Organocatalytic, enantioselective synthesis of VNI: A robust therapeutic development platform for Chagas, a neglected tropical disease

Article abstract of DOI:10.1021/ol303092v

VNI is a potent inhibitor of CYP51 and was recently shown to achieve a parasitological cure of mice infected with T. cruzi in both acute and chronic stages of infection. T. cruzi is the causative parasite of Chagas disease, a neglected tropical disease. The first enantioselective chemical synthesis of VNI (at a materials cost of less than $0.10/mg) is described. Furthermore, the key enantioselective step is performed at the 10 g scale.

Full text of DOI:10.1021/ol303092v

ORGANIC
LETTERS
2012
Vol. 14, No. 24
6322–6325
Organocatalytic, Enantioselective
Synthesis of VNI: A Robust Therapeutic
Development Platform for Chagas, a
Neglected Tropical Disease
Mark C. Dobish,† Fernando Villalta,‡ Michael R. Waterman,§ Galina I. Lepesheva,§ and
Jeffrey N. Johnston*,†
Department of Chemistry & Vanderbilt Institute of Chemical Biology,
Vanderbilt University, Nashville, Tennessee 37235, United States,
Department of Microbiology and Immunology, Meharry Medical College, Nashville,
Tennessee 37208, United States, and Department of Biochemistry, School of Medicine,
Vanderbilt University, Nashville, Tennessee 37232, United States
jeffrey.n.johnston@vanderbilt.edu
Received November 9, 2012
ABSTRACT
VNI is a potent inhibitor of CYP51 and was recently shown to achieve a parasitological cure of mice infected with T. cruzi in both acute and chronic
stages of infection. T. cruzi is the causative parasite of Chagas disease, a neglected tropical disease. The first enantioselective chemical synthesis
of VNI (at a materials cost of less than $0.10/mg) is described. Furthermore, the key enantioselective step is performed at the 10 g scale.
Chagas disease affects more than 8 million people
throughout the Americas,1 and recent evidence suggests
that it is proliferating beyond these traditional geographi-
cal borders, tracking with the lengthening reach of disease
vectors that favor warm climate.2 The causative parasite
of ChagasꢀT. cruziꢀinfects humans in two stages. The
acute stage is treated with benznidazole or nifurtimox,
which have been associated with low efficacy and general
toxicity. There are no established treatments for the
chronic form of infection, but posaconazole is currently
considered the most promising therapeutic and is in phase
II clinical trials. Unfortunately, it is widely regarded as
unaffordable with a cost estimated at over $1000 per
patient. The cost of treatment is an immediate consideration
for drug development since Chagas is endemic to popula-
tions in low resource areas,3 and mere repurposing efforts
for expensive antifungal drugs such as posaconazole are
likely to have little impact. There is insufficient incentive
for industrial drug development, resulting in a neglected
disease status for Chagas.4ꢀ7
Posaconazole was brought to market as an antifungal
therapeutic8 and has been shown to inhibit trypanosomal
sterol 14R-demethylase (CYP51)9ꢀ12 similarly to other
† Department of Chemistry & Vanderbilt Institute of Chemical Biology,
Vanderbilt University.
‡ Department of Microbiology and Immunology, Meharry Medical
College.
(3) Urbina, J. A.; Docampo, R. Trends Parasitol. 2003, 19, 495–501.
Buckner, F. S.; Navabi, N. Curr. Opin. Infect. Dis. 2010, 23, 609–616.
(4) Clayton, J. Nature 2010, 465, S12–S15.
§ Department of Biochemistry, School of Medicine, Vanderbilt University.
(1) Clayton, J. Nature 2010, 465, S4–S5.
(2) Coura, J. R.; Vinas, P. A. Nature 2010, 465, S6–S7.
