Enantioselective synthesis of cyclothiazide analogues: Novel probes of the stereospecific actions of benzothiadiazines at AMPA-type glutamate receptors

Article abstract of DOI:10.1021/ja9525317

The stereospecific interactions of the eight stereoisomers of dihydromethylcyclothiazide, an analogue of cyclothiazide, with AMPA-type glutamate receptors was investigated using electrophysiological methods that measured the ability of each stereoisomer to inhibit AMPA receptor desensitization. The eight stereoisomers were obtained by HPLC separation of four pairs of enantiomerically pure (>95% ee) diastereomers prepared from (1R-exo)-, (1R-endo)-, (1S-exo)-, and (1S-endo)-2-methylbicyclo[2.2.1]heptane-2-carboxaldehyde intermediates. The desensitization process was blocked most potently by [1S-[1α,2α(R*),4α]]-dihydromethylcyclothiazide, one of the stereoisomers prepared from the (1S-endo)-carboxaldehyde. The smallest effects on the desensitization process were found for the four stereoisomers prepared from the (1R-exo)- and (1R-endo)-carboxaldehydes. Significant differences in the ability to inhibit desensitization were observed between all diastereomer pairs except those prepared from the (1S-exo)-carboxaldehyde.

Full text of DOI:10.1021/ja9525317

4550
J. Am. Chem. Soc. 1996, 118, 4550-4559
Enantioselective Synthesis of Cyclothiazide Analogues: Novel
Probes of the Stereospecific Actions of Benzothiadiazines at
AMPA-Type Glutamate Receptors
Yuefei Hu,† Kelvin A. Yamada,‡ David K. Chalmers,† Durga P. Annavajjula,† and
Douglas F. Covey*,†
Contribution from the Departments of Molecular Biology and Pharmacology, Neurology,
and Pediatrics, Washington UniVersity School of Medicine, 660 South Euclid AVenue,
St. Louis, Missouri 63110
ReceiVed July 28, 1995X
Abstract: The stereospecific interactions of the eight stereoisomers of dihydromethylcyclothiazide, an analogue of
cyclothiazide, with AMPA-type glutamate receptors was investigated using electrophysiological methods that measured
the ability of each stereoisomer to inhibit AMPA receptor desensitization. The eight stereoisomers were obtained
by HPLC separation of four pairs of enantiomerically pure (>95% ee) diastereomers prepared from (1R-exo)-, (1R-
endo)-, (1S-exo)-, and (1S-endo)-2-methylbicyclo[2.2.1]heptane-2-carboxaldehyde intermediates. The desensitization
process was blocked most potently by [1S-[1R,2R(R*),4R]]-dihydromethylcyclothiazide, one of the stereoisomers
prepared from the (1S-endo)-carboxaldehyde. The smallest effects on the desensitization process were found for the
four stereoisomers prepared from the (1R-exo)- and (1R-endo)-carboxaldehydes. Significant differences in the ability
to inhibit desensitization were observed between all diastereomer pairs except those prepared from the (1S-exo)-
carboxaldehyde.
Introduction
been used to study this phenomenon,7,8 and its possible
involvement in the processes of neuronal excitotoxicity9-12 and
learning.13 From among the benzothiadiazine derivatives
investigated thus far, the drug cyclothiazide [3-(bicyclo[2.2.1]-
hept-5-en-2-yl)-6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-
7-sulfonamide 1,1-dioxide, Chart 1] is of particular interest
because of its high potency and receptor specificity. This drug
reduces the desensitization of AMPA but not kainate receptors.14
Moreover, cyclothiazide’s effect on AMPA receptor desensitiza-
tion is strongly influenced by alternative splicing of a 38 amino
acid external domain of individual AMPA receptor subunits
called the flip/flop region. Site directed mutation studies
indicate that a single amino acid replacement within the flip/
flop region eliminates cyclothiazide’s effects.15,16 Finally,
cyclothiazide not only affects the desensitization properties of
AMPA receptors but also allosterically reduces the antagonist
actions of competitive and noncompetitive antagonists of AMPA
receptors.17
L-Glutamate is an important excitatory neurotransmitter in
the mammalian central nervous system. Multiple categories of
glutamate receptors have been identified, and each receptor is
composed of combinations of individual protein subunits. The
receptors subserve fast, glutamatergic synaptic signaling between
central neurons and belong in the category of R-amino-2,3-
dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid or AMPA-
type glutamate receptors, which are glutamate-gated ion chan-
nels composed of subunits designated GluRA-D1 or GluR1-
4.2-4 Functional properties such as agonist selectivity, calcium
permeability, desensitization kinetics, and sensitivity to gating
modifiers have been characterized for individual subunits by
combining electrophysiology, pharmacology, and recombinant
receptor expression systems. The differences between these
functional properties suggest that functional diversity among
native AMPA receptors may in part be determined by expression
of various combinations of individual subunits.
For several years there has been sustained interest in the
physiological importance of AMPA receptor desensitization in
synaptic transmission.5,6 Recently some potent and reversible
2H-1,2,4-benzothiadiazine 1,1-dioxide derivatives (Chart 1) have
(7) Yamada, K. A.; Rothman, S. M. J. Physiol. 1992, 458, 385-407.
(8) Yamada, K. A.; Tang, C.-M. J. Neurosci. 1993, 13, 3904-3915.
(9) Moudy, A. M.; Yamada, K. A.; Rothman, S. M. Neuropharmacology
1994, 33, 953-962.
(10) May, P. C.; Robison, P. M. J. Neurochem. 1993, 60, 1171-1174.
(11) David, J. C.; Rosengarten, A. M.; Yamada, K. A.; Goldberg, M. P.
Neurology 1995, 45(Suppl), Abstract 527P, p A309.
† Department of Molecular Biology and Pharmacology.
‡ Departments of Neurology and Pediatrics.
X Abstract published in AdVance ACS Abstracts, April 15, 1996.
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S0002-7863(95)02531-5 CCC: $12.00 © 1996 American Chemical Society

EnantioselectiVe Synthesis of Cyclothiazide Analogues
J. Am. Chem. Soc., Vol. 118, No. 19, 1996 4551
Chart 1
hyde to 4-amino-6-chloro-1,3-benzenedisulfonamide.20 While
it is possible to prepare both exo- and endo-bicyclo[2.2.1]hept-
5-ene-2-carboxaldehyde in optically enriched form using enan-
tioselective Diels-Alder reactions (see below for references),
the potential for enolization of the carboxaldehyde group and
carbonium ion rearrangements of this compound under the acidic
conditions of the coupling reaction suggested to us that the
stereochemical integrity of these carboxaldehydes might not be
maintained in the cyclothiazide stereoisomer products.21 Hence,
we concluded that modifying the structure of the bicyclo[2.2.1]-
hept-5-ene-2-carboxaldehyde to preclude the potential epimer-
ization and racemization of the stereoisomer products during
the coupling reaction was prudent. Thus, we chose to prepare
by an enantiospecific (>95% ee) synthetic route the four
stereoisomers of 2-methylbicyclo[2.2.1]heptane-2-carboxalde-
hyde. These stereoisomers were then subsequently utilized for
the preparation of the four diastereomeric pairs of enantiomeri-
cally pure dihydromethylcyclothiazide stereoisomers (Chart 2).
From among the enantioselective Diels-Alder reactions
catalyzed by chiral Lewis acids,22-26 the best enantioselectivities
are obtained, in most cases, using R,â-unsaturated aldehydes
as dienophiles.23,24 Several chiral catalysts can effect the Diels-
Alder reaction between cyclopentadiene and methacrolein to
yield 2-methylbicyclo[2.2.1]hept-5-ene-2-carboxaldehyde (12 or
25) as a mixture of exo- and endo-isomers with high yields and
excellent enantioselectivities (90-99% ee) under very mild
conditions.24,26 Tartaric acid-derived chiral (acyloxy)borane
(CAB) catalysts were chosen for enantioselective synthesis of
compounds 12 and 25 because they can be prepared conve-
niently from commercially available 99% ee L-tartaric acid ([R-
(R*,R*)]-2,3-dihydroxybutanedioic acid, 5) and 98% ee D-tar-
taric acid ([S-(R*,R*)]-2,3-dihydroxybutanedioic acid, 6).
Since commercial preparations of this drug contain a mixture
of eight stereoisomers (four racemic diastereomers),18 it is
necessary to first separate the four racemic diastereomers from
each other by chromatography and then to separate each
racemate into the enantiomer components using chiral chroma-
tography if one is to obtain each stereoisomer for biological
studies. Thus far, using separation procedures and NMR
spectroscopy, it has been possible to separate, to make structural
assignments, and to identify which racemic diastereomer is most
active as a potentiatior of AMPA-mediated current.19 In
addition, the most active racemic diastereomer has been
separated further into the [1S-[1R,2R(S*),4R]]- and [1R-[1R,2R-
(R*),4R]]-stereoisomers, and it has been shown that there is a
100-fold difference in activity for these enantiomers.19 The
absolute configuration of the most active enantiomer has not
been determined.
Synthesis of Chiral Ligands 9 and 10. The asymmetric
Diels-Alder reactions catalyzed by CAB catalysts have been
developed by Yamamato and colleagues.24c,25,26 CAB catalysts
with [R-(R*,R*)]- and [S-(R*,R*)]-2-[(2,6-dimethoxybenzoyl)-
(20) Whitehead, C. W.; Traverso, J. J.; Sullivan, H. R.; Marshall, F. J.
