Synthesis and biological evaluation of a selective N- and P/Q-Type calcium channel agonist

Article abstract of DOI:10.1021/ml3002083

The acute effect of the potent cyclin-dependent kinase (cdk) inhibitor (R)-roscovitine on Ca²⁺ channels inspired the development of structural analogues as a potential treatment for motor nerve terminal dysfunction. On the basis of a versatile chlorinated purine scaffold, we have synthesized ca. 20 derivatives and characterized their N-type Ca²⁺ channel agonist action. Agents that showed strong agonist effects were also characterized in a kinase panel for their off-target effects. Among several novel compounds with diminished cdk activity, we identified a new lead structure with a 4-fold improved N-type Ca²⁺ channel agonist effect and a 22-fold decreased cdk2 activity as compared to (R)-roscovitine. This compound was selective for agonist activity on N- and P/Q-type over L-type calcium channels.

Full text of DOI:10.1021/ml3002083

Letter
pubs.acs.org/acsmedchemlett
Synthesis and Biological Evaluation of a Selective N- and P/Q-Type
Calcium Channel Agonist†
Mary Liang,‡ Tyler B. Tarr,§ Karla Bravo-Altamirano,‡ Guillermo Valdomir,‡ Gabriel Rensch,‡
Lauren Swanson,‡ Nicholas R. DeStefino,§ Cara M. Mazzarisi,§ Rachel A. Olszewski,§
Gabriela Mustata Wilson,∥ Stephen D. Meriney,*,§ and Peter Wipf*,‡
§
∥
‡Department of Chemistry, Department of Neuroscience and Center for Neuroscience, and Department of Computational and
Systems Biology, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States
S
* Supporting Information
ABSTRACT: The acute effect of the potent cyclin-dependent kinase (cdk) inhibitor
(R)-roscovitine on Ca2+ channels inspired the development of structural analogues as
a potential treatment for motor nerve terminal dysfunction. On the basis of a versatile
chlorinated purine scaffold, we have synthesized ca. 20 derivatives and characterized their
N-type Ca2+ channel agonist action. Agents that showed strong agonist effects were also
characterized in a kinase panel for their off-target effects. Among several novel
compounds with diminished cdk activity, we identified a new lead structure with a 4-fold
improved N-type Ca2+ channel agonist effect and a 22-fold decreased cdk2 activity as
compared to (R)-roscovitine. This compound was selective for agonist activity on N- and
P/Q-type over L-type calcium channels.
KEYWORDS: N/P/Q-type calcium channels, roscovitine, cdk2, selective agonist, Lambert−Eaton myasthenic syndrome, LEMS,
neurological autoimmune disorder
hemical communication in the nervous system is tightly
regulated by the flux of calcium ions through certain
transmitter release in the nervous system.13 The development of
a potent agonist for N- and P/Q-type calcium channels could
have a significant impact on the treatment of LEMS as well as
other neurological diseases. Furthermore, the availability of a
more potent N- and P/Q-selective agonist would be useful as an
experimental tool to facilitate the study of calcium channel gating
and regulation of cellular events. Accordingly, we embarked on
developing analogues of (R)-roscovitine with decreased cdk
activity and increased selectivity for these subsets of voltage-gated
calcium channels.
At the onset of our work, very little was known about
structural modifications of (R)-roscovitine pertaining to its Ca2+
channel modulating properties.14 As summarized in Figure 1,
the available literature activity data on roscovitine analogues
disclosed only three similarly potent N-type calcium channel
agonists among 12 structural analogues. We used a four-zone
approach to analyze the literature results and to guide our initial
round of medicinal chemistry structure−activity relationship
(SAR) studies. In particular, zone 1, the core region, had only
been superficially probed in the inactive analogues 1 and 2,
which, however, differ too greatly from the purine scaffold of
roscovitine to allow for any conclusions regarding the influence
of core scaffold variations.12 Interestingly, translocation of the
nitrogen atom in pyrazolo[1,5-a]-1,3,5-triazine 3,15−17 however,
retained N-type Ca2+ channel agonist activity and confirmed
C
subtypes of voltage-gated channels. A decrease in calcium flux at
synapses can cause neurological diseases. Lambert−Eaton
myasthenic syndrome (LEMS) is a neurological autoimmune
disorder characterized by an autoimmune reduction of pre-
synaptic P/Q-type (Cav2.1) calcium channels at the neuro-
muscular junction and a partial compensatory up-regulation of
N-type (Cav2.2), L-type (Cav1), and R-type (Cav2.3) channels.1,2
N-type and P/Q-type channels appear to be the most relevant for
the control of transmitter release as they selectively bind directly
to and colocalize with transmitter release sites.3 The result is an
overall calcium channel reduction at transmitter release sites that
results in muscle weakness and is associated with compromised
motor function. This disease is estimated to affect 1:100000
individuals in the United States;4 however, the true incidence of
LEMS remains unknown as it is often undiagnosed in patients.5
Current treatment strategies are very limited, and those available
are indirect and sometimes associated with undesirable side
effects.
