Brain-penetrant, orally bioavailable microtubule-stabilizing small molecules are potential candidate therapeutics for Alzheimer's disease and related tauopathies

Article abstract of DOI:10.1021/jm5005623

Microtubule (MT) stabilizing drugs hold promise as potential treatments for Alzheimer's disease (AD) and related tauopathies. However, thus far epothilone D has been the only brain-penetrant MT-stabilizer to be evaluated in tau transgenic mice and in AD p

Full text of DOI:10.1021/jm5005623

Article
pubs.acs.org/jmc
Open Access on 07/03/2015
Brain-Penetrant, Orally Bioavailable Microtubule-Stabilizing Small
Molecules Are Potential Candidate Therapeutics for Alzheimer’s
Disease and Related Tauopathies
Kevin Lou,† Yuemang Yao,‡ Adam T. Hoye,† Michael J. James,‡ Anne-Sophie Cornec,† Edward Hyde,‡
Bryant Gay,† Virginia M.-Y. Lee,‡ John Q. Trojanowski,‡ Amos B. Smith, III,† Kurt R. Brunden,*,‡
and Carlo Ballatore*,†,‡
†Department of Chemistry, School of Arts and Sciences, University of Pennsylvania, 231 South 34th Street, Philadelphia,
Pennsylvania 19104-6323, United States
‡Center for Neurodegenerative Disease Research, Institute on Aging, University of Pennsylvania, 3600 Spruce Street, Philadelphia,
Pennsylvania 19104-6323, United States
S
* Supporting Information
ABSTRACT: Microtubule (MT) stabilizing drugs hold promise as potential
treatments for Alzheimer’s disease (AD) and related tauopathies. However,
thus far epothilone D has been the only brain-penetrant MT-stabilizer to be
evaluated in tau transgenic mice and in AD patients. Furthermore, this natural
product exhibits potential deficiencies as a drug candidate, including an
intravenous route of administration and the inhibition of the P-glycoprotein
(Pgp) transporter. Thus, the identification of alternative CNS-active MT-
stabilizing agents that lack these potential limitations is of interest. Toward this
objective, we have evaluated representative compounds from known classes of
non-naturally occurring MT-stabilizing small molecules. This led to the
identification of selected triazolopyrimidines and phenylpyrimidines that are
orally bioavailable and brain-penetrant without disruption of Pgp function.
Pharmacodynamic studies confirmed that representative compounds from
these series enhance MT-stabilization in the brains of wild-type mice. Thus, these classes of MT-stabilizers hold promise for the
development of orally active, CNS-directed MT-stabilizing therapies.
decreased tau neuropathology, and reduced hippocampal neuron
loss. Furthermore, these effects mediated by 1 led to a significant
improvement of cognitive performance by the tau Tg mice.
Compound 1, however, has thus far been the only brain-
penetrant MT-stabilizer to be evaluated in mouse tauopathy
models.6,7 Furthermore, despite the efficacy and safety displayed
by 1 in Tg mice, this natural product and many other naturally
occurring MT-stabilizing agents or derivatives thereof exhibit
potentially significant deficiencies as drug candidates, including a
generally preferred intravenous (iv) route of administration and,
in the case of 1 and many taxanes, the inhibition of the P-
glycoprotein (Pgp)8 transporter, which upon prolonged dosing
could be responsible for undesired central nervous system
(CNS) toxicities9 and/or drug−drug interactions. As a result, the
development and evaluation of additional CNS-active MT-
stabilizing agents are clearly desirable to identify alternative and
potentially improved clinical candidates. In this context, we
undertook an evaluation of representative compounds from
different classes of non-naturally occurring compounds with
reported MT-stabilizing activities (Figure 1). Among these,
INTRODUCTION
■
Neurodegenerative tauopathies, of which Alzheimer’s disease
(AD) is the most prominent and common example, are
characterized by the misfolding and aggregation of the
microtubule (MT) associated protein tau.1 Normally, tau binds
to and stabilizes MTs, thereby maintaining the network of MTs
essential for axonal transport in neurons.2 In AD and related
diseases, tau becomes hyperphosphorylated, thereby promoting
disengagement from MTs and sequestration into characteristic
aggregates known as neurofibrillary tangles (NFTs) and neuropil
threads.1 The resulting loss of the normal MT-stabilizing
function of tau is believed to perturb the physiological dynamics
of MTs. In neuronal axons, MTs are more stable than in other
cell types, and hyperdynamic, less organized MTs would likely
result in defective axonal transport, with deleterious effects for
the affected neurons.3 Previous studies4−6 have shown that
exogenous MT-stabilizing agents can compensate for loss of tau
function in models of tauopathy and thus may provide a viable
therapeutic strategy for the treatment of AD and related
tauopathies. Indeed, treatment of aged tau transgenic (Tg)
mice, which manifest NFT-like inclusions, with low weekly doses
of the brain-penetrant MT-stabilizing agent epothilone D (1,
Figure 1), improved axonal transport, reduced axonal dystrophy,
Received: April 10, 2014
© XXXX American Chemical Society
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Figure 1. Structures of epothilone D (1) and representative examples from different classes of non-naturally occurring compounds with reported MT-
stabilizing activity.
particularly attractive were the triazolopyrimidines, as a selected
member of this heterocyclic MT-stabilizing series, cevipabulin
(2), has been reported to exhibit excellent pharmaceutical
properties, including oral bioavailability (61%), metabolic
stability (T1/2 = 13 h in female nude mice), and water solubility
(0.89 mg/mL).10 Although we show here that 2 is not a brain-
penetrant compound, other closely related congeners are
identified with significant brain penetration and oral bioavail-
ability, as well as MT-stabilizing properties. Finally, we
demonstrate that selected examples can elicit a profound
pharmacodynamic (PD) response in brain in a manner similar
to what was previously reported for 1.
Next, treatment of the trifluoroaryl compounds 8, 20, and 23
with the appropriate amino alcohol or diol side chain in the
presence of sodium hydride led selectively to the O-arylated
products.10,16 Finally, acylation of 2 and 11 provided amide
derivatives 15−17, while conversion of 12 to the corresponding
tosylate, followed by treatment with tetrabutylammonium
fluoride, furnished 13. Attempted displacement of tosylated 12
with 2,2,2-trifluoroethan-1-amine hydrochloride in the presence
of Hunig’s base, however, resulted in the formation of the chloro
̈
derivative 14 rather than the expected compound 10. The
synthesis of 10 was instead achieved by reacting 8 with 3-[(2,2,2-
trifluoroethyl)amino]propan-1-ol (Scheme 1).
The synthesis of pyridopyrazine 37 and related analogue 38
was achieved as previously described18 (Scheme 3). Thus,
condensation of pyrazine 39 with 2-(2,4,6-trifluorophenyl)acetyl
chloride to form amide 40 followed by base-promoted
cyclization to pyridopyrazine 41 and phosphorus oxychloride
treatment provided the 6,8-dichloro derivative 42, which was
then reacted with isopropylamine to obtain a separable mixture
of pyridopyrazines 37 and 38, as well as byproduct 43, whose
structure was confirmed by single crystal X-ray crystallography.
CHEMISTRY
■
Compounds 311 and 412 (Figure 1) were commercially available.
Phthalimide derivative 513 and pyridazines 614 and 715 (Figure 1)
were synthesized as previously described. The synthesis of 2
(Figure 1) and related triazolopyrimidines (8−19, Scheme 1)
and phenylpyrimidines (20−25, Scheme 1), which included a
series of new analogues (10, 13−17, 19, 24, and 25), was
accomplished following the general synthetic approach depicted
in Scheme 1.10,16 Starting from trifluorophenyl malonate 26,17
condensation with either aminotriazole 27 or the appropriate
amidine 28 or 29 led to the formation of triazolopyrimidine 30,
or phenylpyrimidines 31 and 32, respectively, which were
directly treated with phosphorus oxychloride to furnish the
corresponding dichloro derivatives 33, 34, and 35. Treatment of
33, 34, and 35 with the appropriate fluorinated amine then led to
triazolopyrimidine 8 and phenylpyrimidines 20−23.
EVALUATION OF MICROTUBULE-STABILIZING
ACTIVITY
■
The MT-stabilizing ability of all test compounds was assessed in
QBI293 cells (an HEK293 cell derivative) by monitoring for
compound-induced increase in acetylated α-tubulin (AcTub).
Acetylation of α-tubulin is a post-translational modification that
takes place on polymerized tubulin,8 (i.e., MTs), and as a result,
quantification of AcTub provides a convenient method to
determine the MT-stabilizing activity of test compounds within
the context of a cellular milieu. Previous studies have shown that
50−500 nM concentrations of 1 produce a significant elevation
The chemoselectivity of the nucleophilic displacement leading
to triazolopyrimidine 8 was confirmed by single crystal X-ray
crystallography of azide derivative 36 (Scheme 2).
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a
Scheme 1
a
Reagents and reaction conditions: (a) (i) aminotriazole or the appropriate amidine, tributylamine, 170−180 °C, 2−6 h; (ii) toluene, sodium
hydroxide; (b) phosphorus oxychloride, 110−130 °C, 6−16 h, 46−58% over two steps; (c) appropriate amine, diisopropylethylamine, N,N-
dimethylformamide, rt to 90 °C, 1−18 h, 12−50%; (d) appropriate amino alcohol, sodium hydride, dimethylsulfoxide/tetrahydrofuran, 60 °C, 3−12
h, 8−89%; (e) (i) tosyl chloride, triethylamine, dichloromethane, rt, 16 h; (ii) tetrabutylamonium fluoride, acetonitrile, rt, 4 h, 52% over two steps;
(f) (i) tosyl chloride, triethylamine, dichloromethane, rt, 16 h; (ii) 2,2,2-trifluoroethan-1-amine hydrochloride, diisopropylethylamine, acetonitrile, 80
°C, 4 h, 86% over two steps; (g) acetic anhydride, triethylamine, N,N-dimethylformamide, rt, 30 min, 62−90%; (h) pivaloyl chloride,
diisopropylethylamine, dichloromethane, rt, 30 min, 38%.
of AcTub levels in cells.8 To investigate whether a comparable
increase could be achieved by the triazolopyrimidines and/or
related non-naturally occurring MT-stabilizing heterocyclic
compounds, compound 2, which is believed to be the most
potent compound within the triazolopyrimidine series,10 was
compared with 1 in this assay. Interestingly, as shown in Figure 2,
a
Scheme 2
Figure 2. Comparison of activity of representative compounds in the
a
QBI293 cell MT-stabilization (AcTub) assay.