J. Org. Chem. 1961, 26, 2814-2818.
Further progress in understanding the specific actions of
cyclothiazide or cyclothiazide-like drugs on AMPA receptor
function requires that the absolute configuration of the most
active stereoisomer of cyclothiazide, or a close structural
analogue, be determined. Moreover, additional biological
studies requiring large amounts of each stereoisomer necessitate
the development of new synthetic methods that avoid the need
for extensive separation (particularly chiral chromatography)
techniques. To this end, we report a synthetic strategy that
provides a practical route to each of the eight stereoisomers of
dihydromethylcyclothiazide, a close structural analogue of
cyclothiazide (see Charts 1 and 2). In addition, the results of
electrophysiological studies that establish the absolute config-
uration of the dihydromethylcyclothiazide stereoisomer having
the highest potency as an inhibitor of AMPA receptor desen-
sitization as well as other structure-activity relationships are
described.
(21) A preliminary experiment showed that some epimerization of the
carboxaldehyde group of exo-2-methylbicyclo[2.2.1]hept-5-ene-2-carbox-
aldehyde occurred during the reaction of this compound with 4-amino-6-
chloro-1,3-benzenedisulfonamide.
(22) (a) Hashimoto, S. I.; Komeshima, N.; Koga, K. J. Chem. Soc., Chem.
Commun. 1979, 437-438. (b) Kelly, T. R.; Whiting, A.; Chandrakumar,
N. S. J. Am. Chem. Soc. 1986, 108, 3510-3512. (c) Takemura, H.;
Komeshima, N.; Takahashi, I.; Hashimoto, S. I.; Ikota, N.; Tomiota, K.;
Koga, K. Tetrahedron Lett. 1987, 28, 5687-5690. (d) Bir, G.; Kaufmann,
D. Tetrahedron Lett. 1987, 28, 777-780. (e) Corey, E. J.; Imwinkelried,
R.; Pikul, S.; Xiang, Y. B. J. Am. Chem. Soc. 1989, 111, 5493-5495. (f)
Sartor, D.; Saffrich, J.; Helmchen, G.; Richards, C. J.; Lambert, H.
Tetrahedron: Asymmetry 1991, 2, 639-642. (g) Corey, E. J.; Imai, N.;
Zhang, H. Y. J. Am. Chem. Soc. 1991, 113, 728-729. (h) Devine, P. N.;
Oh, T. J. Org. Chem. 1992, 57, 396-399. (i) Itsuno, S.; Katsuhiro, K.;
Watanabe, K.; Koizumi, T.; Ito, K. Tetrahedron: Asymmetry 1994, 5, 523-
526. (j) Odenkirk, W.; Bosnich, B. J. Chem. Soc., Chem. Commun. 1995,
1181-1182.
(23) (a) Corey, E. J.; Loh, T. P.; J. Am. Chem. Soc. 1991, 113, 8966-
8967. (b) Corey, E. J.; Loh, T. P.; Roper, T. D.; Azimioara, M. D.; Noe,
M. C. J. Am. Chem. Soc. 1992, 114, 8290-8292. (c) Corey, E. J.; Roper,
T. D.; Ishihara, K.; Sarakinos, G. Tetrahedron Lett. 1993, 34, 8399-8402.
(d) Maruoka, K; Murase, N.; Yamamoto, H. J. Org. Chem. 1993, 58, 2938-
2939.
(24) (a) Bao, J.; Wulff, W. D. J. Am. Chem. Soc. 1993, 115, 3814-
3815. (b) Kundig, P.; Bourdin, B.; Bernardinelli, G. Angew. Chem., Int.
Ed. Engl. 1994, 33, 1856-1858. (c) Ishihara, K.; Yamamoto, H. J. Am.
Chem. Soc. 1994, 116, 1561-1562.
(25) (a) Furuta, K.; Miwa, Y.; Iwanaga, K.; Yamamoto, H. J. Am. Chem.
Soc. 1988, 110, 6254-6255. (b) Furuta, K.; Kanematsu, A.; Yamamoto,
H.; Takaoka, S. Tetrahedron Lett. 1989, 30, 7231-7232. (c) Ishihara, K.;
Gao, Q.; Yamamoto, H. J. Org. Chem. 1993, 58, 6917-6919.
(26) (a) Furuta, K.; Shimizu, S.; Miwa, Y.; Yamamoto, H. J. Org. Chem.
1989, 54, 1481-1483. (b) Ishihara, K.; Gao, Q.; Yamamoto, H. J. Am.
Chem. Soc. 1993, 115, 10412-10413.
Results and Discussion
The stereoisomeric composition of cyclothiazide results from
the coupling, under strongly acidic conditions, of a mixture of
racemic exo- and endo-bicyclo[2.2.1]hept-5-ene-2-carboxalde-
(18) Nusser, E.; Banerjee, A.; Gal, G. Chirality 1991, 3, 2-13.
(19) Cordi, A. A.; Serkiz, B.; Hennig, P.; Mahieu, J.-P.; Bobichon, C.;
de Nanteuil, G.; Lepagnol, J. M. Bioorg. Med. Chem. Lett. 1994, 4, 1957-
1960.

4552 J. Am. Chem. Soc., Vol. 118, No. 19, 1996
Hu et al.
Chart 2. The Eight Stereoisomers of Dihydromethylcyclothiazide
Scheme 1a
Scheme 2a
a Reagents: (a) benzyl alcohol, reflux, 2 h; (b) 2,6-dimethoxybenzoic
acid, trifluoroacetic anhydride, room temperature, 1 h; (c) 10% Pd-C,
H2, 100 psi, 3 h.
a Reagents: (a) methacrolein, 9/BH3‚THF, -78 °C, 6 h; (b) Jones
reagent, 0 °C, ∼1 h; (c) I2-KI-NaHCO3-KOH, room temperature,
30 min; (d) Zn-HOAc, room temperature, 3 h.
oxy-3-hydroxybutanedioic acid (Scheme 1, 9 and 10, respec-
tively) as ligands are optimal for the preparation of compounds
12 and 25, respectively (exo:endo ∼9:1, 87% yield and 96%
ee, respectively).26 Compounds 9 and 10 were prepared by a
variation of the method reported for the synthesis of compound
9.27 Heating a mixture of acid 5 (1 equiv) with benzyl alcohol
(4 equiv) in the absence of solvent and acidic catalyst at 200
°C for 1 h and then at 225 °C for another 1 h while removing
water using a Dean-Stark apparatus gave, after chromatographic
purification, a 93% yield of diester 7 as fine white crystals.
Diester 8 was obtained in 95% isolated yield from acid 6 by
using the same procedure. Monoacyloxylation of diesters 7 and
8 with 2,6-dimethylbenzoic acid in the presence of trifluoroacetic
anhydride gave intermediate triesters (55% and 65%, respec-
tively, structures not shown) which were then hydrogenolyzed
on a Pd-C catalyst to give products 9 (99%) and 10 (96%),
respectively.25c
BH3‚THF) yielded a mixture of (1S-exo)- and (1S-endo)-2-
methylbicyclo[2.2.1]hept-5-ene-2-carboxaldehydes (12) which
was immediately oxidized (Jones reagent) without purification
or characterization to the corresponding acids 13 (74% yield
overall for the two steps). Reaction of acids 13 with an aqueous
solution of I2-KI-NaHCO3 (1:4:6 mol ratio) in aqueous NaOH
solution converted acids 13 into a readily separated mixture of
(1S-exo)-acid 14 (77%) and iodolactone 15 (10%).28 Iodolac-
tone 15 was then converted smoothly into (1S-endo)-acid 16
using zinc dust in HOAc at room temperature (92%).29
Acid 14 was found to have [R]22D ) -66.2°, which indicated
an enantiomeric purity of 98.3% ee.30 Although acid 16 is
expected to have the same optical purity as acid 14, this
1
conclusion was verified by a H NMR experiment (Figure 1)
utilizing iodolactone 15 and the chiral alcohol (R)-2,2,2-trifluoro-
(28) (a) Rondestvedt, C. S., Jr.; Ver Nooy, C. D. J. Am. Chem. Soc.
1955, 77, 4878-4883. (b) Meek, J. S.; Trapp, M. B. J. Am. Chem. Soc.
1957, 79, 3909-3912. (c) Berson, J. A.; Remanick, A. J. Am. Chem. Soc.
1961, 83, 4947-4956.
(29) (a) Berson, J. A.; Ben-Efraim, D. A. J. Am. Chem. Soc. 1959, 81,
4083-4087. (b) Kirmse, W.; Siegfried, R. J. Am. Chem. Soc. 1983, 105,
950-956.
(30) Berson, J. A.; Walla, J. S.; Remanick, A.; Suzuki, S.; Reynolds-
Warnhoff, P.; Willner, D. J. Am. Chem. Soc. 1961, 83, 3986-3997.
Synthesis of Dihydromethylcyclothiazide Stereoisomer
Pairs 1a,1b and 2a,2b. As indicated in Scheme 2, the Diels-
Alder reaction between cyclopentadiene (11) and methacrolein
catalyzed at -78 °C by CAB (prepared from compound 9 and
(27) Furuta, K.; Gao, Q.-z.; Yamamoto, H. Org. Synth. 1992, 71, 86-
94.

