Discovery of a series of imidazo[4,5-b ]pyridines with dual activity at angiotensin II Type 1 receptor and peroxisome proliferator-activated receptor-γ

Article abstract of DOI:10.1021/jm200409s

Mining of an in-house collection of angiotensin II type 1 receptor antagonists to identify compounds with activity at the peroxisome proliferator-activated receptor-γ (PPARγ) revealed a new series of imidazo[4,5-b]pyridines 2 possessing activity at these

Full text of DOI:10.1021/jm200409s

ARTICLE
pubs.acs.org/jmc
Discovery of a Series of Imidazo[4,5-b]pyridines with Dual Activity
at Angiotensin II Type 1 Receptor and Peroxisome
Proliferator-Activated Receptor-γ
Agustin Casimiro-Garcia,* Gary F. Filzen,* Declan Flynn, Christopher F. Bigge, Jing Chen, Jo Ann Davis,
Danette A. Dudley, Jeremy J. Edmunds, Nadia Esmaeil, Andrew Geyer, Ronald J. Heemstra, Mehran Jalaie,
Jeffrey F. Ohren, Robert Ostroski, Teresa Ellis, Robert P. Schaum, and Chad Stoner
Pfizer Global Research and Development, Groton Laboratories, Eastern Point Rd, Groton, Connecticut 06340, United States
S Supporting Information
b
ABSTRACT: Mining of an in-house collection of angiotensin II type 1 receptor
antagonists to identify compounds with activity at the peroxisome proliferator-
activated receptor-γ (PPARγ) revealed a new series of imidazo[4,5-b]pyridines 2
possessing activity at these two receptors. Early availability of the crystal structure of
the lead compound 2a bound to the ligand binding domain of human PPARγ
confirmed the mode of interaction of this scaffold to the nuclear receptor and
assisted in the optimization of PPARγ activity. Among the new compounds, (S)-
3-(5-(2-(1H-tetrazol-5-yl)phenyl)-2,3-dihydro-1H-inden-1-yl)-2-ethyl-5-isobutyl-
7-methyl-3H-imidazo[4,5-b]pyridine (2l) was identified as a potent angiotensin II
type I receptor blocker (IC50 = 1.6 nM) with partial PPARγ agonism (EC50 = 212 nM, 31% max) and oral bioavailability in rat. The
dual pharmacology of 2l was demonstrated in animal models of hypertension (SHR) and insulin resistance (ZDF rat). In the SHR,
2l was highly efficacious in lowering blood pressure, while robust lowering of glucose and triglycerides was observed in the male
ZDF rat.
’ INTRODUCTION
that there is a continuous relationship between blood glucose
level and macrovascular disease with no obvious threshold.
Indeed, a recent study showed a clear relationship between
HbA1c and cardiovascular (CV) risk even within a nondiabetic
range.10 Insulin resistance can be treated through diet modifica-
tion and exercise11 and also through pharmaceutical intervention
using, in combination or as standalone therapy, biguanides,
sulfonylureas, glucagon-like peptide-1 (GLP-1) analogues,12
dipeptidyl peptidase 4 (DPP-4) inhibitors,13 and the thiazolidi-
nedione (TZD) class of drugs. TZDs function through insulin
sensitization by activation of the nuclear hormone receptor
peroxisome proliferator activated receptor-γ (PPARγ), though
recent evidence suggest that these drugs may in fact function by
blocking a phosphorylation cascade.14 A consequence of simul-
taneously treating several comorbidities is that a significant pill
burden is placed on the patient, often leading to issues with
compliance and unforeseen drugꢀdrug interactions. Currently,
there are no therapies whereby both hypertension and insulin
resistance can be simultaneously treated with the same pharma-
ceutical agent. ARBs are a highly regarded class of drugs used for
the treatment of hypertension.15 They are safe and effective with
few side effects. One particular ARB, telmisartan (Figure 1), a
potent selective AT1 receptor antagonist, was reported to also
have weak activity at PPARγ.16 Telmisartan’s pharmacokinetic
Hypertension and insulin resistance are intimately linked and
constitute two components of the metabolic syndrome or
Syndrome X. Patients with metabolic syndrome are character-
ized as having three or more of the following comorbidities:
impaired fasting glucose, hypertriglyceridemia, hypertension,
low high-density lipoprotein cholesterol levels, and central
obesity.1 Hypertension is a condition with a well understood
pathophysiology with a number of pharmaceuticals such as
diuretics,2 beta blockers,3 angiotensin converting enzyme inhi-
bitors (ACE inhibitors),4 calcium channel antagonists,5 angio-
tensin receptor blockers6 (ARBs), and more recently renin
inhibitors7 being prescribed for safe and effective treatment. It
has been found that 29% of U.S. adults >18 years of age are
hypertensive with equal prevalence among men and women.8
Approximately 90 million patients exhibit insulin resistance
(prediabetes), and 44 million are both hypertensive and insulin
resistant.
The development of insulin resistance is a critical step in the
evolution of type 2 diabetes, and the increasing prevalence of
central obesity has contributed significantly to this situation.
Patients with diabetes mellitus are at increased risk for premature
disability and death associated with vascular complications.
Additionally, individuals with prediabetes (impaired glucose
tolerance and/or impaired fasting glucose with concomitant
hyperinsulinemia) suffer atherosclerotic vascular events more
frequently than in the general population.9 Evidence suggests
Received: April 6, 2011
Published: May 10, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of New Imidazo[4,5-b]pyridine
ARB programs. Our strategy involved cross screening of known
PPARγ agonists for ARB activity using an AT1 competition
radioligand binding assay, as well as screening of known ARBs for
PPARγ activity in a human PPARγ chimeric receptor transacti-
vation assay. The same treatment was given to marketed ARBs
(losartan, candesartan, valsartan) and marketed PPARγ agonists
(pioglitazone and rosiglitazone). Our screening efforts led to the
unique indanyl-based scaffold 2 derived from a previous ARB
program25,26 that showed robust AT1 activity and partial activa-
tion of PPARγ. In this report, optimization of the novel scaffold
and identification of 2l, a potent AT1 antagonist with partial
PPARγ activity with demonstrated efficacy in animal models of
hypertension and insulin sensitization, are described.
Derivatives 2a
a Reagents and conditions: (a) KOH, MeOH; (b) PhI(OAc)2, KOH,
MeOH, 0 to 25 °C; (c) MgCl2, (R4CO)2O, R4CO2H, 120 °C, 24 h; (d)
7 or 10, DEAD, Ph3P, THF, 0 to 25 °C; (e) 9, Pd(OAc)2, Ph3P, K2CO3,
DME-H2O, 80 °C, 18 h; (f) 3 M HCl, acetone.
Figure 1. Chemical structures of telmisartan (1), new scaffold 2, and
imidazopyridines 2a and 2l.
’ CHEMISTRY
(PK) profile confers 24 h blood pressure (BP) lowering and
reduced BP variability compared to other ARBs. Additionally, small
clinical trials have demonstrated that telmisartan is capable of
improving glycemic parameters in metabolic syndrome patients17
compared to another ARB (losartan) suggesting the effect is not an
AT1 receptor mediated phenomenon. The improvements seen in
the glycemic parameters of metabolic syndrome patients taking
telmisartan versus patients taking other ARBs could be reasonably
attributed to telmisartan’s weak activity at PPARγ. Efforts to identify
the structural features responsible for the PPARγ activity of
telmisartan have been reported.18ꢀ20 In addition, telmisartan ana-
logues have been designed and reported as dual PPARγ agonist/
AT1 antagonists.21ꢀ23 During the course of our research, a novel
chemical scaffold 2 (Figure 1) was discovered which allowed for
interaction with both PPARγ and angiotensin II type 1 (AT1)
receptor.24 A pharmaceutical agent with the dual activity of AT1
inhibition and partial PPARγ agonism could potentially treat several
recognized CV risk factors including hypertension, insulin resistance,
and hypertriglyceridemia in patients with metabolic syndrome.
The novel scaffold 2 was identified by mining our proprietary
collection of compounds and data from previous PPARγ and
The synthesis of the new imidazo[4,5-b]pyridine derivatives 2
is shown in Scheme 1. The imidazo[4,5-b]pyridine headgroup 6
was prepared in three steps from an appropriately substituted
1,3-diketone 3 according to a procedure described by Sena-
nayake and collaborators.27 Condensation of diketone 3 with
amidinoacetamide hydrochloride in the presence of potassium
hydroxide and methanol provided amide 4. In the case of
unsymmetrical 1,3-diketones, this condensation led to a mixture
of regioisomers favoring the product containing the largest R
group at C-6. This mixture was not separated but taken directly
into the next step. Hoffmann rearrangement of amide 4 afforded
imidazolone 5, which was then converted into the corresponding
imidazo[4,5-b]pyridines 6 by reaction with a mixture of an
organic acid and anhydride containing the desired R4 group.
The (R)-5-bromo-1-indanol (7) was prepared via catalytic
enantioselective reduction of 5-bromo-1-indanone using
(S)-methyl-CBS-oxazaborolidine and borane-dimethyl sulfide
complex.28,29 For the determination of enantiomeric purity,
racemic 7 was prepared by sodium borohydride reduction of
5-bromo-1-indanone. Indanol 7 was obtained with >99% ee as
determined by chiral chromatography. Mitsunobu reaction of
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Scheme 2. Synthesis of Carboxylic Acid 14a
a Reagents and conditions: (a) 12, PdCl2(dppf), Na2CO3, dioxane-H2O, 85 °C, 18 h; (b) 1 M NaOH, 100 °C, 24 h.
imidazopyridine 6 with bromo-indanol 7 using diethylazodicar-
boxylate and triphenylphosphine in THF provided 8. Suzuki
cross-coupling of bromide 8 with boronic acid 930 in the presence
of catalytic palladium acetate, triphenylphosphine, and potas-
sium carbonate in DME-water gave 11. Alternatively, trityl-
protected tetrazole 11 was obtained in a single step from
imidazopyridine 6 using the more elaborated indanol 10. This
intermediate was prepared from bromide 7 and boronic acid 9
using the Suzuki cross-coupling conditions mentioned earlier.
Mitsunobu reaction of imidazopyridine 6 with indanol 10 was
accomplished under similar conditions as those described above
to provide 11. To complete the synthesis of the desired compounds,
removal of the trityl group under the conditions of 3 M HCl in
acetone afforded the desired tetrazole derivatives 2. Assesment of
enantiomeric purity of 2l was accomplished by (i) synthesis of
racemic 2l following the same sequence described in Scheme 1, but
departing from racemic 7; (ii) determination of enantiomeric purity
using chiral chromatography with a chiralpak AD-H column and
SFC conditions, and using racemic 2l for the analysis. The
enantiomeric purity of 2l was determined to be 96.5% ee.
The synthesis of carboxylic acid analogue 14 is depicted in
Scheme 2. Suzuki cross-coupling of bromide 8l and boronate
ester 12 under the conditions described previously afforded ester
13. Ester hydrolysis of 13 using sodium hydroxide in methanolꢀ
water afforded 14.
was determined using a chimeric receptor (PPARγ ligand
binding domain (LBD)/ Gal4 DNA binding domain) transacti-
vation assay. The EC50 values and the percent of maximal
activation with darglitazone32 as a reference full agonist
(defined as 100% effect) are presented in Table 1. Telmisartan
and pioglitazone are included as reference compounds.
The novel scaffold 2 was identified from this cross-screening
approach. Imidazopyridine 2a (Figure 1) demonstrated potent
AT1 antagonist activity and partial PPARγ agonism (AT1 IC50
=
7.6 nM; PPARγ EC50 = 591 nM, 24% activation). With this
scaffold in hand, efforts were directed to improve PPARγ activity
while improving or maintaining the inherent AT1 potency. The
SAR associated with the AT1 binding activity of this class of
compounds is well-understood;26,31 however, we had limited
information about how these compounds interacted with
PPARγ. Past experience with nuclear hormone receptors33,34
and initial docking studies suggested that these compounds
might bind in a typical agonist conformation with the carboxylic
acid headgroup or carboxylic acid bioisostere tetrazole forming
a hydrogen bond network with the three charge clamp residues,
Tyr327, His 449, and His323 (Figure 2).35,36 The modeling
studies also suggested that the central aryl core bound within
the hydrophobic pocket formed by helices 3, 6, and 10 and the
lipophilic tail was buried in the hydrophobic cleft in the anterior
ꢀ
of the receptor. To our surprise, the 2.4 Å resolution crystal
structure of 2a complexed with the LBD of human PPARγ
(Figure 3) revealed a completely flipped binding mode (see
Supporting Information S-Table 1 for X-ray data collection and
structure refinement statistics). The lipophilic tail is projected
into the AF-2 domain pocket of the receptor and interacts with
the charge clamp residues through nonpolar, van der Waals
interactions. The crystal structure also revealed that the acidic
tetrazole motif is buried in the hydrophobic anterior of the
receptor and makes a weak H-bond interaction between the
Preparation of 2aꢀc, 2e, and 15 has been previously
described.25,26,31
’ RESULTS AND DISCUSSION
As mentioned earlier, affinity for the AT1 receptor was
determined utilizing human recombinant AT1 receptors in a
competition radioligand binding assay with [125I]Tyr4-Sar1,Ile8-
Angiotensin II. The IC50 values of the imidazo[4,5-b]pyridine
derivatives are displayed in Table 1. Activation of human PPARγ
ꢀ
N-2 of the tetrazole ring and Arg288 (2.7 Å distance).
