Discovery of the First Vitamin K Analogue as a Potential Treatment of Pharmacoresistant Seizures

Article abstract of DOI:10.1021/acs.jmedchem.0c00168

Despite the availability of more than 25 antiseizure drugs on the market, approximately 30% of patients with epilepsy still suffer from seizures. Thus, the epilepsy therapy market has a great need for a breakthrough drug that will aid pharmacoresistant patients. In our previous study, we discovered a vitamin K analogue, 2h, which displayed modest antiseizure activity in zebrafish and mouse seizure models. However, there are limitations to this compound due to its pharmacokinetic profile. In this study, we develop a new series of vitamin K analogues by modifying the structure of 2h. Among these, compound 3d shows full protection in a rodent pharmacoresistant seizure model with limited rotarod motor toxicity and favorable pharmacokinetic properties. Furthermore, the brain/plasma concentration ratio of 3d indicates its excellent permeability into the brain. The resulting data shows that 3d can be further developed as a potential antiseizure drug in the clinic.

Full text of DOI:10.1021/acs.jmedchem.0c00168

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Article
Discovery of the first Vitamin K analog as a
potential treatment of pharmacoresistant seizures
Xiaoyang Li, Richard A. Himes, Lyndsey C. Prosser, Charleston F. Christie, Emma Watt, Sharon
F. Edwards, Cameron Metcalf, Peter West, Karen Wilcox, Sherine S. L. Chan, and C. James Chou
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00168 • Publication Date (Web): 11 May 2020
Downloaded from pubs.acs.org on May 13, 2020
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Discovery of the first Vitamin K analog as a
potential treatment of pharmacoresistant
seizures
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Xiaoyang Li *, Richard A. Himes , Lyndsey C. Prosser , Charleston F. Christie ,
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Emma Watt , Sharon F. Edwards , Cameron S. Metcalf , Peter J. West , Karen S.
Wilcox , Sherine S.L. Chan *, C. James Chou, 3,.5,*
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3,5,
1Ocean University of China, School of Medicine and Pharmacy, Qingdao, Shandong,
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66071, China
2Department of Chemistry and Biochemistry, College of Charleston, 66 George
Street, Charleston, South Carolina 29424, USA
3Neuroene Therapeutics, Mount Pleasant, South Carolina 29464, USA
4Anticonvulsant Drug Development (ADD) Program, Department of Pharmacology &
Toxicology, University of Utah, 84112, USA
5Department of Drug Discovery and Biomedical Sciences, College of Pharmacy,
Medical University of South Carolina, Charleston, South Carolina 29425, USA
Abstract
Despite the availability of more than 25 anti-seizure drugs on the market,
approximately 30% of patients with epilepsy still suffer from seizures. Thus, the
epilepsy therapy market has a great need for a breakthrough drug that will aid
pharmacoresistant patients. In our previous study, we discovered a vitamin K analog,
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2h, which displayed modest anti-seizure activity in zebrafish and mouse seizure models.
However, there were limitations for this compound due to its pharmacokinetic profile.
In this study, we developed a new series of vitamin K analogs by modifying the
structure of 2h. Among these, compound 3d shows full protection in a rodent
pharmacoresistant seizure model with limited rotarod motor toxicity and favorable PK
properties. Furthermore, the brain/plasma concentration ratio of 3d indicates its
excellent permeability to the brain. The resulting data shows 3d can be further
developed as a potential anti-seizure drug in the clinic.
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Introduction
Epilepsy is one of the most common neurological disorders and affects an
1
estimated 70 million people worldwide. Many people with epilepsy have more than
one type of seizure and may have additional neurological issues. Anti-seizure drugs
(ASDs) are the mainstay treatment for epilepsy, effectively controlling seizures for
around 70% patients with epilepsy. Driven by the limited efficacy and toxicity of the
established anti-seizure agents, development of ASD has exploded in the past quarter-
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century, with more than 25 ASDs on the market. Nevertheless, around 30% of patients
have inadequate control of their seizures, a percentage unchanged since 1881.3-4
Currently, there are two main challenges for the treatment of epilepsy; the first and the
most important one is drug resistance, or pharmacoresistance, which refers to patients
who do not achieve adequate seizure control after trials of at least two appropriate ASDs.
Clinically, there are limited therapeutic intervention for pharmacoresistant epilepsy, but
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only for patients with disabling focal epilepsy. The second major concern for ASDs is
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tolerability, as people with epilepsy often require lifelong drug treatment. However,
none of the ASDs currently on the market are free from side effects. In fact, these side-
effects have been found to harm the quality of life of epilepsy patients more than the
seizures themselves, and can lead to low adherence and discontinuation of treatment.6
New third-generation ASDs are marketable due to lowered toxicity and adverse drug
interactions; however, they are only marginally (if at all) more efficacious than older
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drugs. Thus, the epilepsy therapy market is in great need of a breakthrough drug that
would aid medication-resistant patients and be better tolerated.8-9 This leaves room for
new drugs and drug combinations to gain market share quickly.
