Novel sulfenamides and sulfonamides based on pyridazinone and pyridazine scaffolds as CB1 receptor ligand antagonists

Article abstract of DOI:10.1016/j.bmc.2017.11.051

A series of sulfenamide and sulfonamide derivatives was synthesized and evaluated for the affinity at CB₁ and CB₂ receptors. The N-bornyl-S-(5,6-di-p-tolylpyridazin-3-yl)-sulfenamide, compound 11, displayed good affinity and high selectivity for CB₁ receptors (K_i values of 44.6 nM for CB₁ receptors and >40 μM for CB₂ receptors, respectively). The N-isopinocampheyl-sulfenamide 12 and its sulfonamide analogue 22 showed similar selectivity for CB₁ receptors with K_i values of 75.5 and 73.2 nM, respectively. These novel compounds behave as antagonists/inverse agonists at CB₁ receptor in the [³⁵S]-GTPγS binding assays, and none showed adequate predictive blood–brain barrier permeation, exhibiting low estimated LD₅₀. However, testing compound 12 in a supraspinal analgesic test (hot-plate) revealed that it was as effective as the classic CB₁ receptor antagonist rimonabant, in reversing the analgesic effect of a cannabinoid agonist.

Full text of DOI:10.1016/j.bmc.2017.11.051

Accepted Manuscript
Novel sulfenamides and sulfonamides based on pyridazinone and pyridazine
scaffolds as CB1 receptor ligand antagonists.
Gabriele Murineddu, Francesco Deligia, Giulio Ragusa, Laura García-Toscano,
María Gómez-Cañas, Battistina Asproni, Valentina Satta, Elena Cichero, Ruth
Pazos, Paola Fossa, Giovanni Loriga, Javier Fernández-Ruiz, Gerard A. Pinna
PII:
S0968-0896(17)31866-7
https://doi.org/10.1016/j.bmc.2017.11.051
BMC 14104
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 September 2017
24 November 2017
30 November 2017
Please cite this article as: Murineddu, G., Deligia, F., Ragusa, G., García-Toscano, L., Gómez-Cañas, M., Asproni,
B., Satta, V., Cichero, E., Pazos, R., Fossa, P., Loriga, G., Fernández-Ruiz, J., Pinna, G.A., Novel sulfenamides and
sulfonamides based on pyridazinone and pyridazine scaffolds as CB1 receptor ligand antagonists., Bioorganic &
Medicinal Chemistry (2017), doi: https://doi.org/10.1016/j.bmc.2017.11.051
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Novel sulfenamides and sulfonamides based on pyridazinone and pyridazine scaffolds as CB1
receptor ligand antagonists.
Gabriele Murineddu a,*, Francesco Deligia b,§, Giulio Ragusa a,§, Laura García-Toscano c-e, María
f
Gómez-Cañas c-e, Battistina Asproni a, Valentina Satta c-e, Elena Cichero , Ruth Pazos c-e, Paola
Fossa f, Giovanni Loriga g, Javier Fernández-Ruiz c-e, Gerard A. Pinna a
a Department of Chemistry and Pharmacy, University of Sassari, via F. Muroni 23/A, 07100 Sassari,
Italy.
b Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Trav. La Crucca 3, 07100
Sassari, Italy
c Instituto Universitario de Investigación en Neuroquímica, Departamento de Bioquímica y Biología
Molecular, Facultad de Medicina, Universidad Complutense, 28040-Madrid, Spain.
d
Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas
(CIBERNED), Madrid, Spain.
e Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain.
f Department of Pharmacy, University of Genoa, Viale Benedetto XV n. 3, 16132, Genoa, Italy.
g Institute of Translational Pharmacology, National Research Council, 09010 Pula, Cagliari, Italy.
*This author serves as corresponding author:
Gabriele Murineddu: Phone: +39079228740; Fax: +39079228720; E-mail: muri@uniss.it
§FD and GR contributed equally to this work

ABSTRACT
A series of sulfenamide and sulfonamide derivatives was synthesized and evaluated for the
affinity at CB1 and CB2 receptors. The N-bornyl-S-(5,6-di-p-tolylpyridazin-3-yl)-sulfenamide,
compound 11, displayed good affinity and high selectivity for CB1 receptors (Ki values of 44.6 nM
for CB1 receptors and > 40 µM for CB2 receptors, respectively). The N-isopinocampheyl-
sulfenamide 12 and its sulfonamide analogue 22 showed similar selectivity for CB1 receptors with
Ki values of 75.5 and 73.2 nM, respectively. These novel compounds behave as antagonists/inverse
agonists at CB1 receptor in the [35S]-GTPγS binding assays, and none showed adequate predictive
blood-brain barrier permeation, exhibiting low estimated LD50. However, testing compound 12 in a
supraspinal analgesic test (hot-plate) revealed that it was as effective as the classic CB1 receptor
antagonist rimonabant, in reversing the analgesic effect of a cannabinoid agonist.
Keywords: Sulfenamides, sulfonamides, CB1 antagonism, diarylpyridazines, ADMET model,
docking studies

1. Introduction
The discovery and characterization of endogenous cannabinoid system and the implication
of its signalling system in numerous biological processes have drawn the interest of many
pharmaceutical companies and academic researchers. Chemical design has generated numerous new
synthetic ligands able to modulate cannabinoid receptors, acting both as cannabinoid receptor type-
1 (CB1R) or cannabinoid receptor type-2 (CB2R) agonists and as antagonists.1 Unfortunately, very
few synthetic derivatives reached the market and one of them, the promising “magic bullet” against
obesity and weight gain, the CB1R inverse agonist rimonabant, was withdrawn due to its serious
central side effect, as increased incidence of depression and suicides.2 However, considering the
potential of this class of substances, special attention has been paid on the individuation of new
CB1R antagonists with reduced or neglected side effects, i.e. by acting at peripheral level without
effect on Central Nervous System (CNS) or having lower activity or absent inverse agonists.2
Recently, the continuous interest in CBR ligands3-6 prompted us to design novel pyridazinone-based
CBR ligands,7 of general structures A (Fig. 1).
pharmacophoric elements
O
H, CH3, Cl
cycloalkyl
N
H
N
N
O
Aryl
bioisosteric
replacement
A
O O
S
O O
S
structural
rearrangement
R
R
cycloalkyl
cycloalkyl
N
N
H
H
N
N
N
N
O
R
Aryl
C
B

Fig. 1. 2-Alkyl-6-aryl-2,3-dihydropyridazin-3(2H)-one-4-carboxamides (A) and designed
compounds (B,C).
Preliminary biological studies and in-silico ADMET calculations on these derivatives indicated that
the pyridazinone-4-carboxamides could be considered as suitable drug-like cannabinoid
candidates,7 representing a promising scaffold for the development of CBR ligands. Therefore,
keeping the three pharmacophoric elements of A, as two aryl rings and an hydrogen bond acceptor
portion, we planned the synthesis of the bioisosteric series B, and its analogue C, derived by
structural rearrangement, whose general structures are reported in Figure 1. In this paper, binding,
functionality, docking studies and ADMET simulation of derivatives 1,2,7-23 reported in Table 1,
related to the pyridazinone-4-carboxamide scaffold, are presented.
2. Result and discussion
2.1. Chemistry
The synthesis of sulfenamide- and sulfonamide-pyridazinones, derivatives 1 and 2, is
reported in Scheme 1. The acylation of toluene 3 in presence of succinic anhydride gave the γ-
ketoacid 4, whose cyclization with 4-methylbenzylhydrazine in EtOH provided the
dihydropyridazinone 5. The heterocyclic system 5 was converted in mercapto-pyridazinone 6 by
treatment with thionyl chloride. Reaction of 6 with cycloheptylamine in presence of sodium
hypochlorite furnished sulfenamide 1, whose m-chloroperoxybenzoic acid-assisted oxidation in
DCM yielded the sulfonamide 2.

