Metabolites of the angiotensin II antagonist tasosartan: The importance of a second acidic group

Article abstract of DOI:10.1021/jm970690q

Described in this paper is the synthesis and pharmacological activity of five metabolites of the angiotensin II antagonist tasosartan (1). Of particular interest is the effect of the additional acidic group of the enol metabolite (8) on activity. As suggested by the structural-activity relationship of other angiotensin II antagonist series, a second acidic group can improve receptor binding activity but decrease in vivo activity after oral dosing. The metabolic introduction of a second acidic group in tasosartan bypasses this problem and contributes to the excellent profile of the compound. A molecular modeling study provides a rationale for the role of the enol group of 8 in AT₁ receptor binding.

Full text of DOI:10.1021/jm970690q

J . Med. Chem. 1998, 41, 4251-4260
4251
Meta bolites of th e An gioten sin II An ta gon ist Ta sosa r ta n : Th e Im p or ta n ce of a
Secon d Acid ic Gr ou p
J ohn W. Ellingboe,* Michael D. Collini, Dominick Quagliato, J ames Chen, Madelene Antane, J ean Schmid,
Dale Hartupee, Valerie White, C. Hyung Park, Tarak Tanikella, and J ehan F. Bagli
Divisions of Chemical Sciences, Cardiovascular and Metabolic Disorders, and Structural Biology, Wyeth-Ayerst Research,
CN 8000, Princeton, New J ersey 08543, and 401 North Middletown Road, Pearl River, New York 10965
Received April 17, 1997
Described in this paper is the synthesis and pharmacological activity of five metabolites of the
angiotensin II antagonist tasosartan (1). Of particular interest is the effect of the additional
acidic group of the enol metabolite (8) on activity. As suggested by the structural-activity
relationship of other angiotensin II antagonist series, a second acidic group can improve receptor
binding activity but decrease in vivo activity after oral dosing. The metabolic introduction of
a second acidic group in tasosartan bypasses this problem and contributes to the excellent
profile of the compound. A molecular modeling study provides a rationale for the role of the
enol group of 8 in AT1 receptor binding.
Ch a r t 1
The renin-angiotensin system (RAS) plays an im-
portant role in the regulation of blood pressure through
the actions of angiotensin II (A II) (vasoconstriction,
aldosterone secretion, renal sodium reabsorption, and
norepinephrine release) and thus is an appropriate
target for therapeutic intervention in hypertension.1
Inhibition and antagonism of the various components
of the RAS have been the subject of extensive research,
culminating in such drugs as angiotensin converting
enzyme (ACE) inhibitors,2 renin inhibitors,3 and more
recently A II antagonists.4
Many reported A II antagonists have a tetrazole group
which is of particular importance for potency and oral
bioavailability. In addition, most of these antagonists
have a group which can act as a hydrogen bond acceptor,
such as a heterocyclic nitrogen, or as a hydrogen bond
donor, such as a hydroxy group or a carboxylic acid
(Chart 1). In particular, a second acidic group appears
to make a significant contribution to AT1 receptor
binding. However, this second acidic group can have
detrimental effects on oral bioavailability. For example,
the Glaxo compound GR117289 (5) was found to have
only 20% bioavailability in humans due to poor absorp-
tion, which was attributed to the fact that the compound
is a diacid.5 A prodrug approach has been used to mask
the carboxylic acid groups in elisartan (4),6 a prodrug
of EXP3174 (3), and candesartan (6).7 In the case of
losartan (2), a carboxylic acid is introduced metabolically
through oxidation of the hydroxymethyl group, to yield
EXP3174 (3).8 The AT1 receptor IC50 improves from 19
nM to 1.3 nM with the second acidic group.1 However,
oral bioavailability in rats decreases from 33% for
losartan (2) to 12% for EXP3174 (3).9 In normotensive
rats, EXP3174 (3) is 15 times more potent than losartan
by the IV route, but is less potent after oral administra-
tion.10
that may serve as a hydrogen bond acceptor, but lacking
a second acidic group. In Goldblatt (2K-1C) hyperten-
sive rats, 1 is 30 times more potent than losartan (2)
after intragastric dosing.11 The metabolic fate of 1 has
been studied; four metabolites were identified in rats,
and a fifth metabolite was found in man (Chart 2).12
The 5-hydroxy compound (7) and the enol compound (8)
were isolated from rat urine, and the unsaturated
compound (9) and the tetrazole N-2 glucuronide (10)
were found in rat bile. Compounds 7, 8, and 9 were
also identified in rat plasma. A hydroxylated enol
metabolite (11) was identified in human plasma, as were
7, 8, and 9. The enol in compounds 8 and 11 is
relatively acidic, and this functional group appears to
play an important role in binding to the AT1 receptor.
The synthesis and pharmacology of these metabolites
is described in this paper. Also, the role of the enol
group of compounds 8 and 11 in binding to the AT1
receptor is discussed.
Tasosartan (1) is a potent, orally active A II antago-
nist with a tetrazole ring and a heterocyclic nitrogen
* Corresponding author. Current address: Wyeth-Ayerst Research,
401 N. Middletown Rd., Pearl River, NY 10965.
10.1021/jm970690q CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/24/1998

4252 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Ellingboe et al.
Ch a r t 2
metabolite (8). None of the keto tautomer is observed
in the NMR spectrum.
Determination of the pKa of the enol group of 8 was
attempted, but the low solubility of the compound
complicated the experiment. Therefore, intermediate
20 was examined, and a pKa value of 9.73 ( 0.37 for
the enol group was determined by potentiometric titra-
tion. Since the only difference between 8 and 20 is the
tert-butyl protecting group on the tetrazole, the pKa
values for the enols in compounds 8 and 11 are expected
to be very close to the value determined for 20.
The hydroxyenol metabolite (11) was prepared by a
similar route to compound 8, but introduction of the
hydroxy group required four additional steps (Scheme
4). Methoxyacetonitrile (23) was converted into meth-
oxyacetamidine (25) and then reacted with ethyl ac-
etoacetate to yield pyrimidine 26. Iodination, chlori-
nation, and coupling with (1-ethoxyvinyl)tributyltin
gave ethoxyvinylpyrimidine 29. In contrast to the enol
(8) synthesis, the whole biphenyl tetrazole was incor-
porated in one piece. Thus, reaction of 29 with 2′-(2-
tert-butyl-2H-tetrazole-5-yl)-4-methylaminobiphenyl (35)
(prepared according to Scheme 5, by reduction of the
nitrile in compound 34 to an amine) yielded compound
30, which was hydrolyzed to acetylpyrimidine 31. Cy-
clization with diethyl carbonate gave 32, which was
deprotected in two steps. The methyl group was re-
moved with BBr3 and the tert-butyl group was removed
with trifluoromethanesulfonic acid to give the hydroxy-
enol metabolite (11).
Sch em e 1
Metabolite 9, the unsaturated analogue of 1, was
synthesized from pyridopyrimidine 21 (prepared accord-
ing to Sakamoto13). Alkylation of 21 with 2-[4-(bro-
momethyl)phenyl]benzonitrile yielded 22, and conver-
sion of the nitrile into a tetrazole with NaN3/n-Bu3SnCl
gave metabolite 9 (Scheme 6).
Ch em istr y
In Vitr o Bin d in g
Metabolites 7 and 10 were synthesized directly from
compound 1 (Schemes 1 and 2). Thus, oxidation of 1
with KMnO4 gave the hydroxy metabolite (7) in a yield
of 8%. The N-2 glucuronide (10) was prepared in three
steps from 1. Glycosidation of the tetrazole potassium
salt of 1 with bromo-2,3,4-tri-O-acetyl-R-D-glucopyra-
nuronic acid methyl ester gave 34% of the N-2 glucu-
ronide (12a ) and 18% of the N-1 glucuronide (12b).
Removal of the glucuronide acetate groups of 12a with
KCN in MeOH yielded methyl ester 13 (63% yield), and
hydrolysis of the methyl ester with 1 N NaOH in MeOH
gave the glucuronide (10) (62% yield).
