Novel KCNQ2/Q3 agonists as potential therapeutics for epilepsy and neuropathic pain

Article abstract of DOI:10.1021/jm901497b

Current drugs for the treatment of seizure disorders, although effective in many patients, still suffer from a number of failures and are not effective in some forms of resistant epilepsies. Historically,many of these drugs have multiple mechanisms of action including calcium and sodium channel blockade as well as GABAergic activity and thus a number of associated side effects. Modulation of the M-current through opening ofKCNQ channels has been proposed as a way to attenuate neuroexcitability and have a therapeutic benefit for the treatment of seizure disorders. Therefore, as part of our program to identify new treatments for epilepsy, we set out to identify agonists of KCNQ channels. High throughput screening of our corporate collection led to the identification of 1, adamantane-1-carboxylic acid (3-methyl-3H-benzothiazol-2-ylidine) hydrazide, a potent KCNQ2/Q3 agonist. Herein, we describe the syntheses and structure - activity relationships of analogues of 1 as well as their in vivo activity in animal models of epilepsy and neuropathic pain. 2009 American Chemical Society.

Full text of DOI:10.1021/jm901497b

J. Med. Chem. 2010, 53, 887–896 887
DOI: 10.1021/jm901497b
Novel KCNQ2/Q3 Agonists as Potential Therapeutics for Epilepsy and Neuropathic Pain
Paul C. Fritch,* Grant McNaughton-Smith, George S. Amato, James F. Burns, C. Wesley Eargle, Rosemarie Roeloffs,
William Harrison, Leslie Jones, and Alan D. Wickenden
Icagen, Inc., Suite 390, 4222 Emperor Boulevard, Durham, North Carolina 27703
Received October 8, 2009
Current drugs for the treatment of seizure disorders, although effective in many patients, still suffer from a
number of failures and are not effective in some forms of resistant epilepsies. Historically, many of these drugs
have multiple mechanisms of action including calcium and sodium channel blockade as well as GABAergic
activity and thus a number of associated side effects. Modulation of the M-current through opening of KCNQ
channels has been proposed as a way to attenuate neuroexcitability and have a therapeutic benefit for the
treatment of seizure disorders. Therefore, as part of our program to identify new treatments for epilepsy, we set
out to identify agonists of KCNQ channels. High throughput screening of our corporate collection led to the
identification of 1, adamantane-1-carboxylic acid (3-methyl-3H-benzothiazol-2-ylidine) hydrazide, a potent
KCNQ2/Q3 agonist. Herein, we describe the syntheses and structure-activity relationships of analogues
of 1 as well as their in vivo activity in animal models of epilepsy and neuropathic pain.
Introduction
There is also a growing body of evidence supporting the
hypothesis that nociception can be modulated by KCNQ2/
Q3 channels.4 These channels are expressed at a number of key
locations in the pain transmission pathway, including the
thalamus, the cerebral cortex, and the spinal cord and both
KCNQ2 and KCNQ3 are expressed in rat dorsal root ganglia
(DRG) including nociceptive DRG neurons. In fact, studies
have shown that firing in these neurons can be inhibited by a
KCNQ2/Q3 activator, which have been shown to inhibit
nociception in several animal models.17-21 In addition, the
marketed analgesic flupirtine activates KCNQ2/Q3.22 Clearly
the data support the potential of KCNQ2/Q3 channels as
targets for the treatment of disorders associated with hyper-
excitable neurons.
High throughput screening of Icagen’s diverse corporate
collection identified adamantane-1-carboxylic acid (3-methyl-
3H-benzothiazol-2-ylidine)hydrazide 1 (Figure 1) as a potent
KCNQ2/Q3 agonist (see Scheme 1 and Table 1). Further
screening against the cardiac liability channel and family
member KCNQ1 þ KCNE1 revealed that the compound
possessed >100-fold selectivity against this channel. This
initial hit also exhibited anticonvulsant effects in an in vivo
model of epilepsy when administered intraperitoneally (IP)
but not when administered orally (PO) (see Table 2).
Epilepsy and neuropathic pain are disorders characterized by
abnormally excessive or ectopic neuronal discharge. Approved
drugs for the treatment of epilepsy include modulators of
voltage-gated sodium channels, voltage-operated calcium chan-
nels, and those that modulate the GABAergica neurotransmis-
sion system.1-4 Drugs used to treat neuropathic pain include
gabapentin, pregabalin, carbamazepine, and lamotrigine.4-8
Although current treatments for epilepsy and neuropathic pain
provide adequate care for certain patient populations, there
remains a lack of clinical efficacy in a large percentage of pati-
ents. Suboptimal side effect profiles also limit their use.2,9
Although the drugs described above work through different
mechanisms, a common theme is their ability to dampen
neuronal excitability. One approach that has more recently
received attention is the modulation of KCNQ2/Q3 potassium
channels. These channels are the molecular correlate of the
M-current, IM, which is known to play an important role in
neuronal excitability.10 Activation of KCNQ2/Q3 channels
hyperpolarize neuronal membranes, resulting in a dampening
of action potential firing. Therefore, regulation of neuronal
excitability through modulation of KCNQ2/Q3 channels
represents a potentially new way to treat disorders such as
epilepsy and neuropathic pain. In fact, mutations in the
KCNQ2 and KCNQ3 genes have been identified as the genetic
basis of benign familial neonatal convulsions, a generalized
epilepsy syndrome, and potassium channel openers such as
retigabine have anticonvulsant effects in animal models.4,11-16
Our initial medicinal chemistry efforts focused on the
benzothiazolylidene ring system. Specifically, we were inter-
ested in determining the effect of substituting the arylring with
electron-withdrawing groups would have on potency and
more importantly bioavailability. Next, our attention focused
on the N-methyl, thiazolyl, and amide groups in an effort to
understand their contributions to activity.
*To whom correspondence should be addressed. Phone: (919) 941-5206.
Fax: (919) 941-0813. E-mail: pfritch@icagen.com.
aAbbreviations: EBSS, Earle’s balanced salt solution; EC50, concen-
tration required for 50% of efficiency; EtOAc, ethyl acetate; GABA,
γ-aminobutyric acid; HPLC, high performance liquid chromatography;
KCNQ, a class of potassium channel; MES, maximal electroshock
seizure; PTZ, pentylenetetrazol induced seizure; SAR, structure-
activity relationship; SDS, sodium dodecyl sulfate; SNL, spinal nerve
ligation.
Chemistry
The syntheses of the adamantane (benzothiazol-2-ylidene)-
hydrazides and the adamantane (benzooxazol-2-ylidene)-
hydrazide derivatives 4a-m are outlined in Scheme 1 (refer
r
2009 American Chemical Society
Published on Web 12/18/2009
pubs.acs.org/jmc

888 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Fritch et al.
Table 2. Efficacy of Select Compounds in in Vivo Anticonvulsant
Assay, the Mouse Maximal Electroshock Seizure (MES) assay
compd
mouse MES IP 30 mg/kg
mouse MES PO 30 mg/kg
1
5/8a
2/8
6/8
6/8
8/8
3/8
6/8
1/8
0/8
0/8
0/8
8/8
0/8b
1/8
4d
4e
4f
4g
4h
4i
Figure 1. Adamantane-1-carboxylic acid (3-methyl-3H-benzothia-
zol-2-ylidine) hydrazide screening hit.
Scheme 1a
a Number of mice protected from seizure/number of mice tested.
bAssayed at 20 mg/kg dose.
then treated with adamantane-1-carboxylic acid hydrazide to
afford adamantyl derivatives 4a-j.28 Alkylation of 2-(methyl-
thio)benzothiazole with ethyl trifluoromethanesulfonate
followed by treament with adamantane-1-carboxylic acid
hydrazide afforded adamantyl derivative 4k. Methylation of
2-(methylthio)benzothiazole with methyl trifluoromethane-
sulfonate followed by treatment of the resulting triflate salt
with adamantane-1-carboxylic acid N-methylhydrazide, 2,
afforded adamantyl derivative 4l. Compound 4m was pre-
pared from 3-methyl-2(3H)-benzoxazolethione via methyla-
tion with methane trifluoromethanesulfonate and reaction of
the resulting salt with adamantane-1-carboxylic acid hydrazide.
The 3-methyl-6-trifluoromethoxy derivatives were readily
prepared from 2-(methylthio)benzothiazole 3g (Scheme 2,
refer to Table 3 for Y and R groups). Methylation of 3g
afforded the triflate salt, followed by treatment with an
ethanolic solution of hydrazine afforded hydrazide 5.28,29
Acylation of this material with carboxylic acid chlorides,
cyclopentyl 4-nitrophenyl carbonate,30 isopropyl chloro-
formate, or di-tert-butyl dicarbonate proceeded to afford
hydrazides 6a-p.
a Reagents: (a) Methylhydrazine, CH2Cl2, -78 °C, 44%. (b) (1)
EtOCS2K, NMP, 120 °C, 1 to 18 h; (2) MeI, K2CO3, DMF, 23 °C, 2 to
18 h. (c) (1) MeOTf, CH2ClCH2Cl, 23 °C, 4 h or EtOTf, CH2ClCH2Cl,
23 °C, 6 h; (2) adamantane-1-carboxylic acid hydrazide, Et3N, EtOH,
80 °C, 8 h or 2, Et3N, EtOH, 80 °C, 8 h; (d) (1) MeOTf, CH2ClCH2Cl,
23 °C, 1 h; (2) adamantane-1-carboxylic acid hydrazide, Et3N, EtOH,
80 °C, 82% for 2 steps.
Table 1. KCNQ2/Q3 Activation by Adamantyl Derivatives 1, 4a-m
KCNQ2/Q3
R6 (efflux) EC50 (μM)a
compd
Y
R1 R2 R3 R4
R5
Results and Discussion
1
S
S
S
S
S
S
S
S
S
S
S
S
S
O
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
H
F
H
H
H
H
CF3
H
H
H
H
F
H
H
0.027 ( 0.002
0.38 ( 0.06
0.091 ( 0.003
0.16 ( 0.04
0.053 ( 0.005
0.028 ( 0.004
0.16 ( 0.03
0.025 ( 0.003
0.098 ( 0.013
0.021 ( 0.005
2.08 ( 1.04
0.57 ( 0.06
>10b
Compounds were tested to measure their activity against
KCNQ2/Q3.31-35 The EC50 values of compounds 1, 4a-m
are shown in Table 1. The addition of fluoro, trifluoromethyl,
and trifluoromethoxy moieties on the aryl ring were generally
well-tolerated (4a-i) with the exception of trifluoromethyl in
the R6 position (4j). This substitution resulted in a >10-fold
loss in in vitro potency. An increase in size of the N-alkyl
group (4k) was detrimental to potency giving approximately
9-fold less activity. Replacement of sulfur with oxygen re-
sulted in a dramatic loss of activity (4m) and, finally, amide
N-methylationresulted in acompound(4l) thatlacked agonist
activity at 10 μM. This suggests that this part of the molecule
plays a critical role this series ability to increase efflux through
KCNQ2/Q3 channels.
Select compounds were tested for their anticonvulsant
activity in the mouse maximal electroshock seizure (MES)
model.36 The results of this assay are summarized in Table 2.