EnantioselectiVe Synthesis of Cyclothiazide Analogues
J. Am. Chem. Soc., Vol. 118, No. 19, 1996 4553
Scheme 4a
a Reagents: As described in the legend to Scheme 3.
Chart 3
Chart 4
Figure 1. Digitized NMR spectra of the methyl groups of iodolactones
15 and 28 recorded in the presence of the chiral alcohol (R)-2,2,2-
trifluoro-1-(9-anthryl)ethanol in CDCl3. (A) Racemic mixture of
idodolactones 15 and 28 derived from the Diels-Alder reaction of
cyclopentadiene and methacrolein carried out in the absence of a CAB
catalyst. The resonances are at δ 1.14 and δ 1.35. (B) Iodolactone 15
([R]22D ) -103.5°) derived from the same Diels-Alder reaction carried
out in the presence of CAB catalyst prepared from ligand 9. (C)
Iodolactone 28 ([R]22D ) +104.8°) derived from the same Diels-Alder
reaction carried out in the presence of CAB catalyst prepared from
ligand 10. (D) Iodolactone 28 containg 10 mol % of a racemic mixture
of iodolactones 15 and 28. A small peak (indicated by the arrow) for
iodolactone 15 is detectable upfield of the large peak recorded for
iodolactone 28. The scale of the x-axis in panels A and B is expanded
by a factor of 2.3 relative to that shown in panels C and D.
diastereomer products 2a and 2b was obtained from acid 16
utilizing the analogous reaction sequence outlined in Scheme
4.
Synthesis of Dihydromethylcyclothiazide Stereoisomer
Pairs 3a,3b and 4a,4b. Under the same reaction conditions,
but using the catalyst made from ligand 10 and BH3‚THF, a
mixture of (1R-exo) and (1R-endo)-2-methylbicyclo[2.2.1]hept-
5-ene-2-carboxaldehydes (25, Chart 3) was prepared. Oxidation
of carboxaldehydes 25 with Jones reagent followed by iodola-
Scheme 3a
ctone separation gave (1R-exo)-acid 27 (76% yield, [R]22
)
D
+65.6°, indicating 97.5% ee30) and iodolactone 28 (12% yield,
>95% ee as determined by the 1H NMR experiment summarized
in Figure 1). The (1R-exo)-acid 27 was converted in four steps
(27 f 30 f 31 f 32 f 33) into (1S-exo)-carboxaldehyde 33,
and compound 33 was converted into an ∼1:1 ratio of the third
pair of enantiomerically pure diastereomer products 3a and 3b.
Finally, (1R-endo)-acid 29 was converted in four steps (29 f
34 f 35 f 36 f 37) into (1S-endo)-carboxaldehyde 37, and
compound 37 was converted into an ∼1:1 ratio of the fourth
pair of enantiomerically pure diastereomer products 4a and 4b.
a Reagents: (a) CH2N2, room temperature; (b) DIBALH, room
temperature, 4 h; (c) 10% Pd-C, H2, 45 psi, room temperature,
overnight; (d) PCC-NaOAc, room temperature, 2 h; (e) 4-amino-6-
chloro-1,3-benzenedisulfonamide, EtOH-6 N HCl (1:1), room tem-
perature, overnight.
1-(9-anthryl)ethanol in CDCl3.31 Control experiments indicated
that 5% of the enantiomeric iodolactone 28 (vide infra) was
readily detected by this method.
Conformational Analysis and Assignments of Absolute
Configuration
Acid 14 (Scheme 3) was methylated with diazomethane to
give ester 17 (95%) which was then reduced with DIBALH to
give olefinic alcohol 18 (84%). Hydrogenation of the double
bond in alcohol 18 using a Pd-C catalyst yielded saturated
alcohol 19 (83%). Without purification, crude aldehyde 20,
obtained by NaOAc buffered PCC oxidation of compound 19,
was reacted with 4-amino-6-chloro-1,3-benzenedisulfonamide20
to give an ∼1:1 ratio of the first pair of enantiomerically pure
(i.e., >95% ee) diastereomer products 1a and 1b. Similarly,
an ∼1:1 ratio of the second pair of enantiomerically pure
A Monte Carlo conformational search32 was performed on
each of the diastereomers using the simplified model compound
shown in Chart 4. Because high quality parameters were not
available for aryl sulfonamides, the low energy conformations
were reminimized in vacuo using AM1. Figure 2 shows the
minimum energy conformations of compounds 38a and 38b
which are the model compounds with stereochemistries corre-
sponding to those of the dihydromethylcyclothiazide diastere-
omer pair 4a (the most active stereoisomer, Vide infra) and 4b.
(31) (a) Parker, D. Chem. ReV. 1991, 91, 1441-1457. (b) Pirkle, W. H.;
Sikkenga, D. L.; Pavlin, M. S. J. Org. Chem., 1977, 42, 384-387. (c) Pirkle,
W. H.; Sikkenga, D. L. J. Org. Chem., 1977, 42, 1370-1374.
(32) Goodman, J. M.; Still, W. C. J. Comput. Chem. 1991, 12, 1110-
1117.

4554 J. Am. Chem. Soc., Vol. 118, No. 19, 1996
Hu et al.
Table 1. Energies of Dihydromethylcyclothiazide Conformers
Determined in Vacuum Using AM1
compda
conformer
∆Hf (kcal/mol)
θ1
θ2
1a
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
0
174
145
-54
40
128b
2.8
3.4
3.7
0
2.5
3.5
3.8
0
1.9
3.0
5.9
0
2.2
3.1
5.8
145b
23c
128b
1b
2a
2b
177
-130b
-23c
-129b
-132b
-126b
-33c
-136b
-136b
128b
-175
-51
46
-172
-171
-38
83
-174
-174
-41
69
41c
135b
135b
a Data are given for compounds 1a,b and 2a,b. For the enantiomers,
3a,b and 4a,b, the angles are of the opposite sign. b Pseudoaxial.
c Pseudoequatorial.
Because the molecular modeling studies show that each
dihydromethylcylothiazide stereoisomer exists substantially as
a single conformer in solution, the assignment of chirality at
C-3 for the separated diastereomer pairs was based on a
correlation of the calculated minimum energy conformations
with NOE data from 2D NOESY experiments performed on
separated diastereomer pairs 3a and 3b and 4a and 4b. From
modeling each pair of diastereomers, it was observed that
altering the chirality at C-3 alters the relationship between the
atoms in the heterocyclic ring and those in the bicyclo[2.2.1]-
heptyl moiety. In one diastereomer of each pair the short
distance between the bridgehead hydrogen H-1′ and the sul-
fonamide hydrogen H-2 indicates that an NOE between these
two hydrogens is expected, while in the other diastereomer of
the pair an NOE between bridgehead hydrogen H-1′ and the
proximate amino hydrogen H-4 is expected (Figure 2). These
expected NOEs were observed in 2D NOESY experiments
wherein one diastereomer in each pair shows an NOE between
H-1′ and H-2, while the other diastereomer in the pair shows
an NOE between H-1′ and H-4. Accordingly, this NOE data
together with the assignment of the H-2 and H-4 resonances
(made possible by the fact that only H-4 has an expected, and
observed, NOE with aromatic hydrogen H-5) enables the
unambiguous assignment of structure to each component of a
diastereomer pair. In several cases, additional NOEs were also
evident between the amino and sulfonamide hydrogens and the
hydrogens attached to C-3′. These additional NOEs are
consistent with the structural assignments made for the diaste-
reomer pairs. Thus, the absolute configurations of the dihy-
dromethylcyclothiazide stereoisomers shown in Chart 2 are
based on the combination of the results from this NMR analysis,
which was used for the assignment of chirality at C-3, and the
chiralities of the stereocenters established by the enantioselective
synthesis of the bicyclo[2.2.1]heptyl portion of the molecules.
Electrophysiology. Voltage clamp recordings were obtained
from cultured postnatal rat hippocampal neurons using the patch
clamp technique in the whole-cell configuration.34 Pairs of
diastereomers and individual diastereomers >95% pure were
tested for the ability to reduce AMPA receptor desensitization
(Figure 3). AMPA receptor gated currents were elicited by
application of 1 mM L-glutamate for 300 ms, which typically
resulted in a large inward current that desensitized to a much
smaller amplitude (usually about 10% or less of the peak
Figure 2. The global minima, calculated using AM1, of model
compounds 38a and 38b. The diagnostic NOEs used to identify the
chirality of C-3 in the corresponding dihydromethylcyclothiazides 4a
and 4b, respectively, are shown. In acetone-d6 compound 4a shows an
NOE between the sulfonamide hydrogen H-2 and the bridgehead
hydrogen H-1′. Compound 4b shows an NOE between the amino
hydrogen H-4 and the bridgehead hydrogen H-1′. Similary, but not
shown, in the structures assigned to diasteromer pair 3a and 3b an
NOE between H-1′ and H-4 is observed for compound 3a, and an NOE
between H-1′ and H-2 is observed for compound 3b.