The enol metabolite (8) was synthesized in eight steps
from 2,6-dimethyl-4-hydroxypyrimidine (Scheme 3). Io-
dination and chlorination with phosphorus oxychloride
yielded 15, which was coupled with (1-ethoxyvinyl)-
tributyltin to give pyrimidine 16. Reaction of 16 with
4-bromobenzylamine and hydrolysis of the enol ether
yielded 5-acetylpyrimidine 18. Palladium-catalyzed
coupling of 18 with 2-[(2-tert-butyl)-2H-tetrazol-5-yl]-
phenylboronic acid11 gave compound 19. The pyrido
ring was introduced by reaction of 19 with diethyl
carbonate, and the tert-butyl protecting group on the
tetrazole of 20 was removed with trifluoromethane-
sulfonic acid in refluxing toluene to yield the enol
The metabolites (7-11) were tested for the inhibition
of [125I]A II binding in either a rat adrenal or liver
membrane preparation. Results are listed in Table 1.
The IC50 for compound 1 is 1.20 nM (adrenal mem-
brane). The introduction of a 5-hydroxy group as in
metabolite 7 does not have a large effect (IC50 ) 2.30
nM, adrenal membrane), and the unsaturated metabo-
lite 9 is only slightly less potent than 1 (IC50 ) 6.50
nM, liver membrane). Glucuronidation takes place at
the tetrazole N-2 position and the resulting glucuronide
10 does not inhibit binding of A II at 100 nM (liver
membrane). The remaining two compounds, 8 and 11,
contain the enol group and are the most potent com-
pounds in the binding assay, with IC50 values of 0.17
and 0.41 nM (adrenal membrane), respectively. Since
the hydroxy metabolite 7 does not show a similar
increase in potency, the relative acidity of the enol group
appears to contribute to the enhanced binding. The
increase in binding affinity (∼1 order of magnitude)
after the metabolic introduction of the enol group in 8
parallels the increase observed for the carboxylic acid
metabolite (3) of losartan (2). A model for the contribu-
tion of the enol to AT1 receptor binding is discussed
below.

Metabolites of the Angiotensin II Antagonist
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4253
Sch em e 2
Sch em e 3
after both ig and iv dosing. At this dose, the decreases
in MAP were 71 ( 14 (ig) and 53 ( 10 (iv) mmHg.
Higher doses do not produce significantly larger de-
creases in MAP. For compound 7, ig doses of 1.0, 3.0,
and 10.0 mg/kg produced similar decreases in MAP of
55 ( 15, 54 ( 10, and 71 ( 11 mmHg (Table 2, Figure
2), so the ig therapeutic dose is 1.0 mg/kg. Intravenous
doses of 1.0, 3.0, and 10.0 mg/kg of 7 also produced
similar decreases of 51 ( 13, 47 ( 15, and 41 ( 8 mmHg
(Table 2, Figure 2), giving a therapeutic dose of 1.0 mg/
kg. The ig therapeutic dose for compound 8 is 10.0 mg/
kg (66 ( 12 mmHg decrease), and the iv therapeutic
dose is 0.10 mg/kg (67 ( 11 mmHg decrease) (Table 2,
Figure 3). These doses are an order of magnitude higher
than the ig therapeutic dose of 1 and an order of
magnitude lower than the iv therapeutic dose for 1,
respectively. This difference in iv therapeutic doses for
1 and 8 reflects their difference in potency in the AT1
binding assay. The 100-fold difference in the ig and iv
therapeutic doses for 8 (Figure 3) suggests that 8 is not
absorbed as well as 1 after oral administration.
Compound 8 is a major metabolite in mouse, rat, and
monkey plasma (but is not detected in dog plasma)12 as
well as in human plasma. In male rats, after oral
administration of a single dose of 14C-tasosartan (1.0
mg/kg), the metabolites were present in the plasma to
the following extent (% radioactivity in plasma) at 1 h:
(1) 50%, (7) 14%, (8) 30%, and (11) not detected. The
percentage of radioactivity in plasma at 7 h was (1) 4%,
(7) 10%, (8) 76%, and (11) not detected.12 The metabolic
pathway for the plasma metabolites was elucidated as
follows: 1 is hydroxylated to give 7, which is further
oxidized to give 8 and then 11.12 Thus, the in vivo data
reported in Table 2 and Figures 1-3 for compounds 1
and 7 reflects activity due to multiple species (1, 7, and
8). Compound 8 is more slowly eliminated than 1 or
712 and appears to be the major contributor to the
biological activity of tasosartan. It is fortunate that
compound 1 is metabolized to produce 8, since the latter
compound does not appear to be well absorbed after oral
administration yet is far more potent than 1.
In Vivo Testin g
Compounds 1, 7, and 8 were tested in Goldblatt (2K-
1C) hypertensive rats (11 was identified later in clinical
trials and never tested in the model). In this model the
left renal artery of normotensive rats is constricted by
a silver clip. The resultant hypertension is associated
with an elevated plasma renin activity and increased
circulating and/or tissue levels of A II. Mean arterial
pressure (MAP) was monitored for 24 h, and the
maximum decrease in MAP after various intragastric
(ig) and intravenous (iv) doses is reported for all three
compounds in Table 2 and in Figures 1-3. Figures 1
and 3 are plotted on a log scale because of the large dose
ranges, but the x-axis in Figure 2 is linear. The
maximum decrease in MAP is approximately the same
for all compounds and for both routes of administration
(within the normal range of biological variability for the
model), but the therapeutic doses vary significantly. The
therapeutic dose is the minimum dose to produce the
maximum decrease in MAP.
Mod elin g Stu d ies
Since the enolic group of 8 appears to be essential for
the increase in potency over 1, modeling studies were
carried out to determine a role for the enol in receptor
binding. Based on published homology14 and 3D phar-
macophore15 studies depicting the possible bound con-
formations of AT1 antagonists, molecular models of
tasosartan (1) and the enol metabolite (8) were com-
pared with models for losartan (2) and its metabolite
Compound 1 was initially tested ig over a large dose
range (0.1-30.0 mg/kg, Table 2, Figure 1). However,
as the potency of 1 became apparent, subsequent
analogues and metabolites were tested over a narrower
dose range, as seen with the remaining entries in Table
2. Compound 1 has a therapeutic dose of 1.0 mg/kg

4254 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Ellingboe et al.
Sch em e 4
Ta ble 1. In Vitro Data for Metabolites
Sch em e 5
compd
IC50, nM (n)a
1
7
8
9
10
11
1.20 ( 0.6 (3)
2.30 ( 1.6 (2)
0.17 ( 0.06 (3)
6.50 ( 0.26 (3)b
3.5% inhibition at 100 nMb
Sch em e 6
0.41 ( 0.10 (2)
a
IC50 for inhibition of specific binding of [125I]A II to rat adrenal
membrane, mean ( SE, n ) number of experiments. Each
experiment represents an average of triplicate measurements.
b
IC50 for inhibition of specific binding of [125I]A II to rat liver
membrane.
four minimal points of interaction15 are labeled 1-4.
Pharmacophore 1 is an acidic group, represented as a
tetrazole in Figure 4, which is proposed to interact with
Lys 199 in the homology model. Pharmacophore 2
represents a spacer (biphenyl group) which acts as a
scaffold to properly orient the other pharmacophores.
Pharmacophore 3 is an alkyl group that fits into a
lipophilic pocket in the AT1 receptor. In compounds 1
and 8 the lipophilic group is a methyl group whereas
in 2 and 3 it is a butyl chain. Pharmacophore 4 is an
aromatic nitrogen acting as a H-bond acceptor. The
increased potency of metabolites 8 and 11, and of the
losartan metabolite 3, relative to the parent drugs
suggests that a fifth interaction is also important.
Pharmacophore 5 is an acidic group which is proposed
to interact with a basic side chain found on a surface
loop of the AT1 receptor.
