A new and efficient synthetic route for the anxiolytic agent CL285032

Article abstract of DOI:10.1016/j.bmcl.2010.11.017

CL285032 is an anxiolytic compound currently under investigation as a possible treatment for canine noise phobia associated anxiety. A robust scale-up and manufacturing process is essential for the development and marketability of the drug. The current sy

Full text of DOI:10.1016/j.bmcl.2010.11.017

Bioorganic & Medicinal Chemistry Letters 21 (2011) 259–261
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A new and efficient synthetic route for the anxiolytic agent CL285032
Victor P. Ghidu a,b, Marc A. Ilies a,b, Tom Cullen c, Robert Pollet c, Magid Abou-Gharbia a,b,
⇑
a Moulder Center for Drug Discovery Research, Temple University School of Pharmacy, 3307 N Broad Street, Philadelphia, PA 19140, USA
b Department of Pharmaceutical Sciences, Temple University School of Pharmacy, 3307 N Broad Street, Philadelphia, PA 19140, USA
c Fort Dodge Animal Health, 9 Deer Park Drive, Monmouth Junction, NJ 08852, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
CL285032 is an anxiolytic compound currently under investigation as a possible treatment for canine
noise phobia associated anxiety. A robust scale-up and manufacturing process is essential for the devel-
opment and marketability of the drug. The current synthetic route, although reliable, requires seven steps
and has a low overall yield (18%), leaving opportunity for improvement. We are presenting an efficient
alternative approach toward the synthesis of CL285032 and the results thereof.
Received 4 October 2010
Revised 31 October 2010
Accepted 2 November 2010
Available online 5 November 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Triazolopyridazine synthesis
Anxiolytic agent
Noise phobia
Noise and thunderstorm phobias are among the most com-
monly recognized disorders associated with panic or phobic re-
sponses in companion animals such as dogs, cats or horses.
Thunderstorms, fireworks, gunfire, car backfire, etc. frequently in-
duce undesirable nonspecific clinical symptoms in companion ani-
mals, particularly dogs, such as salivating, defecating, urinating,
destroying, escaping, hiding, trembling, vocalizing and the like.
Known treatments for general anxiety behavior in companion ani-
mals generally involve either a long period of onset, that is,
3–4 weeks, or if quick-acting, cause sedation and/or ataxia. How-
ever, most companion animal owners and veterinarians would pre-
fer to treat their animals suffering from noise phobia with a drug
which does not promote sedation or ataxia and which is effective
within an hour or two of administration.1
Advanced investigations have shown that CL285032 possesses
central nervous system activity at nontoxic doses and as such is
useful as an anxiolytic agent. It produced certain responses in stan-
dard tests with laboratory animals which are known to correlate
well with relief of anxiety in man, dogs, cats and horses, being par-
ticularly effective in dogs.1 Therefore CL285032 was advanced for
development as an anxiolytic agent and provides a therapeutically
effective method for the treatment of noise phobia in a companion
animal without undesirable sedative side effect. A robust scale-up
and manufacturing process is essential for the development and
marketability of the drug. The current synthetic route, although
reliable, requires seven steps and has a low overall yield (18%),
leaving opportunity for improvement. We are presenting two
alternative approaches toward the synthesis of CL285032 and the
results thereof.
The reported synthesis of CL285032 and congeners,2,3 involves a
key substitution/cyclization synthetic step of the chloro-pyridazine
compounds of type A into [1,2,4]triazolo[4,3-b]pyridazines of type
B (Scheme 1 a). The method includes various substituents X, for
example, H, p-F, m-CF3, p-OCH3, etc., except for the one of interest
for the synthesis of CL285032, the N-methylated acetamide. The
only nitrogen containing functionality featured in the early reports
was a m-NO2 group, which for the purpose of CL285032 synthesis
was being reduced, then alkylated and acylated respectively, each
additional step adding to the overall decrease of the yield.
Later on, another method emerged in which the key step is an
intramolecular pyridazine ring closure, from an aza-diene interme-
diate of the type C (Scheme 1 b), that has the prerequisite triazole
⇑
Corresponding author. Tel.: +1 215 707 4949.
E-mail address: aboumag@temple.edu (M. Abou-Gharbia).
