Neurotrophic 3,9-bis[(alkylthio)methyl]-and-bis(alkoxymethyl)-K-252a derivatives

Article abstract of DOI:10.1021/jm970031d

A series of 3,9-disubstituted [(alkylthio)methyl]- and (alkoxymethyl)- K-252a derivatives was synthesized with the aim of enhancing and separating the neurotrophic properties from the undesirable NGF (trk A kinase) and PKC inhibitory activities of K-252a. Data from this series reveal that substitution in the 3- and 9-positions of K-252a with these groups reduces trk A kinase inhibitory properties approximately 100- to >500-fold while maintaining or in certain cases enhancing the neurotrophic activity. From this research, 3,9-bis[(ethylthio)methyl]-K-252a (8) was identified as a potent and selective neurotrophic agent in vitro as measured by enhancement of choline acetyltransferase activity in embryonic rat spinal cord and basal forebrain cultures. Compound 8 was found to have weak kinase inhibitory activity for trk A, protein kinase C, protein kinase A, and myosin light chain kinase. On the basis of the in vitro profile, 8 was evaluated in in vivo models suggestive of neurological diseases. Compound 8 was active in preventing degeneration of cholinergic neurons of the nucleus basalis magnocellularis (NBM) and reduced developmentally programmed cell death (PCD) of female rat spinal nucleus of the bulbocavernosus motoneurons and embryonic chick lumbar motoneurons.

Full text of DOI:10.1021/jm970031d

J . Med. Chem. 1997, 40, 1863-1869
1863
Neu r otr op h ic 3,9-Bis[(a lk ylth io)m eth yl]- a n d -Bis(a lk oxym eth yl)-K-252a
Der iva tives
Masami Kaneko,† Yutaka Saito,† Hiromitsu Saito,† Tadashi Matsumoto,† Yuzuru Matsuda,† J effry L. Vaught,‡
Craig A. Dionne,§ Thelma S. Angeles,§ Marcie A. Glicksman,§ Nicola T. Neff,§ David P. Rotella,|
J ames C. Kauer,| J ohn P. Mallamo,| Robert L. Hudkins,*,| and Chikara Murakata*,
Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 1188 Shimotogari, Nagaizumi, Sunto,
Shizuoka 411, J apan, Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6, Asahicho, Machida,
Tokyo 194, J apan, and Departments of Medicinal Chemistry, Cell Biology, and Discovery Research, Cephalon Inc.,
145 Brandywine Parkway, West Chester, Pennsylvania 19380
Received J anuary 14, 1997X
A series of 3,9-disubstituted [(alkylthio)methyl]- and (alkoxymethyl)-K-252a derivatives was
synthesized with the aim of enhancing and separating the neurotrophic properties from the
undesirable NGF (trk A kinase) and PKC inhibitory activities of K-252a. Data from this series
reveal that substitution in the 3- and 9-positions of K-252a with these groups reduces trk A
kinase inhibitory properties approximately 100- to >500-fold while maintaining or in certain
cases enhancing the neurotrophic activity. From this research, 3,9-bis[(ethylthio)methyl]-K-
252a (8) was identified as a potent and selective neurotrophic agent in vitro as measured by
enhancement of choline acetyltransferase activity in embryonic rat spinal cord and basal
forebrain cultures. Compound 8 was found to have weak kinase inhibitory activity for trk A,
protein kinase C, protein kinase A, and myosin light chain kinase. On the basis of the in vitro
profile, 8 was evaluated in in vivo models suggestive of neurological diseases. Compound 8
was active in preventing degeneration of cholinergic neurons of the nucleus basalis magno-
cellularis (NBM) and reduced developmentally programmed cell death (PCD) of female rat
spinal nucleus of the bulbocavernosus motoneurons and embryonic chick lumbar motoneurons.
In tr od u ction
degenerative diseases. Clearly, the discovery and de-
velopment of low molecular weight, CNS permeable
molecules with neurotrophic activity represents a valu-
able therapeutic opportunity.
Neurotrophism may be defined as the ability to slow
or prevent compromised neurons from undergoing ne-
crotic or apoptotic cell death and to assist compromised
neurons to maintain or express a functional phenotype.
The classical example of neurotrophic activity is that
displayed by the protein growth factors such as nerve
growth factor (NGF), neurotrophin-3 (NT-3), and brain-
derived neurotrophic factor (BDNF) which mediate
neuronal survival in a variety of models in vitro and in
vivo.1 NGF has distinct, selective survival-promoting
activities for cholinergic neurons in the central nervous
system (CNS), as well as neurite outgrowth-promoting
properties on sympathetic and sensory neurons of the
dorsal root ganglia (DRG).2 Septal forebrain cholinergic
neurons respond to NGF by increasing choline acetyl-
transferase (ChAT) activity.3 Insulin-like growth factor
I (IGF-I), another neurotrophic factor, prevents the loss
of ChAT activity in rat embryonic spinal cord cultures
and reduces the programmed cell death of motoneurons
in vivo during normal development or following axotomy
or spinal transection.4 Although positive clinical results
have been reported with several neurotrophic proteins
such as NGF5 and IGF-I,6 the therapeutic potential of
these polypeptides remains limited due to their size and
pharmacokinetic characteristics, which prevents their
systemic administration for treatment of central neuro-
The development of small molecule therapeutics will
be greatly aided by the understanding of intracellular
pathways which mediate neurotrophic activity. Neuro-
trophic proteins bind to specific cell surface receptors,
initiating intracellular signaling cascades which result
in the regulation of specific gene transcription and
neuronal survival.7 Despite the initial focus on promo-
tion of neuronal survival by protein growth factors, it
is now evident that several pathways may regulate
survival in a manner dependent upon the activation
state of survival-promoting or death/injury-activated
signaling pathways.8 Thus, neurotrophism may result
from regulation of mechanisms in addition to, and
distinct from, growth factor-activated survival path-
ways.
