In vitro biosynthesis, isolation, and identification of predominant metabolites of 2-(4-(2-hydroxyethoxy)-3,5-dimethylphenyl)-5,7- dimethoxyquinazolin-4(3H)-one (RVX-208)

Article abstract of DOI:10.1016/j.ejmech.2013.03.062

The structures of the two predominant metabolites (M4 and M5) of RVX-208, observed both in in vitro human and animal liver microsomal incubations, as well as in plasma from animal in vivo studies, were determined. A panel of biocatalytic systems was tested to identify biocatalysts suitable for milligram scale production of metabolite M4 from RVX-208. Rabbit liver S9 fraction was selected as the most suitable system, primarily based on pragmatic metrics such as catalyst cost and estimated yield of M4 (～55%). Glucuronidation of RVX-208 catalyzed by rabbit liver S9 fraction was optimized to produce M4 in amounts sufficient for structural characterization. Structural studies using LC/MS/MS analysis and ¹H NMR spectroscopy showed the formation of a glycosidic bond between the primary hydroxyl group of RVX-208 and glucuronic acid. NMR results suggested that the glycosidic bond has the β-anomeric configuration. A synthetic sample of M4 confirmed the proposed structure. Metabolite M5, hypothesized to be the carboxylate of RVX-208, was prepared using human liver microsomes, purified by HPLC, and characterized by LC/MS/MS and ¹H NMR. The structure was confirmed by comparison to a synthetic sample. Both samples confirmed M5 as a product of oxidation of primary hydroxyl group of RVX-208 to carboxylic acid.

Full text of DOI:10.1016/j.ejmech.2013.03.062

European Journal of Medicinal Chemistry 64 (2013) 121e128
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
In vitro biosynthesis, isolation, and identification of predominant
metabolites of 2-(4-(2-hydroxyethoxy)-3,5-dimethylphenyl)-
5,7-dimethoxyquinazolin-4(3H)-one (RVX-208)
Yuri L. Khmelnitsky a, Vadim V. Mozhaev a, Ian C. Cotterill a, Peter C. Michels a,
Sihem Boudjabi b, Vladimir Khlebnikov b, M. Madhava Reddy b, Gregory S. Wagner c,
,
Henrik C. Hansen c
*
a AMRI, 26 Corporate Circle, PO Box 15098, Albany, NY 12212-509, USA
b NAEJA Pharmaceutical Inc., 4290-91A Street, Edmonton, Alberta T6E 5V2, Canada
c Resverlogix Corp. Suite 202, 279 Midpark Way SE, Calgary, Alberta T2X 1M2, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
The structures of the two predominant metabolites (M4 and M5) of RVX-208, observed both in in vitro
human and animal liver microsomal incubations, as well as in plasma from animal in vivo studies, were
determined. A panel of biocatalytic systems was tested to identify biocatalysts suitable for milligram
scale production of metabolite M4 from RVX-208. Rabbit liver S9 fraction was selected as the most
suitable system, primarily based on pragmatic metrics such as catalyst cost and estimated yield of M4
(w55%). Glucuronidation of RVX-208 catalyzed by rabbit liver S9 fraction was optimized to produce M4
in amounts sufficient for structural characterization. Structural studies using LC/MS/MS analysis and 1H
NMR spectroscopy showed the formation of a glycosidic bond between the primary hydroxyl group of
Received 13 March 2013
Received in revised form
27 March 2013
Accepted 28 March 2013
Available online 10 April 2013
Keywords:
Biosynthesis
Metabolite identification
Mass spectrometry
Metabolite synthesis
RVX-208 and glucuronic acid. NMR results suggested that the glycosidic bond has the
b-anomeric
configuration. A synthetic sample of M4 confirmed the proposed structure. Metabolite M5, hypothesized
to be the carboxylate of RVX-208, was prepared using human liver microsomes, purified by HPLC, and
characterized by LC/MS/MS and 1H NMR. The structure was confirmed by comparison to a synthetic
sample. Both samples confirmed M5 as a product of oxidation of primary hydroxyl group of RVX-208 to
carboxylic acid.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
In preliminary in vitro metabolite profiling and identification
studies using liver fractions from various mammalian species, it
2-(4-(2-Hydroxyethoxy)-3,5-dimethylphenyl)-5,7-
was determined that the metabolic pathways of RVX-208 involved
the formation of two significant metabolites, a glucuronidated
metabolite M4 and an oxidized metabolite M5. Metabolites M4
and M5 were also identified as predominant metabolites in
follow-up in vivo animal studies (unpublished results). Therefore,
it was important to establish the exact structures of these me-
tabolites, which required synthesis of authentic M4 and M5 suit-
able for NMR structural characterization. In the absence of prior
knowledge of metabolite structures, one of the most effective
strategies for the production of metabolites is the use of the actual
liver metabolic enzyme systems, typically found in hepatocytes,
microsomes, or S9 fractions [3e5]. We applied this biosynthetic
strategy to synthesize and characterize the two predominant
metabolites (M4 and M5) of RVX-208 to support the ongoing
development program.