Scheme 1. Approaches to [1,2,4]triazolo[4,3-b]pyridazines.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.017

260
V. P. Ghidu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 259–261
Scheme 2. Initial approach toward CL285032.
Scheme 3. Improved synthesis of CL285032.
ring already in place.4 This method has eventually employed an
acetamide substituent X throughout the synthetic sequence, how-
ever, instead of a methyl group, a tert-butyl group was the N-
substituent.5
Our initial attempt at an improved synthesis of CL285032 con-
centrated on the first method (Scheme 1 a) and aimed at having
the required N-methylated acetamide in place from the very begin-
ning while maintaining the established four initial steps of the ori-
ginal sequence that affords the [1,2,4]triazolo[4,3-b]pyridazine.3
To this end, commercially available 30-acetyl-N-methylacetani-
lide 1 was reacted with glyoxylic acid in an acid mediated aldol
Condensation of commercially available 1 with 3-methyl-
4H-1,2,4-triazole-4-amine,6 afforded N-triazolo-imine 6 in near
quantitative yield. Subsequent treatment with tert-butoxy
bis(dimethylamino)methane affords the dimethylamino-aza-diene
7 in quantitative yield, which under standard acidic conditions
generates the [1,2,4]triazolo[4,3-b]pyridazine ring system of
CL285032 in 71% yield.7
Building on previous strategies for synthesizing CL285032 and
related triazolo-pyridazines, our efforts delivered an improved
synthetic sequence leading to CL285032. The overall yield was ele-
vated from the previously moderate 18% to a much improved 65%
and the number of synthetic steps of the process was cut in less
than half. Our strategy is straightforward and amenable to direct
scale-up of this valuable therapeutic agent.
condensation (Scheme 2). The resulting
a,b-unsaturated acid 2
was cyclized with hydrazine under acidic conditions to afford the
desired hydroxy-pyridazine 3 in fair yield. From this point onward
the anticipated sequence of reactions became, somehow expect-
edly, more difficult. Attempts to prepare chloro-pyridazine 4 have
ultimately failed, although milligram amounts of material could be
isolated for characterization purposes. We can only speculate that
the amide reacts with POCl3 to afford Vilsmeier-Haack like reactive
species which then capture the electron-rich hydroxy-pyridazine
ring. Circumventing this problem by preparing triflate 5 did not
provide the desired outcome as well. The attempted substitution/
condensation sequence in presence of acetyl hydrazine did not suc-
ceed, as the triflate hydrolyzed back to hydroxy-pyridazine 3, de-
spite our best efforts to keep the reaction mixture moisture-free.7
Consequently we undertook an alternative, more conservative
approach to access the [1,2,4]triazolo[4,3-b]pyridazine,4,5 by
installing the triazole ring into the molecular framework before
the pyridazine ring closure, which was designed as the last step
(Scheme 3).
Acknowledgments
This work was supported by Fort Dodge Animal Health and
Temple University School of Pharmacy.
References and notes
1. Enos, A.; Eppler, C. M.; Powell, D. W. PCT Int. Appl. 2006, WO 2006127574.
2. Albright, J. D.; Moran, D. B.; Wright, W. B., Jr.; Collins, J. B.; Beer, B.; Lippa, A. S.;
Greenblatt, E. N. J. Med. Chem. 1981, 24, 592.
3. Albright, J. D.; Powell, D. W.; Dusza, J. P. US Patent 4,654,343, 1987.
4. Lieberman, D. F.; Albright, J. D. J. Heterocycl. Chem. 1988, 25, 827.
5. Albright, J. D.; Lieberman, D. F. J. Heterocycl. Chem. 1994, 31, 537.
6. Neunhoeffer, H.; Degen, H. J.; Koehler, J. J. Justus Liebigs Annalen der Chemie 1975,
6, 1120.
7. Experimental: Unless otherwise indicated, all commercial reagents were used as
received without further purification. Commercially available anhydrous grade

V. P. Ghidu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 259–261
261
solvents were used. Reactions were monitored by thin layer chromatography
(TLC) using E. Merck pre-coated silica-gel plates. Visualization was
accomplished with UV light, followed by fixation with alcoholic
phosphomolybdic acid solution or aqueous potassium permanganate solution.