Selective calcium channel agonists that increase the ion flux
through N- and P/Q-type calcium channels represent attractive
potential therapeutics for LEMS and other neuromuscular
diseases; however, to date, no such agonists have been identified.
While (R)-roscovitine was originally developed as a cyclin-
dependent kinase (cdk) inhibitor,6,7 several laboratories have
subsequently demonstrated that it is also a potent agonist for
N- and P/Q-type calcium channels,8−11 showing a concentration
required for 50% efficacy (EC50) = 28−41 μM.12 N- and P/Q-
type channels are the two major subtypes that regulate chemical
Received: July 19, 2012
Accepted: October 1, 2012
Published: October 1, 2012
© 2012 American Chemical Society
985
dx.doi.org/10.1021/ml3002083 | ACS Med. Chem. Lett. 2012, 3, 985−990

ACS Medicinal Chemistry Letters
Letter
Figure 1. Summary of SAR model and literature data on roscovitine analogues evaluated for N-type Ca2+ channel agonist effects.
that at least conservative modifications were tolerable.18 The
literature data on zone 2 analogues were similarly very limited, as
shown for compounds 4, 5, and 6 [e.g., (R)-olomoucine II].8,12
The former two trisubstituted N-6 amine analogues were devoid
of any activity, suggesting that a hydrogen atom was required
at N-6. Because the phenol group in 6 was able to substitute
for roscovitine's benzene ring in zone 2 with minimal loss of
activity, we concluded that zone 2 was amenable to further
modifications. In contrast, analogues 7,12 8 (e.g., bohemine),12
and 9 [e.g., (S)-roscovitine]8,12 demonstrated that zone 3 was
critical for the desired N-type Ca2+ channel agonist activity.
It was also noteworthy and encouraging for the robustness
of the SAR that upon the seemingly minor modification of an
inversion of configuration in zone 3 in 9, the activity dropped ca.
14-fold.
Because the literature data did not present any analogues of
roscovitine with exclusive zone 4 modifications, the conclusions
that could be drawn regarding the combined zone 3/4 altera-
tions shown in Figure 1 were ambiguous. The N-9 methy-
lated compound 10 (e.g., olomoucine) and (dimethylamino)-
olomoucine 11 were devoid of any activity,12 but this deficiency
could also be attributed to the lack of appropriate functionality
in the zone 3 side chain. Similarly, iso-olomoucine 1212 was
inactive either due to the shift in substitution in the imidazole
ring, the lack of a 2-propyl group in zone 4, or, most likely,
once again the absence of the (R)-2-amino-1-butanol side-chain
at C-2 in zone 3.
In summary, the available literature information on the
roscovitine activity profile as it pertained to N-type Ca2+ channel
agonism/antagonism was very limited, and the combination of
all previously observed effects did not yet allow for a rational
second generation lead design and development. On the basis of
the lack of activity resulting from minor modifications in zone
3 and the absence of significant SAR data from zone 1 scaffold
alterations, we focused our studies on interrogating the purine
scaffold further by variations of substituents in zones 2 and 4.
Accordingly, for this first round of systematic SAR studies,
we decided to retain the purine backbone as well as the (R)-
2-amino-1-butanol side chain at C-2.
