Novel sulfamides and sulfamates derived from amino esters: Synthetic studies and anticonvulsant activity

Article abstract of DOI:10.1016/j.ejphar.2016.02.001

We report herein the design and optimization of a novel series of sulfamides and sulfamates derived from amino esters with anticonvulsant properties. The structures were designed based on the pharmacophoric pattern previously proposed, with the aim of imp

Full text of DOI:10.1016/j.ejphar.2016.02.001

European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Novel sulfamides and sulfamates derived from amino esters: Synthetic
studies and anticonvulsant activity
Maria L. Villalba a, Andrea V. Enrique a, Josefina Higgs b, Rocío A. Castaño a,
c
Sofía Goicoechea a, Facundo D. Taborda a, Luciana Gavernet a, , Ileana D Lick ,
n
Mariel Marder b, Luis E. Bruno Blanch a
a Medicinal Chemistry, Department of Biological Sciences, Faculty of Exact Sciences, National University of La Plata, 47 and 115, La Plata B1900BJW, Argentina
b Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956 (C1113AAD), Buenos Aires,
Argentina
c Department of Chemistry, Faculty of Exact Sciences, National University of La Plata, CINDECA (CCT-La Plata-CONICET-UNLP), La Plata B1900BJW, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
We report herein the design and optimization of a novel series of sulfamides and sulfamates derived
from amino esters with anticonvulsant properties. The structures were designed based on the phar-
macophoric pattern previously proposed, with the aim of improving the anticonvulsant action. The
compounds were obtained by a new synthetic procedure with microwave assisted heating and the use of
adsorbents in the isolation process. All the derivatives showed protection against the maximal electro-
shock seizure test (MES test) in mice at the lowest dose tested (30 mg/kg) but they did not show sig-
nificant protection against the chemical induced convulsion by pentylenetetrazole. These results verify
the ability of the computational model for designing new anticonvulsants structures with anti-MES
activity. Additionally, we evaluated the capacity of the synthesized structures to bind to the benzodia-
Received 4 June 2015
Received in revised form
30 January 2016
Accepted 1 February 2016
Keywords:
Epilepsy
Sulfamides
Sulfamates
MES test
Microwave assisted synthesis
zepine binding site (BDZ-bs) of the
medium to low affinity for the BDZ-bs.
γ
-aminobutiric acid receptor (GABAA receptor). Some of them showed
& 2016 Elsevier B.V. All rights reserved.
1. Introduction
2009), with anticonvulsant action evidenced in the Maximal
electroshock seizure test in mice (MES test, Porter et al., 1984).
Epilepsy is not one condition, but a complex set of cerebral
disorders that have in common the occurrence and recurrence of
seizures (Fisher et al., 2014). About 65 million people worldwide
currently live with epilepsy, and only 70% of them control the
seizures with the available medication (Moshé et al., 2015), at
expenses of the significant adverse side effects that increase their
toxic actions when a lifelong medication is required (Bialer et al.,
2013; Löscheran and Schmidt, 2011). The remaining one third of
the patients are still resistant to the current anticonvulsant drugs,
condition known as refractory epilepsy (French, 2007). Under this
scenario, there is a genuine need for new antiepileptic compounds
with more efficacy and safety.
Research from our group and others has focused on sulfamide
derivatives as new targets of anticonvulsant drugs. (Gavernet et al.,
2009, 2007a, 2007b; McComsey et al., 2013). Particularly, our
previous findings allowed us to define a new pharmacophoric
pattern for amino acid derived sulfamides (Fig. 1) (Gavernet et al.,
The most promising compounds were two
derivatives: the methyl [N-(N′-2-propylpentyl)-sulfamoyl]-
ninate and the methyl [N-(N′butyl)-sulfamoyl]- -alaninate (com-
pounds 1 and 2, Fig. 2). They exhibit some structural similarities
with the anticonvulsant drug Valrocemide (Isoherranen et al.,
2001), a valproic acid derivative with a glycinamide moiety linked
to the valpromide function (compound 3, Fig. 2).
β
-Alanine sulfamide
β-ala-
β
We report herein the synthesis and anticonvulsant activity of a
new set of
Additionally, we replaced
fluorobenzyl)-sulfamoyl] -alaninate (compound 9, Fig. 3) by L-
β
-alanine derived sulfamides (compounds 4–9, Fig. 3).
β-alanine moiety of Methyl [N-(p-
β
valine and L-phenyl alanine skeleton (compounds 10 and 11,
Fig. 3). As will be explained in Section 4, compound 9 showed
promising anticonvulsant action, so we synthesized structures 10
and 11 in order to explore the influence of other amino acid chains
to the activity.
To obtain the new structures we design here a new synthetic
protocol by using microwave assisted synthesis. As will be de-
scribed in the next section, the synthetic routes of sulfamides in-
volved the previous preparation of ester sulfamates. Sulfamates
also comply with the pharmacopohore requirements for the polar
n
Corresponding author.
E-mail address: lgavernet@biol.unlp.edu.ar (L. Gavernet).
http://dx.doi.org/10.1016/j.ejphar.2016.02.001
0014-2999/& 2016 Elsevier B.V. All rights reserved.
Please cite this article as: Villalba, M.L., et al., Novel sulfamides and sulfamates derived from amino esters: Synthetic studies and
anticonvulsant activity. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.02.001i

2
M.L. Villalba et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
The synthetic route outlined in Scheme 1 was employed for the
synthesis of -Alanine methyl ester sulfamides. However, our at-
β
tempts to obtain the corresponding sulfamates from L-valine
methyl ester and L-phenylalanine methyl ester were not success-
ful, even when traditional heating and different catechol/amino
ester ratio were used.
Particularly, the synthesis of the sulfamate of L-valine methyl
ester (compound 13) with the traditional heating gave the N,N
′-disubstituted sulfamide, the N,N′-Sulfonyl bis-L-valine dimethyl
ester (Gavernet et al., 2009), as the main product of the reaction;
and small quantities of compound 13 (which was purified and
tested). This result is consistent with the analysis of the products
obtained from the synthesis of other alkyl and aryl sulfamates
(DuBois and Stephenson, 1980). Finally, we prepared the sulfa-
mides 10 and 11 by inverting the synthetic route: first we obtained
the sulfamate of p-fluorobenzyl amine and then we added the
corresponding amino ester (Scheme 2).
The synthetic methods outlined in Schemes 1 and 2 involve the
production of equimolar quantities of sulfamide and catechol. In
our experience, the isolation and purification of the sulfamide
from the relative large amounts of catechol causes difficulties
during the work up process. Traditionally, we washed the crude
product and then we performed at least one column chromato-
graphy process, which require large amounts of silica gel and di-
chloromethane as the elution solvent (Gavernet et al., 2009). In
this investigation we replaced the standard work up process by an
heterogeneous filtration process. We directly added to the reaction
mixture an insoluble inorganic compound with the capacity to
absorb selectively catechol and then filtered the solid.
