Tuning and predicting biological affinity: Aryl nitriles as cysteine protease inhibitors

Article abstract of DOI:10.1039/c2ob00034b

A series of aryl nitrile-based ligands were prepared to investigate the effect of their electrophilicity on the affinity against the cysteine proteases rhodesain and human cathepsin L. Density functional theory calculations provided relative reactivities of the nitriles, enabling prediction of their biological affinity and cytotoxicity and a clear structure-activity relationship.

Full text of DOI:10.1039/c2ob00034b

View Article Online / Journal Homepage / Table of Contents for this issue
Organic &
Biomolecular
Chemistry
This article is part of the
OBC 10th anniversary
themed issue
All articles in this issue will be gathered together
online at
www.rsc.org/OBC10

Dynamic Article Links
Organic &
View Article Online
Biomolecular
Chemistry
Cite this: Org. Biomol. Chem., 2012, 10, 5764
www.rsc.org/obc
COMMUNICATION
Tuning and predicting biological affinity: aryl nitriles as cysteine protease
inhibitors†‡§
Veronika Ehmke,a Jose Enrico Q. Quinsaat,a Pablo Rivera-Fuentes,a Cornelia Heindl,b Céline Freymond,c,d
Matthias Rottmann,c,d Reto Brun,c,d Tanja Schirmeisterb,e and François Diederich*a
Received 5th January 2012, Accepted 23rd January 2012
DOI: 10.1039/c2ob00034b
to react with the catalytic cysteine under formation of a covalent
adduct.
A series of aryl nitrile-based ligands were prepared to inves-
tigate the effect of their electrophilicity on the affinity against
the cysteine proteases rhodesain and human cathepsin
L. Density functional theory calculations provided relative
reactivities of the nitriles, enabling prediction of their bio-
logical affinity and cytotoxicity and a clear structure–activity
relationship.
Different functional groups that irreversibly alkylate or acylate
the active site have been incorporated into a variety of ligands.3
They often show, however, off-target effects and in particular
cross-reactivity towards serine and other cysteine proteases. This
has much hampered the therapeutic development of cysteine pro-
tease inhibitors despite the validation of these enzymes as drug
targets. Only a limited number of electrophilic isosteres have
found application as selective and reversibly-binding ligands.3
The best-studied classes of reversible covalent inhibitors com-
prise aldehydes, certain ketones and few nitriles. Aldehydes and
ketones, however, are more susceptible to side reactions because
of their pronounced electrophilicity, whereas nitriles are gener-
ally less reactive. They form a covalent thioimidate adduct with
the active site cysteine, which resembles the transition state of
the amide bond hydrolysis.4 Several nitrile-bearing cysteine pro-
tease inhibitors are in development and clinical trials, highlight-
ing the relevance of the nitrile group as a pharmacophore in
modern medicinal chemistry.5
Cysteine proteases of protozoan parasites have emerged as
promising targets in drug development due to their indispensable
role in the life cycle of the parasite.6 According to the World
Health Organisation (WHO), 3.5 billion people suffer from para-
sitic diseases, distributed predominantly in tropical areas of the
world.7 Trypanosoma (T.) brucei parasites cause human African
trypanosomiasis (HAT, African sleeping sickness) leading to
50 000–70 000 infections annually. Over 60 million people live
at risk of contracting the disease. Only four drugs are currently
approved for treatment, however, widespread parasite resistance
and severe toxicity issues drastically limit their application.7
Bloodstream T. brucei rhodesiense parasites, causing the most
acute form of the disease, express two papain-family cysteine
proteases, which are directly implicated in the progression of the
disease.8 One of these enzymes is the highly abundant cathepsin
L-like hydrolase rhodesain, which has been identified in all
phases of the life-cycle, particularly during the infective stage of
parasite development.9 Cysteine protease inhibitors have been
shown to cure infections in animal models, validating rhodesain
as an interesting new target against HAT.9,10
Four major classes of proteases selectively catalyse the hydroly-
sis of polypeptide bonds. Their general control over protein syn-
thesis, turnover and function enables them to regulate many
essential physiological processes.1 Cysteine proteases utilise a
catalytic thiolate that mediates protein hydrolysis by nucleophilic
attack on the carbonyl group of the susceptible peptide bond.
Among cysteine proteases, the enzymes of the papain-family use
a catalytic dyad of cysteine and histidine for their proteolytic
activity. They are among the best-characterised eukaryotic pro-
teases and are involved in disease propagation and proliferation.