Pentylenetetrazol (PTZ), a chemical convulsant, induces seizures in various animal
models of epilepsy, including worms, flies, frogs, zebrafish and mice,10-11 among which,
the zebrafish PTZ seizure model is widely used for the initial screen of ASDs, since it
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matches well with rodent models and is amenable to higher-throughput screening. As
epilepsy comes in many forms, it is also prudent that efficacy of the leading candidate
is tested in a wide range of rodent models, particularly in animal models of medication-
resistant epilepsy.7, 13 For over 40 years, the National Institute of Neurological
Disorders and Stroke (NINDS) has supported the Epilepsy Therapy Screening Program
(ETSP), which plays a key role in the identification of novel anti-seizure agents to
address the unmet needs in epilepsy. The current workflow focuses on
pharmacoresistant
and
etiologically
relevant
models
(
https://panache.ninds.nih.gov/CurrentModels.aspx).14-15 Here in this article, our
compounds were screened for acute seizure models including the mouse 6 Hz seizure,
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the maximal electroshock (MES) seizure, and subcutaneous PTZ seizure threshold (s.c.
PTZ) models. The 6 Hz models are believed to mimic partial seizures observed in
humans which are used to screen compound’s ability to block psychomotor seizures.
This model exhibits pharmacoresistance when the stimulation intensity is increased
from 22 to 44 mA.16-17 The 6 Hz 44 mA model is resistant to ASDs that target voltage-
gated ion channels. Levetiracetam and valproic acid, while still effective in this model,
they are significantly less potent at this stimulation intensity.18-19 Therefore, the 6 Hz
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4 mA model provides a high fidelity threshold assay early in the drug identification
process. MES is a model for generalized tonic-clonic seizures and used for screening a
compound’s ability to prevent seizure spread when all neuronal circuits in the brain are
maximally active. These seizures are highly reproducible and are electrophysiologically
consistent with human seizures (https://panache.ninds.nih.gov/CurrentModels.aspx).
The mouse 6 Hz and MES seizure models are included in the current ETSP workflow
for pharmacoresistant epilepsy, and the s.c. PTZ-induced seizure model detects the test
compound’s ability to protect animals from exhibiting clonic, forebrain seizures.
In our previous study, we discovered compound 2h, which was effective in the 6
Hz 32 mA mouse seizure model, but had limitations in the pharmacokinetic (PK) profile
due to short t and limited central nervous system exposure (Figure 1). Moreover, 2h
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did not show any protection in mice when given the higher 44 mA stimulation intensity
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in the 6 Hz model. Therefore, in this study, we modify the structure of 2h to develop
a new series of Vitamin K (VK) analogs. To accelerate the discovery process, a two-
tier whole organism screening process was first set up using the zebrafish PTZ model,
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which is amenable to higher-throughput examination to establish structure activity
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relationship that correlates well with higher animal models. After establishing the
structure-activity relationship (SAR) in the zebrafish model, top candidates were
examined for in vivo anti-seizure activity and PK profiles in mice. Compound 3d shows
titratable full protection in the 6 Hz seizure model at 32 and 44 mA, with limited motor
toxicity and favorable PK properties, making 3d a leading ASD candidate.
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Figure 1. Evolution of a new VK analogs with excellent seizure protection and PK
profile. VK3 and Analog 2h data are previously published.20
Chemistry
Scheme 1 shows the synthesis of 3a-3w. 2-bromonaphthalene-1,4-dione (1) was treated
with various fatty amines (3a-3n), diamines (3o-3s) and alcohol amine (3t-3w) to yield
desired compounds. 2-methylnaphthalene-1,4-dione (4) was reacted with 3,3-
dimethylbutan-1-amine or 3-methylbutan-1-amine to obtain 5a or 5b, respectively
(Scheme 2).
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Scheme 1
O
O
O
H
Br
N
R
R
a
H N
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3a-3w
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O
3
a: R
b: R
3f: R
3g: R
3h: R
3k: R
l: R
3o: R
N
N
3t: R
3u: R
3v: R
OH
3
3
3p: R
3q: R
OH
OH
3
c: R
d: R
3e: R
N
N
3m: R
3
3i: R
3j: R
3r: R
3s: R
3w: R
OH
3
n: R
N
Reagent and condition: (a), different saturated and unsaturated fatty amines, ethanol,
room temperature, overnight, 50-65% yield.
Scheme 2
O
O
O
O
H
N
R
R
a
H N
2
4
2
5a, 5b
5
a: R
5b: R
Reagent and condition: (a), 3-methylbutan-1-amine or 3,3-dimethylbutan-1-amine,
methanol and dichloromethane, room temperature, overnight, 58-65% yield.
Results and Discussion
Discovery of A New VK Analog with Excellent Seizure Protection and PK Profile
In our previous study, we tested VK1, VK2, and VK3, and found that the smallest
VK, VK3, was protective and could reduce PTZ-induced locomotor activity in
zebrafish. However, the lethal dose of VK3 in zebrafish is 7 μM, which is similar to its
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effective does (3-6 μM), therefore, VK3 shows significant toxicity.20 Further
modification achieved VK analog 2h, which exhibited clear anti-seizure activity in
zebrafish at the concentration of 20 μM with no observable toxicity. In 6 Hz 32 mA
mouse models, 2h displayed full protection at 15 min; however, further testing in the
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higher intensity 6 Hz 44 mA model showed that 2h was no longer protective. The
short half-life (1.06 h) of 2h correlates to its acute but short protective duration in the
mouse seizure model (effective within 15 min but the protection drops off sharply in
less than a half-hour). While the compound is stable, displaying no catabolic conversion
even after two hours in mouse serum, its plasma concentration rapidly diminishes in
vivo, suggesting either rapid hepatic metabolism or diminished renal reuptake. This
finding is not surprising, as alkynes are known to be oxidized and excreted rapidly by
the liver and kidney; therefore, we modified alkynes of 2h to produce a new series of
VK analogs, and at the same time, also modified 2-position (R group) (Figure 1). In
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detail, substitution of alkynes with aliphatic chains yields compounds 3a-3h, while with
cyclic aliphatic group yields 3i-3j. The compounds 3o-3s and 3t-3u were the structures
containing tertiary amine and oxhydryl, respectively. Substitution of hydrogen of 3b
and 3d with methyl yields 5a and 5b, respectively (Scheme 1).
