Drug metabolism and pharmacokinetics of 4-substituted methoxybenzoyl-aryl- thiazoles

Article abstract of DOI:10.1124/dmd.110.034348

Tubulins are some of the oldest and most extensively studied therapeutic targets for cancer. Although many tubulin polymerizing and depolymerizing agents are known, the search for improved agents continues. We screened a class of tubulins targeting small

Full text of DOI:10.1124/dmd.110.034348

Supplemental material to this article can be found at:
http://dmd.aspetjournals.org/content/suppl/2010/07/30/dmd.110.034348.DC1.html
0090-9556/10/3811-2032–2039$20.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
DMD 38:2032–2039, 2010
Vol. 38, No. 11
34348/3631113
Printed in U.S.A.
Drug Metabolism and Pharmacokinetics of
4-Substituted Methoxybenzoyl-aryl-thiazoles
□
S
Chien-Ming Li, Yan Lu, Ramesh Narayanan, Duane D. Miller, and James T. Dalton
GTx Inc., Memphis, Tennessee (C.-M.L., R.N., D.D.M., J.T.D.); Division of Pharmaceutics, College of Pharmacy, The Ohio State
University, Columbus, Ohio (C.-M.L., J.T.D.); and Department of Pharmaceutical Sciences, University of Tennessee Health
Science Center, Memphis, Tennessee (Y.L., D.D.M.)
Received May 7, 2010; accepted July 30, 2010
ABSTRACT:
Tubulins are some of the oldest and most extensively studied metabolic processes, including ketone reduction, demethylation,
therapeutic targets for cancer. Although many tubulin polymeriz- combination of ketone reduction and demethylation, and hydroxy-
ing and depolymerizing agents are known, the search for improved lation in human liver microsomes. Metabolite identification studies
agents continues. We screened a class of tubulins targeting small revealed that the ketone and the methoxy groups of SMART-H
molecules and identified 4-(3,4,5-trimethoxybenzoyl)-2-phenyl- were most labile and that ketone reduction was the dominant
thiazole (SMART-H) as our lead compound. SMART-H inhibited the metabolism reaction in human liver microsomes. We designed and
proliferation of a variety of cancer cells in vitro, at subnanomolar tested four derivatives of SMART-H to improve the metabolic sta-
IC50, and in vivo, in nude mice xenografts, with near 100% tumor bility. The oxime and hydrazide derivatives, replacing the ketone
growth inhibition. Metabolic stability studies with SMART-H in liver site, demonstrated a 2- to 3-fold improved half-life in human liver
microsomes of four species (mouse, rat, dog, and human) revealed microsomes, indicating that our prediction regarding metabolic
half-lives between <5 and 30 min, demonstrating an interspecies stability of SMART-H can be extended by blocking ketone reduc-
variability. The clearance predicted based on in vitro data corre- tion. These studies led us to the next generation of SMART com-
lated well with in vivo clearance obtained from mouse, rat, and dog
pounds with greater metabolic stability and higher pharmacologic
in vivo pharmacokinetic studies. SMART-H underwent four major potency.
Introduction
vivo in wild-type and resistant cancer cells (C. M. Li, Z. Wang, Y. Lu,
W. Li, S. Ahn, R. Narayanan, J. D. Kearbey, D. N. Parke, D. D.
Miller, and J. T. Dalton, manuscript submitted for publication).
Pharmacokinetics and metabolism play important roles in lead
optimization as they affect the exposure and thus efficacy of drugs
(Pelkonen et al., 2005). Many molecules that were demonstrated to be
potent in vitro failed in vivo because of poor pharmacokinetic (PK)
properties. A potential drug candidate is expected to be cleared slowly
Cancer is the second leading cause of death in the United States,
with 500,000 people estimated to die from the disease in 2010 (Cancer
Facts and Figures 2010, American Cancer Society Website, http://
www.cancer.org). Despite multiple pathways being used for the de-
velopment of novel therapeutic agents, tubulins remain an attractive
target to treat cancer (Carlson, 2008; Zhao et al., 2009). However,
most of the tubulin polymerizing and depolymerizing agents are
P-glycoprotein substrates, leading to the development of resistance from the system and to have sufficient exposure to elicit its action.
over a prolonged period of treatment (Perez, 2009).
Identification of chemical or functional groups or so-called “soft
spots” is a key first step in the design of compounds with better
metabolic stability (Zhang et al., 2007). Liver microsomes, which
contain several key enzymes such as cytochrome P450s, flavin mono-
oxygenases, and glucuronosyltransferases, all required for drug me-
tabolism, are widely used for in vitro metabolic stability studies. In
vitro microsomal stability assays not only facilitate the selection of
compounds with greater metabolic stability but are also useful to
identify metabolites of the parent molecule (Huang and Ho, 2009).
In this study, we identified the metabolites and PK properties of one
of the SMART compounds, SMART-H, using in vitro liver micro-
somes and PK studies. We also identified the labile sites and devel-
Recently our group developed a series of 4-substituted methoxy-
benzoyl-aryl-thiazole (SMART) compounds that bind to the colch-
icine-binding site and inhibit tubulin polymerization and cancer cell
growth at low nanomolar concentrations (Lu et al., 2009). In addition,
the SMART compounds were not substrates of P-glycoprotein and
retained potent anticancer activity demonstrated both in vitro and in
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.034348.
□S The online version of this article (available at http://dmd.aspetjournals.org)
contains supplemental material.