EXP3174 (3). A pharmacophore model based on these
previous studies is illustrated in Figure 4, utilizing
compound 8 as an example. Potential critical points of
interaction with the AT1 receptor are labeled 1-5. Also
listed are the specific transmembrane domains and
critical side chains based on the homology model.14 The
The key structural difference between compounds 1

Metabolites of the Angiotensin II Antagonist
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4255
Ta ble 2. Dose Response Data for Compounds 1, 7, and 8 in
the Goldblatt Hypertensive Rat after Intragastric and
Intravenous Dosing
compd
(route)
dose
(mg/kg)
max decrease
MAP (mmHg)a
nb
vehicle (n)c
1 (ig)
0.1
0.3
1.0
6 ( 8
6
6
6
9
12
6
7
8
7
6
7
6
7
6
3
6
6
6
6 ( 7 (10)
11 ( 5 (10)
11 ( 5 (10)
11 ( 4 (10)
11 ( 5 (10)
12 ( 3 (10)
6 ( 8 (5)
10 ( 8 (5)
9 ( 8 (5)
11 ( 4 (12)
11 ( 4 (12)
d
45 ( 11e,f
71 ( 14e,f
76 ( 8e,f
81 ( 8e,f
80 ( 11e,f
37 ( 9e
3.0
10.0
30.0
0.1
1.0
3.0
1.0
3.0
10.0
1.0
3.0
10.0
3.0
10.0
0.03
0.10
1 (iv)
7 (ig)
7 (iv)
53 ( 10e,f
54 ( 16e,f
55 ( 15f
54 ( 10e,f
71 ( 11
51 ( 13
47 ( 15
41 ( 8f
F igu r e 3. Change in MAP in Goldblatt hypertensive rats after
ig and iv dosing of compound 8.
d
10 ( 6 (8)
2 ( 7 (8)
4 ( 6 (12)
4 ( 6 (12)
8 ( 5 (8)
8 ( 5 (8)
8 (ig)
8 (iv)
39 ( 12
66 ( 12f
34 ( 5
67 ( 11f,g
6
a
Maximum decrease in mean arterial pressure in mmHg after
intragastric or intravenous dosing in Goldblatt (2K-1C) hyperten-
b
sive rats, mean ( SE. n ) number of rats. c Decrease in mean
arterial pressure in mmHg after intragastric or intravenous dosing
of vehicle in Goldblatt (2K-1C) hypertensive rats, mean ( SE (n
d
) number of rats). Mean arterial pressure for vehicle treated
F igu r e 4. Proposed binding model for compound 8.
rats not recorded. e p < 0.05 versus baseline (time 0). f p < 0.05
versus vehicle control. p < 0.05 versus 0.03 mg/kg group.
g
F igu r e 1. Change in MAP in Goldblatt hypertensive rats after
ig and iv dosing of compound 1.
F igu r e 5. Overlap of compounds 3 and 8.
interaction with a basic side chain should still be
possible. Figure 5 illustrates the final superposition of
compound 8 onto 3, and the pharmacophores defined
in Figure 4 are also indicated. The carboxylic acid of 3
and the enol of 8 occupy the same region so they could
be interacting with the same basic residue. The iso-
electric potential contour maps of compounds 1 and 8
and of 2 and 3 are shown in Figure 6. The isoelectric
potential contours the electrostatic energy at 1 kcal/mol
around each ligand. Red and blue represent the positive
and negative electrostatic fields, respectively. It is
evident that the metabolites 3 and 8 have enhanced
negative electrostatic potentials around the region of
pharmacophore 5. Thus, the carboxylic acid and enol
F igu r e 2. Change in MAP in Goldblatt hypertensive rats after
ig and iv dosing of compound 7.
and 8 as well as between 2 and 3 is the additional acidic
group introduced metabolically. This group serves as
pharmacophore 5 in the model and is in position to
interact favorably with one of the basic side chains (Lys
102 or Arg 167) in the receptor surface loop.14 This
interaction may be responsible for the increase in
potency seen with each pair of compounds. The pKa of
the enol in 8 is assumed to be about 9.7, which is less
acidic than the carboxylic acid in 3. However, an

4256 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Ellingboe et al.
F igu r e 6. Isoelectric potential contour maps of compounds 1, 2, 3, and 8.
groups can display the same type of electrostatic char-
acter to a basic side chain in the receptor.
Exp er im en ta l Section
Melting points were determined with a Thomas-Hoover
apparatus and are uncorrected. The NMR spectra were
recorded on a Varian VXR200, Varian VXR300, or a Bruker
AM-400 instrument. The infrared spectra were recorded on
a Perkin-Elmer diffraction grating or a Perkin-Elmer 784
spectrophotometer. The mass spectra were recorded on a
Hewlett-Packard 5995A or a Finigan 8230 mass spectrometer.
Analyses (C, H, N) were carried out on a modified Perkin-
Elmer Model 240 CHN analyzer. Merck silica gel (70-230
mesh) was used for flash chromatography. Organic extracts
were dried over MgSO4 unless otherwise noted.
5-Hydr oxy-2,4-d im eth yl-8-[2′-(1H-tetr a zol-5-yl)-bip h en -
yl-4-ylmethyl]-6,8-dihydro-5H-pyrido[2,3-d]pyrimidine-7-one
Sesqu ih yd r a te (7). To a cooled (0 °C), mechanically stirred
mixture of 111 (8.2 g, 0.020 mol), MgSO4 (14.4 g, 0.120 mol),
and water (285 mL) was added 2.5 N NaOH (8.78 mL). The
resultant mixture was adjusted to pH 7.0-7.5 with 2 N HCl
(0.7 mL). A solution of KMnO4 (6.3 g, 0.040 mol) in water (362
mL) was added dropwise over 2 h 40 min, and the mixture
was allowed to warm to room temperature. After 24 h,
additional MgSO4 (5.04 g, 0.042 mol) and KMnO4 (2.2 g, 0.014
mol) were added, and stirring was continued for 24 h. The
reaction mixture was filtered through Solka Floc, and the
filtrate was concentrated to about 200 mL. The pH was
adjusted to about 4 with KH2PO4 (13.6 g), and the precipitate
was collected by filtration. The material was taken up in
MeOH and filtered to give 1.96 g. The MnO2 recovered by
filtration through Solka Floc was stirred in water (400 mL)
and filtered. The filtrate was treated with KH2PO4 to bring
the pH to 4, and the precipitate was collected by filtration.
These modeling studies provide support for the idea
that the enol group in compound 8 serves a role similar
to that of the carboxylic acid group in compound 3 in
contributing to the enhanced receptor binding shown
by these compounds relative to their parent drugs.
Su m m a r y
Described in this paper is the synthesis and pharma-
cological activity of five metabolites of the A II antago-
nist tasosartan (1). The hydroxy metabolite 7 and the
glucuronide 10 were synthesized directly from com-
pound 1. The remaining compounds required total
syntheses to incorporate the different oxidation states
of the pyrido ring. Of particular interest is the effect
of the additional acidic group of the enol metabolites 8
and 11 on A II antagonist activity. Modeling studies
suggest that the enol can interact with a basic side chain
in the AT1 receptor, and this additional interaction may
enhance binding. Compound 8 is an order of magnitude
more potent than 1 in the AT1 receptor binding assay.
However, 8 is an order of magnitude less potent than 1
in the Goldblatt hypertensive rat after oral dosing,
probably because of poor absorption. The metabolic
introduction of a second acidic group in 1 bypasses this
problem and contributes to the excellent profile of the
compound.

Metabolites of the Angiotensin II Antagonist
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4257
The material was taken up in methanol and filtered to give
1.36 g. The combined material was purified by flash chroma-
tography (10-30% MeOH/CHCl3), trituration with EtOAc and
MeOH, and trituration with water to give 0.67 g (8%) of
for 2.5 h. The acetone was removed under reduced pressure,
and the aqueous phase was adjusted to pH 4 with 1 N KOH.
The resultant white precipitate was collected by filtration to
give 2.24 g (89%) of product as a white solid. An analytical
sample was recrystallized from hexane: mp 78-80 °C; 1H
NMR (DMSO-d6) δ 2.30 (s, 3 H), 2.36 (s, 3 H), 2.50 (s, 3 H),
4.57 (d, J ) 5.8 Hz, 2 H), 7.25 (d, J ) 8.3 Hz, 2 H), 7.49 (d, J
) 8.3 Hz, 2 H), 8.34 (t, J ) 5.8 Hz, 1 H). Anal. (C15H16BrN3O)
C, H, N.
1
product as a white solid: mp 154-167 °C; H NMR (DMSO-
d6) δ 2.44 (s, 3 H), 2.46 (s, 3 H), 2.79 (dd, J ) 16.3, 2.4 Hz, 1
H), 2.99 (dd, J ) 16.3, 4.0 Hz, 1 H), 4.97 (m, 1 H), 5.12 (d, J
) 15.4 Hz, 1 H), 5.31 (d, J ) 15.4 Hz, 1 H), 5.58 (d, J ) 3.1
Hz, 1 H), 6.98 (d, J ) 8.3 Hz, 2 H), 7.19 (d, J ) 8.3 Hz, 2 H),
7.52 (m, 2 H), 7.63 (m, 2 H). Anal. (C23H21N7O2‚1.5H2O) C,
H, N.