Reagents and reaction conditions: (a) sodium azide, N,N-
dimethylformamide, 70 °C, 24 h, 47%.
compound concentration, suggesting that the activity of this
triazolopyrimidine may be higher than the natural product, 1, in
this cell-based MT-stabilization assay. In addition, the related
triazolopyrimidine, 9, and the phenylpyrimidine, 20, were also
2 elicited a significant increase of cellular AcTub. Moreover, 2
caused an even greater AcTub increase than 1 at 100 nM
a
Scheme 3
a
Reagents and reaction conditions: (a) 2-(2,4,6-trifluorophenyl)acetyl chloride, pyridine, dichloromethane, 4-dimethylaminopyridine, rt, 20 h, 44%;
(b) potassium carbonate, N,N-dimethylformamide, 80 °C, 3 h, 88%; (c) phosphorus oxychloride, N,N-dimethylformamide, 1,2-dichloroethane, 80
°C, 3 h, 80%; (d) isopropylamine, 4-dimethylaminopyridine, potassium carbonate, N,N-dimethylformamide, rt, 24 h, 40% (37), 13% (38), 7% (43).
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found to elicit a large increase in AcTub in the cellular assay.
Again, the increase of cellular AcTub is comparable after
treatment with 100 nM 9 or 1, indicating that both 2 and 9 are of
nearly equal potency as the natural product.
Table 1. Efficacy of Compounds in the QBI293 Cell AcTub
Assay When Tested at 1 μM
a
fold-change relative to vehicle
control
fold-change relative to
compd
0.1 μM 2
To confirm that the observed elevation in AcTub levels caused
by 2 was associated with an increase in MT number and/or
organization, compound-treated QBI293 cells were examined by
AcTub immunofluorescence. As depicted in Figure 3, cells
8
9
1.3 0.6ns
9.1 1.7***
(2.0 1.4)*
2.1 1.1*
0.4 0.1
1.3 0.2
10
11
0.6 0.1
3.4 1.3
26.5 3.0***
(4.2 0.2)**
10.1 5.2***
1.4 0.7ns
1.2 0.7ns
4.5 2.1***
2.4 0.9***
1.3 0.7ns
4.1 1.9***
1.0 0.6ns
2.7 1.3**
1.5 0.9ns
4.2 2.1***
1.6 0.9ns
4.1 2.8*
0.9 0.7ns
1.0 0.8ns
1.0 0.8ns
1.0 0.2ns
0.9 0.6ns
1.0 0.6ns
1.5 0.8ns
1.3 0.8ns
12
13
14
15
16
17
18
19
20
21
22
23
24
25
37
38
3
1.2 0.6
1.5 0.3
0.7 0.1
1.2 0.3
0.9 0.1
0.9 0.1
0.5 0.2
Figure 3. AcTub immunofluorescence of QBI293 cells after 4 h of
incubation with no test compound, colchicine (1 μM), 2 (100 nM), or 1
(100 nM).
4
5
6
7
treated with 100 nM 2 exhibited a large increase in AcTub-
positive structures relative to control cells, with the intensity of
AcTub staining exceeding that produced by 100 nM 1, whereas
the MT-destabilizing agent, colchicine, led to a significant loss of
AcTub staining.
a
Data are expressed as the fold-change relative to vehicle-treated cells
and relative to a positive control (0.1 μM 2). Numbers in parentheses
are the relative activity of compounds that showed a significant effect
at 0.1 μM. ns = not significant. (∗) p < 0.05, (∗∗) p < 0.01, and (∗∗∗)
p < 0.001 by two-tailed t-test.
Several additional examples of non-naturally occurring MT-
stabilizing agents, including a series of triazolopyrimidines,
phenylpyrimidines, pyridazines, pyridopyrazines, and represen-
tative compounds from other classes, such as 3−5 (Figure 1),
underwent testing in the QBI293 AcTub assay at 0.1 or 1 μM
(Table 1). Interestingly, a number of test compounds were found
to elicit significant increases in cellular AcTub at 1 μM (Table 1),
with one additional example (11) causing increased AcTub at 0.1
μM, as observed with 2 and 9. The relative efficacy of each of the
active examples in the AcTub cellular assay is also compared to
that of 0.1 μM 2 in Table 1.
P-GLYCOPROTEIN (Pgp) INTERACTIONS
■
Figure 4. Pgp-interaction assay (calcein-AM). Cyclosporin A is a known
Pgp substrate, whereas verapamil and 1 are known Pgp inhibitors.
Previous bidirectional permeability studies demonstrated that 1
is an inhibitor of the Pgp transporter.8 Compound 2 and several
related congeners have been reported to be cytotoxic against
Pgp-expressing cancer cell lines,10 thus indicating that these
compounds may not be Pgp-substrates. However, these
cytotoxicity data cannot rule out the possibility that these
compounds might be Pgp inhibitors. To determine whether 2
and related analogues inhibit Pgp function, compounds 2, 9, and
20 were evaluated in a standard Pgp-interaction assay that
monitors the ability of test compounds to disrupt Pgp-mediated
transport of a known Pgp substrate, calcein-AM.19 As shown in
Figure 4, both a known Pgp inhibitor (verapamil) and a Pgp
substrate (cyclosporine A) affect Pgp activity in this assay, as both
compete for calcein-AM binding to Pgp. Notably, unlike 1, the
triazolopyrimidines 2 and 9, as well as phenylpyrimidine 20, did
not interfere appreciably with Pgp function.
PHARMACOKINETIC (PK) PROPERTIES
■
A significant hindrance to the development of MT-stabilizing
agents for neurodegenerative disease is the relatively poor
blood−brain barrier (BBB) penetration of many previously
described compounds.20 To evaluate the brain penetration of
active triazolopyrimidines and phenylpyrimidines, the levels of
compound in mouse brain and plasma were evaluated 1 h after
intraperitoneal (ip) administration of 5 mg/kg test compound.
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The majority of CNS-active drugs typically exhibit brain-to-
plasma (B/P) exposure ratios of ≥0.3, although there are
exceptions.21 As summarized in Table 2, compound 2 does not
h after oral administration of the compounds (10 mg/kg). As
shown in Figure 6, both compounds were detected at
micromolar levels in brain and plasma, confirming that these
representative compounds are orally bioavailable.
Table 2. Brain and Plasma Compound Concentration, 1 h
after an ip Injection of 5 mg/kg of Test Compound
compd
brain, nM
plasma, nM
B/P
2
92
8
2500 200
2800 200
4900 500
160 40
0.037 0.003
0.8 0.2
8
2400 600
1300 200
230 30
40 20
9
0.27 0.02
1.5 0.2
10
11
12
14
16
17
18
19
20
22
24
1300 70
0.03 0.01
2.8 0.4
78
4
29
5
160 10
170 10
20 10
178
5
0.88 0.06
0.12 0.03
0.15 0.03
0.49 0.03
1.6 0.2
1500 500
200 100
1200 300
Figure 6. Brain and plasma levels of 9 and 20, 2 h after oral
administration of 10 mg/kg.
600 200
120
3
75
7
2900 100
1500 400
860 130
5000 200
4300 900
2500 200
0.58 0.01
0.34 0.04
0.35 0.06
EVALUATION OF BRAIN PHARMACODYNAMIC
(PD) EFFECTS
■
appear to be readily brain-penetrant, as suggested by the B/P ≪
0.3. However, several compounds were identified that exhibited
B/P ≥ 0.3, with brain concentrations reaching the low
micromolar range for several examples. From these, a
representative compound from each of the triazolopyrimidine
(9) and phenylpyrimidine (20) classes were evaluated in a 16 h
PK study (Figure 5). These studies revealed that the area under
the curve (AUC) B/P ratios for 9 and 20 were 0.37 and 0.72,
Previous studies demonstrated that a single ip injection of 1 (1−6
mg/kg) produces MT-stabilization in the CNS of mice, as
revealed by a significant elevation in AcTub levels in brain
homogenates after dosing.4 To investigate whether the
representative phenylpyrimidine and triazolopyrimidine exam-
ples of orally bioavailable small molecule MT-stabilizing agents
could elicit a comparable PD response in the brain, groups of
mice (n = 3) received daily doses (1 mg/kg, ip) of 9 or 20 for
either 4 or 6 days, followed by a determination of AcTub levels in
brain homogenates, as previously described.8 As shown in Figure
7, both compounds produced a significant PD response,
demonstrating that these small heterocyclic compounds can
stabilize MTs within the brains of mice. Notably, the compound-
induced elevation in brain AcTub levels increased from 4 to 6
days of dosing. Moreover, although 9 shows lesser BBB
permeability than 20, the brain free drug concentrations of the
two compounds are predicted to be relatively comparable during
the first 2−3 h after dosing based on the total brain
concentrations and the observation that the brain Fu value of 9
is ∼3-fold greater than for 20 (see Figure 5). This, coupled with
the greater activity of 9 compared to 20 in the cellular assay
(Figure 2), likely explains the slightly greater PD effect of 9. More
extensive future studies will be required to determine the
minimum doses required to elicit these PD effects, as well as the
dosing frequency and time to reach maximal MT stabilization.
respectively (Figure 5). Moreover, the elimination half-life (T1/2
)
values in plasma and brain for both compounds were found to be
>2 h, with 20 showing particularly long T1/2 values.