Therefore, inhibitors are continuously emerging as promising
therapeutic agents for example for the treatment of parasitic
infections, as well as for inflammatory, immunological, cardio-
vascular and neurodegenerative disorders.2
Cysteine protease inhibitors have been traditionally developed
by screening methods and subsequent lead optimisation.2 They
share as a common feature an electrophilic peptide bond isostere
aLaboratorium für Organische Chemie, ETH Zürich, Hönggerberg,
HCI, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland.
E-mail: diederich@org.chem.ethz.ch; Fax: (+41)44-632-1109
bInstitute of Pharmacy and Food Chemistry, University of Würzburg,
Am Hubland, 97074 Würzburg, Germany
cSwiss Tropical and Public Health Institute, Socinstrasse 57, 4002
Basel, Switzerland
dUniversity of Basel, Petersplatz 1, 4003 Basel, Switzerland
eInstitute of Pharmacy and Biochemistry, Johannes Gutenberg-
University Mainz, Staudinger Weg 5, 55128 Mainz, Germany
†Dedicated to Professor Wilfred van Gunsteren on the occasion of his
65th birthday.
‡This article is part of the Organic & Biomolecular Chemistry 10th
Anniversary issue.
§Electronic supplementary information (ESI) available: Detailed exper-
imental procedures and spectroscopic data for all compounds, biological
assays and additional figures and tables. See DOI: 10.1039/c2ob00034b
Recently, we developed a series of potent peptidomimetic and
triazine nitrile inhibitors against the parasitic cysteine proteases
5764 | Org. Biomol. Chem., 2012, 10, 5764–5768
This journal is © The Royal Society of Chemistry 2012

View Article Online
falcipain-2 of Plasmodium falciparum and rhodesain by molecu-
lar modeling.11 Starting from two available X-ray crystal struc-
tures of rhodesain,9,12 molecular modeling using MOLOC13 led
to a diamino-substituted triazine as the central scaffold (Fig. 1).
Lead structure 1 showed an inhibitory constant (Ki) against
rhodesain in the single-digit nanomolar range. The mainly
hydrophobic S2 pocket was addressed with a cyclohexyl substi-
tuent, and a 1,3-benzodioxol-5-yl group was chosen to stack on
the flat peptide backbone formed by Gly65 and Gly66, thereby
filling the solvent exposed S3 pocket. Some compounds of the
triazine library, however, showed low selectivity against the
structurally closely related human cathepsin L (hCatL, 1:
Ki(hCatL) = 33 nM). More importantly, in vitro assays revealed
cytotoxicity of the active ligands in the low micromolar range.11a
We assumed that the strong electron-withdrawing effect of the
triazine core enhanced the electrophilicity of the nitrile head
group, making it more susceptible to off-target effects. Oballa
et al. proposed a simple computational method to assess the rela-
tive electrophilicity of nitriles, which influences the reversibility
of the adduct formation of nitriles with free cysteine.14,15 In this
work, we sought to establish a generally applicable, predictive
structure–activity relationship (SAR) between experimentally
determined enzyme inhibition and cytotoxicity data with calcu-
lated nitrile electrophilicities.
Starting from lead compound 1, first the effect of various
heteroaromatic nitriles on rhodesain and hCatL inhibition was
explored. A series of ligands 2–12 (Fig. 2) was prepared by
varying the electronic properties of the central aromatic core,
which in turn determine the electrophilicity of the nitrile head
group. The S2 and S3 vectors were kept constant. Triazines 2
and 3 were prepared to evaluate the influence of the morpholine
group of 1 on activation of the nitrile and inhibition. The
number of electron-withdrawing N-atoms was subsequently
reduced to two in ligands 4–9, featuring pyrimidine, quinazoline
and pyrazine cores, and to one in pyridine derivative 10. Ligands
with 5-membered heteroaromatic rings, such as thiazole 11 and
oxazole 12, completed the series. All ligands were designed to
maintain a similar binding geometry at the enzyme active site to
ensure that differences in biological affinity are mainly due to
variation of the electronic properties of the aryl nitriles and not
to other contacts with the enzyme (Fig. S1, ESI§).