Anti-seizure Activity of VK Analogs in Zebrafish
Previous studies showed that zebrafish displays analogous electrophysiological
and molecular features associated with mammalian seizure models when exposed to the
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convulsant agent PTZ. The level of seizure severity in the brains of zebrafish, as
determined by field potential measurements, corresponds with increased swimming
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activity, which is measured as distance traveled. This zebrafish assay has been used to
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screen greater than 500,000 mutagenized fish for seizure-resistant genes, and was
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further validated against rodent models of epilepsy with 13 current ASDs. Therefore,
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we initially used the zebrafish PTZ-induced seizure model to screen our compounds.
Zebrafish larvae (7 days post-fertilization, dpf) were treated with synthesized
candidates at concentrations of 10 μM and 5 μM for 1 h prior to seizure induction with
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5 mM PTZ, 5 mM and 10 mM VPA were used as positive controls. Total swimming
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distance in 15 min was recorded after PTZ-induced seizures. PTZ treatment induced
a 7-fold increase in the total distance traveled compared to the average control zebrafish
(Figure 2a and 2b). The level of seizure protection by compounds at 10 μM are shown
in Figure 2a. Previous lead compound 2h did not significantly reduce distance traveled
at the lower 10 M concentration; which we had previously observed seizure protection
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at 20 μM in our earlier study. Among compounds with aliphatic chains (3a-3h), the
compounds 3b, 3d, 3e, 3f, 3g and 3h protect against PTZ-induced seizures by reducing
the total distance traveled by more than 50%. It should be noted that 10 μM 3d, 3f and
3l display better activity than 10 mM VPA, which was used as positive control.
Compound 3b with substitution of 2,2-dimethylbutyl shows better suppressive activity
than 3a with one carbon shorter and 3c with one carbon longer, which indicates 2
carbons is the optimal length. This is also consistent with the activity of 3d and 3e.
Within compounds bearing cyclic aliphatic group, 3l and 3m, with cyclobutyl and
cyclopentyl respectively, reduce distance traveled greater than 50% as compared to
zebrafish treated with PTZ only. The activity difference between 3k and 3l is also
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consistent with the conclusion drawn from 3a-3c. Unfortunately, compounds with
tertiary amine (3o-3s), oxhydryl (3t-3w) as well as compounds 5a and 5b with methyl
group in 2-position show no protection against PTZ-induced seizures. Among all of the
tested compounds, 3d and 3l display the best activity in zebrafish, by reducing the
distance traveled by 77.4% and 89.0%, respectively. 3d and 3l also show the best
activity at 5 μM (Figure 2b, Table 1). The mean distance traveled per 1 min of the
most active compounds, 3d and 3l, is also shown in Figure 2c, from which we can
observe that these two compounds significantly reduced distance traveled in every
minute. The SAR is summarized in Figure 3.
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Figure 2. (a) and (b) Total mean distance traveled over 15 min. Synthesized compounds
were tested at 10 μM and 5 μM, respectively. 5 mM and 10 mM VPA were used as
positive controls. Data represents the mean distance traveled, error bar represents SEM,
n = 8 for each group. The red color shows > 50% percentage of reduced travel distance
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9
0
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0
(c) Mean distance traveled per 1 min after treatment of 3d and 3l, data are shown as
mean distance traveled, error bar represents SEM, n = 8 for each group. With the
exception of 3d at 10 M + PTZ, all time points are significantly different from the
positive PTZ run p < 0.01. TW = tank water only control.
Table 1. Reduction of PTZ-induced distance traveled after treatment of designed
compounds at the concentration of 10 μM and 5 μM.
O
H
N
R
R1
O
Percentage of reduced travel
Compounds
distance (%)a
R
R1
No.
1
0 μM
5 μM
2
h
H
H
H
H
H
H
H
H
5.4 ± 0.37
18.4 ± 2.30
59.2 ± 5.52
43.5 ± 4.81
77.4 ± 8.97
59.1 ± 6.22
75.8 ± 8.66
52.9 ± 11.10
7.4 ± 0.58
3
a
21.2 ± 2.53
43.4 ± 3.30
-3.0 ± 0.17
51.5 ± 6.79
41.6± 3.20
29.6 ± 2.97
23.0 ± 2.01
3
b
3c
3
d
3
e
3
f
3
g
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5
5
5
5
5
5
6
3h
H
H
H
H
H
H
H
H
H
H
H
65.6 ± 8.83
-4.4 ± 0.26
-10.1 ± 0.79
31.7 ± 2.38
11.4 ± 0.73
70.4 ± 10.50
45.0 ± 4.75
22.2 ± 2.58
-7.2 ± 0.38
-23.9 ± 1.08
-32.4 ± 2.01
-13.4 ± 1.13
3
i
-10.1 ± 0.79
41.6 ± 4.84
16.1 ± 1.14
89.0 ± 11.34
57.4 ± 6.29
24.1 ± 2.47
27.0 ± 1.93
-14.3 ± 1.18
19.1 ± 2.40
-2.90 ± 0.15
3
j
0
1
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8
9
0
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9
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9
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9
0
1
2
3
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8
9
0
3k
3
l
3m
3
n
N
N
3
o
3p
N
3
q
3r
N
N
3
s
H
29.1 ± 2.65
7.3 ± 0.69
3
t
H
18.2 ± 2.64
8.2 ± 0.90
17.4 ± 1.90
-5.6 ± 0.34
26.9 ± 2.55
28.3 ± 3.88
0.78 ± 0.04
-2.4 ± 0.26
20.4 ± 2.28
9.5 ± 0.46
3.3 ± 0.40
-10.8 ± 1.25
OH
OH
3
u
H
OH
3
v
H
3
w
H
OH
5a
CH3
5
b
CH3
a
Assays were performed using eight zebrafish for each group. Values are the mean of
SEM. n = 8 for each group. The negative number indicates increased distance traveled
±
after treatment of designed compounds.
