Discovery of sulfonyl hydrazone derivative as a new selective PDE4A and PDE4D inhibitor by lead-optimization approach on the prototype LASSBio-448: In vitro and in vivo preclinical studies

Article abstract of DOI:10.1016/j.ejmech.2020.112492

Phosphodiesterase 4 (PDE4) inhibitors have emerged as a new strategy to treat asthma and other lung inflammatory diseases. Searching for new PDE4 inhibitors, we previously reported the discover of LASSBio-448, a sulfonamide with potential to prevent and reverse pivotal pathological features of asthma. In this paper, two novel series of sulfonamide (6a-6m) and sulfonyl hydrazone (7a-7j) analogues of LASSBio-448 have been synthetized and evaluated for selective inhibitory activity toward cAMP-specific PDE4 isoforms. From these studies, we have identified 7j (LASSBio-1632) as a new anti-asthmatic lead-candidate associated with selective inhibition of PDE4A and PDE4D isoenzymes and blockade of airway hyper-reactivity (AHR) and TNF-α production in the lung tissue. In addition, it was able to relax guinea pig trachea on non-sensitized and sensitized animals and showed great TGI permeability.

Full text of DOI:10.1016/j.ejmech.2020.112492

Journal Pre-proof
Discovery of sulfonyl hydrazone derivative as a new selective PDE4A and PDE4D
inhibitor by lead-optimization approach on the prototype LASSBio-448: In vitro and in
vivo preclinical studies
Isabelle Karine da Costa Nunes, Everton Tenório de Souza, Italo Rossi Roseno
Martins, Gisele Barbosa, Manoel Oliveira de Moraes Junior, Millena de Melo
Medeiros, Sheyla Welma Duarte Silva, Tatiane Luciano Balliano, Bagnólia Araújo da
Silva, Patrícia Machado Rodrigues Silva, Vinicius de Frias Carvalho, Marco Aurélio
Martins, Lidia Moreira Lima
PII:
S0223-5234(20)30464-5
DOI:
https://doi.org/10.1016/j.ejmech.2020.112492
EJMECH 112492
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 March 2020
Revised Date: 9 May 2020
Accepted Date: 19 May 2020
Please cite this article as: I. Karine da Costa Nunes, E. Tenório de Souza, I.R. Roseno Martins, G.
Barbosa, M. Oliveira de Moraes Junior, M. de Melo Medeiros, S.W. Duarte Silva, T.L. Balliano, B. Araújo
da Silva, P.M. Rodrigues, Silva, V. de Frias Carvalho, M.A. Martins, L.M. Lima, Discovery of sulfonyl
hydrazone derivative as a new selective PDE4A and PDE4D inhibitor by lead-optimization approach
on the prototype LASSBio-448: in vitro and in vivo preclinical studies, European Journal of Medicinal
Chemistry, https://doi.org/10.1016/j.ejmech.2020.112492.
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1
Discovery of sulfonyl hydrazone derivative as a new selective
PDE4A and PDE4D inhibitor by lead-optimization approach on
the prototype LASSBio-448: in vitro and in vivo preclinical
studies
1,2,3
2,4
Isabelle Karine da Costa Nunes , Everton Tenório de Souza , Italo Rossi
5,6
1,2
1,2
Roseno Martins , Gisele Barbosa , Manoel Oliveira de Moraes Junior ,
5
8
Millena de Melo Medeiros , Sheyla Welma Duarte Silva , Tatiane Luciano
8
5,7
Balliano , Bagnólia Araújo da Silva , Patrícia Machado Rodrigues and
1,2,4
4
1,2,4*
Silva , Vinicius de Frias Carvalho , Marco Aurélio Martins
, Lidia Moreira
1
,2
Lima *
1
Instituto Nacional de Ciência e Tecnologia de Fármacos e Medicamentos
INCT-INOFAR; http://www.inct-inofar.ccs.ufrj.br/). Universidade Federal do Rio
(
de Janeiro, Laboratório de Avaliação e Síntese de Substâncias Bioativas
LASSBio®, http://www.farmacia.ufrj.br/lassbio/), CCS, Cidade Universitária,
(
P.O. Box 68024, ZIP: 21941-971, Rio de Janeiro-RJ, Brasil
2
Programa de Pós-graduação em Farmacologia e Química Medicinal, Instituto
de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de
Janeiro, RJ, Brasil
3
Laboratório de Apoio ao Desenvolvimento Tecnológico-LADETEC. Instituto de
Química, Universidade Federal do Rio de Janeiro, RJ, Brasil
4
Laboratório de Inflamação, Instituto Oswaldo Cruz-Fiocruz, Fundação Oswaldo
Cruz, Rio de Janeiro, RJ, 21045-900, Brasil
5
Programa de Pós-graduação em Produtos Naturais e Sintéticos Bioativos,
Centro de Ciências da Saúde, Universidade Federal da Paraíba, João Pessoa,
Brasil.
6
Departamento de Medicina, Campus Senador Helvídio Nunes de Barros,
Universidade Federal do Piauí, Picos, Brasil.
7
Departamento de Ciências Farmacêuticas, Centro de Ciências da Saúde,
Universidade Federal da Paraíba, João Pessoa, Brasil.
8
Laboratório de Cristalografia Bioprocessos e Modelagem Molecular –
LaBioCriMM. Instituto de Química e Biotecnologia, Universidade Federal de
Alagoas, AL, Brasil.
*Address for correspondence: Dr. Lídia Moreira Lima, Universidade Federal do Rio de Janeiro, Centro de Ciências
da Saúde, Bloco B, sala 14, P.O. Box 68024, Rio de Janeiro, RJ, Brasil, ZIP Code: 21941-902

2
PH/FAX 55-21-39386503. Email: lidialima@ufrj.br or lmlima23@gmail.com; and Dr. Marco Aurélio Martins,
Laboratório de Inflamação, Instituto Oswaldo Cruz-Fiocruz, Rio de Janeiro, RJ, Brasil, ZIP Code: 21040-360
PH/FAX 55-21-25621358. Email: mam2856@gmail.com
Abstract
Phosphodiesterase 4 (PDE4) inhibitors have emerged as a new strategy to treat
asthma and other lung inflammatory diseases. Searching for new PDE4
inhibitors, we previously reported the discover of LASSBio-448, a sulfonamide
with potential to prevent and reverse pivotal pathological features of asthma. In
this paper, two novel series of sulfonamide (6a-6m) and sulfonyl hydrazone (7a-
7
j) analogues of LASSBio-448 have been synthetized and evaluated for
selective inhibitory activity toward cAMP-specific PDE4 isoforms. From these
studies, we have identified 7j (LASSBio-1632) as a new anti-asthmatic lead-
candidate associated with selective inhibition of PDE4A and PDE4D
isoenzymes and blockade of airway hyper-reactivity (AHR) and TNF-α
production in the lung tissue. In addition, it was able to relax guinea pig trachea
on non-sensitized and sensitized animals and showed great TGI permeability.
Keywords: asthma; PDE4; LASSBio-448; sulfonyl hydrazone; sulfonamide;
lead-optimization

