Structure-activity relationship study of 2,4-diaminothiazoles as Cdk5/p25 kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.01.140

Cdk5/p25 has emerged as a principle therapeutic target for numerous acute and chronic neurodegenerative diseases, including Alzheimer's disease. A structure-activity relationship study of 2,4-diaminothiazole inhibitors revealed that increased Cdk5/p25 inhibitory activity could be accomplished by incorporating pyridines on the 2-amino group and addition of substituents to the 2- or 3-position of the phenyl ketone moiety. Interpretation of the SAR results for many of the analogs was aided through in silico docking with Cdk5/p25 and calculating protein hydrations sites using WaterMap. Finally, improved in vitro mouse microsomal stability was also achieved.

Full text of DOI:10.1016/j.bmcl.2011.01.140

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2098–2101
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship study of 2,4-diaminothiazoles as Cdk5/p25
kinase inhibitors
Joydev K. Laha a, , Xuemei Zhang b, , Lixin Qiao a, Min Liu a, Snigdha Chatterjee b, Shaughnessy Robinson c,
Kenneth S. Kosik b, Gregory D. Cuny a,
⇑
a Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Brigham & Women’s Hospital and Harvard Medical School, 65 Landsdowne Street,
Cambridge, MA 02139, USA
b Neuroscience Research Institute, University of California at Santa Barbara, Santa Barbara, CA 93106, USA
c Schrödinger, Inc., 120 West Forty-Fifth Street, New York, NY 10036, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Cdk5/p25 has emerged as a principle therapeutic target for numerous acute and chronic neurodegener-
ative diseases, including Alzheimer’s disease. A structure–activity relationship study of 2,4-diaminothiaz-
ole inhibitors revealed that increased Cdk5/p25 inhibitory activity could be accomplished by
incorporating pyridines on the 2-amino group and addition of substituents to the 2- or 3-position of
the phenyl ketone moiety. Interpretation of the SAR results for many of the analogs was aided through
in silico docking with Cdk5/p25 and calculating protein hydrations sites using WaterMap. Finally,
improved in vitro mouse microsomal stability was also achieved.
Received 20 December 2010
Revised 27 January 2011
Accepted 31 January 2011
Available online 3 February 2011
Keywords:
Cdk5
Kinase
Ó 2011 Elsevier Ltd. All rights reserved.
Inhibitor
Alzheimer’s disease
2,4-Diaminothiazoles
Cyclin-dependent kinase 5 (Cdk5) is a member of the serine/
threonine cyclin-dependent kinase (Cdk) family.1 Many Cdks have
emerged as potential therapeutic targets for a variety of diseases,
particularly in oncology.2 Unlike other Cdks that are activated upon
binding to ubiquitously expressed cyclin proteins, Cdk5 activity is
predominantly in postmitotic neurons due to the restricted distri-
bution of its activator protein p35.3 Furthermore, membrane-asso-
ciated p35 can be proteolyticly processed by cysteine proteases,
such as calpains, to generate p25 that similarly activates Cdk5 by
repositioning the activation loop.4 Even though the catalytic effi-
ciencies of Cdk5/p35 and Cdk5/p25 are similar, it is thought that
the altered subcellular compartmentalization of p25 to the cytosol
and nucleus leads to neuronal toxicity.5
Various neuronal insults can initiate a cascade of events (i.e. in-
creased intracellular [Ca2+] followed by calpain activation) leading
to increased phosphorylation of various protein substrates, such as
tau.6 The hyperphosphorylation of tau (and several other proteins)
in many acute and chronic neurodegenerative diseases has high-
lighted the potential role of Cdk5/p25 in a number of these condi-
tions, especially Alzheimer’s disease.7,8 In particular, transgenic
animals producing elevated levels of p25 have increased amounts
of phosphorylated tau and demonstrate Alzheimer’s-like neuronal
lesions.9 In addition to Alzheimer’s disease, Cdk5/p25 has been
implicated in cerebral ischemia,10 multiple sclerosis,11 Hunting-
ton’s disease,12 Parkinson’s disease13 and amyotrophic lateral scle-
rosis (ALS).14 In addition, Cdk5 has been shown to mediate the
phosphorylation of PPAR-c at specific sites, which leads to insulin
resistance. Although the mechanism of Cdk5 activation in adipo-
cytes is unknown, this study potentially extends the therapeutic
scope of Cdk5/p25 inhibition beyond neurological disorders.15
Due to the potential role of Cdk5/p25 in various pathological
conditions, considerable efforts have been expended to identify po-
tent (and ideally selective) inhibitors. A variety of inhibitor struc-
ture classes have been described, including roscovitine (1),16
aloisine-A (2)17 and indirubin-30-oxime (3),18 which are all ATP-
competitive and have also been co-crystallized with Cdk5/
p25.19,20 Previously, we reported a colorimetric enzyme-linked
immunosorbent assay (ELISA) based high throughput screening
protocol for Cdk5 that utilizes full-length tau as substrate.21 Using
this procedure the natural product bellidin (4, IC50 = 0.2
lM) and
the 2,4-diaminothiazole 5 (IC50 = 2.0 M) were discovered as
l
Cdk5/p25 inhibitors (Fig. 1). Both compounds were also co-crystal-
lized with Cdk5/p25 and found to bind at the ATP-site in a similar
manner to 1–3, except that 5 caused significant movement of two
side chain residues (Asn144 and Lys33) in Cdk5 compared to the
other four inhibitors.22 Herein, we report a structure–activity rela-
⇑
Corresponding author. Tel.: +1 617 768 8640; fax: +1 617 768 8606.