SCHEME 1. Reagents and conditions: (a) Succinic anhydride, AlCl3, DCM, 0-60 °C, 3 h. (b) p-
CH3-C6H4CH2NHNH2, AcONa, EtOH, 80 °C, 30 h. (c) SOCl2, RT, 3 h. (d) R-NH2, NaOCl, DCM,
0 °C-RT, 2 h. (e) mCPBA, DCM, RT, 3 h.
The pyridazines (7-23) were prepared following the synthetic route described in scheme 2. The
Friedel-Crafts acylation of toluene with acyl chloride derivative of acid 24 furnished the ketone 25,
which was alkylated with ethyl bromoacetate in presence of NaH to give ketoester 26. Cyclization
of 26 with hydrazine monohydrate gave dihydropyridazinone 27, whose bromine-assisted oxidation
in acetic acid provided compound 28. Pyridazinone 28 was converted in the chloropyridazine 29 by
treatment with phosphoryl chloride, whose reaction with thiourea and sodium carbonate furnished
the key intermediate 30. The reaction of mercaptopyridazine 30 with the appropriate amines in
presence of sodium hypochlorite yielded sulfenamides 7-16. The final m-chloroperoxybenzoic acid
oxidation of compounds 7-13 allowed to obtain the sulfonamides 17-23.

SCHEME 2. Reagents and conditions: (a) Step 1. SOCl2, 100 °C, 24 h; Step 2. AlCl3, toluene, RT,
1 h. (b) NaH, BrCHCOOEt, DMSO, toluene, 0-25 °C, 1 h, N2. (c) NH2-NH2, 80 °C, 7 h. (d) Br2,
CH3COOH, 95 °C, 1 h. (e) POCl3, 60 °C, 3 h. (f) Thiourea, EtOH, Na2CO3, 80 °C, 0,5 h. (g) R-
NH2, NaOCl, DCM, 0 °C-RT, 1-5 h. (h) mCPBA, DCM, RT, 3 h.
2.2. CBRs binding affinities
With the exception of compound 9, which proved to be insoluble under the assay conditions, all
novel sulfenamide (1,7-16) and sulfonamide (2,17-23) derivatives were evaluated in vitro for their

ability to displace the radioligand [3H]-CP55,940 from human CB1R and CB2R transfected into
HEK293 EBNA cells. They were first subjected to a preliminary screening at a concentration of 40
µM. A complete dose-response curve was carried out only for those compounds that displaced the
radioligand to CB1R or CB2R by more than 70% in the preliminary screening. The experimental
binding affinities (Ki) from the respective displacement curves for hCB1R and hCB2R are listed in
Table 1. Furthermore, for comparison, the Ki values of the reference cannabinoid ligand
WIN55,212-2 have been also reported in Table 1.
Table 1. Structures and binding data of pyridazinones 1,2 and pyridazines 7-23.
Ki (nM)a
Compd
Structure
hCB1R
hCB2R
1
> 40000
> 40000
2
7
1193 471
2548 581
R
608.8 250
> 40000
8
9
724.2 314
NDb
> 40000
NDb
10
707.6 222
> 40000

11
44.6 1.4
> 40000
12
13
75.5 14.8
1248 189
> 40000
> 40000
14
246.6 62.4
> 40000
15
16
856.5c
> 40000
> 40000
> 40000
17
800.4c
> 40000
18
19
1462 213
> 40000
> 40000
3604 1520
20
> 10000
> 40000
21
22
1286 710
73.2 5.7
> 40000
> 40000
23
> 10000
> 40000
28.8 4.1
3.7 0.12
WIN55,212-2
aThe Ki values of the new compounds for the CB1R and CB2R were determined using
RBHCB1M400UA and RBXCB2M400UA membranes, respectively, and [3H]CP,55-940 as

radioligand. Ki values were obtained from three independent experiments carried out in triplicate
b
c
and are expressed as mean SEM. Not determined due to poor solubility. Ki value was obtained
from one experiment.
To explore the potentiality of pyridazinone-based system as cannabinoid ligand, we designed the
analogue of lead A, the cycloheptyl-sulfonamide 2. Since the synthesis of sulfonamide was carried
out by oxidation of the corresponding sulfenamide 1, the binding of both compounds 1 and 2 was
determined. The sulfenamide 1 showed no activity towards CBRs, whereas the sulphonamide 2
showed CBRs Ki values in the µmol range (KiCB1 = 1.2 µmol and KiCB2 = 2.5 µmol, respectively).
Due to the low CBRs affinity and selectivity of the pyridazinone sulfen- and sulphonamide based
derivatives we planned the synthesis of the diarylpyridazines, which retained the three structural
elements of 2: the dinitrogen heterocyclic core, two diaryl rings and the sulfen-/sulfonamide group.
Therefore, were prepared two series of diarylpyridazine-derivatives: the sulfenamides 7-16 and
their oxidation products, the sulfonamides 17-23.
Firstly, the effect of three different cycloalkyl residues on amidic portion was investigated; the
introduction of the cycloheptyl ring both on sulfen- (7) and sulfonamide derivative (17), mimicking
derivatives 1 and 2, led to an increase of CB1R affinity and selectivity (KiCB1 = 0.6 µmol and
KiCB2 > 40 µmol for 7 and KiCB1 = 0.8 µmol and KiCB2 > 40 µmol for 17). The introduction of a
cyclopentyl (8,18) and cyclohexyl (9,19) ring in both series resulted in derivatives with no affinity
for CB2R and, in general, with low KiCB1 values (0.7-3.6 µmol).
As is well known, the steric hindrance of the carbonyl group plays a pivotal role in the CBRs
affinity. Therefore, to further evaluate the effect on CBR-affinity of a bulky substituent on sulfen-
and sulfonamide group we synthesized a small collection of nine monoterpene derivatives (10-14
and 20-23). Within this series, the introduction of the N-bornyl (11) and the isopinocampheyl group
(12,22) led to an improvement in CB1R- affinity with KiCB1 values of 44.6 nmol (11), 75.5 nmol
(12) and 73.2 nmol (22), respectively, showing all nine derivatives no affinity for CB2R.

To summarize these results, it is plain that the pyridazine ring is a good template for CB1R affinity,
whether it bears the sulfenamidic group or it has the sulfonamide one; even the introduction of a
monoterpene unit, as expected, led to compounds with improved CB1R affinity. In particular, the
bornyl group on the sulfenamide lead to an improvement of the CB1R-affinity, resulting compound
11 the best derivative of this series, whereas the isopinocampheyl sulfen- (12) and sulfonamide (22)
showed similar CB1R-affinities.
Overall, the introduction of the sulfen- (1) or sulfonamide (2) moiety in the pyridazinone core
showed similar affinity for CBRs, whereas the same modification in the pyridazine ring seems to
play a modest role in enhancing the levels of CB1-affinity.
2.3. CB1R/CB2R-stimulated [35S]-GTPγS binding
Based on their binding profile, compounds 11, 12 and 22 were selected for further biological
characterization. To investigate the functionality of novel compounds as CB1R ligands, GTPγS
experiments have been performed in membranes extracted from HEK293 cells stably expressing
CB1Rs. GTPγS binding assays revealed that the three compounds appear to behave as
antagonists/inverse agonists of CB1R with IC50 values in the nanomolar range (Fig. 2), but being
less potent than the reference compound rimonabant (20-fold more potent than compound 12). With
these differences in receptor activity, it is reasonable to assume that compounds 11, 12 and 22 may
serve better for blocking CB1Rs as they will generate lower inverse agonist activities compared to
rimonabant.