* Address corespondence to: Dr. Robert L. Hudkins, Dept of
Medicinal Chemistry, Cephalon Inc., 145 Brandywine Parkway, West
Chester, PA 19380. E-mail: rhudkins@cephalon.com.
† Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd.
‡ Discovery Research, Cephalon Inc.
K-252a (1, R ) H), an indolocarbazole alkaloid
isolated from Nocardiopsis sp.,9 originally identified as
a protein kinase C (PKC) inhibitor and subsequently
found to inhibit a number of serine/threonine protein
kinases,10 has been reported to possess neurotrophic-
§ Cell Biology, Cephalon Inc.
| Medicinal Chemistry, Cephalon Inc.
Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Co.,
Ltd.
X Abstract published in Advance ACS Abstracts, May 15, 1997.
S0022-2623(97)00031-9 CCC: $14.00 © 1997 American Chemical Society

1864 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Kaneko et al.
Sch em e 1a
a
(a) Ac2O, DMAP, THF, room temperature, 93%; (b) Cl2CHOCH3, TiCl4, CH2Cl2, 66%; (c) NaBH4 CH3OH, CHCl3, 65%; (d) NaOCH3,
CH3OH, ClCH2CH2Cl, room temperature, 90%; (e) ROH, CSA, CH2Cl2; (f) RSH, CSA, CH2Cl2.
like properties.11 K-252a has been demonstrated to
promote neurite outgrowth in human SH-SY5Y neuro-
blastoma cells.12 In primary cultures of embryonic
neurons, K-252a induced survival of dorsal root ganglion
and ciliary ganglion neurons,13 enhanced ChAT activity
in spinal cord14 and basal forebrain cultures,15 and
promoted survival and ChAT activity in striatal cul-
tures.15 The neurotrophic activity demonstrated by
K-252a for enhancing ChAT activity in spinal cord
cultures was comparable to responses elicited by CNTF,
BDNF, and IGF-I. In addition, studies have shown that
K-252a protects neurons against glucose deprivation,16
free-radical-mediated injury, and amyloid â-peptide
toxicity.17 These results suggest that the development
of compounds from the K-252a class as neurotrophic
agents may be effective therapy for the treatment of
peripheral and central neurodegenerative diseases.
In addition to neurotrophic properties, K-252a has a
number of biochemical properties which limit its utility
as a therapeutic agent. K-252a inhibited NGF-induced
neuronal differentiation and survival18a-c and specifi-
cally inhibited the autophosphorylation of the high-
affinity NGF receptor trk A, as well as related neurotro-
phin receptors trk B, trk C, and trk oncogenes at low
nanomolar concentrations.19 A medicinal chemistry
effort was undertaken to enhance and separate neuro-
trophic activity from the undesirable NGF (trk A kinase)
and PKC inhibitory properties of K-252a. This paper
reports on the selective neurotrophic activity of a series
of 3,9-bis-(alkoxymethyl) and bis[(alkylthio)methyl] de-
rivatives of K-252a as assessed by enhancement of
ChAT activity in rat embryonic spinal cord and basal
forebrain cultures, and the subsequent identification of
3,9-bis[(ethylthio)methyl]-K-252a (8), which exhibited
greater neurotrophic potency and efficacy with low
kinase inhibitory properties compared to K-252a.
Ch em istr y
The preparation of the 3,9-bis(alkoxymethyl)- and bis-
[(alkylthio)methyl]-K-252a analogs required the syn-
thesis of 3,9-bis(hydroxymethyl)-K-252a as the key
intermediate (Scheme 1). K-252a was protected as its
diacetyl derivative 2 in high yield. Diacetyl-K-252a (2)
was converted to 3,9-dialdehyde 3 (66% yield) by treat-
ment with 10 equiv of TiCl4 and 20 equiv of R,R-
dichloromethyl methyl ether in CH2Cl2. Reducing the
quantity of R,R-dichloromethyl methyl ether in the
reaction yielded higher amounts of the undesired C-3
monoaldehyde product. The position of the formyl
1
groups was readily assigned from H-NMR spectra. Aryl
protons H-4 and H-8, which appear on 2 as a set of
doublets at δ 9.09 and 8.07, respectively, appear as
singlets at δ 9.53 and 8.66 on dialdehyde 3. Diacetyl-
dialdehyde 3 was reduced to diacetyldiol 4 (65%, NaBH4,
CHCl3-methanol), followed by deprotection to diol 5 in
90% yield using catalytic NaOMe in methanol. Treat-
ment of intermediate diol 5 with various alcohols or
thiols in the presence of camphorsulfonic acid in CH2Cl2
yielded the desired bis(alkoxymethyl)- (6, 7) and bis-
[(alkylthio)methyl]-K-252a derivatives (8-13) in good
yield.

