Novel human metabolites of the angiotensin-II antagonist tasosartan and their pharmacological effects.

Article abstract of DOI:10.1016/S0960-894X(02)00303-7

Three novel metabolites of the angiotensin-II (A-II) receptor antagonist tasosartan have been identified in humans, and the syntheses and pharmacologic profiling of these metabolites are reported. Each metabolite bound the human A-II receptor with IC(50)s

Full text of DOI:10.1016/S0960-894X(02)00303-7

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1967–1971
Novel Human Metabolites of the Angiotensin-II Antagonist
Tasosartan and Their Pharmacological Effects
Hassan M. Elokdah,a,* Gregory S. Friedrichs,b Sie-Yearl Chai,a Boyd L. Harrison,a
John Primeau,a Michael Chlenova and David L. Crandallb
aMedicinal Chemistry, Chemical Sciences, Wyeth Research, CN 8000, Princeton, NJ 08543, USA
bCardiovascular/Women’s Health, Wyeth Research, PO Box 42528, Philadelphia, PA 19101, USA
Received 19 September 2001; accepted 29 April 2002
Abstract—Three novel metabolites of the angiotensin-II (A-II) receptor antagonist tasosartan have been identified in humans, and
the syntheses and pharmacologic profiling of these metabolites are reported. Each metabolite bound the human A-II receptor with
IC50s between 20 and 45 nM. The in vivo effects of these compounds in attenuating the pressor response to angiotensin-II challenge
in anesthetized rats were also investigated. An unsaturated diol metabolite exhibited in vivo efficacy at intravenous doses of 1 and
3 mg/kg, while the other metabolites, both carboxylic acids, had no significant effect at the same doses. # 2002 Elsevier Science Ltd.
All rights reserved.
The development of drugs that act by modulating the
renin–angiotensin systemhas produced a variety of
agents over the past two decades.1 The initial class of
compounds inhibited the enzymatic production of
angiotensin-II by acting on angiotensin converting
enzyme (ACE inhibitors),2 followed by compounds that
blocked the angiotensin-II receptor.3 Importantly, new
data concerning the efficacy of these drugs continue to
be developed. For example, the recently published
HOPE trial indicated that ACE inhibition was unex-
pectedly associated with reduced myocardial infarction,
and renoprotective effects of A-II antagonists indepen-
dent of effects on blood pressure have been reported in
diabetics.4 These reports indicate that this class of com-
pounds will continue to be a focus of both basic
research and clinical studies.
Recently, three additional novel human plasma meta-
bolites were isolated. These human metabolites were
oxidative products of tasosartan characterized by oxi-
dation of one or both methyl groups with or without the
dihydropyridone ring oxidation. Spectroscopic analysis
of these metabolites supported the proposed structures.
Two of the metabolites were assigned the mono-car-
boxylic acid structures (3 and 4) and the third was
assigned the structure of the unsaturated diol (5) (Fig.
1). For structure confirmation, the purpose of this
research report is to describe the syntheses of these
metabolites, followed by their initial characterization in
vitro and in vivo. In order to profile human metabolites,
the in vitro analysis has specifically utilized a well-
characterized human primary cell culture system.7 In
vivo profiling was performed using standard rat pressor
assays.
We have previously reported the development of an
angiotensin-II receptor antagonist, tasosartan, that was
characterized preclinically using both in vitro and in
vivo assays in rodent.5 This agent progressed to clinical
trials, where analysis of plasma from individuals treated
with the compound revealed specific metabolites that
were profiled pharmacologically in an earlier publica-
tion.6 An example of one of these previously described
metabolites is compound 2 (Fig. 1).
