Simultaneous determination of enantiomerization and hydrolysis kinetic parameters of chiral N-alkylbenzothiadiazine derivatives

Article abstract of DOI:10.1002/chir.20751

On-column stopped flow multidimensional HPLC (sfMDHPLC) and dynamic high-performance liquid chromatography were applied to investigate the influence of alkyl substituents at the sulfonamidic and amino moieties of benzothiadiazine 1,1-dioxide derivatives o

Full text of DOI:10.1002/chir.20751

CHIRALITY 22:389–397 (2010)
Simultaneous Determination of Enantiomerization
and Hydrolysis Kinetic Parameters of Chiral
N-Alkylbenzothiadiazine Derivatives
*
MARINA M. CARROZZO, GIUSEPPE CANNAZZA, UMBERTO BATTISTI, DANIELA BRAGHIROLI, AND CARLO PARENTI
Dipartimento di Scienze Farmaceutiche, Universita` degli Studi di Modena e Reggio Emilia, Modena, Italy
ABSTRACT
On-column stopped flow multidimensional HPLC (sfMDHPLC) and
dynamic high-performance liquid chromatography were applied to investigate the influ-
ence of alkyl substituents at the sulfonamidic and amino moieties of benzothiadiazine
1,1-dioxide derivatives on hydrolysis and enantiomerization rate constants. The data
obtained indicate the presence of pyrrolo substituent at the 3,4 positions on benzothia-
diazine rings inhibits the hydrolysis, whereas the enantiomerization occurs in acidic
medium. Hydrolysis rates are quite similar for the two benzothiadiazines methyl substi-
tuted to nitrogen at 2- and 4-positions. Conversely, enantiomerization rate of 4-N-methyl
substituted is significantly higher than 2-N-methyl substituted. Chirality 22:389–397,
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2010.
2009 Wiley-Liss, Inc.
KEY WORDS: N-alkylbenzothiadiazines; stopped-flow multidimensional HPLC; dynamic
HPLC; enantiomerization; hydrolysis
INTRODUCTION
By dynamic methods (DHPLC) it is possible to study
enantiomerization of labile chiral compounds when the
interconversion takes place at the time scale of the separa-
tion process.20,21,23–26 As kinetic parameters of enantiome-
rization are calculated in the presence of chiral stationary
phase (CSP), rate constants of enantiomerization could be
enhanced or depressed by the presence of chiral environ-
ment. Efforts have been made to combine the advantages
of the on-column approach with the option to perform
enantiomerization in an achiral and inert environment.20
With this aim, we have recently developed a multidimen-
sional stopped-flow HPLC method (sfMDHPLC), in which
enantiomerization is performed in an achiral environment.
The technique employs three columns in series: the first
and the third column are chiral and the second column is
achiral. In the first chiral column, the enantiomers are
quantitatively separated and the enantiomerization was
performed in the second achiral column and then, the flow
of the mobile phase introduces the enantiomers to the
third chiral column, in which they are separated.12,13
Previous studies on memory and cognition-enhancing
drugs implicate AMPA (2-amino-2,3-dihydro-5-methyl-3-oxo-4-
isoxazolepropanoic acid) type receptors as their target of
action. These compounds [i.e., (1) diazoxide, (2) cyclothia-
zide, (3) IDRA21, (4) S18986, (5) 7-chloro-5-ethyl-3-methyl-
3,4-dihydro-2H-benzo[1,2,4]thiadiazine 1,1 dioxide, and (6)
4-ethyl-7-fluoro-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-diox-
ide] are thought to work by potentiating glutamate synaptic
currents through removal of AMPA receptor desensitization
(Fig. 1).1–4 As it has been recently suggested that these com-
pounds are useful in the treatment of neurodegenerative dis-
orders such as cognitive disorders, schizophrenia, depres-
sion, and Parkinson’ s disease,5–7 they constitute potent
innovative therapeutic agents. More recently, it has been
reported that a novel N-alkylbenzothiadiazine 1,1-dioxide (6)
(Fig. 1), structurally close to IDRA21, showed an interesting
AMPA modulator activity.8
Previously, we have studied the stability of several ben-
zothiadiazine derivatives and it turned out a rapid intercon-
version of the enantiomers (enantiomerization) in aqueous
solution and a rapid hydrolysis in acidic medium.9–13
Rate constants and free energy barriers of enantiomeri-
zation of configurationally labile chiral compounds were
calculated by several methods, such as dynamic NMR14–16
(DNMR), chiro-optical methods,17,18 off-column and on-col-
umn chromatographic methods [i.e., dynamic gas chroma-
tography (DCG),19 dynamic high-performance liquid chro-
matography (DHPLC),20 dynamic capillary electrophoresis
(DCE),21 and stopped-flow HPLC22(sfHPLC)].
The sfMDHPLC was successfully applied for the determina-
tion of the chiral inversion of (6)-2,3,3a,4-tetrahydro-1H-pyr-
rolo[2,1-c][1,2,4]benzothiadiazine 5,5-dioxide and IDRA21.12,13
Additional Supporting Information may be found in the online version of
this article.
Contract grant sponsor: MIUR (Ministero dell’Istruzione, dell’Universita` e
della Ricerca, Italy).