3
1
. Introduction
Phosphodiesterase (PDE) isoenzymes act by promoting the hydrolysis
and subsequent inactivation of the second-messenger molecules cyclic 3',5'-
adenosine monophosphate (cAMP) and cyclic 3',5'-guanosine monophosphate
(cGMP). They have been organized into at least 11 families (PDE1 to PDE11)
based on a variety of criteria including substrate specificity, inhibitor sensitivity
and sequence homogeneity [1]. Each member within the PDE4 subfamily
(PDE4A to PDE4D) specifically targets cAMP and are mainly expressed in
immunocompetent cells (e.g. T cells, monocytes, macrophages, neutrophils,
dendritic cells and eosinophils), brain, cardiovascular tissues, smooth muscles
and keratinocytes [2].
Targeting PDE4 has therapeutic potential as treatment for several
inflammatory conditions, including acute lung injury, chronic obstructive
pulmonary disease (COPD) and asthma, because cAMP intracellular elevation
following PDE4 blockade strongly correlates with the inhibition of innate and
adaptive immune responses which are over activated in these diseases.
Existing evidence indicates that PDE4 inhibition led to accumulation of cAMP
and anti-inflammatory activity in many cell types, i.e., macrophages, T cells,
neutrophils, monocytes, eosinophils and dendritic cells, mediated by several
downstream key elements, such as PKA, cyclic nucleotide-gated ion channels,
and/or Epac 1/2 [3-6].
Several PDE4 inhibitors have been described in literature [7-10] (Chart
1
). Among them roflumilast is approved to treat severe COPD in patients with
chronic bronchitis and frequent exacerbations. Nevertheless, the clinical dosage
and efficacy of roflumilast is limited by target-related side effects, including
nausea, diarrhea, and headaches (Garnock-Jones, 2015) (doi: 10.1007/s40265-
0
15-0463-1). As the most inflammatory cells abundantly express only PDE4A,
PDE4B, and PDE4D isoforms, it is very important to develop new therapeutic
strategies based on the selective inhibition of PDE4 isoforms to reduce or
prevent the adverse effects caused by non-specific PDE4 inhibition. At the time,
several PDE4B and PDE4D inhibitors with potential clinical efficacy and minimal
systemic adverse effects are under development for the treatment of clinical
inflammatory diseases.

4
As part of a research program aimed at the discovery of new anti-
inflammatory drug candidates, we describe here the attempt to optimize the
prototype LASSBio-448 (5, Chart 1), previously reported to inhibit all PDE4
isoforms, though being about 4-fold less potent concerning blockade of PDE4D
as compared to the other three PDE4 subtypes. Furthermore, given orally,
LASSBio-448 prevented airway hyper-reactivity and lung inflammation triggered
by allergen and LPS [10]. In this context, our goals were to synthetize two
series of sulfonamides (6a-6l) and sulfonyl hydrazones (7a-7j), designed as
LASSBio-448 structural analogues, and to assess their PDE4 inhibitory effect,
drug likeness profile and anti-inflammatory activity in a murine model of
pulmonary inflammation [10].
Chart 1. Examples of PDE4 inhibitors bearing different structural patterns. Data
taken from references: [7-10]
The design conception of the sulfonamides (6a-6l) considered molecular
modifications at the structure of the prototype LASSBio-448 (5). The
modifications were based on bioisosteric replacement of benzodioxole ring (a,
Fig. 1) by ring opening and monovalent group exchange strategies [11]. The
replacement of the ethylene (CH CH ) linker by imine (N=CH) unit allowed the
2
2
design conception of sulfonyl hydrazone series (7a-7j). Further, to investigate

5
3
methyl effect [12], on PDE4 inhibitory activity, the homologation of the N sp (by
the introduction of a methyl group) was proposed (Fig. 1).
Fig. 1. Genesis concept of sulfonamides (6a-6l) and sulfonyl hydrazones (7a-7j)
designed by molecular modification on PDE4 inhibitor LASSBio-448 (5).
2
2
2
. Materials and Methods
.1 Synthesis and Characterization
.1.1 Chemical
Reagents and solvents were purchased from commercial suppliers and
used as received. The progress of all reactions was monitored by thin layer
2
chromatography (TLC), which was performed on 2.5 × 7.5 cm aluminum
sheets precoated with silica gel 60 (HF-254, E. Merck) to a thickness of 0.25
mm. The developed chromatograms were viewed under ultraviolet light (254
−1
nm). IR spectra (cm ) were taken on FTLA spectrometer in KBr discs.
Elemental analyses were carried out on a Thermo Scientific Flash EA 1112
Series CHN-Analyzer. HRMS analyses were carried out on a Q-Exactive
Orbitrap mass spectrometer (MS) (Thermo Fisher Scientific, Bremen, Germany)

6
operating by switching between positive and negative ionization modes and
equipped with an Electrospray Ionization (ESI) source.
Analytical HPLC was used for compound purity and stability determinations
using Shimadzu LC-20AD with or Kromasil 100-5C18 (4.6 mm × 250 mm) and a
Shimadzu SPD-M20A detector at 254 nm wavelength. The solvent system used
for HPLC analyses was acetonitrile: water (70:30 and 60:40), with or without of
0
.1% trifluoroacetic acid. The isocratic HPLC mode was used, and the flow rate
was 1.0 mL/min. The purity of compounds was found to be greater than 95%.
1
13
H e C NMR were determined using a 200/50 MHz Bruker DPX-200/DPX-300
or 500/125 MHz Varian 400-Mr spectrometer, respectively, in DMSO or CDCl3
deuterated containing ca. 1% tetramethylsilane as an internal standard. The
peak positions are given in parts per million (δ ppm), and J values are given in
hertz. Signal multiplicities are represented by: s (singlet), d (doublet), t (triplet),
q (quartet), m (multiplet) and br (broad signal). Melting points were determined
with a Quimis 340 apparatus and are uncorrected. The HPLC solvents
(methanol, acetonitrile and dimethylsulfoxide) were purchased from Sigma-
Aldrich (St. Louis, MO, USA). Water used in the preparations has been
previously purified and filtered using a Milli-Q system (Millipore, St Quentin-en-
Yvelines, France).
X-ray difraction was performed from the diffracted intensity
measurements of the samples obtained using the KappaCCD diffractometer
[
KappaCCD Enraf-Nonius] (IQ-UFAL). Data analysis was performed using the
WINGX program.
Magnesium sulfate (MgSO ), calcium chloride (CaCl ), potassium
4
2
chloride (KCl), sodium chloride (NaCl), glucose, sodium bicarbonate (NaHCO3),

7
hydrochloric acid (HCl), potassium monobasic phosphate (KH PO ) and sodium
2
4
hydroxide (NaOH) were purchased from Vetec Química Fina Ltda. Solutions of
these substances, except for glucose, NaCl and NaHCO , were prepared in
3
distilled water and kept under refrigeration.
2
.2. General procedure for the preparation of sulfonamides
Sulfonamides 6a-l were prepared by a condensation reaction between the
corresponding sulfonyl chloride derivative (1mmol), which was solubilized in
dichloromethane (15 mL), containing 50 µL of triethylamine, with the respective
functionalized amines: 3,4-dimethoxyphenethylamine and N-methyl-3,4-
dimethoxyphenethylamine (1 mmol) to obtain 6a-d, 6i-j and 6e-h, 6k-l,
respectively. The mixture was stirred at room temperature for 2-3h. Afterward,
the isolation was carried out with dilution using 10mL of dichloromethane and
extracted with aqueous HCl 10% (four times with 10 ml each time). The organic
phase was dried with addition of anhydrous sodium sulfate, filtered and
concentrated under reduced pressure to obtain the compounds. Yields and
characterization pattern are described below:
2
.2.1.
N-(3,4-dimethoxyphenethyl)-4-nitrobenzenesulfonamide
(LASSBio-1629, 6a)
The title compound was obtained in 59% yield, by condensing 4-
nitrobenzenesulfonyl chloride with 3,4-dimethoxyphenylethylamine, as a yellow
powder with mp 107-109 °C.
-1
IR (KBr) cm : 3261; 3102; 3037; 3000; 2962; 2933; 2869; 2839; 1606; 1595;
1
9
532; 1515; 1448; 1422; 1353; 1342; 1256; 1233; 1167; 1139; 1092; 1024; 974;
45; 902; 854; 817; 765; 741; 682; 614.

