Notable difference in anti-HIV activity of integrase inhibitors as a consequence of geometric and enantiomeric configurations

Article abstract of DOI:10.1016/j.bmcl.2013.05.050

While some examples are known of integrase inhibitors that exhibit potent anti-HIV activity, there are very few cases reported of integrase inhibitors that show significant differences in anti-HIV activity that result from distinctions in cis- and trans-c

Full text of DOI:10.1016/j.bmcl.2013.05.050

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4112–4116
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Notable difference in anti-HIV activity of integrase inhibitors
as a consequence of geometric and enantiomeric configurations
⇑
Maurice Okello, Sanjay Mishra, Malik Nishonov, Vasu Nair
UGA Center for Drug Discovery, the College of Pharmacy, R.C. Wilson Pharmacy Bldg., Room 320, University of Georgia, Athens, GA 30602, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
While some examples are known of integrase inhibitors that exhibit potent anti-HIV activity, there are
very few cases reported of integrase inhibitors that show significant differences in anti-HIV activity that
result from distinctions in cis- and trans-configurations as well as enantiomeric stereostructure. We
describe here the design and synthesis of two enantiomeric trans-hydroxycyclopentyl carboxamides
which exhibit notable difference in anti-HIV activity. This difference is explained through their binding
interactions within the active site of the HIV-1 integrase intasome. The more active enantiomer 3
(EC50 25 nM) was relatively stable in human liver microsomes. Kinetic data revealed that its impact on
key cytochrome P450 isozymes, as either an inhibitor or an activator, was minor, suggesting a favorable
CYP profile.
Received 9 April 2013
Revised 10 May 2013
Accepted 13 May 2013
Available online 23 May 2013
Keywords:
Integrase
Anti-HIV
Enantiomers
Drug discovery
Cytochrome P450 isozymes
Ó 2013 Elsevier Ltd. All rights reserved.
HIV-1 integrase is a 32 kDa protein, which catalyzes the incor-
poration of HIV DNA into host chromosomal DNA through a specif-
ically defined sequence of reactions, which involves 30-processing
and a key strand transfer (ST) step.1–6 Initiation of integration oc-
curs in the cytoplasm, where a complex is formed between viral
cDNA, previously produced by reverse transcription, and HIV
integrase. Following this is site-specific endonucleatic cleavage of
two nucleotides from each 30-end of double-stranded viral DNA,
which produces truncated viral DNA with terminal CAOH-30 (30-
processing). The next step, ST, involves staggered nicking of chro-
mosomal DNA and joining of each 30-end of the recessed viral
DNA to the 50-ends of the host DNA, followed by repair/ligation.
The ST step is carried out after transport of the processed, preinte-
gration complex from the cytoplasm into the nucleus. Both 30-pro-
cessing and ST steps require divalent metal ions as cofactors.
We recently reported on the synthesis and structure-activity
studies of new anti-HIV-1 integrase inhibitors.7 Among the most
interesting compounds discovered in this study was 4-(5-(2,4-diflu-
orobenzyl)-1-(2-fluorobenzyl)-2-oxo-1,2-dihydropyridin-3-yl)-4-
hydroxy-2-oxobut-3-enoic acid (1, Fig. 1), which exhibited potent
strand transfer (ST) inhibitory activity (IC50 6 nM). However, the
in vitro antiviral data of compound 1 revealed that there was a sig-
nificant disconnect of almost two orders of magnitude between the
anti-HIV-1 activity in cell culture (EC50 500 nM, MAGI cells) and the
low nM ST IC50 data for 1. Because this disconnect appeared to be a
problem associated with the cellular permeability of this ionized
inhibitor, we investigated unionized and somewhat more lipophilic
derivatives of inhibitor 1. Interesting new compounds to emerge
from this study were the hydroxycyclopentyl carboxamides 2
(Fig. 2, clogP 3.41). However, while the racemic trans-compound
showed strong strand transfer inhibition of HIV-1 integrase with
an IC50 of 48 nM, the racemic cis-compound was not an inhibitor
of integrase.8
To explore further whether enantiomers belonging to the trans-
compound related to 2 would show significant differences in anti-
HIV activity in cell culture as a consequence of their specific chiral-
ity, we synthesized their optically pure (S,S) and (RR) enantiomeric
hydroxycarboxamides, 3 and 4 (Fig. 2).