*Correspondence to: Giuseppe Cannazza, Dipartimento di Scienze Farma-
ceutiche, Universita` degli Studi di Modena e Reggio Emilia, Via Campi
183, 4100 Modena, Italy. E-mail: giuseppe.cannazza@unimore.it
Received for publication 21 January 2009; accepted in revised form 5 May
2009; Accepted 13 May 2009
As on-column chromatographic methods required
minute amounts of the unresolved racemic sample, they
are widely used.22
DOI: 10.1002/chir.20751
Published online 2 July 2009 in Wiley InterScience
(www.interscience.wiley.com).
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390
CARROZZO ET AL.
Fig. 1. Structures of benzothiadiazine derivatives.
To gain further insight in understanding the enantiomeri- Rheodyne 7125 manual injector equipped with a 50 ll sam-
zation and hydrolysis mechanisms of chiral 3,4-dihydro-1,2, ple loop. A Merck Hitachi L-7400UV was used as detector.
4-benzothiadiazine 1,1-dioxide type compounds, the Chromatograms were recorded with
a Jasco J-700
sfMDHPLC and DHPLC procedures were applied to program. A Rheodyne 7010 valve was installed after the
study enantiomerization and hydrolysis of three selec- first chiral column 1 and permitted the trapping of the
ted chiral compounds: 7-chloro-2,3,3a,4-tetrahydro-1H-pyrrolo individual enantiomers of (6)-7 or (6)-9 (Fig. 2). Column
[2,1-c][1,2,4]-benzothiadiazine 5,5-dioxide ((6)-7), 7-chloro-3, temperature regulation was achieved with a Haake F3
4-dimethyl-2H-1,2,4-benzothiadiazine 1,1-dioxide ((6)-8), and thermostated water bath.
7-chloro-2,3-dimethyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,
1-dioxide ((6)-9).
The columns used were Chiralcel OD-RH [tris(3,5-di-
methylphenylcarbamate); 150 mm 3 4.6 mm ID; 5 lm]
The results obtained were used to investigate the influ- purchased from Daicel, and Supelcosil LC-18 (250 mm 3
ence of substitution at the nitrogen at 2- and 4-positions of 4.6 mm ID; 5 lm) purchased from Supelco. Melting points
the benzothiadiazine ring on hydrolysis and enantiomeri- were determined with an Electrothermal Apparatus and
zation kinetic constants.
they are uncorrected. IR spectra were recorded on a Perki-
nElmer Model 1600 FT-IR spectrometer and consisted of
the assigned structures. H NMR spectra were recorded
with a Brucker DPX 200 spectrometer using DMSO-d6 as
solvent and tetramethylsilane (TMS) as external standard.
1
EXPERIMENTAL
Instrumentation
The chromatographic apparatus consisted of a Shi- Chemical shifts (d) are in part per million and coupling
madzu LC-10AD Pump, a Merck Hitachi L-6200A Pump, a constant (J) in hertz. Multiplicities are abbreviated as
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ENANTIOMERIZATION OF CHIRAL N-ALKYLBENZOTHIADIAZINE
391
Fig. 2. Schematic representation of stopped-flow multidimensional HPLC (sfMDHPLC). Step 1: Racemic mixture was injected and enantioseparated
on the first chiral OD-RH column at low temperature conditions. Step 2: One of the two eluted enantiomers was trapped into the second achiral C18 col-
umn and enantiomerization and hydrolysis were performed at different pH buffers at 378C for a set time interval. Step 3: The enantiomers and hydrolysis
product were separated in the third chiral OD-RH column at low temperature conditions. E1 and E2 corresponded to the first and second eluted enan-
tiomers. (*) corresponded to hydrolysis product.
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CARROZZO ET AL.
follows: s, singlet; d, doublet; dd, double doublet; m, mul- dead time of the columns (t0) was determined by injection
tiplet. Elemental analyses were performed on a Carlo Erba of 1,3,5-tri-tert-butylbenzene.
Analyzer Model 1106 apparatus.
Stopped-Flow Multidimensional HPLC (sfMDHPLC)
All pH measurements were made using Orion Research
EA940 pH-meter.
Enantiomerization and hydrolysis of (6)-7 and (6)-9
were studied by the sfMDHPLC method described
herein.12,13 Figure 2 shows a schematic representation of
Synthesis
(6)7-Chloro-2,3,3a,4-tetrahydro-1H-pyrrolo[2,1-c] the method used. In the first step, racemic compound was
injected and enantioseparated on column 1. At the appro-
priate time, the individual enantiomers were cut off by
switching the valve into position (b) and trapped into the
second achiral column 2. In the second step, the achiral
column was filled with the buffer at the desired pH and
temperature by pump 2. The enantiomerization and hydro-
[1,2,4]-benzothiadiazine 5,5-dioxide ((6)-7). The
compound was synthesized as previously described by
Cameroni et al.27
Yield 56.4%, mp 212–2148C, FT-IR 3205 cm21 (m NH),
1
1323 cm21 (m SO2 as), 1151 cm21 (m SO2 sim). H NMR
(DMSO-d6) d 5 1.78–2.09 (m, 3H), d 5 2.28–2.37 (m, 1H),
d 5 3.12–3.25 (m, 1H), d 5 3.50–3.3.59 (m, 1H), d 5 4.97– lysis were affected by heating at 37.58C whereas no mobile
phase passed through the column 2 (stopped-flow) for a
set period of time. In the last step, the original mobile
phase was resumed to permit the separation of the
products originated in the second step by the trapping
enantiomer.
The outcome of this protocol resulted in the presence of
three peaks: peak 18 arises from the hydrolysis, peaks 28
and 38 corresponding to the enantiomers of analytes
injected, one arises from enantiomer trapped in the achiral
column and the other one corresponding to its intercon-
verted product.