(5) Apt, W. Drug Des. Devel. Ther. 2010, 4, 243–253.
r
10.1021/ol303092v
Published on Web 12/07/2012
2012 American Chemical Society

small molecules,13 thereby disrupting membrane forma-
tion in T. cruzi. CYP51 has emerged as an effective drug
target since all eukaryotes rely on endogenous sterol
production for membrane biogenesis, and this target has
been further validated in a murine model of acute Chagas
infection.14 VNI15 was recently discovered to inhibit
sterol synthesis in T. cruzi,13 as well as bind to CYP51 in
a manner analogous to posaconazole.11 A contributing
factor to the high cost for posaconazole is its long and
relatively inefficient synthesis;16 however, no enantioselec-
tive synthesis of VNI has been reported. Further develop-
ment of VNI, the more potent enantiomer, and its promise
as a therapeutic of reasonable cost, rests in part upon
the availability of a short, selective, and high yielding
synthesis.17 Herein we report the first enantioselective
chemical synthesis of VNI. This preparation provided
the gram-scale quantities of VNI to establish its efficacy
against T. cruzi in a mouse model of infection, including
evidence for its effectiveness against the chronic infec-
tion.18 The preliminary studies enabled by this material
also suggest that VNI toxicity is low.
Figure 1. Retrosynthetic approach to VNI to maximize conver-
gency and modularity.
The initial quantity of VNI was obtained from a non-
renewable source and without preparative details.13 And
since no preparation of VNI existed in the literature, a
synthesis selective for the more potent enantiomer was
needed. Key structural features of VNI include a chiral
benzylic amine carbon, an amide substituent that projects
a substituted biphenyl into the substrate access channel of
CYP51, and an imidazole that interacts directly with the
iron-heme of CYP51.12 Figure 1 outlines a modular design
for the synthesis route, invoking an enantioselective aza-
Henry reaction to construct the styrenyl diamine backbone
of VNI in the key step. The high degree of convergency
would be reflected in a short longest linear sequence
(LLS).19
(6) Therapeutic development for T. brucei (human African
trypanosomiasis) faces similar challenges: Smithson, D. C.; Lee, J.;
Shelat, A. A.; Phillips, M. A.; Guy, R. K. J. Biol. Chem. 2010, 285,
16771–16781.
(7) Development of serine protease inhibitors targeting cruzain
remains a promising therapeutic development approach: Chen, Y. T.;
Brinen, L. S.; Kerr, I. D.; Hansell, E.; Doyle, P. S.; McKerrow, J. H.;
Roush, W. R. PLoS Negl. Trop. Dis. 2010, 4, e825. Brak, K.; Kerr, I. D.;
Barrett, K. T.; Fuchi, N.; Debnath, M.; Ang, K.; Engel, J. C.; McKerrow,
J. H.;Doyle, P. S.;Brinen, L. S.;Ellman, J. A.J. Med. Chem. 2010, 53, 1763.
(8) Molina, J.; Martins-Filho, O.; Brener, Z.; Romanha, A. J.;
Loebenberg, D.; Urbina, J. A. Antimicrob. Agents Chemother. 2000,
44, 150–155.
Scheme 1. Competitive Double Addition of Imine to
Nitromethane
(9) Lepesheva, G.; Hargrove, T.; Kleshchenko, Y.; Nes, W.; Villalta,
F.; Waterman, M. Lipids 2008, 43, 1117–1125.
(10) Lepesheva, G. I.; Waterman, M. R. Mol. Cell. Endocrinol. 2004,
215, 165–170.
(11) Lepesheva, G. I.; Hargrove, T. Y.; Anderson, S.; Kleshchenko,
Y.; Futak, V.; Wawrzak, Z.; Villalta, F.; Waterman, M. R. J. Biol.
Chem. 2010, 285, 25582–25590.
(12) Lepesheva, G. I.; Park, H.-W.; Hargrove, T. Y.; Vanhollebeke,
B.; Wawrzak, Z.; Harp, J. M.; Sundaramoorthy, M.; Nes, W. D.; Pays,
E.; Chaudhuri, M.; Villalta, F.; Waterman, M. R. J. Biol. Chem. 2009,
285, 1773–1780.
(13) Lepesheva, G. I.; Ott, R. D.; Hargrove, T. Y.; Kleshchenko,
Y. Y.; Schuster, I.; Nes, W. D.; Hill, G. C.; Villalta, F.; Waterman, M. R.
Chem. Biol. 2007, 14, 1283–1293.
(14) Buckner, F. S.; Urbina, J. A. Int. J. Parasitol. Drugs Drug Resist.
