Aminothienopyridazine inhibitors of tau aggregation: Evaluation of structure-activity relationship leads to selection of candidates with desirable in vivo properties

Article abstract of DOI:10.1016/j.bmc.2012.05.027

Previous studies demonstrated that members of the aminothienopyridazine (ATPZ) class of tau aggregation inhibitors exhibit a promising combination of in vitro activity as well as favorable pharmacokinetic properties (i.e., brain-penetration and oral bioavailability). Here we report the synthesis and evaluation of several new analogues. These studies indicate that the thienopyridazine core is essential for inhibition of tau fibrillization in vitro, while the choice of the appropriate scaffold decoration is critical to impart desirable ADME-PK properties. Among the active, brain-penetrant ATPZ inhibitors evaluated, 5-amino-N-cyclopropyl-3-(4-fluorophenyl)-4-oxo-3,4- dihydrothieno[3,4-d]pyridazine-1-carboxamide (43) was selected to undergo maximum tolerated dose and one-month tolerability testing in mice. The latter studies revealed that this compound is well-tolerated with no notable side-effects at an oral dose of 50 mg/kg/day.

Full text of DOI:10.1016/j.bmc.2012.05.027

Bioorganic & Medicinal Chemistry 20 (2012) 4451–4461
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Aminothienopyridazine inhibitors of tau aggregation: Evaluation
of structure–activity relationship leads to selection of candidates
with desirable in vivo properties
Carlo Ballatore a,b, , Alex Crowe b, Francesco Piscitelli a, Michael James b, Kevin Lou a,
⇑
Gabrielle Rossidivito a, Yuemang Yao b, John Q. Trojanowski b, Virginia M.-Y. Lee b,
Kurt R. Brunden b, Amos B. Smith III a
a Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, PA 19104-6323, USA
b Center for Neurodegenerative Diseases Research, Department of Pathology & Laboratory Medicine, University of Pennsylvania, 3600 Spruce Street, Philadelphia, PA 19104-6323, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 March 2012
Revised 8 May 2012
Accepted 12 May 2012
Available online 23 May 2012
Previous studies demonstrated that members of the aminothienopyridazine (ATPZ) class of tau aggrega-
tion inhibitors exhibit a promising combination of in vitro activity as well as favorable pharmacokinetic
properties (i.e., brain-penetration and oral bioavailability). Here we report the synthesis and evaluation of
several new analogues. These studies indicate that the thienopyridazine core is essential for inhibition of
tau fibrillization in vitro, while the choice of the appropriate scaffold decoration is critical to impart desir-
able ADME-PK properties. Among the active, brain-penetrant ATPZ inhibitors evaluated, 5-amino-N-
cyclopropyl-3-(4-fluorophenyl)-4-oxo-3,4-dihydrothieno[3,4-d]pyridazine-1-carboxamide (43) was
selected to undergo maximum tolerated dose and one-month tolerability testing in mice. The latter stud-
ies revealed that this compound is well-tolerated with no notable side-effects at an oral dose of 50 mg/kg/
day.
Keywords:
Alzheimer’s disease
Tauopathy
Aminothienopyridazine
Tau aggregation inhibitor
K18PL
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Recently, we identified a novel class of tau fibrillization inhibi-
tors, the aminothienopyridazines (ATPZs), which exhibit
a
Alzheimer’s disease (AD), which is characterized by the pres-
ence of intraneuronal aggregates of tau proteins (e.g., neurofibril-
lary tangles, or NFTs) and extracellular deposits comprised of Ab
peptide (e.g., senile plaques), remains a challenge for biomedical
intervention. Although the exact neuropathological mechanisms
underlying this disease remain the focus of intense investigation,
several lines of evidence indicate that under pathological condi-
tions tau may act as the mediator of neurodegeneration in re-
sponse to various upstream events.1 As a result, in recent years
there has been an increasing interest in the development of thera-
peutic strategies that could target pathological tau.2 One such
strategy calls for the use of small molecules to inhibit the self-
assembly of tau into oligomeric/polymeric species, including the
well-defined fibrils found within NFTs. Although several classes
of tau assembly inhibitors have demonstrated activity in vitro,3,4
aside from selected cases (e.g., methylene blue5), the majority of
these inhibitors are unlikely to be considered for in vivo efficacy
studies due to potential toxicities and/or limited CNS-uptake.3
favorable combination of efficacy in an in vitro tau fibrillization
assay, as well as drug-like properties (i.e., no violations of Lipinski’s
‘rule of five’).6 Furthermore, prototype compounds from this class
of inhibitors (e.g., 1, Fig. 1) have been developed that can reach
significant concentrations in the brain of mice after intravenous
(i.v.) or oral administration, suggesting that the ATPZs may be
amenable to efficacy studies in animal models.7 However, ATPZ 1
R3
R2
S
5
NH
7
R1
NH2
O
1
N
3
N
O
R4
Cl
1
Figure 1. Representative example of a brain-penetrant ATPZ;7 fragments investi-
gated as part of the SAR studies are marked R1–R4.
⇑
Corresponding author.
E-mail address: bcarlo@sas.upenn.edu (C. Ballatore).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.027

4452
C. Ballatore et al. / Bioorg. Med. Chem. 20 (2012) 4451–4461
S
was found to have limited water solubility (i.e., ꢀ30
lM). Further-
O
R3
O
NH2
O
more, evaluation by equilibrium dialysis revealed that 1 is ꢀ99%
bound in brain homogenates, indicating that relatively high doses
of the compound would be required to achieve efficacious free
drug concentration in the brain.
O
a
b
R3
N
R3
N
O
NH2
O
S
R4
G
Thus, to circumvent these shortcomings and to identify pre-
ferred candidates for future studies, we designed and synthesized
a series of new analogues, including a number of closely related
congeners and a series of compounds bearing a range of modifica-
tions in the ATPZ heterocyclic core (Schemes 1–3).
51: R3 = CH3
52: R3 = Ph
53: R3 = CH3
54: R3 = Ph
55: R3 = CH3, R4 = H
56: R3 = CH3, R4 = Me
57: R3 = CH3, R4 = iPr
58: R3 = CH3, R4 = CH2CF3
59: R3 = CH3, R4 = cyclohexyl
After synthesis, all test ATPZs were evaluated for aqueous
solubility and activity in a heparin-induced tau K18PL fibrillization
assay.6 Analogues that produced full dose-response curves in the
fibrillization assay and whose activity was confirmed in a secondary
centrifugation assay6 progressed to an evaluation of pharmacoki-
netic (PK) properties. Collectively, these studies defined the struc-
ture–activity relationship (SAR) of ATPZs and in turn resulted in
the identification of selected analogues which exhibit, in addition
to excellent brain-penetration and oral bioavailability, more favor-
able properties compared to 1, such as higher aqueous solubility
and higher unbound fraction in brain homogenates. Among these
compounds, ATPZ 43 was evaluated for possible dose-limiting tox-
icities in wild type mice. The results from PK and toxicity studies
suggest that nM free drug levels can be achieved in the brains of
mice after oral administration of well-tolerated doses of 43.
60: R3 = CH3, R4
61: R3 = CH3, R4
=
=
N
N
62: R3 = CH3, R4 = Bn
63: R3 = CH3, R4
=
Cl
64: R3 = Ph, R4 = CH2CF3
65: R3 = Ph, R4 = iPr
Scheme 2. Reagents and conditions: (a) ethyl cyanoacetate, S8, morpholine,
ethanol, 150 °C (microwave irradiation), 15 min; (b) appropriate substituted
hydrazine, 150 °C (microwave irradiation), 0.5–3 h. The insert shows the X-ray
crystal structure of compound 55 (CCDC 853887).
to provide amides of general structure F (Scheme 1). Such an ap-
proach proved both effective and reliable for the synthesis of sev-
eral closely related analogues of 1. However, as stable diazonium
salts can be generally derived from anilines, but not from aliphatic-
or heteroaryl-amines, this method could not be exploited to obtain
analogues bearing R4 – phenyl/substituted phenyl. Thus, to cir-
cumvent this limitation, aminothiophene keto-esters 53 and 54
(Scheme 2) were first prepared by subjecting di-ketones 51 and
52 to the Gewald reaction. Next, condensation of 53 and 54 with
a series of alkyl-, aryl-, and (hetero)aryl-hydrazines, under micro-
wave irradiation-promoted conditions, provided the corresponding
ATPZs (55–65) of general structure G (Scheme 2).
Scheme 3 highlights the synthesis of analogues modified at the
aminothiophene ring, which include examples of N-alkylated
derivatives, such as 66, obtained by treating 29 with methyl iodide
in the presence of potassium carbonate, as well as C7-halogenated
derivatives (67 and 68), obtained from 29 upon treatment with
N-chloro- and N-bromo-succinimmide, respectively. Interestingly,
treatment of 29 with dimethyldioxirane resulted in rapid
(ꢀ15 min) consumption of the starting material with the formation
of the 5,7-dihydroxy-pyrrolopyridazine 69, whose structure was
1.1. Design and synthesis of test aminothienopyridazine (ATPZ)
congeners
Previous SAR studies6,7 indicated that the R3 fragment (Fig. 1) is
relatively well tolerant of structural variations, while N-acylations
(R1) and alkylations at C7 (R2) lead to drastic reduction in the abil-
ity of the derived analogues to interfere with tau fibrillization. The
SAR of R4, on the other hand, remained incompletely defined. Thus,
in addition to a number of congeners of 1 (i.e., compounds of gen-
eral structure F, Scheme 1), a series of ATPZs bearing modifications
at the R3 and R4 fragments (i.e., analogues of general formula G,
Scheme 2), as well as congeners modified at the aminothiophene
ring (Scheme 3), were synthesized.
