Determination of kinetic parameters of enantiomerization of benzothiadiazines by DCXplorer

Article abstract of DOI:10.1002/chir.20838

Benzothiadiazines differently substituted at the sulfonamidic nitrogen atom, at the stereogenic carbon atom and at the anilinic nitrogen atom have been synthesized and fully characterized. Enantioseparation of these compounds has revealed rapid on-column enantiomerization. The recently developed software DCXplorer has been successfully applied to calculate enantiomerization kinetic parameters. Enantiomerization barriers of 3-phenyl substituted benzothiadiazines, calculated in this work, have indicated a higher enantiomerization rate suggesting that the aromatic substituent exerts a strong effect on the enantiomerization process. Methyl substitution on N²position led to higher free energy barriers of enantiomerization, suggesting negative influence of methyl in the N²position on enantiomerization kinetics. However, methylation on N⁴position increases the enantiomerization rates significantly. The results obtained have been employed to postulate an enantiomerization mechanism for chiral benzothiadiazine type compounds.

Full text of DOI:10.1002/chir.20838

CHIRALITY 22:789–797 (2010)
Determination of Kinetic Parameters of Enantiomerization
of Benzothiadiazines by DCXplorer
1
1
1
GIUSEPPE CANNAZZA,1 MARINA M. CARROZZO, UMBERTO BATTISTI, DANIELA BRAGHIROLI,
*
CARLO PARENTI,1 ALESSANDRO TROISI,2 AND LUIGINO TROISI3
1Dipartimento di Scienze Farmaceutiche, Universita` degli Studi di Modena e Reggio Emilia,
4100 Modena, Italy
2Department of Chemistry and Centre of Scientific Computing, University of Warwick,
CV4 7AL Coventry, United Kingdom
3Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, University of Salento,
73100 Lecce, Italy
ABSTRACT
Benzothiadiazines differently substituted at the sulfonamidic nitrogen
atom, at the stereogenic carbon atom and at the anilinic nitrogen atom have been syn-
thesized and fully characterized. Enantioseparation of these compounds has revealed
rapid on-column enantiomerization. The recently developed software DCXplorer has
been successfully applied to calculate enantiomerization kinetic parameters. Enantiome-
rization barriers of 3-phenyl substituted benzothiadiazines, calculated in this work, have
indicated a higher enantiomerization rate suggesting that the aromatic substituent
exerts a strong effect on the enantiomerization process. Methyl substitution on N2 posi-
tion led to higher free energy barriers of enantiomerization, suggesting negative influ-
ence of methyl in the N2 position on enantiomerization kinetics. However, methylation
on N4 position increases the enantiomerization rates significantly. The results obtained
have been employed to postulate an enantiomerization mechanism for chiral benzothia-
C
VV
diazine type compounds. Chirality 22:789–797, 2010.
2010 Wiley-Liss, Inc.
KEY WORDS: benzothiadiazines; HPLC; enantiomerization; enantioseparation
INTRODUCTION
tinuous flow model, peak deconvolution method, approxi-
mation function, and the unified equation.14–19
The U.S. Food and Drug Administration requires that
‘‘the stability protocol for enantiomeric drug substances
and drug products should include a method or methods
capable of assessing the stereochemical integrity of the
drug substance and drug product.’’1 Recently chiral benzo-
thiadiazines type compounds have attracted particular
attention as AMPA receptor positive allosteric modula-
tors.2–9 As there is experimental evidence that indicate the
stereospecific pharmacological action of benzothiadiazine
type compounds, it gains important to evaluate the stereo-
chemical stability of their active enantiomers.
It is possible to study the enantiomerization of chiral la-
bile compounds by different techniques depending on
their interconversion free energy barriers. Among the
most commonly used methods, classical chromatographic
off-column techniques consisting of an examination of
batchwise racemization kinetics of a single enantiomer af-
ter enantioselective separation.
DHPLC is a very attractive technique, because it
requires only minute amounts of the racemic or enan-
tiomer-enriched mixture.
Recently, Trapp et al. have developed the software pro-
gram DCXplorer that permits the calculation of reaction
rate constants of enantiomerization employing the unified
chromatography equation.19–28
As we have demonstrated that some chiral 3,4-
dihydro-1,2,4-benzothiadiazine 1,1-dioxide type compounds
undergo rapid enantiomerization during enantioseparation
in reverse phase conditions,29,30 DCXplorer has been
applied to calculate enantiomerization rate constants of
synthesized benzothiadiazines 1–12 here (Fig. 1). Differ-
ent substituents were placed in the thiadiazine ring to
understand the influence of these substituents on enantio-
merization activation barrier.
