Energy barrier determination of enantiomerization of chiral 3,4-dihydro-1,2,4-benzothiadiazine 1,1-dioxide type compounds by enantioselective stopped-flow HPLC

Article abstract of DOI:10.1016/j.tetasy.2006.11.035

The synthesis and enantioseparation of chiral 3,4-dihydro-1,2,4-benzothiadiazine 1,1-dioxide derivatives are reported herein. A HPLC stopped-flow procedure was applied to the determination of rate constants and free energy barriers of enantiomerization of the compounds synthesized in the presence of achiral stationary phase. The individual enantiomers of the studied compounds were isolated in parallel by preparative HPLC on a Chiraspher NT column. Rate constants and free energy barriers of enantiomerization were determined in the mobile phase. The results were used to determine the influence of the chiral stationary phase on the enantiomerization process.

Full text of DOI:10.1016/j.tetasy.2006.11.035

Tetrahedron: Asymmetry 17 (2006) 3158–3162
Energy barrier determination of enantiomerization of chiral
3,4-dihydro-1,2,4-benzothiadiazine 1,1-dioxide type compounds
by enantioselective stopped-flow HPLC
Giuseppe Cannazza,a,* Daniela Braghiroli,a Piera Iulianib and Carlo Parentia
a
`
Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Modena e Reggio Emilia, Via Campi 183, 4100 Modena, Italy
Dipartimento di Scienze del Farmaco, Universita degli Studi ‘G. d’Annunzio’ Chieti-Pescara, Via dei Vestini, 66100 Chieti, Italy
b
`
Received 11 October 2006; accepted 21 November 2006
Abstract—The synthesis and enantioseparation of chiral 3,4-dihydro-1,2,4-benzothiadiazine 1,1-dioxide derivatives are reported herein.
A HPLC stopped-flow procedure was applied to the determination of rate constants and free energy barriers of enantiomerization of the
compounds synthesized in the presence of achiral stationary phase. The individual enantiomers of the studied compounds were isolated
in parallel by preparative HPLC on a Chiraspher NT column. Rate constants and free energy barriers of enantiomerization were deter-
mined in the mobile phase. The results were used to determine the influence of the chiral stationary phase on the enantiomerization
process.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
O
O
O
O
Cl
S
S
NH
NH
Benzothiadiazines have been shown to be potential drugs
for memory and cognition disorders, such as attention dis-
orders in children and senile dementias, including early
stage Alzheimer diseases.1 In particular, IDRA21 ( )-1
[( )-7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadi-
azine 1,1-dioxide] (Fig. 1) was shown to be a potent cogni-
tion enhancer in animals suggesting the potential
applicability of this drug as a nootropic agent.2–6
N
H
N
H
2
( )-
IDRA21
( )-1
( )-
O
O
O
O
O
O
Cl
S
S
S
H2N
NH
NH
Like other chiral cognition enhancers of the benzothiadi-
azine type, IDRA21 contains a stereogenic carbon atom
in the benzothiadiazine ring.
Cl
N
H
Cl
N
H
( )-4
( )-3
Figure 1. Structures of analytes investigated.
