Potent and cellularly active 4-aminoimidazole inhibitors of cyclin-dependent kinase 5/p25 for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bmcl.2009.08.019

Utilizing structure-based drug design, a 4-aminoimidazole heterocyclic core was synthesized as a replacement for a 2-aminothiazole due to potential metabolically mediated toxicity. The synthetic route utilized allowed for ready synthesis of 1-substituted-4-aminoimidazoles. SAR exploration resulted in the identification of a novel cis-substituted cyclobutyl group that gave improved enzyme and cellular potency against cdk5/p25 with up to 30-fold selectivity over cdk2/cyclin E.

Full text of DOI:10.1016/j.bmcl.2009.08.019

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5703–5707
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potent and cellularly active 4-aminoimidazole inhibitors of
cyclin-dependent kinase 5/p25 for the treatment of Alzheimer’s disease
a
a
a
b
Christopher J. Helal a, , Zhijun Kang , John C. Lucas , Thomas Gant , Michael K. Ahlijanian ,
Joel B. Schachter b, Karl E. G. Richter b, James M. Cook b, Frank S. Menniti b, Kristin Kelly b, Scot Mente c,
Jay Pandit d, Natalie Hosea e
*
a Neuroscience Medicinal Chemistry, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, USA
b Neuroscience Biology, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, USA
c Computational Chemistry, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, USA
d Structural Biology, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, USA
e Pharmacokinetics and Drug Metabolism, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Utilizing structure-based drug design, a 4-aminoimidazole heterocyclic core was synthesized as a
replacement for a 2-aminothiazole due to potential metabolically mediated toxicity. The synthetic route
utilized allowed for ready synthesis of 1-substituted-4-aminoimidazoles. SAR exploration resulted in the
identification of a novel cis-substituted cyclobutyl group that gave improved enzyme and cellular
potency against cdk5/p25 with up to 30-fold selectivity over cdk2/cyclin E.
Received 10 June 2009
Revised 31 July 2009
Accepted 4 August 2009
Available online 8 August 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Cyclin-dependent kinase 5
p25
Cyclin-dependent kinase 2
Alzheimer’s disease
Cyclin dependent kinases (cdk) carry out critical roles in cell cy-
cling, and cdk inhibitors have been extensively researched as ther-
apeutic agents for the treatment of cancer.1 Binding of cdk5 by the
protein p35 results in kinase activation and the ability to phos-
phorylate the cytoskeletal stabilizing protein tau, a critical compo-
nent of cell structure regulation.2 The membrane-bound p35 can
be proteolytically cleaved by calpain to the more stable, cytosolic
p25 which has led to the hypothesis that cdk5/p25 may play an
integral role in Alzheimer’s disease development. The longer-lived
cdk5/p25 complex is believed to over-phosphorylate tau, resulting
in the formation of paired helical filaments and deposition of cyto-
toxic neurofibrillary tangles.3 Thus, inhibition of the aberrant cdk5/
p25 complex is a viable target for treating Alzheimer’s disease by
preventing tau hyperphosphorylation and subsequent neurofibril-
lary tangle formation.4 In addition to being a target for Alzheimer’s
disease, recent evidence suggests that inhibition of cdk5 could also
be relevant for the treatment of type-II diabetes, pain, and stroke,
furthering interest in this enzymatic target.5,6
bind in the ATP binding pocket.7 Selectivity over cdk2 is desired
due to its role in modulating the cell cycle and potential side effects.
This is a challenging task, considering 93% (27/29) of residues are
conserved in the respective ATP pockets of cdk5 and cdk2 and that
the two differing amino acid residues (Cys83 and Asp84 in cdk5;
Leu83 and His84 in cdk2) have sidechains that project away from
the ATP pocket, thereby reducing their impact on inhibitor binding.