Fig. 1. Pharmacophoric pattern proposed by Gavernet et al. (2009). The anti-MES
requirements can be summarized as: (1) a polar moiety (atoms 1–3, in yellow),
(2) a hydrophobic chain (atoms 5–7 in green) placed in a conformation defined by
t1, t2, t3 and d, and connected to the polar moiety through a link atom (atom 4, in
cyan), (3) any group attached to atom 3 should be non polar or H.
group and they present a similar electronic distribution than sul-
famides (Gavernet et al., 2007a). In fact, the anticonvulsant drug
Topiramate presents this function into its chemical structure. For
that reason we included the sulfamates showed in Fig. 4 in the
protocols for biological assays.
It is worth mentioning that sulfamides and sulfamates are
versatile functions that have been employed in medicinal chem-
istry to construct active compounds with different applications.
These functionalities interact with molecular targets of several
diseases, such as aspartic proteases (HIV-1 protease, -secretase),
γ
Details about the synthesis of the new sulfamides and sulfa-
mates, their physicochemical characteristics and their spectro-
scopic characterization are summarized next.
serine proteases, metalloproteinases, steroid sulfatases and 5-HTID
Receptors among others (Nussbaumer and Billich, 2005; Reitz
et al., 2009; Winum et al., 2006). They have also successfully
tested as carbonic anhydrase inhibitors due to their bioisosteric
correspondence with sulfonamide, the classical functional group
that inhibit the active isoforms through its direct interaction with
the active site (Gavernet et al., 2013; McKenna and Supuran, 2014).
2.1.1. General information
Melting points were determined using capillary tubes with an
electrothermal melting point apparatus and were uncorrected.
Thin-layer chromatography (TLC) was performed with aluminum
backed sheets with silica gel 60 F254 (Merck, ref 1.05554), and the
spots were visualized with 254 nm UV light and 5% aqueous so-
lution of ammonium molybdate (VI) tetrahydrate. Column chro-
matography was performed on silica gel 60 (70–230 mesh, Merck,
ref 1.07734.2500). 1H and 13C NMR spectra were recorded for
compounds 3, 4 and 6 on a Varian Gemini 200 spectrometer at 200
and 50 MHz; whereas we employed a BRUKER AVANCE II 500
spectrometer at 500 and 126 MHz for the rest of the structures.
2. Materials and methods
2.1. Chemistry
In previous investigations we synthesized amino acid derived
sulfamides by modifying the synthesis of aril/alkyl sulfamides via
catechol sulfate (DuBois and Stephenson, 1980). Catechol sulfate
(prepared with catechol and sulfuryl chloride) reacts with the salt
of the amino ester under controlled conditions, to yield a sulfa-
mate ester derivative (Scheme 1). Then, the resulting sulfamate
reacts with the alkyl/aryl amine to yield the amino acid derived
sulfamide (Scheme 1, Gavernet et al., 2009). Unlike previous
methodologies, we employed here microwave assisted synthesis
in both steps of the reaction. With this method, we incorporated
reactions under solvent free conditions, which is more benign to
the environment than the use of the traditional reaction media.
The chemical shifts were reported in ppm (
δ scale) relative to
internal TMS, and coupling constants were reported in Hertz (Hz).
FTIR spectra were performed using a Bruker EXINOX 55 equip-
ment. Spectra were recorded at room temperature in the
4000–400 cmꢀ1 range, and the samples were prepared in form of
wafers with KBr. Absorption values were expressed as wave-
numbers (cmꢀ1) and only significant absorption bands are given.
Analytical grade solvents were employed for crystallization, while
pure solvents were used for the reactions, extractions and column
chromatography. Commercial amines were distilled prior to their
Fig. 2. Compounds with anticonvulsant properties analyzed in previous investigations by Gavernet et al. (2009). Atoms are numbered for the centers that define the
pharmacophoric pattern shown in Fig. 1.
Please cite this article as: Villalba, M.L., et al., Novel sulfamides and sulfamates derived from amino esters: Synthetic studies and
anticonvulsant activity. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.02.001i

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3
O
O
S
H
N
OCH3
H
N
HN
S
NH
OCH3
O
O
O
O
4
5
O
S
O
S
O
7
H
N
OCH3
NH
OCH3
HN
NH
O
O
O
6
O
S
O
OCH3
HN
NH
OCH3
HN
S
NH
O
O
O
O
F
9
8
O
O
O
S
O
HN
S
NH
HN
NH
OCH3
OCH3
O
O
F
F
11
10
Fig. 3. Amino acid derived sulfamides synthesized and tested in this investigation.
Fig. 4. Sulfamates synthesized and tested in this investigation.
Scheme 1. Traditional and alternative conditions for the synthetic routes of β-Alanine methyl ester sulfamides. R¼hexyl (compound 4), isobutyl (compound 5), adamantyl
(compound 6), phenethyl (compound 7), α-metilbenzyl (compound 8), p-fluorobenzyl (compound 9).
use. All reagents employed have analytical grade (Sigma-Aldrich,
Fluka, J.Backer). Microwave reactions were carried out in an An-
ton-Paar-monowave-300 reactor (monowave, maximum power
850 W, temperature control via IR-sensor, vial volume: 10–30 ml).
Mass spectra were obtained on an Agilent Technologies LC/MSD
Trap SL. Specific rotations of chiral structures 10, 11 and 13 were
measured on PerKin Elmer 343 at
λ
: 589 nm and 25 °C.
-alanine-methyl ester sul-
2.1.2. Synthesis of 2-Hydroxyphenyl-N-
famate (12)
β
Triethylamine (6.10 ml, 44.6 mmol) was added dropwise to a
solution of H- -Ala-OMe.HCl (1.99 g, 22.30 mmol) and catechol
β
Please cite this article as: Villalba, M.L., et al., Novel sulfamides and sulfamates derived from amino esters: Synthetic studies and
anticonvulsant activity. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.02.001i

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Scheme 2. Synthesis of compound 10 (R´: isopropyl) and 11 (R´: benzyl).
sulfate (4.20 g, 25.12 mmol, prepared with catechol and sulfuryl
chloride according to DuBois and Stephenson, 1980). The resulting
solution was placed into a microwave vial containing a stirrer bar.
The vial was sealed after purging with N2 for some min. The re-
action was then heated to 90 °C for 15 min using the microwave
reactor. After that, dichloromethane (35 ml) was added and the
solution was washed with 15 ml of 5 N hydrochloric acid (2 ꢁ ) and
brine (1 ꢁ ). The combined organic layers were dried over Na2SO4
and concentrated under reduced pressure. The crude product was
then purified by silica column chromatography (15:1
CH2Cl2/MeOH). Crystallization with CH2Cl2 afforded the product as
a white solid (3.60 g, 59.0% yield). Melting point 102–104 °C, Rf:
0.59 (15:1 CH2Cl2/MeOH) (Gavernet et al., 2009).