Previously, we have reported the resolution of IDRA21
enantiomers by liquid chromatography on an optically
active column and found that a rapid interconversion
(enantiomerization) of the IDRA21 enantiomers occurred
in aqueous solution.7 A procedure for the determination
of apparent rate constants, and apparent free energy barri-
ers of enantiomerization without the implementation of
computer simulation has recently been developed.8–21 The
HPLC stopped-flow procedure developed was applied to
study on column enantiomerization of (+)-1 and (ꢀ)-1
and gave a relatively low activation barrier of enantiomer-
ization in aqueous solvents.8
To gain further insight in understanding the enantiomer-
ization mechanism of chiral 3,4-dihydro-1,2,4-benzothiadi-
zine 1,1-dioxide type compounds, the on column HPLC
stopped-flow procedure developed was applied to study
*
Corresponding author. Tel.: +39 059 2055013; fax: +39 059 2055750;
e-mail addresses: cannazza.giuseppe@unimore.it; cannazza.giuseppe@
unimo.it
0957-4166/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2006.11.035

G. Cannazza et al. / Tetrahedron: Asymmetry 17 (2006) 3158–3162
3159
the enantiomerization of three selected chiral compounds:
( )-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-
dioxide ( )-2, ( )-6,7-dichloro-3-methyl-3,4-dihydro-
the peaks 2ꢁ and 3ꢁ belong to the signals corresponding to
the portion of the interconverted sample corresponding to
step 2. Thus it was possible to calculate ka1pp and kapp and
ꢀ1
2H-1,2,4-benzothiadiazine
1,1-dioxide
( )-3
and
apparent free energy barriers of enantiomerization
(DG#app) from the individual chromatographic peak areas,
which are a weighted means of the different enantiomeriza-
tion rates in the mobile phase and on the stationary phase.
( )-6-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine-
7-sulfonamide 1,1-dioxide ( )-4 (Fig. 1).
Subsequently, the individual enantiomer of ( )-2, ( )-3
and ( )-4 was isolated by collecting the respective peak
fractions of HPLC runs employing conditions where no
racemization took place, followed by the determination
of rate constants and free energy barriers of enantiomeriza-
tion in the same solvents used for the on column enantio-
merization. These results were used to investigate the
influence of substitution at the C(6) and C(7) positions of
the benzothiadiazine ring on enantiomerization barriers
and to evaluate the influence of the chiral stationary phase
(CSP) on the enantiomerization process.
The resulted ka1pp and kapp and associated free energies of
enantiomerization (DG#1ꢀap1p and DG#app) for the individual
ꢀ1
enantiomers of ( )-2 and ( )-3 are listed in Table 2.
As can be seen, the interconversion rates of the forward
and reverse reaction of ( )-2 and ( )-3 are practically
the same. This result was quite expected as small values
of separation factors (a) of the ( )-2 and ( )-3 enantiomers
were obtained at 40 ꢁC on a Chiralcel OD-R column.
The mean value of energy of enantiomerization for ( )-2
was about 7 kJ/mol greater than for ( )-3, indicating that
the chlorine substitution at C(6) and C(7) inhibited
enantiomerization.
2. Results and discussion
The results of the analytical enantioseparations of ( )-2,
( )-3 and ( )-4 on Chiralcel OD-R columns are listed in
Table 1. Baseline resolutions without racemization were
obtained at t = 0 ꢁC with the above column.
No ( )-4 enantiomerization occurred on the Chiralcel OD-
R column at 40 ꢁC for 30 min, indicating an energy of
enantiomerization greater than 100 kJ/mol.
The individual enantiomers of ( )-2, ( )-3 and ( )-4,
isolated by semipreparative HPLC on a Chirasphere NT
column (Table 1) (Fig. 3), were used to determine the
enantiomerization rate constants km and the associated
energy barriers of enantiomerization (DG#m) at the same
temperature and in the same solvent used for the on
column enantiomerization. The results are listed in Table 3.
The enantiomeric nature of eluates was confirmed by circu-
lar dichroism indicating, similar to ( )-IDRA21, the (ꢀ)-
enantiomers eluates before the (+)-enantiomers for all
three compounds under investigations.
The on column HPLC stopped-flow procedure previously
developed was used to study the enantiomerization of
( )-2, ( )-3 and ( )-4. As shown in Figure 2, an experi-
mental protocol of a sequence of three steps was followed.
In step 1, the racemic mixture was injected onto the column
and enantiomers began to separate. After a specific time,
the mobile phase flow was stopped. Racemization of the
individual enantiomers occurred in step 2 during column
heating at the desired temperature for a time interval set:
E-(ꢀ)-enantiomer was partially converted into E-(+)-enan-
tiomer, conversely the E-(+)-enantiomer was also trans-
formed into E-(ꢀ)-enantiomer. In step 3 the column was
cooled down to the original low temperature, where practi-
cally no racemization occurred and the enantiomer separa-
tion could be completed.