The data obtained by dynamic HPLC were used to gain
further insight in understanding the enantiomerization
If enantiomerization takes place in the same time-scale
of the chromatographic separation process, a characteris-
tic plateau appears between the two peaks of the intercon-
verting enantiomers.10–13 From the resulting peak shapes
obtained by enantioselective dynamic HPLC (DHPLC), it
is possible to extract the necessary information to calcu-
late racemization kinetic parameters by several methods
*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 1 July 2009; Accepted 5 February 2010
DOI: 10.1002/chir.20838
Published online 30 March 2010 in Wiley Online Library
such as the theoretical plate model, stochastic model, con- (wileyonlinelibrary.com).
C
VV
2010 Wiley-Liss, Inc.

790
CANNAZZA ET AL
on a Bruker Avance 400 apparatus (400.13 and 100.62
MHz, for 1H and 13C, respectively) with CDCl3 and DMSO
1
as solvents and TMS as internal standard (d 5 7.24 for H
spectra; d 5 77.0 for 13C spectra). Chemical shifts (d) are
in part per million and coupling constant (J) in hertz. Mul-
tiplicities are abbreviated as follows: s, singlet; d, doublet;
dd, double doublet; t, triplet; m, multiplet.
The IR spectra were recorded with a Digilab Scimitar Se-
ries FTS 2000 FT-IR spectrophotometer. GC-MS analyses
were performed with an Agilent Technologies 6850 series
II gas chromatograph (with a 5% phenyl-polymethylsilox-
ane capillary column, 30 m, 0.25 mm ID), and a 5973 Net-
work mass selective detector operating at 70 eV. The elec-
trospray ionization (HR-ESI-MS) experiments were carried
out in a hybrid QqTOF mass spectrometer (PE SCIEX-
QSTAR) equipped with an ion spray ionization source. MS
(1) spectra were acquired by direct infusion (5 ml/min) of
a solution containing the appropriate sample (10 pmol/ml),
dissolved in a solution 0.1% acetic acid, methanol:water
50:50 at the optimum ion voltage of 4800 V.
All pH measurements were made with an Orion
Research EA940 pH-meter.
Synthesis
General procedure for the synthesis of derivatives
1–12. A mixture of the appropriate 2-amino-5-chloroben-
zensulfonamide (4 mmol) and
a suitable aldehyde
(40 mmol) in 2-propanol (40 ml) supplemented with 2 ml
of ethyl acetate saturated with dry HCl was refluxed and
stirred for 2 h at 508C. After cooling, the resulting suspen-
sion was concentrated to dryness under reduced pressure.
The solid residue was purified by column chromatography
on silica gel (elution solvent: ethyl acetate:petroleum ether
(40–608C), 1:1 (v/v) for compounds 2–6 and 11–12; 6:4
(v/v) for compounds 7 and 10; and 7:3 (v/v) for com-
pounds 8 and 9).
An alternative pathway was also used for the synthesis
of compound 9. A solution of (6)7-chloro-3-(2-chloro-
phenyl)-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide
((6)-3) (1.0 mmol) in tetrahydrofuran (10 ml) was supple-
mented dropwise at 08C with a solution of lithium diisopro-
pylamide (LDA) (1.0 mmol) in tetrahydrofuran. Methyl
iodide (3.0 mmol) was then added and the mixture was
stirred for 1 h at room temperature. The reaction mixture
was quenched with water (50 ml) and the insoluble mate-
rial was extracted three times with diethyl ether (75 ml).
The combined organic layers were dried over MgSO4 and
filtered. The filtrate was concentrated to dryness under
reduced pressure. The solid residue was purified by col-
umn chromatography on silica gel [elution solvent: ethyl
acetate:petroleum ether (40–608C), 7:3 (v/v)] (Yield 85%).
Fig. 1. Compounds investigated.
mechanism of chiral 3,4-dihydro-1,2,4-benzothiadiazine 1,1-
dioxide type compounds.
EXPERIMENTAL
Instrumentation
The chromatographic apparatus consisted of a Jasco Pu-
2080 Plus pump, a Rheodyne 7725 manual injector
equipped with a 20 ll sample loop and a Jasco UV-2075
Plus detector. Chromatograms were recorded with Jasco
J-700 program. Column temperature regulation was
achieved with a Jasco CO-2067 column oven.