Compounds were initially administered IP and several exhib-
ited pronounced anticonvulsant effects (1, 4e, 4f, 4g, 4i).
However, bioavailability issues were found upon oral dosing.
In fact, only compound 4g, the 3-methyl-6-trifluoromethoxy
analogue, was efficacious when given orally and thus was
chosen for further SAR development.
4a
4b
4c
4d
4e
4f
H
H
H
H
H
H
H
H
F
Me CF3
H
Me
Me
Me
Me
Me
Me
Me
Me
Et
F
F
H
H
H
H
H
H
H
H
H
H
H
F
CF3
OCF3
H
4g
4h
4i
F
H
CF3
H
H
H
4j
H
H
H
H
H
4k
4l
H
Me Me
Me
H
4m
H
H
2.70 ( 0.68
a EC50 values were determined from eight-point, half log concentra-
tion response curves using an KCNQ2/Q3 isotopic efflux assay as
described in the Experimental Section. Data shown with standard error
((SEM) represent the mean of 2-4 separate determinations. All active
compounds (EC50 < 1 μM) exhibited >30-fold selectivity compared to
KCNQ1 þ KCNE1. bAn EC50 value could not be calculated due to low
potency.
to Table 1 for Y and R groups). Adamantane-1-carboxylic
acid N-methylhydrazide, 2, was prepared from adamantane-
carbonyl chloride and methylhydrazine. Commercially avail-
able bromo- or fluoroanilines were condensed with potassium
ethyl xanthate to afford benzothiazole thiones, which were
alkylated with iodomethane to afford methylsulfanylbenzo-
thiazoles 3a-j.23-27 Further alkylation of 3a-j with methyl
trifluoromethanesulfonate afforded triflate salts, which were
The in vitro and in vivo data for compounds 6a-p is
summarized in Table 3. Compound 6d, which contains a small
one-carbon acyl group was not a potent KCNQ2/Q3 agonist.
However, compounds with monocyclic alkyl groups (6a-b)
were approximately as potent as the adamantyl compound 1,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 889
Scheme 2a
a Reagents: (a) (1) MeOTf, CH2ClCH2Cl, 23 °C, 2 h; (2) hydrazine, Et3N, EtOH, 23 °C, 1 h; 81% for 2 steps. (b) RYCOCl, K2CO3, CH3CN-H2O,
23-60 °C, 1-4 h or (1) cyclopentanol, 4-nitrophenyl chloroformate, pyridine, 23 °C, 15 h; (2) 5, NMM, DMF, 75 °C, 6 h, 74% or 1.0 M isopropyl
chloroformate solution in toluene, NMM, THF, 23 °C, 6 h, 64% or Boc2O, THF, 23 °C, 3 h, 69%.
Table 3. KCNQ2/Q3 Activation and Efficacy in the Mouse Maximal Electroshock Seizure (MES) and the Rat Subcutaneous Pentylenetetrazol Induced
Seizure (PTZ) Assays by (3-Methyl-6-trifluoromethoxy-3H-benzothiazol-2-ylidine)hydrazides 6a-p
KCNQ2/Q3
(efflux) EC50 (μM)a
mouse MES PO
20 mg/kg
mouse MES PO
10 mg/kg
mouse MES PO
EC50 (mg/kg)b
rat PTZ PO
EC50 (mg/kg)b
compd
Y
R
6a
6b
6c
6d
6e
6f
chexyl
cpentyl
tBu
0.028 ( 0.004
0.062 ( 0.023
0.71 ( 0.18
>10e
1/8c
0/8
7/8
nt
ntd
nt
nt
nt
nt
nt
0/8
nt
nt
3.8
nt
Me
tBu
nt
CH2
CH2
CH2
0.049 ( 0.001
0.044 ( 0.004
0.22 ( 0.04
5.57 ( 2.84
6.73 ( 2.72
0.10 ( 0.02
0.044 ( 0.004
0.26
7/8
8/8
7/8
nt
8/8
5/8
2/8
nt
4.4
<10
nt
3.8
7.5
nt
cpentyl
iPr
6g
6h
6i
2-THF
3-THF
3-F Ph
4-F Ph
Ph
nt
nt
nt
nt
nt
nt
6j
4/8
7/8
7/8
2/8
nt
nt
nt
nt
6k
6l
5/8
1/8
nt
nt
nt
nt
nt
6m
6n
6o
6p
2-furyl
cpentyl
iPr
0.50 ( 0.01
0.029 ( 0.009
0.095 ( 0.037
0.023 ( 0.009
nt
nt
O
O
O
7/8
5/8
8/8
nt
nt
8/8
nt
8.8
nt
2.6
nt
tBu
a EC50 values were determined from eight-point, half log concentration response curves using an KCNQ2/Q3 isotopic efflux assay as described in the
Experimental Section. Data shown with standard error ((SEM) represent the mean of two to four separate determinations. All active compounds (EC50
< 1 μM) exhibited >30-foldselectivity compared to KCNQ1 þ KCNE1. b EC50 values were determined from latencyto seizure scores as described in the
Experimental Section. c Number of mice protected from seizure/number of mice tested. d Not tested. e An EC50 value could not be calculated due to low
potency.
Table 4. Comparison of Icagen KCNQ2/Q3 Agonists and Reference
Compounds in the Rat Formalin and Spinal Nerve Ligation (SNL)
Models
but these compounds were not efficacious in the MES model
at 20 mg/kg PO dose. Interestingly, the incorporation of
oxygen into the monocyclic alkyl groups resulted in com-
reduction in flinches reduction in tactile
recorded in phase II
(formalin), %
pounds (6h-i) that were approximately 100-fold less active in
dose
mg/kg route
allodynia score
(SNL), %
vitro than 6b. The moderately potent tert-butyl analogue 6c
was efficacious at a dose of 20 mg/kg in the MES model. The
incorporation of aromatic acyl groups produced compounds
that were potent KCNQ2/Q3 agonists and also efficacious in
vivo. Most notable was compound 6k, which protected 5 out
of 8 animals in the mouse MES model (10 mg/kg, po). The
incorporation of a methylene spacer into the branched or
monocyclic alkyl appendages resulted in compounds that
possessed excellent in vitro potency and oral efficacy in the
mouse MES (6e-g) and rat pentylenetetrazol induced seizure
(PTZ) (6e-f) assays.36 As the methylene spacer had proved
beneficial we decided to investigate the possibility of employ-
ing the more enzymatically stable carbamates. The carbazate
analogues 6n-p had good KCNQ2/Q3 potency and were
found to be orally efficacious in the mouse MES model as well
as the rat PTZ model. This suggests that the compounds
possessed broad spectrum anticonvulsant activity. Oral ED50
values were obtained for select compounds in both the mouse
MES and the rat PTZ assays. Compounds 6e, 6f, and 6o
displayed good in vivo efficacy in both of the anticonvulsant
compd
morphine
gabapentin
retigabine
retigabine
6e
3
100
10
sc
38
61a
ntb
49
55
28
47
77
70a
0c
iv
po
po
po
po
po
17
17
nt
nt
6f
6o
34
10
nt
100c
a Data presented by S. Dworetzky, at the IBC 2nd International Ion
Channel Meeting (Boston, MA, October 20-22, 2003). b Not tested.
c Experiments performed at Dr. Frank Porreca’s laboratory, University
of Arizona (Tuscon).
assays with oral ED50 values less than 10 mg/kg. Compounds
6e, 6f, and 6o were also evaluated in pain models (Table 4).
The compounds reducedthe number of flinches in the phase II
portion of the rat formalin model, an effect thought to result
from secondary spinal sensitization.37,38 The percent reduc-
tion in flinches for compounds 6e, 6f, and 6o was similar to
morphine, gabapentin, and retigabine. A 10 mg/kg po dose of

890 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Fritch et al.
compound 6o resulted in a complete reduction of tactile
allodynia in the spinal nerve ligation (SNL) model of neuro-
pathic pain.39 Administration of morphine (3 mg/kg sc) and
gabapentin (100 mg/kg iv) also resulted in a large reduction of
tactile allodynia; however, a 10 mg/kg po dose of retigabine
did not result in any reduction of tactile allodynia. In the
behavioral assays, no overt effects on behavior were noted
that would be considered to confound any findings of efficacy.
Effects on motor coordination were specifically examined in
mice using the rotarod assay.36 In this assay, oral ED50 values
of 20.0 and 16.2 mg/kg were determined for compounds 6e
and 6o, respectively. Inaddition, Cerep receptor panel binding
assays on compounds 6e, 6f, and 6o did not support off-target
mechanisms observed in in vivo efficacy.40 The results of
Cerep receptor panel binding assays are included in the
Supporting Information.
In conclusion, adamantane-1-carboxylic acid (3-methyl-
3H-benzothiazol-2-ylidine)hydrazide 1 was identified as a
potent KCNQ2/Q3 agonist that was >100-fold selective
against cardiac liability channel and family member KCNQ1
þ KCNE1. The SAR of this compound was explored, and an
orally active anticonvulsant (4g) was found. Further optimi-
zation led to novel compounds that were potent, selective
against KCNQ1 þ KCNE1, and Cerep receptor panel binding
assays with oral ED50’s less than 10 mg/kg in two in vivo
anticonvulsant models (6e, 6f, 6o). In addition, these com-
pounds were shown to be orally active in rodent models of pain.
seizure apparatus designed by Walhquist Instrument Co. (Salt
Lake City, UT). Compounds were formulated in 5% dimethyl
sulfoxide/95% (0.5% hydroxypropylmethylcellulose/1% Tween
80) and were administered in a volume of 10 mL/kg 15 min before
electroshock application for ip administration and 30 min before
electroshock application for po administration. The shock level
was set at 50 mA, and the duration was set at 0.2 s. A drop of 1%
proparacaine solution was placed in each eye, electrodes were
placed over the eyes, and shock was administered. Latency to hind
limb extension was measured to the nearest 0.1 s. If extension did
not occur within 6 s, the animal was scored as protected and a
latency score of 6 s was recorded.
Rat Subcutaneous Pentylenetetrazol Seizure (PTZ) Assay.
Compounds were formulated in 0.5% methylcellulose and admi-
nistered po in a volume of 1 mL/kg in male Sprague-Dawley rats
30 min before administration of 85 mg/kg PTZ sc. The latency to
tonic-clonic seizure was recorded in minutes, using administration
of PTZ as time 0. If no seizure was observed within 15 min post-
PTZ the animal was considered protected, and a latency score of
15 min was recorded.
Rat Formalin Assay. One hour prior to assay, male Sprague-
Dawley rats had a small (0.5 g) loose metal band glued to the
right hind paw. Compounds were formulated in 0.5% methyl-
cellulose and administered po. 1 h before assay. Immediately
before testing, 50 μL of 2.5% formalin (Richard-Allen Scienti-
fic) was administered sc into the dorsal surface of the right
hind paw using a 30 gauge needle. Rats were immediately
placed without restraint into a plexiglass observation chamber
situated over an electromagnetic detector system (Automated
Formalin Analyzer, Department of Anesthesiology, University
of California, San Diego). Paw flinches of the formalin-injected
paw are detected by the electromagnetic system and counted for
60 min following formalin injection. Phase I flinches are defined
as flinches occurring within the first 10 min following formalin
injection. Phase II flinches occur from 11 to 60 min following
formalin injection.