E-mail address: gcuny@rics.bwh.harvard.edu (G.D. Cuny).
Equal contribution.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.140

J. K. Laha et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2098–2101
2099
electron donating methoxy (14) resulted in reduced activity. In
an attempt to introduce substituents in place of the aniline that
would impart increased aqueous solubility, several piperidines
were examined. For example, introduction of a 3- or 4-piperidine
(15 and 16) did not increase potency suggesting that retaining
the aryl group may be optimal. Introduction of amino substituents
onto the aryl (17–19) resulted in improved inhibitory activity with
IC50 values <100 nM. However, phenylenediamines raised oxida-
tive liability concerns. Therefore, the phenyl ring was replaced
with a 2-pyridine (20), which demonstrated an IC50 of 33 nM and
lowered the c-Log-D7.4 to 3.35 compared to 4.89 for 5.27
HO
NH
N
n-C4H9
N
O
N
N
N
N
N
NH
OH
N
HN
Et
N
H
i-Pr
H
OH
1
2
3
NH2
O
HO
OH
O
N
S
HN
NO2
O
OH
Given the results obtained with 20, the substituents on the aryl
ketone were more closely examined. The fluorine could be trans-
posed to the 2-position (21) retaining potent inhibitory activity,
but not the 4-position (22). An electron donating methoxy substi-
tuent at the 2-position (23) only resulted in a slight decrease in
activity, but introduction at the 3- or 4-positions (24 and 25)
was much more detrimental. Next, the 2-pyridine was replaced
with a 3-pyridine and various substituents on the arylketone were
examined. Again 2- and 3-F (26 and 27), but not 4-F (28), resulted
in potent inhibition. Removal of the fluorine (29) or replacement
with a 3-nitro (30) was detrimental. Replacement of the fluorine
with a methoxy was only tolerated at the 3-position (31). When
the 2-pyridine was replaced with a 4-pyridine, only the 3-F (35)
or 2-OMe (37) analogs gave potent inhibition. Interestingly, replac-
ing the aryl of the ketone with 2-, 3- or 4-pyridine (40–42) resulted
in significant loss of activity. Finally, introduction of a methyl onto
the thiazole amine (43) abolished inhibitory activity as might be
expected due to the steric clash of the N-methyl group with the
H-bond acceptor carbonyl of GLU81 (Fig. 2a and 2b).28 Disruption
of the apparent internal H-bond between the 4-amino hydrogen
and the ketone oxygen would require >8 kcal/mol based on a quan-
tum mechanical coordinate scan.29
HO
5
4
Cl
Figure 1. Examples of Cdk5/p25 inhibitors.
tionship (SAR) study of the 2,4-diaminothiazole inhibitors with sig-
nificant improvement in Cdk5/p25 inhibitory activity.23,24
The 2,4-diaminothiazoles were prepared according to the route
outlined in Scheme 1.25 An amine 6 was first allowed to react with
thiocarbonyl diimidazole, 7, at room temperature over 1 h to form
the desired isothiocyanate 8, which was generally not isolated.
Several isothiocyanates 8 were commercially available (e.g., 3-
pyridylisothiocyanate) and used directly in the next step. 1-Ami-
dino-3,5-dimethylpyrazole nitrate, 9, and DIPEA were added to
the isothiocyanates and the resulting reaction mixture was heated
at 50 °C for 2–16 h to give 10 in 10–60% overall yield. Next, cycli-
zation of 10 in the presence of alpha-bromoketones in DMF at
50–70 °C for 2–16 h gave the 2,4-diaminothiazoles 11. In certain
cases where R2 contains Boc-protected amine removal of the pro-
tecting group was achieved by treatment with TFA in DCM at room
temperature followed by salt formation with 4 N HCl in 1,4-diox-
ane. In addition, intermediate 10 could be treated with MeNH2 in
methanol to generate 12, which was subsequently cyclized to give
11 (R3 = Me), albeit in only ꢀ10% yield. The remaining material was
11 (R3 = H).