Energy of enantiomerization for ( )-4 was about 6 kJ/mol
greater than for ( )-3 and about 14 kJ/mol greater than for
( )-2.
Moreover, it was possible to calculate the forward ðks1Þ and
backward ðksꢀ1Þ rate constants of enantiomerizations and
the relative energy barriers of enantiomerization ðDG1#s
#s
and DG Þ for ( )-2 and ( )-3 enantiomers in the chiral
ꢀ1
environment of CSP from ka1pp, kapp and km according to
ꢀ1
the following equations:
enant
k
1
ðꢀÞ
ka1pp
¼
¼
enant km þ
enant kS1
ð1Þ
ð2Þ
1 þ kðꢀÞ
1 þ kðꢀÞ
enant
The outcome of this protocol resulted in the presence of
four peaks in the chromatograms: peaks 1ꢁ and 4ꢁ belong
to the signals of the enantiomer separation of step 1, while
k
1
ðþÞ
1 þ kðþÞ
app
k
enant km þ
enant kSꢀ1
ꢀ1
1 þ kðþÞ
Table 1. Chromatographic enantioseparations of ( )-2, ( )-3 and ( )-4
Compounds
Chiralcel OD-Ra
Chiraspher NTb
k0ðꢀÞ
6.92
16.52
8.36
k0ðþÞ
a
RS
k0ðþÞ
k0ðꢀÞ
a
RS
(
(
(
)-2
)-3
)-4
9.59
19.19
11.69
1.39
1.64
1.40
1.92
1.64
1.59
8.91
6.18
19.43
7.73
5.27
17.52
1.15
1.17
1.11
1.86
1.54
1.62
a Column Chiralcel OD-R; mobile phases: water/acetonitrile 70:30 (v/v) for ( )-2 and ( )-3, water/acetonitrile 50:50 (v/v) for ( )-4; t = 0 ꢁC; flow: 0.5 ml/
min for ( )-2 and ( )-3, 0.7 ml/min for ( )-4.
b Column Chiraspher NT; mobile phase: hexane/tetrahydrofurane 70:30 (v/v); t = 25 ꢁC; flow: 3 ml/min.

3160
G. Cannazza et al. / Tetrahedron: Asymmetry 17 (2006) 3158–3162
Figure 2. Computer simulated concept of on-column enantiomerization to be investigated by stopped-flow technique during chromatographic
enantioseparation of studied compounds. Step 1. Compound was injected and a partial elution of enantiomers occurred at low temperature conditions.
Step 2. The mobile phase flow was stopped and the column was kept for a set time at a set higher temperature. Each enantiomer racemized in a time- and
temperature-dependent manner. Step 3. The column was cooled back to the initial temperature and the original flow rate was reinforced, leading to
enantiomer separation.
Table 2. On-column enantiomerization of ( )-2, ( )-3
app
ꢀ1
Compound Eluents
ka1pp ðsꢀ1
Þ
k
ðsꢀ1
Þ
DG#1 app (KJ/mol) DG# app (kJ/mol) t (ꢁC)
ꢀ1
2
3
Water/acetonitrile 80:20 (v/v) 1.45 · 10ꢀ3 0.45 · 10ꢀ3 1.11 · 10ꢀ3 0.05 · 10ꢀ3 93.75 0.71
Water/acetonitrile 60:40 (v/v) 1.29 · 10ꢀ4 0.13 · 10ꢀ4 1.20 · 10ꢀ4 0.12 · 10ꢀ4 99.06 0.57
94.43 0.10
98.31 0.54
40
40
Column: Chiralcel OD-R (25 · 0.46 cm ID, 10 lm) tris(3,5-dimethylphenyl-carbamate); flow rate: 0.5 ml/min, column operation temperature = 12 ꢁC,
n = 4; time intervals for on-column enantiomerization at 40ꢁ = 50, 100, 150, 22.50.
enant
ðꢀÞ
enant
ðþÞ
where k
and k
are the retention factors of the (ꢀ)-
The differences between the energies of enantiomerization
in the mobile phase and in the CSP increase with the reten-
tions of analytes (Table 4).
and (+)-enantiomers, respectively.