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0
Figure 3. Structure and activity relationship study of VK analogs.
Toxicity of VK Analogs in HT-22 Neurons
As most epilepsy patients need to use ASDs throughout their lives, safety is one of
the major concerns of ASDs. Therefore, we tested the toxicity of these compounds in
HT-22 neuronal cells (Table 2). At the concentration of 50 μM, compounds 3o-3r with
tertiary amine exhibit the highest toxicity with about 100% growth inhibition. Even
when the concentration was decreased to 10 μM, 3o-3r still inhibit growth by 74.5%-
8
6.2%. It is interesting that when methyl group of N-substituent of 3r was changed to
phenyl (3s), the percentage of inhibition is reduced to 10.25%. Compounds 3o-3s with
oxhydryl groups also show high toxicity at a concentration of 50 μM with growth
inhibition of 59.7%-76.7%, while inhibition drops to 19.4%-38.6% when the
concentration is reduced to 10 μM. Compounds 3i, 3j and 3k with cyclic aliphatic
groups show modest toxicity at 50 μM (54.1%-62.3%) which also decrease at the
concentration of 10 μM. Compounds 3d and 3l, with the highest seizure protection in
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2
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2
2
2
2
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3
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3
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3
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the PTZ-induced zebrafish seizure model, do not display obvious toxicity in HT-22
cells (Table 2).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Toxicity of VK analogs in HT-22-neurons.
Percentage of inhibition (%)a
Compounds No.
5
0 μM
10 μM
3
a
22.42 ± 6.63
16.97 ± 10.55
6.75 ± 1.29
10.78 ± 1.94
11.91 ± 2.21
3.81 ± 5.08
11.86 ± 9.27
28.77 ± 5.90
2.09 ± 1.41
14.81 ± 4.24
14.96 ± 2.48
16.83 ± 4.98
34.69 ± 5.28
33.70 ± 5.31
27.21 ± 0.82
35.37 ± 4.70
0.07 ± 0.03
83.84 ± 0.33
86.21 ± 0.26
82.63 ± 3.28
74.53 ± 0.62
11.53 ± 1.71
19.39 ± 9.48
25.66 ± 2.59
38.61 ± 0.01
25.52 ± 2.66
3
b
3
c
3
d
17.78 ± 1.41
21.34 ± 3.42
15.01 ± 2.87
41.21 ± 2.98
38.26 ± 0.84
54.10 ± 6.32
56.37 ± 0.79
62.34 ± 0.07
36.29 ± 4.49
43.76 ± 2.83
2.09 ± 2.06
3
e
3
f
3g
3h
3i
3
j
3
k
3
l
3
m
3
n
3
o
100.05 ± 0.21
100.96 ± 0.52
99.78 ± 0.16
96.69 ± 0.69
10.25 ± 6.61
64.21 ± 6.55
76.65 ± 3.41
69.02 ± 2.45
59.67 ± 0.11
3
p
3q
3r
3
s
3
t
3
u
3
v
3
w
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5
a
17.54 ± 2.83
21.39 ± 3.72
10.15 ± 1.34
11.24 ± 2.56
5
b
a
Note: Values are the mean ± SEM, three experiments were performed for each data
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(
n = 3).
In Vivo Anti-seizure Activity in Rodent Seizure Models
The 6 Hz limbic seizure test using a stimulus intensity of 44 mA, is an acute model
for pharmacoresistant seizures. Therefore, the most potent compounds 3d and 3l in
zebrafish were tested for their anti-seizure activity in the 6 Hz rodent seizure models.
Initial screening assays were performed using the lower stimulus intensities: 22 mA
and 32 mA. Unfortunately, the most potent compound, 3l, in the PTZ-induced zebrafish
seizure model shows limited efficacy in the 6 Hz mouse seizure model at 22 mA,
without full seizure protection up to 400 mg/kg (Supporting Information Table S1).
Thus, we did not continue testing 3l in the 6 Hz seizure model at 32 mA and 44 mA.
First, a time course study was performed to establish a time to peak seizure protection
of 1 h in 6 Hz models following ip administration (Supporting Information Table S2).