Compound
IC50 (nM)
249.3 ± 104.1
181.8 ± 145.6
549.6 ± 489.6
9.7 ± 2.0
A
11
12
22
rimonabant
B
C
compound 12
rimonabant
Compound 12
120
25
0
80
40
-25
-50
-12 -10
-8
-6
-4
-2
-12
-10
-8
-6
-4
log compound concentration (M)
log compound concentration (M)
Fig. 2. A: IC50 values of compounds 11, 12 and 22, and the reference CB1R antagonist/inverse
agonist rimonabant, derived from GTPγS binding analyses. Values are means SEM of the data
obtained in three independent analyses each performed in triplicate. B: Representative curve for
compound 12, and C: Comparison with a representative curve for rimonabant.
2.4. ADMET properties in silico
In the search of novel and more druggable compounds, the in silico prediction of descriptors
related to absorption, distribution, metabolism, excretion and toxicity properties (ADMET) allows
to efficiently prioritize the most promising chemical entities to be further developed.8-10
In this work, after the chemical synthesis and biological evaluation of the most promising
derivatives 11, 12, 22, their putative pharmacokinetic and safety profiles have been predicted in
silico, together with those of the reference compound Rimonabant. Calculation of ADMET

properties is thought to be useful for the design and development of further more drug-like
analogues.
In details, we took into account the logarithmic ratio of the octanol-water partitioning coefficient
(cLogP), extent of blood-brain barrier permeation (LogBB), rate of passive diffusion-permeability
(LogPS), human intestinal absorption (HIA), volume of distribution (Vd), the role played by
plasmatic protein binding (%PPB) and by the compound affinity toward the human serum albumin
(LogKaHSA), and an overall perspective of the molecule oral bioavailability (%F).
Furthermore, metabolism and toxicity profiles of any compound were predicted, in terms of ability
to behave as P-glycoprotein substrate, as cytochrome P450 3A4 inhibitor or substrate, to interact
with the endocrine system and calculating the median lethal dose (LD50) by oral administration.
As shown in Table 2, sulfenamides 11 and 12 are characterized by a poor pharmacokinetic profile
in terms of lipophilicity, being the calculated cLogP over 5 (Lipinski rules) and higher than that
displayed by the reference compound Rimonabant. None of them, including rimonabant, showed
adequate blood-brain barrier permeation, showing unfavourable LogBB and/or LogPS
(recommended ranges as follows: 0<LogBB<1.5; -3<LogPS<-1). In addition, the two derivatives
exhibited too high volume of distribution values (Vd) and unproper bioavailability profiles (%F)
with respect to Rimonabant.
On the contrary, the sulfonamide 22 displayed an improved lipophilicity and Vd value, being
predicted as fully able to pass at the central nervous system. In particular, 22 is characterized by
better LogBB and LogPS parameters if compared with Rimonabant. All the newly synthesized
derivatives are able to be fully adsorbed at the human intestinal membrane (HIA).
Table 2. Calculated ADMET descriptors related to absorption and distribution properties for
compounds 11,12 and 22.
HIA
(%)c
Vd
(l/kg)d
LogKa
HSA
%F
(oral)
Compd
cLogP LogBBa LogPSb
%PPB

6.78
7.35
6.52
5.45
0.06
-0.29
0.92
0.07
-1.5
-1.8
-1.5
-1.2
100
100
100
100
30
28
3.6
99.24
99.69
96.78
99.28
5.25
5.59
5.70
6.45
0.5
0.0
36.9
11
12
22
2.9
48.7
Rimonabant
a Extent of brain penetration based on ratio of total drug concentrations in tissue and plasma at
steady-state conditions; bRate of passive diffusion-permeability. PS represents Permeability-Surface
area product and is derived from the kinetic equation of capillary transport; cHIA represents the
human intestinal absorption, expressed as percentage of the molecule able to pass through the
intestinal membrane; dPrediction of Volume of Distribution (Vd) of the compound in the body.
Based on Table 3, none of the compounds here proposed should be substrate of the P-glycoprotein,
or be involved in endocrine system disruption events or in cytochrome P450 3A4 inhibition, being
on the contrary substrate for the enzyme P450 3A4.
Finally, all the derivatives exhibit an acceptable toxicity profile, being the estimated LD50 in the
range of 310-690 mg/Kg for mouse after oral administration, being the most promising (22) also the
most safe (L50 = 690 mg/Kg) and quite comparable with that calculated for the reference compound.
Table 3. Calculated ADMET descriptors related to metabolism, excretion and toxicity properties
for compounds 11, 12 and 22.
Endocrine System Disruptiona
LogRBA > -3 LogRBA > 0
CYP3A4
Inhibitor Substrate
LD50 (mg/Kg)b
(R.I. ≥ 0.30)
Compd
(R.I. ≥ 0.40)
0.19
(R.I. ≥ 0.60) (R.I. ≥ 0.30) (R.I. ≥ 0.30)
0.01
0.02
0.01
0.01
Not
Not
Not
Not
0.96
0.94
0.78
0.94
510
310
690
900
11
0.35
0.23
0.39
12
22
Rimonabant
aRBA represents the relative binding affinity with respect to that of estradiol. Compounds showing
LogRBA > 0 are classified as strong estrogen binders, whereas those showing LogRBA < -3 are
considered as non-binders. bAcute toxicity (LD50) for mouse after oral administration (RI: reliability

index. Borderline-allowed values for reliability parameter are ≥ 0.3, the most predictive fall in the
range 0.50-1.0).
2.5. Hot plate test
With the purpose to confirm or refuse the prediction of a poor penetration of compound 12
through the BBB, we conducted an additional experiment aimed at evaluating its antagonist
properties in a CNS-related response as the supraspinal analgesia. Such response is part of the
classic cannabinoid tetrad that is frequently used to demonstrate cannabinoid effects that are
produced within the CNS. Our hypothesis was that, if peripherally-restricted, compound 12 should
not be able to reverse the analgesic effect of a classic CB1R agonist (∆9-THC), using rimonabant as
a control of this central antagonist. Surprisingly, compound 12 reversed, to the same extent than
rimonabant, the elevated latency response in the hot-plate test exhibited by the CB1R agonist ∆9-
THC (Fig. 3), thus refusing its expected peripherally-restricted properties as CB1R antagonist.
Compound 12 and rimonabant by themselves did not produce any analgesic response (Fig. 3).
Hot plate test
20
vehicle
rimonabant (1 mg/kg)
**
15
12 (2.5 mg/kg)
##
##
∆9-THC (5 mg/kg)
10
5
rimonabant (1 mg/kg) + ∆9-THC (5 mg/kg)
12 (2.5 mg/kg) + ∆9-THC (5 mg/kg)
0
Treatment
Fig. 3. Values of latency to response against a painful stimuli in the hot-plate test measured in mice
treated with the cannabinoid agonist ∆9-THC alone or combined with compound 12 or the reference
CB1R antagonist rimonabant. Values are means SEM of 7-9 subjects per experimental group.