In furthering our exploration of this series, alternative heterocy-
clic cores to the 2-aminothiazole (1) were sought, based upon the
metabolism-induced toxicity potential of this class of heterocycles8
(Fig. 1). Considering the binding mode of inhibitors to cdk5, as
observed in computer modeling in addition to X-ray crystal struc-
tures in cdk2, key H-bond acceptor and donor interactions with Cys
83 in the hinge region (Leu 83 in cdk2) were to be maintained
along with necessary lipophilic groups. The 4-aminoimidazole core
(2) was appropriately disposed to make the requisite hydrogen
bonding and hydrophobic interactions and was thus deemed a
suitable target for biological evaluation.
We reported on the optimization of potency and cdk2/cyclin E
selectivity in a series of 2-aminothiazole cdk5/p25 inhibitors that
The synthesis of 4-aminoimidazole analogs was carried out as
shown in Scheme 1.9 The treatment of 1,4-dinitroimidazole10 (3)
with a primary amine afforded 1-substituted 4-nitroimidazoles 4.
CAUTION: thermodynamic testing showed that 3 was a highly ener-
getic substance with the potential for explosion.11 Catalytic hydroge-
* Corresponding author. Tel.: +1 860 715 5064.
E-mail address: chris.j.helal@pfizer.com (C.J. Helal).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.019

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C. J. Helal et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5703–5707
gested that placement of polar functionality on the 3-position of
H-bonds to hinge region
Cys 83
the cyclobutyl of 7 could possibly lead to interactions with polar
amino acid side chains, Lys33 and Asp144 (Asp145 in cdk2), that
interact with the phosphates of bound ATP.14 These analogs were
synthesized via methods that have been previously described in
detail.15
N
H
N
H
N
N
N
R
S
R1
O
O
Ph
1
2
The initial polar group employed was hydroxyl (Table 3, 15 and
16). A significant effect of stereochemistry was observed with cis-
derivative 15 being ca. 10-fold more potent than trans 16, but with
similar potency to the unsubstituted cyclobutyl analog 7. A similar
cis/trans preference was observed with the methyl esters 17 and
18. An X-ray crystal structure of 15 bound to cdk2 was obtained
(Fig. 2).16 Key observations were that the cyclobutyl appears to
align with Phe80, resulting in a hydrophobic interaction that ex-
plains the observed SAR. Additionally, the hydroxyl group is in po-
sition to make a hydrogen bond with Asp145 (2.7 A) (Asp144 in
cdk5), and is in proximity (3.4 A) to Lys33. Equivalent cdk5 potency
of 15 as the unsubstituted cyclobutyl analog 7 suggests that the
hydroxyl interaction is energetically neutral, potentially due to
desolvation penalties as a result of removing waters associated
with the polar hydroxyl group, the Lys33 and/or Asp144, or with
energetic loss associated with disrupting a favorable salt bridge be-
tween Lys33 and Asp144.
cdk5/p25 IC50=64 nM
1.5 fold selectivity over cdk2
Figure 1.
N
N
N
a
b
H
N
N
N
R
NO2
NO2
R
N
H
O2N
5
3
4
c
N
H
R1
N
R
N
O
2
We thus sought to identify other possible polar groups to forge
interactions with Lys33 and/or Asp144 that would afford increased
potency through enthalpic contributions. The N-acetyl analog 19
gave a 10-fold potency increase (IC50 = 9 nM) relative to 15 and
also showed sevenfold greater potency for cdk5 than cdk2, demon-
strating that productive interactions were in fact being made.
