR data are
reported (Tables 2; Fig. 2; supplementary material) in order to
easily compare differences determined by the spacer or by the
used solvent. We have chosen to present the 1H, 13C and 15
N
chemical shifts (cs) in CD3OD in order to reproduce a polar
medium somewhat reminding a biological environment; other
measurements, made in the more polar CD3OH/H2O 80/20 %
mixture, demonstrated again there are not dramatic changes in
1
the H profile. Calibration was attained using as internal
standard residual proton signal of the solvent (CD2HOD
quintet: d = 3.31 ppm; CHCl3 d = 7.26 ppm and the 13C
solvent septuplet at d = 49.0 ppm and triplet d = 79.0, re-
spectively) [33]. 15N calibration was referred to the absolute
frequency provided that the CH3NO2 as external standard was
also calibrated (90 % CH3NO2 in CD3OH d = 380.5 ppm)
123

Struct Chem
[34]. The average distances are extracted by many 2D-NOSY
and 1D-NOE experiments run at different mixing times [35]
and used according to the two-spin-system approximation;
experimental data are reported in the supplementary material,
whereas the main relevant features are fully discussed in the
experimental section.
Conclusions
Because of the tight relationship between compounds used
in medicinal chemistry and the nature of their functional
and structural features [28, 37], we decided to run specific
structural analysis in solution concerning molecules en-
dowed with pharmaceutical properties. A specific class of
compounds, with the ‘‘privileged’’ BDZ scaffold linked to
the IOX moiety through an AS with two- to four-carbon
atoms, was carefully analysed in solution by NMR tech-
niques. Studies were extended to different solvents in order
to detect possible differences sometimes affecting drug
candidates [29]. In this case though, the pretty rigid con-
formation of the BDZ structure and the imposed quasi-
planar conformation of the IOX, hamper most of the con-
formational arrangements.
Computer-aided analysis
Several NMR simulations, to confirm the correct assignment
related to the specific 3D-structure, were run by the PERCH
NMR software package (v. 2014.1, PERCH Solutions Ltd.,
Kuopio, Finland); as the diastereo-atropisomeric mixture
hamperedtheperfect linefitting, wealsousedthemanualbest
fitting of both PERCH and i-NMR (version 5.4.3 for Win-
dows 7) software packages. All the shown molecular models
are represented using Gaussian View or PERCH MMS be-
longing to Gaussian 03 [36] and PERCH software packages,
respectively. Molecular optimizations well fitting the NMR
data were run by PERCH models with simple molecular
mechanics and later reviewed by Gaussian DFT calculations
with the LSDA method with the G631 basis set. This level of
calculation is considered reasonable according to: (a) the
presence of elements belonging to the first raw except for the
peripheral Bromine atom; (b) the affordable time-consuming
calculations on a normal four processors PC. The output re-
sulting file for structures of 3 (Table 3) are enclosed in the
supplementary material.
Moreover, H line broadening and 13C splitting of the
1
signals around the 5-IOX stereogenic centre clearly ac-
count for the presence of diastereo-atropisomers being the
asymmetric BDZ folded either in the P or in the M mode
against the other traditional 5C-IOX (S or R) stereogenic
centre.
Conformational analysis evidences that the dihedral angle
aC-bC on the AS is pretty locked, whereas two main con-
formations are detected around the N-aC dihedral angle
yielding the completely different pseudo-axial or pseudo-
equatorial orientation of the AS-IOX terminus. This allows
just a limited degree of freedom for 1 and 2 whose short AS
tie causes steric bumping between BDZ and IOX; however, 3
with the butyl AS owns much more flexibility because of the
conformational freedom around AS dihedral angles closer to
the IOX. The drawn structural models fully match all the
NMR data (very big amount of information) unambiguously
confirming the conformational analysis of compound 3.
Starting from the consideration that compound 3 was
shown to possess the best activity against both T. b. brucei
and P. falciparum, respectively, in the low micromolar and
submicromolar range, we can assume that beyond the im-
portance of the BDZ scaffold, which acts as recognition
motif, and of the IOX which behaves as warhead, a key
role in the ability to cross the parasite cell membranes of
inhibitor 3 must be attributed to the AS. On this regard,
while a two- or three-carbon-atom AS is responsible for a
Synthesis
The synthesis of the inhibitors ( )-1–3, was realized as
previously described by our group [21], by treating the
benzodiazepine 4 with the bromo-alkenes 5–7, in the
presence of sodium hydride, to obtain intermediates 8–10.
The terminal olefins 8–10 were, in turn, used as dipo-
larophile to react in a 1,3-dipolar cycloaddition with the
1,3-dipole bromonitrile oxide, generated in situ by dehy-
drohalogenation of the stable precursor dibromoformal-
doxime, to afford the isoxazoline derivatives ( )-1–3
(Scheme 2).
Scheme 2 Reagents and
conditions: a NaH, DMF, 0 °C,
N2, 1 h, then 5–7, rt, 12 h;
b DBF, NaHCO3, EtOAc, 12 h
Br
N
N
N
a
b
N
N
NH
N
Br
+
n
n
O
n
O
O
O
4
5 n=2
8 n=2
9 n=3
10 n=4
( )-1 n=2
( )-2 n=3
( )-3 n=4
6 n=3
7 n=4
123

Struct Chem
16. Micale N, Ettari R, Lavecchia A, Di Giovanni C, Scarbaci K,
limited degree of freedom of homologous compounds 1
and 2, on the contrary, a four-carbon atoms AS contributes
to an enhanced flexibility of compound 3 which accounts
for its great antiparasitic activity reported in Table 1.
Looking at the modelling of similar inhibitors [14], we
might suppose that the pseudo-equatorial conformation is
assumed in the active macromolecular complex; however,
the conformational freedom is kinetically crucial to fit the
molecule in its binding site [28]. Another important role is
probably played by the traditional IOX stereogenic centre
and by the BDZ atropo-stereogenic centre which might
reasonably give to one configurational substrate (over four)
specifically active towards the biological targets. This pa-
per is the beginning molecular approach to get more clues
about the mechanism of action of specific drug candidates;
thus, it might pave the way towards the knowledge of both
the interaction with macromolecules and specific biological
pathways undertaken by these investigated substrates.
`
Troiano V, Grasso S, Novellino E, Schirmeister T, Zappala M
(2013) Eur J Med Chem 64:23–34
17. Scarbaci K, Troiano V, Micale N, Ettari R, Tamborini L, Di
Giovanni C, Cerchia C, Grasso S, Novellino E, Schirmeister T,
`
Lavecchia A, Zappala M (2014) Eur J Med Chem 76:1–9
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`
(2014) Med Res Rev 34:1001–1069
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`
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Acknowledgments We gratefully thank the MIUR (Ministero del-
`
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