(0.46 ml, 3.19 mmol). The reaction mixture was heated to 120 °C
for 15 min in a microwave reactor. Then the reaction medium was
diluted with 20 ml of acetonitrile and 4.00 g of -Al2O3 (x3) was
γ
employed to remove catechol. The product was purified by column
chromatography (CH2Cl2/MeOH 20:1) followed by crystallization
with CH2Cl2/petroleum ether, affording the compound as a white
solid (0.418 g, 54.0%). Melting point: 47–47.5 °C. Rf: 0.72 (20:1
CH2Cl2/MeOH). 1H-NMR (CDCl3)
δ
5.01 (t, J¼6.5 Hz, 1H, NH-
4.50 (t, J¼6.1 Hz, 1H, NH-hexyl), 3.72 (s ,3H, O–CH3 ), 3.31 (AM2X2 ,
J¼6.5, 6.2 Hz, 2H, N-CH2- -Ala), 3.04–2.97 (m, 2H, N–CH2–hexyl),
2.64 (t, J¼6.1 Hz, 2H, CH2-CO- -Ala), 1.58–1.48 (m, 2H, CH3–CH2–
hexyl), 1.29 (m, 6H, CH2–CH2–CH2), 0.92–0.86 (m, 3H, CH3-hexyl).
13C NMR (CDCl3)
172.46 (C¼O ester), 51.96 (O–CH3), 43.45 (N–C–
hexyl), 38.91 (N-C- -Ala), 34.10 (C–CO), 31.45 ( -C-hexyl), 29.86
-C-hexyl), 26.59 C-hexyl), 22.74 –C-hexyl), 14.22
β
-Ala),
β
β
δ
β
α
δ
(β
(
γ–
(
2.1.3. Synthesis of 2-hydroxyphenyl-N-L-valine-methyl ester sulfa-
mate (13)
(CH3-hexyl). IR (KBr): 3465–3296 (N–H), 2956–2852 (C–H, OCH3),
1741 (C ¼O), 1332, 1199 (SO2). Elemental analysis calculated for
The compound was synthesized according to the method pre-
C10H22N2O4S: C 45.1, H 8.3, N 10.5, S 12.0%. Found: C 45.3, H 8.4, N
viously reported for 2-Hydroxyphenyl-N- -alanine-methyl Ester
β
10.4, S 11.9%.
Sulfamate (12) (Gavernet et al., 2009). A solution of catechol sul-
fate (2.80 g, 16.28 mmol) in 10.0 ml of CH2Cl2 was added dropwise
to a solution of L-Val-OMe.HCl (2.72 g, 16.28 mmol) and triethy-
lamine (4.89 ml, 35.2 mmol) in 10.0 ml of CH2Cl2 with vigorous
stirring at 0 °C under N2.The reaction medium was then warmed to
room temperature and stirred for 18 h, concentrated under re-
duced pressure. The crude product was purified by column chro-
2.1.4.2. Methyl [N-(N´isobutyl)-sulfamoyl]β-alaninate (5). The com-
pound was obtained following the general procedure, from sulfa-
mate 12 (1.02 g, 3.64 mmol), acetonitrile and isobutylamine
(0.44 ml, 4.36 mmol). The reaction mixture was heated to 120 °C
for 20 min in a microwave reactor. Then the crude reaction med-
ium was diluted with 20 ml of acetonitrile and 4.24 g of -Al2O3
γ
matography
(CH2Cl2/MeOH
20:1).
Crystallization
with
(x3) was employed to remove catechol. The product was purified
by crystallization with ether, to afford compound 5 as a white solid
(0.416 g, 48.0%). Melting point: 42–43 °C. Rf: 0.66 (20:1 CH2Cl2
CH2Cl2/petroleum ether afforded the product as a white solid
(0.28 g, 28.0% yield). Melting point: 69–71 °C. Rf: 0.57 (20:1
CH2Cl2/MeOH). [
α
]
25: 29.0° (c 0.5, CHCl3).1H NMR (CDCl3)
δ
6.90–
D
/MeOH). 1H NMR (CDCl3)
δ
5.02 (t, J¼6.3 Hz, 1H, NH-
(t, J¼6.2 Hz, 1H, NH-isobutyl), 3.72 (s, 3H, O–CH3), 3.30 (dd, J¼6.3,
6.3 Hz, 2H, N–CH2– -Ala),
-Ala), 2.85 (t, J¼6.5 Hz, 2H, CH2-CO-
β
-Ala), 4.58
7.29 (m, 4H, H–Ar), 6.82 (s, 1H, OH), 5.55 (s, 1H, NH), 4.14 (s, 1H,
CH–N), 3.85 (s, 3H, OCH3), 2.19–2.25 (m, 1H, CH), 0.96–1.06 (dd,
J¼6.8 Hz, 6.9 Hz, 6H, CH3). 13C NMR (CDCl3)
δ
172.36 (C¼O ester),
β
β
148.16 (C–OH), 137.72, 128.30, 122.99, 120.82, 118.24 (C1,3,4,5,6 Ar),
62.74 (C–N), 53.07 (OCH3), 31.48 (CH), 18.79 (CH3), 17.37 (CH3). IR
(KBr) 3272 (O–H), 2983–2881(C–H,OCH3), 1714 (C¼O), 1371, 1168
(SO2). Elemental analysis calculated for C10H22N2O4S: C 47.5, H 5.6,
N 4.6, S 10.6 % Found: C 47.7, H 5.8, N 4.7, S 10.6%.
2.64 (t, J¼6.1 Hz, 2H, CH2-isobutyl), 1.91–1.70 (m, 1H, CH-isobutyl),
0.95 (d, J¼6.3 Hz, 6H, CH3).13C NMR (CDCl3)
δ
β
172.83 (CO), 52.19
(O–CH3 ), 50.78 (N-C-isobutyl), 38.91(N-C-
-Ala), 34.12 (C–CO),
28.49 (CH-isobutyl), 20.23 (CH3). IR (KBr) 3280 (N–H), 2958–2873
(C–H, OCH3),1749 (C¼O), 1311, 1132 (SO2). MS (ESþ): m/z calcu-
lated for C8H18N2O4S: 238.1 [MþH]þ, 499.2 [2MþNa]þ. Found:
239.1, 498.8. Elemental analysis calculated for C8H18N2O4S: C 40.3,
H 7.6, N 11.7, S 13.4%. Found: C 40.5, H 7.6, N 11.5, S 13.0%.