ABBREVIATIONS: SMART, 4-substituted methoxybenzoyl-aryl-thiazole; SMART-H, 4-(3,4,5-trimethoxybenzoyl)-2-phenyl-thiazole; DMSO,
dimethyl sulfoxide; LC, liquid chromatography; MS/MS, tandem mass spectrometry; THF, tetrahydrofuran; MS, mass spectrometry; ESI,
electrospray ionization; SMART-213, pentafluorophenyl-(2-phenyl-thiazol-4-yl)-methanone; SMART-176A, [(2-phenyl-thiazol-4-yl)-(3,4,5-
trimethoxyphenyl)-methylene]-hydrazine; SMART-173A, (2-phenylthiazol-4-yl)(3,4,5-trimethoxyphenyl)methanone oxime; SMART-329,
2-phenyl-4-(3,4,5-trimethoxyphenyl)thiazole.
2032

DRUG METABOLISM AND PHARMACOKINETICS OF SMART COMPOUNDS
2033
jugular vein catheters at 10, 20, and 30 min and 1, 2, 4, 8, 12, 24, and 48 h. All
syringes and vials were heparinized for blood collection. Plasma samples were
obtained as described previously.
A female beagle dog weighing 11.2 kg was used in this study. The animal
was fasted overnight and until 2 h after drug administration. The dog was given
a single intravenous dose of SMART-H (0.25 mg/kg, in PEG300-DMSO, 1:4).
Blood was drawn at 10, 20, and 30 min and 1, 2, 4, 8, 12, 24, 48, and 96 h.
Plasma samples were obtained as described previously.
SMART-H was extracted from 100 ␮l of plasma with 200 ␮l of acetonitrile
containing 100 nM internal standard. The samples were thoroughly mixed and
centrifuged, and the organic extract was transferred to an autosampler for
LC-MS/MS analysis. The PK parameters were determined using noncompart-
mental analysis (WinNonlin; Pharsight, Mountain View, CA).
oped the next generation of SMART compounds having better met-
abolic stability with little or no impact on potency.
Materials and Methods
Metabolic Stability Studies. Incubations for metabolic stability studies
were conducted in a 1-ml reaction volume containing 0.5 ␮M (final concen-
tration) SMART-H or other test compounds and 1 mg/ml microsomal protein
(mouse, rat, dog, and human liver microsomes; XenoTech, LLC, Kansas City,
MO) in reaction buffer [0.2 M phosphate buffer solution (pH 7.4), 1.3 mM
NADPϩ, 3.3 mM glucose 6-phosphate, and 0.4 U/ml glucose-6-phosphate
dehydrogenase] at 37°C in a shaking water bath. SMART-H, at 50 ␮M
concentration, with the above-mentioned conditions, was used for metabolite
identification studies. For glucuronidation studies, 2 mM UDP-glucuronic acid
(Sigma-Aldrich, St. Louis, MO) cofactor in deionized water was incubated
with 8 mM MgCl2, 25 ␮g of alamethicin (Sigma-Aldrich) in deionized water,
and NADPH-regenerating solutions (BD Biosciences, San Jose, CA) as de-
scribed previously. The total DMSO concentration in the reaction solution was
approximately 0.5% (v/v). Aliquots (100 ␮l) from the reaction mixtures were
sampled at 5, 10, 20, 30, 60, and 120 min, and acetonitrile (150 ␮l) containing
100 nM internal standard (an analog of SMART-H) was added to quench the
reaction and to precipitate the proteins. Samples were then centrifuged at
4000g for 15 min at room temperature, and the supernatant was analyzed
directly by LC-MS/MS.
Protein Binding Assay. Plasma protein binding studies of SMART-H were
conducted by the ultrafiltration technique. One milliliter of mouse, rat, dog,
and human plasma (Thermo Fisher Scientific, Waltham, MA) samples was
spiked with 5 ␮l of 100 ␮M SMART-H and incubated at 37°C for 30 min
before ultrafiltration. Samples (400 ␮l) were transferred to Amicon centrifugal
filter devices (30-kDa molecular mass cutoff; Millipore Corporation, Billerica,
MA) and centrifuged at 14,000g for 20 min. An aliquot (50 ␮l) of the
ultrafiltrate was combined with 150 ␮l of acetonitrile containing an internal
standard for LC-MS/MS analysis. Protein binding of SMART-H in mouse, rat,
dog, and human microsomes was performed using 0.5 ␮M SMART-H and 1
mg/ml microsomal proteins in the absence of NADPH. The incubation con-
ditions and sample preparation were the same as those described for plasma
protein binding.