8
1
3
3
H NMR (400 MHz, DMSO-d ) δ (ppm): 2.59 (t, 2H, J 7.1 Hz; 3.05 (q, 2H, J
6
3
4
4
7
6
2
.1 Hz; 3.66 (s, 6H); 6.61 (dd, 1H, J 8,1 Hz, J 1.9 Hz); 6.66 (d, 1H, J 1.9 Hz);
3
3
3
.74 (d, 1H, J 8.1 Hz); 7.92 (d, 2H, J 8.9 Hz); 8.03 (t, 1H, J 5.6 Hz); 8.31 (d,
3
H, J 8.9 Hz);
13
C NMR (125 MHz, DMSO-d ) δ (ppm): 35.2; 44.7; 55.7; 55.8; 112.1; 112.9;
6
1
21; 124.8; 128.3; 131.2; 146.7; 147.7; 148.8, 149.7;
CHN: C H N O S; calculated C 52.45%; H 4.95%; N 7.65% and determinated
16
18
2
6
C 52.77%; H 4.97%; N 7.47%.
HPLC: 60/40 acetonitrile/water; λ 254 nm: 98.8% purity.
2
.2.2. N-(3,4-dimethoxyphenetyl)-2-methylbenzenosulfonamide (LASSBio-
1
613, 6b)
The title compound was obtained in 70% yield, by condensing o-toluenesulfonyl
chloride with 3,4-dimethoxyphenethylamine, as a yellow powder with mp 78-80
°
C.
-1
IR (KBr) (cm ): 3277; 2983; 2966; 2928; 2837; 1594; 1517; 1466; 1443; 1428;
1
360; 1347; 1316; 1262; 1235; 1154; 1102; 1029; 940; 894; 851; 816; 164; 779;
13; 686; 632.
7
1
H NMR (200 MHz, DMSO-d ) δ (ppm): 2.49 (s, 3H); 2.56 (t, 2H, J 7.6 Hz);
6
3
4
2
.96 (q, 2H, J 7.6 Hz); 3.67 (s, 3H); 3.68 (s, 3H); 6.57 (dd, 1H, J 8.1 Hz, J 1.9
4
3
Hz); 6.65 (d, 1H, J 1.8 Hz); 6.77 (d, 1H, J 8.1 Hz); 7.33 (m, 2H); 7.48 (td, 1H,
3
4
3
3
4
J 7.4 Hz, J 1.2 Hz); 7.68 (t, 1H, J 5,7 Hz, NH); 7.35 (dd, 1H, J 8.4 Hz, J 1.4
Hz);
13
C NMR (50 MHz, DMSO-d ) δ (ppm): 20.2; 35.3; 44.4; 55.8; 56; 112.3; 112.8;
6
1
20.9; 126.5; 128.8; 131.5; 132.7; 132.9; 137; 139.1; 147.7; 149.

9
CHN: C H NO S; calculated C 60.87%; H 6.31%; N 4.18% and determinated
17
21
4
C 60.68%; H 6.35%; N 4.26%.
HPLC: 60/40 acetonitrile/water; λ 254 nm: 97.3% purity
2
.2.3.
N-(3,4-dimethoxyphenethyl)-4-methoxybenzenesulfonamide
(LASSBio-1625, 6c)
The title compound was obtained in 73% yield, by condensing 4-
methoxybenzenesulfonyl chloride with 3,4-dimethoxyphenylethylamine, as a
yellow powder with mp 90-92 °C.
-1
IR (KBr) cm : 3249; 2969; 2946; 2839; 1608; 1594; 1519; 1497; 1456; 1442;
1
423; 1327; 1307; 1262; 1237; 1158; 1138; 1094; 1027; 890; 875; 837; 802;
15; 764; 724; 684; 632.
8
1
3
3
H NMR (400 MHz, DMSO-d ) δ (ppm): 2.57 (t, 2H, J 7.6 Hz); 2.9 (q, 2H, J
6
3
4
6
.1 Hz); 3.68 (s, 6H); 3.8 (s, 3H); 6.61 (dd, 1H, J 8.1 Hz, J 1.9 Hz); 6.7 (d, 1H,
4
3
3
3
J 1.8 Hz); 6.79 (d, 1H, J 8.1 Hz); 7.06 (d, 2H, J 8.9 Hz); 7.47 (t, 1H, J 5.7
3
Hz); 7.68 (d, 2H, J 8.9 Hz);
13
C NMR (50 MHz, DMSO-d ) δ (ppm): 35.2; 44.7; 55.8; 56; 112.3; 112.9;
6
1
14.7; 120.9; 129.1; 131.6; 147.7; 149; 162.5;
CHN: C H NO S; calculated C 58.10%; H 6.02%; N 3.99% and determinated
17
21
5
C 58.05%; H 5.99%; N 3.95%.
HPLC: 60/40 acetonitrile/water; λ 254 nm: 97% purity.
2
.2.4.
N-(3,4-dimethoxyphenetyl)-3,4-dimethoxybenzenesulfonamide
(LASSBio-1722, 6d)
The title compound was obtained in 52% yield, by condensing 3,4-
dimethoxybenzenesulfonyl chloride with 3,4-dimethoxyphenylethylamine, as a
white powder with mp 83-85 °C.

1
0
-1
IR (KBr) (cm ): 3284; 3083; 2967; 2940; 2923; 2838; 1608; 1590; 1511; 1470;
1
455; 1421; 1406; 1359; 1318; 1263; 1236; 1188; 1139; 1021; 944; 851; 821;
06; 764; 680; 622.
8
1
3
3
H NMR (200 MHz, DMSO-d ) δ (ppm): 2.59 (t, 2H, J 7.2 Hz); 2.92 (q, 2H, J
6
3
6
.9 Hz); 3.7 (s, 6H); 3.8 (s, 3H); 3.82 (s, 3H); 6.63 (d, 1H, J 8.2 Hz); 6.72 (s,
3
3
3
1
8
H); 6.8 (d, 1H, J 8.2 Hz); 7.07 (d, 1H, J 8.4 Hz); 7.27 (s, 1H); 7.35 (d, 1H, J
3
.4 Hz); 7.49 (t, 1H, J 5.4 Hz);
13
C NMR (50 MHz, DMSO-d ) δ (ppm): 35.3; 44.7; 55.9; 56.0; 56.3; 109.9;
6
1
11.6; 112.4; 113.1; 120.7; 121; 131.7; 132.6; 147.8; 149.1; 149.2; 152.3.
CHN: C H NO S; calculated C 56.68%; H 6.08%; N 3.67% and determinated
18
23
6
C 56.56%; H 6.10%; N 3.70%.
HPLC: 60/40 acetonitrile/water; λ 254nm: 97.1% purity.
2
.2.5.
N-(3,4-
dimethoxyphenethyl)-[1,1'-biphenyl]-4-sulfonamide
(LASSBio-1622, 6i)
The title compound was obtained in 70% yield, by condensing 4-biphenyl-4-
sulfonyl chloride with 3,4-dimethoxyphenylethylamine, as a yellow powder with
mp 84-86 °C.
1
3
3
H NMR (400 MHz, DMSO-d ) δ (ppm): 2.63 (t, 2H, J 7.3 Hz); 3.01 (q, 2H, J
6
3
3
6
.5 Hz); 3.68 (s, 6H); 6.65 (d, 1H, J 8.1 Hz); 6.74 (s, 1H); 6.81 (d, 1H, J 8.1
Hz); 7.49 (m, 3H); 7.74 (m, 3H); 7.88 (s, 4H);
13
C NMR (125 MHz, DMSO-d ) δ (ppm): 35.4; 44.8; 55.9; 56.1; 112.4; 113.1;
6
1
21.1; 127.6; 127.7; 127.9; 129; 131.7; 139.1; 139.8; 144.3; 147.9; 149.1;
HPLC: 60/40 acetonitrile/water; λ 254 nm: 98% purity.
+
HRMS (ESI) (m/z) calcd for [C H NO S + H] 398,4953 found 398.14164
22
23
4