dimethoxyquinazolin-4(3H)-one (RVX-208) (Fig. 1) is a novel BET
bromodomain inhibitor. It is an orally active small molecule drug
candidate for the treatment of cardiovascular diseases. It is pro-
posed to work by increasing the levels of apolipoprotein A1 (Apo-
A1), resulting in an increase in high-density lipoprotein cholesterol
(HDL-c), thereby potentially reducing the risk for cardiovascular
disease [1,2]. RVX-208 is currently in phase 2 clinical trials for the
treatment of atherosclerosis. Identification of the main metabolites
is an essential component of understanding the elimination of the
parent compound from the body, as well as understanding the
safety profile for the drug candidate.
* Corresponding author. Tel.: þ1 403 254 9252; fax: þ1 403 256 8495.
E-mail address: henrik@resverlogix.com (H.C. Hansen).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.062

122
Y.L. Khmelnitsky et al. / European Journal of Medicinal Chemistry 64 (2013) 121e128
Table 2
Estimated yields (%) of metabolite M4 in incubations of RVX-208 with S9 Fractions.a
Source of S9 Fraction
Reaction time (hours)
1
6
24
7.3
2.2
2.1
0.6
30.9
0.7
Male beagle dog
0.9
0.4
0.3
0.4
4.1
0
1.9
1.1
1.1
0.8
16.0
0.4
Male cynomolgus monkey
Male SpragueeDawley rat
Male ICR/CD-1 mouse
Male New Zealand white rabbit
Pooled human mixed sexes
(10 individuals)
Male Yucatan minipig
1.7
6.5
14.2
a
Estimated from the HPLC peak area.
<1%). Based on results of the screen, microsomes and S9 fraction
from rabbit liver and microsomes from calf liver all had an esti-
mated yield above 30% and were selected for further optimization.
2.1.2. Synthesis, purification and structural characterization of
metabolite M4
The yield in the glucuronide metabolite synthesis primarily
depends on the concentrations of the parent compound and
UDPGA (the cofactor for glucuronidation), and the amount of a
catalyst added. In the optimization screen, these parameters were
varied over a broad range of values at a fixed reaction time of 24 h.
The results are shown in Table 3.
In the majority of reaction conditions tested in the optimization
screen, the estimated yield of M4 exceeded 20%, and in several
reaction systems it was greater than 50%. Overall, calf microsomes
were better catalysts than rabbit microsomes and S9 fraction.
However, because rabbit liver S9 fraction offers the most cost
effective option compared to other catalysts in Table 3, they were
selected as a practical catalyst for synthesis of M4. Based on the
results of the optimization study, the following reaction conditions
were selected for scale-up synthesis of metabolite M4: 1 mM RVX-
208, 2 mg/mL S9 protein (rabbit), and 10 mM UDPGA.
After 24 h of incubation LC/MS analysis of the reaction mixture
showed a 22% yield of M4. Following work-up of the reaction and
purification by preparative HPLC, a total of 2.6 mg of M4 was ob-
tained as a white solid with >99% purity as determined by HPLC
(see Supporting information).
Fig. 1. Structures of RVX-208 and its metabolites M4 and M5.
2. Results and discussion
2.1. Synthesis and structural characterization of glucuronidated
metabolite M4
2.1.1. Screening of biocatalytic systems for production of metabolite
M4
Initially, a broad spectrum of mammalian liver microsomes
(human, mouse, rat, dog, rabbit, monkey, minipig, calf), S9 fractions
(from human, mouse, rat, dog, rabbit, monkey, minipig), and twelve
human recombinant glucuronyl transferases were screened to
identify a system that would be practical for the biosynthesis of M4.
The initial selection criteria was the estimated yield after 24 h of
incubation. Estimated yields of M4 in reactions catalyzed by
mammalian liver microsomes and S9 fractions are shown in
Tables 1 and 2, respectively. Metabolite yields were estimated from
integrated areas of corresponding peaks in HPLC chromatograms
with UV detection at 230 nm. In reactions with UGT enzymes,
metabolite M4 was formed only in trace amounts (estimated yield
Table 3
Optimization of reaction conditions for synthesis of metabolite M4.
