Reductions in log P improved protein binding and clearance predictions enabling the prospective design of cannabinoid receptor (CB1) antagonists with desired pharmacokinetic properties

Article abstract of DOI:10.1021/jm4010835

Several strategies have been employed to reduce the long in vivo half-life of our lead CB1 antagonist, triazolopyridazinone 3, to differentiate the pharmacokinetic profile versus the lead clinical compounds. An in vitro and in vivo clearance data set revealed a lack of correlation; however, when compounds with <5% free fraction were excluded, a more predictable correlation was observed. Compounds with log P between 3 and 4 were likely to have significant free fraction, so we designed compounds in this range to give more predictable clearance values. This strategy produced compounds with desirable in vivo half-lives, ultimately leading to the discovery of compound 46. The progression of compound 46 was halted due to the contemporaneous marketing and clinical withdrawal of other centrally acting CB1 antagonists; however, the design strategy successfully delivered a potent CB1 antagonist with the desired pharmacokinetic properties and a clean off-target profile.

Full text of DOI:10.1021/jm4010835

Article
pubs.acs.org/jmc
Reductions in log P Improved Protein Binding and Clearance
Predictions Enabling the Prospective Design of Cannabinoid
Receptor (CB1) Antagonists with Desired Pharmacokinetic Properties
Bruce A. Ellsworth,* Philip M. Sher, Ximao Wu, Gang Wu, Richard B. Sulsky, Zhengxiang Gu,
Natesan Murugesan, Yeheng Zhu, Guixue Yu, Doree F. Sitkoff, Kenneth E. Carlson, Liya Kang, Yifan Yang,
Ning Lee, Rose A. Baska, William J. Keim, Mary Jane Cullen, Anthony V. Azzara, Eva Zuvich,
Michael A. Thomas, Kenneth W. Rohrbach, James J. Devenny, Helen E. Godonis, Susan J. Harvey,
Brian J. Murphy, Gerry G. Everlof, Paul I. Stetsko, Olafur Gudmundsson, Susan Johnghar,
Asoka Ranasinghe, Kamelia Behnia, Mary Ann Pelleymounter, and William R. Ewing
Research and Development, Bristol-Myers Squibb, Co., P.O. Box 5400, Princeton, New Jersey 08543-5400, United States
*
S Supporting Information
ABSTRACT: Several strategies have been employed to reduce
the long in vivo half-life of our lead CB1 antagonist,
triazolopyridazinone 3, to differentiate the pharmacokinetic
profile versus the lead clinical compounds. An in vitro and in
vivo clearance data set revealed a lack of correlation; however,
when compounds with <5% free fraction were excluded, a
more predictable correlation was observed. Compounds with
log P between 3 and 4 were likely to have significant free
fraction, so we designed compounds in this range to give more
predictable clearance values. This strategy produced com-
pounds with desirable in vivo half-lives, ultimately leading to
the discovery of compound 46. The progression of compound
4
6 was halted due to the contemporaneous marketing and
clinical withdrawal of other centrally acting CB1 antagonists; however, the design strategy successfully delivered a potent CB1
antagonist with the desired pharmacokinetic properties and a clean off-target profile.
INTRODUCTION
metabolism within their chemical series via reduction in log P6
from 5.6 to 4.6, resulting in an improved microsomal stability.
Griffith et al. reported the discovery of otenabant via reductions
in log P relative to rimonabant (e log D = 4.0 vs 5.8) that led to7
greater microsomal stability and low metabolic clearance.
While those efforts to reduce log P were employed to increase
half-life, we wished to reduce the long in vivo half-life of our lead
CB1 antagonist 3 in an attempt to provide a differentiated
pharmacokinetic (PK) profile versus other CB1 antagonists.
The most clinically advanced CB1 antagonists, rimonabant,
taranabant, and otenabant, exhibit long elimination half-lifes
■
The cannabinoid receptor 1 (CB1) is a G-protein-coupled
receptor (GPCR) with high expression in the central nervous
system (CNS) that promotes feeding when activated by
endogenous cannabinoid agonists such as anandamide and 2-
1
AG. The CB1 receptor antagonist, rimonabant (SR141716),
which had been marketed in Europe as Acomplia for the
treatment of obesity, binds to the human CB1 receptor with
nanomolar affinity and blocks signaling of the receptor. In the
2
RIO clinical trials in North America and Europe, 20 mg qd of
rimonabant was shown to reduce body weight of obese subjects
by ∼4.7% over placebo during the first year of treatment. This
weight loss was then maintained over the second year of
8
−10
(
T1/2 of 9−15 days, 3−4 days, and 5−8 days, respectively)
in humans. Compounds with long elimination half-lives may
add significant time and expense to early clinical trials, and
there may be significant delay in drug withdrawal for patients
3
treatment. Rimonabant was withdrawn by Sanofi SA from
consideration for regulatory approval by the FDA in the U.S.,
and its marketing was voluntarily suspended in Europe. The
European Commision later withdrew marketing authorization
due to psychiatric side effects.
The structures of CB1 antagonists have been reviewed;
11
experiencing adverse events. Drug accumulation to steady-
12
state and drug washout studies typically require 4 times T1/2
,
4
as demonstrated by the reported 13 days required for
taranabant to reach steady state and 28 day monitoring of
5
however, there are only a few reports that describe efforts to
affect in vivo clearance of CB1 antagonists via changes in log P.
Letourneau et al. have described their efforts to reduce
Received: July 17, 2013
©
XXXX American Chemical Society
A
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Figure 1. Evolution of the triazolopyridazine series of CB1 receptor antagonists.
9
,10
drug elimination for otenabant in human PK studies.
Scheme 1. Methyl Grignard Addition to
Triazolopyridazinone 3
Whereas a single-dose PK assessment can be made for
compound with a 12 h half-life with 4 days of monitoring, it
may take as much as 120 days of monitoring for each dose level
of a compound with 15 day half-life. Furthermore, there is a
significant delay in drug clearance in the event of an adverse
event. For example, diazepam (washout half-life of 79 h)
exhibited fatigue and sedation side effects 2 weeks after drug
withdrawal while oxazepam (washout half-life of 10.7 h) side
1
2
13
effects subsided rapidly after drug withdrawal. By developing
a CB1 receptor antagonist with a shortened elimination half-
life, it was hoped that our early clinical program would move
quickly and that rapid drug washout would improve safety in
the case of reversible adverse events. To accomplish this goal,
herein we describe efforts to discover potent and selective CB1
antagonists with predictable half-life using medicinal chemistry
principles that may be applicable beyond the field of CB1.
These strategies may be useful when applied to other lipophilic
receptor ligands that have high MW or log P, leading to long in
vivo half-life.
found in compound 7. For example, compound 8 (Table 1)
provided encouragement that there may be a new binding
pocket that could be exploited. Having extensively searched for
polar moieties to replace the N2-lipophilic CF -benzyl group of
3
16
compound 2 with limited success, we hoped we could exploit
this new pocket to introduce a polar substituent at N5. We
started this exploration with two central tenets: (1) the C8-
pyridine moiety of compound 3 had brought several favorable
traits, including polarity, so we wished to incorporate a pyridine
or a suitable substitute and (2) we chose to minimize the
substituent in the N2-pharmacophoric space because large
hydrophobic moieties counteracted the benefits of the polar
C8-pyridine moiety. Transposition of the C8 pyridine and the
C7 chlorophenyl groups, a product that was synthesized as the
result of a nonregioselective route, led to greater affinity for
compound 11 as compared to compound 10 (Figure 2).
As we explored the SAR of the N5-benzyl substituent in this
transposed series, we found similar activity trends to that
exhibited by the N2-benzyl compounds in the original series,
suggesting that the two isomeric structures may be occupying
the same binding pockets in the CB1 receptor, as depicted in
Figure 3. As further support for this hypothesis, mutagenesis
studies reveal that compound 11 and compound 3 lose 20−40-
fold binding affinity against mutant F200A while the affinity is
recovered in mutant F200L, suggesting similar interactions with
RESULTS AND DISCUSSION
■
Our lead series of CB1 antagonists originated from high-
throughput screening (HTS) hits and evolved through a series
of pyrazine carboxamides (1, Figure 1) as reported previously.
To lower log P and improve physicochemical properties (e.g.,
aqueous solubility), we explored alternative cores, resulting in
14
15
16
the discovery of the triazolopyridazine (TZP) series. An effort
to replace two chlorophenyl substituents and the benzyl group
of compound 2 revealed that pyridine groups in compound 3
conferred the desired solubility and log P improvement while
maintaining good human CB1 receptor (hCB1) binding
affinity. Compound 3 was found to have a long half-life in all
species tested (T1/2 = 15, 108, and 26 h in rat, dog, and
17
monkey, respectively), so the medicinal chemistry effort
focused on an attempt to maintain the compound’s positive
characteristics while decreasing in vivo half-life.
21
the CB1 receptor. While that observation argued against the
likelihood of finding more potent polar substituents at N5 of
compound 12, it was immediately recognized that the
properties of the transposed triazolopyridazin-diones (TZP-
diones) were favorable despite bearing the same substituents.
With some sacrifice of in vitro affinity, we found significant
improvement in crystalline aqueous solubility (40 vs 1 μg/mL
for 12 and 7, respectively) as well as desired reductions of in
vivo half-life in rats (6 vs 35 h for 12 and 7, respectively).
Capitalizing upon the SAR of the earlier TZP series,
incorporation of a 6-methyl-2-trifluoromethylpyrid-3-yl side
chain increased affinity by ∼2−3-fold (compound 13) and a
slightly larger N2-alkyl substituent (compound 15) further
improved CB1 binding affinity (Table 2). Compound 15 was
In an effort to introduce a methyl group as a metabolic soft
spot, methyl magnesium bromide was found to add to the 6-
position of the TZP ring which, upon rearomatization by air or
DDQ, led to the formal replacement of methyl for hydrogen at
1
8
the C6-position (Scheme 1). Unfortunately, an alkyl
substituent at this position significantly decreased in vitro
affinity (compound 4 vs 3, Table 1). In contrast, a methoxy
group at this position (compound 5, Table 1) was equipotent
in vitro to compound 3. Compound 7, an O- to N-methyl
isomer of 5, was also a potent CB1 antagonist. These
compounds, however, did not yield desired improvements of
in vivo efficacy or half-life in rats vs compound 3.