1
1
2
.2.6. N-(3,4-dimethoxyphenethyl) naphthalene-2-sulfonamide (LASSBio-
1
611, 6j)
The title compound was obtained in 70% yield, by condensing 2-
naphthalenesulfonyl chloride with 3,4-dimethoxyphenylethylamine, as a yellow
powder with mp 84-86 °C.
-1
IR (KBr) cm : 3284; 3071; 3059; 2997; 2965; 2943; 2921; 2874; 2838; 1605;
1
591; 1521; 1472; 1421; 1353; 1318; 1262; 1236; 1157; 1145; 1134; 1073;
019; 954; 936; 863,816; 757; 677; 637; 621.
1
1
3
3
H NMR (400 MHz, DMSO-d ) δ (ppm): 2.58 (t, 2H, J 7.2 Hz); 2.98 (q, 2H, J
6
3
4
7
.1 Hz); 3.6 (s, 6H); 6.6 (dd, 1H, J 8.1 Hz, J 1.8 Hz); 6.68 (s, 1H); 6.75 (d, 1H,
3
3
3
J 8.1 Hz); 7.66 (m, 2H); 7.77 (m, 2H); 8.01 (d, 1H, J 7.9 Hz); 8.09 (d, 1H, J
3
8
.9 Hz); 8.11 (d, 1H, J 7.7 Hz); 8.4 (s,1H);
13
C NMR (50 MHz, DMSO-d ) δ (ppm): 35.3; 44.7; 55.8; 55.9; 112.2; 112.9;
6
1
1
20.9; 122.7; 127.7; 128; 128.25; 129.1; 129.6; 129.8; 131.5; 132.2; 134.5;
37.9; 147.7; 149.1;
CHN: C H NO S; calculated 56.68%; H 6.08%; N 3.67% and determinated C
20
21
4
5
6.48%; H 6.06%; N 3.66%.
HPLC: 60/40 acetonitrile/water; λ 254 nm: 96.3% purity.
.2.7. N'-(3,4-dimethoxyphenethyl)-N-methyl-4-nitrobenzenesulfonamide
LASSBio-1630, 6e)
2
(
The title compound was obtained in 71% yield, by condensing 4-
nitrobenzenesulfonyl chloride with N-methyl-3,4-dimethoxyphenethylamine, as a
white powder with mp 139-141°C.

1
2
-1
IR (KBr) cm : 3104; 2976; 2963; 2858; 2839; 1608; 1591; 1532; 1518; 1467;
1
7
453; 1422; 1349; 1311; 1166; 1147; 1116; 1023; 941; 927; 856; 819; 782; 765;
37; 705; 684; 617; 602.
1
3
H NMR (400 MHz, DMSO-d ) δ (ppm): 2.72 (t, 2H, J 7.6 Hz); 2.78 (s, 3H);
6
3
3
4
3
1
8
.27 (t, 2H, J 7.7 Hz); 3.70 (s, 6H); 6.71 (dd, 1H, J 8.1 Hz, J 1.9 Hz); 6.77 (d,
4
3
3
3
H, J 1.9 Hz); 6.82 (d, 2H, J 8.1 Hz); 7.98 (d, 2H, J 8.8 Hz); 8.36 (d, 2H, J
.8 Hz);
13
C NMR (50 MHz, DMSO-d ) δ (ppm): 33.5; 34.92; 51.60; 55.8; 55.9; 112.2;
6
1
13; 121.1; 125; 128.9; 131; 143.5; 147.8; 149.0, 150.1;
CHN: C H N O S; calculated C 53.67%; H 5.30%; N 7.36% and determinated
17
23
2
6
C 53.93%; H 5.32%; N 7.21%.
HPLC: 60/40 acetonitrile/water; λ 254 nm: 97.8 % purity.
2
.2.8. N'- (3,4-dimethoxyphenethyl) -N, 2-methylbenzenesulfonamide
(LASSBio-1623, 6f)
The title compound was obtained in 68% yield, by condensing o-toluenesulfonyl
chloride with N-methyl-3,4-dimethoxyphenethylamine, as a white powder with
mp 54-55 °C.
-1
IR (KBr) cm : 3080; 2970; 2949; 2936; 2917; 2834; 1605; 1590; 1515; 1464;
1
443; 1417; 1359; 1309; 1263; 1236; 1156; 1141; 1132; 1062; 1025; 950; 933;
56; 814; 788; 789; 755; 676; 630.
8
1
H NMR (400 MHz, DMSO-d ) δ (ppm): 2.43 (s, 3H); 2.71 (t, 2H, J 7.8 Hz);
6
3
2
.78 (s, 3H); 3.3 (q, 2H, J 7.8 Hz); 3.68 (s, 3H); 3.7 (s, 3H); 6.54 (dd, 1H, J 8.1
4
4
3
Hz, J 1.9 Hz); 6.72 (d, 1H, J 1.8 Hz); 6.8 (d, 1H, J 8.1 Hz); 7.37 (m, 2H); 7.5
3
3
(
m, 1H); 7.68 (t, 1H, J 5.7 Hz); 7.75 (d, 1H, J 7.8 Hz);

1
3
13
C NMR (50 MHz, DMSO-d ) δ (ppm): 20.3; 33.6; 34.2; 51.1; 55.8; 56.0;
6
1
12.3; 112.8; 120.9; 126.7; 129.2; 131.2; 133.2; 137.4; 137.4; 147.8; 149.1;
CHN: C H NO S; calculated C 61.87%; H 6.63%; N 4.01% and determinated
18
23
4
C 62.04%; H 5.99%; N 4.09%.
HPLC: 60/40 acetonitrile/water; λ 254 nm: 97.7% purity.
2
.2.9.
N'-(3,4-dimethoxyphenethyl)-4-methoxy-N-methylbenzene-
sulfonamide (LASSBio-1628, 6g)
The title compound was obtained in 78% yield, by condensing 4-
methoxybenzenesulfonyl chloride with N-methyl-3,4-dimethoxyphenethylamine,
as a white powder with mp 70-72°C.
-1
IR (KBr) cm : 3040; 2943; 2923; 2845; 2830; 1596; 1579; 1518; 1496; 1464;
1
8
449; 1422; 1336; 1298; 1255; 1236; 1152; 1109; 1090; 1028; 1022; 956; 833;
15; 756; 707; 658; 631.
1
3
H NMR (400 MHz, DMSO-d ) δ (ppm): 2.64 (s, 3H); 2.68 (t, 2H, J 7.9 Hz); 3.1
6
3
3
4
(
t, 2H, J 8 Hz); 3.7 (s, 6H); 3.82 (s, 3H); 6.7 (dd, 1H, J 8.1 Hz, J 1.3 Hz); 6.78
3
3
3
(
s, 1H); 6.83 (d, 1H, J 8.1 Hz); 7.09 (d, 2H, J 8.7 Hz); 7.66 (d, 2H, J 8.7 Hz);
13
C NMR (50 MHz, DMSO-d ) δ (ppm): 33.5; 35.1; 51.6; 55.8; 55.9; 56.1;
6
1
12.3; 113; 114.9; 121; 129.1; 129.7; 131.3; 147.8; 149.1, 162.9;
CHN: C H NO S; calculated C 59.16%; H 6.34%; N 4.01% and determinated
18
23
5
C, 59.16%; H 6.34%; N 4.09%.
HPLC: 60/40 acetonitrile/water; λ 254 nm: 98.7 % purity.
2
.2.10.
N'-(3,4-dimethoxyphenethyl)-3,4-dimethoxy-N-methylbenzene-
sulfonamide (LASSBio-1610, 6h)

