Structure-activity relationship with pyrazoline-based aromatic sulfamates as carbonic anhydrase isoforms I, II, IX and XII inhibitors: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2019.111638

Four new series of aromatic sulfamates were synthesized and investigated for the inhibition of four human (h) isoforms of zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), hCA I, II, IX, and XII. The reported derivatives, obtained by a sulfamoylation reaction of the corresponding phenolic precursors, bear 3,5-diarylpyrazoline moieties as spacers between the benzenesulfamate fragment which binds the zinc ion from the active site, and the tail of the inhibitor. Pyrazolines are biologically privileged scaffolds, endowed with versatile biological activity, such as an anti-proliferative action. The derivatives were tested for the inhibition of the cytosolic, hCA I and II (off target isoforms) and the trans-membrane, tumor-associated hCA IX and XII enzymes (anticancer drug targets). Generally, hCA I was not effectively inhibited, whereas many low nanomolar inhibitors were evidenced against hCA II (KIs in the range of 0.42–90.1 nM), IX (KIs in the range of 0.72–63.6 nM), and XII (KIs in the range of 0.88–85.2 nM). The best substitution fragments at the pyrazoline ring included for CA II a 4-sulfamic group on the 3-aryl and halogens on the 5-aryl or a methoxy group on the 3-aryl and a 4-sulfamate group on the 5-aryl; for CA IX and CA XII they included the sulfamic group on the 3- or 4-position of the 5-aryl and an electronwithdrawing group on the 4-postion of the 3-aryl ring.

Full text of DOI:10.1016/j.ejmech.2019.111638

Journal Pre-proof
Structure-activity relationship with pyrazoline-based aromatic sulfamates as carbonic
anhydrase isoforms I, II, IX and XII inhibitors: Synthesis and biological evaluation
Davide Moi, Alessio Nocentini, Alessandro Deplano, Gianfranco Balboni, Claudiu T.
Supuran, Valentina Onnis
PII:
S0223-5234(19)30772-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.111638
EJMECH 111638
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 July 2019
Revised Date: 21 August 2019
Accepted Date: 21 August 2019
Please cite this article as: D. Moi, A. Nocentini, A. Deplano, G. Balboni, C.T. Supuran, V. Onnis,
Structure-activity relationship with pyrazoline-based aromatic sulfamates as carbonic anhydrase
isoforms I, II, IX and XII inhibitors: Synthesis and biological evaluation, European Journal of Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111638.
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Structure-activity relationship with pyrazoline-based aromatic sulfamates as carbonic anhydrase
isoforms I, II, IX and XII inhibitors: synthesis and biological evaluation.
Davide Moi,a† Alessio Nocentini,b,† Alessandro Deplano,c Gianfranco Balboni,a Claudiu T. Supuran,b*
Valentina Onnis,a*
aDepartment of Life and Environmental Sciences, Unit of Pharmaceutical, Pharmacological and
Nutraceutical Sciences, University of Cagliari, via Ospedale 72, Cagliari I-09124, Italy
bDepartment NEUROFARBA – Pharmaceutical and Nutraceutical Section, University of Firenze, via Ugo
Schiff 6, I-50019 Sesto Fiorentino, Firenze, Italy
cPharmacelera, Placa Pau Vila, 1, Sector 1, Edificio Palau de Mar, Barcelona 08039, Spain
*To whom correspondence should be addressed. E-mail: vonnis@unica.it; claudiu.supuran@unifi.it
†These authors equally contribute to the present paper
Highlights
* Pyrazoline aryl sulfamates were designed and synthesized.
* Sulfamates were assayed in vitro as human Carbonic Anhydrase (CA) isoforms inhibitors.
* Synthesized compound inhibited selectively CAII, CAII, CAIX and CAXII isoforms.
* Sulfamates 8h, 9e, 9i, 17d, 18e show CAIX and CAXII Ki in the nanomolar to sub-nanomolar range.
* Docking revealed CA isoform selective inhibition related to distinguish interactions.
