Discovery of brain-penetrant, orally bioavailable aminothienopyridazine inhibitors of Tau aggregation

Article abstract of DOI:10.1021/jm100138f

Agents capable of preventing the misfolding and sequestration of the microtubule-stabilizing protein tau into insoluble fibrillar aggregates hold considerable promise for the prevention and/or treatment of neurodegenerative tauopathies such as Alzheimer's

Full text of DOI:10.1021/jm100138f

J. Med. Chem. 2010, 53, 3739–3747 3739
DOI: 10.1021/jm100138f
Discovery of Brain-Penetrant, Orally Bioavailable Aminothienopyridazine Inhibitors
of Tau Aggregation
Carlo Ballatore,*,†,‡ Kurt R. Brunden,‡ Francesco Piscitelli,† Michael J. James,‡ Alex Crowe,‡ Yuemang Yao,‡ Edward Hyde,‡
John Q. Trojanowski,‡ Virginia M.-Y. Lee,‡ and Amos B. Smith, III†
†Department of Chemistry, School of Arts and Sciences, University of Pennsylvania, 231 South 34th Street, Philadelphia,
Pennsylvania 19104-6323, and ‡Center for Neurodegenerative Diseases Research, Institute on Aging, University of Pennsylvania,
3600 Spruce Street, Philadelphia, Pennsylvania 19104-6323
Received February 2, 2010
Agents capable of preventing the misfolding and sequestration of the microtubule-stabilizing protein
tau into insoluble fibrillar aggregates hold considerable promise for the prevention and/or treatment of
neurodegenerative tauopathies such as Alzheimer’s disease. Because tauopathies are characterized by
amyloidosis that is restricted to the central nervous system (CNS), plausible candidate compounds for in
vivo evaluation must both prevent tau fibrillization and achieve significant brain levels. Recently, we
reported the discovery of the aminothienopyridazine (ATPZ) class of tau aggregation inhibitors and
now describe a series of new analogues that are both effective inhibitors of tau fibrillization and display
significant brain-to-plasma exposure ratios after administration to mice. Further, two of the most
promising examples, 15 and 16, were found to reach significant brain exposure levels following oral
administration. Taken together, these results suggest that examples from the ATPZ class hold promise
as candidates for in vivo efficacy studies in animal models of neurodegenerative tauopathies.
Introduction
ultimately results in the formation of characteristic structures,
such as neurofibrillary tangles (NFTs) and neuropil threads,
that constitute the diagnostic signatures of neurodegenerative
tauopathies. Although the exact mechanisms of tau-mediated
neurodegeneration have not been completely defined, aggre-
gation of tau can lead to neuropathology at least in part by
causing or contributing to axonal transport deficits arising
from the loss of the normal MT-stabilizing function of tau.5
Further, different forms of aggregated tau may also contri-
bute to neuropathology by other less defined mechanisms.6
Agents capable of preventing the self-assembly of tau thus
comprise an attractive strategy for the prevention and/or
treatment of neurodegenerative tauopathies.7,8
The misfolding and aggregation within neurons of the
microtubule (MTa) associated protein (MAP) tau comprise
a key pathogenic phenomenon shared by different neuro-
degenerative conditions, collectively known as tauopathies,
which include Alzheimer’s disease (AD), Pick’s disease, and
certain forms of frontotemporal dementia (FTD) including
FTD with Parkinsonism linked to chromosome 17 (FTDP-
17).1 The protein tau, expressed predominantly in neurons,
stabilizes the MT network within axons, thereby facilitating
axonal transport of proteins, trophic factors, and other
cellular constituents, including neurotransmitters. Tau is in
dynamic equilibrium between the MT-bound and -unbound
states in the cytosol. This equilibrium is believed to be con-
trolled predominantly by the action of tau kinases and
phosphatases, as the phosphorylation state of tau is known
to modulate the affinity of this protein for the MTs.2 Under
physiological conditions, the vast majority (i.e., ∼99%) of tau
is associated to MTs.3,4 Conversely, under pathological con-
ditions, tau is abnormally disengaged from the MTs and as
such becomes considerably more prone to misfolding.5 Once
tau is misfolded, a self-assembly reaction can ensue that
In recent years, several compound classes have been dis-
closed that can effectively inhibit the assembly of tau into
filaments in vitro.9-19 However, with the exception of a
member of the phenothiazines, methylene blue (1, Figure 1),
*To whom correspondence should be addressed. Phone: (215) 898-
4891. Fax: (215) 898-5129. E-mail: bcarlo@sas.upenn.edu.
Figure 1
a Abbreviations: CNS, central nervous system; ATPZ, aminothieno-
pyridazine; MT, microtubule; AD, Alzheimer’s disease; FTD, frontotem-
poral dementia; FTDP-17, frontotemporal dementia with Parkinsonism
linked to chromosome 17; NFT, neurofibrillary tangle; BBB, blood-
brain barrier; SAR, structure-activity relationship; PSA, polar surface
area; HBD, hydrogen-bond donor; HBA, hydrogen-bond acceptor; ThT,
thioflavine-T; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel
electrophoresis;ip, intraperitoneal;iv, intravenous;PK, pharmacokinetic;
AUC, area under the curve.
which has progressed to human clinical trials, no tau assembly
inhibitor has been assessed for efficacy in vivo. Notably, the
results from a phase 2 study, involving 321 patients with mild
or moderate AD revealed a significant reduction in cognitive
decline in patients that received methylene blue compared to a
placebo. These promising findings, if confirmed, validate the
idea that tau aggregation inhibitors can be therapeutically
r
2010 American Chemical Society
Published on Web 04/14/2010
pubs.acs.org/jmc

3740 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Ballatore et al.
tau fibrillization assays as well as druglike physical-chemical
properties (Figure 2).21 To evaluate better the potential of the
ATPZs as possible candidates for future in vivo efficacy
studies, we designed and synthesized a set of derivatives
focused on possible BBB permeability. These compounds
were evaluated for efficacy against tau aggregation in vitro,
as well as for brain penetration in vivo. Notably, these experi-
ments (vide infra) led to the identification of prototype com-
pounds (i.e., 15, and 16; Scheme 1), which are both effective
in preventing the fibrillization of tau in vitro and capable
of reaching significant brain levels in mice after oral admini-
stration.
Compound Design and Synthesis
Figure 2. Summary of structure tau antifibrillization activity of
ATPZs.21
The design of the ATPZs employed in these studies took
into account the structure-activity relationships (SARs) for
this class,21 as well as key physical-chemical properties such
as lipophilicity (i.e., calculated partition coefficient between
n-octanol and water, or ClogP), polar surface area (PSA), the
number of hydrogen-bond donors (HBD) and acceptors
(HBA), and molecular weight, all of which are known to play
an important role in determining the ability of small molecules
to permeate biological membranes via passive diffusion.22
Thus, on the basis of our understanding of the SAR summar-
ized in Figure 2, we designed analogues modified in the X and
Y fragments, as these regions of the molecule were known to
be relatively tolerant to a series of structural variations.
With respect to physical-chemical properties, we consid-
useful for the treatment of neurodegenerative tauopathies.