Spinal Nerve Ligation (SNL) Assay. A neuropathic pain
condition was induced in male Sprague-Dawley rats using a
modification of the nerve injury model described by Kim and
Chung.39 Rats were anesthetized with halothane, and the L5
spinal nerve was exposed, carefully isolated, and tightly ligated
with 4.0 silk suture distal to the dorsal root ganglia. The wound
was sutured, and the rats were allowed to recover for 2-3 weeks.
To test the effect of compounds on tactile allodynia following
L5 nerve ligation, Von Frey tactile withdrawal thresholds were
determined. Test animals were placed in a box separated by
walls with a wire mesh floor allowing access to the plantar
surface of the paw. Tactile testing was conducted using a set of
calibrated nylon fibers (Von Frey hairs), each approximately
3 cm long and sequentially increasing in diameter and stiffness,
mounted on handles. Beginning with a medium hair, the tip of
the fiber was placed on the plantar surface of the rat paw and
applied with a pressure to make it slightly bend. If the rat
responded by lifting its paw, the next descending hair was tested.
Failure to lift the hind paw after 4 s was scored a negative
response and the next ascending hair was applied. Dixon’s
Up-Down Method was applied for a total of six responses
following and including the first change in response to determine
50% paw withdrawal thresholds. A top threshold was set at 15 g.
Baseline measurements of allodynia were performed immedi-
ately prior to subject selection and compound administration.
Compounds were formulated in 0.5% methylcellulose and were
administered po. Behavioral testing was conducted one and two
hours following administration.
Experimental Section
General Procedures. All reagents and solvents were obtained
from commercial sources and used as received. 1H and 13C NMR
spectra were obtained with a Varian Inova spectrometer at 300 and
75 MHz, respectively, in the solvent indicated. Coupling constants
(J) are in hertz (Hz). Mass spectrometric identification of com-
pounds was performed using standard electrospray ionization
methods, MS (ESI), with a Perkin-Elmer SCIEX API 150 EX.
Melting points were obtained with a Electrothermal IA9000 digital
melting point apparatus. All compounds were assessed as greater
than 95% pure by two HPLC columns (C18 and Cyano) on a
Michrom Bioresources Magic 2002 system (solvent A = 100%
water/1.0 mL/L formic acid; solvent B = 99% CH3OH/1% water/
1.0 mL/L formic acid; gradient of 0% to 100% B over 10 min).
CMA80 is solvent mixture that was used as an eluent in chromato-
graphy; it is a 2/18/80 mixture of NH4OH/CH3OH/CHCl3.
KCNQ2/Q3 Isotopic Efflux Assay. Cells expressing voltage-
gated Kþ channels, such as KCNQ2-like channels were loaded
with 86 Rbþ by culture in media containing 86 RbCl. Following
loading, culture media were removed and the cells were washed
in EBSS to remove residual traces of 86RBþ. Cells were pre-
incubated with drug (0.01-30 μM in EBSS) and then 86Rbþ
efflux was stimulated by exposing cells to EBSS solution sup-
plemented with a submaximal concentration of KCl (generally
7-20 mM) in the continued presence of drug. After a suitable
efflux period, the EBSS/KCl solution was removed from the
cells and the 86Rbþ content determined by Cherenkov counting
(Wallac Trilux). Cells were then lysed with a SDS solution and
the 86Rbþ content of the lysate determined. Percent 86Rbþ efflux
was calculated according to:
þ
þ
86
86
ð Rb content in EBSS=ð Rb content in EBSS
86Rbþ content of the lysateÞÞ ꢀ 100
Adamantane-1-carboxylic Acid N-Methylhydrazide (2). A so-
lution of methylhydrazine (1.064 mL, 20.0 mmol) in 20 mL of
CH2Cl2 at -78 °C was treated over 30 min with adamantane-
carbonyl chloride (1.99 g, 10.0 mmol). After addition was
complete, the reaction mixture was warmed to rt over a 2 h
period. The reaction mixture was diluted with CH2Cl2 (100 mL),
þ
Efflux was normalized to the maximal 86Rbþ efflux (i.e., that
induced by a high concentration of KCl, generally 30-135 mM).
Mouse Maximal Electroshock Seizure (MES) Assay. Male
CD-1 mice were tested in the MES assay using the electroshock

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 891
washed with 1:1 H2O/ NaHCO3 (satd aq solution) (2 ꢀ 100 mL),
dried (Na2SO4), and concentrated. Chromatography on silica
(elution with a gradient of CHCl3 to 10% CMA80/CHCl3) afforded
916 mg (4.40 mmol, 44%) of 2 as a white powder; mp 146-147 °C.
1H NMR (CDCl3) δ: 1.74 (s, C(CH2CHCH2)3), 2.03 (s, C(CH2-
CHCH2)3), 2.12 (s, C(CH2CHCH2)3), 3.25 (s, 3 H, C(O)N(CH3)-
NH2), 3.88 (b, 2 H, C(O)N(CH3)NH2). 13C NMR (DMSO-d6) δ:
28.7 (C(CH2CHCH2)3), 37.2 (C(CH2CHCH2)3), 38.7 (C(CH2-
CHCH2)3), 40.8 (C(CH2CHCH2)3), 41.6 (C(O)N(CH3)NH2),
177.3 (C(O)N(CH3)NH2). MS (ESI) m/z 209 (M þ H)þ.
1H NMR (CDCl3) δ: 2.75 (s, SCH3), 7.11 (ddd, J =2.6, 8.9,
8.9 Hz, CHCHCF), 7.40 (dd, J=2.5, 8.1 Hz, CFCHCS), 7.77
(dd, J =4.8, 8.9 Hz, CNCHCH). 13C NMR (CDCl3) δ: 16.0
(SCH3), 107.5 (d, J=27.2 Hz, CFCHCS), 114.4 (d, J=24.7 Hz,
CHCHCF), 122.1 (d, J = 9.1 Hz, CNCHCH), 136.1 (d, J =
11.0 Hz, CHCSCN), 150.0 (d, J=1.6 Hz, CSCNCH), 159.8 (d,
J=244.7 Hz, CF), 167.6 (d, J=2.8 Hz, SC(N)SCH3). MS (ESI)
m/z 200 (M þ H)þ.
6-Trifluoromethyl-2-(methylthio)benzothiazole (3f). Prepared
from 2-fluoro-4-(trifluoromethyl)aniline according to mMethod
A; mp 55.8-57.0 °C. 1H NMR (CDCl3) δ: 2.82 (s, SCH3), 7.65
(d, J=8.5 Hz, CHCHC(CF3), 7.94 (d, J=8.6 Hz, CNCHCH),
8.04 (s, C(CF3)CHCS). 13C NMR (CDCl3) δ: 16.0 (SCH3),
118.6 (q, J=4.3 Hz, ArCH), 121.6 (CNCHCH), 123.3 (q, J=
3.4 Hz, ArCH), 124.3 (q, J = 272.2 Hz, CF3), 126.3 (q, J =
33.0 Hz, C(CF3)), 135.4 (CHCSCN), 155.4 (CSCNCH), 171.9
(SC(N)SCH3). MS (ESI) m/z 250 (M þ H)þ.
Representative Procedure for Conversion of 2-Fluoro Anilines
to 2-(Methylthio)benzothiazoles (Method A). 4-Fluoro-2-(methyl-
thio)benzothiazole (3a). A solution of 2,6-difluoroaniline (2.16 g,
16.73 mmol) in 16.7 mL of NMP was treated dropwise with a
solution of potassium ethyl xanthate (2.81 g, 17.57 mmol) in
16.7 mL of NMP and the mixture was heated at 120 °C for 4 h.
The reaction mixture was cooled and partitioned between
EtOAc (300 mL) and brine (200 mL). The organic phase was
washed with brine (2 ꢀ 200 mL), dried (Na2SO4), and con-
centrated. Chromatography on silica (elution with CHCl3)
followed by recrystallization from CHCl3 afforded 2.02 g
(10.9 mmol) of the intermediate 4-fluorobenzothiazole-2(3H)-
thione. The intermediate was dissolved in 40 mL of DMF and
treated with K2CO3 (1.81 g, 13.1 mmol) and iodomethane
(0.81 mL, 13.1 mmol). The reaction mixture was stirred over-
night and partitioned between EtOAc (300 mL) and H2O (200
mL). The organic phase was washed with brine (2 ꢀ 200 mL),
dried (Na2SO4), and concentrated. Chromatography on silica
(elution with 5% EtOAc/hexane) afforded 1.99 g (10.0 mmol,
60% for 2 steps) of 3a as an oil, which becomes a waxy solid
upon standing for a few days; mp 37.1-37.4 °C. 1H NMR
(CDCl3-CD3OD) δ: 2.72 (s, SCH3), 7.04 (ddd, J = 0.8, 8.0,
8.1 Hz, ArH), 7.16 (ddd, J=4.6, 8.1, 8.1 Hz, ArH), 7.43 (d, J=
8.0 Hz, ArH). 13C NMR (CDCl3-CD3OD) δ: 16.0 (SCH3),
111.9 (d, J =18.0 Hz, CFCH), 116.7 (d, J =4.3 Hz, CFCH-
CHCH), 124.9 (d, J = 7.4 Hz, CFCHCHCH), 137.6 (d, J =
3.7 Hz, CHCSCN), 141.8 (d, J=13.7 Hz, CSCNCF), 155.7 (d,
J = 254.9 Hz, CF), 169.7 (SC(N)SCH3). MS (ESI) m/z 200
(M þ H)þ.
6-Trifluoromethoxy-2-(methylthio)benzothiazole (3g). Prepared
from 2-fluoro-4-(trifluoromethoxy)aniline according to method A;
mp 35.6-35.9 °C. 1H NMR (DMSO-d6) δ: 2.78 (s, SCH3),
7.35-7.42 (m, CHCHC(OCF3)), 7.88 (d, J=8.8 Hz, CNCHCH),
8.08-8.16 (m, C(OCF3)CHCS). 13C NMR (DMSO-d6) δ: 16.2
(SCH3), 115.6 (C(OCF3)CHCS), 120.6 (CHCHC(OCF3)), 120.9
(q, J=254.4 Hz, OCF3), 122.6 (CNCHCH), 136.5 (CHCSCN),
145.3 (CHC(OCF3)CH), 152.5 (CSCNCH), 170.8 (SC(N)SCH3).
MS (ESI) m/z 266 (M þ H)þ.
7-Fluoro-2-(methylthio)benzothiazole (3h). Prepared from
2,3-difluoroaniline according to method A; mp 50.4-50.8 °C.