The elution order was validated by the use of a circular
dichroism spectroscopy detector: whereas the second and
the third peaks have opposite cotton effect, the first peak
was not detected by CD detection because benzensulfona-
mide is an achiral compound.
The kinetic parameters (rate constants of enantiomeriza-
tion k) and the enantiomerization barriers (DG#) were cal-
culated from the corresponding peak areas, from the enan-
tiomerization time, and from the enantiomerization tem-
perature as described in ‘‘Calculation of kinetic rate
constants and free energy barriers of enantiomerization’’
section. It is assumed that the precision of DG# is not influ-
enced within the experimental standard deviation by the
warm-up times on the achiral column for stopped-flow mul-
tidimensional experiments, in which separation is carried
out at low temperature (08C) and the enantiomerization at
higher temperature (378C). However, in case of very short
enantiomerization times, this systematic error increases
and it cannot be ignored.28–30
5.06 (m, 1H), d 5 6.77 (d, 1H, J 5 8.98), d 5 7.44 (dd, 1H,
J 5 2.55, J 5 8.94), d 5 7.53 (d, 1H, J 5 2.53). The micro-
analyses results were within 60.4%.
(6)7-Chloro-3,4-dimethyl-2H-1,2,4-benzothiadia-
zine 1,1-dioxide ((6)-8). The compound was synthe-
sized as previously described by Francotte et al.8
Yield 86%, mp 129–1308C, FT-IR 3205 cm21 (m NH),
1377 cm21 (m SO2 as), 1154 cm21 (m SO2 sim). 1H
NMR(DMSO-d6) d 5 1.45 (d, 3H), d 5 2.87 (s, 3H), d 5
4.75–4.80 (m, 1H), d 5 6.89 (d, 1H, J 5 8.97), d 5 7.39–
7.42 (dd, 1H, J 5 2.64, J 5 8.96), d 5 7.45 (d, 1H, J 5
2.17). The microanalyses results were within 60.4%.
(6)7-Chloro-2,3-dimethyl-3,4-dihydro-2H-1,2,4-
benzothiadiazine 1,1-dioxide ((6)-9). The compound
was synthesized as previously described by Francotte
et al.8
Yield 73%, mp 74–758C, FT-IR 3341 cm21 (m NH), 1321
cm21 (m SO2 as), 1152 cm21 (m SO2 sim). 1H NMR
(DMSO-d6) d 5 1.44 (d, 3H), d 5 2.46 (s, 3H), d 5 5.21–
5.26 (m, 1H), d 5 6.86 (d, 1H, J 5 8.93), d 5 7.35–7.39
(dd, 1H, J 5 2.24, J 5 8.87), d 5 7.49 (d, 1H, J 5 2.32).
The microanalyses results were within 60.4%.
Chromatography
Separation of enantiomers of (6)-7, (6)-8, and (6)-9
were carried out isocratically at different temperatures on
Chiralcel OD-RH column. The mobile phase consisted of
water: acetonitrile at different percentage. Separation of
compounds (6)-8 and (6)-9 from their hydrolysis prod-
ucts [5-chloro-2-(methylamino)benzensulfonamide (11)
and 2-amino-5-chloro-N-methylbenzensulfonamide (12)]
were carried out in the same conditions. The compounds
were dissolved in acetonitrile and subsequently diluted
1:10 (v/v) with mobile phase at final concentration of 100
lg/ml. The injection volume was 50 ll. The detectors
were set at 254 nm.
Off-Column Hydrolysis
Pure (6)-8 was dissolved in acetonitrile and subse-
quently diluted 1:10 (v/v) with individual solvent (1 ml) at
final concentration of 100 lg/ml and kept thermostated at
378C. The hydrolysis was monitored by chromatography
on the Chiralcel OD-RH column using a mobile phase
water and acetonitrile 60:40 (v/v). Four repeats of each
Chromatographic parameters. The separation factor experiment were made. The two peaks, corresponding to
(a) was calculated as k2/k1 and retention factors (k1 and (6)-8 and hydrolysis product (5-chloro-2-(methylamino)-
k2) as k1 5 (t1 2 t0)/t0, where t1 and t2 refer to the reten- benzensulfonamide (11)), were identified by injecting pure
tion times of the first and second eluted enantiomers. The compounds in the same experimental conditions.
resolution factor (Rs) was calculated by the formula Rs 5
Buffer solution at pH 1.20 was prepared by mixing 407
2(t2 2 t1)/(w1 1 w2), where w1 and w2 are the peak widths ml of 0.2 M hydrochloric acid with 93 ml of 0.2 M potas-
at base for the first and second eluted enantiomer. The sium chloride and diluting with water to one liter. Chloroa-
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ENANTIOMERIZATION OF CHIRAL N-ALKYLBENZOTHIADIAZINE
393
cetate buffer solution at pH 2.20 was prepared by mixing
50.99 ml of 0.1 M chloroacetic acid with 2.76 ml of 0.1 M
KOH and diluting with water to 100 ml. Acetate buffer so-
lution at pH 4.20 was prepared by mixing 43.03 ml of 0.1
M acetic acid with 9.93 ml of 0.1 M KOH and diluting
with water to 100 ml. Phosphate buffer solution at pH
6.40 was prepared by mixing 30.21 ml of 0.02 M KH2PO4
with 13.19 ml of 0.01 Na2HPO4 and diluting with water to
100 ml.