The results showed that the interactions of the enantiomers
with the CSP have an inhibitory effect on enantiomeriza-
The finding that the complexation of enantiomers with the
CSP increased the enantiomerization barrier was expected,
tion being DG1#s and DG#s greater than DG#m (Table 3).
ꢀ1

G. Cannazza et al. / Tetrahedron: Asymmetry 17 (2006) 3158–3162
3161
3. Experimental
The chromatographic apparatus consisted of Jasco PU-980
intelligent Pump, and a 7125 Reodhyne manual injector
equipped with a 20 ll or 200 ll sample loop. A model Jasco
J-710 spectropolarimeter or a UV a Jasco 875-UV was used
as a detector. Chromatograms were recorded with J-700 sys-
tem program or a Spectra Physics Integrator. A 7000 Reo-
dhyne valve installed in a post column mode permitted the
trapping of peak fractions for the acquisition of CD and
UV spectra with an on-line installed spectropolarimeter
detector. Column temperature regulation was achieved by a
thermostated water bath Haake F3. The columns used were
a Chiralcel OD-R column (cellulose tris-3,5-dimethylphen-
ylcarbammate; 250 · 4.6 mm ID; 10 lm) purchased from
Daicel and a Chiraspher NT (poly(N-acryloyl-S-phenylala-
ninethylester); 250 · 4 mm. ID; 5 lm)purchasedfrom Merck.
Melting points were determined with an Electrothermal
apparatus and are uncorrected. IR spectra were recorded
on a Perkin Elmer Model 1600 FT-IR spectrometer (KBr
pellets) and were consistent with the assigned structures.
1H NMR spectra were recorded with a Brucker DPX 200
spectrometer using DMSO-d6 as solvent and tetramethyl-
silane (TMS) as the external standard. Chemical shifts (d)
are in parts per million and coupling constants (J) in hertz.
Multiplicities are abbreviated as follows: s, singlet; d, dou-
blet; dd, double doublet, m, multiplet. Elemental analyses
were performed on a Carlo Erba Elemental Analyzer Mod-
el 1106 apparatus and the results are within 0.4% of the
theoretical values.
3.1. Chemistry
3.1.1. General procedure for the synthesis of derivatives
2–4. A solution of 20 mmol of the corresponding benz-
enesulfonamide in dioxane, containing 1.1 equiv of acetal-
dehyde and few drops of concentrated H2SO4, was heated
at reflux for 3 h. The solvent was evaporated and the crude
was crystallized from acetone/petroleum benzine at a tem-
perature of 90–100 ꢁC.22
3.1.2. ( )-3-Methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine
1
1,1-dioxide ( )-2. Yield: 92%, mp 212–213 ꢁC, H NMR
(DMSO-d6) d = 1.44 (d, J = 6.2, 3H), d = 4.76–4.91 (m,
1H), d = 6.67–6.75 (m, 1H), d = 6.77 (dd, 1H, J = 0.6;
0.8), d = 7.08 (s, 1H), d = 7.25–7.33 (m, 1H), d = 7.43 (d,
1H, J = 11.9), d = 7.45 (dd, 1H, J = 1.4; 11.9).
3.1.3. ( )-6,7-Dichloro-3-methyl-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide ( )-3. Yield: 91%, mp 222–
225 ꢁC, 1HNMR (DMSO-d6) d = 1.42 (d, J = 6.2, 3H),
d = 4.75–4.90 (m, 1H), d = 7.00 (s, 1H), d = 7.49 (s, 1H),
d = 7.66 (s, 1H), d = 7.45 (d, 1H, J = 1.4; 11.7).