For the synthesis of analogues of general structure F, we em-
ployed the reaction sequence highlighted in Scheme 1.7 Central
to this strategy is the Gewald reaction protocol8,9 to construct
the ATPZ heterocyclic core from pyridazines of general structure
C. Next, saponification of esters D to the corresponding acids (E)
was followed by coupling reactions with the appropriate amine
S
S
OEt
Y
S
OEt
O
OEt
OH
CN
O
NH2
NH2
NH2
O
O
O
O
O
a
c
b
NH2
d
N
N
N
N
e
N
N
N
O
NH
N
O
N
O
X
X
X
X
X
X
A
B
C
D
E
F
2: X = 4-F
10: X = 4-F
11: X = 2-Cl
12: X = 3-Cl
13: X = 4-Cl
14: X = -CF3
15: X = -OCF3
16: X = -OH
17: X = -iPr
18: X = 4-F
26: X = 4-F
27: X = 2-Cl
28: X = 3-Cl
29: X = 4-Cl
34: X = 4-F
35: X = 2-Cl
36: X = 3-Cl
37: X = 4-Cl
42: X = 4-F; Y = -NHiPr
43: X = 4-F; Y = -NHcPr
44: X = 4-F; Y = -NHCH2CF3
45: X = 2-Cl; Y = -NHcPr
46: X = 3-Cl; Y = -NHiPr
1: X = 4-Cl; Y = -NHcPr
47: X = 4-CF3; Y = -NHcPr
48: X = 4-OCF3; Y = -NHiPr
49: X = -4OH; Y = -NHiPr
50: X = 4iPr; Y = -NHcPr
3: X = 2-Cl
4: X = 3-Cl
5: X = 4-Cl
6: X = 4-CF3
7: X = 4-OCF3
8: X = 4-OH
9: X = 4-iPr
19: X = 2-Cl
20: X = 3-Cl
21: X = 4-Cl
22: X = 4-CF3
23: X = 4-OCF3
24: X = 4-OH
25: X = 4-iPr
30: X = 4-CF3
31 : X = 4-OCF3
32: X = 4-OH
33: X = 4-iPr
38: X = 4-CF3
39: X = 4-OCF3
40: X = 4-OH
41: X = 4-iPr
Scheme 1. Reagents and conditions: (a) (i) NaNO2, 37% HCl, ethanol, water, 0 °C, 20 min; (ii) ethyl acetoacetate, sodium acetate, ethanol, water, 0 °C, 2 h; (b) ethyl
cyanoacetate, ammonium acetate, acetic acid, 170 °C (microwave irradiation), 4 min; (c) S8, morpholine, ethanol, 150 °C (microwave irradiation), 15 min; (d) LiOHꢁH2O,
tetrahydrofuran, water, rt, 16 h; (e) amine, BOP reagent, N,N-diisopropylethylamine, dimethylsulfoxide, rt, 4–16 h.

C. Ballatore et al. / Bioorg. Med. Chem. 20 (2012) 4451–4461
4453
S
OEt
compounds were evaluated in a secondary assay, which consisted
of SDS-PAGE analysis of the soluble and insoluble fractions ob-
tained after centrifugation of the ATPZ-treated fibrillizing mixtures
(centrifugation assay).6,13 The results of these experiments, sum-
marized in Table 1, in general reveal a good correlation between
the primary and secondary assays, although higher % inhibitions
are typically observed in the ThT assay compared to the centrifuga-
tion assay. The different % inhibitions seen in the two assays may
not be necessarily indicative of some degree of interference of the
ATPZ analogues with ThT binding and/or fluorescence, but could
be explained considering the differences between the ThT- and cen-
trifugation assay. While the former is dependent on protein-confor-
mation, as it is based on the characteristic red-shift in excitation
spectrum of ThT that occurs upon ThT binding to pleated b-sheets
found in amyloid fibrils,14 the latter is based on the total amount
of pelletable material, regardless of protein-conformation. Consis-
tent with previous results, the majority of active ATPZs were found
N
H
O
a
29
N
N
O
66
Cl
R2
S
OEt
NH2
O
b or c
29
N
N
O
67: R2 = Cl
68: R2 = Br
Cl
HO
OEt
to exhibit IC50 values in the 1–10
l
M range. Given that the tau con-
M, these results sug-
NH
centration in the in vitro assay is kept at 20
l
O
O
gest that ATPZ inhibitors may be most effective when present in a
ꢀ1:1 ratio with tau protein. Interestingly, the activity data illus-
trated in Table 1 indicate that all compounds lacking the thienopy-
ridazine heterocycle, such as pyridazine 21, aminothiophene 54,
and pyrrolopyridazine 69, were essentially devoid of any activity
against K18PL fibrillization as determined by centrifugation assay.
Conversely, minimally substituted ATPZs, such as 55 and 56, as well
as variously decorated derivatives, including hydroxythienopyrida-
zines (70 and 72), and ATPZs halogenated at C7 (67 and 68), all pro-
duced near full inhibition in the K18PL fibrillization assay. These
results strongly suggest that the thienopyridazine heterocycle is
essential for inhibition of tau fibrillization, while the nature of the
R1-R4 substituents may play a role in modulating the potency of
the active thienopyridazine, possibly by affecting ATPZ-tau interac-
tions and/or the physicochemical properties of the ATPZs. For
example, the relatively lower inhibition observed in the centrifuga-
d
29
N
N
O
69
Cl
S
S
R3
R3
OH
O
e
34 or 37
N
N
N
O
N
O
X
X
preferred tautomer
tion assay, which is conducted at 50 lM compound concentration,
70: X = F, R3 = COOH
71: X = Cl, R3 = COOH
with ATPZ esters of structure D relative to the corresponding acids
(E) and amides (F) may be attributed in part to the generally lower
water solubility of these esters.
f
72: X = Cl, R3 = CONHiPr
Scheme 3. Reagents and conditions: (a) MeI, K2CO3, 65 °C, 10 h.; (b) N-chlorosucc-
inimmide; (c) N-bromosuccinimmide; (d) DMDO, acetone, rt, 5 min; (e) 1 N HCl,
acetonitrile, 120 °C (microwave irradiation), 30 min; (f) isopropyl amine, BOP
reagent, N,N-diisopropylethylamine, dimethylsulfoxide, rt, 4 h. The insert shows the
X-ray crystal structure of compound 69 (CCDC 853892).
2.2. Determination of brain-penetration
Among all active tau aggregation inhibitors shown in Table 1, a
representative subset having aqueous buffer solubility >50 lM
confirmed by single crystal X-ray analysis. Treatment of acids 34 and
37 with hydrochloric acid under microwave-promoted conditions10
then produced, respectively, thienopyridazines 70 and 71, in which
the amine is replaced by a hydroxyl. Compound 71 was then con-
verted to the corresponding cyclopropyl amide 72. In agreement
with previous reports,10 the NMR spectra of 70–72 are consistent
with the thiolactone tautomeric forms shown in Scheme 3.
were selected for studies consisting of determination of brain
and plasma levels 1 h after i.p. administration of 5 mg/Kg (Table
2). For each of the ATPZs found to be brain-penetrant, equilibrium
dialysis experiments were also performed to determine the un-
bound fraction in plasma and brain homogenates (Table 2). These
values provide for an estimate of the percentage of drug that is
freely available in these tissues. The results from the PK studies
suggest that substitution of the amino group with an hydroxyl re-
sults in derivatives that are not brain-penetrant (cf., 1 and 72). Fur-
thermore, the observation that smaller derivatives lacking the R4
fragment (e.g., 55) exhibit low plasma levels 1 h after dosing sug-
gests that a phenyl group in R4, although not strictly required for
biological activity (cf., Table 1), may be an important determinant
of metabolic stability and possibly brain-penetration. Notably,
among all brain-penetrant ATPZs, 42, 43 and 44 were found to be
able to partition freely between plasma and brain with B/P ratio
near unity (Table 2). Equally important, evaluation of non-specific
plasma/brain protein binding revealed that the unbound fractions
in brain homogenates are ꢀ4.3% (43) and 5.0% (44), respectively.
Finally, a full PK evaluation of 43 confirms that there is good
brain exposure to the compound (Fig. 2). Moreover, the oral bio-
availability of 43 was found to be ꢀ60% (see Supplementary data).
2. Results
2.1. Evaluation of aminothienopyridazine (ATPZ) analogues in
the tau fibrillization assay
Initially, each test compound was evaluated for aqueous solubil-
ity in the sodium acetate buffer employed in the fibrillization as-
say.7 Next, ATPZ analogues were evaluated in vitro for their
ability to disrupt the heparin-induced fibrillization of the K18 tau
fragment (amino acids 244–369)11 bearing the P301L12 mutation
found in inherited frontotemporal lobar degeneration (K18PL).
The activity of the ATPZs was determined by a thioflavin-T (ThT)
fluorescence assay, which monitors the formation of ThT-positive
cross-beta fibrils in the fibrillization reaction.13 In addition, all

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C. Ballatore et al. / Bioorg. Med. Chem. 20 (2012) 4451–4461
Table 1
Activity and Solubility of Test Compounds
R2
X
R3
R1
Solubility in assay buffer
K18PL ThT assay
K18PL centrifugation assay
N
N
O
R4
Compd number R1
–NH2
R2
R3
R4
X
Upper solubility limit (
l
M) IC50
(
l
M)
Max % inhib. % Soluble
26
a29
27
28
30
31
32
33
66
67
68
69
34
a37
35
36
38
39
40
70
42
43
44
a1
45
46
47
48
49
50
72
55
56
57
58
59
60
61
62
63
64
65
–H
–H
–H
–H
–H
–H
–H
–H
–COOEt
–COOEt
–COOEt
–COOEt
–COOEt
–COOEt
–COOEt
–COOEt
–COOEt
–COOEt
–COOEt
4-F-Ph
S
S
S
S
S
S
S
S
S
S
S
>200
13
12.8
32
1.0 1.11
4.0 2.99b
2.0 1.42b
3.2 2.02b
DNC
77
71
75
82
48
41
81
8
63
47
71
73
27
29
69
17
51
65
62
15
83
74
64
73
83
71
70
87
77
78
75
69
44
73
83
42
84
86
85
81
76
17
78
86
78
84
73
70
82
92
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
4-Cl-Ph
2-Cl-Ph
3-Cl-Ph
4-CF3-Ph
4-OCF3-Ph
4-OH-Ph
4-iPr-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-F-Ph
4-Cl-Ph
2-Cl-Ph
3-Cl-Ph
4-CF3-Ph
4-OCF3-Ph
4-OH-Ph
4-F-Ph
32
12.8
12.8
32
80
32
DNCb
5.0 1.29
DNCb
–NHCH3 –H
DNC
39
88
92
57
84
84
86
92
88
92
84
92
89
88
83
83
68
82
64
67
86
82
96
87
82
95
81
65
85
74
89
85
103
89
–NH2
–NH2
–OH
–Cl
–Br
2.0 1.53
4.0 1.10
DNC
12.8
–OH –COOEt
NH >200
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–OH
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–OH
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–COOH
–COOH
–COOH
–COOH
–COOH
–COOH
–COOH
–COOH
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
>200
>200
>200
32
>200
>200
>200
>200
>200
>200
>200
32
80
32
80
80
>200
80
ND
>200
>200
80
>200
>200
>200
>200
80
2.0 1.27
5.0 2.15b
3.2 1.58
1.6 1.22
19.9 1.10
1.6 1.53
4.0 1.58b
2.5 1.26b
1.6 1.39
2.5 2.93b
8.0 1.28
8.0 2.48b
DNC
–CONH-iPr
–CONH-cPr
–CONH–CH2CF3 4-F-Ph
4-F-Ph
4-F-Ph
–CONH-cPr
–CONH-cPr
–CONH-iPr
–CONH-cPr
–CONH-iPr
–CONH-iPr
–CONH-cPr
–CONH-iPr
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–Ph
–Ph
4-Cl-Ph
2-Cl-Ph
3-Cl-Ph
4-CF3-Ph
4-OCF3-Ph
4-OH-Ph
4-iPr-Ph
4-Cl-Ph
–H
–CH3
iPr
CH2CF3
Cyclohexyl
2-Py
3.2 2.02b
6.3 2.28
15.8 1.95
15.8 1.38
4.0 2.88
10.0 1.25
8.0 1.87b
2.5 1.19
19.9 1.16
10 1.18b
31.6 1.12
2.0 1.27
4.0 1.41
1.6 1.24
2.0 1.23
6.3 1.63b
19.9 1.63b
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
OEt
3-Py
–Bn
4-Cl-Ph
CH2CF3
iPr
80
>200
>200
CN
O
O
N
N
21
12.8
DNCb
4.4
21
Cl
O
COOEt
Ph
54
>200
DNC
29
6.5
NH2
S
aPreviously reported data;7 DNC = did not calculate due to absence of asymptote; ND = not determined; all IC50 values were determined through sigmoidal curve fitting of
compound concentration-response analyses, with each concentration tested in triplicate. All results are from a single curve fit, except where indicated byb, where the mean of
2 or more separate analyses are reported.