Synthesis of the enantiomeric carboxamides was achieved from
1, which was prepared from 5-bromo-2-methoxypyridine 5 via the
acetylpyridinone 7,7 as summarized in Scheme 1. Conversion of
diketo acid 1 to produce the optically active carboxamide 3 re-
quired the coupling amine, (1S,2S)-(+)-trans-2-aminocyclopentanol
(Sigma–Aldrich). Treatment of 1 in DMF with hydroxybenzotriazole
(HOBt) followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiim-
ide hydrochloride (EDCI–HCl) produced the activated intermediate
of 1, which was coupled using (1S,2S)-(+)-trans-2-aminocyclopent-
anol hydrochloride in the presence of NaHCO3. Purification of the
resulting crude product using silica gel chromatography gave
highly purified (1S,2S)-4-(5-(2,4-difluorobenzyl)-1-(2-fluoroben-
zyl)-2-oxo-1,2-dihydropyridin-3-yl)-4-hydroxy-N-(2-hydroxycycl-
opentyl)but-3-enamide (3) in 66% yield (½a D20
ꢀ
+41.8, c 0.01,
CH3OH).9 The enantiomer of 3, that is, compound 4, was prepared
by a similar route, but using (1R,2R)-2-aminocyclopentanol hydro-
chloride as the coupling reagent.10 This coupling reagent was
⇑
Corresponding author. Tel.: +1 (706) 542 6293; fax: +1 (706) 583 8283.
E-mail address: vnair@rx.uga.edu (V. Nair).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.050

M. Okello et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4112–4116
4113
Table 1
In vitro anti-HIV-1 data of compounds 3 and 4 in MAGI cells
Inhibitor
EC50 (SD) nM
EC90 (SD) nM
CC50 (lM)
3 (S,S-Enantiomer)
4 (R,R-Enantiomer)
25.1 ( 3.9)
42.2 ( 1.3)
415.1 ( 39.1)
871.4 ( 23.5)
40.4 ( 1.9)
137.0 ( 6.0)
EC50 = concentration for 50% inhibition of virus replication. Mean of three
determinations.
EC90 = concentration for 90% reduction of virus replication. Mean of three
determinations.
CC50 = concentration to reduce cell viability by 50%.
Figure 1. Structures of HIV-1 integrase inhibitor 1 and four possible hydroxycycl-
opentyl carboxamides 2.
green; hydroxyl hydrogen—cyan. Notable interactions common to
both compounds include: non-polar interactions involving
Pro145; polar interactions with two Mg2+ ions, that is, chelation
with diketo chromophore in both compounds; pi–pi stacking inter-
action of DC16 with the o-fluorophenyl ring and pi stacking of DA17
with the diketo chromophore. However, molecular modeling
images in Figure 3C and D (partial structures shown) clearly show
the difference in spatial orientation of the cyclopentyl carboxamide
moiety of compounds 3 and 4 within the HIV-1 integrase intasome
active site. This difference is in the interactions involving amino
acid residues 143 and 118 within the active site. Compound 3 inter-
acts with Tyr143 through the cyclopentylhydroxyl hydrogen and
the aromatic pi-electrons of Tyr143. This interacting distance is
about 2.8 Å.14 The corresponding interaction is absent in the case
of enantiomer 4, which apparently forms a hydrogen bond with
Gly118 in which the amide carbonyl oxygen of Gly118 is the accep-
tor.15,16 The tyrosine ring is significantly further away (>6.0 Å) from
the cyclopentyl OH in 4 to allow for the hydroxyl hydrogen–pi
interaction seen in 3. Our docking studies also indicated that the
protein–ligand binding free energy favored compound 3.
Because of the importance of establishing early, the behavior of
a potentially promising new anti-HIV compound in phase I metab-
olism, our next studies involved the more active compound 3 and
its in vitro microsome stability as well as its cytochrome P450
(CYP) profile.17–19 The microsome stability studies were performed
through incubations using pooled, mixed-gender human liver
microsomes.20 Analyses were performed by HPLC separation com-
bined with UV detection and further bioanalytical identification of
compound 3 and its metabolites. The results showed that com-
pound 3 was relatively stable in pooled human liver microsomes
(Fig. 4), exhibiting a half-life (t1/2) of approximately 2 h.