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Table 1. AT1 and PPARγ Transactivation Activity of Imidazo[4,5-b]pyridine Derivatives 2aꢀo, 14, and 15
compound
R1
R2
R3
R4
AT1 IC50 (nM)
h-PPARγ EC50 (nM)a (% max)b
Telmisartan
0.49
-
1520 (33)
1280 (80)
591 (24)
574 (16)
2620 (20)
1320 (24)
2200 (28)
295 (19)
762 (24)
1820 (23)
1340 (25)
494 (26)
264 (23)
212 (31)
89 (25)
Pioglitazone
2a (S)c
2b (rac)
2c (R)c
2d
Me
H
H
H
H
H
H
H
Me
Et
H
H
H
H
H
H
Me
Et
Et
Et
7.6
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
i-butyl
Me
96
Me
661
13.3
238
10.2
8.2
Me
n-propyl
n-butyl
i-propyl
c-propyl
Et
2e (rac)
2f
Me
Me
2g
Me
2h
Me
202
15.7
6.8
2i
Me
Et
2j
Et
Et
2k
Et
i-propyl
Et
16.9
1.6
2l
i-butyl
i-butyl
Me
2m
i-propyl
Et
3.5
2n
748
5.2
165 (28)
90 (27)
2o
benzyl
Et
14
10.5
2.7
175 (33)
>20000 (7)
15
a TA (transactivation assay). Mean value of at least two determinations. b The maximal efficacy of darglitazone in the PPARγ activation assay was defined
as 100%. c Stereochemistry of the chiral center. Unless specified, all compounds were prepared as the (S)-enantiomer.
Interestingly, despite the lack of direct engagement with the
charge clamp and the AF-2 activation motif this and other
compounds in this series retain appreciable PPARγ agonist
activity as discussed below.
Analysis of the complex of 2a bound to human PPARγ-LBD
revealed areas that could be utilized to improve interaction with
this receptor. Modification of R4 (Figure 1) with both linear and
branched alkyl groups appeared attractive to improve interaction
with PPARγ, as there are several hydrophobic residues that
surround this region of the molecule, as well as a large volume
which could potentially accommodate larger substituents. In-
creasing the length of R4 (Table 1) from ethyl in 2a to either
propyl in 2d (AT1 IC50 = 13.3 nM; PPARγ EC50 = 1320 nM,
24% activation) or butyl in 2e (AT1 IC50 = 238 nM; PPARγ
EC50 = 2200 nM, 24% activation) led to loss of potency in both
AT1 and PPARγ. It should be noted that 2e was racemic and no
efforts to obtain the (S)-enantiomer were pursued due to poor
AT1 activity. Modification of R4 to cyclopropyl in 2g (AT1
IC50 = 8.2 nM; PPARγ EC50 = 762 nM, 24% activation) or
isopropyl in 2f (AT1 IC50 = 10.2 nM; PPARγ EC50 = 295 nM,
19% activation) was well tolerated. Data from this set of compounds
showed that both ethyl and isopropyl are suitable R4 substituents in
order to obtain potent dual AT1 and PPARγ activity.
Availability of the set of compounds 2aꢀc in our compound
collection provided an opportunity to examine the effect of the
stereochemistry at C-1 of the indane ring on the AT1 and PPARγ
activity. The (S)-enantiomer 2a exhibited potent activity for both
receptors (AT1 IC50 = 7.6 nM; PPARγ EC50 = 591 nM, 24%
activation), while the (R)-enantiomer 2c exhibited a significant
drop in AT1 activity with only a moderate change in PPARγ
potency (AT1 IC50 = 661 nM; PPARγ EC50 = 2620 nM, 20%
activation). AT1 potency for racemic 2c was reduced when
compared to (S)-enantiomer 2a, while it retained similar potency
for PPARγ (AT1 IC50 = 96 nM; PPARγ EC50 = 574 nM, 16%
activation). The effect of opening the indane ring was investi-
gated with the known benzyl analogue 15.31 This compound was
reported as a potent AT1 receptor antagonist and this was
corroborated in our screen (IC50 = 2.7 nM). Interestingly, 15
was completely devoid of PPARγ activity (EC50 > 20 000 nM,
7% activation) demonstrating that conformational restriction
derived from the indane ring is required for PPARγ activity.
Guided by this data, synthetic efforts were focused on indane
derivatives in the (S)-enantiomer series.
The effect of modifying the R2 group on the AT1 activity was
uncertain as this position was not previously modified. On the
basis of the complex of 2a bound to human PPARγ-LBD, it
appeared to be a less attractive location to improve interaction with
PPARγ. As such, only a limited set of analogues were prepared with
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Figure 2. (A) Simplified ligand topology and simplified binding interaction map for a typical PPARγ full-agonist.34,37 (B) Initial docking poses of 2a
bound in an agonist conformation to the PPARγ LBD suggested that the tetrazole moiety might interact with the charge clamp residues His323, His449,
and Tyr327. This binding mode was subsequently shown by X-ray crystallography to be flipped 180° in the horizontal plane.
Figure 3. Three-dimensional representation of 2a (gold) bound in the ligand binding pocket of human PPARγ (green ribbons and β-sheets with gray
residues). The AF-2 helix which is partially disordered due to the lack of direct interactions between the ligand and the charge clamp residues is shown on
the left-hand side of the figure. The compound wraps around helix 3 that lies in the middle. Formation of an H-bond between N-2 of the tetrazole ring
ꢀ
and Arg288 (2.7 Å distance) was observed as was multiple hydrophobic interactions within the pocket.
modifications at this position. Substitution with methyl in 2h (AT1
IC50 = 202 nM; PPARγ EC50 = 1820 nM, 23% activation), or ethyl
in 2i (AT1IC50 = 16 nM; PPARγEC50 = 1340 nM, 25% activation)
was detrimental for activity at both receptors and no further
expansion at this position was performed.
On the basis of the complex of 2a bound to human PPARγ-
LBD, two areas that looked promising to improve interaction
with PPARγ were substitutions at R1 and R3 on the imidazopyr-
idine ring. The substitution of R1 appeared optimal to increase
interaction with PPARγ as both lipophilic and hydrophilic
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pockets were identified that could be reached with appropriate
R1 substitution. With the above observations in hand, modifica-
tions of the R1 group were evaluated. Increasing the size of R1
with ethyl as seen in 2j (AT1 IC50 = 6.8 nM; PPARγ EC50 = 494
nM, 26% activation) and 2k (AT1 IC50 = 16.9 nM; PPARγ
EC50 = 264 nM, 23% activation) was well tolerated. Further
increasing the size of R1 to isobutyl led to 2l (AT1 IC50 = 1.6 nM;
PPARγ EC50 = 212 nM, 31% activation), a compound with
improved potency at both receptors that maintained partial
PPARγ agonism. Combining isobutyl at R1 and isopropyl at
R4 provided 2m (AT1 IC50 = 3.5 nM; PPARγ EC50 = 89 nM,
31% activation), a potent analogue with markedly improved
potency at PPARγ. To further examine the space surrounding
the R1 substituent, a larger benzyl group was incorporated in 2o
(AT1 IC50 = 5.2 nM; PPARγ EC50 = 90 nM, 27% activation) that
also showed potent activity at both receptors. Not surprisingly,
large hydrophobic substituents led to marked improvements in
PPARγ activity as there are several lipophilic residues in this
region. The data obtained with 2jꢀm and 2o suggested that
potency for both receptors can be modulated through appro-
priate selection of the R1 group. Modification of R3 substituents was
briefly examined with 2n (AT1 IC50 = 748 nM; PPARγ EC50 = 165
nM, 28% activation) which incorporated an isobutyl at such position.
Results with this compound suggested that there was a limited
opportunity to modify R3 without compromising AT1 activity.
Tetrazoles are common bioisosteres of carboxylic acids,38 and
both moieties have been used extensively in the field of AT1
antagonists.39,40 The impact of replacing tetrazole with a benzoic
acid moiety in the current series was assessed with 14 (AT1
IC50 = 10.5 nM; PPARγ EC50 = 175 nM, 33% activation) that
incorporated isobutyl for R1 and ethyl for R4. The tetrazole to
carboxylic acid change appeared to mainly affect AT1 activity as
observed with the approximately 7-fold drop in potency when
comparing AT1 IC50 values for 14 to 2l.
Imidazopyridine 2l was identified with potent AT1 binding
affinity (Ki = 0.69 nM) and showed an IC50 value (IC50 = 1.6
nM) that was only 3-fold lower than telmisartan. Other com-
pounds in this series exhibited >5-fold reduction in AT1 potency
when compared to telmisartan as assessed by their IC50 values.
Retaining AT1 potency similar to that of telmisartan was an
important criterion for advancing compounds into further stud-
ies. Furthermore, imidazopyridine 2l exhibited improved PPARγ
potency (PPARγ EC50 = 212 nM, 31% activation) when
compared to that of telmisartan and retained partial agonism.
On the basis of this potency, 2l was selected for additional
profiling. Imidazopyridine 2l exhibited a high degree of selec-
tivity over AT2 (Ki >10 000 nM). The AT1 receptor antagonist
class can be divided into surmountable and insurmountable
antagonists based upon their functional effects in assays such as
isolated rabbit aorta.41,42 Examples include losartan,42 which is a
classic competitive antagonist (surmountable) and telmisartan,
which is an insurmountable antagonist,43 wherein the observed
pseudoirreversible blockade is driven by a very slow off rate from
the AT1 receptor. It is believed that the very slow dissociation
rate from the AT1 receptor contributes to the enhanced efficacy
seen with this cohort of compounds. In order to characterize 2l as
a surmountable or insurmountable antagonist, a 96-well plate
based method was developed. 2l was incubated with the receptor
at 10 μM to achieve complete receptor occupancy at equilibrium.
Following washout of unbound compound, the receptor-com-
pound complex was coincubated with the [125I] radioligand at
3 ꢁ Kd to measure displacement of compound over time. As
Figure 4. Dissociation of 2l and reference ARBs from the AT1 receptor
in a 96-well plate based assay (n = 3).
Table 2. Pharmacokinetic Properties of 2l in Rat
dose
AUC
t1/2
Cl
Vss
route (mg/kg) (ng h/mL) (h) F (%) (mL/min/kg) (L/kg)
3
IV
1
5
1220
4950
2.1
14
2.9
PO
80
shown in Figure 4, imidazopyridine 2l did not demonstrate any
significant dissociation from the receptor over the time course of
the assay. This profile was similar to that of telmisartan and
strongly suggested an insurmountable profile.
Imidazopyridine 2l was also screened in human PPARR and
PPARβ chimeric receptor transactivation assays, and no detect-
able activity could be observed, thus demonstrating selectivity
over these receptors. As the next step in this investigation was
to perform studies in animal models of hypertension and
diabetes, the pharmacokinetic properties of 2l were evaluated
in rat (Table 2) to determine if this compound would be
suitable for oral administration. Bioavailability was high (80%)
following a single 5 mg/kg dose. In vivo clearance and
volume of distribution were moderate (Cl = 14 mL/min/kg;
Vss =2.9 L/kg). The measured half-life value was 2.1 h. The
profile of 2l was considered appropriate to progress into in vivo
studies.
The dual pharmacology exhibited by 2l required the use of two
in vivo models to adequately assess this compound and define
efficacy. First, 2l was evaluated in the spontaneously hypertensive
rat (SHR), a model that has been shown over decades to predict
human antihypertensive efficacy. 2l was administered orally to
SHRs in a dose range from 0.3 to 10 mg/kg (n = 8 animals/
group). After a single oral dose, 2l demonstrated a dose-
dependent sustained lowering of mean arterial BP with a maximal
drop of 36 mmHg at 10 mg/kg (Figure 5). This was associated
with a modest reflex tachycardia at the highest dose (þ37 beats/
min at 4ꢀ8 h post dose). Determination of systemic drug
exposure for 2l showed Cmax and AUC of 74/283/3073 ng/mL
and 320/1285/6833 ng hr/mL, respectively, for the 1, 3, and
3
10 mg/kg doses. Telmisartan showed a similar doseꢀresponse
profile (data not shown). Interestingly, BP remained suppressed
for >20 h after a single dose of 2l, and this was despite plasma
levels of 2l being undetectable at 20 h post dose. Upon cessation
of dosing with 2l, BP returned to predose levels in all animals.
The magnitude of BP lowering seen with 2l after single dose was
consistent with historical in-house data for compounds in this
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structural class. Further studies showed improved efficacy upon
repeat dosing with once-a-day dosing for 7 days producing
reductions in BP of >60 mmHg at 10 mg/kg. This was not due
to compound accumulation but is consistent with the mechanism
requiring 3ꢀ5 days to achieve maximal effects in rodents.
Imidazopyridine 2l was also evaluated in the male Zucker
diabetic fatty rat (ZDF), a model widely used to demonstrate
insulin sensitization. Animals were placed on study at 6 weeks of
age when they were insulin resistant but not yet diabetic as
evident by euglycemia with concomitant plasma hyperinsuline-
mia. Control rats, when left untreated, will develop marked
hyperglycemia, elevated triglycerides, and eventually loss of beta
cell function and insulin insufficiency as the disease progresses.
Compound 2l was administered orally across a dose range of
0.1ꢀ100 mg/kg/day (n = 8 animals/group) for 40 days with
pioglitazone (3 mg/kg) and telmisartan (100 mg/kg) included in
the study as comparators. Insulin, glucose, triglycerides (TG),
free fatty acids (FFA), adiponectin, HDL and VDL cholesterol,
and body weight (BW) were monitored in this study. The effect
of 2l on the development of hyperglycemia in the ZDF rat is
shown in Figure 6. Systemic exposure of 2l in this strain of rat was
determined in satellite animals dosed at 3, 10, and 100 mg/kg p.o.