2.3. In vivo tolerability evaluation of ATPZ 43
signs of toxicity were seen until a dose of 43 of 100 mg/kg, at
which point only mild and transient lethargy was observed.
Subsequently, 43 was administered in drinking water (containing
3% PEG400 and 0.5% methylcellulose) to normal mice for a
1-month period at a dose equivalent of 50 mg/kg/day (assuming
To evaluate potential dose-limiting toxicities of ATPZ 43 in
mice, we determined the maximum tolerated dose (MTD) after a
single oral administration of the compound. Notably, no overt

C. Ballatore et al. / Bioorg. Med. Chem. 20 (2012) 4451–4461
4455
Table 2
Brain and plasma level of test compounds
Compd
Brain (ng/g)
Plasma (ng/mL)
B/Pa (total)
Fub (%) brain
Fub (%) plasma
B/P (fu)c
1e
43
42
72
49
44
55
56
60
62
1300 110
670 100
280 410
16.3 8.8
14.1 9.4
637 75
BLDd
820 150
610 27
110 160
4700 1000
150 100
401 14
BLDd
10 18
61 20
78 16
1.61 0.28
1.09 0.15
2.68 0.47
0.0035 0.0015
0.098 0.007
1.59 0.21
—
1.1 0.6
4.3 0.9
2.9 0.4
DNC
DNC
3.2 0.7
DNC
DNC
DNC
5.1 1.8
1.9 0.2
6.9 0.4
5.7 0.5
DNC
DNC
2.2 0.2
DNC
DNC
DNC
4.0 1.0
0.92 0.16
0.68 0.09
1.30 0.23
—
—
2.31 0.31
—
—
—
BLDd
—
7.5 5.3
48 18
0.116 0.039
0.60 0.12
0.77 0.15
a
b
c
Brain-to-plasma ratio.
Fraction unbound.
Ratio of free drug in brain and plasma.
Below limit of detection.
d
e
Previously reported data7; DNC = did not calculate due to low B/P.
bioavailability and safety that might be suitable for longer-term
in vivo studies. Based on the observation that the IC50 values of
all active ATPZs in our in vitro assay are typically in the range of
stoichiometric equivalence with tau, it seems plausible that this
chemotype may be interfering with the fibrillization reaction by
interacting with tau monomers. If this is the case, similar stoichi-
ometric requirement of tau:ATPZ may be expected in vivo for
effective inhibition of tau assembly. The intraneuronal tau concen-
tration is estimated to be ꢀ2
lM. However, under normal circum-
stances, tau is believed to be almost completely (>99%) bound to
microtubules (MTs).15 Thus, the free intraneuronal tau fraction
available to enter the fibrillization cascade may be <20 nM, even
under pathological conditions.4 Although the values of free tau
are only estimates, they suggest that candidate ATPZs should pref-
erably achieve free brain levels in this concentration range to be
effective if there is indeed a requirement for stoichiometric
amounts of the drug. From the results displayed in Table 1, the
pharmacophore of ATPZs would appear to be the thienopyridazine
heterocycle, while the R1–R4 substituents may be important deter-
minants of ADME-PK properties. In addition, our results suggest
that the presence of a para-fluorophenyl residue at the N3, a pri-
mary amino group at the C5 position, and a carboxylamide moiety
at C1 of the thienopyridazine heterocycle, comprise the optimal
substitution pattern to achieve the desired combination of activity
and ADME-PK properties. Indeed, based on the results from our PK
studies, administration of a dose of 50 mg/kg/day of compound 43
to mice in drinking water resulted in total brain compound levels
of ꢀ140 nM and a predicted brain free drug concentration of
6 nM. Furthermore, since the MTD for compound 43 was found
to be >50 mg/kg, it is likely that even higher doses could be utilized
during long-term in vivo testing. Taken together, these results indi-
cate that 43 and possibly other closely related congeners, such as
42 and 44, are suitable candidates for further investigating the
in vivo properties and potential biological activity of the ATPZ class
of tau fibrillization inhibitors.
Figure 2. Brain and plasma level of 43 after i.p. administration of 5 mg/kg.
5 ml water consumption/day/mouse). The amount of water con-
sumed by the compound-treated mice was normal, and no visible
adverse events were noted. Importantly, no differences in body
weight gain were observed between the compound- and vehicle-
treated (PEG400/methylcellulose) groups (n = 5/group; 9.7% and
8.2% increases of body weight, respectively). Moreover, there were
no differences in organ weights, including heart, liver, kidney,
spleen and brain, between the mice that received compound 43
or vehicle only (see Supplementary data). Similarly, there were
no alterations in any blood cell counts in the mice that were
administered the ATPZ (see Supplementary data). An evaluation
of brain levels of 43 after 3 days of dosing via drinking water at
50 mg/kg/day revealed total brain levels of 139 8 nM, with a cal-
culated free compound level of 6.0 0.4 nM. Notably, the plasma
and brain levels of 43 in mice that received compound in drinking
water for 30 days was comparable to the levels in mice that re-
ceived the compound in water for 3 days. This reveals that re-
peated dosing of the compound over a prolonged interval did not
result in the induction of liver enzymes that might alter the metab-
olism of the compound.
4. Conclusions
To investigate the SAR of ATPZs and to identify viable candi-
dates for in vivo studies, a series of new analogues were synthe-
sized and evaluated. Among the active, brain-penetrant ATPZ
analogues, 43 exhibited good oral bioavailability, as well as accept-
able water solubility and non-specific brain tissue binding, such
that oral administration of doses lower than the MTD are expected
to achieve potentially efficacious free drug concentrations in the
mouse brain after oral administration. These results indicate that
43 may be suitable for long-term in vivo testing.
3. Discussion
That ATPZ inhibitors of tau fibrillization hold promise as
potential candidates for in vivo testing was suggested by earlier
studies from our laboratory.6 The purpose of this study was to
investigate further the SAR of this class of compounds and to iden-
tify selected candidates that have solubility, brain-penetration, oral

4456
C. Ballatore et al. / Bioorg. Med. Chem. 20 (2012) 4451–4461
5.1.1.3. Ethyl 2-(2-(3-chlorophenyl)hydrazono)-3-oxobutanoate
(12).
Yield: 70%. 1H NMR (CDCl3): d 1.42 (t, J = 7.5 Hz, 3H),
2.60 (s, 3H), 4.36 (q, J = 7.2 Hz, 2H), 7.14 (d, J = 8.0 Hz, 1H), 7.24
(d, J = 9.0 Hz, 2H), 7.30 (t, J = 8.2 Hz, 1H), 7.48 (d, J = 3.2 Hz, 1H)
ppm. 13C NMR (CDCl3): d 14.4, 30.8, 61.1, 114.6, 116.4, 125.6,
5. Experimental
5.1. Materials and methods
All solvents were reagent grade. All reagents were purchased
from Aldrich or Acros and used as received. Thin layer chromatog-
raphy (TLC) was performed with 0.25 mm E. Merck pre-coated sil-
ica gel plates. Flash chromatography was performed with silica gel
60 (particle size 0.040–0.062 mm) supplied by Silicycle and Sor-
bent Technologies. Spots were detected by viewing under a UV
light. Yields refer to chromatographically and spectroscopically
pure compounds. Infrared spectra were recorded on a Jasco Model
FT/IR-480 Plus spectrometer. All melting points were obtained on a
Thomas-Hoover apparatus. Proton (1H) and carbon (13C) NMR
126.8, 130.6, 135.7, 143.0, 164.8, 197.4 ppm. IR:
m 3345, 1725,
1686 cmꢃ1. MS [ESI]+: Calcd for C12H14ClN2O3 269.07. Found
269.16.
+
5.1.1.4. Ethyl 2-(2-(4-chlorophenyl)hydrazono)-3-oxobutanoate
(13).
See Ref. 7.
5.1.1.5. Ethyl 3-oxo-2-(2-(4-(trifluoromethyl)phenyl)hydrazon-
o)butanoate (14).
Yield: 94%; 1H NMR (CDCl3): d 1.39 (t,
J = 7.1 Hz, 3H), 2.50 (s, 3H), 4.38 (q, J = 7.1 Hz, 2H), 7.40 (d,
J = 8.5 Hz, 2H), 7.62 (t, J = 6.6 Hz, 2H) ppm.
spectra were recorded on
a Bruker AMX-500 spectrometer.
Chemical shifts were reported relative to solvents. High-resolution
mass spectra were measured at the University of Pennsylvania,
Mass Spectrometry Service on a Waters LCT Premier XE LC–MS sys-
tem. Single-crystal X-ray structure determinations were performed
at the University of Pennsylvania. X-ray intensity data were
collected on a Bruker APEXII CCD area detector employing graph-
ite-monochromated Mo-Ka radiation (l = 0.71073 Å) at a tempera-
ture of 143(1) K. Analytical reversed-phased (Sunfire™ C18;
4.6 ꢂ 50 mm, 5 mL) high-performance liquid chromatography
(HPLC) was performed with a Water binary gradient module
2525 equipped with Waters 2996 PDA and Water micromass ZQ.
All samples were analyzed employing a linear gradient from 10%
to 90% of acetonitrile in water over 8 min and flow rate of 1 mL/
min, and unless otherwise stated, the purity level was >95%. Pre-
parative reverse phase HPLC purification was performed employ-
5.1.1.6. Ethyl 3-oxo-2-(2-(4-(trifluoromethoxy)phenyl)hydraz-
ono)butanoate (15).
Yield: 96%; 1H NMR (CDCl3): d 1.41 (m,
3H), 2.50 (s, 2H), 2.61 (s, 1H), 4.37 (m, 2H), 7.24–7.26 (m, 2H),
7.24–7.26 (m, 1H), 7.34–7.37 (m, 1H), 7.42–7.45 (m, 1H) ppm.
13C NMR (CDCl3): d 14.2, 27.0, 61.7, 116.6, 117.5, 122.49, 122.65,
128.0, 140.5, 163.8, 197.4 ppm.
5.1.1.7. Ethyl 2-(2-(4-hydroxyphenyl)hydrazono)-3-oxobutano-
ate (16).
Yield: 72%, mp: 106–108 °C. 1H NMR (CD3OD): d
1.36–1.41 (m, 3H), 2.46 (s, 2H), 2.53 (s, 1H), 4.30–438 (m, 2H),
6.84–6.87 (m, 2H), 7.31–7.34 (m, 1H) ppm. MS [ESI]+: Calcd for
+
C
12H15N2O4 251.10. Found 251.23.
