Synthesis and structure-activity relationship of 4-(1,3-benzothiazol-2-yl)- thiophene-2-sulfonamides as cyclin-dependent kinase 5 (cdk5)/p25 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.07.068

4-(1,3-Benzothiazol-2-yl)thiophene-2-sulfonamide (4a) was found to be a moderately potent inhibitor of cyclin-dependent kinase 5 (cdk5) from a HTS screen. The synthesis and SAR around this hit is described. The X-ray coordinates of ligand 4a with cdk5 are also reported, showing an unusual binding mode to the hinge region via a water molecule.

Full text of DOI:10.1016/j.bmcl.2012.07.068

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5919–5923
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure-activity relationship of 4-(1,3-benzothiazol-2-yl)-
thiophene-2-sulfonamides as cyclin-dependent kinase 5 (cdk5)/p25 inhibitors
Jonas Malmström a, , Jenny Viklund a, Can Slivo a, Ana Costa b, Mickaël Maudet c, Catrin Sandelin a,
⇑
Gösta Hiller d, Lise-Lotte Olsson e, Anna Aagaard e, Stefan Geschwindner e, Yafeng Xue e, Mervi Vasänge f
a Medicinal Chemistry, CNSP iMed Science, AstraZeneca R&D, Innovative Medicines, SE-15185 Södertälje, Sweden
b Syntagon, Tallvägen 2, S-15102 Södertälje, Sweden
c Oncology iMed, AstraZeneca R&D Reims, Chemin de Vrilly, Parc Industriel Pompelle, BP1050, 51689 Reims Cedex, France
d Project Management, CNSP iMed, AstraZeneca R&D S-15185 Södertälje, Sweden
e Discovery Sciences, AstraZeneca R&D Mölndal, SE-43183 Mölndal, Sweden
f Neuroscience, CNSP iMed Science, AstraZeneca R&D, Innovative Medicines, SE-15185 Södertälje, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
4-(1,3-Benzothiazol-2-yl)thiophene-2-sulfonamide (4a) was found to be a moderately potent inhibitor of
cyclin-dependent kinase 5 (cdk5) from a HTS screen. The synthesis and SAR around this hit is described.
The X-ray coordinates of ligand 4a with cdk5 are also reported, showing an unusual binding mode to the
hinge region via a water molecule.
Received 25 May 2012
Revised 17 July 2012
Accepted 18 July 2012
Available online 25 July 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Kinase
cdk5
cdk2
Alzheimer disease
4-(1,3-Benzothiazol-2-yl)thiophene-2-
sulfonamide
Cyclin-dependent kinase 5 (cdk5) belongs to a family of proline-
directed serine/threonine kinases. It plays an important role in
many physiological functions, including the development of the
central nervous system.1–4 Cdk5 is not only an important partici-
pant in neuronal death, but also a key player in neuronal survival,5
and a key regulator of synaptic transmission and synaptic plastic-
ity.6 Cdk5 is mainly active in postmitotic neurons due to the selec-
tive neuronal expression of its non-cyclin activators p35 and p39.
The discovery that the short-lived, membrane bound p35 can be
proteolytically cleaved by calpain to the more stable, cytosolic
form p25 led to the hypothesis that cdk5/p25 plays an important
role in Alzheimer disease (AD) etiology. The longer-lived cdk5/
p25 complex is thought to be one of a group of kinases that hyper-
phosphorylate tau, leading to the formation of paired helical
filaments and deposition of cytotoxic neurofibrillary tangles.7–9
Thus, deregulation of cdk5 clearly has a role in AD.10–12
Deregulation of cdk5 activity has also been observed in several
neurodegenerative diseases other than AD, for example, amyotro-
phic lateral sclerosis (ALS), Parkinson disease, Huntington disease,
and abuse.1,13
Selectivity over cyclin-dependent kinase 2 (cdk2) is desired due
to its role in modulating the cell cycle, possibly leading to unde-
sired side effects.14 However, selectivity is difficult to achieve
due to the close homology between cdk5 and cdk2 (60% sequence
identity).15 There is also a 85% similarity and 78% identity of the
amino acids between the two kinases within 10 Å from ATP in
the active site. Nevertheless, there are a few references in the liter-
ature reporting compounds showing selectivity for cdk5 over
cdk2.16,17 Two examples (1 and 2) are shown in Figure 1 with a
18- and 34-fold selectivity, respectively.18,19
The crystal structure of the cdk5/p25 complex has previously
been reported.20 A number of ligands, such as (R)-Roscovitine, Aloi-
sine-A and Indirubin-30-oxime have been co-crystallized with cdk5
(compounds 3a–c in Fig. 2).21–24 A common feature of these com-
pounds is the planar, substituted heterocyclic ring system that is
highly complementary to the ATP-binding cavity.