4-(7-Chloro-1,1-dioxido-3,4-dihydro-2H-1,2,4-ben-
zothiadiazin-3-yl)phenol ((6)-1). The title compound
was synthesized as previously reported.4
The column used was Chiralcel OD-RH [cellulose
tris(3,5-dimethylphenylcarbamate); 150 mm 3 4.6 mm ID; 1,2,4-benzothiadiazine 1,1-dioxide ((6)-2). Yield
5 lm] purchased from Daicel. Melting points were deter- 71%, mp 172–173.28C. H NMR (CDCl3) d 5 3.85 (s, 3H),
(6)7-Chloro-3-(2-methoxyphenyl)-3,4-dihydro-2H-
1
mined with an electrothermal apparatus and are uncor- d 5 6.15 (d, 1H, J 5 12.2 Hz), d 5 6.92 (d, 1H, J 5 8.9
1
rected. The H and the 13C NMR spectra were recorded Hz), d 5 7.06 (t, 1H, J 5 7.5 Hz), d 5 7.12 (d, 1H,
Chirality DOI 10.1002/chir

DETERMINATION OF KINETIC PARAMETERS BY DCXplorer
791
J 5 8.3 Hz), d 5 7.36 (dd, 1H, J 5 2.5 Hz, J 5 8.9 Hz), d 5
7.43 (t, 1H, J 5 8.3 Hz), d 5 7.47 (s, 1H, broad, exchange
with D2O), d 5 7.55 (d, 1H, J 5 2.5 Hz), d 5 7.71 (d, 1H, J
5 7.5 Hz), d 5 7.95 (d, 1H, J 5 12.2 Hz, exchange with
D2O). 13C NMR (CDCl3) d 55.7, 61.7, 111.3, 118.2, 119.8,
120.4, 122.1, 122.9, 124.5, 128.0, 130.5, 132.8, 142.9, 156.1.
FTIR (KBr) 3440, 3330, 3020, 1484, 1215, 1167 cm21. GC-
MS (70 eV) m/z 324(82) [M1], 259(20), 206 (38), 126
(95), 119 (100), 91 (50). HRMS-ESI: calcd. for C14H14Cl
N2O3S [M1H]1 325.0415; found: 325.0412.
(6)7-Chloro-3-(2-chlorophenyl)-3,4-dihydro-2H-1,2,
4-benzothiadiazine 1,1-dioxide ((6)-3). Yield 76%, mp
211.2–212.38C. 1H NMR (CDCl3) d 5 6.18 (s, 1H), d 5
6.95 (d, 1H, J 5 8.9 Hz), d 5 7.40 (dd, 1H, J 5 2.2 Hz, J
5 8.9 Hz), d 5 7.47–7.59 (m, 4H), d 5 7.68 (s, 1H), d 5
7.87–7.89 (m, 1H), d 5 8.16 (s, 1H, broad). 13C NMR
(CDCl3) d 64.8, 118.4, 120.4, 122.4, 122.9, 127.6, 129.1,
129.5, 130.9, 132.3, 133.0, 133.9, 142.7. FTIR (KBr) 3369,
3201, 3320, 2925, 2828, 1607, 1485, 1318, 1155 cm21. GC-
MS (70 eV) m/z 328 (50) [M1] 247 (55), 207 (100), 126
(75). HRMS-ESI: calcd. for C13H11Cl2 N2O2S [M1H]1
328.9920; found: 328.9922.
(6)7-Chloro-3-(4-chlorophenyl)-3,4-dihydro-2H-1,2,
4-benzothiadiazine 1,1-dioxide ((6)-4). Yield 64%, mp
227.3–228.58C. 1H NMR (CDCl3) d 5 5.82 (s, 1H), d 5
6.93 (d, 1H, J 5 8.9 Hz), d 5 7.38 (d, 1H, J 5 8.9 Hz), d
5 7.53–7.55 (m, 3H), d 5 7.62 (s, 1H), d 5 7.69 (d, 2H, J
5 8.2 Hz), d 5 8.1 (s, 1H, broad, exchange with D2O). 13
C
NMR (CDCl3) d 67.5, 118.4, 120.2, 122.3, 122.8, 128.5,
129.4, 132.9, 133.8, 135.8, 142.6. FTIR (KBr) 3387, 3252,
3020, 2924, 2854, 1606, 1487, 1325, 1159 cm21. GC-MS (70
eV) m/z 328 (40) [M1] 281 (27), 247 (25), 207 (100), 126
(42). HRMS-ESI: calcd. for C13H11Cl2 N2O2S [M1H]1
328.9920; found: 328.9921.