N-methylsulfonyl derivative 20 maintained a similar potency pref-
erence for cdk5 over cdk2 (sixfold), but cdk5 potency dropped off
considerably (IC50 = 178 nM) compared to 19. With the 1-naphthyl
acetamide on the 4-aminoimidazole, N-acetyl analog 21 had simi-
lar potency (IC50 = 8 nM) to 19 and was more selective for cdk5
over cdk2 (17-fold). The placement of polar oxygen and nitrogen
atoms was demonstrated to be critical as the reversed amide 22
lost significant cdk5 potency (IC50 = 391 nM) and selectivity over
cdk2 (twofold). Interestingly, the N-methyl-N-acetyl analog 23 lost
cdk5 potency (IC50 = 107 nM), but maintained a preference for
cdk5 potency over cdk2 (15-fold). Importantly, this indicates that
the amide carbonyl was a primary contributor to selectivity and
the amide N–H contributed more to potency. Compound 19 was
profiled against a panel of 49 kinases and was found to inhibit four
Scheme 1. Reagents and conditions: (a) RNH2, MeOH, 23 °C, 42–75% yield; (b) H2
(50 psi), Pd/C, EtOAc; (c). R0CO2H, tri-n-propylphosphonic anhydride, Et3N, CH2Cl2,
ꢀ10 °C, 32–72% yield.
nation gave an unstable 4-aminoimidazole 5 that, following filtra-
tion through Celite, was immediately acylated with an activated
carboxylic acid to afford compounds of formula 2.
Extensive SAR of the amide side chain (not shown) revealed that
the aryl acetamides were the most potent substituents. As shown
in Table 1, phenylacetamide 6 was ca. eightfold less potent than
the corresponding aminothiazole 1.12 Addition of a 4-methoxy
group increased potency ca. fourfold (7). The 1-napthyl acetamide
8 afforded the best potency with IC50 = 46 nM. Activity against
cdk2 was essentially equivalent to cdk5 activity.
The ability to readily synthesize N-1 group analogs as shown in
Scheme 1 allowed for a rapid and broad exploration of SAR at this
position(Table 2). The role of the N-1 group is critical as methyl 9
was inactive. Isopropyl analog 10 and cyclopropyl analog 11
showed improved activity, whereas cyclobutyl 7 and cyclopentyl
12 were preferred groups with IC50 <200 nM. Cyclohexyl 13 lost
activity as compared to 7 and 12, as did benzyl 14. Compound 7
was subsequently profiled for selectivity against 20 diverse kinases
>30% at 10
carried out at 1
(100% @ 10 M, 94% @ 1 lM), MAPK1/ERK2 (89%, 47%), CK1-d
lM. Subsequent profiling against these four kinases was
lM to ascertain potency at lower doses; GSK3b
l
at 10 lM and showed >30% inhibition against three (GSK3b, 34%;
(39%, 7%), CDK2/Cyclin A (101%, 94%), and CLK1 (92%, 55%).17 In
particular, the significant increase in potency for GSK3b activity
is striking with the addition of the acetyl group in 19 as compared
AMPK, 48%; PHK, 32%).13
At this point, modeling of the cyclobutyl derivative 7 in a cdk5
homology model based upon a cdk2 X-ray crystal structure sug-
to the unsubstituted cyclobutyl analog 7 (94% @ 1 lM vs 34% @
Table 1
SAR of 4-N-acyl group
Table 2
SAR of N-1 substituent
N
H
N
N
OMe
N
R1
H
O
N
R
N
R1
cdk5 IC50 nM (std dev)
500
O
R
cdk5 IC50 nM (std dev)
6
7
8
CH2Ph
9
10
11
7
12
13
14
Me
i-Pr
c-Pr
c-Bu
c-Pentyl
c-Hexyl
Benzyl
>10,000
425
814 (161)
145 (8)
154 (27)
748 (192)
8640 (1490)
145 (8)
46 (18)
CH2-4-MeOPh
CH2-1-Napthyl

C. J. Helal et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5703–5707
5705
Table 3
SAR of cyclobutyl 3-substituent
N
H
N
N
B
A
O
A
cis (c)
trans (t)
B
cdk5 IC50 nM
(std dev)
Selecta
(K2/K5)
15
16
17
18
c
t
PMP
PMP
PMP
PMP
95 (42)
0.7
0.7
0.7
0.2
HO
1090 (516)
145 (57)
HO
Figure 2. X-ray crystal structure of 15 bound to cdk2. The aminoimidazole core
makes hydrogen-bonds (indicated by dashed lines) with Ile 83 in the hinge region.
