Dibenzo[1,2,5]thiadiazepines are non-competitive GABAA receptor antagonists

Article abstract of DOI:10.3390/molecules18010894

A new process for obtaining dibenzo[c,f][1,2,5]thiadiazepines (DBTDs) and their effects on GABAA receptors of guinea pig myenteric neurons are described. Synthesis of DBTD derivatives began with two commercial aromatic compounds. An azide group was obtain

Full text of DOI:10.3390/molecules18010894

Molecules 2013, 18, 894-913; doi:10.3390/molecules18010894
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Dibenzo[1,2,5]thiadiazepines Are Non-Competitive GABAA
Receptor Antagonists
Juan F. Ramírez-Martínez 1,2, Rodolfo González-Chávez 2, Raquel Guerrero-Alba 1,
Paul E. Reyes-Gutiérrez 2, Roberto Martínez 3, Marcela Miranda-Morales 4,
Rosa Espinosa-Luna 1, Marco M. González-Chávez 2,* and Carlos Barajas-López 1,*
1
División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica,
San Luis Potosí 78216, Mexico; E-Mails: francisco.martinez@uaslp.mx (J.F.R.-M.);
biogigio@hotmail.com (R.G.-A.); respinosa@ipicyt.edu.mx (R.E.-L.)
2
Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, San Luis Potosí 78210,
Mexico; E-Mails: rodolfo.gonzalez@uaslp.mx (R.G.-C.); pa_edu3@yahoo.com.mx (P.E.R.-G.)
3
Instituto de Química, Universidad Nacional Autónoma de México, Coyoacán 04510, Mexico;
E-Mail: robmar@servidor.unam.mx
4
Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología,
Universidad Nacional Autónoma de México, Querétaro 76230, Mexico;
E-Mail: mmirandam@unam.mx
* Authors to whom correspondence should be addressed; E-Mails: gcmm@uaslp.mx (M.M.G.-C.);
cbarajas@ipicyt.edu.mx (C.B.-L.); Tel.: +52-444-826-2440 (ext. 526) (M.M.G.-C.);
Tel.: +52-444-834-2035 (C.B.-L.); Fax: +52-444-834-2010 (C.B.-L.).
Received: 14 December 2012; in revised form: 31 December 2012 / Accepted: 5 January 2013 /
Published: 11 January 2013
Abstract: A new process for obtaining dibenzo[c,f][1,2,5]thiadiazepines (DBTDs) and
their effects on GABAA receptors of guinea pig myenteric neurons are described. Synthesis
of DBTD derivatives began with two commercial aromatic compounds. An azide group
was obtained after two sequential reactions, and the central ring was closed via a nitrene to
obtain the tricyclic sulfonamides (DBTDs). Whole-cell recordings showed that DBTDs
application did not affect the holding current but inhibited the currents induced by GABA
(IGABA), which are mediated by GABAA receptors. These DBTDs effects reached their
maximum 3 min after application and were: (i) reversible, (ii) concentration-dependent
(with a rank order of potency of 2c = 2d > 2b), (iii) mediated by a non-competitive
antagonism, and (iv) only observed when applied extracellularly. Picrotoxin (which binds
in the channel mouth) and DBTDs effects were not modified when both substances were

Molecules 2013, 18
simultaneous applied. Our results indicate that DBTD acted on the extracellular domain of
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GABAA channels but independent of the picrotoxin, benzodiazepine, and GABA binding
sites. DBTDs used here could be the initial model for synthesizing new GABAA receptor
inhibitors with a potential to be used as antidotes for positive modulators of these receptors
or to induce experimental epilepsy.
Keywords: dibenzothiadiazepines; GABAA receptor antagonists; patch clamp;
neurochemistry; biological activity; enteric neurons; electrophysiology
1. Introduction
The synthesis of tricyclic compounds with a central thiadiazepine ring (see Figure 1, 1 ring B) were first
described by Weber [1], followed by a description of the compounds anti-depressive effects (compound
1a) [2,3]. In 1991, Giannotti et al. [4] prepared DBTD (compound 1b) structural variants at nitrogen 11
(N-11) with the purpose of increasing the antidepressive effects previously observed by Weber, while
reducing possible side effects. In addition to the effects of DBTDs on the central nervous system, these
substances were found to act as non-nucleosidic reverse transcriptase inhibitors of HIV-1
(compound 1c) [5] and to have anti-proliferative activity on leukemia cell lines (compound 1d) [6],
effects that increased the attention toward these tricyclic compounds.
Figure 1. General chemical structure of the dibenzo[c,f][1,2,5]thiadiazepines 1, and several
DBTDs reported to have biological activity (compounds 1a, 1b, 1c, and 1d).
The central ring of DBTDs has been synthesized via the Goldberg method, which involves an
Ullmann intra-molecular condensation reaction (N-C) (Figure 2, route a) [7]. This reaction is limited
by the fact that ortho-haloanilines significantly reduce the number of possible substituents that may be
included in the DBTDs. Only one report has described the use of this methodology for obtaining
compounds 2a and 2d [8]. As an alternative approach, N-C bonds may be formed via intra-molecular
reactions of aryl azides with benzene derivatives (Figure 2, route b), which has been described during
the formation of carbazoles through thermolysis [9], photolysis [10], and recently, via metal

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catalysis [11,12]. The advantage of using aryl azides is that C-N bonds are formed directly. Therefore,
the use of monosubstituted anilines with diverse functional groups can lead us toward obtaining
DBTDs with functional variations in the C ring. Known DBTDs and triheterocyclic analogous
compounds include diverse substituents on the nitrogen of the thiadiazepine ring (Figure 1); however, no
studies have examined the biological activities of the parent compound and 9-substituted derivatives
(Figure 2).
Figure 2. Retrosynthetic analysis of the non-substituted DBTDs. (a) Classical method for
obtaining 2 by the Goldberg methodology; (b) Obtaining 2 from an aryl azide.
This work is the first report to consider a biological study of DBTDs without substituents on
nitrogens 6 and 11 of B ring, which were obtained through a process distinct from the Ullmann
method. The presence of a hydrogen atom at N-6 and N-11 in a DBTD can determine the compound’s
affinity toward proteins via hydrogen bond interactions [13], as shown in Figure 3.
Figure 3. The dotted lines in DBTDs indicate probable interactions between hydrogen
bonds and proteins.