Inhibition of the anomalous cdk5/p25 complex is a viable target
for treating Alzheimer disease by preventing hyperphosphoryla-
tion of tau and neurofibrillary tangle formation.
As part of a drug discovery program designed to identify cdk5
inhibitors, a high-throughput screen was conducted. One of the
hits was compound 4a (Fig. 3) which showed moderate inhibition
⇑
Corresponding author. Tel.: +46 8 553 26891.
E-mail address: Jonas.Malmstrom@astrazeneca.com (J. Malmström).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.068

5920
J. Malmström et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5919–5923
N
H
O
N
N
O
N
O
O
N
H
N
H
N
N
O
N
2
1
Cl
IC50 cdk5: 6 nM
cdk2: 204 nM
IC50 cdk5: 30 nM
cdk2: 540 nM
Figure 1. Examples of cdk5 selective compounds.
H
O
NH
N
N
O
N
N
N
NH
N
OH
H
N
N
H
N
OH
3a
3b
3c
(R)-Roscovitine
Aloisine-A
Indirubin-3'-oxime
Figure 2. Ligands that have been co-crystallized with cdk5.
O
van der Waals interactions with the gatekeeper residue amino acid,
NH2
S
Phe80. To date we are not aware of any kinase inhibitor in the lit-
erature that binds via a water molecule in this way. Tan et al. have
demonstrated, by simulations, the significance of water molecules
in other locations in the inhibition of cdk5 and cdk2 complexes.26
The overall kinase selectivity for compound 4a was good, as
evaluated against a wide range of kinases, and exhibited no signif-
N
S
O
S
4a
Figure 3. 4-(1,3-Benzothiazol-2-yl)thiophene-2-sulfonamide (4a), cdk5 HTS hit
icant activity at 10 l
M except against cdk2 (IC50 4500 nM).27 Be-
(IC50 551 nM).
cause of the selectivity over other kinases and the unusual
binding mode, it was interesting to explore this class of kinase
inhibitors further. A follow-up program was initiated to investigate
the SAR around this structure and explore its potential.
of cdk5 (IC50 551 nM). Furthermore, compound 4a also exhibited a
desirable clogP (2.4) and good experimentally determined phys-
ico-chemical and permeability properties (solubility 24 lM;
The analysis of the X-ray structure and the synthetic feasibility
indicated two possible sites for further optimization, namely posi-
tion 6 (R1) on the benzothiazole ring, and the substituents on the
sulfonamide moiety (R2). A number of compounds (4a–o in Table 1)
were designed, prepared and tested against cdk5 and cdk2.
The in vitro potencies for cdk5/p2529,30 and cdk2/cyclin E31
were determined using primary scintillation proximity assays
(SPA).32 The IC50 values were determined from dose–response
curves as the average of at least two replications. The two assays
are robust and reproducible with Z0-values33 of more than 0.8
and 0.6, respectively.
Caco-2 permeability 32
l
cm/s, and BBB 18 ꢀ 10ꢁ3 cm/min).
