Ligand-based design and synthesis of novel sodium channel blockers from a combined phenytoin-lidocaine pharmacophore

Article abstract of DOI:10.1016/j.bmc.2008.10.031

The voltage-gated sodium channel remains a rich area for the development of novel blockers. In this study we used comparative molecular field analysis (CoMFA), a ligand-based design strategy, to generate a 3D model based upon local anesthetics, hydantoins

Full text of DOI:10.1016/j.bmc.2008.10.031

Bioorganic & Medicinal Chemistry 17 (2009) 7064–7072
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Ligand-based design and synthesis of novel sodium channel blockers
from a combined phenytoin–lidocaine pharmacophore
Yuesheng Wang a, Paulianda J. Jones a, Timothy W. Batts b, Victoria Landry a, Manoj K. Patel b,
Milton L. Brown c,d,e,
*
a Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA
b Department of Anesthesiology, University of Virginia, Charlottesville, VA 22904, USA
c Department of Oncology, Georgetown University Medical Center, 3970 Reservoir Road, Washington, DC 20057, USA
d Department of Neuroscience, Georgetown University Medical Center, 3970 Reservoir Road, Washington, DC 20057, USA
e Drug Discovery Program, 3970 Reservoir Road, Washington, DC 20057, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The voltage-gated sodium channel remains a rich area for the development of novel blockers. In this
study we used comparative molecular field analysis (CoMFA), a ligand-based design strategy, to generate
a 3D model based upon local anesthetics, hydantoins, and a-hydroxyphenylamides to elucidate a SAR for
Received 22 April 2008
Revised 3 October 2008
Accepted 3 October 2008
Available online 17 October 2008
their binding site in the neuronal sodium channel. Correlation by partial least squares (PLS) analysis of
in vitro sodium channel binding activity (expressed as pIC50) and the CoMFA descriptor column gener-
ated a final non-cross-validated model with q2 = 0.926 for the training set. The CoMFA steric and electro-
static maps described a binding site predominately hydrophobic in nature. This model was then used to
design and predict a series of novel sodium channel blockers that utilized overlapping structural features
of phenytoin, hydroxy amides, and the local anesthetic lidocaine. Synthesis and evaluation of these com-
pounds for their ability to inhibit [3H]-batrachotoxin revealed that these compounds have potent sodium
channel blockade. Furthermore, the CoMFA model was able to accurately predict the binding of these
compounds to the neuronal sodium channel. Synthesis and subsequent sodium channel evaluation of
Keywords:
Sodium channels
CoMFA
Antagonist
Electrophysiology
compound 37 (predicted IC50 = 7
overlapping regions of phenytoin and lidocaine are better binders to the sodium channel than phenytoin
itself (IC50 = 40 M).
lM, actual IC50 = 6 lM), established that novel compounds based on
l
Ó 2009 Published by Elsevier Ltd.
1. Introduction
deadly neurotoxin of the puffer fish. The binding site for batracho-
toxin (BTX), an alkaloid from poison dart frogs, binds to the inacti-
vated state of the NVSC, thereby disrupting the equilibrium of
protein states. Substitution with a para-[3H] benzoate ester at C20
on BTX resulted in the development of a very useful radioligand
[3H]-BTX-B.2 The binding of [3H]-BTX-B to the NVSC protein is
allosterically linked3 to and overlaps with the binding sites of both
anticonvulsants (phenytoin) and local anesthetics (lidocaine).
Functionally, BTX stabilizes the NVSCs in their open conformation,
whereas local anesthetics block Na+ conductance.
Neuronal voltage-gated sodium channels (NVSC) play an impor-
tant role in the generation and propagation of action potentials in
neurons and other excitable cells. Thus, NVSC blocking agents rep-
resent a clinically important class of drugs used in the treatment of
pain, seizures and arrhythmia.
Cloning, purification and sequence analysis of NVSC’s have
identified at least ten highly conserved
composed of six transmembrane segments (S1–S6) that form the
260 kDa -subunit complex and at least four important accessory
b-subunits.1
Information concerning channel topography has been obtained
a-subunits. The NVSC is
a
The use of [3H]-BTX-B as a NVSC screening tool has indicated an
important relationship between molecular structure and pharma-
cological effects for interactions of small molecule inhibitors. Using
this information, a ligand-based molecular modeling study was
successful in the design of a new small molecule which binds po-
through the use of neurotoxins. In fact, voltage-sensitivity is char-
acterized based on the channel’s sensitivity to tetrodotoxin (TTX), a
tently (IC50 = 9 l
M) to the NVSC4–6 and a relationship between
[3H]-BTX-B inhibition and anticonvulsant activity in mice has been
established.7
* Corresponding author. Address: Departments of Oncology and Neuroscience,
Georgetown University Medical Center, 3970 Reservoir Road, Washington, DC
20057, USA. Tel.: +1 202 687 8603.
Site-directed mutagenesis studies have identified clusters of
common residues at D1–S6, D3–S6, and D4–S6 segments within
E-mail address: mb544@georgetown.edu (M.L. Brown).
0968-0896/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.10.031

Y. Wang et al. / Bioorg. Med. Chem. 17 (2009) 7064–7072
7065
posed
a
series of novel grafted phenytoin/lidocaine and
O
N
hydroxyamide/lidocaine compounds (Fig. 1) that were identi-
fied and predicted by CoMFA to have potent NVSC activity.
We have validated our CoMFA alignment by synthesizing these
proposed compounds and evaluating them for inhibition of
[3H]-BTX-B.