Figure 2. Structures of the (S,S) and (R,R) enantiomers (3 and 4) of the trans-isomer
of compound 2.
synthesized using a modification of a literature procedure.11 The
yield of pure compound 4 from 1 was 42% (½a D20
ꢁ41.2, c 0.01,
ꢀ
CH3OH).
A summary of the in vitro anti-HIV-1 NL4-3 data of 3 and 4 in
MAGI cells are shown in Table 1.8 With respect to the EC50 data,
it is evident that both compounds are active and show low nM cell
culture activity. However, the data also clearly indicate that the
(S,S)-isomer 3 was more active than its (R,R)-enantiomer 4, in
terms of both the EC50 and EC90 anti-HIV data. The question then
was to see if this difference in viral replication inhibition could
be explained through the molecular details of the interaction of
these compounds with the HIV-1 intasome (i.e., HIV-1 integrase–
processed viral DNA complex) and this is discussed below.
Comparison of the binding interactions of compounds 3 and 4
within the modeled active site of HIV-1 integrase intasome was car-
ried out through docking experiments in order to provide an expla-
nation for the difference in antiviral activity.12 The results are
shown in Figure 3. The amino acid residues are designated with
HIV-1 numbering and are color coded by hydrophobicity with the
orange color indicating more hydrophobicity and the blue color
indicating less hydrophobicity. Atoms of DNA residues and of inhib-
itors 3 and 4 (ball and stick representation) are colored according to
atom type: carbon—grey; nitrogen—blue; oxygen—red; fluorine—
The CYP isozyme profile study also utilized pooled mixed-gen-
der human liver microsomes and two substrates for CYP 3A4 (tes-
tosterone and triazolam) and one substrate for CYP 2D6
(dextromethorphan).21 These two CYP isozymes account for the
metabolism of about 80% of the drugs that are metabolized in
Scheme 1. Methodology for the synthesis of the enantiomeric trans-carboxamides 3 and 4.

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M. Okello et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4112–4116
Figure 3. Docking of HIV integrase inhibitor 3: A and C and 4: B and D with the integrase catalytic site of the HIV-1 integrase–DNA intasome (PDB code 3OAY).13 DNA residues
and inhibitor atom types: carbon—grey, nitrogen—blue, oxygen—red, fluorine—green, hydroxyl hydrogen—cyan. R and S are stereocenter designations.
served were generally within expected experimental variations.
Thus, compound 3 is neither a significant inhibitor nor an activator
of these key CYP isozymes and its CYP profile appears to be favor-
able with respect to these key isozymes.
100.0
80.0
60.0
40.0
20.0
0.0
In summary, there are very few examples known of integrase
inhibitors that show significant differences in anti-HIV activity that
result from distinctions in cis- and trans-configurations as well as
enantiomeric stereostructure. We have described herein the ste-
reospecific synthesis of two enantiomeric of trans-hydroxycycl-
opentyl carboxamides. The (S,S)-enantiomer 3 was notably more
active than its (R,R)-counterpart 4 on the basis of both the EC50
and EC90 data in anti-HIV-1 assays in cell culture. This difference
in antiviral activity may be explained by examining the molecular
level interactions of these compounds with the HIV-1 integrase
intasome. The more active (S,S)-compound, on which further study
was continued, was moderately stable in pooled, mixed-gender
human liver microsomes. Kinetic data showed that its behavior to-
wards key cytochrome P450 isozymes, either as an inhibitor or an
activator, was relatively minor, suggesting that the compound is
expected to possess a generally favorable CYP profile. Further stud-
ies are in progress on the molecular design and synthesis, antiviral
evaluations and CYP studies of novel integrase-based anti-HIV
compounds, in which preferred configurational geometry and
enantiomeric structure are both critical for significant antiviral
activity.