Keywords
Carbonic anhydrase, Zinc-binding group, Aromatic sulfamates, Pyrazolines, Enzyme inhibition, Molecular
docking, Sulfamoylation
Graphical abstract
Abstract
Four new series of aromatic sulfamates were synthesized and investigated for the inhibition of four human
(h) isoforms of zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), hCA I, II, IX, and XII. The reported
derivatives, obtained by a sulfamoylation reaction of the corresponding phenolic precursors, bear 3,5-
diarylpyrazoline moieties as spacers between the benzenesulfamate fragment which binds the zinc ion
from the active site, and the tail of the inhibitor. Pyrazolines are biologically privileged scaffolds, endowed
with versatile biological activity, such as an anti-proliferative action. The derivatives were tested for the
inhibition of the cytosolic, hCA I and II (off target isoforms) and the trans-membrane, tumor-associated hCA
IX and XII enzymes (anticancer drug targets). Generally, hCA I was not effectively inhibited, whereas many
low nanomolar inhibitors were evidenced against hCA II (KIs in the range of 0.42–90.1 nM), IX (KIs in the
range of 0.72–63.6 nM), and XII (KIs in the range of 0.88–85.2 nM). The best substitution fragments at the
pyrazoline ring included for CA II a 4-sulfamic group on the 3-aryl and halogens on the 5-aryl or a methoxy
group on the 3-aryl and a 4-sulfamate group on the 5-aryl; for CA IX and CA XII they included the sulfamic
group on the 3- or 4-position of the 5-aryl and an electronwithdrawing group on the 4-postion of the 3-aryl
ring.

1. Introduction
Pyrazoline ring is a privileged structure in medicinal chemistry because of its wide spectrum of
pharmacological activities. A number of pyrazoline derivatives have been reported for their antibacterial
[1], antimalarial [2, 3], anti-inflammatory [4], MAO inhibitory [5, 6], antioxidant [7, 8], neuroprotective [9],
antidepressant [10] and anticancer activity [11-18]. Furthermore, several series of pyrazoline derivatives
displayed carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity [19-25]. CAs are a superfamily of
metalloenzymes that catalyze the CO2 hydration/dehydration reaction, and are classified into seven
genetically distinct families, named α-, β-, γ-, δ-, ζ-, η-, and ɵ-CAs [26-28]. All human (h) CAs are α-class
enzymes [28]. Fifteen different human isoforms have been identified and characterized to date, among
which twelve are catalytically active (hCAs I-IV, VA, VB, VI, VII, IX, XII-XIV). Human CAs can be further
categorized into four different subsets depending on their subcellular localization. Among those identified,
hCA I, II, III, VII, VIII, X, XI, XIII are cytosolic proteins, hCA VA and VB are present in the mitochondrial matrix,
hCA VI is a secreted enzyme, hCA IV is a glycosylphosphatidylinositol (GPI)-anchored protein and hCA IX, XII
and XIV are trans-membrane isoforms [26, 28]. These enzymes are widely distributed in many tissues and
organs where they are implicated in a wealth of pivotal physiological processes. Dysregulated expression
and/or abnormal activity of hCAs can result into severe pathological conditions [26, 29-32]. hCA IX and XII
have been validated as markers of disease progression in many hypoxic tumors and their targeted
inhibition has been associated with a significant reduction of the growth of both primary tumors and
metastases [26]. In contrast, ubiquitous isoforms hCA I and II are the main off-target isoforms because their
promiscuous inhibition might lead to undesired side effects [26]. CA inhibitors (CAIs) can be clustered into
several different groups considering their binding mode to the enzyme active site, among which the zinc-
binders are the most effective and thus most investigated for drug-design purposes [33]. Within this subset,
sulfonamides are the ideal zinc-binding group (ZBG) owing to a peculiar combination of interactions that
this moiety can solely establish with the zinc ion and the residues nearby [33]. Pyrazoline sulfonamides
were recently reported as CA inhibitors [34] and as dual CA and AchE inhibitors [35-39]. Bioisosteric
replacement of the sulfonamide with the sulfamate group, one of sulfonamide most related congeners,
produced interesting examples of CAs selective inhibitors [40-43]. These considerations led us to report a
small library of pyrazoline sulfamates displaying interesting profile and selectivity as CA inhibitors [44].
Here, we have extended these studies to have full SAR of this class of compounds.