1
4
The title compound was obtained in 70% yield, by condensing 3,4-
dimethoxybenzenesulfonyl chloride with N-methyl-3,4-
dimethoxyphenethylamine, as a white powder with mp 78-80°C.
-1
IR (KBr) cm : 3114; 3087; 2996; 2959; 2933; 2836; 1588; 1517; 1507; 1465;
1
454; 1403; 1424; 1374; 1326; 1261;1145; 806; 721; 699; 654; 578.
1
3
H NMR (400 MHz, DMSO-d ) δ (ppm): 2.7 (m, 5H); 3.15 (t, 2H, J 7.4 Hz);
6
3
3
.72 (s, 6H); 3.82 (s, 6H); 6.71 (d, 1H, J 8 Hz); 6.81-6.87 (m, 2H); 7.1-1.19 (m,
3
2
H); 7.33 (d, 1H, J 8.4 Hz);
13
C NMR (50 MHz, DMSO-d ) δ (ppm): 33.6; 35.2; 51.6; 55.9; 56.0; 56.3; 56.4;
6
1
1
10.4; 111.9; 112.4; 113.2; 121.1; 121.4; 129.3; 131.5; 147.9; 149.2; 149.3;
52.8;
HPLC: 60/40 acetonitrile/water; λ 254 nm: 96.1% purity.
+
HRMS (ESI) (m/z) calcd for [C H NO S + H] 396,4778 found 396.14703
19
25
6
2
.2.11.
N'-(3,4-dimethoxyphenethyl)-N-methyl-[1,1'-biphenyl]-4-
sulfonamide (LASSBio-1631, 6k)
The title compound was obtained in 55% yield, by condensing biphenyl-4-
sulfonyl chloride with N-methyl-3,4-dimethoxyphenethylamine, as a white
powder with mp 68-70°C.
-1
IR (KBr) cm : 3049; 3026; 2992; 2953; 2935; 2873; 2834; 1609; 1591; 1516;
1
1
460; 1449; 1363; 1335; 1301; 1258; 1245; 1158; 1139; 1127; 1091; 1027;
015; 996; 947; 924; 884; 864; 834; 807; 763; 772; 725; 704; 696; 661; 636.
1
3
H NMR (400 MHz, DMSO-d ) δ (ppm): 2.72 (m, 5H); 3.2 (t, 2H, J 7.9 Hz); 3.7
6
3
4
4
3
(
s, 6H); 6.72 (dd, 1H, J 8.1 Hz, J 1.9 Hz); 6.8 (d, 1H, J 1.9 Hz); 6.83 (d, 1H, J
8
.1 Hz); 7.42 (m, 1H); 7.49 (m, 2H); 7.71 (m, 2H); 7.79 (m, 2H); 7.87 (m, 2H);

1
5
13
C NMR (50 MHz, DMSO-d ) δ (ppm): 33.6; 35.1; 51.66; 55.8; 55.9; 112.3;
6
1
1
13.1; 121.1; 127.5; 127.9; 128.2; 129; 129.6; 131.3; 136.4; 138.9; 144.7;
47.8; 149.1;
HPLC: 60/40 acetonitrile/water; λ 254 nm: 97 % purity.
+
HRMS (ESI) (m/z) calcd for [C H NO4S + H] 412,5218 found 412.15733
23
25
2
.2.12. N'-(3,4-dimethoxyphenethyl)-N-methylnaphthalene-2-sulfonamide
(LASSBio-1612, 6l)
The title compound was obtained in 73% yield, by condensing 2-
naphthalenesulfonyl chloride with N-methyl-3,4-dimethoxyphenethylamine, as a
white powder with mp 78-80°C.
-1
IR (KBr) cm : 3049; 2993; 2933; 2834; 1606; 1590; 1517; 1464; 1455; 1439;
1
8
421; 1337; 1290; 1247; 1234; 1196; 1149; 1129; 1122; 1073; 1028; 946; 927;
54; 821; 747; 716; 653; 637; 618.
1
3
H NMR (400 MHz, DMSO-d ) δ (ppm): 2.72 (m, 5H); 3.2 (t, 2H, J 7.9 Hz); 3.7
6
3
4
4
(
s, 6H); 6.71 (dd, 1H, J 8.1 Hz, J 1.8 Hz); 6.83 (d, 1H, J 1.8 Hz); 6.84 (d, 1H,
3
3
3
J 8.1 Hz); 7.43 (t, 1H, J 7.3 Hz); 7.5 (t, 2H, J 7.1 Hz); 7.72 (); 7.8 (m, 2H);
3
7
.87 (d, 1H, J 8.6 Hz);
13
C NMR (50 MHz, DMSO-d ) δ (ppm): 33.9; 35.1; 51.6; 55.8; 55.9; 112.3;
6
1
1
13.1; 121.1; 127.5; 128; 128.2; 129; 129.6; 131.3; 136.4; 138.8; 144.7; 147.7;
49.1;
HPLC: 60/40 acetonitrile/water; λ 254 nm: 97.5% purity.
+
HRMS (ESI) (m/z) calcd for [C H NO S + H] 386,4846 found 386.14172
21
23
4
2
.3. Procedure to prepare of sulfonyl hydrazides (9a-e)

1
6
The key intermediates sulfonyl hydrazides 9a and 9c-b were obtained in
accordance to the methodology previously described in the literature [13,14,15].
2
.3.1. 6-methyl-3,4-methylenedioxy-benzenesulfonyl hydrazide (9a)
The title compound was obtained in 84% yield, by reaction with 6-methyl-3,4-
methylenedioxy-benzenesulfonyl chloride and hydrazine hydrate, as a white
powder with mp 121-123°C [13].
2
.3.2. 2-methylbenzenesulfonyl hydrazide intermediate (9b)
The title compound was obtained in 92% yield, by reaction with 2-
methylbenzene-1-sulfonyl chloride and hydrazine hydrate, as a colorless oil with
mp 144-146°C [14,15].
2
.3.3. 4-methoxybenzenesulfonyl hydrazide (9c)
The title compound was obtained in 83% yield, by reaction with 4-
methoxybenzene-1-sulfonyl chloride and hydrazine hydrate, as a white powder
with mp 150-152°C [14,15].
2
.3.4. 4-(trifluoromethyl)benzenesulfonyl hydrazide (9d)
The title compound was obtained in 71% yield, by reaction with 4-
trifluoromethyl) benzenesulfonyl chloride and hydrazine hydrate, as a white
powder with mp 149-151°C [14,15].
.3.5. Benzenesulfonyl hydrazide intermediate (9e)
(
2
The title compound was obtained in 98% yield, by reaction with benzenesulfonyl
chloride and hydrazine hydrate, as a white powder with mp 138-140°C [14,15].
2
.4. General procedure for the synthesis of sulfonyl hydrazones and N-
methyl sulfonyl hydrazones
The sulfonyl hydrones 7a-e were prepared by interconversion of
functional groups as previously described by Zapata-Sudo and coworkers [14].