N
O
H
O
NH
R
Lidocaine
HO
NH
2
HN
O
R
O
2. Results
O
NH
R
HO
NH
HN
2
O
O
R
O
Our previous sodium channel SAR studies suggested that logP
was one of the parameters important for enhanced binding to
the NVSC.5 In the training set, logP correlation to pIC50 gave
an R2 of 0.59 (Fig. 3). This is poor correlation, in fact, the groups
of compounds at logP ꢀ 2.5 and logP ꢀ 3.0 have IC50 values that
span 2 log units. It is clear that local anesthetic binding to the
NVSC is dependent on other factors besides lipophilicity. Com-
parisons within the present model of local anesthetics and hyd-
antoins with the same logP and different structures often show
very different binding affinities to the NVSC (e.g. 3 and pipero-
caine). We were thus encouraged to use CoMFA to investigate
the differences in structural and electrostatic features of these
analogues in an effort to formulate an improved model. The so-
dium channel binding affinities and lipophilicities for the CoMFA
training set are presented in Table 1. PLS correlation of in vitro
N
N
N
H
N
H
Figure 1. Design of novel grafted phenytoin/lidocaine compounds.
the NVSC
(phenytoin) and local anesthetics (lidocaine).8,9 Mutations of ami-
no acid residues in specific segments of the -subunit have re-
a-subunit that are critical for binding of hydantoins
a
vealed important voltage dependence and modification of protein
conformational states (open, inactivated, closed).10 Altogether,
these studies suggest that phenytoin and lidocaine share a non-
identical but overlapping receptor11 on the extracellular side of
the NVSC.12
Considering the accepted overlapping nature of the binding
sites of phenytoin and lidocaine, we have employed the use
of comparative molecular field analysis (CoMFA) to assist in
identifying important overlapping structural features and to
provide insight into essential SAR for the development of novel
NVSC inhibitors. CoMFA, a 3D ligand-based discovery program
is useful in correlating biological activity with structural fea-
tures and has become a valuable tool in the design of new
inhibitors of biological receptors. In this study, we have pro-
sodium channel binding, expressed as –logIC50, and CoMFA
descriptors generated a CoMFA model with q2 = 0.926 for the
training set (Fig. 4). Further more, the CoMFA model provided
better correlation than that for the logP model (R2 = 0.59) from
the same data set.
Investigations of the CoMFA steric map revealed that a long al-
kyl chain at the C5 position appears to be required for tight binding
to the hydantoin site. The steric contour map in Figure 5 reveals a
O
O
O
N
NH
R
O
O
R
O
3
NH
NH
HN
HN
N
O
1
HN
HO
NH
H C
2
3
O
R
N
2
O
O
N
n
-C H
7
15
R
n
O
1
R
R
4
2
n
R
14-317
20
18-19
H C
13
1-8
9-12
O
3
H C
3
S
O
N
OCH
3
NH
H C
3
2
5
2
O
6
1
4
3
OCH3
NH
7
H C
3
O
N
O
10
8
O
N
9
CH
3
H
O
CH
3
O
HN
Bupivacaine (22)
Benzocaine (21)
Cocaine (24)
Carticaine (23)
CH
CH
3
3
N
O
H C
3
H C
3
O
5
2
O
6
1
4
3
O
H C
3
N
7
N
9
N
H
3
N
H
8
10
N
N
H
O
N
H
CH
CH O
3
3
CH
3
CH
Hexylcaine (27)
Lidocaine(28)
Etidocaine (26)
Dibucaine (25)
H C
3
O
CH
3
O
H
N
CH
3
O
N
O
O
H C
3
N
H
N
N
O
N
O
CH
3
O
Prilocaine (31)
Parethoxycaine (29)
Procaine (32)
Piperocaine (30)
NH
2
H
N
O
O
NH
2
CH
3
CH
H C
3
3
•HCl
O
O
O
O
HCl
N
N
O
O
Proparacaine (33)
Propoxycaine (34)
Tetracaine(35)
Figure 2. Test set compounds used in developing the CoMFA model.

7066
Y. Wang et al. / Bioorg. Med. Chem. 17 (2009) 7064–7072
Table 1
Sodium channel binding activities for the training set used in the final non-cross-validated CoMFA modela
3H-BTX IC50
(lM)
Final model, ÀpIC50
b
Compound
R1
R2
R3
R4
n
LogP
Obsd
Pred
Res
1
2
3
4
5
6
7
8
9
n-Propyl
n-Butyl
n-Butyl
n-Pentyl
n-Hexyl
n-Heptyl
n-Nonyl
Methyl
H
H
H
H
H
H
H
H
H
H
1.96
2.46
3.01
2.96
3.40
3.96
4.96
2.02
0.96
1.46
1.96
2.46
1.52
2.96
2.96
2.96
2.96
2.01
2.51
3.70
1.39
3.86
162
103
285
39
13
5
À2.21
À2.01
À2.45
À1.59
À1.11
À0.70
À0.70
À2.86
À3.32
À2.93
À2.40
À2.40
À2.40
À2.35
À1.76
À1.98
À1.76
À2.85
À2.63
À0.95
À2.96
À0.73
À1.71
À1.69
À0.15
À0.54
À0.85
À2.38
À1.38
À1.11
À1.73
À2.04
À0.28
À1.38
À0.53
À1.60
À2.10
À1.91
À2.16
À1.55
À1.17
À0.82
À0.54
À2.87
À2.87
À2.66
À2.52
À2.43
À2.46
À2.03
À1.78
À2.22
À1.91
À3.11
À2.79
À0.96
À3.10
À0.95
À1.65
À1.71
0.22
À0.99
À0.81
À1.94
À1.12
À0.86
À1.81
À1.96
À0.72
À1.50
À0.73
À1.95
À0.11
À0.10
À0.30
À0.04
0.06
Methyl
H
H
H
H
0.12
5
À0.16
Ethyl
720
2112
851
251
251
250
225
58
0.01
1
2
3
4
À0.46
À0.27
0.12
0.03
0.06
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
n-Butyl
n-Butyl
n-Butyl
Cyclohexyl
Methyl
H
H
H
À0.33
H
H
H
Methyl
H
H
0.02
Methyl
H
95
58
0.25
0.14
0.27
0.16
0.01
0.14
0.22
2
3
700
427
9
910
5.4
51
À0.06
1.82
3.77
3.87
3.34
2.70
2.74
3.01
2.18
2.53
2.42
2.42
3.27
2.46
49
0.02
1.4
3.5
7.1
240
24
12.9
53.7
109.6
2
À0.37
0.44
À0.04
À0.44
À0.26
À0.25
0.08
À0.08
0.44
24
3.4
40
0.12
0.20
0.35
a
Number of components = 4; s = 0.245; q2 = 0.926.