During the course of the SAR study it was noted that the analog
subset of R1 as 2- or 3-pyridyl, and the R2 as 2- or 4-substituted
phenyl exhibits a trend of the 4-substitued phenyl analogs being
less active compared to the 2-substituents phenyl analogs. In order
to probe these observations further an analysis of the protein
hydration sites was conducted. The compounds in Table 1 were
docked using Glide30a in XP mode with a core constraint based
on the crystallized analog 5. Using WaterMap30b,c to calculate the
Compounds were evaluated for Cdk5/p25 inhibition using a
radiometric assay.5,26 The goals of the SAR study were to increase
potency, to replace the aromatic nitro, to increase in vitro meta-
bolic stability, and to increase aqueous solubility.
The aromatic nitro in 5 was not necessary for activity. For exam-
ple, it was initially replaced with an electron withdrawing fluorine
(13) without diminishing activity, whereas replacement with an
S
b
a
ArNH2
ArNCS
+
N
N
N
N
N
6
7
8
H
N
R3
NH
N
R1
Me
H
N
c
H
N
S
Me
N
R1
d
11
O
R2
(if necessry)
S
NH
10
c
e
H
N
H
N
H
N
R1
Me
S
NH
12
Scheme 1. Reagents and condition: General synthetic approach to 2,4-diamino-
thiazoles. (a) DIPEA, DMF, rt, 1–6 h; (b) 1-amidino-3,5-dimethylpyrazoleꢁHNO3 (9),
DIPEA or KOH, DMF, 50 °C, 2–16 h; (c) R2C(@O)CH2Br, TEA or DIPEA, DMF, 50–70 °C,
2–16 h; (d) TFA, DCM, rt and then 4 N HCl in 1,4-dioxane; (e) MeNH2, MeOH, rt.
Figure 2a. A view of Cdk5/p25 complexed with 5 (in CPK rendering with green C,
red O, dark green Cl, blue N and yellow S). Cdk5 is shown with white ribbons and
p25 in yellow ribbons with 5 occupying the ATP binding site.

2100
J. K. Laha et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2098–2101
protein hydration sites in the vicinity of the ATP binding pocket re-
veals site 16 as having a stable
G = ꢂ3.2 kcal/mol as shown in Fig-
ure 3. In contrast, site 48 has an unstable G = 6.2 kcal/mol.
D
D
Using the listed activities, a binding DDG = ꢂ2.6 kcal/mol was
calculated for the exemplar pair of 26 and 28. Both ligands displace
site 48 and the unlabeled sites with a favorable effect on binding
affinity. In addition, the 4-fluorophenyl of 28 also displaces site
16. The displacement of a stable hydration site results in a pre-
dicted DDG in agreement with the observed value within experi-
mental and computational error. Similar DD
G values were
observed for the other ortho/para pairs in the subset. For com-
pounds in Table 1 where R2 is 4-pyridyl and for 15 – 19 the SAR
is possibly due to a different binding orientation. Further studies
will be necessary to address this possibility.
The selectivity of 26 was briefly examined against Cdk2 and
GSK-3b, two structurally related serine/threonine kinases belong-
ing to the CMGC kinase subgroup. Compound 26 displayed potent
activity against both kinases with IC50 values of 25 nM and 45 nM
([ATP] = 60 lM) for Cdk2 and GSK-3b, respectively.
Next, the inhibitory effect of 26 was studied in primary neurons.
Rat brain cells were incubated with compound at five different
concentrations for 4 h at 37 °C, lysed and immunoblotted with
antibodies against Cdk5 and phosphorylated tau at Serine 235
(Ptau235), a predominant phosphorylation site.31 Roscovitine, 1,
was used as a positive control. As shown in Figure 4, 26 displayed
a dose-dependent inhibition of tau phosphorylation with an EC50
Figure 2b. A close view of 5 (ball and stick rendering) showing the H-bonds (yellow
dashed lines) to the hinge residues in Cdk5 and of the NO2 group with the catalytic
Lys33.
value of 5.5 lM.