For the synthesis of the ligands, triazine nitriles 2 and 3 were
prepared according to the protocol reported for 1 (for details see
ESI§).11 Pyrimidine and quinazoline derivatives 4–8 were
obtained following the sequence shown exemplarily for pyrimi-
dines 5 and 7 in Scheme 1a. Nucleophilic aromatic substitution
of amine 13 and 5-bromo-2,4-dichloropyrimidine gave pyrimi-
dine 14, which was converted into nitrile 5 using potassium
cyanide and 1,4-diazabicyclo[2.2.2]octane (DABCO) in excel-
lent yield. Methyl fluorosulfonyldifluoroacetate and copper(I)
iodide were used to in situ generate trifluoromethylcopper16 as
the nucleophilic trifluoromethylating agent giving 7 in good
yield. The syntheses of pyrazine 9 and pyridine 10 relied on
Buchwald–Hartwig amination protocols17 followed by
Fig. 1 Structure and binding mode of inhibitor 1 in the active site of
rhodesain (PDB code: 2P86, resolution 1.16 Å) as generated with
MOLOC. Colour code: Cenzyme grey, Cligand green, O red, N blue, S
yellow. Distance is given in Å.
Fig. 2 Nitrile scale of calculated electrophilicities (level of theory: B3LYP/6-311G(d,p)) in kJ mol−1 of simplified aryl nitriles (top). Inhibition con-
stants Ki of the corresponding ligands 1–12 are given in nanomolar against rhodesain and hCatL (bottom).
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 5764–5768 | 5765

View Article Online
Scheme 2 Model reaction of a simplified aromatic nitrile with metha-
nethiol resulting in a thioimidate adduct. Relative electrophilicities are
calculated as ΔHthioimidate − (ΔHnitrile + ΔHmethanethiol). ΔH values were
obtained at the B3LYP/6-311G(d,p) level of theory.
(Scheme 2).14 The aryl nitriles were structurally based on
ligands 1–12, but simplified concerning the S2 and S3 substitu-
ents (Fig. 2, top). The structures of methanethiol, the simplified
nitriles and the corresponding thioimidate adducts were opti-
mised in water (PCM solvation model) at the B3LYP/6-311G
(d,p) level of theory using Gaussian 09.20 The resulting electro-
philicity index corresponds to the differences in formation
enthalpies ΔHthioimidate − (ΔHnitrile + ΔHmethanethiol) and is given
in kJ mol−1. The values obtained give only thermodynamic
information within the model reaction and therefore should only
be used to qualitatively arrange and rank the aryl nitriles.
i
i
Scheme 1 (a) Synthesis of inhibitors 5 and 7: (i) Pr2NEt, PrOH,
80 °C, 18 h, 84%; (ii) KCN, DABCO, Me2SO–H2O 9 : 1, 120 °C, 2 h,
89%; (iii) methyl fluorosulfonyldifluoroacetate, CuI, HMPA, 80 °C,
2.5 h, 73%; (b) synthesis of inhibitor 11: (iv) thiourea, EtOH, 85 °C,
t
1.5 h, 98%; (v) CuCl2, BuONO, MeCN, 65 °C, 5.5 h, 27%; (vi) 13,
1,4-dioxane, 120 °C, 7 d, 9%; (vii) LiOH·H2O, THF–MeOH–H2O
2 : 2 : 1, 40 °C, 3 h; (viii) SOCl2, DMF, toluene, 80 °C, 1.5 h; (ix) NH3–
MeOH, toluene, 0→25 °C, 1 h, 66% (over 3 steps); (x) Burgess reagent
(methyl N-(triethylammonium-sulfonyl)carbamate), CH2Cl2, 25 °C, 2 h,
82%; DABCO = 1,4-diaza-bicyclo[2.2.2]octane, HMPA = hexamethyl-
phosphoramide.
The scale of electrophilicity values of the designed aromatic
nitriles as obtained by DFT calculations is shown in Fig. 2 (top).