We found that the N5-position (X = N) could tolerate
significantly larger substituents than the simple methyl group as
20
efficacious in reducing food intake by 43% acutely, and in a 4-
B
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Table 1. Pharmaceutical Properties of Pyridazinone Core Substitutions
a
19 b
The term “e log P” refers to experimentally measured log P. MED represents the minimally efficacious dose that produces statistically significant
20 c
food reduction in nonfasted rats feeding ad libidum over a 20 h period. Terminal half-life (T1/2) and clearance [Cl] were measured in Sprague−
Dawley rats upon iv administration of 3 mg/kg of test compound. Log P was not measured for this compound; calculated log P of compound 8 is
d
4
.6.
concentrations of 4 μM at Cmax and AUC0−24 = 48 μM h). In
the same study, rimonabant gave 5% body weight reduction
when administered at 10 mg/kg qd. Several liabilities had been
resolved at this point: compound 15 displayed good in vitro
affinity and in vivo efficacy while maintaining relatively low log
P and shortened in vivo half-life relative to compound 3.
Compound 15 was synthesized according to the sequence
depicted in Scheme 2. Commercially available 4-pyridylacetic
acid was O-alkylated with bromide 17, followed by cyclization
to butenolide 18. Base-catalyzed air oxidation generated
anhydride 19 and reaction with hydrazine gave pyridazin-
dione 20. Dehydrative chlorination of 20 was achieved with
POCl3 to give pyridazine dichloride 9. Displacement of
Figure 2. SAR of 4-pyridine positional isomers.
2
2
day study it reduced body weight by 4% when administered
qd at 10 mg/kg to ad libidum fed rats (resulting in plasma
C
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Figure 3. Comparison of proposed binding modes and of hCB1 K values for N-2-CF -pyridyl- vs N-5-CF -pyridyl-triazolopyridazinediones 7 and
i
3
3
1
2.
Table 2. SAR of Pyridyl-Containing TZP-diones
a
b
R
compd
hCB1 K (nM)
SD (nM)
9.3
MED in rats @ 8 h (% redn)
rat T1/2 [Cl] (mL/min/kg)
CYP isoform IC50 (μM)
i
R1 = H
12
28
9.0
15
3 mg/kg
(21%)
6 h
2C19 = 4.3
2C9 = 1.9
R2 = Me
[13]
3
A4 = 6.6
R1 = Me
R2 = Me
13
14
15
4.3
6.9
1.5
3 mg/kg
(33%)
3.3 h
[6.3]
2C19 = 0.7
2C9 = 2.9
3
A4 = 6.8
R1 = H
R2 = Et
n.d.
2.4 h
[37]
2C19 = 11
2C9 = 4.9
3
A4 = 18
R1 = Me
R2 = Et
4.5
10 mg/kg
(51%)
5 h
2C19 = 2.6
2C9 = 4.2
[9.9]
c
3
A4 = 9.1
a
b
Due to shorter in vivo half-life, MED was determined at 8 h instead of 20 h. Materials and methods for CYP inhibition assays are found in the
c
Supporting Information. CYP 3A4 inhibition study in human liver microsomes. Upon re-evaluation in hepatocytes, compound 15 CYP 3A4 IC
=
5
0
3
μM.
dichloride 9 was effected with NaOH, resulting in a separable
mixture of regioisomers. Alkylation of 21a with 3-(chlor-
omethyl)-2-methyl-6-(trifluoromethyl)pyridine gave chloride
compound 3), compound 15 was predicted to overshoot this
goal.
Confident that a sufficiently short half-life was possible, we
set out to identify a compound with appropriate half-life and
bioavailability for predicted qd administration in humans while
eliminating the CYP inhibition liability observed for compound
2
3. The triazolo-ring was formed via chloride displacement
with hydrazine and cyclization with 1,1′-carbonyldiimidazole to
give bicyclic intermediate 24. Alkylation with ethyl iodide
completes the 9-step linear sequence, enabling the synthesis of
several grams of compound 15 for advanced efficacy and safety
studies.
15. We discovered SAR (Table 3), suggesting that the 4-pyridyl
group of compound 15 was associated with CYP inhibition, but
a 4-cyanophenyl derivative (28) displayed increased in vitro
affinity as well as reduced CYP inhibition. However,
substitution of a 4-cyanophenyl for a 4-pyridyl group at C7
ran counter to the program goals of decreased log P (4.6 vs 3.9)
Several liabilities were discovered during the preclinical in
vitro and in vivo characterization of compound 15. CYP
inhibition was a concern because efficacy in humans was
predicted to require micromolar plasma exposures of 15. In
higher species PK studies, compound 15 was also shown to
have variable (15−50%) bioavailability and medium to high
24
and increased solubility, and this compound also displayed
unacceptably long elimination half-lives in dogs (70 h) and
cynomologous monkeys (40 h).
In an attempt to introduce a metabolic soft spot to reduce its
in vivo half-life, we incorporated a methyl group at various
positions on compound 28. Most of the derivatives in Table 4
had undesirable levels of hCB2 binding IC s from 77 to 400
2
3
clearance. Allometric scaling led to a predicted high clearance
39 mL/min/kg) in humans. While the goal of this program
was to deliver a compound with shortened half-life (relative to
(
50
D
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a
Scheme 2. Synthesis of Compound 15
a
Conditions: (a) KOtBu, DMF, 88%; (b) air, KOtBu, THF; (c) hydrazine hydrate, EtOH, HCl, 32% (2 steps); (d) POCl , 85 °C, 72%; (e) NaOH,
3
CH CN, 75%; (f) K CO , DMF, 66%; (g) hydrazine (anhyd) pyridine; (h) 1,1′-carbonyldiimidazole, THF, 73% (2 steps); (i) EtI, K CO , DMF,
3
2
3
2
3
6
7%.
Table 3. CB1 and CYP Isoform SAR of C7-Aryl-
triazolopyridazinediones
half-life, making it difficult to overcome a major program goal
without performing an untenable number of in vivo PK studies.
On the basis of this observation, we analyzed the entire
program data set to develop correlations that could be used to
predict compounds with shortened in vivo half-life based on in
vitro data. There seemed to be little correlation between
clearance in vitro and in vivo, as demonstrated in Figure 4A. In
vitro and in vivo clearance correlations that factor in free
fraction have been reported, and they express in vitro clearance
25
as unbound Clint. These correlations were developed for
2
6,27
compounds with log P < 3 and MW <500,
but many of our
28
hCB1 Ki
SD
CYP isoform
potent CB1 antagonists are outside of these limits. To
improve our correlation, we factored in free fraction by using
unbound Cl instead of Cl and we filtered the data set to only
Ar
R1
H
compd
(nM)
(nM) e log P
IC50 (μM)
4
4
3
-pyridyl
14
15
6.9
1.5
3.5
4.0
3.9
4.2
2C19 = 11
29
2
C9 = 5
A4 = 18
2C19 = 3
C9 = 6
A4 = 3
2C19 = 32
consider compounds with protein binding ≤95%. A trend
emerged (Figure 4B) between unbound in vitro metabolism
and in vivo clearance in rats. In the process of culling
compounds based on free fraction, we plotted e log P vs protein
binding (Figure 5) and found that compounds in the range of e
log P 3−4 have a much higher probability of being ≤95%
protein bound than compounds with e log P > 4.5.
3
-pyridyl
Me
Me
15
27
4.5
8.4
2
3
-methyl-4-
pyridyl
2
C9 = 5
A4 = >40
2C19 > 40
To test the predictive correlation of our model between in
vitro clearance and in vivo clearance and half-life, we mined our
compound collection to find a derivative in the desired log P
range for which we had not yet measured free fraction or
clearance. Compound 11 has a measured log P = 3 with 5% free
fraction in rat plasma. The unbound in vitro metabolism
3
4
-
Me
28
1.4
0.58
4.6
cyanophenyl
2
C9 > 40
A4 > 40
3
(
0.0063 nmol/min/mg) and in vivo clearance values (iv Cl = 7
nM, making them undesirable for progression, but two
compounds within the table (28 and 32) highlight a
disconnection that became apparent (vide infra, Figure 4A)
more broadly for the chemotype: that in vitro metabolic
stability determinations did not predict in vivo clearance or
mL/min/kg) were in line with predictions and gratifyingly
exhibited an anticipated half-life of 3.5 h in rat. To verify the
efficacy of the PK/affinity profile of 11 as a CB1 antagonist,
compound was administered at 10 mg/kg to rats in an acute
20
feeding model wherein reduced feeding was observed at a
E
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Table 4. Methyl Scan of Compound 28 in an Attempt to Introduce a Metabolic Soft-Spot
a
In vitro rat clearance was determined in rat liver microsomes, as described in the Supporting Information. Rat T1/2 was not determined for
compounds 29−31, 33, and 34.
similar level to 10 mg/kg of rimonabant, the positive control in
the study.
7 h, 9 h) relative to 28. Unfortunately, both compounds had
liabilities that made them unsuitable for advancement.
Compound 35 had poor physicochemical properties, making
it difficult to formulate and administer to rats. Compound 36
displayed weak efficacy in the 20 h feeding experiment due to a
combination of its short half-life and low plasma and brain
exposure over the course of the in vivo experiment. There are
many factors beyond clearance that affect in vivo half-life, but
these results provided encouragement that compounds with log
Using the above criteria, we prospectively considered two
compounds with the e log P range of 3−4. Compound 35, with
e log P = 4.2, had 1% free fraction in rat plasma and
demonstrated a shortened half-life (rat, dog, cyno = nd, 45 h, 8
h) relative to benchmark compound 28 (Figure 6). Compound
3
6, with e log P = 3.6, had 9% free fraction in rat plasma and it
also demonstrated a shortened half-life (rat, dog, cyno = 1.1 h,
F
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Figure 4. (A) In vitro metabolism rate (rat liver microsomes (RLM)) vs rat in vivo Cl (mL/min/kg). (B) Unbound RLM Cl (mL/min/kg) vs rat in
vivo Cl (mL/min/kg), filtered to show compounds with ≤95% protein binding.
P between 3 to 4 could meet the goal of higher free fraction
that would bring the possibility of predictable half-life in
preclinical species.
With the N2 and N5-substituents of the TZP-dione
extensively explored, due in part to their late introduction in
the synthesis (steps 5 and 9, Scheme 2), the C7-position
remained underexplored despite the experience that polar
moieties such as the C7-pyridyl group of compound 15 were
tolerated by the CB1 receptor. The synthetic sequence required
commitment to the C7 and C8-aryl groups in the first step of
the 9-step synthetic sequence (Scheme 2); however, a revised
synthesis was developed for late C7 and C8-aryl group
introduction, as depicted in Scheme 3, to facilitate the
exploration of these substituents.