Table 1
Cdk5/p25 inhibitory result for 5 and 13–44
H
N
R3
NH
N
R1
S
O
R2
Compound
R1
R2
R3
IC50, nM
5
4-ClPh
4-ClPh
4-ClPh
3-Piperidyl
4-Piperidyl
3-NH2Ph
3-NHMePh
4-NMe2Ph
2-Py
2-Py
2-Py
2-Py
2-Py
2-Py
3-Py
3-Py
3-Py
3-Py
3-Py
3-Py
3-Py
3-Py
4-Py
4-Py
4-Py
4-Py
4-Py
4-Py
3-Py
3-Py
3-Py
3-Py
3-(6-CF3)Py
3-NO2Ph
3-FPh
3-OMePh
3-FPh
3-FPh
3-FPh
3-FPh
3-FPh
3-FPh
2-FPh
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
750
700
4000
500
6200
31
44
68
33
38
1200
80
400
1100
30
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
Figure 3. A view showing 26 (brown carbons) and 28 (green carbons) docked into
the Cdk5/p25 binding site. The spheres represent protein hydration sites as
computed by WaterMap with a
Only overlapped sites are shown for clarity.
D
G: ꢂ3.2 (bright green) to 6.2 kcal/mol (bright red).
4-FPh
2-OMePh
3-OMePh
4-OMePh
2-FPh
3-FPh
4-FPh
62
4100
341
130
418
11
3500
360
18
Ph
3-NO2Ph
2-OMePh
3-OMePh
4-OMePh
2-FPh
3-FPh
4-FPh
2-OMePh
3-OMePh
4-OMePh
2-Py
400
60
200
600
800
100
1710
>10000
160
3-Py
4-Py
2-FPh
2-FPh
Figure 4. The inhibition effect of 26 on tau phosphrylation in primary neurons.
Western blot is presented where tau phosphorylation was probed by antibody
Ptau235 and Cdk5 was used as the control. Relative band intensity is shown in the
bar graph. Roscovitine, 1, at 30 lM was used as a positive control.

J. K. Laha et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2098–2101
2101
16. Meijer, L.; Borgne, A.; Mulner, O.; Chong, J. P.; Blow, J. J.; Inagaki, N.; Inagaki,
M.; Delcros, J. G.; Moulinoux, J. P. Eur. J. Biochem. 1997, 243, 527.
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lM and a t1/2 = 29 min was determined. Based on computa-
10
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tional models predicting the intrinsic oxidative stability to
CYP450 3A4 and 2D6, the 6-position of the 3-pyridyl was predicted
to be a site of oxidation. Since this area of the molecule is also pre-
dicted not to make close contacts with Cdk5 when bound, assum-
ing a similar binding mode as 5, an increase in metabolic stability
was anticipated with additional of a trifluoromethyl at this posi-
tion (44). Although the Cdk5 inhibitory IC50 increased to 160 nM,
the mouse microsomal half-life increased to 66 min.
In summary, a series of potent 2,4-diaminothiazoles were de-
signed and synthesized based upon the previously identified
Cdk5/25 inhibitor 5. Incorporation of pyridines in place of the 4-
chlorophenyl and addition of substituents to the 2- or 3-position
of the phenyl ketone moiety resulted in increased inhibitory po-
tency, such as 26 (LDN-193594). Interpretation of the SAR results
for many of the analogs was aided through in silico docking with
Cdk5/p25 and calculating protein hydrations sites with Water-
Map.30b,c Furthermore, addition of a substituent at the 6-position
of the incorporated 3-pyridyl resulted in increased in vitro mouse
microsomal stability. The results of this study provide probes to
study Cdk5/p25-mediated biology and should prove useful in the
further optimization of this series of Cdk5 inhibitors.
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T.; Shanahan, F.; Davis, N.; Prabhavalkar, D.; Wiswell, D.; Seghezzi, W.; Paruch,
K.; Dwyer, M. P.; Doll, R.; Nomeir, A.; Windsor, W.; Fischmann, T.; Wang, Y.;
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M.; Volpi, D.; Vulpetti, A.; Ciomei, M. Bioorg. Med. Chem. 2010, 18, 1844; (c)
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Anizon, F.; Moreau, P. Bioorg. Med. Chem. 2009, 17, 4420; (e) Helal, C. J.; Kang,
Z.; Lucas, J. C.; Gant, T.; Ahlijanian, M. K.; Joel B. Schachter, J. B.; Richter, K. E. G.;
Cook, J. M.; Menniti, F. S.; Kelly, K.; Mente, S.; Pandit, J.; Hosea, N. Bioorg. Med.