However, since methylene blue is known to modulate a
multitude of biological targets,20 whether the outcome ob-
served in the phase 2 study can be ascribable solely to a direct
inhibition of tau aggregation is not yet clear. As a result,
further evaluation of the therapeutic potential of tau aggrega-
tion inhibitors is likely to require in vivo efficacy studies
involving additional candidate compounds. To this end and
in light of the fact that tauopathies are characterized by
amyloidosis that is restricted to the central nervous system
(CNS), candidate compounds for in vivo testing will have to
be brain-penetrant. Although numerous classes of tau fibrilli-
zation inhibitors have been reported in recent years, including
some that exhibit calculated physical-chemical properties
potentially appropriate for blood-brain barrier (BBB) per-
meation,17 to date, there are no reports demonstrating brain
penetration of any of these candidates.
2
˚
ered the following criteria: MW e 450 Da, PSA e 90 A ,
HBA e 7, HBD e 3, and ClogP e 3. On the basis of these
restrictions in terms of SAR and physical-chemical proper-
ties, compounds of general structure F (Scheme 1), bearing a
lipophilic p-chloro orp-bromophenyl(X= Clor Br) as well as
an amide moiety in the Y fragment, were identified as
potentially promising (cf., Table 1 for calculated physical-
chemical properties). Furthermore, as part of these studies,
Recently, we reported the discovery of a novel class of tau
aggregation inhibitors, known as the aminothienopyridazines
(ATPZ), which exhibit a promising combination of activity in
Scheme 1. General Synthetic Schemea
a Reagents and reaction conditions: (a) (i) NaNO2, 37% HCl, ethanol, water, 0 °C, 20 min; (ii) ethyl acetoacetate, sodium acetate, ethanol, water, 0 °C,
2 h. (b) X = Cl: ethyl cyanoacetate, ammonium acetate, acetic acid, 170 °C (microwave irradiation), 4 min. (c) X = Br: ethyl cyanoacetate, 4-aminobutyric
acid, 160 °C, 2.5 h. (d) S8, morpholine, ethanol, 150 °C (microwave irradiation), 15 min; (e) titanium(IV) isopropoxide, isopropanol, 170 °C (microwave
irradiation), 40 min; (f) LiOH H2O, tetrahydrofuran, water, room temp, 16 h; (g) appropriate amine, BOP reagent, N,N-diisopropylethylamine,
3
dimethylsulfoxide, room temp, 4 h.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3741
Table 1. Physical-Chemical Properties, Solubility, Activity, and Brain and Plasma Levelsg
a Physical-chemical properties (MW, molecular weight; PSA, polar surface area; ClogP, calculated partition coefficient between n-octanol and
water). b Solubility in the sodium acetate buffer employed in the tau fibrillization assay, as determined by light scattering. c Tau (K18PL) fibrillization
assay monitored by thioflavine (ThT) binding and fluorescence (all test compounds tested in triplicate). d Maximal percent inhibition of the K18PL
fibrillization as determined by SDS-PAGE analyses of the soluble and insoluble fractions obtained after centrifugation of the fibrillizing K18PL
mixtures. e Maximal percent inhibition of the tau40 fibrillization as determined by SDS-PAGE analyses of the soluble and insoluble fractions obtained
after centrifugation of the fibrillizing tau40 mixtures. f Ratio between brain/plasma drug level, as determined by LC-MS/MS, 1 h after ip administration
of 5 mg/kg test compound. gSymbols indicate the following: (§) ClogP of the neutral form; (ø) half maximal inhibitory concentration (IC50); (/) maximal
percent inhibition achieved in the assay; (†) detected amount of hydrolysis product (i.e., 11).
we included two representative ester derivatives (i.e., 8 and 10)
to evaluate the potential of these congeners to be brain-
penetrant, possibly acting as lipophilic precursors of the
corresponding acid (11). Depending on the rate of esterase-
mediated hydrolysis in brain and plasma, we reasoned that
such derivatives may potentially lead to an accumulation of
the corresponding acid inside the CNS.
The synthesis of these compounds was achieved via the reac-
tion sequence illustrated in Scheme 1, which entails: (a) a diazo-
nium coupling reaction to form hydrazones of general structure
B, (b) a Knovenagel-type condensation between B and ethyl
cyanoacetate to form pyridazines C,23,24 and (c) a Gewald ami-
nothiophene synthesis25 to furnish the desired ATPZs of general
structure D. Compound 8 was then trans-esterified to the
corresponding isopropyl ester 10 upon treatment with Ti(iPrO)4
in isopropanol. Alternatively, compounds 8 and 9 were saponi-
fied and the resulting acids (11 and 12, respectively) employed in
a series of benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (BOP) coupling reactions with various
primary and secondary amines to furnish compounds 13-19.
by turbidimetric measurements (Table 1). Compounds were
then evaluated in a heparin-induced tau assembly assay, in
which the fibrillization of the truncated K18 tau fragment
(comprising four MT-binding repeats) bearing the P301L
mutation (K18PL) found in FTDP-17 was monitored by
thioflavine-T (ThT) binding and fluorescence (Figure 3A).21
The inhibitory activity of test compounds was then con-
firmed with an orthogonal sedimentation assay in which
compound-treated fibrillizing mixtures were centrifuged and
quantitation of K18PL tau in the soluble and insoluble
fraction was performed by densitometric analyses of
SDS-PAGE (Figure 3B). As summarized in Table 1, the
ATPZs exhibited IC50 values in the 2-32 μM range in the
primary ThT assay, with maximal percent inhibition of
70-85%. The activity against tau fibrillization was confirmed
by sedimentation assay, where the majority of compounds
exhibited >50% reduction in pelletable material compared to
the untreated control. Although many of the ATPZ com-
pounds were found to reach a solubility limit within the upper
concentration range used in the biochemical assay, maximal
inhibitory effect was typically achieved at compound con-
centrations below 30 μM, where the majority of compounds
appeared to be fully soluble (cf., solubility data in Table 1 with
representative dose-response curves shown in Figure 3A).
Compound Evaluation in Vitro
The solubility of all test compounds in the sodium acetate
buffer used for subsequent fibrillization reactions was determined

3742 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Ballatore et al.
Figure 4. (A) Comparison of maximal percent inhibition of K18PL
fibrillization caused by test compounds in which the fibrillizing
mixtures did (sup) or did not (tot) undergo centrifugation at time
zero to remove insoluble material. Compounds were added at
50 μM to the tau fibrillization mixture and incubated for 30 min.
Samples were then divided into two halves, one of which was
incubated without further treatment (“tot”) and one that was
centrifuged at 186000g for 30 min. After centrifugation of the latter
sample, the supernatants (“sup”) were collected and incubated as
per the fibrillization protocol.21 (B) Representative examples of
SDS-PAGE analysis of supernatant (S) or pellet (P) samples
obtained after centrifugation of fibrillizing mixtures prior to in-
cubation. (C) Representative dose-response curves with (“sup”)
and without (“tot”) initial centrifugation.
Figure 3. (A) Representative dose-response curves in the heparin-
induced K18PL fibrillization assay; (B) SDS-PAGE analysis of
supernatant (S) or pellet (P) samples obtained after centrifugation
of fibrillizing mixtures incubated in the presence of DMSO vehicle
or 100 μM test compound (11 and 16).