Data were assessed by the one-way analysis of variance followed by the Student-Newman- Keuls
test (**p<0.01 versus vehicle, rimonabant or 12 given alone; ##p<0.01 versus ∆9-THC given
alone).
2.6. Molecular docking
Nowadays, the rational design and discovery of CB1 ligands was efficiently driven by
deepening computational studies, including homology modelling of the biological target,11-13 as
well as ligand-based analyses.14-16 In particular, molecular docking studies within cannabinoid
receptor models allowed to clarify some structural features involved in the pharmacodynamics
process of the related ligands.
Concerning CB2, a number of homology models were specifically built to analyze the
binding mode of agonists and antagonists, making the design of further new molecules much more
focused and reliable.13,17 On the other hand, in the search of selective CB1 antagonists, the recently
available X-Ray crystallographic structures of the human CB1R (pdb code = 5U09; resolution = 2.6
Å)18 (pdb code = 5TGZ; resolution = 2.8 Å) definitively guide for a more effective rational design
process.19 Indeed, these experimental data explored the binding mode of the co-crystallized
propanamide-based (pdb code = 5U09) and pyrazole-containing (pdb code = 5TGZ) derivatives,
giving useful insights for the further development of novel series of analogues and/or isosteres.
In this work, we performed two series of docking studies within CB1R and CB2R, aimed at
clarifying the different responsiveness experienced by the two proteins in presence of the
pyridazinone and pyridazine scaffolds (See Tables in Supplementary material).
In particular, based on the most interesting biological data observed at the CB1R, the newly
synthesized compounds were deeply analysed and compared with the CB1 antagonist Rimonabant.
These calculations were performed using the experimental structure of hCB1 in complex with
pyrazole reference antagonist AM6538. Indeed, the pyridazinone and pyridazine chemotypes
resemble as bioisosteres of the pyrazole core of the co-crystallized AM6538.

Briefly, at CB1 X-ray data the co-crystallized ligand AM6538 exhibited one H-bond
between the hydrogen atom of the carboxamide group and the side-chain of S383, moving the
cyclohexyl substituent towards I119, F174, A380, M384. The two aromatic centers occupied a deep
hydrophobic pocket including I169, L193, V196, F268, W279, L359, W356, M363, L387. Notably,
the docking mode proved by Rimonabant was highly comparable with the positioning displayed by
the co-crystallized compound AM6538 (Fig. 4).
Fig. 4. Docking mode of Rimonabant (C atom; magenta) within the X-Ray crystallographic
coordinates of hCB1R in complex with AM6538 (C atom; green). The most important residues are
labelled. H-bonds detected by Rimonabant are shown by dot lines (in yellow).
Concerning the CB2 receptor, we performed our work using the protein homology model we
previously described, followed by molecular dynamics simulations (MD) including the well-known
CB2 agonist WIN-55,212-2. The collected results underlined three crucial H-bonds involving the
reference agonistsand the S112, N188 and S285 residues of the protein binding site.17

According to our calculations, in silico CB1 and CB2 complexes including compound 2,
bearing the pyridazinone chemotype featuring a sulfonamide moiety, displayed poor and
comparable binding affinity values, while the sulfenamide analogue 1 proved to be inactive (see
Tables in Supporting Information S19). Indeed, all of them lack most of the aforementioned key
contacts discussed for the reference compounds AM6538 and WIN-55,212-2.
Docking calculations of the newly synthesized pyridazines at the X-ray data of CB1 allowed
us to explore the structure-activity relationship for the novel more promising compounds here
discussed. In particular, we clarified the role played by the variation concerning the R substituent,
when it is coupled with the sulfenamide (7-16) and the sulfonamide moieties (17-23).
All of them displayed a common positioning, moving the two phenyl rings towards the
aforementioned hydrophobic pocket, in agreement with the binding mode of the aromatic moieties
in AM6538. Moreover, the pyridazine core proved to mimic the role played by the pyrazole center
of the co-crystallized antagonist, whereas the specific sulfenamide and sulfonamide moieties
exhibited a comparable docking mode, turning sometimes in effective contacts with S383.
In particular, the sulfenamide derivative 12 was H-bonded to the S383 side-chain in
presence of one acid hydrogen atom onto the nitrogen one (Fig. 5).

Fig. 5. Docking mode of 12 (C atom; violet) within the X-Ray crystallographic coordinates of hCB1
receptor in complex with AM6538 (C atom; green). The most important residues are labelled. H-
bonds detected by the pyridazine compound are shown by dot lines (in yellow).
On the other hand, oxygen atoms of the sulfonamide moiety exhibited the same key H-bond,
especially in presence of a branched hydrophobic chain in R, as for the derivatives 22 (Fig. 6).

Fig. 6. Docking mode of 22 (C atom; magenta) within the X-Ray crystallographic coordinates of
hCB1 receptor in complex with AM6538 (C atom; green). The most important residues are labelled.
H-bonds detected by the pyridazine compound are shown by dot lines (in yellow).
Thus, it is expected that proper H-bonding features, such as sulfen- and sulphonamides, linked to
the pyridazine nucleus, coupled with branched and hydrophobic groups, could represent an
adequate scaffold able to fulfil the minimal pharmacophore requirements to exert CB1 antagonism.
3. Conclusions
We synthesized a series of compounds, as dihydropyridazinone-4-sulfenamide (1),
dihydropyridazinone-4-sulfonamide (2), diarylpyridazin-3-sulfenamides (7-16) and 3-sulfonamides
(17-23). The new derivatives were evaluated in competition studies for their ability to bind CB1R or
CB2Rs. All compounds showed an extremely poor affinity for the CB2R with Ki values in the
micromolar range and frequently > 40 µM. The same trend was observed for their affinities for the
CB1R, with the exception of N-bornyl-(5,6-di-p-tolylpyridazin-3-yl)-sulfenamide (11, Ki = 44.6

nM), its N-isopinocampheyl-analogue (12, Ki = 75.5 nM) and the N-isopinocampheyl-(5,6-di-p-
tolylpyridazin-3-yl)-sulfonamide (22, Ki = 73.2 nM) having Ki for the CB1R in the nanomolar
range. Other derivatives bearing a C3 sulfenamide or sulfonamide moiety exhibited lower affinities
for the CB1R, but still in the low micromolar range. The most active compounds at the CB1Rs (11,
12 and 22) were also analyzed in [35S]-GTPγS binding assays to determine their agonist or
antagonist/inverse agonist functionalities at the CB1R. All behaved as antagonists/inverse agonists
with the N-isopinocampheyl-(5,6-di-p-tolylpyridazin-3-yl)-sulfenamide (12) being the most active
(IC50 = 181.8 nM). In silico ADMET studies on these compounds predicted that they may be
restricted to the periphery, exhibiting a good toxicity profile, therefore they could be represent
useful tools to obesity and metabolic syndrome investigations, without the reported problems for
those antagonists able to penetrate in the brain. However, in vivo analysis of the capability of
compound 12 to reverse agonist-induced analgesia in the hot-plate test, a centrally-mediated
cannabinoid effect, revealed that this compound was active, to a similar extent than rimonabant,
then refusing its predicted peripherally-restricted properties.
In summary, we have developed a new series of selective CB1R antagonists/inverse agonists based
on diarylpyridazine scaffold, functionalized with different sulfen- and sulfonamides in which,
according to a perspective of the docking results, the substituents on the nitrogen of the amine
portion showed crucial steric requirements to make the compound well-suited in CB1R-targeting.
Indeed, these portions in any case fall within a protein narrow cavity delimited by I119, F174, A380
and M384.
4. Experimental
4.1. Chemistry
All reactions involving air or moisture-sensitive compounds were performed under nitrogen
atmosphere. Solvents and reagents were obtained from commercial suppliers and were used without
further purification. p-Tolylacetic acid 24 and amines for the synthesis of final compounds were