0
15
30
45
60
Time, min
Figure 4. Stability of compound 3 in pooled, mixed-gender human liver micro-
somes. Data bars indicate the mean from three determinations. The error bars are
standard deviations ( S.Ds.) from the mean.
phase I metabolism by CYP isozymes.17 Summarized in Table 2 is
the effect of compound 3 on the substrate activity of these key
CYP isozymes (CYP3A4–hydroxylation of testosterone and triazo-
lam, and CYP2D6–demethylation of dextromethorphan). The data
indicate only relatively small changes in the level of substrate
activity compared to the control study in each case. Changes in
compound 3 concentration also did not influence significantly
the inhibition or activation of the probe substrates. The data ob-

M. Okello et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4112–4116
4115
Table 2
Influence of compound 3 on CYP isozyme substrate activity in pooled human liver microsomes
CYP isozyme
Substrate
Probe substrate concentrationa
(l
M)
Compound 3 concentration (
lM)
Substrate activity % S.D.b
CYP 3A4
CYP 3A4
CYP 3A4
CYP 3A4
CYP 3A4
CYP 3A4
CYP 3A4
CYP 3A4
CYP 2D6
CYP 2D6
CYP 2D6
CYP 2D6
Testosterone
Testosterone
Testosterone
Testosterone
Triazolam
Triazolam
Triazolam
Triazolam
Dextromethorphan
Dextromethorphan
Dextromethorphan
Dextromethorphan
100
100
100
100
200
200
200
200
200
200
200
200
Control
109 13
97 18
112 15
Control
124 23
12.5
25
50
12.5
25
50
98
7
100
8
Control
86
12.5
25
50
7
87 17
87 13
a
The concentration of each probe substrate was determined by the Km value for that substrate.17
Change in substrate activity (%) relative to the control value (100%). Standard deviations ( S.D.) were determined from the mean of three determinations. Substrate and
b
metabolite peaks were monitored by HPLC–UV bioanalytical methods.21
a chilled solution of 4-(5-(2,4-difluorobenzyl)-1-(2-fluorobenzyl)-2-oxo-1,2-
dihydropyridin-3-yl)-4-hydroxy-2-oxobut-3-enoic acid (1) (150 mg,
Acknowledgments
0.338 mmol) in dimethylformamide (DMF) (2.0 mL), was added
hydroxybenzotriazole (HOBT) (50 mg, 0.372 mmol), followed by 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI–HCl, 71 mg,
0.372 mmol) and the mixture was stirred for 30 min. A solution of (1S,2S)-(+)-
trans-2-aminocyclopentanol hydrochloride and NaHCO3 (31 mg, 0.372 mmol)
was added followed by stirring for 2 h at 0–5 °C. Cold water was added to the
reaction mixture, which was then extracted with ethyl acetate (2 ꢂ 20 mL). The
combined organic phase was separated, washed with water twice, then once
with 1 N HCl solution, and finally with saturated aqueous NaHCO3 solution.
Concentration in vacuo afforded the crude product which was passed through a
short silica gel column with chloroform as the eluting solvent. The eluent
containing the product was concentrated and the resulting residue was
triturated with hexanes, which afforded the product as a yellow solid in
This project was supported by research Grant RO1 AI 43181
(NIAID) and shared equipment Grant IS10RR016621 (NCRR) from
the National Institutes of Health. The contents of this submission
are solely the responsibility of the authors and do not necessarily
represent the official views of the NIH. We thank the Terry Endow-
ment, the Georgia Research Alliance and the University of Georgia
for additional support. Some of the data cited here were deter-
mined at Inhibitex, Inc., Alpharetta, GA and at the Southern Re-
search Institute, Frederick, MD and we express our thanks to them.