K-252a Derivatives
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1865
Ta ble 1. Neurotrophic Activity and trk A Inhibitory Activity of K-252a Derivatives
% of controla
spinal cord ChAT
basal forebrain ChAT
compound
R
30 nM
<120
<120
300 nM
5 0 nM
250 nM
trk A IC50 (nM)c
1 (K-252a)
H
186 ( 3
<120
148 ( 10
ntb
325 ( 22d
2.4
ntb
270
5
6
7
8
9
CH2OH
CH2OMe
CH2OEt
CH2SEt
CH2SnPr
CH2SiPr
ntb
193 ( 11
218 ( 14
188 ( 20
280 ( 14
315 ( 20
289 ( 17
302 ( 12
289 ( 6
168 ( 12
340 ( 21
237 ( 22e
363 ( 26
180 ( 6
224 ( 24
191 ( 12
200 ( 19
158 ( 15
153 ( 17
140 ( 8
<120
<120
<120
<120
<120
<120
210
143 ( 15
>1000
>1000
>1000
>1000
>1000
ntb
<120
10
ntb
11
12
13
CH2SCH2CHdCH2
<120
CH2SnBu
143 ( 8
CH2SCH2CH2NMe2
208 ( 8
<120
a
b
Enhancement of ChAT activity versus untreated control cultures. nt ) not tested. c Concentration required to inhibit 50% of trk
kinase. >200 nM concentration decreases ChAT below basal levels. e Maximum efficacy of 340 ( 26 at 500 nM.
d
Resu lts a n d Discu ssion
ChAT activity presumably due to toxicity.15 The history
of K-252a in these assays serves as a reliable and
effective reference standard. Analogs were evaluated
for efficacy at 300 nM in the spinal cord and at 250 nM
in the basal forebrain ChAT assays. Relative potency
was assessed by screening at 30 and 50 nM in the two
assays, respectively. Compounds which increased ChAT
activity at least 20% above basal (120%) levels were
significant and considered active.
The trk A inhibitory and neurotrophic activity of the
bis-substituted K-252a derivatives (5-13) are shown in
Table 1. Compounds were tested for their ability to
inhibit trk A tyrosine kinase using an ELISA-based
enzyme assay.20 Compounds were assessed for neuro-
trophic activity as measured by the ability to enhance
ChAT activity in embryonic rat spinal cord14 and basal
forebrain cultures.15 ChAT catalyzes the synthesis of
the neurotransmitter acetylcholine and is considered a
specific biochemical marker for functional cholinergic
neurons. In the spinal cord, motor neurons are cholin-
ergic and express ChAT.21 ChAT activity has been used
extensively to study the effects of neurotrophins (e.g.,
NGF or NT-3) on the survival and/or function of cho-
linergic neurons.2-4,14,15 During embryonic development
in the rat there is a wave of motoneuron death between
embryonic day 14 (E14) and E19. It is hypothesized
that neuronal death occurs due to limited amounts of
required trophic support and the overproduction of
neurons that do not form functional synapses.22 In
spinal cord cultures (E14-E19), a significant number
of cholinergic neurons would be expected to die in the
absences of a motoneuron survival factor. A continual
decline in ChAT activity is observed with increasing
culture time.14 In addition to spinal cord, basal fore-
brain neurons have also been identified as a K-252a-
responsive neuronal population, showing increasing
survival and ChAT activity.15
Bis(hydroxymethyl) compound 5 was found to be
inactive in the ChAT assays, whereas, in the spinal cord
ChAT assay, the methyl (6) and ethyl (7) ethers dem-
onstrated efficacy at 300 nM approximately equal to
K-252a and, unlike K-252a, were effective at 30 nM. In
the basal forebrain ChAT assay 6 demonstrated activity
equal to that of K-252a while the ethyl derivative 7 was
inactive at 50 nM; equal efficacy was observed at higher
concentrations (500 nM). Similar to the ether analogs,
the alkylthio derivatives retain neurotrophic activity in
both the basal forebrain and spinal cord ChAT assays.
The (ethylthio)methyl derivative 8 was the only sulfur
analog to show activity at the lower concentrations in
both the spinal cord and basal forebrain ChAT assays.
Alkyl groups larger than ethyl (8) produced a decrease
in potency in the basal forebrain and/or the spinal cord
assay (see 9-12). The alkyoxy and alkylthio derivatives
show different structure-activity relationships based
on efficacy in the spinal cord and basal forebrain ChAT
models. The size of the alkylthio group does not appear
to affect efficacy in the spinal cord ChAT assay, whereas
alkyl groups larger than ethyl (9-12) display reduced
efficacy in the basal forebrain ChAT assay. 3,9-Bis-
[(alkylthio)methyl] substitution enhances efficacy over
K-252a (unsubstituted R ) H, 186%) in the spinal cord
ChAT assay, whereas, in basal forebrain cultures, a
reduced efficacy is observed except for bis[(ethylthio)-
methyl] 8, which was equal in activity to K-252a at 250
nM. As described above, K-252a shows a decrease in
ChAT activity above 200 nM concentrations, presum-
ably due to toxicity in the low-density culture15 whereas
K-252a was used as an internal control for direct
comparison in all ChAT assay experiments. The ex-
perimental data represent the mean ( standard devia-
tion from three independent experiments. K-252a is
ineffective in the spinal ChAT assay at concentrations
less than 100 nM and shows a maximum enhancement
of ChAT activity of 186 ( 3% above basal levels (100%)
at 300 nM. In the basal forebrain assay, K-252a
demonstrated good activity at 50 nM (175%) and
exhibited a maximum effect on ChAT of 325 ( 22% at
200 nM. Above 200 nM, K-252a shows a decrease in

1866 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Kaneko et al.
Ta ble 2. Inhibitory Effect of Compound 8 on Various Kinases
IC50 (µM)
of the motoneurons that would die during this period.
Lower does of 1.2 and 1.8 µg/day rescued 25%.23b,c
Similar microgram doses of neurotrophic proteins (e.g.