Chem. 2009, 17, 5703; (f) Potterat, O.; Puder, C.; Bolek, W.; Wagner, K.; Ke, C.;
Ye, Y.; Gillardon, F. Pharmazie 2005, 60, 637; (g) Moreau, P.; Gaillard, N.;
´
Marminon, C.; Anizon, F.; Dias, N.; Baldeyrou, B.; Bailly, C.; Pierre, A.; Hickman,
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Walter, J.; Cleverley, K. E.; Salinas, P. C.; Wu, Y.-Z.; Biernat, J.; Mandelkow, E.-
M.; Kim, S.-H.; Pettit, G. R. Chem. Biol. 2000, 7, 51; (j) Leost, M.; Schultz, C.; Link,
A.; Wu, Y.-Z.; Biernat, J.; Mandelkow, E.-M.; Bibb, J. A.; Snyder, G. L.; Greengard,
P.; Zaharevitz, D. W.; Gussio, R.; Senderowicz, A. M.; Sauville, E. A.; Kunick, C.;
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Acknowledgements
We are grateful to the National Institutes of Health (U01
AG033931) and the Harvard NeuroDiscovery Center for financial
support.
21. Ahn, J. S.; Musacchio, A.; Mapelli, M.; Ni, J.; Scinto, L.; Stein, R.; Kosik, K. S.; Yeh,
L. A. J. Biomol. Screen. 2004, 9, 122.
22. Ahn, J. A.; Radhakrishnan, M. L.; Mapelli, M.; Choi, S.; Tidor, B.; Cuny, G. D.;
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Supplementary data
23. For other thiazole containing Cdk5/p25 inhibitors see: (a) Rzasa, R. M.; Kaller,
M. R.; Liu, G.; Magal, E.; Nguyen, T. T.; Osslund, T. D.; Powers, D.; Santora, V. J.;
Viswanadhan, V. N.; Wang, H. L.; Xiong, X.; Zhong, W.; Norman, M. H. Bioorg.
Med. Chem. 2007, 15, 6574; (b) Shiradkar, M. R.; Akula, K. C.; Dasari, V.; Baru, V.;
Chiningiri, B.; Santosh Gandhi, S.; Kaur, R. Bioorg. Med. Chem. 2007, 15, 2601;
(c) Zhong, W.; Liu, H.; Kaller, M. R.; Henley, C.; Magal, E.; Nguyen, T.; Osslund,
T. D.; Powers, D.; Rzasa, R. M.; Wang, H. L.; Wang, W.; Xiong, X.; Zhang, J.;
Norman, M. H. Bioorg. Med. Chem. Lett. 2007, 17, 5384; (d) 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; (e) Larsen, S. D.; Stachew, C. F.; Clare, P. M.;
Cubbage, J. W.; Leach, K. L. Bioorg. Med. Chem. Lett. 2003, 13, 3491.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.01.140.
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24. For thiazole containing Cdk1, 2 and 4 inhibitors see: Chen, L.; Chu, X.-J.; Lovey,
A.J.; Zhao, C. PCT Int. Patent Appl. WO 2006005508, 2006.
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26. The phosphorylation of H1P (Histone H1-derived peptide PKTPKKAKKL) by ATP
was conducted in buffer containing 20 mM MOPS (pH 7.5), 10 mM MgCl2,
1 mM DTT, BSA 0.5 mg/ml, 60 lM ATP, 40 lM H1P, and [c-
33P]-ATP. The
reactions were conducted in duplicate, initiated by the addition of Cdk5/p25
(6.4 nM), and incubated at room temperature for 45 min. The reaction was
stopped by the addition of 20 mM EDTA, the mixture was then transferred to a
multiscreen PH filtration plate (Millipore, Billerica, MA) and washed six times
with 75 mM H3PO4. The plate was dried, filters were removed and samples
were counted with
a scintillation counter. The background reaction was
conducted in the absence of Cdk5/p25. Roscovitine, 1, was used as a positive
control and demonstrated an IC50 value of 270 nM.
27. Calculations were made using Molinspiration (http://www.molinspiration.
com/).
28. X-ray coordinates (file number: 3O0G) have been deposited with the RCSB
Protein Data Bank. These coordinates were used for molecular modeling.
29. QM relaxed coordinate scans were done with Jaguar, Schrödinger, Inc. using a
6-31Gꢃꢃ basis set.
30. (a) Glide XP5.0, Schrödinger, Inc.; (b) WaterMap, Schrödinger, Inc.; (c) Abel, R.;
Young, T.; Farid, R.; Berne, B. J.; Friesner, R. A. J. Am. Chem. Soc. 2008, 130, 2817.
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