Catalyst
[UDPGA]
mM
[Total protein]
mg/mL
[RVX-208]
mM
Estimated
yield, %
Rabbit microsomes
5
25
5
4
2
0.5
2
0.5
0.2
2
0.5
2
0.5
0.2
2
0.5
2
0.5
0.2
2
0.5
2
30.0
24.1
38.4
39.5
34.6
48.9
54.3
39.7
49.4
49.7
30.3
33.0
17.5
23.2
22.3
35.2
41.3
16.7
25.7
21.4
50.1
40.9
56.0
1
4
1
Table 1
Estimated yields (%) of metabolite M4 in incubations of RVX-208 with liver
microsomes.a
Rabbit S9 fraction
4
1
Source of microsomes
Reaction time (hours)
1
6
24
Male Beagle Dog
0.9
0.2
0.1
0.1
6.4
1.1
0.5
0.3
15.5
2.9
0.7
0.5
30.3
1.2
25
5
4
1
Male Cynomolgus Monkey
male Sprague-dawley rat
Male ICR/CD-1 Mouse
0.5
0.2
2
2
0.5
Male New Zealand White Rabbit
Pooled Human Mixed Sexes (10 individuals)
Calf
12.2
0.1
3.7
22.4
0.6
27.7
11.8
Calf microsomes
4
1
35.9
14.3
Male Yucatan Minipig
2.5
a
Estimated from the HPLC peak area.

Y.L. Khmelnitsky et al. / European Journal of Medicinal Chemistry 64 (2013) 121e128
123
Structural characterization of metabolite M4 was conducted
using LC/MS/MS analysis and 1H NMR spectroscopy. Results of LC/
MS/MS analysis suggested that glucuronidation occurred at the
hydroxyl group of RVX-208 (Fig. 2). 1H NMR data analysis
confirmed the formation of a glycosidic bond between the hydroxyl
group of RVX-208 and glucuronic acid (See Supporting
information). Analysis of the 1H NMR spectrum in narrow spec-
tral ranges (Fig. 3) allowed determination of the anomeric config-
uration of the glycosidic bond. Based on the coupling constant for
the anomeric proton, which is approximately 8 Hz (panel B in
Fig. 3), the glycosidic bond was determined to be in the b-config-
uration [6]. The proposed structure of metabolite M4, derived from
LC/MS/MS and NMR data, is shown in Fig. 1.
To verify the structure of metabolite M4, it was synthesized
chemically from RVX-208 using the synthetic route shown in
Scheme 1 (see Experimental section for details). For the synthesis,
the initial protection of the amide nitrogen was required to avoid
formation of the N-glucuronide. Having the 2-O-acetate glycosyl
chemically using the synthetic route shown in Scheme 2 (See
Experimental for details). The LC/MS/MS and 1H NMR properties of
synthetic M5 are identical to those of the metabolite M5 prepared
using human liver microsomes, thus confirming the structural
identity of M5.
3. Conclusion
In conclusion, we successfully applied biosynthetic strategy to
synthesize the predominant metabolites (M4 and M5) of the clin-
ical drug candidate RVX-208 on a preparative scale. A wide variety
of biocatalytic systems were tested for synthetic suitability, the
most effective catalysts were identified, and conditions were opti-
mized to achieve practical yields of the target metabolites. The
structures of the metabolites were proposed from LC/MS/MS and
1H NMR spectroscopy data, and they were verified by direct
chemical synthesis.
donor supported the formation of the b-anomer. Both chemically
4. Experimental procedures
and biosynthetically prepared samples of M4 exhibit identical LC/
MS/MS and 1H NMR data, confirming the structure shown in Fig. 1.
4.1. General procedures and materials
2.2. Synthesis and structural characterization of oxidized
metabolite M5
Calf liver microsomes were obtained from CellzDirect (Pittsboro,
NC). All other mammalian liver microsomes and mammalian S9
fractions were obtained from In Vitro Technologies, Inc. (Baltimore,
MD). Metabolic competency of liver microsomes and S9 fractions
was confirmed in a separate control experiment using a standard
substrate, testosterone. Components of the NADPH regeneration
system and human recombinant UDP-glucuronyl transferases were
obtained from BD Gentest (Woburn, MA). RVX-208 was supplied by
Resverlogix Corp. (Calgary, Canada). Solvents and reagents for
chemical syntheses were used as purchased from commercial
suppliers, unless otherwise noted. Synthetic chemical reactions
were monitored by thin-layer chromatography (TLC) carried out on
For structural identification of metabolite M5, conditions of the
initial screening protocol used for microsomal incubations were
found to be adequate to generate sufficient quantities of M5. Using
human liver microsomes an analytical amount of M5 was produced
from RVX-208 (See Experimental section for details). After purifi-
cation by HPLC, the material was subjected to LC/MS/MS charac-
terization. Results of this analysis (Fig. 4) suggested that M5 is the
result of oxidation of the primary alcohol in RVX-208 to a carboxylic
acid. To verify the structure of metabolite M5, it was synthesized
+MS2 (547.00) CE (50): 63 MCA scans from Sample 1 (01-26-07 #3) of 01-26-07 #3.wiff (Turbo Spray)
Max. 4.6e6 cps.