5.1.1.8. (E/Z) Ethyl 2-(2-(4-isopropylphenyl)hydrazono)-3-oxo-
butanoate (17).
Yield: 94%; 1H NMR (CDCl3): d 1.25–1.27 (m,
12H, 2 ꢂ CH(CH3)2), 1.41 (m, 6H, 2 ꢂ CH2CH3), 2.49, 2.59 (s, 6H,
2 ꢂ CH3), 2.92 (m, 2H, 2 ꢂ CH), 4.32–4.40 (m 4H, 2 ꢂ CH2CH3),
7.24–7.30 (m, 6H), 7.36 (d, J = 8.5 Hz, 2H) ppm.
ing Waters SunFire™ prep C18 OBD™ columns (5
l
m 19 ꢂ 50 or
19 ꢂ 100 mm). All samples were purified employing a linear gradi-
ent from 10% to 90% of acetonitrile in water over 15 min and flow
rate of 20 mL/min. The preparative HPLC system was equipped
with Gilson 333 pumps, a 215 Liquid Handler, 845Z injection mod-
ule, and UV detector. Unless otherwise stated, all final compounds
were found to be >95% as determined by HPLC/MS and NMR.
5.1.1.9. General procedure for the synthesis of pyridazines
C.
Method A: A mixture of the appropriate hydrazone B
(1.0 mmol), ethyl cyanoacetate (0.113 g, 1.0 mmol), and sodium
acetate (0.17 g, 2.4 mmol) in acetic acid (3.3 mL) was heated to
170 °C using microwave irradiation for 4 min. After cooling, the
precipitate was collected and crystallized in ethanol to give the de-
sired pyridazine C. Method B: A mixture of the appropriate hydra-
5.1.1. General procedure for the synthesis of hydrazones B
To a solution of the appropriate aniline (39.2 mmol) and 37%
hydrochloridric acid (9.93 mL) in water (4.96 mL) and ethanol
(4.96 mL), a solution of sodium nitrite (2.93 g, 42.5 mmol) in water
(13 mL) was added dropwise at 0 °C. The reaction mixture was stir-
red at 0 °C for 30 min and then a solution of sodium acetate
(12.65 g, 154 mmol) in water (20 mL) and a solution of ethyl aceto-
acetate (5.10 g, 4.96 mL, 39.2 mmol) in ethanol (10 mL) were
added maintaining the temperature below 5 °C. The reaction mix-
ture was stirred at 0 °C for 4 h. Water was then added and the
resultant mixture was extracted with ethyl acetate. The combined
organic extracts were washed with brine, dried over MgSO4, fil-
tered and concentrated in vacuo. Finally, crystallization from etha-
nol provided the desired hydrazone B, which was directly used
without further purification.
zone
B
(6.29 mmol), ethyl cyanoacetate (1.06 g, 1.0 mL,
9.44 mmol), and 4-aminobutyric acid was heated to 160 °C for
2.5 h. After cooling, the residue was purified by silica gel column
chromatography using ethyl acetate-hexanes 1:3 as eluant to pro-
vide the desired pyridazine C.
5.1.1.10. Ethyl 5-cyano-1-(4-fluorophenyl)-4-methyl-6-oxo-1,6-
dihydropyridazine-3-carboxylate (18).
Method A. Yield:
55%, mp: 146–147 °C. 1H NMR (CDCl3): d 1.40 (t, J = 7.0 Hz, 3H),
2.76 (s, 3H), 4.43 (q, J = 7.2 Hz, 2H), 7.19 (t, J = 8.7 Hz, 2H), 7.63
(dd, J = 9.0 and 5.0 Hz, 2H) ppm. 13C NMR (CDCl3): d 14.2, 19.3,
62.9, 112.4, 115.9, 116.1 (d, JCF2 = 23.0 Hz), 127.1 (d, JCF3 = 8.6 Hz),
136.1, 137.4, 150.8, 155.9, 162.0, 162.7 (d, JCF1 = 243.9 Hz). IR
5.1.1.1. Ethyl 2-(2-(4-fluorophenyl)hydrazono)-3-oxobutanoate
(10).
Yield: 98%, mp: 81–83 °C. 1H NMR (CDCl3): d 1.40 (t,
(film):
m .
3123, 1727, 1683, 1602 cmꢃ1
J = 7.0 Hz, 3H), 2.59 (s, 3H), 4.34 (q, J = 7.2 Hz, 2H), 7.09 (t,
J = 8.7 Hz, 2H), 7.39 (dd, J = 9.0 and 5.0 Hz, 2H) ppm. 13C NMR
(CDCl3): d 14.4, 30.8, 61.1, 116.5 (d, JCF2 = 22.9 Hz), 118.0 (d,
JCF3 = 8.1 Hz), 126.1, 138.1, 161.3 (d, JCF1 = 243.9 Hz), 165.0,
5.1.1.11. Ethyl 1-(2-chlorophenyl)-5-cyano-4-methyl-6-oxo-1,6-
dihydropyridazine-3-carboxylate (19).
Method B. Yield:
67%, mp: 93–95 °C (from ethanol). 1H NMR (CDCl3): d 1.38 (t,
J = 7.1 Hz, 3H), 2.78 (s, 3H), 4.41 (q, J = 7.1 Hz, 2H), 7.40–7.49 (m,
3H), 7.57 (dd, J = 7.9 and 1.4 Hz, 1H) ppm. 13C NMR (CDCl3): d
14.3, 19.6, 63.0, 112.3, 115.9, 128.1, 128.8130.8, 131.5, 131.6,
197.2 ppm. IR (film):
for C12H14FN2O3 253.10. Found 253.25.
m
3125, 1701, 1620 cmꢃ1. MS [ESI]+: Calcd
+
5.1.1.2. Ethyl 2-(2-(2-chlorophenyl)hydrazono)-3-oxobutanoate
137.6, 137.8, 151.5, 155.4, 162.0 ppm. IR:
m .
2235, 1731, 1690 cmꢃ1
(11).
See Ref. 16.

C. Ballatore et al. / Bioorg. Med. Chem. 20 (2012) 4451–4461
4457
5.1.1.12. Ethyl 1-(3-chlorophenyl)-5-cyano-4-methyl-6-oxo-1,6-
5.1.2.3. Ethyl 5-amino-3-(3-chlorophenyl)-4-oxo-3,4-dihydro-
thieno-[3,4-d]pyridazine-1-carboxylate (28).
Yield: 72%. 1H
dihy-dropyridazine-3-carboxylate (20).
Method B. Yield:
90%. 1H NMR (CDCl3): d 1.41 (d, J = 7.0 Hz, 6H), 2.76 (s, 3H), 4.45
(q, J = 7.1 Hz, 2H), 7.44–7.45 (m, 2H), 7.57–7.59 (m, 1H), 7.67 (s,
1H) ppm. 13C NMR (CDCl3): d 14.2, 19.3, 63.0, 112.3, 116.1 123.3,
125.4, 129.7, 130.1, 134.9, 137.6, 140.8, 150.9, 155.7, 161.9 ppm.
NMR (CDCl3): d 1.44 (t, J = 7.2 Hz, 3H), 4.45 (q, J = 7.1 Hz, 2H),
6.20 (br s, 2H), 7.26 (s, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.39 (t,
J = 8.1 Hz, 1H), 7.54 (d, J = 8.1 Hz, 1H), 7.66 (s, 1H) ppm. 13C NMR
(CDCl3): d 14.4, 62.2, 105.0, 106.7, 124.1, 126.2, 127.0, 127.8,
129.7, 134.1, 134.3, 141.7, 159.4, 161.9, 163.0 ppm. MS [ESI]+:
Calcd for C15H14N3O4S+ 350.04. Found 350.09.
IR:
m
3429, 3221, 1690, 1634 cmꢃ1
.
MS [ESI]+: Calcd for
+
C15H13ClN3O3 318.06. Found 318.08.
5.1.1.13. Ethyl 5-amino-3-(2-chlorophenyl)-4-oxo-3,4-dihydro-
thieno-[3,4-d]pyridazine-1-carboxylate (21). See Ref. 7.
5.1.2.4. Ethyl 5-amino-3-(4-chlorophenyl)-4-oxo-3,4-dihydro-
thieno-[3,4-d]pyridazine-1-carboxylate (29).
See Ref. 7.
5.1.1.14. Ethyl 5-cyano-4-methyl-6-oxo-1-(4-(trifluoromethyl)
5.1.2.5. Ethyl 5-amino-4-oxo-3-(4-(trifluoromethyl)phenyl)-3,4-
phenyl)-1,6-dihydropyridazine-3-carboxylate (22).
Method
dihydrothieno[3,4-d]pyridazine-1-carboxylate (30).
Yield:
A. Yield: 35%; 1H NMR (CDCl3): d 1.41 (t, J = 7.1 Hz, 3H), 2.77 (s,
3H), 4.44 (q, J = 7.1 Hz, 2H), 7.77 (d, J = 8.6 Hz, 2H), 7.83 (d,
J = 8.6 Hz, 2H) ppm. 13C NMR (CDCl3): d 14.2, 19.4, 63.0, 112.3,
116.3, 125.5, 123.7 (q, JCF1 = 272.5 Hz), 126.36 (q, JCF2 = 3.7 Hz),
131.38 (q, JCF2 = 37.8 Hz), 137.9, 142.7, 151.1, 155.7, 161.9 ppm
90%. 1H NMR (CDCl3): d 1.43 (t, J = 7.1 Hz, 2H), 4.45 (q, J = 7.1 Hz,
1H), 7.27 (s, 1H), 7.72 (d, J = 8.6 Hz, 1H), 7.80 (d, J = 8.4 Hz, 1H),
7.72 (d, J = 8.6 Hz, 1H), 7.80 (d, J = 8.4 Hz, 1H) ppm. 13C NMR
(CDCl3): d 14.4, 62.2, 100.1, 105.3, 106.7, 123.0, 125.2, 125.85,
125.89, 125.91, 127.0, 129.2, 129.5, 134.4, 143.6, 159.4, 162.0,
162.9 ppm. IR (film):
m
3438, 3333, 1658, 1604 cmꢃ1. MS [ESI]+:
5.1.1.15. Ethyl 5-cyano-4-methyl-6-oxo-1-(4-(trifluoromethoxy)
Calcd for C16H13F3N3O3S+ 384.06. Found 384.15.
phenyl)-1,6-dihydropyridazine-3-carboxylate (23).
Method
B. Yield: 43%; 1H NMR (CDCl3): d 1.41 (t, J = 7.1 Hz, 3H), 2.77 (s,
3H), 4.44 (q, J = 7.1 Hz, 2H), 7.36 (d, J = 8.3 Hz, 2H), 7.72 (d,
J = 9.1 Hz, 2H) ppm. 13C NMR (CDCl3): d 14.3, 19.4, 63.0, 112.3,
116.2, 121.5, 126.7, 137.7, 138.3, 150.9, 155.8, 162.0 ppm.