The synthetic strategy used for SAR investigations toward
selective N-type Ca2+ channel agonists based on purines 13 is
summarized in Scheme 1. Starting with the commercially available
2,6-dichloropurine 14,16 N-9 alkylation of the purine with primary
alkyl halides would lead to intermediate 15. Preferential nucleo-
philic aromatic substitution (SNAr) reaction at the more reactive
C-6 position followed by displacement of the C-2 chloride in 16
with primary amines would provide target analogues 13.25
986
dx.doi.org/10.1021/ml3002083 | ACS Med. Chem. Lett. 2012, 3, 985−990

ACS Medicinal Chemistry Letters
Letter
Scheme 1. Synthetic Strategy for Purine Side Chain Variations
Table 1. Chemical Structures and N-Type Ca2+ Channel Agonist Properties of Roscovitine Analogues 13 [R3 = (R)-2-Amino-1-
butanol]
structure
R2
yields (%)
N-type Ca2+ channel activity
Cdk2 activity
a
b
compd
R1
14 → 15
15 → 16
16 → 13
EC50 SEM (μM)
EC50 SEM (μM)
(R)-roscovitine
13a
i-Pr
Pr
Bn
27.58 1.65
>100
0.151 0.004
CH2(biphenyl)
CH(Ph)2
78
78
78
78
78
78
78
64
64
64
64
64
64
64
78
65
65
65
65
65
65
65
64
78
91
65
85
81
59
71
95
86
71
83
68
65
95
78
61
52
82
86
76
72
94
60
86
89
59
67
32
66
49
66
62
62
63
64
61
54
59
37
67
85
34
61
71
64
81
25
93
91
ND
13b
13c
Pr
>100
ND
Pr
(CH2)2Ph
>100
ND
13d
13e
Pr
Bn
14.23 2.71
>100
3.34 0.05
ND
Pr
Ph
13f
Pr
CH2(3-Py)
>100
ND
13g
Pr
CH2CH(CH2)2
CH2(biphenyl)
CH(Ph)2
>100
3.63 0.42
ND
13h
13i
Me
Me
Me
Me
Me
Me
Me
Pr
>100
>100
ND
13j
(CH2)2Ph
>100
ND
13k
13l
Bn
>70
1.44 0.02
ND
Ph
>100
13m
13n
13o
13p
13q
13r
CH2CH(CH2)2
CH2(3-Py)
>100
ND
>100
ND
CH2[(p-trifluoromethyl)phenyl]
CH2CH(CH2)2
(CH2)2Ph
>100
ND
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
Me
Pr
>100
ND
>100
ND
CH2(3-Py)
>100
ND
13s
CH2(biphenyl)
CH(Ph)2
>100
ND
13t
>100
ND
13u
13v
CH2[(2-methyl)5-thiophenyl]
CH2[(m-trifluoromethyl)phenyl]
CH2[(2-methyl)5-thiophenyl]
CH2[(2-methyl)5-thiophenyl]
11.29 1.48
>100
0.262 0.0002
ND
13w
30.02 1.87
7.21 0.86
3.04 0.17
3.29 0.43
13x
a
Ca2+ channel agonist EC50 values were determined by whole cell perforated patch clamp recordings of deactivation currents from N-type channels
as described in the Experimental Procedures. Cpd 13k was only soluble up to 200 μM in saline with 1% DMSO, thus limiting our ability to
b
accurately determine agonist properties. Cdk1 cyclinB(h) inhibitory EC50 values were determined in duplicate at 0.2, 2, and 20 μM agent
concentrations, with roscovitine as the positive control. ND = not determined.
Zone 4 modifications R1 were readily achieved by subjecting
dichloropurine 14 to deprotonation in DMSO in the presence
of a mild base such as potassium carbonate, followed by the
addition of alkyl bromides or iodides at 16−18 °C. Primary
alkyl halides (R1 = Me, Pr) provided a similar yield (64−78%)
to the secondary alkyl halide (R1 = i-Pr, 65%). Microwave
irradiation at 120 °C for 20 min in n-BuOH and triethylamine
in the presence of various aryl- and alkylamines converted
intermediates 15 to the C-6 aminated purines 16 in moderate-
to-good yields (52−95%). Finally, aminolysis at C-2 under
forcing conditions (heating neat in a sealed flask at 170 °C in
the presence of the R3-amine for 8−15 h) introduced the (R)-
2-amino-1-butanol side chain to give target molecules 13 in
variable yields (25−93%).26
N-type Ca2+ channel activity, an encouraging result was ob-
tained with 13d, which showed a ca. 2-fold increased agonism
and a 22-fold decreased cdk2 kinase activity versus the
standard, (R)-roscovitine. We attributed the decreased kinase
activity of 13d to the replacement of the i-propyl side chain
at R1 with the more flexible n-propyl group, a hypothesis that
was supported by the similarly decreased cdk2 activity of
13g. In addition, previous docking and experimental studies of
roscovitine−cdk2 interactions also convey the significant effect
of N-9 substituents on active site binding.14
The preference of the R1 group for the branched i-propyl
with regard to cdk2 activity is quite pronounced, as shown for
the methylated 13k, which also reduced the kinase activity ca.