c
t
MeO2C
MeO2C
4380 (247)
HN
O
19
20
c
c
PMP
PMP
9 (8)
7
6
HN
178 (46)
SO2
HN
O
21
22
23
c
c
c
1-Napth
1-Napth
1-Napth
8 (3)
17
2
391 (118)
107 (6)
H2N(O)C
Figure 3. X-ray crystal structure of 19 bound to the ATP binding site of cdk2.
Dashed lines indicate hydrogen bonds.
N
O
15
PMP = para-methoxyphenyl; 1-napth = 1-naphthyl.
ried out (Table 4). In addition to cdk5 enzyme inhibition and selec-
tivity for cdk5 over cdk2, inhibition of cellular cdk5/p25-
mediatated tau phosphorylation was measured.19
a
Select = ratio of cdk2 IC50/cdk5 IC50
.
The N-acetyl analog 21 showed a >1000-fold loss in whole cell
activity versus enzyme activity. Determination of permeability in
Caco-2 cells showed significant asymmetry between the BA Papp
(17.5 ꢁ 10ꢀ6 cm/s) and AB Papp (1.5 ꢁ 10ꢀ6 cm/s), suggestive of
impaired membrane permeability. While a reduction in whole cell
activity was expected, based upon the higher ATP concentration in
cells than in the enzyme inhibition assay, it was hoped that a re-
duced shift in activity could be realized with improved permeabil-
ity. In a particular sub-series of interest, the 4- and 3-pyridyl
derivatives (24 and 25, respectively) were less active than the 2-
pyridyl derivative 26 in the whole cell assay. The Caco-2 profile
of 26, with BA Papp = 21 ꢁ 10ꢀ6 cm/s and AB = 26 ꢁ 10ꢀ6 cm/s,
was indicative of the relation between permeability and whole cell
activity. The 6-chloro pyridine 27 gave improved enzyme potency
(IC50 = 6 nM), cdk5 selectivity over cdk2 (34-fold), and more potent
whole cell activity (IC50 = 673 nM) with an improved ratio of whole
cell/enzyme activity (ca. 100-fold). The 6-methyl derivative 2820
had similar enzyme activity, an 18-fold preference for cdk5 versus
cdk2, and the best whole cell activity (IC50 = 230 nM) with a whole
cell/enzyme ratio of 38. The Caco-2 profile was also good with BA
Papp = 14 ꢁ 10ꢀ6 cm/s and AB Papp = 13.9 ꢁ 10ꢀ6 cm/s.
10 lM). Considering the potential role of GSK3b in tau phosphory-
lation and thus Alzheimer’s disease, the ability to combine both
cdk5/p25 and GSKb inhibition into a single molecule could allow
for enhanced efficacy in treating disease.18
N-Acetyl analog 19 was crystallized with cdk2 in order to deter-
mine key interactions and better understand the increased selec-
tivity for cdk5 over cdk2 (Fig. 3). The amide carbonyl appears to
accept a hydrogen bond from Lys33 (3.1 A), which has shifted 1.6
angstroms relative to Figure 2. Additionally, the amide carbonyl
interacts with a water molecule (2.7 A) that bridges to the NH of
Asp145. The acetyl NH is making a hydrogen bond to a water mol-
ecule, but that water is not clearly in position to bind to the protein
and may thus be a crystallographic artifact.
Lys33 and Asp145 are positioned in a region of both cdk2 and
cdk5 (ASP 144) where ATP phosphate binding and thus phosphor-
ylation substrate binding happen. In cdk2 X-ray crystal structures,
disorder has been observed in this region of the protein, suggesting
the existence of some flexibility to accommodate the native sub-
strate, which has been corroborated by the difference in Lys33
positioning. Thus, one hypothesis regarding preferential cdk5 inhi-
bition versus cdk2 is that subtle differences exist between the two
kinases in this flexible ATP/substrate binding region that are
exploited by compounds such as 19 and 21.