Calculation of Kinetic Rate Constants and Free Energy
Barriers of Enantiomerization
Kinetic rate constants and free energy barriers of enan-
tiomerization of (6)-7 and (6)-9 were calculated by
sfMDHPLC.
The kinetic rate constants can be calculated by fitting
the data to eq. 1
Fig. 3. Enantioresolution of (6)-7. Column: Chiralcel OD-RH
(15 3 0.46 I.D., 5 lm); mobile phase: water:acetonitrile 60:40 (v/v);
temperature: 258C.
8
9
a0
>
:
>
;
ln
¼ 2ktenant
ð1Þ
2at ꢀ a0
RESULTS AND DISCUSSION
Chromatography
where k is the rate constant of forward or backward enan-
tiomerization [s21], ao is the peak of the initial enantiomer
before enantiomerization [5 100%], at is the relative peak
area of the enantiomer remaining after enantiomerization
time t [%] (i.e., at 5 100-conversion), and tenant the enantio-
merization time(s).
From the kinetic rate constants, the corresponding
activation energies of enantiomerization (rotational energy
barriers) DG#(T) can be calculated by the Eyring
equation:
Chromatographic analysis of (6)-7 were performed on
OD-RH column with a mobile phase of water:acetonitrile
60:40 (v/v). The enantioseparation was conducted at differ-
ent temperatures between 2108C and 378C. As expected
the retention times and enantioseparation increase with
the decreasing of temperature analysis (2108C: k01 5 7.02,
k02 5 10.98, a 5 1.56, Rs 5 3.70; 08C: k01 5 6.17, k02
5
10.24, a 5 1.66, Rs 5 2.97; 258C: k01 5 3.67, k02 5 5.8, a 5
1.58, and Rs 5 2.90; 378C: k01 5 2.93, k02 5 4.50, a 5 1.53,
Rs 5 2.85). Baseline resolution of (6)-7 with acceptable
analysis time was obtained at 258C (Fig. 3). These results
indicate that hardly any enantiomerization of the two enan-
tiomers of (6)-7 occurred during chromatographic enan-
tioseparation at temperature between 2108C and 378C
using water:acetonitrile as mobile phase.
8
9
kh
#
>
:
>
;
DG ðTÞ ¼ ꢀRT ln
ð2Þ
jkBT
where k is the kinetic rate constant, kB is the Boltzmann
constant (kB 5 1.380662 3 10223 J K21), h is the Planck’s
constant (h 5 6.626176 3 10234 J s), R is the universal gas
constant (R 5 8.31441 J/(K mol)21), j the transmission
coefficient (j 5 0.5 for the reversible microscopic inter-
conversion), and T the temperature (K).
Kinetic rate constants and free energy barriers of enan-
tiomerization of (6)-8 were calculated by DCXplorer soft-
ware, developed by Trapp (DCXplorer software is available
from Trapp as executable programs, running under Micro-
soft Windows 2000, XP, and Vista).31
Chromatographic analysis of (6)-8 was conducted on
OD-RH column and with a mobile phase of water:acetoni-
trile 60:40(v/v) at different temperatures between 2108C
and 258C. Chromatograms in Figure 4 show that at tem-
perature of 08C a pronounced plateau was observed
between the two peaks corresponding to enantiomers. A
single peak was observed at 258C indicating the coales-
cence of the two enantiomers. Chromatogram correspond-
ing to the enantioresolution in the same chromatographic
conditions [column OD-RH, mobile phase water:acetoni-
trile 60:40 (v/v)] but at temperatures of 2108C indicate
Calculation of Kinetic Rate Constants of Hydrolysis
an inhibition of enantiomerization process with
a
The kinetic rate constants can be calculated by fitting
the data to eq. 3:
dramatic reduction of plateau between the peaks (Fig. 4).
These results indicate that a rapid thermal enantiomeriza-
tion of (6)-8 occurred in water:acetonitrile with DG#
barriers lower than 90 kJ mol21 32
.
The free energy barrier
At ¼ A0 expðꢀkitiÞ
ð3Þ
of enantiomerization of (6)-8 is not high enough to pre-
where ki is the rate constant of hydrolysis [s21], Ao is the vent racemization during chromatographic enantioresolu-
peak of the (6)-8 and (6)-9 before hydrolysis [5 100%], tion, at 08 and 258C, and thereby does not allow isolation
At is the relative peak area of the injected compound of single enantiomers. Consequently, the temperature
remaining after hydrolyzation time ti [%] (i.e., at 5 100- has been lowered (2108C) to decrease the rate of
conversion), and ti the hydrolysis time(s).
enantiomerization.
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Fig. 5. Enantioresolution of (6)-9. Column: Chiralcel OD-RH (15 3
0.46 I.D., 5 lm); mobile phase: water:acetonitrile 70:30 (v/v); tempera-
ture : 08C.
recognition of the OD-RH stationary phase. Indeed, a good
enantioresolution has been obtained for (6)-8 with nitro-
gen atom of the sulfonamidic moiety not substituted,
whereas N-methylated (6)-9 exhibits a partial optical
resolution with a 5 1.05.
Enantiomerization and Hydrolysis of (6)-7
Enantiomerization and hydrolysis of (6)-7 were studied
by sfMDHPLC method (see experimental part for details)
at 378C and in the pH range over 1.20–6.40.
The sfMDHPLC method consists in trapping each enan-
tiomer, previously separated by a chiral column, in a C18
achiral column that is subsequently filled with the selected
buffers and left to react for certain time. Afterward, the
products of enantiomerization and hydrolysis of the
trapped enantiomer will be separated by a chiral column.