5.1.2.6. Ethyl 5-amino-4-oxo-3-(4-(trifluoromethoxy)phenyl)-
3,4-dihydrothieno[3,4-d]pyridazine-1-carboxylate
(31).
Yield: 55%. 1H NMR (CDCl3): d 1.43 (t, J = 7.1 Hz, 2H),
4.45 (q, J = 7.1 Hz, 2H), 7.26 (d, J = 6.5 Hz, 1H), 7.31 (d, J = 8.5 Hz,
2H), 7.67 (d, J = 2.0 Hz, 2H) ppm. 13C NMR (CDCl3): d 14.4, 62.2,
105.1, 119.6, 121.3, 127.1, 127.3, 133.8, 134.1, 139.1, 148.2,
159.4, 161.8, 163.0 ppm. MS [ESI]+: Calcd for C16H13F3N3O4S+
400.06. Found 400.07.
5.1.1.16. Ethyl 5-cyano-1-(4-hydroxyphenyl)-4-methyl-6-oxo-
1,6-dihydropyridazine-3-carboxylate (24).
See Ref. 17.
5.1.1.17. Ethyl 5-cyano-1-(4-isopropylphenyl)-4-methyl-6-oxo-
1,6-dihydropyridazine-3-carboxylate (25).
Method B. Yield:
5.1.2.7. Ethyl 5-amino-3-(4-hydroxyphenyl)-4-oxo-3,4-dihydro-
thieno[3,4-d]pyridazine-1-carboxylate (32).
85%; 1H NMR (CDCl3): d 1.28 (d, J = 7.0 Hz, 6H), 1.40 (t, J = 7.0 Hz,
3H), 2.76 (s, 3H), 2.98 (sept, J = 6.8 Hz, 1H), 4.42 (q, J = 7.1 Hz,
2H), 7.35 (d, J = 8.5 Hz, 2H), 7.54 (d, J = 8.5 Hz, 2H) ppm. 13C NMR
(CDCl3): d 14.3, 19.3, 24.0, 34.1, 62.8, 112.6, 115.8, 124.9, 127.2,
137.1, 137.9, 150.5, 150.6, 156.0, 162.2 ppm.
Yield: 46%. 1H
NMR (DMSO-d6): d 1.29 (t, J = 7.2 Hz, 3H), 4.31 (q, J = 7.0 Hz, 2H),
6.83 (d, J = 8.5 Hz, 2H), 7.07 (s, 1H), 7.25 (d, J = 9.0 Hz, 2H), 7.57
(br s, 2H), 9.68 (br s, 1H) ppm. 13C NMR (DMSO-d6): d 14.7, 61.9,
103.5, 104.8, 115.5, 126.8, 128.3, 132.8, 157.3, 159.1, 163.1,
163.5. IR (film):
C
m .
3404, 1639 cmꢃ1 MS [ESI]+: Calcd for
15H14N3O4S+ 332.07. Found 332.04.
5.1.2. General procedure for synthesis of
aminothienopyridazines D
A mixture of the appropriate pyridazine C (0.31 mmol), sulfur
(15 mg, 0.46 mmol), and morpholine (54 mg, 54 lL, 0.62 mmol)
in ethanol (1.5 mL) was heated to 150 °C using microwave irradia-
tion for 15 min. After cooling, the precipitate was collected and
purified by silica gel column chromatography using ethyl acetate
and hexane 1:3 as eluant to obtain the desired aminothienopyrid-
azine D.
5.1.2.8. Ethyl 5-amino-3-(4-isopropylphenyl)-4-oxo-3,4-dihy-
dro–thieno[3,4-d]pyridazine-1-carboxylate
(33).
Yield:
72%. 1H NMR (CDCl3): d 1.28 (d, J = 7.0 Hz, 6H), 1.42 (t, J = 7.5 Hz,
3H), 2.96 (sept, J = 6.9 Hz, 1H), 4.43 (q, J = 7.0 Hz, 2H), 6.20 (br s,
2H), 7.23 (s, 1H), 7.31 (d, J = 8.0 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H)
ppm. 13C NMR (CDCl3): d 14.1, 24.1, 34.0, 62.0, 104.4, 107.2,
126.0, 126.9, 127.4, 133.5, 138.3, 148.6, 159.7, 161.5, 163.2 ppm.
IR (film):
m .
3426, 3322, 3179, 1705, 1658, 1601 cmꢃ1
5.1.2.1. Ethyl 5-amino-3-(4-fluorophenyl)-4-oxo-3,4-dihydro-
thieno[3,4-d]pyridazine-1-carboxylate (26).
Yield: 70%,
5.1.3. General procedure for ester hydrolysis
mp: 178–180 °C. 1H NMR (CDCl3): d 1.42 (t, J = 6.9 Hz, 3H), 4.43
(q, J = 7.1 Hz, 2H), 6.06 (br s, 2H), 7.13 (t, J = 8.3 Hz, 2H), 7.22 (s,
1H), 7.54–7.57 (m, 2H) ppm. 13C NMR (CDCl3): d 14.4, 62.1,
104.7, 106.2, 115.7 (d, JCF2 = 22.8 Hz), 127.9, (d, JCF3 = 8.6 Hz),
133.9, 136.7, 159.5, 161.7 (d, JCF1 = 269.8 Hz), 163.1 ppm. IR (film):
To a solution of the appropriate aminothienopyridazine of gen-
eral structure D (0.43 mmol) in tetrahydrofuran (3 mL) and water
(2 mL), lithium hydroxide monohydrate (0.063 g, 1.29 mmol) was
added. The reaction mixture was stirred at rt for 16 h. The reaction
was quenched with 1 N hydrochloric acid (pH ꢀ2) and filtered.
Purification by reverse phase preparative HPLC afforded the de-
sired acid of general structure E.
m
3423, 3302, 3128, 1718, 1641 cmꢃ1
15H13FN3O3S+ 334.07. Found 334.11.
.
MS [ESI]+: Calcd for
C
5.1.2.2. Ethyl 5-amino-3-(2-chlorophenyl)-4-oxo-3,4-dihydro-
thieno-[3,4-d]pyridazine-1-carboxylate (27).
Yield: 87%. 1H
5.1.3.1. 5-Amino-3-(4-fluorophenyl)-4-oxo-3,4-dihydrothieno
[3,4-d]pyridazine-1-carboxylic acid (34).
Yield: 77%. 1H
NMR (CDCl3): d 1.28 (t, J = 7.0 Hz, 3H), 4.31 (q, J = 7.0 Hz, 2H),
7.15 (s, 1H), 7.47 (m, 2H), 7.53–7.60 (m, 3H), 7.64 (s, 1H) ppm.
13C NMR (CDCl3): d 14.0, 54.9, 61.4, 103.5, 104.1, 126.0, 128.0,
129.7, 130.4, 130.6, 131.7, 133.0, 138.3, 157.9, 162.2, 163.1 ppm.
NMR (DMSO-d6): d 7.12 (s, 1H), 7.29 (t, J = 8.7 Hz, 2H), 7.55–7.58
(m, 4H) ppm. 13C NMR (DMSO-d6): d 104.3, 104.5, 115.8 (d,
JCF2 = 22.5 Hz), 126.9, 128.9 (d, JCF3 = 8.5 Hz), 134.2, 137.7, 159.0,
161.3 (d, JCF1 = 242.4 Hz), 163.7, 164.6 ppm. MS [ESI]+: Calcd for
IR:
C
m
3425, 3303, 1710, 1648, 1587 cmꢃ1. HRMS [ESI]+: Calcd for
C
13H9FN3O3S+ 306.03. Found 306.07.
15H12ClNaN3O3S+ 372.0186. Found 372.0190.

4458
C. Ballatore et al. / Bioorg. Med. Chem. 20 (2012) 4451–4461
5.1.3.2.
5-Amino-3-(2-chlorophenyl)-4-oxo-3,4-dihydrothie-
Yield: 91%. 1H
161.2 (d, JCF1 = 246.7 Hz), 163.4, 164.4 ppm. IR (film):
3105 (br band), 1660, 1643, 1587 cmꢃ1. MS [ESI]+: Calcd for
16H14FN4O2S+ 345.08. Found 345.14.
m 3298,
no[3,4-d]pyridazine-1-carboxylic acid (35).
NMR (DMSO-d6): d 7.17 (s, 1H), 7.50 (m, 2H), 7.55–7.59 (m, 3H),
7.63–7.65 (m, 1H) ppm. 13C NMR (DMSO-d6): d 103.7, 104.4,
126.4, 128.0, 129.7, 130.4, 130.7, 131.8, 134.0, 138.4, 158.2,
163.0, 164.0 ppm.
C
5.1.4.3. 5-Amino-3-(4-fluorophenyl)-4-oxo-N-(2,2,2-trifluoro-
ethyl)-3,4-dihydrothieno[3,4-d]pyridazine-1-carboxamide
(44).
Yield 54%; 1H NMR (CDCl3): d 4.04 (qd, J = 8.9, 6.8 Hz,
5.1.3.3.
5-Amino-3-(3-chlorophenyl)-4-oxo-3,4-dihydrothie-
Yield: 89%. 1H
2H), 6.19 (s, 2H), 7.16–7.19 (m, 2H), 7.38 (t, J = 6.4 Hz, 1H), 7.52–
7.49 (m, 2H), 7.55 (s, 1H) ppm. 13C NMR (CDCl3): d 40.4, 40.7,
106.5, 106.8, 115.79, 115.97, 123.0, 126.4, 127.92, 127.99, 134.2,
136.48, 136.51, 159.6, 160.9, 161.6, 162.92, 162.97 ppm. HRMS
[ESI]ꢃ: Calcd for C15H9F4N4O2Sꢃ 385.0382. Found 385.0397.
no[3,4-d]pyridazine-1-carboxylic acid (36).
NMR (DMSO-d6): d 7.13 (s, 1H), 7.44 (d, J = 8.4 Hz, 1H), 7.50 (t,
J = 8.5 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.65–7.68 (m, 3H) ppm.
13C NMR (DMSO-d6): d 104.3, 104.7, 125.2, 126.4, 126.8, 127.6,
130.6, 133.1, 142.6, 148.9, 157.7, 158.9, 164.0 ppm. IR:
v 3439,
3322, 3065 (br, acid band), 1712, 1646, 1590 cmꢃ1. MS [ESI]+: Calcd
for C13H9ClN3O3S+ 322.00. Found 322.10.
5.1.4.4. 5-Amino-3-(2-chlorophenyl)-N-cyclopropyl-4-oxo-3,4-
dihydrothieno[3,4-d]pyridazine-1-carboxamide (45).
Yield
62%; 1H NMR (CDCl3): d 0.59–0.62 (m, 2H), 0.82 (dd, J = 6.9,
5.4 Hz, 2H), 2.79 (m, 1H), 6.12 (bs, 2H), 7.12 (d, J = 4.2 Hz, 1H),
7.46–7.40 (m, 3H), 7.56 (dd, J = 5.8, 3.5 Hz, 1H), 7.64 (s, 1H) ppm.