Using the conformational search procedure, several low
energy conformations were identified for each compound. The
structural similarity of the eight compounds results in similar
minima being observed for each compound. Rotation of the
bicyclo[2.2.1]heptyl moiety relative to the heterocyclic ring
results in three low energy, staggered conformations which can
be characterized using the dihedral angle θ1 (H-3, C-3, C-2′,
methyl C).33 The low energy conformations were also found
to include two different ring puckers which can be characterized
by the angle θ2 (H-2, N-2, C-3, H-3). When θ2 is near (130°
(the sign of the angle depends on the chirality of C-3) the
bicyclo[2.2.1]heptyl moiety is in a pseudoequatorial orientation
and near (35° this moiety is in a pseudoaxial orientation. The
energy difference between the pseudoaxial and pseudoequatorial
conformations is between 1.9 and 3.4 kcal/mol.
Table 1 lists the energy differences between the low energy
states. For compounds 1a-4a and 1b-4b the minimum energy
conformation of each stereoisomer contained the bicyclo[2.2.1]-
heptyl moiety in a pseudoequatorial orientation with θ1 near
180°. In each case, the energy difference between the global
minimum and the next lowest conformer is between 1.9 and
2.8 kcal/mol as calculated using AM1.
(33) The ring carbons in the bicyclo[2.2.1]heptyl group are represented
as primed numbers to avoid confusion. The names given for the dihydro-
methylcyclothiazide stereoisomers in the Experimental Section do not
contain the primed numbering of these carbons because labeling of the
carbons in this manner is not in conformance with correct rules of
nomenclature.
(34) Hamill, O. P.; Marty, A.; Neher, E.; Sakmann, B.; Sigworth, F. J.
Pflu¨egers Arch. 1981, 391, 85-100.

EnantioselectiVe Synthesis of Cyclothiazide Analogues
J. Am. Chem. Soc., Vol. 118, No. 19, 1996 4555
current), reaching a steady-state well within the first 100 ms of
glutamate application. Reduction of desensitization was mani-
fested by slowing the rate of desensitization and by potentiation
of the steady-state current amplitude. The steady-state current
was defined as the average current measured over the last 30
ms of the 300 ms application of glutamate, and potentiation by
a compound was quantitated as a percentage of the control
response without compound. The results demonstrate that the
absolute configuration of the bicyclo[2.2.1]heptyl portion of the
molecules had the largest effect upon pharmacological activity.
Thus, diastereomer pairs 1a and 1b and 2a and 2b prepared
from (1R-exo)-carboxaldehyde 20 and (1R-endo)-carboxalde-
hyde 24, respectively, were less potent inhibitors of desensitiza-
tion than diastereomer pairs 3a and 3b and 4a and 4b prepared
from (1S-exo)-carboxaldehyde 33 and (1S-endo)-carboxaldehyde
37, respectively (Figure 3A,B). Moreover, 1 µM diastereomer
pair 4a and 4b had a potency comparable to that of 1 µM of a
commercial preparation of cyclothiazide (Figure 3B), suggesting
that the absence of the double bond and the presence of the
2-methyl substituent in the bicyclo[2.2.1]heptyl group of the
dihydromethylcyclothiazide diastereomers does not markedly
diminish their potency as inhibitors of AMPA receptor desen-
sitization.
The diastereomer pairs were also tested at 10 µM, and greater
reduction of desensitization was observed than at 1 µM.
Notably, the superiority of pair 4a and 4b as seen at 1 µM was
much more obvious at the higher concentration (Figure 3C, open
bars). Then, each diastereomer pair was separated into its
components (>95% purity by NMR criteria for each HPLC
purified diastereomer), and the individual diastereomers were
tested at 10 µM. For each diastereomer pair, each diastereomer
component was evaluated on the same cells to reduce variability
in what were relatively small, but significant differences in
activity between the components of the 1a and 1b pair and the
2a and 2b pair. In contrast, 3a and 3b were not significantly
different in activity, while the largest difference was between
4a and 4b (Figure 3C, hatched bars). The most active
compound was 4a, and representative traces comparing 4a to
its enantiomer 2a emphasize the dramatic difference in activity
between these compounds at low micromolar concentrations
(Figure 3D,E).
At 10 µM, the potentiation of steady-state currents by the
diastereomers was quickly and completely reversible, except
for compound 4a. The average halftime to recovery to the
control steady-state to peak ratio (usually about 0.10 for control
responses) was 163 ( 29 s for compound 4a (five cells)
compared to 62 ( 10 s for 4b (four cells, P < 0.01). The slow
recovery from potentiation is a characteristic of commercially
available cyclothiazide8,17a,35 and suggests that the activity of
cyclothiazide is dominated by the stereoisomer component
having a structure analogous to that of compound 4a. For
cyclothiazide, this stereoisomer is 1R-[1R,2R(R*),4R]]-3-bicyclo-
[2.2.1]hept-5-en-2-yl-6-chloro-3,4-dihydro-2H-1,2,4-benzothia-
diazine-7-sulfonamide 1,1-dioxide. As observed with pair 4a
and 4b, the slow off rate for compound 4a may explain why
the activity of the pair is similar to that of compound 4a alone,
instead of giving activity intermediate between compounds 4a
and 4b. In fact, some of the activity observed for compound
4b may be accounted for by the presence of a few percent
(<5%) of compound 4a.
Figure 3. Stereoisomers of dihydromethylcyclothiazide differentially
affect AMPA receptor desensitization. (A) Recordings from a repre-
sentative voltage-clamped neuron at -60 mV. L-Glutamate (GLU, 1
mM) applied for the duration of the bar produces a large inward current
that diminishes (desensitizes) to a much smaller steady-state amplitude
(defined as the average measured between the arrows) in spite of the
continued presence of glutamate. Successive traces from left to right
are GLU currents after preapplication of 1 µM of the diastereomer pairs
1a and b, 2a and b, 3a and b, and 4a and b. Current desensitization
was slowest and thus most obvious with 4a and b, but potentiation of
the steady-state current also reflects reduction of desensitization and
was evident for pairs 2a and b and 3a and b. (B) Cumulative data
from 13 cells upon which all four pairs were tested showed that 4a
and b potentiates the steady-state current the most, comparable to
cyclothiazide (CYZ, 9 of 13 cells, trace not shown in A). (C) In a
different set of cells, 10 µM of each of the diastereomer pairs potentiated
GLU currents (open bars), and the superiority of 4a and b over the
other three pairs was more obvious. Each pair of hatched bars in (C)
represent comparisons between 10 µM of the separated a and b
diastereomer components. The four diastereomer pairs were evaluated
on four separate sets of cells (n ) 11-14), with each a and b pair
tested on the same set of cells. Except for 3a versus 3b, a significantly
different (*P < 0.05) potentiation of the GLU current was observed
for the separated stereoisomers of each diastereomer pair. (D and E)
Representative tracings from two different cells exemplifying the
stereospecific reduction of desensitization by dihydromethylcyclothiaz-
ide isomers. Compounds 2a and 4a are an enantiomeric pair. Stereoi-
somer 2a (10 µM) produced modest potentiation of the steady-state
current (D), while stereoisomer 4a (10 µM) produced marked steady-
state current potentiation and obvious reduction of desensitization (E).
example, these compounds will be useful pharmacological
probes of AMPA receptor subtype specificity. Studies of the
stereospecific effects of the compounds on glutamate excito-
toxicity and the memory enhancing effects of each stereoisomer
are also warranted. It is also likely that the dihydromethylcy-
clothiazide stereoisomers will be of use in pharmacological
studies of the stereospecificity of benzothiadiazine actions on
the sodium-chloride cotransporter found in mammalian kidney.
Historically, this cotransporter was the original therapeutic target
of benzothiadiazine or “thiazide-like” diuretics, and the inhibi-
The availability of these pure dihydromethylcyclothiazide
stereoisomers will make it possible to address many additional
issues related to glutamate physiology and pharmacology. For
(35) Patneau, D. K.; Vyklickly, L. J.; Mayer, M. L. J. Neurosci. 1993,
13, 3496-3509.

4556 J. Am. Chem. Soc., Vol. 118, No. 19, 1996
Hu et al.
saturated aqueous NaHCO3 and extracted with dichloromethane (2 ×
300 mL). The combined organic layers were washed with water (300
mL) and dried over Na2SO4. The solvent was removed to give an oil
which was purified by chromatography (silica gel, hexane-Et2O-CH2-
Cl2 ) 6:2.5:1.5) to give an intermediate triester (16.5 g, 63%) as a
colorless oil. This triester (16 g, 30 mmol) in EtOAc (150 mL) was
hydrogenolyzed over a 10% Pd-C catalyst (1.6 g) under hydrogen
(100 psi, room temperature) for 2 h. The catalyst was filtered and
washed with EtOAc (30 mL). The solvent was removed to give
compound 9 as a colorless oil which, after the addition of EtOAc (30
mL), became a white solid (9.5 g, 99%): mp 178-181 °C (lit.27 mp
tion of this cotransporter explains the clinical use of these
compounds for the treatment of hypertension and edematous
states resulting from heart, kidney, and liver disease.36 The
recent cloning and characterization of this cotransporter37-39
greatly enhances the attractiveness of the suggested stereochem-
ical studies.