Dichloropyridazin-one 37 was brominated under basic
conditions, followed by selective N-alkylation with 22, to give
pyridazinone 39. Chlorine displacement with sodium meth-
oxide led to a single regioisomer, 40. Palladium-catalyzed N-
arylation of Boc-hydrazine gave chloride 41 that underwent
Suzuki coupling to introduce the C-8 substituent at an
intermediate stage in the synthesis. Deprotection of 42 and
cyclization with 1,1′-carbonyldiimidazole led to triazolopyr-
idazin-dione 43. Alkylation of the triazolo-nitrogen with ethyl
iodide gave protected triazolopyridazin-dione 44. Demethyla-
Figure 5. Graph of e log P vs rat plasma protein binding.
tion of the methoxy-group with BBr followed by triflation gave
3
intermediate 45, enabling introduction of the C7-group in the
final step via Suzuki coupling to give 46.
This revised synthesis enabled a more rapid exploration of
the C-7 substituent, targeting compounds with log P in the
range that gave a higher likelihood of having higher free
fraction. Compound 47 (Table 5) exhibited significant CYP
inhibition, and compounds 48 and 49 exhibited reduced hCB1
affinity; however, methylpyrimidine 46 was found to be a
potent and selective CB1 antagonist with e log P = 3.8. The
compound exhibited reasonable aqueous solubility (17 μg/mL)
in crystalline form and excellent DMSO/water solubility to
enable an accurate in vitro assessment of off-target activities.
Figure 6. Potent CB1 antagonists (hCB1 K = 5 nM for both 35 and
i
3
2
6) with lowered log P and shortened half-life relative to compound
8.
G
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a
Scheme 3. Revised Synthesis of TZP-diones to Enable Late Introduction of C7 and C8-Substituents
a
(
a) Br , LiOH, MeOH; (b) 22, K CO , DMF, 80% (2 steps); (c) NaOMe, dioxane, 72%; (d) Pd(dppf)Cl , BocNHNH , Cs CO , toluene, 78%;
2 2 3 2 2 2 3
(
8
e) 4-ClPhB(OH) , Pd(PPh ) , Na CO , toluene, 92%; (f) HCl, dioxane; (g) 1,1′-carbonyldiimidazole, THF, 70% (2 steps); (h) EtI, K CO , DMF,
2
3
4
2
3
2
3
4%; (i) BBr , CH Cl ; (j) Tf O, TEA, CH Cl ; (k) Pd(dppf)Cl , K CO , THF, 2-methylpyrimidinyl-5-boronic acid, 14% (3 steps).
3 2 2 2 2 2 2 2 3
Compound 46 was devoid of CYP inhibition up to 40 μM,
meeting another criterion for advancement. The compound
demonstrated high (13% and 7%) free fraction respectively in
rat and human plasma, in line with the expectation of significant
free fraction. Compound 46 demonstrated low clearance in rat
liver microsomes (unbound Cl = 0.0022 nmol/min/mg).
However, in vivo clearance was unexpectedly high (43 mL/
min/kg), which may have been predicted by its subsequently
measured extensive metabolism in rat hepatocytes (5%
remaining). Fortunately, this metabolism appeared rat-specific
and the compound exhibited the predicted clearance in higher
species, leading to a desirable half-life (T1/2 = 5, 42, 12 h) and
bioavailabilty (73%, 75%, 51%) in the rat, dog, and
cynomologous monkey, respectively.
mg/kg. Upon multiple day administration at 3 and 10 mg/kg,
46 caused a statistically significant reduction of body weight
over 4 days of 2.8 and 7.2% respectively. Treatment of 46 in a
28-day study in diet-induced obese rats demonstrated body
weight reductions of 2.4, 4.1, and 10.2% when administered po
at 1, 3, and 10 mg/kg, respectively. In this same study,
administration of 10 mg/kg of rimonabant produced a 6.2%
body weight reduction.
22
The safety of compound 46 was evaluated both in vitro and
in vivo. No off-target liabilities were found despite extensive
screening against a panel of GPCRs and ion channels; this may
15
be an added benefit of the reduced log P of the compound.
The potential toxicity profile of 46 was evaluated when
administered orally to dogs for a 12-day period. The results of
the toxicological studies will be described elsewhere; however,
the effects observed were consistent with known effects of other
14
Metabolism was the major route of elimination of [ C]-
labeled compound 46 when administered orally at 5 mg/kg to
bile duct-cannulated rats. Two major oxidative metabolites
containing 14C-label were observed by LC/MS ([M + 2O −
30
CB1 receptor antagonists and were not attributed to any off-
target liabilities. The findings from the toxicological study, in
conjunction with the contemporaneous marketing withdrawal
of rimonabant and withdrawals of taranabant and otenabant
from clinical development, led to the decision that clinical
evaluation of 46 or other centrally acting compounds in this
series would not be pursued.
2H] and [M + O + glucuronide], structures undetermined),
and these metabolites accounted for 36% and 26% of the dose,
respectively. In comparison, excretion of parent was a minor
route of elimination (less than 1% of the dose). Within 48 h
post dose, 76% and 7% of the radioactivity was recovered in
bile and urine, respectively, with 0.8% of parent radioactivity
observed in bile and no parent drug in urine. In rats, therefore,
the disposition of compound 46 appears to be primarily
mediated by oxidative metabolism, with minimal contribution
from direct renal and biliary excretion of the parent compound.
The favorable pharmacokinetic properties of 46, combined
CONCLUSIONS
■
We report the discovery of a potent, selective CB1 antagonist,
compound 46, which also demonstrates significantly lower log
P as compared to other compounds in its class. The lower log P
was a critical element in conferring greater free fraction and
allowing better predictive in vivo clearance values based on in
vitro microsomal metabolism. The introduction of a metabolic
with its potent hCB1 K , result in significant reduction in food
i
intake when administered acutely to ad libidum fed rats at 1
H
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Table 5. CB1 and CYP Isoform SAR of C7-Aryl-triazolopyridazinediones
soft spot did not result in compounds with the desired PK
profile. A key improvement in the physicochemical properties
was achieved when pharmacophoric groups were transposed
across an earlier TZP core. A synthetic route was developed
that facilitated the introduction of a 2-methyl pyrimidine group
that conferred greater polarity to the series while maintaining
CB1 antagonist affinity and selectivity. The progression of 46,
and our entire centrally mediated CB1 antagonism program,
was halted due to undesired class-related CB1 effects in vivo.
We have described herein the methods that accelerated the
discovery of potent CB1 antagonists in this series with lower
log P and greater free fraction, resulting in more predictable
pharmacokinetics and an improved profile against off-target
liabilities.
Elemental analyses were performed by the Bristol-Myers Squibb
Discovery Analytical Science Department. Melting points were taken
on a Hoover Unimelt melting point apparatus and were uncorrected.
LC/MS data were recorded on a Shimadzu LC-10AT equipped with a
SIL-10A injector, a SPD-10AV detector normally operating at 220 nm,
and interfaced to a Micromass ZMD mass spectrometer. LC/MS or
HPLC retention times were reported using a Phenomenex Luna C-18
4
.6 mm × 50 mm column eluting with a 4 min gradient of 0−100%
solvent B, where solvent A was 10:90:0.1 CH OH−H O−TFA and
solvent B was 90:10:0.1 CH OH−H O−TFA. Reactions were
3
2
3
2
monitored by TLC using 0.25 mm E. Merck silica gel plates (60
F254) and were visualized using UV light and 5% phosphomolybdic
acid in 95% EtOH. All compounds were found to be >95% pure by
HPLC analysis unless otherwise noted.
7-(4-Chlorophenyl)-6-methyl-8-(pyridin-4-yl)-2-((6-
(
trifluoromethyl)pyridin-3-yl)methyl)-[1,2,4]triazolo[4,3-b]-
pyridazin-3(2H)-one (4). To a solution of 100 mg (0.2 mmol) of
1
7
EXPERIMENTAL SECTION
compound 3 in 2 mL of THF at −20 °C was added 0.2 mL of a 3 M
■
solution of methylmagnesium bromide (0.6 mmol). The mixture was
Chemistry. All anhydrous reactions were carried out using oven-
dried glassware under an atmosphere of argon or nitrogen. All reagents
and solvents were obtained from commercial vendors and used
without further purification unless otherwise indicated. NMR spectra
stirred for 30 min, and 2 mL of NH Cl (satd aq) was added to quench
4
the reaction. The solution was diluted with 40 mL each of EtOAc and
satd aq NH
dried (Na SO
The residue was dissolved in 2 mL of CH
Cl, and the layers were extracted. The organic layer was
), filtered, and evaporated to give 100 mg of a yellow oil.
Cl , and 47 mg of 2,3-
4
1
13
(
H, C) were recorded on JEOL JNM-ECP500, JEOL GSX400, and
2
4
Bruker 400 spectrometers. Chemical shifts were given in parts per
million (ppm) downfield from internal reference tetramethylsilane
standard; coupling constants (J values) were given in hertz (Hz).
2
2
dichloro-5,6-dicyano-p-benzoquinone (DDQ, 0.2 mmol) was added.
After 15 min at room temperature, the mixture was stored overnight at
I
dx.doi.org/10.1021/jm4010835 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
−
40 °C. The crude reaction mixture was loaded onto silica gel for
purification to give 18 mg of a yellow oil that was further purified via
preparative RP-HPLC to give 12 mg of compound 4 as a yellow solid
mixture was cooled to room temperature, and the solution was poured
over 600 mL of ice. The slurry was agitated for 30 min, and the pH of
the resulting solution was carefully adjusted to pH 5 with cooling as
needed. The resulting aqueous slurry was diluted with 400 mL of
CH Cl , and the layers were extracted. The aqueous layer was re-
(
12%). Analytical HPLC (4 min gradient): 2.94 min (97.1%
homogeneity index (HI)). HRMS anal. calcd for C H ClF N O
24
17
3
6
2
2
1
[
M + H]: 497.1104, found 497.1104. H NMR (400 MHz, CD OD) δ
extracted with 200 mL of CH Cl , and the resulting organic layers
3
2 2
ppm 8.75 (d, J = 1.8 Hz, 1H), 8.49 (dd, J = 1.7, 6.2 Hz, 2H), 8.04 (dd,
J = 1.8, 8.2 Hz, 1H), 7.80 (d, J = 8.2 Hz, 1H), 7.40−7.23 (m, 6H), 4.85
were combined, washed with brine, dried (Na SO ), filtered, and
2 4
evaporated to give a black oil. The residue was purified via flash
(
s, 2H), 2.29 (s, 3H). 13C NMR (100 MHz, CD OD) δ ppm 155.26,
chromatography on silica gel that had been pretreated with 2% TEA/
3
1
1
1
50.84, 150.50, 150.17, 141.34, 139.19, 136.87, 136.50, 136.21, 133.87,
hexanes to give 1.7 g of 9 in 46% yield from 4-pyridylacetic acid. H
33.12, 132.56, 130.04, 126.26, 121.79, 22.14. 19F NMR (151 MHz,
NMR (500 MHz, CDCl ) δ 8.6 (dd, 2H, J = 1.7, 4.4 Hz), 7.29 (dt, 2H,
3
CD OD) δ ppm −69.28.