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The linear synthetic route to the DBTDs 2a–g comprises four stages and proceeds as described in
Scheme 1. The thiadiazepine ring is formed through direct amination of the C ring via intramolecular
thermal cyclization of 4 (Figure 2, route b). This methodology provides an alternative to the classical
amination of Goldberg approach, with respect to the formation of ring B in the substituted DBTDs
(Figure 2, route a) [2,4,5,8,14]. DBTDs with a modified B ring were shown to have biological effects.
Giannotti et al. synthesized a series of DBTDs with substituents in the thiadiazepine ring, and they
showed that the compounds displayed a potential antidepressive effect using the apomorphine-induced
hypothermia test [4]. However, these authors found no binding of DBTDs with receptors to dopamine,
serotonin, histamine, benzodiazepine, GABA, acetylcholine, and adrenaline, and reported that DBTDs
lack of effect on serotonin and noradrenaline uptake. However, such observations does not rule out that
DBTDs might be modulating any of these receptor proteins through a different binding site than the
one directly activated by a given agonist or modulator. At least three observations indicated us that
GABAA channels might be the target for DBTDs: (i) the tricyclic sulfonamide 2 is structurally similar
to the 1,4-benzodiazepines, which are major positive modulators of these channels [15]; (ii) DBTDs
have antidepressive actions [4] and (iii) GABAA channels have been implicated in mood disorders,
including depression [16,17]. Therefore, the aim of the present study was to further investigate the
effects of DBTDs on GABAA channels and to report a new synthetic platform for obtaining DBTD
compounds that do not include the thiadiazepine ring substitutions. We found that these compounds
inhibit directly GABAA channels by a mechanism that is independent of the binding sites for GABA,
picrotoxin, and benzodiazepine.
Scheme 1. Reagents and conditions: (a) Anhydrous pyridine, dry acetone, N2(g), 24 h;
(b) SnCl2·2H2O, ethyl acetate, 4 h; (c) first step: NaNO2, F3CCO2H, 1 h; second step: NaN3,
1 h; (d) (C6H5)2O, 208 °C, 5 min. Compound 2g was obtained from 2f: first step: KOH 10%, 1 h.
2. Results and Discussion
2.1. Chemistry
The synthetic route begins with the reaction of 4-substituted-anilines with 2-nitrobenzenesulfonyl
chloride under reflux using pyridine as the base and acetone as solvent. The 2-nitrosulfonamides 5a–f

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were obtained in good yields (66%–88%). The subsequent catalytic hydrogenation of the nitro group
using tin(II) chloride under reflux with ethyl acetate yielded the amines 6a–f in yields above 88%. The
amino compounds were transformed to the corresponding 2-azidobenzensulfonamides through their
diazotization with sodium nitrite in trifluoroacetic acid (note that the transformation of 6e was
accomplished using hydrochloric acid). The in situ substitution of the diazo group with sodium azide
induced conversion to 4a–f with a 78%–87% yield. In the final step, the thiadiazepine formation
reaction proceeded via thermolysis above 208 °C in diphenyl ether, this temperature favoured the
formation of an intermediate nitrene reagent [9]. A direct N-C-type amination of the C ring provided
the DBTDs 2a–f with yields of 67%–85%, except for compound 2f, which was isolated in a yield of
12%. Compound 2g was obtained by basic hydrolysis of 2f after a hydrochloric acid treatment.
2.2. Biological Results
The inhibitory effects of DBTDs on the native GABAA receptors of guinea pig myenteric neurons
were studied here for the first time. The Cl− concentrations outside and inside the neurons were similar,
and a holding potential was −60 mV. At this potential, GABA (0.03–3 mM) induced inward currents
(IGABA) in 86% of myenteric neurons. The amplitude of the currents was concentration-dependent
(EC50 = 115 ± 10 μM) and varied among the different neurons with a range of 0.1–6 nA, in response to
300 μM GABA. Most neurons maintained a stable value of IGABA during repeated GABA applications.
Otherwise, the data were rejected. In order to test that these GABA currents are mediated by GABAA
channels bicuculline (0.1–100 μM) and picrotoxin (3–1,000 μM) were used, inhibitors of these
receptors [18–20]. Both substances inhibited IGABA in a concentration-dependent manner (data not shown)
with an IC50 of 10 ± 2 and 6 ± 1 μM, respectively. Maximal concentrations used for both antagonists
virtually abolished IGABA, as it was previously reported [18–20].
Figure 4 shows that application of DBTDs (100 μM) inhibited the currents induced by GABA
(300 μM) in a time-dependent manner (3–180 s) with time constants (t) of 4.9, 2.6, and 29.3 s for 2b,
2c, and 2d, respectively. These constants were calculated by fitting the data using the
Michaelis–Menten equation (R2 = 0.98, 0.88, and 0.99, respectively). The maximum inhibition induced
by 2b and 2c (100 μM) was reached 3 min after initial exposure. For 2d, the time required to reach the
maximum inhibition was calculated to be 17 min; however, the experimental maximum inhibition
observed after a 3 min treatment was 74.2 ± 2.4% (n = 10), similar to the calculated maximum
inhibition (87.0 ± 3.0%). In all subsequent experiments, a DBTD treatment time of 3 min was used.
The current inhibition induced by 2b, 2c, and 2d was fully reversed within five minutes of washing.
The holding current remained constant in the presence of the compounds at all concentrations tested,
indicating that the compounds could not open the GABAA receptors or any other neuronal ion channel
under the experimental conditions.
Control experiments were conducted using DMSO, the solvent used for all DBTDs, demonstrating
that DMSO did not modify the properties of IGABA alone at the maximum concentration used here,
0.33% V/V (data not shown). The inhibitory activities of the DBTDs were likely to be use-independent
because their effects did not require active GABAA receptors and the inhibitory activity increased over
time, despite the absence of the agonist (GABA) [21]. This indicates that DBTDs bind to these
channels during their closed stage.

Molecules 2013, 18
Figure 4. Halogenated DBTDs inhibited the IGABA in a time-depended manner. (A) IGABA
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was recorded before, during application of 100 μM 2b, and after removal of the inhibitor of
a given myenteric neuron for various lengths of time. The horizontal bars above the traces
indicate the application profiles of the indicated substances. (B) Time course of IGABA
(induced by 300 μM GABA) inhibition induced by 2 over 3–180 s. Michaelis–Menten fits.
Each data point represents the mean value from 3–12 different experiments. The lines
represent the SEM.
The inhibitory effects of seven DBTDs at a 100 μM concentration are listed in Table 1. The data
indicate that the rank order of potency for these inhibitors was 2c = 2d > 2b; we were unable to
calculate the potency of 2f, 2e, 2a, 2g due to solubility problems. Figure 5 shows the
concentration–response curves for the inhibitory effects of three DBTDs (3–1,000 μM) on the currents
induced by GABA (300 μM). The maximum effect of 2b was achieved at 1 mM (inducing a complete
inhibition of the GABA-activated currents), and an IC50 value (104 ± 9.2 μM). We did not reach the
maximum inhibition for 2c and 2d because the compounds were insoluble at concentrations exceeding
300 μM under our experimental conditions. Curve fits were obtained in both cases with IC50 values of
50.4 ± 6.4 μM and 47.6 ± 30.3 μM for 2c and 2d, respectively.
Table 1. Percent inhibition in the presence of 100 μM compounds on GABA-induced
inward currents, p IC50, cLog P, and physical data.
No.
2a
2b
2c
2d
2e
2f
Percentage of inhibition [a]
28.4 ± 1.4 (5)
p IC50/M
ND
cLog P [b]
2.18
Yield/%
69
mp/°C
198
50.8 ± 2.1 (12)
74.2 ± 3.6 (6)
3.98
4.30
4.32
ND
1.50
70
202
2.69
79
248
74.2 ± 2.4 (10)
43.7 ± 3.8 (3)
2.97
85
250
1.92
67
171
47.1 ± 3.7 (7)
ND
2.25
12
227
2g
16.0 ± 2.4 (6)
ND
1.87
100
350
[a] Values are given as the mean ± SEM, with the number of experiments in parentheses; [b] Data generated
using HyperChem.