Then, a dose-response quantitation study was performed at the 1 h peak response time
to determine the ED . Compound 3d is highly effective at both 32 mA and 44 mA,
5
0
with full protection at the concentration of 400 mg/kg without obvious motor/sedative
effects. EC values of 3d in the 6 Hz model at 32 mA and 44 mA are 152.7 and 263.7
5
0
mg/kg, respectively (Figure 4a, Table 3). Previously, 2h did not show any protection
2
0
in 6 Hz 44 mA model, therefore, 3d is more potent than both 3l and 2h. This is
consistent with the established pharmacoresistant seizures; most drugs lose seizure
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protection effectiveness with increased mA, thus making this increased 44 mA voltage
intensity a model of pharmacoresistance. Furthermore, only four FDA approved anti-
seizure medications to date (valproic acid, levetiracetam, felbamate and cannabidiol)
have shown activity in this particular model and are all used clinically to treat refractory
or hard-to-control seizures seizures.22-23 We also compared the EC and TD (median
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50
toxic dose, a toxicology term that relates to the median toxic dose of a substance in
which toxicity occurs in 50% of a species) of 3d with these four drugs (Table 4). The
reported EC value of levetiracetam is over 1000 mg/kg in this model and it is one of
5
0
the most commonly prescribed medicine for refractory seizures, compound 3d’s EC50
value is comparable to all other anti-seizure medicines with EC values ranging from
5
0
100 mg/kg to 300 mg/kg. It should be noted that 3d is well tolerated with a TD value
5
0
>
800 mg/kg, which is much higher than the other FDA approved drugs. Then 3d was
further screened in MES model and produced full protection at 350 mg/kg with EC50
value of 108.12 mg/kg (Figure 4b, Table 3). Result shows 3d blocks seizures in the
mouse 6 Hz and MES models in a dose-dependent manner, reaching full seizure
protection at doses that do not produce motor impairment, which indicates its potential
in the treatment of pharmacoresistant seizures. To provide more data of 3d’s efficacy
against clonic seizures, the ED value was also determined in the s.c. PTZ mouse
5
0
model at the 1h peak point. 3d protects 6 out of 8 animals at the concentration of 450
mg/kg with ED value of 349.22 mg/kg (Figure 4c, Table 3). Of note, 3d does not
5
0
produce major motor impairment at the time of seizure testing, as the rotarod assay was
performed immediately prior to 6 Hz stimulation (1 h after administration). However,
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other effects of treatment are noted following administration with 3d that will be the
subject of ongoing investigation. Namely, 3d administration is associated with writhing
(abdominal constriction) both at the time of treatment and during the ~1 h observation
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period that followed. In addition, some animals also show discolored urine and lethargy
following treatment (data not shown). Subsequent studies also reveal that higher doses
of 3d (e.g. greater than 500 mg/kg) are lethal in some animals after electric stimulation
after 24h (data not shown). The mechanisms contributing to the lethality will be the
subject of future studies. Nonetheless, 3d does not show signs of major motor
impairment during the acute (~1h) observation period with a treatment dose up to 800
mg/kg in mice (mouse rotarod assay) (Supporting Information Table S2).
Figure 4. Compound 3d in the acute seizure models, 3d was injected via ip
administration 1 h prior to electrical stimulation or injection of Metrazol. The
percentage of mice protected is shown for each of the four dose levels for 3d (n = 8).
(a) Dose-dependent effect of 3d in mouse 6 Hz 32 mA and 44 mA seizure models. (b)
Dose-dependent effect of 3d in the mouse MES seizure model. (c) Dose-dependent
effect of 3d in the s.c. PTZ seizure model.
Table 3. Effect of 3d in 6 Hz mouse seizure models.
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Time of
test
ED value
95% confidence
interval (mg/kg)
5
0
Seizure model
(mg/kg)
152.7
263.7
108.1
349.2
6 Hz 32mA mouse model
1 h
1 h
1 h
1 h
98.6-211.0
198.9-321.8
70.4-152.9
256.0-432.1
0
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6
Hz 44mA mouse model
MES model
s.c. PTZ mouse model
Table 4. Comparison of 3d and four FDA approved ASDs tested in the 6 Hz 44 mA
seizure model.
6
Hz 44 mA ED50
Compound
TD (mg/kg)
5
0
(mg/kg)
3
d
263.7
289.1
1089.3
97.5
>800
390
Valproic Acid23
Levetiracetam23
Felbamate22
>500
452
Cannabidiol22
173.5
272
Vitamin K Analogs Increase ATP Levels
The exact molecular mechanisms of seizures are unknown and may involve
multiple different factors. The 6 Hz 44 mA seizure model is particularly resistant to
ASDs that target voltage gated sodium and potassium channels. To eliminate that our
inhibitors are targeting these ion channels, recurrent epileptiform discharges (REDs) in
brain slices derived from the kainite-induced status epilepticus model of temporal lobe
2
4
epilepsy were examined in vitro. Compound 3d does not attenuate spontaneous REDs
(
Supporting Information Figure S1), which is consistent with the hypothesis that it has
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novel mechanism of action other than voltage gated sodium and potassium ion channel
inhibitors. Importantly, neurons have high metabolic energy demands, as well as a low
capacity to store ATP. A sudden change in ATP levels in neurons can impair Na/K
ATPase activity and decrease neuronal membrane potential, which can give rise to an
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increase in neuronal excitability resulting in seizures. In addition, heightened neuronal
excitability impairs calcium sequestration resulting in an increased glutamate release
2
5
into synaptic clefts, which can result in neuronal injury. The ketogenic diet and
valproic acid (VPA) are commonly used in combination for relapse seizures and the