From the peak areas it is possible to calculate kinetics of
enantiomerization and hydrolysis simultaneously.
The results indicate that any hydrolysis of compound
(6)-7 occurred for at least 2 h at 378C and at pH between
1.20 and 6.40.
The kinetic rate constants and Gibbs free energies of
activation of enantiomerization of (6)-7 at 378C and at pH
1.20, 2.20, 4.20, and 6.40 were reported in Table 1.
The data obtained indicate that enantiomerization rates
strongly depend on pH, increasing with the decreasing of
pH (1.20–2.20), demonstrating that enantiomerization of
(6)-7 was an acid catalyzed process.
Fig. 4. Enantioresolution of (6)-8. Column: Chiralcel OD-RH (15 3
0.46 I.D., 5 lm); mobile phase: water:acetonitrile 60:40 (v/v); temperature:
(a) 2108C, (b) 08C, (c) 258C.
Chromatographic analysis of the racemic (6)-9 was
performed on OD-RH column with water:acetonitrile at dif-
ferent percentage compositions and at different tempera-
tures. No baseline enantiomeric resolution was obtained in
chromatographic conditions used. Partial enantioresolu-
tion was obtained using OD-RH column with water:aceto-
nitrile 70:30 (v/v) at 08C (k01 5 16.2, k02 5 17, a 5 1.05,
Rs 5 1.28; Fig. 5). The poor enantioresolution obtained for
the compound (6)-9 indicates that hydrogen bonding
between the nitrogen atom of the sulfonamidic moiety
adjacent to the chiral center has a great influence on chiral
The results suggest that the pyrrolo substituent on the
benzothiadiazine ring allowed enantiomerization in acidic
medium whereas hydrolysis was inhibited.
Previous studies performed on S18986, structurally
related to (6)-7, demonstrated that any hydrolysis
occurred, whereas the enantiomerization is an acid cata-
lyzed process.12 The kinetic parameters obtained for (6)-
7 and S18986 are quite similar, indicating that the pres-
ence of chlorine atom at the 7-position of benzothiadiazine
ring not significantly influence the rates of enantiomeriza-
tion and hydrolysis.12
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ENANTIOMERIZATION OF CHIRAL N-ALKYLBENZOTHIADIAZINE
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TABLE 1. Rate constants and free energy barriers of
enantiomerization of (6)-7 determinated by sfMDHPLC
TABLE 3. Rate constants and free energy barriers of
enantiomerization of (6)-8 at different percentage
of acetonitrile determinated in DHPLC by DCXplorer
pH
k (sec21
)
DG# (kJ mol21
)
Water:acetonitrile (v/v)
k (sec21
)
DG# (kJ mol21
)
1.20
2.20
4.20
6.40
10.50 6 1.44 3 1024a
6.56 6 0.62 3 1024a
92.12 6 0.34b
93.33 6 0.23b
60:40
70:30
75:25
9.63 3 1025
8.20 3 1025
6.81 3 1025
86.13
86.49
86.91
0.22 6 0.05 3 1024a
102.09 6 0.49b
c
c
Columns: Chiralcel OD-RH, Supelcosil LC-18. Column operation tempera-
ture: 08C. Eluent (Pump 1): water:acetonitrile 60:40 (v/v). Buffers (Pump
2): buffer solution at pH 1.20, chloroacetate buffer solution at pH 2.20,
acetate buffer at pH 4.20, and phosphate buffer at pH 6.40. Time intervals
for enantiomerization at 37.58C 5 50, 100, 150, 200, 300, 600, 1200 (n 5 4).
aRate constants in C18 column.
Column: Chiralcel OD-RH; temperature 08C. Eluents:water:acetonitrile
60:40 (v/v), 70:30 (v/v), and 75:25 (v/v).
(off-column method) in a series of buffers over a pH range
of 1.20–6.40 at 378C as described in the experimental part.
The calculated kinetic rate constants of hydrolysis were
reported in Table 4.
bFree energy barriers in C18 column.
cNo enantiomerization occurred in 2 h.
The data obtained show that hydrolysis rate constants
depend strongly on pH, increasing with the decreasing of
pH, indicating that hydrolysis of (6)-8 was an acid cata-
lyzed process.
Enantiomerization and Hydrolysis of (6)-8
The chromatograms relative to the enantioseparation of
(6)-8 have been demonstrated that a rapid thermal enan-
tiomerization occurs during chromatographic enantioreso-
lution (Fig. 4). As the applicability of sfMDHPLC requires
that the enantiomerization process is suppressed during
the chromatographic separation (initial and final separa-
tion steps), it was not possible to apply the method to
determinate the enantiomerization and hydrolysis kinetic
parameters of (6)-8. Accordingly, the enantiomerization
rate constants were calculated by dynamic chromatogra-
phy experiments (DHPLC) using DCXplorer software.31
The program employs the unified equation of chromatog-
raphy that can evaluate reaction rate constants of all kinds
of first order reactions taking place during a separation
process.33–41 The data obtained were reported in Tables 2
and 3.
Enantiomerization and Hydrolysis of (6)-9
Chromatogram in Figure 5 of (6)-9 indicates that only a
partial enantioresolution has been obtained. As dynamic
chromatography methods need at least a baseline resolu-
tion of the peaks corresponding to enantiomers, it was not
possible to apply the DHPLC procedure to calculate enan-
tiomerization kinetic parameters. The enantiomerization
and hydrolysis of (6)-9 were studied by sfMDHPLC pro-
cedure because it was possible to trap pure enantiomer by
switching the valve at appropriate time in the achiral col-
umn, where occurs enantiomerization and hydrolysis proc-
esses (Fig. 6).