Compound 4a was co-crystallized with cdk5 and its structure
solved to a resolution of 1.90 Å. The ligand was shown to be situ-
ated in the ATP binding site as outlined in Figure 4.25 The X-ray
structure showed that the ligand is not directly bound to the back-
bone (Glu 81 and Cys83) in the ATP site of the kinase, as is the
usual case. Intriguingly, a water molecule was found to form three
bridging interactions between the ligand and the hinge backbone,
involving Glu81, Cys83 and the nitrogen in the benzothiazole ring.
Furthermore, the sulfonamide interacts through hydrogen bonding
with Asp86 and Ile10, and the benzothiazole ring appears to form
The in vitro potencies for cdk5/p25 and cdk2/cyclin E are shown
in Table 1. When the hydrogen in 4a is replaced by a fluorine (4b)
or chlorine (4c) atom the cdk5 potency was increased. However
when a slightly bigger electron donating methyl group was intro-
duced (4d), a twofold drop in cdk5 potency compared to com-
pound 4a was observed. The strong electron withdrawing nitro
substituent (4e) was equipotent with compound 4d in the cdk5 as-
say. Increasing the size of the substituent to an acetyl group (4f)
gave a low potency inhibition at both cdk5 and cdk2. In summary,
only small substituents such as hydrogen or halogens were toler-
ated in position 6 (R1) on the benzothiazole ring. Unfortunately,
they also became more active against cdk2. One explanation could
be that the larger substituents clash with the gatekeeper residue
Phe80, leading to unfavorable binding and less potent compounds.
The effect sulfonamide (R2) substitution on SAR was also ex-
plored (compounds 4g–o, Table 1). When small substituents like
a hydroxyl group (4g) or an alkyl group (4h) were introduced, a de-
crease in potency as compared with the unsubstituted analogue
Figure 4. X-ray crystal structure of compound 4a in the cdk5 ATP binding site. The
figures were made using PyMOL.28

J. Malmström et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5919–5923
5921
Table 1
In vitro potencies for compounds 4a–o against cdk5 and cdk2
H
N
O
S
R2
N
S
O
S
R1
R1
R2
cdk5/p25 IC50 (nM)
cdk2/cyclin E IC50 (nM)
a
a
Cmpd
4a34
4b
4c
H
F
Cl
H
H
H
551
331
355
4500
718
861
4d
4e
4f
4g
4h
CH3
NO2
COCH3
H
H
H
H
OH
1260
1272
6312
7415
>10,000
2256
4524
>10,000
nd
H
CH2CH2F
nd
N
4i
4j
H
H
1764
8220
5484
nd
S
N
S
N
CH2CO2Et
4k
4l
H
H
5088
5472
nd
S
6720
N
N
N
4m
H
8274
nd
4n
4o
H
H
9936
7704
nd
nd
H
O
N
O
N
nd: Not determined.
a
Values are geometric means of n P 2 experiments, with a range of less than 20% of mean value.
(4a) was observed. Unexpectedly, when an unsubstituted thiazole
ring was introduced (4i) it gave a moderate inhibitor of cdk5.
Unfortunately, when the thiazole ring was substituted a drop in
potency was observed (4j–k). The introduction of a benzylpyridine
(4l) gave a moderate inhibitor. Substituted pyridines (4m–o) were
not tolerated.
The loss of activity observed when substituting the sulfonamide
is probably due to the loss of favorable hydrogen-bonding of the
sulfonamide to Ile10 and Asp86 (Fig. 5).
Unfortunately, the selectivity against cdk2 was generally low in
the series (2- to 3-fold), except for 4a (eightfold).
In addition to compounds 4a–o, a tertiary sulfonamide (5,
Fig. 6) was also prepared, but it had low potency at both cdk5
and cdk2, probably due to poor hydrogen-bonding possibilities to
Asp86.
A few variations through scaffold hopping were also made, both
for the bicyclic core and the thiophene, but they gave less potent
compounds as compared to 4a.