Reaction progress and purity of intermediates was also assessed by LC–MS
analysis using an Agilent Technologies 1200 Series LC system coupled to a 6130
Quadrupole MS. Chromatographic separation was performed using the
indicated solvent system on a Teledyne Isco CombiFlash Rf system. Proton
nuclear magnetic resonance (1H NMR) spectra were recorded on a 400 MHz
Bruker Avance III spectrometer at ambient temperature. Chemical shifts (d,
ppm) are reported relative to tetramethylsilane internal standard or residual
proton signal of the deuterated solvent used.
and the volatiles were removed in vacuo. Flash chromatography using EtOAc/
Hexanes (0–100%) afforded approximately 5 mg (<5%) of 4 as a yellow oil: 1H
NMR (CDCl3, d, ppm): 7.09–7.96 (m, 2H), 7.85 (d, J = 8.8 Hz, 1H), 7.62 (d,
J = 8.8 Hz, 1H), 7.60–7.57 (m, 1H), 7.36 (d, J = 8.0 Hz, 1H), 3.34 (s, 3H), 1.94 (s,
3H). LC–MS (ESI): 262.1.
6-(3-(N-Methylacetamido)phenyl)pyridazin-3-yl trifluoromethanesulfonate 5:
A
solution of 3 (0.31 g, 1.27 mmol) in CH2Cl2 (5 mL) was cooled at 0 °C and
pyridine (0.52 mL, 6.37 mmol) was added drop wise, followed by Tf2O (0.43 mL,
2.55 mmol). After stirring for 1 h at 0 °C the reaction mixture was diluted with
CH2Cl2 and quenched with excess ice cold water. Extraction with CH2Cl2 was
followed by drying the combined organic fractions over MgSO4. After removal of
volatiles in vacuo the resulting residue was transferred to silica gel. Flash
chromatography using EtOAc/Hexanes (60–100%) afforded 5 (0.19 g, 40%) as a
light-yellow solid: 1H NMR (CDCl3, d, ppm): 8.10 (d, J = 9.2 Hz, 1H), 8.02 (t,
J = 0.8 Hz, 1H), 7.99 (d, J = 7.6 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H), 7.54 (d, J = 9.2 Hz,
1H), 7.40 (d, J = 7.6 Hz, 1H), 3.34 (s, 3H), 1.94 (s, 3H). LC–MS (ESI): 376.1.
(E)-N-Methyl-N-(3-(1-((3-methyl-4H-1,2,4-triazol-4-
(E)-4-(3-(N-Methylacetamido)phenyl)-4-oxobut-2-enoic acid 2: To a solution of 1
(2 g, 10.46 mmol) in anhydrous formic acid (2 mL) glyoxylic acid monohydrate
(1.01 g, 10.98 mmol) was added portion wise. The mixture was warmed up to
110 °C and refluxed overnight. After cooling to room temperature the reaction
mixture was injected on top of
a silica gel chromatographic column and
separated using CH2Cl2/MeOH (0–50%). Evaporation of solvent in vacuo afforded
2.52 g (97%) of 3 as a light-yellow solid: 1H NMR (CDCl3, d, ppm): 8.08–7.98 (m,
1H), 7.94 (d, J = 15.6 Hz, 1H), 7.90–7.80 (m, 1H), 7.61 (q, J = 8 Hz, 1H), 7.50 (d,
J = 8.4 Hz, 1H), 6.93 (d, J = 15.6 Hz, 1H), 3.32 (s, 3H), 1.93 (s, 3H). LC–MS (ESI):
248.1.
yl)imino)ethyl)phenyl)acetamide 6: To a solution of the starting material 1
(0.65 g, 3.40 mmol) in toluene (40 mL) 3-methyl-4H-1,2,4-triazol-4-amine
(0.33 g, 3.36 mmol) was added and the reaction mixture was refluxed
overnight (22 h) with water removal via a Dean-Stark trap. After cooling to
room temperature the reaction mixture was transferred to silica gel and the
volatiles were removed in vacuo. Flash chromatography using MeOH/CH2Cl2 (0–
10%) afforded 7 (0.85 g, 92%) as a light-yellow solid: 1H NMR (CDCl3, d, ppm):
8.63 (s, 1H), 7.95 (br s, 2H), 7.61–7.59 (m, 2H), 3.20 (s, 3H), 2.37 (s, 3H), 2.32 (s,
3H), 1.82 (s, 3H). LC–MS (ESI): 272.2.
