MAOS protocols for the general synthesis and lead optimization of 3,6-disubstituted-[1,2,4]triazolo[4,3-b]pyridazines

Article abstract of DOI:10.1016/j.tetlet.2008.10.127

General, high-yielding MAOS protocols for the expedient synthesis of functionalized 3,6-disubstituted-[1,2,4]triazolo[4,3-b]pyridazines are described amenable to an iterative analog library synthesis strategy for the lead optimization of an M1 antagonist

Full text of DOI:10.1016/j.tetlet.2008.10.127

Tetrahedron Letters 50 (2009) 212–215
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
MAOS protocols for the general synthesis and lead optimization
of 3,6-disubstituted-[1,2,4]triazolo[4,3-b]pyridazines
Leslie N. Aldrich a, , Evan P. Lebois b, , L. Michelle Lewis d, Natalia T. Nalywajko d,
Colleen M. Niswender b,c, C. David Weaver b,c,d, P. Jeffrey Conn b,c,d, Craig W. Lindsley a,b,c,d,
*
a Department of Chemistry, Vanderbilt University and Medical Center, Nashville, TN 37232, USA
b Department of Pharmacology, Vanderbilt University and Medical Center, Nashville, TN 37232, USA
c Vanderbilt Program in Drug Discovery, Vanderbilt University and Medical Center, Nashville, TN 37232, USA
d Vanderbilt Specialized Chemistry Center for Accelerated Probe Development, Vanderbilt University and Medical Center, Nashville, TN 37232, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
General, high-yielding MAOS protocols for the expedient synthesis of functionalized 3,6-disubstituted-
[1,2,4]triazolo[4,3-b]pyridazines are described amenable to an iterative analog library synthesis strategy
for the lead optimization of an M1 antagonist screening hit. Optimized compounds proved to be highly
selective M1 antagonists.
Received 30 September 2008
Accepted 24 October 2008
Available online 31 October 2008
Ó 2008 Elsevier Ltd. All rights reserved.
In the course of our program in small molecule probe develop-
ment for the Molecular Library Screening Center Network
(MLSCN),1 a high-throughput screen identified the 3,6-disubsti-
tuted-[1,2,4]triazolo[4,3-b]pyridazine scaffold 1 as an attractive
hit for a CNS target (Fig. 1). While numerous reports describe syn-
theses of 1, yields are typically moderate (<50%) with prolonged
reaction times at high temperatures (steps requiring 18- to >60 h
at reflux).2–5 In order to employ an iterative analog library synthe-
sis approach for the lead optimization of 2, a weak, but selective
heterocyclic templates employing microwave-assisted organic
synthesis (MAOS).6–12 In recent reports, we have described general,
high-yielding MAOS protocols for the expedient synthesis of
1,2,4-triazines 3,6 imidazoles 4,7 quinoxalines 5,8 pyrazinone 6,9
5-aminooxazoles 7,10 quinoxalinones 8,11 pyrazolo[1,5-a]pyrimi-
dines 9,12 and pyrazolo[3,4-d]pyrimidines 1012 from simple start-
ing materials (Fig. 2). Therefore, application of MAOS to develop
a general, high-yielding, and expedient synthesis of the 3,6-di-
substituted-[1,2,4]triazolo[4,3-b]pyridazine scaffold
1
seems
muscarinic acetylcholine receptor antagonist (M1 IC50 = 22
lM,
warranted (see Scheme 1).
M4 IC50 > 150 M), significant refinements were required in the
l
synthetic protocols for delivering analogs 1, with diversity at both
C3 and C6.2–5
H
R1
R2
R1
R2
N
N
N
N
R1
R2
N
N
R3
As many of the leads identified from HTS campaigns are small
heterocyclic compounds, our laboratory has devoted significant ef-
fort to develop efficient protocols for the preparation of diverse
R3
N
H
R3
3
4
5
CO2R1
NR2R3
O
O
R1
R2
N
R3
O
N
R1
O
N
R2
R1
N
6
N
5
N
Br
Ar
N
R4
N
H
N
H
O
3
N
N
R2
N
N
N
2
N
1
N
6
7
8
8
NR1R2
1
2
Ar2
N
N
N
N
Figure 1. Generic structure of 3,6-disubstituted-[1,2,4]triazolo[4,3-b]pyridazine 1
and our M1 antagonist screening lead 2.
N
N
N
Ar1
Ar
9
10
* Corresponding author. Tel.: +1 615 322 8700; fax: +1 615 343 6532.
E-mail address: craig.lindsley@vanderbilt.edu (C. W. Lindsley).
These authors contributed equally to this work.