Prediction of In Vivo Clearance of SMART-H in Mouse, Rat, Dog, and
Human. In vivo clearance was predicted using the data obtained from meta-
bolic stability (half-life in liver microsomes) and protein binding in plasma and
liver microsomes studies. The intrinsic hepatic clearance (Cli, in vitro) of
SMART-H was determined using the equation: Cli, in vitro ϭ [0.693/(t1/2 in
minutes ꢀ protein concentration in milligrams per milliliter)]. The intrinsic
clearance was then scaled to predict clearance that would occur in the liver in
vivo. Scaling factors (milligrams of protein per gram of liver ꢀ gram of liver per
kilogram body weight) are 2400, 1815, and 1980 for rat, dog, and human,
respectively (Baarnhielm et al., 1986; Smith et al., 2008). In vivo intrinsic
hepatic clearance (Cli, h, milliliters per minute per kilogram body weight) in
liver was estimated by multiplying Cli, in vitro by the scaling factors. In vivo
hepatic clearance (Clh) was estimated by incorporating estimates of Cli, h, Qh,
Analytical Method. Sample solution (10 ␮l) was injected into an high-
performance liquid chromatography system (Agilent 1100 Series Chemstation;
Agilent Technologies, Santa Clara, CA). SMART-H and its metabolites were
separated on a narrow-bore C4 column (2.1 ϫ 150 mm, 5 ␮m; Varian Inc.,
Palo Alto, CA). Two gradient modes were used. For metabolic stability, the
gradient mode was used to achieve the separation of analytes using mixtures of
mobile phase A [acetonitrile-H2O (5:95%) containing 0.1% formic acid] and
mobile phase B (acetonitrile-H2O (95:5%) containing 0.1% formic acid) at a
flow rate of 300 ␮l/min. Mobile phase A was used at 55% from 0 to 0.5 min
followed by a linearly programmed gradient to 100% of mobile phase B within
2.5 min; 100% of mobile phase B was maintained for 1 min before a quick
ramp up to 55% mobile phase A. Mobile phase A was continued for another
8 min toward the end of analysis. For metabolite identification studies, a
slower gradient mode was used to achieve the separation of analytes with the
same flow rate and mobile phase A and B as described. Mobile phase A was
used at 20% from 0 to 1 min followed by a linearly programmed gradient to
100% of mobile phase B within 17 min; 100% of mobile phase B was
maintained for 2 min before a quick ramp up to 20% mobile phase A. Mobile
phase A was continued for another 25 min toward the end of analysis.
A triple-quadruple mass spectrometer (API Qtrap 4000; Applied Biosys-
tems/MDS SCIEX, Concord, ON, Canada) operating with a TurboIonSpray
source was used. The spraying needle voltage was set at 5 kV for positive
mode. Curtain gas was set at 10; gas 1 and gas 2 were set at 50, collision-
assisted dissociation gas at medium, and the source heater probe temperature
at 500°C. Data acquisition and quantitative processing were accomplished
using Analyst software (version 1.4.1; Applied Biosystems).
Cell Culture and Cytotoxicity Assay of Prostate Cancer. We examined
the antiproliferative activity of the SMART compounds in four human prostate
cancer cell lines, LNCaP, DU 145, PC-3, and PPC-1 (American Type Culture
Collection, Manassas, VA). Cells were cultured in RPMI 1640 (Mediatech,
Inc., Manassas, VA) supplemented with 10% fetal bovine serum (Mediatech)
and were maintained at 37°C in a humidified atmosphere containing 5% CO2.
Depending on cell types, 1000 to 5000 cells were plated into each well of
96-well plates and exposed to different concentrations of the compound of
interest for 96 h. At the end of the treatments, cell viability was measured using
the sulforhodamine B assay. Percentage of cell survival was plotted against
drug concentrations and the IC50 values (concentration that inhibited cell
growth by 50% of untreated control) were obtained by nonlinear regression
analysis using WinNonlin.
and fu into the well stirred model (venous equation): Clh ϭ [Qh ꢀ fu, p
ꢀ
(Cli, h/fu, m)]/[Qh ϩ fu, p ꢀ (Cli, h/ fu, m)] (Chiba et al., 2009). Qh, fu, p, and fu, m
represent hepatic blood flow, fraction unbound in plasma, and fraction un-
bound in microsomes, respectively.
Synthesis of SMART Compounds. All reagents were purchased from
Sigma-Aldrich, Fisher Scientific, AK Scientific (Mountain View, CA), and
Oakwood Products (West Columbia, SC) and were used without further
purification. Moisture-sensitive reactions were performed under an argon
atmosphere. Routine thin-layer chromatography was performed on aluminum-
backed Uniplates (Analtech, Newark, DE). NMR spectra were obtained on an
Bruker AX 300 (Bruker, Newark, DE) spectrometer or an Inova 500 spec-
trometer (Varian, Inc., Palo Alto, CA). Chemical shifts are reported as parts per
million relative to tetramethylsilane in CDCl3. Mass spectral data were col-
lected on a Bruker ESQUIRE electrospray/ion trap instrument in positive and
negative ion modes.
Pharmacokinetic Studies. All animal studies were conducted under the
auspices of a protocol reviewed and approved by the Institutional Laboratory
Animal Care and Use Committee of The University of Tennessee. SMART-H
(15 mg/kg) was dissolved in PEG300-DMSO (1:4) and administered once
intravenously into the tail vein of 6- to 8-week-old ICR mice (n ϭ 3 per each
time point; Harlan Inc., Indianapolis, IN). Blood samples were collected in
heparinized tubes via cardiac puncture under isoflurane anesthesia at 2, 5, 15,
and 30 min and 1, 2, 4, 8, 16, and 24 h after administration. Plasma samples
were collected by centrifugation at 8000g for 5 min and stored immediately at
Ϫ80°C for further analysis.
SMART-H was administered intravenously into the thoracic jugular vein
(catheters from Braintree Scientific Inc., Braintree, MA) of male Sprague-
Synthesis of Putative Metabolites (Reduction of the Carbonyl Group in
SMART-H). Synthesis of (2-phenyl-thiazol-4-yl)-(3,4,5-trimethoxyphenyl)-
Dawley rats (n ϭ 4; 254 Ϯ 4 g; Harlan, Indianapolis, IN) at 2.5 mg/kg (in methanol. SMART-H (355 mg, 1 mmol) was dissolved in anhydrous THF and
PEG300-DMSO, 1:4). An equal volume of heparinized saline was injected to cooled to Ϫ78°C. Lithium aluminum hydride (1 M solution in THF; 1 ml) was
replace the removed blood, and blood samples (250 ␮l) were collected via the added dropwise under argon protection. After completion of the reaction,

2034
LI ET AL.