Cmax values of 267/2730/176667 ng/mL and AUC of 4395/
27973/2599853 ng hr/mL, respectively, were calculated.
3
The results show a time dependent increase in hyperglycemia
in the vehicle treated rats with concomitant increases in TG, FFA
and reduction in adiponectin (Table 3). Compound 2l prevented
the changes in a dose dependent fashion with the lower doses
delaying the progression to overt diabetes and the higher doses
preventing it altogether. At doses of 10 mg/kg and higher, 2l
maintained euglycemia, kept TG at baseline values, and doubled
adiponectin levels relative to control by the end of the study. The
improvement in metabolic phenotype was marked and at least
equivalent to that of pioglitazone 3 mg/kg in this study. Inter-
estingly, this efficacy was associated with a lower degree of
bodyweight gain than was seen in the TZD group (Table 3
and Figure 7). Compared to vehicle controls, body weight gain
was much greater for the pioglitazone group with significant (p <
0.05) increases in weight gain seen as early as day 6. The growth
curves continued to diverge throughout the study with total
weight gain in these animals being double that seen in vehicle
controls. Telmisartan (100 mg/kg) induced modest increases of
weight that achieved statistical significance from control at day
32. Since there are no reports of weight gain associated with the
clinical use of telmisartan,44 this is more likely a reflection of a
“failure to thrive” in the control animals rather than a PPARγ
effect of telmisartan. Certainly, the weight gain seen in control
animals starts to plateau at day 28 (10 weeks old) and might
reflect significant loss of calories through glycosuria. Compound
2l showed increases in weight gain, which were statistically
significant at 3 mg/kg with this dose causing gains similar to
telmisartan. Doses of 10ꢀ100 mg/kg produced a greater degree
of weight gain although this was not dose dependent and
Figure 5. Effect of 2l (0.3ꢀ10 mg/kg p.o.) on mean blood pressure in
the SHR. All data represented as mean ( SEM, n = 8 animals/group.
Figure 6. Effect of 2l (0.1ꢀ100 mg/kg p.o. once daily for 40 days) on
blood glucose in the male ZDF rat. All data represented as mean ( SEM,
n = 8 animals/group. * p < 0.05 vs vehicle control.
Table 3. Evaluation of 2l in Male ZDF Ratsa
treatment
dose (mg/kg/day)
glucose (mg/dL)
triglycerides (mg/dL)
BW gain (g)b
adiponectin (μg/mL)
Vehicle
771 ( 60d
118 ( 4c
618 ( 52
316 ( 29c
935 ( 76
559 ( 62
725 ( 69
734 ( 73
393 ( 41
288 ( 43c
151 ( 11c
133 ( 9
269 ( 4c
157 ( 4c
128 ( 10
150 ( 7
159 ( 7c
200 ( 5c
202 ( 6c
202 ( 6c
11.9 ( 1.5
30.5 ( 1.2c
13.2 ( 1.5
9.9 ( 1.0
Pioglitazone
Telmisartan
2l
3
100
0.1
1
510 ( 97c
771 ( 100
612 ( 57
520 ( 37c
159 ( 34c
140 ( 25c
116 ( 5c
10.2 ( 0.9
11.4 ( 1.5
24.4 ( 2.0c
24.3 ( 1.8c
27.5 ( 1.0c
3
10
30
100
a All data mean ( SEM, n = 8 animals/group. Tested compounds were administered orally once a day for 40 days. Glucose, triglycerides, and adiponectin
results are day 40 values at end of study. b BW gain calculated from change in BW in each animal from day 0 to day 40. c Significantly different from time
matched vehicle control, p < 0.05. d Significantly different from pioglitazone, p < 0.05.
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often termed “metabolic syndrome” highlights the fact that
hypertension rarely occurs in isolation and is often associated
with other risk factors such as obesity, hypertriglyceridemia, and
insulin resistance. The notion that a single molecule could start to
address multiple risk factors provides a point of differentiation
and potentially significant benefits to the “at risk” patient.
Compound 2l has demonstrated highly potent activity at two
distinct molecular targets, a GPCR (AT1 receptor) and a nuclear
hormone receptor (PPARγ). The combined beneficial effect of
targeting these receptors has been demonstrated in two well-
validated in vivo models of hypertension (SHR) and diabetes
(ZDF). Compound 2l demonstrated dual pharmacology with
potent selective antagonism of the AT1 receptor that translated
into highly effective BP lowering in the SHR. A single dose of 10
mg/kg produced BP lowering for a full 24 h equivalent to the
most potent marketed ARB, telmisartan. It also displayed efficacy
via partial agonism of the PPARγ receptor as demonstrated in
cell based assays where Emax was only 20ꢀ30% of that seen with a
reference TZD, darglitazone. This potent, partial activation
translated into dose-dependent efficacy in the male ZDF rat
with glycemic improvements equivalent to that of pioglitazone
with a significantly reduced incidence of weight gain. A com-
pound with a profile similar to 2l should prove to be an attractive
candidate for the treatment and prevention of type 2 diabetes and
associated comorbidities such as hypertension and diabetic
dyslipidemia.
Figure 7. Effect of chronic (40 days) dosing of 2l (0.1ꢀ100 mg/kg p.
o.), telmisartan (100 mg/kg p.o.), and pioglitazone (3 mg/kg p.o.) on
body weight gain in the 7 week old male ZDF rat. All data represented as
mean ( SEM, n = 8 animals/group.
appeared to indicate a maximum effect on bodyweight despite
improved efficacy on glucose and lipid end points. The weight
gain due to drug, even at 100 mg/kg, was only 51% of that
induced by pioglitazone (69 vs 136 g) reflecting the compound’s
partial agonist profile at the PPARγ receptor. The increase in
adiponectin levels at doses of 10ꢀ100 mg/kg was equivalent to
the effect of pioglitazone and, when viewed temporally, reflected
a maintenance of baseline values rather than an acute increase.
Therefore, as glycemic control decreased in vehicle control and
low dose animals, adiponectin levels also declined whereas higher
doses of 2l and pioglitazone maintained both adiponectin levels
and glycemic control. The relationship between adiponectin
levels and glycemic control, either causal or association, was
not investigated further in this model but did highlight the
potential prognostic value of adiponectin as a biomarker for this
mechanism of action.
A key question generated as a result of using two in vivo
models was whether the projected PK/PD for the two biological
activities overlapped, meaning that efficacy could be seen for
both targets at similar plasma drug concentrations. In the SHR,
the key steady-state plasma concentration required to lower BP
was derived by comparing preclinical to clinical translation for
telmisartan. The highest dose registered for telmisartan is 80 mg
and was shown clinically to produce 90% of the maximal BP
lowering observed in clinical trials. This provided a reference
point that allowed translation of preclinical PK/PD and led us to
model the preclinical EC90 for both telmisartan and 2l. The
steady-state free EC90 for 2l in SHR was 7.2 nM. In the male ZDF
rat, pioglitazone at 3 mg/kg was shown to produce a similar %
maximal response in this preclinical model as the clinically used
dose of 45 mg in human. Again, this dose and associated steady-
state plasma concentration were used as the benchmark, and the
steady-state free plasma concentration of 2l that achieved similar
efficacy was calculated to be 2.6 nM. On the basis of this
modeling in two different disease models, plasma concentrations
achieving significant efficacy were within 3-fold in the SHR and
ZDF models for 2l demonstrating the potential utility of this
compound in treating multiple risk factors at a single dose level.
’ EXPERIMENTAL SECTION
General. All chemicals, reagents, and solvents were purchased from
commercial sources (e.g., Aldrich Chemical Co., Inc., Milwaukee, WI;
Mallinckrodt Baker, Inc., Paris, KY, etc.) where available and used
without further purification. All intermediates were characterized by
proton nuclear magnetic spectroscopy (1H NMR) and mass spectro-
metry (MS) using atmospheric pressure chemical ionization (APCI) or
electron scatter (ES) ionization sources. All final compounds were
determined to be consistent with the proposed structure by 1H NMR
and MS. All final compounds were purified by flash chromatography
except where listed. HPLC conditions: method A: Symmetry C18, 4.6 ꢁ
150 mm; mobile phase: A: water þ 0.1%TFA; B: CH3CN þ 0.1% TFA;
flow rate: 1 mL/min; gradient: 90% A to 10% A in 15 min, hold for
5 min, go back to 90%A in 1 min and maintain at 90% A for 4 min;
detection: DAD at 220 nm; injection volume: 10 uL; method A1:
Phenomenex C18, 4.6 ꢁ 150 mm flow rate: 1 mL/min; gradient: 90% A
to 10% A in 10 min; detection: DAD at 210 nm; injection volume: 10 uL.
Chiral HPLC conditions (method B): chiralcel AS, 4.6 mm ꢁ 250 mm;
mobile phase A: hexanes; B: isopropanol; isocratic 80:20; flow rate:
0.8 mL/min; injection volume: 10 μL; detection: DAD 214 nm. Purity
of final compounds was determined by elemental analysis and HPLC. All
final compounds were within theoretical limits for elemental analysis
(CHN), unless noted.
Synthesis of (S)-3-(5-(2-(1H-Tetrazol-5-yl)phenyl)-2,3-dihydro-1H-in-
den-1-yl)-5,7-dimethyl-2-propyl-3H-imidazo[4,5-b]pyridine (2d). Step 1.
2-Amino-4,6-dimethylnicotinamide (4d). General procedure A. A solu-
tion of KOH (6.16 g, 110 mmol) in MeOH (100 mL) was treated
sequentially with 2-amidino-acetamide hydrochloride (13.74 g, 99.88 mmol)
and pentane-2,4-dione (10.29 mL, 99.88 mmol) and then stirred at room
temperature for 24 h. Mixture diluted with water (50 mL) and cooled in ice
bath to give white precipitate. Solid separated by filtration, washed with
water, and dried in air overnight to give 4d as a white solid (13.1 g, 79%
yield): 1H NMR (DMSO-d6, 400 MHz) δ 7.58 (s, 1 H), 7.44 (s, 1 H), 6.24
(s, 1 H), 5.59 (s, 2 H), 2.14 (s, 3 H), 2.12 (s, 3 H); CIMS: 166.0 (APCI)þ;
164.0 (APCI)-.
’ CONCLUSION
The current hypertension market is highly competitive and
populated with many efficacious agents that have already or will
soon lose market exclusivity. This makes the ability for an NCE
to differentiate itself in such a market not just desirable but
essential. The clustering of cardiovascular morbidities in what is
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Step 2. 5,7-Dimethyl-1H-imidazo[4,5-b]pyridin-2(3H)-one
(5d). General procedure B. A mixture of 2-amino-4,6-dimethylnicoti-
namide (4d) (9 g, 54.48 mmol) was added to solution of KOH (6.1 g,
56.106 mmol) in MeOH (150 mL). After cooling the mixture in an ice
bath, PhI(OAc)2 (17.55 g, 54.48 mmol) was added as a solid in small
portions over 2 min. The mixture was stirred in an ice bath for 3 h, then
warmed to room temperature and stirred overnight. The precipitate was
collected by filtration, washed with ethyl ether, and air-dried to give 5d as
an off-white solid (8.39 g, 90% yield): 1H NMR (DMSO-d6, 400 MHz) δ
6.57 (s, 1 H), 2.26 (s, 3 H), 2.16 (s, 3 H); CIMS: 164.0 (APCI)þ; 162.0
(APCI)-.
Step 3. 5,7-Dimethyl-2-propyl-3H-imidazo[4,5-b]pyridine (6d). General
procedure C. 5,7-Dimethyl-1H-imidazo[4,5-b]pyridin-2(3H)-one (5d)
(2.5 g, 15 mmol) was added to butyric anhydride (10 mL, 61.28 mmol) at
room temperature under nitrogen. Butyric acid(5.6 mL, 61.28 mmol) was
added followed by magnesium chloride (1.46 g, 15.32 mmol) as a solid.
The mixture was then heated at 120 °C for 24 h. The reaction mixture was
allowed to cool, and then MeOH (25 mL) was added. The mixture was
heated at reflux for 4 h. Mixture allowed to cool, diluted with water (20 mL),
and basified to approximately pH = 8. Mixture was partitioned between
ethyl acetate (50 mL) and brine (50 mL). Phases separated. Aqueous phase
was extracted with ethyl acetate (3 ꢁ 50 mL). The combined organic
extracts were dried over magnesium sulfate and filtered and the solvent was
removed. The residue was purified by MPLC on silica gel eluting with a
gradient of ethyl acetate in hexanes (0% to 100%) to provide 6d as an white
solid (1.89 g, 65% yield): 1H NMR (DMSO-d6, 400MHz) δ12.40 (s, 1 H),
6.78 (s, 1 H), 2.71ꢀ2.68 (m, 2 H), 2.40 (s, 6 H), 1.71 (dt, J = 7.3 Hz, 2 H),
0.88 (t, J = 7.3 Hz, 3 H); CIMS: 190.0 (APCI)þ; 188.1 (APCI)-. HPLC
(method A): >99% purity; tR = 7.404 min.
(11d). General procedure D. Indanol 10 (0.300 g, 0.576 mmol),
imidazopyridine 6d (0.22 g, 1.2 mmol), and triphenylphosphine
(0.23 g, 0.86 mmol) were stirred in dry THF (20 mL) under a
nitrogen atmosphere. Mixture cooled to 0 °C and a solution of
DEAD (0.136 mL, 0.864 mmol) in THF (2 mL) was added dropwise.