Step 6: 2,6-Dim eth yl-4-[N-(2′-(2-ter t-bu tyl-2H-tetr a zole-
5-yl)-4-(m eth yla m in o)bip h en yl)]-5-a cetylp yr im id in e (19).
To a heated solution of 18 (1.67 g, 5.00 mmol) and tetrakis-
(triphenylphosphine)palladium(0) (0.29 g, 0.25 mmol) in tolu-
ene (20 mL) was added a partial solution of 2-[(2-tert-butyl)-
2H-tetrazol-5-yl]phenylboronic acid11 (1.50 g, 6.10 mmol) and
Na2CO3 (1.06 g, 10.00 mmol) in water (10 mL) and EtOH (5
mL) in several portions over 1 h. Heating was continued for
2.5 h. The mixture was diluted with EtOAc, and the layers
were separated. The organic phase was washed with 0.1 N
NaOH and brine, dried, and concentrated. Purification by
flash chromatography (40-50% EtOAc/hexane) and trituration
with hexane gave 1.71 g (75%) of product as a white solid. An
analytical sample was recrystallized from ether: mp 131-132
2,4-Dim eth yl-5-h yd r oxy-8-[2′-(1H-tetr a zol-5-yl)bip h en -
yl-4-ylm eth yl]-8H-p yr id o[2,3-d ]p yr im id in -7-on e (8). Step
1: 2,6-Dim eth yl-5-iod o-4-h yd r oxyp yr im id in e (14). A me-
chanically stirred mixture of 2,6-dimethyl-4-hydroxypyrimi-
dine (50.0 g, 0.403 mol), iodine (102.2 g, 0.403 mol), and 1 N
NaOH (503 mL) was heated under reflux for 2 h. The mixture
was extracted with CHCl3 (some product fell out of solution
and was collected by filtration: 24.5 g). The extracts were
dried and concentrated to give 40.9 g of an orange solid which
contained starting material and product. Trituration with
EtOAc gave 26.0 g of product. The total yield of product was
50.5 g (50%): mp 208-210 °C (dec). An analytical sample was
recrystallized from EtOH: mp 216-218 °C; 1H NMR (DMSO-
d6) δ 2.21 (s, 3 H), 2.39 (s, 3 H), 12.58 (br s, 1 H). Anal. (C6H7-
IN2O) C, H, N.
Step 2: 4-Ch lor o-2,6-d im eth yl-5-iod op yr im id in e (15).
A mixture of 14 (24.5 g, 0.098 mol), phosphorus oxychloride
(30.0 g, 0.196 mol), and toluene (200 mL) was heated under
reflux for 1 h. The mixture was concentrated, and ice water
(100 mL) was added. The pH was adjusted to 5 with 2.5 N
NaOH and the mixture was extracted with CH2Cl2. The
combined extracts were washed with brine, dried, and con-
centrated to give 24.6 g of a brown solid. The crude product
was combined with 26.0 g of similarly prepared material and
filtered through a short column of silica gel eluted with CH2-
Cl2 to give 33.8 g (62%) of product as a yellow solid. An
analytical sample was recrystallized from hexane/CH2Cl2: mp
62-64 °C; 1H NMR (CDCl3) δ 2.61 (s, 3 H), 2.73 (s, 2 H). Anal.
(C6H6ClIN2) C, H, N.
Step 3: 4-Ch lor o-2,6-d im eth yl-5-(1-eth oxyvin yl)p yr i-
m id in e (16). A mixture of 15 (13.5 g, 0.050 mol), (1-
ethoxyvinyl)tributyltin (20.0 g, 0.055 mol), lithium chloride (6.4
g, 0.150 mol), tetrakis(triphenylphosphine)palladium(0) (1.7
g, 0.0015 mol), and dioxane (100 mL) was heated under reflux
for 23 h. Additional tetrakis(triphenylphosphine)palladium-
(0) (1.1 g, 0.0010 mol) was added, and heating was continued
for 25 h. The mixture was cooled and diluted with EtOAc,
and 1 N KF (100 mL) was added. The solids were removed
by filtration through Celite, and the layers were separated.
The organic phase was washed with pH 7 phosphate buffer
(100 mL) and brine (100 mL), dried, and concentrated.
Purification by flash chromatography (twice; 5-10% EtOAc/
hexane) gave 6.44 g (61%) of product as a colorless oil: 1H
NMR (CDCl3) δ 1.35 (t, J ) 7.0 Hz, 3 H), 2.49 (s, 3 H), 2.65 (s,
3 H), 3.91 (q, J ) 7.0 Hz, 3 H), 4.21 (d, J ) 2.8 Hz, 1 H), 4.52
(d, J ) 2.8 Hz, 1 H). Anal. (C10H13ClN2O) C, H, N.
1
°C; H NMR (CDCl3) δ 1.54 (s, 9 H), 2.51 (s, 3 H), 2.56 (s, 3
H), 2.64 (s, 3 H), 4.71 (d, J ) 5.7 Hz, 2 H), 7.12 (d, J ) 8.3 Hz,
2 H), 7.22 (d, J ) 8.3 Hz, 2 H), 7.45 (m, 3 H), 7.88 (m, 1 H),
9.0 (br s, 1 H). Anal. (C26H29N7O) C, H, N.
Step 7: 2,4-Dim eth yl-5-h yd r oxy-8-[2′-(2-ter t-bu tyl-2H-
tetr a zol-5-yl)bip h en yl-4-ylm eth yl]-8H-p yr id o[2,3-d ]p yr i-
m id in -7-on e (20). To a heated suspension of NaH (60%
dispersion in mineral oil; 0.24 g, 5.93 mmol) in diethyl
carbonate (1.40 g, 11.85 mmol) and THF (10 mL) was added a
solution of 19 (1.08 g, 2.37 mmol) in THF (10 mL) over 10 min.
Heating was continued for 1.5 h. The mixture was concen-
trated, taken up in water, and extracted twice with ether
(discarded). The aqueous phase was acidified to pH 4, and
the precipitate was collected by filtration to give 965 mg (85%)
of product. An analytical sample was recrystallized from
acetone: mp 235-237 °C; 1H NMR (CDCl3) δ 1.47 (s, 9 H),
2.72 (s, 3 H), 2.89 (s, 3 H), 5.52 (s, 2 H), 6.18 (s, 1 H), 7.04 (d,
J ) 8.2 Hz, 2 H), 7.31 (d, J ) 8.2 Hz, 2 H), 7.44 (m, 3 H), 7.84
(m, 1 H). Anal. (C27H27N7O2) C, H, N.
Step 8: 2,4-Dim eth yl-5-h yd r oxy-8-[2′-(1H-tetr a zol-5-yl)-
b ip h en yl-4-ylm et h yl]-8H -p yr id o[2,3-d ]p yr im id in -7-on e
Sesqu ih yd r a te (8). To a refluxing mixture of 20 (10 g, 0.020
mol) in toluene (250 mL) was added dropwise trifluoromethane-
sulfonic acid (10 mL). After 2 h, water (125 mL) was added
dropwise and the mixture was cooled to room temperature.
The toluene and water were decanted, and the residue was
washed three times with water (decanted) and dried under a
stream of N2. The material was dissolved in THF and filtered
through a pad of silica gel, eluting with 2.5% AcOH in THF.
The mixture was concentrated, EtOAc was added, and the
mixture was concentrated to give an oil. Water was added,
and the mixture was concentrated (twice). After the second
addition of water and concentration the compound crystallized.
The material was filtered and washed with water to give 7.4
g (84%) of product. Recrystallization of 520 mg of similarily
prepared product from EtOH/water gave 424 mg of product
as an off-white solid: mp 278-280 °C (dec); 1H NMR (DMSO-
d6) δ 2.55 (s, 3 H), 2.80 (s, 3 H), 5.43 (s, 2 H), 5.86 (s, 1 H),
6.99 (d, J ) 8.1 Hz, 2 H), 7.14 (d, J ) 8.1 Hz, 2 H), 7.42 (m, 1
H), 7.47 (dd, J ) 7.7, 1.5 Hz, 1 H), 7.54 (m, 1 H), 7.58 (dd, J
) 7.5, 1.5 Hz, 1 H). Anal. (C23H19N7O2‚1.5H2O) C, H, N.
