Efficient synthesis of nevirapine analogs to study its metabolic profile by click fishing

Article abstract of DOI:10.1016/j.bmcl.2009.09.011

Knowledge of the biotransformation and pharmacokinetics of the antiretroviral agent nevirapine is still insufficient. In order to trace rash inducing metabolites of nevirapine, we devised a short and efficient multi-gram synthesis of a nevirapine analog that can be coupled to azide containing compounds by click chemistry.

Full text of DOI:10.1016/j.bmcl.2009.09.011

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6127–6130
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Efficient synthesis of nevirapine analogs to study its metabolic profile
by click fishing
b,
Sylvain Bernard a, Daniel Defoy b, Yves L. Dory a, , Klaus Klarskov
*
*
a Département de Chimie, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, 3001 12e Avenue nord, Sherbrooke, Québec, Canada J1H 5N4
b Département de Pharmacologie, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, 3001 12e Avenue nord, Sherbrooke, Québec, Canada J1H 5N4
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 July 2009
Revised 2 September 2009
Accepted 4 September 2009
Available online 9 September 2009
Knowledge of the biotransformation and pharmacokinetics of the antiretroviral agent nevirapine is still
insufficient. In order to trace rash inducing metabolites of nevirapine, we devised a short and efficient
multi-gram synthesis of a nevirapine analog that can be coupled to azide containing compounds by click
chemistry.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Nevirapine
Synthesis
5-Exo-dig cyclization
Click coupling
Nevirapine (NVP) is a non-nucleoside reverse transcriptase
inhibitor (Fig. 1) currently used in the treatment of HIV.1 It is
known that NVP causes cutaneous adverse reactions with an
incidence of 16% with occasional serious side effects such as
Stevens–Johnson syndrome (SJS) or SJS/toxic epidermal necrolysis
transition syndrome occurring with a probability of 0.3%.2 In rat
and human, NVP is metabolized into several metabolites of which
12-hydroxynevirapine has been proposed to be converted into a
quinone methide reactive metabolite.3
Two ways were originally envisaged to synthesize 1 and other
analogs substituted at position 11 (Fig. 3).6 Either the 2,20-dihaloa-
mide could react (addition then cyclization) with the suitable
amine or the diazepine-like core could be alkylated at position
N11.
Propargyl alcohol 2 was transformed quantitatively into its TMS
derivative 37 by means of n-BuLi and TMSCl (Scheme 1). Replace-
ment of the alcohol by a bromide yielded 4 (78%),8 whose bromide
In order to determine which reactive metabolites could be in-
volved in adverse reactions and to identify which macromolecules
are targeted by reactive metabolites, we have synthesized a NVP
derivative that can be used to trace nevirapine metabolites in bio-
logical systems. With a tethering application in mind, a NVP analog
containing an anchorage site was designed. In a previous study it
was demonstrated that Et-NVP (Fig. 1), a NVP analog in which
the cyclopropyl was substituted by an ethyl group did not affect
the ability of the drug to cause cutaneous rash similar to NVP.4
For this reason, we expect that the alkyne NVP analog 1 (Fig. 1) will
behave like its isosteric Et-NVP and thus retain the in vivo adverse
side effect. Moreover, the terminal alkyne group is an appropriate
site for reaction with azide conjugated probes using click chemis-
try adapted to biological media (Fig. 2).5 This article describes the
synthesis of the nevirapine propargyl analog 1 and demonstrates
its reactivity towards azides.
Figure 1. Structures of Nevirapine, Et-NVP and 1.
* Corresponding authors. Tel.: +1 819 564 5299 (Y.L.D.).
E-mail address: Yves.Dory@USherbrooke.ca (Y.L. Dory).
Figure 2. Click fishing principle of metabolites of 1.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.011