Mixture stirred overnight while allowing mixture to warm to RT.
Mixture concentrated and purified by MPLC eluting with ethyl
acetate in hexanes (0% to 20%) to afford as a colorless solid (0.208 g,
48% yield): CIMS: 692 (APCI)þ; 448 (APCI)-. Product was taken
to the next step without further characterization.
Step 7. (S)-3-(5-(2-(1H-Tetrazol-5-yl)phenyl)-2,3-dihydro-1H-inden-
1-yl)-5,7-dimethyl-2-propyl-3H-imidazo[4,5-b]pyridine (2d). General
procedure E. Trityl-protected tetrazol 11d (0.205 g, 0.296 mmol) was
dissolved in acetone (6 mL) and 3 M HCl (1 mL) was added. The mixture
was stirred at room temperature for 3 h. Mixture was concentrated to give an
aqueous residue that was cooled in an ice bath and basified with 2 N KOH to
approximately pH 13. The resulting solid was separated by filtration and
washed with water. The filtrate was extracted with diethyl ether (2 ꢁ
15 mL). The aqueous layer was cooled in an ice bath, and the pH adjusted to
approximately pH 6.5 with 1.0 M HCl to give a cloudy solution. Mixture was
extracted with chloroform (4 ꢁ 20 mL). The combined organic extracts
were dried over magnesium sulfate, filtered, and the solvent evaporated. The
resulting solid was dried under high vacuum to give 2d as an off-white solid
(25 mg, 19% yield): 1H NMR (DMSO-d6, 400 MHz) δ 7.66ꢀ7.60 (m, 2
H), 7.55ꢀ7.50 (m, 2 H), 7.11 (s, 1 H), 6.76 (d, J = 7.6 Hz, 1 H), 6.67 (d, J =
7.8 Hz, 1 H), 6.31 (bs, 1 H), 4.06ꢀ3.97 (m, 1 H), 3.23ꢀ3.18 (m, 1 H),
3.01ꢀ2.93 (m, 1 H), 2.64ꢀ2.63 (m, 3 H), 2.46 (s, 3 H), 2.43 (s, 3H),
1.87ꢀ1.66 (m, 3 H), 1.14 (m, 2 H), 0.84 (m, 3 H); CIMS: 450.2 (APCI)þ;
448.1 (APCI)-; HPLC (method A): 98.57% purity; tR = 10.62 min.
Synthesis of (S)-3-(5-(2-(1H-Tetrazol-5-yl)phenyl)-2,3-dihydro-1H-
inden-1-yl)-2-isopropyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridine
(2f). Step 1. 2-Isopropyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridine (6f).
Prepared from 5d (2.0 g, 12.3 mmol), isobutyric acid (4.54 mL, 49.03
mmol), and isobutyric anhydride (8.13 mL, 49.03 mmol) following General
procedure C. 6f was obtained as a pale yellow solid (0.92 g, 36% yield): 1H
NMR (DMSO-d6, 400 MHz) δ12.37 (s, 1 H), 6.78 (s, 1 H), 3.08ꢀ3.03 (m,
1 H), 2.44 (s, 3 H), 2.40 (s, 3 H); CIMS: 190.0 (APCI)þ; 188.0 (APCI)-.
Step 2. (S)-3-(5-Bromo-2,3-dihydro-1H-inden-1-yl)-2-isopropyl-
5,7-dimethyl-3H-imidazo[4,5-b]pyridine (8f). General procedure
F. A mixture of indanol 7 (0.4 g, 1.88 mmol), imidazopyridine 6f
(0.53 g, 2.82 mmol), and triphenylphosphine (0.74 g, 2.82 mmol) in dry
THF (20 mL) was stirred under nitrogen in an ice bathꢀbrine in a
Dewar flask (approximately ꢀ4 °C). A solution of DEAD (0.44 mL, 2.82
mmol) in THF (2 mL) was added slowly. The mixture was stirred in ice
bathꢀbrine for 5ꢀ6 h and then allowed to warm to room temperature
and stirred overnight. Solvents were removed and the residue purified by
MPLC on silica gel using a gradient of ethyl acetate in hexanes (0ꢀ14%).
Pure fractions were combined and the solvent evaporated to give 8f as
a colorless foam (0.44 g, 61% yield): 1H NMR (DMSO-d6, 400 MHz)
δ 7.52 (s, 1 H), 7.22 (d, J = 7.6 Hz, 1 H), 6.82 (s, 1 H), 6.68 (d, J = 8.1 Hz,
1 H), 6.24 (bs, 1 H), 3.23ꢀ3.20 (m, 2 H), 3.05ꢀ2.97 (m, 1 H), 2.44 (s,
3 H), 2.62ꢀ2.58 (m, 2 H), 2.44 (s, 3 H), 2.42 (s, 3 H), 1.28 (d, J = 6.6 Hz,
3 H), 1.12 (m, 3 H); CIMS: 385.9 (APCI)þ; 383.9 (APCI)-; HPLC
(method A): 98.2% purity; tR = 12.2 min.
Step 4. (R)-5-Bromo-2,3-dihydro-1H-inden-1-ol (7). A solution of
5-bromo-1-indanone (15 g, 71.1 mmol) in anhydrous THF (120 mL)
was stirred under nitrogen. A 1.0 M solution of (S)-methyl-CBS-
oxazaborolidine in toluene (10.7 mL, 10.7 mmol) was added and the
solution was warmed to 35 °C. A 2.0 M solution of borane-dimethyl-
sulfide (46 mL, 92 mmol) was added dropwise over 90 min while
maintaining the temperature at 35 °C for 1 h, then cooled to room
temperature and stirred overnight. The mixture was cooled in ice bath,
quenched with water (30 mL), and solvent removed. The mixture was
extracted with ethyl acetate (3 ꢁ 150 mL). The combined organic
extracts were washed with brine (100 mL), dried over magnesium
sulfate, filtered, and concentrated. The residue was dissolved in chloro-
form (35 mL) at 50 °C. Hexanes (100 mL) were added slowly. Mixture
was allowed to cool slowly to room temperature, and then further cooled
in an ice bath. The white precipitate was separated by filtration, washed
1
with hexanes, and dried to give 7 as white powder (8.95 g, 59%): H
NMR (DMSO-d6, 400 MHz) δ 7.36 (s, 1 H), 7.30 (d, J = 8 Hz, 1 H),
7.20 (d, J = 8 Hz, 1 H), 4.93 (q, J = 6.3 Hz, 1 H), 2.88ꢀ2.82 (m, 1 H),
2.69ꢀ2.61 (m, 1 H), 2.30ꢀ2.22 (m, 1 H), 1.75ꢀ1.66 (m, 1 H). Chiral
HPLC (method B): tR (major enantiomer) = 6.583 min, tR (minor
enantiomer) = 5.672 min; >99% ee.
Step 5. (R)-5-(2-(1-Trityl-1H-tetrazol-5-yl)phenyl)-2,3-dihydro-
1H-inden-1-ol (10). To a degassed solution of triphenylphosphine
(0.12 g, 0.40 g, 0.44 mmol) in DME was added Pd(OAc)2 (0.025 g,
0.11 mmol) and stirred for 10 min. To the reaction mixture was added
boronic acid 930 (0.50 g, 1.11 mmol), K2CO3 (0.38 g, 2.78 mmol),
indanol 7 (0.24 g, 1.11 mmol), and water (0.05 mL, 2.78 mmol). The
reaction mixture was heated at 110 °C overnight in a sealed tube. The
solvent was removed under vacuum, and the residue was subjected to
column chromatography (25% ethyl acetate in hexanes) to afford 10
(0.45 g, 89%): 1H NMR (400 MHz, CDCl3) δ 8.00 (d, J = 7.6 Hz, 1H),
7.59ꢀ6.90 (m, 22 H), 5.21ꢀ5.19 (m 1H), 2.85ꢀ2.79 (m, 1H),
2.62ꢀ2.55 (m, 1H), 2.44ꢀ2.38 (m, 1H), 1.90ꢀ1.80 (m, 1H).
Step 6. (S)-5,7-Dimethyl-2-propyl-3-(5-(2-(1-trityl-1H-tetrazol-5-yl)-
phenyl)-2,3-dihydro-1H-inden-1-yl)-3H-imidazo[4,5-b]pyridine
Step 3. (S)-2-Isopropyl-5,7-dimethyl-3-(5-(2-(1-trityl-1H-tetrazol-
5-yl)phenyl)-2,3-dihydro-1H-inden-1-yl)-3H-imidazo[4,5-b]pyridine
(11f). General procedure G. Palladium(II) acetate (25.4 mg, 0.113
mmol) was added to a degassed solution of triphenylphosphine (118
mg, 0.452 mmol) in dry DME (10 mL). The mixture was stirred at room
temperature for 20 min. Boronic acid 9 (0.63 g, 1.5 mmol), a solution of
bromide 8f (0.43 g, 1.13 mmol) in dry DME (5 mL), K2CO3 (0.39 g,
2.82 mmol), and water (0.051 mL) were added. The mixture was
degassed for an additional 30 min, and then heated at 80 °C overnight.
Mixture was allowed to cool, diluted with ethyl acetate, and silica gel was
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ARTICLE
added. Solvents were removed and the residue was purified by MPLC on
silica gel using a gradient of ethyl acetate in hexanes (0ꢀ20%). Pure
fractions were combined and evaporated to give 11f as an off-white foam
(0.65 g., 81% yield): CIMS: 692.3 (APCI)þ; 448.2 (APCI)-; HPLC
(method A): 97% purity; tR = 16.31 min; product was taken to the next
step without further characterization.
Step 5. (S)-2-Ethyl-5,6,7-trimethyl-3-(5-(2-(1-trityl-1H-tetrazol-5-yl)-
phenyl)-2,3-dihydro-1H-inden-1-yl)-3H-imidazo[4,5-b]pyridine (11h).
Prepared from bromide 8h (0.42 g, 1.09 mmol) following general
procedure G. 11h was obtained as a foam (0.67 g., 85% yield): CIMS:
692.4 (APCI)þ; 448.2 (APCI)-. Product was taken to the next step
without further characterization.
Step 6. (S)-3-(5-(2-(1H-Tetrazol-5-yl)phenyl)-2,3-dihydro-1H-in-
den-1-yl)-2-ethyl-5,6,7-trimethyl-3H-imidazo[4,5-b]pyridine (2h).
Prepared from trityl-protected tetrazole 11h (0.67 g, 0.973 mmol)
following general procedure E. Tetrazole 2h was obtained as a white
solid (0.325 g, 77% yield): 1H NMR (DMSO-d6, 400 MHz) δ
7.63ꢀ7.59 (m, 2 H), 7.57ꢀ7.48 (m, 2 H), 7.08 (s, 1 H), 6.72 (d, J =
7.8 Hz, 1 H), 6.64 (d, J = 8 Hz, 1 H), 6.26 (bs, 1 H), 2.98ꢀ2.90 (m, 1 H),
2.64ꢀ2.57 (m, 2 H), 2.44 (s, 3 H), 2.36 (s, 3 H), 2.15 (s, 3 H), 1.17 (t, J =
7.3 Hz, 3 H); CIMS: 450.2 (APCI)þ; 448.1 (APCI)-; HPLC (method
Step 4. (S)-3-(5-(2-(1H-Tetrazol-5-yl)phenyl)-2,3-dihydro-1H-in-
den-1-yl)-2-isopropyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridine (2f).
Prepared from trityl-protected tetrazole 11f (0.64 g, 0.93 mmol)
following general procedure E to provide 11f as a white solid (0.35 g,
1
86% yield): H NMR (DMSO-d6, 400 MHz) δ 7.64ꢀ7.57 (m, 2 H),
7.52ꢀ7.48 (m, 2 H), 7.12 (s, 1 H), 6.83 (s, 1 H), 6.71 (d, J = 7.8 Hz, 1 H),
6.63 (d, J = 7.8 Hz, 1 H), 6.35 (bs, 1 H), 3.26ꢀ3.10 (m, 1 H), 3.00ꢀ2.92
(m, 1 H), 2.64ꢀ2.58 (m, 1 H), 2.44 (s, 3 H), 2.42 (s, 3 H), 2.35 (bs, 2 H),
1.26 (bs, 3 H), 1.04 (m, 3 H); CIMS: 448.2 (APCI)-; HPLC (method
A): >98% purity; tR = 10.73 min. Anal. Calcd for C27H27N7 0.15
A): >99% purity; tR = 10.59 min; Anal. Calcd for C27H27N7 1.0 H2O: C,
3
3
EtOAc: C, 71.63; H, 6.14; N, 21.19. Found: C, 71.26; H, 6.05; N, 21.01.
Synthesis of (S)-3-(5-(2-(1H-Tetrazol-5-yl)phenyl)-2,3-dihydro-1H-
inden-1-yl)-2,6-diethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridine
(2i). Step 1. 2-Amino-5-ethyl-4,6-dimethylnicotinamide (4i).