mechanism of action is hypothesized to be involved in modulating mitochondrial
activity.26-29 In the 12 marked ASDs tested in the pharmacoresistant seizure models,
only VPA and levetiracetam displayed protection and both have mitochondrial
3
0
effects. VK is also known to play a role in the mitochondrial electron transfer chain
and is capable of maintaining mitochondrial homeostasis. What’s more, in our previous
study, VK3 and its analog 2h can increase ATP levels in HT-22 cells at the
2
0
concentration of 5 μM. Hence, effects of 3d and 3l on ATP levels were determined
for both zebrafish and neuronal cells (HT-22). In zebrafish, 3d and 3l increase ATP
levels by 19.0% and 21.4% at the concentration of 1 μM, respectively (Figure 5). In
HT-22 cells, 3d and 3l increase ATP levels by 25.2% and 11.0% at the concentration
of 0.5 μM, respectively (Figure 5). Therefore, the most potent compounds at preventing
seizures in zebrafish, 3d and 3l, significantly increase ATP levels in zebrafish as well
as HT-22 cells, which indicates that maintaining mitochondrial homeostasis and ATP
levels are likely to play a role in its mechanism of action. The VK analogs’ molecular
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target likely involves proteins in the quinone oxidation/reduction process in the electron
transport chain (ETC). VK in combination with vitamin C has been used in the clinic
to enhance the oxidative phosphorylation process for the treatment of rare
mitochondrial diseases with complex III mutations.31-32 VK has also been shown to play
a key role as an alternative electron carrier that can rescue pink1 knock-out phenotype
0
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in Drosophila, and the VK synthase is a dominant enhancer of the phenotype. VK is
also involved in maintaining calcium ion homeostasis in cells through modulating -
glutamyl carboxylation of VK-dependent proteins. Proteins such as growth arrest
specific gene 6 (Gas-6) and protein S regulate calcium ion signaling in the central
nervous system. Gas-6 activation prevents amyloid beta protein and phospholipase
A(2)-IIA induced calcium ion influx and attenuates neuronal death.34-35 Calcium ions,
in turn, play a key role in the induction of seizures. Thus, the ability of VK to modulate
the levels of calcium ion in neurons could be a major contributing factor in its ability
to attenuate seizures.
Figure 5. 3d and 3l significantly increase ATP concentrations (nmole/mg) in zebrafish
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and HT-22 cells. Mean concentrations are plotted ± SEM, n = 7-13. Treatment with
compounds significantly increased ATP concentrations compared to controls (p < 0.05).
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Tissue Distribution and PK Profile of 3d
Compound 3d displays promising activity in rodent models and was chosen for
further evaluation. As epilepsy is a neurological disorder, effective drug candidates
need to cross the blood-brain barrier. 3d was expected to have excellent lipophilicity
and blood-brain barrier permeation as shown by its predicted cLogP of 3.11 and a tPSA
of 46.17 (molinspiration software http://www.molinspiration.com/). To verify the
blood-brain barrier permeation of our compounds, 3d was assessed for brain/plasma
concentration ratio at the 0.5, 1, 2, 4, and 8 h time points, respectively. The brain/plasma
ratio for 3d is 2.64 at 0.5 h, and the ratio declines to 0.816 at 8 h, which indicates the
excellent permeability of 3d (Figure 6). The brain/plasma concentration ratio of 2h is
0
.325 at 0.5 h (Supporting information Table S3), which is much lower than 3d.
Figure 6. Brain/plasma concentration ratio of 3d with i.p. administration at
concentration of 400 mg/kg.
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Improvement of the PK profile of 2h is one of our goals to develop the new VK
analogs as potential ASDs; therefore, we accessed the PK of 3d in mice. As we used ip
administration in rodent models, we initially studied the PK profile of 3d with ip
administration and compared with 2h. Compound 2h rapidly reaches its mean peak
plasma concentration (Cmax) within 15 min. After that, its plasma concentration declines
quickly with a short t of 1.10 h (Table 5, Figure 7a). Unlike 2h, 3d reaches its C
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within 60 min, and its plasma concentration declines more slowly with a much longer
t of 3.38 h (Table 5, Figure 7b). The area under the plasma concentration versus time
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/2
curve (AUC) of 3d is 1,726 hr*ng/mL, which is 1.8 times higher than 2h (978 hr*ng/mL)
Table 5). Therefore, 3d displays a more favorable PK profile with longer t1/2 and
(
higher AUC. A detailed PK study of 3d was also conducted by i.v. and oral
administration; parameters are shown in Figure 8 and Table 6. With i.v. and oral
administration, t1/2 of 3d are 4.47 and 5.28 h, respectively, and oral bioavailability of
3d is 35.9%. The PK profile indicates 3d is also suitable for oral administration.
Table 5. PK parameters of 2h and 3d.a
Cpd Admini
Dose
(mg/kg)
tmax
(h)
Cmax
(ng/kg)
AUClast
(hr*ng/mL)
t (h)
1
/2
No.
stration
2h
ip
ip
20
20
1.10 ± 0.08
3.38 ± 0.16
0.25 1490 ± 79.3 978 ± 111.5
1.00 320 ± 91.7 1716 ± 264.9
3
d
a
Note: ip intraperitoneal administration. Three mice were used for each compound.
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Figure 7. Comparison of PK profile of 2h and 3d by ip administration at 20mg/kg. 3
mice were used for each compound.
Figure 8. PK profile of 3d with iv and oral administration three mice were used for
each compound.
Table 6. PK parameters of 3d with iv and oral administration.a
Admini
stration
Dose
(mg/kg)
Cmax
(ng/kg)
AUClast
(hr*ng/mL)
t (h)
F (%)
1
/2
iv
5
4.47±0.54 1907±84.65
5.28±0.55 1025±273.3
636±25.5
ND
po
20
903±175.5
35.9±6.7
a
Note: iv: intravenous injection administration; po: oral administration. Three mice
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were used for each experiment.