J = 1.7, 8.3 Hz), 7.02−6.99 (m, 4H).
3
7
-(4-Chlorophenyl)-6-methoxy-8-(pyridin-4-yl)-2-((5-
trifluoromethyl)pyridin-2-yl)methyl)-[1,2,4]triazolo[4,3-b]-
pyridazin-3(2H)-one (5). To a solution of compound S6 (71 mg,
.14 mmol) in methanol (0.3 mL) was added NaOMe (25 wt % in
4-((7-(4-Chlorophenyl)-2-methyl-3,6-dioxo-8-(pyridin-4-yl)-
2,3-dihydro-[1,2,4]triazolo[4,3-b]pyridazin-5(6H)-yl)methyl)-
benzonitrile (10). To a solution of compound S4 (8.0 mg, 0.023
mmol) in DMF (0.3 mL) was added K CO (5.0 mg, 0.036 mmol),
(
0
2
3
methanol solution) (0.7 mL, 3.2 mmol). The reaction mixture was
heated at 50 °C for 20 min, followed by cooling to room temperature
and concentration. The residue was diluted with 50 mL each of ethyl
acetate and water, and the layers were extracted. The organic layer was
followed by 4-(bromomethyl)benzonitrile (5.0 mg, 0.025 mmol). After
5 min, the reaction mixture was diluted with ethyl acetate, and the
resulting solution was washed with water. The organic layer was dried
1
over Na SO , filtered, and concentrated under reduced pressure. The
2
4
separated and dried (Na SO ). The dried organic layer was filtered and
2
4
crude product was purified by reverse phase HPLC to give 4.2 mg of
-((7-(4-chlorophenyl)-3,6-dioxo-8-(pyridin-4-yl)-2,3-dihydro-[1,2,4]-
concentrated under reduced pressure to give 71 mg (100%) of the title
compound as a yellow foam. Analytical HPLC (4 min grad): 3.23 min
4
triazolo[4,3-b]pyridazin-5(6H)-yl)methyl)benzonitrile as a yellow
foam. Analytical HPLC (4 min gradient) retention time = 2.70 min
(96.0% HI). LC-MS anal. calcd for C H ClN O : 454.1, found [M +
(
+
99.1% HI). LC-MS anal. calcd for C H ClF N O : 512.1, found [M
H] 512.9. H NMR (500 MHz, CDCl ) δ ppm 8.78 (d, J = 1.6 Hz,
3
24 16 3 6 2
1
2
4
15
6
2
1
8
2
H), 8.60−8.56 (m, 2H), 7.95 (dd, J = 8.0, 1.9 Hz, 1H), 7.67 (d, J =
.2 Hz, 1H), 7.38−7.27 (m, 2H), 7.17−7.09 (m, 2H), 7.06−6.98 (m,
H), 5.28 (s, 2H), 4.04 (s, 3H).
1
H] 455.1. H NMR (400 MHz, CD OD) δ ppm 8.69 (d, J = 6.6 Hz,
3
2H), 7.84−7.65 (m, 6H), 7.35−7.27 (m, 2H), 7.25−7.07 (m, 2H),
5.95 (s, 2H). A solution of 4-((7-(4-chlorophenyl)-3,6-dioxo-8-
7
-(4-Chlorophenyl)-8-(pyridin-4-yl)-2-((5-(trifluoromethyl)-
pyridin-2-yl)methyl)-[1,2,4]triazolo[4,3-b]pyridazine-3,6-
2H,5H)-dione (6). To a solution of compound S6 (170 mg, 0.33
(
pyridin-4-yl)-2,3-dihydro-[1,2,4]triazolo[4,3-b]pyridazin-5(6H)-yl)-
methyl)benzonitrile (2 mg, 0.004 mmol), methyl iodide (0.001 mL, 1
mg, 0.006 mmol), and K CO (2.5 mg, 0.02 mmol) in DMF (0.2 mL)
(
2
3
mmol) in THF (10 mL) was added potassium trimethylsilanol (169
mg, 1.3 mmol). The reaction mixture was heated to reflux at 85 °C for
was heated at 80 °C for 2 h. The mixture was cooled to room
temperature, and it was filtered and purified by reverse phase HPLC to
give 2 mg (90%) of compound 10 as a colorless foam. Analytical
HPLC (4 min gradient) retention time = 2.78 min (95.0% HI). LC-
1
h. The mixture was cooled to room temperature, and it was
concentrated to a thick oil. The residue was diluted with ethyl acetate,
and the resulting solution was extracted with water. The organic layer
was dried (Na SO ), filtered, and concentrated under reduced
1
MS anal. calcd for C H ClN O : 468.1, found [M + H] = 469.1. H
25
17
6
2
2
4
NMR (400 MHz, CDCl ) δ ppm 8.83 (br s, 2H), 7.82−7.64 (m, 4H),
3
pressure. The residue was purified by reverse phase HPLC to give
9 mg (17%) of compound 6 as a yellow solid. Analytical HPLC (4
7
.61 (d, J = 4.4 Hz, 2H), 7.36−7.29 (m, 2H), 7.07 (d, J = 8.8 Hz, 2H),
2
5
.98 (s, 2H), 3.58 (s, 3H).
min gradient) retention time = 3.15 min (97.2% HI). LC-MS anal.
1
4-((8-(4-Chlorophenyl)-2-methyl-3,6-dioxo-7-(pyridin-4-yl)-
calcd for C H ClF N O : 498.1, found [M + H] 499.2. H NMR
23
14
3
6
2
2
,3-dihydro-[1,2,4]triazolo[4,3-b]pyridazin-5(6H)-yl)methyl)-
(
8
7
500 MHz, DMSO-d ) δ ppm 12.93−12.85 (m, 1H), 8.71 (s, 1H),
6
benzonitrile (11). To solution of compound S3 (26 mg, 0.064
.58−8.52 (m, 2H), 7.99−7.88 (m, 2H), 7.40−7.34 (m, 2H), 7.32−
.27 (m, 2H), 7.23−7.17 (m, 2H), 5.31 (s, 2H).
mmol) in THF (1 mL) was added potassium trimethylsilanol (25 mg,
0
.19 mmol), and the mixture was heated to 85 °C for 10 min. The
solution was cooled to room temperature, and the pH was adjusted to
with 1N HCl. The resulting solution was extracted with ethyl acetate,
7
-(4-Chlorophenyl)-5-methyl-8-(pyridin-4-yl)-2-((5-
(
trifluoromethyl)pyridin-2-yl)methyl)-[1,2,4]triazolo[4,3-b]-
7
pyridazine-3,6(2H,5H)-dione (7). To a solution of compound 6 (61
and the organic layer was extracted with brine. The organic layer was
mg, 0.12 mmol) and Li CO (28 mg, 0.38 mmol) in DMF (1 mL) was
2
3
dried (MgSO ), filtered, and concentrated to give 26 mg of a yellow
added iodomethane (2.3 mg, 0.16 mmol), and the mixture was stirred
at room temperature for 1 h. The resulting mixture was diluted with
ethyl acetate, and the resulting solution was washed with water. The
organic layer was dried (Na SO ), filtered, and concentrated under
4
solid that was used as is. A solution of the yellow solid (26 mg, 0.051
mmol), 4-(bromomethyl)benzonitrile (11 mg, 0.056 mmol), and
K CO (21 mg, 0.15 mmol) in DMF (1 mL) was heated at 80 °C for
2
3
2
4
1
0 min, and the mixture was cooled to room temperature. The
solution was diluted with ethyl acetate, and the organic layer was
extracted with water. The organic layer was dried (Na SO ), filtered,
reduced pressure. The residue was purified by reverse phase HPLC to
give 29 mg (46%) of compound 7 as a light-yellow foam. Analytical
HPLC (4 min gradient) retention time = 2.71 min (98.6% HI). LC-
2
4
1
and concentrated under reduced pressure. The crude product was
purified by reverse phase HPLC, then treated with 1N HCl to give 15
mg (63%) of compound 11 as yellow solid. Analytical HPLC (4 min
gradient) retention time = 2.30 min (98.3% HI). LC-MS anal. calcd
MS anal. calcd for C H ClF N O : 512.1, found [M + H] 513.0. H
24
16
3
6
2
NMR (500 MHz, CDCl ) δ ppm 8.74 (s, 1H), 8.68 (br s, 2H), 7.90
3
(
d, J = 7.7 Hz, 1H), 7.68 (d, J = 7.7 Hz, 1H), 7.39 (br s, 2H), 7.27 (br
s, 2H), 7.02 (d, J = 8.2 Hz, 2H), 5.14 (s, 2H), 4.12 (s, 3H).
1
for C H ClN O : 468.1, found [M + H] 469.0. H NMR (500 MHz,
4
,4′-(7-(4-Chlorophenyl)-3,6-dioxo-8-(pyridin-4-yl)-[1,2,4]-
25 17
6
2
triazolo[4,3-b]pyridazine-2,5(3H,6H)-diyl)bis(methylene)-
dibenzonitrile (8). The title compound (3.6 mg, 22.5% yield) was
isolated as a yellow solid as a side-product in the synthesis of
compound 10. Analytical HPLC (4 min gradient) retention time =
CD OD) δ ppm 8.67 (d, J = 6.0 Hz, 2H), 7.81 (d, J = 6.0 Hz, 2H),
3
7.61 (q, J = 8.2 Hz, 4H), 7.32−7.05 (m, 4H), 5.84 (s, 2H), 3.44 (s,
3H).
8-(4-Chlorophenyl)-2-methyl-7-(pyridin-4-yl)-5-((6-
(trifluoromethyl)pyridin-3-yl)methyl)-[1,2,4]triazolo[4,3-b]-
pyridazine-3,6(2H,5H)-dione (12). Compound 12 was synthesized
from compound S5 and 2-trifluoromethyl-5-bromomethylpyridine
according to the procedure of compound 10. Analytical HPLC (4
min gradient) retention time = 2.67 min (99.1% HI). HRMS anal.
3
.15 min (90.0% HI). LC-MS anal. calcd for C H ClN O : 569.1,
32 20 7 2
1
found [M + H] 570.1. H NMR (500 MHz, CD OD) δ ppm 8.68 (d, J
3
=
2
6.6 Hz, 2H), 7.69 (m, 8H), 7.45 (d, J = 8.2 Hz, 2H), 7.35−7.27 (m,
H), 7.23−7.10 (m, 2H), 5.95 (s, 2H), 5.12 (s, 2H).