Metabolite 3 was synthesized directly fromtasosartan
(1) in two steps (Scheme 1). Selenium dioxide oxidation
in aqueous dioxane afforded the aldehyde (6). Further
oxidation (benzeneseleninic acid, hydrogen peroxide)
furnished the carboxylic acid (3). Contrary to that
which was previously reported,5 when the selenium
dioxide oxidation reaction was carried out in anhydrous
dioxane as a solvent, the main product isolated was the
des-methyl pyridopyrimidine derivative (7) rather than
the aldehyde (6). Compound 7 was erroneously reported
to be the aldehyde 6.5
*Corresponding author. Tel.: +1-732-274-4504; fax: +1-732-274-
4505; e-mail: elokdah@wyeth.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00303-7

1968
H. M. Elokdah et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1967–1971
The 2-carboxylic acid metabolite (4) was prepared in
three steps from8-(4-bromobenzyl)-2-(hydroxymethyl)-
4-methyl-5,8-dihydropyrido[2,3-d]pyrimidin-7(6H)-one
(8)6 (Scheme 2). Oxidation of 8 with MnO2 afforded the
carboxylic acid (9). Palladium-catalyzed coupling of the
bromo derivative 9 with 2-[(2-t-butyl)-2H-tetrazol-5-yl]-
phenylboronic acid furnished compound 10. Deprotection
of the tetrazole with hydrochloric acid and purification
by HPLC9 afforded metabolite 4.
The unsaturated bishydroxymethyl metabolite (5) was
synthesized in eight steps (Scheme 3). Condensation of
methoxymethyl acetamidine hydrochloride 11 with
methyl methoxy acetoacetate yielded the pyrimidine
derivative 12. Iodination of 12 afforded the iodo pyr-
imidine 13 that was coupled with ethyl acrylate, under
the Heck conditions to give the pyrimidinyl acrylate 14.
Conversion of the hydroxy group of 14 to the chloro
group (15) with POCl3 followed by reaction with 4-
bromo-benzylamine afforded 16. Cyclization of 16 at a
temperature of 240 ꢀC provided the pyridopyrimidinone
17. Palladium-catalyzed coupling of 17 with 2-[(2-t-
butyl)-2H-tetrazol-5-yl]-phenylboronic acid furnished
compound 18. Deprotection of the methoxy groups
with BBr3, followed by removal of the t-butyl protecting
group of the tetrazole with hydrochloric acid, afforded
metabolite 5.
Tasosartan and its metabolites were tested for the inhi-
bition of [125I]A-II binding (AT1 receptors). For the
determination of angiotensin-II receptor binding by the
compounds, studies were performed in cultured human
preadipocytes. The test compounds were initially sus-
pended in 100% DMSO at a concentration of 10 mM,
then serially diluted with assay buffer to yield a final
concentration from10 ꢁ7 to 10ꢁ9 M.11 Results are shown
in Table 1.
Figure 1. Structures of tasosartanTM and its human metabolites.
Rats weighing 343ꢂ8 g were anesthetized with sodium
pentobarbital (50 mg/kg, ip), placed in the supine position
Scheme 1. Synthesis of metabolite 3: (a) SeO2, dioxane/water;8 (b)
PhSeO2H, H2O2, THF; (c) SeO2, dioxane.
Scheme 3. Synthesis of metabolite 5:10 (a) NaOEt, EtOH; (b) I2,
K2CO3, THF; (c) ethyl acrylate, Pd(OAc)2, Et3N, sealed tube, 130 ꢀC;
(d) POCl3, benzene, heat; (e) 4-bromo-benzylamine, n-BuOH, Et3N;
(f) heat; (g) 2-[(2-t-butyl)-2H-tetrazol-5-yl]-phenylboronic acid,
Pd(PPh3)4, Na2CO3, EtOH, H2O, toluene, heat; (h) BBr3, CH2Cl2;
(i) HCl, heat.
Scheme 2. Synthesis of metabolite 4: (a) MnO2, acetone; (b)
(Ph3P)4Pd, Na2CO3, toluene, EtOH, H2O, 2-[(2-t-butyl)-2H-tetrazol-
5-yl]-phenylboronic acid; (c) HCl, heat.

H. M. Elokdah et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1967–1971
Table 1. In vitro data for tasosartan and its metabolites
1969
Compd
IC50, nMa
1
3
4
5
38ꢂ0.75
44ꢂ0.30
23ꢂ1.30
44ꢂ0.54
aDetermination of the IC50 for each compound was based upon
linear regression curve fitting fromdata obtained from2–3 separate
experiments.
on a heating pad (K-model 100, Baxter Laboratories),
and a longitudinal incision made on the neck to expose
the right carotid artery and jugular vein. The jugular
vein was cannulated with a saline filled polyethylene
catheter (PE 10) and used for intravenous drug infusion.