2.1.4. General procedure for the synthesis of sulfamides 4 to 9
The corresponding amine was added to one solution of the
sulfamate 12 in acetonitrile (5.00 ml). The mixture was placed into
a microwave vial containing a stirrer bar, which was sealed after
purging with N2 for some min. The resulting solution was heated
to 110–130 °C for 15–35 min (depending on the amine). The re-
action medium was then diluted with acetonitrile (20 ml) and
2.1.4.3. Methyl [N-(N´1-adamantyl)-sulfamoyl]
β-alaninate (6). The
compound was obtained following the general procedure. Trie-
thylamine (1.10 ml, 8.0 mmol) was added dropwise to a solution of
the sulfamate 12 (1.14 g, 4.00 mmol), amantadine hydrochloride
(0.76 g, 4.00 mmol) and 5.00 ml acetonitrile. The reaction medium
was then heated to 120 °C for 20 min in a microwave reactor. After
that, the crude product was diluted with acetonitrile (20 ml) and
γ
-Al2O3 was added to remove catechol. The acetonitrile solution
was filtered and the solvent was removed under reduced pressure.
The products were purified by column chromatography and/or
crystallization.
9.25 g of
γ
-Al2O3 (x3) were used to remove catechol. Then the
acetonitrile solution was filtered, and the solvent was removed
under reduced pressure. The product was purified by column
chromatography (CH2Cl2/MeOH 15:1) and crystallization with
CH2Cl2/hexane, affording compound 6 as a white solid (0.534 g,
2.1.4.1. Methyl [N-(N´hexyl)-sulfamoyl]β-alaninate (4). The com-
pound was obtained following the general procedure, from sulfa-
mate 12 (0.800 g, 2.90 mmol), acetonitrile, and hexylamine
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5
42.0%). Melting point: 86–87 °C. Rf: 0.54 (40:1 CH2Cl2/ MeOH). 1H
NMR (CDCl3) 4.82 (s, 1H, NH- -Ala), 3.73 (s, 3H, O–CH3), 3.33 (t,
J¼6.0 Hz, 2H, CH2–NH), 2.68 (t, J¼6.0 Hz, 2H, N–CH2– -Ala), 2.12
(s, 3H
3CH-adamantyl), 1.96 (d, J¼2.3 Hz, 6H,
–CH2-adamantyl), 1.68 (t, 6H ,3
–CH2-adamantyl).13C NMR
(CDCl3),
172.32 (C¼O ester), 55.74 (O–CH3), 51.91 (N-C-ada-
mantyl), 42.42(C- -adamantyl), 38.95 (N-C- -Ala), 35.89 (CH–
adamantyl), 33.76 (C–CO), 29.58 (C- -Adamantyl). IR (KBr) 3330–
(s, 3H, O–CH3), 3.29 (t, J¼6.0 Hz, 2H, N-CH2-
J¼6.0 Hz, 2H ,CH2–CO
-Ala).13C NMR (CDCl3)
ter), 163.47–161.70 (C–F), 132.46, 129.78, 115.62 (C3,2,1–Ar), 51.77
(O–CH3), 46.59 (C-benzyl), 38.69 (N-C- -Ala), 33.67 (C–CO). IR
β
-Ala), 2.60 (t,
δ 172.57 (C¼O es-
δ
β
β
β
,
3
β
α
β
(KBr) 3488–3288 (N–H), 2960–2860 (C–H, OCH3), 1743 (C¼O),
δ
1510 (C–F), 1310, 1141 (SO2). Elemental analysis calculated for
α
β
C11H15FN2O4S: C 45.5, H 5.2, N 9.6, S 11.0 %. Found: C 45.5, H 5.3, N
9.5, S 11.1 %.
β
3284 (N–H), 2921-2861 (C–H, OCH3), 1737 (C¼O), 1321, 1143 (SO2).
Elemental analysis calculated for C14H24N2O4S : C 53.1, H 7.6, N 8.8,
S 10.0 %. Found: C 52.9, H 7.7, N 8.8, S 9.8%.
2.1.5. Synthesis of 2-hydroxyphenyl-N-4-fluorobenzyl sulfamate (14)
Triethylamine (2.30 ml, 16.8 mmol) was added dropwise to a
cold solution of 4-fluorobenzylamine (1.70 ml, 16.83 mmol) and
catechol sulfate (2.91g, 16.3 mmol, prepared with catechol and
sulfuryl chloride). The reaction mixture was placed into a micro-
wave vial containing a stirrer bar. The vial was sealed after purging
with N2 for some min. The reaction was then heated to 55 °C for
3 min in a microwave reactor. The crude product was purified by
column chromatography (40:1 CH2Cl2/MeOH). Crystallization with
CH2Cl2 afforded the product as a white solid (1.91 g, 38.3% yield).
Melting point 129–130 °C. Rf: 0.53 (40:1 CH2Cl2/MeOH). 1H NMR
2.1.4.4. Methyl [N-(N´phenethyl)-sulfamoyl]β-alaninate (7). The
compound was obtained following the general procedure from
sulfamate 12 (0.80 g, 2.90 mmol), acetonitrile and phenethylamine
(0.36 ml, 2.92 mmol). The reaction mixture was heated to 130 °C
for 15 min in a microwave reactor. Then the crude reaction med-
ium was diluted with 20 ml of acetonitrile, and 4.00 g of -Al2O3
γ
(x3) was employed to remove catechol. The product was purified
by crystallization with CH2Cl2/petroleum ether, to afford com-
pound 7 as a white solid (0.39 g, 47.0%). Melting point: 72–73 °C .
(DMSO)
δ
7.39–6.77 (m, 8H, Ar), 6.97 (s, 1H ,OH), 4.28 (s, 1H, NH),
163.55, 160.33 (C–F), 150.31
Rf: 0.57 (20:1 CH2Cl2 /MeOH). 1H-NMR (CDCl3)
δ
7.36–7.18 (m, 5H,
3.31 (S, 2H, CH2). 13C NMR (DMSO)
δ
Ar–H), 4.86 (s, broad, 1H, NH-
nethyl), 3.70 (s, 3H, O-CH3), 3.31 (t, J¼6.5 Hz, 2H,
β
Ala), 4.26 (s, broad, 1H, NH-phe-
(COH), 138.18 (C–O), 134.32, 130.27, 127.70 (C3,2,1–Ar–F) 123.95,
119.52, 117.87,115.40 (C3,4,5,6–Ar–OH), 39.97 (CH2). IR (KBr) 3423
(O–H), 3282 (N–H), 2958–2860 (C–H, OCH3), 1363, 1155 (SO2),
1220 (C–F). Elemental analysis calculated for C13H12FNO4S: C 52.5,
H 4.1, N 4.7, S 10.8%. Found: C 52.3, H 4.2, N 4.8, S 10.9%.
N-CH2-phenethyl), 3.17 (s, broad, 2H, CH2-benzyl), 2.87 (t,
J¼6.5 Hz, 2H, N-CH2-
β
-Ala), 2.55 (t, J¼6.1 Hz, 2H ,CH2-CO-
β
-Ala).