371.1
4.6e6
4.4e6
326
165
4.2e6
4.0e6
3.8e6
3.6e6
3.4e6
3.2e6
3.0e6
2.8e6
2.6e6
2.4e6
2.2e6
2.0e6
1.8e6
1.6e6
1.4e6
1.2e6
1.0e6
8.0e5
6.0e5
4.0e5
2.0e5
HO
O
HO
O
326
OH
O
OH
OH
O
OH
O
O
165
O
OH
O
N
O
N
OH
NH
NH
O
O
O
371
371
311
O
311
138
138
547.1
326.2
165.2
297.1
311.1
138.2
283.3
281.4
148.0
122.2
240.2
250
94.9
180.0
50
100
150
200
300
m/z, am u
350
400
450
500
550
Fig. 2. MS/MS spectrum and fragmentation analysis for metabolite M4.

124
Y.L. Khmelnitsky et al. / European Journal of Medicinal Chemistry 64 (2013) 121e128
Fig. 3. 1H NMR spectra of purified metabolite M4, showing narrow spectral ranges. Panels: A, spectrum in the range from 6.0 to 8.0 ppm; B, spectrum in the range from 3.8 to
4.5 ppm; C, spectrum in the range from 3.35 to 3.8 ppm.

Y.L. Khmelnitsky et al. / European Journal of Medicinal Chemistry 64 (2013) 121e128
125
OAc
AcO
OAc
O
O
OH
OH
MeO
N
O
MeO
N
O
Br
O
3
CO2Me
N
O
NH
a
b
MeO
O
MeO
1
2
RVX-208
OH
OAc
O
HO
OH
CO2H
AcO
OAc
O
O
O
O
O
CO2Me
MeO
N
O
MeO
N
NH
O
N
c
MeO
O
MeO
O
M4
4
Scheme 1. Chemical synthesis of metabolite M4 (see Materials and methods for details). a: NaH, DMF, chloromethylpivalate; b: methylene chloride, AgO; c: NaOMe, MeOH.
MachereyeNagel (MN) Alugram SIL G/UV254 silica gel 60 plates
with fluorescent indicator UV254 (catalog #818 133) or Sigmae
Aldrich silica gel plates (catalog #Z193291-1PAK) using phospho-
molybdic acid in absolute ethanol as a developing agent. NMR
spectra were recorded on a 400 MHz Varian Mercury spectrometer
or a 500 MHz Bruker Avance System, using residual undeuterated
solvent as an internal reference. The low resolution mass spectra
(MS) were recorded on a Water Micromass ZQ using electrospray
ionization-ion trap (ESI) unless otherwise stated. MS/MS analysis
was carried out on an API 3200 system (Applied Biosystems). Q1
was set up to select a precursor ion at desired m/z, pass the ion
through a hexapole collision cell, and the Q3 analyzer then scanned
the product ions in the mass range from 0 to 600 amu. Nitrogen was
used as the collision gas, and the collision energy was scanned to
100 V.
4.1.1. Reactions with mammalian liver microsomes and S9 fractions
A 10 mM stock solution of RVX-208 [7] in DMSO was prepared
and used in these experiments. The incubation mixtures, prepared
in duplicate, consisted of 0.50 mg microsomal protein, 100 mM
potassium phosphate buffer, pH 7.4, 100 mM test compound, and
the NADPH regenerating system (1.3 mM NADPþ, 3.3 mM glucose-
6-phosphate, 0.4 U/mL glucose-6-phosphate dehydrogenase, and
3.3 mM magnesium chloride), UDPGA (5 mM), and D-saccharic acid
lactone (5 mM) in a final volume of 0.5 mL. The reaction mixture
minus the microsomal protein or S9 fraction was pre-warmed for
5 min at 37 ꢀC. In experiments with S9 fractions, 0.1 mL of the S9
fraction suspension was added directly to the reaction mixture. In
experiments with microsomes, prior to addition to the reaction
mixture, ice-cold microsomes were pre-incubated for 20 min with
alamethicin (4 mL of alamethicin stock solution in DMSO, 1.5 mg/
Fig. 4. LC/MS/MS and fragmentation analysis of purified metabolite M5. Panels: A, UV trace (detection at 230 nm); B, MS/MS spectrum.

126
Y.L. Khmelnitsky et al. / European Journal of Medicinal Chemistry 64 (2013) 121e128
to 100% B in 20 min, followed by an isocratic hold for 1 min at a
O
flow rate of 0.35 mL/min. The column was re-equilibrated for
10 min after programming back to the starting solvent mixture
over 0.5 min. UV data were acquired at 230 nm with an injection
O
OH
OH
a
OHC
N
OHC
MeO
volume of 10 mL of sample solution. The mass spectrometer was
5
6
operated in positive ion mode with spray voltage set at 5300 V.
The ion source temperature was set at 425 ꢀC. The 0.35 mL/min
effluent from the HPLC column was directed to an ESI ion source
without splitting after UV detection. The mass scan was performed
in the range from 200 to 750 amu. The retention time of RVX-208
was 12.3 min.