2. Results and discussion
2.1 Chemistry
The synthetic pathway to obtain N1-acetyl-3,5-diaryl-4,5-dihydropyrazole bearing a sulfamate group at 4- or
3-position on 3-aryl ring (compounds 8a-m and 9a-m, Scheme 1) and on 5-aryl ring (compounds 17a-h and
18 a-h, Scheme 2) started with the preparation of chalcones (4, 5, 14 and 15) through the Claisen-Schmidt
condensation [45-47] between substituted acetophenone (1, 2 and 10) and substituted benzaldehydes (3,
11 and 12) in methanol in the presence 50% aqueous NaOH. Chalcones were then treated with hydrazine
hydrate in boiling AcOH [48] to afford the cyclization into 4,5-dihydropyrazoles (6, 7, 15 and 16). Finally, the
desired compounds were obtained by sulfamoylation of the phenolic hydroxy groups by treatment with
freshly prepared sulfamoyl chloride in N,N-dimethylacetamide. Structures were confirmed based on
analytical and spectral data which are consistent with results of reported studies [48, 49].

Scheme 1. General synthetic procedure for sulfamates subsets 8 and 9. Reagents and conditions: (i) MeOH,
50% aqueous NaOH, r.t., 12 h; (ii) NH2NH2·H2O, AcOH, 3 h, reflux; (iii) ClSO2NH2, DMA, r.t. 12 h.
Scheme 2. General synthetic procedure for sulfamates subsets 17 and 18. Reagents and conditions: (i)
MeOH, 50% aqueous NaOH, r.t., 12 h; (ii) NH2NH2·H2O, AcOH, 3 h, reflux; (iii) ClSO2NH2, DMA, r.t. 12 h.
2.2. Carbonic anhydrase inhibition
The inhibitory activity against hCA I, hCA II, hCA IX and hCA XII of sulfamate derivatives 8a-m, 9a-m, 17a-h
and 18 a-h was tested by a stopped flow CO2 hydrase assay in the presence of acetazolamide as standard
inhibitor [50].
Table 1. Inhibition data of human CA isoforms hCA I, II, IX and XII with sulfamates subsets 8, 9, 17 and 18
reported here and the standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped flow CO2 hydrase
assay [50].
KI (nM)a
Cmpd
8ab
R1
R2
CA I
CA II
8.9
6.5
5.3
1.2
CA IX
34.1
7.1
CA XII
4-OSONH2
4-OSONH2
4-OSONH2
4-OSONH2
H
n.d.
1308.4
817.1
4-CH3
4-OCH3
4-NO2
8b
8c
8db
22.3
42.6
n.d.
1607.4
3208.5
25.0
74.3

8eb
8f
8gb
4-OSONH2
4-OSONH2
4-OSONH2
4-OSONH2
4-OSONH2
4-OSONH2
4-OSONH2
4-OSONH2
4-OSONH2
3-OSONH2
3-OSONH2
3-OSONH2
3-OSONH2
3-OSONH2
3-OSONH2
3-OSONH2
3-OSONH2
3-OSONH2
3-OSONH2
3-OSONH2
3-OSONH2
3-OSONH2
H
4-CF3
4-F
n.d.
2741.9
666.5
0.83
1.5
29.3
5.9
11.8
n.d.
2-Cl
8253.1
2313.3
1137.1
3752.5
4752.2
4083.0
3298.5
188.4
9.0
25.9
0.72
6.9
8h
3-Cl
0.87
6.0
9.8
27.3
26.2
n.d.
8i
4-Cl
8j
8kb
2,4-Cl2
9.3
11.2
25.5
33.4
49.0
29.9
29.4
8.7
2,5-Cl2
12.4
13.2
27.5
39.4
71.2
52.4
133.9
22.2
38.0
90.1
56.9
14.7
194.7
92.6
223.4
215.7
44.8
9.9
8l
2,6-Cl2
43.7
56.4
55.1
55.9
48.6
20.8
8.9
8m
9°
naphthyl
H
9b
4-CH3
558.2
9c
4-OCH3
4-NO2
172.2
9d
1458.2
951.8
10.1
3.7
9e
4-CF3
9f
4-F
67.4
8.4
13.5
74.4
44.6
9.3
9g
2-Cl
939.5
43.6
7.3
9h
3-Cl
805.2
9i
4-Cl
437.7
6.9
9j
2,4-Cl2
1413.2
2035.7
1782.5
2244.1
644.7
13.8
58.9
63.6
51.1
45.1
22.4
22.8
15.0
8.3
33.8
34.2
61.3
462.6
10.6
7.7
9k
2,5-Cl2
9l
2,6-Cl2
9m
17°
17b
17c
17d
17f
17g
17h
18°
18b
18d
18eb
18f
18g
18h
AAZ
naphthyl
4-OSONH2
4-OSONH2
4-OSONH2
4-OSONH2
4-OSONH2
4-OSONH2
4-OSONH2
3-OSONH2
3-OSONH2
3-OSONH2
3-OSONH2
3-OSONH2
3-OSONH2
3-OSONH2
4-CH3
2338.4
2977.2
3818.1
970.2
4-OCH3
0.42
9.5
9.5
4-NO2
0.88
29.5
42.0
12.9
22.9
49.8
20.5
n.d.