2012, 2, 236ꢀ242. Doyle, P. S.; Chen, C.-K.; Johnston, J. B.; Hopkins,
S. D.; Leung, S. S. F.; Jacobson, M. P.; Engel, J. C.; McKerrow, J. H.;
Podust, L. M. Antimicrob. Agents Chemother. 2010, 54, 2480. Kraus,
J. M.; Tatipaka, H. B.; McGuffin, S. A.; Chennamaneni, N. K.; Karimi,
M.; Arif, J.; Verlinde, C. L. M. J.; Buckner, F. S.; Gelb, M. H. J. Med.
Chem. 2010, 53, 3887.
(15) The abbreviation ‘VNI’ refers specifically to the (R)-enantiomer,
the more potent inhibitor of CYP51. An X-ray crystal structure of VNI
bound to CYP51 has been reported (ref 11). VNI is the dextrorotatory
(þ) enantiomer.
N-Boc imine 3 was prepared using a standard proce-
dure from 2,4-dichlorobenzaldehyde.20 Feasibility for
the key stereochemistry-determining step was first evalu-
ated by analysis of the enantioselective addition of com-
mercially available nitromethane to N-Boc imine 3 using
(16) See the Supporting Information for an outline of the synthesis
reported in: Saksena, A. K.; Girijavallabhan, V. M.; Lovey, R. G.; Pike,
R. E.; Wang, H.; Liu, Y.-T.; Ganguly, A. K.; Bennett, F. Schering Corp.,
USA, 1995; p 82.
(17) A synthesis of rac-1 was recently reported: Hargrove, T. Y.;
ꢀ
Kim, K.; de Nazare Correia Soeiro, M.; da Silva, C. F.; da Gama Jaen
Batista, D.; Batista, M. M.; Yazlovitskaya, E. M.; Waterman, M. R.;
Sulikowski, G. A.; Lepesheva, G. I. Int. J. Parasitol. Drugs Drug Resist.
2012, 2, 178–186.
(18) Villalta, F.; Dobish, M. C.; Nde, P. N.; Kleshchenko, Y. Y.;
Hargrove, T. Y.; Johnson, C. A.; Waterman, M. R.; Johnston, J. N.;
Lepesheva, G. I. J. Infect. Dis. 2012, in press.
(19) Selected recent applications of organocatalysis to therapeutic
development platforms: Coulthard, G.; Erb, W.; Aggarwal, V. K.
Nature 2012, 489, 278–281. Davis, T. A.; Danneman, M. W.; Johnston,
J. N. Chem. Commun. (Cambridge, U. K.) 2012, 48, 5578–5580. Davis,
T. A.; Johnston, J. N. Chem. Sci. 2011, 2, 1076–1079.
Org. Lett., Vol. 14, No. 24, 2012
6323

Scheme 2. Multigram-Scale Enantioselective Synthesis of VNI for Preclinical Studies
bis(amidine) catalysis (Scheme 1).21,22 Using our first
generation catalyst23 to form this adduct would have
required a higher catalyst loading and longer reaction
time, as well as the use of nitromethane as solvent. To
circumvent this problem, we selected a more Brønsted
basic version from our second generation of catalysts for
the reaction.19,24 This approach delivered a disappointing
2.7:1 ratio of 4:5, the latter resulting from an unexpectedly
competitive addition of adduct 4 to a second equivalent of
imine 3. In principle, this 2:1 adduct could be minimized
through the use of excess nitromethane; however, the ratio
was improved only to the 6.5:1 level and at the cost of a
drop in enantioselection (Scheme 1). As a result, commer-
cially available bromonitromethane was investigated using
the hypothesis that the bromine substituent would slow the
second addition as a result of its size. In the event, the
desired adduct (9) was prepared in excellent yield (90%),
using only 2 equiv of bromonitromethane (Scheme 2).25
This reaction was catalyzed by (þ)-PBAM (6),26 which
delivered the addition product in 97ꢀ99% ee for each
diastereomer. Immediate treatment of the mixture with
cobalt boride formed in situ27 provided the fully reduced
product (amine 2). Although adduct 9 is a mixture of dia-
stereomers, they are homochiral at the benzylic position,
thereby producing the same enantiomer after reduction to
diamine 2. Formation of the imidazole was achieved by
standard condensation of amine 2 with glyoxal, formalin,
and ammonium acetate.28 Deprotection and subsequent
acylation of the amine with carboxylic acid 1229 provided
milligram quantities of the desired dextrorotatory product.