purchased from Sigma-Aldrich®: fenchylamine was synthesized according to the literature
procedure.20 Flash column chromatography was performed automatically on Flash-master
(Biotage®) with pre-packed Biotage® SNAP silica gel cartridges or manually on silica gel
−0.063 mm, Merck®).
(Kieselgel 60, 0.040
Thin layer chromatography (TLC) was performed with
Polygram SIL N-HR/HV254 pre-coated plastic sheets (0.2 mm) on aluminum sheets (Kieselgel 60
F254, Merck®). Melting points were obtained on a Köfler melting point apparatus and are
uncorrected. NMR experiments were run on a Bruker Avance III Nanoboy 400 system (400.13
1
MHz for H, and 100.62 MHz for 13C). Spectra were acquired using deuterated chloroform
(Chloroform-d) or deuterated dimethylsulfoxide (DMSO-d6) as solvents. Chemical shifts (δ) for 1H-
and 13C-NMR spectra are reported in parts per million (ppm) using the residual non-deuterated
solvent resonance as the internal standard (for Chloroform-d: 7.26 ppm, 1H and 77.16 ppm, 13C; for
1
13
DMSO-d6: 2.50 ppm, H, 39.52 ppm, C). Data are reported as follows: chemical shift (sorted in
descending order), multiplicity (s for singlet, br s for broad singlet, d for doublet, dd for double
doublet, t for triplet, q for quadruplet, m for multiplet), integration and coupling constants (J) in
Hertz (Hz). LC/MS analyses were run on an Agilent 1100 LC/MSD system consisting of a single
quadrupole detector (SQD) mass spectrometer (MS) equipped with an electrospray ionization (ESI)
−550 nm. ESI in positive
interface and a photodiode array (PDA) detector; PDA range was 120
mode was applied; mobile phases: (A) ACN in H2O (8:2). Analyses were performed with a flow
rate of 0.9 mL/min; and at room temperature All final compounds displayed ≥ 95% purity as
determined by elemental analysis on a Perkin-Elmer 240-B analyser, for C, H, and N.
4.1.1. 4-Oxo-4-(p-tolyl)butanoic acid (4). To a suspension of succinic anhydride (1 g, 10.2 mmol,
1eq) in toluene (4.6 mL, 44.8 mmol, 4.4 eq ) kept at 0 °C, aluminum chloride (2.7 g, 20.4 mmol, 2
eq) was added in small portions. The resulting suspension was stirred at room temperature for 3 h,
then poured onto ice and acidified with HCl conc. The aqueous solution was extracted with EtOAc
and the combined organic layers were dried (Na2SO4) and concentrated under reduced pressure.
The residue was triturated with petroleum ether to furnish 4 as a white solid (1.35 g, 69 %). Rf =

0.36 (petroleum ether/EtOAc 4:6), mp 125-128 °C; 1H NMR (400 MHz, Chloroform-d) δ 7.88 (d, J
= 8.0 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H), 3.29 (t, J = 6.6 Hz, 2H), 3.00 (br s, 1H, OH, exch. with
D2O), 2.80 (t, J = 6.0 Hz, 2H), 2.41 (s, 3H).
4.1.2. 2-(4-Methylbenzyl)-6-(p-tolyl)-4,5-dihydropyridazin-3(2H)-one (5). 4-Methylbenzyl-
hydrazine (0.99 g, 5.73 mmol, 1.1 eq) and sodium acetate (3.88 g, 47.3 mmol, 9 eq) were added to a
stirred solution of acid 4 (1 g, 5.2 mmol, 1eq) in EtOH (20 mL). The mixture was refluxed for 30 h,
then the solvent was removed under reduced pressure and the resulting residue diluted with H2O.
Afterwards, K2CO3 was added until pH 8, and basic aqueous solution extracted with EtOAc. The
organic phase was washed with brine, dried (Na2SO4) and concentrated under reduced pressure to
give 5 as a light yellow solid (0.79 g, 52 %), Rf = 0.27 (petroleum ether/EtOAc 8:2), mp 106-109
°C; 1H NMR (400 MHz, Chloroform-d) δ 7.62 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 7.6 Hz, 2H), 7.19 (d,
J = 8.0 Hz, 2H), 7.12 (d, J = 7.6 Hz, 2H), 4.98 (s, 2H), 2.91 (t, J = 8.4 Hz, 2H), 2.59 (t, J = 8.4 Hz,
2H), 2.37 (s, 3H), 2.31 (s, 3H).
4.1.3. 4-Mercapto-2-(4-methylbenzyl)-6-(p-tolyl)pyridazin-3(2H)-one (6). A solution of 5 (1.3
g, 4.4 mmol, 1 eq) and thionyl chloride (8.8 mL, 122 mmol, 27.7 eq) was stirred at room
temperature for 3 h. Then, excess of thionyl chloride was removed under reduced pressure and the
resulting residue was purified by column flash chromatography (petroleum ether/EtOAc 7:3) to
obtain 6 as a white solid (0.64 g, 45 %), Rf = 0.81 (petroleum ether/EtOAc 6:4), mp 128-130 °C; 1H
NMR (400 MHz), Chloroform-d) δ 7.42 (d, J = 8.0 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 7.28 (d, J =
13
8.0 Hz, 2H), 7.12 (d, J = 7.6 Hz, 2H), 6.89 (s, 1H), 5.28 (s, 2H), 2.42 (s, 3H), 2.31 (s, 3H); C
NMR (101 MHz, CDCl3) δ 158.59 (C=O), 143.80 (C), 141.46 (C), 140.36 (C), 137.76 (C), 132.82
(C), 130.53 (C), 129.48 (CH x 2), 129.30 (CH x 2), 128.88 (CH x 2), 128.80 (CH x 2) 122.72 (CH),
54.83 (CH2), 21.43 (CH3), 21.17 (CH3).
4.1.4. p-Methylbenzyl-p-tolylketone (25). p-Tolylacetic acid 24 (1 g, 6.63 mmol, 1eq) and
thionyl chloride ( 2.5 mL, 19.89 mmol, 3 eq) were stirred at 100 °C for 24 h. Then the solution was
cooled at room temperature and excess of thionyl chloride was removed under reduced pressure.

The obtained residue was slowly added to a cold suspension of aluminum chloride (1.33 g, 9.94
mmol, 1.5 eq) in Toluene (1 mL). The resulting mixture was allowed to stand at room temperature,
then stirred for 1 h. After, HCl conc. was added, and the aqueous solution was extracted with
EtOAc. The organic phase was washed with 5% KHCO3 sol. and brine, then dried (Na2SO4) and
concentrated under reduced pressure to give a solid which was purified by gradient column flash
chromatography (petroleum ether/EtOAc 99:1 to 97:3) to obtain 25 as a yellow solid (1.2 g, 80.7
1
%). Rf = 0.51 (petroleum ether/EtOAc 95:5); mp 85-88 °C; H NMR (400 MHz, Chloroform-d) δ
8.00 (d, J = 8.1 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 7.27 – 7.20 (m, 4H), 4.31 (s, 2H), 2.49 (s, 3H),
2.41 (s, 3H); 13C NMR (101 MHz, Chloroform-d) δ 197.47 (C=O), 143.87 (C), 136.38 (C), 134.17
(C), 131.69 (C), 129.36 (CH x2), 129.29 (CH x2), 129.27 (CH x2), 128.77 (CH x2), 45.07 (CH2),
21.64 (CH3), 21.06 (CH3).
4.1.5. Ethyl 4-oxo-3,4-di-p-tolylbutanoate (26). To a solution of ketone 25 (1 g, 4.46 mmol, 1 eq)
in DMSO (6.4 mL) was added portionwise NaH (0.2 g, 13.4 mmol, 3 eq) at 0 °C, under N2. To the
resulting suspension was added dropwise a solution of ethyl bromoacetate (0.54 mL, 4.90 mmol,
1.1 eq) in dry toluene (4 mL). The mixture of reaction was stirred at room temperature for 2 h, then
poured onto ice-water, acidified with HCl 10%, and extracted with Et2O. The organic layer was
dried (Na2SO4) and concentrated under reduce pressure to obtain a residue which was purified by
column flash chromatography (petroleum ether/EtOAc 95:5) to obtain 26 as a yellow oil (1.19 g, 86
%). Rf = 0.37 (EtPet/AcOEt 95:5); 1H NMR (400 MHz, Chloroform-d) δ 7.88 (d, J = 8.2 Hz, 2H),
7.21 – 7.12 (m, 4H), 7.08 (d, J = 7.8 Hz, 2H), 5.03 (dd, J = 9.6, 5.1 Hz, 1H), 4.09 (q, J = 7.1, 6.4 Hz,
2H), 3.33 (dd, J = 16.8, 9.6 Hz, 1H), 2.68 (dd, J = 16.8, 5.1 Hz, 1H), 2.33 (s, 3H), 2.26 (s, 3H), 1.19
13
(t, J = 7.1 Hz, 3H); C NMR (101 MHz, Chloroform-d) δ 198.29 (C=O), 172.11 (C=O), 143.65
(C), 137.01 (C), 135.34 (C), 133.73 (C), 129.79 (CH x2), 129.15 (CH x2), 128.99 (CH x2), 127.97
(CH x2), 60.58 (CH2), 49.01 (CH), 38.67 (CH2), 21.57 (CH3), 21.00 (CH3), 14.13 (CH3).
4.1.6. 5,6-di-p-Tolyl-4,5-dihydropyridazin-3(2H)-one (27). A mixture of ketoester 26 (2 g, 6.44
mmol) and hydrazine hydrate (13.7 mL, 44 eq) was stirred at 80 °C for 7 h. After cooling at room