O
NH2
O
OH
NH2
MeO
MeO
O
7
NH
b
M5
MeO
O
4.1.4. LC/MS analysis for monitoring testosterone-based reactions
LC/MS analysis was performed on a PE SCIEX API 2000 system
with a PDA detector. Chromatography was done at room temper-
Scheme 2. Chemical synthesis of metabolite M5 (see Materials and methods for de-
tails). a: Bromoacetic acid, NaOH, water; b: NaHSO3, pTSA, DMAC.
ature using a Waters SunFire C18 column (2.1 ꢁ 50 mm, 3.5
mm)
30 mL, were mixed with 0.2 mL of microsomal suspension) and then
with the mobile phase initially composed of 10% of solvent A (0.1%
formic acid in water) and 90% of solvent B (0.1% formic acid in
acetonitrile). Elution was performed with a linear gradient from 10
to 30% B in 12 min, then from 30 to 100% B in 0.5 min, and isocratic
hold for 2 min at a flow rate of 0.35 mL/min. The column was re-
equilibrated for 8 min after programming back to the starting sol-
vent mixture over 0.5 min. UV data were acquired with total
added to initiate the reaction. After 1 h, 6 h, or 24 h incubation in a
shaking water bath at 37 ꢀC, the reactions were stopped by the
addition of an equal volume of methanol. After incubation on ice for
15 min, samples were centrifuged (10,000g, 15 min, 4 ꢀC) to remove
precipitated protein. The supernatants were filtered using a TeflonÒ
syringe filter (4 mm PTFE, 0.45 mm) from Whatman, Inc. (Clifton, NJ)
followed by evaporation to dryness in a Centrivap concentrator and
Freeze Dry system (both Labconco, Kansas City, MO). Sample resi-
dues were resuspended in 80 mL of 50% (v/v) methanol, mixed for
wavelength count (TWC) and the injection volume was 20 mL of
sample solution. The mass spectrometer was operated in positive
ion mode with spray voltage set at 5400 V. The ion source tem-
perature was set at 450 ꢀC. The 350
mL/min effluent from the HPLC
15 s on a vortex mixer, and sonicated for 30 s followed by centri-
fugation (20,000g, 30 s, room temperature). The supernatant was
then transferred to HPLC vials for LC/MS analysis.
In experiments to optimize the reaction conditions for the
synthesis of metabolite M4, stock solutions of RVX-208 in DMSO
with concentrations of 0.2 M, 0.05 M, and 0.02 M were prepared
and used to obtain final substrate concentrations of 2 mM, 0.5 mM,
and 0.2 mM, respectively, after 100-fold dilution. The concentration
of microsomal protein in the reaction mixture was 1 mg/mL or
4 mg/mL; the concentration of UDPGA was 5 mM or 25 mM.
column was directed to an ESI ion source without splitting after UV
detection. The retention time of 6
13.8 min.
b-hydroxytestosterone was
4.1.5. Biocatalytic synthesis and purification of metabolite M4
All experiments were carried out with the 0.1 M stock solution
of RVX-208 in DMSO. Six identical incubations were performed in
parallel in 20 mL scintillation vials, each containing 10 mL of the
reaction mixture. Each reaction consisted of 20 mg rabbit liver S9
protein, 100 mM potassium phosphate buffer, pH 7.4, 1 mM test
compound, the NADPH regenerating system (1.3 mM NADPþ,
3.3 mM glucose-6-phosphate, 0.4 U/mL glucose-6-phosphate de-
hydrogenase, and 3.3 mM magnesium chloride), and 10 mM
UDPGA in a final volume of 10 mL. After 24 h incubation in a shaker
at 37 ꢀC, the reaction mixtures were combined, diluted 2-fold with
cold methanol, centrifuged, and the supernatant was diluted five-
fold with water and passed over a solvated and equilibrated All-
tech C18 SPE (4 g) cartridge. The column was washed with 20 mL of
acetonitrile and then equilibrated with 20 mL of water. Following
sample loading, the absorbent was washed with 40 mL of water.
The elution was then performed using acetonitrile (3 ꢁ 20 mL),
which allowed complete desorption of RVX-208, whereas metab-
olite M4 was not eluted. Final elution was performed using 8 mL of
50% (v/v) acetonitrile. The eluate was then injected in 4 mL portions
on a Shimadzu LC8A preparative HPLC system consisting of two
Shimadzu LC8A pumps, a Shimadzu SPD-10A UV detector, and a
SCL-10A system controller. Chromatography was accomplished at
room temperature using a Waters Sunfire Prep C18 OBD column
4.1.2. Reactions with recombinant UDP-glucuronyl transferases
A 10 mM stock solution of RVX-208 in methanol was prepared
and used in these experiments. Incubation mixtures consisted of
100 mM potassium phosphate buffer, pH 7.4, 100
mM test com-
pound, 3.3 mM magnesium chloride, 5 mM UDPGA, and protein
(from 0.025 mg/mL to 1 mg/mL, depending on the enzyme) in a
final volume of 0.5 mL. The reaction mixture minus the enzyme was
pre-warmed for 5 min at 37 ꢀC and the enzyme suspension was
added. After 1 h, 6 h, or 24 h incubation in a shaking water bath at
37 ꢀC, the reactions were stopped by the addition of an equal vol-
ume of methanol. After incubation on ice for 15 min, samples were
centrifuged (10,000g, 15 min, 4 ꢀC) to remove precipitated protein.
