A convergent, scalable and stereoselective synthesis of azole CYP51 inhibitors

Article abstract of DOI:10.1016/j.tetlet.2017.09.070

The study and development of azole-based CYP51 inhibitors is an active area of research across disciplines of biochemistry, pharmacology and infectious disease. Support of in vitro and in vivo studies require the development of robust asymmetric routes to single enantiomer products of this class of compounds. Herein, we describe a scalable and enantioselective synthesis to VNI and VFV, the two potent inhibitors of protozoan sterol 14α-demethylase (CYP51) that are currently under consideration for clinical trials for Chagas disease. A key transformation is the Jacobsen Hydrolytic Kinetic Resolution (HKR) reaction. The utility of the synthetic route is illustrated by the preparation of >25 g quantities of single enantiomers of VNI and VFV.

Full text of DOI:10.1016/j.tetlet.2017.09.070

Accepted Manuscript
A convergent, scalable and stereoselective synthesis of azole CYP51 inhibitors
Galina Lepesheva, Plamen Christov, Gary A. Sulikowski, Kwangho Kim
PII:
S0040-4039(17)31206-6
https://doi.org/10.1016/j.tetlet.2017.09.070
TETL 49340
DOI:
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 September 2017
20 September 2017
24 September 2017
Please cite this article as: Lepesheva, G., Christov, P., Sulikowski, G.A., Kim, K., A convergent, scalable and
stereoselective synthesis of azole CYP51 inhibitors, Tetrahedron Letters (2017), doi: https://doi.org/10.1016/j.tetlet.
2017.09.070
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Tetrahedron Letters
journal homepage: www.els evier.com
A convergent, scalable and stereoselective synthesis of azole CYP51 inhibitors
Galina Lepesheva, Plamen Christov, Gary A. Sulikowski and Kwangho Kimꢀ
Departments of Chemistry and Biochemistry, Institute of Chemical Biology, Vanderbilt University, Nashville, TN 77842–3012, U.S.A
ARTICLE INFO
ABSTRACT
Article history:
Received
The study and development of azole-based CYP51 inhibitors is an active area of research across
disciplines of biochemistry, pharmacology and infectious disease. Support of in vitro and in vivo
studies require the development of robust asymmetric routes to single enantiomer products of
this class of compounds. Herein, we describe a scalable and enantioselective synthesis to VNI
and VFV, the two potent inhibitors of protozoan sterol 14α-demethylase (CYP51) that are
currently under consideration for clinical trials for Chagas disease. A key transformation is the
Jacobsen Hydrolytic Kinetic Resolution (HKR) reaction. The utility of the synthetic route is
illustrated by the preparation of >25 gram quantities of single enantiomers of VNI and VFV.
Received in revised form
Accepted
Available online
Keywords:
Kinetic Resolution
Stereospecific
Enzyme inhibitor
Azole
2009 Elsevier Ltd. All rights reserved.
The cytochrome P450 sterol 14α-demethylase (CYP51) is a
conserved metabolic enzyme required for sterol biosynthesis in
eukaryotes, validated target for clinical and agricultural
antifungals, and a target in trials for human infections with
protozoan parasites.1 Inhibition of CYP51 is effective for the
treatment of these infections as the product of the pathway
ergosterol is an essential component of membrane assembly in
these pathogens.2 Recently, CYP51 inhibition has attracted
interest as a potential anticancer target as rapidly proliferating
tumor cells have a high demand for cholesterol to support their
membrane assembly.3 The design, synthesis and evaluation of
small molecule CYP51 inhibitors is an active area of research.4
Lepesheva and co-workers have pursued the development of
several azole-based CYP51 inhibitors including VNI and VFV
(Figure 1) using both biochemical5 and in vivo models.6 This
work benefits from a series of crystal structures of sterol 14α-
demethylase (CYP51) co-crystalized with VNI and its analogs5
which resulted in the structure-based design of VFV, a compound
with the broader antiprotozoan spectrum of activity. Furthermore,
co-crystal structures provided an explanation of the significant
difference in potency of enantiomeric forms of the chiral scaffold
(cf. Figure 1).5c In addition to compound potency, drug
metabolism and pharmacokinetic properties (DMPK) have been
evaluated using animal models and were found to be very
favorable.6b
In order to provide support for Structure-Activity Relationship
(SAR) studies
a convergent synthesis enabling structural
modification at three key regions of the VNI scaffold was
required; the azole iron binding heterocycle, the hydrophobic
aromatic residue and the polycyclic ring system occupying the
enzyme substrate entrance tunnel. In addition, selective access to
the more potent (R)-configured isomer was needed. Finally, in
vivo animal studies required VNI and VFV to be available in
multi-gram quantities and the material to be of >98% purity.
In earlier work we developed a racemic and scalable synthesis
of VNI readily allowing variation at the azole and the
hydrophobic aromatic residue (Scheme 1).5b In general, azole (2)
opening of aromatic epoxide 1 provides secondary alcohol 3. The
latter is converted to the corresponding amine by way of simple
three-step reaction sequence. Primary amine 4 is then condensed
with carboxylic acid 5 using a HATU coupling. As this five-step
synthetic route allows variation of all three regions of the
Figure 1. Structures of CYP51 inhibitors VNI and VFV.
———
ꢀ
Corresponding author.
E-mail address: kwangho.kim@vanderbilt.edu