Prepared from 2-amidino-acetamide hydrochloride (3.22 g, 23.41
mmol) and 3-ethylpentane-2,4-dione (3 g, 23.41 mmol) following
general procedure A. Nicotinamide 4i was obtained as an off-white solid
(1.39 g, 31% yield): 1H NMR (DMSO-d6, 400 MHz) δ 7.62 (s, 1 H),
7.42 (s, 1 H), 5.20 (s, 2 H), 2.42 (q, J = 7.4 Hz, 2 H), 2.21 (s, 3 H), 2.10 (s,
3 H), 0.96 (t, J = 7.4 Hz, 3 H); CIMS: 194.0 (APCI)þ; 192.0 (APCI)-.
Step 2. 6-Ethyl-5,7-dimethyl-1H-imidazo[4,5-b]pyridin-2(3H)-one
(5i). Prepared from nicotinamide 4i (1.2 g, 6.2 mmol) following general
procedure B. 5i was obtained as a solid (1.11 g, 94% yield): 1H NMR
(DMSO-d6, 400 MHz) δ 2.51 (q, J = 7.6 Hz, 2 H), 2.31 (s, 3 H), 2.17 (s,
3 H), 0.98 (q, J = 7.6 Hz, 3 H); CIMS: 192.0 (APCI)þ; 190.0 (APCI)-.
Step 3. 2,6-Diethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridine (6i).
Prepared from 5i (0.6 g, 3.138 mmol), propionic acid (0.94 mL, 12.55
mmol), and propionic anhydride (1.62 mL, 12.55 mmol) following
general procedure C. 6i was obtained as an off-white solid (0.335 g, 52%
yield): 1H NMR (DMSO-d6, 400 MHz) δ 12.24 (s, 1 H), 2.72 (q, J = 7.6
Hz, 2 H), 2.62 (q, J = 7.6 Hz, 2 H), 2.44 (s, 6 H), 1.23 (t, J = 7.6 Hz, 3 H),
1.02 (t, J = 7.6 Hz, 3 H); CIMS: 204.0 (APCI)þ; 202.1 (APCI)-.
Step 4. (S)-3-(5-Bromo-2,3-dihydro-1H-inden-1-yl)-2,6-diethyl-
5,7-dimethyl-3H-imidazo[4,5-b]pyridine (8i). Prepared from imida-
zopyridine 6i (0.335 g, 1.648 mmol) following general procedure F to
69.36; H, 6.25; N, 20.97. Found: C, 69.71; H, 5.89; N, 20.57.
Synthesis of (S)-3-(5-(2-(1H-Tetrazol-5-yl)phenyl)-2,3-dihydro-1H-inden-
1-yl)-2-cyclopropyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridine (2g). Step 1.
(S)-2-Cyclopropyl-5,7-dimethyl-3-(5-(2-(1-trityl-1H-tetrazol-5-yl)phenyl)-
2,3-dihydro-1H-inden-1-yl)-3H-imidazo[4,5-b]pyridine (11g). Prepared
from imidazopyridine 6g45 (0.108 g, 0.576 mmol) following general
procedure D to provide 11g as a white solid (76 mg, 35%): CIMS: 690.1
(APCI)þ; 446.1 (APCI)-. Product was taken to the next step without
further characterization.
Step 2. (S)-3-(5-(2-(1H-Tetrazol-5-yl)phenyl)-2,3-dihydro-1H-inden-
1-yl)-2-cyclopropyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridine (2g). Pre-
pared from trityl-protected tetrazole 11g (72 mg, 0.104 mmol) following
General procedure E to provide 2g as a white solid (35 mg, 83% yield): 1H
NMR (DMSO-d6, 400 MHz) δ 7.65ꢀ7.59 (m, 2 H), 7.53ꢀ7.50 (m, 2 H),
7.11 (s, 1 H), 6.84 (s, 1 H), 6.74 (s, 2 H), 6.49 (t, J= 8.4 Hz, 1 H), 3.16ꢀ3.14
(m, 1 H), 3.02ꢀ2.94 (m, 1 H), 2.71ꢀ2.61 (m, 2 H), 2.53ꢀ2.48 (m, 1 H),
2.44 (s, 3 H), 2.38 (s, 3 H), 0.94ꢀ0.90 (m, 3 H), 0.89ꢀ0.74 (m, 1 H);
CIMS: 448.0 (APCI)þ, 446.1 (APCI)-; HPLC (method A): 99.39% purity;
tR = 10.51 min.
Synthesis of (S)-3-(5-(2-(1H-Tetrazol-5-yl)phenyl)-2,3-dihydro-1H-in-
den-1-yl)-2-ethyl-5,6,7-trimethyl-3H-imidazo[4,5-b]pyridine (2h). Step 1.
2-Amino-4,5,6-trimethylnicotinamide (4h). Prepared from 2-amidino-
acetamide hydrochloride (12.05 g, 87.61 mmol) and 3-methylpentane-2,4-
dione (10 g, 87.61 mmol) following general procedure A. Nicotinamide 4h
was obtained as an off-white solid (7.48 g, 77% yield): 1H NMR (DMSO-d6,
400 MHz) δ 7.60 (s, 1 H), 7.42 (s, 1 H), 5.20 (s, 2 H), 2.19 (s, 3 H), 2.06 (s,
3 H), 1.95 (s, 3 H); CIMS: 180.0 (APCI)þ; 178.0 (APCI)-.
1
give 8i as a yellow oil (0.36 g, 54% yield): H NMR (DMSO-d6, 400
MHz) δ 7.52 (s, 1 H), 7.22 (d, J = 8 Hz, 1 H), 6.72 (d, J = 8 Hz, 1 H), 6.18
(bs, 1 H), 3.11ꢀ2.96 (m, 1 H), 2.74ꢀ2.70 (m, 1 H), 2.62ꢀ2.58 (m,
2 H), 2.44 (s, 3 H), 1.20 (t, J = 7.3 Hz, 3 H), 1.01 (t, J = 7.6 Hz, 3 H);
CIMS: 398.1 (APCI)þ.
Step 2. 5,6,7-Trimethyl-1H-imidazo[4,5-b]pyridin-2(3H)-one (5h).
Prepared from nicotinamide 4h (7.48 g, 41.7 mmol) following general
procedure B. 5h was obtained as an greyish solid (3.16 g, 43% yield): 1H
NMR (DMSO-d6, 400 MHz) δ 10.83 (bs, 1 H), 10.60 (bs, 1 H), 2.28 (s, 3
H), 2.14 (s, 3 H), 2.04 (s, 3 H); CIMS: 178.0 (APCI)þ; 176.0 (APCI)-.
Step 3. 2-Ethyl-5,6,7-trimethyl-3H-imidazo[4,5-b]pyridine (6h).
Prepared from 5h (1.65 g, 9.3 mmol), propionic acid (2.8 mL, 37.2
mmol), and propionic anhydride (4.80 mL, 37.2 mmol) following
general procedure C. 6h was obtained as an off-white solid (1.24 g,
70% yield): 1H NMR (DMSO-d6, 400 MHz) δ 12.23 (s, 1 H), 2.72 (q,
J = 7.6 Hz, 2 H), 2.44 (s, 3 H), 2.40 (s, 3 H), 2.15 (s, 3 H), 1.23 (t, J =
7.6 Hz, 3 H); CIMS: 190.0 (APCI)þ; 188.0 (APCI)-.
Step 5. (S)-2,6-Diethyl-5,7-dimethyl-3-(5-(2-(1-trityl-1H-tetrazol-
5-yl)phenyl)-2,3-dihydro-1H-inden-1-yl)-3H-imidazo[4,5-b]pyridine
(11i). Prepared from bromide 8i (0.36 g, 0.898 mmol) following general
procedure G. 11i was obtained as a white foam (0.389 g, 61% yield):
CIMS: 706.3 (APCI)þ; 462.1 (APCI)-. Product was taken to the next
step without further characterization.
Step 6. (S)-3-(5-(2-(1H-Tetrazol-5-yl)phenyl)-2,3-dihydro-1H-in-
den-1-yl)-2,6-diethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridine (2i).
Prepared from trityl-protected tetrazole 11i (0.389 g, 0.551 mmol)
following general procedure E. Tetrazole 2i was obtained as a white foam
(61 mg, 24% yield): 1H NMR (DMSO-d6, 400 MHz) δ 7.63ꢀ7.58 (m,
2 H), 7.52ꢀ7.48 (m, 2 H), 7.08 (s, 1 H), 6.73 (d, J = 7.8 Hz, 1 H), 6.67 (d,
J = 8 Hz, 1 H), 6.26 (bs, 1 H), 2.98ꢀ2.90 (m, 1 H), 2.63 (q, J = 7.3 Hz,
4 H), 2.44 (s, 3 H), 1.17 (t, J = 7.3 Hz, 3 H), 1.02 (t, J = 7.3 Hz, 3 H);
CIMS: 450.2 (APCI)þ; 448.1 (APCI)-; HPLC (method A): 92.79%
purity; tR = 11.232 min.
Step 4. (S)-3-(5-Bromo-2,3-dihydro-1H-inden-1-yl)-2-ethyl-5,6,7-
trimethyl-3H-imidazo[4,5-b]pyridine (8h). Prepared from imidazo-
pyridine 6h (0.53 g, 2.82 mmol) following general procedure F to give
8h (0.42 g, 58% yield): 1H NMR (DMSO-d6, 400 MHz) δ 7.51 (s, 1 H),
7.22 (d, J = 8 Hz, 1 H), 6.69 (d, J = 8.5 Hz, 1 H), 6.17 (bs, 1 H),
3.04ꢀ2.96 (m, 1 H), 2.73 (bs, 1 H), 2.63ꢀ2.55 (m, 3 H), 2.44 (s, 3 H),
2.32 (s, 3 H), 2.13 (s, 3 H), 1.20 (t, J = 7.6 Hz, 3 H); CIMS: 386.1
(APCI)þ; 384 (APCI)-; HPLC (method A): 97.95% purity; tR
=
Synthesis of (S)-3-(5-(2-(1H-Tetrazol-5-yl)phenyl)-2,3-dihydro-1H-in-
den-1-yl)-2,5-diethyl-7-methyl-3H-imidazo[4,5-b]pyridine (2j). Step 1.
12.338 min.
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Journal of Medicinal Chemistry
ARTICLE
2-Amino-6-ethyl-4-methylnicotinamide (4j). Prepared from 2-amidino-
acetamide hydrochloride (12.05 g, 87.61 mmol) and hexane-2,4-dione
(10 g, 87.61 mmol) following general procedure A. Nicotinamide 4j was
obtained as an off-white solid (12.98 g, 83% yield): 1H NMR (DMSO-d6,
400 MHz, major isomer) δ 7.59 (s, 1 H), 7.44 (s, 1 H), 6.25 (s, 1 H), 5.57
(s, 2 H), 2.44 (q, J = 7.6 Hz, 2 H), 2.13 (s, 3 H), 1.08 (d, J = 7.6 Hz, 3 H);
MS: 180.0 (APCI)þ; 178.0 (APCI)-.
off-white solid (0.21 g, 60% yield): 1H NMR (DMSO-d6, 400 MHz) δ
7.63ꢀ7.57 (m, 2 H), 7.52ꢀ7.44 (m, 2 H), 7.10 (s, 1 H), 6.81 (s, 1 H),
6.71 (d, J = 7.8 Hz, 1 H), 6.64 (d, J = 7.8 Hz, 1 H), 6.29 (bs, 1 H),
3.00ꢀ2.92 (m, 1 H), 2.61ꢀ2.45 (m, 3 H), 2.44 (s, 3 H), 1.29 (d, J = 6.3
Hz, 3 H), 1.22ꢀ0.95 (m, 6 H); CIMS: 464.3 (APCI)þ; 462.2 (APCI)-;
HPLC (method A): >99% purity; tR = 11.26 min. Anal. Calcd for
C29H29N7 0.14 EtOAc: C, 72.06; H, 6.38; N, 20.60. Found: C, 72.35;
3
H, 6.47; N, 20.23.
Step 2. 5-Ethyl-7-methyl-1H-imidazo[4,5-b]pyridin-2-(3H)-one
(5j). Prepared from nicotinamide 4j (7.85 g, 43.8 mmol) following
general procedure B. 5j was obtained as an off-white solid (6.74 g, 87%
yield): 1H NMR (DMSO-d6, 400 MHz) δ 10.90 (bs, 2 H), 6.58 (s, 1 H),
2.54 (q, J = 7.6 Hz, 2 H), 2.17 (s, 3 H), 1.12 (t, J = 7.6 Hz, 3 H); CIMS:
178.0 (APCI)þ; 176.0 (APCI)-.
Synthesis of (S)-3-(5-(2-(1H-Tetrazol-5-yl)phenyl)-2,3-dihydro-1H-inden-
1-yl)-2-ethyl-5-isobutyl-7-methyl-3H-imidazo[4,5-b]pyridine (2l). Step 1.
2-Amino-6-isobutyl-4-methyl-nicotinamide (4l). Prepared from 6-methyl-
heptane-2,4-dione (5 g, 35.2 mmol) following general procedure A. These
conditions provided an approximately 86:14 mixture of 2-amino-6-
isobutyl-4-methyl-nicotinamide (major isomer) and 2-amino-4-isobutyl-
6-methyl-nicotinamide (minor isomer) as determined by 1H NMR that
was not separated. Mixture was obtained as an off-white solid (3.83 g, 53%
yield): 1H NMR (DMSO-d6, 400 MHz, major isomer) δ 7.60 (s, 1 H),
7.44 (s, 1 H), 6.21 (s, 1 H), 5.57 (s, 2 H), 2.25 (d, J = 7.2 Hz, 2 H), 2.13 (s,
3 H), 1.96ꢀ1.89 (m, 1 H), 0.81 (d, J = 6.6 Hz, 6 H); CIMS: 208.0
(APCI)þ; 206.1 (APCI)-.