BDNF, IGF-I, NT-3) were also effective, with ciliary
neurotrophic factor (CNTF) demonstrating the greatest
efficacy (40-45%).25 In female rats, motoneurons of the
spinal nucleus of the bulbocarvernosus (SNB) die post-
natal due to the absence of steroid hormone, a required
non-protein survival factor. During the early prenatal
life of female rats (to postnatal day (PN) 4) more than
50% of the motoneurons of the sexually dimorphic SNB
are eliminated by PCD. Testicular secretion of andro-
gen steroids reduce SNB motoneuron death in males,
and when administered to females, testosterone results
in a fully masculine number of SNB motoneurons.26
Administration of 8 (1 mg/kg sc) to female rats attenu-
ated motoneuron death with efficacy equal to that in
testosterone controls, demonstrating that peripherally
administered 8 was biologically active in promoting in
vivo survival of SNB motoneurons.23b,d No effects on
testosterone levels in rats treated with 8 were observed.
Compound 8 displayed significant effects in screens
for enhancement of ChAT activity in the basal forebrain
cultures. Compound 8 was assessed for neurotrophic-
like activity in an in vivo model of basal forebrain
cholinergic degeneration.27 Basal forebrain cholinergic
neurons degenerate in Alzheimer’s disease (AD), and
their loss may contribute to cognitive deficits of the
disease. Several animal models of AD attempt to mimic
basal forebrain cholinergic deficits by damaging the
cortically projecting cholinergic neurons of the nucleus
basalis magnocellularis (NBM). Ibotenate infusion into
the NBM produced a 30% deficit in cortical ChAT
activity and a 45% decrease in ChAT positive neurons
in the NBM. Systemically administered 8 (0.03-1.0 mg/
kg effective dose range) reduced the lesion-induced loss
of cortical ChAT activity and ChAT-positive neurons to
9% and 5%, respectively. Moreover, 8 ameliorated
cortical ChAT loss if administration was initiated 1 day,
but not 7 days postlesion.27 These results suggest that
8 protects cortically projecting basal forebrain cholin-
ergic neurons from degeneration in vivo and that this
compound may have utility in preventing the degenera-
tion of cholinergic neurons in AD.
kinase
K-252a
compound 8
protein kinase Ca
0.028
0.016
0.02
16.3
>10
>10
cAMP-dependent protein kinaseb
Myosin light chain kinasec
a
b
C-kinase was prepared from rat brain. A-kinase was pre-
pared from rabbit skeletal muscle. c MLCK was prepared from
chicken gizzard.
8 did not show this inverted dose-response curve up to
2 µM (data not shown). The addition of a basic amine
function (13) in the alkyl side chain retained neuro-
trophic activity only in the spinal cord ChAT assay.
The bis-substituted ether derivatives 6 (IC50 ) 270
nM) and 7 (IC50 ) 210 nM) were ca. 100-fold weaker
than K-252a (IC50 ) 2.4 nM) at inhibiting trk A tyrosine
kinase, whereas in general the thio series (8-12) had
IC50 values greater than 1 µM. These data suggest that
proper substitution in the 3- and 9-positions with
alkylthio groups reduces trk kinase inhibitory properties
>500-fold while enhancing the neurotrophic activity of
K-252a.
K252a has been reported to potently inhibit not only
trk A tyrosine kinase but also additional serine/threo-
nine kinases such as protein kinase C (PKC, IC50 ) 28
nM), cyclic AMP dependent protein kinase (PKA, IC50
) 16 nM), and myosin light chain kinase (MLCK, IC50
) 20 nM).10 Compound 8 was screened for inhibition
of these additional serine/threonine kinases (Table 2).
3,9-Bis[(ethylthio)methyl]-K-252a (8) only weakly in-
hibited protein kinase C (IC50 ) 16.3 µM), protein kinase
A (IC50 > 10 µM), and myosin light chain kinase (IC50
> 10 µM).
The increase in ChAT activity promoted by 8 in spinal
cord cultures was found associated with a promotion of
motoneuronal survival in low-density spinal cord cul-
tures enriched for motoneurons. Compound 8 which
was more efficacious than K-252a in the enhancement
of ChAT activity in heterogeneous spinal cord cultures
was also more efficacious than K-252a in promotion of
motoneuronal survival.23a,b At maximum effective con-
centrations, K-252a (150 nM) and compound 8 (300 nM)
enhanced motoneuron survival 128% and 169%, respec-
tively. The enhancement of spinal cord ChAT activity
and motoneuronal survival by 8 is comparable to that
elicited by optimal concentrations of protein growth
factors.23b Again, at the higher concentrations, K-252a
was cytotoxic.
Neurodegeneration, necrotic death, or loss of function
of neurons (phenotypic expression) is a feature of many
human neurodegenerative disorders. On the basis of
its in vitro profile, analog 8 was further evaluated in in
vivo models of neuronal degeneration and/or death. The
enhancement of ChAT activity and motoneuronal sur-
vival in spinal cord cultures suggests potential utility
in motoneuron disorders such as amyotrophic lateral
sclerosis (ALS) and certain peripheral neuropathies.