Our first generation approach relied on chromatogra-
phy to purify adduct 9, in addition to chromatographic
purification in three subsequent steps. However, we first
optimized the key enantioselective step, with respect to
both catalyst loading and scale. Although decreasing the
catalyst loading was favorable for enantioselection (Table 1,
entries 1ꢀ3), a diminished yield was observed as the scale
increased. This was partly due to issues associated with
column chromatography on a large scale. Fortunately, the
catalyst could be removed with straightforward filtration,
delivering over 6 g of pure product (Table 1, entry 4). An
almost 500-fold increase in scale from initial experiments
(from 0.1 mmol scale) returned favorable results (Table 1,
entry 5): filtration provided 19.2 g (nearly quantitative
yield) of analytically and enantiomerically pure product
using 1 mol % PBAM. The use of this highly reactive or-
ganocatalyst (>100 turnovers) in a large scale preparation
to deliver the scaffold is punctuated by the ability to recover
and recycle the catalyst without loss of activity.
(20) Petrini, M. Chem. Rev. 2005, 105, 3949–3977. Petrini, M.;
Torregiani, E. Tetrahedron Lett. 2006, 47, 3501–3503. Marianacci, O.;
Micheletti, G.; Bernardi, L.; Fini, F.; Fochi, M.; Pettersen, D.; Sgarzani,
V.; Ricci, A. Chem.;Eur. J. 2007, 13, 8338–8351. Yin, B.; Zhang, Y.;
Xu, L.-W. Synthesis 2010, 3583–3595.
(21) Shen, B.; Makley, D. M.; Johnston, J. N. Nature 2010, 465,
1027–1032. Singh, A.; Johnston, J. N. J. Am. Chem. Soc. 2008, 130,
5866–5867. Shen, B.; Johnston, J. N. Org. Lett. 2008, 10, 4397–4400.
Singh, A.; Yoder, R. A.; Shen, B.; Johnston, J. N. J. Am. Chem. Soc.
2007, 129, 3466–3467. Hess, A. S.; Yoder, R. A.; Johnston, J. N. Synlett
2006, 147–149.
(22) Selected examples of enantioselective aza-Henry reactions:
Marques-Lopez, E.; Merino, P.; Tejero, T.; Herrera, R. P. Eur. J. Org.
Chem. 2009, 2401–2420. Arrayas, R. G.; Carretero, J. C. Chem. Soc. Rev.
2009, 38, 1940–1948. Wang, C. J.; Dong, X. Q.; Zhang, Z. H.; Xue, Z. Y.;
Teng, H. L. J. Am. Chem. Soc. 2008, 130, 8606. Rueping, M.; Antonchick,
A. P. Org. Lett. 2008, 10, 1731–1734. Rampalakos, C.; Wulff, W. D. Adv.
Synth. Catal. 2008, 350, 1785–1790.
The overall synthesis route (Scheme 2) provided VNI
analogs for evaluation withsimilar brevity. Among thoseis
(23) Nugent, B. M.; Yoder, R. A.; Johnston, J. N. J. Am. Chem. Soc.
2004, 126, 3418–3419.
(24) Davis, T. A.; Wilt, J. C.; Johnston, J. N. J. Am. Chem. Soc. 2010,
132, 2880. Dobish, M. C.; Johnston, J. N. Org. Lett. 2010, 12, 5744.
(25) Shen, B.; Makley, D. M.; Johnston, J. N. Nature 2010, 465,
1027–1032.
(27) Heinzman, S. W.; Ganem, B. J. Am. Chem. Soc. 1982, 104, 6801–
6802. Satoh, T.; Suzuki, S.; Suzuki, Y.; Miyaji, Y.; Imai, Z. Tetrahedron
Lett. 1969, 4555–4558.
(28) Matsuoka, Y.; Ishida, Y.; Sasaki, D.; Saigo, K. Tetrahedron
2006, 62, 8199–8206.