Very negative values indicate high electrophilicity in accordance
with the equilibrium of the model reaction, whereas small nega-
tive values are attributed to poor electrophilicity. As expected,
triazine nitriles are predicted to be highly reactive towards
nucleophiles due to their pronounced electron-withdrawing
effect (−42.3 to −36.8 kJ mol−1). Electrophilicities of pyrimi-
dine and quinazoline-based nitriles span a wide range from
strongly activated in the trifluoromethylated analogue (−39.8
kJ mol−1) to intermediate activation for the quinazoline com-
pound (−33.1 kJ mol−1). Pyrazine and pyridine nitriles are only
moderately reactive towards nucleophiles (−27.2 and −22.6
kJ mol−1). Finally, 5-membered heteroaromatic systems are in a
similar intermediate range due to their electron-rich aromatic
palladium-catalysed cyanation with zinc(II) cyanide (see ESI§).18
The 5-membered heteroaromatic nitriles 11 and 12 were obtained
as outlined for thiazole 11 in Scheme 1b. A condensation reac-
tion of thiourea and ethyl bromopyruvate gave quantitatively
aminothiazole 15, which was converted into 2-chlorothiazole 16
by a Sandmeyer reaction. Subsequent nucleophilic aromatic sub-
stitution by amine 13 gave ester 17 in poor yield due to the low
electrophilicity of the 2-chlorothiazole ring. The ethyl ester was
transformed into the corresponding amide 18, and dehydration
using Burgess reagent19 gave nitrile 11 in high yield. Methyl
fluorosulfonyldifluoroacetate and copper(I) iodide were used
to in situ generate trifluoromethylcopper16 as the nucleophilic
trifluoromethylating agent giving 7 in good yield. The syntheses
of pyrazine 9 and pyridine 10 relied on Buchwald–Hartwig
amination protocols17 followed by palladium-catalysed cyanation
with zinc(II) cyanide (see ESI§).18 The 5-membered heteroaro-
matic nitriles 11 and 12 were obtained as outlined for thiazole 11
in Scheme 1b. A condensation reaction of thiourea and ethyl
bromopyruvate gave quantitatively aminothiazole 15, which was
converted into 2-chlorothiazole 16 by a Sandmeyer reaction.
Subsequent nucleophilic aromatic substitution by amine 13 gave
ester 17 in poor yield due to the low electrophilicity of the 2-
chlorothiazole ring. The ethyl ester was transformed into the cor-
responding amide 18, and dehydration using Burgess reagent19
gave nitrile 11 in high yield.
system. The unactivated benzonitrile analogue (−2.5 kJ mol−1
)
and other heterocyclic aromatic nitriles covering the range from
−16.5 to −1.2 kJ mol−1 (not shown in Fig. 2) were included to
complete and verify the scale (for the extended electrophilicity
scale see Table S1, ESI§). This approach provides a useful and
straightforward measure of the relative electrophilicities and can
be generally applied to aryl nitrile systems.
Nitriles 1–12 were subsequently tested against rhodesain from
T. brucei rhodesiense and hCatL (see ESI§) in order to correlate
the calculated electrophilicities to the biological affinities of the
covalent reversible nitrile ligands. Inhibitory constants against
rhodesain and hCatL span from single-digit nanomolar for tria-
zine ligands 1 and 2 to >80 μM for nitriles 9–12. In Fig. 2
(bottom), all ligands are arranged according to their inhibitory
constants. Noticeably, this alignment is in accordance with the
nature of the central core and leads to grouping of the corre-
sponding heteroaromatic rings (triazines > pyrimidines/quinazo-
We used density functional theory (DFT) calculations to
assess the relative reactivities of the nitrile groups attached to
different heteroaryl scaffolds towards a sulfur nucleophile, in
order to predict the biological affinities of the new nitrile-based
inhibitors against rhodesain and hCatL. The electrophilicities
were calculated as the formation enthalpies of the thioimidate
adducts formed in a theoretical reaction between methanethiol
and aryl nitriles similarly to the method reported by Oballa et al.
lines
> pyrazine/pyridine/thiazole/oxazole). Moreover, the
biological results correlate very well with the calculated electro-
philicities of our model system. Strongly activated nitriles
present in triazines 1–3 lead to highly potent inhibition against
5766 | Org. Biomol. Chem., 2012, 10, 5764–5768
This journal is © The Royal Society of Chemistry 2012

View Article Online
Table 1 Cytotoxicity of compounds 1–12 and calculated
electrophilicities.a The compounds are arranged according to their
theoretical electrophilicities
Fig. 3 Inhibition constants Ki in nanomolar of nitrile ligands 1–12
against rhodesain (black squares) and hCatL (grey spheres) plotted
against the calculated electrophilicities in kJ mol−1. The y-axis is shown
as a logarithmic scale. The threshold at −33 kJ mol−1 (green) marks the
limit for nanomolar inhibition.
a All values represent the mean of at least two independent
measurements, each performed in duplicate. b Rat skeletal myoblasts
were used to assess cytotoxicity. Podophyllotoxin served as positive
control in the assay (IC50 = 0.0082 μM).