Compound 28 was evaluated for brain penetration in both
wild-type FV/B mice and MDR1A/1B knockout mice that lack the
p-glycloprotein transporter (Table 5).21 In wild-type mice, brain/
plasma = 0.12, whereas an order of magnitude increase in brain/
With the advent of increased selectivity and potency, further
SAR exploration via modification of the cyclobutyl amide was car-

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C. J. Helal et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5703–5707
Table 4
References and notes
cdk5 enzyme and whole cell potency, and cdk2 selectivity
1. McInnes, C. Drug Discovery Today 2008, 13, 875.
2. Lau, L.-F.; Schachter, J. B.; Seymour, P. A.; Sanner, M. A. Curr. Top. Med. Chem.
2002, 2, 395.
3. Lau, L.-F.; Seymour, P. A.; Sanner, M. A.; Schachter, J. B. J. Mol. Neurosci. 2002, 19,
267.
N
O
H
N
N
R
N
H
O
4. (a) Cruz, J. C.; Tsai, L.-H. Trends Mol. Med. 2004, 10, 452; (b) Churcher, I. Curr.
Top. Med. Chem. 2006, 6, 579.
5. (a) Wei, F.-Y.; Tomizawa, K. Mini-Rev. Med. Chem. 2007, 7, 1070; (b) Pareek, T.
K.; Kulkarni, A. B. Cell Cycle 2006, 5, 585; (c) Slevin, M.; Krupinski, J. Curr. Opin.
Pharmacol. 2009, 9, 119.
6. For recent reviews of cdk5/p25 inhibitors, see: (a) Glicksman, M. A.; Cuny, G.
D.; Liu, M.; Dobson, B.; Auerbach, K.; Stein, R. L.; Kosik, K. S. Curr. Alzheimer Res.
2007, 4, 547; (b) Sridhar, J.; Akula, N.; Pattabiraman, N. AAPS J. 2006, 8. Article
25.
7. Helal, C. J.; Sanner, M. A.; Cooper, C. B.; Gant, T.; Adam, M.; Lucas, J. C.; Kang, Z.;
Kupchinsky, S.; Ahlijanian, M. K.; Tate, B.; Menniti, F. S.; Kelly, K.; Peterson, M.
Bioorg. Med. Chem. Lett. 2004, 14, 5521.
8. Kalgutkar, A. S.; Driscoll, J.; Zhao, S. X.; Walker, G. S.; Shepard, R. M.; Soglia, J. R.;
Atherton, J.; Yu, L.; Mutlib, A. E.; Munchhof, M. J.; Reiter, L. A.; Jones, C. S.; Doty,
J. L.; Trevena, K. A.; Shaffer, C. L.; Ripp, S. L. Chem. Res. Toxicol. 2007, 20, 1954.
9. For synthesis details, see Supplementary data and Ahlijanian, M. K.; Cooper, C.
B.; Helal, C. J.; Lau, L.-F.; Menniti, F. S.; Sanner, M. A.; Seymour, P. A.; Villalobos,
A. WO 2002010141 A1; Chem. Abstr. 2002, 107322.
10. Bulusu, S.; Damavarapu, R.; Autera, J. R.; Behrens, R., Jr.; Minier, L. M.;
Villanueva, J.; Jayasuriya, K.; Axenrod, T. J. Phys. Chem. 1995, 99, 5009.
11. Thermodynamic testing showed that 3 was a highly energetic substance with
the potential for self-heating from 35 °C that could lead to explosion. This
material should be used in small amounts and stored in a freezer. Solutions of 3
in methanol (1 M or less) were found to have reduced explosive potential and
thus reactions at 23 °C were deemed to be of reduced risk. Nonetheless,
appropriate safety procedures should be strictly observed when handling this
compound.