CYP450 Isoform and hERG Inhibition
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Cytochrome P450 (CYP450) isozymes play an important role in metabolism of
drugs. Assessment of the potential of a compound to inhibit a specific CYP450 enzyme
allows prediction of potential drug-to-drug interactions that could occur in humans as
the compound may alter the metabolism of a co-administered drug. Among all of the
CYP450 isoforms, there are six major CYP450s that metabolize 90 percent of drugs.
3
6
The two most significant are CYP3A4 and CYP2D6. According to the FDA Drug-
Drug Interaction (DDI) Studies Guidance for Industry (www.fda.gov/media/108130/),
we evaluated compound 3d’s potential to inhibit CYP isoforms including CYP1A2,
CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. Among all the
tested isoforms, only CYP1A2 is moderately inhibited by 3d with IC of 5.57 μM, and
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other CYP450 isoforms are all weakly inhibited by 3d with IC > 10 μM (Figure 9).
5
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Therefore, 3d does not significantly influence CYP450 enzymatic activity, which
indicates it can be safely co-administered with other drugs, with specific attention paid
to potential CYP1A2 interaction. We have also examined 3d’s inhibitory activity
against hERG (human ether-à-go-go-related gene, a key potassium ion channel in the
heart) to determine potential cardiac-toxicity. hERG inhibitory IC of 3d is higher than
5
0
3
0 μM comparing to the positive control verapamil at 400 nM (Supporting Information
Figure S2). As plasma tissue binding ratio of 3d in mouse is 98.5% (Supporting
Information Table S3), the free EC in mice is approximately 123 nM according to the
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PK study (Supporting Information Figure S3) and give a free safety margin of 243
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folds.
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Figure 9. IC curves of 3d in the inhibition of CYP450 isoforms.
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PAINS Exclusion Assay
Promiscuous compounds appear as “frequent hitters” and display misleading assay
readouts. Thus, many of these compounds have been classified as Pan Assay
Interference Compounds (PAINS).37 The quinone structure of VK3 contains the
potential reactive “Michael acceptor” and can be considered as a promiscuous PAINS
compound.37-38 Although, pharmacology studies above have shown that our analogs are
not promiscuous compounds (weak and differential CYP inhibition, little hERG
inhibition, and clear SAR in a whole organism), since 3d is a VK3 analog, we tested
3
d for its ability for Michael reaction. VK3 and 3d were subjected to 2-mercaptoethanol,
respectively, VK3 rapidly reacts with 2-mercaptoethanol, while our lead compound 3d
does not react with thiol, even over a 24 h period (Supporting Information Figure S4),
indicating 3d is not a PAINS compound. The deactivation of the “Michael acceptor” in
3
d may be due to amidation of the naphthoquinone. In addition, unlike VK3, amidated
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naphthoquinone does not increase oxygen consumption in cells unlike electron donors
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such as VK3 and methylene blue from our previous publication, further indicating
that an amidated naphthoquinone ring is also less likely to undergo redox cycle under
physiological conditions.
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Conclusions
In this study, we discover a new series of VK analogs with anti-seizure activity by
the modification of VK3 analog 2h in the group of alkynes. Among these, the best
compounds, 3d and 3l, as assessed by the zebrafish PTZ-induced seizure model showed
significantly improved seizure protection compared to our previous lead compound 2h
at the 10 μM and 5 μM concentrations. Compound 3d attenuates seizures in a dose-
dependent manner in the mouse 6 Hz and MES models. Full seizure protection is also
attained at doses that do not produce motor impairment in mice, which indicates its
potential in the treatment of pharmacoresistant epilepsy. Moreover, 3d is also effective
in the s.c. PTZ mouse model with an ED of 349.22 mg/kg, which indicates its efficacy
5
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against clonic seizures. 3d is well tolerated without obvious toxicity at the tested
concentrations. Further PK studies show 3d has a longer t1/2 and higher AUC than
previous lead compound 2h with ip administration. With iv and oral administration, t1/2
of 3d are 4.47 and 5.28 h, respectively; oral bioavailability of 3d is 35.9%, which
indicates 3d is suitable for oral administration. In addition to the in vivo anti-seizure
efficacy and favorable PK profile, 3d also shows excellent brain penetration.
For the clinical safety and tolerability concern, 3d does not show obvious toxicity
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at concentration of 50 μM in HT-22 neuronal cells. In mice seizure models, the LD50
for 3d is greater than 800 mg/kg, which is extremely high for a small molecule and has
sufficient safety index even for its EC at approximately 200 mg/kg. What is more, 3d
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did not unduly influence CYP450 enzyme activity, it is expected to be safe with a low
incidence of potential drug-to-drug interaction when co-administered with other
therapeutic agents. Furthermore, 3d has a predictive free hERG-safety margin of 243
folds suggesting a high cardiac-safety index. All of our results indicate that 3d can be
further developed as a potential ASD with good tolerability for patients with
pharmacoresistant epilepsy.