both rhodesain (1: Ki(rhodesain) = 8 nM, 2: Ki(rhodesain) =
9 nM, 3: Ki(rhodesain) = 19 nM) and hCatL (1: Ki(hCatL) =
33 nM, 2: Ki(hCatL) = 2 nM, 3: Ki(hCatL) = 38 nM). For pyrimi-
dine and quinazoline ligands 4–8, moderate to high inhibition is
predicted according to their electrophilicities. Indeed, nitriles
4–8 inhibited rhodesain and hCatL with Ki values ranging from
87 nM to low micromolar. Less activated nitriles, such as pyra-
zine 9, pyridine 10, thiazole 11 and oxazole 12, showed no or
only low inhibition, as predicted by the calculated low electro-
philicities. This SAR validates the results obtained by the DFT
calculations and confirms the initial hypothesis that biological
affinity is strongly influenced by the electronic nature of the
nitrile. In order to obtain potent ligands with nanomolar or very
low micromolar inhibitory constants, a certain electrophilicity of
the head group is required (Fig. 3). The threshold for both rhode-
sain and hCatL lies below an activation of the nitrile of around
−33 kJ mol−1. The data indicate that the calculated electrophili-
city is useful in reliably predicting the order of magnitude of
inhibitory constants against the investigated cysteine proteases.
The only exemption in the ligand series comprised trifluoro-
methyl-substituted pyrimidine 7, for which higher inhibition
would have been expected. This might be due to unfavourable,
allyl 1,3-strain-type intramolecular repulsions between the steri-
cally demanding CF3 substituent and the benzylic CH2 group,
leading to a loss in binding affinity (Fig. S2, ESI§).
therefore also be applied to give an indication of the cytotoxicity
of cysteine protease inhibitors, even though other aspects such as
pharmacokinetic properties of the compounds need to be
considered.
In conclusion, we studied the SAR of aryl nitriles as covalent
reversible inhibitors of rhodesain and hCatL. A series of ligands
were designed and synthesised with varying aromatic cores to
investigate the influence of the electronic properties of the nitrile
on inhibition and cytotoxicity. DFT calculations of the nitrile
electrophilicities were used to predict the affinity of the ligands
against cysteine proteases and also their cytotoxicity. The
method proved to be powerful in its simplicity, allowing the
direct comparison of electrophilicities of related nitrile-bearing
compounds. This nitrile scan was validated by a clear correlation
of the activation of the head group to the inhibitory constants
and cytotoxicity. Furthermore, the electrophilicity scale enabled
us to predict biological affinities according to the electronic
nature of the nitrile and will guide and simplify future optimis-
ations of cysteine protease inhibitors. The further development
towards a safe drug for HAT treatment in our laboratory will take
into account these findings and rely on a multidimensional
approach, optimising molecular recognition properties of the
ligands to gain additional affinity from binding to the S2 and S3
sub-pockets and reducing in parallel the electrophilicity of the
nitrile head group.
Cell proliferation inhibition was assessed for compounds 1–12
using rat skeletal myoblast (L-6) cells in order to study the direct
influence of the electrophilicity on cytotoxicity. The aryl nitrile
ligands showed in general low cytotoxicity with IC50 values
between 5.6 to 43.0 μM and revealed an excellent correlation to
the calculated electrophilicities (Table 1). Aryl nitriles with high
electrophilicity such as triazines 2 and 3 and pyrimidine 7
showed the lowest IC50 values against L-6 cells (2: IC50
=
Acknowledgements
5.6 μM, 3: IC50 = 6.7 μM, 7: IC50 = 6.0 μM). Thiazole 11 and
oxazole 12, featuring a much less reactive nitrile group, exhib-
ited a 5-fold lower cytotoxicity with IC50 values of 31.5 and
43.0 μM, respectively. These data clearly indicate that reduced
electrophilicity leads to reduced cytotoxicity, which is in agree-
ment with the correlation found for the inhibition constants
against rhodesain and hCatL. The electrophilicity scale can
V.E. is grateful for a Kekulé fellowship of the German Fonds der
Chemischen Industrie and P.R.-F. for a doctoral fellowship of the
Stipendienfonds der Schweizerischen Chemischen Industrie
(SSCI). Financial support by the Deutsche Forschungs-
gemeinschaft (SFB 630, TP A4, for T.S.) and the Swiss National
Science Foundation is gratefully acknowledged. We thank
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 5764–5768 | 5767

View Article Online
11 (a) V. Ehmke, C. Heindl, M. Rottmann, C. Freymond, W. B. Schweizer,
R. Brun, A. Stich, T. Schirmeister and F. Diederich, ChemMedChem,
2011, 6, 273–278; (b) V. Ehmke, F. Kilchmann, C. Heindl, K. Cui,
J. Huang, T. Schirmeister and F. Diederich, Med. Chem. Commun., 2011,
2, 800–804.