To more accurately estimate the BBB permeability of these
compounds, the relative B/P ratio of free (unbound) compound
was estimated after determining the amount of nonspecific
binding to plasma proteins and brain tissue by equilibrium
dialysis, as previously described.8 The equilibrium dialysis results
revealed that both compounds had high plasma protein binding
and brain homogenate binding, with fraction unbound (Fu)
values as highlighted in Figure 5. This resulted in estimated free
compound B/P ratios of 0.09 0.01 for 9 and 1.7 0.6 for 20.
Thus, these data would suggest that whereas 20 can readily
partition across the BBB, the passage of 9 is relatively limited,
although an appreciably greater brain exposure is obtained with
this compound than with 2 (cf. 2 and 9, Table 2).
Finally, to ascertain whether these compounds could be orally
absorbed, brain and plasma levels of 9 and 20 were determined 2
Figure 5. Brain and plasma pharmacokinetics of 9 and 20 after 5 mg/kg ip dosing to CD1 mice.
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binding site on β-tubulin, this compound did affect vinblastine
binding to β-tubulin.28 It is not clear yet whether 2 binds directly
at the vinca alkaloid site on β-tubulin or rather at a distinct site
that regulates vinblastine binding allosterically.28 Notably,
cytotoxicity studies in cancer cell lines10 revealed that 2 exhibits
potent antimitotic effects that are similar to that observed with
classical MT-stabilizing molecules, such as paclitaxel and 1.
Furthermore, 2 was found to be active against Pgp-expressing
cancer cell lines, suggesting that the compound is not a substrate
for this transporter. Finally, 2 was found to exhibit desirable
druglike properties, including good water solubility, oral
bioavailability, and metabolic stability.10 Thus, because of these
features of 2, we evaluated a series of triazolopyrimidines and
related heterocycles as potential leads for the development of
orally bioavailable, CNS-active MT-stabilizing agents. Interest-
ingly, an evaluation of such molecules in a cell-based assay that
employs AcTub as a surrogate marker for compound-induced
MT-stabilization revealed that several triazolopyrimidines and
phenylpyrimidines produce a significant elevation in AcTub
levels that appeared to be comparable to or higher than the
response caused by 1. In contrast, other related heterocycles with
reported MT-stabilizing activity, including pyridopyridazine 37,
pyridazines 6 and 7, and nonrelated compounds 3−5, were
found to be essentially inactive in this assay at 1 μM.
Evaluation of representative examples of active triazolopyr-
imidines and phenylpyrimidines reveals that these small
molecules, unlike 1, do not interfere with Pgp function.
Furthermore, although 2 was found to exhibit negligible brain
penetration, possibly due to the presence of an ionizable
secondary amine in the side chain, relatively simple structural
modifications consisting of a removal of the side chain (e.g., 8,
20) or substitutions at the terminal amine that reduce the
solvation (e.g., 9) and/or ionization of the amino group in
plasma (e.g., 10, 19) led to a significant improvement in brain
penetration. Although these modifications generally resulted in a
moderate attenuation of the MT-stabilizing activity of these
compounds relative to 2, several examples were identified that
retained significant MT-stabilizing properties (Table 1). Among
these compounds, the representative triazolopyrimidine 9 and
phenylpyrimidine 20 underwent further PK analyses, which
revealed that both of these compounds achieve meaningful brain
exposure after ip or oral administration, although in the case of 9,
correction of the B/P ratio for the unbound fractions in plasma
and brain homogenates highlights a limited BBB permeability.
Moreover, the elimination T1/2 in plasma and brain for both
compounds was found to be >2 h, with 20 showing particularly
long T1/2 values. Importantly, an evaluation of the brain PD
activity of these two brain-penetrant, orally bioavailable
compounds revealed that relatively low daily dosing of either 9
or 20 resulted in a marked time-dependent elevation in AcTub
levels in the brain of WT mice. The extent of AcTub elevation
elicited upon repeated dosing of these compounds is comparable
to what was previously observed with the natural product, 1,
although the latter required less frequent dosing due to a very
prolonged CNS retention.8 The observation that a marked PD
effect could be elicited by relatively low doses of these small
heterocycles is clearly promising, and future efficacy studies in
tau Tg mice in comparison with 1 will more fully determine the
potential of these and related compounds as candidates for the
treatment of neurodegenerative tauopathies.
Figure 7. Relative increase in AcTub level in the brain of WT mice that
received 9 or 20, 1 mg/kg daily ip for 4 or 6 days: ∗, p < 0.05; ∗∗, p <
0.01, as determined by ANOVA with a Dunnett’s multiple comparison
test.
DISCUSSION
■
The rationale for treating AD and related neurodegenerative
tauopathies with MT-stabilizing agents is to compensate for the
MT dysfunction that likely results from a loss of tau MT-
stabilizing function caused by tau hyperphosphorylation and
sequestration into inclusions, with a consequent impairment of
axonal transport.22 Initial proof-of-concept for this therapeutic
strategy23 was obtained in 2005, when paclitaxel was found to
improve the neurodegenerative phenotype of tau Tg mice that
develop brain stem and spinal cord tau pathology.5 However,
paclitaxel, which does not readily cross the BBB, presumably
provided benefit in these mice through uptake at peripheral
neuromuscular junctions with consequent drug delivery to motor
neurons. Recent studies have further validated this therapeutic
strategy when it was observed that once-weekly doses of 1 far
below levels used in cancer clinical trials were effective in treating
both the neuropathology and cognitive deficits of PS19 tau Tg
mice,4,6 which develop age-dependent AD-like tau pathology in
the brain.24 Furthermore, subsequent studies confirmed that 1
was effective in two additional tau Tg animal models,7 and a
clinical trial was subsequently initiated with this agent (referred
to as BMS-241027) in AD patients. Collectively, these studies
provide important validation that brain-penetrant MT-stabilizing
agents may be useful for the treatment of AD and related
tauopathies. However, as noted previously, 1 exhibits deficiencies
as a drug candidate, including the inhibition of the Pgp
transporter and a preferred iv route of administration. Therefore,
as part of our efforts toward the identification of alternative and
potentially improved clinical candidates, we complemented our
ongoing investigations of MT-stabilizing natural products with
an evaluation of known non-naturally occurring MT-stabilizing
small molecules that are endowed with potentially favorable
druglike properties, with the objective of identifying brain-
penetrant, orally bioavailable MT-stabilizing candidates. Among
these classes of compounds are certain triazolopyrimidines,
typified by 2,10 as well as some structurally related phenyl-
pyrimidines,16 pyridopyridazines,18 and pyridazines.15 The first
examples of these synthetic small molecule MT-stabilizing agents
were members of the triazolopyrimidines discovered as
antifungal agents.25 Since then, improved analogues, as well as
related scaffolds, have been reported that appear to share similar
MT-stabilizing properties, antimitotic activity, and structure−
activity relationships.10,15,16,18,26,27 Interestingly, radioligand
binding studies suggested that the mode of action of these
heterocyclic MT-stabilizing agents may be rather unique.28,29
Whereas 2 was found not to compete for the taxane/epothilone
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2-(2,6-Diisopropylphenyl)-5-hydroxyisoindoline-1,3-dione
(5). 5 was prepared as described in Li et al.13 Reverse-phase HPLC
purification of the crude product afforded the title compound. 1H NMR
(500 MHz, CDCl3) δ 7.89 (s, 1H), 7.80 (d, J = 8.2 Hz, 1H), 7.47 (t, J =
7.8 Hz, 1H), 7.37 (d, J = 2.2 Hz, 1H), 7.31 (d, J = 7.8 Hz, 2H), 7.06 (dd, J
= 8.2, 2.2 Hz, 1H), 2.74 (p, J = 6.8 Hz, 2H), 2.16 (s, 1H), 1.18 (dd, J =
6.8, 1.9 Hz, 11H) ppm. 13C NMR (126 MHz, CDCl3) δ 168.70, 168.68,
162.47, 147.35, 134.35, 130.33, 126.71, 126.11, 124.07, 122.94, 121.07,
111.18, 29.35, 23.99, 23.97 ppm. HRMS [ESI]+ calculated for
C20H22NO3, 324.1600; found, 324.1563.
5-(4-Chlorophenyl)-6-methyl-4-(2,4,6-trifluorophenyl)-
pyridazin-3(2H)-one (6). 6 was prepared as described in Manabe et
al.14 Reverse-phase HPLC purification of the crude product afforded the
title compound. 1H NMR (500 MHz, CDCl3) δ 11.40 (bs, 1H), 7.34−
7.28 (m, 2H), 7.07−7.00 (m, 2H), 6.57 (dd, J = 8.7, 7.2 Hz, 2H), 2.11 (s,
3H) ppm. 13C NMR (125 MHz, CDCl3) δ 159.86, 147.46, 144.87,
135.21, 133.26, 129.08, 129.00, 100.69 (d, J = 25.2 Hz), 20.91 ppm.
HRMS [ESI]+ calculated for C17H10Cl2F3N2, 369.0173; found,
369.0179.
CONCLUSIONS
■
Several lines of investigation suggest that CNS-directed MT-
stabilizing therapies may be effective in the treatment of
neurodegenerative tauopathies, including AD, as well as other
neurodegenerative diseases in which axonal transport deficits
appear to be caused by hyperdynamic axonal MTs.30 However, in
spite of the large number of MT-stabilizing agents that have been
developed to date for cancer chemotherapy, thus far only a few
examples have been identified that exhibit the required PK/PD
properties to be considered as potential candidates for the
treatment of AD and related neurodegenerative diseases.20 The
studies presented herein demonstrate that representative
members of the triazolopyrimidine and related phenylpyrimidine
MT-stabilizing small molecules exhibit promising PK properties,
including oral bioavailability and brain penetration, without
significant interference of Pgp function. Furthermore, PD studies
confirmed that examples from these series produce an increase in
a biomarker of MT-stabilization in the brain of mice. Thus, these
classes of MT-stabilizing agents exhibit properties that provide
potential advantages over the existing MT-stabilizing natural
products. As a result, these compounds may be promising leads
for the development of orally active, CNS-directed MT-
stabilizing therapies.