This indicates that the inhibition of tau aggregation is likely to
be caused by the ATPZs present in solution. However, to
further ensure that the observed inhibitory activity of these
ATPZs may not be artifactually generated by the presence of
insoluble or aggregated test compound,26 we conducted addi-
tional experiments in which both DMSO- and compound-
treated fibrillizing mixtures were initially centrifuged for
30 min prior to incubation (i.e., at time zero of the fibrilliza-
tion reaction) to remove any pelletable material. As shown in
Figure 4A, no significant differences in compound-mediated
inhibition of tau assembly were observed with and without the
precentrifugation step. Furthermore, the IC50 values of the
centrifuged and control samples did not appear to differ
appreciably (Figure 4C). Moreover, SDS-PAGE analysis
of the soluble and pelletable material after the initial centri-
fugation step revealed no significant difference between com-
pound- and vehicle-treated mixtures; in all cases K18PL
remained largely in the soluble fraction (Figure 4B) and thus
was not precipitated by the test compound. Collectively, the
activity and SDS-PAGE data confirm that the inhibition of
K18PL fibrillization produced by the ATPZ analogues is not
caused by insoluble or aggregated material.
All test compounds also inhibited the aggregation of full-
length tau (tau40) as determined by sedimentation assay,
which revealed efficacy values in the 50-60% range (cf.,
Table 1). Both K18PL and tau40 activity data are in keeping
with prior SAR that suggested that the presence of halogens in
the para position of the phenyl ring, as well as carboxylic acid,
esters, and amides in the Y fragment, would be generally well
tolerated.21 Also consistent with our previous studies is the
observation that all ATPZs tested (i.e., 8, 10, 11, 13-19) did
not appear to interfere with the normal MT-stabilizing func-
tion of tau in a MT-polymerization assay (Figure 5).
Finally, toevaluatepossible major toxicities associatedwith
the ATPZs, all test compounds were evaluated in a cyto-
toxicity assay that employs rapidly dividing HEK-293 cells.
All compounds were found to be nontoxic at 100 μM.
Figure 5. MT-assembly assay. Tubulin (30 μM) and tau40 (20 μM)
were incubated with the test compound (50 μM, e.g., 16 [green
triangles]) or DMSO (open and black squares) to evaluate possible
interference in the tau-promoted MT-polymerization that may be
caused by the ATPZ inhibitors of tau aggregation. As control
experiment, MT-polymerization assay was also conducted in the
absence of tau40 (open blue circles). Additional results are pre-
sented in the Supporting Information.
Pharmacokinetic Studies
Test compounds underwent preliminary evaluations of
brain penetration, in which 5 mg/kg of each compound was

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3743
administered intraperitoneally (ip) to a group of three normal
mice; drug levels in brain and plasma were determined at a
single time point (1 h) by LC-MS/MS using prevalidated
calibration curves. The results of these experiments, summari-
zed in Table 1, revealed that with the exception of 11, all other
test compounds displayed significant brain uptake as demon-
strated by the brain-to-plasma exposure ratios (B/P) above
0.3. The lack of brain penetration of 11 was not unexpected
given that the carboxylic acid moiety of this compound would
be mostly negatively charged at physiological pH and thus
likely to result in limited passive diffusion of the compound
across the BBB. Conversely, the more lipophilic ester deriva-
tives (8 and 10) exhibited comparatively higher B/P ratio. In
particular, 10 was found to reach significantly higher brain
concentrations compared to the corresponding acid 11. How-
ever, both esters appeared to have relatively short half-lives in
plasma, as indicated by the limited amount of parent drug
detected after 1 h from administration of the compounds.
Moreover, monitoring for the hydrolyzed metabolite (i.e., 11)
in both brain and plasma revealed a considerable amount of
acid 11 in plasma but not in the brain 1 h after administration
of either ester. This observation suggests that while lipophilic
esters may be able to gain access to the CNS (e.g., 10), these
derivatives may not produce an accumulation of the corres-
ponding ATPZ acid in the CNS at the rate observed in plasma
because of significantly less esterase-mediated hydrolysis in
the brain. Finally, among the amide derivatives, 14, 15 and 16
appeared to be particularly interesting because of favorable
B/P ratios combined with promising indications of good
metabolic stability. Compound 16 was selected for full PK
analysis in which brain and plasma drug levels were deter-
mined atsix time points (i.e., 30 min, 1 h, 2 h, 4 h, 8 h, and 16 h)
after intravenous (iv) administration of 2 mg/kg test com-
pound to a group of 18 normal mice (3 mice per time point).
The results from this experiment, illustrated in Figure 6A,
confirmed that the total B/P exposure ratio over 16 h is ∼1.6.
Furthermore, these data reveal that 16 exhibits good meta-
bolic stability as indicated by an elimination half-life of g2.5 h
in plasma.
Finally, in order to investigate the potential for ATPZ
congeners to be orally administered, we evaluated brain
and plasma level of 16 and 15 after oral administration (oral
gavage) of 5 mg/kg. In both cases, significant concentrations
of each test compound were achieved in the brain after oral
administration (cf., Figure 6B and Figure 6C). Comparison of
the integrated areaunder the curve (AUC) in plasmaafter oral
and iv administration of 16 revealed an oral bioavailability (F)
of ∼70% (cf., Figure 6A and Figure 6B).
Figure 6. (A) Brain and plasma level of 16 after iv administration of
2 mg/kg; (B) brain and plasma level of 16 after oral (po) adminis-
tration of 5 mg/kg; (C) brain and plasma level of 15 after oral
administration of 5 mg/kg.
efficacy, we conducted a study in which selected analogues,
designed for improved BBB-permeability, were evaluated for
in vitro activity as well as for brain penetration. Results from
the in vitro efficacystudies appeared tobefullyconsistentwith
our previous results and confirmed that the ATPZs are most
effective in preventing tau fibrillization when present in ∼1:1
molar ratio with tau (i.e., 15 μM in the fibrillization assay).21
Assuming that similar stoichiometric requirements also exist
in vivo, any ATPZ candidate for in vivo efficacy study will
have to reach free brain concentrations thatare comparable to
that of the unbound fraction of tau. The total intraneuronal
tau concentration (i.e., MT-bound and MT-unbound tau) is
estimated to be in the low micromolar range,3 and as pre-
viously noted, >99% of the protein is bound to MTs under
physiological conditions.3,4 Although the concentration of the
MT-unbound fraction of tau is likely to increase in diseased
neurons, this may be in the submicromolar range. Thus, only
compounds that readily cross the BBB would be viable
candidates for in vivo evaluations of efficacy. Interestingly,
Discussion
Despite the hypothesis that tau aggregation inhibitors may
be therapeutically useful for AD and related tauopathies, to
date, only one compound of this type, methylene blue, has
entered clinical trials. Numerous additional classes of com-
pounds have been shown to inhibit tau assembly in vitro, but
there are no reports of these molecules being evaluated in vivo
for pharmacokinetic (PK) properties or efficacy in models of
tauopathy. Because BBB permeability is known to be a major
bottleneck that hampers the development of new CNS-active
drugs,27 an early evaluation of the brain penetration of
candidate compounds is important, as such studies would
permitfocus onthe most promisingcompound type. For these
reasons and to assess the potential of ATPZ inhibitors as
possible candidate compounds for in vivo evaluation of

3744 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Ballatore et al.
preliminary evaluation of brain exposures of the ATPZ test
compounds revealed that with the exception of the acid
derivative 11, all other ATPZ congeners exhibited B/P ratios
above 0.3. Considering that most CNS-active drugs typically
exhibit B/P > 0.3-0.5,28 these results indicate that ATPZs
have the potential to achieve appreciable brain concentra-
tions. Furthermore, selected amide derivatives, such as 14, 15,
and 16, were found to reach brain concentrations above
800 ng/g (i.e., >2 μM) 1 h after ip administration of 5 mg/kg.