Experimental Section
Material and Methods
All chemical starting materials including reagents and solvents were purchased
from Sigma-Aldrich, Fisher Scientific, or other chemical vendors and used as received
without purification, unless otherwise noted. H and 13C NMR spectras were
1
characterized using a Bruker Nanobay 400 MHz instrument in DMSO-d with TMS as
6
an internal standard. Chemical shifts (δ) in parts per million, and coupling constants (J)
in hertz (Hz) are reported. Mass spectral were determined using Thermo LCQ Fleet
mass spectrometer via electrospray ionization. Column purification was performed via
Teledyne Isco Combiflash 200 on prepacked C18-Aq columns. All target compounds’
purities were at least 95% detected at 254 nM via ESI-LCMS on an Agilent 1100 HPLC
instrument using an ODS HYPERSIL column (5 μm, 4.6 mm × 250 mm) with
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water/methanol gradient plus 0.1% formic acid (3a-3w: 0-13 mins from 0% to 100%
methanol, 13-14 mins: from 100% to 0% methanol. 5a and 5b: 0-3minutes from 0% to
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0% methanol, 3-17 mins from 90% to 100% methanol, and 17-18 mins from 100% to
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0% methanol).
General Procedure A:
2
-bromonaphthalene-1,4-dione (1, 0.24g, 1mmol) was suspended in 20 mL anhydrous
ethanol, then different fatty amine was added (2 mmol). The mixture was stirred
overnight at room temperature. The ethanol solvent was removed via rotovap in vacuum,
and the crude product was purified by recrystallization or combi-flash column.
Synthesis of 3a-3x
2
-(neopentylamino)naphthalene-1,4-dione (3a)
Using the General procedure A, 2-bromonaphthalene-1,4-dione and 2,2-
1
dimethylpropan-1-amine gave 3c as an orange-yellow solid, 56% yield. H NMR (400
MHz, DMSO-d ) δ 7.99 (dd, J = 7.7, 1.3 Hz, 1H), 7.93 (dd, J = 7.6, 1.3 Hz, 1H), 7.82
6
(td, J = 7.5, 1.4 Hz, 1H), 7.72 (td, J = 7.5, 1.4 Hz, 1H), 7.18 (t, J = 6.8 Hz, 1H), 5.83 (s,
13
1
H), 3.03 (d, J = 6.9 Hz, 2H), 0.94 (s, 9H). C NMR (101 MHz, DMSO-d ) δ 182.00,
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181.93, 149.68, 135.31, 133.60, 132.58, 130.77, 126.38, 125.73, 100.09, 53.10, 34.06,
2
2
7.91 . ESI-MS m/z: 244.25 [M + H]+.
-((3,3-dimethylbutyl)amino)naphthalene-1,4-dione (3b)
Using the General procedure A, 2-bromonaphthalene-1,4-dione and 3,3-dimethylbutan-
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1-amine gave 3d as an orange-yellow solid, 53% yield. H NMR (400 MHz, DMSO-
d6) δ 7.97 (dd, J = 7.7, 1.3 Hz, 1H), 7.94 (dd, J = 7.7, 1.3 Hz, 1H), 7.82 (td, J = 7.5, 1.3
Hz, 1H), 7.72 (td, J = 7.5, 1.4 Hz, 1H), 7.52 (t, J = 6.0 Hz, 1H), 5.62 (s, 1H), 3.24 –
1
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3.09 (m, 2H), 1.55 – 1.44 (m, 2H), 0.94 (s, 9H). C NMR (101 MHz, DMSO-d ) δ
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82.01, 181.54, 148.79, 135.29, 133.69, 132.56, 130.84, 126.33, 125.79, 99.45, 40.99,
38.97, 30.19, 29.58 . ESI-MS m/z: 258.25 [M + H]+.
2
-((4,4-dimethylpentyl)amino)naphthalene-1,4-dione (3c)
Using the General procedure A, 2-bromonaphthalene-1,4-dione and 4,4-
1
dimethylpentan-1-amine gave 3e as an orange-yellow solid, 63% yield. H NMR (400
MHz, DMSO-d ) δ 7.97 (dd, J = 7.7, 1.3 Hz, 1H), 7.94 (dd, J = 7.6, 1.3 Hz, 1H), 7.82
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(
td, J = 7.5, 1.4 Hz, 1H), 7.71 (td, J = 7.5, 1.4 Hz, 1H), 7.55 (t, J = 6.2 Hz, 1H), 5.66 (s,
1
3
1H), 3.15 (q, J = 6.9 Hz, 2H), 1.58-1.51 (m, 2H), 1.22-1.18 (m, 2H), 0.86 (s, 10H). C
NMR (101 MHz, DMSO-d ) δ 182.03, 181.63, 148.94, 135.26, 133.69, 132.54, 130.85,
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1
26.32, 125.77, 99.59, 43.09, 41.15, 30.42, 29.63, 23.20. ESI-MS m/z: 272.25 [M +
H]+.
2-(isopentylamino)naphthalene-1,4-dione (3d)
Using the General procedure A, 2-bromonaphthalene-1,4-dione and 3-methylbutan-1-
1
amine gave 3a as an orange-yellow solid, 63% yield. H NMR (400 MHz, DMSO-d )
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δ 7.96 (ddd, J = 14.0, 7.7, 1.3 Hz, 2H), 7.82 (td, J = 7.6, 1.4 Hz, 1H), 7.72 (td, J = 7.5,
1.3 Hz, 1H), 7.56 (t, J = 6.1 Hz, 1H), 5.65 (s, 1H), 3.18 (q, J = 6.9 Hz, 2H), 1.68-1.51
1
3
(
m, 1H), 1.50-1.45 (m, 2H), 0.91 (d, J = 6.6 Hz, 6H). C NMR (101 MHz, DMSO-d )
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