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Figure 5. DBTDs inhibited IGABA in a concentration-dependent manner. (A) Representative
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IGABA recordings from a myenteric neuron in the presence of various concentrations of 2b,
which was added 3 min before GABA application. (B) Concentration—response curves for
the effects of the DBTDs on the amplitude of IGABA. The lines indicate fits to the
experimental data using a two-parameter logistic function, [22] assuming an inhibition of
100%. Each data point represents the mean ± SEM from 3–12 individual experiments.
We considered the possibility that because these novel substances were highly lipophilic
(log P ~ 2.0 for all compounds, Table 1), the DBTDs could permeate the neuron membrane and
interact with the inner part of the GABAA channel, thereby inhibiting IGABA. To investigate this
possibility, we added 100 μM 2b to the pipette solution (internal) and monitored IGABA, and we applied
2b to the outside of the cells and monitored the inhibitory effects (Figure 6). Experiments were
performed using 2b, even though cLog P was less than 2, because 2c and 2d were insoluble under the
conditions employed. The amplitude of IGABA (300 μM) for neurons with 2b (100 μM) applied inside
(−1408 ± 344 pA; n = 4) and measured 2 to 3 min after obtaining the whole cell configuration did not
differ from the amplitude of the control IGABA (−1510 ± 333 pA; n = 12) of experiments in which 2b
was tested extracellularly. Consistent with these findings, in recordings with 2b in the pipette, the
amplitude of IGABA was the same 5 min (−1536 ± 379 pA) and 10 min (−1604 ± 409 pA; n = 4) after
obtaining the whole-cell configuration. In addition, the presence of 2b inside the neurons did not affect
the magnitude of the inhibition induced by the extracellular application of 2b (100 μM). Thus, such an
inhibition was as large as that observed without 2b inside the cells (Figure 6B). Altogether, these data
rule out that DBTDs inhibitory effects on GABAA channels are mediated by an intracellular target and
therefore, they must be acting on the extracellular domain.
Figure 7 shows two concentration-response curves for the effects of GABA, one in the absence and
the other in the presence of 100 μM 2b. As shown, the antagonistic effect of 2b is not surmounted by
increasing the GABA concentration. Indeed, the EC50 values for these curves were 127 ± 16 and
123 ± 84 μM in the absence and in the presence of 2b, whereas, the maximum inhibition clearly
decreased in the presence of 2b (~50%) across the full GABA concentration–response curve. Our data
demonstrate that the pharmacological antagonism by which 2b inhibits the GABAA receptors is non-
competitive and therefore, it is unlikely acting at the GABA binding site. This is in agreement with a
previous study [4] that shows no binding of DBTDs to GABA receptors.

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Figure 6. The inhibitory effects of 2b on GABAA receptors were mediated by an
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extracellular binding site. A) IGABA for a 100 μM concentration of 2b in the pipette (2b-/i),
10 min after obtaining the whole cell. IGABA was recorded before (-/i) and in presence of
extracellular (o/i) 2b (100 μM for 3 min). B) Bars indicate the average amplitude of IGABA
,
and the lines above indicate the SEM. IGABA amplitude or the inhibitory effect of 2b did not
differ significantly (NS) by the presence of 2b inside the pipette. Statistical comparison of
the data was done using the unpaired Student’s t-test.
Figure 7. 2b inhibits IGABA in a non-competitive manner. (A) Concentration–response
curves for GABA in the absence (Control) and in the presence of 2b. Responses were
normalized with respect to the curves obtained in the presence of 3 mM GABA in each cell
and in the absence of 2b. Each point represents the mean ± SEM for 5–12 neurons. The
lines indicate fits of experimental data to a three-parameter logistic function.
We further investigated if the inhibitory effect of DBTD on IGABA was voltage dependent by
conducting experiments at two holding membrane potentials, −60 mV and +40 mV in the same
neurons. IGABA (300 μM) was recorded in the absence or presence of 100 μM 2b, 2c, and 2d. As shown
in Figure 8, the inhibitory effects induced by any of the three substances were identical for an inward
IGABA (recorded at −60 mV) than for the outward IGABA (recorded at +40 mV). These results indicate
that the DBTDs inhibit IGABA via a voltage-independent mechanism.

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Figure 8. The inhibitory effects of compounds 2 on GABAA channels were voltage
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independent. A) IGABA induced by 300 μM GABA without (Control) or in presence of 2c
(100 μM) at −60 mV (upper traces) and +40 mV (lower traces) from the same neuron.
I
GABA was recorded at 5 min intervals, and 2c was applied 3 min before the second GABA
application. B) The average (bars) inhibitory effect of 2b, 2c, and 2d was the same at both
membrane potentials. Lines over the bars indicate the SEM.
The fact that binding of compounds 2 on GABAA channels can occur during the close stage and is
voltage-independent suggests that its binding site is not within the channel pore. In order to further
investigate this, we tested if picrotoxin interacts with the binding of 2c. Picrotoxin is known to bind
into a site within the channel formed by the second transmembrane domains of the five subunits
constituting the GABAA receptors [23,24]. We found that neither picrotoxin effect was modified by 2c
nor the inhibitory effect of 2c was affected by picrotoxin (Figure 9), which would indicate that 2c does
not bind into the picrotoxin binding site.
Figure 9. 2c-induced inhibition of GABAA channels was unaffected by picrotoxin (PTx).
Bars and lines on their top, are means and SEM (n = 4). Statistical comparison of each pair
of bars was done using the paired Student’s t-test. P values and non significant (NS)
differences are indicated.

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903
The tricyclic sulfonamide 2 is also structurally similar to the 1,4-benzodiazepines, hence, the
inhibitory actions of DBTDs may be related to the benzodiazepine modulator sites. The inhibitory
effects of 2b, 2c, and 2d remained constant in the absence and presence of flumazenil, a known
antagonist of the benzodiazepine site on the GABAA receptors (Figure 10). Inhibition by 100 μM 2b,
2c, and 2d without flumazenil (50.8 ± 2.1%, 74.2 ± 3.6%, 74.2 ± 2.4%, respectively) was not blocked
by the co-application of 10 μM flumazenil (47.8 ± 5.8%, 69.6 ± 5.2%, 68.4 ± 6.8%, respectively). The
same concentration of flumazenil, applied for 3 min, did not induce changes in the holding current or
I
GABA in experiments carried out using five different neurons (data not shown). We showed that 2b did
not act through the benzodiazepine binding site, which is in agreement with the lack of binding with
receptors to benzodiazepine, previously reported [4].
Figure 10. The inhibitory effects of compounds 2 on the GABAA channels were
independent of the benzodiazepine binding site. (A) First and last traces represent control
currents induced by GABA (300 μM). The two middle traces were recorded in 2d
(100 μM) alone or plus flumazenil (10 μM), all traces are from the same neuron. (B) Each
pair of bars represents the mean inhibition of IGABA induced by DBTDs, before (Control)
and in the presence of flumazenil. Lines over the bars indicate the SEM.
3. Experimental
3.1. Chemistry
3.1.1. General
All reagents and solvents were reagent-grade and were used as received from Sigma-Aldrich Co.
(St. Louis, MO, USA). Flash chromatography was performed using Merk Kiesegel 60 silica gel
(230–400 mesh). Melting points are reported uncorrected. The FTIR spectra were recorded on a
Thermo Nicolet Nexus 470 FTIR as thin films on a KBr disk (for solids) or a germanium ATR crystal
(for liquids). 1H-NMR and 13C-NMR spectra were obtained using an Eclipse Jeol (operating at 300 and
75 MHz, respectively) and a Varian-Gemini (operated at 200 MHz and 50 MHz, respectively), and the
signals are reported in ppm relative to TMS. All mass spectra (MS) were recorded on a Jeol AX505HA
mass spectrometer. Elemental analyses were performed on a CE-440 Exeter Analytical Inc.