2 ml of ethanol was added an aqueous solution (0.5 ml) of hydroxylamine
120
100
80
60
40
20
0
hydrochloride (34 mg, 0.49 mmol). Then 0.5 ml of 1 N NaOH was added
dropwise to the reaction mixture, and the mixture was stirred at 55°C for 3 h.
After completion of the reaction, the residue was absorbed on silica gel and
purified by column chromatography to give compound SMART-173A (26 mg;
50% yield). 1H NMR (300 MHz, DMSO-d6): ␦ 11.95 (s, 1H), 8.35, 8.34 (s, s,
1H), 7.91–7.89 (m, 2H), 7.50–7.44 (br, 3H), 7.34 (s, 1H), 6.85, 6.85 (s, s, 1H),
5.93 (d, 1H), 3.73, 3.73 (s, s, 6H), 3.71, 3.70 (s, s, 3H). MS (ESI) m/z 393.1
[M ϩ Na]ϩ, 368.9 [M Ϫ H]Ϫ.
SMART-329: synthesis of 2-phenyl-4-(3,4,5-trimethoxyphenyl)thiazole.
2-Bromo-1-(3,4,5-trimethoxyphenyl)ethanone was prepared as reported previ-
ously (Sun et al., 2004). To a 10-ml round-bottom flask with a magnetic stir bar
was added 2-bromo-1-(3,4,5-trimethoxyphenyl)ethanone (0.5 mmol) and eth-
anol (2.5 ml). Benzothioamide (0.5 mmol) was then added, and the mixture
was refluxed for 1 h. After completion of the reaction, the residue was
absorbed on silica gel and purified by flash chromatography on silica gel to
give SMART-329 (167 mg; 51% yield). 1H NMR (300 MHz, DMSO-d6): ␦
8.05–8.03 (m, 2H), 7.48–7.44 (m, 3H), 7.41 (s, 1H), 7.22 (s, 2H), 3.97 (s, 6H),
3.89 (s, 3H). MS (ESI) m/z 350.1 [M ϩ Na]ϩ.
0
20
40
60
80
100
120
minutes
FIG. 1. Phase I metabolic stability of SMART-H in human liver microsome in the
presence of NADPH (F) and in the absence of NADPH (E). ꢀ, phase I ϩ II
metabolic stability in the presence of NADPH and UDP-glucuronic acid. Values
represent the mean Ϯ S.D. of triplicate experiments.
Results
excess lithium aluminum hydride was destroyed with ethyl acetate and then
quenched with 20% H2SO4 solution. The organic layer was washed with brine,
dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure.
The residue was purified by column chromatography to give the compound as
light yellow crystals (252 mg; 71% yield). 1H NMR (300 MHz, CDCl3): ␦
7.96–7.93 (q, 2H), 7.44–7.42 (m, 3H), 6.97 (s, 1H), 6.76 (s, 2H), 5.93 (d, 1H),
3.86 (s, 9H). MS (ESI) m/z 380.1 [M ϩ Na]ϩ, 355.9 [M Ϫ H]Ϫ.
SMART-213: synthesis of pentafluorophenyl-(2-phenyl-thiazol-4-yl)-
methanone. To a solution of pentafluorophenylmagnesium bromide (0.5 M,
2.7 ml) in 2 ml of THF was charged a solution of 2-phenyl-4, 5-dihydrothia-
zole-4-carboxylic acid methoxymethylamide (227 mg, 0.915 mmol) in 3 ml of
THF at 0°C. The mixtures were stirred for 30 min until amides disappeared on
thin-layer chromatography plates. The reaction mixture was quenched with
saturated NH4Cl, extracted with ethyl ether, and dried with MgSO4. The
solvent was removed under reduced pressure to yield a crude product, which
was purified by column chromatography to obtain pure compound SMART-
213 (147 mg) as a yellow solid. Yield: 45.2%. 1H NMR (300 MHz, CDCl3):
␦ 8.30 (s, 1H), 7.82–7.80 (m, 2H), 7.40–7.37 (m, 3H). 13C NMR (75.5 MHz,
CDCl3): ␦ 178.7, 169.0, 154.3, 146.3, 144.8, 142.6, 140.2, 132.5, 131.1, 129.1,
128.3, 126.9. 19F NMR (282.4 MHz, CDCl3): ␦ Ϫ33.81, Ϫ44.89, Ϫ44.96. MS
(ESI) m/z 378.0 [M ϩ Na]ϩ.
SMART-176A: synthesis of [(2-phenyl-thiazol-4-yl)-(3,4,5-trimethoxyphe-
nyl)-methylene]-hydrazine. A solution of SMART-H (230 mg, 0.65 mmol) in 2
ml of CH2Cl2 was added to a hot solution of hydrazine in 3 ml of ethanol and
refluxed overnight. After completion of the reaction, the residue was absorbed on
silica gel and purified by column chromatography to give compound SMART-
176A as a mixture of isomers (136 mg; 57% yield). 1H NMR (500 MHz,
CDCl3): ␦ 8.01–7.99 (m, 2H), 7.50–7.48 (m, 3H), 7.46 (br, 2H), 7.34 (s, 1H),
6.82 (s, 2H), 5.93 (d, 1H), 3.87 (s, 3H), 3.85 (s, 6H). MS (ESI) m/z 370.1 [M ϩ H]ϩ.