The results indicate that enantiomerization rates
depend poorly by temperature (2108C: k 5 2.66 3 1025
sec21 and DG# 5 85.71 kJ mol21, 08C: k 5 9.63 3 1025
sec21 and DG# 5 86.13 kJ mol21). Moreover, it has been
observed that the pH does not significantly influence the
enantiomerization rates, as only small differences were
obtained between pH 1.20–6.40 (Table 2). Similarly, the
variation of percentage of acetonitrile in the mobile phase
causes only small changes in enantiomerization rate
constants.
Single enantiomer of (6)-9 hydrolyzed completely to 2-
amino-5-chloro-N-methylbenzensulfonamide in 5 min at
378C and at pH 1.20 and 2.20.
The kinetic rate constants of enantiomerization and hy-
drolysis at pH 4.20 and at 378C were 1.66 6 0.11 3 1024
sec21 (DG# 5 96.88 6 0.09 kJ mol21) and 4.15 6 0.33 3
1024 sec21, respectively.
Conversely, at pH 6.40 and at 378C, any enantiomeriza-
tion and hydrolysis were obtained for at least 2 h.
The data indicate that the enantiomerization and hydro-
lysis processes depend strongly by pH, increasing with
the decreasing of pH.
The rate constants of hydrolysis of (6)-8 were
calculated by the classical batch-wise kinetic method
TABLE 4. Rate constants of hydrolysis of (6)-8 at
TABLE 2. Rate constants and free energy barriers of
enantiomerization of (6)-8 at pH 1.20–6.40 determinated in
DHPLC by DCXplorer
pH 1.20–6.40 determinated by off-column method
pH
ki (sec21
)
1.20
2.20
4.20
6.40
(*)
Eluent
k (sec21
)
DG# (kJ mol21
)
54.40 6 19.60 3 1024
6.99 6 1.14 3 1024
1.89 6 0.12 3 1024
Buffer 1.20:ACN
Buffer 2.20:ACN
Buffer 4.20:ACN
Buffer 6.40:ACN
1.61 3 1024
2.04 3 1024
1.80 3 1024
1.35 3 1024
84.96
84.42
84.70
85.36
Column: Supelcosil LC-18; flow 0.5 ml min21; temperature: 378C; buffers:
chloride buffer pH 1.20, chloroacetate buffer pH 2.20, acetate buffer pH
4.20, and phosphate buffer pH 6.40. Time intervals for off-column hydroly-
sis at 37.58C 5 50, 100,150, 200, 300, 600, and 1200 (n 5 4).
Column: Chiralcel OD-RH; temperature: 08C; eluent: buffer:acetonitrile
60:40 (v/v); buffers: chloride buffer pH 1.20, chloroacetate buffer pH 2.20,
acetate buffer pH 4.20, and phosphate buffer pH 6.40.
(*) Hydrolysis too fast to be calculated.
Chirality DOI 10.1002/chir

396
CARROZZO ET AL.
benzothiadiazines methyl substituted to N atom at 2- and
4-positions. Conversely, enantiomerization rate of 4-N-
methyl substituted is significantly higher than 2-N-methyl
substituted.
Recently, N-alkylbenzothiadiazine derivatives have
attracted particular attention as they act as potent AMPA
potentiators. The data obtained indicate that benzothiadia-
zine derivatives methyl-substituted to N atom at 2- and
4-positions rapidly hydrolyzed in acidic medium to the cor-
responding benzensulfonamide. Thus, it is possible that
the structurally similar N-alkylbenzothiadiazine could be
hydrolyzed when administered orally in the acidic medium
of the stomach in pharmacological tests.
ACKNOWLEDGMENTS
The authors thank Ms. S. Lucchi for the technical help-
ful gave in enantiomerization experiments.
Fig. 6. Chromatogram of enantiomerization and hydrolysis experiments
of (6)-9 performed by sfMDHPLC procedure: peaks 18 (2-amino-5-chloro-N-
methylbenzensulfonamide (12)) arises from hydrolysis process and peaks
28 and 38 corresponding to the enantiomers of (6)-9, one arise from enan-
tiomer trapped in the achiral column and the other one corresponding to its
interconverted product. Columns: Chiracel OD-RH (15 3 0.46 I.D., 5 lm)
Supelcosil LC-18 (25 3 0.46 I.D.); mobile phase: water:acetonitrile 70:30
(v/v); time intervals 30 min.; temperature 378C; buffer pH 4.20.
LITERATURE CITED
1. Lestage P, Danober L, Lockart B, Roger A, Lebrun C, Robin JL, Desos
P, Cordi A. S18986, positive allosteric modulator of AMPA receptors
as a novel cognition enhancer in rodents. Res Pract Alzheimer’s Dis
2002;6:253–259.
2. Philips D, Sonnenberg J, Arai AC, Vaswani R, Krutzik PO, Kleisli T,
Kessler M, Granger R, Lynch G, Chamberline AR. 50-Alkyl-benzothia-
diazides:
a new subgroup of AMPA Receptor modulators with
CONCLUSION
improved affinity. Bioorg Med Chem 2002;10:1229–1248.