13C NMR (CDCl3): d 6.6, 22.5, 106.8, 107.1, 126.7, 127.7, 130.06,
130.23, 130.5, 132.9, 135.3, 138.2, 159.5, 161.3, 164.3 ppm. HRMS
[ESI]+: Calcd for C16H13N4O2SCl+ 361.0526. Found 361.0537.
5.1.3.4.
5-Amino-3-(4-chlorophenyl)-4-oxo-3,4-dihydrothie-
See Ref. 7.
no[3,4-d]pyridazine-1-carboxylic acid (37).
5.1.3.5. 5-Amino-4-oxo-3-(4-(trifluoromethyl)phenyl)-3,4-dihy-
drothieno[3,4-d]pyridazine-1-carboxylic acid (38).
Yield:
81%. 1H NMR (CD3OD): d 7.21 (s, 1H), 7.76 (d, J = 8.3 Hz, 2H), 7.86
(d, J = 8.3 Hz, 2H) ppm.
5.1.4.5.
5-Amino-3-(3-chlorophenyl)-N-isopropyl-4-oxo-3,4-
dihydrothieno[3,4-d]pyridazine-1-carboxamide (46).
Yield:
5.1.3.6.
5-Amino-4-oxo-3-(4-(trifluoromethoxy)phenyl)-3,4-
acid
Yield: 51%. 1H NMR (CD3OD): d 7.17 (s, 1H), 7.35 (d,
53%. 1H NMR (CD3OD): d 1.27 (d, J = 6.6 Hz, 6H), 4.12–4.16 (m, 1H),
7.28 (s, 1H), 7.38 (dt, J = 8.0, 0.9 Hz, 1H), 7.46 (t, J = 8.0 Hz, 1H), 7.58
(dt, J = 8.0, 0.9 Hz, 1H), 7.71 (s, 1H) ppm. 13C NMR (CD3OD): d 19.7,
40.0, 103.13, 103.28, 122.9, 124.6, 124.9, 125.6, 128.1, 132.2, 135.2,
dihydrothieno[3,4-d]pyridazine-1-carboxylic
(39).
J = 8.5 Hz, 3H), 7.69 (d, J = 8.9 Hz, 3H) ppm. IR (film):
m 3428, 3329,
1725, 1637, 1598 cmꢃ1
.
13C NMR (CD3OD): d 102.7, 103.1, 118.1,
140.6, 157.9, 161.4, 162.2 ppm. IR (film): m 3413, 3305, 2971, 1652,
119.3, 120.2, 125.0, 126.1, 132.8, 138.1, 146.4, 157.9, 162.4, 162.8 ppm.
1591 cmꢃ1. MS [ESI]+: Calcd for C16H16ClN4O2S+ 363.84. Found
363.16.
5.1.3.7. 5-Amino-3-(4-hydroxyphenyl)-4-oxo-3,4-dihydrothie-
no[3,4-d]pyridazine-1-carboxylic acid (40).
NMR (DMSO-d6): d 6.82 (d, J = 8.6 Hz, 2H), 7.09 (s, 1H), 7.26 (d,
J = 8.6 Hz, 2H), 7.55 (s, 2H), 9.63 (s, 1H) ppm. IR (film): 3425
(br, acid band), 1725, 1635 cmꢃ1
Yield: 76%. 1H
5.1.4.6. 5-Amino-3-(4-chlorophenyl)-N-cyclopropyl-4-oxo-3,4-
dihydrothieno[3,4-d]pyridazine-1-carboxamide
(1).
See
m
Ref. 7.
.
5.1.4.7. 5-Amino-N-cyclopropyl-4-oxo-3-(4-(trifluoromethyl)-
phenyl)-3,4-dihydrothieno[3,4-d]pyridazine-1-carboxamide
(47).
5.1.4. General procedure for the synthesis of compounds of
general structure F
Yield: 53%. 1H NMR (DMSO-d6): d 0.57–0.60 (m, 2H),
To a mixture of the appropriate acid of general structure E
(0.15 mmol), the appropriate amine (0.22 mmol), and benzotriazole-
1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate
(0.097 g, 0.22 mmol) in anhydrous dimethylsulfoxide (2 mL), N,N-
0.66–0.70 (m, 2H), 2.79 (m, 1H), 7.21 (s, 1H), 7.64 (s, 2H), 7.80 (d,
J = 8.6 Hz, 2H), 7.91 (d, J = 8.5 Hz, 2H), 8.32 (d, J = 4.1 Hz, 1H) ppm.
13C NMR (DMSO-d6): d 6.4, 23.2, 104.3, 105.5, 125.85, 125.88,
125.91, 126.4, 126.8, 137.4, 144.6, 158.9, 164.0, 164.3 ppm. MS
[ESI]+: Calcd for C17H14F3N4O2S+ 395.08. Found 394.98.
diisopropyl-ethylamine (0.028 g, 38 lL, 0.23 mmol) was added. The
reaction mixture was stirred at room temperature for 4 h. The reaction
was quenched with water and extracted with ethyl acetate. The com-
bined organic extracts were washed with brine, dried over MgSO4, fil-
tered, and concentrated in vacuo. Purification by preparative reverse
phase HPLC furnished the desired amide of general structure F.
5.1.4.8.
phenyl)-3,4-dihydrothieno[3,4-d]pyridazine-1-carboxamide
(48).
Yield 50%; 1H NMR (CDCl3): d 1.25 (d, J = 6.6 Hz, 6H),
5-Amino-N-isopropyl-4-oxo-3-(4-(trifluoromethoxy)-
4.18–4.25 (m, 1H), 6.19 (s, 2H), 6.89 (d, J = 7.9 Hz, 1H), 7.34 (d,
J = 8.2 Hz, 2H), 7.60–7.62 (m, 3H) ppm. 13C NMR (CDCl3): d 22.8,
41.6, 106.89, 106.98, 121.4, 126.7, 127.4, 135.8, 139.2, 159.6,
5.1.4.1.
5-Amino-3-(4-fluorophenyl)-N-isopropyl-4-oxo-3,4-
dihydrothieno[3,4-d]pyridazine-1-carboxamide (42).
Yield:
161.5, 161.9 ppm. IR (film): m
3411, 3281, 3176, 1647 cmꢃ1. MS
74%. 1H NMR (CDCl3): d 1.24 (d, J = 7.0 Hz, 6H), 4.17–4.24 (m, 1H),
5.83 (br s, 2H), 6.89 (br s, 1H), 7.15–7.18 (m, 2H), 7.50–7.58 (m, 3H)
ppm. 13C NMR (CDCl3): d 22.8, 41.5, 106.6, 106.9, 115.7 (d,
JCF2 = 22.6 Hz), 126.8, 128.0 (d, JCF3 = 8.5 Hz), 135.5, 136.8, 159.7,
[ESI]+: Calcd for C17H16F3N4O3S+ 413.09. Found 413.20.
5.1.4.9. 5-Amino-3-(4-hydroxyphenyl)-N-isopropyl-4-oxo-3,4-
dihydrothieno[3,4-d]pyridazine-1-carboxamide (49).
Yield:
161.5, 161.7 (d, JCF1 = 245.5 Hz), 162.0 ppm. IR (film):
m
3405,
49%. 1H NMR (DMSO-d6): d 1.14 (d, J = 6.6 Hz, 6H), 4.02–4.07 (m,
1H), 6.82 (d, J = 8.8 Hz, 2H), 7.16 (s, 1H), 7.34 (d, J = 8.8 Hz, 2H),
3301, 1651, 1598 cmꢃ1
347.10. Found 347.11.
.
MS [ESI]+: Calcd for C16H16FN4O2S+
7.50 (s, 2H), 7.85 (d, J = 8.1 Hz, 1H), 9.61 (s, 1H) ppm. IR (film):
3429, 3323, 3269, 2975, 1640, 1507 cmꢃ1. MS [ESI]+: Calcd for
16H17N4O3S+ 345.10. Found 345.23.
m
5.1.4.2. 5-Amino-N-cyclopropyl-3-(4-fluorophenyl)-4-oxo-3,4-
C
dihydrothieno[3,4-d]pyridazine-1-carboxamide (43).
Yield:
71%. 1H NMR (DMSO-d6): d 0.54–0.72 (m, 4H), 2.74–2.81 (m, 1H),
7.20 (s, 1H), 7.23–7.31 (m, 2H), 7.53–7.71 (m, 4H), 8.25 (br s, 1H)
ppm. 13C NMR (DMSO-d6): d 6.3, 23.2, 104.6, 104.9, 115.5 (d,
JCF2 = 22.2 Hz), 126.6, 128.7 (d, JCF3 = 9.2 Hz), 136.7, 137.5, 159.0,
5.1.4.10. 5-Amino-N-cyclopropyl-3-(4-isopropylphenyl)-4-oxo-
3,4-dihydrothieno[3,4-d]pyridazine-1-carboxamide
(50).
0.65–0.69 (m, 2H), 2.79 (td, J = 7.3, 4.0 Hz, 1H), 2.94 (dt, J = 13.8,
Yield: 55%. 1H NMR (DMSO-d6): d 0.57–0.60 (m, 2H),

C. Ballatore et al. / Bioorg. Med. Chem. 20 (2012) 4451–4461
4459
6.9 Hz, 1H), 7.19 (s, 1H), 7.30 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.4 Hz,
2H), 7.54 (s, 2H), 8.20 (d, J = 4.3 Hz, 1H) ppm. 13C NMR (DMSO-d6):
d 6.3, 23.2, 24.5, 33.8, 104.64, 104.81, 126.58, 126.68, 126.75,
136.5, 139.1, 147.8, 159.1, 163.3, 164.5 ppm. MS [ESI]+: Calcd for
105.0, 131.4, 141.2, 159.0, 162.0 ppm. MS [ESI]+: Calcd for
10H14N3OS+ 224.08. Found 224.00.
C
5.1.5.4. 7-Amino-4-methyl-2-(2,2,2-trifluoroethyl)thieno[3,4-d]
pyridazin-1(2H)-one (58).
Yield: 47%. 1H NMR (DMSO-d6): d
C
19H19N4O2S+ 369.14. Found 369.13.
2.23 (s, 3H), 4.65 (q, J = 9.2 Hz, 2H), 6.75 (s, 1H), 7.46 (s, 2H)
ppm. 13C NMR (DMSO-d6): d 18.5, 48.13 (d, JCF2 =33.1 Hz), 103.0,
103.4, 131.0, 142.9, 159.5, 163.2 ppm. HRMS [ESI]ꢃ: Calcd for
C9H7N3OF3Sꢃ 262.0262. Found 262.0272. X-ray crystal structure
shown in Supplementary data.
5.1.4.11.
(53).