117 mg (66%), mp 61–63 °C, ½a D20
ꢀ
+41.8 (c 0.01, methanol), UV kmax 396 nm (e
References and notes
14,455, methanol). 1H NMR (CDCl3, 500 MHz): d 15.33 (br s, 1H), 8.16 (d, 1H,
J = 2.0 Hz), 8.08 (s, 1H), 7.61–6.85 (m, 9H), 5.22 (s, 2H), 4.11 (m, 1H), 3.94 (m,
1H), 3.78 (s, 1H), 2.23 (m, 1H), 2.10 (m, 1H), 1.88 (m, 1H), 1.78 (m, 2H), 1.59 (m,
1H). 13C NMR (CDCl3, 125 MHz): d 181.3, 180.6, 163.4, 163.3, 162.4, 162.1,
162.0, 161.4, 161.3, 160.4, 160.1, 160.0, 159.2, 145.5, 143.9, 142.3, 141.6, 132.4,
132.4, 132.3, 131.5, 131.4, 131.3, 131.3, 131.2, 130.7, 130.6, 125.0, 124.9, 124.8,
122.8, 122.7, 122.5, 122.2, 122.2, 122.1, 122.1, 117.1, 115.9, 115.7, 115.6, 111.9,
111.9, 111.8, 111.7, 104.7, 104.5, 104.3, 98.3, 79.5, 79.3, 61.0, 60.6, 51.5, 47.7,
47.3, 32.9, 32.8, 32.7, 30.8, 30.7, 30.5, 21.7, 21.5. HRMS: calcd for C28H26F3N2O5
(M+H), 527.1794; found 527.1799.
1. Frankel, A. D.; Young, J. A. T. Annu. Rev. Biochem. 1998, 67, 1.
2. Asante-Appiah, E.; Skalka, A. M. Adv. Virus Res. 1999, 52, 351.
3. Nair, V.; Chi, G. Rev. Med. Virol. 2007, 17, 277.
4. Krishnan, L.; Engleman, A. J. Biol. Chem. 2012, 287, 40858.
5. Nair, V.; Chi, G. In HIV-1 Integrase: Mechanism and Inhibitor Design; Neamati, N.,
Ed.; Wiley: Hoboken, New Jersey, 2011; pp 379–388.
6. Liao, C.; Marchand, C.; Burke, T. R.; Pommier, Y.; Nicklaus, M. C. Future Med.
Chem. 2010, 2, 1107.
7. Seo, B. I.; Uchil, V. R.; Okello, M.; Mishra, S.; Ma, X.-H.; Nishonov, M.; Shu, Q.;
Chi, G.; Nair, V. ACS Med. Chem. Lett. 2011, 2, 877.
10. Synthesis
dihydropyridin-3-yl)-4-hydroxy-N-(2-hydroxycyclopentyl)but-3-enamide
Compound was synthesized using the procedure described above for
of
(1R,2R)-4-(5-(2,4-difluorobenzyl)-1-(2-fluorobenzyl)-2-oxo-1,2-
(4):
4
8. Integrase inhibition and antiviral assays. Integrase inhibition assay: DNA substrate
(final concentration 20 nM), serial dilutions of test compounds (starting from
compound 3. The coupling compound, (1R,2R)-2-aminocyclopentanol
hydrochloride, was synthesized by modification of a literature procedure.10
The crude product was purified on silica gel to give 4 as a yellow solid (42%),
1.5
were combined in a reaction buffer (10 mM MgCl2, 20 mM HEPES (pH 7.5), 5%
PEG, 0.1 mg/mL BSA, 5 mM DTT) at a final volume of 40 L/well of a 96 well
lM) and HIV-1 integrase (final concentration 1 lg/well) (Bioproducts, MD)
mp 55–57 °C,
methanol). 1H NMR (CDCl3, 500 MHZ): d 15.41 (br s, 1H), 8.16 (s, 1H), 8.07 (s,
1H), 7.60–6.84 (m, 9H), 5.21 (s, 2H), 4.10–3.76 (m, 5H), 2.25–1.73 (m, 6H). 13
½
a 2D0
ꢀ
ꢁ41.2 (c 0.1, methanol), UV kmax 396 nm
(e 14,100,
l
polypropylene plate. The mixture is incubated for 1 h at 37 °C. After the
reaction was completed, the mixtures were adjusted to 20 mM Tris–Cl (pH 8.0),
400 mM NaCl, 10 mM EDTA + 0.1 mg/mL sonicated salmon sperm DNA at
100 lL/well final volume. The samples were transferred to a 96 well
Streptavidin coated ELISA plates (Nunc). The plates were incubated for 1 h at
room temperature. Following the incubation, the plate was washed 3 times
C
NMR (CDCl3, 125 MHz): d 181.1, 163.0, 162.1, 160.2, 159.0, 143.7, 141.4, 132.1,
132.1, 132.1,131.2, 131.1, 131.1, 131.0, 130.5, 130.4, 124.7, 124.7, 122.5, 122.4,
122.3, 121.8, 116.9, 115.5, 115.3, 111.7, 111.7, 111.5, 111.5, 104.4, 104.2, 104.0,
98.1, 79.1, 60.2, 47.5, 32.5, 30.5, 30.4, 21.4. HRMS: calcd for C28H26F3N2O5
(M+H), 527.1794; found 527.1804.