Dr Daniel Bur (Actelion Pharmaceuticals Ltd., Basel) for
helpful discussions.
12 I. D. Kerr, J. H. Lee, C. J. Farady, R. Marion, M. Rickert, M. Sajid, K.
C. Pandey, C. R. Caffrey, J. Legac, E. Hansell, J. H. McKerrow,
C. S. Craik, P. J. Rosenthal and L. S. Brinen, J. Biol. Chem., 2009, 284,
25697–25703.
13 P. R. Gerber and K. Müller, J. Comput.-Aided Mol. Des., 1995, 9, 251–
268.
14 R. M. Oballa, J.-F. Truchon, C. I. Bayly, N. Chauret, S. Day, S. Crane
and C. Berthelette, Bioorg. Med. Chem. Lett., 2007, 17, 998–1002.
15 For recent examples on experimentally determined electrophilic reactivity
scales, as opposed to the electrophilicity scale described herein, see:
(a) R. Appel and H. Mayr, J. Am. Chem. Soc., 2011, 133, 8240–8251;
(b) M. Horn and H. Mayr, Eur. J. Org. Chem., 2011, 6470–6475.
16 M. A. McClinton and D. A. McClinton, Tetrahedron, 1992, 48, 6555–
6666.
References
1 (a) D. Leung, G. Abbenante and D. P. Fairlie, J. Med. Chem., 2000, 43,
305–341; (b) B. Turk, Nat. Rev. Drug Discovery, 2006, 5, 785–799.
2 (a) F. Lecaille, J. Kaleta and D. Brömme, Chem. Rev., 2002, 102, 4459–
4488; (b) H.-H. Otto and T. Schirmeister, Chem. Rev., 1997, 97, 133–
171; (c) M. Drag and G. S. Salvesen, Nat. Rev. Drug Discovery, 2010, 9,
690–701.
3 R. Vicik, M. Busemann, K. Baumann and T. Schirmeister, Curr. Top.
Med. Chem., 2006, 6, 331–353.
4 E. Dufour, A. C. Storer and R. Ménard, Biochemistry, 1995, 34, 9136–
9143.
5 F. F. Fleming, L. Yao, P. C. Ravikumar, L. Funk and B. C. Shook, J.
Med. Chem., 2010, 53, 7902–7917.
6 M. Klemba and D. E. Goldberg, Annu. Rev. Biochem., 2002, 71, 275–
305.
7 R. Brun, J. Blum, F. Chappuis and C. Burri, Lancet, 2010, 375, 148–159.
8 J. H. McKerrow, E. Sun, P. J. Rosenthal and J. Bouvier, Annu. Rev.
Microbiol., 1993, 47, 821–853.
17 J. M. Coterón, D. Catterick, J. Castro, M. J. Chaparro, B. Díaz,
E. Fernández, S. Ferrer, F. J. Gamo, M. Gordo, J. Gut, L. de las Heras,
J. Legac, M. Marco, J. Miguel, V. Muñoz, E. Porras, J. C. de la Rosa,
J. R. Ruiz, E. Sandoval, P. Ventosa, P. J. Rosenthal and J. M. Fiandor, J.
Med. Chem., 2010, 53, 6129–6152.
9 I. D. Kerr, P. Wu, R. Marion-Tsukamaki, Z. B. Mackey and L. S. Brinen,
PLoS Negl. Trop. Dis., 2010, 4, e701.
10 C. R. Caffrey, S. Scory and D. Steverding, Curr. Drug Targets, 2000, 1,
155–162.
18 F. Jin and P. N. Confalone, Tetrahedron Lett., 2000, 41, 3271–3273.
19 S. Khapli, S. Dey and D. Mal, J. Indian Inst. Sci., 2001, 81, 461–476.
20 M. J. Frisch, et al., GAUSSIAN 09 (Revision A.02), Gaussian, Inc.,
Wallingford CT, 2009 (for the full reference, see ESI§).
5768 | Org. Biomol. Chem., 2012, 10, 5764–5768
This journal is © The Royal Society of Chemistry 2012