8-fold. Unfortunately, all larger substituents introduced at this
position ablated the agonist activity or decreased compound
solubility to the extent that we were unable to measure activities
(results not shown). We were hoping to use the R2 substituent
in zone 2 to gain further channel selectivity, but unfortunately,
Table 1 presents an overview of the synthetic analogues
prepared using this pathway, as well as their N-type Ca2+
channel agonist and cdk2 kinase inhibitory properties. While
most of the new derivatives were ineffective at enhancing
987
dx.doi.org/10.1021/ml3002083 | ACS Med. Chem. Lett. 2012, 3, 985−990

ACS Medicinal Chemistry Letters
Letter
all modifications of the benzyl group at this site, including the
cyclo-propylmethyl group in 13g, led to complete loss of N-type
Ca2+ channel activity. These negative results were hard to predict,
given the dearth of information about zone 2 modifications prior
to our work (Figure 1).
parameters for interactions with (R)-roscovitine analogues and
to lay the groundwork for future second generation SAR studies.
The MVD algorithm for docking combines an algorithm for
cavity detection in the 3D structure of the target protein with
an optimization algorithm that evaluates different poses for the
protein−ligand complex. Because the crystal structure of cdk2
(PDB ID: 3DDQ)23 contains (R)-roscovitine bound to the
active site, it was possible to identify critical residues surrounding
the binding pocket. The MolDock scoring function was in com-
bination with the MolDock SE search algorithm and Tabu
clustering.24 A search space volume of 25 Å radius encompassing
the (R)-roscovitine binding domain was chosen for docking, and
the ligands and catalytic pocket residues were allowed to be
flexible during the simulation. Each ligand was docked iteratively
into the chosen cavity in 10 independent runs, each of which
consisted of 1500 steps. Poses generated from each run were
subjected to Tabu clustering whereby the lowest energy pose
below an energy threshold of 100 was generated as output. Thus,
there were 10 poses per ligand ranked by energy. The lowest
energy pose of the 10 poses per ligand was selected for visual
inspection.
For validation purposes, docking was first applied to
(R)-roscovitine, and the docked pose was computed to be
very close to the position of the ligand in the X-ray structure
(i.e., RMSD = 0.27 Å). The binding free energy, as estimated by
the MolDock Score (arbitrary units) was −140, which indicated
a strong interaction with cdk2 binding site. The same protocol
was then applied to all (R)-roscovitine analogues. The docking
studies demonstrated that analogues 13d, 13g, 13k, 13w, and
13x bound with a MolDock Score value between −127 and
−143 and in a different orientation, less favorable than
(R)-roscovitine. Compound 13u bound similarly to (R)-roscovitine
with a MolDock Score of −142 (Figures 2 and 3). These results
are consistent with the experimental data that show this com-
pound to retain cdk2 activity, while 13d, 13g, 13k, 13w, and 13x
displayed reduced cdk2 activities.
The first group at R2 that proved to be an effective mimic
of the benzyl group in roscovitine was the methylthiophenyl-
substituted 13u. This compound had almost 3-fold improved
N-type Ca2+ channel agonism, even though it was still a low
nanomolar cdk2 inhibitor. Because 13u was substituted with an
i-propyl group at R1, we speculated that replacing this group
with methyl or n-propyl while maintaining the methylthiophenyl
group at R2 should give us reduced kinase inhibitory properties
analogous to 13d, 13g, and 13k. Indeed, this turned out to be
the case. Both 13w and 13x had an EC50 ≈ 3 μM against cdk2,
but the R1 = methyl substitution in 13w also decreased the
channel activity ca. 3-fold versus 13u. In contrast, 13x proved
to be a considerably more potent N-type Ca2+ channel agonist
with an EC50 = 7.2 μM for N-type channels. The effects of
small changes in the R1 substitution on the N-channel affinity are
quite remarkable. For 13x, we also examined agonist effects on
P/Q-type (EC50 = 8.8 1.1 μM) and L-type calcium channels
(EC50 > 100 μM). Accordingly, 13x showed selectivity for
N- and P/Q-type over L-type calcium channels. Furthermore, we
profiled 13x against cdk1, cdk5, mitogen-activated protein kinase
1 (MAPK1), and myosin light chain kinase (MLCK) kinase
targets and found inhibitory effects with EC50 = 20.56 0.96,
3.03
0.32, >20, and >20 μM, respectively, further illustrat-
ing the quite favorable low-activity kinase profile of this lead
structure. In light of the large cellular ATP concentrations (in
the 1−10 mM range),27 single-digit micromolar activities of 13x
against some kinases are likely readily compensated for in vivo
and are therefore not considered significant impediments from
possible therapeutic applications of 13x as N/P/Q-type calcium
channel agonist in LEMS.