Arterial pressure and heart rate were monitored via a
saline filled polyethylene catheter (PE 10) introduced
into the carotid artery and connected to a P23 ID Sta-
tham/Gould pressure transducer. Subcutaneous needles
were positioned in the limbs for ECG recordings. All
data outputs were recorded on a Gould Model 6600
series recorder with a Po-ne-mah data acquisition sys-
temand displayed on a physiology platformCRS800W/
CRS400W recorder.
Figure 2. Pressor response to angiotensin-II administration in four
groups of rats (n=3–5 each). Each rat was subjected to four separate
bolus injections of angiotensin-II following the introduction of either
the vehicle or graded doses of tasosartan (1) (panel A), metabolite 3
(panel B), metabolite 4 (panel C) and metabolite 5 (panel D) (0.3, 1.0,
or 3.0 mg/kg, iv). Data are presented as percent change (meanꢂSEM)
in the pressor response. *p<0.05 versus the vehicle response in the
respective treatment group.
Metabolite 3
The experimental protocol for assessing in vivo A-II
antagonist activity involved compound dosing followed
by measurement of the pressor response to A-II chal-
lenge. Each rat froman individual treatment group
(n=3–5) was initially dosed intravenously (iv) with
either vehicle (2% ethanol in saline solution) or test
compound (1, 3, 4, or 5). The test compounds were
administered initially at a dose of 0.3 mg/kg (iv) fol-
lowed 2 min later by angiotensin-II challenge (3 mg/kg,
iv), and the maximal increase in mean arterial pressure
was recorded. Twenty minutes later, baseline pressure
values were re-established and the next dose of test
compound (1.0 mg/kg, iv) was administered followed by
angiotensin-II challenge. This regimen was repeated for
the 3.0 mg/kg dose for each test compound. All animals
were euthanized at the conclusion of the protocol.
There was a 38ꢂ7% increase in arterial pressure to an
angiotensin-II challenge in the absence of any test com-
pound in the group of rats assigned to this treatment
group. Administration of metabolite 3 at doses of 0.3,
1.0, or 3.0 mg/kg (iv) did not attenuate the pressor
response to angiotensin-II (Fig. 2, panel B).
Metabolite 4
There was a 41ꢂ4% increase in arterial pressure to an
angiotensin-II challenge in the absence of any test com-
pound in the group of rats assigned to this treatment
group. Administration of metabolite 4 at doses of 0.3,
1.0, and 3.0 mg/kg (iv) failed to significantly attenuate
the pressor response to angiotensin-II (Fig. 2, panel C).
Each rat was randomly assigned to one of four drug
treatment groups in an effort to evaluate the in vivo
pressor response to an angiotensin-II challenge in the
presence of one of the test compounds listed above. A
dose–response protocol was established and the maximal
change in arterial pressure following angiotensin-II was
recorded for each dose of the compound. The results were
represented as a percent change in maximal pressure
recorded before and after the angiotensin-II challenge.
Metabolite 5
There was a 47ꢂ6% increase in arterial pressure to an
angiotensin-II challenge in the absence of any test com-
pound in the group of rats assigned to this treatment
group. Administration of metabolite 5 at doses of 1.0
and 3.0 mg/kg (iv) significantly (p<0.05) attenuated the
pressor response to angiotensin-II. The 0.3 mg/kg dose
did not alter the pressor response to the bolus injection
of angiotensin-II (Fig. 2, panel D).
Tasosartan (1)
There was a 32ꢂ0.2% increase in arterial pressure to an
angiotensin-II challenge in the absence of any test com-
pound in the group of rats assigned to this treatment
group. Administration of tasosartan at doses of 1.0 and
3.0 mg/kg (iv) significantly (p<0.05) attenuated the
pressor response to angiotensin-II (Fig. 2, panel A).
We describe the syntheses of three previously unidenti-
fied human metabolites of tasosartan (1) and their in
vitro inhibition of A-II receptor binding in human cell
cultures. While all three metabolites bound the A-II
receptor, the 2-carboxylic acid metabolite 4 showed the
greatest in vitro activity with an IC50 of 23ꢂ1.3 nM.