Figure 2. Heterocyclic templates accessed through MAOS in our laboratory.
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.10.127

L. N. Aldrich et al. / Tetrahedron Letters 50 (2009) 212–215
213
By varying solvent and temperature parameters, microwave
conditions were rapidly developed to accelerate and generalize
the synthesis of the 3-phenyl-6-chloro-[1,2,4]triazolo[4,3-b]pyrid-
azine 15 core employing 3,6-dichloropyridazine 11 and acylhyd-
razide 14 (Table 1). When HOAc was employed as a solvent or
catalyst, a corresponding acetylated phenyl acylhydrazide 16 was
obtained in varying quantities. Optimal HOAc conditions employed
5% HOAc/EtOH at 150 °C for 10 min to afford the desired 15, along
with 16 in an 85:15 ratio (Table 1, entry 9). Despite the side prod-
uct, the conversion to 15/16 was quantitative and isolated yields of
15 exceeded 82%. Application of the same MAOS conditions, but
replacement of HOAc with 5% 4 N HCl in dioxane, afforded 100%
conversion to 15 in 95% isolated yield without producing 16 (Table
1, entry 13). Thus, a reaction that previously required up to 60 h of
conventional heating to provide <50% yield,3–5 now afforded 95%
yield of the desired product 15 in 10 min by virtue of MAOS—a
360-fold reduction in reaction time.13
Attention was now directed at the application of these new
MAOS conditions to a diverse array of acylhydrazides to ensure
that this new protocol would indeed be general. As shown in Table
2, the MAOS protocol, employing either catalytic HOAc (Method A)
or HCl (Method B), proved to be general with respect to a wide
range of electron-rich (entry 13g), electron-deficient (entry 13f),
and hindered acylhydrazides (entry 13a) 17 as well as heterocyclic
O
Ar
N
H2NHN
12
Ar
N
N
N
Cl
N
Cl
Cl
toluene, 110 oC
16-60 hours
<50%
N
13
11
R1
R2
R1
N
N
Ar
H
N
R2
N
N
neat, or
steel bomb
N
100 oC-140 oC
8-24 hours
1
Scheme 1. Classical synthesis of 3,6-disubstituted-[1,2,4]triazolo 4,3-b]pyridazines
1.
Classical conditions for the synthesis of 3,6-disubstituted-
[1,2,4]triazolo[4,3-b]pyridazines 1 involve refluxing 3,6-dichloro-
pyridazine 11 with an acylhydrazide 12 in toluene for 16 h, or
more typically for 60 h, to provide the 3-aryl-6-chloro-[1,2,4]triaz-
olo[4,3-b]pyridazine 13 in yields less than 50%.3–5 Introduction of
the amino moiety in the 6-position was accomplished through an
SNAr reaction employing either neat or steel bomb conditions at
100–140 °C for 8–30 h to deliver analogs 1 in yields ranging from
40% to 70%.3–6 Moreover, previous efforts were focused on tradi-
tional medicinal chemistry approaches and the development of
structure–activity relationships (SARs), with little concern for
achieving high chemical yields or reaction generality for either
the heterocycle synthesis or the SNAr reaction. Indeed, the 3,6-
disubstituted-[1,2,4]triazolo[4,3-b]pyridazine scaffold 1 has been
an important pharmacophore for the development GABAA receptor
Table 2
Generality of the MAOS protocol to deliver analogs 13
Cl
O
Ar(Het)
N
Cl
N
N
N
H2NHN
R
N
17
N
150 oC, mw, 10 min
Cl
5% H+/EtOH
11
13
Yielda (%)
Yieldb (%)
92
agonists at the
a2/a
3-subunit.3–5 Interestingly, microwave-
assisted organic synthesis has never before been applied to this
heterocyclic system, and even more surprising when one considers
a 1–6 day reaction time to deliver a single derivative of 1.
Compound
R
13a
79
Cl
13b
13c
77
81
93
91
F
Table 1
Optimization of MAOS conditions to produce 15
F
Cl
O
Ph
N
O
Cl
N
N
N
H2NHN
Ph
N
14
+
AcHNHN
Ph
N
13d
13e
13f
87
80
75
74
70
72
96
95
97
96
88
86
conditions
Cl
F
11
16
15
Entry
T (°C)
Solvent
Time (min)
15:16a
CH3
1
2
3
4
5
6
7
8
9
10
11
12
13
140
160
180
200
150
170
150
170
150
170
135
135
150
HOAc
HOAc
HOAc
HOAc
10
10
10
10
10
10
10
10
10
10
20
10
10
42:58
28:72
24:76
13:87
78:22
64:36
79:21
74:26
85:15
77:23
80:20
—
CF3
50% HOAc/EtOH
50% HOAc/EtOH
10% HOAc/EtOH
10% HOAc/EtOH
5% HOAc/EtOH
5% HOAc/EtOH
5% HOAc/EtOH
EtOHb
OCH3
13g
13h
N
5% HCI/EtOHc
100:0
13i
N
S
a
Ratio determined by analytical LC–MS and 1H NMR; conversion >95%.