St ep 4: 4-(4-Br om ob en zyla m in o)-2,6-d im et h yl-5-(1-
eth oxyvin yl)p yr im id in e (17). A mixture of 16 (3.00 g,
0.0141 mol), 4-bromobenzylamine hydrochloride (3.45 g, 0.0155
mol), NaHCO3 (2.37 g, 0.0282 mol), and n-BuOH (20 mL) was
heated under reflux for 70 h. The mixture was concentrated,
taken up in EtOAc, washed with water, dried, and concen-
trated. Trituration with ether/hexane gave 2.84 g (56%) of
product as a white solid. An analytical sample was recrystal-
2,4-Dim eth yl-8-[2′-(1H-tetr azol-5-yl)-biph en yl-4-ylm eth -
yl]-8H-p yr id o[2,3-d ]p yr im id in -7-on e (9). Step 1: 2,4-Di-
methyl-8-[[2’-cyano[1,1-biphenyl]-4-yl]methyl]-7H-pyrido[2,3-
d ]p yr im id in 7-on e (22). To a suspension of NaH (60%
dispersion in mineral oil; 0.123 g, 3.08 mmol) in DMF (10 mL)
was added 2,4-dimethyl-7H-pyrido[2,3-d]pyrimidin-7-one (21)
(0.54 g, 3.08 mmol), prepared according to Sakamoto et al.13
The reaction mixture was stirred at room temperature for 1
h, and 2-[4-(bromomethyl)phenyl]benzonitrile (1.00 g, 3.7
mmol) was added. Stirring was continued for 5 h, and the
1
lized from ether/hexane: mp 111-113 °C; H NMR (DMSO-
d6) δ 1.24 (t, J ) 6.9 Hz, 3 H), 2.15 (s, 3 H), 2.24 (s, 3 H), 3.84
(q, J ) 6.9 Hz, 2 H), 4.18 (d, J ) 1.7 Hz, 1 H), 4.50 (d, J ) 6.2
Hz, 2 H), 4.53 (d, J ) 1.7 Hz, 1 H), 6.91 (t, J ) 6.2 Hz, 1 H),
7.22 (d, J ) 8.3 Hz, 2 H), 7.46 (d, J ) 8.3 Hz, 2 H). Anal.
(C17H20BrN3O) C, H, N.
Step 5: 2,6-Dim eth yl-4-[N-(4-br om oben zyla m in o)]-5-
a cetylp yr im id in e (18). A solution of 17 (2.72 g, 7.51 mmol),
1 N HCl (8.3 mL), and acetone (8 mL) was heated under reflux

4258 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Ellingboe et al.
mixture was concentrated and taken up in water. The aqueous
layer was extracted with CH2Cl2, and the combined extracts
were dried and concentrated. Purification by flash chroma-
tography (2% MeOH/CH2Cl2) and trituration with ether gave
0.65 g (58%) of product as a yellow solid: mp 177-179 °C; 1H
NMR (DMSO-d6) δ 2.63 (s, 3 H), 2.69 (s, 3 H), 5.61 (s, 2 H),
6.72 (d, J ) 9.6 Hz, 1 H), 7.50 (m, 6 H), 7.76 (m, 1 H), 7.91 (d,
J ) 7.6 Hz, 1 H), 8.20 (d, J ) 9.6 Hz, 1 H).
Step 2: 2,4-Dim eth yl-8-[2′-(1H-tetr a zol-5-yl)-bip h en yl-
4-ylm eth yl]-8H-p yr id o[2,3-d ]p yr im id in -7-on e (9). A mix-
ture of 22 (0.65 g, 1.8 mmol), n-Bu3SnCl (0.75 g, 2.3 mmol),
NaN3 (0.15 g, 2.3 mmol), and xylene (8.0 mL) was heated under
reflux for 48 h. The reaction mixture was cooled, and 1 N HCl
was added. The mixture was extracted with EtOAc, and the
layers were separated. The organic phase was extracted with
1 N NaOH, and the combined basic layers were acidified to
pH 4 and extracted with CH2Cl2. The combined extracts were
dried and concentrated to give a yellow oil. The oil was
triturated and recrystallized from EtOH to give 0.26 g (55%)
of product as an off-white solid: mp 253-255 °C; 1H NMR
(DMSO-d6) δ 2.60 (s, 3 H), 2.68 (s, 3 H), 5.51 (s, 2 H), 6.71 (d,
J ) 9.6 Hz, 1 H), 7.00 (d, J ) 8.0 Hz, 2 H), 7.20 (d, J ) 8.0 Hz,
2 H), 7.59 (m, 4 H), 8.20 (d, J ) 9.6 Hz, 1 H). Anal.
(C23H19N7O) C, H. N: calcd, 23.95; found, 23.42.
1-Deoxy-1-{5-[4′-(7-oxo-6,7-d ih yd r o-5H -p yr id o[2,3-d ]-
p yr im id in -8-ylm eth yl)-bip h en yl-2-yl]-tetr a zol-2-yl}-â-D-
glu cu r on ic Acid Sod iu m Sa lt Mon oh yd r a te (10). Step
1: 1-Deoxy-2,3,4-tr i-O-a cetyl-1-{5-[4′-(7-oxo-6,7-d ih yd r o-
5H -p yr id o[2,3-d ]p yr im id in -8-ylm et h yl)-b ip h en yl-2-yl]-
tetr a zol-2-yl}-â-D-glu cu r on ic Acid Meth yl Ester (12a ). To
a solution of 1 (1.27 g, 3.1 mmol) in MeOH (10 mL) was added
1 N KOH (3.1 mL). The mixture was concentrated, and the
potassium salt was dissolved in acetone (14 mL). Bromo-2,3,4-
tri-O-acetyl-R-D-glucopyranuronic acid methyl ester (1.4 g, 3.55
mmol) was added, and the mixture was heated under reflux
for 18 h. The mixture was cooled, concentrated, and purified
by flash chromatography (20% acetone/Et2O) to give 0.77 g
(34%) of N-2 glucuronide product (12a ) and 0.40 g (18%) of
N-1 glucuronide product (12b). 12a : 1H NMR (CDCl3) δ 2.04
(s, 3 H), 2.05 (s, 3 H), 2.40 (s, 3 H), 2.57 (s, 3 H), 2.73 (t, J )
6.5 Hz, 2 H), 2.84 (t, J ) 6.9 Hz, 2 H), 3.74 (s, 3 H), 4.28 (d, J
) 9.5 Hz, 1 H), 5.29 (s, 2 H), 5.40 (m, 2 H), 5.80 (t, J ) 9.3 Hz,
1 H), 6.02 (d, J ) 9.3 Hz, 1 H), 7.04 (d, J ) 6.5 Hz, 1 H), 7.50
(m, 5 H), 7.78 (dd, J ) 7.3, 1.5 Hz, 1 H). 12b: 1H NMR (CDCl3)
δ 1.71 (s, 3 H), 1.98 (s, 3 H), 1.99 (s, 3 H), 2.40 (s, 3 H), 2.56
(s, 3 H), 2.70 (t, J ) 7.9 Hz, 2 H), 2.84 (t, J ) 8.0 Hz, 2 H),
3.75 (s, 3 H), 3.77 (d, J ) 7.5 Hz, 1 H), 5.20 (m, 5 H), 5.66 (t,
J ) 8.9 Hz, 1 H), 7.04 (d, J ) 8.3 Hz, 2 H), 7.35 (d, J ) 8.3 Hz,
1 H), 7.55 (m, 3 H), 7.65 (m, 1 H).
Step 2: 1-Deoxy-1-{5-[4′-(7-oxo-6,7-d ih yd r o-5H-p yr id o-
[2,3-d ]p yr im id in -8-ylm et h yl)-b ip h en yl-2-yl]-t et r a zol-2-
yl}-â-D-glu cu r on ic Acid Meth yl Ester (13). To a solution
of 12a (1.1 g, 1.50 mmol) in MeOH (30 mL) was added KCN
(50 mg, 0.75 mmol), and the mixture was stirred at room
temperature for 5 h. The mixture was concentrated and
purified by flash chromatography (5% MeOH/CH2Cl2) to give
0.57 g (63%) of product as a white foam: 1H NMR (DMSO-d6)
δ 2.36 (s, 3 H), 2.44 (s, 3 H), 2.72 (t, J ) 7.9 Hz, 2 H), 2.88 (t,
J ) 7.7 Hz, 2 H), 3.51 (m, 2 H), 3.68 (s, 3 H), 3.94 (m, 1 H),
4.26 (d, J ) 9.1 Hz, 1 H), 5.19 (s, 2 H), 5.47 (d, J ) 5.2 Hz, 1
H), 5.60 (d, J ) 5.2 Hz, 1 H), 5.66 (d, J ) 5.5 Hz, 1 H), 6.04 (d,
J ) 9.2 Hz, 1 H), 7.03 (d, J ) 8.0 Hz, 2 H), 7.18 (d, J ) 8.1 Hz,
2 H), 7.55 (m, 2 H), 7.76 (d, J ) 7.5 Hz, 1 Hz).
H), 7.05 (d, J ) 8.3 Hz, 2 H), 7.19 (d, J ) 8.3 Hz, 2 H), 7.38 (br
s, 1 H), 7.55 (m, 3 H), 7.78 (dd, J ) 7.5, 1.2 Hz, 1 Hz). Anal.