Ethyl
4-acetyl-2-aminothiophene-3-carboxylate
mixture of 2,3-butanedione (20 mg, 20 L,
L, 0.23 mmol), and
sulfur (11 mg, 0.35 mmol) in N,N-dimethylformammide (0.5 mL)
morpholine (40 mg, 40 L, 0.46 mmol) was added. The reaction
To
a
l
0.23 mmol), ethyl cyanoacetate (26 mg, 25
l
l
mixture was stirred at rt for 16 h. A saturated solution of sodium
chloride was added and the resulting mixture was extracted with
ethyl acetate. The combined organic extracts were washed with
brine, dried over sodium sulfate, filtered and concentrated in va-
cuo. Crystallization from acetate-hexanes furnished 53. Yield:
32%, mp: 105–106 °C. 1H NMR (CDCl3): 1.32 (t, J = 7.2 Hz, 3H),
2.42 (s, 3H), 4.28 (q, J = 7.2 Hz, 2H), 6.07 (br s, 2H), 6.42 (s, 1H)
ppm. 13C NMR (CDCl3): d 14.2, 30.6, 60.5, 104.6, 109.3, 143.0,
5.1.5.5. 7-Amino-2-cyclohexyl-4-methylthieno[3,4-d]pyridazin-
1(2H)-one (59).
Yield: 64%. 1H NMR (DMSO-d6): d 1.10–1.18
(m, 1H), 1.34 (qt, J = 13.1, 3.4 Hz, 2H), 1.61–1.73 (m, 6H), 1.79 (d,
J = 13.3 Hz, 2H), 2.22 (s, 3H), 4.58 (m, 1H), 6.61 (s, 1H), 7.32 (s,
2H) ppm. 13C NMR (DMSO-d6): d 18.9, 25.7, 26.0, 31.5, 53.0,
100.7, 105.0, 131.3, 140.9, 159.0, 162.1 ppm. MS [ESI]+: Calcd for
C
13H18N3OS+ 264.12. Found 264.07.
163.7, 164.7, 198.6 ppm. IR (film):
m
3429, 3324, 1677, 1593 cmꢃ1
.
5.1.5.6. 7-Amino-4-methyl-2-(pyridin-2-yl)thieno[3,4-d]pyrida-
zin-1(2H)-one (60).
Yield: 53%, mp: 227–229 °C. 1H NMR
MS [ESI]+: Calcd for C9H12NO3S+ 214.05. Found 214.22.
(CDCl3): 2.41 (s, 3H), 6.02 (br s, 2H), 6.46 (s, 1H), 7.29 (t,
J = 8.5 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.83 (t, J = 8.5 Hz, 1H), 8.66
(d, J = 4.0 Hz, 1H) ppm. 13C NMR (CDCl3): d 18.7, 102.2, 106.5,
121.5, 122.5, 131.7, 138.0, 143.3, 148.9, 153.0, 159.8, 161.8 ppm.
5.1.4.12. Ethyl 2-amino-4-benzoylthiophene-3-carboxylate
(54). A mixture of the 1-phenylpropane-1,2-dione (500 mg,
0.45 mL, 3.37 mmol), sulfur (162 mg, 5.05 mmol), ethyl cyanoace-
tate (382 mg, 0.36 mL, 3.37 mmol), and triethylamine (682 mg,
940 mL, 6.00 mmol) in N,N-dimethylformamide (10 mL) was stir-
red at rt for 12 h. The reaction was quenched with water and ex-
tracted with ethyl acetate. The combined organic extracts were
washed with brine, dried over sodium sulfate, filtered and concen-
trated in vacuo. Purification by silica gel column chromatography
using ethyl acetate-hexanes 2:3 as eluant provided 54. Yield:
95%, mp: 103–105 °C. 1H NMR (CDCl3): d 0.78 (t, J = 7.2 Hz, 3H),
3.90 (q, J = 7.2 Hz, 2H), 5.95 (br s, 2H), 6.44 (s, 1H), 7.42 (t,
J = 7.7 Hz, 2H), 7.53 (t, J = 7.2 Hz, 1H), 7.84 (d, J = 8.5 Hz, 2H) ppm.
13C NMR (CDCl3): d 13.5, 60.1, 106.0, 109.3, 128.4, 129.4, 133.0,
IR:
C
m .
3415, 3113, 1637, 1594 cmꢃ1 MS [ESI]+: Calcd for
12H11N4OS+ 259.06. Found 259.11.
5.1.5.7. 7-Amino-4-methyl-2-(pyridin-3-yl)thieno[3,4-d]pyrida-
zin-1(2H)-one (61).
Yield: 43%. 1H NMR (DMSO-d6): d 2.29 (s,
3H), 6.80 (s, 1H), 7.45–7.52 (m, 3H), 7.95 (dd, J = 8.1, 1.3 Hz, 1H),
8.46 (d, J = 0.7 Hz, 1H), 8.78 (d, J = 0.8 Hz, 1H) ppm. 13C NMR
(DMSO-d6): d 18.7, 102.9, 103.9, 123.9, 131.0, 133.2, 143.5, 146.9,
147.4, 149.9, 159.2, 163.7 ppm. MS [ESI]+: Calcd for C12H11N4OS+
259.06. Found 259.05.
138.0, 140.0, 163.5, 164.7, 193.0 ppm. IR (film):
m
3431, 3327,
5.1.5.8.
1(2H)-one (62).
7-Amino-2-benzyl-4-methylthieno[3,4-d]pyridazin-
Yield: 97%, mp: 123–125 °C. 1H NMR (CDCl3):
1667, 1595 cmꢃ1
.
2.30 (s, 3H), 5.17 (s, 2H), 5.99 (br s, 2H), 6.35 (s, 1H), 7.25 (t,
J = 8.5 Hz, 1H), 7.32 (t, J = 8.2 Hz, 2H), 7.41 (d, J = 8.5 Hz, 2H) ppm.
13C NMR (CDCl3): d 18.5, 52.2, 101.0, 107.3, 127.4, 128.2, 128.6,
5.1.5. General procedure for the synthesis of compounds of
general structure G
A mixture of the appropriate aminothiophene keto-ester (53 or
54, 0.23 mmol), the appropriate hydrazine (0.23 mmol) in ethanol
was stirred for 0.5–3 h at 150 °C (microwave irradiation). After
cooling, the solvent was removed and the residue was purified
by either preparative reverse phase HPLC, or silica gel column
chromatography using gradients of ethyl acetate in hexanes 1:3
as eluent, to give the desired compound of general structure G.
132.3, 138.1, 141.9, 159.9, 160.3 ppm. IR (film):
m 3397, 3112,
1600 cmꢃ1. MS [ESI]+: Calcd for C14H14N3OS+ 272.09. Found 272.17.
5.1.5.9.
d]pyridazin-1(2H)-one (63).
7-Amino-2-(4-chlorophenyl)-4-methylthieno[3,4-
Yield: 73%. 1H NMR (CDCl3): d
2.38 (s, 3H), 6.09 (br s, 2H), 6.47 (s, 1H), 7.40 (d, J = 9.0 Hz, 2H),
7.56 (d, J = 9.0 Hz, 2H) ppm. 13C NMR (CDCl3): d 18.6, 101.7,
107.2, 127.1, 128.8, 132.1, 132.5, 139.8, 142.7, 159.6, 162.3 ppm.
5.1.5.1.
(55).
7-Amino-4-methylthieno[3,4-d]pyridazin-1(2H)-one
Yield: 77%. 1H NMR (DMSO-d6): d 2.18 (s, 3H), 6.62 (s,
IR:
C
m .
3411, 3329, 1633, 1590 cmꢃ1 MS [ESI]+: Calcd for
13H11ClN3OS+ 292.03. Found 292.12.
1H), 7.26 (br s, 2H), 11.04 (br s, 1H) ppm. 13C NMR (DMSO-d6): d
31.3, 101.4, 104.9, 31.8, 141.5, 160.9, 162.0 ppm. IR:
m
3422, 3260,
5.1.5.10. 7-Amino-4-phenyl-2-(2,2,2-trifluoroethyl)thieno[3,4-
d]pyridazin-1(2H)-one (64).
Yield: 46%. 1H NMR (DMSO-d6):
1642 cmꢃ1. MS [ESI]+: Calcd for C7H8N3OS+ 182.04. Found 182.20.
d 4.77 (q, J = 9.2 Hz, 2H), 6.69 (s, 1H), 7.51–7.53 (m, 3H), 7.59 (s, 2H),
7.67–7.69 (m, 2H) ppm. 13C NMR (DMSO-d6): d 48.2, 48.5, 103.8,
104.3, 128.4, 128.9, 129.3, 130.1, 135.4, 144.5, 159.2, 163.6 ppm.
HRMS [ESI]+: Calcd for C14H11N3OF3S+ 326.0575. Found 326.0571.
5.1.5.2. 7-Amino-2,4-dimethylthieno[3,4-d]pyridazin-1(2H)-one
(56).
Yield: 79%, mp: 158–160 °C. 1H NMR (CDCl3): d 2.29 (s,
3H), 3.57 (s, 3H), 6.13 (br s, 2H), 6.36 (s, 1H) ppm. 13C NMR (CDCl3):
d 18.4, 36.6, 100.9, 107.3, 132.3, 141.5, 159.9, 160.1 ppm. IR:
m
3386, 3266, 1604 cmꢃ1. MS [ESI]+: Calcd for C8H10N3OS+ 196.05.
Found 196.17.
5.1.5.11. 7-Amino-2-isopropyl-4-phenylthieno[3,4-d]pyridazin-
1(2H)-one (65).
Yield: 36%. 1H NMR (DMSO-d6): d 1.27 (d,
J = 6.6 Hz, 6H), 5.11 (dt, J = 13.3, 6.6 Hz, 1H), 6.62 (s, 1H), 7.51–
7.45 (m, 5H), 7.71 (dd, J = 7.9, 1.5 Hz, 2H) ppm. 13C NMR (DMSO-
d6): d 21.6, 45.8, 101.9, 105.4, 128.3, 129.13, 129.21, 129.6, 136.5,
142.5, 158.8, 162.5 ppm. MS [ESI]+: Calcd for C15H16N3OS+
286.10. Found 285.96.
5.1.5.3. 7-Amino-2-isopropyl-4-methylthieno[3,4-d]pyridazin-
1(2H)-one (57).
Yield: 59%. 1H NMR (DMSO-d6): d 1.20 (d,
J = 6.7 Hz, 6H), 2.22 (s, 3H), 5.01 (7, J = 6.7 Hz, 1H), 6.61 (s, 1H),
7.31 (s, 2H) ppm. 13C NMR (DMSO-d6): d 19.0, 21.5, 45.2, 100.8,

4460
C. Ballatore et al. / Bioorg. Med. Chem. 20 (2012) 4451–4461
5.1.5.12. Ethyl 3-(4-chlorophenyl)-5-(methylamino)-4-oxo-3,4-
dihydrothieno[3,4-d]pyridazine-1-carboxylate (66). A mix-
and the desired product was filtered and washed with water. Yield:
58%. 1H NMR (DMSO-d6): d 4.93 (s, 2H), 7.61–7.66 (m, 4H) ppm.
ture of 29, potassium carbonate and iodometane in acetonitrile
was stirred at 70 °C for 10 h. The reaction was quenched by addi-
tion of water and then extracted with ethyl acetate. The combined
organic extracts were washed with brine, dried over MgSO4, fil-
tered and concentrated in vacuo. Purification by silica gel column
chromatography using ethyl acetate/hexanes 1:3 as eluant pro-
vided 68. Yield 27%; 1H NMR (CDCl3): d 1.43 (t, J = 7.0 Hz, 3H),
3.07 (d, J = 5.5 Hz, 3H), 4.44 (q, J = 7.0 Hz, 2H), 7.19 (s, 1H), 7.42
(d, J = 9.0 Hz, 2H), 7.54 (bs, 1H), 7.58 (d, J = 9.0 Hz, 2H) ppm. 13C
NMR (CDCl3): d 14.39, 33.99, 62.05, 102.83, 104.32, 127.07,
127.60, 128.82, 133.07, 134.08, 139.34, 159.39, 163.15,
5.1.5.18. 3-(4-Chlorophenyl)-N-isopropyl-4,5-dioxo-3,4,5,7-tet-
rahydrothieno[3,4-d]pyridazine-1-carboxamide (72).