13C NMR (50 MHz, CDCl3)
δ
172.75 (C¼O ester) 138.45 (C1 Ar),
129.29, 129.01, 127.23 (C2,3,4 Ar), 51.90 (O–CH3), 44.81(CH2–NH),
39.35 (N-C-
β
-Ala), 35.64 (CH2-benzyl), 34.39 (C–CO). IR (KBr)
2.1.6. Synthesis of methyl [N-(N´-4-fluorobenzyl)]-sulfamoyl]L-vali-
nate (10)
Triethylamine (0.65 ml, 5.4 mmol) was added dropwise to a
solution of H-Val-OMe.HCl (0.390 g, 2.37 mmol) and 2-Hydro-
xyphenyl-N-4-fluorobenzyl sulfamate 14 (0.705 mg, 2.37 mmol) in
acetonitrile (5 ml). The reaction mixture was stirred and heated to
3477-3269 (N–H), 2954–2896 (C–H, OCH3), 1757 (C¼O), 1319,
1145 (SO2). Elemental Analysis calculated for C12H18N2O4S: C
50.4, H 6.3, N 9.8, S 11.2 %. Found: C 50.7, H 6.5, N 9.6, S 11.1%.
2.1.4.5. Methyl [N-(N´
compound was obtained following the general procedure from
-methyl-
α-methylbenzyl)-sulfamoyl]β-alaninate (8). The
120 °C for 20 min in
a microwave reactor. After that, di-
sulfamate 12 (0.961g, 3.49 mmol), acetonitrile, and L(þ)
α
chloromethane (35 ml) was added and the solution was washed
with 15 ml of 2.5 N hydrochloric acid (1 ꢁ ) and brine (1 ꢁ ). The
combined organic layers were dried over Na2SO4 and concentrated
under reduced pressure. Then the crude product was diluted with
benzylamine (0.49 ml, 3.84 mmol). The reaction mixture was he-
ated to 120 °C for 25 min in a microwave reactor. Then the crude
reaction medium was diluted with 20 ml of acetonitrile, and 4.28g
of
γ
-Al2O3 (x3) was employed to remove catechol. The crude pro-
20 ml of acetonitrile, and 3.75 g of -Al2O3 (x2) was employed to
γ
duct was purified by chromatography (Hexane/ethyl acetate 1:1)
remove catechol. Finally the product was purified by crystal-
lization with CH2Cl2/hexane, to afford compound 10 as a white
solid (0.338g, 44.7 %). Melting point: 70–71 °C. Rf: 0.56 (40:1
affording compound 8 as colorless oil (0.401 g, 40.0%). Rf: 0.62
(CH2Cl2/MeOH 20:1). 1H NMR (CDCl3)
δ
7.40–7.29 (m, 5H, C–Ar),
4.53–4.57 (m, 1H, CH-benzyl), 3.68 (s, 3H, O–CH3) 3.18–2.96 (m, 2H,
CH2Cl2/MeOH). [α D
]
25: ꢀ9.0° (c 0.5, MeOH). 1H NMR (CDCl3)
δ
N–CH2-
β
-Ala), 2.49–2.34 (m, 2H, CH2-CO
β
-Ala), 1.56 (d, J ¼ 6.3 Hz,
7.32–7.29 (m, 4H, H–Ar), 5.10 (d, J¼6.8 Hz, 1H, NH-Val), 4.57 (s, 1H,
NH), 4.23 – 4.16 (s, 2H, CH2 benzyl), 3.87–3.85 (m, 1H, CH), 3.75 (s,
3H, OCH3), 2.17–2.10 (m, 1H, CH-isopropyl), 1.04-0.93 (dd,
J¼6.8 Hz, 6.9 Hz, 6H). IR (KBr) 3313 (N–H), 2973–2881 (C–H,
OCH3), 1743 (C¼O), 1321, 1143 (SO2), 1234 (C–F). Elemental ana-
lysis calculated for C13H19FN2O4S: C 49.0, H 6.0, N 8.8, S 10.1.
Found: C 49.3, H 6.1, N 8.5, S 10.1%.
3H, CH3).13C NMR (CDCl3)
δ
172.07 (C¼O ester) 142.25 (C1 Ar),
128.30, 127.63, 126.43 (C2,3,4 Ar), 53.41 (N–C–Ar), 51.57 (O–CH3),
38.81 (N-C- -Ala), 33.80 (C–CO), 23.55 (CH3). IR (KBr) 3311 (N–H),
β
2977 (C–H, OCH3), 2370 (C–H Ar) 1735 (C¼O), 1440 (C–C Ar), 1330,
1161(SO2) . MS (ESþ): m/z calculated for C12H18N2O4S: 286.3
[MþHþ], 595.7 [2MþNa]þ. Found 286.9, 594.8. Elemental analysis
calculated for C12H18N2O4S: C 50.3, H 6.3, N 9.8, S 11.2%. Found: C
50.2, H 6.3, N 9.7, S 10.9%.
2.1.7. Synthesis of Methyl [N-(N´-4-fluorobenzyl)]-sulfamoyl] L-phe-
nylalaninate (11)
2.1.4.6. Methyl [N-(p-fluorobenzyl)-sulfamoyl]
β
-alaninate (9). The
Triethylamine (0.93 ml, 6.76 mmol) was added dropwise to a
solution of H-Phe-OMe.HCl (0.730 g, 3.38 mmol) and 2-Hydro-
xyphenyl-N-4-fluorobenzyl sulfamate (1.0g, 3.38 mmol), in acet-
onitrile (5 ml). The reaction mixture was placed into a microwave
vial containing a stirrer barand it was heated to 120 °C for 25 min.
After that, dichloromethane (35 ml) was added, and the solution
was washed with 5 ml of 2.5 N hydrochloric acid (1 ꢁ ) and brine
(1 ꢁ ). The combined organic layers were dried over Na2SO4 and
concentrated under reduced pressure. Then the crude reaction
medium was diluted with 30 ml of acetonitrile and 5.58 g of
compound was obtained following the general procedure, from
sulfamate 12 (0.904 g, 3.42 mmol), acetonitrile and p-fluor-
obenzylamine (0.76 ml, 3.76 mmol). The reaction was heated to
120 °C for 35 min in a microwave reactor. Then the crude reaction
medium was diluted with 20 ml of acetonitrile, and 5.28 g of
γ
-Al2O3 (x4) was employed to remove catechol. The crude product
was purified by column chromatography (Hexane/ethyl acetate
6:4) followed by crystallization with CH2Cl2/hexane; to give the
product as a white solid (0.449 g, 45.0%). Melting point 87–88 °C.
Rf: 0.58 (20:1 CH2Cl2/MeOH). 1H NMR (CDCl3)
δ
7.33–7.36 (m, 2H,
γ
-Al2O3 (x2) was employed to remove catechol. The product was
purified by crystallization with CH2Cl2/ether, to afford compound
C4,5–Ar), 7.04–7.08 (m, 2H, C2,6–Ar), 4.20 (s, 2H, CH2-benzyl), 3.71
Please cite this article as: Villalba, M.L., et al., Novel sulfamides and sulfamates derived from amino esters: Synthetic studies and
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M.L. Villalba et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
11 as a white solid (0.495 g, 41.3%). Melting point: 64–65 °C. Rf:
drugs. This test is designed to detect minimal neurological deficit.