with 200
with 200
l
l
L/well 30 mM NaOH, 200 mM NaCl, 1 mM EDTA followed by 3ꢂ
11. Overman, L. E.; Sugai, S. J. Org. Chem. 1985, 50, 4154.
L/well 10 mM Tris–Cl (pH 8.0), 1 mM EDTA. The wash solutions
12. Molecular modeling and ligand docking on intasome: Molecular modeling of the
crystal structure of prototype foamy virus (PFV) integrase intasome (PDB code
3OYA) with compounds 3 and 4 docked within the catalytic site was achieved
by using the Surflex-Dock package within Sybyl–X [Sybyl–X 1.3 (winnt_os5x)
version] (Tripos, St. Louis, MO, 2011). Processing was done according to default
conditions of Surflex-Dock and Biopolymer. The prepared ligands were docked
to the intasome active site as guided by an appropriately generated protomol.
The modeling was validated by screening a ligand set for compounds 3, 4 and a
number of known anti-HIV integrase inhibitors and all of the active
compounds were recognized, including compounds 3 and 4, and all showed
significantly high total scores.
13. Hare, S.; Gupta, S. S.; Valkov, E.; Engelman, A.; Cherepanov, P. Nature 2010, 464,
232.
14. Bissantz, C.; Kuhn, B.; Stahl, M. J. Med. Chem. 2010, 53, 5061.
15. Rademacher, P.; Khelashvili, L.; Kowski, K. Org. Biomol. Chem. 2005, 3,
2620.
were incubated on the plate for 5 min between each wash. The final product
was detected using Sheep anti-Digoxigenin-POD, Fab fragments (1:200
dilution; 1 h incubation at 37 °C) (Roche). Unbound detection antibody was
removed with 6 washes of 1ꢂ PBS, 0.1% Tween-20 (350
washer). 100 L of TMB substrate (KPL) was incubated on the plate for 10 min
at room temperature followed by the addition of 100 L/well of TMB stop
lL/wash using a plate
l
l
solution (KPL). The absorbance values were read at 450 nm using the
SpectraMax M2 Plate Reader from Molecular Devices. Antiviral assay. MAGI
cells are infected with HIV-1 in the presence of test compound. If the virus is
able to infect and replicate in the cells, it will proceed through reverse
transcription and integration and begin transcription from the integrated
provirus. One of the first virus proteins produced is HIV-1 Tat, which
transactivates the HIV-1 LTR promoter, driving expression of b-galactosidase
from an LTR-b-galactosidase reporter construct engineered into the cells. As a
result, infected cells begin to overproduce the b-galactosidase enzyme. After 6
days post infection, the cells are lysed and b-galactosidase enzyme activity is
measured using chemiluminescence detection (Perkin–Elmer Applied
Biosystems). Compound toxicity is monitored on replicate plates using MTS
dye reduction (CellTiter 96Ò Reagent, Promega, Madison, WI).
16. Steiner, T.; Koellner, G. J. Mol. Biol. 2001, 305, 535.
17. Cytochrome P450: Structure, Mechanism, and Biochemistry; Ortiz de Montellano,
P. R., Ed., 3rd ed.; Kluwer Academic/Plenum: New York, 2005.
18. Baranczewski, P.; Stanczak, A.; Sundberg, K.; Svensson, R.; Wallin, A.; Jansson,
J.; Garberg, P.; Postlind, H. Pharmacol. Rep. 2006, 58, 453.