SMART-173A: synthesis of (2-phenylthiazol-4-yl)(3,4,5-trimethoxyphe-
Metabolic Stability. Figure 1 shows the metabolic stability of
SMART-H in the presence and absence of NADPH and UDP-glucuronic
acid. In the absence of NADPH, more than 85% of the parent SMART-H
remained after 120 min, suggesting that metabolism of SMART-H was
NADPH-dependent. In the presence of NADPH, SMART-H had a half-
life of 17 min by phase I reaction, suggesting that SMART-H was rapidly
metabolized by phase I metabolic processes. The half-life (17 min) in the
presence of UDP-glucuronic acid was identical to that observed in its
absence (Fig. 1). Thus, SMART-H exhibited moderate to high in vitro
clearance in human liver microsomes exclusively through a phase I reaction.
Prediction of the In Vivo Clearance of SMART-H in Mouse,
Rat, Dog, and Human. Table 1 summarizes in vitro half-lives (in
liver microsomes), protein binding (in plasma and liver microsomes),
and clearance predictions in mouse, rat, dog, and human samples.
Half-lives were Ͻ5, 31, 19, and 17 min for mouse, rat, dog, and
human, respectively, indicating that SMART-H exhibited interspecies
variability in its metabolism. The protein-binding results indicated
that SMART-H is highly protein bound (Ͼ90% bound) in plasma and
liver microsomes of all four species. With high protein binding,
SMART-H was predicted to have low hepatic clearances and extraction
rates (Յ0.3) in rat, dog, and human. Mouse was an exception. SMART-H
was extremely unstable in mouse liver microsomes. Therefore, a high
clearance and extraction rate were predicted in this species. Encourag-
ingly, in vitro protein binding and metabolic stability further suggested
that humans will have a low hepatic extraction ratio and low clearance.
Pharmacokinetic Studies of SMART-H. A single dose intrave-
nyl)methanone oxime. To a suspension of SMART-H (50 mg, 0.14 mmol) in nous bolus of SMART-H was administered to ICR mice, Sprague-
TABLE 1
Prediction of in vivo hepatic clearance of SMART-H in mouse, rat, dog, and human from in vitro data
Hepatic blood flow values were referenced from Davies and Morris (1993).
Mouse
Rat
Dog
30
Human
Hepatic blood flow (ml/min/kg)
fu, p in plasma (%)
fu, m in liver microsomes (%)
t1/2, in liver microsomes (min)
Scaling factor
Scaling factor (mg of protein/g liver ꢀ g of liver/kg b.wt.)
Cli, in vitro
Cli, h
90
55
21
0.64
6.14
ϽϽ5
N.A.
N.A.
N.A.
N.A.
N.A.
High
0.55
6.20
31
2400
(54 ꢀ 45)
0.022
53.65
4.38
0.50
2.28
19
1815
(55 ꢀ 33)
0.036
66.20
9.78
0.22
3.55
17
1980
(77 ꢀ 25.7)
0.041
80.71
4.04
Clh (ml/min/kg)
Extraction rate, prediction
0.1
0.3
0.2
N.A., not available.

DRUG METABOLISM AND PHARMACOKINETICS OF SMART COMPOUNDS
2035
TABLE 2
Dawley rats, and a beagle dog to characterize the pharmacokinetics in
these species (Fig. 2). Their PK parameters are summarized in Table 2. In
vivo clearances were 130, 5.3, and 2.7 ml/min/kg for mouse, rat, and
dog, respectively (Table 2), indicating a high hepatic extraction ratio
(Ͼ0.7) for mouse and low extraction ratios (Ͻ0.3) for rat and dog.
These data were consistent with our predictions based on in vitro data,
suggesting that prediction of in vivo clearance based on in vitro
protein binding and metabolic stability was reliable. In mouse,
SMART-H clearance was higher than 90 ml/min/kg (the hepatic blood
flow rate in mice), suggesting that in addition to hepatic removal,
other degradation routes may be involved in the elimination of
SMART-H in this species. Although SMART-H exhibited high pro-
tein binding in mouse, it did not compensate for instability in mouse
liver microsomes. These data suggest that metabolic stability of
Pharmacokinetic parameters of SMART-H in mouse, rat, and dog
The half-life is presented as harmonic mean Ϯ pseudo-S.D. in rat.
Mouse
Rat
Dog
n
3
15
4.9
1.9
4
2.5
11 Ϯ 4
5.8 Ϯ 1.1
1
0.25
0.4
1.6
0.0004
Dose (mg/kg)
Volume of distribution, Vss (l/kg)
AUC (h ꢀ mg/ml)
Ke (minϪ1
)
0.0049 0.00032 Ϯ 0.000045
140
130
90
Ͼ1
Half-life, t1/2 (min)
2143 Ϯ 292
5.3 Ϯ 0.9
55
1866
2.7
30
0.1
Clearance (ml/min/kg)
Hepatic blood flow (ml/min/kg)
Extraction rate
0.1
AUC, area under the concentration-time curve.