Pre-
pared following the general procedure described for the
synthesis of compounds of general structure F. 1H NMR (DMSO-
d6): d 1.17 (d, J = 6.6 Hz, 6H), 4.07–4.13 (m, 1H), 4.96 (s, 2H),
7.62–7.65 (m, 2H), 7.70–7.72 (m, 2H), 8.36 (d, J = 8.2 Hz, 1H)
ppm. 13C NMR (DMSO-d6): d 22.6, 35.1, 41.6, 114.4, 128.8, 129.4,
131.1, 133.9, 136.8, 155.3, 157.5, 161.2 ppm.
5.1.6. Pharmacokinetic studies
165.50 ppm. IR (film):
m
3370, 2923, 1718, 1653, 1567 cmꢃ1. MS
Test compounds were administered to 2-month old female
B6C3F1 mice (20–25 g average body weight). Typical studies in-
volved i.p. injection of test ATPZ at a dose of 5 mg/kg, followed
by sacrifice 1 h later. For full PK analysis of compound 43, mice
(n = 3/time point) received i.p. injections at a dose of 5 mg/kg, fol-
lowed by sacrifice at times ranging from 0.5–16 h post-dosing. Per-
fused whole brain hemispheres, obtained from mice euthanized
according to protocols approved by the University of Pennsylvania
Institutional Animal Care and Use Committee, were homogenized
in 10 mM ammonium acetate, pH 5.7 (50% w/v), using a hand-held
sonic homogenizer. Plasma was obtained from blood that was col-
lected into a 1.5 mL tube containing 0.5 M EDTA solution and sub-
jected to centrifugation for 10 min at 4500g at 4 °C. Aliquots
[ESI]+: Calcd for C16H15ClN3O3S+ 364.05. Found 364.07.
5.1.5.13. Ethyl 5-amino-7-chloro-3-(4-chlorophenyl)-4-oxo-3,4-
dihydrothieno[3,4-d]pyridazine-1-carboxylate (67).
A mix-
ture 30 (30 mg, 0.086 mmol) and N-chlorosuccinimide (11 mg,
0.086 mmol) in anhydrous N,N-dimethylformamide (0.5 mL) was
heated to 80 °C for 30 minutes. After cooling, the reaction was
quenched with water and extracted with ethyl acetate. The com-
bined organic extracts were washed with brine, dried over sodium
sulfate, filtered and concentrated in vacuo. Purification by prepara-
tive reverse phase HPLC furnished 50. Yield: 79%. 1H NMR (CDCl3): d
1.42 (t, J = 7.5 Hz, 3H), 4.45 (q, J = 7.2 Hz, 2H), 6.25 (br s, 2H), 7.40 (d,
J = 9.0 Hz, 2H), 7.51 ppm (d, J = 9.0 Hz, 2H). 13C NMR (CDCl3): d 14.1,
62.8, 105.7, 106.7, 122.7, 127.1, 128.9, 133.3, 136.2, 138.7, 158.5,
(50 lL) of brain homogenate or plasma were mixed with 0.2 mL
of acetonitrile and centrifuged at 15000g, and the resulting super-
natants were used for subsequent LCꢃMS/MS analysis. The
LCꢃMS/MS system comprised an Aquity UPLC and a TQ MS that
was controlled using MassLynx software (Waters Corporation, Mil-
ford, MA). Compounds were detected using multiple reaction mon-
itoring (MRM) of their specific collision-induced ion transitions.
159.1, 162.9 ppm. IR (film):
m .
3435, 3324, 1725, 1656, 1594 cmꢃ1
MS [ESI]+: Calcd for C15H12Cl2N3O3S+ 384.00. Found 384.01.
5.1.5.14. Ethyl 5-amino-7-bromo-3-(4-chlorophenyl)-4-oxo-
3,4-dihydrothieno[3,4-d]pyridazine-1-carboxylate
(68).
Prepared as 67 from 29 and N-bromosuccinimmide.
Samples (5
lL) were separated on an Aquity BEH C18 column
Yield: 75%. 1H NMR (CDCl3): d 1.43 (t, J = 7.1 Hz, 3H), 4.46 (q,
J = 7.2 Hz, 2H), 6.34 (br s, 2H), 7.40 (d, J = 8.5 Hz, 2H), 7.52 (d,
(1.7
l
m, 2.1 mm ꢂ 50 mm) at 35 °C. Operation was in positive
electrospray ionization mode, with mobile phase A of 0.1% (v/v)
formic acid and mobile phase B of acetonitrile with 0.1% (v/v) for-
mic acid at a flow rate of 0.6 mL/min using a gradient from 5% to
95% B over 2 min, followed by wash and re-equilibration steps.
The mass spectrometer was operated with a desolvation tempera-
ture of 450 °C and a source temperature of 150 °C. Desolvation and
source nitrogen gas flows were 900 and 50 L/h, respectively. Source
and MS/MS voltages were optimized for each compound using the
MassLynx autotune utility. Standard curves were generated for
each compound from brain homogenate and plasma samples and
extracted as above. Peak areas were plotted against concentration
and a linear regression curve was used to quantify the unknowns.
In all cases, the tissue-derived sample peak areas fell within the
linear portion of standard curves that were prepared and analyzed
concurrently with the samples.
J = 9.0 Hz, 2H) ppm. IR:
m
3425, 3317, 1732, 1656, 1589 cmꢃ1. MS
[ESI]+: Calcd for C15H12BrClN3O3S+ 427.95. Found 427.73.
5.1.5.15. Ethyl 2-(4-chlorophenyl)-5-hydroxy-1,7-dioxo-2,5,6,7-
tetrahydro-1H-pyrrolo[3,4-d]pyridazine-4-carboxylate
(69).
A solution of 29 in acetone (2 mL) was added at room
temperature with a freshly prepared solution of dimethyldioxirane
in acetone (0.07–0.09 M solution prepared as described in Organic
Syntheses, Coll. Vol 9, p.288 (1998); Vol 74, p.91 (1997)). The reac-
tion was monitored by TLC and after ꢀ20 minutes the starting
material was completely consumed. The reaction mixture was then
evaporated to dryness and the resulting material purified by pre-
parative HPLC obtaining 71. Yield: 20%. 1H NMR tautomeric mix-
ture (DMSO-d6): d 1.31 (t, J = 7.1 Hz, 3H), 3.30 (d, J = 11.8 Hz, 1H),
4.33–4.40 (m, 2H), 6.12 (d, J = 7.1 Hz, 1H), 6.65 (d, J = 8.6 Hz,
0.5H), 7.60–7.65 (m, 4H), 9.22 (s, 0.5H) ppm. IR (film):
m
3359,
5.1.7. Determination of unbound fraction in plasma and brain
homogenates
The unbound fractions of drug in mouse plasma and brain were
determined in vitro using rapid equilibrium dialysis (Pierce Bio-
technology, Rockford, IL). Test compounds were mixed at a concen-
2923, 2852, 1734 cmꢃ1. HRMS [ESI]+: Calcd for C15H12ClN3NaO5
372.0363. Found 372.0374. See X-ray crystal structure report in
the Supplementary data.
+
5.1.5.16. 3-(4-Fluorophenyl)-4,5-dioxo-3,4,5,7-tetrahydrothie-
tration of 1
diluted threefold with 100 mM sodium phosphate, pH 7.4. Tripli-
cate 100 L aliquots were placed in sample chambers and dialyzed
against 300 L of 100 mM sodium phosphate, pH 7.4, for 5 h at
lM with mouse plasma or brain homogenate that was
no[3,4-d]pyridazine-1-carboxylic
acid (70).
1H
NMR
(DMSO-d6): d 4.93 (s, 2H), 7.39–7.43 (m, 2H), 7.62–7.64 (m, 2H)
l
ppm. MS [ESI]+: Calcd for C13H8FN2O4S+ 307.02. Found 306.97.
l
37 °C with shaking at 100 rpm. After incubation, 20uL aliquots
were removed from each side of the dialysis membrane and an
equal volume of plasma, brain homogenate, or buffer added in or-
der to create identical matrix compositions. Compounds were sub-
5.1.5.17. 3-(4-Chlorophenyl)-4,5-dioxo-3,4,5,7-tetrahydrothie-
no[3,4-d]pyridazine-1-carboxylic acid (71).
To a solution of
41 (50 mg, 0.15 mmol) in acetonitrile (1 mL) 1 N hydrochloric acid
(1.5 mL) was added. The reaction mixture was stirred at 130 °C
(microwave irradiation) for 30 minutes. Water was then added
sequently extracted with 120
lL acetonitrile, vortexed and
centrifuged at 15,000g for 15 min. Supernatants were analyzed

C. Ballatore et al. / Bioorg. Med. Chem. 20 (2012) 4451–4461
4461
by LC–MS/MS. Unbound fraction (fu) was calculated as the ratio of
Supplementary data
the buffer chamber response to the sample chamber response.
Brain unbound fraction was calculated using the following equa-
tion, adjusting for tissue dilution (D).
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.05.027.
Undiluted f u ¼ ð1=DÞ=½ð1=fumeasuredÞ ꢃ 1 þ ð1=DÞꢄ
References and notes
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5.1.8. Mouse tolerability studies
To determine the maximum tolerated dose of ATPZ 43, 2-month
old female B6C3F1 mice (n = 5) were administered compound by
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were carefully monitored for the next 48 h for signs of ataxia, leth-
argy, piloerection, salivation or other unusual behaviors. If no signs
of intolerance were observed, doing was increased in gradual steps
(2–3 fold increases) until a noticeable change occurred in at least 2
mice. For the 1-month tolerability testing of compound 43, groups
of 2-month old female B6C3F1 mice (n = 5) were administered
compound in drinking water containing 3% PEG400 and 0.5% meth-
ylcellulose. Control mice received the vehicle only. Water con-
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were determined. The mice then underwent perfusion and eutha-
nization as described above, and organs weights were recorded
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Acknowledgments
17. Ferguson, G. N.; Valant, C.; Horne, J.; Figler, H.; Flynn, B. L.; Linden, J.; Chalmers,
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Financial support for this work was provided in part by the
Alzheimer’s Drug Discovery Foundation, Coins for Alzheimer’s
Research Trust, Marian S. Ware Drug Discovery Program, and the
NSF (Grant CHE-0840438, X-ray facility).