1H NMR (CDCl3) δ: 2.82 (s, SCH3), 7.03 (dd, J=8.4, 8.8 Hz,
CHCHCF), 7.38 (ddd, J=5.5, 8.0, 8.2 Hz, CHCHCH), 7.68 (d,
J = 8.0 Hz, CNCHCH). 13C NMR (CDCl3) δ: 16.2 (SCH3),
109.8 (d, J = 18.6 Hz, CHCHCF), 117.2 (d, J = 3.6 Hz,
CNCHCH), 122.1 (d, J = 16.8 Hz, CFCSCN), 127.1 (d, J =
7.3 Hz, CHCHCH), 156.2 (d, J=2.7 Hz, CSCNCH), 156.6 (d,
J=249.1 Hz, CHCFCS), 169.5 (d, J=1.8 Hz, SC(N)SCH3). MS
(ESI) m/z 200 (M þ H)þ.
5,6-Difluoro-2-(methylthio)benzothiazole (3i). Prepared from
2,4,5-trifluoroaniline according to method A; mp 80.7-81.2 °C.
1H NMR (CDCl3) δ: 2.77 (s, SCH3), 7.50 (dd, J=7.5, 9.3 Hz,
CHCFCF), 7.62 (dd, J=7.1, 10.6 Hz, CHCFCF). 13C NMR
(CDCl3) δ: 16.1 (SCH3), 108.7 (d, J=22.2 Hz, CHCF), 109.3
(d, J=19.5 Hz, CHCF), 130.2 (d, J=8.8 Hz, CHCSCN), 148.4
(dd, J = 14.7, 247.8 Hz, CHCFCF), 149.5 (d, J = 12.2 Hz,
CSCNCH), 150.0 (dd, J = 14.5, 246.3 Hz, CHCFCF), 169.8
(SC(N)SCH3). MS (ESI) m/z 218 (M þ H)þ.
4-Trifluoromethyl-2-(methylthio)benzothiazole (3b). Prepared
from 2-fluoro-6-(trifluoromethyl)aniline according to method
A; mp 64.9-65.2 °C. 1H NMR (CDCl3) δ: 2.82 (s, SCH3), 7.30
(dd, J = 7.8, 8.0 Hz, C(CF3)CHCH), 7.68 (d, J = 7.7 Hz,
C(CF3)CH), 7.90 (d, J = 8.0 Hz, CHCHCS). 13C NMR
(CDCl3) δ: 16.2 (SCH3), 122.5 (q, J=32.0 Hz, CCF3), 123.4
(ArCH), 123.7 (q, J=4.9 Hz, C(CF3)CH), 123.9 (q, J=273.1 Hz,
CF3), 124.8 (ArCH), 137.3 (CHCSCN), 149.6 (CSCNC(CF3)),
171.0 (SC(N)SCH3). MS (ESI) m/z 250 (M þ H)þ.
7-Trifluoromethyl-2-(methylthio)benzothiazole (3j). Prepared
from 2-fluoro-3-(trifluoromethyl)aniline according to method
A; mp 41.3-41.7 °C. 1H NMR (CDCl3) δ: 2.83 (s, SCH3),
7.51 (ddd, J=0.6, 7.8, 7.8 Hz, CHCHCH), 7.59 (dd, J=0.4, 7.5
4,6-Difluoro-2-(methylthio)benzothiazole (3c). Prepared from
2,4,6-trifluoroaniline according to mMethod A; mp 55.9-
Hz, CHCHC(CF3)), 8.03 (dd, J=0.3, 7.8 Hz, CNCHCH). 13
C
1
56.5 °C. H NMR (CDCl3) δ: 2.78 (s, SCH3), 6.90 (ddd, J=
NMR (CDCl3) δ: 16.0 (SCH3), 121.5 (q, J=4.3 Hz, CHCHC-
(CF3)), 123.7 (q, J = 272.5 Hz, CF3), 124.2 (q, J = 33.9 Hz,
CHC(CF3)CS), 124.7 (ArCH), 126.1 (ArCH), 131.9 (C(CF3)-
CSCN), 154.7 (CSCNCH), 170.4 (SC(N)CH3). MS (ESI) m/z
250 (M þ H)þ.
2.3, 10.0, 10.0 Hz, CFCHCF), 7.24 (ddd, J=1.4, 2.3, 7.6 Hz,
CFCHCS). 13C NMR (CDCl3-CD3OD) δ: 16.1 (SCH3), 102.0
(dd, J=21.9, 28.0 Hz, CFCHCFCH), 103.3 (dd, J=14.5, 26.5
Hz, CFCHCFCH), 138.0 (dd, J=4.8, 12.5 Hz, CSCNCF), 138.9
(J=1.8 Hz, CHCSCN), 153.7 (dd, J=13.4, 258.2 Hz, CF), 159.4
(dd, J=10.4, 247.3 Hz, CF), 168.3 (d, J=1.8 Hz, SC(N)SCH3).
MS (ESI) m/z 218 (M þ H)þ.
Representative Procedure for Conversion of 2-(Methylthio)-
benzothiazoles to Adamantane-1-carboxylic Acid (3-Methyl-3H-
benzothiazol-2-ylidine)hydrazides (Method B). Adamantane-1-
carboxylic Acid (3-methyl-3H-benzothiazol-2-ylidine)hydrazide (1).
A solution of 2-(methylthio)benzothiazole (399 mg, 2.20 mmol) in
7.0 mL of 1,2-dichloroethane was treated with methyl trifluoro-
methanesulfonate (266 μL, 2.42 mmol) and the reaction was stirred
at rt for 1 h. The reaction mixture was concentrated in vacuo and
dried under vacuum. The crude triflate salt was dissolved in 11 mL of
ethanol and added to a solution of adamantane-1-carboxylic
acid hydrazide (513 mg, 2.64 mmol) and triethylamine (552 μL,
3.96 mmol) in 11 mL of ethanol. The reaction mixture was heated at
80 °C for 8 h then concentrated in vacuo. The residue was
partitioned between EtOAc (100 mL) and H2O (60 mL). The
organic phase was washed with brine (60 mL), dried (Na2SO4),
5-Trifluoromethyl-2-(methylthio)benzothiazole (3d). Prepared
from 2-fluoro-5-(trifluoromethyl)aniline according to method
A; mp 67.8-68.2 °C. 1H NMR (CDCl3) δ: 2.78(s, SCH3),
7.48 (d, J = 8.3 Hz, C(CF3)CHCH), 7.78 (d, J = 8.3 Hz,
C(CF3)CHCHCS), 8.09 (s, CNCHC(CF3)). 13C NMR
(CDCl3) δ: 15.9 (SCH3), 118.4 (q, J=4.2 Hz, CNCHC(CF3)),
120.5 (q, J=3.5 Hz, C(CF3)CHCH), 121.5 (CHCHCS), 124.3
(q, J=272.2 Hz, CF3), 128.7 (q, J=32.6 Hz, C(CF3)), 138.7
(CHCSCN), 153.0 (CSCNCH), 170.6 (SC(N)SCH3). MS (ESI)
m/z 250 (M þ H)þ.
6-Fluoro-2-(methylthio)benzothiazole (3e). Prepared from 2,4-
difluoroaniline according to mMethod A; mp 69.9-70.2 °C.

892 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Fritch et al.
and concentrated. Recrystallization from EtOAc afforded 588 mg
(1.72 mmol, 78%) of 1 as a white solid; mp 236.8-237.2 °C.
1H NMR (DMSO-d6) δ: 1.67 (s, C(CH2CHCH2)3), 1.85 (s,
C(CH2CHCH2)3), 1.98 (s, C(CH2CHCH2)3), 3.47 (s, NCH3),
7.05(ddd, J=1.0, 7.6, 7.7 Hz, ArH),7.20(d, J=7.8 Hz, CNCHCH),
7.30 (ddd, J=1.1, 8.2, 8.3 Hz, ArH), 7.54 (dd, J=1.0, 7.6 Hz, 1 H,
CHCHCS), 9.83 (s, NNHC(O)). 13C NMR (DMSO-d6) δ: 28.3
(C(CH2CHCH2)3), 31.2 (NCH3), 36.8 (C(CH2CHCH2)3), 39.4
(C(CH2CHCH2)3), 40.4 (C(CH2CHCH2)3), 110.4 (CNCHCH),
122.2 (ArCH), 122.4 (CHCSCN), 123.0 (ArCH), 127.2 (ArCH),
141.6 (CSCNCH), 166.8 (SC(N)N), 173.7 (NHC(O)C). MS (ESI)
m/z 342 (M þ H)þ.
1.86 (s, C(CH2CHCH2)3), 1.99 (s, C(CH2CHCH2)3), 3.46 (s,
NCH3), 7.10-7.22 (m, CHCHCF and CFCHCS), 7.54 (dd, J=
2.5, 8.8 Hz, CNCHCH), 9.82 (s, NNHC(O)). 13C NMR (DMSO-
d6) δ: 28.2 (C(CH2CHCH2)3), 31.1 (NCH3), 36.7 (C(CH2CH-
CH2)3), 39.2 (C(CH2CHCH2)3), 40.3 (C(CH2CHCH2)3), 110.2
(d, J=27.7 Hz, CHCHCF), 110.6 (d, J=8.6 Hz, CNCHCH), 113.6
(d, J=23.6 Hz, CFCHCS), 123.8 (d, J=10.0 Hz, CHCSCN), 138.1
(CSCNCH), 157.8 (d, J=237.4 Hz, CHCFCH), 166.4 (SC(N)N),
173.5 (NHC(O)C). MS (ESI) m/z 360 (M þ H)þ.
Adamantane-1-carboxylic Acid (6-Trifluoromethyl-3-methyl-
3H-benzothiazol-2-ylidine)hydrazide (4f). Prepared from 6-tri-
fluoromethyl-2-(methylthio)benzothiazole (3f) according to
1
method B; mp 206.1-206.6 °C. H NMR (DMSO-d6) δ: 1.67
Adamantane-1-carboxylic Acid (4-Fluoro-3-methyl-3H-benzo-
thiazol-2-ylidine)hydrazide (4a). Prepared from 4-fluoro-2-(methyl-
thio)benzothiazole (3a) according to method B; mp 211.0-
211.8 °C. 1H NMR (DMSO-d6) δ: 1.70 (s, C(CH2CHCH2)3),
1.90 (s, C(CH2CHCH2)3), 2.00 (s, C(CH2CHCH2)3), 3.66 (d, J=
2.9 Hz, NCH3), 7.03(ddd, J=3.7, 7.8, 8.2 Hz, CHCHCH), 7.15(dd,
J=8.3, 13.0 Hz, CFCHCH), 7.38 (d, J=7.7 Hz, CHCHCS), 9.89
(s, NNHC(O)). 13C NMR (DMSO-d6) δ: 28.2 (C(CH2CHCH2)3),
33.5 (d, J = 8.9 Hz, NCH3), 36.7 (C(CH2CHCH2)3), 39.2
(C(CH2CHCH2)3), 40.4 (C(CH2CHCH2)3), 114.5 (d, J=19.5 Hz,
CFCHCH), 119.1 (CHCSCN), 122.5 (d, J=7.0 Hz, CHCHCH),
125.1 (d, J=3.6 Hz, CHCHCS), 128.8 (d, J=9.2 Hz, CSCNCF),
148.0 (d, J=243.3 Hz, CF), 166.2 (SC(N)N), 173.5 (NHC(O)C).
MS (ESI) m/z 360 (M þ H)þ.
