C. Yoakim et al. / Bioorg. Med. Chem. Lett. 14 (2004) 739–742
741
Table 2. SAR at position 2 and 11
EC50 against wild type HIV. Following intravenous
administration (1 mg/kg), inhibitor 6 exhibited high
clearance (57.0Æ0.9 mL/min/kg), a volume of dis-
tribution at steady state (V ) of 4.68Æ0.16 L/kg, and
SS
1
3
a short half life (T =1.5 h), consistent with the
1
/2
observed metabolic rate. The apparent bioavailability
was found to be 76%.
Compd
R
2
R
11
IC50 (nM)9
EC50 (nM)10
In summary, nevirapine-like inhibitors with excellent
activity against wild type and the key K103N/Y181C
mutant virus were identified. In particular, the 2,6-dime-
thylpyridine derivative (6) has shown a good pharmaco-
kinetic profile in spite of poor metabolic stability in rat
liver microsomes. Unfortunately, the metabolic instabil-
ity of this series in a human liver microsome preparation
precludes further development. Although not all meta-
bolites were characterized, the two major metabolites
were identified as the corresponding sulfoxide and sul-
fone. Unfortunately, upon synthesis and evaluation,
these metabolites were found to be much less potent
then the parent compounds. Based on these findings, the
design of new derivatives having similar potency but
with improved metabolic stability is in progress and the
results will be reported shortly.
WT K103N/Y181C WT K103N/Y181C
6
Cl
F
H
Et
Et
Et
7.0
6.9
4.2
60
120
170
280
1.2
1.0
0.46
2.2
14
21
12
104
16
17
18
Cl c-Propyl 12
Having on hand this interesting 2,6-dimethyl-4-mercap-
topyridine derivative, we decided to further optimize
5
,6
positions 2 and 11 based on previous knowledge. As
shown in Table 2, 2-chloro (6), 2-fluoro (16) and
2-hydrogen (17) derivatives exhibited similar potencies
against WT and K103N/Y181C RT. Compound 17 had
the best antiviral activity against the WT virus
(
EC =0.46 nM). Although the chloro derivative 6 was
50
the most potent in the enzymatic assay against
K103N/Y181C mutant, this improved activity was not
seen in the antiviral assay. Modifications at the N-11
position indicated that only small alkyl groups such as
ethyl or cyclopropyl were tolerated. The cyclopropyl
derivative (18) proved to be significantly less potent
against the critical double mutant.
Acknowledgements
We would like to thank our colleagues at Boehringer
Ingelheim (Canada) Ltd, Research & Development for
their valuable contribution in compound characteriza-
tion and biological evaluation.
Antiretroviral treatment includes a triple combination
therapy, thus drug–drug interaction is an important
concern. One of the mechanisms underlying drug–drug
interaction is cytochrome P450 inhibition. As high-
lighted in Table 3, all three inhibitors had IC50 values
significantly greater than our minimum requirement of 1
mM. Compounds were also evaluated for first pass
References and notes
1
2
. De Clerq, E. Trends Pharmacol. Sci. 1990, 11, 198.
. Kohlstaedt, L. A.; Wang, J.; Friedman, J. M.; Rice, P. A.;
Steiz, T. A. Science 1992, 256, 1783.
. Campiani, G.; Ramunno, A.; Maga, G.; Nacci, V.; Fat-
toruso, C.; Catalanotti, B.; Morelli, E.; Novellino, E.
Curr. Pharm. Des. 2002, 8, 615.
1
1
metabolism using male human and rat liver micro-
3
1
2
somes. All three compounds were found to be rapidly
metabolized resulting in short half-life (see Table 3).
4
. Schinazi, R.; Larder, B. A.; Mellors, J. W. Int. Antiviral
News 1997, 5, 129.
Due to the excellent intrinsic and antiviral activity
against the K103N/Y181C mutant virus and, in spite of
the disappointing metabolic stability results, compounds
were selected for in vivo pharmacokinetic profiling in
rats. Compound 6 was found to have the best profile.
After oral gavage of 6 at 5 mg/kg we observed an
AUCpo of 2 mMh and a C max of 0.8 mMwhich corre-
sponds to approximately ꢁ700-fold higher then the
5
. Klunder, J. M.; Hoermann, M.; Cywin, C. L.; David, E.;
Brickwood, J. R.; Schwartz, R.; Barringer, K. J.; Pauletti,
D.; Shih, C.-K.; Erickson, D. A.; Sorge, C. L.; Joseph,
D. P.; Hattox, S. E.; Adams, J.; Grob, P. M. J. Med.
Chem. 1998, 41, 2960.
. Cywin, C. L.; Klunder, J. M.; Hoermann, M.; Brickwood,
J. R.; David, E.; Grob, P. M.; Schwartz, R.; Pauletti, D.;
Barringer, K. J.; Shih, C.-K.; Sorge, C. L.; Erickson,
D. A.; Joseph, D. P.; Hattox, S. E. J. Med. Chem. 1998,
6
4
1, 2972.
Table 3. Cyp450 inhibition and metabolic stability
7
8
. Evans, R. F.; Brown, H. C. J. Org. Chem. 1962, 27, 1665.
. Profft, E.; Rolle, W. Wissenschaftliche Zeitschrift der
Technishen Hochschule fu¨r Chemie Leuna-Merseburg 1959,
Cyp450 Inhibition
IC50 (mM)
Metabolic rate
1/2(min)
T
2, 187.
Compd 1A2 2C9 2C19 2D6 3A4-BFCa 3A4-BQa Rat Human
9. Enzymatic assays: IC50 values for wild-type and mutant
RTs were obtained from a scintillation proximity assay
6
1
1
23 2.6 4.2
26 4.5 5.4
>30 7.4 26
27
27
>30
2.3
2.5
1.6
8.1
10
10
7
5
9
4
5
9
3
ꢀ
using poly rC/biotin-dG15 and H-dGTP at 37 C. Each
value represents the mean of at least three determinations
The reproducibility of the assay was gauged using an
internal standard.
6
7
a
7-Benzyloxy-4-(trifloromethyl)-coumarin and 7-benzyloxyquinoline
(BQ) are fluorogenic substrates for cyp3A4.
10. Replication assays: For EC50 determinations, C8166 cells