(6)7-Chloro-3-(3-chlorophenyl)-3,4-dihydro-2H-1,2,
4-benzothiadiazine 1,1-dioxide ((6)-5). Yield 72%, mp
1
201.6–202.88C. H NMR (CDCl3) d 5 5.83 (s, 1Hz), d 5
6.94 (d, 1H, J 5 8.9 Hz), d 5 7.39 (dd, 1H, J 5 2.5 Hz, J 5
8.9 Hz), d 5 7.50–7.65 (m, 5Hz), d 5 7.78 (s, 1H, broad,
exchange with D2O), d 5 8.10 (s, 1H, broad, exchange
with D2O). 13C NMR (CDCl3) d 67.5, 118.4, 120.3, 122.3,
122.9, 126.4, 127.4, 129.1, 130.4, 132.9, 133.1, 139.1, 142.6.
FTIR (KBr) 3381, 3210, 3064, 2924, 2869, 1605, 1498, 1318,
1162 cm21. GC-MS (70 eV) m/z 328 (95) [M1] 247 (49),
142 (52), 126 (100). HRMS-ESI: calcd. for C13H11Cl2N2O2S
[M1H]1 328.9920; found: 328.9922.
(6)7-Chloro-3-[4-(trifluoromethyl)phenyl]-3,4-dihy-
dro-2H-1,2,4-benzothiadiazine
1,1-dioxide
((6)-
1
6). Yield 83%, mp 205.3–206.88C. H NMR (CDCl3) d 5
5.93 (s, 1Hz), d 5 6.94 (d, 1H, J 5 8.9 Hz), d 5 7.39 (dd,
1H, J 5 2.5 Hz, J 5 8.9 Hz), d 5 7.56 (d, 1H, J 5 2.5 Hz), d
5 7.68 (s, 1H, broad, exchange with D2O), d 5 7.90 (d,
2H, J 5 8.5 Hz), d 5 8.20 (s, 1H, broad, exchange with
D2O), d 5 8.85 (d, 2H, J 5 8.5 Hz). 13C NMR (CDCl3) d
Fig. 2. General synthesis procedure for compounds 1–12.
67.6, 73.8, 118.4, 120.3, 122.4, 122.8, 125.3, 125.4, 128.5, 362 (100) [M1] 281 (75), 173 (25), 142 (30), 126 (80).
132.9, 141.2, 142.6. FTIR (KBr) 3390, 3216, 3050, 2950, HRMS-ESI: calcd. for C14H11 ClF3N2O2S [M1H]1
2854, 1606, 1491, 1326, 1160 cm21. GC-MS (70 eV) m/z 363.0183; found: 363.0181.
Chirality DOI 10.1002/chir

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CANNAZZA ET AL
TABLE 1. Chromatographic enantioseparation of racemic benzothiadiazine derivatives 1–12
(6)-1
(6)-2
(6)-3
(6)-4
(6)-5
(6)-6
(6)-7
(6)-8
(6)-9
(6)-10
(6)-11
(6)-12
k1
k2
a
2.38
3.52
1.48
5.75
5.75
1.00
10.26
14.20
1.38
8.94
11.64
1.30
9.07
12.84
1.41
7.07
9.88
1.39
12.58
21.01
1.67
10.57
11.38
1.07
11.6
13.23
1.14
10.47a
11.10a
1.06a
1.69
2.34
1.77
2.84
3.76
1.77
Column: Chiralcel OD-RH; mobile phase: water:acetonitrile 50:50 (v/v); T 5 258C; flow: 0.5 ml/min.
aAnalysis temperature 48C.