3-Chloro-5-(5-chlorothiophen-2-yl)-6-methyl-4-(2,4,6-
trifluorophenyl)pyridazine (7). 7 was prepared as described in
Lamberth et al.15 Reverse-phase HPLC purification of the crude product
afforded the title compound. 1H NMR (500 MHz; CDCl3) δ 6.83 (d, J =
3.8 Hz, 1H), 6.73−6.70 (m, 3H), 2.71 (s, 3H) ppm. HRMS [ESI]+
calculated for C15H8Cl2F3N2S, 374.9737; found, 374.9715.
(S)-5-Chloro-6-(2,4,6-trifluorophenyl)-N-(1,1,1-trifluoropro-
pan-2-yl)[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (8). 8 was pre-
pared as described in Zhang et al.10 Reverse-phase HPLC purification
afforded the title compound as a colorless solid (25% yield). 1H NMR
(500 MHz, CDCl3) δ 8.41 (s, 1H), 6.95−6.89 (m, 2H), 5.88 (d, J = 9.2
Hz, 1H), 4.69 (bm, 1H), 1.43 (d, J = 6.8 Hz, 3H) ppm. 13C NMR (126
MHz, CDCl3) δ 164.55 (dt, J = 255.0, 15.1 Hz), 161.62 (ddd, J = 253.3,
14.9, 8.1 Hz), 161.26 (ddd, J = 251.8, 15.0, 8.2 Hz), 157.91, 155.47,
154.23, 146.04, 124.57 (q, J = 282.0 Hz), 105.46 (td, J = 20.6, 4.8 Hz),
101.74 (td, J = 25.9, 4.2 Hz), 101.36 (td, J = 25.4, 4.5 Hz), 91.54, 51.01
(q, J = 32.2 Hz), 15.18 (q, J = 1.9 Hz) ppm. IR: ν 3393, 3334, 3258, 3112,
2953, 1620 cm−1. HRMS [ESI]+ calculated for C14H8N5F6Cl, 396.0445;
found, 396.0450.
General Procedure A (Installment of Side Chain). According to
reported procedures,10,16 to a suspension of sodium hydride (60% in
mineral oil, 4.0 equiv) in a 2:1 mixture of dimethylsulfoxide and
tetrahydrofuran (0.35 M) was added the appropriate amino alcohol (4.0
equiv), and the mixture was heated to 60 °C for 1 h. The resulting yellow
turbid solution was treated with a solution of trifluoroarene (1.0 equiv)
in a 1:1 mixture dimethylsulfoxide and tetrahydrofuran (0.5 M). The
reaction mixture was stirred at 60 °C for 3 h and monitored by LCMS. If
the starting material was not fully consumed within 3 h, additional amino
alcohol (4.0 equiv) and sodium hydride (4.0 equiv) were added
sequentially, and the reaction mixture was heated for 12 h. Following
complete consumption of the starting material, the reaction mixture was
cooled to room temperature and diluted with water and ethyl acetate.
The organic layer was washed with water and brine, and the combined
aqueous layers were extracted with ethyl acetate (×3). The combined
organic layers were dried (MgSO4), filtered, and concentrated. The
crude products were purified by reverse-phase HPLC.
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 chromatography 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 spectrometer. 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 Spectrometry Center
on either a VG Micromass 70/70H or VG ZAB-E spectrometer. Single-
crystal X-ray structure determinations were 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 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%. 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. Unless otherwise stated, all final compounds were found to be
>95% pure as determined by HPLC/MS and NMR.
(S)-5-Chloro-6-(4-(3-(dimethylamino)propoxy)-2,6-difluoro-
phenyl)-N-(1,1,1-trifluoropropan-2-yl)[1,2,4]triazolo[1,5-a]-
pyrimidin-7-amine (9). General procedure A was followed, using 3-
dimethylamino-1-propanol and 8. Reverse-phase HPLC purification
afforded the title compound as a colorless, hygroscopic solid (38%
yield). 1H NMR (500 MHz, DMSO-d6) δ 8.51 (bs, 1H), 8.18 (s, 1H),
6.89 (dd, J = 4.0, 9.9 Hz, 2H), 5.89−5.83 (m, 1H), 4.10 (t, J = 6.3 Hz,
2H), 2.55 (t, J = 7.6 Hz, 2H), 2.29 (s, 6H), 1.93 (p, J = 6.8 Hz, 2H), 1.37
(d, J = 6.8 Hz, 3H) ppm. 13C NMR (126 MHz, MeOD) δ 170.42, 164.32
(t, J = 14.2 Hz), 163.66 (dd, J = 246.9, 9.2 Hz), 163.01 (dd, J = 246.4, 9.2
Hz), 159.57, 156.12, 155.87, 149.34, 126.71 (q, J = 281.7 Hz), 102.27 (t,
J = 21.3 Hz), 100.52 (dd, J = 25.9, 3.3 Hz), 100.18 (dd, J = 25.9, 3.2 Hz),
94.91, 68.32, 52.37 (q, J = 31.8 Hz), 27.77, 14.05 ppm. HRMS [ESI]+
calculated for C19H21N6OF5Cl, 479.1386; found, 479.1384.
(S)-5-Chloro-6-(2,6-difluoro-4-(3-(methylamino)propoxy)-
phenyl)-N-(1,1,1-trifluoropropan-2-yl)[1,2,4]triazolo[1,5-a]-
pyrimidin-7-amine (2). 2 was prepared as described in Zhang et al.10
Reverse-phase HPLC purification of the crude product afforded the title
compound (35% yield). 1H NMR (500 MHz, MeOD) δ 8.48 (s, 1H),
6.84 (d, J = 9.5 Hz, 2H), 5.50 (s, 1H), 4.32−4.06 (m, 2H), 3.23 (t, J = 7.5
Hz, 2H), 2.75 (s, 3H), 2.34−2.12 (m, 2H), 1.43 (d, J = 6.9 Hz, 3H) ppm.
13C NMR (126 MHz, MeOD) δ 163.82 (t, J = 14.3 Hz), 163.65 (dd, J =
247.6, 8.8 Hz), 163.00 (dd, J = 246.7, 9.2 Hz), 162.96, 162.68, 159.51,
156.07, 155.91, 149.28, 126.68 (q, J = 281.7 Hz), 119.35, 117.03, 102.80
(t, J = 21.2 Hz), 100.66 (dd, J = 26.2, 3.3 Hz), 100.29 (dd, J = 26.0, 3.4
Hz), 94.67, 67.48, 52.36 (q, J = 31.6 Hz), 34.01, 27.05, 14.06 ppm.
HRMS [ESI]+ calculated for C18H19N6OF5Cl, 465.1229; found,
465.1230.
G
dx.doi.org/10.1021/jm5005623 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(S)-5-Chloro-6-(2,6-difluoro-4-(3-((2,2,2-trifluoroethyl)-
amino)propoxy)phenyl)-N-(1,1,1-trifluoropropan-2-yl)[1,2,4]-
triazolo[1,5-a]pyrimidin-7-amine (10). General procedure A was
followed, using 3-((2,2,2-trifluoroethyl)amino)propan-1-ol and 8.
Reverse-phase HPLC purification afforded the title compound (89%
yield). 1H NMR (500 MHz, CDCl3) δ 8.40 (s, 1H), 6.69−6.65 (m, 2H),
5.92 (d, J = 10.3 Hz, 1H), 4.77 (bs, 1H), 4.15 (t, J = 6.1 Hz, 2H), 3.25 (q,
J = 9.4 Hz, 2H), 2.99 (t, J = 6.7 Hz, 2H), 2.04 (quintet, J = 6.4 Hz, 2H),
1.42 (d, J = 6.8 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3)
characteristic signals δ 162.39 (t, J = 13.8 Hz), 161.89 (dd, J = 250.3, 8.8
Hz), 161.62 (dd, J = 248.7, 8.7 Hz), 158.71, 155.34, 154.16, 146.19,
124.73 (q, J = 281.9 Hz), 124.57 (q, J = 280 Hz), 101.09 (t, J = 21.1 Hz),
99.52 (dd, J = 25.6, 3.4 Hz), 99.25 (dd, J = 25.6, 3.2 Hz), 92.63, 66.64,
50.96 (q, J = 32.2 Hz), 49.81 (q, J = 32.6, 31.8 Hz), 46.25, 28.20, 15.32
ppm. HRMS [ESI]+ calculated for C19H18ClF8N6O, 533.1103; found,
533.1199.
HRMS [ESI]+ calculated for C17H15N5OF6Cl, 454.0864; found,
454.0858.
(S)-5-Chloro-6-(4-(3-chloropropoxy)-2,6-difluorophenyl)-N-
(1,1,1-trifluoropropan-2-yl)[1,2,4]triazolo[1,5-a]pyrimidin-7-
amine (14). To a solution of 12 (0.05 g, 0.12 mmol) in anhydrous
dichloromethane (1.20 mL) were added tosyl chloride (0.07 g, 0.36
mmol) and triethylamine (0.05 mL, 0.36 mmol). The reaction mixture
was stirred at room temperature for 16 h. The reaction was then
quenched by addition of water, and the resulting mixture was washed
with saturated aqueous solution of sodium bicarbonate and brine. The
organic layer was then dried over sodium sulfate, filtered, and
concentrated. Purification by silica gel column chromatography, using
a 1:1 mixture of ethyl acetate/hexanes, provided the tosylated
intermediate (0.05 g, 0.07 mmol). To a solution of the latter
intermediate (0.01 g, 0.02 mmol) in acetonitrile (0.24 mL) were
added 2,2,2-trifluoroethan-1-amine hydrochloride (0.01 g, 0.06 mmol)
and diisopropylethylamine (0.01 mL, 0.08 mmol). The reaction mixture
was stirred at 80 °C for 4 h. The reaction mixture was then diluted with
water and ethyl acetate, and the organic layer was washed with brine.