(C29H28N7NaO7‚H2O) H, N. C: calcd, 55.45; found, 54.90.
5-Hyd r oxy-2-h yd r oxym eth yl-4-m eth yl-8-[2′-(1H-tetr a -
zol-5-yl)-biph en yl-4-ylm eth yl]-8H-pyr ido[2,3-d]pyr im idin -
7-on e (11). Step 1: 2-Eth oxy-3-m eth oxy Im id a te Hyd r o-
ch lor id e (24). To a solution of methoxyacetonitrile (120 g,
1.6882 mol) in EtOH (99 mL) and Et2O (500 mL) at -15 °C
(ice/MeOH bath) was added HCl gas rapidly for 20 min, until
the solution was at or near saturation. This solution was
stirred overnight at room temperature under a nitrogen
atmosphere. The mixture was recooled to -15 °C, and the
solid was vacuum-filtered, washed with ether, and dried by
drawing dry nitrogen gas through the solid for 1 h using a
vacuum filtration apparatus. The yield of white solid was
216.1 g (83.3%): 1H NMR (300 MHz, DMSO-d6) δ 1.38 (t, J )
7.0 Hz, 3 H), 3.42 (s, 3 H), 4.42 (s, 2 H), 4.54 (q, J ) 7.0 Hz, 2
H).
St ep 2: 1-Met h oxya cet a m id in e H yd r och lor id e (25).
Dry EtOH (1.0 L) was cooled to -15 °C, and anhydrous
ammonia (65.0 g) was added followed by 24 (200.0 g, 1.302
mol); a small amount of EtOH was used to wash in any
remaining solid. This mixture was stirred at room tempera-
ture overnight. It was recooled to -15 °C, and a small amount
of solid was filtered. The filtrate was placed on an evaporator
to remove the solvent and then under high vacuum to remove
the last traces of solvent. The clear oil spontaneously crystal-
lized to give 162.2 g of a white solid (100%): 1H NMR (300
MHz, DMSO-d6) δ 3.39 (s, 3 H), 4.26 (s, 2 H), 9.00 (br s).
Step 3: 2-Meth oxym eth yl-6-m eth yl-4-p yr im id on e (26).
To a solution of NaOEt/EtOH, made from EtOH (1419 mL)
and sodium (55.4 g), was added 25 (150.0 g, 1.204 mol) and
ethylacetoacetate (156.71 g, 1.204 mol). This mixture was
heated to reflux for 18 h. The reaction mixture was cooled,
and the EtOH was removed on a rotary evaporator. The solids
were dissolved in water, and the pH was adjusted to 5.5 using
concentrated HCl. The aqueous mixture was extracted with
CH2Cl2 and ethyl acetate. The organics were combined,
washed with brine, dried (Na2SO4), filtered and evaporated
to leave a light-yellow solid. The solid was triturated using
EtOAc/hexane (2/3), collected via vacuum filtration, and placed
under high vacuum for 3 h. The yield of white solid was 126.0
g (68%): 1H NMR (300 MHz, DMSO-d6) δ 2.15 (s, 3 H), 3.31
(s, 3 H), 4.21 (s, 2 H), 6.08 (s, 1 H); IR (KBr), cm-1 1680 (s),
910 (vs); MS (EI) M+ m/z 154, (M - OCH3)+ m/z 124.
Step 4: 2-Meth oxym eth yl-5-iod o-6-m eth yl-4-p yr im i-
d on e (27). To a well-stirred solution of 26 (15.0 g, 0.0973 mol)
in 1.25 N NaOH (160 mL) was added iodine (25.4 g, 0.10 mol).
When the iodine was in solution the mixture was heated to
reflux (110 °C oil bath) for 2 h. The solution was then cooled
to 0 °C and held there for 1 h. During this time, a solid
precipitated and it was collected via vacuum filtration and
washed with a little cold water. The filtrate was extracted
with CHCl3, dried (Na2SO4), filtered, and evaporated to leave
a solid that was a mixture of starting material and product.
Flash chromatography (silica gel, EtOAc as eluant) was used
to separate these components and afford 10.5 g of pure product
(both crystalline precipitate and chromatographed material).
The yield was 52.5% (corrected for 4.0 g of starting material
recovered): 1H NMR (300 MHz, CDCl3) δ 2.57 (s, 3 H), 3.54
(s, 3 H), 4.38 (s, 2 H), 10.70 (br s, 1 H); IR (KBr), cm-1 1645
(vs), 1000 (s); MS (EI) M+ m/z 280, (m - OCH3)+ m/z 250.
Step 5: 2-Meth oxym eth yl-4-ch lor o-5-iod o-6-m eth ylp y-
r im id in e (28). To a well-stirred solution of 27 (50.0 g, 0.1785
mol) in toluene at room temperature was added phosphorus
oxychloride (2 equiv, 34.2 mL). The mixture was heated to
reflux (115 °C oil bath) for 1.5 h, then cooled to approximately
10 °C, and quenched with ice water. The toluene was removed
on an evaporator, and 150 mL of cold water were added. The
pH was adjusted to 5.5 with 2.5 N aqueous NaOH, and the
mixture was extracted with CH2Cl2. The combined extracts
were washed with brine, dried (Na2SO4), filtered, and evapo-
rated. Final purification by flash chromatography (short silica
column, CH2Cl2/EtOAc as eluant) afforded 48.5 g (91%) of the
Step 3: 1-Deoxy-1-{5-[4′-(7-oxo-6,7-d ih yd r o-5H-p yr id o-
[2,3-d ]p yr im id in -8-ylm et h yl)-b ip h en yl-2-yl]-t et r a zol-2-
yl}-â-D-glu cu r on ic Acid Sod iu m Sa lt Mon oh yd r a te (10).
To a solution of (13) (0.53 g, 0.89 mmol) in MeOH (5 mL) was
added 1 N NaOH (0.89 mL), and the mixture was stirred at
room temperature for 3 h. The mixture was filtered to give
1
0.35 g (62%) of a white solid: mp 226-228 °C (dec); H NMR
(DMSO-d6) δ 2.36 (s, 3 H), 2.45 (s, 3 H), 2.72 (t, J ) 8.3 Hz, 2
H), 2.88 (t, J ) 7.5 Hz, 2 H), 3.23 (m, 1 H), 3.37 (m, 1 H), 3.56
(d, J ) 10.0 Hz, 1 H), 3.83 (m, 1 H), 5.18 (d, J ) 5.0 Hz, 1 H),
5.19 (s, 2 H), 5.40 (d, J ) 5.8 Hz, 1 H), 5.72 (d, J ) 9.1 Hz, 1

Metabolites of the Angiotensin II Antagonist
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4259
product as a light tan oil: 1H NMR (300 MHz, CDCl3) δ 2.82
phase was filtered. The aqueous filtrate was extracted with
CH2Cl2 and EtOAc; the filtered solid was treated with EtOAc/
MeOH (3/1, 160 mL) and filtered, and the filtrate was
combined with the other organic extracts. The organic solution
was dried (Na2SO4), filtered, and evaporated. The product was
purified by flash chromatography (silica gel; CH2Cl2/MeOH,
25/1, as eluant) to give 5.575 g (72%) of a white solid. Four
identical experiments of the above were run to give 22.3 g of
product: 1H NMR (400 MHz, DMSO-d6) δ 1.36 (s, 9 H), 2.84
(s, 3 H), 3.32 (s, 3 H), 4.52 (s, 2 H), 5.46 (s, 2 H), 5.91 (s, 1 H),
6.96 (d, J ) 8.35 Hz, 2 H), 7.25 (d, J ) 8.35 Hz, 2 H), 7.42 (m,
1 H), 7.51(m, 1 H), 7.58 (m, 1 H), 7.74 (m, 1 H), 11.98 (br, 1
(s, 3 H), 3.53 (s, 3 H), 4.59 (s, 2 H).