Figure 3. Chromatographic resolution of (a) ( )-3-methyl-3,4-dihydro-2H-
1,2,4-benzothiadiazine 1,1-dioxide ( )-2, (b) ( )-6,7-dichloro-3-methyl-
3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide
( )-(3), (c) ( )-6-
chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide
1,1-dioxide ( )-4 on Chiraspher NT column monitored by absorption
(below) and circular dichroism (CD) detection (above). Eluent: hexane:
tetrahydrofurane 70:30 (v/v); t = 25 ꢁC; flow: 3 ml/min.
3.1.4. ( )-6-Chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzo-
thiadiazine-7-sulfonamide 1,1-dioxide ( )-4. Yield: 44%,
mp 238–240 ꢁC, 1H NMR (DMSO-d6) d = 1.45 (d,
J = 6.2, 3H), d = 4.85–4.94 (m, 1H), d = 6.93 (s, 1H),
d = 7.45 (s, 2H), d = 7.80 (d, J = 11.6), d = 7.96 (s, 1H),
d = 7.98 (s, 1H).
as interactions with the CSP may have to be broken to
reach the transition state.

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G. Cannazza et al. / Tetrahedron: Asymmetry 17 (2006) 3158–3162
Table 3. Calculated values of enantiomerization of IDRA21 on OD-CSP
Compound Solvents
Ks1 ðsꢀ1
Þ
Ksꢀ1 ðsꢀ1
Þ
DG#1 s (kJ/mol) DG#s (kJ/mol) Temperatures (ꢁC)
ꢀ1
2
Water/acetonitrile 80:20 4.93 · 10ꢀ4 2.62 · 10ꢀ4 6.95 · 10ꢀ5 8.13 · 10ꢀ6
96.55 1.10
101.64 0.29
40
(v/v) (flow 0.5 ml/min)
3
Water/acetonitrile 70:30 6.90 · 10ꢀ5 1.68 · 10ꢀ5 8.13 · 10ꢀ5 1.59 · 10ꢀ5 101.72 0.57
101.29 0.46
40
(v/v) (flow 0.5 ml/min)
ks1 and ksꢀ1 were calculated according to Eqs. 1 and 2.
Table 4. Off-column enantiomerization of ( )-(2), ( )-(3), ( )-(4) enantiomers in water/acetonitrile
Compound Solvents
km1 ðsꢀ1
Þ
kmꢀ1 ðsꢀ1
Þ
DG#1 m (kJ/mol) DG#m (kJ/mol) t (ꢁC)
ꢀ1
2
3
4
Water/acetonitrile 80:20 (v/v) 3.65 · 10ꢀ3 0.12 · 10ꢀ3 4.20 · 10ꢀ4 0.42 · 10ꢀ4
Water/acetonitrile 70:30 (v/v) 6.36 · 10ꢀ4 0.54 · 10ꢀ4 6.39 · 10ꢀ4 0.63 · 10ꢀ4
91.34 0.10
95.84 0.21
90.98 0.25
95.88 0.24
104.73 0.05
40
40
60
Water/acetonitrile 70:30 (v/v) 3.31 · 10ꢀ4 0.11 · 10ꢀ4 2.54 · 10ꢀ4 0.04 · 10ꢀ4 103.99 0.09
Column: Chiralcel OD-R (25 · 0.46 cm ID, 10 lm) tris(3,5-dimethylphenyl-carbamate); flow rate: 0.5 ml/min, column operation temperature = 12 ꢁC,
n = 4; time intervals for on-column enantiomerization at 40ꢁ = 50, 100, 150, 22.50.
3.2. Chromatographic enantioseparations
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As shown in Figure 2, a racemic mixture of IDRA21 was
first injected on column, kept at low temperature and
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the mobile phase flow was stopped and the column was
kept at a certain temperature for a set period of time to
effect enantiomerization of the separated individual enantio-
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to the initial low temperature conditions and continued to
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´
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