R
cdk5 IC50 nM
(std dev)
Selecta
(K2/K5)
cdk5 whole cell IC50 nM
(std dev)
21
24
25
26
27
28
8 (3)
17
11
15
20
34
18
>30,000
—
H3C
28 (5)
46 (14)
14 (9)
6 (1.8)
6 (2)
N
>30,000
1300
N
N
673 (321)
230 (98)
N
Cl
N
12. Kinase inhibition was measured by the use of scintillation proximity assays
a
Select = ratio of cdk2 IC50/cdk5 IC50
.
(SPA). The ATP final concentration of about 0.5
cdk5/p25 and cdk2/cyclin E assay. The Km of ATP was measured to be 12
for cdk5/p25 and 19 M for cdk2/cyclin E. Unless otherwise indicated, for
compounds with IC50 < 1 M, the IC50s reported are the mean of at least two
independent measurements with standard deviation (std dev) calculated.
lM was the same in both the
lM
l
l
Table 5
In vivo exposure for 28 at 1 h in FV/B wild-type (WT) and MDR1A/1B knockout micea
Each IC50 was determined from
triplicate.
a six-point dose-response curve run in
FV/B WT
MDR1A/B KO
13. Compound 7 was profiled against the following 20 kinases at a concentration
of 10 M and [ATP] at Km for the respective kinase: MKK1, JNK, p38, p38
p38 , p38d, MAPKAP-K2, MSK1, PKC PDK1, PKB SGK, P70SK6, GSK3b,
ROCKII, AMPK, CK2, MAPKAP-K1b, MAPK1, PHK.
l
a,
Dose (mg/kg)
Plasma (ng/mL)
Brain (ng/g)
2
280
34
2
c
a,
a,
336
383
1.1
14. The cdk5 homology model was built on in-house X-ray structures of cdk2-
inhibitor complexes, and due to the high homology between the two, is very
similar to the cdk2 X-ray structures themselves. Retrospectively, comparing
the homology model to that of the reported cdk5-inhibitor complexes (Mapelli,
M.; Massimiliano, L.; Crovace, C.; Seeliger, M. A.; Tsai, L.-H.; Meijer, L.;
Musacchio, A. J. Med. Chem., 2005, 48, 671) reveals nearly identical residue-by-
residue similarity.
15. (a) Helal, C. J.; Kang, Z. K.; Lucas, J. C.; Bohall, B. R. Org. Lett. 2004, 6, 1853; (b)
See Supplementary data for representative experimental conditions, yields,
and spectral data.
16. (a) Human recombinant cdk2 was expressed, purified and crystallized as
described previously (Brown, N. R.; Noble, M. E.; Lawrie, A. M.; Morris, M.
C.; Tunnah, P.; Divita, G.; Johnson, L. N.; Endicott, J. A. J. Biol. Chem. 1999,
274, 8746). Structures were derived from cdk2 crystals that were soaked
in solutions containing inhibitor.; (b) The X-ray crystal structures have
been placed in the PDB with code numbers 3IGG (Fig. 2) and 3IG7
(Fig. 3).
Brain/plasma
0.12
a
Subcutaneous dosing; vehicle = 10% DMSO, 10% Emulphor, 80% saline.