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3.1.2. General Procedures for Synthesizing N-(4-(R)-Phenyl)-2-nitrobenzenesulfonamides 5a–f
Anhydrous pyridine (1.10 mL, 13.6 mmol) and 4-R-aniline (1.24 mL, 13.6 mmol) in dry acetone
were added, via cannula, to a stirred solution of 2-nitrobenzenesulfonyl chloride (3.01 g, 13.6 mmol) in
dry acetone (25 mL) under nitrogen, and the reaction mixture was stirred at room temperature. After
24 h the mixture was neutralized with a saturated sodium bicarbonate solution, and the resulting solid
was collected, washed with water and ethanol, and dried under vacuum. The solid was purified by
flash chromatography (silica gel, eluting with 90:10 hexane–ethyl acetate), then recrystallized from
ethyl acetate–hexane (30:70) to obtain 5a as a white solid (11.97 mmol, 88%); m.p.: 118–119 °C. The
same procedure was used for the synthesis of 5b–5f. Products 5, 6, and 4 (except for series f) were first
reported by Saeed and Rama [25]. However, 5 and 6 compounds were not characterized and 4 was
only partially characterized by IR and MS spectroscopies.
1
N-(Phenyl)-2-nitrobenzenesulfonamide (5a). H-NMR (300 MHz, CDCl3 + DMSO-d6): δ = 7.08 (m,
1H), 7.21 (m, 4H), 7.63 (ddd, Jo = 7.5 Hz, Jm = 1.6 Hz, 1H), 7.70 (ddd, Jo = 7.5 Hz, Jm = 1.6 Hz, 1H),
7.76 (dd, Jo = 8.0 Hz, Jm = 1.6 Hz, 1H), 7.92 (dd, Jo = 7.9 Hz, Jm = 1.6 Hz, 1H), 9.9 ppm (s, 1H); IR
(KBr): ν = 3324, 1380, 1180 cm−1; MS (EI, 70 eV): m/z: 278 [M]+.
N-(4-Fluorophenyl)-2-nitrobenzenesulfonamide (5b). Yellow crystals. Yield 79%; m.p.: 106 °C;
1H-NMR (200 MHz, DMSO-d6): δ = 7.13 (d, Jo = 6.8 Hz, 4H), 7.88 (m, 4H), 10.7 ppm (s, 1H); IR
(KBr): ν = 3293, 1360, 1160 cm−1; MS (EI, 70 eV): m/z: 296 [M]+.
N-(4-Chlorophenyl)-2-nitrobenzenesulfonamide (5c). Yellow crystals. Yield 66%; m.p.: 122 °C;
1H-NMR (200 MHz, DMSO-d6): δ = 7.13 (d, Jo = 9.0 Hz, 2H), 7.35 (d, Jo = 9.0 Hz, 2H), 7.89 (m, 4H),
10.9 ppm (s, 1H); IR (KBr): ν = 3309, 1334, 1162 cm−1; MS (EI, 70 eV): m/z: 312 [M]+.
N-(4-Bromophenyl)-2-nitrobenzenesulfonamide (5d). Colorless crystals. Yield 79%; m.p.:
118 °C; 1H-NMR (200 MHz, DMSO-d6): δ = 7.06 (d, Jo = 9.0 Hz, 2H), 7.47 (d, Jo = 8.8 Hz, 2H), 7.91
(m, 4H), 10.9 ppm (s, 1H); IR (KBr): ν = 3297, 1363, 1164 cm−1. MS (EI, 70 eV): m/z: 358/356 [M]+.
N-(4-Methoxyphenyl)-2-nitrobenzenesulfonamide (5e). Yellow needle crystals. Yield 72%; m.p.:
90 °C; 1H-NMR (200 MHz, DMSO-d6): δ = 3.67 (s, 3H), 6.83 (d, Jo = 9.0 Hz, 2H), 7.03 (d, Jo = 9.0 Hz,
2H), 7.87 (m, 4H), 10.4 ppm (s, 1H); IR (KBr): ν = 3259, 1361, 1172 cm−1; MS (EI, 70 eV): m/z: 308 [M]+.
Ethyl-4-(2-nitrophenylsulfonamido)benzoate (5f). Brown solid. Yield 82%; m.p.: 172 °C;
1H-NMR (300 MHz, DMSO-d6): δ = 1.25 (t, Jo = 7.1 Hz, 3H), 4.22 (q, Jo = 7.1 Hz, 2H), 7.13 (BB',
Jo = 8.7 Hz, 2H), 7.75 (ddd, Jo = 7.5 Hz, 1H), 7.78 (AA', Jo = 8.7 Hz, 2H), 7.79 (ddd, Jo = 7.5 Hz, 1H),
7.91 (dd, Jo = 6.6 Hz, 1H), 7.98 ppm (dd, Jo = 7.0 Hz, 1H); IR (KBr): ν = 3200, 1690, 1365,
1162 cm−1; MS (EI, 70 eV): m/z: 350 [M]+.
3.1.3. General Procedures for the Synthesis of 2-Amino-N-(4-(R) phenyl)benzenesulfonamides 6a–f
N-(4-(R) phenyl)-2-nitrobenzenesulfonamide 5 (4.3 g, 15.6 mmol) and tin (II) chloride dehydrate
(14.82 g, 65.7 mmol) were heated in ethyl acetate under reflux for 4 h. The mixture was stirred, and a
saturated sodium bicarbonate solution was added to a pH of 6. The solution was extracted with ethyl