SMART-H was critical for hepatic clearance. Intermediate volumes of liver microsomes to identify metabolically labile sites (i.e., soft spots)
distribution of 4.9 and 11 l/kg were obtained for mouse and rat. in the SMART-H pharmacophore. LC-MS/MS was used for metabo-
However, a smaller value of 0.4 l/kg was obtained in the dog, lite identification based on mass shifts compared with the molecular
suggesting that SMART-H also exhibited species difference in vol- ion [M ϩ H]ϩ and retention time shifts to the parent SMART-H. To
ume of distribution. SMART-H exhibited long half-lives (more than identify the potential metabolites, the product ion scan for each peak
24 h) in both rat and dog, whereas a short half-life was obtained in of interest was examined to obtain structural information. Four major
mouse because of its high clearance.
metabolites of SMART-H were identified in human liver microsomes.
Identification of Metabolites in Human Liver Microsomes. In Figure 3A shows the multiple reaction monitoring chromatography of
vitro metabolite identification studies were performed using human SMART-H with its four metabolites (M1–M4) after the 2-h incuba-
tion. Because of the high polarity exhibited by the hydroxyl groups,
all of the metabolites had a shorter retention time than SMART-H.
Two metabolites, M2 and M3, were present as isomers and were
separated based on the gradient chromatographic condition. The prod-
uct ion spectrum of SMART-H (Fig. 3B) had an abundant product ion
m/z 188 (loss of trimethoxybenzene). The major and most abundant
metabolite, M1 (m/z 358, mass shifts ϩ 2 Da, ketone reduction) (Fig.
3C), resulted in the fragment ion m/z 340 (loss of H2O). This metab-
olite was synthesized, and the retention time and product ion spectrum
were confirmed (data not shown). This metabolite demonstrated al-
tered fragment ion spectra compared with SMART-H and thus could
not be detected by either precursor ion or neutral loss scans. M2 (m/z
342, mass shifts Ϫ 14 Da, demethylation) (Fig. 3D) was the second
most abundant metabolite of SMART-H in human liver microsomes
with the same fragment ion pathway and product ion m/z as the parent
SMART-H. M2 was present as both the 3- and 4-demethylated me-
tabolites of SMART-H, which were separated by our chromatographic
conditions. However, we were unable to distinguish which of the
methoxy groups were demethylated. M3 (m/z 344, mass shift Ϫ 12
Da, ketone reduction and demethylation) (Fig. 3E) demonstrated a
fragmentation pattern similar to M1 and was apparently formed after
M1. The major fragment ion of M3 was m/z 326 (loss of H2O). M3
exhibited the shortest retention time because of the presence of two
hydroxyl groups. M4 (m/z 374, mass shift ϩ 16 Da, hydroxylation)
(Fig. 3F) was an oxidized metabolite, having a hydroxyl group on the
B-ring and fragmentation pattern similar to that of M1. The demeth-
ylated metabolite (M2) was the only one to be detected by the
precursor ion scan (precursor of m/z 188). On the contrary, other
metabolites exhibited different fragmentation patterns and could be
detected by neither precursor ion (precursor of 188) nor by natural
loss scan (loss of m/z 170) based on the fragmentation pattern of
SMART-H fragmentation pattern. The metabolite pathway of
SMART-H is summarized in Fig. 4.
Species-Specific Metabolism of SMART-H. We determined the
metabolite kinetics of SMART-H (0.5 ␮M) in the presence of liver
microsomes from mouse, rat, dog, and human. The parent,
SMART-H, and the four metabolites identified in human liver
microsomes were simultaneously monitored in each sample (Fig. 5). The
half-lives of SMART-H in microsomes from different species
FIG. 2. Pharmacokinetic studies of SMART-H in mouse (A), rat (B), and dog (C).
A single intravenous bolus administration was given with 15, 2.5, and 0.25 mg/kg
for mouse (n ϭ 3), rat (n ϭ 4), and dog (n ϭ 1), respectively. Bars, S.D.

2036
LI ET AL.
FIG. 3. Metabolite identification. SMART-H (50 ␮M) was incubated with 1 mg/ml human liver microsomes for 2 h. A, chromatography. B, MS/MS spectrum of parent
SMART-H. Four major MS/MS spectra were identified in M1 (C), M2 (D), M3 (E), and M4 (F).
varied over a broad range from Ͻ5 to 30 min (Fig. 5; Table 1). We pounds (SMART-329, -173A, and -176A) with modified or direct
found that the metabolite arising from ketone reduction (M1) was
dominant only in human liver microsomes. We then increased the
SMART-H concentration from 0.5 to 50 ␮M and incubated with 1
mg/ml human and rat liver microsomes to saturate the enzymes and
maximize the metabolite signals (Fig. 6). The demethylated me-
tabolite, M2, was found in microsomal incubation using both rat
and human liver microsomes. A significant difference in M1 and M3
levels was observed between the two species. In rat liver microsomes
(Fig. 6B), the primary metabolite was the demethylated metabolite (M2).
M1 could be found in rat liver microsomes only when a higher concen-
tration of SMART-H was used. However, the amount of M1 in rat liver
microsomes was much less than that in human liver microsomes (Fig. 6).
In addition, the relative amount of the demethylated metabolite (M2) in
rat liver microsomes was greater than that in the human liver microsomes.