1970
H. M. Elokdah et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1967–1971
The 4-carboxylic acid metabolite 3 and the bis-
hydroxymethyl metabolite 5 were equipotent (IC50=
44ꢂ0.30 nM and IC50=44ꢂ0.54 nM, respectively).
However, in vivo testing of the three metabolites did not
yield parallel results. At test doses of 1 and 3 mg/kg (iv),
metabolite 5 produced a significant attenuation of the
hypertensive response to A-II challenge similar to that
observed for tasosartan. Metabolites 3 and 4 had no
significant effect at the same doses. These data indicate
that while all metabolites have similar in vitro inhibitory
activity, only the unsaturated diol exhibits in vivo
activity at the doses tested. Future studies examining the
pharmacokinetics of each compound, including plasma
stability and protein binding, need to be undertaken to
better understand the diversity between in vitro and in
vivo results.
material was purified by flash chromatography on a silica gel
column using 18% THF in methylene chloride as the eluent.
The fraction containing the desired material was concentrated
under reduced pressure. The residue was stirred in ether and
the solid was collected by filtration and dried to give the
desired aldehyde as an off-white solid (0.75 g): mp 203–206 ꢀC
(decomposition); 1H NMR (500 MHz, DMSO-d6) d 2.57 (s,
3H), 2.75 (t, J=7.7 Hz, 2H), 3.31 (t, J=7.7 Hz, 2H), 5.22 (s,
2H), 7.0 (d, J=8.1 Hz, 2H), 7.22 (d, J=8.1 Hz, 2H), 7.50–7.56
(m, 2H), 7.62–7.67 (m, 2H), 9.96 (s, 1H), 12.27 (br. s, 1H); IR
(KBr), cmꢁ1 1700, 1715. HRMS [M+H]+ Exact mass:
.
426.16730, Experimental: 426.16756. Anal. for C23H19N7O2
1/2H2O. Calcd: C, 63.59; H, 4.64; N, 22.57. Found: C, 63.83;
H, 4.50; N, 22.45.
9. Compounds 3 and 4 were purified by reverse phase HPLC
on a Primesphere C18 (5 ꢃ 25 cm) column with 0.1% TFA in
acetonitrile as the mobile phase and detection at 254 nm.
10. Synthesis of 2,4-bis-hydroxymethyl-8-[20(1H-tetrazol-5-
yl)-biphenyl-4-ylmethyl]-8-[20 -(1H-tetrazol-5-yl)-bipheny-4-
ylmethyl]-8H-pyrido[2,3-d]pyrimidin-7-one (5). Step 1: 2,6-Bis-
(methoxymethyl)-4-pyrimidinol (12). To a solution of NaOEt/
EtOH, made from EtOH (200 mL) and sodium (3.5 g), was
added 1-methoxyacetamidine hydrochloride (13.7 g, 0.11 mol)
and methyl methoxyethylacetoacetate (14.6 g, 0.1 mol). This
mixture was heated to reflux for 4 h. The reaction mixture was
cooled, and acidified with hydrochloric acid to a pH of ꢄ6.
The solvent was evaporated under vacuum. The residue was
extracted with hot ethanol (3ꢃ300 mL). The ethanolic extracts
were combined and evaporated to dryness. The product was
purified by flash chromatography (silica gel; MeOH/CHCl3,
3:97) to afford 15.8 g (85%) of a white solid. Mp 99–100 ꢀC;
1H NMR (400 MHz, DMSO-d6) d 3.32 (s, 3H), 3.36 (s, 3H),
4.22 (s, 2H), 4.24 (s, 2H), 6.15 (s, 1H), 12.35 (br. s, 1H); IR
(KBr), cmꢁ1 1680; MS (+ESI, [M+H]+) m/z 185. Anal. for
C8H12N2O3. Calcd: C, 52.17; H, 6.57; N, 15.21. Found: C,
52.17; H, 6.32; N, 15.14. Step 2: 5-Iodo-2,6-bis(methoxy-
methyl)-4-pyrimidinol (13). To a well-stirred solution of 12
(8.28 g, 0.045 mol) in THF (300 mL) were added iodine (22.86 g,
0.09 mol) and potassium carbonate (13.0 g, 0.095 mol). The
mixture was heated at reflux for 6 h then cooled to ambient
temperature. The mixture was neutralized with hydrochloric
acid and solvent evaporated. The product was purified by flash
chromatography (silica gel; MeOH/CHCl3, 3:97) to afford
3.3 g of a white solid. Mp 112–114 ꢀC; 1H NMR (400 MHz,
DMSO-d6) d 3.33 (s, 3H), 4.27 (s, 2H), 4.39 (s, 2H), 12.80 (br s,
1H); IR (KBr), cmꢁ1 1650; MS (+ESI, [M+H]+) m/z 311.