temperature, ice was added and the resulting precipitate was filtered off and washed with water. The
obtained solid dried to air furnished the pure analytically product 27 as a white solid (1.74 g, 97 %).
Rf = 0.33 (petroleum ether/EtOAc 7:3); mp 183-185 °C; 1H NMR (400 MHz, Chloroform-d) δ 8.57
(br s, 1H, NH, exch. with D2O), 7.59 (d, J = 7.9 Hz, 2H), 7.15 (d, J = 7.9 Hz, 2H), 7.12 – 7.08 (m,
4H), 4.43 (d, J = 7.8 Hz, 1H), 2.98 (dd, J = 16.9, 7.8 Hz, 1H), 2.77 (d, J = 16.8 Hz, 6.1 Hz, 1H),
2.34 (s, 4H), 2.29 (s, 3H); 13C NMR (101 MHz, Chloroform-d) δ 165.90 (C=O), 151.78 (C), 140.05
(C), 137.44 (C), 134.61 (C), 132.46 (C), 130.01 (CH x2), 129.33 (CH x2), 126.90 (CH x2), 126.11
(CH x2), 39.39 (CH), 35.34 (CH2), 21.31 (CH3), 21.01 (CH3).
4.1.7. 5,6-di-p-Tolylpyridazin-3(2H)-one (28). A solution of dihydropyridazinone 27 (1.7 g, 6.1
mmol, 1 eq) in acetic acid (150 mL) was heated at 95 °C. Then, bromine (0.31 mL, 6.1 mmol, 1 eq)
was added and the resulting solution was stirred at the same temperature for 1 h. After cooling at
room temperature, the orange solution was poured onto ice-water and extracted with Et2O. The
combined organic phases dried (Na2SO4) and concentrated under reduce pressure gave the pure
analytically product 28 as beige solid (1.6 g, 95 %). Rf = 0.18 (petroleum ether/EtOAc 7:3); mp
1
190-192 °C; H NMR (400 MHz, Chloroform-d) δ 7.12 – 7.07 (m, 4H), 7.06 – 7.00 (m, 4H), 6.96
(s, 1H), 2.35 (s, 3H), 2.32 (s, 3H); 13C NMR (101 MHz, Chloroform-d) δ 161.69 (C=O), 147.38
(C), 147.11 (C), 139.14 (C), 138.54 (C), 133.28 (C), 132.50 (C), 129.18 (CH x2), 129.05 (CH x2),
128.83 (CH), 128.76 (CH x2), 128.57 (CH x2), 21.27 (CH3), 21.26 (CH3).
4.1.8. 6-Chloro-3,4-di-p-tolylpyridazine (29). A mixture of pyridazinone 28 (2.3 g, 8.3 mmol, 1
eq) and phosphoryl chloride (19 mL, 200.7 mmol, 24 eq) was stirred at 60 °C for 3 h. After cooling
at room temperature the solution was slowly poured onto ice-water. The resulting precipitate was
filtered off under vacuum, washed with water and air-dried to yield the pure analytically product 29
as a light brown solid (2.2 g, 90 %). Rf = 0.77 (petroleum ether/EtOAc 7:3), mp 83-84 °C; 1H NMR
(400 MHz, Chloroform-d) δ 7.47 (s, 1H), 7.29 (d, J = 7.8 Hz, 2H), 7.17 – 7.05 (m, 6H), 2.35 (s,
13
3H), 2.34 (s, 3H); C NMR (101 MHz, Chloroform-d) δ 159.27 (C=O), 155.13 (C), 141.60 (C),

139.43 (C), 139.13 (C), 132.99 (C), 132.70 (C), 129.78 (CH x2), 129.56 (CH x2), 128.99 (CH x2),
128.83 (CH x2), 128.27 (CH), 21.33 (CH3), 21.30 (CH3).
4.1.9. 5,6-di-p-Tolylpyridazine-3-thiol (30). To a stirred solution of chloropyridazinone 29 (2.13
g, 7.2 mmol, 1 eq) in EtOH (30 mL), thiourea (0.55 g, 7.2 mmol, 1 eq) was added. The resulting
mixture was refluxed for 30 min, then the solvent was removed under reduced pressure and the
residue was treated with a solution of Na2CO3 (0.35 g) in H2O (40 mL). The resulting precipitate
was filtered off under vacuum, washed with water and air dried to give 30 as a pale yellow solid
1
(1.7 g, 81 %). Rf = 0.62 (petroleum ether/EtOAc 8:2), mp 185-188 °C; H NMR (400 MHz,
Chloroform-d) δ 7.71 (s, 1H), 7.11-7.08 (m, 6H), 7.03 (d, J = 8.0 Hz, 2H), 2.35 (s, 3H), 2.33 (s,
3H); 13C NMR (101 MHz, Chloroform-d) δ 178.91 (C), 152.06 (C), 140.43 (C), 139.75 (C),
139.47 (C), 132.13 (C), 131.49 (C), 129.82 (CH), 129.38 (CH x2), 128.99 (CH x2), 128.95 (CH
x2), 128.66 (CH x2), 21.32 (CH3), 21.27 (CH3).
4.1.10. General procedure for the synthesis of sulfenamides 1, 7-16.
To an ice-bath cooled solution of mercapto derivative 6 or 30 (1 mmol, 1 eq) in DCM (10
mL) was added the appropriate amine (3 mmol, 3 eq) followed by sodium hypochlorite (0.2 mL, 3
mmol, 3 eq) and the resulting mixture was stirred at room temperature for 1-5 h. Then, solution was
diluted with EtOAc, washed with water and brine. The organic solution was dried (Na2SO4) and
concentrated under reduced pressure to afford the crude product, which was purified by column
flash chromatography using the appropriate eluents.
4.1.11. N-Cycloheptyl-2-(4-methylbenzyl)-6-(p-tolyl)pyridazin-3(2H)-one-4-sulfenamide
(1).
General procedure for the synthesis of sulfenamides was used to convert thiol 6 and
cycloheptylamine into the title product. The mixture of reaction was stirred for 2 h and the resulting
crude product was purified by column flash chromatography (petroleum ether/EtOAc 8:2) to afford
1 (324 mg, 75 %) as a light yellow solid. Rf = 0.39 (petroleum ether/EtOAc 8:2); mp 121-123 °C;
1H NMR (400 MHz, Chloroform-d) δ 7.38 (dd, J = 8.0, 4.0 Hz, 4H), 7.24 (d, J = 7.9 Hz, 2H), 7.13
(d, J = 7.6 Hz, 2H), 7.05 (s, 1H), 5.30 (s, 2H), 2.90 – 2.81 (m, 1H), 2.49 (d, J = 4.6 Hz, 1H, NH,