Experimental Section
General Methods. All melting points were determined with a
capillary melting point apparatus and are uncorrected. NMR spectra
were recorded at ambient temperature in CDCl3 (unless noted otherwise)
with a 5 mm probe on either a Varian Gemini-300 operating at 300
184-186 °C); [R]22 ) -75.1° (EtOH); IR 3402 (br), 3031, 1790,
D
1
1741, 1723, 1601, 1478, 1259, 1103 cm-1; H NMR (acetone-d6) δ
1
MHz (1H) or 75 MHz (13C). For H NMR and 13C NMR spectra the
7.36 (t, J ) 8.5 Hz, 1H), 6.67 (d, J ) 8.6 Hz, 2H), 5.41 (d, J ) 2.1
Hz, 1H), 4.57 (s, 1H), 3.72 (s, 6H); 13C NMR (DMSO-d6) δ 171.62,
168.27, 164.78, 157.29, 131.77, 111.74, 104.23, 73.77, 70.12, 55.97.
Anal. Calcd for C13H14O9: C, 49.69; H, 4.49. Found: C, 49.49; H,
4.75.
internal references were TMS (δ 0.00) and CDCl3 (δ 77.00), respec-
tively. IR spectra were recorded as films on a NaCl plate with a Perkin-
Elmer 1710 FT-IR spectrophotometer. Optical rotations were recorded
on a Perkin-Elmer Model 241 polarimeter. Elemental analyses were
carried out by M-H-W Laboratories, Phoenix, AZ. Solvents were used
either as purchased or dried and purified by standard methodology.
The 4-amino-6-chloro-1,3-benzenedisulfonamide was purchased from
Aldrich Chemical Co., Milwaukee, WI. Flash chromatography was
performed using silica gel (32-63 microns) purchased from Scientific
Adsorbants, Atlanta, GA.
[S-(R*,R*)]-2-[(2,6-Dimethoxybenzoyl)oxy-3-hydroxybutanedio-
ic Acid (10). Using the same procedure described immediately above,
dibenzyl ester 8 (14.9 g, 41.1 mmol) gave an intermediate triester (14.0
g, 65%) as a colorless oil which was hydrogenolyzed to give compound
10 (8.0 g, 96%) as a white solid: mp 173-176 °C; [R]22 ) +72.2°
D
1
(EtOH). The IR, H NMR, and 13C NMR spectra were identical to
those described for compound 9. Anal. Calcd for C13H14O9: C, 49.69;
H, 4.49. Found: C, 49.65; H, 4.31.
Computer Modeling and 2D NOESY. Monte-Carlo conforma-
tional searches32 were performed using Macromodel 5.040 with the
MM3* derivative of the MM3 forcefield.41 The low energy conformers
were reminimized with Mopac 9342 using the AM1 method.43 The 2D
NOESY experiments were performed at 600 MHz in acetone-d6.
[R-(R*,R*)]-2,3-Dihydroxybutanedioic Acid Bis(phenylmethyl)
Ester (7). A mixture of [R-(R*,R*)]-2,3-dihydroxybutanedioic acid
(5, 10 g, 66.6 mmol) and benzyl alcohol (28.8 g, 266.4 mmol) was
heated at 200 °C (oil bath temperature) in a flask equipped with a Dean-
Stark apparatus for 1 h under nitrogen. Then, the temperature was
raised to 225 °C (oil bath temperature) for another 1 h. The mixture
was cooled to room temperature and purified by chromatography (silica
gel, hexane-Et2O-CH2Cl2 ) 6:2.5:1.5) to give dibenzyl ester 7 (20.5
g, 93%) as a white solid: mp 62-64 °C (lit.27,44 mp 49-50 °C; mp
67-69 °C); [R]22D ) +8.3° (EtOH); [R]22D ) -10.1° (CHCl3); IR 3483,
(1S-exo)-2-Methylbicyclo[2.2.1]hept-5-ene-2-carboxylic Acid (14)
and [3S-(3r,3aâ,5r,6â,6aâ)]-Hexahydro-6-iodo-3-methyl-3,5-methano-
2H-cyclopenta[b]furan-2-one (15). To a stirred solution of crude
product 12 (a mixture of (1S-endo)- and (1S-exo)-diastereomers) in
acetone (150 mL), which was obtained by a literature procedure26a from
methacrolein (3.3 mL, 40 mmol) and cyclopentadiene (11, 9.8 mL,
120 mmol) in the presence of CAB catalyst (10% by mol) made from
compound 9 (1.3 g, 4 mmol) and BH3‚THF (1.0 M solution in THF,
4.0 mL, 4.0 mmol), was added dropwise Jones reagent until an orange
color persisted at 0 °C. Isopropyl alcohol (2 mL) was added to destroy
any excess reagent. After most solvent was removed, the residue was
diluted with water (100 mL) and extracted with EtOAc (2 × 150 mL).
The combined organic layers were washed with brine (150 mL) and
dried over Na2SO4. The solvent was removed to give an oil which
was dissolved in 10% aqueous K2CO3 (30 mL) and extracted with ether
(2 × 30 mL). The aqueous layer was acidified with 6 N aqueous HCl
and extracted with EtOAc (2 × 100 mL). The combined organic layers
were washed with brine (100 mL) and dried over Na2SO4. The solvent
was removed to give a mixture of acids 13 (4.5 g, 74% overall for the
two steps) as a yellowish solid.
3032, 2961, 1747, 1498, 1456, 1378, 1265, 1128, 1090, 738, 698 cm-1
;
1H NMR δ 7.37 (s, 10H), 5.28 (dd, J ) 12.3 Hz, J ) 20.3 Hz, 4H),
4.63 (d, J ) 6.9 Hz, 2H), 3.34-3.30 (m, 2H); 13C NMR δ 171.33,
134.71, 128.64, 128.36, 72.08, 68.02. Anal. Calcd for C18H18O6: C,
65.45; H, 5.49. Found: C, 65.69; H, 5.32.
[S-(R*,R*)]-2,3-Dihydroxybutanedioic Acid Bis(phenylmethyl)
Ester (8). Using the same procedure described immediately above,
[S-(R*,R*)]-2,3-dihydroxybutanedioic acid (6, 10 g, 66.6 mmol) gave
To a solution of acids 13 (4.5 g, 30 mmol) and NaOH (1.2 g, 30
mmol) dissolved in water (10 mL) was added a solution of I2-KI-
NaHCO3 (1:4:6 by mol) in water (60 mL/10 mmol of I2) at 0 °C until
a deep orange color persisted. After stirring for another 40 min, the
mixture was extracted with Et2O (2 × 50 mL). The aqueous layer
was acidifed with 6 N aqueous HCl at 0 °C and then was extracted
with Et2O (2 × 100 mL). The combined organic layers were washed
with water (100 mL), 10% Na2S2O3 (20 mL), and brine (100 mL) and
dried over Na2SO4. The solvent was removed to give crude product
as a yellow semisolid which was purified by recrystallization from
hexane to give product 14 (3.4 g, 76%) as white crystals: mp 39-41
1
dibenzyl ester 8 (20.8 g, 95%) as a white solid with the same IR, H
NMR, and 13C NMR data as 7. Compound 8 had mp 66-68 °C (lit.44
mp 67.5-69 °C); [R]22D ) -8.0° (EtOH). Anal. Calcd for C18H18O6:
C, 65.45; H, 5.49. Found: C, 65.60; H, 5.55.
[R-(R*,R*)]-2-[(2,6-Dimethoxybenzoyl)oxy-3-hydroxybutanedio-
ic Acid (9). A stirred solution of compound 7 (18 g, 50 mmol) and
2,6-dimethoxybenzoic acid (9.3 g, 51 mmol) in dry dichloromethane
(150 mL) was treated with trifloroacetic anhydride (11.6 g, 55 mmol)
added dropwise within 10 min at 0 °C under nitrogen. After an
additional 60 min at room temperature, the mixture was poured into
°C; [R]22 ) -66.2° (95% EtOH); IR 2978, 2877, 1695, 1578, 1458,
D
(36) Stokes, J. B. Kidney Int. 1989, 36, 427-433.
(37) Gamba, G.; Saltzberg, S. N.; Lombardi, M.; Miyanoshita, A.; Lytton,
J.; Hediger, M. A.; Brenner, B. M.; Hebert, S. C. Proc. Natl. Acad. Sci.
U.S.A. 1993, 90, 2749-2753.
(38) Gamba, G.; Miyanoshita, A.; Lombardi, M.; Lytton, J.; Lee, W.-
S.; Hediger, M. A.; Hebert, S. C. J. Biol. Chem. 1994, 269, 17713-17722.
(39) Hebert, S. C.; Gamba, G. Clin. InVestig. 1994, 72, 692-694.
(40) Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.;
Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput.
Chem. 1990, 11, 440-467.
(41) Allinger, N. L.; Yuh, Y. H.; Lii, J.-H. J. Am. Chem. Soc. 1989,
111, 8551-8556.
(42) Mopac 93. Fujitsu Limited obtained through QCPE, Creative Arts
Building 181, Bloomington, IN, 47405.
(43) Stewart, J. J. P. J. Comput. Aided Mol. Design 1990, 4, 1-105.
(44) Smith, H. L.; Brown, E. S.; Smith, J. D.; Andrako, J. J. Pharm.
Sci. 1965, 54, 1269-1273.
1
1405 cm-1; H NMR δ 6.26-6.23 (m, 1H), 6.12-6.09 (m, 1H), 3.06
(s, 1H), 2.85 (s, 1H), 2.46 (dd, J ) 3.9 Hz, J ) 12.1 Hz, 1H), 1.47 (s,
2H), 1.17 (s, 3H), 0.88 (d, J ) 12.1 Hz, 1H); 13C NMR δ 185.84,
138.74, 133.53, 50.41, 49.50, 49.04, 42.84, 37.35, 24.21. Anal. Calcd
for C9H12O2: C, 71.03; H, 7.95. Found: C, 70.88; H, 8.13.