Approximately 50% of vertebrate motoneurons undergo
programmed cell death (PCD) during embryogenesis. In
the chick, 40-50% of the lumbar motoneurons die
between E6 and E10.24 To examine the effect on
neuronal PCD, doses of 8 were delivered locally onto
the chorioallantoic membrane surrounding the embryo
from E6 through E9 and sacrificed on E10. Maximally
effective doses of 2.3 and 7 µg/day/egg of 8 rescued 40%
In conclusion, a series of 3,9-disubstituted K-252a
derivatives was synthesized and evaluated for neuro-
trophic activity. Our data suggests that substitution
with alkylthio groups in the 3- and 9-positions of K-252a
reduces trk kinase inhibitory properties while enhancing
neurotrophic activity. 3,9-Bis[(ethylthio)methyl]-K-
252a (8) was identified as a potent neurotrophic agent
in vitro as measured by enhancement of choline acetyl-
transferase activity in embryonic rat spinal cord and
basal forebrain cultures. Moreover, compound 8 was
found to have weak kinase inhibitory activity for trk A,
PKC, PKA, and MLCK.
Exp er im en ta l Section
Ch em istr y. Melting points were determined on Laboratory
Devices capillary melting point apparatus and are uncorrected.
IR spectra were recorded on a J ASCO IR-810 spectrometer.
1H-NMR spectra were recorded on J EOL a400 (400 MHz) and
Brucker AM-500 (500MHz) spectrometers. Fast atom bom-
bardment mass spectra (FABMS) were recorded on a J EOL
J MS-HX110 spectrometer. Elemental analyses were per-
formed using a Yamato MT-5 analyzer at Kyowa Hakko
laboratories.

K-252a Derivatives
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1867
6-N,3′-O-Dia cetyl-K-252a (2). To a solution of 1 (51.4 g,
110 mmol) in THF (1.1 L) were added Ac2O (110 mL, 1.17 mol)
and DMAP (67.2 g, 550 mmol). After stirring overnight, the
reaction mixture was poured into 2 N HCl at 0 °C and
extracted with CHCl3. The organic layer was washed with
brine, dried over MgSO4, and concentrated. Recrystallization
from a mixture of MeOH-CHCl3 gave 56.3 g (93%) of 2: mp
208-210 °C; FAB-MS m/z 552 (MH+); 1H-NMR (500 MHz,
DMSO-d6) δ 1.71 (s, 3H), 2.22 (s, 3H), 2.24 (dd, 1H, J ) 5.0,
14.7 Hz), 2.67 (s, 3H), 3.89 (dd, 1H, J ) 7.5, 14.7 Hz), 3.95 (s,
3H), 5.36 (d, 1H, J ) 17.7 Hz), 5.40 (d, 1H, J ) 17.7 Hz), 7.30
(dd, 1H, J ) 5.0, 7.5 Hz), 7.33 (m, 1H), 7.44 (m, 1H), 7.54 (m,
1H), 7.60 (m, 1H), 8.01 (d, 2H), 8.07 (d, J ) 7.6, 1H), 9.09 (d,
J ) 7.5, 1H); HRFAB-MS calcd for C31H25N3O7 552.1770, found
552.1791.
6-N,3′-O-Dia cetyl-3,9-d ifor m yl-K-252a (3). To a solution
of 2 (5.00 g, 9.07 mmol) in CH2Cl2 (100 mL) were added TiCl4
(10.0 mL, 91.2 mmol) and R,R-dichloromethyl methyl ether
(16.4 mL, 181 mmol). After 2 h of stirring, the reaction
mixture was poured into ice-cooled saturated NaHCO3. The
precipitate was filtered, and the filtrate was extracted with
CHCl3. The organic layer was washed with brine, dried over
MgSO4, and concentrated. Recrystallization from CHCl3 gave
3.66 g (66%): mp 267-269 °C dec; FAB-MS m/z 608 (MH+);
1H-NMR (500 MHz, DMSO-d6) δ 1.71 (s, 3H), 2.27 (s, 3H), 2.39
(dd, 1H, J ) 5.0, 14.9 Hz), 2.65 (s, 3H), 3.93 (dd, 1H, J ) 7.6,
14.9 Hz), 3.97 (s, 3H), 5.40 (d, 1H, J ) 17.7 Hz), 5.46 (d, 1H,
J ) 17.7 Hz), 7.40 (dd, 1H, J ) 5.0, 7.6 Hz), 8.03 (m, 1H), 8.14
(m, 1H), 8.19 (d, 1H, J ) 8.8 Hz), 8.21 (d, 1H, J ) 8.8 Hz),
8.66 (d, 1H, J ) 1.1 Hz), 9.53 (d, 1H, J ) 1.3 Hz), 10.09 (s,
1H), 10.24 (s, 1H); HRFAB-MS calcd for C33H25N3O9 608.1669,
found 608.1687. Anal. (C33H25N3O9‚H2O) C, H, N.
at room temperature. After 4 days of stirring at room
temperature, the reaction mixture was poured into saturated
NaHCO3 solution and extracted with CHCl3. The organic layer
was washed with brine, dried over Na2SO4, and concentrated
to give the crude product. Purification by column chromatog-
raphy (CHCl3/MeOH/NH3OH, 9/1/0.1) gave 28.8 mg of 13
(42%): mp 224-226 °C dec; IR (KBr) 1732, 1670 cm-1; FAB-
MS m/z 702 (MH+); 1H-NMR (400 MHz, DMSO-d6) δ 2.00 (dd,
1H, J ) 4.9, 14.2 Hz), 2.13 (s, 3H), 2.14 (s, 6H), 2.15 (s, 6H),
2.46-2.59 (m, 8H), 3.38 (dd, 1H, J ) 7.3, 14.2 Hz), 3.92 (s,
3H), 3.95 (s, 2H), 3.98 (s, 2H), 4.95 (d, 1H, J ) 17.7 Hz), 5.01
(d, 1H, J ) 17.7 Hz), 6.32 (s, 1H), 7.11 (dd, 1H, J ) 4.9, 7.3
Hz), 7.44-7.95 (m, 5H), 8.62 (s, 1H), 9.13 (d, 1H, J ) 1.4 Hz);
HRFAB-MS calcd for C37H43N5O5S2 702.2789, found 702.2773.