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S. Bernard et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6127–6130
ative 17 (45%). This alkylation of 15 at the wrong position was con-
firmed by theoretical calculations (GAMESS).13 The N5 anion is
more stable than the N11 anion by as much as 9.1 kcal molꢀ1
(B3LYP/6-31+Gd).14
Without doubts 15 would never yield the desired compound 1,
unless its dianion was the reactive nucleophile. Since such direct
transformation had been achieved in very low yields on related
systems, it was abandoned.6a It became obvious that the N5 site
had to be protected to alkylate selectively at the desired N11 posi-
tion. The Boc protective group was introduced on amide 13 (88%).
The PMB protective group located at position 11 could be removed
by means of DDQ,15 but the yield was moderate (47%) and the reac-
tion was excessively time consuming (2 days without even reach-
ing completion). Owing to our desire to establish a multi-gram
scale synthesis for target 1, this latter step was much too low-
yielding to be really useful. Therefore, we looked for an alternative
N11 protective group that would be easily cleaved.
Figure 3. Retrosynthetic routes to 1 and 11-substituted analogs.
The 3,4-dimethoxy benzyl amine 20 (DMB-NH2) was intro-
duced in much the same way as PMB-NH2 11 was coupled to 9
(Scheme 3). The substituted aniline 21 was obtained with an excel-
lent yield (85%) and was further cyclized by means of NaHMDS in
THF to yield the seven-membered ring 22 (90%) without traces of
its five-membered ring competitor. Compound 19 was then pre-
pared from 22 following the same two-step sequence as that used
to make 19 from 13. Cleavage of the DMB protective group occured
with ease with DDQ.11 Compound 19 was coupled with 16 using
K2CO3 as a base (92%) to produce propargylamine 24.16 Carbamate
cleavage with aqueous 6 N HCl, immediately followed by NaHCO3
provided target 1 neatly (98%).
We had recourse to these atypical Boc cleavage conditions be-
cause the usual procedure (TFA in CH2Cl2) did not produce the ex-
pected product 1 from 24. Instead, a 85:15 mixture of two very
polar and unseparable yellow compounds 25 and 26 (Scheme 4).
Compound 25 slowly crystallized from the oily residue obtained
after removal of solvent (Fig. 4).
Upon addition of TFA in CH2Cl2, a medium of low dielectric con-
stant, the two pyridine nitrogen atoms of 24 get hydrogen bonded
to TFA.17 Consequently, both nitrogen atoms remain sufficiently
nucleophilic; they can attack the neighboring alkyne group in a
5-exo-dig fashion18 with concomitant proton delivery. The reaction
is very quick as observed by the yellow coloration that starts
appearing almost immediately after addition of TFA. It is likely that
Boc cleavage remains faster since the outcome of the reaction is the
same whenever starting from 1 or 24. In all cases N10 undergoes
cyclization preferentially over N1. This behavioral difference can
be explained in terms of residual nucleophilicity of these nitrogen
Scheme 1. Tentative synthesis of 10.
atom was SN2 displaced with potassium phtalamide to give 5
(90%).9 Hydrazine treatment of 5 gave the ammonium salt 6 quan-
titatively. Acid 7 was activated as its acid chloride and reacted with
aniline 8 to give amide 9 (88%).5a,10 All attempts to prepare 10, by
addition of 6 to 9 remained fruitless.
Since route A (Fig. 3) via a SNAr reaction—Chichibabin cycliza-
tion11 sequence had failed, route B remained the only alternative.
PMB-NH2 11 added swiftly to 9 to yield 12 (49%) whose cyclization
produced 13 (88%) in THF and with NaHMDS as a base (Scheme 2).
When the reaction was run with NaH in diglyme instead of
NaHMDS, a tricyclic compound 14 resulted, although NaH in dig-
lyme was a known procedure to avoid this unwanted cyclization.12
Tricycle 15 was prepared from 13 by simple TFA treatment (67%) to
check if it was possible to find selective N11 alkylation conditions.
However, only N5 alkylation occurred when the anion of 15 was
left to react with propargyl bromide 16 to give the propargyl deriv-
Scheme 2. First synthesis of 19.
Scheme 3. Final synthesis of 1.