The combined organic layers separated from the above acidified
aqueous layer were washed with 10% aqueous Na2S2O3 (20 mL) and
brine (50 mL) and dried over Na2SO4. The solvent was removed to
give a solid which was purified by chromatography (silica gel, 10%
EtOAc in hexane) to give iodolactone 15 (0.8 g, 9.7%) as colorless
needles (from Et2O-hexane): mp 81-82 °C; [R]22 ) -103.5°
D
(CHCl3); IR 2968, 2881, 1776, 1443, 1380, 1084, 1043 cm-1; 1H NMR
δ 5.04 (d, J ) 5.5 Hz, 1H), 3.82 (d, J ) 2.5 Hz, 1H), 2.77 (d, J ) 4.4
Hz, 1H), 2.64 (s, 1H), 2.34 (dd, J ) 1.8 Hz, J ) 11.6 Hz, 1H), 1.92

EnantioselectiVe Synthesis of Cyclothiazide Analogues
J. Am. Chem. Soc., Vol. 118, No. 19, 1996 4557
(dd, J ) 2.2 Hz, J ) 13.5 Hz, 1H), 1.86 (dd, J ) 1.7 Hz, J ) 9.9 Hz,
1H), 1.54 (dd, J ) 4.2 Hz, J ) 13.6 Hz, 1H), 1.15 (s, 3H); 13C NMR
δ 180.85, 86.90, 51.55, 47.39, 42.67, 41.75, 36.21, 29.86, 19.29. Anal.
Calcd for C9H11IO2: C, 38.87; H, 3.99; I, 45.63. Found: C, 39.06; H,
3.77; I, 45.49.
(1R-exo)-2-Methylbicyclo[2.2.1]hept-5-ene-2-carboxylic Acid (27)
and [3R-(3r,3aâ,5r,6â,6aâ)]-Hexahydro-6-iodo-3-methyl-3,5-methano-
2H-cyclopenta[b]furan-2-one (28). Using the same procedure de-
scribed immediately above, acids 26 were converted into compounds
27 and 28.
56-57 °C; [R]22D ) -63.5° (CHCl3); IR 3250, 3060, 2960, 2925, 2869,
1572, 1451, 1039, 1012 cm-1; 1H NMR δ 6.15-6.08 (m, 2H), 3.57 (s,
2H), 2.77 (s, 1H), 2.56 (s, 1H), 1.55 (d, J ) 8.6 Hz, 1H), 1.44 (dd, J
) 3.6 Hz, J ) 11.7 Hz, 1H), 1.36 (d, J ) 8.5 Hz, 1H), 0.92 (s, 3H),
0.78 (dd, J ) 2.6 Hz, J ) 11.7 Hz, 1H); 13C NMR δ 136.76, 135.37,
72.21, 47.76, 47.51, 43.61, 43.05, 37.24, 22.75. Anal. Calcd for
C9H14O: C, 78.21; H, 10.21. Found: C, 78.15; H, 10.15.
(1R-exo)-2-Methylbicyclo[2.2.1]hept-5-ene-2-methanol (31). Com-
pound 31 (86% yield) was obtained from ester 30 using the same
procedure described immediately above as colorless crystals (from
hexane): mp 56-57 °C; [R]22D ) +63.0° (CHCl3). The IR, 1H NMR,
and 13C NMR spectra were identical to those described for compound
18. Anal. Calcd for C9H14O: C, 78.21; H, 10.21. Found: C, 78.00;
H, 10.42.
Compound 27 (76% yield) was obtained as white crystals: mp 38-
41 °C; [R]22 ) +65.6° (95% EtOH, lit.30 [R]22 ) +67.3° in 95%
D
D
EtOH). The IR, 1H NMR, and 13C NMR spectra were identical to those
described for compound 14. Anal. Calcd for C9H12O2: C, 71.03; H,
7.95. Found: C, 70.88; H, 7.76.
(1R-exo)-2-Methylbicyclo[2.2.1]heptane-2-methanol (19). A solu-
tion of compound 18 (1.3 g, 9.4 mmol) in EtOAc (30 mL) was
hydrogenated (45 psi, room temperature, overnight) over 10% Pd-C
catalyst (130 mg). The catalyst was filtered and washed with EtOAc
(30 mL). The combined organic layers were evaporated to give a
colorless oil which was purified by chromatography (silica gel, 20%
EtOAc in hexane) to give compound 19 (1.1 g, 83%) as colorless
Compound 28 (12% yield) was obtained as colorless needles: mp
1
81-82 °C; [R]22 ) +104.8° (CHCl3). The IR, H NMR, and 13C
D
NMR spectra were identical to those described for compound 15. Anal.
Calcd for C9H11IO2: C, 38.87; H, 3.99; I, 45.63. Found: C, 39.00; H,
4.04; I, 45.45.
(1S-endo)-2-Methylbicyclo[2.2.1]hept-5-ene-2-carboxylic Acid (16).
To a solution of iodolactone 15 (6.0 g, 21.6 mmol) in HOAc (25 mL)
was added portions of zinc dust (2.1 g, 32 mmol) with stirring in a
water bath (22 °C). After stirring at room temperature for another 3
h, the mixture was filtered and washed with Et2O (2 × 30 mL). The
combined organic layers were washed with water (3 × 50 mL) and
dried over Na2SO4. The solvent was removed to give a solid which
was purifed by recrystallization from 25% aqueous EtOH to give acid
16 (3.0 g, 91%) as white crystals: mp 96-100 °C; [R]22D ) +109.7°
crystals: mp 28-30 °C (from hexane); [R]22 ) -27.9° (CHCl3); IR
D
1
3350, 2950, 2869, 1451, 1048, 1027 cm-1; H NMR δ 3.40 (d, J )
10.6 Hz, 1H), 3.21 (d, J ) 10.6 Hz, 1H), 2.18 (t, J ) 4.3 Hz, 1H),
1.98 (d, J ) 3.9 Hz, 1H), 1.65-1.13 (m, 7H), 1.04 (s, 3H), 0.82 (dd,
J ) 2.7 Hz, J ) 12.2 Hz, 1H); 13C NMR δ 70.94, 41.70, 42.34, 41.85,
37.64, 37.51, 28.47, 24.46, 21.58. Anal. Calcd for C9H16O: C, 77.09;
H, 11.50. Found: C, 77.14; H, 11.41.
(1S-exo)-2-Methylbicyclo[2.2.1]heptane-2-methanol (32). Com-
pound 32 (96% yield) was obtained from alcohol 31 using the same
procedure described immediately above as colorless crystals (from
hexane): mp 28-30 °C; [R]22D ) +28.2° (CHCl3). The IR, 1H NMR,
and 13C NMR spectra were identical to those described for compound
19. Anal. Calcd for C9H16O: C, 77.09; H, 11.50. Found: C, 77.02;
H, 11.66.
1
(CHCl3); IR 2983, 1696, 1573, 1470, 1374 cm-1; H NMR δ 6.18-
6.11 (m, 2H), 2.84 (s, 1H), 2.79 (s, 1H), 1.88 (dd, J ) 2.6 Hz, J )
12.0 Hz, 1H), 1.57 (d, J ) 8.7 Hz, 1H), 1.48 (s, 3H), 1.46-1.15 (m,
2H); 13C NMR δ 184.45, 137.88, 135.45, 50.72, 49.98, 46.87, 42.55,
37.62, 26.47. Anal. Calcd for C9H12O2: C, 71.03; H, 7.95. Found:
C, 71.10; H, 7.76.
[1R-[1r,2â(R*),4r]]-6-Chloro-3,4-dihydro-3-(2-methylbicyclo-
[2.2.1]hept-2-yl)-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-Di-
oxide (1a) and [1R-[1r,2â(S*),4r]]-6-Chloro-3,4-dihydro-3-(2-
methylbicyclo[2.2.1]hept-2-yl)-2H-1,2,4-benzothiadiazine-7-
sulfonamide 1,1-Dioxide (1b). To a stirred suspension of PCC (6.5
g, 30 mmol) and NaOAc (2.5 g, 30 mmol) in dichloromethane (125
mL) was added a solution of alcohol 19 (2.8 g, 20 mmol) in
dichloromethane (25 mL) at 10 °C under nitrogen. After the mixture
was stirred for another 2 h at room temperature, it was filtered through
a short column filled with silica gel and washed with Et2O (2 × 25
mL). The combined organic layers were washed with brine (2 × 50
mL) and dried over Na2SO4. The solvent was removed to give (1R-
exo)-2-methylbicyclo[2.2.1]heptane-2-carboxaldehyde (20, 2.2 g, 80%)
as an oil which was used immediately without further purification or
characterization.