Step 6: 2-Meth oxym eth yl-4-ch lor o-5-(1-eth oxyvin yl)-
6-m eth ylp yr im id in e (29). A mixture of 28 (39.80 g, 0.1333
mol), (1-ethoxyvinyl)tributyltin (48.15 g, 0.1333 mol), lithium
chloride (19.5 g, 0.46 mol), and copper(I) iodide (1.0 g) in dry
dioxane (250 mL) was deoxygenated for 20 min using a stream
nitrogen gas. To this mixture was then added tetrakis-
(triphenylphosphine)palladium(0) (8.1 g, 0.007 mol) and tris-
(dibenzylideneacetone)dipalladium(0) (3.0 g, 0.0033 mol), and
it was heated to mild reflux (105 °C oil bath) for 40 h. The
mixture was cooled, diluted with EtOAc (250 mL), and treated
with 1 N aqueous KF (200 mL). This was stirred for 20 min
and then filtered through Celite. The layers were separated,
the aqueous phase was extracted with EtOAc, the organics
were combined, washed with brine, dried (Na2SO4), filtered,
and evaporated. The product was purified by flash chroma-
tography (silica gel, EtOAc/hexane (9/1) as eluant) to afford
24.3 g (75%) of product as a light-yellow oil: 1H NMR (300
MHz, CDCl3) δ 1.38 (t, J ) 7.0 Hz, 3 H), 2.58 (s, 3 H), 3.53 (s,
3 H), 3.94 (t, J ) 7.0 Hz, 2 H), 4.25 (d, J ) 2.9 Hz, 1 H), 4.55
(d, J ) 2.9 Hz, 1 H), 4.62 (s, 2 H).
Step 7: 2′-(2-ter t-Bu tyl-2H-tetr a zole-5-yl)-4-m eth yla m i-
n obip h en yl (35). A mixture of 2′-(2-tert-butyl-2H-tetrazole-
5-yl)-4-cyanobiphenyl (34) (30.70 g, 0.1012 mol), Raney nickel
(9.0 g), concentrated ammonium hydroxide (90 mL), and EtOH
(425 mL) was placed into a 1 L vessel under 50 psig of
hydrogen gas on a Parr apparatus. The vessel was shaken
for 22 h while maintaining a pressure of 50 psig. The mixture
was then filtered through Celite and evaporated. The residue
was dissolved in CH2Cl2, washed with brine, dried (Na2SO4),
filtered, and evaporated to leave a colorless oil. High vacuum
for 4 h afforded 31.1 g (100%) of pure product that was used
as is in the next step: 1H NMR (300 MHz, CDCl3) δ 1.60 (s, 9
H), 3.86 (s, 2 H), 7.14-7.90 (series of m, 8 H).
Step 8: 2-Meth oxym eth yl-4-[N-(2′-(2-ter t-bu tyl-2H-tet-
r azole-5-yl)-4-(m eth ylam in o)biph en yl)]-5-(1-eth oxyvin yl)-
6-m eth ylp yr im id in e (30). A mixture of 35 (28.28 g, 0.092
mol), 29 (20.30 g, 0.0836 mol), and NaHCO3 (7.73 g, 0.092 mol)
in n-butyl alcohol (105 mL) was heated to 120 °C for 48 h.
The cooled mixture was evaporated in vacuo, and the residue
was dissolved in EtOAc and washed with dilute brine (2×).
The organic phase was dried (Na2SO4), filtered, evaporated,
and placed under high vacuum. The product was used as is
in the next step. The yield was 42.0 g (98%) of unpurified
material: 1H NMR (300 MHz, CDCl3) δ 1.33 (t, J ) 7.0 Hz, 3
H), 1.56 (s, 9 H), 2.38 (s, 3 H), 3.50 (s, 3 H), 3.87 (q, J ) 7.0
Hz, 2 H), 4.27 (d, J ) 2.2 Hz, 1 H), 4.46 (s, 2 H), 4.53 (d, J )
2.2 Hz, 1 H), 4.68 (d, J ) 5.6 Hz, 2 H), 5.59 (m, 1 H), 7.15-
7.87 (series of m, 8 H).
Step 9: 2-Meth oxym eth yl-4-[N-(2′-(2-ter t-bu tyl-2H-tet-
r a zole-5-yl)-4-(m eth yla m in o)bip h en yl)]-5-a cetyl-6-m eth -
ylp yr im id in e (31). To a solution of 30 (41.5 g, 0.0808 mol)
in acetone (125 mL) was added 1 N aqueous HCl (125 mL).
This mixture was heated to reflux (65 °C oil bath) for 2.5 h.
The mixture was then cooled, the acetone evaporated, the pH
adjusted to 5.0, and the mixture extracted with CH2Cl2. The
combined extracts were dried (Na2SO4), filtered, and evapo-
rated. The product was purified by flash chromatography
(silica gel; Et2O/EtOAc first 3/1, then 1/1 as eluant) to afford
30.1 g (74%, two steps) of a yellow oil: 1H NMR (300 MHz,
CDCl3) δ 1.56 (s, 9 H), 2.58 (s, 3 H), 2.68 (s, 3 H), 3.52 (s, 3 H),
4.47 (s, 2 H), 4.73 (d, J ) 5.6 Hz, 2 H), 7.15-7.90 (series of m,
8 H), 8.86 (br m, 1 H); MS (+FAB), [M + H]+ m/z 486.
1
H); H NMR (400 MHz, CDCl3) δ 1.49 (s, 9 H), 2.91 (s, 3 H),
3.52 (s, 3 H), 4.67 (s, 2 H), 5.58 (s, 2 H), 6.21 (s, 1 H), 7.04 (d,
J ) 8.35 Hz, 2 H), 7.33 (s, 1 H), 7.36 (d, J ) 8.35 Hz, 2 H),
7.45 (m, 2 H), 7.83 (m, 1 H), 11.60 (br s, 1 H); MS (EI) M+ m/z
511.
Step 11: 2-Hyd r oxym eth yl-5-h yd r oxy-8-[N-(2′-(2-ter t-
bu tyl-2H-tetr azole-5-yl)-4-yl-m eth ylbiph en yl)]-8H-pyr ido-
[2,3-d ]p yr im id in e-7-on e (33). To a well-stirred solution of
32 (11.4 g, 0.0223 mol) in CH2Cl2 (100 mL) at 0 °C was added
BBr3 (55.75 mL of a 1 M solution in CH2Cl2). A solid formed
immediately. The reaction mixture was warmed slowly to 45
°C and held there for 2.5 h. TLC indicated the absence of
starting material. The reaction mixture was cooled to 0 °C,
and 1.25 N aqueous NaOH (175 mL) was added rapidly
dropwise. This was stirred for 2 h at 0 °C and allowed to come
to room temperature. The pH was adjusted to 6.0, the layers
were separated, and the aqueous layer was extracted with
CH2Cl2. The combined organic extracts were dried (Na2SO4),
filtered, and evaporated. The product was purified by flash
chromatography (silica gel; CH2Cl2/MeOH, 20/1, as eluant) to
afford 8.05 g (72.5%) of white solid. Another smaller scale
experiment was run for an additional 6.85 g: 1H NMR (300
MHz, CDCl3) δ 1.51 (s, 9 H), 2.83 (s, 3 H), 3.74 (br s, 1 H),
4.81 (s, 2 H), 5.51 (s, 2 H), 6.13 (s, 1 H), 7.06 (d, J ) 8.35 Hz,
2 H), 7.27 (d, J ) 8.35 Hz, 2 H), 7.33 (m, 1 H), 7.45 (m, 2 H),
1
7.84 (m, 1 H), 12.20 (br s, 1 H); H NMR (400 MHz, DMSO-
d6) δ 1.37 (s, 9 H), 2.88 (s, 3 H), 4.49 (s, 2 H), 5.12 (br s, 1 H),
5.45 (s, 2 H), 5.47 (s, 1 H), 6.93 (d, J ) 8.13 Hz, 2 H), 7.10 (br,
1 H), 7.27 (d, J ) 8.13 Hz, 2 H), 7.43 (m, 1 H), 7.50 (m, 1 H),
7.56 (m, 1 H), 7.75 (m, 1 H); MS (EI), M+ m/z 497.