,6-Dichloro-4-(4-chlorophenyl)-5-(pyridin-4-yl)pyridazine
9). Compound 20 (4.9 g crude) was dissolved in 30 mL of POCl ,
3
1
(
calcd for C H ClF N O [M + H]: 513.1054, found: 513.1046. H
3
24 17
3
6
2
and the resultant solution was heated to 85 °C for 2 h. The reaction
NMR (500 MHz, CDCl ) δ ppm 8.96 (d, J = 1.7 Hz, 1H), 8.56 (dd, J
3
J
dx.doi.org/10.1021/jm4010835 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
=
1.7, 16.1 Hz, 2H), 8.14 (dd, 1.7, 8.3 Hz, 1H), 7.69 (d, J = 8.2 Hz,
solution was stirred for 16 h, followed by evaporation. The residue was
1
6
1
1
H), 7.31−7.27 (m, 2H), 7.15−7.12 (m, 2H), 7.07−7.05 (m, 2H),
dissolved in 200 mL of Et O, and the organic layer was extracted with
2
.03 (s, 2H), 3.59 (s, 3H). 13C NMR (100 MHz, CDCl ) δ ppm 158.0,
10% Na CO (100 mL), then brine (100 mL). The organic phase was
3
2
3
50.9, 149.7, 147.0, 140.4, 138.7, 136.6, 135.9, 134.6, 131.3, 131.2,
29.0, 127.8, 125.2, 120.4, 44.2, 33.7.
dried (Na SO ), filtered, and concentrated to give 13.7 g (48%) of
2 4
1
compound 22 as a tan oil. H NMR (400 MHz, CD CN) δ 7.96 (d,
3
8
- ( 4 - C h l o r o p h e n y l ) - 2 - m e t h y l - 5 - ( ( 2 - m e t h y l - 6 -
1H, J = 7.7 Hz), 7.64 (d, 1H, J = 7.9 Hz), 4.78 (s, 2H), 2.67 (s, 3H).
13
(
trifluoromethyl)pyridin-3-yl)methyl)-7-(pyridin-4-yl)-[1,2,4]-
C NMR (100 MHz, CD CN) δ 158.4, 146.0 (q, J = 34.0 Hz), 138.3,
3
triazolo[4,3-b]pyridazine-3,6(2H,5H)-dione (13). To a solution of
compound S2 (0.17 g, 0.45 mmol) in 0.5 mL of 1,4-dioxane was added
a solution of LiOH (38 mg, 0.9 mmol) in 0.5 mL of water. The
mixture was stirred, and Bu NOH (0.03 mL of 40% solution in water,
.04 mmol) was added. The resulting mixture was heated in a
microwave at 150 °C for 30 min. The mixture was evaporated to
dryness and was used as-is in the next step. To a solution of crude 6-
chloro-8-(4-chlorophenyl)-2-methyl-7-(pyridin-4-yl)-[1,2,4]triazolo-
4,3-b]pyridazin-3(2H)-one (35 mg, 0.07 mmol) in DMF (0.37 mL)
was added compound 22 (15 mg, 0.07 mL) and K CO (20 mg, 0.15
mmol). The resulting mixture was heated at 65 °C for 1 h, and then it
was cooled to room temperature. The crude reaction mixture was
diluted with MeOH, and it was purified via preparative RP-HPLC to
give 16 mg of the O-alkylated regioisomer, 8-(4-chlorophenyl)-2-
methyl-6-((2-methyl-6-(trifluoromethyl)pyridin-3-yl)methoxy)-7-(pyr-
idin-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3(2H)-one, and 15 mg
1
34.9, 124.1 (q, CF3, J = 275 Hz), 118.2, 42.5, 20.8.
4-(4-Chlorophenyl)-3-(pyridin-4-yl)furan-2(5H)-one (18). Po-
tassium tert-butoxide (40 mmol; 4.7 g) dissolved in MeOH (20 mL)
was added to 4-pyridylacetic acid hydrochloride (20 mmol; 3.5 g)
slurried in MeOH (20 mL). After stirring for 15 min, the solvent was
removed under reduced pressure and the residue was dissolved in dry
DMF (30 mL). To this solution was slowly added 2-bromo-4′-
chloroacetophenone (20 mmol; 4.8 g), and the solution turned to a
reddish-brown color. After 1 h at room temperature, the reaction
mixture was poured into water (200 mL) and this solution was
extracted with dichloromethane (2 × 100 mL). The combined organic
4
0
[
2
3
extracts were dried (MgSO ), filtered, and concentrated to provide 5.5
4
g of a dark-green semisolid. The product was slurried in EtOH (∼10
mL), and then it was isolated by vacuum filtration and drying under
high vacuum to give 3.2 g of 18 (60% yield) as a granular green solid.
Analytical HPLC (4 min gradient) retention time = 1.23 min (100.0%
HI). LCMS anal. calcd for C H ClNO 271.04, found [M + H] 272.
(
38%) of compound 13 as a yellow solid. Analytical HPLC (4 min
gradient) retention time = 2.65 min (100% HI). LCMS anal. calcd for
1
5
10
2
1
1
H NMR (400 MHz, CDCl
3
) δ 8.6 (dd, 1.6, 4.6 Hz, 2H), 7.38−7.23
C H ClF N O 526.11, found [M + H] 527.2. H NMR (400 MHz,
2
5
18
3
6
2
13
(
m, 6H), 5.18 (s, 2H). C NMR (100 MHz, CDCl ) δ 171.9, 157.7,
3
CD CN) δ 8.45 (dd, 2H, J = 1.8, 4.4 Hz), 7.79 (d, 1H, J = 7.9 Hz),
3
1
50.4, 137.9, 137.6, 129.7, 128.8, 128.4, 124.3, 123.6, 114.6, 70.6.
-(4-Chlorophenyl)-5-(pyridin-4-yl)pyridazine-3,6-diol (20).
7
2
2
1
.59 (d, 1H, J = 7.9 Hz), 7.34 (dd, 2H, J = 2.6, 8.4), 7.22 (dd, 2H, J =
4
.7, 8.3), 7.11 (dd, 2H, J = 1.8, 4.4 Hz), 5.79 (s, 2H), 3.39 (s, 3H),
.65 (s, 3H). 13C NMR (100 MHz, CD CN) δ 149.6, 136.1, 135.5,
Compound 18 (3 g, 11 mmol) was dissolved in 550 mL of THF,
and potassium tert-butoxide (2.5 g, 28 mmol, 2 equiv) was added. Air
was bubbled through the solution with vigorous stirring, and the
reaction mixture turned from red to dark-green within a few minutes.
When starting material had disappeared, as monitored by HPLC, the
reaction was quenched via addition of excess (>3 equiv) 1N HCl. The
mixture was stirred for 1 h and evaporated to give 19 as a dark-green
oil. The residue was dissolved in 50 mL of ethanol, and 0.35 mL of
hydrazine (11 mmol, 1 equiv) was added, followed by 3 mL of conc
HCl. The reaction mixture was heated to 72 °C for 18 h and, after
cooling, the mixture was evaporated to give 4.9 g of 20 as a red foam
that was used as-is in the next step.
3
31.9, 128.7, 125.6, 45.1, 33.1, 21.8.
8
-(4-Chlorophenyl)-2-ethyl-7-(pyridin-4-yl)-5-((6-
trifluoromethyl)pyridin-3-yl)methyl)-[1,2,4]triazolo[4,3-b]-
pyridazine-3,6(2H,5H)-dione (14). A solution of 5-(chloromethyl)-
-(trifluoromethyl)pyridine (32 mg, 0.16 mmol) and NaI (27 mg, 0.18
(
2
mmol) in DMF (0.5 mL) was stirred at 50 °C for 20 min. The mixture
was transferred into a solution of 8-(4-chlorophenyl)-2-ethyl-7-
pyridin-4-yl)-[1,2,4]triazolo[4,3-b]pyridazine-3,6(2H,5H)-dione (70
(
mg, 0.15 mmol) and LiOH (4 mg, 0.2 mmol) in DMF (0.5 mL).
The reaction mixture was heated at 70 °C for 30 min, and then it was
cooled to room temperature and diluted with ethyl acetate. The
resulting organic layer was extracted with water, and the organic layer
was dried (Na SO ), filtered, and concentrated under reduced
pressure. The crude product was purified by reverse phase HPLC
and silica gel chromatography. The product-containing fractions were
evaporated and dissolved in ethyl acetate. The organic layer was
extracted with sodium bicarbonate, and then it was dried (Na SO ),
6-Chloro-5-(4-chlorophenyl)-4-(pyridin-4-yl)pyridazin-3(2H)-
one (21, As a Mixture of Isomers). To a solution of compound 9
(11 g, 32.7 mmol) in 100 mL of acetonitrile was added 100 mL of 1N
NaOH (aq). The mixture was heated at 65 °C for 2 h, and the reaction
was quenched via addition of 1N HCl to pH 6. A precipitate formed
that was collected and suction dried to give 7.8 g (75%) of a beige
solid as a 8:1 mixture of regioisomers. Analytical HPLC (4 min
2
4
2
4
filtered, and evaporated to give 6.6 mg (6.6%) of compound 14 as a
yellow foam. Analytical HPLC (4 min gradient) retention time = 2.96
min (98.0% HI). LC-MS anal. calcd for C H ClF N O : 526.1,
1
gradient) retention time = 2.07 min (88.3% HI). H NMR (400 MHz,
CD CN) δ major isomer: 8.60 (d, J = 6.8 Hz, 2H), 7.78 (d, J = 6.8 Hz,
25
18
3
6
2
3
1
found [M + H] 527.2. H NMR (500 MHz, CDCl ) δ ppm 9.01 (s,
2H), 7.58−7.36 (m, 2H), 7.22−7.17 (m, 2H), 2.05 (br s, 1H); minor
isomer: 8.70 (d, J = 6.8 Hz, 2H), 7.87 (d, J = 6.8 Hz, 2H), 7.36−7.26
(m, 2H), 7.14−7.12 (m, 2H), 2.05 (br s, 1H).
6 - C h l o r o - 5 - ( 4 - c h l o r o p h e n y l ) - 2 - ( ( 2 - m e t h y l - 6 -
(trifluoromethyl)pyridin-3-yl)methyl)-4-(pyridin-4-yl)-
pyridazin-3(2H)-one (23). To a solution of compound 21 (7.6 g, 24
3
1
H), 8.82 (d, J = 4.9 Hz, 2H), 8.21 (d, J = 7.7 Hz, 1H), 7.76 (d, J = 8.2
Hz, 1H), 7.65 (d, J = 4.4 Hz, 2H), 7.38 (d, J = 7.7 Hz, 2H), 7.17 (d, J
8.2 Hz, 2H), 6.07 (s, 2H), 4.25−3.82 (m, 2H), 1.38 (t, J = 7.4 Hz,
H).
-(Chloromethyl)-2-methyl-6-(trifluoromethyl)pyridine (22).