In parallel to our synthetic studies, docking analyses to
the cdk2/roscovitine complex were used to analyze structural
Figure 2. Docking of 13d, 13g, 13k, 13w, 13x, and 13u to the cdk2/roscovitine complex. The electrostatic interaction surface at the binding site
region is displayed and colored red for negative charge and blue for positive charge. Docking simulations were performed using Molegro Virtual
Docker, taking into account side chain flexibility for all residues in the binding region.
988
dx.doi.org/10.1021/ml3002083 | ACS Med. Chem. Lett. 2012, 3, 985−990

ACS Medicinal Chemistry Letters
Letter
To assess the biological effects of (R)-roscovitine derivatives, whole-
cell currents through Ca2+ channels were recorded using perforated
patch methods as previously described.20−22 Briefly, the pipet solution
consisted of 70 nM Cs2SO4, 60 mM CsCl, 1 mM MgCl2, and 10 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) at pH
7.4. Cultured cells were bathed in a saline composed of 130 mM
choline chloride (ChCl), 10 mM tetraethylammonium chloride
(TEA-Cl), 2 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES at pH
7.4. Patch pipettes were fabricated from borosilicate glass, and cap-
acitive currents and passive membrane responses to voltage commands
were subtracted from the data. Currents were amplified by an
Axopatch 200B amplifier, filtered at 5 kHz, and digitized at 10 kHz for
subsequent analysis using pClamp software (Axon Instruments/
Molecular Devices, Sunnyvale, CA). A liquid junction potential of
−11.3 mV was subtracted during recordings. To measure effects on
calcium channel tail currents, the tail current integral was measured
before and after application of a derivative, with the integral of each
trace being normalized to its peak. All experiments were carried out
at room temperature (22 °C). All (R)-roscovitine derivatives were
dissolved in DMSO as a 100 mM stock and stored at −20 °C. For
whole-cell recordings, (R)-roscovitine derivatives were diluted on
the day of use into saline at a final concentration of 1−100 μM, and
were bath-applied via a glass pipet in a ∼0.5 mL static bath chamber.
Control recordings performed with 0.1−1% DMSO alone added
to the drug delivery pipet solution revealed no significant effects on
whole-cell Ca2+ currents. All other salts and chemicals were obtained
from Sigma-Aldrich Chemical Company (St. Louis, MO).
Cdk1 cyclinB(h), cdk2 cyclinA(h), cdk5 p35(h), MAPK1(h), and
MLCK(h) inhibitory activities were determined by Millipore UK Ltd. at 0.2,
2, and 20 μM agent concentration, with roscovitine as the positive control.
Flexible docking studies were performed using Molegro Virtual
Docker (MVD, University of Aarhus, Denmark) to evaluate if the
analogues bound to the (R)-roscovitine binding site of cdk2.23 The
basis of these docking studies was a new hybrid search algorithm, that
is, a guided differential evolution (DE), which combines DE opti-
mization with a cavity prediction algorithm, which is dynamically
used during the docking process. Briefly, all individual ligands were
initialized, evaluated, and scored (Escore/MolDock Score) according to
the fitness function, which is the sum of the intermolecular interaction
energy between the ligand and the protein and the intramolecular
interaction energy of the ligand: Escore = Einter + Eintra, with Einter being
the ligand−protein interaction energy and Eintra being the internal
energy of the ligand.24
Figure 3. Hydrogen bond interactions between analogues 13d, 13g,
13k, 13u, 13w, 13x, and cdk2. Interacting chemical groups of the
analogues are shown in blue ellipses on the (R)-roscovitine scaffold.