These results were further confirmed by complete PK studies
on 16, which displayed an average brain-to-plasma AUC
exposure ratio of ∼1.6. Equally important, significant brain
concentrations were also observed after oral administration of
15 or 16, with the oral bioavailability of the latter compound
being ∼70%. Collectively, the results from the PK studies
combined with the promising in vitro activity and safety data
suggest that the ATPZ class of tau aggregation inhibitors holds
considerable promise as candidate compounds for efficacy
testing in transgenic mouse models of tauopathies.
Ethyl 2-(2-(4-Chlorophenyl)hydrazono)-3-oxobutanoate (4). 4
was prepared as previously reported.29 Yield: 83%. Mp: 84-
85 °C (from ethanol, lit.29 81-84 °C). 1H NMR (CDCl3): δ 1.41
(t, J = 7.0 Hz, 3H), 2.60 (s, 3H), 4.34 (q, J = 7.2 Hz, 2H),
7.34-7.36 ppm (m, 4H). 13C NMR (CDCl3): δ 14.5, 30.9, 61.2,
117.6, 126.5, 129.8, 131.0, 140.4, 164.9, 197.4 ppm. IR:
ν 3359, 1706, 1617 cm-1. HRMS (ESIþ): calculated for
C12H13ClN2NaO3þ 291.0518, found 291.0512.
Ethyl 2-(2-(4-Bromophenyl)hydrazono)-3-oxobutanoate (5).
4-Bromoaniline (2.0 g, 11.63 mmol) was dissolved in 37% hydro-
chloric acid (2.94 mL), ethanol (1.50 mL), and water (1.50 mL).
The reaction mixture was cooled to 0 °C in an ice-water bath
before a solution of sodium nitrite (0.88 g, 12.75 mmol) in water
(2.0 mL) was added dropwise. The resulting mixture was stirred
at 0 °C for 20 min. Sodium acetate (3.72 g, 45.3 mmol) in water
(5.9 mL) and ethyl acetoacetate (1.51 g, 1.43 mL, 11.63 mmol)
were added, and the reaction mixture was stirred at 0 °C for 2 h.
The precipitated solid was then filtered, washed with water, and
dried under high vacuum for 16 h to provide 5 as yellow solid that
was crystallized in ethanol and used without further purification in
the next step. Yield: 98%.
Ethyl 1-(4-Chlorophenyl)-5-cyano-4-methyl-6-oxo-1,6-dihydro-
pyridazine-3-carboxylate (6). 6 was prepared as previously re-
ported.30 Yield: 47%. Mp: 160-161 °C (from ethanol, lit.30
Conclusions
Although the preliminary phase 2 clinical data obtained
with methylene blue suggest that tau aggregation inhibitors
may be therapeutically useful, further validation of the thera-
peutic potential of compounds of this type for the treatment
and/or prevention of neurodegenerative tauopathies is likely
to require additional in vivo efficacy studies involving differ-
ent candidate compounds. To this end, the ATPZs presented
here appear to be very promising candidates because of a
favorable combination of biological activity in vitro and
desirable PK properties, including excellent brain penetration
and oral bioavailability.
1
190 °C). H NMR (CDCl3): δ 1.41 (t, J = 7.0 Hz, 3H), 2.76
(s, 3H), 4.43 (q, J = 7.2 Hz, 2H), 7.48 (d, J = 8.5 Hz, 2H), 7.62
ppm (d, J = 8.5 Hz, 2H). 13C NMR (CDCl3): δ 14.2, 19.4, 63.0,
112.4, 116.0, 126.3, 129.4, 135.5, 137.5, 138.5, 150.8, 155.8, 162.0
ppm. IR: ν 2235, 1726, 1685 cm-1. MS (ESIþ): calculated for
C15H13ClN3O3þ 318.06, found 318.07.
Ethyl 1-(4-Bromophenyl)-5-cyano-4-methyl-6-oxo-1,6-dihydro-
pyridazine-3-carboxylate (7). A mixture of 5 (2.0 g, 6.39 mmol),
4-aminobutyric acid (1.33 g, 12.99 mmol), and ethyl cyanoacetate
(1.1 g, 1.0 mL, 9.44 mmol) was stirred neat at 160 °C for 2.5 h.
After the mixture was cooled, the residue was purified by silica gel
flash chromatography (using an ethyl acetate/n-hexane gradient
from 5% to 80% of ethyl acetate) to provide 7 as a white solid.
Yield: 76%. Mp: 174-176 °C (from ethanol). 1H NMR (CDCl3):
δ 1.41 (t, J = 7.0 Hz, 3H), 2.76 (s, 3H), 4.43 (q, J = 4.43 Hz, 7.2
Hz, 2H), 7.56 (d, J = 9.0 Hz, 2H), 7.64 ppm (d, J = 9.0 Hz, 2H).
13C NMR (CDCl3): δ 14.2, 19.4, 62.9, 112.4, 116.0, 123.5, 126.6,
132.3, 137.6, 139.1, 150.8, 155.7, 162.0 ppm. IR: ν 22þ30, 1727,
1681 cm-1. MS (ESIþ): calculated for C15H13BrN3O3 362.02,
found 362.01.
Experimental Section
Materials and Methods. All solvents were reagent grade. All
reagents were purchased from Aldrich or Acros and used as
received. Thin layer chromatography (TLC) was performed with
0.25 mm E. Merck precoated silica gel plates. Flash chromato-
graphy was performed with silica gel 60 (particle size 0.040-
0.062 mm) supplied by Silicycle and Sorbent Technologies. TLC
spots were detected by viewing under a UV light. Infrared (IR)
spectra were recorded on a Jasco model FT/IR-480 Plus spectro-
meter. All melting points were obtained on a Thomas-Hoover
apparatus. Proton (1H) and carbon (13C) NMR 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 Spectro-
metry Center on either a VG Micromass 70/70H or VG ZAB-E
spectrometer. Single-crystalX-raystructure determinationswere
performed at the University of Pennsylvania with an Enraf
Nonius CAD-4 automated diffractometer. Analytical reverse-
phased (Sunfire C18; 4.6 mm ꢀ 50 mm, 5 mL) high-performance
liquid chromatography (HPLC) was performed with a Waters
binary gradient module 2525 equipped with Waters 2996 PDA
and Waters micromass ZQ. All samples were analyzed employ-
ing a linear gradient from 10% to 90% of acetonitrile in water
over 8 min and a flow rate of 1 mL/min, and unless otherwise
stated, the purity level was >95%. Preparative reverse phase
HPLC purifications were performed on a Gilson instrument (i.e.,
Gilson 333 pumps, a 215 liquid handler, 845Z injection module,
and PDA detector) employing Waters SunFire preparative C18
OBD columns (5 μm; 19 mm ꢀ 50 mm, or 19 mm ꢀ 100 mm).