Anal. (C37H43N5O5S2‚H2O) C, H, N.
Compounds 6-12 were prepared by the general procedure
described for 13.
3,9-Bis(m eth oxym eth yl)-K-252a (6): mp 203-205 °C dec;
FAB-MS m/z 555 (M+); IR (KBr) 1732, 1663 cm-1 1H-NMR
;
(500 MHz, DMSO-d6) δ 2.010 (dd, 1H, J ) 4.9, 14.1 Hz), 2.14
(s, 3H), 3.34 (s, 3H), 3.36 (s, 3H), 3.38 (dd, 1H, J ) 7.4, 14.1
Hz), 3.93 (s, 3H), 4.58 (s, 2H), 4.62 (s, 2H), 4.98 (d, 1H, J )
16.9 Hz), 5.03 (d, 1H, J ) 16.9 Hz), 6.33 (s, 1H), 7.13 (dd, 1H,
J ) 4.9, 7.4 Hz), 7.44-7.46 (m, 2H), 7.87 (d, 1H, J ) 8.4 Hz),
7.92 (d, 1H, J ) 8.7 Hz), 7.97 (d, 1H, J ) 1.1 Hz), 8.61 (s, 1H),
9.17 (d, 1H, J ) 1.0 Hz); HRFAB-MS calcd for C31H29N3O7
555.2006, found 555.2015. Anal. (C31H29N3O7‚0.3CHCl3) C,
H, N.
3,9-Bis(eth oxym eth yl)-K-252a (7): mp 160-162 °C; IR
1
(KBr) 1732, 1657 cm-1; FAB-MS m/z 583 (M+); H-NMR (500
MHz, DMSO-d6) δ 1.19 (t, 3H, J ) 7.0 Hz), 1.20 (t, 3H, J )
7.0 Hz), 2.00 (dd, 1H, J ) 4.9, 14.1 Hz), 2.14 (s, 3H), 3.39 (dd,
1H, J ) 7.4, 14.1 Hz), 3.55 (q, 2H, J ) 7.0 Hz), 3.56 (q, 2H, J
) 7.0 Hz), 3.93 (s, 3H), 4.62 (s, 2H), 4.66 (s, 2H), 4.98 (d, 1H,
J ) 16.9 Hz), 5.03 (d, 1H, J ) 16.9 Hz), 6.33 (s, 1H), 7.124
(dd, 1H, J ) 4.9, 7.4 Hz), 7.45-7.47 (m, 2H), 7.86 (d, 1H, J )
8.3 Hz), 7.91 (d, 1H, J ) 8.7 Hz), 7.97 (d, 1H, J ) 1.2 Hz),
8.60 (s, 1H), 9.16 (d, 1H, J ) 1.0 Hz); HRFAB-MS calcd for
C33H33N3O7 583.2319, found 583.2332. Anal. (C33H33N3O7‚
1.5H2O) C, H, N.
3,9-Bis[(eth ylth io)m eth yl]-K-252a (8): mp 163-165 °C;
IR (KBr) 1725, 1680 cm-1; FAB-MS m/z 615(M+); 1H-NMR (400
MHz, DMSO-d6) δ 1.23 (t, 6H, J ) 7.3 Hz), 1.99 (dd, 1H, J )
4.8, 14.1 Hz), 2.132 (s, 3H), 2.489 (q, 2H, J ) 7.3 Hz), 2.505
(q, 2H, J ) 7.3 Hz), 3.37 (dd, 1H, J ) 7.6, 14.1 Hz), 3.92 (s,
3H), 3.94 (s, 2H), 3.98 (s, 2H), 4.95 (d, 1H, J ) 17.6 Hz), 5.02
(d, 1H, J ) 17.6 Hz), 6.32 (s, 1H), 7.10 (dd, 1H, J ) 4.8, 7.6
Hz), 7.450 (m, 2H), 7.84 (d, 1H, J ) 8.5 Hz), 7.88 (d, 1H, J )
8.8 Hz), 7.95 (d, 1H, J ) 1.0 Hz), 8.60 (s, 1H), 9.13 (d, 1H, J
) 0.7 Hz); HRFAB-MS calcd for C33H33N3O5S2 615.1862, found
615.1869. Anal. (C33H33N3O5S2‚0.5H2O) C, H, N.
6-N,3′-O-Dia cetyl-3,9-bis(h yd r oxym eth yl)-K-252a (4).
To a suspension of 3 (10.0 g, 16.5 mmol) in MeOH-CHCl3 (80
mL-500 mL) was added NaBH4 (1.88 g, 49.7 mmol) at 0 °C.