(s, C(CH2CHCH2)3), 1.86 (s, C(CH2CHCH2)3), 1.97 (s, C(CH2-
CHCH2)3), 3.50 (s, NCH3), 7.32 (d, J=8.4 Hz, CNCHCH), 7.60
(d, J=8.4 Hz, CHCHC(CF3)), 7.97 (s, C(CF3)CHCS), 9.90 (s,
NNHC(O)). 13C NMR (DMSO-d6) δ: 28.2 (C(CH2CHCH2)3),
31.3 (NCH3), 36.7 (C(CH2CHCH2)3), 39.2 (C(CH2CHCH2)3),
40.4 (C(CH2CHCH2)3), 110.1 (CNCHCH), 120.0 (d, J=4.0 Hz,
CHCHC(CF3)), 122.3 (q, J=32.0 Hz, C(CF3)), 123.6 (CHC-
SCN), 124.4 (d, J = 4.0 Hz, C(CF3)CHCS), 124.9 (q, J =
271.3 Hz, CF3), 144.5 (CSCNCH), 166.1 (SC(N)N), 173.5
(NHC(O)C). MS (ESI) m/z 410 (M þ H)þ.
Adamantane-1-carboxylic Acid (6-Trifluoromethoxy-3-methyl-
3H-benzothiazol-2-ylidine)hydrazide (4g). Prepared from 6-tri-
fluoromethoxy-2-(methylthio)benzothiazole (3g) according to
1
method B; mp 170.1-170.5 °C. H NMR (DMSO-d6) δ: 1.70
Adamantane-1-carboxylic Acid (4-Trifluoromethyl-3-methyl-
3H-benzothiazol-2-ylidine)hydrazide (4b). Prepared from 4-trifluo-
romethyl-2-(methylthio)benzothiazole (3b) according to method
(s, C(CH2CHCH2)3), 1.89 (s, C(CH2CHCH2)3), 2.00 (s, C(CH2-
CHCH2)3), 3.50 (s, NCH3), 7.24-7.30 (m, CNCHCH and
CHCHC(OCF3)), 7.72 (s, C(OCF3)CHCS), 9.88 (s, NNHC(O)).
13C NMR (DMSO-d6) δ: 28.3 (C(CH2CHCH2)3), 31.4 (NCH3),
36.8 (C(CH2CHCH2)3), 39.4 (C(CH2CHCH2)3), 40.4 (C(CH2-
CHCH2)3), 110.8 (CHCHC(OCF3)), 116.7 (CNCHCH), 120.4
(C(OCF3)CHCS), 120.9 (q, J=255.8, OCF3), 124.1 (CHCSCN),
140.8 (CSCNCH), 143.1 (C(OCF3)), 166.4 (SC(N)N), 173.6
(NHC(O)C). MS (ESI) m/z 426 (M þ H)þ.
1
B; mp 203.5-204.4 °C. H NMR (CDCl3-CD3OD) δ: 1.66 (s,
C(CH2CHCH2)3), 1.89 (s, C(CH2CHCH2)3), 1.98 (s, C(CH2CH-
CH2)3), 3.65 (s, NCH3), 7.01 (dd, J=8.0, 8.0 Hz, CHCHCH), 7.42
(d, J=7.6 Hz, C(CF3)CHCH), 7.48 (d, J=8.0 Hz, CHCHCS). 13
C
NMR (CDCl3-CD3OD) δ: 28.0 (C(CH2CHCH2)3), 34.6 (q, J=
7.0 Hz, NCH3), 36.4 (C(CH2CHCH2)3), 39.0 (C(CH2CHCH2)3),
40.6 (C(CH2CHCH2)3), 113.3 (q, J = 32.6 Hz, C(CF3)), 120.9
(CHCHCH), 123.5 (q, J= 271.8 Hz, CF3), 125.2 (ArC), 125.3
(ArC), 125.4 (ArC), 125.8 (ArC), 138.5 (CSCNC(CF3)), 167.8
(SC(N)N), 175.2 (NHC(O)C). MS (ESI) m/z 410 (M þ H)þ.
Adamantane-1-carboxylic Acid (4,6-Difluoro-3-methyl-3H-
benzothiazol-2-ylidine)hydrazide (4c). Prepared from 4,6-di-
fluoro-2-(methylthio)benzothiazole (3c) according to method B;
Adamantane-1-carboxylic Acid (7-Fluoro-3-methyl-3H-benzo-
thiazol-2-ylidine)hydrazide (4h). Prepared from 7-fluoro-2-(methyl-
thio)benzothiazole (3h) according to method B; mp 223.7-
1
224.0 °C. H NMR (DMSO-d6) δ: 1.70 (s, C(CH2CHCH2)3),
1.89 (s, C(CH2CHCH2)3), 2.01 (s, C(CH2CHCH2)3), 3.51
(s, NCH3), 6.97 (dd, J=8.9, 8.7 Hz, CHCHCF), 7.10 (d, J=
8.1 Hz, CNCHCH), 7.36 (ddd, J=8.2, 8.2, 6.0 Hz, CHCHCH),
9.95 (s, NNHC(O)). 13C NMR (DMSO-d6) δ: 28.2 (C(CH2-
CHCH2)3), 31.7 (NCH3), 36.7 (C(CH2CHCH2)3), 39.2 (C(CH2-
CHCH2)3), 40.4 (C(CH2CHCH2)3), 106.7 (d, J = 2.8 Hz,
CNCHCH), 108.3 (d, J = 24.3 Hz, CFCSCN), 108.5 (d, J =
18.6 Hz, CHCHCF), 128.9 (d, J=7.9 Hz, CHCHCH), 143.9 (d,
J=7.0 Hz, CSCNCH), 156.5 (d, J=242.3 Hz, CHCFCS), 165.5
(SC(N)N), 173.6 (NHC(O)C). MS (ESI) m/z 360 (M þ H)þ.
Adamantane-1-carboxylic Acid (5,6-difluoro-3-methyl-3H-
benzothiazol-2-ylidine)hydrazide (4i). Prepared from 5,6-di-
fluoro-2-(methylthio)benzothiazole (3i) according to method B;
mp 145.0-145.9 °C. NMR (DMSO-d6) δ: 1.68 (s, C(CH2CH-
CH2)3), 1.85 (s, C(CH2CHCH2)3), 1.99 (s, CCH2CHCH2)3), 3.45
(s, NCH3), 7.43 (dd, J=6.7, 11.6 Hz, CNCHCF), 7.75 (dd, J=7.8,
10.1 Hz, CFCHCS), 9.85 (s, NNHC(O)). 13C NMR (CDCl3-
CD3OD) δ: 28.2 (C(CH2CHCH2)3), 31.1 (NCH3), 36.5 (C(CH2-
CHCH2)3), 39.1 (C(CH2CHCH2)3), 40.6 (C(CH2CHCH2)3), 99.1
(d, J=23.8 Hz, CNCHCF), 110.9 (d, J=22.6 Hz, CFCHCS), 117.2
(d, J=4.9 Hz, CHCSCN), 137.5 (d, J=9.7 Hz, CSCNCH), 145.8
(dd, J=14.0, 243.5 Hz, CFCFCHCS), 149.7 (dd, J=14.0, 245.8
Hz, CHCFCF), 167.1 (SC(N)N), 175.2 (NHC(O)C). MS (ESI)
m/z 378 (M þ H)þ.
1
mp 166.3-167.3 °C. H NMR (CDCl3) δ: 1.76 (s, C(CH2CH-
CH2)3), 1.99 (s, C(CH2CHCH2)3), 2.08 (s, C(CH2CHCH2)3), 3.75
(s, NCH3), 6.79 (d,d,d, J=1.9, 9.7, 9.7 Hz, CFCHCF), 6.89 (d,
J=7.0 Hz, CFCHCS), 7.94 (s, NNHC(O)). 13C NMR (CDCl3-
CD3OD) δ: 28.2 (C(CH2CHCH2)3), 33.5 (d, J=9.2 Hz, NCH3),
36.6 (C(CH2CHCH2)3), 39.1 (C(CH2CHCH2)3), 40.7 (C(CH2-
CHCH2)3), 103.1 (dd, J=24.4, 27.1 Hz, CFCHCF), 105.5 (dd,
J = 4.0, 26.6 Hz, CFCHCS), 125.6 (ArC), 125.7 (ArC), 147.7
(dd, J=12.2, 248.4 Hz, CNCFCH), 157.3 (dd, J=11.0, 244.5 Hz,
CHCFCH), 167.1 (SC(N)N), 175.5 (NHC(O)C). MS (ESI) m/z
410 (M þ H)þ.
Adamantane-1-carboxylic Acid (5-Trifluoromethyl-3-methyl-
3H-benzothiazol-2-ylidine)hydrazide (4d). Prepared from 5-trifluo-
romethyl-2-(methylthio)benzothiazole (3d) according to method
B; mp 239.8-240.1 °C. 1H NMR (DMSO-d6) δ: 1.70 (s, C(CH2-
CHCH2)3), 1.89 (s, C(CH2CHCH2)3), 2.01 (s, C(CH2CHCH2)3),
3.56 (s, NCH3), 7.39 (d, J = 8.2 Hz, C(CF3)CHCH), 7.53
(s, CNCHC(CF3)), 7.79 (d, J = 8.1 Hz, CHCHCS), 9.94 (s,
NNHC(O)). 13C NMR (DMSO-d6) δ: 28.2 (C(CH2CHCH2)3),
31.3 (NCH3), 36.7 (C(CH2CHCH2)3), 39.2 (C(CH2CHCH2)3),
40.9 (C(CH2CHCH2)3), 106.7 (CNCHC(CF3)), 118.5 (C(CF3)-
CHCH, 123.5 (CHCSCN), 124.9 (q, J=272.3 Hz, CF3), 127.5
(CHCHCS), 127.8 (q, J=31.7 Hz, (C(CF3)), 142.0 (CSCNCH),
166.0 (SC(N)N), 173.5 (NHC(O)C). MS (ESI) m/z 410 (M þ H)þ.