Anal. for C8H11IN2O3. Calcd: C, 30.99; H, 3.58; N, 9.03.
Found: C, 30.83; H, 3.38; N, 8.86. Step 3: Ethyl (E)-3-[4-
hydroxy-2,6-bis(methoxy-methyl)-5-pyrimidinyl]-2-propenoate
(14). A mixture of the iodo-pyrimidine (13) (3.1 g, 0.01 mol),
ethyl acrylate (2.2 mL, 0.02 mol), palladium acetate (0.1 g) and
triethyl amine (10 mL) was heated under nitrogen in a sealed
tube at 130 ꢀC for 45 min. The mixture was cooled to ambient
temperature, diluted with chloroform (300 mL), and filtered
through Celite1. The filtrate was evaporated to dryness and
the product was purified by flash chromatography (silica gel;
20–40% ethyl acetate in hexane) to afford 1.8 g (64%) of an
Acknowledgements
We would like to thank Dr. Brent Kleintop, Ms. Claire
Canonico, and Mr. Stephen Sears for metabolite iden-
tification; Mr. James Mattes for NMR spectroscopy;
Mr. Dennis Busler, Mr. Robert Swillo, Mr. Tom
Antrilli, and Ms. Gwen Morgan for technical support in
pharmacologic profiling of the metabolites.
References and Notes
1. Jackson, E. K.; Garrison, J. C. Renin and Angiotensin. In
The Pharmacological Basis of Therapeutics, 9th ed.; Hardman,
J., Limbird, L., Molinoff, P., Ruddon, R., Eds.; McGraw-Hill:
New York, 1996; p 733.
2. McAreavey, D.; Robertson, J. I. S. Drugs 1990, 40, 326.
3. Wexler, R. R.; Greenlee, W. J.; Irvin, J. D.; Goldberg,
M. R.; Prendergast, K.; Smith, R. D.; Timmermans,
P. B. W. M. J. Med. Chem. 1996, 39, 625.
4. (a) Dagenais, G. R.; Yusuf, S.; Bourassa, M. G.; Yi, Q.;
Bosch, J.; Lonn, E. M.; Kouz, S.; Grover, J. Circulation 2001,
104, 522. (b) Lewis, E. J.; Hunsicker, L. G.; Clarke, W. R.;
Berl, T.; Pohl, M. A.; Lewis, J. B.; Ritz, E.; Atkins, R. C.;
Rohde, R.; Raz, I. N. Engl. J. Med. 2001, 345, 910.
5. Ellingboe, J. W.; Antane, M.; Nguyen, T. T.; Collini, M. D.;
Antane, S.; Bender, R.; Hartupee, D.; White, V.; McCallum,
J.; Park, C. H.; Russo, A.; Osler, M. B.; Wojdan, A.; Dinish,
J.; Ho, D. M.; Bagli, J. F. J. Med. Chem. 1994, 37, 542.
6. Ellingboe, J. W.; Collini, M. D.; Quagliato, D.; Chen, J.;
Antane, M.; Schmid, J.; Hartupee, D.; White, V.; Park, C.;
Tanikella, T.; Bagli, J. F. J. Med. Chem. 1998, 41, 4251.
7. (a) Crandall, D. L.; Herzlinger, H. E.; Saunders, B. D.;
Zolotor, R. C.; Feliciano, L.; Cervoni, P. Metabolism 1993, 42,
511. (b) Crandall, D. L.; Herzlinger, H. E.; Saunders, B. D.;
Armellino, D. C.; Kral, J. G. J. Lipid Res. 1994, 35, 1378. (c)
Crandall, D. L.; Armellino, D. C.; Busler, D. E.; McHendry-
Rinde, B.; Kral, J. G. Endocrinology 1999, 140, 154.