(6)7-Chloro-3-(4-isopropylphenyl)-3,4-dihydro-2H- 5 2.4 Hz, J 5 9.0 Hz), d 5 7.56 (d, 1H, J 5 2.4 Hz), d 5
1,2,4-benzothiadiazine 1,1-dioxide ((6)-7). Yield 8.21 (d, 3H, J 5 6.1 Hz), d 5 8.63 (d, 2H, J 5 10.6 Hz). 13C
1
68%, mp 204–205.68C. H NMR (CDCl3) d 5 1.22 (d, 6H, J NMR (CDCl3) d 66.2, 118.7, 120.7, 122.4, 122.7, 125.2,
5 6.9 Hz), d 5 2.92 (septet, 1H, J 5 6.9 Hz), d 5 5.74 (d, 133.3, 142.2, 142.9, 154.9. FTIR (KBr) 3267, 3047, 2828,
1H, J 5 10.9 Hz), d 5 6.93 (d, 1H, J 5 9.0 Hz), d 5 7.33– 1602, 1486, 1324, 1174 cm21. GC-MS (70 eV) m/z 293 (80)
7.38 (m, 3H), d 5 7.54–7.59 (m, 4H), d 5 7.99 (d, 1H, J 5 [M1] 189 (75), 125 (100), 105 (46). HRMS-ESI: calcd. for
10.9 Hz). 13C NMR (CDCl3) d 23.8, 33.3, 68.1, 118.3, 119.9, C12H11ClN3O2S [M1H]1 295.0262; found: 296.0261.
122.1, 122.8, 126.4, 127.5, 132.8, 134.4, 142.7, 149.6. FTIR
(6)7-Chloro-3-pyridin-2-yl-3,4-dihydro-2H-1,2,4-
(KBr) 3393, 3228, 3051, 2957, 1608, 1496, 1320, 1150
benzothiadiazine 1,1-dioxide ((6)-12). Yield 98%,
cm21. GC-MS (70 eV) m/z 336 (100) [M1],255 (40), 147
1
mp 179.6–180.88C. H NMR (CDCl3) d 5 6.08 (d, 1H, J 5
(41), 127 (95). HRMS-ESI: calcd. for C16H18Cl N2O2S
10.3 Hz), d 5 7.12 (d, 1H, J 5 8.9 Hz), d 5 7.42 (dd, 1H, J
5 2.4 Hz, J 5 8.9 Hz), d 5 7.50 (s, 1H, broad, exchange
[M1H]1 337.0779; found: 337.0777.
with D2O), d 5 7.54 (d, 1H, J 5 2.4 Hz), d 5 7.70 (dd, 1H,
J 5 5.7 Hz, J 5 7.1 Hz), d 5 7.91 (d, 1H, J 5 8.0 Hz), d 5
8.20 (t, 1H, J 5 8.0 Hz), d 5 8.47 (d, 1H, J 5 10.3 Hz), d 5
8.75 (d, 1H, J 5 5.7 Hz). 13C NMR (CDCl3) d 67.2, 118.8,
120.4, 122.2, 122.7, 123.1, 125.2, 133.1, 140.6, 142.1, 146.5,
153.8. FTIR (KBr) 3212, 3051, 2990, 2918, 2830, 1612,
1506, 1475, 1458, 1331, 1160 cm21. GC-MS (70 eV) m/z
295 (15) [M1] 230 (18), 215 (100), 203 (14), 126 (18), 79
(22). HRMS-ESI: calcd. for C12H11ClN3O2S [M1H]1
296.0262; found: 296.0260.
(6)7-Chloro-2-methyl-3-phenyl-3,4-dihydro-2H-1,2,
4-benzothiadiazine 1,1-dioxide ((6)-8).31 Yield 80%,
mp 208–2108C H NMR (DMSO-d6) d 5 2.30 (s, 3H), d 5
1
6.29 (s, 1H), d 5 7.13 (d, 1H, J 5 9.0 Hz), d 5 7.46 (m,
3H), d 5 7.57(m, 4H), d 5 7.77(s, 1H). FTIR (KBr) 3378,
3276, 3053, 2952, 1605, 1318,1156 cm21
.
HRMS-ESI: calcd. for C14H14ClN2O2S [M1H]1
309.0466; found: 309.0466.
(6)7-Chloro-3-(2-chlorophenyl)-2-methyl-3,4-dihy-
dro-2H-1,2,4-benzothiadiazine 1,1-dioxide ((6)-
1
Enantioselecive dynamic HPLC
9). Yield 91%, mp 179.8–180.98C. H NMR (CDCl3) d 5
2.34 (s, 3H), d 5 6.61 (s, 1H), d 5 7.10 (d, 1H, J 5 9.0
Hz), d 5 7.47 (dd, 1H, J 5 2.4 Hz, J 5 9.0 Hz), d 5 7.51–
7.54 (m, 2H), d 5 7.58–7.62 (m, 2H), d 5 7.78–7.81 (m,
2H). 13C NMR (CDCl3) d 29.1, 68.3, 118.8, 119.2, 121.1,
124.0, 127.1, 129.7, 130.2, 131.2, 131.6, 133.3, 133.4, 142.1.