1
7
Then, a regioselective alkylation reaction was performed in the presence of
K CO (1.91 mmol), acetone (5mL) as solvent and methyl iodide (1.91 mmol) as
2
3
alkyl halide. The reaction was heated at 40 °C and maintained under stirring for
.5h under reflux. Afterward, the reaction was evaporated under reduced
1
pressure; the residual solid was suspended in 2 mL of ethanol and then poured
into cold water. Purification was achieved by simple filtration to obtain the N-
methyl target compounds 7f-j [14]. Yields and characterization patterns are
described below:
2
.4.1. (E) -N’- (3,4-dimethoxybenzylidene)-6-methylbenzo [d] [1,3] dioxole-
-sulfonyl hydrazone (LASSBio-1624, 7a)
The title compound was obtained in 72% yield, as a white powder with mp 178-
80 °C.
IR (KBr) cm : 3201; 3122; 3084; 3044; 2980; 2952; 2912; 2845; 1612; 1601;
5
1
-1
1
1
580; 1515; 1498; 1480; 1441; 1424; 1350; 1334; 1321; 1269; 1249; 1233;
162; 1145; 1043; 1021; 992; 957; 930; 909; 887; 867; 816; 774; 748; 730; 705;
58; 625; 615.
6
1
H NMR (200 MHz, DMSO-d ) δ (ppm): 2.55 (s, 3H); 3.72 (s, 3H); 3.74 (s, 3H);
6
3
6
.08 (s, 2H); 6.94 (m, 2H); 7.04 (d, 1H, J 8.2 Hz); 7.08 (s, 1H); 7.32 (s, 1H);
7
.83 (s, 1H); 11.3 (s, 1H).
13
C NMR (50 MHz, DMSO-d ) δ (ppm): 20.7; 55.9; 56.1; 102.8; 109.1; 109.5;
6
1
27.1; 130.6; 133.8; 146.0; 149.5; 151.1; 151.3.
CHN: C H N O S; calculated C 53.96%; H 4.79%; N 7.40% and determinated
17
18
2
6
C 53.71%; H 4.67%; N 7.50%.
HPLC: 60/40 acetonitrile/water; λ 306 nm: 99.0 % purity.

1
8
2
.4.2.
(E)
-N'-
(3,4-dimethoxybenzylidene)-2-methylbenzenesulfonyl
hydrazone (LASSBio-1847, 7b)
The title compound was obtained in 70% yield, as a yellow powder with mp 141-
1
43 °C.
-1
IR (KBr) cm : 3184; 3077; 3065; 3004; 2983; 2937; 2839; 1600; 1580; 1517;
1
1
462; 1440; 1410; 1369; 1360; 1320; 1262; 1240; 1177; 1166; 1131; 1060;
043; 1021;1016; 898; 873; 821; 809; 763; 707; 694; 623; 607.
1
H NMR (200 MHz, CDCl ) δ (ppm): 2.76 (s, 3H); 3.86 (s, 3H); 3.88 (s, 3H); 6.8
3
3
3
(
d, 1H, J 8.2 Hz); 6.96 (d, 1H, J 8.2 Hz); 7.18 (s, 1H); 7.33 (m, 2H); 7.46 (d,
3
3
1
H, J 8.6 Hz); 7.72 (s, 1H); 7.98 (s, 1H); 8.08 (d, 1H, J 7.4 Hz).
13
C NMR (50 MHz, CDCl ) δ (ppm): 21.0; 56.0; 56.1; 108.4; 110.7; 122.4;
3
1
26.4; 126.5; 130.9; 132.8; 133.5; 136.9; 138.2; 147.7; 149.5; 151.4.
CHN: C H N O S; calculated C 57.47%; H 5.43%; N 8.83%and determinated
16
18
2
4
C 57.41%; H 5.31%; N 8.43%.
HPLC: 60/40 acetonitrile/water; λ 306 nm: 98.5 % purity.
2
.4.3. (E) -N'- (3,4-dimethoxybenzylidene) -4-methoxybenzenesulfonyl
hydrazone (LASSBio-1845, 7c).
The title compound was obtained in 70% yield, as a yellow powder with mp 141-
1
43 °C.
-1
IR (KBr) cm : 3198; 3098; 3082; 3085; 3013; 2977; 2943; 2839; 1598; 1578;
1
1
521; 1499; 1453; 1429; 1407; 1353; 1317; 1271; 1263; 1242; 1162; 1132;
095; 1021; 970; 941; 901; 837; 803; 781; 765; 703; 629; 615.

1
9
1
3
H NMR (200 MHz, CDCl ) δ (ppm): 3.82 (s, 3H); 3.86 (s, 6H); 6.79 (d, 1H, J
3
3
8
1
.2 Hz); 6.96 (m, 3H); 7.2 (s, 1H); 7.75 (s, 1H); 7.92 (d, 2H, J 8.9 Hz), 8.31 (s,
H).
13
C NMR (50 MHz, CDCl ) δ (ppm): 55.7; 56.0; 56.1; 108.7; 110.8; 114; 122.3;
3
1
26.5; 130; 130.2; 148.7;
CHN: C H N O S; calculated C 54.85%; H 5.18%; N 7.99% and determinated
16
18
2
5
C 54.74%; H 5.11%; N 8.00%.
HPLC: 60/40 acetonitrile/water; λ 306 nm: 99.4 % purity.
2
.4.4. N'-(3,4-dimethoxybenzylidene)-4-(trifluoromethyl) benzenesulfonyl
hydrazide (LASSBio-1849, 7d).
The title compound was obtained in 66% yield, as a white powder with mp 87-
8
9°C.
1
3
H NMR (200 MHz, CDCl ) δ (ppm): 3.87 (s, 6H); 6.81 (d, 1H, J 8.1 Hz); 7.02
3
3
3
(
d, 1H, J 8 Hz); 7.2 (s, 1H); 7.77 (m, 3H); 8.13 (d, 2H, J 8.1 Hz); 8.39 (s, 1H).
13
C NMR (50 MHz, CDCl ) δ (ppm): 56.1; 56.2; 108.6; 110.9; 120.6; 122.6;
3
2
1
1
1
26; 126.3 (q, J 3.6 Hz); 128.6; 135.1 (q, J 33 Hz); 138.8; 142.1; 149.5;
CF CF
51.7.
HPLC: 60/40 acetonitrile/water; λ 306 nm: 99.6 % purity.
+
HRMS (ESI) (m/z) calcd for [C H F N O S + H] 389,3694 found 389.07748
16
15
3
2
4
2
.4.5. (E) -N'- (3,4-dimethoxybenzylidene) benzenesulfonyl hydrazone
LASSBio-1851, 7e).
The title compound was obtained in 66% yield, as a white powder with mp 87-
9 °C.
(
8

2
0
1
3
H NMR (200 MHz, CDCl ) δ (ppm): 3.77 (s, 6H); 6.71 (d, 1H); 6.92 (d, 1H, J
3
3
8
1
.2 Hz); 7.1 (s, 1H); 7.44 (m, 3H); 7.68 (s, 1H); 7.92 (d, 2H, J 8.1 Hz); 8.39 (s,
H).
13
C NMR (50 MHz, CDCl ) δ (ppm): 56.0; 108.5; 110.8; 122.4; 126.4; 128;
3
1
29.1; 133.4; 138.5; 148.8; 149.4; 151.4.
CHN: C H N O S; calculated C 56.24%; H 5.03%; N 8.74% and determinated
15
16
2
4
C 56.27%; H 5.04%; N 8.96%.
HPLC: 60/40 acetonitrile/water; λ 306 nm: 99.5 % purity
2
.4.6. (E) -N’- (3,4-dimethoxybenzylidene)-N, 6-methylbenzo [d] [1,3]
dioxole-5-sulfonyl hydrazone (LASSBio-1632, 7j)
The title compound was obtained in 64% yield, as a white powder with mp 156-
1
58 °C.
1
H NMR (200 MHz, CDCl ) δ (ppm): 2.55 (s, 3H); 3.23 (s, 3H); 3.81 (s,s, 6H);
3
5
.93 (s, 2H); 6.62 (s, 1H); 6.74-6.76 (d, 1H); 6.93-6.95 (d, 1H); 7.17 (s, 1H);
.40 (s, 1H); 7.45 (s, 1H).
7
13
C NMR (50 MHz, CDCl ) δ (ppm): 21.1; 32.5; 55.7-55.9; 102.1; 106.1 –
3
1
51.3.
CHN: C H N O S; calculated C 55.09%; H 5.14%; N 7.14% and determinated
18
20
2
6
C 55.17%; H 5.14%; N 7.22%.
HPLC: 60/40 acetonitrile/water; λ 306 nm: 98% purity.
2
.4.7.
N'-(3,4-dimethoxybenzylidene)-N,2-methylbenzenesulfonyl
hydrazone (LASSBio-1848, 7f).
The title compound was obtained in 70% yield, as a white powder with mp 84-
8
6 °C.