The mouse is placed on a 1 inch diameter knurled plastic rod ro-
tating at 6 rpm. Normal mice remain indefinitely on a rod rotating
at this speed. Neurological deficit (e.g., ataxia, sedation, hyper-
excitability) is indicated by the inability of the animal to maintain
equilibrium on the rod for at least 1 min in each of three trials
(Dunham and Miya,1957).
0.71 (40:1 CH2Cl2 /MeOH). [
α
]
25: ꢀ16.8° (c 0.5, MeOH). 1H NMR
D
(DMSO) 7.33–6.96 (m, 9H, H–Ar), 4.95 (d, J¼6.4 Hz, 1H, NH), 4.42
(broad, 1H, NH), 4.30–4.23 (m, 1H, CH), 4.01–3.84 (dd, 2H, CH2–Ar–
F), 3.60 (s, 3H, O–CH3), 3.15–2.94 (m, 2H, CH2-benzyl).13C NMR
(DMSO)
δ 173.17 (C ¼O ester), 162.73, 160.60 (C–F), 134.75, 129.91,
115.23 (C3,2,1–Ar–F), 137.43, 129.09, 127.24, 115.40 (C5,6,7,8–Ar),
57.63 (CH), 52.24 (OCH3), 44.81 (CH2–Ar–F), 38.41 (CH2-benzyl). IR
(KBr) 3280 (N–H), 2962–2873 (C–H, OCH3), 1726 (C¼O), 1326,
1145 (SO2), 1216 (C–F). Elemental analysis calculated for
2.2.5. GABAA binding assay
The binding of [3H]-flunitrazepam (81.8 Ci/mmol; obtained
from PerkinElmer Life and Analytical Sciences, Boston, MA, USA) to
the benzodiazepine binding site was performed in washed crude
synaptosomal membranes from rat cerebral cortex. Membranes
were prepared according to Wasowski et al. (2012). The com-
pounds were added to 0.2–0.3 mg membrane protein suspended
in 1 ml of 25 mM Tris–HCl buffer in the presence of
[3H]-flunitrazepam 0.3 nM. In the screening assays each com-
C
18H21FN2O4S: C 55.7, H 5.2, N 7.6, S 8.7%. Found: C 55.4, H 5.2, N
7.5, S 8.5%.
2.2. Pharmacology
Biological evaluation was performed following the standard
procedures proposed by The NIH anticonvulsant drug develop-
ment (ADD) program, via the anticonvulsant screening project
(Krall et al., 1978; see Experimental section). We employed the most
popular seizure models, the MES test and the PTZ test in mice
(Porter et al., 1984). The MES test (maximal electroshock seizure
test) is associated with the electrical induction of the seizure,
whereas PTZ test (pentilenetetrazol test) involves a chemical in-
duction to generate the convulsion. Toxicity was also evaluated by
using the standardized RotoRod test, which is also included in the
primary phase of ADD program (Dunham and Miya, 1957; Porter
et al., 1984). Additionally, a radioligand binding assay was used to
evaluate the putative action of the compounds on the benzodia-
zepine binding site of the GABAA receptor complex. Details of the
experiments are given in the next section.
pound was tested at 300
says, the incubations were done with 3–900
pound. Diazepam was used as positive control in concentrations
μ
M in triplicate. In the competition as-
μ
M of each com-
between 1 and 100 nM. Non-specific binding was measured in the
presence of flunitrazepam 10 M and represented 5–15% of the
μ
total binding. The incubations were carried out at 4 °C for 1 h.
After incubation, the assays were terminated by filtration under
vacuum through Whatman GF/B glass-fiber filters followed by
washing three times with 3 ml each of incubation medium. In-
dividual filters were incubated overnight with scintillation cocktail
(OptiPhase ‘HiSafe’ 3) before measuring radioactivity in a Wallac
Rackbeta 1214 liquid scintillation counter.
2.2.1. Animals for MES, PTZ and Rototod test
3. Results
Adult male Swiss mice (18–25 g) were employed in the phar-
macological assays. They were obtained from the Faculty of Ve-
terinary, National University of La Plata. The animals were main-
tained on a 12 h light/dark cycle and allowed free access to food
and water, except during the time they were removed from their
cages for testing. They were daily manipulated during one week
before the experiment, to minimize stress caused by handling at
the time of trial. Housing, handling, and experimental procedures
complied with the recommendations provided by the National
Institutes of Health Guide for Care and Use of Laboratory Animals.
3.1. Chemistry
We first prepared the intermediate 2-hydroxyphenyl-N- -ala-
β
nine-methyl ester sulfamate (Scheme 1). The best conditions were
achieved after several experiments that differ from each other in
the reaction temperatures, reaction times and presence/absence of
solvent. The best yield, 59%, was obtained by stirring the reactants
(without solvent) under microwave radiation at 90 °C for 15 min
(traditional synthesis needs 6 h of reaction according to Gavernet
et al., 2009). The yield obtained from MW heating is lower than
the one achieved from traditional heating, but the reaction is
faster and cleaner.
2.2.2. MES test
To evaluate protection in the MES test, Maximal Electroshock
Seizures were elicited in mice by delivering a 60 Hz/50 mA elec-
trical-stimulus for 0.2 s via ear clip electrodes, at 0.5 and 4 h after
the drug injection, using an UGO Basile equipment. A drop of
saline solution was applied on each ear before placing the elec-
trodes to ensure adequate electrical contact. In these conditions,
maximal seizures are produced in virtually all normal mice. The
maximal seizure typically consists of a short period of initial tonic
flexion and a prolonged period of tonic extension (especially of the
hind limbs) followed by terminal clonus. The typical seizure lasts
approximately 22 s. Failure to extend the hind limbs to an angle
with the trunk greater than 90° is defined as protection.
Regarding the sulfamide products synthesized from 2-hydro-
xyphenyl-N- -alanine-methyl ester sulfamate, they also were
β
prepared via microwave assisted heating. However, compounds 4,
5 and 7 were also obtained following the standard procedures, to
compare the performance of the methodologies. Table 1 sum-
marizes the conditions employed for the preparation of the com-
pounds and the results obtained. Microwave assisted preparation
over performs the traditional procedure in both yields and
Table 1
Comparison of the result obtained from conventional and microwave assisted
synthesis of sulfamide derivatives. γ-Al2O3 was used for the work up processes in
all cases. The yields were calculated from the pure products.
2.2.3. PTZ test
The induced seizure entailed the subcutaneous (sc) adminis-
tration of 85 mg/kg of PTZ in 0.9% saline in the posterior midline of
the mice. The animals were observed for 30 min. Protection was
defined as the failure to observe a single episode of clonic spasms
of at least 5 s duration.