Molecules 2013, 18
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acetate. The solvent was removed, and the residue was purified by flash chromatography (eluting with
a 90:10 solution of hexane–ethyl acetate).
2-Amino-N-phenylbenzenesulfonamide (6a). Yellow powder (14.04 mmol, 90%); m.p.: 123–124 °C;
1H-NMR (300 MHz, DMSO-d6): δ = 5.99 (s, 2H), 6.54 (ddd, Jo = 7.6 Hz, Jm = 1.2 Hz, 1H), 6.75 (dd,
Jo = 8.1 Hz, Jm = 0.9 Hz, 1H), 6.97 (ddd, Jo = 8.2 Hz, 1H), 7.04 (dd, Jo = 8.6 Hz, 2H), 7.2 (m, 3H),
7.49 (dd, Jo = 8.5 Hz, Jm = 1.4 Hz, 1H), 10.2 ppm (s, 1H); IR (KBr): ν = 3457, 3368, 3240, 1360,
1180 cm−1; MS (EI, 70 eV): m/z: 248 [M]+.
2-Amino-N-(4-fluorophenyl) benzenesulfonamide (6b). Brown liquid. Yield 99%; 1H-NMR (200 MHz,
DMSO-d6): δ = 5.98 (s, 2H), 6.53 (ddd, Jo = 7.05 Hz, Jm = 1.2 Hz, 1H), 6.74 (dd, Jo = 8.3 Hz,
Jm = 1.2 Hz, 1H), 7.05 (d, Jo = 6.4 Hz, 4H), 7.21 (ddd, Jo = 7.0 Hz, Jm = 1.6 Hz, 1H), 7.43 (dd, Jo = 8.2 Hz,
Jm = 1.6 Hz, 1H), 10.2 ppm (s, 1H); IR (KBr): ν = 3469, 3382, 3284, 1313, 1147 cm−1; MS (EI, 70 eV):
m/z: 266 [M]+.
2-Amino-N-(4-chlorophenyl) benzenesulfonamide (6c). Brown liquid. Yield 99%; 1H-NMR (200 MHz,
DMSO-d6 ): δ = 6.0 (s, 2H), 6.53 (ddd, Jo = 8.0 Hz, Jm = 1.1, 1H), 6.73 (dd, Jo = 8.3 Hz, Jm = 0.9 Hz,
1H), 7.03 (d, Jo = 8.8 Hz, 2H), 7.2 (ddd, Jo = 8.5 Hz, Jm = 1.6, 1H), 7.26 (d, Jo = 8.8 Hz, 2H), 7.47 (dd,
Jo = 8.0 Hz, Jm = 1.6, 1H), 10.4 ppm (s, 1H); IR (KBr): ν = 3467, 3378, 3245, 1313, 1135 cm−1;
MS (EI, 70 eV): m/z: 282 [M]+.
2-Amino-N-(4-bromophenyl) benzenesulfonamide (6d). Brown liquid. Yield 99%; 1H-NMR (200 MHz,
DMSO-d6): δ = 6.0 (s, 2H), 6.55 (ddd, Jo = 7.8 Hz, 1H), 6.76 (dd, Jo = 8.3 Hz, Jm = 0.9 Hz, 1H), 7.0 (d,
Jo = 8.8 Hz, 2H), 7.21 (ddd, Jo = 7.0 Hz, Jm = 1.6 Hz, 1H), 7.39 (d, Jo = 8.8 Hz, 2H), 7.49 (dd,
Jo = 7.9 Hz, Jm = 1.5 Hz, 1H), 10.4 ppm (s, 1H); IR (KBr): ν = 3482, 3384, 3268, 1319, 1139 cm−1; MS
(EI, 70 eV): m/z: 328/326 [M]+.
2-Amino-N-(4-methoxyphenyl) benzenesulfonamide (6e). Brown liquid. Yield 99%; 1H-NMR
(200 MHz, DMSO-d6): δ = 3.65 (s, 3H), 5.9 (s, 2H), 6.5 (ddd, Jo = 7.7 Hz, Jm = 1.2 Hz, 1H), 6.72 (dd,
Jo = 8.7 Hz, 1H), 6.77 (d, Jo = 9.0 Hz, 2H), 6.95 (d, Jo = 9.0 Hz, 2H), 7.19 (ddd, Jo = 8.2 Hz,
Jm = 1.6 Hz, 1H), 7.36 (dd, Jo = 8.1 Hz, Jm = 1.6 Hz, 1H), 9.84 ppm (s, 1H); IR (KBr): ν = 3480, 3380,
3266, 1321, 1147 cm−1; MS (EI, 70 eV): m/z: 278 [M]+.
Ethyl-4-(2-aminophenylsulfonamido)benzoate (6f). Yellow crystals. Yield 88%; m.p.:163 °C; 1H-NMR
(200 MHz, DMSO-d6): δ = 1.25 (t, Jo = 6.3 Hz, 3H), 4.22 (q, Jo = 7.0 Hz, 2H), 6.0 (s, 2H), 6.56 (ddd,
Jo = 7.6 Hz, 1H), 6.73 (dd, Jo = 7.8 Hz, 1H), 7.14 (BB', Jo = 8.8 Hz, 2H), 7.22 (ddd, Jo = 7.7 Hz, 1H),
7.57 (dd, Jo = 8.1 Hz, Jm = 1.6 Hz, 1H), 7.79 (AA', Jo = 8.8 Hz, 2H), 10.8 ppm (s, 1H); IR (KBr):
ν = 3470, 3380, 3230, 1690, 1322, 1144 cm−1; MS (EI, 70 eV): m/z: 320 [M]+.
3.1.4. General Procedures for Synthesizing 2-Azido-N-(4-(R)phenyl)benzenesulfonamides 4a–f
An aqueous solution of sodium nitrite (3.6 g, 51.8 mmol) was added to 2-amino-N-(4-
(R)phenyl)benzenesulfonamide 6 (2.86 g, 11.5 mmol) in trifluoroacetic acid, and the reaction mixture
was stirred for 1 h. Sodium azide (1.9 g, 28.8 mmol) was added, and the solution was stirred for an

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additional 1 h. The mixture was neutralized with saturated sodium bicarbonate solution and extracted
with ethyl acetate. The reaction mixture was concentrated and purified by flash chromatography
(70:30, hexane–ethyl acetate). The obtained solid was recrystallized in ethyl acetate–hexane (30:70).
1
2-Azido-N-phenylbenzenesulfonamide (4a). Brown powder (9.78 mmol, 85%); m.p.:139 °C; H-NMR
(200 MHz, DMSO-d6): d = 6.97 (ddd, Jo = 7.0 Hz, 1H), 7.1 (dd, Jo = 7.4 Hz, 2H), 7.2 (ddd, Jo = 7.4 Hz,
2H), 7.28 (ddd, Jo = 7.6 Hz, 1H), 7.5 (dd, Jo = 8.1 Hz, 1H), 7.64 (ddd, Jo = 7.8 Hz, 1H), 7.86 (dd,
Jo = 7.8 Hz, 1H), 10.4 ppm (s, 1H); IR (KBr): ν = 3253, 2133, 1340, 1190 cm−1; MS (EI, 70 eV): m/z:
274 [M]+.
2-Azido-N-(4-fluorophenyl) benzenesulfonamide (4b). White solid. Yield 83%; m.p.: 129 °C; 1H-NMR
(200 MHz, DMSO-d6): δ = 7.09 (m, 4H), 7.27 (ddd, Jo = 7.3 Hz, Jm = 1.4 Hz, 1H), 7.52 (dd,
Jo = 8.0 Hz, Jm = 1.0 Hz, 1H), 7.62 (ddd, Jo = 8.4 Hz, Jm = 1.6 Hz, 1H), 7.8 (dd, Jo = 7.8 Hz,
Jm = 1.6 Hz, 1H), 10.2 ppm (s, 1H); IR (KBr): ν = 3249, 2140, 1334, 1166 cm−1; MS (EI, 70 eV): m/z:
292 [M]+.
2-Azido-N-(4-chlorophenyl) benzenesulfonamide (4c). White crystals. Yield 80%; m.p.: 134 °C;
1H-NMR (200 MHz, DMSO-d6): δ = 7.11 (d, Jo = 8.8 Hz, 2H), 7.27 (d, Jo = 8.8 Hz, 2H), 7.31 (ddd,
Jm = 1.1 Hz, 1H), 7.51 (dd, Jo = 8.0 Hz, Jm = 1.4 Hz, 1H), 7.66 (ddd, Jo = 7.7 Hz, Jm = 1.6 Hz, 1H),
7.86 (dd, Jo = 7.8 Hz, Jm = 1.4 Hz, 1H), 10.5 ppm (s, 1H); IR (KBr): ν = 3345, 2132, 1338, 1164 cm−1;
MS (EI, 70 eV): m/z: 308 [M]+.
2-Azido-N-(4-bromophenyl) benzenesulfonamide (4d). Yellow powder. Yield 87%; m.p.:
1
124 °C; H-NMR (200 MHz, DMSO-d6): δ = 7.05 (d, Jo = 8.8 Hz, 2H), 7.29 (ddd, Jo = 7.0 Hz,
Jm = 1.2 Hz, 1H), 7.4 (d, Jo = 8.8 Hz, 2H), 7.52 (dd, Jo = 8.0 Hz, Jm= 1.2 Hz, 1H), 7.67 (ddd,
Jo = 6.8 Hz, Jm = 1.4 Hz, 1H), 7.86 (dd, Jo = 7.8 Hz, Jm = 1.6 Hz, 1H), 10.5 ppm (s, 1H); IR (KBr):
ν = 3338, 2132, 1338, 1164 cm−1; MS (EI, 70 eV): m/z: 354/352 [M]+.
2-Azido-N-(4-methoxyphenyl) benzenesulfonamide (4e). Brown crystals. Yield 78%; m.p.:
130 °C; 1H-NMR (200 MHz, DMSO-d6): δ = 3.63 (s, 3H), 6.76 (d, Jo = 9.3 Hz, 2H), 7.01 (d, Jo = 8.7 Hz,
2H), 7.23 (ddd, Jo = 7.4, Jm = 1.1 Hz, 1H), 7.51 (dd, Jo = 7.9 Hz, Jm = 1.1 Hz, 1H), 7.62 (ddd,
Jo = 7.8 Hz, Jm = 1.6 Hz, 1H), 7.73 (dd, Jo = 7.9 Hz, Jm = 1.2 Hz, 1H), 9.87 ppm (s, 1H); IR (KBr):
ν = 3274, 2138, 1336, 1164 cm−1; MS (EI, 70 eV): m/z: 304 [M]+.
Ethyl-4-(2-azidophenylsulfonamido) benzoate (4f). Brown crystals. Yield 78%; m.p.:
182–184 °C; 1H-NMR (200 MHz, DMSO-d6): δ = 1.23 (t, Jo = 7.0 Hz, 3H), 4.20 (q, Jo = 7.1 Hz, 2H),
7.19 (BB', Jo = 8.7 Hz, 2H), 7.30 (ddd, Jo = 7.8 Hz, 1H), 7.48 (dd, Jo = 8.1 Hz, 1H), 7.65 (ddd, Jo = 7.8 Hz,
1H), 7.77 (AA', Jo = 8.7 Hz, 2H), 7.94 (dd, Jo = 7.8 Hz, 1H), 10.9 ppm (s, 1H); IR (KBr): ν = 3230,
2110, 1690, 1300, 1165 cm−1; MS (EI, 70 eV): m/z: 346 [M]+.