After 30 min of stirring at that temperature, the reaction
mixture was poured into water. The resultant suspension was
filtered. The precipitate was washed with water and dried in
vacuo at 80 °C to give the crude 4 (6.53 g, 65%). 4 was used
in the next step without further purification. A portion of
crude 4 was purified by column chromatography (MeOH/Et3N/
CHCl3, 2/0.5/98) to obtain physicochemical data: mp 297-299
1
°C dec; FAB-MS m/z 612(MH+); H-NMR (400 MHz, DMSO-
d6) δ 1.72 (s, 3H), 2.21 (s, 3H), 2.22 (dd, 1H, J ) 4.6, 14.6 Hz),
2.72 (s, 3H), 3.89 (dd, 1H, J ) 7.2, 14.6 Hz), 3.95 (s, 3H), 4.70
(d, 2H, J ) 5.1 Hz), 4.76 (d, 2H, J ) 5.1 Hz), 5.22 (t, 1H, J )
5.1 Hz), 5.35 (t, 1H, J ) 5.1 Hz), 5.42 (s, 2H), 7.28 (dd, 1H, J
) 4.6, 7.2 Hz), 7.53 (m, 2H), 7.97 (d, 1H, J ) 8.3 Hz), 7.96 (d,
1H, J ) 8.8 Hz), 8.05 (s, 1H), 9.07 (d, 1H, J ) 0.6 Hz); HRFAB-
MS calcd for C33H29N3O9 612.1983, found 612.1978. Anal.
(C33H29N3O9‚1.5H2O) C, H, N.
3,9-Bis(h yd r oxym eth yl)-K-252a (5). 4 (6.53 g, 10.7 mmol)
was dissolved in MeOH-ClCH2CH2Cl (100-300 mL). To the
mixture was added 25 wt % NaOMe in MeOH (0.500 mL, 2.19
mmol). After 30 min of stirring min at room temperature, the
reaction mixture was poured into ice-cooled water. The
resultant suspension was filtered. The precipitate was washed
with water and dried in vacuo at 80 °C to give crude 5 (5.06 g,
90%), which was used in the next reaction without further
purification. A portion of crude 5 was purified by preparative
TLC (MeOH/CHCl3, 1/9) to obtain physicochemical data: mp
280-282 °C dec; IR (KBr) 1724, 1682 cm-1; FAB-MS m/z 528
(MH+); 1H-NMR (400 MHz, DMSO-d6) δ 1.99 (dd, 1H, J ) 4.9,
14.1 Hz), 2.14 (s, 3H), 3.38 (dd, 1H, J ) 7.4, 14.1 Hz), 3.92 (s,
3H), 4.67 (s, 2H), 4.71 (s, 2H), 4.96 (d, 1H, J ) 17.0 Hz), 5.01
(d, 1H, J ) 17.0 Hz), 5.20 (br s, 2H), 7.11 (dd, 1H, J ) 4.9, 7.4
Hz), 7.44-7.48 (m, 2H), 7.84 (d, 1H, J ) 8.4 Hz), 7.88 (d, 1H,
J ) 8.7 Hz), 7.96 (s, 1H), 8.59 (s, 1H), 9.14 (d, 1H, J ) 0.9
Hz); HRFAB-MS calcd for C29H26N3O7 528.1770, found 528.1763.
Anal. (C29H26N3O7‚1.5H2O) C, H, N.
3,9-Bis[(p r op ylth io)m eth yl]-K-252a (9): mp 138-140 °C;
IR (KBr) 1727, 1664 cm-1; FAB-MS m/z 643 (M+); 1H-NMR
(400 MHz, DMSO-d6) δ 0.94 (t, 3H, J ) 7.3 Hz), 0.95 (t, 3H, J
) 7.3 Hz), 1.56-1.66 (m, 4H), 2.00 (dd, 1H, J ) 4.8, 14.1 Hz),
2.13 (s, 3H), 2.46 (t, 2H, J ) 7.3 Hz), 2.47 (t, 2H, J ) 7.3 Hz),
3.38 (dd, 1H, J ) 7.4, 14.1 Hz), 3.92 (s, 5H), 3.96 (s, 2H), 4.95
(d, 1H, J ) 17.1 Hz), 5.01 (d, 1H, J ) 17.1 Hz), 6.32 (s, 1H),
7.10 (dd, 1H, J ) 4.8, 7.4 Hz), 7.43-7.46 (m, 2H), 7.84 (d, 1H,
J ) 8.3 Hz), 7.88 (d, 1H, J ) 8.6 Hz), 7.94 (d, 1H, J ) 1.5 Hz),
8.60 (s, 1H), 9.12 (d, 1H, J ) 1.5 Hz); HRFAB-MS calcd for
C35H37N3O5S2 643.2174, found 643.2192. Anal. (C35H37N3O5S2‚
0.5CH3OH) C, H, N.
3,9-Bis[(isop r op ylth io)m eth yl]-K-252a (10): mp 158-160
°C; IR (KBr) 1730, 1666 cm-1; FAB-MS m/z 643 (M+); 1H-NMR
(300 MHz, DMSO-d6) δ 1.26 (d, 3H, J ) 6.6 Hz), 1.27 (d, 9H,
J ) 6.6 Hz), 1.99 (dd, 1H, J ) 5.0, 14.1 Hz), 2.13 (s, 3H), 2.88
(m, 2H), 3.38 (dd, 1H, J ) 7.3, 14.1 Hz), 3.92 (s, 3H), 3.96 (s,
2H), 4.002 (s, 2H), 4.95 (d, 1H, J ) 17.1 Hz), 5.02 (d, 1H, J )
17.1 Hz), 7.10 (dd, 1H, J ) 5.0, 7.3 Hz), 7.44-7.47 (m, 2H),
7.83 (d, 1H, J ) 8.1 Hz), 7.88 (d, 1H, J ) 8.8 Hz), 7.96 (d, 1H,
J ) 1.7 Hz), 8.592 (s, 1H), 9.14 (d, 1H, J ) 1.2 Hz); HRFAB-
MS calcd for C35H37N3O5S2 643.2175, found 643.2205. Anal.
(C35H37N3O5S2‚0.5CH3OH) C, H, N.