=
3
3
A solution of 1 M LAH in THF (40 mL, 40 mmol) was added
mmol) in 100 mL of DMF was added K CO , and the mixture was
2 3
dropwise to a cooled (0 °C) solution of methyl 2-methyl-6-
heated to 65 °C. To the heated mixture was added a solution of
compound 22 (6.0 g, 29 mmol) in DMF (10 mL) in three portions
over 2 h. After 45 min, the mixture was poured into 800 mL of water
and 500 mL of EtOAc was added. The layers were extracted, and the
organic layer was successively washed with 500 mL each of water and
31
(
trifluoromethyl)nicotinate 25 (8.8 g, 40 mmol) in 40 mL of dry
THF at 0 °C under argon atmosphere. The solution was warmed to
room temperature over 1 h. The reaction was slowly quenched with
1
0% aq Rochelle’s salt solution (20 mL) over 10 min, followed by
stirring for 1 h. The resulting mixture was diluted with 50 mL of
EtOAc and water, and the layers were extracted. The organic phase
was dried (Na SO ), filtered, and concentrated to obtain 7.5 g of (2-
brine. The organic layer was dried (Na SO ), filtered, and evaporated
2 4
to give a red oil. The residue was purified via silica gel chromatography
to give 7.8 g (66%) of 23 as beige crystals (the minor regioisomer is
2
4
methyl-6-(trifluoromethyl)pyridin-3-yl)methanol as a colorless oil,
which was greater than 98% purity by HPLC/MS. To a solution of (2-
methyl-6-(trifluoromethyl)pyridin-3-yl)methanol (14.5 g, 71 mmol) in
removed during silica purification). Analytical HPLC (4 min gradient)
1
retention time = 3.37 min (100.0% HI). H NMR (400 MHz, CDCl
)
3
δ 8.57−8.43 (m, 2H), 7.96 (d, J = 7.9 Hz, 1H), 7.54 (d, J = 7.9 Hz,
1H), 7.35−7.19 (m, 2H), 7.07−6.91 (m, 4H), 5.45 (s, 2H), 2.83 (s,
3H).
1
00 mL of CH Cl at room temperature under argon atmosphere was
2 2
added SOCl (16.8 g, 142 mmol) and 1.5 mL of DMF. The reaction
2
K
dx.doi.org/10.1021/jm4010835 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
8
-(4-Chlorophenyl)-5-((2-methyl-6-(trifluoromethyl)pyridin-
6.99 (d, J = 7.1 Hz, 1H), 5.93 (s, 2H), 3.92 (q, J = 7.3 Hz, 2H), 2.74
(s, 3H), 2.47 (s, 3H), 1.33 (t, J = 7.2 Hz, 3H).
3
3
-yl)methyl)-7-(pyridin-4-yl)-[1,2,4]triazolo[4,3-b]pyridazine-
,6(2H,5H)-dione (24). To a solution of compound 23 (6.6 g, 13
4
- ( 8 - ( 4 - C h l o r o p h e n y l ) - 2 - e t h y l - 5 - ( ( 2 - e t h y l - 6 -
mmol) in pyridine (25 mL) was added 6 mL of hydrazine (130
mmol), and the mixture was heated in a microwave at 200 °C for 30
min. The reaction mixture was evaporated to give a light-yellow foam
that was dissolved in THF (140 mL). To this solution was added 1,1′-
carbonyldiimidazole (2.2 g, 13 mmol), and the solution was heated to
5 °C for 1 h. The crude reaction mixture was purified over silica gel
to give 5.0 g of compound 24 as an orange foam (73% from 23).
Analytical HPLC (4 min gradient) retention time = 2.37 min (100.0%
(trifluoromethyl)pyridin-3-yl)methyl)-3,6-dioxo-2,3,5,6-tetra-
32
hydro-[1,2,4]triazolo[4,3-b]pyridazin-7-yl)benzonitrile (30).
Compound 30 was isolated in 47% yield as a light-yellow solid.
Analytical HPLC (4 min gradient) retention time = 3.82 min (98.6%
HI). LCMS anal. calcd C H ClF N O : 578.14, found [M + H] =
2
9
22
3
6
2
1
6
579. H NMR (400 MHz, CDCl ) δ 7.61 (d, J = 7.91 Hz, 1H), 7.57
3
(d, J = 8.79 Hz, 2H), 7.49 (d, J = 7.91 Hz, 1H), 7.31 (d, J = 8.79 Hz,
2H), 7.23−7.28 (m, 2H), 7.18 (d, J = 8.79 Hz, 2H), 5.98 (s, 2H), 3.93
(q, J = 7.18 Hz, 2H), 3.03 (q, J = 7.47 Hz, 2H), 1.35 (td, J = 7.31,
12.63 Hz, 6H).
1
HI). H NMR (400 MHz, CD CN) δ 8.56−8.40 (m, 2H), 7.85 (d, J =
3
7
2
.8 Hz, 1H), 7.69−7.49 (m, 1H), 7.43−7.32 (m, 2H), 7.32−7.21 (m,
H), 7.20−7.09 (m, 2H), 7.04 (s, 1H), 5.83 (s, 2H), 2.69 (s, 3H).
4-(8-(4-Chlorophenyl)-2-isopropyl-5-((2-methyl-6-
8
-(4-Chlorophenyl)-2-ethyl-5-((2-methyl-6-(trifluoromethyl)-
(trifluoromethyl)pyridin-3-yl)methyl)-3,6-dioxo-2,3,5,6-tetra-
32
pyridin-3-yl)methyl)-7-(pyridin-4-yl)-[1,2,4]triazolo[4,3-b]-
hydro-[1,2,4]triazolo[4,3-b]pyridazin-7-yl)benzonitrile (31).
pyridazine-3,6(2H,5H)-dione (15). To a mixture of compound 24
Compound 31 was isolated in 66% yield as a light-yellow solid.
Analytical HPLC (4 min gradient) retention time = 3.81 min (95.6%
HI). LCMS anal. calcd C H ClF N O : 578.14, found [M + H] =
(
5.0 g, 9.8 mmol) and K CO (1.6 g, 12 mmol) in DMF (50 mL) was
2 3
added a solution of ethyl iodide (0.8 mL) in DMF (3 mL), and the
mixture was stirred at room temperature for 3 h. The reaction mixture
was diluted with 1 L of water, and the resulting milky solution was
extracted with 2 × 500 mL of EtOAc. The combined organic layers
were washed successively with water, 10% Na S O , and brine. The
organic layer was dried (Na SO ), filtered, and evaporated to give 4.0 g
of a brown foam. The material was purified via silica gel
chromatography to give 3.6 g of compound 15 as a yellow foam
67%). Recrystallization of 1.4 g of this material provided 1.2 g (90%
2
9
22
3
6
2
1
579. H NMR (400 MHz, CDCl ) δ 7.66 (d, J = 8.35 Hz, 1H), 7.57
3
(d, J = 8.35 Hz, 2H), 7.51 (d, J = 8.35 Hz, 1H), 7.30 (d, J = 8.79 Hz,
2H), 7.23−7.27 (m, 2H), 7.13−7.21 (m, 2H), 5.93 (s, 2H), 4.53
(quin, J = 6.70 Hz, 1H), 2.74 (s, 3H), 1.35 (d, J = 7.03 Hz, 6H).
4-(8-(4-Chlorophenyl)-5-((2-methyl-6-(trifluoromethyl)-
pyridin-3-yl)methyl)-3,6-dioxo-2-propyl-2,3,5,6-tetrahydro-
2
2
3
2
4
3
2
[1,2,4]triazolo[4,3-b]pyridazin-7-yl)benzonitrile (32).
Com-
(
pound 32 was isolated in 45% yield as a light-yellow solid. Analytical
recovery) of off-white crystals. Analytical HPLC (4 min gradient)
retention time = 2.89 min (100.0% HI). HRMS anal. calcd for
C H N O ClF [M + H]: 541.1370, found: 541.1361. H NMR (600
6
HPLC (4 min gradient) retention time = 3.82 min (100% HI). LCMS
1
anal. calcd C29
H22ClF
N
O
2
: 578.14, found [M + H] = 579. H NMR
3
6
1
(400 MHz, CDCl
) δ 7.64 (d, J = 7.91 Hz, 1H), 7.57 (d, J = 8.35 Hz,
2
21
6
2
3
3
MHz, DMSO-d ) δ ppm 8.49 (d, 2H, J = 4.9 Hz), 7.95 (d, 1H, J = 8.1
Hz), 7.73 (d, 1H, J = 8.1 Hz), 7.44 (d, 2H, J = 8.5 Hz), 7.30 (d, 2H, J
2H), 7.50 (d, J = 7.91 Hz, 1H), 7.31 (d, J = 8.79 Hz, 2H), 7.24−7.28
(m, 2H), 7.17 (d, J = 8.35 Hz, 2H), 5.94 (s, 2H), 3.78−3.87 (m, 2H),
2.74 (s, 3H), 1.76 (sxt, J = 7.38 Hz, 2H), 0.91 (t, J = 7.47 Hz, 3H).
4-(8-(4-Chlorophenyl)-2-ethyl-5-(4-methylbenzyl)-3,6-
dioxo-2,3,5,6-tetrahydro-[1,2,4]triazolo[4,3-b]pyridazin-7-yl)-
6
=
8.5 Hz), 7.16 (d, 2H, J = 4.9 Hz), 5.72 (s, 2H), 3.80 (q, 2H, J = 7.2
13
Hz), 2.64 (s, 3H), 1.18 (t, 3H, J = 7.2 Hz). C NMR (150 MHz,
DMSO-d ) δ ppm 158.5, 156.6, 149.6, 146.5, 144.5 (q, J = 33.9 Hz),
6
3
2
benzonitrile (33). Compound 33 (40 mg, 100%) was isolated as a
yellow solid. Analytical HPLC (4 min gradient) retention time = 3.90
min (97.5% HI). LCMS anal. calcd for C H ClN O : 495.1, found
1
1
2
41.5, 141.6, 136.2, 135.8, 135.6, 135.5, 134.7, 132.2, 131.9, 129.7,
28.9, 125.8, 122.2 (q, J = 274 Hz), 118.7 (q, J = 2 Hz), 45.7, 41.3,
2.3, 14.1. 19F NMR (376.46 MHz, DMSO-d ) δ ppm −66.68.
28 22
5
2
6
1
[
2
4
(
M + H] 496.4. H NMR (400 MHz, CD CN) δ ppm 7.64−7.57 (m,
Elemental Anal. Calcd for C H N O Cl F : C 57.71, H 3.76, N
3
26
20
6
2
3
H), 7.34 (d, J = 7.9 Hz, 2H), 7.32−7.26 (m, 4H), 7.21−7.13 (m,
H), 5.80 (s, 2H), 3.84 (q, J = 7.2 Hz, 2H), 2.30 (s, 3H), 1.29−1.19
1
1
5.48, Cl 6.53, F 10.50. Found: C 57.73, H 3.69, N 15.53, Cl 6.60, F
1.12.