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3.1.5. General Procedures for Synthesizing 9-(R)-6,11-Dihydrodibenzo[c,f][1,2,5]thiadiazepine-5,5-
dioxides 2a–f
2-Azido-N-(4-(R)phenyl) benzenesulfonamide 4 (0.2 g, 0.73 mmol) was added to a solution of diphenyl
ether (10 mL, 63 mmol) at 208 °C. The solution was stirred for 5 min then cooled to room temperature.
The reaction mixture was purified by flash chromatography (70:30, ethyl acetate–hexane). The resulting
residue was recrystallized in ethyl acetate–hexane (30:70). Compounds 2a and 2d were previously
reported by Altamura et al. [8], and spectroscopic data are in agreement with those reported here.
6,11-Dihydrodibenzo[c,f][1,2,5]thiadiazepine-5,5-dioxide (2a). Brown crystals (0.50 mmol, 69%);
m.p.: 198 °C; 1H-NMR (300 MHz, DMSO-d6): δ = 6.82 (ddd, Jo = 7.8 Hz, Jm = 0.9 Hz, 1H), 6.88 (dd,
Jo = 7.4 Hz, Jm = 1.5 Hz, 1H), 7.03 (ddd, Jo = 7.7 Hz, Jm = 1.5 Hz, 1H), 7.07 (dd, Jo = 8.1 Hz, Jm = 1.5 Hz,
1H), 7.14 (ddd, Jo = 7.5 Hz, Jm = 1.5 Hz, 1H), 7.18 (dd, Jo = 8.2 Hz, Jm = 1.0 Hz, 1H), 7.37 (ddd,
Jo = 7.7 Hz, Jm = 1.6 Hz, 1H), 7.61 (dd, Jo = 8.0 Hz, Jm = 1.6 Hz, 1H), 8.98 (s, 1H), 9.80 ppm (s, 1H);
13C-NMR (75 MHz, DMSO-d6): δ = 117.5, 119.5, 119.6, 121.1, 125.2, 125.9, 127.3, 128.4, 128.8,
132.9, 139.5, 139.9 ppm; IR (KBr): ν = 3380, 3301, 1313, 1160 cm−1; MS (EI, 70 eV): m/z: 246 [M]+;
Anal. calculated for C12H10N2O2S: C 58.52%, H 4.09%, N 11.37%, found: C 58.11%, H 4.11%, N 11.13%.
9-Fluoro-6,11-dihydrodibenzo[c,f][1,2,5]thiadiazepine-5,5-dioxide (2b). Colorless needle crystals.
1
Yield 70%; m.p.: 202 °C; H-NMR (300 MHz, DMSO-d6): δ = 6.69 (ddd, Jo= 8.2 Hz,
JmH-F = 3.0 Hz, 1H), 6.86 (dd, Jo = 7.2 Hz, 1H), 6.89 (d, Jm = 2.7 Hz, 1H), 7.05 (dd, Jo = 7.5 Hz, 1H),
7.16 (dd, Jo = 8.1 Hz, Jm = 0.6 Hz, 1H), 7.41 (ddd, Jo = 7.6 Hz, Jm = 1.5 Hz, 1H), 7.63 (dd, Jo = 8.0 Hz,
13
Jm = 1.8 Hz, 1H), 9.14 (s, 1H), 9.78 ppm (s, 1H); C-NMR (75 MHz, DMSO-d6): δ = 105.6, 107.6,
119.0, 121.4, 126.1, 129.3, 130.4, 133.1, 139.2, 141.3, 159.2, 162.5 ppm; IR (KBr): ν = 3365, 3226,
1295, 1159 cm−1; MS (EI, 70 eV): m/z: 264 [M]+; Anal. calculated for C12H9N2O2SF: C 54.54%,
H 3.43%, N 10.60%, found: C 54.05%, H 3.32%, N 10.34%.
9-Chloro-6,11-dihydrodibenzo[c,f][1,2,5]thiadiazepine-5,5-dioxide (2c). White powder. Yield 79%;
m.p.: 248 °C; 1H-NMR (300 MHz, DMSO-d6): δ = 6.87 (ddd, Jo = 7.8 Hz, Jm = 1.2 Hz, 1H), 6.88 (dd,
Jo = 8.4 Hz, Jm = 2.1 Hz, 1H), 7.01 (d, Jo = 8.4 Hz, 1H), 7.13 (d, Jm = 2.1 Hz, 1H), 7.15 (dd,
Jo = 8.1 Hz, Jm = 0.6 Hz, 1H), 7.41 (ddd, Jo = 7.7 Hz, Jm = 1.5 Hz, 1H), 7.62 (dd, Jo = 8.0 Hz,
13
Jm = 1.6 Hz, 1H), 9.11 (s, 1H), 9.95 ppm (s, 1H); C-NMR (75 MHz, DMSO-D6): δ = 118.3, 118.7,
119.7, 120.4, 124.2, 125.8, 129.2, 129.5, 131.1, 133.3, 139.2, 140.5 ppm; IR (KBr): ν = 3369, 3269,
1304, 1156cm−1; MS (EI, 70 eV): m/z: 280 [M]+; Anal. calculated for C12H9N2O2SCl: C 51.34%,
H 3.23%, N 9.98%, found: C 51.03%, H 3.22%, N 9.81%.
9-Bromo-6,11-dihydrodibenzo[c,f][1,2,5]thiadiazepine-5,5-dioxide (2d). Brown powder. Yield 85%;
m.p.: 250 °C; 1H-NMR (300 MHz, DMSO-d6): δ = 6.87 (ddd, Jo = 7.4 Hz, 1H), 6.94 (d, Jo = 8.4 Hz, 1H),
7.01 (dd, Jo = 8.2 Hz, Jm = 2.0 Hz, 1H), 7.15 (d, Jo = 8.4 Hz, 1H), 7.28 (d, Jm = 1.8 Hz, 1H), 7.41 (ddd,
Jo = 7.8 Hz, 1H), 7.62 (dd, Jo = 7.8 Hz, Jm = 1.2 Hz, 1H), 9.10 (s, 1H), 9.96 ppm (s, 1H);
13C-NMR (75 MHz, DMSO-d6): δ = 118.3, 119.2, 119.7, 121.6, 123.3, 124.6, 125.8, 129.2, 129.7,
133.3, 139.2, 140.7 ppm; IR (KBr): ν = 3371, 3268, 1308, 1160 cm−1; MS (EI, 70 eV): m/z: 326/324