The syntheses of compounds 4a–o were conducted as outlined
in Scheme 1. For all the examples, the starting benzothiazole 7 was
commercially available except, for example, 4f. In that case, aniline
6 was treated with sodium thiocyanate (NaSCN) and bromine in
acetic acid according to a literature procedure, giving the desired
benzothiazole in a 41% isolated yield.35 The benzothiazole 7 was
Figure 5. Compound 4a in the active site showing the hydrogen bonds to Ile10 and
then deaminated via
a
Sandmayer protocol using copper(II)
Asp86.

5922
J. Malmström et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5919–5923
NH2
N
S
O
a
N
S
S
R1
SH
R1
N
S
O
OH
S
13
10
Scheme 2. Reagents: (a) 3-Thiophenecarboxylic acid, PPA, 55% (R1 = H).
5
cdk5/p25 IC50 8880 nM,
cdk2/cyclin E IC50 >10000 nM
Figure 6. A tertiary sulfonamide.
SO2NH2
N
S
S
SO2NH2
N
S
bromide, isoamylnitrite (isoAmONO) in acetonitrile/PEG-200 to
yield the corresponding bromide 9.36,37 When R1 is hydrogen, the
chloride 8 was commercially available. The chloride 8 or bromide
9 was then subjected to a Suzuki–Miyaura coupling using thio-
phene-3-boronic acid and PdCl2(dppf) as the catalyst to give com-
pound 10.38 When R1 is hydrogen, compound 10 could also be
obtained in 55% isolated yield by treating thiol 13 and 3-thiophen-
ecarboxylic acid with polyphosphoric acid (PPA) as outlined in
Scheme 2. The sulfonic acid 11 was obtained by treating compound
10 with chlorosulfonic acid. The sulfonic acid 11 was treated with a
mixture of phosphorus pentachloride and phosphorus oxychloride
to yield the sulfonyl chloride 12. The final compound 4 was ob-
tained by reacting sulfonyl chloride 12 with the desired amine in
pyridine or ammonia in methanol.
At this point in our investigation it was interesting to introduce
a substitutent in the 7-position on the benzothiazole ring to deter-
mine if the potency and selectivity could be influenced by interact-
ing with the salt bridge or the glycine-rich loop. In order to explore
the space, it was decided to prepare compounds 14 and 15 (Fig. 7).
As shown in Figure 7, a small chlorine substituent in position 7
was tolerated (14), but it gave a twofold drop in potency as com-
pared to compound 4b. Compound 14 was equipotent in the
cdk5 and cdk2 assays with IC50 values of 672 nM and 492 nM,
respectively. However, when a fluorine-substituted phenyl ring
was introduced in position 7 it gave a weak inhibitor (15). The
IC50 for compound 15 was determined to be 6780 nM in the cdk5
assay but could not be determined in the cdk2 assay. The low activ-
ity for compound 15 is probably due to a steric clash between the
large substituent in position 7 and the salt bridge. In conclusion,
with the limited data in hand, the introduction of substituents in
S
F
Cl
F
14
cdk5/p25 IC50 672 nM,
15
cdk5/p25 IC50 6780 nM,
cdk2/cyclin E IC50
cdk2/cyclin E IC50 492 nM
Could not be determined (>10 µM)
Figure 7. (Benzothiazol-2-yl)-thiophene derivatives substituted in position 7.
position 7 on the benzothiazole ring seems to give limited oppor-
tunities for improving the selectivity against cdk2.
Compound 14 was prepared as outlined in Scheme 1. The syn-
thesis of compound 15 is outlined in Scheme 3. The starting aniline
16 was commercially available and brominated in the 7-position to
yield bromide 17 in 26% yield.39 This compound was then con-
verted to the bromide 18 with hypophosphorous acid (H3PO2) un-
der Sandmayer conditions. Compound 18 was then subjected to a
Suzuki–Miyaura coupling with 4-fluorophenylboronic acid and
PdCl2(dppf) as the catalyst as described for compound 8 to give
compound 19 in moderate yield. The bromide 20 could then be ob-
tained in 39% yield by treating compound 19 with n-butyl lithium
and carbon tetrabromide.40 The bromide 20 was then subjected to
a Suzuki coupling as described above giving the thiophene deriva-
tive 21 in 69% isolated yield.38 The final sulfonamide 15 was then
synthesized via the method previously described for compound 4a.