Step 12: 2-Hyd r oxym eth yl-5-h yd r oxy-8-[N-(2′-(2H-tet-
r a zole-5-yl)-4-yl-m eth ylbip h en yl)]-8H-p yr id o[2,3-d ]p yr i-
m id in e-7-on e (11). A mixture of 33 (0.005 g, 0.01 mmol),
toluene (0.25 mL), and trifluoromethanesulfonic acid (0.01
mL), under nitrogen atmosphere, was heated in an oil bath at
115 °C for 2 h. The mixture was cooled to approximately 20
°C, methanol (1 mL) and water (0.5 mL) were added, and the
pH was adjusted to 5.5. The organic solvents were removed
on an evaporator, and an aqueous suspension resulted. The
solids were collected by vacuum filtration, washed with water,
then with ethyl acetate/ether (1/1), and sucked dry in a small
fine sintered glass funnel under a flow of nitrogen gas for 1 h.
The solid was purified by prep-layer chromatography using
silica gel, and the eluant was dichloromethane/methanol/acetic
acid (91/9.5/0.5). The material was placed under high vacuum
at 70 °C for 22 h to yield 0.003 g (67%) of a white solid: 1H
NMR (400 MHz, DMSO-d6) δ 2.83 (s, 3 H), 4.55 (s, 2 H), 5.10
(br, 1 H), 5.50 (s, 2 H), 5.89 (s, 1 H), 6.97 (d, J ) 8.35 Hz, 2 H),
7.25 (d, J ) 8.35 Hz, 2 H), 7.51 (m, 2 H), 7.62 (m, 2 H), 11.97
(s, 1 H); MS (+FAB), [M + H]+ m/z 442. A second batch of
11.8 g was prepared in a similar fashion. HPLC (inertsil,
5/95-80/20 CH3CN/10 mM KH2PO4, pH ) 3.50) showed the
compound to be 99.92% pure. Anal. (C23H19N7O3‚1.5H2O) H,
N. H: calcd, 4.73; found, 4.14.
Step 10: 2-Meth oxym eth yl-5-h yd r oxy-8-[N-(2′-(2-ter t-
bu tyl-2H-tetr azole-5-yl)-4-yl-m eth ylbiph en yl)]-8H-pyr ido-
[2,3-d ]p yr im id in e-7-on e (32). To a heated suspension (60
°C oil bath) of NaH (60% in oil, 1.51 g, 0.0377 mol) and diethyl
carbonate (9.14 mL, 0.0754 mol) in THF (65 mL) was added,
over 20-30 min, a solution of 31 in THF (40 mL). After the
addition, the solution was heated to reflux for 2 h. The cooled
mixture (10 °C) was quenched with water (5 mL), and pH 4
buffer was added until the pH of the reaction mixture was 5.0.
The THF was removed on an evaporator, and the aqueous
p Ka Deter m in a tion . The pKa values for compound 20
were determined by potentiometric titration with a Sirius
GLpKa titrator. A sample of 20 (2-5 mg) was dissolved in
0.15 M KCl with 15-57% methanol and titrated with 0.5 M
HCl from pH 1.8 to pH 11.5. All of the samples were sonicated
at pH 1.8 before titration to ensure complete dissolution. No
precipitation appeared during the titration at this pH range
as monitored by a turbidity probe. The aqueous pKa values

4260 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Ellingboe et al.
were obtained from extrapolation of the five apparent pKa
values in the presence of methanol to 0% methanol. Three
pKa values were obtained for 20 at 0% methanol: 9.73 ( 0.37,
6.20 ( 0.17, and 2.48 ( 0.18. The slope of the linear plot of
the apparent pKa values in the presence of methanol as a
function of the solvent dielectric constant is positive for the
value of 9.73, which indicates that the pKa value is due to an
acidic group. The negative slopes for the values of 6.20 and
2.48 are a result of ionization from basic groups.
23, and 24 h following dosing. At each time point following
dosing, averages were determined for absolute MAP, change
in MAP (in mmHg), and heart rate for all animals, and the
standard error of the mean was calculated.
Ack n ow led gm en t. We express our appreciation to
the Analytical Department of Wyeth-Ayerst for elemen-
tal analyses and spectral data.
Molecu la r Mod elin g. Molecular models were generated
using Sybyl (v6.4) molecular modeling software from Tripos
Associates.16 The conformations of the ligand models used in
this study were set as close as possible to the conformations
found in recent studies14,15 based on stereo diagrams and
specific distance constraints. Each model was minimized to
its local minimum using MAXIMIN2.16 Solvent conditions
were represented implicitly using a distance-dependent di-
electric constant along with a nonbonded cutoff set to 15 Å;
atom partial charges were generated using the semiempirical
method MOPAC.16 All ligands were superimposed for com-
parison using a rigid body fitting algorithm called FIT16 where
specific pharmacophore atoms represented in all of the ligands
were superimposed.
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P h a r m a cology:
[
125I]A II Bin d in g Assa y. Rat liver
membrane was obtained as a kit from New England Nuclear
(NED-014A, Boston, MA). The membrane was diluted and the
A II reconstituted with the diluent buffer provided with the
kit. The final concentrations of A II were 10-10 to 10-7 M for
the standard and 10-4 M for determination of the nonspecific
binding. The [125I]A II was diluted in deionized water. The
test compounds were dissolved in 50% DMSO as concentrates.
For the assay, 25 µL of a standard, reference, test compound
or vehicle was pipetted into appropriate test tubes. In addi-
tion, 25 µL of diluted [125I]A II and 200 µL of membrane
suspension were pipetted into each test tube. The contents
of the test tube were mixed and incubated for 3 h at room
temperature. The incubation was stopped by addition of saline
and filtration on to a glass fiber filter. The filter disks were
counted in a gamma counter to determine the [125I]A II binding
to the rat liver membrane. The IC50 values were determined
as the concentration of the compound required to inhibit the
specific [125I]A II binding by 50% of the vehicle control.
Gold bla tt (2K-1C) Hyp er ten sive Ra ts. Sprague-Dawley
rats weighing 150 g were anesthetized with pentobarbital (50
mg/kg ip), and the left renal artery was clipped with a silver
wire bent to an internal diameter of 0.2 mm. The hypertension
was established 4-7 weeks after clipping. At that time, the
animals were anesthetized with pentobarbital (50 mg/kg ip),
and the right carotid artery was cannulated. The catheter was
passed subcutaneously to the dorsal side of the neck and
exteriorized. The animals were fasted overnight but allowed
access to water, and the experiment was performed the next
morning. The carotid catheter was connected to a blood
pressure transducer which was linked to a MI2 computer data
acquisition system or to a Grass or Beckman recorder for the
recording of mean arterial pressure and heart rate. The rats
were given either compound 1, 7, 8, or vehicle (0.5% methyl
cellulose in distilled water) by gastric gavage in a volume of 5
mL/kg. In the experiments with 1, 7, and 8, saline was infused
into the arterial cannula at 5 µL/min throughout the experi-
ment to maintain patency of the cannula. A separate vehicle
control group was run with a saline infusion into the arterial
cannula. Each compound was administered to a separate
group of 6-12 rats and MAP and heart rate were recorded for
24 h. Data were recorded every 15 min for the first hour, every
30 min for the second hour, hourly up to 8 h, and then at 22,
(15) Prendergast, K.; Adams, K.; Greenlee, W. J .; Nachbar, R. B.;
Patchett, A. A.; Underwood, D. J . Derivation of a 3D pharma-
cophore model for the angiotensin-II site one receptor. J .
Comput.-Aided Mol. Des. 1994, 8, 491-512.
(16) Sybyl, v6.4, Tripos Associates, St. Louis, Missouri.
J M970690Q