b
ÀpIC50 = ÀlogIC50
.
favorable steric volume near the 5-alkyl side chain. In addition, ste-
ric bulk in the amide portion of the local anesthetics appears to
also be favorable as revealed by comparison of binding by benzo-
phenytoin (hydantoin) pharmacophore or the hydroxyamide
pharmacophore and the local anesthetic lidocaine would be
potent binders to the neuronal sodium channel as they
would potentially have the ability to bind to an expanded
binding region encompassing both the phenytoin and local
anesthetic binding sites. Using the CoMFA model described
above, our newly developed grafted analogues were pre-
caine (IC50 = 910
anesthetic site.
lM) and etidocaine (IC50 = 3.5 lM) to the local
An important part of a 3D-QSAR model is the ability to
accurately predict the relative binding potencies of novel
analogues before synthesis. Based upon the model described
here, we proposed that novel analogues containing both the
dicted to have IC50 values of 3
ble 3).
lM (40) to 66 lM (38) (Ta-
4
y = -0.7786x + 3.8464, R2 = 0.5936
9
21
3
10
13
188
19
12 28
11
1
3
14
16
2
32
2
1
0
31
15, 17
4
24
36
34
29
30
5
27
21
22
7
6
35
26
25
33
0
1
2
3
4
5
6
Log P
Figure 3. Plot of logP versus pIC50 for compounds in the training set.

Y. Wang et al. / Bioorg. Med. Chem. 17 (2009) 7064–7072
7067
4
3.5
3
q2= 0.9213, s = 0.245 for noncrossvalidation
18
21
19 8
9
11
10
12
2.5
2
13
1
16
2
14
3
36 17
31
32
28
24 15
34
29
23
4
1.5
1
5
26
35
22 20
30
6
27
33
0.5
0
7
0.5
1
1.5
2
2.5
3
3.5
4
-0.5
Observed pIC50
Figure 4. Plot of observed pIC50 versus predicted pIC50 values for the non-cross-validated CoMFA model in Table 1.
Figure 5. Electrostatic and steric CoMFA fields for the model developed from compounds in Table 1. For electrostatic contours, increased binding results from placing more
positive (+) charge near blue and negative (À) charge near red. For steric contours increased binding results from placing more bulk near green and less bulk near yellow. (A)
Overlay of compounds used in the training set. (B) Active analogue 44. (C) Active analogue 44 with electronic contour overlay. (D) Active analogue 44 with steric contour
overlay.
After successfully completing the syntheses of analogues 37–45
Observed [3H]-BTX displacement revealed that compound 37
(Scheme 1) we evaluated them for activity using [3H]-BTX-B bind-
displaced 83% [3H]-BTX at 40
lM. Compounds 41 and 42, the direct
ing assay with each compound at a concentration of 40
lM (IC50 of
grafts of phenytoin and lidocaine, displaced 50 and 49% [3H]-BTX,
respectively. As a comparison between the hydroxyamides and
hydantoins, compound 44 (hydantoin analogue of 37) displaced
phenytoin in this assay). This series of analogues encompasses a
range of lipophilicities (logP = 1.4–5.3) as well as phenyl ring sub-
stitutions (R2 = H or CH3), pharmacophore changes (hydroxya-
71% [3H]-BTX at 40
l
M. The latter data is shown in Table 3, and
these four analogues were chosen based on their potent inhibition
at 40 M and to allow comparisons between direct phenytoin/lido-
mides vs hydantoins), and chain length (R1 = Ph, C6H13, C7H15
,
and C9H19). The predictive ability of the CoMFA model was evalu-
ated by comparing the observed versus predicted activities of the
novel hybrid phenytoin/lidocaine and hydroxyamide/lidocaine
analogues.
l
caine grafts and hydroxyamide/hydantoin pharmacophores. As
previously mentioned, compounds 37 and 44 represent optimal
binders based upon initial [3H]-BTX data when tested at a concen-

7068
Y. Wang et al. / Bioorg. Med. Chem. 17 (2009) 7064–7072
O
O
O
R
R2
R
2O
R1
R1
2O
R1
b
a
c
N
Br
R2COOH
N
H
NH2
N
H
R2
R1
R2
R2
R2
R2
R1
R1
R2
H
CH3
H
CH3
82% yield
H
CH3
CH3
CH3
C6H5
C6H5
C6H13
C7H15
C6H5
C6H5
C6H13
C7H15
65% yield
C6H5 85% yield
C6H5
C6H13
C7H15
88% yield
82% yield
75% yield
75% yield
69% yield
70% yield
69% yield
52% yield
46% yield
CH3
CH3
CH3
CH3
f
d, e
O
HN
NH2
O
NH
O
OH
R
2O
R
2O
R1
R1
N
N
N
N
H
H
R2
R2
R2
CH3 84% yield (37)
R2
R1
C7H15
C6H5
R1
C6H5
C6H5
C6H13
40% yield (41)
61% yield (42)
H
CH3
83% yield (38)
H
CH3
C6H5
86% yield (39)
CH3 57% yield (43)
C7H15
C9H19
C9H19
CH3 48% yield (44)
CH3 78% yield (45)
CH3
71% yield (40)
Scheme 1. Reagents and conditions: (a) PPA, 180–200 °C; (b) bromoacetyl bromide, DMAP, THF; (c) diethylamine, NaHCO3, KI, MeOH; (d) (1) TMSCN, KCN, 18-Crown-6, (2)
15% HCl; (e) 1,4-dioxane, HCl, HCl (g); (f) KCN, (NH)4CO3, EtOH, 60–65 °C.