No product formed without acid catalysis.
5% 4 N HCl/dioxane.
b
c
a
5% HOAc/EtOH, remaining mass balance congeners or 16.
5% 4 N HCI/dioxane. All yields for isolated, analytically pure materials.
b

214
(I)
L. N. Aldrich et al. / Tetrahedron Letters 50 (2009) 212–215
Developing a general MAOS-mediated SNAR protocol for the
R1
R2
R1
N
N
H
Ar
N
Ar
N
reaction of diverse amines with analogs 13 to deliver 3-aryl, 6-ami-
no-[1,2,4]triazolo[4,3-b]pyridazines 1 proved more difficult. Nucleo-
philic amines (benzyl, aliphatic, piperidines, and piperazines)
reacted smoothly in EtOH at 170 °C for 10 min to produce analogs
1 in yields ranging from 73% to 92% (Scheme 2). Less nucleophilic
amines, such as anilines, required K2CO3 in DMF with micorwave
irradiation for 15 min at 180 °C to produce analogs 1 in yields
exceeding 65%.14 Furthermore, analogs 13 readily participated in
general microwave-assisted Sonogashira and Suzuki couplings to
afford analogs 18 and 19 in yields exceeding 80% in every case
examined (Scheme 2).
Utilizing these new MAOS protocols, we resynthesized the M1
versus M4 selective antagonist HTS hit 2 (Scheme 3). Beginning
with 15, delivered in 95% yield (Table 1), an SNAr reaction with
Boc-piperazine provided 20, which was then deprotected using
1:1 TFA:DCM to afford 21 in 80% yield for the two steps. 21 was
then acylated employing standard polymer-supported reagents
and scavengers to generate the original HTS hit 2 in 70% yield.15
Evaluation of 2 against M1–M5 indicated that 2 was indeed a selec-
Cl
Cl
Cl
Cl
N
N
N
N
N
N
N
2.0 equiv.
170 oC, 10 min
N
N
R2
N
N
EtOH
73-92%
1
13
(II)
Ar
N
Ar
N
ArNH2, K2CO3
ArHN
N
N
180 oC, 10 min
DMF
N
N
1
13
>65%
(III)
R
Ar
N
R
Ar
N
CuI, Pd(tBuP
)
2
N
N
60 oC, 30 min
THF,Et2NH
>80%
N
N
18
13
tive M1 antagonist (M1 IC50 = 23 lM, M2–M5 IC50 >> 50 lM). Prior
to this discovery there was only one other M1 selective small
molecule antagonist,16 and prior to its discovery, the only M1
selective antagonist was MT7, a 71 amino acid peptide toxin from
the green mamba snake.17 Encouraged by this result, we employed
an iterative parallel synthesis approach, employing our new MAOS
protocols, to rapidly develop structure–activity relationships in an
attempt to improve the M1 antagonist potency while maintaining
selectivity for M2–M5.
(IV)
Ar
N
(Het)ArB(OH)2
10% Pd(tBuP)2
Ar
N
(Het)Ar
N
N
N
160 oC,10 min
1:1 THF:1 M Cs2CO
N
N
3
19
13
>80%
Scheme 2. MAOS protocols to functionalize the 3-aryl-6-chloro-[1,2,4]triazolo[4,3-
b]pyridazine 13.
As shown inFigure 3, we simultaneously varied the substituents
at the C-3 and C-6 positions, synthesizing small 12- to 24-member
libraries employing the synthetic routes depicted in Schemes 2 and
3. Analogs of 2 were triaged in a single point 10 lM screen for the
compound’s ability to decrease an EC80 concentration of
congeners (entries 13h, 13i) affording the desired 3-aryl/
heteroaryl-6-chloro-[1,2,4]triazolo[4,3-b]pyridazines 13 in iso-
lated yields ranging from 74% to 97% in 10 min at 150 °C.
acetylcholine.