Step 3. 2,5-Diethyl-7-methyl-3H-imidazo[4,5-b]pyridine (6j). Pre-
pared from 5j (2.0 g, 11.3 mmol), propionic acid (3.37 mL, 45.15 mmol),
and propionic anhydride (5.82 mL, 45.15 mmol) following general
procedure C. 6j was obtained as a yellow solid (1.40 g, 65% yield): 1H
NMR (DMSO-d6, 400 MHz) δ 12.41 (s, 1 H), 6.79 (s, 1 H), 2.74 (q, J =
7.6 Hz, 2 H), 2.68 (q, J = 7.6 Hz, 2 H), 2.41 (s, 3 H), 1.25 (t, J = 7.6 Hz,
3 H), 1.18 (t, J = 7.6 Hz, 7 H); CIMS: 190.0 (APCI)þ; 188.0 (APCI)-.
Step 4. (S)-2-Ethyl-5,7-dimethyl-3-(5-(2-(1-trityl-1H-tetrazol-5-yl)-
phenyl)-2,3-dihydro-1H-inden-1-yl)-3H-imidazo[4,5-b]pyridine (11j).
Prepared from imidazopyridine 6j (0.172 g, 0.910 mmol) following general
procedure D to provide 11j as colorless solid (0.107 g, 31% yield): CIMS:
629.1 (APCI)þ; 448.1 (APCI)-. Product was taken to the next step
without further characterization.
Step 5. (S)-3-(5-(2-(1H-Tetrazol-5-yl)phenyl)-2,3-dihydro-1H-in-
den-1-yl)-2,5-diethyl-7-methyl-3H-imidazo[4,5-b]pyridine (2j). Pre-
pared from trityl-protected tetrazole 11j (0.103 g, 0.149 mmol)
following general procedure E. Tetrazole 2j was obtained as a white
solid (44 mg, 73% yield): 1H NMR (DMSO-d6, 400 MHz) δ 7.64ꢀ7.57
(m, 2 H), 7.53ꢀ7.46 (m, 2 H), 7.00 (s, 1 H), 6.82 (s, 1 H), 6.72 (d, J = 9.0
Hz, 1 H), 6.67 (d, J = 7.8 Hz, 1 H), 6.28 (bs, 1 H), 2.99ꢀ2.91 (m, 1 H),
2.77ꢀ2.48 (m, 4 H), 2.43 (s, 3 H), 1.19 (t, J = 7.3 Hz, 3 H), 1.07 (t, J =
7.3 Hz, 3 H); CIMS: (APCI)þ; (APCI)-. HPLC (method A): 95.42%
purity; tR = 10.979 min. Anal. Calcd for C27H27N7-0.31 EtOAc: C,
71.13; H, 6.23; N, 20.56. Found: C, 70.83; H, 6.24; N, 20.18.
Step 2. 5-Isobutyl-7-methyl-1,3-dihydro-imidazo[4,5-b]pyridin-2-
one (5l). Prepared from the mixture of 2-amino-6-isobutyl-4-methyl-
nicotinamide (4l) and 2-amino-4-isobutyl-6-methyl-nicotinamide (3.8
g, 18.3 mmol) following general procedure B. These conditions
provided an approximately 86:14 mixture of 5-isobutyl-7-methyl-1,3-
dihydro-imidazo[4,5-b]pyridin-2-one (5l, major isomer) and 7-isobutyl-
5-methyl-1,3-dihydro-imidazo[4,5-b]pyridin-2-one (minor isomer) that
was not separated. Mixture was obtained as an off-white solid (2.62 g,
70% yield): 1H NMR (DMSO-d6, 400 MHz, major isomer) δ 10.98 (s,
1 H), 10.71 (s, 1 H), 6.54 (s, 1 H), 2.38 (d, J = 7.3 Hz, 2 H), 2.17 (s, 3 H),
1.95ꢀ1.88 (m, 1 H), 0.79 (d, J = 6.8 Hz, 6 H); CIMS: 206.0 (APCI)þ;
204.0 (APCI)-.
Step 3. 2-Ethyl-5-isobutyl-7-methyl-3H-imidazo[4,5-b]pyridine
(6l). Prepared from the approximately the mixture of 5-isobutyl-7-
methyl-1,3-dihydro-imidazo[4,5-b]pyridin-2-one (5l) and 7-isobutyl-
5-methyl-1,3-dihydro-imidazo[4,5-b]pyridin-2-one (1.0 g, 4.87 mmol),
propionic acid (1.45 mL, 19.5 mmol), and propionic anhydride
(2.51 mL, 19.5 mmol) following general procedure C. These conditions
provided an approximately 86:14 mixture of 2-ethyl-5-isobutyl-7-meth-
yl-3H-imidazo[4,5-b]pyridine (6l, major product) and 2-ethyl-7-isobu-
tyl-5-methyl-3H-imidazo[4,5-b]pyridine as an off-white solid (0.93 g,
88% yield): 1H NMR (DMSO-d6, 400 MHz, major isomer) δ 12.41 (s,
1 H), 6.75 (s, 1 H), 2.80ꢀ2.71 (m, 2 H), 2.52 (d, J = 7.2 Hz, 2 H), 2.41 (s,
3 H), 2.03ꢀ1.96 (m, 1 H), 1.24 (t, J = 7.6 Hz, 3 H), 0.82 (d, J = 6.6 Hz,
6 H); CIMS: 218.0 (APCI)þ; 216.1 (APCI)-. HPLC (method A)
showed two compounds: (major component): 86% area; tR = 9.095 min;
(minor component): 14% area; tR = 8.735 min.
Step 4. (S)-3-(5-Bromo-2,3-dihydro-1H-inden-1-yl)-2-ethyl-5-iso-
butyl-7-methyl-3H-imidazo[4,5-b]pyridine (8l). Prepared from the
mixture of imidazopyridine 6l and corresponding regioisomer (0.61 g,
2.82 mmol) following general procedure F to give 8l (0.4 g, 52% yield):
1H NMR (DMSO-d6, 400 MHz) δ 7.49 (s, 1 H), 7.18 (d, J = 8.05 Hz,
1 H), 6.74 (s, 1 H), 6.70 (d, J = 8.05 Hz, 1 H), 6.15 (bs, 1 H), 3.04ꢀ2.96
(m, 1 H), 2.83ꢀ2.48(m, 5 H), 2.44 (s, 3 H), 1.83ꢀ1.76 (m, 1 H), 1.25 (t,
J = 7.6 Hz, 3 H), 0.67 (dd, J = 15.4, 6.6 Hz, 6 H); CIMS: 412.0 (APCI)þ;
412.0 (APCI)-; HPLC (method A): 93.89% purity; tR = 13.925 min.
Step 5. (S)-2-Ethyl-5-isobutyl-7-methyl-3-(5-(2-(1-trityl-1H-tetrazol-5-
yl)phenyl)-2,3-dihydro-1H-inden-1-yl)-3H-imidazo[4,5-b]pyridine (11l).
Prepared from bromide 8l (0.39 g, 0.948 mmol) following general procedure
G. 11l was obtained as a pale yellow oil (0.56 g., 72% yield): CIMS: 720.4
(APCI)þ; 476.2 (APCI)-. Product was taken to the next step without further
characterization.
Synthesis of (S)-3-(5-(2-(1H-Tetrazol-5-yl)phenyl)-2,3-dihydro-1H-inden-
1-yl)-5-ethyl-2-isopropyl-7-methyl-3H-imidazo[4,5-b]pyridine (2k). Step 1.
5-Ethyl-2-isopropyl-7-methyl-3H-imidazo[4,5-b]pyridine (6k). Prepared
from 5j (2.0 g, 11.3 mmol), isobutyric acid (4.19 mL, 45.15 mmol), and
isobutyric anhydride (7.48 mL, 45.15 mmol) following general procedure
1
C. 6k was obtained as a yellow solid (1.85 g, 81% yield): H NMR
(DMSO-d6, 400 MHz) δ 12.41 (s, 1 H), 6.80 (s, 1 H), 3.10ꢀ3.01 (m,
1 H), 2.68 (q, J = 7.6 Hz, 2 H), 2.44 (s, 3 H), 1.27 (d, J = 6.8 Hz, 6 H), 1.18
(t, J = 7.6 Hz, 3 H); CIMS: 204.0 (APCI)þ; 202.1 (APCI)-.
Step 2. (S)-3-(5-Bromo-2,3-dihydro-1H-inden-1-yl)-5-ethyl-2-iso-
propyl-7-methyl-3H-imidazo[4,5-b]pyridine (8k). Prepared from imi-
dazopyridine 6k (0.57 g, 2.82 mmol) following general procedure F to
give 8k (0.37 g, 50% yield): 1H NMR (DMSO-d6, 400 MHz) δ 7.51 (s,
1 H), 7.20 (d, J = 7.3 Hz, 1 H), 6.80 (s, 1 H), 6.68 (d, J = 8.0 Hz, 1 H), 6.21
(bs, 1 H), 3.07ꢀ2.97 (m, 1 H), 2.67ꢀ2.47 (m, 5 H), 2.44 (s, 3 H), 1.31 (d,
J = 6.6 Hz, 3 H), 1.17 (s, 3 H), 1.00 (s, 3 H); CIMS: 400.0 (APCI)þ; 398.1
(APCI)-; HPLC (method A): >99% purity; tR = 13.038 min.
Step 3. (S)-5-Ethyl-2-isopropyl-7-methyl-3-(5-(2-(1-trityl-1H-tetrazol-5-
yl)phenyl)-2,3-dihydro-1H-inden-1-yl)-3H-imidazo[4,5-b]pyridine (11k).
Prepared from bromide 8k (0.38 g, 0.944 mmol) following general
procedure G. 11k was obtained as a pale yellow oil (0.548 g., 81% yield):
CIMS: 706.4 (APCI)þ; 462.3 (APCI)-. Product was takentothe next step
without further characterization.
Step 4. (S)-3-(5-(2-(1H-Tetrazol-5-yl)phenyl)-2,3-dihydro-1H-in-
den-1-yl)-5-ethyl-2-isopropyl-7-methyl-3H-imidazo[4,5-b]pyridine
(2k). Prepared from trityl-protected tetrazole 11k (0.548 g, 0.776
mmol) following general procedure E. Tetrazole 2k was obtained as an
Step 6. (S)-3-(5-(2-(1H-Tetrazol-5-yl)phenyl)-2,3-dihydro-1H-in-
den-1-yl)-2-ethyl-5-isobutyl-7-methyl-3H-imidazo[4,5-b]pyridine
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Journal of Medicinal Chemistry
ARTICLE
(2l). Prepared from trityl-protected tetrazole 11l (0.55 g, 0.764 mmol)
following general procedure E. Tetrazole 2l was obtained as an off-white
solid (0.24 g, 75% yield): 1H NMR (DMSO-d6, 400 MHz) δ 7.64ꢀ7.57
(m, 2 H), 7.52ꢀ7.43(m, 2 H), 7.07 (s, 1H), 6.76 (s, 1 H), 6.71 (d, J= 8.05
Hz, 1 H), 6.65 (d, J = 7.8 Hz, 1 H), 6.24 (bs, 1 H), 2.98ꢀ2.90 (m, 1 H),
2.75 (m, 1 H), 2.66ꢀ2.58 (m, 2 H), 2.41 (s, 3 H), 1.86ꢀ1.83 (m, 1 H),
1.21 (t, J = 7.3 Hz, 3 H), 0.71 (t, J = 6.8 Hz, 6 H); CIMS: 478.2 (APCI)þ;
476.3 (APCI)-; HPLC (method A): >99% purity; tR = 12.04 min.
Chiral HPLC (SFC conditions): chiralpak AD-H, 4.6 mm ꢁ 250 mm;
mobile phase: 80/20 CO2/MeOH; gradient: isocratic; flow rate:
2.5 mL/min; injection volume: 10 μL; detection: UV at 210 nm; tR
(major enantiomer) = 4.97 min, tR (minor enantiomer) = 3.89 min;
11n (1.3 g, 1.8 mmol) following general procedure E. Tetrazole
2n was obtained as a white solid (45 mg, 5% yield): 1H NMR (DMSO-
d6, 400 MHz) δ 7.64ꢀ7.58 (m, 2 H), 7.53ꢀ7.49 (m, 2 H), 7.09 (s,
1 H), 6.80 (s, 1 H), 6.73 (d, J = 7.8 Hz, 1 H), 6.67 (d, J = 8.05 Hz, 1 H),
6.31 (bs, 1 H), 3.18ꢀ3.15 (m, 1 H), 2.98ꢀ2.90 (m, 1 H), 2.69 (d,
J = 7.3 Hz, 2 H), 2.63ꢀ2.61 (m, 2 H), 2.43 (s, 3 H), 2.18ꢀ2.07 (m,
1 H), 1.15 (t, J = 7.3 Hz, 3 H), 0.84 (d, J = 6.6 Hz, 6 H); CIMS: 478.2
(APCI)þ; 476.3 (APCI)-; HPLC (method A): 96.12% purity; tR
=
11.79 min.
Synthesis of (S)-3-(5-(2-(1H-Tetrazol-5-yl)phenyl)-2,3-dihydro-1H-
inden-1-yl)-5-benzyl-2-ethyl-7-methyl-3H-imidazo[4,5-b]pyridine
(2o). Step 1. 2-Amino-6-benzyl-4-methylnicotinamide (4o). Pre-
pared from 2-amidino-acetamide hydrochloride (3.20 g, 23.3 mmol) and
1-phenylpentane-2,4-dione (4.20 g, 23.3 mmol) following general proce-
dure A. An approximate 60:40 mixture of 2-amino-6-benzyl-4-methylni-
cotinamide (4o) and 2-amino-4-benzyl-6-methylnicotinamide was
obtained (2.44 g, 43%): 1H NMR (400 MHz, DMSO-d6) δ 2.14, 2.16
(s, 3 h), 3.79, 3.86 (s, 2 H), 5.60, 5.67 (s, 1 H), 6.16, 6.32 (s, 1 H),
7.13ꢀ7.31 (m, 5 H), 7.50, 7.56 (bs, 1 H), 7.67, 7.85 (bs, 1 H); CIMS:
242.1 (APCI)þ, 240.0 (APCI)-.