In summary, in ca. 20 synthetic analogues, we were able to
generate a new lead structure 13x with a 4-fold improved
N-type Ca2+ channel agonist efficacy and a 22-fold decreased
cdk2 activity as compared to (R)-roscovitine. The SAR followed
a logical trend, with small structural changes at R1 greatly
influencing the cdk2 activity. Replacing the benzyl group at R2
with bioisosteric functions mostly led to compounds lacking
N-type Ca2+ channel activity, with the notable exception of the
5-methylthiophene group, which considerably increased the
desired agonism. Molecular docking studies were used to predict
and analyze the structural trends leading to reduced cdk2
activity. We are now studying the in vivo effects of 13x as well as
further improving its N- and P/Q-type Ca2+ channel activity
profile. The long-term goal of our program is to develop selective
voltage-gated N- and P/Q-type calcium channel agonists for the
therapy of LEMS and other neurological diseases. The potential
side effects of a use-dependent N- and P/Q-type calcium channel
agonist that enhances calcium flux through calcium channels that
are normally activated by action potential activity are expected to
be few and manageable (assuming it does not cross the blood−
brain barrier). This expectation is based on extensive clinical
literature that uses an indirect method of achieving a similar
outcome with potassium channel blockers such as 3,4-
diaminopyridine (DAP).28,29 DAP prolongs the duration of the
presynaptic action potential, and this indirectly increases the
activation of all voltage-gated calcium channels in the peripheral
nervous system. DAP is generally well tolerated by LEMS
patients,28,29 with many of the side effects reported likely due to
either broadening of action potentials in all neurons or the
nonselective indirect effect of increasing calcium flux through all
voltage-gated channels. On the basis of these previous reports,
selective use-dependent N- and P/Q-type calcium channel
agonists are predicted to be well tolerated.
EPLP is a piecewise linear potential using two different sets of
parameters: one set for approximating the steric van der Waals term
between atoms and the other stronger potential for hydrogen bonds.
Eclash assigns a penalty of 1000 if the distance between two atoms
(more than two bonds apart) is less than 2.0 Å. Thus, the Eclash term
punishes infeasible ligand conformations.
Offspring were created using a weighted difference of the parent
solutions, which were randomly selected from the population. If, and only
if, the offspring was more fit, it would replace the parent. Otherwise, the
parent survived and was passed on to the next generation, representing
an iteration of the algorithm. The search process was terminated when
the number of fitness evaluations exceeded the maximum number of
evaluations permitted.
EXPERIMENTAL PROCEDURES
■
For the synthesis of 15, 16, and 13, see the Supporting Information.
Biological evaluations of the effects of (R)-roscovitine derivatives on
N-type calcium channels were initially performed using a tsA201 cell
line19 that stably expresses all of the subunits of the N-type Ca2+
channel splice variant predominantly present in mammalian brain and
spinal cord: Cav2.2 rnα1B‑c (Cav 2.2 e[24a,Δ31a]), Cavβ3, and Cavα2δ1.
For subsequent evaluation of effects on N-, P/Q-, or L-type channels,
tsA-201 cells were transiently transfected with Cav2.2, Cav2.1, or
Cav1.3, in combination with Cavβ3 and Cavα2δ1 (Addgene, Cambridge,
MA) using FuGENE 6 (Promega, Madison, WI). All cells were
maintained in DMEM supplemented with 10% fetal bovine serum.
For the stable cell line expressing N-type channels, 25 μg/mL zeocin,
5 μg/mL blasticidin, and 25 μg/mL hygromycin were added as selection
agents.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures, assay results, and spectral data for
new compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 412-624-8283. Fax: 412-624-9198. E-mail: meriney@pitt.
edu(S.D.M.). Tel: 412-624-8606. Fax: 412-624-0787. E-mail:
pwipf@pitt.edu (P.W.).
989
dx.doi.org/10.1021/ml3002083 | ACS Med. Chem. Lett. 2012, 3, 985−990

ACS Medicinal Chemistry Letters
Letter
(13) Sheng, Z.-H.; Westenbroek, R. E.; Catterall, W. A. Physical link
and functional coupling of presynaptic calcium channels and the
synaptic vesicle docking/fusion machinery. J. Bioenerg. Biomembr.
1998, 30, 335−345.
(14) For a discussion of SAR effects pertaining to the cdk2 activity
profile of roscovitine, see Otyepka, M.; Krystof, V.; Havlicek, L.;
Siglerova, V.; Strnad, M.; Koca, J. Docking-based development of
purine-like inhibitors of cyclin-dependent kinase-2. J. Med. Chem.