Adamantane-1-carboxylic Acid (6-Fluoro-3-methyl-3H-benzo-
thiazol-2-ylidine)hydrazide (4e). Prepared from 6-fluoro-2-(methyl-
thio)benzothiazole (3e) according to method B; mp 227.5-
229.0 °C. 1H NMR (DMSO-d6) δ: 1.68 (s, C(CH2CHCH2)3),
Adamantane-1-carboxylic Acid (7-Trifluoromethyl-3-methyl-
3H-benzothiazol-2-ylidine)hydrazide (4j). Prepared from 7-trifluo-
romethyl-2-(methylthio)benzothiazole (3j) according to method B;
1
mp 189.2-189.4 °C. H NMR (CDCl3) δ: 1.77 (s, C(CH2CH-
CH2)3), 2.01 (s, C(CH2CHCH2)3), 2.09 (s, C(CH2CHCH2)3), 3.59
(s, NCH3), 7.11 (d, J=7.9 Hz, CHCHC(CF3)), 7.29 (d, J=7.7 Hz,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 893
CNCHCH), 7.39 (dd, J = 7.8, 7.9 Hz, CHCHCH), 7.90 (s,
NNHC(O)). 13C NMR (CDCl3) δ: 28.2 (C(CH2CHCH2)3), 31.3
(NCH3), 36.5 (C(CH2CHCH2)3), 39.2 (C(CH2CHCH2)3), 40.7
(C(CH2CHCH2)3), 112.2 (CNCHCH), 118.9 (q, J = 4.3 Hz,
CHCHC(CF3)), 120.5 (C(CF3)CSCN), 123.5 (q, J = 273.1 Hz,
CF3), 124.5 (q, J=33.6 Hz, C(CF3)), 127.1 (CHCHCH), 142.5
(CSCNCH), 164.9 (SC(N)N), 174.7 (NHC(O)C). MS (ESI) m/z
410 (M þ H)þ.
heated at 80 °C for 7 h then concentrated in vacuo. The residue
was partitioned between EtOAc (50 mL) and H2O (30 mL). The
organic phase was washed with brine (30 mL), dried (Na2SO4),
and concentrated. Recrystallization from EtOAc afforded
268 mg (0.823 mmol, 82%) of 4m as a white solid; mp 157.3-
1
159.7 °C. H NMR (CDCl3-CD3OD) δ: 1.67 (s, C(CH2CH-
CH2)3), 1.88 (s, C(CH2CHCH2)3), 1.99 (s, C(CH2CHCH2)3),
3.28 (s, NCH3), 6.81 (d, J=7.6 Hz, CNCHCH), 6.91 (dd, J=7.7,
7.8 Hz, CNCHCHCH), 7.05-7.10 (m, CHCHCHCO and
CHCHCO). 13C NMR (CDCl3-CD3OD) δ: 28.0 (C(CH2CH-
CH2)3), 28.4 (NCH3), 36.4 (C(CH2CHCH2)3), 39.0 (C(CH2CH-
CH2)3), 40.3 (C(CH2CHCH2)3), 107.4 (ArCH), 109.2 (ArCH),
120.9 (CHCHCH), 124.6 (CHCHCH), 133.1 (COCNCH),
144.7 (CHCOCN), 152.0 (OC(N)N), 173.4 (NHC(O)C). MS
(ESI) m/z 326 (M þ H)þ.
Adamantane-1-carboxylic Acid (3-Ethyl-3H-benzothiazol-
2-ylidine)hydrazide (4k). A solution of 2-(methylthio)benzothi-
azole (1.24 g, 6.84 mmol) in 20.0 mL of 1,2-dichloroethane
was treated with ethyl trifluoromethanesulfonate (0.975 mL,
7.52 mmol) and the reaction was stirred at rt for 6 h. The
reaction mixture was concentrated in vacuo and dried under
vacuum. The crude triflate salt was dissolved in 20 mL of
ethanol and added to a solution of adamantane-1-carboxylic
acid hydrazide (1.59 g, 8.21 mmol) and triethylamine (1.72 mL,
12.3 mmol) in 20 mL of ethanol. The reaction mixture was
heated at 80 °C for 8 h and then concentrated in vacuo. The
residue was partitioned between EtOAc (200 mL) and H2O
(120 mL). The organic phase was washed with brine (120 mL),
dried (Na2SO4), and concentrated. Chromatography on silica
(4:1 EtOAc/hexane eluent) afforded 1.65 g (4.64 mmol, 68%) of
(6-Trifluoromethoxy-3-methyl-3H-benzothiazol-2-ylidine)-
hydrazone (5). A solution of 6-trifluoromethoxy-2-(methyl-
thio)benzothiazole (3g) (3.32 g, 12.53 mmol) in 25 mL of
1,2-dichloroethane was treated with methyl trifluoromethane-
sulfonate (1.70 mL, 15.04 mmol) and the reaction was stirred at
rt for 2 h. The reaction mixture was concentrated in vacuo and
dried under vacuum. The crude triflate salt was dissolved in
50 mL of ethanol and slowly added dropwise to a solution of
hydrazine (1.97 mL, 62.6 mmol) in 50 mL of ethanol. After
addition was complete, the reaction mixture was stirred at rt for
1 h. Most of the ethanol was removed in vacuo and the residue
was dissolved in CHCl3 (500 mL), washed with satd NaHCO3
solution (250 mL), brine (250 mL), dried (Na2SO4), and con-
centrated in vacuo. Chromatography on silica (1/4 CH3CN/
CHCl3 eluent) afforded 2.80 g (10.64 mmol, 85%) of 5 as a white
solid; mp 113.4-113.6. 1H NMR (DMSO-d6) δ: 3.34 (s, NCH3),
5.10 (s, NNH2), 7.04 (d, J = 8.7 Hz, CNCHCH), 7.18 (ddd,
J = 0.8, 2.3, 8.6 Hz, CHCHC(OCF3)), 7.58 (d, J = 1.7 Hz,
C(OCF3)CHCS). 13C NMR (DMSO-d6) δ: 30.9 (NCH3), 109.0
(CHCHC(OCF3)), 116.4 (CNCHCH), 119.8 (C(OCF3)CHCS),
120.8 (q, J = 256.1 Hz, C(OCF3)), 124.8 (CHCSCN), 141.4
(CSCNCH), 142.5 (q, J=2.1 Hz, C(OCF3)), 157.0 (SC(N)N).
MS (ESI) m/z 264 (M þ H)þ.
1
4k as a white solid; mp 162.6-164.2 °C. H NMR (CDCl3-
CD3OD) δ: 1.24 (t, J=7.1 Hz, NCH2CH3), 1.66 (s, C(CH2CH-
CH2)3), 1.88 (s, C(CH2CHCH2)3), 1.97 (s, C(CH2CHCH2)3),
3.99 (q, J=7.2 Hz, NCH2CH3), 6.87-6.96 (m, CNCHCH and
CHCHCS), 7.18 (dd, J = 7.8, 7.8 Hz, CNCHCH), 7.24 (d,
J = 7.6 Hz, CHCHCS). 13C NMR (CDCl3-CD3OD) δ: 11.7
(NCH2CH3), 28.1 (C(CH2CHCH2)3), 36.4 (C(CH2CHCH2)3),
39.0 (C(CH2CHCH2)3), 39.1 (NCH2CH3), 40.5 (C(CH2CH-
CH2)3), 109.4 (CNCHCH), 121.6 (ArCH), 122.1 (ArCH), 122.4
(CHCSCN), 126.6 (CNCHCHCH), 140.2 (CSCNCH), 166.4
(SC(N)N), 175.0 (NHC(O)C). MS (ESI) m/z 356 (M þ H)þ.
Adamantane-1-carboxylic Acid (3-Methyl-3H-benzothiazol-
2-ylidine)hydrazide (4l). A solution of 2-(methylthio)benzothia-
zole (181 mg, 1.00 mmol) in 3.0 mL of 1,2-dichloroethane
was treated with methyl trifluoromethanesulfonate(121 μL,
1.10 mmol) and the reaction was stirred at rt for 1 h. The
reaction mixture was concentrated in vacuo and dried under
vacuum. The crude triflate salt was dissolved in 5 mL of ethanol
and added to a solution of adamantane-1-carboxylic acid
N-methylhydrazide (2) (250 mg, 1.20 mmol) and triethylamine
(250 μL, 1.80 mmol) in 5.0 mL of ethanol. The reaction mixture
was heated at 80 °C for 8 h then concentrated in vacuo. The
residue was partitioned between EtOAc (50 mL) and H2O
(30 mL). The organic phase was washed with brine (30 mL),
dried (Na2SO4), and concentrated. Chromatography on silica
(5-33% EtOAc/hexane eluent) afforded 78.7 mg (0.22 mmol,
22%) of 4l as a white solid; mp 199.0-203.6 °C. 1H NMR
(CDCl3-CD3OD) δ: 1.57 (s, C(CH2CHCH2)3), 1.87 (s, C(CH2-
CHCH2)3), 1.94 (s, C(CH2CHCH2)3), 3.08 (s, NN(CH3)C(O)),
3.53 (s, NCH3), 6.99-7.06 (m, CNCHCH and CHCHCS), 7.27
(ddd, J = 7.7, 7.8 Hz, CNCHCH), 7.34 (d, J = 7.8 Hz, CH-
CHCS). 13C NMR (CDCl3-CD3OD) δ: 28.3 (C(CH2CHCH2)3),
30.7 (NCH3), 36.7 (C(CH2CHCH2)3), 36.9 (NN(CH3)C(O)), 37.5
(C(CH2CHCH2)3), 42.3 (C(CH2CHCH2)3), 109.7 (CNCHCH),
122.1 (ArCH), 122.3 (ArCH), 122.4 (CHCSCN), 126.8 (ArCH),
141.1 (CSCNCH), 168.8 (SC(N)N), 178.4 (NHC(O)C). MS (ESI)
m/z 356 (M þ H)þ.
Representative Procedure for Conversion of (6-Trifluoro-
methoxy-3-methyl-3H-benzothiazol-2-ylidine)hydrazone (5) to
Carboxylic Acid (6-Trifluoromethoxy-3-methyl-3H-benzothiazol-
2-ylidine)hydrazides (Method C). Cyclohexylcarboxylic Acid
(6-Trifluoromethoxy-3-methyl-3H-benzothiazol-2-ylidine)-
hydrazide (6a). A solution of (6-trifluoromethoxy-3-methyl-
3H-benzothiazol-2-ylidine)hydrazone (5) (80.5 mg, 0.306 mmol)
and K2CO3 (47 mg, 0.336 mmol) in 6 mL of 9:1 CH3CN/
H2O was treated with cyclohexanecarbonyl chloride (45 μL,
0.336 mmol) and stirred at rt for 1 h. The reaction mixture was
concentrated in vacuo. The residue was partitioned between
EtOAc (15 mL) and water (15 mL). The organic phase was
washed with 1 M HCl solution (15 mL) and brine (15 mL) and
then dried (Na2SO4) and concentrated in vacuo. Chromato-
graphy on silica (15-25% CH3CN/CHCl3 eluent) afforded
82.7 mg (0.221 mmol, 72%) of 6a as a white solid; mp 198.6-
199.3. 1H NMR (DMSO-d6) δ:1.03-1.50(m,5AlkylH), 1.56-1.78
(m, 5 AlkylH), 2.20 (m, NHC(O)CH), 3.43 (s, NCH3), 7.19-7.29
(m, CNCHCH and CHCHC(OCF3)), 7.68 (s, C(OCF3)CHCS),
10.11 (s, NNHC(O)). 13C NMR (DMSO-d6) δ:25.9(C(O)CHCH2-
CH2), 26.1 (C(O)CHCH2CH2CH2), 30.0 (C(O)CHCH2), 31.3
(NCH3), 43.3 (NHC(O)CH), 110.7 (CHCHC(OCF3)), 116.6
(CNCHCH), 120.4 (C(OCF3)CHCS), 120.9 (q, J=255.5, OCF3),
124.0 (CHCSCN), 140.8 (CSCNCH), 143.1 (C(OCF3)), 164.9
(SC(N)N), 172.0 (NHC(O)C). MS (ESI) m/z 374 (M þ H)þ.