Purifications were carried out employing a linear gradient from
10% to 90% of acetonitrile in water for 15 min with a flow rate of
20 mL/min. Yields refer to chromatographically and spectro-
scopically pure compounds.
Synthesis of Thienyl Derivatives with General Structure D
(Gewald Reaction). Representative Example: Ethyl 5-Amino-
3-(4-chlorophenyl)-4-oxo-3,4-dihydrothieno[3,4-d]pyridazine-1-
carboxylate (8). A mixture of 6 (0.100 g, 0.31 mmol), sulfur
(0.015 g, 0.47 mmol), and morpholine (0.55 g, 0.55 mL,
0.63 mmol) was heated to 150 °C using microwave irradiation
for 15 min. After the mixture was cooled, the precipitate that
formed was collected and purified by preparative HPLC to give
8 as a yellow solid. HPLC-MS retention time: 8.20 min. Yield:
64%. Mp: 189-191 °C (from ethanol). 1H NMR (CDCl3):
δ 1.43 (t, J = 7.3 Hz, 3H), 4.44 (q, J = 7.2 Hz, 2H), 6.20
(broad s, 2H), 7.25 (s, 1H), 7.42 (d, J = 8.5 Hz, 2H), 7.56 ppm
(d, J = 8.5 Hz, 2H). 13C NMR (CDCl3): δ 14.4, 62.2, 104.9,
106.9, 127.1, 127.2, 128.9, 133.3, 134.0, 139.2, 159.4, 161.8, 163.0
ppm. IR: ν 3411, 3303, 1724, 1709, 1660 cm-1. HRMS (ESIþ):
calculated for C15H13ClN3O3Sþ 350.0366, found 350.0355.
Ethyl 5-Amino-3-(4-bromophenyl)-4-oxo-3,4-dihydrothieno[3,4-
d]pyridazine-1-carboxylate (9). 9 was synthesized in the same
manner as 8 starting with 7. Yield: 55%. Mp: 198-200 °C (from
ethanol). 1H NMR (CDCl3): δ 1.42 (t, J = 7.0 Hz, 3H), 4.44 (q,
J = 7.2 Hz, 2H), 6.12 (broad s, 2H), 7.25 (s, 1H), 7.51 (d, J = 8.5
Hz, 2H), 7.58 ppm (d, J = 8.5 Hz, 2H). 13C NMR (CDCl3): δ 14.1,
62.1, 105.9, 106.7, 121.3, 127.1, 127.5, 131.8, 134.0, 139.7,
159.4, 161.9, 163.0 ppm. IR: ν 3407, 3310, 1709, 1657 cm-1
.
HRMS (ESIþ): calculated for C15H13BrN3O3Sþ 393.9861, found
393.9879.

Article
Isopropyl 5-Amino-3-(4-chlorophenyl)-4-oxo-3,4-dihydrothieno-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3745
the same manner as 13 starting with 11 using isopropylamine.
HPLC-MS retention time: 6.58 min. Yield: 89%. Mp:
[3,4-d]pyridazine-1-carboxylate (10). A mixture of 8 (0.050 g, 0.143
mmol) and titanium(IV) isopropoxide (0.002 g, 2.1 μL, 7 μmol) in
isopropanol (0.5 mL) was heated to 170 °C using microwave
irradiation for 40 min. After the mixture was cooled, the solvent
was evaporated and the residue was purified by preparative HPLC
to provide 10 as yellow solid. HPLC-MS retention time: 8.73 min.
Yield: 77%. Mp: 118-120 °C (from ethanol). 1H NMR (CDCl3):
δ 1.41 (d, J = 6.5 Hz, 6H), 5.26-5.33 (m, 1H), 6.25 (broad s, 2H),
7.17 (s, 1H), 7.38 (d, J = 8.5 Hz, 2H), 7.57 ppm (d, J = 8.5 Hz,
2H). 13C NMR (CDCl3): δ 22.0, 70.1, 104.7, 106.8, 127.1, 127.2,
128.8, 133.1, 134.4, 139.2, 159.4, 161.9, 162.5 ppm. IR: ν 3408,
3294, 3145, 1716, 1663, 1585 cm-1. HRMS (ESIþ): calculated for
C16H14ClN3O3NaSþ 386.0342, found 386.0331.
1
197-199 °C (from ethanol). H NMR (CD3OD): δ 1.22 (d,
J = 6.5 Hz, 6H), 4.09-4.19 (m, 1 H), 7.25 (s, 1H), 7.46 (d, J =
8.5 Hz, 2H), 7.59 ppm (d, J = 8.5 Hz, 2H). 13C NMR
(CD3OD): δ 19.6, 39.9, 102.9, 103.3, 124.9, 126.1, 126.9,
131.2, 135.1, 138.1, 158.0, 161.4, 162.1 ppm. IR: ν 3404,
3294, 3178, 1651, 1590 cm-1. HRMS (ESIþ): calculated for
C16H15ClN4O2NaSþ 385.0502, found 385.0492.
5-Amino-3-(4-chlorophenyl)-N-cyclopropyl-4-oxo-3,4-dihydro-
thieno[3,4-d]pyridazine-1-carboxamide (16). 16 was prepared in
the same manner as 13 starting with 11 using cyclopropylamine.
HPLC-MS retention time: 7.43 min. Yield: 41%. Mp: 188-
190 °C (from ethanol). 1H NMR (DMSO-d6): δ 0.56-0.60 (m,
2H), 0.66-0.70(m, 2H), 2.76-2.81 (m, 1H), 7.19 (s, 1H), 7.50, (d,
J = 9.0 Hz, 2H), 7.67 (d, J = 9.0 Hz, 2H), 8.28 ppm (broad s,
1H). 13C NMR (DMSO-d6): δ 5.7, 22.6, 103.8, 104.4, 125.9,
127.5, 128.1, 131.0, 136.3, 139.4, 158.3, 163.0, 163.7 ppm. IR:
ν 3408, 3302, 1654, 1593 cm-1. HRMS (ESIþ): calculated for
C16H13Cl4O2N4NaSþ 383.0345, found 383.0333.
5-Amino-3-(4-chlorophenyl)-N,N-dimethyl-4-oxo-3,4-dihydro-
thieno[3,4-d]pyridazine-1-carboxamide (17). 17 was prepared in
the same manner as 13 starting with 11 using dimethylamine
hydrochloride. HPLC-MS retention time: 6.43 min. Yield:
62%. Mp: 242-244 °C (from ethanol). 1H NMR (CDCl3):
δ 3.14 (s, 3H), 3.16 (s, 3H), 6.18 (broad s, 2H), 6.73 (s, 1H),
7.41 (d, J = 9.0 Hz, 2H), 7.52 ppm (d, J = 9.0 Hz, 2H). 13C
NMR (CDCl3): δ 35.7, 39.0, 104.0, 106.9, 127.0, 128.1, 128.8,
132.9, 139.2, 139.3, 159.1, 161.7, 164.3 ppm. IR: ν 3414, 3300,
1646, 1598 cm-1. HRMS (ESIþ): calculated for C14H13ClN4-
NaO2Sþ 371.0345, found 371.0366.
Synthesis of Compounds with General Structure E (Esters Hy-
drolysis). Representative Example: 5-Amino-3-(4-chlorophenyl)-
4-oxo-3,4-dihydrothieno[3,4-d]pyridazine-1-carboxylic Acid (11).