To a stirred suspension of 4-amino-6-chloro-1,3-benzenedisulfona-
mide (4.7 g, 16.5 mmol) in 6 N aqueous HCl (50 mL) and EtOH (50
mL) at 50 °C was added carboxaldehyde 20 (2.2 g, 16 mmol). After
30 min at 50 °C, the reaction mixture was stirred at room temperature
overnight. The solid product mixture was diluted with water (100 mL),
cooled in an ice-water bath for 1 h, filtered, and washed with water
until the solid was free of mineral acid. The solid was purifed by
recrystallization from 50% aqueous EtOH to give a mixture of 1a and
1b (∼1:1, 4.0 g, 60%) as white crystals: mp 288-291 °C. Anal. Calcd
for C15H20ClN3O4S2: C, 44.38; H, 4.97; Cl, 8.73; N, 10.35; S, 15.80.
Found: C, 44.68; H, 5.25; Cl, 8.95; N, 10.40; S, 15.66.
(1R-endo)-2-Methylbicyclo[2.2.1]hept-5-ene-2-carboxylic Acid (29).
Compound 29 (96% yield) was obtained from iodolactone 28 using
the same procedure described immediately above as white crystals
(hexane): mp 95-99 °C; [R]22D ) -107.1° (CHCl3). The IR, 1H NMR,
and 13C NMR spectra were identical to those described for compound
16. Anal. Calcd for C9H12O2: C, 71.03; H, 7.95. Found: C, 70.79;
H, 7.83.
(1S-exo)-2-Methylbicyclo[2.2.1]hept-5-ene-2-carboxylic Acid Meth-
yl Ester (17). Acid 14 (3.2 g, 21 mmol) was treated with diazomethane
in Et2O to give the corresponding methyl ester as a yellowish oil.
Purification by chromatography (silica gel, 10% EtOAc in hexane) gave
ester 17 (3.3 g, 94.6%) as a colorless oil: [R]22 ) -63.2° (CHCl3);
D
1
IR 3062, 2973, 2876, 1731, 1576 cm-1; H NMR δ 6.10-6.01 (m,
1H), 5.96-5.94 (m, 1H), 3.57 (s, 3H), 2.90 (s, 1H), 2.69 (s, 1H), 2.32
(dd, J ) 3.9 Hz, J ) 12.0 Hz, 1H), 1.33-1.23 (m, 2H), 0.97 (s, 3H),
0.72 (dd, J ) 2.5 Hz, J ) 11.5 Hz, 1H); 13C NMR δ 178.77, 138.32,
133.25, 51.60, 50.11, 49.26, 48.76, 42.55, 37.33, 23.96. Anal. Calcd
for C10H14O2: C, 72.26; H, 8.49. Found: C, 72.41; H, 8.33.
(1R-exo)-2-Methylbicyclo[2.2.1]hept-5-ene-2-carboxylic Acid Meth-
yl Ester (30). This compound was prepared from acid 27 using the
same procedure described immediately above. Compound 30 (92%
yield) was obtained as a colorless oil: [R]22D ) +59.9° (CHCl3). The
1
IR, H NMR, and 13C NMR spectra were identical to those described
for compound 17. Anal. Calcd for C10H14O2: C, 72.26; H, 8.49.
Found: C, 72.24; H, 8.59.
(1S-exo)-2-Methylbicyclo[2.2.1]hept-5-ene-2-methanol (18). To
a solution of compound 17 (2.0 g, 12 mmol) in dichloromethane (100
mL) was added a solution of DIBALH in THF (1.0 solution in THF,
30 mL, 30 mmol) at 0 °C under nitrogen. After the mixture was stirred
for another 4 h at room temperature (monitored by TLC, 30% EtOAc
in hexane), isopropyl alcohol (2 mL) was added at 0 °C to destroy any
excess reagent, and then 10% aqueous HCl (30 mL) was added. The
organic layer was separated, and the aqueous layer was extracted with
dichloromethane (2 × 30 mL). The combined organic layers were
washed with brine (2 × 50 mL) and dried over Na2SO4. The solvent
was removed to give a solid, which was purified by recrystallization
from hexane to give product 18 (1.4 g, 84%) as colorless crystals: mp
This mixture was separated by reversed phase HPLC (Alltech
Econosil C18 10U column, 250-mm × 10-mm, 1:1 acetone in water,
1.5 mL/min). Compound 1a was obtained as white crystals: mp 285-
286.5 °C; [R]22D ) -120.5° (EtOH); IR 3371, 3261, 2954, 2873, 1597,
1555, 1496, 1334, 1272, 1161, 1069, 1033, 952 cm-1; 1H NMR (CD3-
OD) δ 8.16 (s, 1H), 7.12 (s, 1H), 4.72 (s, 1H) 1.12 (s, 3H); 13C NMR
(CD3OD) δ 149.29, 136.40, 129.38, 127.78, 120.60, 118.87, 73.51,
44.76, 44.42, 44.05, 39.62, 39.03, 28.88, 25.35, 17.98.
Compound 1b was obtained as white crystals: mp 279-281 °C;
[R]22D ) +93.9° (EtOH); IR 3381, 3282, 2953, 2874, 1597, 1555, 1497,
1458, 1344, 1167, 1069, 960 cm-1; 1H NMR (CD3OD) δ 8.17 (s, 1H),

4558 J. Am. Chem. Soc., Vol. 118, No. 19, 1996
Hu et al.
7.10 (s, 1H), 4.65 (s, 1H) 1.10 (s, 3H); 13C NMR (CD3OD) δ 148.84,
136.46, 129.49, 127.76, 120.75, 118.70, 73.39, 44.58, 44.10, 43.70,
39.03, 38.67, 28.88, 25.55, 17.70.
sulfonamide 1,1-Dioxide (2b). Using the same procedure described
for preparation of diastereomers 1a and 1b, alcohol 23 (1.4 g, 9.8 mmol)
was first converted into crude (1R-endo)-2-methylbicyclo[2.2.1]heptane-
2-carboxaldehyde (24, 1.34 g, 10 mmol containing traces of pyridine),
which was reacted with 4-amino-6-chloro-1,3-benzenedisulfonamide
(2.9 g, 10 mmol) to give diastereomers 2a and 2b (∼1:1, 2.0 g, 46%)
as white crystals (from 50% aqueous EtOH): mp 284-286 °C. Anal.
Calcd for C15H20ClN3O4S2: C, 44.38; H, 4.97; Cl, 8.73; N, 10.35; S,
15.80. Found: C, 44.19; H, 5.23; Cl, 8.62; N, 10.51; S, 15.90.
This mixture was separated by reversed phase HPLC (Alltech
Econosil C18 10U column, 250-mm × 10-mm, 47% acetone in water,
1.2 mL/min). Compound 2a was obtained as white crystals: mp 284-
286 °C; [R]22D ) -109.1° (EtOH); IR 3273, 2955, 2873, 1597, 1535,
[1S-[1r,2â(S*),4r]]-6-Chloro-3,4-dihydro-3-(2-methylbicyclo-
[2.2.1]hept-2-yl)-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-Di-
oxide (3a) and [1S-[1r,2â(R*),4r]]-6-Chloro-3,4-dihydro-3-(2-
methylbicyclo[2.2.1]hept-2-yl)-2H-1,2,4-benzothiadiazine-7-
sulfonamide 1,1-Dioxide (3b). Using the same two step procedure
described immediately above, alcohol 32 gave a mixture of diastere-
omers 3a and 3b (∼1:1, 47% yield) as white crystals (from 50%
aqueous EtOH): mp 287-289 °C. Anal. Calcd for C15H20ClN3O4S2:
C, 44.38; H, 4.97; Cl, 8.73; N, 10.35; S, 15.80. Found: C, 44.28; H,
5.18; Cl, 8.49; N, 10.32; S, 15.65.
1
1479, 1426, 1338, 1256, 1175, 1064, 946, 731 cm-1; H NMR (CD3-
This mixture was separated by reversed phase HPLC (Alltech
Econosil C18 10U column, 250-mm × 10-mm, 1:1 acetone in water,
OD) δ 8.17 (s, 1H), 7.09 (s, 1H), 4.75 (s, 1H) 1.08 (s, 3H); 13C NMR
(CD3OD) δ 149.21, 136.45, 129.28, 127.75, 120.54, 118.67, 73.14,
47.34, 44.83, 44.68, 39.02, 38.12, 28.96, 24.51, 21.17.
1.5 mL/min). Compound 3a was obtained as white crystals and had:
1
mp 279-280 °C; [R]22 ) +116.8° (EtOH). The IR, H NMR, and
D
13C NMR spectra were identical to those described for compound 1a.
Compound 3b was obtained as white crystals and had mp 277-278
°C; [R]22D ) -90.2° (EtOH). The IR, 1H NMR, and 13C NMR spectra
were identical to those described for compound 1b.
Compound 2b was obtained as white crystals: mp 281-283 °C;
[R]22 ) +118.6° (EtOH); IR 3366, 2960, 1598, 1554, 1497, 1337,
D
1
1173, 1067, 1039, 952 cm-1; H NMR (CD3OD) δ 8.19 (s, 1H), 7.17
(s, 1H), 4.62 (s, 1H) 1.06 (s, 3H); 13C NMR (CD3OD) δ 148.75, 136.50,
129.71, 127.76, 121.03, 118.87, 73.05, 45.49, 44.92, 44.58, 38.72, 38.31,
29.19, 25.22, 21.06.
(1S-endo)-2-Methylbicyclo[2.2.1]hept-5-ene-2-methanol (22). Acid
16 (3.0 g, 23.9 mmol) was treated with diazomethane in Et2O to give
a yellowish oil which was purified by chromatography (silica gel, 10%
EtOAc in hexane) to give the corresponding methyl ester 21 (3.3 g,
67%) as a colorless oil which was used without characterization.