Cyclopentylcarboxylic Acid (6-Trifluoromethoxy-3-methyl-
3H-benzothiazol-2-ylidine)hydrazide (6b). Prepared from (6-tri-
fluoromethoxy-3-methyl-3H-benzothiazol-2-ylidine)hydrazone
(5) and cyclopentanecarbonyl chloride according to method
C; mp 215.5-216.0. 1H NMR (DMSO-d6) δ: 1.50-1.82 (m,
8 AlkylH), 2.63-2.72 (m, NHC(O)CH), 3.47 (s, NCH3), 7.23-
7.30 (m, CNCHCH and CHCHC(OCF3)), 7.72 (d, J=0.9 Hz,
Adamantane-1-carboxylic Acid (3-methyl-3H-benzoxazol-
2-ylidine)hydrazide (4m). A solution of 3-methyl-2(3H)-benzox-
azolethione (165 mg, 1.00 mmol) in 3.0 mL of 1,2-dichloro-
ethane was treated with methyl trifluoromethanesulfonate
(136 μL, 1.20 mmol) and the reaction was stirred at rt for 1 h.
The reaction mixture was concentrated in vacuo and dried under
vacuum. The crude triflate salt was dissolved in 5 mL of ethanol
and added to a solution of adamantane-1-carboxylic acid
hydrazide (233 mg, 1.20 mmol) and triethylamine (250 μL,
1.80 mmol) in 5.0 mL of ethanol. The reaction mixture was

894 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Fritch et al.
C(OCF3)CHCS), 10.2 (s, NNHC(O)). 13C NMR (DMSO-d6) δ:
26.3 (C(O)CHCH2CH2), 30.6 (C(O)CHCH2CH2), 31.2 (NCH3),
43.3 (NHC(O)CH), 110.6 (CHCHC(OCF3)), 116.5 (CNCHCH),
120.3 (C(OCF3)CHCS), 120.7 (q, J = 255.7 Hz, OCF3), 123.9
(CHCSCN), 140.6 (CSCNCH), 142.9 (C(OCF3)), 164.7 (SC(N)-
N), 172.1 (NHC(O)C). MS (ESI) m/z 360 (M þ H)þ.
123.8 (CHCSCN), 140.7 (CSCNCH), 142.9 (C(OCF3), 164.6, 168.9
(SC(N)N and NHC(O)C). MS (ESI) m/z 348 (M þ H)þ.
2-Tetrahydrofuroic Acid (6-Trifluoromethoxy-3-methyl-3H-
benzothiazol-2-ylidine)hydrazide (6h). Prepared from (6-trifluoro-
methoxy-3-methyl-3H-benzothiazol-2-ylidine)hydrazone (5) and
2-tetrahydrofuroyl chloride40 according to method C; mp 169.7-
170.0 °C. 1H NMR (CDCl3) δ: 1.96-2.05 (m, OCH2CH2),
2.23-2.42 (m, OCH2CH2CH2), 3.59 (s, NCH3), 3.96 (m, OCHH0-
CH2), 4.07 (m OCHH0CH2), 4.58 (dd, J=5.3, 8.4 Hz, C(O)CH),
6.97 (d, J = 8.8 Hz, CNCHCH), 7.20 (d, J = 8.8 Hz, CHCH-
C(OCF3)), 7.29 (s, C(OCF3)CHCS), 8.60 (s, NNHC(O)). 13C
NMR (CDCl3-CD3OD) δ: 25.2 (OCH2CH2), 30.2 (NCH3),
30.6 (OCH2CH2CH2), 69.4 (OCH2CH2), 77.8 (C(O)CH), 109.7
(CHCHC(OCF3), 115.5 (CNCHCH), 120.0 (C(OCF3)CHCS),
120.3 (q, J = 256.6 Hz, OCF3), 123.0 (CHCSCN), 139.8
(CSCNCH), 143.5 (C(OCF3), 165.9, 170.3 (SC(N)N and NHC-
(O)C). MS (ESI) m/z 362 (M þ H)þ.
2,2-Dimethylpropanoic Acid (6-Trifluoromethoxy-3-methyl-
3H-benzothiazol-2-ylidine)hydrazide (6c). Prepared from (6-tri-
fluoromethoxy-3-methyl-3H-benzothiazol-2-ylidine)hydrazone
(5) and trimethylacetyl chloride according to method C except
that the reaction mixture was heated at 60 °C for 4 h; mp
214.7-216.9. 1H NMR (CDCl3) δ: 1.29 (s, C(O)C(CH3)3),
3.50 (s, NCH3), 6.89 (d, J = 8.8 Hz, CNCHCH), 7.11 (d,
J = 8.9 Hz, CHCHC(OCF3)), 7.19 (s, C(OCF3)CHCS), 7.85
(s, NNHC(O)). 13C NMR (CDCl3) δ: 27.5 (C(O)C(CH3)3),
31.2 (NCH3), 38.9 (C(O)C(CH3)3), 109.6 (CHCHC(OCF3)),
115.9 (CNCHCH), 120.2 (C(OCF3)CHCS), 120.7 (q, J =
256.9 Hz, OCF3), 123.8 (CHCSCN), 140.1 (CSCNCH), 143.8
(C(OCF3)), 166.2 (SC(N)N), 175.1 (NHC(O)C). MS (ESI) m/z
348 (M þ H)þ.
3-Tetrahydrofuroic Acid (6-Trifluoromethoxy-3-methyl-3H-
benzothiazol-2-ylidine)hydrazide (6i). Prepared from (6-trifluoro-
methoxy-3-methyl-3H-benzothiazol-2-ylidine)hydrazone (5) and
3-tetrahydrofuroyl chloride40 according to method C; mp 199.4-
199.6 °C. 1H NMR (DMSO-d6, 60 °C) δ: 2.00-2.08 (m, OCH2-
CH2), 2.96-3.12 (m, C(O)CH), 3.45 (s, NCH3), 3.65-3.80
(m, OCH2CH2 and OCHH0CH), 3.90 (dd, J = 8.1, 16.3 Hz,
OCHH0CH), 7.19-7.25 (m, CNCHCH and CHCHC(OCF3)),
7.65 (s, C(OCF3)CHCS), 10.2 (s, NNHC(O)). 13C NMR (DMSO-
d6, 60 °C) δ: 30.6 (NCH3), 31.4 (OCH2CH2), 43.3 (C(O)CH), 68.3
(OCH2CH2), 70.9 (OCH2CH), 110.8 (CHCHC(OCF3), 116.4
(CNCHCH), 120.3 (C(OCF3)CHCS),120.9(q,J=256.0 Hz, OCF3),
124.1 (CHCSCN), 140.8 (CSCNCH), 143.3 (C(OCF3), 164.7, 169.5
(SC(N)N and NHC(O)C). MS (ESI) m/z 362 (M þ H)þ.
Acetic Acid (6-Trifluoromethoxy-3-methyl-3H-benzothiazol-
2-ylidine)hydrazide (6d). Prepared from (6-trifluoromethoxy-
3-methyl-3H-benzothiazol-2-ylidine)hydrazone (5) and acetyl
1
chloride according to method C; mp 209.0-209.2 °C. H NMR
(DMSO-d6, 60 °C) δ: 1.93 (s, NHC(O)CH3), 3.48 (s, NCH3),
7.22-7.30 (m, CNCHCH and CHCHC(OCF3)), 7.70 (s, C(OCF3)-
CHCS), 10.02 (s, NNHC(O)). 13C NMR (DMSO-d6, 60 °C) δ: 21.7
(C(O)CH3), 31.4 (NCH3), 110.7 (CHCHC(OCF3)), 116.4 (CNCH-
CH), 120.3 (C(OCF3)CHCS), 120.9 (q, J=256.0 Hz, OCF3), 124.1
(CHCSCN), 140.8 (CSCNCH), 143.2 (C(OCF3)), 163.9, 166.0
(SC(N)N and NHC(O)C). MS (ESI) m/z 306 (M þ H)þ.
3-Fluorobenzoic Acid (6-Trifluoromethoxy-3-methyl-3H-
benzothiazol-2-ylidine)hydrazide (6j). Prepared from (6-trifluoro-
methoxy-3-methyl-3H-benzothiazol-2-ylidine)hydrazone (5) and
3-fluorobenzoyl chloride according to method C; mp 151.2-
3,3-Dimethylbutanoic Acid (6-Trifluoromethoxy-3-methyl-
3H-benzothiazol-2-ylidine)hydrazide (6e). Prepared from (6-tri-
fluoromethoxy-3-methyl-3H-benzothiazol-2-ylidine)hydrazone
(5) and tert-butylacetyl chloride according to method C; mp
1
1
151.6 °C. H NMR (CDCl3-CD3OD) δ: 3.39 (s, NCH3), 6.83
209.0-209.4 °C. H NMR (DMSO-d6) δ: 1.01 (s, C(O)CH2-
(d, J=8.9 Hz, CNCHCH), 7.01-7.12 (m, 3 ArH), 7.26 (ddd, J=
2.3, 7.8, 7.9 Hz, ArH), 7.51 (d, J=9.3 Hz, ArH), 7.58 (d, J=7.8 Hz,
ArH). 13C NMR (CDCl3-CD3OD) δ: 30.8 (NCH3), 109.6
(CHCHC(OCF3), 114.5 (d, J = 23.2 Hz, C(O)CCHCF), 115.6
(CNCHCH), 118.5 (d, J = 21.3 Hz, C(O)CCHCFCH), 120.1
(C(OCF3)CHCS), 120.4 (q, J=256.9 Hz, OCF3), 123.0 (d, J=
2.7 Hz, C(O)CCHCH), 123.4 (CHCSCN), 130.1 (d, J=7.9 Hz,
C(O)CCHCFCHCH), 135.3 (d, J = 7.0 Hz, C(O)C), 139.7
(CSCNCH), 143.6 (q, J = 2.2 Hz, C(OCF3), 162.5 (d, J =
247.2 Hz, CF), 164.4, 166.6 (SC(N)N and NHC(O)C). MS (ESI)
m/z 386 (M þ H)þ.
C(CH3)3), 2.03 (s, C(O)CH2C(CH3)3), 3.44 (s, NCH3), 7.18-
7.32 (m, CNCHCH and CHCHC(OCF3)), 7.72 (s, C(OCF3)-
CHCS), 10.1 (s, NNHC(O)). 13C NMR (DMSO-d6) δ: 30.5
(C(O)CH2C(CH3)3), 31.3 (C(O)CH2C(CH3)3), 31.4 (NCH3),
47.9 (C(O)CH2C(CH3)3), 110.8 (CHCHC(OCF3), 116.7 (CNC-
HCH), 120.5 (C(OCF3)CHCS), 120.9 (d, J=256.7 Hz, OCF3),
124.0 (CHCSCN), 140.8 (CSCNCH), 143.1 (C(OCF3)), 164.5,
167.7 (SC(N)N and NHC(O)C). MS (ESI) m/z 362 (M þ H)þ.