Lithium hydroxide monohydrate (0.063 g, 1.29 mmol) was added
to a solution of 8 (0.150 g, 0.43 mmol) in tetrahydrofuran (3 mL)
and water (2 mL). The reaction mixture was stirred at room
temperature for 16 h. Then 1 N HCl was added (pH ∼ 2) and
the formed precipitate was filtered and purified by preparative
HPLC to afford 11 as a yellow solid. HPLC-MS retention time:
1
6.63 min. Yield: 67%. Mp: >300 °C (from ethanol). H NMR
(CD3OD): δ 7.19 (s, 1H), 7.44 (d, J = 9.0 Hz, 2H), 7.58 ppm (d,
J = 9.0 Hz, 2H). 13C NMR (DMSO-d6): δ 104.4, 104.5, 126.8,
128.3, 128.9, 132.0, 134.5, 140.2, 158.9, 163.9, 164.6 ppm. IR:
ν 3439, 3322, 3065, 1712, 1646, 1590 cm-1. HRMS (ESIþ):
calculated for C13H9ClN3O3Sþ 322.0053, found 322.0062.
5-Amino-3-(4-bromophenyl)-4-oxo-3,4-dihydrothieno[3,4-d]pyri-
dazine-1-carboxylic Acid (12). 12 was prepared in the same manner
as 11 starting with 9. HPLC-MS retention time: 6.76 min. Yield:
77%. Mp: >300 °C (from ethanol). 1H NMR (DMSO-d6): δ 7.12
(s, 1H), 7.52 (d, J = 9.0 Hz, 2H), 7.63 (broad s, 2H), 7.66 ppm (d,
J = 8.5 HZ, 2H). 13C NMR (DMSO-d6): δ 104.4, 104.5, 120.4,
126.8, 128.6, 131.9, 134.5, 140.6, 158.8, 163.9, 164.5 ppm. IR:
ν 3439, 3322, 3065, 1712, 1646 cm-1. HRMS (ESIþ):calculatedfor
C13H7BrN3NaO3Sþ 387.9367, found 387.9378.
5-Amino-3-(4-chlorophenyl)-N-ethyl-N-methyl-4-oxo-3,4-di-
hydrothieno[3,4-d]pyridazine-1-carboxamide (18). 18 was pre-
pared in the same manner as 13 starting with 11 using ethyl-
methylamine. HPLC-MS retention time: 6.85 min. Yield:
1
47%. Mp: 148-150 °C (from ethanol). H NMR (CDCl3): δ
1.19-1.26 (m, 3H), 3.46-3.63 (m, 2H), 6.16 (broad s, 2H), 6.70
(s, 1H), 7.40-7.42 (m, 2H), 7.51-7.54 ppm (m, 2H). 13C NMR
(CDCl3): δ 12.2, 14.0, 32.9, 36.3, 42.9, 46.1, 103.5, 106.4, 111.3,
117.0, 126.7, 126.8, 127.0, 127.1, 127.8, 127.9, 128.8 (ꢀ2),
132.8, 132.9, 159.1 (ꢀ2), 162.2, 164.0, 164.5 ppm. IR: ν 3409,
Synthesis of Compounds with General Structure F. Represen-
tative Example: 5-Amino-3-(4-chlorophenyl)-N-methyl-4-oxo-
3,4-dihydrothieno[3,4-d]pyridazine-1-carboxamide (13). DIPEA
(0.028 g, 38 μL, 0.23 mmol) was added to a mixture of 11 (0.050
g, 0.15 mmol), methylamine (2.0 M solution in THF, 110 μL,
0.22 mmol), and BOP reagent (0.097 g, 0.22 mmol) in anhydrous
DMSO (2 mL). The reaction mixture was stirred at room
temperature for 4 h. Water was added, and the resulting mixture
was extracted with ethyl acetate. The organic layer was washed
with brine, dried over MgSO4, filtered, and evaporated. The
residue was purified by preparative HPLC to give 13 as yellow
solid. HPLC-MS retention time: 6.83 min. Yield: 52%. Mp:
3290, 3175, 1635 cm-1
.
HRMS (ESIþ): calculated for
C
16H15ClN4NaO2Sþ 385.0502, found 385.0491.
5-Amino-3-(4-bromophenyl)-N-isopropyl-4-oxo-3,4-dihydro-
thieno[3,4-d]pyridazine-1-carboxamide (19). 19 was prepared in
the same manner as 13 starting with 12. HPLC-MS retention
time: 8.20 min. Yield: 68%. Mp: 222 °C dec (from ethanol). 1H
NMR (DMSO-d6): δ 1.15 (d, J = 6.6 Hz, 6H), 4.02-4.10 (m,
1H), 7.20 (s, 1H), 7.60 (broad s, 2H), 7.62 (d, J = 8.9 Hz, 2H),
7.65 (d, J = 8.9 Hz, 2H), 7.99 ppm (broad d, 1H). 13C NMR
(DMSO-d6): δ 22.0, 40.1, 103.8, 104.5, 119.4, 125.9, 127.9,
131.1, 136.5, 139.9, 158.3, 161.6, 162.9 ppm. IR: ν 3412, 3305,
1655, 1598 cm-1. HRMS (ESIþ): calculated for C16H15BrN4-
NaO2Sþ 428.9997, found 428.9986.
Solubility Studies. Compounds were serially diluted in
DMSO to achieve a 2.5-fold dilution series from 20 mM down
8 points to 3 μM on a 384-well master plate (Costar 3672).
Aliquots (2 μL) were transferred to a clear 96-well assay plate
(Fisher 12-565-501) containing 198 μL of fibrillization buffer
(100 mM NaOAc, pH 7.0) for a 100-fold dilution (200 μM down
to 0.03 μM). The plate was incubated with agitation for 2.5 h,
and the absorbance at 550, 600, 650, and 700 nm was measured
on a Spectramax M5 spectrophotometer. DMSO (1%) in
fibrillization buffer served as a control that was subtracted from
each data point. The absorbances were averaged, and the
concentration at which the average absorbance rose above
0.03 AU was an indication of insoluble compound, with solu-
bility limits reported as the previous concentration in the dilu-
tion series. Absorbance scans of compounds in DMSO indicated
that the compounds tested here did not have any absorbance
1
192-194 °C (from ethanol). H NMR (DMSO-d6): δ 2.75 (d,
J = 5.0 Hz, 3H), 7.24 (s, 1H), 7.51 (d, J = 9.0 Hz, 2H), 7.60
(broad s, 2H), 7.69 (d, J = 9 Hz, 2H), 8.27 ppm (broad s, 1H).
13C NMR (DMSO-d6): δ 25.7, 103.9, 104.6, 126.0, 127.5, 128.1,
131.0, 131.6, 136.1, 139.5, 158.3, 162.9, 163.0 ppm. IR: ν 3418,
3302, 1656, 1593 cm-1. HRMS (ESIþ): calculated for C14H10-
ClN4O2Sþ 333.0213, found 333.0210.