N-(3-(6-Hydroxypyridazin-3-yl)phenyl)-N-methylacetamide 3: To a solution of 2
(2.52 g, 10.2 mmol) in 15 mL water at room temperature Na2SO3 (1.41 g,
11.2 mmol) was added portion wise and the reaction mixture was warmed up
with stirring to 90 °C. After 1 h LC–MS analysis shows complete consumption of
the starting material. The reaction flask was allowed to cool for 10 min then HCl
6 N (2.2 mL, 13.46 mmol) was added drop wise. After stirring for another 10 min
the reaction mixture was re-heated to 90 °C and hydrazine hydrate (1.1 mL,
22.42 mmol) was added drop wise. Stirring was continued for another 2 h at
90 °C. The reaction mixture was cooled to room temperature, diluted with brine
and extracted with CH2Cl2. Combined organic phases were dried over MgSO4.
Evaporation of volatiles in vacuo afforded 1.34 g (54%) of crude product as a
light-yellow solid, suitable for the next synthetic step: 1H NMR (CDCl3, d, ppm):
12.38 (s, 1H), 7.78 (d, J = 10 Hz, 1H), 7.77–7.69 (m, 2H), 7.53 (t, J = 7.8 Hz, 1H),
7.31–7.26 (m, 1H), 7.12 (d, J = 10 Hz), 3.31 (s, 3H), 1.92 (s, 3H). LC–MS (ESI):
244.1.
N-(3-((1E,2E)-3-(Dimethylamino)-1-((3-methyl-4H-1,2,4-triazol-4-
yl)imino)allyl)phenyl)-N-methylacetamide 7: To
a solution of 6 (0.77 g,
2.84 mmol) in THF (5 mL) tert-butoxy bis(dimethylamino)methane (1.19 mL,
5.68 mmol) was added drop wise. After stirring at room temperature for 4.5 h
the reaction mixture was transferred to silica gel and the volatiles were removed
in vacuo. Flash chromatography using MeOH/CH2Cl2 (0–15%) afforded 8 (0.95 g,
quantitative) as a light-yellow solid: 1H NMR (CDCl3, d, ppm): 8.39 (s, 1H), 7.55–
7.47 (m, 4H), 6.88 (d, J = 12.4 Hz, 1H), 4.52 (d, J = 12.8 Hz, 1H), 3.20 (s, 3H), 2.95
(s, 3H), 2.67 (s, 3H), 2.22 (s, 3H), 1.84 (s, 3H). LC–MS (ESI): 327.2.
CL285032: A solution of 7 (0.2 g, 0.61 mmol) in AcOH (3 mL) was refluxed (bath
temperature 140 °C) overnight (20 h). After cooling to room temperature the
reaction mixture was transferred to silica gel and the volatiles were removed in
vacuo. Flash chromatography using MeOH/CH2Cl2 (0–15%) afforded CL285032
(0.122 g, 71%) as a light-yellow solid: 1H NMR (CDCl3, d, ppm): 8.42 (d, J = 9.6 Hz,
1H), 8.11 (br s, 2H), 7.98 (d, J = 9.6 Hz, 1H), 7.66 (t, J = 7.8 Hz, 1H), 7.57 (d,
J = 6.8 Hz, 1H), 3.24 (s, 3H), 2.78 (s, 3H), 1.85 (s, 3H). LC–MS (ESI): 282.1.
N-(3-(6-Chloropyridazin-3-yl)phenyl)-N-methylacetamide 4: To
a
refluxing
solution of (0.1 g, 0.41 mmol) in CHCl3 (3 mL) freshly distilled POCl3
3
(0.11 mL, 1.23 mmol) was added drop wise. After 3 h reflux the reaction
mixture was cooled to room temperature and carefully quenched with excess
MeOH (safety warning: HCl evolution). The mixture was transferred to silica gel