Gen er a l P r oced u r e for F or m a tion of (Alk yoxym eth yl)-
a n d [(a lk ylth io)m eth yl]- K-252a Der iva tives. 3,9-Bis-
[[(d im eth yla m in o)th io]m eth yl]-K-252a (13). To a suspen-
sion of 5 (51.7 mg, 0.0981 mmol) and 2-(dimethylamino)-
ethanethiol hydrochloride (142.9 mg, 1.01 mmol) in CH2Cl2 (2
mL) was added camphorsulfonic acid (206.5 mg, 0.889 mmol)
3,9-Bis[(a llylth io)m eth yl]-K-252a (11): mp 125-127 °C;
IR (KBr) 1726, 1670 cm-1; FAB-MS m/z 639 (M+); 1H-NMR

1868 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Kaneko et al.
(500 MHz, DMSO-d6) δ 2.00 (dd, 1H, J ) 4.9, 14.1 Hz), 2.14
(s, 3H), 3.13 (d, 2H, J ) 7.0 Hz), 3.17 (d, 2H, J ) 7.1 Hz), 3.38
(dd, 1H, J ) 7.4, 14.1 Hz), 3.86 (s, 2H), 3.92 (s, 2H), 3.92 (s,
3H), 4.96 (d, 1H, J ) 16.8 Hz), 5.02 (d, 1H, J ) 16.8 Hz), 5.16-
5.19 (m, 2H), 5.22 (dd, 1H, J ) 1.6, 17.0 Hz), 5.33 (dd, 1H, J
) 1.5, 17.0 Hz), 5.81-5.92 (m, 2H), 6.32 (s, 1H), 7.11 (dd, 1H,
J ) 4.9, 7.4 Hz), 7.43-7.45 (m, 2H), 7.85 (d, 1H, J ) 8.4 Hz),
7.89 (d, 1H, J ) 8.7 Hz), 7.92 (d, 1H, J ) 1.4 Hz), 8.60 (s, 1H),
9.12 (d, 1H, J ) 1.4 Hz); HRFAB-MS calcd for C35H33N3O5S2
639.1862, found 639.1835. Anal. (C35H33N3O5S2‚1.4H2O) C,
H, N.
3,9-Bis[(bu tylth io)m eth yl]-K-252a (12): mp 119-121 °C;
IR (KBr) 1729, 1662 cm-1; FAB-MS m/z 671 (M+); 1H-NMR
(500 MHz, DMSO-d6) δ 0.87 (t, 3H, J ) 7.4 Hz), 0.88 (t, 3H, J
) 7.4 Hz), 1.33-1.41 (m, 4H), 1.54-1.60 (m, 4H), 2.00 (dd,
1H, J ) 4.9, 14.1 Hz), 2.13 (s, 3H), 2.48 (t, 2H, J ) 7.4 Hz),
2.49 (t, 2H, J ) 7.4 Hz), 3.38 (dd, 1H, J ) 7.5, 14.1 Hz), 3.92
(s, 5H), 3.96 (s, 2H), 4.95 (d, 1H, J ) 16.9 Hz), 5.00 (d, 1H, J
) 16.9 Hz), 6.31 (s, 1H), 7.10 (dd, 1H, J ) 4.9, 7.4 Hz), 7.43-
7.46 (m, 2H), 7.83 (d, 1H, J ) 8.4 Hz), 7.88 (d, 1H, J ) 8.7
Hz), 7.94 (d, 1H, J ) 1.5 Hz), 8.60 (s, 1H), 9.12 (d, 1H, J ) 1.4
Hz); HRFAB-MS calcd for C37H41N3O5S2 671.2488, found
671.2494. Anal. (C37H41N3O5S2‚0.3H2O) C, H, N.
Sp in a l Cor d Ch AT Assa y. The spinal cord ChAT assay
was performed as previously described and references therein.14
K-252a was used as an internal control for direct comparison
in all ChAT assay experiments. The experimental data
represent the mean ( standard deviation from three independ-
ent experiments. Briefly, fetal rat spinal cord cells were
dissociated, and experiments were performed as described.
Dissociated cells were prepared from spinal cords dissected
from rats (embryonic day 14 or 15) by standard trypsin
dissociation techniques. Cells were plated at 6 × 105 cells/
cm2 on poly-L-ornithine-coated plastic tissue culture wells in
serum-free N2 medium supplemented with 0.05% bovine
serum albumin (BSA). Cultures were incubated at 37 °C in a
humidified atmosphere of 5% CO2/95% air for 48 h. ChAT
activity was measured after 2 days in vitro.
Ba sa l F or ebr a in Ch AT Assa y. The basal forebrain ChAT
assay was performed as previously described and references
therein.15 K-252a was used as an internal control for direct
comparison in all ChAT assay experiments. The experimental
data represent the mean ( standard deviation from three
independent experiments. Briefly, the basal forebrain was
dissected from rat embryos (day 17 or 18 embryos), and the
cells were dissociated with a neutral protease (Dispas, Col-
laborative Research). Neurons were plated at a density of 5
× 104 cells/well (1.5 × 105 cells/cm2) in poly-L-ornithine- and
laminin-coated plates. Cells were cultured in serum-free N2
medium containing 0.05% BSA at 37 °C in a humidified
atmosphere, 5% CO2/95% air. ChAT activity was assessed 5
days after plating by using the ChAT assay as described in
ref 14.
spinal cord and basal forebrain ChAT assays. We
express our gratitude to Dr. Mayumi Yoshida and her
staff for recording the NMR spectra and the elemental
analyses.
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