13
exch with D2O), 2.40 (s, 3H), 2.32 (s, 3H), 1.98 – 1.86 (m, 2H), 1.68 – 1.32 (m, 10H); C NMR
(101 MHz, Chloroform-d) δ 158.26 (C), 150.81 (C), 141.97 (C), 138.56 (C), 136.43 (C), 132.63
(C), 130.33 (C), 128.14 (CH x 2), 128.07 (CH x 2), 127.86 (CH x 2), 127.42 (CH x 2), 118.03
(CH), 60.10 (CH), 53.36 (CH2), 34.25 (CH2 x 2), 26.96 (CH2 x 2), 22.78 (CH2 x 2), 20.37 (CH3),
20.14 (CH3). MS (ESI): C26H31N3OS requires m/z 433.2, found 434.1 [M + 1]+; Anal. calcd for
C26H31N3OS: C, 72.02; H, 7.21; N, 9.69; Found: C, 72.17; H, 7.19; N, 9.70.
4.1.12. N-Cycloheptyl-(5,6-di-p-tolylpyridazin-3-yl)-sulfenamide (7). General procedure for the
synthesis of sulfenamides was used to convert thiol 30 and cycloheptylamine into the title product.
The mixture of reaction was stirred for 4 h and the resulting crude product was purified by column
flash chromatography (petroleum ether/EtOAc 8:2) to afford 7 (274 mg, 67%) as a white solid. Rf =
1
0.46 (petroleum ether/EtOAc 8:2); mp 111-112 °C; H NMR (400 MHz, Chloroform-d) δ 7.40 (s,
1H), 7.30 (d, J = 7.7 Hz, 2H), 7.16 – 7.05 (m, 6H), 3.34 (br s, 1H, NH, exch. with D2O), 3.20 – 3.02
(m, 1H), 2.38 (s, 3H), 2.35 (s, 3H), 2.13 – 1.96 (m, 2H), 1.78 – 1.62 (m, 2H), 1.52 (m, 6H), 1.47 –
1.33 (m, 2H). 13C NMR (101 MHz, Chloroform-d) δ 166.08 (C), 157.07 (C), 138.90 (C), 138.66
(C), 138.35 (C), 134.16 (C), 133.97 (C), 129.76 (CH x 2), 129.34 (CH x 2), 128.89 (CH x 2),
128.79 (CH x 2), 122.64 (CH), 61.33 (CH), 34.89 (CH2 x 2), 28.27 (CH2 x 2), 23.88 (CH2 x 2),
21.26 (CH3 x 2). MS (ESI): C25H29N3S requires m/z 403.2, found 403.2 [M + 1]+; Anal. calcd for
C25H29N3S: C, 74.40; H, 7.24; N, 10.41; Found: C, 74.58; H, 7.21; N, 10.39.
4.1.13. N-Cyclopentyl-(5,6-di-p-tolylpyridazin-3-yl)-sulfenamide (8). General procedure for the
synthesis of sulfenamides was used to convert thiol 30 and cyclopentylamine into the title product.
The mixture of reaction was stirred for 2 h and the resulting crude product was purified by column
flash chromatography (petroleum ether/EtOAc 9:1) to afford 8 (263 mg, 70%) as a white solid. Rf =
1
0.52 (petroleum ether/EtOAc 8:2); mp 55-57 °C; H NMR (400 MHz, Chloroform-d) δ 7.37 (s,
1H), 7.31 (d, J = 7.7 Hz, 2H), 7.15 – 7.06 (m, 6H), 3.68 (br s, 1H, NH, exch. with D2O), 3.46 (s,
1H), 2.36 (s, 3H), 2.34 (s, 3H), 1.89 – 1.71 (m, 4H), 1.69 – 1.58 (m, 4H). 13C NMR (101 MHz,
Chloroform-d) δ 165.60 (C), 157.09 (C), 138.89 (C), 138.69 (C), 138.39 (C), 134.13 (C), 133.93

(C), 129.75 (CH x 2), 129.35 (CH x 2), 128.86 (CH x 2), 128.79 (CH x 2), 122.78 (CH), 61.64
(CH), 33.21 (CH2 x 2), 23.87 (CH2 x 2), 21.26 (CH3 x 2). MS (ESI): C23H25N3S requires m/z 375.2,
found 376.2 [M + 1]+; Anal. calcd for C23H25N3S: C, 73.56; H, 6.71; N, 11.19; Found: C, 73.39; H,
6.72; N, 11.22.
4.1.14. N-Cyclohexyl-(5,6-di-p-tolylpyridazin-3-yl)-sulfenamide (9). General procedure for the
synthesis of sulfenamides was used to convert thiol 30 and cyclohexylamine into the title product.
The mixture of reaction was stirred for 2 h and the resulting crude product was purified by column
flash chromatography (petroleum ether/EtOAc 9:1) to afford 9 (230 mg, 60%) as a white solid. Rf =
1
0.55 (petroleum ether/EtOAc 8:2); mp 116-117 °C; H NMR (400 MHz, Chloroform-d) δ 7.43 (s,
1H), 7.30 (d, J = 7.8 Hz, 2H), 7.21 – 6.98 (m, 6H), 3.34 (br s, 1H, NH, exch. with D2O), 2.93 (s,
1H), 2.37 (s, 3H), 2.34 (s, 3H), 2.12 – 2.02 (m, 2H), 1.81 – 1.68 (m, 2H), 1.65 – 1.54 (m, 2H), 1.34
13
– 1.14 (m, 4H). C NMR (101 MHz, Chloroform-d) δ 166.30 (C), 157.04 (C), 138.89 (C), 138.66
(C), 138.35 (C), 134.18 (C), 133.97 (C), 129.76 (CH x 2), 129.34 (CH x 2), 128.89 (CH x 2),
128.79 (CH x 2), 122.56 (CH), 59.00 (CH), 33.39 (CH2 x 2), 25.83 (CH2), 24.70 (CH2 x 2), 21.26
(CH3 x 2). MS (ESI): C24H27N3S requires m/z 389.2, found 390.1 [M + 1]+; Anal. calcd for
C24H27N3S: C, 74.00; H, 6.99; N, 10.79; Found: C, 73.86; H, 6.98; N, 10.82.
4.1.15. N-Menthyl-(5,6-di-p-tolylpyridazin-3-yl)-sulfenamide (10). General procedure for the
synthesis of sulfenamides was used to convert thiol 30 and menthylamine into the title product. The
mixture of reaction was stirred for 3 h and the resulting crude product was purified by column flash
chromatography (petroleum ether/EtOAc 8:2) to afford 10 (270 mg, 61%) as a white solid. Rf =
1
0.53 (petroleum ether/EtOAc 8:2); mp 164-165 °C; H NMR (400 Mhz, Chloroform-d) δ 7.62 (s,
1H), 7.30 (d, J = 8.4 Hz, 2H), 7.18-7.06 (m, 6H), 3.08 – 3.00 (br s, 1H, NH, exch. with D2O), 2.81-
2.61 (m, 1H), 2.36 (s, 3 H), 2.34 (s, 3H), 2.31 – 2.20 (m, 1H), 1.72 – 1.55 (m, 3H), 1.42 – 1.27 (m,
1H), 1.25 – 1.20 (m, 2H), 1.20 – 1.02 (m, 2H), 0.97 – 0.87 (m, 6H), 0,81 (d, J = 6.8 Hz, 3H); 13C
NMR (101 MHz, Chloroform-d) δ 163.99 (C), 154.22 (C), 137.80 (C), 137.48 (C), 137.09 (C),
133.22 (C), 132.79 (C), 129.01 (CH2 x 2), 128.35 (CH2 x 2), 127.69 (CH2 x 2), 127.70 (CH2 x 2),