Compound R Heating
Time reaction Temperature(°C) Yield(%)
Hexyl
Conventional 10 h
MW 15 min
Conventional 22 h
80-100
120
45
40
54
0
Isobutyl
Phenetyl
MW
20 min
120
90
130
48
41
46
Conventional 22 h
2.2.4. Rotorod test
MW
15 min
It was used to determine possible neurotoxic effects of the test
Please cite this article as: Villalba, M.L., et al., Novel sulfamides and sulfamates derived from amino esters: Synthetic studies and
anticonvulsant activity. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.02.001i

M.L. Villalba et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
7
Table 2
equimolar samples of catechol. The quantification was performed
by gas chromatography with a Shimadzu GC-2014 chromatograph
equipped with a FID detector. We employed a semi-polar Supelco
SPB-5 column. It was heated from room temperature to 210 °C
with a temperature ramp program of 10° Cmꢀ1 and linear velocity
of 30 cm sꢀ1. The capacity of the absorption was expressed as the
percent of catechol adsorbed in each experiment. We performed
more than one adsorption experiments over the same sample of
Results obtained from the adsorption experiments. In all cases we employed a
solution of 0.0166 mM of catechol in dichloromethane as initial sample.
Adsorbent
Mass of adsorvent (mg)
% Adsortion
α-Al2O3
ZrO2
ZrO2
ZrO2
100
100
200
100–100
2
57
68
69
catechol for the most promising solids (ZrO2 and
According to the results shown in Table 2, the best adsorbent
for catechol is -Al2O3, which quantitatively retains the catechol
γ
-Al2O3).
2 adsorptions
ZrO2
3 adsorptions
γ-Al2O3
100–100–100
86
γ
100
93
after two adsorption processes. This solid present the highest
capability of adsorption for the tested adsorbents, and it can be
handled without modification with cupric acetate. It presents a
high specific area and it is a low priced and accessible commercial
reactant.
γ-Al2O3
100–100
100
2 adsorptions
Cu(OAc)2–ZrO2
Cu(OAc)2–α–Al2O3
100
100
28
61
reaction times.
3.2. Pharmacology
We employed heterogeneous filtration to eliminate the ca-
techol produced in the last step of the reactions. In order to find
the optimal conditions, we initially tested the capacity of several
The results of the biological evaluation of the synthesized
compounds are shown in Table 3. We also incorporated into the
table the biological profile of compounds 1, 2 and 12 for compar-
ison. All compounds of the set showed positive response in the
MES test. In fact, all sulfamides presented protection at the lowest
dose proposed by the ADD program (30 mg/kg), with at least 1/3
animal protected at 0.5 and/or 4 h after administration. Moreover,
compounds 5, 6 and 10 showed anticonvulsant activity for all
tested animals at 30 mg/kg. No sedative effects were observed for
most of the molecules with exception of compound 4 (1/3 mice at
solids to retain catechol. We evaluated zirconia (ZrO2),
α
–alumina
(α
-Al2O3) and -alumina ( -Al2O3) because these solids are widely
γ
γ
studied as adsorbents suitable for effluent treatment in industrial
processes (Lin and Juang, 2009). Additionally, we impregnate the
selected oxides with cupric acetate (to obtain oxide supported
cupric acetate), since the cupric salt itself was used to absorb ca-
techol in old synthetic procedures (DuBois and Stephenson, 1980).
Characteristics of the solids are given as Supporting information.
Table 2 shows the results obtained from the absorption of
Table 3
Pharmacological profile (Phase I) of the synthesized compounds.
Compound
Doses (mg/kg)
aMES activity time (h)
bPTZ activity time (h)
cTOX time (h)
dClass
Binding inhibition [Ki (μM)]e
0.5
4
0.5
4
0.5
4
g
1
30f
100f
30f
100f
30
100
30
100
30
100
30
100
30
100
30
100
30
100
30
100
30f
100f
30
100
30
100
1/3
0/3
1/3
1/3
1/3
0/3
0/3
1/3
0/3
2/3
2/3
3/3
1/3
1/3
0/3
2/3
1/3
3/3
1/2
0/2
0/3
1/3
1/4
1/4
1/2
2/4
3/3
0/3
1/3
1/3
2/3
0/3
3/3
3/3
3/3
2/3
1/3
1/3
1/3
1/3
1/3
2/3
3/3
1/3
2/4
3/4
0/3
1/3
0/4
2/4
0/2
3/4
1/2
2/2
0/3
0/3
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
nt
0/2
0/2
1/3
0/3
0/2
0/2
0/2
0/2
0/2
0/2
0/3
0/3
0/2
0/2
0/2
0/2
0/2
0/2
0/2
1/2
0/2
0/2
0/2
0/2
0/2
nt
0/2
0/2
0/3
0/3
0/2
0/2
0/2
0/2
0/5
0/5
0/6
0/3
1/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/2
1/2
0/3
0/3
0/2
0/2
0/4
2/4
0/5
0/5
0/6
0/6
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/2
2/4
0/3
0/3
0/2
0/2
0/2
1/4
1
1
4
1
1
1
1
1
1
1
1
1
1
þ
2
nt
4
þ þ
[140731]
5
–
6
–
7
þ þ
[85721]
8
–
9
þ þ
[123715]
þ þ
10
11
12
13
14
[61721]
þ þ þ
[1471]
þ
–
–
a
Maximal Electroshock Seizure test.
Pentilenetetrazol test
Toxicity evaluated in Rotorod test.
b
c
d
e
The compounds can be classified into four classes according to their activity in MES test.
nt: Not tested. eCapacity of the compounds, at 300 μM, to inhibit the binding of [3H]-flunitrazepam to the benzodiazepine binding site of the GABAA receptor indicated
as: inhibition480% (þ þ þ); inhibition; 40–80% (þ þ); inhibition 20–40% (þ) and inhibition o20% (ꢀ). Ki7 standard error of the mean values are means of 2–3
independent determinations. Diazepam, used as a control, gave a Ki value of 0.007070.0005 μM (n¼5).
f
Anticonvulsant data already reported by Gavernet et al (2009)
Value previously reported by Wasowski et al (2012).
g
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30 mg/Kg), 11 and 13 (at higher doses). The absence of toxic effects
of most structures at this early stage of the evaluation process
supports the use of amino acids as suitable functionalities for the
design of anticonvulsant compounds.
Gavernet et al., 2009).
In relation to Valrocemide (which ED50 in MES test is
755 mol/Kg, according to Isoherranen et al., 2001) compound
μ
9 increases 5 times the antiepileptic action at this preclinical stage.
For that reason, we synthesized other amino acid derived sulfa-
mides that retains the p-fluorobenzyl substituent. We replaced the
According to the biological results, the molecules can be
grouped into one of the four classes proposed to classify the ac-
tivity at the initial evaluation (Malawska et al., 2004): Class 1 in-
volves those compounds with anticonvulsant activity at 100 mg/kg
or less; whereas class 2 represents structures with anticonvulsant
activity at doses higher than 100 mg/kg. Class 3 covers inactive
molecules at any doses up to 300 mg/kg and class 4 implicates
inactive compounds at 300 mg/kg and toxic at 30 mg/kg or less.