S. Bernard et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6127–6130
6129
Scheme 5. Click reaction between 1 and 29.
warrant that 1 can be used safely since no strongly acidic anhy-
drous conditions comparable to TFA in CH2Cl2 exist physiologically
and 1 is stable towards vigorous aqueous acids as can be found in
the digestive tract.
On the whole, the seven-step DMP route (7–9–21–22–23–19–
24–1) is very efficient (49% overall yield). Moreover, only two
products, 23 and 24, must be purified by chromatography along
the way; all other compounds are either triturated or simply fil-
tered or washed. This very convenient synthesis is also versatile,
alkylating agent other than 16 may be used.
Finally, we wanted to test the post-metabolism chemical step,
the click reaction.19 Compound 1 will likely experience several
metabolic transformations; all those that modify the alkyne moiety
will remain unreactive towards azides. However, this is of little
importance because substituents at position N11 are known not
to be responsible for the rash side effect provoked by NVP.4,6 On
the other hand, all metabolites whose N11 propargyl group is still
present will be trapped by click fishing (Fig. 2). Azide 2920 reacted
extremely well with alkyne 1 (97%) in water and MeCN to give the
corresponding triazole 30 (Scheme 5). This result insures that all
alkyne metabolites will be collected from plasma extracts.19c
In summary, we have designed a nevirapine analog 1 in which
the cyclopropyl group is replaced with an alkyne anchor for further
click harvesting of metabolites. This compound was synthesized
very efficiently following a seven-step sequence suitable for large
scale production. We believe that compound 1 will be of value
for identification and deducing the behavior of nevirapine-like
metabolites.21 We are currently evaluating its pharmacokinetics
and biotransformation profile.
Scheme 4. 5-exo-dig cyclization of 1 and 24 under anhydrous acidic conditions.
Strong and weak hydrogen bonds are shown as short and long dashed bonds,
respectively.
Acknowledgments
Figure 4. Crystal structure of 25 (ORTEP 33% probability level).
We thank NSERC Canada for financial support.
Supplementary data
atoms after their partial protonation (Scheme 4). In compounds 1
and 24, N1 hydrogen binds more tightly to TFA since it benefits
from methyl-12 and N5 electron donation, contrarily to N10 whose
electrons are pulled out by the amide carbonyl. This is in excellent
agreement with calculations (B3LYP/6-31Gd)13 which show that
protonation at positions N1, N10 and N11 yield ammonium cations
with relative energies of 0, 3.8 and 12.2 kcal molꢀ1, respectively.
Therefore, partially protonated N10 remains the most nucleophilic
center and yields the preferred adduct 25 as expected. In order to
prove this mechanism, 1 was treated with CF3CO2D in CDCl3. This
experiment led to the formation of two deuterated compounds 27
and 28 (85:15) equivalent to 25 and 26. The position of the D atom
on each of these molecules indicates that its delivery was anti to
pyridine nitrogen attack. The most likely explanation for such a re-
sult is that the D atom originates from a CF3CO2D molecule initially
not hydrogen bound to the attacking pyridine nitrogen atom.
The logical way to bypass the unwanted cyclization reaction is
to carry out Boc cleavage with an easily ionized acid, in a high
dielectric constant solvent, to provoke clean protonation of both
pyridine nitrogen atoms17 and prevent them from acting as nucle-
ophiles. Aqueous 6N HCl proved very efficient for this purpose
since 1 was the only isolated product. Moreover, these results
Supplementary data (experimental procedures, spectral char-
acterizations, calculation and crystallographic details. CCDC
742668 contains the supplementary crystallographic data for
compound 25) associated with this article can be found, in the
online version, at doi:10.1016/j.bmcl.2009.09.011.
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