2000, 43, 2506−2513.
(15) Popowycz, F.; Fournet, G.; Schneider, C.; Bettayeb, K.;
Ferandin, Y.; Lamigeon, C.; Tirado, O. M.; Mateo-Lozano, S.;
Notario, V.; Colas, P.; Bernard, P.; Meijer, L.; Joseph, B. Pyrazolo[1,5-
a]-1,3,5-triazine as a purine bioisostere: Access to potent cyclin-
dependent kinase inhibitor (R)-roscovitine analogue. J. Med. Chem.
2009, 52, 655−663.
Funding
This study was supported in part by the National Institutes of
Health, the NIH P50 CMLD Program (GM067082 to P.W.),
and a National Science Foundation Grant 0844604 (S.D.M.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Diane Lipscombe (Brown University) for kindly
providing the stable N-type calcium channel tsA201 cell line
and Michael Frasso (University of Pittsburgh) for the resynthesis
of 13v.
(16) Oumata, N.; Bettayeb, K.; Ferandin, Y.; Demange, L.; Lopez-
Giral, A.; Goddard, M.-L.; Myrianthopoulos, V.; Mikros, E.; Flajolet,
M.; Greengard, P.; Meijer, L.; Galons, H. Roscovitine-derived, dual-
specificity inhibitors of cyclin-dependent kinases and casein kinases 1.
J. Med. Chem. 2008, 51, 5229−5242.
(17) Bettayeb, K.; Sallam, H.; Ferandin, Y.; Popowycz, F.; Fournet,
G.; Hassan, M.; Echalier, A.; Bernard, P.; Endicott, J.; Joseph, B.;
Meijer, L. N-&-N, a new class of cell death-inducing kinase inhibitors
derived from the purine roscovitine. Mol. Cancer Ther. 2008, 7, 2713−
2724.
DEDICATION
■
†This paper is dedicated to the memory of Dr. Hiroyuki
Nemoto.
ABBREVIATIONS
■
cdk, cyclin-dependent kinase; LEMS, Lambert−Eaton myasthenic
syndrome; EC50, concentration required for 50% efficacy; SAR,
structure−activity relationship; SNAr, nucleophilic aromatic sub-
stitution; MAPK1, mitogen-activated protein kinase 1; MLCK,
myosin light chain kinase
(18) Meriney, S. D. Unpublished results.
(19) Lin, Y.; McDonough, S. I.; Lipscombe, D. Alternative splicing in
the voltage-sensing region of N-type Cav2.2 channels modulates
channel kinetics. J. Neurophys. 2004, 92, 2820−2830.
(20) White, M. G.; Crumling, M. A.; Meriney, S. D. Developmental
changes in calcium current pharmacology and somatostatin inhibition
of calcium current in chick parasympathetic neurons. J. Neurosci. 1997,
17, 6302−6313.
(21) Yazejian, B.; DiGregorio, D. A.; Vergara, J. L.; Poage, R. E.;
Meriney, S. D.; Grinnell, A. D. Direct measurements of presynaptic
calcium and calcium-activated potassium currents regulating neuro-
transmitter release at cultured Xenopus nerve-muscle synapses. J.
Neurosci. 1997, 17, 2990−3001.
(22) Cho, S.; Meriney, S. D. The effects of presynaptic calcium
channel modulation by roscovitine on transmitter release at the adult
frog neuromuscular junction. Eur. J. Neurosci. 2006, 23, 3200−3208.
(23) Bettayeb, K.; Oumata, N.; Echalier, A.; Ferandin, Y.; Endicott, J.
A.; Galons, H.; Meijer, L. CR8, a potent and selective, roscovitine-
derived inhibitor of cyclin-dependent kinases. Oncogene 2008, 27,
5797−5807.
(24) Thompson, E. E.; Kornev, A. P.; Kannan, N.; Kim, C.; Ten
Eyck, L. F.; Taylor, S. S. Comparative surface geometry of the protein
kinase family. Protein Sci. 2009, 18, 2016−2026.
(25) For selective replacements of heterocyclic chloride substituents
with carbon nucleophiles, see, for example, Wipf, P.; George, K. M.
Regioselective palladium-catalyzed cross-coupling reactions of 2,4,7-
trichloroquinazoline. Synlett 2010, 644−648.