The sfMDHPLC is a novel method to calculate kinetic
parameters of enantiomerization and hydrolysis processes
of chiral labile compounds in an achiral and inert environ-
ment simultaneously.12,13 Moreover, the method can be
applied to partial enantioresolved compounds because it is
possible to trap in the achiral column pure enantiomers by
cutting the peaks at the appropriate time. The sfMDHPLC
has been successfully used to calculate kinetic parameters
of enantiomerization and hydrolysis of compounds (6)-7
and (6)-9, demonstrating the applicability of the method
developed.
Anyway, as sfMDHPLC method requires that enantio-
merization is suppressed during the chromatographic sep-
aration process, the method cannot be applied to the
determination of kinetic parameters of compounds that
interconverted during chromatographic enantioresolution
also at low temperature. Accordingly, the rate constants
and free energy barriers of resolvable enantiomers that
interconverted rapidly on the separation time scale can be
calculated by DHPLC. As we were unable to suppress
enantiomerization of (6)-8 during chromatographic enan-
tioresolution, its kinetic enantiomerization parameters
were calculated by DHPLC with the novel software pro-
gram DCXplorer that permits quick evaluation of elution
profiles of interconverting stereoisomers, utilizing the uni-
fied equation of chromatography.31,33–41
3. Braghiroli D, Puia G, Cannazza G, Tait A, Parenti C, Losi G, Baraldi
M. Synthesis of 3,4-Dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide
derivatives as potential allosteric modulators of AMPA/Kainate recep-
tors. J Med Chem 2002;45:2355–2357.
4. Pirotte B, Podona T, Diouf O, de Tullio P, Lebrun P, Dupont L, Som-
ers F, Delarge J, Morain P, Lestage P, Lepagnol J, Spedding M. 4H-
1,2,4-pyridothiadiazine 1,1-dioxides and 2,3-dihydro-4H-1,2,4-pyrido-
thiadiazine 1,1-dioxides chemically related to diazixide and cyclothia-
zide as powerful positive allosteric modulators of (R/S)-2-amino-3-(3-
hydroxy-5-methylisoxazol-4-yl) propionic acid receptors: design, syn-
thesis, phatmacology, and structure-activity relationships.
Chem 1998;41:2946–2959.
J Med
5. Graindorge E, Francotte P, Boverie S, de Tullio P, Pirotte B. New
trends in the development of positive allosteric modulators. Curr Med
Chem 2004;4:95–103.
6. Hashimoto K, Okamura N, Shimizu E, Iyo M. Glutamate hypothesis
of schizophrenia and approach for possible therapeutic drugs. Curr
Med Chem 2004;4:147–154.
7. Black MD. Therapeutic potential of positive AMPA modulators and
their relationship to AMPA receptor subunits. A review of preclinical
data. Psychopharmacology 2005;179:154–163.
8. Francotte P, de Tullio P, Goffin E, Dintilhac G, Graindgorge E, Fraikin
P, Lestage P, Danober L, Thomas JY, Caignard DH, Pirotte B. Design,
synthesis and pharmacology of novel 7-substituted 3,4-dihydro-2H-
1,2,4-benzothiadiazine 1,1-dioxide as positive allosteric modulators of
AMPA receptors. J Med Chem 2007;50:3153–3157.
9. Cannazza G, Braghiroli D, Baraldi M, Parenti C. Chiral resolution of
the enantiomers of 7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothia-
diazine 1,1-dioxide using high performance liquid chromatography on
cellulose-based chiral stationary phases.
2000;23:117–125.
J Pharm Biomed Anal
The enantiomerization and hydrolysis of three benzo-
thiadiazine different substituted to heterocyclic moiety has
been studied by sfMDHPLC and DHPLC procedures.
The results indicate that the presence of pyrrolo substit-
uent at the 3,4 positions on benzothiadiazine ring inhibits
the hydrolysis, whereas the enantiomerization occurs in
acidic medium. Hydrolysis rates are quite similar for the
10. Cannazza G, Braghiroli D, Tait A, Baraldi M, Parenti C, Lindner W.
Studies of enantiomerization of chiral 3,4-dihydro-1,2,4-benzothiadia-
zine 1,1-dioxide type compounds. Chirality 2001;13:94–101.
11. Cannazza G, Braghiroli D, Iuliani P, Parenti C. Energy barrier deter-
minationof enantiomerization of chiral 3,4-dihydro-1,2,4-benzothiadia-
zine 1,1-dioxide type compounds by enantioselective stopped-flow
HPLC. Tetrahedron Asymmetry 2006;17:3158–3162.
Chirality DOI 10.1002/chir

ENANTIOMERIZATION OF CHIRAL N-ALKYLBENZOTHIADIAZINE
397
12. Cannazza G, Carrozzo MM, Braghiroli D, Parenti C. Enantiomeriza-
tion of chiral 2,3,3a,4-tetrahydro-1H-pyrrolo[2,1-c][1,2,4]benzothiadia-
zine 5,5-dioxide by stopped-flow multidimensional HPLC. J Chroma-
togr B 2008;875:192–199.
26. D’Acquarica I, Gasparrini F, Pierini M, Villani C, Zappia G. Dynamic
HPLC on chiral stationary phases: a powerful tool for the investigation
of stereosimulation processes. J Sep Sci 2006;29:1508–1516.
27. Cameroni R, Ferioli V, Bernabei MT. Sistemi eteropoliciclici-2,2-dii-
dro-1H-pirrolo[2,1-c][1,2,4]benzotiadiazin-5,5-diossido e suoi derivati.