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[M]+; Anal. calculated for C12H9N2O2SBr: C 44.32%, H 2.79%, N 8.61%, found: C 44.09%, H 2.81%,
N 8.74%.
9-Methoxy-6,11-dihydrodibenzo[c,f][1,2,5]thiadiazepine-5,5-dioxide (2e). Yellow crystals. Yield
1
67%; m.p.: 171 °C; H-NMR (300 MHz, DMSO-d6): δ = 3.73 (s, 3H), 6.49 (dd, Jo = 8.7 Hz,
Jm = 2.7 Hz, 1H), 6.66 (d, Jm = 2.7 Hz, 1H), 6.83 (ddd, Jo = 7.5 Hz, Jm = 0.9 Hz, 1H), 6.96 (d, Jo = 8.7 Hz,
1H), 7.17 (d, Jo = 7.8 Hz, 1H), 7.37 (ddd, Jo = 7.7 Hz, Jm = 1.5 Hz, 1H,), 7.62 (dd, Jo = 7.8 Hz, Jm = 1.5 Hz,
13
1H), 8.99 (s, 1H), 9.51 ppm (s, 1H); C-NMR (75 MHz, DMSO-D6): δ = 55.2, 104.2, 107.4, 117.7,
118.1, 119.5, 126.3, 129.0, 130.4, 132.8, 139.7, 141.2, 158.6 ppm; IR (KBr): ν = 3374, 3228, 1322,
1149 cm−1; MS (EI, 70 eV): m/z: 276 [M]+; Anal. calculated for C13H12N2O3S: C 56.51%, H 4.38%,
N 10.14%, found: C 56.56%, H 4.40%, N 9.89%.
Ethyl 6,11-dihydrodibenzo[c,f][1,2,5]thiadiazepine-9-carboxylate-5,5-dioxide (2f). Brown crystals.
1
Yield 12%; m.p.: 227 °C; H-NMR (300 MHz, DMSO-d6): δ = 1.31 (t, Jo = 7.0 Hz, 3H), 4.30 (q,
Jo = 7.2 Hz, 2H), 6.85 (ddd, Jo = 7.5 Hz, 1H), 7.07 (d, Jo = 8.1 Hz, 1H), 7.19 (d, Jo = 7.8 Hz, 1H), 7.40
(dd, Jo = 8.1 Hz, 1H), 7.40 (ddd, Jo = 7.6 Hz, 1H), 7.61 (dd, Jo = 7.8 Hz, Jm = 1.5 Hz, 1H), 7.73 (d,
13
Jm = 1.8 Hz, 1H), 9.20 (s, 1H), 10.3 ppm (s, 1H); C-NMR (75 MHz, DMSO-d6): δ = 14.2, 60.7,
118.1, 119.6, 120.4, 121.3, 125.3, 126.8, 128.2, 128.9, 129.6, 133.4, 138.3, 139.6, 165.1 ppm; IR
(KBr): ν = 3360, 3234, 1700, 1328, 1170 cm−1; MS (EI, 70 eV): m/z: 318 [M]+; Anal. calculated for
C15H14N2O4S: C 56.59%, H 4.43%, N 8.80%, found: C 56.67%, H 4.44%, N 8.64%.
3.1.6. Procedure for Synthesizing 6,11-Dihydrodibenzo[c,f][1,2,5]thiadiazepine-9-carboxylic acid 5,5-
dioxide (2g)
A solution of potassium hydroxide 10% (w/v) was added to ethyl 6,11-dihydrodibenzo
[c,f][1,2,5]thiadiazepine-9-carboxylate-5,5-dioxide (2f, 0.5 g, 1.57 mmol). The reaction mixture was
heated under reflux and stirred for 60 min, after which a chloride acid solution was added to a pH of 6.
The resulting solid was collected, washed with water, and dried under vacuum. The resulting yellow
1
solid residue was obtained in a quantitative yield; m.p.: 350 °C; H-NMR (300 MHz, DMSO-d6):
δ = 6.84 (ddd, Jo = 7.6 Hz, Jm = 1.0 Hz, 1H), 7.05 (d, Jo = 8.1 Hz, 1H), 7.18 (d, Jo = 8.1 Hz, 1H), 7.39
(dd, Jo = 8.1 Hz, Jm = 1.8 Hz, 1H), 7.39 (ddd, Jo = 7.6 Hz, Jm = 1.8 Hz, 1H), 7.61 (dd, Jo = 8.0 Hz,
13
Jm = 1.6 Hz, 1H), 7.72 (d, Jm = 1.8 Hz, 1H), 9.15 (s, 1H), 10.7 ppm (s, 2H); C-NMR (75 MHz,
DMSO-d6): δ = 118.0, 119.6, 120.7, 121.6, 125.4, 126.8, 128.9, 129.3, 129.4, 133.4, 138.3, 139.7,
166.7 ppm; IR (KBr): ν = 3360, 3234, 1700, 1328 cm−1; MS (EI, 70 eV): m/z: 290 [M]+; Anal.
calculated for C13H10N2O4S: C 53.79%, H 3.47%, N 9.65%, found: C 53.73%, H 3.47%, N 9.28%.
3.2. Biological Methods
3.2.1. Primary Cultures of the Myenteric Neurons
Guinea pigs (100–200 g; either male or female) were sacrificed by cervical dislocation and carotid
exsanguination. These methods have been approved by the Animal Care Committee of the IPICYT and
are in agreement with the published Guiding Principles in the Care and Use of Animals, approved by
the American Physiological Society. A segment of ~10 cm of the jejunum was removed and placed in