Compound 4c was also evaluated in a cell-based assay. It was
demonstrated that compound 4c dose-dependently inhibits non-
muscle myosin heavy chain phosphorylation in HEK293 cells. The
NH2
N
N
S
c
a
b
NH2
X
R1
R1
R1
S
6
7
8 X= Cl when R1=H
9 X=Br
O
OH
S
N
S
N
S
d
e
O
S
R1
S
R1
10
11
H
O
O
N
R2
Cl
S
N
S
S
N
S
f
O
O
S
R1
S
R1
4
12
Scheme 1. Reagents: (a) NaSCN, bromine, acetic acid, 47% (R1 = COCH3) (b) CuBr2, IsoAmONO, MeCN-PEG200, 71–92%; (c) thiophene-3-boronic acid, PdCl2(dppf), aq Na2CO3,
toluene/EtOH 10/1, 67%; (d) ClSO3H, CHCl3, 90% crude; (e) PCl5, POCl3, 91% crude; (f) R2NH2, pyridine or ammonia in methanol (R2 = H, 48%). The yields in steps c–f are given
for compound 4a.

J. Malmström et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5919–5923
5923
6. Cheung, Z. H.; Fu, A. K. Y.; Ip, N. Y. Neuron 2006, 50, 13.
7. Ballatore, C.; Lee, V. M. Y.; Trojanowski, J. Q. Nat. Rev. Neurosci. 2007, 8, 663.
8. Gong, C.-X.; Iqbal, K. Curr. Med. Chem. 2008, 15, 2321.
9. Froelich-Fabre, S.; Bhat, R. V. Drug Discov. Today Dis. Mech. 2004, 1, 391.
10. Cruz, J. C.; Tsai, L.-H. Trends Mol. Med. 2004, 10, 452.
11. Tsai, L.-H.; Lee, M.-S.; Cruz, J. Biochim. Biophys. Acta Proteins and Proteomics
2004, 1697, 137.
N
S
N
S
N
S
c
b
a
H2N
H2N
Br
Br
17
18
16
12. Monaco, E. A. I. Curr. Alzheimer Res. 2004, 1, 33.
13. Shelton, S. B.; Johnson, G. V. W. J. Neurochem. 2004, 88, 1313.
14. Shapiro, G. I. J. Clin. Oncol. 2006, 24, 1770.
15. Zhang, B.; Tan, V. B. C.; Lim, K. M.; Tay, T. E. J. Comput. Aided Mol. Des. 2006, 20,
395.
16. Larsen, S. D.; Stachew, C. F.; Clare, P. M.; Cubbage, J. W.; Leach, K. L. Bioorg. Med.
Chem. Lett. 2003, 13, 3491.
N
S
N
Br
d
e
S
17. Shiradkar, M. R.; Akula, K. C.; Dasari, V.; Baru, V.; Chiningiri, B.; Gandhi, S.;
Kaur, R. Bioorg. Med. Chem. 2007, 15, 2601.
18. Booth, R. J.; Chatterjee, A.; Malone, T. C. WO 2001055148.
19. Helal, C. J.; Kang, Z.; Lucas, J. C.; Gant, T.; Ahlijanian, M. K.; Schachter, J. B.;
Richter, K. E. G.; Cook, J. M.; Menniti, F. S.; Kelly, K.; Mente, S.; Pandit, J.; Hosea,
N. Bioorg. Med. Chem. Lett. 2009, 19, 5703.
19
20
F
F
20. Tarricone, C.; Dhavan, R.; Peng, J.; Areces, L. B.; Tsai, L.-H.; Musacchio, A. Mol.
Cell 2001, 8, 657.