SAR for this series was rather ‘flat’, with subtle changes leading
to a complete loss of M1 inhibitory activity. Out of ꢀ60 analogs,
only four demonstrated significant M1 antagonism; however, we
managed to improve upon HTS hit 2. As shown in Figure 4, explo-
ration of the C3 position identified both the 3-OMe phenyl deriva-
tive 22 and the 4-Me phenyl congener 23 as engendering more
Replace Amide linkage
O
Alternative Aryl
moieties
O
N
Ph
N
Br
N
N
potency (M1 IC50 = 3.59
lM and 4.09
lM, respectively), while
N
maintaining selectivity (M2–M5 IC50 >> 50
lM). When holding
N
the 3-OMe phenyl moiety constant at C3 and exploring alterna-
tively functionalized piperazines for the bromofuranoic amide at
C6, we identified two piperazinyl piperazine analogs, 24 and 25,
Alternative Amides
2
Figure 3. Synthetic plan to optimize 2 for M1 antagonist potency, while maintain-
ing selectivity versus M2–M5.
which maintained M1 antagonism (M1 IC50 = 3.99
l
M
and
M).
6.64 M, respectively) and selectivity (M2–M5 IC50 >> 50
l
l
BocN
Ph
Ph
N
HN
NBoc
Cl
N
N
N
N
N
N
K2CO3, DMF
N
N
180 oC, mw, 15 min
1:1 TFA:DCM
rt, 2 h
15
20
(80%, 2 steps)
O
O
O
O
OH
N
HN
Br
Ph
N
Br
Ph
N
N
N
N
N
N
TFA
N
DCC, HOBt
N
N
DCM, DIEA
(70%)
2
21
Scheme 3. Application of MAOS protocols for the resynthesis of the M1 antagonist HTS hit 2.

L. N. Aldrich et al. / Tetrahedron Letters 50 (2009) 212–215
215
Me
MeO
O
O
O
O
N
N
Br
Br
N
N
N
N
N
N
N
N
N
N
22
23
M1 IC50 = 4.09 μM
μ
M2-M5 IC50 >> 50 M
M1 IC50 = 3.59 μM
M2-M5 IC50 >> 50
μ
M
MeO
MeO
N
N
N
N
N
N
N
N
N
N
N
N
N
N
24
25
M1 IC50 = 6.64 μM
M1 IC50 = 3.99 μM
M2-M5 IC50 >> 50
μ
M
μ
M
M2-M5 IC50 >> 50
Figure 4. Optimized analogs of 2 as highly selective M1 antagonists with improved M1 inhibitory activity as compared to HTS hit 2.
6. Zhao, Z.; Leister, W. H.; Strauss, K. A.; Wisnoski, D. D.; Lindsley, C. W.
Tetrahedron Lett. 2003, 44, 1123–1127.
7. Wolkenberg, S. E.; Wisnoski, D. D.; Leister, W. H.; Zhao, Z.; Wang, Y.; Lindsley,
C. W. Org. Lett. 2004, 6, 1453–1456.
8. Zhao, Z.; Wisnoski, D. D.; Wolkenberg, S. E.; Leister, W.; Wang, Y.; Lindsley, C.
W. Tetrahedron Lett. 2004, 45, 4873–4876.
9. Lindsley, C. W.; Zhao, Z.; Leister, W. H.; Robinson, R. G.; Barnett, S. F.; Defeo-
Jones, D.; Jones, R. E.; Hartman, G. D.; Huff, J. R.; Huber, H. E.; Duggan, M. E.
Bioorg. Med. Chem. Lett. 2005, 15.
10. Nolt, M. B.; Smiley, M. A.; Varga, S. L.; McClain, R. T.; Wolkenberg, S. E.;
Lindsley, C. W. Tetrahedron 2006, 62, 4698–4704.
11. Shipe, W. D.; Yang, F.; Zhao, Z.; Wolkenberg, S. E.; Nolt, M. B.; Lindsley, C. W.
Heterocycles 2006, 70, 665–689.
12. Daniels, R. N.; Kim, K.; Hughes, M. A.; Lebois, E. P.; Muchalski, H.; Lindsley, C.
W. Tetrahedron Lett. 2008, 49, 305–310.
13. Typical MAOS experimental for 6-chloro-3-p-tolyl-[1,2,4]triazolo[4,3-b]-
Moreover, these latter analogs, with basic amines, afforded
improved solubility and physiochemical characteristics.
In summary, we have applied MAOS to the preparation of 3,6-
disubstituted-[1,2,4]triazolo[4,3-b]pyridazines 1, and developed
general and high-yeilding protocols with over a 360-fold accelera-
tion in reaction rate. For both the heterocyclic synthesis and the
subsequent SNAr steps, reaction time, yield, and overall reaction
generality were dramatically improved under these MAOS proto-
cols; more importantly, these new protocols allow for an iterative
analog library synthesis approach for lead optimization to be em-
ployed for the rapid synthesis of large numbers of analogs of 1.