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a modified Krebs solution (in mM: NaCl, 126; NaH2PO4, 1.2; MgCl2, 1.2; CaCl2, 2.5; KCl, 5;
NaH2CO3, 25; glucose, 11. The sample was gassed under 95% O2 and 5% CO2) and opened
longitudinally. A dissecting microscope was used to dissect the mucosa and submucosa layers prior to
removing most of the circular muscle layer, leaving the myenteric plexus embedded in a longitudinal layer.
The cell isolation procedure has been described elsewhere [26]. The myenteric preparation was
dissociated by sequential treatment with two enzymatic solutions: the first solution contained papain
(0.01 mL·mL−1 activated with 0.4 mg·mL−1 L-cysteine), and the second solution contained collagenase
(1 mg·mL−1) and dispase (4 mg·mL−1). The enzymes were removed by washing the neurons with L15
medium, and the neurons were placed on round coverslips coated with sterile rat-tail collagen. The
culture medium was varied from minimal medium to essential medium 97.5% containing 2.5% guinea
pig serum, 2 mM L-glutamine, 10 U·mL−1 penicillin, 10 μg·mL−1 streptomycin, and 15 mM glucose.
3.2.2. Whole-Cell Recordings of the Membrane Currents Induced by GABA
To reduce the effects of the membrane currents other than those mediated by the activation of
LGIC, experiments were conducted in the presence of Cs+ (a potassium channel blocker). This was
important because GABA modulates the membrane ion channels of the central neurons (enteric
neurons) via G-protein linked receptors [27–29]. Membrane currents induced by GABA were recorded
using a Gene Clamp 500B amplifier (Molecular Devices, CA, USA). The holding potential was
−60 mV (unless otherwise stated), and the short-term (4–50 h) primary cultures of the myenteric
neurons were used to prevent space-clamp problems due to neurite growth. Glass pipettes with a
resistance of 2–5 M were prepared as described previously . This low resistance and slight
suction inside the pipette during the recordings maintained a low series resistance (around 6 M).
All experiments were conducted using standard solutions with the following compositions (in mM);
inside the pipette: CsCl, 160; EGTA, 10; HEPES, 5; NaCl, 10; ATPMg, 3 and GTP, 0.1; external
solution: NaCl, 160; CaCl2, 2; glucose, 11; HEPES, 5 and CsCl, 3. The pH of all solutions was
adjusted to 7.3–7.4 using either CsOH (pipette solution) or NaOH (external solution). The seal
resistance in the whole-cell mode ranged from 1 to 10 G. The whole-cell current data were recorded
on a PC using the AxoScope software (Axon Instruments, Inc.) and were analyzed using the
AXOGRAPH software (Molecular Devices, CA, USA). The recording chamber was superfused with
an external solution at ~2 mL·min−1. The solution around the neuron was quickly exchanged during
recordings using an eight-tube device. Each tube was connected to a syringe (10 mL) containing either
the control or the experimental solution. A control tube was positioned ~300 μm in front of the
recorded neuron, and substances were applied externally by abruptly interchanging the tube for another
tube containing the control solution plus the drug(s). Desensitization of the GABAA receptors was
prevented by applying GABA at intervals of at least 5 min, in between cells were continuously
superfused with extracellular solution. Experimental substances were removed by returning to the
control solution. External solutions were applied by gravity, and the height of the syringes was
continuously adjusted to minimize changes in the flow rate. The experiments were performed at room
temperature (24 ± 1 °C).

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3.2.3. Solutions and Reagents
L15 medium, minimum essential medium, Hanks solution, penicillin-streptomycin, and L-glutamine
were purchased from GIBCO (Life Technologies Corp., Carlsbad, CA, USA). Collagenase and papain
were purchased from Worthington (Worthington Biochemical Corp., Lakewood, NJ, USA), and
dispase was purchased from Roche (Indianapolis, IN, USA). Cesium chloride, sodium chloride, ethylene
glycol-bis(2-aminoethylether)-N,N,N',N'-tetra-acetic acid (EGTA), HEPES, adenosine-5'-triphosphate
magnesium salt (ATP magnesium salt), guanosine-5'-triphosphate sodium salt (GTP sodium salt),
cesium hydroxide, flumazenil, GABA, picrotoxin, and dimethyl sulfoxide were purchased from
Sigma-Aldrich (St. Louis, MO, USA). Pentobarbital-Na was purchased from Lab Ttokkyo, S.A.
(México, D.F., Mexico). Stock solutions (0.01–1 M) were prepared using de-ionized distilled water
and were stored frozen, except for picrotoxin and the DBTD stock solutions, which were prepared in
ethanol (50% v/v) and DMSO, respectively. The desired final drug concentration was obtained by
diluting the stock solutions in an external solution prior to application.
3.2.4. Data Analysis
The concentration–response data were fit to a logistic model:
I = Imax/[1 + (EC50/[A])nH]
(1)
where [A] is the agonist concentration, I is the current, and Imax is the maximum current. EC50 is the
concentration of drug that elicits a half-maximum response, and nH is the Hill coefficient.
Experimental data were reported as ± SEM, and n represents the number of cells used. The unpaired
Student’s t-test was applied to data obtained from two different groups of cells. One-way ANOVA and
the Bonferroni tests were used to compare multiple means. The two-tailed P values of 0.05 or less
were considered to be statistically significant.
3.2.5. Theoretical Calculations
Quantum chemical calculations of the DBTDs structures 2a–g in the gas phase were performed
using GAUSSIAN 03 [30] in conjunction with density functional theory (DFT) calculations. Geometry
optimization of the DBTDs was followed by frequency calculations performed at the B3LYP/6-311
++G(d,p) level. Table 1 reports the cLog P, generated using HyperChem, of each optimized structure
obtained from Gaussian V03.
4. Conclusions
We described the preparation of novel DBTDs via a nitrene radical that inserted into the C on
the aromatic ring. Seven derivatives 2a–g were generated and their effects on GABAA neuronal
receptors were tested. It was shown, for the first time, that DBTDs inhibited IGABA in a time- and
concentration-dependent manner.
The DBTDs displayed an inhibitory effect on the GABAA channel of myenteric neurons, and this
antagonism was non-competitive indicating that it does not bind to the GABA receptor and their effect
is likely allosteric. Inhibition was mediated by an extracellular binding site that was most likely not in

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the mouth of the channel and therefore, it is unlikely that this effect is mediated by channel blockage.
Their effect was also independent of the benzodiazepine binding site. The DBTDs described here
could be used as a model to explore new GABAA receptor inhibitors with a potential to be used as
antidotes for substances known to positively modulate GABAA channel activity or as a new drugs to
induce experimental epilepsy. These compounds appear to bind on a different site than picrotoxin;
therefore, they could be used alone or in combination with picrotoxin. Future experiments will be
aimed to molecularly identify DBTDs binding sites on GABAA receptors.
Acknowledgments
The authors wish to thank Estela Nuñez-Pastrana and María Guadalupe Ortega-Salazar (Facultad de
Ciencias Químicas, UASLP) for their technical assistance. This work was supported by CONACYT,
México (Project no. 134687). Support for this work by the UASLP via FAI (C03-FAI-11-21.56) and
PIFI 2.0 is gratefully acknowledged. We also thank A. Peña, E. Huerta, E. Hernández, L. Velasco, and
J. Pérez from the Instituto de Química (UNAM) for technical support. J.F. Ramírez Martínez was
supported by a CONACYT scholarship (181001).
Conflict of Interest
The authors declare no conflict of interest.
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