The organic layer was dried over sodium sulfate, filtered, and
concentrated. Purification by silica gel column chromatography, using
a 30% mixture of ethyl acetate in hexanes, afforded the title compound
(86% yield). 1H NMR (500 MHz; CDCl3) δ 8.41 (s, 1H), 6.68 (m, 2H),
5.89 (d, J = 9.8 Hz, 1H), 4.79 (bs, 1H), 4.21 (t, J = 5.8 Hz, 2H), 3.79 (t, J
= 6.2 Hz, 2H), 2.32 (m, 2H), 1.43 (d, J = 6.8 Hz, 3H) ppm. 13C NMR
(126 MHz, CDCl3) δ 162.50 (t, J = 13.7 Hz), 161.84 (dd, J = 250.3, 8.8
Hz), 161.57 (dd, J = 248.8, 9.0 Hz), 158.58, 155.50, 146.11, 124.70 (q, J
= 282.0 Hz), 100.99 (t, J = 21.1 Hz), 99.54 (dd, J = 25.5, 3.5 Hz), 99.25
(dd, J = 25.4, 3.4 Hz), 92.45, 65.52, 50.86 (q, J = 32.2 Hz), 41.23, 31.97,
29.92, 15.42 ppm. IR: ν 3341, 2919, 2851, 1617 cm−1. HRMS [ESI]+
calculated for C17H15N5OF5Cl2, 470.0568; found, 470.0578.
(S)-6-(4-(3-Aminopropoxy)-2,6-difluorophenyl)-5-chloro-N-
(1,1,1-trifluoropropan-2-yl)[1,2,4]triazolo[1,5-a]pyrimidin-7-
amine (11). General procedure A was followed, using 3-aminopropan-
1-ol and 8. Reverse-phase HPLC purification afforded the title
1
compound (22% yield). H NMR (500 MHz; DMSO-d6) δ 8.30 (s,
1H), 8.09 (d, J = 0.3 Hz, 1H), 6.76−6.74 (m, 2H), 5.78 (bs, 1H), 4.13 (t,
J = 5.9 Hz, 2H), 3.36 (bs, 2H), 2.98 (t, J = 7.2 Hz, 2H), 2.03 (t, J = 6.6 Hz,
2H), 1.21 (d, J = 6.5 Hz, 3H) ppm. 13C NMR (126 MHz, MeOD) δ
170.43, 163.95 (t, J = 14.2 Hz), 163.67 (dd, J = 247.3, 9.1 Hz), 163.02
(dd, J = 246.7, 9.0 Hz), 159.50, 156.08, 155.90, 149.28, 126.70 (q, J =
281.6 Hz), 102.74 (t, J = 21.3 Hz), 100.62 (dd, J = 26.0, 3.3 Hz), 100.25
(dd, J = 26.0, 3.3 Hz), 94.69, 67.64, 52.37 (q, J = 31.7 Hz), 38.61, 28.97,
14.08 ppm. HRMS [ESI]+ calculated for C17H17ClF5N6, 451.1073;
found, 451.1076.
(S)-3-(4-(5-Chloro-7-((1,1,1-trifluoropropan-2-yl)amino)-
[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorophenoxy)-
propan-1-ol (12). General procedure A was followed, using propane-
1,3-diol and 8. Reverse-phase HPLC purification afforded the title
compound (60% yield). 1H NMR (500 MHz; CDCl3) δ 8.40 (s, 1H),
6.70−6.67 (m, 2H), 5.91 (d, J = 8.7 Hz, 1H), 4.77 (bs, 1H), 4.21 (t, J =
6.1 Hz, 2H), 3.91 (t, J = 5.9 Hz, 2H), 2.18−2.09 (m, 2H), 1.70 (s, 1H),
1.42 (d, J = 6.8 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3) δ 162.73 (t,
J = 13.8 Hz), 161.79 (dd, J = 249.7, 9.2 Hz), 161.53 (dd, J = 248.3, 9.0
Hz), 158.64, 155.44, 154.14, 146.06, 124.67 (q, J = 281.8 Hz), 100.70 (t,
J = 21.1 Hz), 99.49 (dd, J = 25.5, 3.3 Hz), 99.21 (dd, J = 25.4, 3.4 Hz),
92.51, 66.28, 59.53, 50.81 (q, J = 32.1 Hz), 31.85, 15.37 ppm. HRMS
[ESI]+ calculated for C17H16ClF5N5O2, 452.0913; found, 452.0902.
(S)-5-Chloro-6-(2,6-difluoro-4-(3-fluoropropoxy)phenyl)-N-
(1,1,1-trifluoropropan-2-yl)[1,2,4]triazolo[1,5-a]pyrimidin-7-
amine (13). To a solution of 12 (0.05 g, 0.12 mmol) in anhydrous
dichloromethane (1.20 mL) were added tosyl chloride (0.07 g, 0.36
mmol) and triethylamine (0.05 mL, 0.36 mmol). The reaction mixture
was stirred at room temperature for 16 h. The reaction was quenched by
addition of water, and the resulting mixture was washed with saturated
aqueous solution of sodium bicarbonate and brine. The organic layer
was then dried over sodium sulfate, filtered, and concentrated.
Purification by silica gel column chromatography, using a 1:1 mixture
of ethyl acetate/hexanes, provided the tosylated intermediate (0.05 g,
0.07 mmol). To a solution of the latter intermediate (0.03 g, 0.05 mmol)
in acetonitrile (0.65 mL) was added tetrabutylammonium fluoride (1 M
in tetrahydrofuran, 0.06 mmol). The reaction mixture was stirred at
room temperature for 4 h. The reaction mixture was then diluted with
dichloromethane and washed with water and brine. The organic layer
was dried over sodium sulfate, filtered, and concentrated. Purification by
silica gel column chromatography, using a 30% mixture of ethyl acetate
(S)-N-(3-(4-(5-Chloro-7-((1,1,1-trifluoropropan-2-yl)amino)-
[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorophenoxy)-
propyl)acetamide (15). To a solution of 11 (0.08 g, 0.08 mmol) in
N,N-dimethylformamide (2.52 mL) was added triethylamine (0.01 mL,
0.08 mmol). The reaction mixture was stirred at room temperature for
10 min, and then acetic anhydride (0.01 mL, 0.08 mmol) was added.
The reaction mixture was stirred at room temperature for 1 h, and was
then diluted with ethyl acetate and washed with water. The organic layer
was dried over sodium sulfate, filtered, and concentrated. Purification by
silica gel column chromatography, using a 5% mixture of methanol in
1
dichloromethane, afforded the title compound (90% yield). H NMR
(500 MHz; CDCl3) δ 8.37 (s, 1H), 6.64 (d, J = 8.9 Hz, 2H), 6.11 (s, 1H),
5.98 (d, J = 10.6 Hz, 1H), 4.85 (s, 1H), 4.10 (t, J = 6.1 Hz, 2H), 3.48 (q, J
= 6.3 Hz, 2H), 2.08 (t, J = 6.4 Hz, 2H), 2.01 (s, 3H), 1.42 (d, J = 6.8 Hz,
3H) ppm. 13C NMR (126 MHz, CDCl3) δ 170.57, 161.74 (dd, J = 241.2,
8.7 Hz), 161.50 (dd, J = 248.3, 9.4 Hz), 158.46, 155.31, 154.19, 146.17,
124.66 (q, J = 282.4 Hz), 100.77 (t, J = 20.9 Hz), 99.42 (dd, J = 25.6, 3.2
Hz), 99.16 (dd, J = 26.0, 3.3 Hz), 92.54, 67.08, 53.53, 50.86 (q, J = 32.3
Hz), 36.84, 29.24, 23.35, 15.22 ppm. IR: ν 3298, 3103, 2925, 2853, 1642,
1616 cm−1. HRMS [ESI]+ calculated for C19H19ClF5N6O2, 493.1178;
found, 493.1180.
(S)-N-(3-(4-(5-Chloro-7-((1,1,1-trifluoropropan-2-yl)amino)-
[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorophenoxy)-
propyl)pivalamide (16). To a solution of 11 (0.01 g, 0.02 mmol) in
dichloromethane (0.20 mL) were added pivaloyl chloride (3 μL, 0.02
mmol) and diisopropylethylamine (4 μL, 0.02 mmol). The reaction
mixture was stirred for 30 min at room temperature, and then it was
diluted with dichloromethane and washed with water. The organic layer
was dried over sodium sulfate, filtered, and concentrated. Purification by
silica gel column chromatography, using a 5% mixture of methanol in
1
in hexanes, afforded the title compound (52% yield). H NMR (500
1
dichloromethane, afforded the title compound (38% yield). H NMR
MHz; CDCl3) δ 8.39 (s, 1H), 6.76−6.56 (m, 2H), 5.89 (d, J = 11.0 Hz,
1H), 4.77 (s, 1H), 4.68 (dt, J = 47.0, 5.6 Hz, 2H), 4.18 (t, J = 6.1 Hz, 2H),
2.24 (dt, J = 26.3, 5.8 Hz, 2H), 1.41 (d, J = 6.8 Hz, 3H) ppm. 13C NMR
(126 MHz, CDCl3) δ 162.56 (t, J = 13.8 Hz), 161.82 (dd, J = 250.1, 8.8
Hz), 161.56 (dd, J = 248.5, 9.0 Hz), 158.59, 155.48, 154.20, 146.08,
124.69 (q, J = 282.0 Hz), 100.92 (t, J = 20.9 Hz), 99.51 (dd, J = 25.4, 3.3
Hz), 99.21 (dd, J = 25.4, 3.4 Hz), 92.45, 80.35 (d, J = 165.3 Hz), 64.81
(d, J = 4.7 Hz), 50.84 (q, J = 32.1 Hz), 30.24 (d, J = 20.1 Hz), 15.39 (q, J
= 1.9, 1.5 Hz) ppm. IR: ν 3340, 2953, 2920, 2851, 1642, 1618 cm−1.