(26) Oumata, N.; Ferandin, Y.; Meijer, L.; Galons, H. Practical
synthesis of roscovitine and CR8. Org. Process Res. Dev. 2009, 13, 641−
644.
REFERENCES
■
(1) Urbano, F. J.; Pagani, M. R.; Uchitel, O. D. Calcium channels,
neuromuscular synaptic transmission and neurological diseases. J.
Neuroimmunol. 2008, 201−202, 136−144.
(2) Motomura, M.; Lang, B.; Johnston, I.; Palace, J.; Vincent, A.;
Newsom-Davis, J. Incidence of serum anti-P/Q-type and anti-N-type
calcium channel autoantibodies in the Lambert-Eaton myasthenic
syndrome. J. Neurol. Sci. 1997, 147, 35−42.
́ ̂ ̀
(3) Seagar, M.; Leveque, C.; Charvin, N.; Marqueze, B.; Martin-
Moutot, N.; Boudier, J. A.; Boudier, J. L.; Shoji-Kasai, Y.; Sato, K.;
Takahashi, M. Interactions between proteins implicated in exocytosis
and voltage-gated calcium channels. Philos. Trans. R. Soc. B, 1999, 354,
289−97.
(4) Lindquist, S.; Stangel, M. Update on treatment options for
Lambert-Eaton myasthenic syndrome: Focus on use of amifampridine.
Neuropsychiatr. Dis. Treat. 2011, 7, 341−349.
(5) Sanders, D. B. Lambert-Eaton myasthenic syndrome: Diagnosis
and treatment. Ann. N.Y. Acad. Sci. 2003, 998, 500−508.
(6) Meijer, L.; Raymond, E. Roscovitine and other purines as kinase
inhibitors. From starfish oocytes to clinical trials. Acc. Chem. Res. 2003,
36, 417−425.
(7) Meijer, L.; Bettayeb, K.; Galons, H. (R)-Roscovitine (cyc202,
seliciclib). In Inhibitors of Cyclin-Dependent Kinases as Anti-Tumor
Agents; Smith, P. J., Yue, E. W., Eds.; CRC/Taylor & Francis: Boca
Raton, 2007; pp 187−225.
(8) Cho, S.; Meriney, S. D. The effects of presynaptic calcium
channel modulation by roscovitine on transmitter release at the adult
frog neuromuscular junction. Eur. J. Neurosci. 2006, 23, 3200−3208.
(9) De Stefino, N. R.; Pilato, A. A.; Dittrich, M.; Cherry, S. V.; Cho,
S.; Stiles, J. R.; Meriney, S. D. (R)-Roscovitine prolongs the mean
open time of unitary N-type calcium channel currents. Neuroscience
2010, 167, 838−849.
(10) Yan, Z.; Chi, P.; Bibb, J. A.; Ryan, T. A.; Greengard, P.
Roscovitine: A novel regulator of P/Q-type calcium channels and
transmitter release in central neurons. J. Physiol. 2002, 540, 761−70.
(11) Buraei, Z.; Anghelescu, M.; Elmslie, K. S. Slowed N-type
calcium channel (CaV2.2) deactivation by the cyclin-dependent kinase
inhibitor roscovitine. Biophys. J. 2005, 89, 1681−1691.
(12) Buraei, Z.; Elmslie, K. S. The separation of antagonist from
agonist effects of trisubstituted purines on Cav2.2 (N-type) channels. J.
Neurochem. 2008, 105, 1450−1461.
(27) Kemp, G. J.; Meyerspeer, M.; Moser, E. Absolute quantification
of phosphorus metabolite concentrations in human muscle in vivo by
31P MRS: A quantitative review. NMR Biomed. 2007, 20, 555−565.
(28) Oh, S. J.; Claussen, G. G.; Hatanaka, Y.; Morgan, M. B. 3,4-
Diaminopyridine is more effective than placebo in a randomized,
double-blind, cross-over drug study in LEMS. Muscle Nerve 2009, 40,
795−800.
(29) Titulaer, M. J.; Lang, B.; Verschuuren, J. J. Lambert-Eaton
myasthenic syndrome: From clinical characteristics to therapeutic
strategies. Lancet Neurol. 2011, 10, 1098−1107.
990
dx.doi.org/10.1021/ml3002083 | ACS Med. Chem. Lett. 2012, 3, 985−990