tration of 40
l
M (IC50 of phenytoin). Compounds 41 and 42 repre-
tion in this region, as increasing the chain length to 9 carbons
(37 vs 40) actually results in a less potent compound. Interest-
ingly, in the lidocaine/hydantoin analogue, the series of 6, 7, to
9 carbon chains at R1 showed no difference in potency. This result
appears to support our previous findings that hydantoins within
the same class having similar logP values exhibit similar binding
potencies.7
We determined the effects of compound 37 on sodium currents
recorded from HEK cells stably expressing human Nav1.2. At a con-
centration of 100 lM compound 37 exhibited a 67.7% 5.6 (n = 4)
sent grafts of phenytoin and lidocaine with and without the phenyl
ring methyl groups of lidocaine. It is evident from this data that the
CoMFA model has excellent predictive ability. Residual values (the
difference between the observed IC50 and the predicted IC50) ran-
ged from 0.05 to 0.26 for these novel compounds. As expected,
compounds 37 and 44, which were designed to take advantage of
the steric requirement at C5 with a long alkyl chain demonstrating
the best activity with IC50 values of 6.0 and 13.5
Compounds 41 and 42 showed identical IC50 values of about
39.8 M (predicted = 44.7 and 50.1 M, respectively). It should
be noted that when tested at 40
M in the preliminary [3H]-BTX
lM, respectively.
l
l
block of sodium channel current (Fig. 6) and were fully reversible
on washout. These results demonstrate that the newly designed
compound 37 has significant functional effects on inhibiting so-
dium channel currents.
l
assay, these two compounds showed [3H]-BTX displacement of
50% and 49%, further validating the predictive ability of the CoMFA
model. Compound 42 was designed as a direct graft of the anticon-
vulsant phenytoin and the local anesthetic lidocaine. The sodium
channel binding potency of this analogue was correctly predicted
by the CoMFA model (observed IC50 = 39.8
IC50 of lidocaine in this assay is 240 M and that of phenytoin is
40 M, thus this analogue increases the binding potency of the
lidocaine moiety 6-fold.
lM). Interestingly, the
l
l
In comparing the inhibition of BTX binding to Na+ channels be-
tween the hydroxyamides (37–40) and hydantoins (41–45), it is
apparent that when R1 is a phenyl group, hydantoins are much
more active than the corresponding hydroxyamides. This differ-
ence cannot be solely attributed to lipophilicity as the logP values
for these lidocaine analogues are all within one log unit of one an-
other (1.4–2.5).
100 μM Compound 37
Substitution of a heptyl group for the phenyl group at R1 in the
lidocaine/hydroxyamide analogues (37 vs 39) resulted in a great-
er than 5-fold increase in inhibition (83% vs 16%). Similarly, the
lidocaine/hydantoin analogue showed an increase in inhibition
(49% for R1 = Ph vs 71% for R1 = heptyl) but it was not nearly as
dramatic as that seen in the other analogues. The substitution
of a heptyl group for a phenyl group corresponds to both an in-
crease in size as well as lipophilicity (ꢁ1.5-fold increase in logP).
The heptyl substitution appears to remain the optimal substitu-
2 nA
Washout
500 m s
Control
Figure 6. Sample current traces shows block by compound 37 of the human Nav1.2
sodium channel isoform stably expressed in HEK 293 cells. Currents were elicited
by a step depolarization to +10 mV for 12 ms from a holding potential of À100 mV.
Compound 37 blocked 67.7 5.6% (n = 4) of the recorded current which was
partially reversible upon washout.

Y. Wang et al. / Bioorg. Med. Chem. 17 (2009) 7064–7072
7069
5.2. General procedure for the preparation of lidocaine-
ketones
3. Discussion
Voltage-gated sodium channels are important in many physio-
logical processes and their dysregulation are known to play a key
role in several pathological human diseases.13 In view of this, volt-
age-gated sodium channels remain an attractive target for medic-
inal chemistry. Since the X-ray structure of the voltage-gated
sodium channel remains unsolved, the development of computer
based strategies that contribute to the design of new sodium chan-
nel ligands are critical in our pursuit of targeted inhibitors. The
CoMFA analysis presented in this study provides such a model
and allowed us to encompass a range of new structures. The pre-
dictive sodium channel binding affinities for four novel compounds
not present in the training set and not previously reported pro-
vided a focused rationale for targeted synthesis. The novel syn-
thetic compounds were evaluated for direct displacement of a
sodium channel specific radioligand and function block of cellular
sodium channel currents was confirmed for one lead. Both the 3H-
BTX and the electrophysiology confirms our hypothesis for target-
ing site 2 of the sodium channel and our study provides validation
of this technique with functional sodium channel block. Finally,
this study expands our understanding concerning the binding re-
gion encompassing both the local anesthetic and anticonvulsant
binding sites within site 2 of the sodium channel.
To a solution of bromoacetyl bromide (1.5 equiv) in THF at 0 °C
was added dropwise, under stirring and nitrogen atmosphere, a
solution of R–NH2 (1.0 equiv) and 4-(dimethylamino)pyridine
(0.5 equiv) in THF. The reaction mixture was stirred at 0 °C for
1.5 h, then at room temperature for 1 h. The reaction was
quenched with water and extracted with CH2Cl2, dried over MgSO4,
filtered, and concentrated by rotary evaporator. The crude product
was purified by silica gel column chromatography, eluting with
EtOAc/hexane.