(500 MHz, CDCl3) δ 8.39 (s, 1H), 6.64 (m, 2H), 5.96 (m, 2H), 4.77 (bs,
1H), 4.10 (t, J = 5.9 Hz, 2H), 3.49 (q, J = 6.3 Hz, 2H), 2.07 (m, 2H), 1.41
(d, J = 6.8 Hz, 3H), 1.22 (s, 9H) ppm. 13C NMR (126 MHz, CDCl3) δ
178.92, 162.51 (t, J = 13.8 Hz), 161.86 (dd, J = 250.3, 8.7 Hz), 161.59
(dd, J = 248.1, 8.8 Hz), 158.55, 155.52, 154.20, 146.08, 124.69 (q, J =
281.9 Hz), 100.97 (t, J = 21.0 Hz), 99.46 (dd, J = 25.6, 3.3 Hz), 99.15
(dd, J = 25.5, 3.4 Hz), 92.42, 67.62, 50.85 (d, J = 32.1 Hz) 38.93, 37.27,
29.27, 27.82, 15.40 ppm. IR: ν 3343, 2961, 2931, 2816, 1614, 1581 cm−1.
H
dx.doi.org/10.1021/jm5005623 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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HRMS [ESI]+ calculated for C22H25N6O2F5Cl, 535.1642; found,
535.1641.
3134, 1695, 1634, 1580, 1557 cm−1. HRMS [ESI]+ calculated for
C16H9ClF6N5, 420.0451; found, 420.0459.
(S)-N-(3-(4-(5-Chloro-7-((1,1,1-trifluoropropan-2-yl)amino)-
[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorophenoxy)-
propyl)-N-methylacetamide (17). 17 was prepared as for 15, starting
(S)-6-Chloro-2-(pyridin-2-yl)-5-(2,4,6-trifluorophenyl)-N-
(1,1,1-trifluoropropan-2-yl)pyrimidin-4-amine (22). 22 was pre-
pared as described in Zhang et al.16 Reverse-phase HPLC purification of
the crude product afforded the title compound (50% yield). 1H NMR
(500 MHz; CDCl3) δ 8.86 (d, J = 4.6 Hz, 1H), 8.43 (d, J = 7.9 Hz, 1H),
7.90−7.87 (m, 1H), 7.44 (dd, J = 7.2, 5.0 Hz, 1H), 6.92−6.86 (m, 2H),
5.41−5.35 (m, 1H), 4.60 (d, J = 9.3 Hz, 1H), 1.41 (d, J = 7.0 Hz, 3H)
ppm. 13C NMR (126 MHz, CDCl3) δ 164.22 (dt, J = 253.7, 15.0 Hz),
162.87, 161.41, 161.11, 160.88 (ddd, J = 252.8, 14.9, 8.9 Hz), 153.24,
149.82, 137.62, 125.69, 125.49 (q, J = 281.8 Hz), 124.29, 105.10 (td, J =
20.5, 20.0, 4.4 Hz), 104.17, 101.82 (td, J = 25.8, 4.1 Hz), 101.68 (td, J =
25.8, 4.1 Hz), 48.22 (q, J = 31.3 Hz), 14.65 (q, J = 1.8 Hz) ppm. HRMS
[ESI]− calculated for C18H10ClF6N4, 431.0498; found, 431.0508.
6-Chloro-2-(pyridin-2-yl)-N-(2,2,2-trifluoroethyl)-5-(2,4,6-
trifluorophenyl)pyrimidin-4-amine (23). 23 was prepared as
described in Zhang et al.16 Purification by silica gel chromatography,
using gradients of ethyl acetate in hexanes, afforded the title compound
(12% yield). 1H NMR (500 MHz; CDCl3) δ 8.83 (d, J = 4.0 Hz, 1H),
8.43 (d, J = 7.9 Hz, 1H), 7.88 (t, J = 7.3 Hz, 1H), 7.44 (t, J = 5.8 Hz, 1H),
6.84−6.81 (m, 2H), 5.04 (t, J = 6.0 Hz, 1H), 4.38 (dd, J = 15.4, 8.0 Hz,
2H) ppm. 13C NMR (126 MHz, CDCl3) δ 164.31 (dt, J = 253.8, 15.1
Hz), 161.59, 161.51, 161.06 (dd, J = 252.8, 9.0 Hz), 160.94 (dd, J =
253.0, 9.0 Hz), 153.37, 149.79, 137.82, 125.77, 124.38 (q, J = 279.3 Hz),
124.37, 124.35, 105.19 (td, J = 21.2, 5.3 Hz), 104.46, 101.75 (td, J = 26.0,
3.4 Hz), 42.72 (q, J = 34.8 Hz) ppm. HRMS [ESI]+ calculated for
C17H10ClF6N4, 419.0498; found, 419.0496.
1
from 2 (62% yield). H NMR (500 MHz, CDCl3) mixture of E/Z
isomers δ 8.39 (apparent d, J = 3.0 Hz, 1H), 6.81−6.54 (m, 2H), 5.92 (d,
J = 10.8 Hz, 1H), 4.78 (s, 1H), 4.16−3.96 (m, 2H), 3.57 (m, 2H), 3.06−
2.96 (apparent d, 3H), 2.11 (m, 5H), 1.41 (apparent dd, J = 7.0, 1.7 Hz,
3H) ppm. 13C NMR (126 MHz, CDCl3) mixture of E/Z isomers δ
171.07, 170.76, 162.84, 162.76, 162.65, 162.57, 162.54, 162.50, 162.27,
162.17, 162.06, 160.86, 160.78, 160.60, 160.52, 158.57, 158.51, 155.52,
155.48, 154.21, 146.12, 146.05, 128.06, 128.03, 125.82, 125.79, 123.58,
123.55, 121.34, 121.30, 101.47, 101.30, 101.14, 100.92, 100.76, 100.59,
99.62, 99.59, 99.42, 99.39, 99.29, 99.26, 99.19, 99.09, 99.06, 98.98, 92.52,
92.27, 67.13, 65.63, 51.22, 50.97, 50.71, 50.46, 47.37, 44.94, 36.71, 33.33,
32.13, 29.90, 29.86, 29.81, 29.67, 29.57, 29.52, 29.49, 27.77, 27.20, 22.90,
22.14, 21.33, 15.38, 14.34 ppm. IR: ν 3411, 3207, 2923, 2853, 1638,
1616 cm−1. HRMS [ESI]+ calculated for C20H21ClF5N6O2, 507.1335;
found, 507.1335.
(S)-5-Chloro-6-(2,6-difluoro-4-(3-morpholinopropoxy)-
phenyl)-N-(1,1,1-trifluoropropan-2-yl)[1,2,4]triazolo[1,5-a]-
pyrimidin-7-amine (18). 18 was prepared as described in Zhang et
al.10 Reverse-phase HPLC purification afforded the title compound
1
(68% yield). H NMR (500 MHz; CDCl3) δ 8.39 (s, 1H), 6.67−6.63
(m, 2H), 5.95 (d, J = 10.5 Hz, 1H), 4.81 (bs, 1H), 4.11 (t, J = 6.1 Hz,
2H), 3.82 (t, J = 4.7 Hz, 4H), 2.75 (m, 6H), 2.13 (m, 2H), 1.41 (d, J = 6.8
Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3) δ 165.76, 162.46 (t, J =
13.9 Hz), 161.78 (dd, J = 250.0, 8.9 Hz), 161.50 (dd, J = 248.8, 9.0 Hz),
158.50, 155.39, 154.15, 146.08, 130.76, 124.65 (q, J = 282.1 Hz), 100.82
(t, J = 20.9 Hz), 99.43 (dd, J = 25.5, 3.3 Hz), 99.17 (dd, J = 25.4, 3.3 Hz),
92.47, 66.93, 66.03, 55.08, 53.15, 50.80 (q, J = 32.0 Hz), 25.20, 15.28
ppm. IR: ν 3425, 2958, 2861, 2817, 1640, 1615, 1578 cm−1. HRMS
[ESI]+ calculated for C21H23N6O2F5Cl, 521.1486; found, 521.1488.
(S)-5-Chloro-6-(2,6-difluoro-4-(3-(phenylamino)propoxy)-
phenyl)-N-(1,1,1-trifluoropropan-2-yl)[1,2,4]triazolo[1,5-a]-
pyrimidin-7-amine (19). General procedure A was followed, using 3-
(phenylamino)propan-1-ol and 8. Reverse-phase HPLC purification
afforded the title compound (53% yield). 1H NMR (500 MHz; CDCl3)
δ 8.40 (s, 1H), 7.20 (m, 2H), 6.73 (m, 1H), 6.67 (m, 4H), 5.89 (d, J =
10.1 Hz, 1H), 4.77 (bs, 1H), 4.16 (t, J = 5.9 Hz, 2H), 3.40 (t, J = 6.6 Hz,
2H), 2.17 (quintet, J = 6.2 Hz, 2H), 1.41 (d, J = 6.8 Hz, 3H) ppm. 13C
NMR (126 MHz, CDCl3) δ 162.59 (t, J = 14.0 Hz), 161.77 (dd, J =
250.1, 8.8 Hz), 161.51 (dd, J = 248.4, 8.9 Hz), 158.56, 155.41, 154.11,
148.08, 146.00, 129.51, 124.63 (q, J = 281.4, 280.7 Hz), 117.87, 112.94,
100.77 (t, J = 20.9 Hz), 99.41 (dd, J = 25.6, 3.2 Hz), 99.14 (dd, J = 25.4,
3.5 Hz), 92.43, 67.03, 50.79 (q, J = 32.3 Hz), 40.83, 29.02, 15.33 ppm.
IR: ν 3338, 3209, 3113, 3055, 2950, 1614, 1575 cm−1. HRMS [ESI]+
calculated for C23H21N6OF5Cl, 527.1380; found, 527.1385.