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scaffold [azole, hydrophobic (aryl) and polycylic (carboxylic
acid)] we favored its modification to an enantioselective
synthetic process. We also required a process that would scale up
in order to enable access to multi-gram quantities of the azole
products for in vivo animal studies. To this end, we required
epoxide 1 as a single enantiomer and a stereospecific (i.e. no loss
of ee) interconversion of the secondary alchol to the
corresponding primary amine (3 to 4).
concentration (minimal solvent waste), uses low catalyst
loading (5 mol%), has broad substrate scope and readily proceeds
on large reaction scales (>25 g). In this way application of the
HKR reaction to epoxide (+)-7 proceeded in 43% yield on a 30
gram scale to provide (R)-7 in >98% enantiomeric excess. The
epoxide product was reacted with imidazole under basic
conditions to afford azole alcohol 8, the product of epoxide
opening at the less hindered primary carbon. Stereospecific
conversion of alcohol 8 to amine 10 was achieved using a two
step process. First, reaction of 8 with diphenylphosphoryl azide11
provided azide 9 followed by its reduction with lithiumaluminum
hydride to give amine 10 (>98% ee). In the racemic synthesis
series the conversion of alcohol 8 to azide 9 proceeded by way of
an intermediate mesylate (cf. 3 to 4, Scheme 1), however this
transformation occured with complete racemization when
conducted with optically active alcohol (8, Scheme 2). The
synthesis of (+)-VNI was completed using previously described
HATU coupling of amine 5 and carboxylic acid 5.5b Using this
route we prepared over 20 grams of (+)-VNI of >95% purity and
enantiomeric excess.
Scheme 1. Racemic route to VNI and derivatives.5b
Scheme 3. Synthesis of (+)-VFV.
Scheme 2. Multi-gram synthesis of (+)-VNI.
The synthesis of (+)-VFV (Scheme 3) illustrates the flexibility
of our enantioselective and enabled the preparation of greater
than 20 grams of (+)-VFV (Scheme 3). The reaction sequence
started with a Suzuki coupling of aryl bromide 11 and boronic
acid 12. Aldehyde 13 was methylenated using the Corey-
Chaykovsky reaction to give racemic epoxide 14. The latter was
resolved on 28 gram scale using the Jacobsen HKR to give (R)-
Single enantiomer azole agents such as VNI have been
accessed by asymmetric reductions7, (aza) Henry reactions8, and
Lipase mediated kinetic resolutions.9 We found the use of the
Jacobsen hydrolytic kinetic resolution (HKR)10 of epoxide 7 to be
most convenient as the reaction is conducted at high

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14 in >98% ee. Epoxide (R)-14 was reacted with imidazole and
the product, alcohol 15, converted to amine 17 by way of azide
16 without loss of optical acvtivity. Installation of the oxadiazole
proceeded using established HATU coupling conditions. Using
this synthetic route 28 grams of optically pure (+)-VFV was
prepared.
Supplementary Material
Supplementary data associated with this article can be found
in the online version, at do:XXXXX.
In conclusion, we have developed a robust and reliable
synthetic route to azole antimicrobial agents as illustrated by
multi-gram synthesis of (+)-VNI and (+)-VFV. The purity
(>98%) of these products was verified by LC-MS analysis using
multiple wavelengths and 1H and 13C NMR analysis of high
concentration analytyical solutions. The synthetic products were
employed in animal studies by the Lepesheva group6 and
demonstrated their effectiveness as antiprotozoan agents and are
under consideration for clinical trials for Chagas disease.
Acknowledgments This research was supported by the
National Institutes of General Health (GM 067871) and the
Vanderbilt Institute of Chemical Biology.
References and notes
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Highlights
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Jacobsen Hydrolytic Kinetic Resolution
Stereospecific
Enzyme inhibitor
Azole heterocycle

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A convergent, scalable and stereoselective synthesis of azole
CYP51 inhibitors
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Galina Lepesheva, Plamen Christov, Gary A. Sulikowski and
Kwangho Kim*