Table 3 shows the classification for the new structures. Most of
them belong to class 1, except for compound 3 which presents
anticonvulsant activity but we decided to classify it as class 4 due
to its sedative effects at the lower doses.
β
-alanine moiety of 9 by L-valine and L-phenyl alanine skeleton
(compounds 10 and 11) in order to explore the influence of other
amino acid chains to the activity. Compound 11 showed similar
anticonvulsant profile in MES test than 9 with higher activity at
4 h after administration, but it presented neurotoxic effects.
Structure 10 showed an interesting anticonvulsant action in MES
test with 3/3 mice protected at 30 mg/Kg (at 4 h) and 3/3 mice
protected at 100 mg/Kg (at 0.5 h). However, 75% of the animals
tested with compound 10 against PTZ assay died after the ex-
periment. For that reason we did not complete the biological
profile at 100 mg/Kg.
Sulfamates also showed anticonvulsant activity against MES
test but, according to the preliminary results obtained in this in-
vestigation, they give the impression of having less potency than
sulfamides. However, more biological studies (and more struc-
tures) will be necessary to confirm this hypothesis. Toxicity was
observed for the sulfamate with p-fluorobenzyl substituent.
Regarding the PTZ assays, the new compounds did not show
significant protection against this chemically induced convulsion.
Similar results were found in previous investigations for other
sulfamides and sulfamates (Gavernet et al., 2009, 2007a, 2007b).
As the PTZ test could provide equivocal results in contrast to
clinical studies (Löscher et al., 1991), we included an in vitro test to
evaluate the capacity of the structures to bind the BDZ-bs of the
GABAA receptor in washed crude synaptosomal membranes of rat
cerebral cortex (Table 3). The best results were achieved from
4. Discussion
The anticonvulsant activity found in MES-test justifies further
evaluations for the most promising compounds (i.e. phase 2 of the
ADD program: median effective doses calculations). However, with
the biological results obtained in phase 1 we can arrive to some
preliminary conclusions.
Compound 4 shows a similar profile than its homologous 2 at
the lower dose tested (Gavernet et al., 2009), showing low influ-
ence of the length of the alkyl chain to the activity (at least with
2 more carbon atoms). However, the evidence of sedative effects
found for structure 4 suggests that it is not the most promising
candidate and more experiments might be performed at lower
doses to see if the anticonvulsant action prevails without these
adverse effects.
On the other hand, the branched isomer of 2, compound 5,
presents a better profile than structures 1, 2 and 4, with all animals
protected at 4 h. With these results in mind, an adamantane sul-
famide derivative was also included in the set (compound 6),
which has a very bulky aliphatic substituent near the polar end.
The results at this preliminary phase showed good activity, with
100% protection at the lowest dose tested.
Interesting results were also obtained for the aromatic sulfa-
mide derivatives, compared with the ones previously reported that
showed weak (or none) protection in phase 1 (Gavernet et al.,
2009). Signs of anticonvulsant activity in MES test were detected
compounds 10 and 11; which suggest that -alanine is not the best
β
amino acid to promote the binding to BDZ-bs. Again, more biolo-
gical studies and new structures will be necessary to verify this
hypothesis.
Additionally, we tested the anxiolytic effect of compounds
9 and 11, since previous results from our laboratories demon-
strated the combined anticonvulsant and anxiolytic like activities
of some other sulfamide derivatives (Wasowski et al., 2012).
However, compounds 9 and 11 showed no effect in the plus maze
in mice (1 mg/kg i.p.), revealing no anxiolytic effect (data not
shown).
before only for the methyl [N-(N’benzyl)-sulfamoyl]- -alaninate,
β
which was active at 100 mg/Kg in 1/3 mice tested (Gavernet et al.,
2009). Here we evaluated a homolog of this compound (com-
pound 7), its branched isomer (compound 8) and a p-fluorine-
substituted benzyl sulfamide derivative (compound 9). All new
structures showed protection at the lowest doses (30 mg/Kg).
Based on the results obtained from Phase 1, compounds 5 and
9 were selected for further analysis. Phase 2 of the ADD program
includes the calculations of median effective doses (ED50). ED50
measures the dose of drug that is effective in 50% of the tested
animals. This value is calculated at the time of peak effect (TPE),
which has to be previously identified (Porter et al., 1984). The TPE
was identified for compound 5 (1 h) but no apparent dose–re-
sponse relationship was found, so the ED50 value cannot be cal-
culated. On the contrary, ED50 was determined for 9 with a final
5. Conclusions
The structures tested as anticonvulsants in this investigation
were selected based on one pharmacophoric pattern previously
defined, with the aim of improving the knowledge about the in-
fluence of the second substitution of the sulfamide moiety for the
anticonvulsant action. The results of this investigation support the
strategy of designing active compounds that include the -alanine
β
sulfamide as main structure. All the derivatives tested showed
protection against MES test, confirming the ability of the phar-
macophore model for designing new anticonvulsants compounds.
The set of new structures were obtained after optimizing syn-
thetic methodologies in both chemical reactions and work up
stages, and these results could be useful for future preparations of
new derivatives of the family.
value of 152
interesting, since compound 9 at least duplicates the protection
found for other aromatic sulfamate derivative of -Alanine (2-
hydroxyphenyl-N-
-alanine-methyl ester sulfamate, ED50¼
374 mol/Kg according to Gavernet et al., 2009). However, it is less
active than aliphatic sulfamides of the family (compound 1,
ED50¼79 mol/Kg and compound 2, ED50¼71 mol/Kg;
μmol/Kg (TPE¼4 h). This pharmacological results are
Based on the biological results, we suggest that the second
substitution of the sulfamide function by branched alkyl groups
would increase the anti-MES activity. However more biological
test will be performed in future investigations, to confirm our
hypothesis.
β
β
μ
μ
μ
Regarding the binding to BDZ-bs, some of the compounds
Please cite this article as: Villalba, M.L., et al., Novel sulfamides and sulfamates derived from amino esters: Synthetic studies and
anticonvulsant activity. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.02.001i

M.L. Villalba et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
9
showed medium to low affinity for this receptor active site. We
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onvulsant action of the studied compounds.
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Acknowledgment
L.E. Bruno-Blanch and A. Enrique are members of the Facultad
de Ciencias Exactas, Universidad Nacional de La Plata; L. Gavernet,
M. Marder and I. D. Lick are members of Consejo Nacional de In-
vestigaciones Científicas y Técnicas de la República Argentina
(CONICET); M.L Villalba is a fellowship holder of Agencia Nacional
de Promoción Científica y Tecnológica and J. Higgs from CONICET.
This research was supported in part from Agencia de Promoción
Científica y Tecnológica (PICT 2010-1774, PICT 2013-3175), CON-
ICET, and Universidad Nacional de La Plata, Argentina.
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Please cite this article as: Villalba, M.L., et al., Novel sulfamides and sulfamates derived from amino esters: Synthetic studies and
anticonvulsant activity. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.02.001i