A
solution of above product (1.0 equiv), diethylamine
(1.5 equiv), sodium bicarbonate (1.5 equiv), and potassium iodide
(catalytic amount) in methanol was stirred at 50 °C for 4 h. The
reaction mixture was cooled to room temperature and diluted with
ethyl acetate and water, then the mixture was extracted with
CH2Cl2, dried over MgSO4, filtered, and concentrated. The residue
was purified by silica gel column chromatography, eluting with
EtOAc/hexane.
5.3. General procedure for the preparation of
(37–40)
a-hydroxyamides
To a solution of the ketone (1.0 equiv) in CH2Cl2 were added
KCN and 18-Crown-6 (10 mg per mol of ketone) and trim-
ethylsilylcyanide (2.5 equiv) by syringe under nitrogen atmo-
sphere. The reaction mixture was stirred for 24 h at 25 °C. After
24 h. the solvent was removed in vacuo.
To the residue was added 1,4-dioxane (5 ml per mmol of ke-
tone) and cooled to 0 °C. Previously cooled concentrated HCl
(1 ml per 1 mmol of ketone) was added and HCl gas (from H2SO4
and NH4Cl) was then passed through the reaction mixture for 1 h
at 0 °C. The mixture was allowed to stand at room temperature
overnight then extracted with CH2Cl2. The extracts were dried over
MgSO4, filtered, concentrated, and purified by silica gel column
chromatography, eluting with EtOAc/hexane.
4. Conclusion
One of the most challenging facets of ion channel drug discov-
ery is the design of new classes of small molecule leads with appro-
priate potency without the direct assistance of 3D protein X-ray
structures. In this study we have used ligand-based drug design
through CoMFA and validated the model by synthesis and evalua-
tion of human sodium channel interactions. This finding continues
to support the use of CoMFA in transmembrane drug discovery
paradigms where little or no target structural data is known.
5. Experimental
5.3.1. 2-[4-(2-Diethylamino-acetylamino)-3,5-dimethyl-
phenyl]-2-hydroxy-nonanoic acid amide (37)
All reaction requiring anhydrous conditions were carried out in
flame-dried glassware under nitrogen atmosphere. Solvents were
purified by pressure filtration through activated alumina. Melting
points were determined in open capillary tubes with an electro-
thermal Mel-temp melting point apparatus and are uncorrected.
1H and 13C NMR spectra were measured on a General Electric
300 MHz spectrometer. Chemical shifts are reported in ppm rela-
tive to resonances of the solvent CDCl3 (unless specified other-
wise): 7.25 ppm in the 1H spectra and 77.08 ppm in the 13C spectra.
Thin-layer chromatography was performed on silica gel 60 F 254
aluminum sheets with UV detection. Chromatography separations
Isolated as a light brown oil. 1H NMR: d 9.20–8.39 (s, 1H), 7.78–
7.23 (s, 1H), 7.04–6.94 (s, 1H), 6.74–6.64 (s, 1H), 6.30–6.20 (s, 1H),
2.65–2.48 (s, 2H), 2.49–2.33 (m, 4H), 1.65–1.49 (m, 2H), 1.91–1.75
(s, 6H), 1.02–0.84 (m, 14H), 0.84–0.71 (t, J = 7.5 Hz, 6H), 0.58–0.46
(t, J = 6 Hz, 3H); 13C NMR: d 177.97, 162.83, 142.56, 134.60, 133.18.
125.66, 78.42, 49.14, 36.59, 32.07, 31.45, 30.08, 29.44, 22.84, 18.93,
14.32, 12.30; HRMS (EI): calcd for C23H39N3O3 405.5809; found,
405.5811.
5.3.2. 2-[4-(2-Diethylamino-acetylamino)-phenyl]-2-hydroxy-
2-phenyl-acetamide (38)
were carried out on silica gel columns (silica gel 60, 40–63 lm).
Isolated as a light brown oil (40%). 1H NMR: d 9.69–8.91 (s, 1H),
7.52–6.47 (m, 9H), 3.03–2.82 (s, 2H), 2.62–2.41 (m, 4H), 1.06–0.86
(t, J = 6 Hz, 6H); 13C NMR: d 176.93, 170.96, 143.78, 139.45, 137.43,
128.87, 128.43, 128.13, 119.34, 70.52, 58.32, 49.31, 12.86; HRMS
(EI): calcd for C20H25N3O3 355.4358: found 355.4357.
Mass spectra were obtained on Thermofinnigan LCQ iontrap
(Classic) instrument. High-resolution mass spectrometry was per-
formed at the University of Illinois at Urbana-Champaign, School of
Chemical Science.
5.1. General procedure for the preparation of 4-
aminobenzophenones
5.3.3. 2-[4-(2-Diethylamino-acetylamono)-3,5-dimethyl-
phenyl]-2-hydroxy-2-phenylacetamide (39)
Isolated as a light brown oil (61%). 1H NMR: d 9.42–8.20 (s, 1H),
7.53–7.39 (m, 2H), 7.39–7.34 (m, 3H), 7.20–7.07 (s, 2H), 6.63–6.35
(s, 1H), 6.08–5.80 (s, 1H), 3.29–2.97 (s, 2H), 2.80–2.54 (m, 4H),
2.23–2.06 (s, 6H), 1.25–1.00 (t, J = 6 Hz, 6H); 13C NMR: d 176.8,
171.18, 143.15, 142.14, 135.45, 134.19, 128.61, 128.36, 128.03,
127.99, 81.47, 57.79, 49.44, 19.22, 13.11; HRMS (EI): calcd for
C22H29N3O3 383.4906; found 383.4905.