8
m, 3H).
-(4-Chlorophenyl)-2-ethyl-5-((2-methyl-6-(trifluoromethyl)-
pyridin-3-yl)methyl)-7-(2-methylpyridin-4-yl)-[1,2,4]triazolo-
4,3-b]pyridazine-3,6(2H,5H)-dione (27). Compound 27 was
4-(2-Ethyl-5-((2-methyl-6-(trifluoromethyl)pyridin-3-yl)-
[
methyl)-3,6-dioxo-8-(p-tolyl)-2,3,5,6-tetrahydro-[1,2,4]-
32
triazolo[4,3-b]pyridazin-7-yl)benzonitrile (34). Compound 34
was isolated in 50% yield as a light-yellow solid. Analytical HPLC (4
min gradient) retention time = 3.64 min (96.7% HI). LCMS anal.
synthesized according to the method of compound 15. Analytical
HPLC (4 min gradient) retention time = 2.462 min (98% HI), LC-MS
1
anal. calcd for C H ClF N O : 554.1, found [M + H] = 555.2. H
27
22
3
6
2
1
calcd for C H F N O : 544.18, found [M + H] 545. H NMR (400
NMR (500 MHz, CDCl ) δ 8.34 (d, J = 5.5 Hz, 1H), 7.64−7.42 (m,
29 23
3
6
2
3
MHz, CDCl ) δ 7.65 (d, J = 7.91 Hz, 1H), 7.54 (d, J = 8.35 Hz, 2H),
2
H), 7.24 (d, J = 8.2 Hz, 2H), 7.13 (d, J = 8.8 Hz, 2H), 6.91 (s, 1H),
.74 (d, J = 4.9 Hz, 1H), 5.86 (s, 2H), 3.85 (q, J = 7.1 Hz, 2H), 2.67
3
7
2
7
.51 (d, J = 7.91 Hz, 1H), 7.20−7.31 (m, 2H), 7.12 (s, 4H), 5.93 (s,
H), 3.92 (q, J = 7.03 Hz, 2H), 2.74 (s, 3H), 2.35 (s, 3H), 1.33 (t, J =
.03 Hz, 3H).
6
(
s, 3H), 2.44 (s, 3H), 1.26 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz,
CDCl ) δ 158.5, 158.1, 157.0, 148.9, 146.5, 146.3, 136.5, 136.1, 135.2,
3
4
-(8-(4-Chlorophenyl)-5-(4-cyanobenzyl)-2-ethyl-3,6-dioxo-
1
35.0, 133.0, 131.3, 128.9, 128.0, 124.8, 122.3, 118.0, 45.0, 41.8, 24.4,
2
,3,5,6-tetrahydro-[1,2,4]triazolo[4,3-b]pyridazin-7-yl)-
2
2.4, 13.8.
32
benzonitrile (35). Compound 35 (70 mg, 52%) was isolated as a
4
-(8-(4-Chlorophenyl)-2- ethyl-5-((2-methyl-6-
yellow solid. Analytical HPLC (4 min gradient) retention time = 3.55
(
trifluoromethyl)pyridin-3-yl)methyl)-3,6-dioxo-2,3,5,6-tetra-
32
min (98.3% HI). LC-MS anal. calcd for C H ClN O : 506.1, found
hydro-[1,2,4]triazolo[4,3-b]pyridazin-7-yl)benzonitrile (28).
28 19
6
2
1
[
2
M + H] 507.2. H NMR (400 MHz, CD CN) δ ppm 7.79−7.69 (m,
Compound 28 (35 mg, 80% yield) was isolated as a light-yellow
solid. Analytical HPLC (4 min gradient) retention time = 3.67 min
3
H), 7.65−7.56 (m, 4H), 7.36−7.26 (m, 4H), 7.24−7.15 (m, 2H),
(
98.7% HI). LCMS anal. calcd C H ClF N O : 564.13, found [M +
5.85 (s, 2H), 3.84 (q, J = 7.3 Hz, 2H), 1.31−1.19 (m, 3H).
8-(4-Chlorophenyl)-2-ethyl-7-(6-(hydroxymethyl)pyridin-3-
yl)-5-((2-methyl-6-(trifluoromethyl)pyridin-3-yl)methyl)-
28
20
3
6
2
1
H] = 565. H NMR (400 MHz, CDCl ) δ 7.65 (d, J = 7.91 Hz, 1H),
3
7
.57 (d, J = 8.35 Hz, 2H), 7.51 (d, J = 7.91 Hz, 1H), 7.31 (d, J = 8.35
Hz, 2H), 7.22−7.28 (m, 2H), 7.18 (d, J = 8.79 Hz, 2H), 5.93 (s, 2H),
.92 (q, J = 7.18 Hz, 2H), 2.74 (s, 3H), 1.34 (t, J = 7.25 Hz, 3H).
-(8-(4-Chlorophenyl)-2- ethyl-5-((2-methyl-6-
trifluoromethyl)pyridin-3-yl)methyl)-3,6-dioxo-2,3,5,6-tetra-
[
1,2,4]triazolo[4,3-b]pyridazine-3,6(2H,5H)-dione (36). To a
stirred solution of 8-(4-chlorophenyl)-2-ethyl-5-((2-methyl-6-
trifluoromethyl)pyridin-3-yl)methyl)-7-(6-methylpyridin-3-yl)-
3
(
[
4
3
2
1,2,4]triazolo[4,3-b]pyridazine-3,6(2H,5H)-dione (140 mg, 0.24
(
hydro-[1,2,4]triazolo[4,3-b]pyridazin-7-yl)-2-methylbenzoni-
mmol) in CH Cl (2 mL) was added mCPBA (65 mg of 77%
2
2
3
2
trile (29). Analytical HPLC (4 min gradient) retention time = 3.79
mCPBA, 0.29 mmol) at room temperature. After stirring for 1 h, the
solution was diluted with ethyl acetate and the organic layer was
min (98.7% HI). LCMS anal. calcd C H ClF N O : 578.14, found
2
9
22
3
6
2
1
[
M + H] = 579. H NMR (400 MHz, CDCl ) δ ppm 7.63 (d, J = 8.1
extracted with satd aq NaHCO . The organic layer was separated and
3
3
Hz, 1H), 7.58−7.42 (m, 2H), 7.38−7.28 (m, 2H), 7.22−7.11 (m, 3H),
dried (MgSO ). The dried organic layer was filtered and concentrated
4
L
dx.doi.org/10.1021/jm4010835 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
to give a colorless foam that was dissolved in CH Cl (2 mL), and
155.6, 155.4, 151.8, 146.7, 144.5, 137.4, 133.1, 118.0, 81.8, 60.7, 51.1,
28.2, 28.0, 22.4.
tert-Butyl 2-(4-(4-Chlorophenyl)-5-methoxy-1-((2-methyl-6-
(trifluoromethyl)pyridin-3-yl)methyl)-6-oxo-1,6-dihydropyri-
dazin-3-yl)hydrazinecarboxylate (42). To a solution of compound
2
2
trifluoroacetic anhydride (2 mL) was added. The vessel was sealed and
heated to 50 °C for 2 h. The reaction mixture was cooled to room
temperature and was evaporated to an oil. The residue was purified via
preparative RP-HPLC; product-containing fractions were evaporated
and redissolved in CH Cl and water that was adjusted to pH 8 with
4
1 (1.5 g, 3.23 mmol) in 16 mL of toluene was added 0.76 g of 4-
chlorophenyl boronic acid (4.9 mmol) and 1.9 mL of 2 M Na CO
3
2
2
solid NaHCO . The layers were extracted and the organic layer was
2
3
(3.9 mmol). The mixture was degassed via vacuum/Ar purge sequence
dried (MgSO ), filtered, and evaporated to give 56 mg (36%) of
4
three times, and Pd(PPh ) (0.19 g, 0.16 mmol) was added. The
compound 36 as a yellow solid. Analytical HPLC (8 min gradient)
3 4
resulting mixture was heated at 110 C for 85 min. After cooling to
room temperature, the mixture was diluted with 300 mL each of ethyl
acetate/water, and the layers were extracted. The organic layer was
retention time = 4.0 min (100% HI). anal. calcd for C H ClF N O
27
22
3
6
3
1
5
1
70.14, found [M + H] 571.5. H NMR (400 MHz, CDCl ) δ 8.26 (d,
H, J = 2.2 Hz), 7.64 (d, 1H, J = 7.7), 7.55 (dd, 1H, J = 2.2, 8.2), 7.50
3
washed with brine, then it was dried (MgSO ), filtered, and
(
(
d, 1H, J = 7.7), 7.33−7.20 (m, 6H), 5.94 (s, 2H), 4.73 (s, 2H), 3.93
4
13
evaporated. The residue was purified via silica gel chromatography
to give 1.8 g of compound 42 as a beige solid. Analytical HPLC (4 min
q, 2H, J = 7.1), 2.74 (s, 3H), 1.33 (t, 3H, J = 7.1 Hz). C NMR (100
MHz, CDCl ) δ 159.4, 158.6, 156.9, 149.9, 146.4, 139.1, 136.4, 135.9,
3
1
6
35.3, 135.0, 133.0, 131.5, 130.2, 129.0, 128.2, 127.1, 119.6, 118.0,
4.0, 53.4, 45.0, 41.8, 22.3, 13.7.
gradient) retention time = 1.90 min (94.1% HI). LCMS anal. calcd for
1
C
H25ClF
N
3
O
4
539, found [M + H] 540. H NMR (400 MHz,
24
5
3
3
6-Bromo-4,5-dichloropyridazin-3(2H)-one (38). To a sol-
CDCl ) δ 7.73 (d, 8 Hz, 1H), 7.49−7.46 (m, 4H), 7.33 (d, 7.4 Hz,
3
ution of 20 g of 4,5-dichloropyridazin-3(2H)-one (121 mmol) in
methanol at 70 °C was added 5 g of LiOH (121 mmol), followed by
slow portionwise addition of bromine (14 mL, 270 mmol). To the
heated solution was added 3 g of LiOH until the solution became
light-red/orange. The volatile components were removed in vacuo,
and the resulting slurry was suspended in 150 mL of water. The solids
were collected via filtration, and the filter cake was washed with water
and suction dried to give 30 g of a beige solid that was used without
further purification.
1H), 6.1 (br s, 1H), 5.5 (d, 4.1 Hz, 1H), 5.3 (s, 2H), 4.0 (s, 3H), 2.8
(s, 3H), 1.48−1.39 (m, 9H).