plasma to 1.1 was observed in the MDR1A/B knockout mice. This
result shows that 28 is a substrate for p-glycoprotein22 with limited
brain penetration in mouse and highlights the challenges of generat-
ing potent, cellularly active kinase inhibitors that can readily enter
brain.18c
In summary, we have developed a novel series of 4-aminoimidaz-
ole cdk5/p25 inhibitors with potent enzyme and good whole cell
activity, with good permeability in Caco-2 cell lines. Utilizing struc-
ture-based drug design, the systematic effort to build interactions
with Lys 33 and Asp144, residues that are involved in binding to
the ATP phosphates and thus in close proximity to substrate binding,
has resulted in up to 30-fold selectivity for cdk5 over cdk2. This
serves to demonstrate that selectivity is in fact possible for cdk5 over
cdk2. Concurrently, we have initial data to suggest that this strategy
also increases inhibition of GSK3b, a related tau kinase, thus poten-
tially providing an opportunity to merge two key tau kinase inhibi-
tory profiles into one molecule. As shown in this study, the
preparation of cellularly active and selective kinase inhibitors with
acceptable brain penetration remains a challenge for the field of
medicinal chemistry and demonstrates a continued need for novel
design strategies to access centrally located biological targets.
17. Compound 19 was profiled against the following 49 kinases with [ATP] = Km for
the respective kinase: ABL, CK1d, GSK3b, IKKi, IKKb, LCK, MAPKAP-K2, p38a,
PKA, PKCf, CDK2/Cyclin A, CHK1, CHK2, FGFR1, MET, PAK4, PDK1, PIM2, SRC,
CK-II, EGFR, TRK-A, VEGFR-2, AKT1, INSR, Aurora-A, MAPK1/ERK2, SGK, BTK,
TAOK3, CAMK1, ECK, PKCb, CLK1, MARK1, NEK2, JAK3, MYLK2, MAP3K9, MASK,
S6K-T389E-D3E, ROCK1, SYK, MST2, CDK6/Cyclin D3, MAP4K4, MEK1, TGFR-1,
BRAF.
18. (a) Gong, C.-X.; Iqbal, K. Curr. Med. Chem. 2008, 15, 2321; (b) Engmann, O.;
Giese, K. P. Front. Mol. Neurosci. 2009, 2, 2; (c) Mazanetx, M. P.; Fischer, P. M.
Nat. Rev. Drug Discovery 2007, 6, 464.
19. cdk5 whole cell assay: A tetracycline-regulated expression system was used to
produce a stable CHO cell line carrying inducible expression of human Cdk5,
P25 and tau driven from modified pRevTRE Tet-Off vectors containing distinct
antibiotic resistance genes. The cell line was maintained in the uninduced state
in media containing doxycycline and then transgene induction was initiated by
removal of doxycycline from the medium for 24 h. Following full induction of
CDK5, P25, and tau, cells were incubated for 2 h with test compounds before
lysis. Phosphorylated tau was measured in detergent-soluble extracts of cells
using AlphaLISA (Perkin–Elmer). A capture antibody to total tau (HT7, Pierce)
was conjugated to Acceptor beads, and a biotinylated phospho-epitope specific
detection antibody (AT8, Pierce) was bound to streptavidin donor beads
(Perkin–Elmer).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.08.019.

C. J. Helal et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5703–5707
5707
20. Compound 28 characterization data: 1H NMR (400 MHz, CDCl3) d 9.15 (s, 1H),
8.28 (d, J = 8.3 Hz, 1H), 8.01 (d, J = 7.5 Hz, 1H), 7.97 (d, J = 7.5 Hz, 1H), 7.96 (d,
J = 7.5 Hz, 1H), 7.81 (dd, J = 2.4, 6.6 Hz, 1H), 7.72 (m, 1H), 7.5 (m, 5H), 7.2 (m,
1H), 7.16 (s, 1H), 4.45 (m, 1H), 4.25 (m, 1H), 4.18 (s, 2H), 2.98 (m, 2H), 2.60 (s,
3H), 2.40 (m, 2H); MS (AP/CI): 440.3 (M+H)+.
21. Shinkel, A. H.; Wagenaar, E.; Mol, C. A.; van Deemter, L. J. Clin. Invest. 1996, 97,
2517.
22. For an analysis of commercial drugs’ brain penetration in wild type versus
MDR1A/1B knockout mice, see: Doran, A. et al Drug Metab. Dispos. 2005, 33,
165.