96.5%ee. Anal. Calcd for C29H31N7 0.48 H2O: C, 71.63; H, 6.62;
3
N, 20.16. Found: C, 71.58; H, 6.43; N, 19.77.
Synthesis of (S)-3-(5-(2-(1H-Tetrazol-5-yl)phenyl)-2,3-dihydro-1H-in-
den-1-yl)-5-isobutyl-2-isopropyl-7-methyl-3H-imidazo[4,5-b]pyridine
(2m). Step 1. 5-Isobutyl-2-isopropyl-7-methyl-3H-imidazo[4,5-b]-
pyridine (6m). Prepared from the mixture of 5-isobutyl-7-methyl-1,3-
dihydro-imidazo[4,5-b]pyridin-2-one (5l) and 7-isobutyl-5-methyl-1,3-
dihydro-imidazo[4,5-b]pyridin-2-one (1.0 g, 4.87 mmol), isobutyric
acid (1.81 mL, 19.49 mmol), and isobutyric anhydride (3.23 mL,
19.49 mmol) following general procedure C. These conditions provided
6m as an off-white foam (0.68 g, 56% yield): 1H NMR (DMSO-d6, 400
MHz, major isomer) δ 6.75 (s, 1 H), 3.10ꢀ3.01 (m, 1 H), 2.52 (d, J = 7.2
Hz, 2 H), 2.44 (s, 3 H), 2.04ꢀ1.97 (m, 1H), 1.27 (d, J = 7 Hz, 6 H), 0.82
(d, J = 6.6 Hz, 6 H); CIMS: 232.1 (APCI)þ; 230.1 (APCI)-.
Step 2. 5-Benzyl-7-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-one
(5o). Prepared from the mixture of 2-amino-6-benzyl-4-methylnicoti-
namide (4o) and 2-amino-4-benzyl-6-methylnicotinamide (1.80 g, 7.46
mmol) following general procedure B. An approximate 3:2 mixture of
5-benzyl-7-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-one (5o) and
7-benzyl-5-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-one was obtained
1
(1.62 g, 91% yield): H NMR (400 MHz, DMSO-d6) δ 2.22, 2.29 (s,
Step 2. (S)-3-(5-Bromo-2,3-dihydro-1H-inden-1-yl)-5-isobutyl-2-
isopropyl-7-methyl-3H-imidazo[4,5-b]pyridine (8m). Prepared from
imidazopyridine 6m (0.42 g, 1.80 mmol) following general procedure F
to afford 8m as a white solid (0.24 g, 41% yield): 1H NMR (DMSO-d6,
400 MHz) δ 7.49 (s, 1 H), 7.17 (d, J = 7.6 Hz, 1 H), 6.73 (s, 3 H), 6.65
(d, J = 8 Hz, 1 H), 6.18 (bs, 1 H), 3.06ꢀ2.96 (m, 1 H), 2.66ꢀ2.40 (m,
6 H), 2.41 (s, 3 H), 1.76 (m, 1 H), 1.34ꢀ1.21 (m, 6 H), 0.67ꢀ0.61 (m,
6 H); CIMS: 428.1 (APCI)þ; 426.1 (APCI)-; HPLC (method A):
>99% purity; tR = 14.27 min.
Step 3. (S)-5-Isobutyl-2-isopropyl-7-methyl-3-(5-(2-(1-trityl-1H-
tetrazol-5-yl)phenyl)-2,3-dihydro-1H-inden-1-yl)-3H-imidazo[4,5-b]-
pyridine (11m). Prepared from bromide 8m (0.24 g, 0.56 mmol)
following general procedure G. 11m was obtained as a colorless solid
(0.35 g., 85% yield): CIMS: 734.2 (APCI)þ; 492.0 (APCI)-. Product
was taken to the next step without further characterization.
3 H), 3.92, 3.93 (s, 2 H), 6.60, 6.68 (s, 1 H), 7.12ꢀ7.32 (m, 5 H), 11.11
(bs, 2 H); CIMS: 240.0 (APCI)þ, 238.0 (APCI)-.
Step 3. 5-Benzyl-2-ethyl-7-methyl-3H-imidazo[4,5-b]pyridine (6o).
Prepared from the mixture of 5-benzyl-7-methyl-1H-imidazo[4,5-b]-
pyridin-2(3H)-one (5o) and 7-benzyl-5-methyl-1H-imidazo[4,5-b]pyri-
din-2(3H)-one (1.50 g, 6.27 mmol), propionic acid (2.81 mL, 37.61
mmol), and propionic anhydride (4.85 mL, 37.61 mmol) following
general procedure C. An approximate 3:2 mixture of 5-benzyl-2-ethyl-7-
methyl-3H-imidazo[4,5-b]pyridine (6o) and 7-benzyl-2-ethyl-5-methyl-
3H-imidazo[4,5-b]pyridine was obtained (1.07 g, 68% yield): 1H NMR
(400 MHz, DMSO-d6) δ 1.26ꢀ1.36 (m, 3 H), 2.45, 2.43 (s, 3 H),
2.74ꢀ2.89 (m, 2 H), 3.92, 4.05, 4.15, 4.20 (s, 2 H), 6.78, 6.87, 6.90 (s,
1 H), 7.13ꢀ7.21 (m, 1 H), 7.22ꢀ7.35 (m, 4 H); CIMS: 252.1 (APCI)þ,
250.1 (APCI)-.
Step 4. (S)-3-(5-(2-(1H-Tetrazol-5-yl)phenyl)-2,3-dihydro-1H-in-
den-1-yl)-5-isobutyl-2-isopropyl-7-methyl-3H-imidazo[4,5-b]pyridine
(2m). Prepared from trityl-protected tetrazole 11m (0.35 g, 0.48 mmol)
following general procedure E. Tetrazole 2m was obtained as a white solid
(0.19 g, 79% yield): 1H NMR (DMSO-d6, 400 MHz) δ 7.63ꢀ7.56 (m,
2 H), 7.51ꢀ7.47 (m, 1 H), 7.42 (d, J = 7.6 Hz, 1 H), 7.09 (s, 1 H), 6.74 (s,
1 H), 6.69 (d, J = 7.8 Hz, 1 H), 6.61 (d, J = 7.8 Hz, 1 H), 6.26 (bs, 1 H),
2.99ꢀ2.91 (m, 1 H), 2.61 (m, 1 H), 2.41 (s, 3 H), 1.80 (m, 1 H), 1.31ꢀ1.10
(m, 6 H), 0.69 (m, 6 H); CIMS: 492.0 (APCI)þ; 490.1 (APCI)-; HPLC
Step 4. (S)-5-Benzyl-3-(5-bromo-2,3-dihydro-1H-inden-1-yl)-2-
ethyl-7-methyl-3H-imidazo[4,5-b]pyridine (8o). Prepared from the
mixture of 5-benzyl-2-ethyl-7-methyl-3H-imidazo[4,5-b]pyridine (6o)
and 7-benzyl-2-ethyl-5-methyl-3H-imidazo[4,5-b]pyridine (1.06 g, 4.22
mmol) following general procedure F to provide 8o as a white solid
(1.06 g, 56% yield): 1H NMR (400 MHz, DMSO-d6) δ 7.60 (d,
J = 1.7 Hz, 1 H), 7.26 (dd, J = 7.9, 1.8 Hz, 1 H), 7.08ꢀ7.21 (m, 3 H),
7.04 (d, J = 7.1 Hz, 2 H), 6.89 (s, 1 H), 6.76 (d, J = 8 Hz, 1 H), 6.19 (t, J =
8.2 Hz, 1 H), 3.88 (s, 2 H), 3.00ꢀ3.13 (m, 1 H), 2.72ꢀ3.00 (m, 2 H),
2.54ꢀ2.73 (m, 2 H), 2.44 (s, 3 H), 1.31 (t, J = 7.6 Hz, 3 H); CIMS: 448.1
(APCI)þ.
(method A): >99% purity; tR = 12.32 min. Anal. Calcd for C30H33N7 0.09
3
CH2Cl2: C, 72.39; H, 6.70; N, 19.64. Found: C, 72.51; H, 6.37; N, 19.27.
Synthesis of (S)-3-(5-(2-(1H-Tetrazol-5-yl)phenyl)-2,3-dihydro-1H-inden-
1-yl)-2-ethyl-7-isobutyl-5-methyl-3H-imidazo[4,5-b]pyridine (2n). Step 1.
(S)-2-Ethyl-7-isobutyl-5-methyl-3-(5-(2-(1-trityl-1H-tetrazol-5-yl)phenyl)-
2,3-dihydro-1H-inden-1-yl)-3H-imidazo[4,5-b]pyridine (11n). Pre-
pared from the approximately 86:14 mixture of 2-ethyl-5-isobutyl-7-meth-
yl-3H-imidazo[4,5-b]pyridine (6l) and 2-ethyl-7-isobutyl-5-methyl-3H-
imidazo[4,5-b]pyridine (6n) (2.5 g, 12 mmol) following general procedure
D. Mixture purified by MPLC to give a mixture of 11l and 11n (1.94 g) that
was taken directly to the next step without further characterization.
Step 2. (S)-3-(5-(2-(1H-Tetrazol-5-yl)phenyl)-2,3-dihydro-1H-in-
den-1-yl)-2-ethyl-7-isobutyl-5-methyl-3H-imidazo[4,5-b]pyridine
(2n). Prepared from the mixture of trityl-protected tetrazole 11l and
Step 5. (S)-5-Benzyl-2-ethyl-7-methyl-3-(5-(2-(1-trityl-1H-tetrazol-
5-yl)phenyl)-2,3-dihydro-1H-inden-1-yl)-3H-imidazo[4,5-b]pyridine
(11o). Prepared from bromide 8o (0.600 g, 1.34 mmol) following
general procedure G to provide 11o as a foam (0.479 g, 47% yield):
CIMS: 754.3 (APCI)þ, 510.2 (APCI)-. Product was taken to the next
step without further characterization.
Step 6. (S)-3-(5-(2-(1H-Tetrazol-5-yl)phenyl)-2,3-dihydro-1H-in-
den-1-yl)-5-benzyl-2-ethyl-7-methyl-3H-imidazo[4,5-b]pyridine
(2o). Prepared from trityl-protected tetrazole 11o (0.479 g, 0.635
mmol) following general procedure E to afford 2o as a pale yellow
solid (0.146 g, 45%): 1H NMR (400 MHz, DMSO-d6) δ 7.62ꢀ7.74
(m, 2 H), 7.51ꢀ7.62 (m, 2 H), 7.06ꢀ7.21 (m, 6 H), 6.89 (s, 1 H),
4230
dx.doi.org/10.1021/jm200409s |J. Med. Chem. 2011, 54, 4219–4233

Journal of Medicinal Chemistry
ARTICLE
6.70ꢀ6.82 (m, 2 H), 6.30 (bs, 1 H), 3.96 (bs, 2 H), 2.92ꢀ3.05 (m,
1 H), 2.82 (bs, 1 H), 2.61ꢀ2.73 (m, 2 H), 2.52ꢀ2.59 (m, 1 H), 2.45
(s, 3 H), 1.27 (t, J = 7.2 Hz, 3 H); CIMS: 512.2 (APCI)þ, 510.2
(APCI)-; HPLC (method A): 98.27% purity; tR = 11.854 min.
Synthesis of (S)-2-(1-(2-Ethyl-5-isobutyl-7-methyl-3H-imidazo[4,5-
b]pyridin-3-yl)-2,3-dihydro-1H-inden-5-yl)benzoic Acid (14). Step 1.
(S)-Ethyl 2-(1-(2-ethyl-5-isobutyl-7-methyl-3H-imidazo[4,5-b]pyri-
din-3-yl)-2,3-dihydro-1H-inden-5-yl)benzoate (13). A solution of 8l
(148 mg, 0.36 mmol), 2-(methoxycarbonyl)phenylboronic acid (78 mg,
0.43 mmol), and 0.36 mL of 2 M Na2CO3 in 2 mL dioxane were
degassed and then treated with PdCl2(dppf) (26 mg, 0.036 mmol). The
mixture was then heated at 85 °C under nitrogen for 18 h. The reaction
was then cooled, filtered through a bed of Celite, and concentrated in
vacuo. Purification by MPLC on silica gel eluting with a gradient of ethyl
acetate in heptane (0% to 70%) provided 13 as an off-white solid (78 mg,
46% yield): 1H NMR (400 MHz, CDCl3) δ 7.78 (dd, J = 1.1, 7.7 Hz,
1H), 7.42ꢀ7.55 (m, 1H), 7.30ꢀ7.41 (m, 2H), 7.26 (s, 1H), 7.03 (d, J =
7.8 Hz, 1H), 6.87 (d, J = 7.8 Hz, 1H), 6.78 (s, 1H), 6.50 (br s, 1H),
3.56ꢀ3.65 (m, 3H), 3.25ꢀ3.41 (m, 1H), 3.11 (td, J = 8.4, 16.3 Hz, 1H),
2.67ꢀ2.89 (m, 2H), 2.43ꢀ2.66 (m, 6H), 2.02 (td, J = 6.6, 13 Hz, 1H),
1.31 (t, J = 7.4 Hz, 3H), 0.74ꢀ0.93 (m, 6H); CIMS: 468.4 (ESI)þ.