9. Synthesis
of
(1S,2S)-4-(5-(2,4-difluorobenzyl)-1-(2-fluorobenzyl)-2-oxo-1,2-
dihydropyridin-3-yl)-4-hydroxy-N-(2-hydroxycyclopentyl)but-3-enamide (3): To

4116
M. Okello et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4112–4116
19. Chauret, N.; Gauthier, A.; Martin, J.; Nicoll-Griffith, D. A. Drug Metab. Dispos.
1997, 25, 1130.
solvent concentration was kept at or below 1.5% by volume. Each reaction was
terminated by adding ice-cold acetonitrile (100 L). Proteins were precipitated
l
20. Stability of compound
contained 0.5 mg/mL protein, 50
G-6-P-DH, 5 mM MgCl2, and 5 mM G-6-P in a final volume of 400
3
in pooled human liver microsomes: Incubations
M test compound, 2 mM NADPH, 0.5 U/mL
L in
from the samples by centrifugation at 5000ꢂg for 10 min at room temperature
in a microcentrifuge tube. The supernatant was analyzed by HPLC-UV for
detection of substrates and metabolites. Reversed-phase HPLC analyses were
performed on a Beckman Coulter Gold 127 solvent system with a Gold 166 UV
l
l
100 mM potassium phosphate buffer (pH 7.4). The reaction mixture was pre-
incubated for 3 min at 37 °C before initializing by addition of NADPH solution,
analytical detector. The data were collected and processed using a Gold
software package. CYP HPLC analytical assays: Testosterone and 6b-
followed by incubation for 60 min. A solution of 60
lL was withdrawn at 0, 15,
30, 45 and 60 min and quenched with 60 L of ice-cold acetonitrile. The
l
hydroxytestosterone analysis involved analytical column Delta Pak C18
precipitated proteins were removed by centrifugation at 5000ꢂg for 5 min at
room temperature before analysis of the supernatant by HPLC. Analysis was
done using a reversed-phase HPLC column on a Beckman Coulter Gold 127
solvent system and Gold 166 UV analytical detector. The data were collected
and processed using Gold software. The elution process involved: analytical
(15 lm, 300 ꢂ 3.9 mm, 100 Å) eluting with (A) 10 mM potassium phosphate
buffer (pH 4.5) and (B) acetonitrile, in a gradient mixture starting with 25% (B)
at time 0 min to 100% (B) at time 23 min. at a flow rate of 1.2 mL/min, with
detection wavelength set at 254 nm. Testosterone and 6b-hydroxytestosterone
were detected at 14.01 and 8.90 min rt, respectively. The analytical column
column Delta Pak C18 (15
l
m, 300 ꢂ 3.9 mm, 100 Å) with a mobile phase of
Novapak C18 (4 lm, 150 ꢂ 3.9 mm, 60 Å) mounted on the above HPLC system
(A) 10 mM potassium phosphate buffer (pH 6.5) and (B) acetonitrile, which
were used as follows: 0–2 min, 20% B, 2–8 min, 20–80% B 8–18 min, 80% B, 18–
20 min, 20% B. The flow rate was kept at 1.5 mL/min, with UV detection at
360 nm. Retention times for compound 3 and its slowly produced metabolite
were 10.48 and 9.1 min, respectively.
was used in the analysis of triazolam and 4-hydroxytriazolam as well as
dextromethorphan and dextrophan respectively. The elution solvent for both
assay system was similar to testosterone above with variation in gradient
system. Triazolam and hydroxytriazolam were separated with
a gradient
solvent mixture of 30% (B) at 0 min to 100% B at 16 min, at a flow rate of
1.0 mL/min while detecting at 260 nm wavelength. The rt for Triazolam and
hydroxytriazolam were 8.46 and 5.48 min, respectively. Dextromethorphan
and dextrorphan were similarly separated with gradient solvent elution
system of 28% (B) at 0 min to 100% (B) after 16 min, at a flow rate of 1.0 mL/
min, with UV detection set at 278 nm. RT for dextromethorphan and
dextrorphan were 11.96 and 5.96 min, respectively.
21. Cytochrome P450 studies: Incubation mixtures contained potassium phosphate
buffer (100 mM, pH 7.4), MgCl2 (5 mM), the requisite substrate dissolved in an
appropriate solvent, compound 3 (range of concentrations) and pooled human
liver microsomes. The reaction mixture (final volume 100 lL) was pre-
incubated for 3 min at 37 °C before initiation of the reaction by the addition
of NADPH (final concentration 2 mM). In each incubation mixture, the organic