A mixture of 2,6-dimethylaniline (1.0 equiv), RCOOH (2.0 equiv)
in excess PPA was heated with vigorous stirring at 180–200 °C. for
1 h. Then, 2N hydrochloric acid and water was added and stirred
for an additional hour. The reaction was cooled to 25 °C and ex-
tracted with CH2Cl2, dried over MgSO4, filtered, and concentrated.
The residue was purified by silica gel column chromatography,
eluting with EtOAc/hexane.

7070
Y. Wang et al. / Bioorg. Med. Chem. 17 (2009) 7064–7072
5.3.4. 2-[4-(2-Diethylamono-acetylamono)-3,5-dimethyl-
phenyl]-2-hydroxy-undecanoic acid amide (40)
2H), 3.33–3.02 (t, J = 9 Hz, 2H), 2.80–2.48 (m, 4H), 2.33–2.09 (s,
6H), 1.87–1.83 (m, 2H), 1.36–1.13 (m, 14H), 1.19–1.10 (t,
J = 7.5 Hz, 6H), 0.92–0.68 (t, J = 6 Hz, 3H); 13C NMR: d 176.58,
171.57, 164.61, 137.39, 135.83, 134.27, 125.66, 68.88, 60.78,
57.50, 53.90, 50.47, 49.30, 32.24, 31.97, 29.90, 29.74, 23.04,
19.10, 14.52, 12.76; HRMS (EI): calcd for C26H42N4O3 458.6445;
found, 458.6444.
Isolated as a light yellow oil 71%). 1H NMR: d 9.21–8.41 (s, 1H),
7.55–6.92 (s, 2H), 6.99–6.41 (s, 1H), 6.32–5.89 (s, 1H), 3.56–3.30 (s,
2H), 3.16–2.90 (t, J = 9 Hz, 2H), 2.68–2.42 (m, 4H), 2.19–1.99 (s,
6H), 1.35–1.10 (m, 14H), 1.09–1.049 (t, J = 6 Hz, 6H), 0.89–0.63 (t,
J = 6 Hz, 3H); 13C NMR: d 178.01, 170.93, 142.48, 134.92, 133.42,
125.94, 78.61, 57.83, 49.38, 39.58, 32.31, 32.20, 30.36, 30.07,
29.97, 29.74, 23.07, 19.16, 14.52, 13.04. HRMS (EI): calcd for
C25H43N3O3 433.6352; found, 433.6353.
5.5. Molecular modeling conformational analysis
The X-ray coordinates for phenytoin (DPH) were utilized in this
study.14 The conformations of training set compounds 1–36 (Fig. 2)
were determined as previously described.6 An additional 15 com-
mon local anesthetics were added to this model and modified from
a representative X-ray structure. These modified structures were
energy-minimized with the Tripos force field,15 without solvent,
using default bond distances and angles and neglecting electrostat-
ics. The minimization was completed by aggregating using the
SYBYL/AGGREGATE module for only the X-ray structure atoms
and allowing the modified portion to minimize. For internal consis-
tency, we used only the R-configuration for all chiral compounds.
The test set analogues 37–45 (Table 3) were modified in SYBYL
using the X-ray conformation of lidocaine and representative
low-energy conformations were obtained using the Tripos force
field. To determine the low-energy conformations for 37–45, we
utilized GRIDSEARCH on rotatable bonds over 360° in 1° incre-
ments. The atomic charges for all analogues were calculated using
AM1 (MOPAC).
5.4. Generalprocedure for the preparation ofhydantoins (41–45)
To a stirring solution of the ketone (1.0 equiv) in 50% ethanol
were added KCN (2.0 equiv) and ammonium carbonate (4.0 equiv).
The reaction mixture was stirred at 60–65 °C for more than 24 h
(using TLC to monitor reaction progress). When the reaction was
completed, the reaction mixture was cooled to room temperature
and extracted with CH2Cl2. The organic extracts were dried over
MgSO4, filtered, concentrated, and purified by silica gel column
chromatography, eluting with EtOAc/hexane.
5.4.1. 2-Diethylamino-N-[4-(2,5-dioxo-4-phenyl-imidazolidin-
4-yl)-phenyl]-acetamide (41)
Isolated as a white solid. mp 253–244 °C; 1H NMR: d 9.74–9.17 (s,
1H), 8.62–8.47 (s, 1H), 8.10–7.96 (s, 1H), 7.56–7.17 (m, 9H), 3.29–
2.93 (s, 1H), 2.81–2.40 (m, 4H), 1.22–0.81 (t, J = 6 Hz, 6H); 13C NMR
(CDCl3): d 175.47, 171.34, 164.91, 139.63, 138.02, 135.32, 129.17,
128.86, 128.22, 127.34, 120.11, 71.72, 58.37, 49.35, 12.86; HRMS
(EI): calcd for C21H24N4O3 380.4466; found, 380.4465.
5.6. Molecular alignment
5.4.2. 2-Diethylamino-N-[4-(2,5-dioxo-4-phenyl-imidazolidin-
4-yl)-2,6-dimethyl-phenyl]-acetamide (42)
Benzocaine, lidocaine, butacaine, cocaine, hexylcaine, pipero-
caine, parethoxycaine, and piperocaine utilized in the training set
were fit to overlap the C1 and C4 carbons of the phenyl ring and
the adjacent carbon or atom to C1. All hydantoins were fit as pre-
viously reported.6 Similarly, for analogues 41–45 in the test set, we
aligned the C1 and C4 carbons of the phenyl ring and the adjacent
nitrogen and the hydantoin ring was fit as previously described.6
The hydroxyamides 37–40 were aligned such that the OH group
was superimposed with N1 and the carboxyamide was aligned
with C4 and N3. The n-alkyl groups at R1 in analogues 37, 40,
and 43–45 in the test set approximated a fully extended conforma-
tion following energy minimization, which was arbitrarily selected
for this study.