Cyclopentylacetic Acid (6-Trifluoromethoxy-3-methyl-3H-
benzothiazol-2-ylidine)hydrazide (6f). Prepared from (6-trifluoro-
methoxy-3-methyl-3H-benzothiazol-2-ylidine)hydrazone (5) and
cyclopentylacetyl chloride according to method C; mp 214.7-
215.3 °C. 1HNMR(DMSO-d6, 80°C) δ:1.16-1.32 (m, 2 AlkylH),
1.45-1.70 (m, 4 AlkylH), 1.73-1.85 (m, 2 AlkylH), 2.18-2.30 (m,
3 AlkylH), 3.47 (s, NCH3), 7.18-7.30 (m, CNCHCH and CHCH-
C(OCF3)), 7.65 (s, C(OCF3)CHCS), 9.90 (bs, NNHC(O)). 13C
NMR (DMSO-d6, 80 °C) δ: 25.3 (C(O)CH2CHCH2CH2), 31.5
(NCH3), 32.8 (C(O)CH2CHCH2CH2), 37.4 (C(O)CH2CHCH2-
CH2), 41.5 (C(O)CH2CHCH2CH2), 110.7 (CHCHC(OCF3),
116.4 (CNCHCH), 120.2 (C(OCF3)CHCS), 121.0 (q, J =
255.9 Hz, OCF3), 124.3 (CHCSCN), 140.9 (CSCNCH), 143.4
(C(OCF3), 164.3, 168.7 (SC(N)N and NHC(O)C). MS (ESI) m/z
374 (M þ H)þ.
4-Fluorobenzoic Acid (6-Trifluoromethoxy-3-methyl-3H-
benzothiazol-2-ylidine)hydrazide (6k). Prepared from (6-trifluoro-
methoxy-3-methyl-3H-benzothiazol-2-ylidine)hydrazone (5) and
4-fluorobenzoyl chloride according to method C; mMp 166.9-
168.5 °C. 1H NMR (CDCl3) δ: 3.56 (s, NCH3), 6.94 (d, J=8.8 Hz,
CNCHCH), 7.07-7.21 (m, 3 ArH), 7.24 (s, C(OCF3)CHCS),
7.82-7.90 (m, 2 ArH), 8.25 (s, NNHC(O)). 13C NMR (CDCl3-
CD3OD) δ: 30.8 (NCH3), 109.8 (CHCHC(OCF3), 115.4 (d, J=
22.0 Hz, CFCH), 115.7 (CNCHCH), 120.1 (C(OCF3)CHCS),
120.6 (q, J=256.7 Hz, OCF3), 123.5 (CHCSCN), 129.4 (d, J=
3.1 Hz, C(O)C), 129.8 (d, J = 8.9 Hz, CFCHCH), 139.9
(CSCNCH), 143.7 (q, J = 2.1 Hz, C(OCF3), 164.8 (d, J =
252.1 Hz, CF), 165.0, 166.8 (SC(N)N and NHC(O)C). MS (ESI)
m/z 386 (M þ H)þ.
3-Methylbutanoic Acid (6-Trifluoromethoxy-3-methyl-3H-
benzothiazol-2-ylidine)hydrazide (6g). Prepared from (6-trifluoro-
methoxy-3-methyl-3H-benzothiazol-2-ylidine)hydrazone (5) and
isovaleryl chloride according to method C; mp 189.1-189.4 °C.
1H NMR (DMSO-d6) δ: 0.95 (d, J=15.7, C(O)CH2CH(CH3)2),
1.98-2.10 (m, C(O)CH2CH(CH3)2 and C(O)CH2CH(CH3)2),
3.47 (s, NCH3), 7.24-7.34 (m, CNCHCH and CHCHC(OCF3)),
7.75 (s, C(OCF3)CHCS), 10.2 (s, NNHC(O)). 13C NMR (DMSO-
d6) δ: 22.9 (C(O)CH2CH(CH3)2), 26.2 C(O)CH2CH(CH3)2), 31.3
(NCH3), 43.7 (C(O)CH2CH(CH3)2), 110.7 (CHCHC(OCF3), 116.5
(CNCHCH), 120.4 (C(OCF3)CHCS), 120.5 (d, J=255.6 Hz, OCF3),
Benzoic Acid (6-Trifluoromethoxy-3-methyl-3H-benzothiazol-
2-ylidine)hydrazide (6l). Prepared from (6-trifluoromethoxy-
3-methyl-3H-benzothiazol-2-ylidine)hydrazone (5) and benzoyl
chloride according to method C; mp 158.0-158.2 °C. 1H NMR
(DMSO-d6) δ: 3.55 (NCH3), 7.27-7.38 (m, 2 ArH), 7.43-7.57
(m, 3 ArH), 7.74 (d, J=1.2 Hz, C(OCF3)CHCS), 7.88 (d, J=
6.7 Hz, C(O)C(CH)2), 10.96 (s, NNHC(O)). 13C NMR (DMSO-
d6) δ: 31.0 (NCH3), 110.6 (CHCHC(OCF3), 116.2 (CNCHCH),
120.1 (C(OCF3)CHCS), 120.3 (q, J=255.8 Hz, OCF3), 123.5

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 895
(CHCSCN), 127.4 (ArCH), 128.5 (ArCH), 131.4 (C(O)-
CCH), 133.8 (C(O)C), 140.3 (CSCNCH), 142.7 (q, J=1.9 Hz,
C(OCF3)), 163.3, 165.7 (SC(N)N and NHC(O)C). MS (ESI) m/z
368 (M þ H)þ.
methoxy-3-methyl-3H-benzothiazol-2-ylidine)hydrazone (5)
(127 mg, 0.482 mmol) in 2.0 mL of THF was treated with a
solution of di-tert-butyldicarbonate (116 mg, 0.531 mmol) in
1.0 mL of THF. The reaction mixture was stirred at rt for 3 h
and then concentrated. Chromatography on silica (CHCl3 to
15% CH3CN/CHCl3 eluent) afforded 120.5 mg (0.332 mmol,
2-Furoic Acid (6-Trifluoromethoxy-3-methyl-3H-benzothiazol-
2-ylidine)hydrazide (6m). Prepared from (6-trifluoromethoxy-3-
methyl-3H-benzothiazol-2-ylidine)hydrazone (5) and furoyl
chloride41 according to method C; mp 199.4-201.0 °C. 1H
NMR (CDCl3) δ: 3.57 (s, NCH3), 6.56 (dd, J = 1.7, 3.5 Hz,
C(O)CCHCHCH), 6.94 (d, J = 8.9 Hz, CNCHCH), 7.13-
7.27 (m, CHCHC(OCF3), C(O)CCHCHCH and C(O)CCHCH-
CH), 7.48 (s, C(OCF3)CHCS), 8.40 (s, NNHC(O)). 13C NMR
(CDCl3) δ: 30.9 (NCH3), 109.8 (CHCHC(OCF3), 112.1 (C(O)-
CCHCHCH), 115.1 (C(O)CCHCHCH), 115.7 (CNCHCH),
120.2 (C(OCF3)CHCS), 120.4 (q, J=257.0 Hz, OCF3), 123.4
(CHCSCN), 139.9 (CSCNCH), 143.7 (q, J=1.8 Hz, C(OCF3)),
144.6 (C(O)CCHCHCH), 146.6 (C(O)CCHCHCH), 156.4,
166.4 (SC(N)N and NHC(O)C). MS (ESI) m/z 358 (M þ H)þ.
Cyclopentyl (6-Trifluoromethoxy-3-methyl-3H-benzothiazol-
2-ylidine)hydrazine carboxylate (6n). A solution of cyclopenta-
nol (0.290 mL, 3.20 mmol) in 15 mL of CH2Cl2 was treated with
4-nitrophenyl chloroformate (645 mg, 3.20 mmol) and pyridine
(0.259 mL, 3.20 mmol) and stirred at rt for 15 h. The reaction
mixture was washed with brine (2 ꢀ 10 mL), dried (Na2SO4),
and concentrated. Chromatography on silica gel (hexanes to
20% EtOAc/hexanes eluent) afforded 508 mg (2.02 mmol, 63%)
of cyclopentyl 4-nitrophenyl carbonate as an colorless oil.
1H NMR (CDCl3) δ: 1.60-2.00 (m, 8 H), 5.20-5.28 (m, 1 H),
7.39 (d, J=9.0 Hz, 1 H), 8.28 (d, J=9.0 Hz, 1 H). A solution of
cyclopentyl 4-nitrophenyl carbonate (403 mg, 1.60 mmol) and
4-methylmorpholine (0.882 mL, 8.02 mmol) in 5.0 mL of DMF
was treated with a solution of (6-trifluoromethoxy-3-methyl-
3H-benzothiazol-2-ylidine)hydrazone (5) (422 mg, 1.60 mmol)
in 5.0 mL of DMF. The reaction mixture was stirred at 75 °C for
15 h, concentrated, and partitioned between EtOAc and water.
The organic phase was washed with brine, dried (Na2SO4),
and concentrated. Chromatography on silica (CHCl3 to 15%
CH3CN/CHCl3 eluent) afforded 448 mg (1.193 mmol, 74%) of
6n as an off-white solid; mp 124.4-125.2 °C. 1H NMR (CDCl3)
δ: 1.53-1.95 (m, OCH(CH2CH2)2), 3.52 (s, NCH3), 5.17-5.26
(m, (OCH(CH2CH2)2), 6.76 (s, NNHC(O)O), 6.91 (d, J =
8.8 Hz, CNCHCH), 7.15 (d, J=2.2, 8.8 Hz, CHCHC(OCF3)),
7.25 (d, J=2.2 Hz, C(OCF3)CHCS). 13C NMR (CDCl3) δ: 23.5
(OCH(CH2CH2)2), 30.9 (NCH3), 32.6 (OCH(CH2CH2)2), 78.5
(OCH(CH2CH2)2), 109.1 (CHCHC(OCF3), 115.6 (CNCHCH),
119.8 (C(OCF3)CHCS), 120.4 (q, J=256.7 Hz, OCF3), 123.7
(CHCSCN), 140.0 (CSCNCH), 143.4 (C(OCF3)), 155.6 (NHC-
(O)O), 165.5 (SC(N)N). MS (ESI) m/z 376 (M þ H)þ.
1
69%) of 6p as a white solid; mp 133.3-133.5 °C. H NMR
(CDCl3) δ: 1.52 (s, OC(CH3)3), 3.59 (s, NCH3), 6.74 (s,
NNHC(O)O), 6.95 (d, J=8.8 Hz, CNCHCH), 7.17 (dd, J=
2.4, 8.8 Hz, CHCHC(OCF3)), 7.29 (d, J=2.2 Hz, C(OCF3)-
CHCS). 13C NMR (CDCl3) δ: 28.2 (OC(CH3)3), 30.8 (NCH3),
80.8 (OC(CH3)3), 109.0 (CHCHC(OCF3), 115.6 (CNCHCH),
119.7 (C(OCF3)CHCS), 120.4 (q, J=256.6 Hz, OCF3), 123.7
(CHCSCN), 140.0 (CSCNCH), 143.3 (C(OCF3)), 154.7
(NHC(O)O), 165.1 (SC(N)N). MS (ESI) m/z 308 (M-C4H8)þ.
Acknowledgment. We acknowledge our late friend and
scientific adviser, Henry Rapoport, for thoughtful ideas and
discussions.
Supporting Information Available: Data for Cerep receptor
binding assays on compounds 6e, 6f, and 6o. This material is
available free of charge via the Internet at http://pubs.acs.org.
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