21. Ahn, J. S.; Radhakrishnan, M. L.; Mapelli, M.; Choi, S.; Tidor, B.; Cuny, G. D.;
Musacchio, A.; Yeh, L.-A.; Kosik, K. S. Chem. Biol. 2005, 12, 811.
22. Mapelli, M.; Massimiliano, L.; Crovace, C.; Seeliger, M. A.; Tsai, L.-H.; Meijer, L.;
Musacchio, A. J. Med. Chem. 2005, 48, 671.
SO2NH2
N
S
N
S
f, g, h
S
S
23. Polychronopoulos, P.; Magiatis, P.; Skaltsounis, A.-L.; Myrianthopoulos, V.;
Mikros, E.; Tarricone, A.; Musacchio, A.; Roe, S. M.; Pearl, L.; Leost, M.;
Greengard, P.; Meijer, L. J. Med. Chem. 2004, 47, 935.
21
24. Meijer, L.; Skaltsounis, A.-L.; Magiatis, P.; Polychronopoulos, P.; Knockaert, M.;
Leost, M.; Ryan, X. P.; Vonica, C. A.; Brivanlou, A.; Dajani, R.; Crovace, C.;
Tarricone, C.; Musacchio, A.; Roe, S. M.; Pearl, L.; Greengard, P. Chem. Biol. 2003,
10, 1255.
25. The experimental details are described in the attached Supplementary data.
The coordinates and structure factors for the complex of cdk5 with 4a have
been deposited in the Protein data bank (www.rcsb.org) under accession code
4au8.
26. Zhang, B.; Tan, V. B. C.; Lim, K. M.; Tay, T. E. J. Chem. Inf. Model. 1877, 2007, 47.
27. A panel of 29 kinases screened in the AZ-Dundee collaboration: AMPK, Chk1,
CKII, GSK3b, JNK, Lck, MAPK2/ERK2, MAPKAP-K2, MEK1, MSK1, p70 S6K, PDK1,
Phos kinase, PKA, PKBa, PKCa, PRAK, ROCK-II, SAPK2a, SAPK2b, SAPK3, SAPK4,
SGK, CSK, CDK2/cyclin A, DYRK1A, CK1, MAPKAP-K1a(RSK1), PP2a.
28. Delano, W. L. The PyMOL Molecular Graphis System; Delano Scientific: Palo Alto,
CA, USA, 2002.
15
F
F
Scheme 3. Reagents: (a) Bromine, acetic acid, 26%; (b) Sodium nitrite, H2SO4,
H3PO2, 75%; (c) p-F-Ph-B(OH)2, PdCl2(dppf), aq Na2CO3, toluene/EtOH, 48%; (d) n-
BuLi, CBr4, 39%; (e) Thiophene-3-boronic acid, PdCl2(dppf), aq Na2CO3, toluene/
EtOH, 69%; (f) ClSO3H, CHCl3, 63%; (g) PCl5, POCl3, not isolated; (h) ammonia in
MeOH, 63%.
IC50 value was determined to be 4.36 lM, which gave a 12-fold
drop-off (IC50 in the primary assay: 355 nM).41
In summary, we have developed a novel series of benzothiazol-
2-yl-thiophene-2-sulfonamides as cdk5/p25 inhibitors with mod-
erately potent enzyme activity. A novel kinase pharmacophore
was identified and an X-ray crystallographic structure demon-
strated an unprecedented ligand interaction with the hinge back-
bone via a bridging water molecule. The SAR around the original
hit 4a was explored, and it was found that halogen substituted
benzothiazoles showed increased potency against cdk5 (com-
pounds 4b–c), but decreased selectivity against cdk2. Furthermore,
we demonstrated that substituting the sulfonamide (compounds
4g–o) gave lower potency. Compound 4c also showed moderate
potency in a secondary cell-based assay.
29. Malmström, J.; Viklund, J. WO 2006004507.
30. Jämsä, A.; Bäckström, A.; Gustafsson, E.; Dehvari, N.; Hiller, G.; Cowburn, R. F.;
Vasänge, M. Biochem. Biophys. Res. Commun. 2006, 345, 324.