5-Amino-3-(4-chlorophenyl)-N-ethyl-4-oxo-3,4-dihydrothieno-
[3,4-d]pyridazine-1-carboxamide (14). 14 was prepared in the
same manner as 13 starting with 11 using ethylamine hydro-
chloride. HPLC-MS retention time: 7.43 min. Yield: 58%. Mp:
203-205 °C (from ethanol). 1H NMR (CDCl3): δ 1.23 (t, J = 7.3
Hz, 3H), 3.41-3.47 (m, 2H), 6.18 (broad s, 2H), 7.07 (s, 1H), 7.45
(d, J = 9.0 Hz, 2H), 7.51 (d, J = 9.0 Hz, 2H), 7.60 ppm (broad s,
1H). 13C NMR (CDCl3): δ 14.9, 34.3, 106.8, 106.9, 126.7, 127.3,
129.0, 133.3, 135.6, 139.2, 159.5, 161.4, 162.6 ppm. IR: ν 3405,
3297, 1652, 1600 cm-1
.
HRMS (ESIþ): calculated for
C15H13ClN4NaO2Sþ 371.0345, found 371.0341.
5-Amino-3-(4-chlorophenyl)-N-isopropyl-4-oxo-3,4-dihydro-
thieno[3,4-d]pyridazine-1-carboxamide (15). 15 was prepared in

3746 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Ballatore et al.
peaks in the above range; therefore, all absorbance was due to
light scattering by insoluble material.
Tau K18PL Fibrillization Assay. Activity evaluations of test
compounds in the K18PL fibrillization assay were performed as
previously described.21
Secondary Sedimentation Assay (K18PL and tau40). The
determination of compound-induced effects on the amount of
tau (K18PL or tau40) that remains soluble upon centrifugation
following incubation with test compounds was conducted as
previously described.9
the average peak area from triplicate injections. Drug half-life
was calculated using the elimination rate constant determined
from the slope of a line plotted through natural log of the plasma
concentration versus time for the 4, 8, and 16 h time points using
Microsoft Excel. Area under the brain or plasma concentration
curve from 0.5 to 16 h was calculated using the trapezoid rule
with GraphPad Prism.
Acknowledgment. Financial support for this work was
provided in part by the University of Pennsylvania Institute
on Aging (IOA Pilot Research Grant) and by the NIH/NIA
(AG029213-AI).
MT-Assembly Assay. The MT assembly assay was conducted
in a 384-well plate essentially as described in Hong et al.31 Wild-
type full-length human brain tau (tau40, 2N4R) was employed
in this assay. Lyophilized bovine brain tubulin (Cytoskeleton
Inc., TL238) was reconstituted in 1ꢀ RAB (100 mM MES, pH
6.9, 1 mM EDTA, 0.5 mM MgSO4), pH 6.9, to a concentration
of 10 mg/mL. ATPZ compounds (62.5 μM) were added to tau
(25 μM) in RAB, pH 6.9, and preincubated at room temperature
for 120 min. To initiate the MT assembly reaction, 8.25 μL per
well of 10 mg/mL tubulin was dispensed on a UV-clear 384-well
plate (NUNC 265196) followed by 1.0 μL of 100 mM GTP (1ꢀ
RAB pH 6.9) and 40 μL of the ATPZ/tau mixture. The final
reaction mixture was 30 μM tubulin, 20 μM tau, 50 μM ATPZ,
and 2 mM GTP. The plate was then incubated in a Spectramax
M5 plate reader at 37 °C, and the absorbance at 340 nm was
measured every minute for 45 min.
Cytotoxicity Assay. Cells were plated in each well of a 96-well
plate in 100 μL of media (20 000 cells; DMEM plus 10% fetal
calf serum with antibiotics). After 24 h, half-log serial dilutions
of the test compounds, starting at 200 μM, were added to an
additional 100 μL of media. The cells were cultured for an
additional 72 h, and then Alamar blue (Biosource, Camarillo,
CA) was added, with cell viability at each drug concentration
measured using a SpectraMax M5 fluorescence plate reader
(Molecular Devices, Sunnyvale, CA). CC50 values were gener-
ated using the Prism software.
Supporting Information Available: HPLC chromatograms
and mass, IR, 1H NMR, and 13C NMR spectra for compounds
8, 10, 11, 13-19; X-ray crystal structures for compounds 8, and
16; MT-assembly assay results for compounds 8, 10, 11, 13-19.
This material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Lee, V. M.-Y.; Goedert, M.; Trojanowski, J. Q. Neurodegenerative
tauopathies. Annu. Rev. Neurosci. 2001, 24, 1121–1159.
(2) Mazanetz, M. P.; Fischer, P. M. Untangling tau hyperphospho-
rylation in drug design for neurodegenerative diseases. Nat. Rev.
Drug Discovery 2007, 6, 464–479.
(3) Congdon, E. E.; Kim, S.; Bonchak, J.; Songrug, T.; Matzavinos,
A.; Kuret, J. Nucleation-dependent tau filament formation: the
importance of dimerization and an estimation of elementary rate
constants. J. Biol. Chem. 2008, 283, 13806–13816.
(4) Drubin, D. G.; Feinstein, S. C.; Shooter, E. M.; Kirschner,
M. W. Nerve growth factor-induced neurite outgrowth in PC12
cells involves the coordinate induction of microtubule assembly
and assembly-promoting factors. J. Cell Biol. 1985, 101, 1799–
1807.
(5) Ballatore, C.; Lee, V. M.-Y.; Trojanowski, J. Q. Tau-mediated
neurodegeneration in Alzheimer’s disease and related disorders.
Nat. Rev. Neurosci. 2007, 8, 663–672.
Determination of Plasma and Brain Drug Concentrations. Test
compounds were administered to 2 month old female B6C3F1
mice (20 g average body weight). Whole brain hemispheres,
obtained from mice euthanized according to protocols ap-
proved by the University of Pennsylvania Institutional Animal
Care and Use Committee, were homogenized in 10 mM ammo-
nium acetate, pH 5.7 (1:2, w/v), using a hand-held sonic homo-
genizer. Plasma was obtained from blood that was collected into
a 1.5 mL tube containing 0.5 M EDTA solution and subjected to
centrifugation for 10 min at 4500g at 4 °C. Aliquots (50 μL) 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 LC-MS/MS analysis. The
LC-MS/MS system comprised an Aquity UPLC and a TQ MS
that was controlled using MassLynx software (Waters Corpora-
tion, Milford, MA). Compounds were detected using multiple
reaction monitoring (MRM) of their specific collision-induced
ion transitions. Samples (5 μL) were separated on an Aquity
BEH C18 column (1.7 μm, 2.1 mm ꢀ 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 either
acetonitrile or methanol with 0.1% (v/v) formic 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 temperature 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 com-
pound using the MassLynx autotune utility. Standard curves
were generated for each compound from brain homogenate and
plasma samples that had compound added at 4, 40, 400, and
4000 ng/mL and extracted as above. Peak areas were plotted
against concentration, and a 1/x weighted linear regression
curve was used to quantify the tissue-derived samples using
(6) Brunden, K. R.; Trojanowski, J. Q.; Lee, V. M.-Y. Evidence that
non-fibrillar tau causes pathology linked to neurodegeneration and
behavioral impairments. J. Alzheimer’s Dis. 2008, 14, 393–399.