8. Synthesis of 2-methyl-7-oxo-8-{[20-(2H-1,2,3,4-tetraazol-5-
yl)[1,10-biphenyl]-4-yl]methyl}-5,6,7,8-tetrahydropyrido[2,3-d]-
pyrimidine-4-carboxaldehyde (6). 5,8-Dihydro-2,4-dimethyl-8-
[(20-(1H-tetrazol-5-yl)-[1,10-biphenyl]-4-yl)methyl]pyrido[2,3-d]-
pyrimidin-7(6H)-one (1) (1.2 g, 2.92 mmol) was added under
nitrogen to selenium dioxide (0.33 g, 2.97 mmol) in 14 mL of
dioxane and 0.25 mL of water and the mixture was heated at
reflux for 4 h then cooled to room temperature. The mixture
was filtered through Celite1 and the filtrate was concentrated
under reduced pressure to give 1.42 g of an orange glass. The
off-white solid. Mp 162–163 ꢀC; H NMR (400 MHz, DMSO-
1
d6) d 1.25 (t, J=7.2 Hz, 3H), 3.31 (s, 3H), 3.34 (s, 3H), 4.17 (q,
J=7.2 Hz, 2H), 4.31 (s, 2H), 4.46 (s, 2H), 7.26 (d, J=15.6 Hz,
1H), 7.69 (d, J=15.6 Hz, 1H), 12.95 (br. s, 1H); IR (KBr),
cmꢁ1 1700, 1670, 1625; MS (+ESI, [M+H]+) m/z 283. Anal.
for C13H18N2O5. Calcd: C, 55.31; H, 6.43; N, 9.92. Found: C,
55.24; H, 6.15; N, 9.73. Step 4: Ethyl (E)-3-[4-chloro-2,6-bis-
(methoxy-methyl)-5-pyrimidinyl]-2-propenoate (15). A mixture
of the hydroxy-pyrimidine (14) (1.2g, 0.004 mol), phosphorus
oxychloride (1.0mL), and benzene (20 mL) was heated at reflux
for 2 h. The mixture was cooled to ambient temperature,
poured over ice, neutralized with sodiumbicarbonate, and

H. M. Elokdah et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1967–1971
1971
.
extracted with ethyl acetate. The organic phase was washed
with water, dried and evaporated. Purification by flash chro-
matography (silica gel; 20% ethyl acetate in hexane) afforded
Anal. for C29H31N7O3 H2O. Calcd: C, 64.07; H, 6.12; N,
18.04. Found: C, 63.67; H, 5.73; N, 18.12. Step 8: 8-({20-[2-
(tert-Butyl)-2H-1,2,3,4-tetrazol-5-yl][1,10-biphenyl]-4-yl}methyl)-
2,4-bis(hydroxymethyl)pyrido[2,3-d]pyrimidin-7(8H)-one (19).
Boron tribromide (6 mL of 1 M solution in methylene chloride,
0.0060 mol) was added slowly to the solution of 18 (0.8 g,
0.0015 mol) in methylene chloride (50 mL) at ambient tem-
perature. The mixture was stirred for 3 h. Methanol (5 mL)
was added dropwise. The mixture was stirred for 30 min and
water (2 mL) was added. The solvent was evaporated and the
product was purified by flash chromatography (silica gel; 1–
3% MeOH in CHCl3) to afford 0.6 g (80%) of a white solid.