31. CDK2 assay: The assay experiments were carried out in duplicate with 10
different concentrations of the inhibitors in clear-bottom 384-well microtiter.
Recombinant human Cdk2/Cyklin E (Cyklin dependent kinase 2) (AstraZeneca
Biotech Laboratory) was diluted 1:80 in an assay buffer containing 50 mM
hydroxyethylpiperazineethanesulfonic acid (HEPES), pH 7.35 10 mM
manganese cloride MnCl2 100 M sodium-orto-vanadate Na-orto-Vanadate
1 mM dithioerythritol DTT 100 M sodium fluoride NaF 10 mM sodium-
glycerophosphate. After incubation for 15 min the reaction was initiated by
the addition of 37.5 ng/l of a GST-tagged retinoblastoma peptide substrate
AstraZeneca Biotech Laboratory 0.07 Ci
unlabelled ATP and in an final assay volume of 21
40 min at room temperature each reaction was terminated by the addition of
30 l stop solution containing 10 mM EDTA 84 M ATP and 6.00 mg/ml anti-
c
33P ATP Amersham UK. 0.3 M
l. After incubation for
l
l
GST-tagged streptavidine coated scintillation proximity assay SPA beads
Amersham UK The microtiter plates were centrifuged for 2 min at 200 g and
Acknowledgments
the radioactivity was determined in
MicroBeta Trilux Wallac Finland. The inhibition curves were analysed by
non-linear regression using XL-fit.
a liquid scintillation counter 1450
The authors wish to thank Dr Istvan Macsari for valuable dis-
cussions during the preparation of the manuscript.
32. Evans, D. B.; Rank, K. B.; Sharma, S. K. J. Biochem. Biophys. Methods 2002, 50,
151.
33. Z0-factor is
a characteristic parameter for the quality of the assay itself,
Supplementary data
meaningful in a range of ꢁ1<Z0003E1.
34. Analytical data for compound 4a: 1H NMR (DMSO-d6, 400 MHz) d 8.54 (d, 1H),
8.12 (d, 1H), 8.08 (d, 1H), 7.97 (d, 1H), 7.82 (br s, 2H), 7.50 (t, 1H), 7.42 (t, 1H).
35. McDonald, F. E.; Burova, S. A.; Huffman, L. G. J. Synthesis 2000, 970.
36. Suzuki, N.; Nomoto, T.; Toya, Y.; Kanamori, N.; Yoda, B.; Saeki, A. Biosci.
Biotechnol. Biochem. 1993, 57, 1561.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
07.068.
37. Suzuki, N.; Nomoto, T.; Toya, Y.; Yoda, B.; Saeki, A. Chem. Express. 1992, 7, 717.
38. Majo, V. J.; Prabhakaran, J.; Mann, J. J.; Kumar, J. S. D. Tetrahedron Lett. 2003, 44,
8535.
References and notes
39. Jeon, Y. T.; Gluchowski, C. WO 9731636.
40. Boga, C.; Del Vecchio, E.; Forlani, L.; Todesco, P. E. J. Organomet. Chem. 2000,
601, 233.
41. Jämsä, A.; Agerman, K.; Radesäter, A.-C.; Ottervald, J.; Malmström, J.; Hiller, G.;
Liu, G.; Vasänge, M. J. Biomed. Sci. 2009, 16, 55.
1. Dhariwala, F. A.; Rajadhyaksha, M. S. Cell. Mol. Neurobiol. 2008, 28, 351.
2. Dhavan, R.; Tsai, L.-H. Nat. Rev. Mol. Cell Biol. 2001, 2, 749.
3. Camins, A.; Verdaguer, E.; Folch, J.; Canudas, A. M.; Pallas, M. Drug News
Perspect. 2006, 19, 453.
4. Cruz, J. C.; Tsai, L.-H. Curr. Opin. Neurobiol. 2004, 14, 390.
5. Cheung, Z. H.; Ip, N. Y. Neurosci. Lett. 2004, 361, 47.