(26) Davis, T. A.; Dobish, M. C.; Schwieter, K. E.; Chun, A. C.;
Johnston, J. N. Org. Synth. 2012, 89, 380–393.
(29) Rostamizadeh, S.; Ghamkhar, S. Chin. Chem. Lett. 2008, 19,
639–642.
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Org. Lett., Vol. 14, No. 24, 2012

quantities of VNI; and (3) nearly all steps benefit from
solid intermediates, resulting in either filtration or re-
crystallization for most manipulations. This synthesis
is therefore an immediate platform for VNI drug devel-
opment, including further structureꢀactivity relation-
ship studies.
Table 1. Reaction Optimization of Key Bond Forming Stepa
yieldb
entry
cat. (mol %)
scale (mmol)
(g)
(%)
eec (%)
1
10
5
1.0
0.37
0.37
1.99
6.05
19.2
90
95
96
96
97
98
2
1.0
90
3
2
5.8
83
4d
5d
1.3
1.0
14.9
47.0
98
>98
a All reactions were run at ꢀ20 °C for 2 d. b Yields are isolated yields.
c Enantiomer ratios measured by HPLC using chiral stationary phase and
are average of both diastereomers. d No column chromatography, only
filtration to remove catalyst. See Supporting Information for details.
Figure 2. VNI analog preparation: FF-VNI.
In summary, VNI is prepared through total chemical
synthesis using generally inexpensive materials. The salient
features of the synthesis, including length (LLS = 7 steps),
selectivity (>99% ee) in the key step, and nature of
the intermediates (all crystalline solids) bode well for its
potential scalability beyond its current multigram scale.
The current materials cost of less than $ 0.10/mg31 for VNI
highlights the promise of a small molecule therapeutic for a
disease endemic to low resource areas.32 Furthermore, the
convergencyofthe approach providesimmediate, straight-
forward access to derivatives to more rapidly progress
through lead optimization and into preclinical candidate
selection.
FF-VNI (13), prepared from the 2,4-difluoro-derivative of
9 (Figure 2). The halogenated aromatic ring is buried deep
in the CYP51 pocket while the amide chain is projected
into the substrate access channel. The exchange of both
chlorines with fluorines within the VNI backbone resulted
in a 4-fold improvement in binding to CYP51 (T. cruzi)
relative to VNI.17,30
The final part of this study involved the preparation of
gram quantities of VNI for evaluation in murine models of
both acute and chronic Chagas infection. Inthis campaign,
several chromatographic manipulations were targeted for
replacementwithfiltrationoractiveprecipitation. Thefirst
three steps were completed by straightforward filtrations,
and the first chromatography was applied to the borohy-
dride reduction product 2. The final three steps could also
be completed with a single chromatographic step of the
final product, aided by precipitation steps.
Acknowledgment. The work was supported by National
Institutes of Health (NIH) GM084333 (J.N.J.), Vanderbilt
Institute of Chemical Biology (VICB) for fellowship sup-
port (M.C.D.), VICB Pilot Project Grant 2011 (G.I.L.), and
in part by GM067871 (G.I.L., M.R.W.), AI080580 and U54
MD007593 (F.V.).
The route illustrated in Scheme 2 establishes that (1) the
catalyzed addition has been scaled to the 20 g level using
1 mol % of the organocatalyst PBAM,26 providing the
desired product in essentially enantiomerically pure form;
(2) the overall process can be used to prepare multigram
Supporting Information Available. Procedures (gram-
scale) and analytical data for all new compounds, cost of
materials analysis to produce VNI, and comparative
analysis of posaconazole and VNI synthesis schemes.
This material is available free of charge via the Internet
at http://pubs.acs.org.
(30) Relative efficacy of CYP51 ligands is estimated through the use
of several measurements in addition to Kd; see refs 12, 13, and 17. For
this reason, VNI remains the best current lead.
(31) Unfortunately, the materials cost to produce posaconazole is
unavailable, thereby preventing a direct comparison at this stage.
However, the low materials cost for VNI at the gram scale bodes well
for the development of an inexpensive small molecule.
(32) Zhu, C.; Cook, S. P. J. Am. Chem. Soc. 2012, 134, 13577–13579.
The authors declare no competing financial interest.
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