Mp 107–109 ꢀC; 1H NMR (400 MHz, DMSO-d6) d 1.34 (s,
9H), 4.64 (d, J=6.12 Hz, 2H), 4.84 (d, J=5.72 Hz, 2H), 5.36
(t, 6.26 Hz, 1H), 5.58 (s, 2H), 5.68 (t, J=5.92 Hz, 1H), 6.75 (d,
J=9.88 Hz, 1H), 6.97 (d, J=8.32 Hz, 2H), 7.34 (d, J=8.36 Hz,
2H), 7.43 (d, 7.44 Hz, 1H), 7.51 (t, J=7.48 Hz, 1H), 7.58 (t,
J=7.48 Hz, 1H), 7.77 (d, J=7.68 Hz, 1H), 8.31 (d, J=9.68 Hz,
1H); IR (KBr), cmꢁ1 1670, 1565; MS (+APCI, [M+H]+) m/z
498. Anal. for C27H27N7O3. Calcd: C, 65.18; H, 5.47; N, 19.71.
Found: C, 64.91; H, 5.45; N, 19.36. Step 9: 2,4-Bis(hydroxy-
methyl)-8-{[20 -(1H-1,2,3,4-tetrazol-5-yl)[1,10 -biphenyl]-4-yl]-
methyl}pyrido[2,3-d]pyrimidin-7(8H)-one hydrochloride (5). A
mixture of 19 (0.2 g, 0.0004 mol) and hydrochloric acid
(10 mL) was heated at reflux for 2 h. The mixture was cooled
to ambient temperature, diluted with water (30 mL), and fil-
tered. Evaporation of the solvent under vacuumand drying of
the residue under high vacuumafforded the product as a solid
1
0.8 g (67%) of an oil. H NMR (400 MHz, DMSO-d6) d 1.26
(t, J=7 Hz, 3H), 3.31 (s, 3H), 3.38 (s, 3H), 4.22 (q, J=7 Hz,
2H), 4.54 (s, 2H), 4.58 (s, 2H), 6.51 (d, J=16.24 Hz, 1H), 7.65
(d, J=16.24 Hz, 1H); IR (KBr), cmꢁ1 1720; MS (+ESI,
[M+H]+) m/z 301. Anal. for C13H17 ClN2O4 1=4H2O. Calcd: C,
.
51.15; H, 5.78; N, 9.18. Found: C, 51.32; H, 5.53; N, 9.25. Step 5:
Ethyl (E)-3[4-(4-bromo-benzylamino)-2,6-bis(methoxymethyl)-
5-pyrimidinyl]-2-propenoate (16). A mixture of the chloro-
pyrimidine (15) (0.6 g, 0.002 mol), 4-bromo-benzylamine (0.4 g,
0.004 mol), triethylamine (2 mL), and butanol (25 mL) was
heated at reflux for 2 h. The solvent was evaporated under
vacuum. The residue was dissolved in ethyl acetate (100 mL)
and water (50 mL). The organic phase was washed with water,
dried and evaporated to dryness. The product was purified by
flash chromatography (silica gel; 40–50% ethyl acetate in
1
hexane) to afford 0.85 g (94%) of an oil. H NMR (400 MHz,
DMSO-d6) d 1.27 (t, J=7.14 Hz, 3H), 3.27 (s, 3H), 3.31 (s,
3H), 4.20 (q, J=7.14 Hz, 2H), 4.28 (s, 2H), 4.29 (s, 2H), 4.57
(d, J=5.92 Hz, 2H), 6.38 (d, J=16.04 Hz, 1H), 7.29 (d, J=
8.56 Hz, 1H), 7.47 (d, J=8.56 Hz, 2H), 7.63 (d, J=16.04 Hz,
1H), 7.88 (t, J=5.92 Hz, 1H); IR (KBr), cmꢁ1 3350, 1710; MS
(+ESI, [M+H]+) m/z 450/452. Anal. for C20H24BrN3O4.
Calcd: C, 53.34; H, 5.37; N, 9.33. Found: C, 52.95; H, 5.41; N,
9.16. Step 6: 8-(4-Bromo-benzyl)-2,4-bis(methoxymethyl)pyr-
ido[2,3-d]pyrimidin-7(8H)-one (17). The pyrimidinyl-propeno-
ate (16) was heated under vacuumat 240 ꢀC for 4 h. The
mixture was cooled to ambient temperature and the product
was purified by flash chromatography (silica gel; 60% ethyl
acetate in hexane) to afford 0.4 g (60%) of an off-white solid.