FTIR (KBr) 3379, 3067, 2999, 2933, 1605, 1487, 1320, 1152
cm21. GC-MS (70 eV) m/z 342 (9) [M1], 249 (100), 231
(21), 214(62), 177 (24), 152 (27). HRMS-ESI: calcd. for
C14H13Cl2N2O2S [M1H]1 343.0076; found: 343.0075.
Separation of enantiomers of (6)-1-(6)-12 were carried
out isocratically at different temperatures (4–408C) on
Chiralcel OD-RH column. The mobile phase consisted of
water:acetonitrile 50:50 (v/v). The compounds were dis-
solved in acetonitrile (for compounds 1–10) or ethanol
(for compounds 11 and 12) and subsequently diluted
1:100 (v/v) with mobile phase to a final concentration of
10 lg/ml. The injection volume was 20 ll. The detector
was set at 254 nm.
The enantiomerization kinetic parameters of benzothia-
diazine derivatives 1–12 have been investigated by
dynamic chromatography experiments (DHPLC) by using
the DCXplorer software developed by Trapp.19 The pro-
gram employs the unified chromatography equation to
directly evaluate elution profiles in a graphical user inter-
face.19–28 Chromatographic row data in ASCII have been
opened with the DCxplorer software and the elution pro-
files have been evaluated by zooming in on the area of the
interconverting peaks. All chromatographic parameters
have been directly determined by integration and have
been used to calculate reaction rate constants.19–28
(6)7-Chloro-3-(2-chlorophenyl)-4-methyl-3,4-dihy-
dro-2H-1,2,4-benzothiadiazine 1,1-dioxide ((6)-
1
10). Yield 97%, mp 189.3–190.58C. H NMR (DMSO-d6)
d 5 2.76 (s, 3H), d 5 6.11 (s, 1H), d 5 7.08 (d, 1H, J 5 9.0
Hz), d 5 7.37–7.45 (m, 3H), d 5 7.54–7.63 (m, 3H), d 5
8.70 (s, 1H, broad, exchange with D2O). 13C NMR
(CDCl3) d 36.1, 71.4, 116.8, 120.4, 122.7, 124.5, 127.3,
129.0, 129.8, 130.4, 132.5, 133.3, 133.6, 143.5. FTIR (KBr)
3232, 3060, 2923, 2854, 1598, 1486, 1319, 1161 cm21. GC-
MS (70 eV) m/z 342 (28) [M1], 203 (45), 140 (100),
111(18). HRMS-ESI: calcd. for C14H13Cl2N2O2S [M1H]1
343.0076; found: 343.0074.
#
#
#
Evaluation of Activation Parameters DG , DH , and DS
The Gibbs free activation energy DG#(T) has been cal-
(6)7-Chloro-3-pyridin-4-yl-3,4-dihydro-2H-1,2,4-
benzothiadiazine 1,1-dioxide ((6)-11). Yield 97%,
1
mp 206.2–207.98C. H NMR (CDCl3) d 5 6.21 (d, 1H, J 5 culated from the kinetic rate constants by fitting the data
10.6 Hz), d 5 7.09 (d, 1H, J 5 9.0 Hz), d 5 7.44 (dd, 1H, J to the Eyring equation:
Chirality DOI 10.1002/chir

DETERMINATION OF KINETIC PARAMETERS BY DCXplorer
793
Fig. 3. Enantioresolution of (6)-4. Column: Chiralcel OD-RH (15 3
0.46 I.D., 5 lm); mobile phase: water:acetonitrile 50:50 (v/v); temperature:
(a) 158C, (b) 258C.
ꢀ
ꢁ
kh
DG#ðTÞ ¼ ꢀRT ln
ð1Þ
jkBT
where k is the kinetic rate constant, kB the Boltzmann con-
stant (kB 5 1,380,662 3 10223 J K21), h Planck’s constant
(h 5 6,626,176 3 10234 J s), R the universal gas constant
(R 5 831,441 J K mol21), j the transmission coefficient (j
5 0,5 for the reversible microscopic interconversion), and
T the temperature (K).
As the experiments on compounds 1–12 were per-
formed at different temperatures (4–408C), it was possible
to evaluate the enantiomerization activation enthalpy DH#
from the slope and the enantiomerization activation en-
tropy DS# from the intercept of the Eyring plot [ln(k1/T
versus T21)].