Employing these new MAOS protocols, a lead optimization cam-
paign centered on the selective, but weak M1 antagonist hit 2
pyridazine (13e). (Method A) To
a 5 mL microwave reaction vessel were
added 3,6-dichloropyridazine (100 mg, 0.671 mmol) and p-toluic hydrazide
(111 mg, 0.738 mmol) in a 3 mL solution of 5% AcOH/EtOH. The vial was
irradiated in a microwave synthesizer at 150 °C for 10 min. LC–MS (single peak,
2.91 min, m/e, 245.1 (M+1)) indicated that all starting material had been
consumed affording 131 mg (80%) of 6-chloro-3-p-tolyl-[1,2,4]triazolo[4,3-b]-
pyridazine as a white solid following column purification. (Method B) To a
5 mL microwave reaction vessel were added 3,6-dichloropyridazine (100 mg,
0.671 mmol) and p-toluic hydrazide (111 mg, 0.738 mmol) in a 3 mL solution
of 5% 4 N HCl/EtOH. The vial was irradiated in a microwave synthesizer at
150 °C for 10 min. LC–MS (single peak, 2.91 min, m/e, 245.1 (M+1)) indicated
that all starting material had been consumed affording 156 mg (95%) of 6-
chloro-3-p-tolyl-[1,2,4]triazolo[4,3-b]pyridazine as a white solid following a
silica plug and concentration in vacuo. 1H NMR (DMSO-d6, 600 MHz) d (ppm):
2.41 (s, 3H), 7.43 (d, J = 8 Hz, 2H), 7.53 (d, J = 9.7 Hz, 1H), 8.19 (d, J = 8.2 Hz, 2H),
8.52 (d, J = 9.7 Hz, 1H); 13C NMR (DMSO-d6, 150 MHz) d (ppm): 21.1, 122.5,
122.8, 127.1, 127.3, 129.5, 140.3, 143.8, 146.8, 149.1; LC–MS: single peak,
2.91 min, m/e, 245.1 (M+1).
(M1 IC50 = 23
6-fold increase in M1 inhibitory activity (M1 IC50 = 3.99
6.64 M, respectively) while maintaining selectivity versus M2–
M5 (IC50 >> 50 M). These compounds represent a novel chemo-
type of selective, small molecule M1 antagonists, and hold promise
as leads for potential new therapeutic agents for Parkinson’s
Disease and dystonia.
l
M) delivered two analogs, 22 and 24, with over a
l
M and
l
l
Acknowledgments
The authors warmly thank Department of Pharmacology and
the NIH/MLSCN (3U54 MH074427-02S1, Chemistry Supplement)
for support of this research. Vanderbilt is a member of the produc-
tion phase of the MLCSN, termed the MLPCN, and houses the Van-
derbilt Specialized Chemistry Center for Accelerated Probe
Development (1U54MH084659-01).
14. Typical MAOS experimental for N-(4-methoxybenzyl)-3-p-tolyl-[1,2,4]tri-
azolo[4,3-b]pyridazin-6-amine. To a 5 mL microwave reaction vessel were
added 6-chloro-3-p-tolyl- [1,2,4]triazolo[4,3-b]pyridazine (50 mg, 0.205
mmol) and 4-methoxy-benzyl amine (35 lL 6 mmol) in 3 ml of ethanol. The
vial was initially heated in a microwave synthesizer to 170 °C for 25 min.
Preparative LC–MS afforded 51.6 mg (73%) of N-(4-methoxybenzyl)-3-p-tolyl-
[1,2,4]triazolo[4,3- b]pyridazin-6-amine as a white solid. 1H NMR (DMSO-d6,
600 MHz) d (ppm): 2.38 (s, 3H), 3.71 (s, 3H), 4.41 (d, J = 5.5 Hz, 2H), 6.88 (d,
J = 9.9 Hz, 1H), 6.92 (d, J = 8.6 Hz, 2H), 7.33 (d, J = 6.7 Hz, 2H), 7.36 (d, J = 8.5 Hz,
2H), 7.97 (d, J = 9.8 Hz, 1H), 8.21 (d, J = 8.2 Hz, 2H). 13C NMR (DMSO-d6,
150 MHz) d (ppm): 21.5, 44.7, 55.5, 114.2, 117.0, 124.4, 124.6, 127.0, 129.3,
129.6, 130.9, 139.4, 143.8, 146.3, 154.1, 158.8; LC–MS: single peak, 3.00 min,
m/e, 346.2 (M+1).
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