These studies confirmed that M1 was a dominant metabolite only in
human liver microsomes.
linkage between the trimethoxyphenyl ring and thiazole ring were
made and tested, and a single compound (SMART-213) with a pen-
tafluoro phenyl ring was made to replace the trimethoxy substituents
(Fig. 7). The inclusion of a pentafluoro phenyl ring (SMART-213)
failed to increase metabolic stability in human liver microsomes (Fig. 8),
suggesting that demethylation may not be critical for metabolic sta-
bility or that SMART-213 was susceptible to other new metabolic
pathways. We also examined the metabolic stability of analogs con-
taining electron-withdrawing substituents such as 4-fluorophenyl or
3,4,5-trifluorophenyl to replace the trimethoxyphenyl ring. All of
these derivatives failed to increase the metabolic stability (data not
shown). A dramatic increase in the metabolic stability was observed
with compound SMART-176A which contains CAN–NH2 instead of
CAO found in SMART-H. This compound exhibited 3-fold longer
stability than that of our lead compound, SMART-H, in human liver
microsomes. SMART-173A and SMART-329 also demonstrated an
increase in metabolic stability by 2-fold. These data indicate that the
enhanced metabolic stability observed in human liver microsomes
correlated with the modification of the labile ketone site of
SMART-H.
Blockage of Soft Spots of SMART-H to Increase the Metabolic
Stability in Human Liver Microsomes. The two major metabolites
in human liver microsomes, M1 (ketone reduction) and M2 (demeth-
ylation), identified the carbonyl and methoxy groups as the most labile
sites (i.e., soft spots) to metabolic conversion. We thus designed the
next generation of SMART compounds to include substitutions aimed
Table 3 shows IC50 values of SMART-213, -173A, -176A, and
to improve the metabolic stability of these soft spots. Three com- -329 for antiproliferative effects on four prostate cancer cell lines,

DRUG METABOLISM AND PHARMACOKINETICS OF SMART COMPOUNDS
2037
FIG. 4. Metabolite profile of SMART-H in human liver microsome.
LNCaP, PC-3, DU 145, and PPC-1. SMART-173A retained potent and dog to study the in vitro-in vivo relationship of SMART-H
activity with an average IC50 value of 143 nM. SMART-176A had metabolism. The in vitro metabolic stability of SMART-H was ex-
less potent ability (IC50 value of 1250 nM) to inhibit cell prolif-
eration. With the removal of the labile functional group (CAO),
SMART-329 demonstrated increased metabolic stability; however,
it lost the ability to inhibit cell growth (IC50 value was Ͼ10,000
nM). SMART-213 failed to either inhibit cell growth or improve
metabolic stability. These results indicate that blocking ketone
reduction increased metabolic stability in human liver microsomes
but that in vitro anticancer activity could only be partially retained
by substituting with the oxime (SMART-173A).
tremely poor in mouse liver microsomes, resulting in the high clear-
ance value obtained during in vivo pharmacokinetic studies in mice.
The in vivo total clearance in rats was 5.3 ml/min/kg, which is close
to our prediction based on in vitro microsomal stability studies. The in
vivo total clearance in dog was 2.7 ml/min/kg, which was somewhat
smaller than our prediction. In mouse, rat, and dog pharmacokinetic
studies, the distributional kinetics, not the long terminal phase, seem
to define most of the area under the concentration-time curve (Fig. 2).
The in vitro value for dogs may have been overestimated as a result
of errors in the scaling factor or determination of fraction unbound in
plasma and liver microsomes, whereas the predicted clearance value
Discussion
In vitro microsomal stability assays represent a high-throughput for mice slightly underestimated the in vivo clearance apparently as a
method to predict in vivo hepatic clearance. Here, we used mice, rats, result of extrahepatic drug clearance. The in vivo clearance values for
FIG. 5. Metabolite profile in species. Substrate
(0.5 ␮M) was incubated with 1 mg/ml microso-
mal protein of human (A), rat (B), mice (C), and
dog (D). Four metabolites with the parent were
measured by LC-MS/MS (n ϭ 3). Bars, SD.

2038
LI ET AL.
TABLE 3
Antiproliferation of SMART compounds on prostate cancer cell lines
n ϭ 3.
IC50
LNCaP
PC-3
DU 145
PPC-1
nM
SMART-H
33
26
73
41
SMART-213
SMART-329
SMART-173A
SMART-176A
Ͼ10,000
Ͼ10,000
189
Ͼ10,000
Ͼ10,000
120
Ͼ10,000
Ͼ10,000
102
Ͼ10,000
Ͼ10,000
160
1800
1120
1210
872
SMART-H reduced its in vivo clearance. However, high protein
binding did not preclude high clearance when the metabolic stability
was poor, as seen in mice.
Although SMART-H demonstrated reasonable metabolic stability
during incubation with rat, dog, and human liver microsomes, notable
differences in the relative quantities and identity of the metabolites
formed in different species were observed (Fig. 6). Because such
differences would complicate future toxicological assessment and
interspecies comparisons, we sought to define these differences and
evaluate structurally optimized SMART compounds with improved
metabolic stability and potent anticancer activity.
In metabolite identification studies in rat liver microsomes, the
O-demethylated metabolite (M2) was the major metabolite. M2 was
found in a time-dependent manner when 50 ␮M substrate was used.
The level of the O-demethylated metabolite was much greater than
that of the ketone-reduced (M1) metabolite. This phenomenon was
confirmed by in vivo experiments. In rat PK (dose of 2.5 mg/kg
i.v.) studies, we compared the metabolite levels in plasma samples
(supplemental data). Only the O-demethylated (M2) and ketone-
reduced metabolites (M1) could be detected in rat plasma. The M2
metabolite exhibited approximately 10-fold higher exposure than
M1 metabolite in rat plasma. These data further corroborate the
conclusion that in vitro metabolic stability is a good predictor of in
vivo pharmacokinetics.