The supernatants were filtered using a TeflonÒ syringe filter (4 mm
PTFE, 0.45
mm) from Whatman, Inc. (Clifton, NJ) followed by
evaporation to dryness in a Centrivap concentrator and Freeze Dry
system (both Labconco, Kansas City, MO). Sample residues were
resuspended in 80 mL of 50% (v/v) methanol, mixed for 15 s on a
vortex mixer, and sonicated for 30 s followed by centrifugation
(20,000g, 30 s, room temperature). The supernatant was then
transferred to HPLC vials for LC/MS analysis.
(150 ꢁ 19 mm, 5
mm) with the mobile phase initially composed of
98% of solvent A (water with 0.1% TFA) and 2% of solvent B
(acetonitrile with 0.1% TFA). Elution was performed with a linear
gradient from 2 to 100% B in 20 min followed by an isocratic hold at
100% B for 1 min at a flow rate of 20 mL/min. The column was re-
equilibrated for 10 min after programming back to the starting
solvent mixture over 0.5 min. UV data were acquired at 230 nm.
Fractions were collected for the peak eluting at 9.5 min. The pooled
sample was concentrated to dryness first by evaporating acetoni-
trile under reduced pressure and then removing the residual water
4.1.3. LC/MS analysis for monitoring RVX-208 and its metabolites
LC/MS analysis was performed on a PE SCIEX API 150 system
with a PDA detector. Chromatography was done at room temper-
ature using a Waters SunFire C18 column (100 ꢁ 2.1 mm, 3.5
mm)
with the mobile phase initially composed of 98% of solvent A (0.1%
formic acid in water) and 2% of solvent B (0.1% formic acid in
acetonitrile). Elution was performed using a linear gradient from 2

Y.L. Khmelnitsky et al. / European Journal of Medicinal Chemistry 64 (2013) 121e128
127
by lyophilization on a Labconco freeze dryer to give 2.6 mg of M4 as
3H), 3.93 (s, 3H), 3.88e3.85 (m, 1H), 3.77 (s, 3H), 2.36 (s, 6H), 2.05 (s,
a white solid.
3H), 2.03 (s, 6H), 1.21 (s, 9H).
4.1.6. Biocatalytic synthesis and purification of metabolite M5
The reaction of RVX-208 with human liver microsomes was set
up as described above in the screening protocol. After 4 h incuba-
tion at 37 ꢀC, the reaction mixture was diluted 2-fold with cold
methanol, incubated on ice for 15 min, and centrifuged (10,000g,
15 min, 4 ꢀC) to remove precipitated protein. The supernatant was
4.2.3. 6-(2-(4-(5,7-Dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)-
2,6-dimethylphenoxy)-ethoxy)-3,4,5-trihydroxytetrahydropyran-2-
carboxylic acid (M4)
A 0.05 M solution of sodium methoxide in methanol (129 mL,
6.40 mmol) was added to 3,4,5-triacetoxy-6-(2-(4-(3-(2,2-
dimethylpropionyloxymethyl)-5,7-dimethoxy-4-oxo-3,4-dihydro
quinazolin-2-yl)-2,6-dimethylphenoxy)ethoxy)tetrahydropyran-2-
carboxylic acid methyl ester (4) (0.90 g, 1.1 mmol) at 0 ꢀC under
nitrogen. The reaction progress was monitored by TLC and mass
spectroscopy. After disappearance of the starting material, water
(0.5 mL) was added to hydrolyze the methyl ester over 2e3 h at
room temperature. The reaction mixture was treated with amberlite
Hþ until pH became neutral. The amberlite was filtered off and
washed with methanol. The crude material was purified by Biotage
filtered using a TeflonÒ syringe filter (4 mm PTFE, 0.45
Whatman, Inc. (Clifton, NJ) and injected (20 L) on an analytical
mm) from
m
HPLC using the same gradient as described above for LC/MS anal-
ysis of RVX-208 and its metabolites. The fractions corresponding to
metabolite M5 (retention time 13.5 min) were collected and sub-
mitted to MS/MS analysis.