(S)-6-Chloro-5-(4-(3-(dimethylamino)propoxy)-2,6-difluoro-
phenyl)-2-(pyrazin-2-yl)-N-(1,1,1-trifluoropropan-2-yl)-
pyrimidin-4-amine (24). To a solution of 20 (0.03 g, 0.06 mmol) and
3-(dimethylamino)propan-1-ol (0.04 mL, 0.321 mmol) in dimethylsulf-
oxide (0.32 mL) was added sodium hydride (60% in mineral oil, 0.01 g,
0.321 mmol) at room temperature. The reaction mixture was stirred at
60 °C for 2 h, cooled to room temperature, dissolved in additional
dimethylsulfoxide, filtered, and purified by preparative reverse phase
1
HPLC to obtain the title compound as a white solid (8% yield). H
NMR (500 MHz; DMSO-d6) δ 9.58 (d, J = 1.2 Hz, 1H), 8.86−8.84 (m,
2H), 7.70 (d, J = 8.8 Hz, 1H), 6.95 (t, J = 10.0 Hz, 2H), 5.53 (dd, J = 15.3,
7.6 Hz, 1H), 4.12 (t, J = 6.2 Hz, 2H), 2.38 (t, J = 7.1 Hz, 2H), 2.17 (s,
6H), 1.90 (quintet, J = 6.7 Hz, 2H), 1.36 (d, J = 7.1 Hz, 3H) ppm. 13C
NMR (126 MHz, DMSO-d6) δ 165.05, 162.01, 161.70 (t, J = 14.4 Hz),
161.07 (dd, J = 244.9, 10.1 Hz), 160.73, 160.72 (dd, J = 244.1, 10.4 Hz),
160.14, 148.65, 146.28, 145.01, 144.57, 125.95 (q, J = 283.1 Hz), 104.77,
100.36 (t, J = 21.7 Hz), 99.37 (dd, J = 25.4, 2.4 Hz), 99.27 (dd, J = 25.5,
2.5 Hz), 66.93, 55.45, 47.21 (q, J = 29.2 Hz), 45.21, 26.69, 13.13 ppm.
HRMS [ESI]+ calculated for C22H23ClF5N6O, 517.1542; found,
517.1536.
6-Chloro-5-(4-(3-(dimethylamino)propoxy)-2,6-difluoro-
phenyl)-2-(pyridin-2-yl)-N-(2,2,2-trifluoroethyl)pyrimidin-4-
amine (25). 25 was prepared as for 24 from 23 and 3-(dimethylamino)-
propan-1-ol (10% yield). 1H NMR (500 MHz; MeOD) δ 8.72 (dd, J =
4.7, 0.7 Hz, 1H), 8.54−8.52 (m, 1H), 8.03 (td, J = 7.8, 1.7 Hz, 1H), 7.58
(ddd, J = 7.5, 4.9, 1.0 Hz, 1H), 6.79 (d, J = 9.3 Hz, 2H), 4.38 (q, J = 9.1
Hz, 2H), 4.14 (t, J = 6.0 Hz, 2H), 2.73 (t, J = 7.6 Hz, 2H), 2.45 (s, 6H),
2.07 (dd, J = 13.9, 7.3 Hz, 2H) ppm. 13C NMR (126 MHz, MeOD) δ
170.36, 164.16, 163.70 (t, J = 14.2 Hz), 163.67, 162.82 (dd, J = 246.7,
10.0 Hz), 161.93, 154.88, 150.44, 139.20, 127.18, 126.34 (q, J = 279.2
Hz), 125.50, 106.57, 102.34 (t, J = 21.6 Hz), 100.48−100.17 (m),
100.56−100.17 (m), 68.10, 57.15, 45.20, 42.91 (q, J = 34.6 Hz), 27.54
ppm. HRMS [ESI]+ calculated for C22H22ClF5N5O, 502.1433; found,
502.1425.
(S)-6-Chloro-2-(pyrazin-2-yl)-5-(2,4,6-trifluorophenyl)-N-
(1,1,1-trifluoropropan-2-yl)pyrimidin-4-amine (20). 20 was pre-
pared as described in Zhang et al.16 Purification by silica gel
chromatography, using gradients of ethyl acetate in hexanes, afforded
the title compound (18% yield). 1H NMR (500 MHz; DMSO-d6) δ 9.57
(d, J = 1.4 Hz, 1H), 8.85−8.83 (m, 2H), 7.70 (d, J = 8.8 Hz, 1H), 7.48−
7.42 (m, 2H), 5.52 (m, 1H), 1.35 (d, J = 7.1 Hz, 3H) ppm. 13C NMR
(126 MHz, DMSO-d6) δ 163.59 (dt, J = 248.3, 16.2 Hz), 161.84 (ddd, J
= 247.9, 16.2, 9.5 Hz), 161.63, 161.14, 160.48 (ddd, J = 248.2, 16.1, 9.4
Hz), 159.98, 148.51, 146.39, 145.07, 144.59, 125.91 (q, J = 282.8 Hz),
105.42 (td, J = 21.4, 4.6 Hz), 103.73, 101.73 (td, J = 26.8, 3.8 Hz). 101.65
(td, J = 26.7, 3.8 Hz). 47.24 (q, J = 30.6 Hz), 13.14 ppm. IR: ν 3282,
2920, 1638, 1580, 1556 cm−1. HRMS [ESI]+ calculated for
C17H11ClF6N5, 434.0607; found, 434.0616.
6-Chloro-2-(pyrazin-2-yl)-N-(2,2,2-trifluoroethyl)-5-(2,4,6-
trifluorophenyl)pyrimidin-4-amine (21). 21 was prepared as
described in Zhang et al.16 Purification by silica gel chromatography,
using gradients of ethyl acetate in hexanes, afforded the title compound
(31% yield). 1H NMR (500 MHz; CDCl3) δ 9.65 (d, J = 1.4 Hz, 1H),
8.79 (dd, J = 2.4, 1.5 Hz, 1H), 8.72 (d, J = 2.4 Hz, 1H), 6.89−6.85 (m,
2H), 5.21−5.18 (m, 1H), 4.37 (qd, J = 8.7, 6.6 Hz, 2H) ppm. IR: ν 3255,
5,7-Dichloro-6-(2,4,6-trifluorophenyl)[1,2,4]triazolo[1,5-a]-
pyrimidine (33). Following a reported procedure,10 a slurry of 2-
(2,4,6-trifluorophenyl)malonate (1.19 g, 4.10 mmol) and aminotriazole
(0.36 g, 4.31 mmol) in tributylamine (1.03 mL) was heated to 170 °C
for 2 h. The resulting homogeneous brown mixture was cooled to 130
°C, and toluene (4.00 mL) was added before cooling to 50 °C. A
solution of sodium hydroxide (0.36 mL, 50% aqueous) was added, and
the precipitated solids were collected by vacuum filtration, washed with
I
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Journal of Medicinal Chemistry
Article
cold toluene, and dried to afford the bis-sodium salt of 30 as a beige
powder (1.24 g, 3.80 mmol, 93% yield).
After overnight growth at 37 °C in 5% CO2, test compounds, a known
Pgp substrate (cyclosporin A), or known Pgp inhibitors (verapamil and
epoD) that were dissolved in dimethylsulfoxide and diluted in PBS were
added in triplicate (50 μL volume) over a range of concentrations such
that the final dimethylsulfoxide concentration was 0.25%. Vehicle
control wells were also included. After incubation for ∼20 min at 37 °C
in 5% CO2, Calcein AM was added to a final concentration of 0.25 nM
(50 μL at a dimethylsulfoxide concentration of 0.2%) and allowed to
incubate an additional 25 min at 37 °C in 5% CO2. The medium was
then removed, and the cells were washed twice with 0.2 mL of cold PBS,
followed by analysis of calcein fluorescence with excitation at 485 nm
and emission at 530 nm. Data were fit with a nonlinear regression
sigmoidal dose−response, with the buffer control defining no inhibition
and the average signal obtained with 0.1 mM verapamil defining 100%
inhibition.
Determination of Plasma and Brain Drug Concentrations.
Test compounds were administered to 2-month old CD1 mice (20 g
average body weight) under a protocol approved by the University of
Pennsylvania Institutional Animal Care and Use Committee. Whole
brain hemispheres, obtained from mice euthanized according to
approved protocols, were homogenized in 10 mM ammonium acetate,
pH 5.7 (1:2, w/v), using a hand-held sonic homogenizer. 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, centrifuged at 15 000g, and the resulting
supernatants were used for subsequent LC−MS/MS analysis. The LC−
MS/MS system comprised an Aquity UPLC instrument and a TQ MS
instrument that was controlled using MassLynx software (Waters
Corporation, Milford, MA, USA). 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 water with 0.1%
(v/v) formic acid and mobile phase B of acetonitrile 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 MS
instrument 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 compound using the MassLynx auto tune
utility. Standard curves were generated for each compound from brain
homogenate and plasma samples that had compound added at
concentrations ranging from 0.005 to 5 μM and extracted as above.
Drug half-life was calculated using the elimination rate constant
determined from the slope of a line plotted through natural log plasma
concentration versus time for the 4, 8, and 16 h time points using
GraphPad Prism. Area under the brain or plasma concentration curve
from 0.5 to 16 h was calculated using the trapezoid rule with GraphPad
Prism.