Isolated as a white solid, (61%); mp 164–167 °C; 1H NMR: d
10.46–9.82 (s, 1H), 9.56–8.76 (s, 2H), 7.70–6.47 (m, 7H) 3.42–3.04
(s, 2H), 2.90–2.52 (m, 4H), 2.53–2.16 (s, 6H), 1.29–0.92 (t,
J = 7.5 Hz, 6H); 13C NMR (CDCl3):
d 174.39, 173.13, 157.58,
141.43, 140.1, 128.55, 128.45, 128.04, 126.73, 126.63,70.86,
57.44, 49.60, 19.35, 13.28; HRMS (EI): calcd for C23H28N4O3
408.5003: found 408.5003.
5.4.3. 2-Diethylamino-N-[4-(4-hexyl-2,5-dioxo-imidazolidin-4-
yl)-2,6-dimethyl-phenyl]-acetamide (43)
Isolated as a white solid (57%); mp 107–110 °C; 1H NMR: d9.85–
10.00 (b, 1H), 9.24–8.63 (s, 1H), 7.90–8.00 (b, 1H), 7.54–6.86 (s,
2H), 3.35–3.13 (s, 2H), 2.78–2.53 (m, 4H), 2.31–2.11 (s, 6H),
1.37–1.29 (m, 10H), 1.28–1.14 (t, J = 9.6 Hz), 0.94–0.67 (t,
J = 6 Hz, 3H); 13C NMR: d 176.48, 171.74, 158.08, 135.91, 133.99,
125.96, 125.42, 68.79, 57.67, 49.47, 31.95, 29.49, 23.09, 23.00,
19.20, 19.07, 14.62, 14.20; HRMS (EI): calcd for C23H36N4O3
416.5636; found, 416.5633.
5.7. CoMFA calculations
CoMFA, using default parameters except where noted, was cal-
culated in the QSAR option of SYBYL 6.8 on a Silicon Graphics with
an Octane II R12000 dual processor. The CoMFA grid spacing was
2.0 Å in the x, y, and z directions, and the grid region was automat-
ically generated by the CoMFA routine to encompass all molecules
with an extension of 4.0 Å in each direction. An sp3 carbon and a
charge of +1.0 were used as probes to generate the interaction
energies at each lattice point. The default value of 30 kcal/mol
was used as the maximum electrostatic and steric energy cutoff.
5.4.4. 2-Diethylamino-N-[4-(4-heptyl-2,5-dioxo-imidazolidin-
4-yl)-2,6-dimethyl-phenyl]-acetamide (44)
Isolated as a light yellow solid (48%); mp 97–100 °C; 1H NMR: d
9.36–8.57 (s, 1H), 7.60–6.80 (s, 2H), 3.35–3.07 (s, 2H), 2.82–2.54
(m, 4H), 2.35–2.07 (s, 6H), 2.03–1.75 (m, 2H), 1.37.1.14 (m, 10H),
1.13–0.97 (t, J = 7.5 Hz, 6H), 0.95–0.67 (t, J = 6 Hz, 3H); 13C NMR:
d 176.44, 164.51, 157.93, 136.04, 136.01, 125.88, 125.77, 68.86,
57.72, 49.51, 32.23, 29.84, 29.48, 24.17, 23.06, 19.24, 14.54,
13.15; HRMS (EI): calcd for C24H38N4O3 430.5907; found, 430.5905.
5.8. Partial least squares (PLS) regression analysis
A single conformer for each compound was selected by the
smallest cross-validated residual value. Single conformers of each
training set compound were used in both the non-cross-validated
model (Table 1) and the cross-validated model (Table 2). We also
used the final non-cross-validated CoMFA model to predict the so-
dium channel binding activities for all low-energy conformations
of the test set compounds 37–45 (Table 3).
5.4.5. 2-Diethylamino-N-[2.6-dimethyl–4-(4-nonyl-2,5-dioxo-
imidazolidin-4-yl)-phenyl]-acetamide (45)
Isolated as a white solid (78%); mp 95–98 °C; 1H NMR: d 9.23–
8.79 (s, 1H), 8.11–7.86 (s, 1H), 7.81–7.47 (s, 1H), 7.31–6.98 (s,

Y. Wang et al. / Bioorg. Med. Chem. 17 (2009) 7064–7072
7071
Table 2
Cross-validated PLS analysis
5.10. Biological data
The structures and literature reference [3H]-BTX displacement
data for compounds 1–20, the 15 local anesthetics (21–35), and
phenytoin (36) that form the training set, are listed in Figure 2
and Table 1. Table 3 describes the structures and sodium channel
binding activities of compounds 37–45, which form the test set.
Components
s
q2
1
2
3
4
5
6
0.746
0.663
0.654
0.644
0.691
0.674
0.242
0.418
0.452
0.485
0.426
0.473
5.11. [3H]-BTX-B experiment
s, standard error for the estimate of logIC50; q2, correlation coefficient or press
value = 1.0 – {
R
(Y predicted – Y actual)2 Ä (Y actual – Y mean)2}. Optimum number
In the sodium channel evaluation, the IC50, which represents the
micromolar concentration of compound required to displace 50%
of specifically bound [3H]-BTX) was determined using rat cerebral
cortex synaptoneurosomes. To calculate the IC50 value, the percent
inhibition at 5–7 concentrations, which spanned the IC50 value,
was determined in triplicate and the IC50 value was obtained by
a Probit analysis of the concentration versus percent binding curve.