RESULTS AND DISCUSSION
Chemistry
The synthesis of compounds 1–12 was achieved by clo-
sure of the appropriate 2-amino-5-chlorobenzensulfonamide
and suitable aldehyde in the presence of hydrochloric acid
as the catalyst (Fig. 2). An alternative pathway was used
for the synthesis of compound 9. In previous work, sele-
Chirality DOI 10.1002/chir

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CANNAZZA ET AL
Chirality DOI 10.1002/chir

DETERMINATION OF KINETIC PARAMETERS BY DCXplorer
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Fig. 4. Enantiomerization mechanism postulated.
ctive alkylation of heterocyclic ring at N2 of benzothiadia-
Enantiomerization
zine ring was obtained with potassium carbonate as base in
refluxing acetonitrile.3,5 (6)7-Chloro-3-(2-chlorophenyl)-
3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide ((6)-3)
was selectively alkylated with LDA and methyl iodide to
give compound 9. Selective methylation occurred in the 2-
position of (6)7-Chloro-3-(2-chlorophenyl)-3,4-dihydro-2H-
1,2,4-benzothiadiazine 1,1-dioxide ((6)-3). This second
method proved to be an effective alternative to the potas-
sium carbonate procedure and may be adopted for further
synthesis of 2-alkylbenzothiadiazines.
In this work DCXplorer was employed to calculate ki-
netic enantiomerization parameters of compounds 1, 3–7,
and 10–12 at temperatures between 4 and 378C.
The results obtained are reported in Table 2. The enan-
tiomerization rate constants obtained were used to calcu-
late free energy barriers of enantiomerization (DG#) apply-
ing the Eyring equation and the results are reported in Ta-
ble 3. The activation enthalpies DH# and activation
entropies DS# of the tested compounds were obtained by
plotting ln(k1/T) as a function of T21 according to the Eyr-
ing equation and are summarized in Table 4.
The obtained benzothiadiazines were fully characterized
by 1H and 13C NMR spectroscopy, FTIR, and GC-MS.
It was possible to calculate enantiomerization rate con-
stants of compound 1 at 4, 15, and 258C but not at 378C
because of peak coalescence. As shown in Table 3, the
Chiral Separation
Among different commercial available chiral stationary free energy barrier of 1 was lower than those of the other
phases (CSP), cellulose 3,5-dimethylphenylcarbamate benzothiadiazines tested except for 10 indicating that the
(Chiracel OD-RH) was chosen, because it was previously o-hydroxyphenyl substituent at C3 position dramatically
employed successfully for the enantioseparation of chiral increases the enantiomerization rate.
benzothiadiazine-type compounds.4,29,30,32–34
The enantiomerization rate constants of compound 5
The enantioseparation of chiral benzothiadiazines 1–12 are slightly lower than those of compounds 3 and 4, indi-
was performed in reversed phase conditions using a Chira- cating that m-chloro phenyl substituent possesses an in-
cel OD-RH column with mobile phases of water:acetoni- hibitory effect on enantiomerization rate in comparison
trile at different percentages. Among the mobile phases with o- and p-chloro phenyl substituents.
used, water:acetonitrile 50:50 (v/v) was chosen, as it gave
Compounds 6 and 7, bearing respectively a p-trifluoro-
the higher enantioresolution with acceptable analysis methylphenyl and a p-isopropylphenyl substituent at C3
times (Table 1). Although good enantioseparation at 258C position, show quite similar enantiomerization rate con-
was obtained for all compounds with the exception of 2 stants indicating that the two substituents have similar
and 10, baseline enantiomeric resolution was achieved influences on enantiomerization kinetics. With the aim of
only for 8 and 9 (Table 1). A pronounced plateau was evaluating the influence of methyl substituents on N2- and
observed between the peaks corresponding to the two N4 positions, enantiomerization rate constants were calcu-
enantiomers, indicating that rapid enantiomerization of lated for compounds 8, 9, and 10.
compounds 1, 3–7, and 10–12 occurred in water:acetoni-
No enantiomerization occurred for compounds 8 and 9
trile during enantioseparation at 258C (Fig. 3). Conversely, between 4 and 378C, indicating that N2-methyl substituent
any plateau observed in the chromatograms correspond- dramatically increases free energy barrier of enantiomeri-
ing to enantioseparation of compounds 8 and 9 at temper- zation.
atures between 4 and 378C using water:acetonitrile 50:50
As the rapid thermal enantiomerization of compound 10
(v/v) as mobile phase indicated that any racemization results in peak coalescence between 15 and 378C, it was
occurs in the experimental conditions used (Table 1). At not possible to calculate enantiomerization rate constants.