To a solution of ester 21 (2.8 g, 16.8 mmol) in dichloromethane
(100 mL) was added a solution of DIBALH (1.0 M solution in THF,
54 mL, 54 mmol) at 0 °C under nitrogen. After stirring at room
temperature for another 3 h, the reaction was quenched by the addition
of 6 N aqueous HCl at 0 °C. The organic layer was separated, and the
aqueous layer was extracted with dichloromethane (2 × 50 mL). The
combined organic layers were washed with water (100 mL) and brine
(100 mL) and dried over Na2SO4. The solvent was removed to give a
solid which was recrystallized from hexane to give alcohol 22 (1.78 g,
[1S-[1r,2r(R*),4r]]-6-Chloro-3,4-dihydro-3-(2-methylbicyclo-
[2.2.1]hept-2-yl)-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-Di-
oxide (4a) and [1S-[1r,2r(S*),4r]]-6-Chloro-3,4-dihydro-3-(2-
methylbicyclo[2.2.1]hept-2-yl)-2H-1,2,4-benzothiadiazine-7-
sulfonamide 1,1-Dioxide (4b). Using the same two step procedure
described immediately above, alcohol 36 gave a mixture of diasteromers
4a and 4b (∼1:1, 39% yield) as white crystals (from 50% aqueous
EtOH): mp 278-281 °C. Anal. Calcd for C15H20ClN3O4S2: C, 44.38;
H, 4.97; Cl, 8.73; N, 10.35; S, 15.80. Found: C, 44.58; H, 4.78; Cl,
8.87; N, 10.35; S, 16.06.
This mixture was separated by reversed phase HPLC (Alltech
Econosil C18 10U column, 250-mm × 10-mm, 47% acetone in water,
1.2 mL/min). Compound 4a was obtained as white crystals: mp 277-
278.5 °C; [R]22D ) +108.4° (EtOH). The IR, 1H NMR, and 13C NMR
spectra were identical to those described for compound 2a.
75%) as white crystals: mp 90-92 °C; [R]22 ) +31.6° (CHCl3); IR
D
3277, 3066, 2967, 2933, 2870, 1572, 1464, 1443, 1034 cm-1; 1H NMR
δ 6.13-6.07 (m, 2H), 3.27 (q, J ) 10.5 Hz, 2H), 2.79 (s, 1H), 2.50 (d,
J ) 1.2 Hz, 1H), 1.25 (s, 3H); 13C NMR δ 136.40, 135.22, 71.01,
49.56, 47.57, 43.69, 42.53, 36.91, 24.87. Anal. Calcd for C9H14O:
C, 78.21; H, 10.21. Found: C, 78.31; H, 10.36.
Compound 4b was obtained as white crystals: mp 276-278 °C;
1
[R]22 ) -118.3° (EtOH). The IR, H NMR, and 13C NMR spectra
D
were identical to those described for compound 2b.
Cell Cultures. Primary cultures of dissociated hippocampal neurons
from P1 neonatal rat pups were prepared via methods originally
described by Huettner and Baughman,45 with slight modifications.8 In
brief, hippocampi from Sprague-Dawley P1 neonatal rat pups were
removed, minced, and incubated with papain. The tissue was then
mechanically dissociated by gentle trituration, centrifuged through a
solution of albumin/trypsin inhibitor, and resuspended in growth
medium. Neurons were plated on a layer of glial cells in 35 mm plastic
dishes, which were prepared by preplating cortical glia from P1-4 rats
4 days earlier and allowing them to grow to confluence. 5-Fluoro-2-
deoxyuridine and uridine were added 2 days after the neurons were
plated to prevent glial overgrowth. Cells were used 1-7 days after
plating.
Electrophysiology. Growth medium was replaced by an external
recording solution which contained (in mM) 140 NaCl, 3.0 KCl, 1.0
MgCl2, 2.0 CaCl2, 10 HEPES, Phenol Red 10 mg/L, 0.6 µM
tetrodotoxin, and 20 µM MK-801; pH 7.3. The internal recording
solution contained (in mM) CsCl 130, 10 TEACl, 10 HEPES, 1.1
EGTA, 5.5 glucose; pH 7.2. Aqueous L-glutamate stock solutions (50
mM) were diluted in external recording solution to a final L-glutamate
concentration of 1 mM. Stock solutions of dihydromethylcyclothiazide
stereoisomers were made in DMSO and diluted in external recording
solution; the final DMSO concentration was 0.1% by volume. Record-
ings were performed at room temperature on the stage of an inverted
microscope. Patch electrodes were pulled in three stages (Sutter
Instruments, Novato, California) and had DC resistances of 5-10 MΩ.
These electrodes were connected to a patch amplifier in voltage-clamp
mode using the whole-cell configuration.34 Compensation was made
for ∼60-80% of the series resistance. The holding potential in all
experiments was -60 mV.
(1R-endo)-2-Methylbicyclo[2.2.1]hept-5-ene-2-methanol (35). Us-
ing the same two step procedure described immediately above, acid
29 was converted to methyl ester 34 (67% yield, colorless oil) and
then reduced to give alcohol 35 (77% yield) as white crystals (from
hexane): mp 90-92 °C; [R]22D ) -31.3° (CHCl3). The IR, 1H NMR,
and 13C NMR spectra were identical to those reported for compound
22. Anal. Calcd for C9H14O: C, 78.21; H, 10.21. Found: C, 78.43;
H, 10.12.
(1R-endo)-2-Methylbicyclo[2.2.1]heptane-2-methanol (23). A so-
lution of compound 22 (1.7 g, 12.3 mmol) in EtOAc (50 mL) was
hydrogenated (45 psi, room temperature, overnight) over 10% Pd-C
catalyst (170 mg). The catalyst was filtered and washed with EtOAc
(2 × 50 mL). The combined organic layers were evaporated to give
a colorless oil which was purified by chromatography (20% EtOAc in
hexane) to give compound 23 (1.4 g, 78%) as colorless crystals (from
hexane): mp 70-72 °C; [R]22 ) +12.9° (CHCl3); IR 3304, 2954,
D
2870, 1472, 1454, 1033 cm-1; 1H NMR 3.52-3.37 (m, 2H), 2.20 (t, J
) 4.3 Hz, 1H), 1.94 (d, J ) 3.2 Hz, 1H), 1.67-1.00 (m, 7H), 1.04 (s,
3H), 0.85 (dd, J ) 2.5 Hz, J ) 12.0 Hz, 1H); 13C NMR δ 69.90, 43.85,
42.75, 42.26, 37.63, 37.10, 28.36, 25.38, 24.41. Anal. Calcd for
C9H16O: C, 77.09; H, 11.50. Found: C, 77.32; H, 11.30.
(1S-endo)-2-Methylbicyclo[2.2.1]heptane-2-methanol (36). Using
the same procedure described immediately above, alcohol 35 gave
compound 36 (87% yield) as colorless crystals (from hexane): mp 70-
1
72 °C; [R]22 ) -12.5° (CHCl3). The IR, H NMR, and 13C NMR
D
spectra were identical to those reported for compound 23. Anal. Calcd
for C9H16O: C, 77.09; H, 11.50. Found: C, 76.88; H, 11.44.
[1R-[1r,2r(S*),4r]]-6-Chloro-3,4-dihydro-3-(2-methylbicyclo-
[2.2.1]hept-2-yl)-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-Di-
oxide (2a) and [1R-[1r,2r(R*),4r]]-6-Chloro-3,4-dihydro-3-(2-
methylbicyclo[2.2.1]hept-2-yl)-2H-1,2,4-benzothiadiazine-7-
(45) Huettner, J. E.; Baughman, R. W. J. Neurosci. 1986, 6, 3044-3060.

EnantioselectiVe Synthesis of Cyclothiazide Analogues
J. Am. Chem. Soc., Vol. 118, No. 19, 1996 4559
Drug Application. Continuous perfusion of the dish with drug-
free recording solution was accomplished via gravity flow at a rate of
2-3 mL/min. The cell recorded from was also locally perfused via a
large-bore fused silica-quartz tube (internal diameter 320 µm) connected
to four separate reservoirs, each with a separate solenoid valve to control
its flow. Only one valve could be opened at a time, allowing control
buffer, or various stereoisomers to bathe the cell from this first tube.
L-Glutamate (1 mM) was applied via a second identical tube by opening
its solenoid valve while simultaneously closing the valve to the first
tube. Flow occurred via gravity.
Scott Williams for technical assistance with the HPLC separation
of the stereoisomers. We thank Dr. D. Andre d’Avignon for
his assistance with the 2D NOSEY NMR experiments. Cy-
clothiazide was a generous gift from Eli Lilly and Co. This
work was supported by Grants NS 01443 (KAY) and NS 14834
(DFC) from the National Institutes of Health. Support was also
received from Sigma Tau Pharmaceuticals by their funding of
the Child Neurology Society Young Investigator Award to
K.A.Y. Assistance was also provided by the Washington
University High Resolution NMR Facility supported in part by
NIH 1 S10 RR 00204 and a gift from the Monsanto Co.
Acknowledgment. We are grateful to Nancy Lancaster for
preparing and maintaining hippocampal cell cultures and Mr.
JA9525317