(7) Brunden, K. R.; Ballatore, C.; Crowe, A.; Smith, A. B., III; Lee,
V. M.-Y.; Trojanowski, J. Q. Tau-directed drug discovery
for Alzheimer’s disease and related tauopathies: a focus on tau
assembly inhibitors. Exp. Neurol. 2009, DOI: 10.1016/j.expneurol.
2009.08.031.
(8) Brunden, K. R.; Trojanowski, J. Q.; Lee, V. M.-Y. Advances in
tau-focused drug discovery for Alzheimer’s disease and related
tauopathies. Nat. Rev. Drug Discovery 2009, 8, 783–793.
(9) Crowe, A.; Ballatore, C.; Hyde, E.; Trojanowski, J. Q.; Lee,
V. M.-Y. High throughput screening for small molecule inhibitors
of heparin-induced tau fibril formation. Biochem. Biophy. Res.
Commun. 2007, 358, 1–6.
(10) Pickhardt, M.; von Bergen, M.; Gazova, Z.; Hascher, A.; Biernat,
J.; Mandelkow, E. M.; Mandelkow, E. Screening for inhibitors of
tau polymerization. Curr. Alzheimer Res. 2005, 2, 219–226.
(11) Pickhardt, M.; Larbig, G.; Khlistunova, I.; Coksezen, A.; Meyer,
B.; Mandelkow, E. M.; Schmidt, B.; Mandelkow, E. Phenylthia-
zolyl-hydrazide and its derivatives are potent inhibitors of tau
aggregation and toxicity in vitro and in cells. Biochemistry 2007,
46, 10016–10023.
(12) Pickhardt, M.; Gazova, Z.; von Bergen, M.; Khlistunova, I.; Wang,
Y.; Hascher, A.; Mandelkow, E. M.; Biernat, J.; Mandelkow, E.
Anthraquinones inhibit tau aggregation and dissolve Alzheimer’s
paired helical filaments in vitro and in cells. J. Biol. Chem. 2005,
280, 3628–3635.
(13) Larbig, G.; Pickhardt, M.; Lloyd, D. G.; Schmidt, B.; Mandelkow, E.
Screening for inhibitors of tau protein aggregation into Alzheimer
paired helical filaments: a ligand based approach results in successful
scaffold hopping. Curr. Alzheimer Res. 2007, 4, 315–323.
(14) Khlistunova, I.; Biernat, J.; Wang, Y.; Pickhardt, M.; von Bergen,
M.; Gazova, Z.; Mandelkow, E.; Mandelkow, E.-M. Inducible
expression of tau repeat domain in cell models of tauopathy:
aggregation is toxic to cells but can be reversed by inhibitor drugs.
J. Biol. Chem. 2006, 281, 1205–1214.
(15) Bulic, B.; Pickhardt, M.; Khlistunova, I.; Biernat, J.; Mandelkow,
E. M.; Mandelkow, E.; Waldmann, H. Rhodanine-based tau

Article
aggregation inhibitors in cell models of tauopathy. Angew. Chem.,
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3747
Scammells, P. J. 2-Aminothienopyridazines as novel adenosine A1
receptor allosteric modulators and antagonists. J. Med. Chem.
2008, 51, 6165–6172.
Int. Ed. 2007, 46, 9215–9219.
(16) Taniguchi, S.; Suzuki, N.; Masuda, M.; Hisanaga, S.; Iwatsubo, T.;
Goedert, M.; Hasegawa, M. Inhibition of heparin-induced tau
filament formation by phenothiazines, polyphenols, and porphyr-
ins. J. Biol. Chem. 2005, 280, 7614–7623.
(17) Chang, E.; Congdon, E. E.; Honson, N. S.; Duff, K. E.; Kuret, J.
Structure-activity relationship of cyanine tau aggregation inhibi-
tors. J. Med. Chem. 2009, 52, 3539–3547.
(24) Abdelhamid, I. A.; Ghozlan, S. A. S.; Elnagdi, M. H. Alkyl-
substituted heteroaromatics as precursors to polycyclic heteroaro-
matics: recent developments. ARKIVOC 2008, 2008, 54–84.
(25) Gewald, K. Heterocycles from CH-acidic nitriles. VII. 2-Amino-
thiophene from alpha-oxo mercaptans and methylene-active
nitriles. Chem. Ber. 1965, 98, 3571–3577.
(18) Chirita, C.; Necula, M.; Kuret, J. Ligand-dependent inhibition and
reversal of tau filament formation. Biochemistry 2004, 43, 2879–
2887.
(19) Wischik, C. M.; Edwards, P. C.; Lai, R. Y. K.; Roth, M.;
Harrington, C. R. Selective inhibition of Alzheimer disease-like
tau aggregation by phenothiazines. Proc. Natl. Acad. Sci. U.S.A.
1996, 93, 11213–11218.
(26) McGovern, S. L.; Helfand, B. T.; Feng, B.; Shoichet, B. K. A
specific mechanism of nonspecific inhibition. J. Med. Chem. 2003,
46, 4265–4272.
(27) Pardridge, W. M. The blood-brain barrier: bottleneck in brain
drug development. NeuroRx 2005, 2, 3–14.
(28) Reichel, A. The role of blood-brain barrier studies in the pharma-
ceutical industry. Curr. Drug Metab. 2006, 7, 183–203.
(20) Wainwright, M.; Crossley, K. B. Methylene blue—a therapeutic
(29) Nagakura, M.; Ota, T.; Shimidzu, N.; Kawamura, K.; Eto, Y.;
Wada, Y. Syntheses and antiinflammatory actions of 4,5,6,7-tetra-
hydroindazole-5-carboxylic acids. J. Med. Chem. 1979, 22, 48–52.
(30) Sotelo, E.; Mocelo, R.; Suarez, M.; Loupy, A. Synthesis of poly-
functional pyridazine derivatives using a solvent-free microwave
assisted method. Synth. Commun. 1997, 27, 2419–2423.
(31) Hong, M.; Zhukareva, V.; Vogelsberg-Ragaglia, V.; Wszolek, Z.;
Reed, L.; Miller, B. I.; Geschwind, D. H.; Bird, T. D.; McKeel, D.;
Goate, A.; Morris, J. C.; Wilhelmsen, K. C.; Schellenberg, G. D.;
Trojanowski, J. Q.; Lee, V. M. Y. Mutation-specific functional
impairments in distinct tau isoforms of hereditary FTDP-17.
Science 1998, 282, 1914–1917.
dye for all seasons? J. Chemother. 2002, 14, 431–443.
(21) Crowe, A.; Huang, W.; Ballatore, C.; Johnson, R. L.; Hogan, A.
M.; Huang, R.; Wichterman, J.; McCoy, J.; Huryn, D.; Auld, D. S.;
Smith, A. B., III; Inglese, J.; Trojanowski, J. Q.; Austin, C. P.;
Brunden, K. R.; Lee, V. M.-Y. Identification of aminothienopyr-
idazine inhibitors of tau assembly by quantitative high-throughput
screening. Biochemistry 2009, 48, 7732–7745.
(22) Pajouhesh, H.; Lenz, G. R. Medicinal chemical properties of
successful central nervous system drugs. NeuroRx 2005, 2, 541–553.
(23) Ferguson, G. N.; Valant, C.; Horne, J.; Figler, H.; Flynn, B. L.;
Linden, J.; Chalmers, D. K.; Sexton, P. M.; Christopoulos, A.;