A mixture of bis-sodium 30 (0.30 g, 0.9 mmol) and phosphorus
oxychloride (1.50 mL, 16 mmol) was heated to 130 °C for 6 h. The
reaction mixture was cooled to room temperature and was carefully
quenched with H2O (2 mL). This aqueous mixture was extracted with
ethyl acetate (3 × 5 mL), and the combined organic layers were dried
(MgSO4), filtered, and concentrated to give the title compound as an
orange oil that was used without further purification and without full
1
characterization (50% yield). H NMR (500 MHz, CDCl3) δ 8.62 (s,
1H), 6.94−6.89 (m, 2H) ppm. MS (ESI+) 319.00 [M + H+].
4,6-Dichloro-2-(pyrazin-2-yl)-5-(2,4,6-trifluorophenyl)-
pyrimidine (34). 34 was prepared as described in Zhang et al.16
Purification by silica gel chromatography, using gradients of ethyl
acetate in hexanes, afforded the title compound that was used without
full characterization (32% yield). 1H NMR (500 MHz; CDCl3) δ 9.73 (s,
1H), 8.84 (s, 1H), 8.77 (s, 1H), 6.88−6.84 (m, 2H) ppm.
4,6-Dichloro-2-(pyridin-2-yl)-5-(2,4,6-trifluorophenyl)-
pyrimidine (35). 35 was prepared as 34 from picolinimidamide
hydrochloride and 2-(2,4,6-trifluorophenyl)malonate (58% yield). The
1
title compound was used without full characterization. H NMR (500
MHz, CDCl3) δ 9.16 (d, J = 5.4 Hz, 1H), 9.00−8.98 (m, 1H), 8.78 (td, J
= 7.9, 1.2 Hz, 1H), 8.36 (t, J = 6.7 Hz, 1H), 6.88 (dd, J = 8.4, 7.5 Hz, 2H)
ppm.
(S)-5-Azido-6-(2,4,6-trifluorophenyl)-N-(1,1,1-trifluoropro-
pan-2-yl)[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (36). To a sol-
ution of 8 (0.013 g, 0.032 mmol) in N,N-dimethylformamide (1 mL)
under nitrogen was added sodium azide (0.002 g, 0.032 mmol). The
reaction mixture was heated at 70 °C for 24 h. The reaction mixture was
cooled to room temperature and then concentrated. After addition of
water (5 mL), the mixture was extracted with ethyl acetate (3 × 5 mL)
and the combined organic layers were washed with water (5 mL), dried
(MgSO4), filtered, and concentrated. Purification by silica gel
preparative thin layer chromatography (hexanes/ethyl acetate 7/3)
afforded the title compound as a brown solid (47% yield). 1H NMR (500
MHz, CDCl3) δ 8.33 (s, 1H), 6.87 (dtd, J = 8.9, 6.5, 2.0 Hz, 2H), 5.92 (d,
J = 10.9 Hz, 1H), 4.50 (s, 1H), 1.41 (d, J = 6.9 Hz, 3H) ppm. 13C NMR
(126 MHz, CDCl3) δ 164.34 (dt, J = 254.4, 15.3 Hz), 161.59 (ddd, J =
252.9, 14.9, 8.3 Hz), 161.26 (ddd, J = 250.7, 14.8, 8.3 Hz), 160.01,
154.79, 154.44, 145.64, 124.65 (q, J = 282.0 Hz), 103.99 (td, J = 21.1,
20.5, 4.5 Hz), 101.63 (td, J = 25.8, 4.2 Hz), 101.12 (td, J = 25.9, 4.1 Hz),
83.28, 50.69 (q, J = 32.1 Hz), 15.32 ppm. IR: ν 2918, 2852, 2136, 1624
cm−1. MS (ESI+) calculated for C14H9F6N8 [M+H+], 403.27; found,
403.12.
6-Chloro-N-isopropyl-7-(2,4,6-trifluorophenyl)pyrido[2,3-b]-
pyrazin-8-amine (37). 37 was prepared as described in Crowley et
al.18 Purification by silica gel preparative thin layer chromatography
using a 1:1 mixture of ethyl acetate in hexanes as eluent furnished the
title compound, as well as 38 and byproduct 43. C-8 alkylated
pyridopyrazine 3718: 1H NMR (500 MHz, CDCl3) δ 8.99 (d, J = 1.9 Hz,
1H), 8.66 (d, J = 1.8 Hz, 1H), 6.93 (bs, 1H), 6.83 (dd, J = 8.6, 7.6 Hz,
2H), 3.36 (m, 1H), 1.11 (d, J = 6.3 Hz, 6H) ppm. HRMS [ESI]+
calculated for C16H13ClF3N4, 353.0781; found, 353.0793. 3818: 1H
NMR (500 MHz, CDCl3) δ 8.84 (d, J = 2.0 Hz, 1H), 8.63 (d, J = 2.0 Hz,
1H), 6.93 (dd, J = 8.6, 6.9 Hz, 2H), 4.72−4.60 (m, 1H), 4.54 (s, 1H),
1.24 (d, J = 6.5 Hz, 6H) ppm. HRMS [ESI]+ calculated for
Determination of Compound Unbound Fractions in Plasma
and Brain. The assessment of nonspecific compound binding to plasma
proteins and brain homogenates was as previously described.8
ASSOCIATED CONTENT
■
S
1
* Supporting Information
C16H13ClF3N4, 353.0781; found, 353.0782. Byproduct 43: H NMR
NMR spectra of test compounds and X-ray crystallographic
information and structures for compounds 23, 36, and 43. This
material is available free of charge via the Internet at http://pubs.
acs.org.
(500 MHz, CDCl3) δ 8.84 (d, J = 1.7 Hz, 1H), 8.60 (d, J = 1.7 Hz, 1H),
7.13 (dd, J = 9.5, 2.1 Hz, 1H), 6.77 (ddd, J = 12.7, 9.4, 2.1 Hz, 1H), 5.76
(s, 1H), 5.61−5.42 (m, 1H), 1.68 (d, J = 7.1 Hz, 6H), 1.38 (d, J = 6.3 Hz,
6H) ppm. HRMS [ESI]+ calculated for C19H20F2N5, 356.1687; found,
356.1688.
Acetyl α-Tubulin (AcTub) Assay. Compound-induced changes in
QBI293 cell AcTub levels were determined as previously described.8,31
P-Glycoprotein (Pgp) Assay. The ability of test compounds to
affect the Pgp transporter was assessed in NCI/ADR-RES cells
(ATCC), which express the human Pgp. The NCI/ADR-RES cells
were plated at 35 000 cells/well into 96-well flat-bottomed clear plates in
0.1 mL of minimal essential medium supplemented with 10% fetal
bovine serum, 50 units/mL penicillin, and 50 μg/mL streptomycin.
AUTHOR INFORMATION
■
Corresponding Authors
*K.R.B.: e-mail, kbrunden@upenn.edu; phone, 215-615-5262.
*C.B.: e-mail, bcarlo@sas.upenn.edu; phone, 215-898-4983.
Notes
The authors declare no competing financial interest.
J
dx.doi.org/10.1021/jm5005623 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(11) Haggarty, S. J.; Mayer, T. U.; Miyamoto, D. T.; Fathi, R.; King, R.
W.; Mitchison, T. J.; Schreiber, S. L. Dissecting cellular processes using
small molecules: identification of colchicine-like, Taxol-like and other
small molecules that perturb mitosis. Chem. Biol. 2000, 7, 275−286.
(12) Yang, W. S.; Shimada, K.; Delva, D.; Patel, M.; Ode, E.; Skouta, R.;
Stockwell, B. R. Identification of simple compounds with microtubule-
binding activity that inhibit cancer cell growth with high potency. ACS
Med. Chem. Lett. 2012, 3, 35−38.
ACKNOWLEDGMENTS
■
Financial support for this work has been provided by the NIH/
NIA (Grant AG044332-01), the Comprehensive Neuroscience
Center Pilot Grant (University of Pennsylvania), the Marian S.
Ware Alzheimer Program, and the NSF (Grant CHE-0840438,
X-ray facility).
(13) Li, P. K.; Pandit, B.; Sackett, D. L.; Hu, Z.; Zink, J.; Zhi, J.;
Freeman, D.; Robey, R. W.; Werbovetz, K.; Lewis, A.; Li, C. A
thalidomide analogue with in vitro antiproliferative, antimitotic, and
microtubule-stabilizing activities. Mol. Cancer Ther. 2006, 5, 450−456.
(14) Manabe, A.; Morishita, H. Pyridazine compound and use thereof.
WO2005121104 A, 2005.
(15) Lamberth, C.; Trah, S.; Wendeborn, S.; Dumeunier, R.; Courbot,
M.; Godwin, J.; Schneiter, P. Synthesis and fungicidal activity of tubulin
polymerisation promoters. Part 2: Pyridazines. Bioorg. Med. Chem. 2012,
20, 2803−2810.
(16) Zhang, N.; Ayral-Kaloustian, S.; Nguyen, T.; Hernandez, R.;
Lucas, J.; Discafani, C.; Beyer, C. Synthesis and SAR of 6-chloro-4-
fluoroalkylamino-2-heteroaryl-5-(substituted)phenylpyrimidines as
anti-cancer agents. Bioorg. Med. Chem. 2009, 17, 111−118.
(17) Meyer, O.; Eisenacht, R. Process for the Preparation of
halogenated phenylmalonates. US 6,156,925, 2000.
ABBREVIATIONS USED
■
AcTub, acetylated α-tubulin; AD, Alzheimer’s disease; AUC, area
under the curve; BBB, blood−brain barrier; B/P, brain-to-plasma
ratio; CNS, central nervous system; FTD, frontotemporal
dementia; ip, intraperitoneal; iv, intravenous; MT, microtubule;
NFT, neurofibrillary tangle; PD, pharmacodynamic; Pgp, P-
glycoprotein; PK, pharmacokinetic; Tg, transgenic
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