4.2. Chemical synthesis
reverse phase column chromatography (KP-C18-HS, 35e70 mm,
90 A; 25/75 to 40/60 gradient of MeOH/H2O as an eluent; column
eluted until compound was isolated as monitored by TLC) to give 6-
(2-(4-(5,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)-2,6-dim
ethylphenoxy)ethoxy)-3,4,5-trihydroxytetrahydropyran-2-carbox
ylic acid (M4) as a white solid. Yield: 186 mg (34%). Mp 208.6e
4.2.1. 2,2-Dimethylpropionic acid 2-(4-(2-hydroxyethoxy)-3,5-
dimethylphenyl)-5,7-dimethoxy-4-oxo-4H-quinazolin-3-ylmethyl
ester (2)
To a stirred solution of 2-(4-(2-hydroxyethoxy)-3,5-dimeth
ylphenyl)-5,7-dimethoxy-3H-quinazolin-4-one (1; RVX-208) (3.0 g
, 8.1 mmol) in DMF (75 mL) under nitrogen atmosphere, sodium
hydride (60% in mineral oil, 0.39 g, 9.7 mmol) was added in small
portions at room temperature. The reaction mixture was stirred for
1 h, then chloromethylpivalate (1.76 mL,12.1 mmol) was added, and
stirring was continued at room temperature for 24 h. The reaction
was quenched with water and the mixture was extracted with ethyl
acetate. The crude material was purified bycolumn chromatography
(Silica Gel 230e400 mesh; 7/3 methylene chloride/ethyl acetate as
eluent; column eluted until compound was isolated as monitored by
TLC) to give 2,2-dimethylpropionic acid 2-(4-(2-hydroxyethoxy)-
3,5-dimethylphenyl)-5,7-dimethoxy-4-oxo-4H-quinazolin-3-
ylmethyl ester (2) as a white solid. Yield: 2.2 g (56%). 1H NMR
210.8 ꢀC; UV (
l
, log ε): 211 (1.70), 258 (1.68) nm; Purity by HPLC:
97.7%; 1H NMR (400 MHz, CD3OD):
d
7.70 (s, 2H), 6.79 (d, J ¼ 2.2 Hz,
1H), 6.55 (d, J ¼ 2.2 Hz,1H), 4.42 (d, J ¼ 7.7 Hz,1H), 4.32e4.25 (m,1H),
4.16e4.09 (m, 1H), 4.08e4.03 (m, 1H), 4.02e3.95 (m, 1H), 3.92 (s,
3H), 3.91 (s, 3H), 3.59 (d, J ¼ 9.0 Hz, 1H), 3.50e3.40 (m, 1H), 3.28 (d,
J ¼ 9.0 Hz, 1H), 2.40 (s, 6H). Exchangeable protons (CO2H, OH and
NH) are not detected. MS (ESþ) m/z: 547.24 (Mþ1). Analysis calcu-
lated for C26H30N2O11$4H2O (618.52), %: C 50.48; H 6.19; N 4.53.
Found, %: C 50.11; H 5.55, N 4.80. The material (M4) was hydroscopic
and prolonged drying did not reduce water content. Water content
by Karl Fisher varied between 5.5 and 10.7%.
4.2.4. 4-Formyl-2,6-dimethylphenoxyacetic acid (6)
(400 MHz, CDCl3):
J ¼ 2.4 Hz, 1H), 6.39 (s, 2H), 3.97 (m, 4H), 3.95 (s, 3H), 3.93 (s, 3H),
2.40 (s, 6H), 2.22 (t, J ¼ 5.8 Hz, 1H), 1.21 (s, 9H).
d
8.20 (s, 2H), 6.95 (d, J ¼ 2.4 Hz, 1H), 6.47 (d,
A solution of sodium hydroxide (2.5 g, 63 mmol) in water
(65 mL) was added to a mixture of bromoacetic acid (5.3 g,
38 mmol) and 3,5-dimethyl-4-hydroxybenzaldehyde (5) (1.9 g,
13 mmol) in water (30 mL). The reaction mixture was stirred at
100 ꢀC for 24 h, then the solution was acidified (pH w2) with conc.
HCl. The resulting brown solid was isolated, washed with water,
dried, and purified by column chromatography (Silica Gel 230e
400 mesh; 0e10% gradient of methanol in methylene chloride as an
eluent; column eluted until compound was isolated as monitored
by TLC) to give 4-formyl-2,6-dimethylphenoxyacetic acid (6) as a
light brown solid. Yield 0.40 g (15%). 1H NMR (400 MHz, CDCl3):
4.2.2. 3,4,5-Triacetoxy-6-(2-(4-(3-(2,2-
dimethylpropionyloxymethyl)-5,7-dimethoxy-4-oxo-3,4-
dihydroquinazolin-2-yl)-2,6-dimethylphenoxy)ethoxy)-
tetrahydropyran-2-carboxylic acid methyl ester (4)
To a stirred solution of 2,2-dimethylpropionic acid 2-(4-(2-
hydroxyethoxy)-3,5-dimethylphenyl)-5,7-dimethoxy-4-oxo-4H-qui
nazolin-3-ylmethyl ester (2) (3.10 g, 6.40 mmol) in methylene chlo-
ꢀ
ride (76 mL), 4 A molecular sieves (15.4 g) were added under nitro-
d 9.90 (s, 1H), 7.59 (s, 2H), 4.54 (s, 2H), 2.39 (s, 6H).