258C, enantioseparation was achieved for compounds 2
Plateau formation was observed only at 48C, indicating
and 10 using mobile phases with different percentages of that rapid enantiomerization takes place during chromato-
acetonitrile. Compound 10 was enantioseparated at 48C graphic separation. This data suggests that a methyl sub-
with a separation factor of 1.06. Compound 2 has shown a stituent at N4 considerably increases enantiomerization
partial enantioseparation only at 2108C. We were unable rates. Compound 11 has a free energy barrier of enantio-
to ascribe the poor enantioseparation of 2 and 10 to a fast merization slightly greater than for compound 12 indicat-
on-column enantiomerization or to weak stereoselectivity ing that a 2-pyridine substituent at C3 increases enantiome-
of the CSP.
rization rate.
Chirality DOI 10.1002/chir

796
CANNAZZA ET AL
Fig. 5. Pathway example of ring opening.
Previous studies regarding enantiomerization of 3-alkyl sulfon-N-alkylamides or 2-aminobenzensulfon-N-benzyla-
substituted benzothiadiazines suggested an enantiomeriza- mides were obtained, suggesting the formation and subse-
tion barrier of about 94 kJ/mol under similar experimental quently reduction of an imine intermediate.37
conditions.32–35 Enantiomerization barriers of 3-phenyl
Therefore, all the observed effect of substituents at N2,
substituted benzothiadiazines, calculated in the present C3, and N4 on enantiomerization rates, the negative enan-
work, indicated a higher enantiomerization rates suggest- tiomerization entropy, and other evidence cited supported
ing that aromatic substituents exert a strong effect on the the postulated enantiomerization mechanism.
enantiomerization process. The methyl substitution on N2
ACKNOWLEDGMENTS
position (compounds 8 and 9) led to higher free energy
barriers of enantiomerization, suggesting a negative influ-
ence of this methyl group on enantiomerization kinetics.
However, methylation on N4 significantly increases enan-
tiomerization rates.
The authors thank Dr. L. Costa for the technical help he
gave for synthesis and enantiomerization experiments.
This research was partly financially supported by MIUR
(Ministero dell’Istruzione, dell’Universita` e della Ricerca,
Italy).
The resultant free energy barriers of enantiomerization
of compounds with aromatic substituents at C3 are consist-
ent with an interconversion mechanism that involves an
imine intermediate with the double bond between C3 and
N2 (Fig. 4). Because aromatic imines are more stable than
aliphatic imines, aromatic substituents at the stereogenic
carbon atom decrease free energy barriers of enantiomeri-
zation. The formation of imine intermediates can occur via
several pathways and one example is given in (Fig. 5). As
the observed negative entropy of enantiomerization sug-
gests, a bimolecular reaction mechanism can occur, prob-
ably protonation of N4, followed by deprotonation of N2
and simultaneous imine bond formation between N2 and
C3 (Fig. 5). This pathway has been proposed to account
for the observed enantiomerization behavior of the benzo-
thiadiazine-type compounds tested. The electron donating
effect of a methyl substituent at N4 (10) accelerates enan-
tiomerization presumably as a consequence of a more
favorable protonation of nitrogen. However, the methyl
substituent at N2 in 8 and 9 accounts for their higher
energy enantiomerization barriers, because the replace-
ment of the acidic proton suppresses ring opening. This
enantiomerization mechanism proposed is supported by
previous studies. Mollica et al. suggested that hydrolysis
of benzothiadiazine compounds (e.g. hydrochlorthiazide)
can occur via an imine intermediate that undergoes attack
by water to form a carbinolamine, which subsequently
decomposes.36 Moreover, we have recently disclosed the
rapid enantiomerization and reversible hydrolysis in acidic
conditions of (6)-7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-
benzothiadiazine 1,1-dioxide (IDRA21) to 2-amino-5-chloro-
benzensulfonamide and acetaldehyde by a mechanism
similar to that observed for hydrolysis of imine com-
pounds.30 Ghelardoni et al. have reported the action of
KBH4 on the 3-alkyl or 3-aryl substituted 3,4-dihydro-1,2,4-
benzothiadiazines 1,1-dioxyde in water-ethanol as sol-
vent.37 Compounds with the structure of 2-aminobenzen-
Chirality DOI 10.1002/chir
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