4-Cl
19.5
5.3
2,4-Cl2
3518.6
2898.4
755.0
49.9
7.7
3,4-Cl2
27.6
16.5
25.4
22.9
6.5
H
13.1
8.9
4-CH3
1539.2
2546.8
5225.3
2140.9
6936.0
8174.1
250
4-NO2
9.4
4-F
15.8
0.81
48.7
15.5
25
4-Cl
32.7
12.0
54.8
12.5
22.3
30.8
85.2
5.7
2,4-Cl2
3,4-Cl2
-
aMean from 3 different assays, by a stopped flow technique (errors were in the range of ± 5-10 % of the
reported values); bfrom reference [44].
The following structure–activity relationships (SAR) were gathered from the inhibition data reported in
Table 1.
1) CA I: all pyrazoline sulfamates 8a-m, 9a-m, 17a-g and 18a-g showed low inhibitory activity except for
compounds 9a, 9c and 9f that displayed inhibitory values better the reference AAZ.

2) CA II: 5-Aryl substituted pyrazoline sulfamates 8a-l showed high activity against this CA isoform (KI in the
0.8-13.2 nM range). Compound 8e bearing a 4-trifluoromethylphenyl ring showed the best activity (KI 0.8
nM). The replacement of the 4-trifluoromethyl moiety with a chlorine atom (compound 8i) led to a
reduction in activity. On the contrary the displacement of the chlorine from the 4-position to the 3-position
repristinate the activity (compound 8h, KI 0.87 nM). The displacement of the chlorine into 2-position
(compound 8g) as well as the introduction of two chlorine atoms (compounds 8j-8l) led to a reduction in
activity as compared to the analog 8h. The comparison of the sulfamates 8 and 9 inhibitory activity
revealed that the displacement of the 4-sulfamate group into 3-position produced a reduction in activity.
However, sulfamates 9e and 9i bearing 4-trifluoromethyl and 4-chlorine moieties maintained high
inhibitory activity. In the pyrazoline sulfamates 17 series the inhibitory activity is related to the presence of
substituents on the 3-aryl ring; as matter of fact derivatives 17a showed the lowest activity in this series.
The best compound was 17c bearing a 4-methoxyphenyl group. Interestingly the shift of the methoxy and
sulfamate moieties from the 3-aryl to 5-aryl, as can be seen by the comparison of compounds 8c and 17c
KIs, generated a different inhibition profile. This trend is confirmed by the other compounds of the series.
The presence of 4-methyl, 4-nitro and 2,4-dichloro substituents was favorable for the activity in the
pyrazoline 17 series, while the presence of the same groups on the 5-aryl of pyrazolines 8 did not increase
the activity. Pyrazoline sulfamates 18 showed high activity as compared to their analogs 9 except for 18f. As
general trend the displacement of the 4-sulfamate group of pyrazolines 17 into 3-position to give
pyrazolines 18 led to a slight reduction in activity when substituents are present on the 5-aryl ring.