gen. The resulting mixture was stirred for 5 min before commercially
available (2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-bromotetrahydropyra
n-2-carboxylic acid methyl ester (3) (2.54 g, 6.40 mmol) was added
followed by silver (I) oxide (4.50 g,19.3 mmol). The reaction mixture
was stirred at room temperature in the dark for 3 days. The solid was
filtered off and was washed with methylene chloride. The combined
filtrates were concentrated and the crude material was purified by
column chromatography (Silica Gel 230e400 mesh; 7/3 methylene
chloride/ethyl acetate as eluent; column eluted until compound was
isolated as monitored by TLC) followed by crystallization from
acetone/hexanes (15 mL/25 mL) to give 3,4,5-triacetoxy-6-(2-(4-(3-
(2,2-dimethylpropionyloxymethyl)-5,7-dimethoxy-4-oxo-3,4-dihy
droquinazolin-2-yl)-2,6-dimethylphenoxy)ethoxy)tetrahydropyran
-2-carboxylic acid methyl ester (4) as a white solid. Yield: 1.7 g (34%).
4.2.5. 2-(4-(5,7-Dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)-
2,6-dimethylphenoxy)-acetic acid (M5)
To a solution of 2-amino-4,6-dimethoxybenzamide (7) (0.15 g,
0.76 mmol) in DMAC (5 mL) were added 4-formyl-2,6-dimethyl-
phenoxyacetic acid (6) (0.16 g, 0.76 mmol), sodium hydrogen sulfite
(Assay > 58.5%, 0.150 g, 0.84 mmol) and p-toluenesulfonic acid
monohydrate (15 mg, 0.076 mmol). The reaction mixture was
stirred at 150 ꢀC for 3 h, cooled to room temperature, and water
(40 mL) was added. The yellow precipitate was filtered, washed
with water and small amount of methanol, then triturated with 10%
methanol in diethyl ether to give 0.084 g of compound which was
further purified by preparative HPLC to give 47 mg (16%) of M5 as a
white solid. Selected data for M5: Mp 281e282 ꢀC; UV (
(2.58), 261 (2.80) nm; Purity by HPLC: 96.2%; 1H NMR (400 MHz,
DMSO-d6): 12.96 (br s, 1H), 11.85 (s, 1H), 7.90 (s, 2H), 6.74 (d,
J ¼ 2.2 Hz, 1H), 6.52 (d, J ¼ 2.2 Hz, 1H), 4.46 (s, 2H), 3.89 (s, 3H), 3.84
l, log ε): 209
1H NMR (400 MHz, CDCl3):
d
8.18 (s, 2H), 6.95 (d, J ¼ 2.2 Hz,1H), 6.47
(d, J ¼ 2.2 Hz,1H), 6.39 (s, 2H), 5.33e5.24 (m, 2H), 5.14e5.07 (m,1H),
d
4.79 (d, J ¼ 7.6 Hz,1H), 4.21e4.12 (m,1H), 4.04e3.96 (m, 3H), 3.95 (s,

128
Y.L. Khmelnitsky et al. / European Journal of Medicinal Chemistry 64 (2013) 121e128
(s, 3H), 2.31 (s, 6H). 13C NMR (100 MHz, DMSO-d6):
d
170.8, 164.9,
References
161.7, 160.4, 158.6, 153.8, 153.1, 131.3 (2 carbons), 129.0 (2 carbons),
128.3, 105.4, 101.9, 98.3, 69.5, 56.6, 56.3, 16.8 (2 carbons).
[1] D. Bailey, R. Jahagirdar, A. Gordon, A. Hafiane, S. Campbell, S. Chatur,
G.S. Wagner, H.C. Hansen, F.S. Chiacchia, J. Johansson, L. Krimbou, N.C.W. Wong,
J. Genest, J. Am. Coll. Cardiol. 55 (2010) 2580e2589.
[2] S.J. Nicholls, A. Gordon, J. Johansson, K. Wolski, C.M. Ballantyne,
J.J.P. Kastelein, A. Taylor, M. Borgman, S.E. Nissen, J. Am. Coll. Cardiol. 57
(2011) 1111e1119.
Acknowledgments
This research was sponsored by Resverlogix Corp. We thank Dr.
Charles Jensen for critically reviewing the manuscript.
[3] V.V. Mozhaev, L.V. Mozhaeva, P.C. Michels, Y.L. Khmelnitsky, Drug Metab. Dis-
pos. 36 (2008) 1998e2004.
[4] J.F. Levesque, E. Templeton, L. Trimble, C. Berthelette, N. Chauret, Anal. Chem. 77
(2005) 3164e3172.
[5] Q. Wang, R. Jia, C. Ye, M. Garcia, J.B. Li, I.J. Hidalgo, In Vitro Cell. Dev. Biol. Anim.
41 (2005) 97e103.
[6] M.D. Green, T.R. Tephly, Drug Metab. Dispos. 24 (1996) 356e363.
[7] H.C. Hansen, US Patent 8,053,440.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.03.062.