Mp 113–115 ꢀC; 1H NMR (400 MHz, DMSO-d6) d 3.36 (s,
3H), 3.37 (s, 3H), 4.62 (s, 2H), 4.79 (s, 2H), 5.51 (s, 2H), 6.81
(d, J=9.64 Hz, 1H), 7.32 (d, J=8.34 Hz, 2H), 7.48 (d,
J=8.34 Hz, 2H), 8.26 (d, J=9.64 Hz, 1H); IR (KBr), cmꢁ1
1675, 1570; MS (+ESI, [M+H]+) m/z 404/406. Anal. for
C18H18BrN3O3. Calcd: C, 53.48; H, 4.49; N, 10.39. Found: C,
53.35; H, 4.38; N, 10.10. Step 7: 8-({20-[2-(tert-Butyl)-2H-
1,2,3,4 -tetrazol - 5 - yl] [1,10 -biphenyl] - 4 - yl}methyl) - 2,4 -bis-
(methoxy methyl) pyrido[2,3-d]pyrimidin-7(8H)-one (18). A
mixture of 17 (0.81 g, 0.0020 mol), 2-[(2-tert-butyl)-2H-tetra-
zol-5-yl]-phenylboronic acid (0.59 g, 0.0024 mol), tetrakis-tri-
phenylphosphine palladium(150gm), sodiumcarbonate
(0.8 g), ethanol (3 mL), toluene (20 mL) and water (2 mL) was
heated at reflux for 18 h. The solvent was evaporated. The
residue was treated with ethyl acetate (200 mL) and filtered.
The filtrate was evaporated to dryness and the residue was
purified by flash chromatography (silica gel; 60% ethyl acetate
in hexane) to afford 0.92 g (88%) of an oil. 1H NMR
(400 MHz, DMSO-d6) d 1.32 (s, 9H), 3.36 (s, 3H), 3.37 (s, 3H),
4.61 (s, 2H), 4.79 (s, 2H), 5.54 (s, 2H), 6.80 (d, J=9.84 Hz,
1H), 6.98 (d, J=8.24 Hz, 2H), 7.31 (d, J=8.24 Hz, 2H), 7.43
(d, J=7.68 Hz, 1H), 7.51 (t, 5.92 Hz, 1H), 7.58 (t, J=5.92 Hz,
1H), 7.77 (d, J=7.60 Hz, 1H), 8.23 (d, J=10.0 Hz, 1H); IR
(KBr), cmꢁ1 1670, 1570; MS (+APCI, [M+H]+) m/z 526.
(0.162 g). Mp 195–197 ꢀC; H NMR (400 MHz, DMSO-d6) d
1
4.64 (s, 2H), 4.84 (d, 2H), 5.58 (s, 2H), 6.75 (d, J=9.64, 1H),
6.99 (d, J=8.32 Hz, 2H), 7.29 (d, J=8.32 Hz, 2H), 7.44–7.58
(m, 2H), 7.60–7.66 (m, 2H), 8.32 (d, J=9.8, 8 Hz, 1H); IR
(KBr), cmꢁ1 16650, 1570; MS (+ESI, [M+H]+) m/z 442.
.
Anal. for C23H19N7O3 HCl. Calcd: C, 57.80; H, 4.22; N,
20.52. Found: C, 57.47; H, 4.16; N, 20.13.
11. Between passages 2 and 5, preadipocytes fromthe sub-
cutaneous depot (Zen-Bio, Research Triangle Park, NC, USA)
were plated in 24-well plates at a density of 25,000 cells/well.
Cells were maintained in Medium 199 supplemented with 10%
heat-inactivated fetal bovine serumand 1% antibiotic-anti-
mycotic (Life Technologies, Grand Island, NY, USA). The
experiment was initiated by aspirating the medium and rinsing
the cells twice with phenol red-free Medium199. The binding
assay was then begun by adding 160 mL of binding buffer
(50 mM Tris, 5 mM MgCl2, 0.25% bovine serumalbumin, pH
7.4; Sigma, St. Louis, MO), 20 mL of 10 mM unlabelled A-II
(nonspecific binding), 20 mL of buffer (total binding), or 20 mL
of test compound. The cells were incubated at room tempera-
ture for 10 min, followed by addition of 20 mL of 125I-[Sar1,-
Ile8]A-II (Amersham, Arlington Heights, IL, USA). The
culture plate was shaken gently for 1 h at roomtemperature,
followed by aspiration of the medium. Each well was rinsed
twice with 300 mL of the assay buffer, followed by addition of
100 mL of 0.5 M NaOH/0.5% Triton to solubilize the cells. The
radioactivity fromeach well was then determined in an
appropriate detector.