FIG. 6. Kinetics of four metabolites of SMART-H in human liver microsome (A) and
rat liver microsome (B). SMART-H (50 ␮M) was incubated with 1 mg/ml microsomal
proteins. f, M1; ꢁ, M2; Œ, M3; E, M4.
Our ultimate goal is to develop an anticancer drug for use in
humans with appropriate PK properties. In this study, SMART-H
exhibited a half-life of 17 min in human liver microsomes, suggesting
that additional measures could be taken to improve its metabolic
stability. The metabolic kinetic profiles in rat and human suggested
that ketone reduction was an important metabolic pathway in humans
but not in rats, because of the apparent presence of M1 in humans
(Fig. 6). We speculated that modifying the ketone functional group
would improve the metabolic stability in human liver microsomes.
Blocking or removing labile sites is a common strategy to increase
metabolic stability. In this study, we synthesized three analogs
(SMART-173A, -176A, and -329) to prevent the ketone reduction
reaction. The half-lives were longer (35, 55, and 32 min, respectively)
in human liver microsomes, indicating that the ketone site is a critical
soft spot for improving metabolic stability in humans. The in vitro
metabolic stability of SMART-176A, a hydrazine derivative, was not
tested using conditions suitable for acetylation (perhaps resulting in an
overestimate of its stability) but failed to retain anticancer activity.
SMART-173A retained its potency, with an average IC50 value of 143
nM in four prostate cancer cell lines.
FIG. 7. Blockage of soft spots.
rat and dog were lower than expected on the basis of their in vitro
metabolic stability without considering protein binding. These low
clearance data are reasonable when the high protein binding of
SMART-H is considered, demonstrating that high protein binding of
In summary, the studies presented here predict that SMART-H will
exhibit low clearance in humans based on in vitro data. However, our
studies also suggest that the metabolic stability of SMART-H and its
analogs can be further optimized to obtain prolonged half-life and
FIG. 8. Metabolic stability of SMART-H (F), SMART-213 (E), SMART-173A
(ꢂ), SMART-176A (ꢀ), and SMART-329 (f) in human liver microsome. Bar, S.D. increased exposure in humans. Metabolism studies demonstrate that

DRUG METABOLISM AND PHARMACOKINETICS OF SMART COMPOUNDS
2039
Davies B and Morris T (1993) Physiological parameters in laboratory animals and humans.
Pharm Res 10:1093–1095.
SMART-H was rapidly metabolized under phase I reactions in human
liver microsomes. Ketone reduction and O-demethylation reactions
were the primary metabolic pathways for SMART-H. The metabolic
stability (half-life) was successfully improved two to three times in
vitro by blocking the ketone group. Although SMART-173A, the
oxime derivative with CAN–OH instead of CAO of SMART-H,
demonstrated greater metabolic stability, the in vitro potency was
compromised as evidenced by a nearly equal increase in IC50 in
prostate cancer cell lines. Studies to identify additional analogs with
improved metabolic stability that retain high potency are ongoing in
our laboratory.
Huang M and Ho PC (2009) Identification of metabolites of meisoindigo in rat, pig and human
liver microsomes by UFLC-MS/MS. Biochem Pharmacol 77:1418–1428.
Lu Y, Li CM, Wang Z, Ross CR 2nd, Chen J, Dalton JT, Li W, and Miller DD (2009) Discovery
of 4-substituted methoxybenzoyl-aryl-thiazole as novel anticancer agents: synthesis, biological
evaluation, and structure-activity relationships. J Med Chem 52:1701–1711.
Pelkonen O, Turpeinen M, Uusitalo J, Rautio A, and Raunio H (2005) Prediction of drug
metabolism and interactions on the basis of in vitro investigations. Basic Clin Pharmacol
Toxicol 96:167–175.
Perez EA (2009) Microtubule inhibitors: differentiating tubulin-inhibiting agents based on
mechanisms of action, clinical activity, and resistance. Mol Cancer Ther 8:2086–2095.
Smith R, Jones RD, Ballard PG, and Griffiths HH (2008) Determination of microsome and
hepatocyte scaling factors for in vitro/in vivo extrapolation in the rat and dog. Xenobiotica
38:1386–1398.
Sun L, Vasilevich NI, Fuselier JA, and Coy DH (2004) Abilities of 3,4-diarylfuran-2-one analogs
of combretastatin A-4 to inhibit both proliferation of tumor cell lines and growth of relevant
tumors in nude mice. Anticancer Res 24:179–186.
Zhang H, Zhang D, Li W, Yao M, D’Arienzo C, Li YX, Ewing WR, Gu Z, Zhu Y, Murugesan
N, et al. (2007) Reduction of site-specific CYP3A-mediated metabolism for dual angiotensin
and endothelin receptor antagonists in various in vitro systems and in cynomolgus monkeys.
Drug Metab Dispos 35:795–805.
Acknowledgments. We thank Terrence A. Costello, Katie N. Kail,
and Stacey L. Barnett for providing technical support for animal
studies at GTx Inc.
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Address correspondence to: Dr. James T. Dalton, GTx Inc., 3 N. Dunlap St.,
Memphis TN 38163. E-mail: jdalton@gtxinc.com