2
1
1
H NMR (200 MHz, CDCl ) δ (ppm): 2.64 (s, 3H); 3.24 (s, 3H); 3.77 (s, 3H); 3.8
3
(s, 3H); 6.73 (d, 1H, J 7 Hz); 6.92 (d, 1H, J 7 Hz); 7.1 (s, 1H, J 7 Hz); 7.21 (m,
2
H); 7.35 (d, 1H, J 7 Hz); 7.44 (s, 1H); 7.92 (d, 1H, J 8 Hz).
13
C NMR (50 MHz, CDCl ) δ (ppm): 21.1; 32.4; 55.8; 56.1; 108.2; 110.8; 122;
3
1
26.1; 127.5; 130.8; 132.8; 133.2; 135.9; 138.8; 142.1; 149.4; 150.9.
CHN: C H N O S; calculated C 58.60%; H 5.79%; N 8,04% and determinated
17
20
2
4
C 58.31%; H 5.86%; N 8.18%.
HPLC: 60/40 acetonitrile/water; λ 306 nm: 99.6 % purity
2
.4.8. N'-(3,4-dimethoxybenzylidene)-4-methoxy-N-methylbenzene-sulfonyl
hydrazone (LASSBio-1846, 7g).
The title compound was obtained in 70% yield, as a white powder with mp 141-
1
43 °C.
1
H NMR (200 MHz, CDCl ) δ (ppm): 3.18 (s, 3H); 3.84 (s, 3H); 3.9 (s, 3H); 3.93
3
(s, 3H); 6.85 (d, 1H, J 7 Hz ); 6.96 (d, 2H, J 7 Hz); 7.07 (d, 1H, J 7 Hz); 7.29 (s,
1
H); 7.61 (s, 1H); 7.83 (d, 2H, J 7.8 Hz).
13
C NMR (50 MHz, CDCl ) δ (ppm): 34.1; 55.7; 56.0; 108.7; 110.8; 114.1;
3
1
22.1; 127.4; 128; 130.5; 145.5; 149.9; 151.1; 163.4.
CHN: C H N O S; calculated C 56.03%; H 5.53%; N 7.69% and determinated
17
20
3
4
C 55.73%; H 5.52%; N 7.69%.
HPLC: 60/40 acetonitrile/water; λ 306 nm: 99.6 % purity
2
.4.9.
trifluoromethyl)benzenesulfonyl hydrazone (LASSBio-1850, 7h).
The title compound was obtained in 66% yield, as a white powder with mp 87-
9 °C.
N'-(3,4-dimethoxybenzylidene)-4-(trifluoromethyl)-N--methyl-4-
(
8

2
2
1
H NMR (200 MHz, CDCl ) δ (ppm): 3.15 (s, 1H); 3.83 (s, 3H); 3.84 (s, 3H);
3
3
3
6
.78 (d, 1H, J 8.2 Hz); 7.01 (d, 1H, J 8.2 Hz); 7.17 (s, 1H); 7.57 (s, 1H); 7.69
3
3
(
d, 2H, J 8.1 Hz); 7.95 (d, 2H, J 8.1 Hz).
13
C NMR (50 MHz, CDCl ) δ (ppm): 34.1; 56.1; 56.1; 108.7; 111; 120.6; 122.3;
3
2
1
1
1
26 (q, J 3.6 Hz); 126.9; 129; 134.8 (q, J 33 Hz); 140.1; 146.7; 149.5;
CF CF
51.5.
CHN: C H F N O S; calculated C 50.74%; H 4.26%; N 6.96% and
17
17
3
2
4
determinated C 50.53%; H 4.31%; N 7.04%.
HPLC: 60/40 acetonitrile/water; λ 306 nm: 98.5 % purity.
2
.4.10.
N'-(3,4-dimethoxybenzylidene)-N-methylbenzenesulfonyl
hydrazone (LASSBio-1852, 7i).
The title compound was obtained in 60% yield, as a white powder with mp 108-
1
10 °C.
1
H NMR (200 MHz, CDCl ) δ (ppm): 3.13 (s, 3H); 3.83 (s, 3H); 3.85 (s, 3H);
3
3
3
6
.77 (d, 1H, J 8.2 Hz); 7.01 (d, 1H, J 8.2 Hz); 7.2 (s, 1H); 7.44 (m, 3H); 7.54
3
(
s, 1H); 7.82 (d, 2H, J 8.1 Hz).
13
C NMR (50 MHz, CDCl ) δ (ppm): 34.1; 56.1; 56.1; 108.7; 110.9; 122.2;
3
1
27.3; 128.4; 128.9; 133.3; 136.6; 145.7; 149.5; 151.2.
HPLC: 60/40 acetonitrile/water; λ 306 nm: 99% purity.
+
HRMS (ESI) (m/z) calcd for [C H N O S + H] 335,3981 found 335.10567
16
18
2
4
2
.5. X-ray diffraction experiments
The crystals were obtained by the slow evaporation method from 7j and
a under a saturating condition at 298K. After X days, a suitable yellow single
7
crystal was observed for both samples.

2
3
Intensity data were collected at room temperature (293K) with graphite
monochromated MoKa radiation (l=0.71073 Å), using an Enraf-Nonius Kappa-
CCD diffractometer. The unit cell was refined using the software Collect [36]
and Scalepack [37], and the final cell parameters were obtained on all
°
reflections. Datawere collected up to 24.41° in θ for Lassbio-1624 and 27.88 for
7
j in θ, giving 12,489 and 16, 238 Bragg reflections respectively. Data reduction
was carried out using the software Denzo-SMN and Scalepack, and the
program XdisplayF was used for visual representation of the data. No significant
-1
-1
absorption coefficient (0.227 mm for 7a and 0.211 mm for 7a) was observed.
Thus, a semi-empirical absorption correction based on equivalents was applied
[
38]. The structure was solved using the software SHELXS-97 [39], and refined
using the software SHELXL-97, where the C, N and O atoms were clearly
solved, and full-matrix least square refinement of these atoms with anisotropic
thermal parameters was carried out. The hydrogen atoms were positioned
stereochemically and were refined with the riding model. The details concerning
the data collection and structure refinement were prepared using WinGX
(
version 1.80.05) [40]. The ORTEP-3 [41] program was used to prepare the
figures.
The complete crystallographic data were deposited at the Cambridge
Crystallographic Data Centre as supplementary publication no. CCDC 1944285
and no. CCDC 1944286 for LASSBio-1624 and LASSBio-1632 respectively.
Copies of the data are available on application to The Director, CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK (e-mail: deposit@ccdc.cam.ac.uk or
http://www.ccdc.cam.ac.uk).
2
.6. Solubility Assay

2
4
The solubility assay was performed considering the absorptivity of compounds
under ultraviolet spectroscopy as described by Schneider and coworkers. The
assay wavelength was determined by the λmax characteristic of each
compound. Saturated aqueous solutions were prepared (0.8-1.0 mg/mL) and
were kept under stirring for 4 hours at 37 °C. The supernatant was filtered in
0
.45 mm filters and transferred to a quartz cuvette (10 mm) to spectra
acquisition. Solubility was determined by linear regression using as excel graph
plots, solutions prepared by dilutions of the original solution in methanol. The
data were obtained in triplicates and the mean values were used to the graph
2
plots. The correlation coefficient (R ) values were between 0.9982 and 1.
2
.7. Chemical Stability Assay
The chemical stability studies were conducted at two different pH (2.0
and 7.4). The stock solutions of compounds were prepared at 5 mM to 10 mM
concentrations and solubilized in DMSO. Standard solutions were prepared
adding 2 μL of the stock solution in 249 μL acid buffer (KCl 0.2 M and HCl 0.2
M; pH = 2.0) or neutral buffer (PBS, pH = 7.4) in eppendorf microtube. The
mixture was placed in a water bath at 37 °C under vigorous stirring for 0, 30, 60
and 120 minutes. At the end of each time reaction, 249 µL of basic buffer
(phosphate buffer, pH = 8.4) was added to neutralize the pH of the medium in
experiments using acidic buffer. Extraction of the compound was performed by
adding 1.0 mL of acetonitrile. The organic phase was separated, filtered and
analyzed by HPLC-PDA (acetonitrile / water mobile phase and 50% to 60%).
2
2
.8. Pharmacological Evaluation
.8.1 Animals