Briefly, rat forebrain membranes were incubated with [3H]-
Batrachotoxin (30–60 Ci/mmol). Reactions are carried out in
50 mM HEPES (pH 7.4) containing 130 mM choline chloride at
37 °C for 60 min. The reaction was terminated by rapid vacuum fil-
tration of the reaction contents onto glass fiber filters. Radioactiv-
ity trapped onto the filters was determined and compared to
control values in order to ascertain any interactions of the test
compound with the sodium channel site 2 binding site. Aconitine
of components is 4 with q2 = 0.485.
Cross-validated (preliminary and final models) and non-cross-
validated PLS analyses (final model) were performed within the
SYBYL/QSAR routine. Cross-validation of the dependent column
(pIC50) and the CoMFA column was performed with 2.0 kcal/mol
column filtering. Scaled by the CoMFA standard deviation, the final
cross-validated analysis generated an optimum number of compo-
nents equal to 4 and q2 = 0.485 (Table 2). PLS analysis with non-
cross-validation, performed with 4 components, gave a standard
error of estimate of 0.245, a probability of R2 = 0 (n1 = 4, n2 = 31)
equal to 0.000, an F value (n1 = 4, n2 = 31) of 96, and a final
q2 = 0.926. The relative steric (0.624) and electrostatic (0.376) con-
tributions to the final model were contoured as the standard devi-
ation multiplied by the coefficient at 80% for favored steric
(contoured in green) and favored positive electrostatic (contoured
in blue) effects and at 20% for disfavored steric (contoured in yel-
low) and favored negative electrostatic (contoured in red) effects,
as shown in Figure 5. On the basis of this analysis, the sodium
channel binding activities for low-energy conformers of analogues
37–45, from the test set (Table 3), were predicted.
[1 lM] was used as a positive control.
5.12. Cell culture and electrophysiology
Human embryonic cells (HEK) cells stably expressing human
Nav1.2 were grown in DMEM/F12 media (Invitrogen, Corp., CA,
USA) supplemented with 10% fetal bovine serum, penicillin
(100 U/ml), streptomycin (100 lg/ml) and G418 (500 lg/ml; Sig-
ma, MO, USA). Cells were grown in a humidified atmosphere of
5.9. Chemistry
5% CO2 and 95% air at 37 °C.
Sodium currents were recorded using the whole-cell configura-
tion of the patch clamp recording technique with an Axopatch
200B amplifier (Axon Instruments, Foster City, CA). All voltage pro-
tocols were applied using pCLAMP 8 software (Axon, USA) and a
Digidata 1322A (Axon, USA). Currents were amplified and low pass
filtered (2 kHz) and sampled at 33 kHz. Borosilicate glass pipettes
were pulled using a Brown–Flaming puller (model P87, Sutter
The synthesis of compounds 37–45 is shown in Scheme 1 and
detailed in the experimental section. Briefly, the aminobenz-
ophenones were generated by reaction of substituted anilines
and acids in polyphosphoric acid (PPA). Subsequent reaction
with diethylamine, NaHCO3, and KI in MeOH generated the lido-
caine-ketones which were carried on to final product amides and
hydantoins.
Table 3
Predicted and actual sodium channel binding activities for novel grafted analogues forming the test set
O
O
NH
HO
NH
2
HN
O
R
2
R
2
O
R
1
O
R
1
N
N
N
H
N
H
R
2
R
2
37-40
41-45
Compound
R1
R2
LogPa
[3H]-BTX Inhibition at 40
l
M (%)
ÀpIC50
Predb
Obsd
Res
37
38
39
40
41
42
43
44
45
n-Heptyl
Phenyl
Phenyl
n-Nonyl
Phenyl
Phenyl
n-Hexyl
n-Heptyl
n-Nonyl
Methyl
H
Methyl
Methyl
H
Methyl
Methyl
Methyl
Methyl
3.6
1.4
2.4
4.4
1.6
2.5
3.3
3.7
4.5
83 1.53
À0.78
nd
nd
À0.85
À1.82
À1.74
À0.51
À1.65
À1.70
À1.09
À0.87
À0.59
À0.07
4
4.56
16 5.23
63 1.01
50 0.92
49 2.72
68 4.26
71 0.30
69 0.89
nd
À1.60
À1.60
nd
À0.05
À0.10
À1.13
À0.26
nd
a
Calculated using the chemical properties tool of ChemDraw (CambridgeSoft, Inc.).
Data generated from the CoMFA non-cross-validated run (see QSAR methods).
b

7072
Y. Wang et al. / Bioorg. Med. Chem. 17 (2009) 7064–7072
Instruments Co., Novato, CA) and heat polished to produce elec-
trode resistances of 0.5–1.5 M when filled with the following
References and notes
X
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electrode solution (in mM); CsCl 130, MgCl2 1, MgATP 5, BAPTA
10, HEPES 5 (pH adjusted to 7.4 with CsOH). Cells were plated on
glass coverslips and superfused with solution containing the fol-
lowing composition; (in mM) NaCl 130, KCl 4, CaCl2 1, MgCl2 5,
HEPES 5, and glucose 5 (pH adjusted to 7.4 with NaOH).
Compounds were prepared as 100 mM stock solutions in Dim-
ethly sulfoxide (DMSO) and diluted to desired concentration in per-
fusion solution. The maximum DMSO concentration used was 0.3%
and had no effect on current amplitude. All experiments were per-
formed at room temperature (20–22 °C). After establishing whole-
cell, a minimum series resistance compensation of 75% was applied.
10. Yarov-Yarovoy, V.; Brown, J.; Sharp, E. M.; Clare, J. J.; Scheuer, T.; Catterall, W.
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We thank the NIH R01 NIH CA105435-0, Jeffress Trust Fund and
the Paul Mellon Fund for financial support. We appreciate the tech-
nical support provided on the [3H]-BTX-B studies from Novascreen,
Inc., (Tammy Ranson and Dr. Charles Bauer).