Farmaco 1971;27:574–581.
13. Cannazza G, Carrozzo MM, Braghiroli D, Parenti C. Enantiomeriza-
tion and hydrolysis of (6)-7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide by stopped-flow multidimensional high-per-
formance liquid chromatography. J Chromatogr A 2008;1212:41–47.
28. Schurig V, Reich S. Determination of the rotational barriers of atropi-
someric polychlorinated biphenyls (PCBs) by a novel stopped-flow
multidimensional gas chromatographic technique. Chirality 1998;
10:316–320.
14. Schurig V, Keller F, Reich S, Fluck M. Dynamic phenomena involving
chiral dimethyl-2,3-pentadienedioate in enantioselective gas chroma-
tography and NMR spectroscopy. Tetrahedron Asymmetry
1997;8:3475–3480.
29. Reich S, Schurig V. Stopped-flow multidimensional gas chromatogra-
phy: a new method for the determination of enantiomerization bar-
riers. J Microcolumn Sep 1999;11:475–479.
15. Kiesswetter R, Burgemeister T, Mannschreck A. Chiral 2H-pyrans,
8.10,30,30-trimethyl-6,8-dinitrospiro[2H-1-benzopyran-2,20-indoline]: fast
thermal enantiomerization and slow thermal equilibration with a ring-
opened isomers. Enantiomer 1999;4:289–296.
30. Reich S, Trapp O, Schurig V. Enantioselective stopped-flow multidimen-
sional gas chromatography: determination of interconversion barrier of
1-chloro-2,2-dimethylaziridine. J Chromatogr A 2000;892:487–498.
16. Bringmann G, Heubes M, Breuning M, Gobel L, Ochse M, Schoner
B, Schupp O. Atropisomerization barrier of configurationally unstable
biaryl compounds, useful substrates for atroposelective conversionsa
to axially chiral byiaryls. J Org Chem 2000;65:722–728.
31. Trapp O. DCXplorer software. Available from http://www.mpi-
muelheim.mpg.de/kofo/institut/arbeitsbereiche/trapp/downloads_
e.html Accessed 6/26/2009.
32. Allenmark SG. Chromatographic enantioseparation. New York: Wiley;
1988. p 184.
17. Mannschreck A, Andert D, Eiglsperg E, Gmahl E, Buchner H. Chirop-
tical detection. Novel possibilities of its application to enantiomers.
Chromatographia 1988;25:182–188.
33. Trapp O. A unified equation for fast and precise access to rate con-
stants of first order reactions in dynamic and on-column reaction chro-
matography. Anal Chem 2006;78:189–198.
18. Mannschreck A, Kiessel A. Enantiomerization during HPLC on an
optically active sorbent. Deconvolution of experimental chromato-
grams. Chromatographia 1989;28:263–266.
34. Trapp O. Fast and precise access to enantiomerization rate constants
in dynamic chromatography. Chirality 2006;18:489–497.
19. Trapp O, Schoetz G, Schurig V. Determination of enantiomerization
barriers by dynamic and stopped-flow chromatographic methods.
Chirality 2001;13:403–414.
35. Trapp O. The unified equation for the evaluation of first order reac-
tions in dynamic electrophoresis. Electrophoresis 2006;27:534–541.
20. Krupcik J, Oswald P, Majek P, Sandra P, Armstrong DW. Determina-
tion of the interconversion energy barrier of enantiomers by separa-
tion methods. J Chromatogr A 2003;1000:779–800.
36. Trapp O. A dynamic molecular probe to investigate catalytic effects
and Joule heating in enantioselective micellar electrokinetic chroma-
tography. Electrophoresis 2007;28:691–696.
21. Wolf C. Stereolabile chiral compounds: analysis by dynamic chroma-
tography and stopped-flow methods. Chem Soc Rev 2005;34:595–608.
37. Trapp O, Schurig V. Stereointegrity of Troger’s base: gas chromato-
graphic determination of the enantiomerization barrier. J Am Chem
Soc 2000;122:1424–1430.
22. Schurig V. Contributions to the thoery and practice of the chromato-
graphic separation of enantiomers. Chirality 2005;17:205–226.
38. Trapp O, Weber SK, Bauch S, Hofstadt W. High-throughput screening
of catalysts by combining reactions and analysis. Angew Chem Int Ed
Engl 2007;46:7307–7310.
23. Mannschreck A, Zinner H, Pustet N. The significance of the HPLC
time scale: an example of interconvertible enantiomers. Chimia
1989;43:165–166.
39. Trapp O. Gas-chromatographic high-throughput screening techniques
in catalysis (review article). J Chromatogr A 2008;1184:160–190.
24. Veciana J, Crespo MI. Dynamic HPLC: a method for determination
rate constants, energy barriers, and equilibration constants of molecu-
lar dynamic processes. Angew Chem Int Ed Engl 1991;30:74–77.
40. Trapp O, Schurig V. ChromWin—a computer program for the deter-
mination of enantiomerization barriers in dynamic chromatography.
Comput Chem 2001;25:187–195.
25. Jung M, Schurig V. Determination of enantiomerization barriers by
computer simulation of interconversion profiles: enantiomerization of
diaziridines during chiral inclusion gas chromatography. J Am Chem
Soc 1992;114:529–534.
41. Trapp O. A novel software tool for high-throughput measurements of
interconversion barriers: DCXplorer. J Chromatogr B 2008;875:42–47.
Chirality DOI 10.1002/chir