8-(4-Chlorophenyl)-7-methoxy-5-((2-methyl-6-
(
trifluoromethyl)pyridin-3-yl)methyl)-[1,2,4]triazolo[4,3-b]-
pyridazine-3,6(2H,5H)-dione (43). Compound 42 (1.8 g, 3.3
mmol) was dissolved in 16 mL of CH Cl , and 2 mL of TFA was
2
2
added. After stirring at room temperature for 4.5 h, the mixture was
evaporated to dryness to give an orange oil. The residue was dissolved
in 100 mL of CH Cl , and the mixture was extracted successively with
2
2
1
N aq NaOH and brine. The organic layer was dried (MgSO4),
6
-Bromo-4,5-dichloro-2-((2-methyl-6-(trifluoromethyl)-
pyridin-3-yl)methyl)pyridazin-3(2H)-one (39). To a heated (70
C) solution of compound 38 (17 g, 69 mmol) in 100 mL of DMF
containing 19 g of K CO (140 mmol) was added a solution of
filtered, and evaporated to a yellow foam. The solids were dissolved in
6 mL of CH Cl , and triphosgene (3 g, 10 mmol) was added. The
1
°
2
2
slurry was further diluted with 1 mL of CH Cl to re-establish stirring,
2
2
2
3
and 0.3 g of triphosgene was added. After 2 h, the mixture was diluted
with CH Cl and water, and the layers were extracted. The organic
compound 22 (14 g, 69 mmol) in DMF (100 mL). The solution was
heated for 2 h, and the mixture was cooled to room temperature. The
resulting solution was diluted into 1.5 L of water. The mixture was
extracted with 2 × 600 mL of ethyl acetate. The combined organic
2
2
layer was washed with brine, and then it was dried (MgSO ), filtered,
4
1
and evaporated to give 1.2 g of compound 42 as a white solid. H
NMR (400 MHz, CD CN) δ 10.2 (s, 1H), 7.75 (d, 8.0 Hz, 1H),
layers were washed with brine, and then it was dried (MgSO ), filtered,
3
4
1
3
and evaporated to give 25 g of a red solid that was used without further
purification.
7.65−7.54 (m, 5H), 5.78 (s, 2H), 3.89 (s, 3H), 2.69 (s, 3H). C NMR
(125 MHz, CD CN) δ 162.1, 161.9, 153.4, 140.3, 136.9, 133.7, 132.6,
3
6
- B r o m o - 5 - c h l o r o - 4- m e t h o x y - 2 - ( ( 2 - m e t h yl - 6-
128.5, 66.1, 50.2, 26.8.
(
trifluoromethyl)pyridin-3-yl)methyl)pyridazin-3(2H)-one (40).
8-(4-Chlorophenyl)-2-ethyl-5-((2-methyl-6-(trifluoromethyl)-
pyridin-3-yl)methyl)-7-(2-methylpyrimidin-5-yl)-[1,2,4]-
triazolo[4,3-b]pyridazine-3,6(2H,5H)-dione (46). To a solution of
The residue (25 g of crude compound 39) was dissolved in NaOMe/
MeOH (6.5 g of a 25% w/w solution, 30 mmol) at room temperature.
After 15 min, the mixture was diluted into 500 mL of EtOAc/water.
The layers were extracted, and the organic layer was washed with
compound 43 (1.0 g 2 mmol) in 2 mL of CH Cl at −40 °C was
2
2
added 2 mL of 1N BBr in CH Cl (2 mmol). After 15 min, the
3
2
2
mixture was diluted with 100 mL of CH Cl and the mixture was
brine. The organic layer was separated and dried (MgSO ), filtered,
2
2
4
extracted with an aq soln of Rochelle’s salt. The layers were extracted,
and the organic layer was washed with brine. The organic layer was
and evaporated to give a red oil that was purified via silica gel
chromatography to give 4.0 g (35%) compound 40 as a white solid.
Analytical HPLC (4 min gradient) retention time = 3.66 min (97.6%
HI). LCMS anal. calcd for C H BrClF N O : 411, 413, 415, found
dried (MgSO ), and then it was filtered and evaporated to give 0.62 g
4
of a brown oil. The residue was dissolved in 6.5 mL of CH Cl at 0 °C,
2
2
1
3
10
3
3
2
1
and to this solution was added triethylamine (0.36 mL, 2.6 mmol) and
triflic anhydride (0.33 mL, 1.9 mmol). After 15 min, the mixture was
diluted into 50 mL of CH Cl and 50 mL of 0.1 N aq HCl. The layers
[
M + H] = 412, 414, 416. H NMR (400 MHz, CDCl ) δ 7.7 (d, 8.0
3
Hz, 1H), 7.5 (d, 8.0 Hz, 1H), 5.3 (s, 2H), 4.31 (s, 3H), 2.75 (s, 3H).
1
3
C NMR (100 MHz, CDCl ) δ 158.2, 155.8, 152.1, 147.3, 138.0,
2
2
3
were extracted, and the organic layer was washed with brine. The
1
2
32.0, 129.8, 125.7, 123.0 (app q, CF ), 118.1 (d, 2.8 Hz), 61.1, 52.1,
2.5.
3
organic layer was dried (MgSO ), and it was filtered and evaporated to
4
give 0.7 g of compound 45 as a red oil that was used as-is in the next
step. Compound 45 (0.15 g, 0.24 mmol), 2-methylpyrimidine-5-
tert-Butyl 2-(4-Chloro-5-methoxy-1-((2-methyl-6-
trifluoromethyl)pyridin-3-yl)methyl)-6-oxo-1,6-dihydropyri-
(
dazin-3-yl)hydrazinecarboxylate (41). To an argon flushed vessel
containing 3.7 g of compound 40 (9 mmol), boc-hydrazine (4.7 g, 36
mmol), and cesium carbonate (3.5 g, 11 mmol) in toluene (45 mL)
boronic acid (0.05 g, 0.36 mmol), Pd(dppf)Cl
2
−CH
2 2
Cl complex
(0.04 g, 0.05 mmol), and K PO (0.16 g, 0.73 mmol) were placed in a
3
4
flask that was then evacuated and purged with argon three times. To
the vessel was added 1.2 mL of THF, and the mixture was heated at 80
°C for 3 h. The mixture was allowed to stand at room temperature
overnight, followed by dilution with 50 mL each of ethyl acetate and
water. The layers were extracted, and the organic layer was washed
was added Pd(dppf)Cl -methylene chloride complex (0.37 g, 0.45
2
mmol), and the mixture was heated at reflux for 4 h. The cooled
mixture was diluted with 200 mL each of ethyl acetate and water. The
layers were extracted, and the organic layer was washed successively
with 1N HCl and brine. The organic layer was dried (MgSO ), and the
with brine. The organic layer was dried (MgSO ), and then it was
4
4
mixture was filtered and evaporated to give a red oil that was purified
via silica gel chromatography to give 3.2 g (77%) of compound 41 as
filtered and evaporated to give a red oil. The residue was purified via
silica gel chromatography (using silica that had been pretreated with
2% triethylamine/hexanes) to give 0.03 g of the title compound as a
light-yellow solid. Analytical HPLC (4 min gradient) retention time =
3.42 min (100% HI). LCMS Anal. calcd for C H N O F Cl 555.14,
an orange foam. Analytical HPLC (4 min gradient) retention time =
1
3
1
2
.21 min (91.3% HI). H NMR (500 MHz, CDCl ) δ 7.6 (d, 7.7 Hz,
3
H), 7.44 (d, 8.0 Hz, 1H), 6.47 (br s, 1H), 5.24 (s, 2H), 4.29 (s, 3H),
26
21
7
2 3
.74 (s, 3H), 1.48 (m, 9H). 13C NMR (125 MHz, CDCl ) δ 157.8,
found 556.2 [M + H]. H NMR (400 MHz, DMSO-d ) δ ppm 8.45 (s,
1
3
6
M
dx.doi.org/10.1021/jm4010835 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2
H), 7.94 (d, 1H, J = 8.0 Hz), 7.72 (d, 1H, J = 8.0 Hz), 7.50 (d, 2H, J
8.8 Hz), 7.37 (d, 2H, J = 8.6 Hz), 5.75 (s, 2H), 3.81 (q, 2H, J = 7.3
ABBREVIATIONS USED
■
=
13
CB1, cannabinoid receptor 1; hCB1, human CB1 receptor;
GPCR, G-protein coupled receptor; 2-AG, 2-arachidonoylgly-
cerol; PK, pharmacokinetic; HTS, high-throughput screening;
TZP, triazolopyridazine; TZP-dione, triazolopyridazin-dione;
MED, minimally efficacious dose; Cl, clearance; CYP,
cytochrome P450
Hz), 2.65 (s, 3H), 2.57 (s, 3H), 1.18 (t, 3H, J = 7.2 Hz). C NMR
(
100 MHz, CDCl ) δ ppm 166.4, 158.3, 157.9, 156.1, 145.9, 144.0 (q,
3
J = 33.6 Hz), 135.6, 135.4, 135.1, 134.8, 134.3, 131.9, 129.1, 128.5,
1
2
3
3
6
27.9, 124.3, 121.6 (q, J = 273.6), 118.2 (d, J = 3.1 Hz), 45.2, 40.8,
5.3, 21.7, 13.5. 19F NMR (471 MHz, DMSO-d ) δ ppm −66.10 (br s,
6
F). Elemental analysis: Anal. calcd for C H N O F Cl C 56.17, H
26
21
7
2 3
.81, N 17.64, Cl 6.38, F 10.25. Found: C 56.01, H 4.01, N 17.24, Cl
.16, F 10.25.
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■
In Vitro CB1 Binding Assay. The source of CB1 receptor was a
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5
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d
i
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3
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d
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values obtained with both radioligands are similar to those reported in
34
the literature.
ASSOCIATED CONTENT
Supporting Information
Experimental procedures for the synthesis of compounds 47−
9 and S1−S6, methods for in vitro and in vivo biological
■
*
S
(8) Rimonabant: Acomplia EPAR Product Information; European
4
Agency for The Evaluation of Medicinal Products: London, 2009; http://
www.ema.europa.eu/ema/index.jsp?curl=pages/medicines/human/
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WC0b01ac058001d124.
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This material is available free of charge via the Internet at
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AUTHOR INFORMATION
Corresponding Author
Phone: +1-609-818-4965. Fax: +1-609-818-3550. E-mail:
Bruce.Ellsworth@BMS.com.
■
*
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The authors declare no competing financial interest.
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preparation of this manuscript.
■
N
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O
dx.doi.org/10.1021/jm4010835 | J. Med. Chem. XXXX, XXX, XXX−XXX