Step 2. (S)-2-(1-(2-Ethyl-5-isobutyl-7-methyl-3H-imidazo[4,5-
b]pyridin-3-yl)-2,3-dihydro-1H-inden-5-yl)benzoic Acid (14). A solu-
tion of ester 13 (50 mg, 0.11 mmol) in 10 mL methanol was treated with
3 mL 1 M NaOH (3 mmol). The reaction was then heated at 100 °C for
24 h. The reaction was cooled and adjusted to pH 2 with 1 M HCl
resulting in the formation of a precipitate. The precipitate was collected
by filtration, washed with water, and air-dried to give the title compound
(46 mg, 95%) as a beige solid: 1H NMR (400 MHz, CDCl3) δ 7.93 (dd,
J = 1.3, 7.7 Hz, 1H), 7.51ꢀ7.57 (m, 1H), 7.42 (dt, J = 1.2, 7.6 Hz, 1H),
7.34 (s, 1H), 7.30 (dd, J = 1.2, 7.6 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H), 7.02
(s, 1H), 6.89 (d, J = 8.0 Hz, 1H), 6.65 (br s, 1H), 3.19 (td, J = 8.3, 16.3
Hz, 2H), 3.00 (br s, 2H), 2.87ꢀ3.00 (m, 2H), 2.79 (s, 3H), 2.64 (d, J =
7.0 Hz, 2H), 2.49 (br s, 2H), 1.33ꢀ1.44 (m, 3H), 1.22ꢀ1.25 (m, 1H),
1.19ꢀ1.22 (m, 1H), 0.84 (t, J = 5.8 Hz, 6H); CIMS: 454.4 (ESI)þ;
HPLC (method A1): 97.29 purity; tR = 6.954 min.
anesthetized with Telazol (1:1 tiletamine HCl and zolazepam HCl)
20ꢀ40 mg/kg IM and the descending aorta exposed via a midline
incision. Radiotelemetry transmitters with polyethylene tubing
(TA11PA-C40) were inserted into the aorta via an undersize hole below
the renal arteries. The cannula was secured in the aorta using a “purse
string” suture (6ꢀ0 suture) to allow anterograde blood flow to the
hindlimb vascular bed. The body of the transmitter was secured on the
inside of left abdominal wall and the cannula was anchored to the psoas
muscle. The midline incision was closed in two steps using continuous
over and over sutures. Each rat was then given penicillin 30 000 units IM,
returned to cages, and allowed to recover postoperatively for a minimum
of 1 week. During that recovery week, patency and quality of the BP and
HR signals from the transmitter were tested to ensure that animals were
viable for study inclusion. On the day before the study, blood pressure
data were collected and mean average BP over a 2 h period was
determined. Rats were subsequently randomized based on BP and
allocated to treatment groups to ensure consistency in baseline record-
ings. On the first day of the study (termed Day 0), all animals received
the vehicle treatment via oral gavage and BP was followed for 20 h
postdose. On the following day (Day 1), rats received the allocated
treatment and again BP was followed for 20 h postdose. Each animal was
used as its own control to derive changes in BP by calculating differences
between Day 0 and Day 1. All experiments utilizing animals were
reviewed and approved by Pfizer’s Institutional Animal Care and Use
Committee.
In Vivo ZDF Rat Studies. Male Zucker Diabetic Fatty (ZDF/
Leprfa- Crl) rats were obtained from Charles River Laboratories
(Portage, MI). The ZDF male rat normally presents with NIDDM,
transient hyperinsulinemia, and hypertriglyceridemia. Rats were pair
housed under a 12 h light/dark cycle with free access to water and Purina
5008 rat chow (protein 26.8%, fat 16.7%, carbohydrates 56.5% kcal/vol;
Purina Mills, Richmond, IN). Prior to the onset of diabetic hyperglyce-
mia (approximately 6 weeks of age, fed blood glucose <200 mg/dL), rats
were allocated into groups by following a postprandial, conscious tail
venipuncture. Tail venipuncture in nonanesthetized, postprandial ani-
mals was performed weekly to determine blood glucose, insulin,
adiponectin, triglycerides, and free fatty acid measurements. Glucose
levels were determined with a HemoCue Glucose Monitor (Ryan
Diagnostics), insulin, and adiponectin were determined by ELISA
(Alpco), triglycerides were determined by Cobas Mira Analyzer
(Roche) and FFA by enzymatic assays (Wako). Rats were administered
a once daily oral dose for 7 weeks with suspensions of vehicle alone
(1.5% carboxymethylꢀcellulose, 0.2% Tween 20), or vehicle plus test
compound at the specified dose. All experiments utilizing animals were
reviewed and approved by Pfizer’s Institutional Animal Care and Use
Committee.
All PPAR in vitro assays and pharmacokinetic study in rat were carried
out as previously reported.33,34
Characterization of Dissociation Rate of Angiotensin II
Receptor Antagonists from the Type 1 Receptor. The radi-
oligand 125I-angiotensin II (125I-Sar1-Ile8-Angiotensin II), and human
angiotensin II receptor, subtype-1(hAT1) coated Flashplates were both
purchased from PerkinElmer Life Sciences (Boston, MA). Test com-
pounds were prepared as 10 μM solutions in binding buffer (50 mM
Tris-HCl, pH 7.4, 5 mM MgCl2, 1 mM EDTA, and 0.2% BSA) and
incubated in Flashplates at room temperature for 60 min. For non-
specific binding (N) determination, 10 μM of AT II was spiked into the
binding buffer during the incubation. After incubation, the plate was
washed 3 times with binding buffer and 2 nM of 125I-AT II (3 x Kd) in
binding buffer was added to the plate and the radioactivity was counted
in TopCount immediately and at regular intervals out to 8 h. As
compound dissociated from the receptor, Ang II radioligand was
available in excess to bind to the free receptor, so allowing dissociation
to be measured as the appearance of counts over time. The percent
binding was calculated as ([B ꢀ N]/[B0 ꢀ N]) ꢁ 100%, where (B) is the
binding activity in the presence of each compound and (B0) is in the
absence of any drug. The percent inhibition was calculated by subtract-
ing the percent bound from 100. The dissociation curve was generated
by plotting percent inhibition against incubation time after radioligand
was added. Dissociation rate was calculated in PRISM (GraphPad, San
Diego, CA) using the formula Y = 100 ꢁ exp[ꢀK ꢁ x].
X-ray Crystallography. A truncated construct of human PPARγ
ligand binding domain (PPARγ-LBD) containing residues Glu207 to
Tyr477 was recombinantly expressed in E. coli, purified, and crystallized
as previously described.33,34 Crystals were soaked overnight in a 0.8 mM
solution of 2a at room temperature prior to flash-cooling in 25%
glycerol. X-ray diffraction data were collected at a wavelength of 1.00
Å at 100 K on the Industrial Macromolecular Crystallographer Associa-
tion (IMCA) beamline 17-ID at the Advanced Photon Source, Argonne
National Laboratories. Diffraction data were processed using Denzo and
Scalepack in the HKL2000 program suite.46 The space group was
determined to be centered monoclinic C2 with two molecules per
asymmetric unit, corresponding to a solvent content of ∼50% The
structure was determined by the method of Fourier difference using the
homodimer of human PPARγ-LBD (3IA6) in the CCP4i suite.47,48
Structural refinement calculations and electron density maps were
calculated with the program Refmac5 in the CCP4i suite or the
AutoBuster program using the complete data with no resolution or
sigma cutoff.49,50 Manual fitting and real space refinement of the model
Spontaneously Hypertensive Rat Studies. SHR rats arrived
from suppliers (SHR/NCrl, Charles River, Wilmington MA) at 12ꢀ16
weeks old and allowed 1 week to acclimate. Once acclimated, rats were
4231
dx.doi.org/10.1021/jm200409s |J. Med. Chem. 2011, 54, 4219–4233

Journal of Medicinal Chemistry
ARTICLE
was performed with the program Coot.51 The final model of PPARγ-
LBD consists of two monomers of PPARγ-LBD, 2 molecules of 2a and
(4) Bertrand, M. E. Provision of cardiovascular protection by ACE
inhibitors: a review of recent trials. Curr. Med. Res. Opin. 2004,
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93 water molecules with the refinement statistics, Rwork = 26.7%, Rfree
=
(5) Guazzi, M. D.; Polese, A.; Fiorentini, C.; Bartorelli, A.; Moruzzi,
P. Treatment of hypertension with calcium antagonists. Hypertension
1983, 5, II85–90.
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a new approach in the treatment of hypertension. Curr. Vasc. Pharmacol.
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Ferguson, T. B.; Flegal, K.; Ford, E.; Furie, K.; Go, A.; Greenlund, K.;
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31.4% (see Supporting Information S-Table 1). The coordinates and
structure factors may be found in the Protein Data Bank47 under the ID
codes: 3R8A.pdb and 3R8A.sf.
’ ASSOCIATED CONTENT
S
Supporting Information. Crystallographic data collec-
b
tion and structure refinement statistics. This material is available
free of charge via the Internet at http://pubs.acs.org.
Accession Codes
†Structure deposited in the RCSB Protein Data Bank under PDB
ID:3R8A.
(9) Saydah, S. H.; Eberhardt, M. S.; Loria, C. M.; Brancati, F. L.
Subclinical stales of glucose intolerance and risk of death in the US.
Diabetes Care 2001, 24, 447–453.
’ AUTHOR INFORMATION
Corresponding Author
(10) Khaw, K. T.; Wareham, N.; Bingham, S.; Luben, R.; Welch, A.;
Day, N. Association of hemoglobin A1c with cardiovascular disease and
mortality in adults: the European prospective investigation into cancer in
Norfolk. Ann. Intern. Med. 2004, 141, 413–420.
*(A. C.-G.) Phone: (860) 686-9155. Fax: (860) 686-5256. E-mail:
agustin.casimiro-garcia@pfizer.com. (G. F. F.) Phone: (860) 686-
3546. E-mail: gary.filzen@pfizer.com.
(11) Klein, S.; Sheard, N. F.; Pi-Sunyer, X.; Daly, A.; Wylie-Rosett, J.;
Kulkarni, K.; Clark, N. G. Weight management through lifestyle
modification for the prevention and management of type 2 diabetes:
rationale and strategies: a statement of the American Diabetes Associa-
tion, the North American Association for the Study of Obesity, and the
American Society for Clinical Nutrition. Diabetes Care 2004, 27,
2067–2073.
(12) Chia, C. W.; Egan, J. M. Role and development of GLP-1
receptor agonists in the management of diabetes. Diabetes Metab. Syndr.
Obes. 2009, 2, 37–49.
(13) Drab, S. R. Incretin-based therapies for type 2 diabetes mellitus:
current status and future prospects. Pharmacotherapy 2010, 30, 609–624.
(14) Choi, J. H.; Banks, A. S.; Estall, J. L.; Kajimura, S.; Bostrom, P.;
Laznik, D.; Ruas, J. L.; Chalmers, M. J.; Kamenecka, T. M.; Bluher, M.;
Griffin, P. R.; Spiegelman, B. M. Anti-diabetic drugs inhibit obesity-
linked phosphorylation of PPARγ by Cdk5. Nature 2010, 466, 451.
(15) de Gasparo, M.; Catt, K. J.; Inagami, T.; Wright, J. W.; Unger, T.
International union of pharmacology. XXIII. The angiotensin II recep-
tors. Pharmacol. Rev. 2000, 52, 415–472.
(16) Benson, S. C.; Pershadsingh, H. A.; Ho, C. I.; Chittiboyina, A.;
Desai, P.; Pravenec, M.; Qi, N.; Wang, J.; Avery, M. A.; Kurtz, T. W.
Identification of telmisartan as a unique angiotensin II receptor antago-
nist with selective PPARγ-modulating activity. Hypertension 2004,
43, 993–1002.
’ ACKNOWLEDGMENT
Use of the IMCA-CAT beamline 17-ID at the Advanced
Photon Source was supported by the companies of the Industrial
Macromolecular Crystallography Association through a contract
with Illinois Institute of Technology. Use of the Advanced
Photon Source was supported by the U.S. Department of Energy,
Office of Science, Office of Basic Energy Sciences, under Con-
tract No. W-31-109-Eng-38. Special thanks to Dr. Jack Bikker for
technical assistance in rendering Figure 3.
’ ABBREVIATIONS
ACE, angiotensin converting enzyme; ARB, angiotensin receptor
blocker;AT1, angiotensin II type 1 receptor;BP, blood pressure;
BW, body weight; CV, cardiovascular; DPP-4, dipeptidyl
peptidase 4; FFA, free fatty acids; GLP-1, glucagon-like pep-
tide-1; LBD, ligand binding domain; PK, pharmacokinetic;
PPARγ, peroxisome proliferator-activated receptor-γ; SFC,
supercritical fluid chromatography; SHR, spontaneously
hypertensive rat; TG, triglycerides; TZD, thiazolidinedione;
ZDF, Zucker diabetic fatty rat
(17) Vitale, C.; Mercuro, G.; Castiglioni, C.; Cornoldi, A.; Tulli, A.;
Fini, M.; Volterrani, M.; Rosano, G. M. C. Metabolic effect of telmisartan
and losartan in hypertensive patients with metabolic syndrome. Cardi-
ovasc. Diabetol. 2005, 4, 6–13.
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