2
5
Male A/J mice (18-20 g) were obtained from the Oswaldo Cruz
Foundation breeding colony and used in accordance with the guidelines of the
Committee on Use of Laboratory Animals of the Oswaldo Cruz Institute (CEUA-
IOC/FIOCRUZ, license L-027/2016). Mice were housed in groups of four in a
temperature-, humidity-, and light-controlled (12 h light: 12 h darkness cycle)
colony room. Mice were given ad libitum access to food and water.
Adult guinea-pigs (Cavia porcellus) from the Professor Thomas George
Bioterium at Federal University of Paraíba (UFPB), weighing 300 – 500 g, were
used. Animals were maintained on a 12-h light-dark cycle, under controlled
ventilation and temperature with free access to food and water. Actions on
reducing pain, stress, and any suffering were taken in accordance to the local
ethical guidelines for animal usage. All experimental procedures had been
approved and performed in accordance to the Animal Research Ethic
Committee of UFPB guidelines (CEPA/UFPB license 0610/11).
2
.8.2. Drugs
Methacholine, LPS (Escherichia coli, serotype 0127:B8) and nembutal
were purchased from Sigma Chemical Co. Pancuronium bromide, isoflurane
and sodium thiopental were purchased from Cristalia (São Paulo, Brazil). LPS,
pancuronium bromide, nembutal and sodium thiopental were diluted in 0.9%
NaCl sterile solution. Methacholine was diluted in PBS. All work solutions were
freshly prepared by dissolving the compounds in DMSO 0.1% and further
diluting them in 0.9% NaCl. All solutions were freshly prepared immediately
before use.
Arachidonic acid (AA), carbamylcholine hydrochloride (CCh), ovalbumin
(
OVA) (grade V), rolipram (1), aluminum hydroxide (Al(OH) ) and Cremophor
3

2
6
TM
EL
were purchased from Sigma-Aldrich (Brazil). CCh was dissolved in
distilled water and AA was dissolved in ethanol (95%). Compounds were
solubilized in Cremophor EL™ (3%) and distilled water at the concentration of
-2
1
0 M, being again diluted in distilled water as required for each experimental
protocol. The final content of Cremophor EL™ in the organ solution never
exceeded 0.01% (v/v). The carbogen mixture (95% O and 5% CO ) was
2
2
acquired from White Martins (Brazil).
.8.3. PDE4 activity evaluation in vitro
PDE4A, 4B, 4C and 4D activities were measured by employing an IMAP
2
TR-FRET protocol (kit from Molecular Devices, Sunnyvale, CA, USA). Briefly,
the enzymatic reactions were carried at room temperature in a 96-well black
plate by co-incubating 25 μL of 200 nM FAM-cAMP (R7513), 5 μL of putative
inhibitory compounds and 20 μL of the PDE4 isoform dissolved in assay buffer
(
R7364) for 1 h. All enzymes were obtained from human recombinant sources
MDS PHARMA), whereas the other reagents were purchased from Molecular
(
Devices. Fluorescence polarization intensity was measured at 485 nm
excitation and 520 nm emission using a microplate reader, SpectraMax M5
(Molecular Devices, Sunnyvale, CA, USA). Cilomilast was dissolved in dimethyl
sulfoxide (DMSO) at a final concentration of 0.1%. At this condition, the vehicle
had no significant effect on PDE4 activity. The concentration of drugs that
produced 50% inhibition of substrate hydrolysis (IC ) was calculated by
50
nonlinear regression analysis from concentration response curves, using the
GraphPad Prism Software, version 5.0 (USA).
2
.8.4. LPS-induced lung inflammation

2
7
Mice were anesthetized with isoflurane aerosol and then stimulated with
LPS (25μg/25 μL) or 0.9% NaCl sterile solution by intranasal instillation.
Treatment with test compounds (6.25 and 25 µmol/kg) or cilomilast (3 µmol/kg)
was performed by gavage, 1 h before LPS provocation. Untreated mice
received vehicle (0.1% DMSO) by gavage. The analyses were carried out 24 h
after LPS stimulation.
2
.8.5. Evaluation of airway hyper-reactivity
Following general anesthesia with nembutal (60 mg/kg, i.p.), and
®
neuromuscular blockade (pancuronium bromide, 1 mg/kg, i.v.), lung elastance
of mice was measured using an invasive whole-body plethysmography (Buxco
Electronics, United States) as previous described [12]. Briefly, after the
mechanical ventilator was connected to the mouse through an endotracheal
tube, it was stabilized for 5 min and increasing concentrations of methacholine
(3, 9, and 27 mg/ml) were aerosolized for 5 min each. Baseline lung elastance
was assessed with aerosolized phosphate-buffered saline (PBS).
2
.8.6. Lung TNF-α quantification
Tumour necrosis factor (TNF)-α was quantified in the right lung samples
using a commercial ELISA kit according to the manufacturer’s instructions (R&D
Systems).
2
.8.7. Relaxant effect in non-sensitized and sensitized guinea pig’s
trachea
The animals were randomly divided into two groups: non-sensitized and
st
sensitized. On the 1 day, guinea pigs were treated with OVA (60 μg/mL, i.p.
and s.c.), as a sensitizing agent, and with Al(OH) (1 mg/mL, i.p. and s.c.), as
3
an adjuvant. Both were dissolved on saline solution (NaCl 0,9 %). After, on the

2
8
th
8
day, the animals were individually placed in a closed polyacrylic box coupled
to an ultrasonic nebulizer and nebulized with an OVA solution (OVA 3 mg/mL +
th
saline) for 5 minutes. Then, on the 15 day, guinea pigs were nebulized again
with another OVA solution (OVA 1 mg/mL + saline) for 1 minute. Lastly, from
st
th
the days 21 to 25 the animals were euthanized by cervical dislocation
followed by sectioning of cervical vessels for experimentation. Non-sensitized
animals were subjected to the same treatment procedure but received only
saline (adapted from Espinoza et al., 2013) [42].
After evaluation of epithelium integrity using arachidonic acid [34], the
tracheal rings from non-sensitized and sensitized animals were contracted with
CCh (1.0 mM) and when a stable contraction was obtained. Rolipram,
LASSBio-448 (1) and LASSBio-1632 (3j) were individually and cumulatively
added to the preparation. Relaxation was expressed as pEC50 and Emax
,
calculated from the obtained concentration-response curves.
2
.9. Statistical analysis
All data are presented as means ± standard error of the mean (SEM) and
statistical analysis involving two groups was done, with Student’s t test,
whereas ANOVA followed by the Newman-Keuls-Student’s t test or by
Bonferroni’s test were used to compare more than 2 groups. P values of 0.05 or
less were considered significant (the null hypothesis was rejected when p <
0
.05).
2
.10. Parallel artificial membrane permeability assay (PAMPA)
To determine the permeability profile (passive diffusion) of the target,
Parallel artificial membrane permeability assay (PAMPA) was selected as
passive permeation prediction method. Commercial drugs (obtained from