121.12 (CH2), 55.72 (CH), 49.49 (CH), 43.35 (CH), 34.55 (CH2), 26.48 (CH), 23.86 (CH2), 22.15
(CH2), 21.40 (CH3), 21.25 (CH3 x 2), 21.16 (CH3), 19.35 (CH3). MS (ESI): C28H35N3S requires m/z
445.3, found 446.3 [M + 1]+; Anal. calcd for C28H35N3S: C, 75.46; H, 7.92; N, 9.43; Found: C,
75.60; H, 7.93; N, 9.45.
4.1.16. N-Bornyl-(5,6-di-p-tolylpyridazin-3-yl)-sulfenamide (11). General procedure for the
synthesis of sulfenamides was used to convert thiol 30 and bornylamine into the title product. The
mixture of reaction was stirred for 2 h and the resulting crude product was purified by column flash
chromatography (petroleum ether/EtOAc 9:1) to afford 11 (314 mg, 71%) as a white solid. Rf =
1
0.59 (petroleum ether/EtOAc 8:2); mp 150-151 °C; H NMR (400 MHz, Chloroform-d) δ 7.47 (s,
1H), 7.30 (d, J = 7.7 Hz, 2H), 7.14 – 7.05 (m, 6H), 3.35 (br s, 1H, NH, exch wih D2O), 3.28 – 3.18
(m, 1H), 2.35 (s, 3H), 2.33 (s, 3H) 2.31 – 2.25 (m, 1H), 1.96 – 1.83 (m, 1H), 1.81 – 1.69 (m, 1H),
1.68 – 1.59 (m, 1H), 1.41 – 1.28 (m, 1H), 1.28 – 1.16 (m, 1H), 1.11 – 1.02 (m, 1H), 0.96 (s, 3H),
13
0.87 (s, 3H), 0.85 (s, 3H); C NMR (101 MHz, Chloroform-d) δ 165.93 (C), 156.00 (C), 137.91
(C), 137.60 (C), 137.28 (C), 133.18 (C), 132.99 (C), 128.75 (CH2 x 2), 128.31 (CH2 x 2), 127.84
(CH2 x 2), 127.77 (CH2 x 2), 121.31 (CH2), 66.27 (CH), 48.83 (C), 47.78 (C), 43.94 (CH), 37.19
(CH2), 27.38 (CH2), 26.16 (CH2), 20.27 (CH3), 20.23 (CH3), 18.91 (CH3), 17.65 (CH3), 13.16
(CH3). MS (ESI): C28H33N3S requires m/z 443.2, found 444.3 [M + 1]+; Anal. calcd for C28H33N3S:
C, 75.80; H, 7.50; N, 9.47; Found: C, 75.59; H, 7.49; N, 9.45.
4.1.17. N-Isopinocampheyl-(5,6-di-p-tolylpyridazin-3-yl)-sulfenamide (12). General procedure
for the synthesis of sulfenamides was used to convert thiol 30 and isopinocampheylamine into the
title product. The mixture of reaction was stirred for 3 h and the resulting crude product was
purified by column flash chromatography (petroleum ether/EtOAc 9:1) to afford 12 (292 mg, 67%)
1
as a white solid. Rf = 0.52 (petroleum ether/EtOAc 8:2); mp 168-169 °C; H NMR (400 MHz,
Chloroform-d) δ 7.35 (s, 1H), 7.31 (d, J = 7.9 Hz, 2H), 7.17 – 7.05 (m, 6H), 3.34 (br s, 1H, NH,
exch wih D2O), 3.17 – 3.06 (m, 1H), 2.35 (s, 3H), 2.34 (s, 3H), 2.22 – 2.13 (m, 1H), 1.97 – 1.83 (m,
4H), 1.62 – 1.47 (m, 1H), 1,21 (s, 3H) 1.17 (d, J = 7.2 Hz, 3H), 0.96 (s, 3H), 0.93 – 0.86 (m, 1H);

13C NMR (101 MHz, Chloroform-d) δ 165.51 (C), 157.10 (C), 138.93 (C), 138.69 (C), 138.40 (C),
134.11 (C), 133.94 (C), 129.76 (CH x 2), 129.34 (CH x 2), 128.86 (CH x 2), 128.79 (CH x 2),
122.71 (CH), 44.06 (CH), 42.00 (CH ), 41.46 (CH x 2), 40.23 (C), 33.22 (CH2), 27.96 (CH3 x 2),
26.12 (CH2), 23.31 (CH3), 21.30 (CH2), 21.26 (CH3 x 2). MS (ESI): C28H33N3S requires m/z 443.2,
found 444.2 [M + 1]+; Anal. calcd for C28H33N3S: C, 75.80; H, 7.50; N, 9.47; Found: C, 75.99; H,
7.52; N, 9.48.
4.1.18. N-Myrtanyl-(5,6-di-p-tolylpyridazin-3-yl)-sulfenamide (13). General procedure for the
synthesis of sulfenamides was used to convert thiol 30 and myrtanylamine into the title product.
The mixture of reaction was stirred for 5 h and the resulting crude product was purified by column
flash chromatography (petroleum ether/EtOAc 85:15) to afford 13 (257 mg, 58%) as a white solid.
1
Rf = 0.50 (petroleum ether/EtOAc 8:2); mp 166-168 °C; H NMR (400 Mhz, Chloroform-d) δ 7.35
(s, 1H), 7.31 (d, J = 8.0 Hz, 2H), 7.15 – 7.03 (m, 6H), 3.31 (br s, 1H, NH, exch with D2O), 3.18 –
3.08 (m, 2H), 2.36 (s, 3H), 2.34 (s, 3H), 2.04 – 1.96 (m, 2H), 1.94 – 1.79 (m, 3H), 1.61 – 1.44 (m,
13
2H), 1.23 (d, J = 9.0 Hz, 1H), 1.17 (s, 3H), 0.97 (s, 3H), 0.92 (d, J = 9.0 Hz, 1H); C NMR (101
MHz, Chloroform-d) δ 162.20 (C), 152.12 (C), 139.50 (C), 138.22 (C), 138.23 (C), 137.39 (C),
133.70 (C), 128.99 (CH x 2), 129.11 (CH x 2), 128.92 (CH x 2), 128.30 (CH x 2), 119.03 (CH),
56.23 (CH2), 43.80 (CH), 40.63 (CH), 40.39 (CH), 37.70 (C), 32.30 (CH2), 27.00 (CH3), 25.04
(CH2), 22.18 (CH3), 21.25 (CH3), 21.10 (CH3), 20.03 (CH2). MS (ESI): C28H33N3S requires m/z
443.2, found 444.2 [M + 1]+; Anal. calcd for C28H33N3S: C, 75.80; H, 7.50; N, 9.47; Found: C,
76.03; H, 7.52; N, 9.45.
4.1.19. N-Fenchyl-(5,6-di-p-tolylpyridazin-3-yl)-sulfenamide (14). General procedure for the
synthesis of sulfenamides was used to convert thiol 30 and fenchylamine into the title product. The
mixture of reaction was stirred for 5 h and the resulting crude product was purified by column flash
chromatography (petroleum ether/EtOAc 9:1) to afford 14 (292 mg, 66 %) as a pale yellow solid. Rf
1
= 0.77 (petroleum ether/EtOAc 8:2); mp 154-156 °C; H NMR (400 MHz, Chloroform-d) δ 7.65 (s,
1H), 7.30 (d, J = 7.7 Hz, 2H), 7.18 – 7.03 (m, 6H), 3.00 (d, J = 8.8 Hz, 1H, NH, exch with D2O),