3) CA IX: Among pyrazoline sulfamates 8 subset, the 3-chlorine derivative 8h showed the best activity (KI
0.72 nM). The displacement of the chlorine atom into 4-position (compound 8i) led to a 10-fold reduction in
activity. The replacement of the 4-chlorine of compound 8i with a methyl group (compound 8b) maintained
the same activity level, while the replacement with a 4-fluorine atom (sulfamate 8f) produced an increase
in activity. On the contrary the presence of two chlorine atoms (compounds 8j-8l) produced a reduction in
activity when compared to both 8h and 8i analogs. The displacement of the 4-sulfamate group into 3-
position to give the isomeric pyrazolines 9 induced similar or better activity in comparison to the
corresponding analogs 8 except for compounds 9g, 9k and 9l. In the pyrazoline sulfamates 17 series the
best activity was showed by the chlorine substituted compound 17g whose KI is very similar to the analog 8i
as well as for the compounds 17a and 17c as compared to their analogs 8a and 8c. Interestingly, the
transposal of the nitro and sulfamate groups on the aryl rings of 8d to give the isomeric 17d caused about a
5-fold increase in activity. The shift of the sulfamate moiety of pyrazolines 9 on the 5-aryl ring to give the
analogs 18 produced improvement in activity especially in compound 18f that was the best of the series
showing an 8.5-fold higher activity than analog 9i. The same compound 18f was about 10-fold more active
as compared to the analog 17e. The same activity trend is showed by the pyrazoline 18a that was about 3-
fold more active than the analog 17a.
4) CA XII: Pyrazoline sulfamates 8f, 8h, 9c, 9f, and 9i confirmed their good inhibitory activity against the
other CA isoform expressed in hypoxic tumoral cells. Thus, the presence of a halogen atom or a
trifluoromethyl group in 4-position enhanced the activity in the series 9, while in the series 8 the best
activity was correlated to the presence of 4-fluorine or 3-chlorine atom on the 5-aryl ring. The pyrazoline
17d showed the best activity of all four series (KI 0.88 nM). The removal of the 4-nitro group of 17d or its
replacement with a methoxy group led to about a 10-fold reduction in activity. Pyrazoline sulfamates of
series 18 showed reduced activity as compared to both pyrazolines 17 and 9.
2.3 Molecular Docking
To better understand the binding patterns of our compounds they were docked in the four CA isoforms
evaluated in this study, CA I (PDBID: 3w6h), CA II (PDBID: 4g0c), CA IX (PDBID: 3iai), CA XII (PDBID: 1jd0).
These crystal structures were selected for the presence of AAZ as co-crystallized ligand used as a reference
for discussing the experimental activities.
A selection of the most interesting compounds 8f, 8h, 9e, 17c, 17d, 17g, 18f were used to deeply analyze
the ligand-protein interactions (the complete docking scores of all compounds can be found on Table 1S of
supplementary material).
Just by a first look at the binding site, we can retrieve some useful information. The main difference among
the isoforms is the size of the binding sites (Figure S1). The smaller binding site of CA I can explain the lower

activity of the compounds on this isoform. A second evident information is about hydrophobic properties.
In fact, CA XII has a more polar binding site in contrast with other isoforms. Finally, another important
structural difference between CA I and the other isoforms pertains residue 200, which is a histidine in CA I,
while a threonine in CA II, IX and XII. As in most of the poses of the docked compounds there is an
interaction between the ligand and Thr200, the Thr/His mutation is likely to induce the ligands lower
activity against CA I. Unfortunately, we could not find a clear correlation between the docking score and the
in-vitro results. However, some useful information to better understand the reasons behind activity and
selectivity can be obtained looking at the best scored poses.
The sulfamate moiety of all compounds reproduced almost the same interactions with the sulfonamide
group of AAZ. In detail the sulfamate group fit deeply into the active site with the negatively charged
nitrogen coordinating the Zinc. Moreover, the hydrogen of the sulfamate establish an H-Bond with the
T199 oxygen of the hydroxy group and the amidic hydrogen of the same residue binds the S=O oxygens
and/or the Ph-O-S oxygen (Figure S2).
About the other portion of the molecules, for what concerns CA I no other important interaction was found
except for the compound 9e which establishes an H-Bond between one fluorine of the trifluoromethyl
group and Asn69 side chain.
Speaking about CA II, all selected compounds perform a π-π stacking with Phe131. Moreover, other
common H-bonds performed by the compounds are with residues Asn62, Asn67 (8f, 8h, 17f). Moreover, for
the most active compound of the series against this isoform, 17c, as well as compounds 17d and 18f, a H-
bond exists between their carbonyl moiety and the hydroxyl group of Thr200 (Figure S3).
Within CA IX active site, the four most active compounds 8f, 8h, 9e, and 18f show potential H-bond with
Asn67 and Gln92 (Figure S4A). Conversely, compounds 17c, 17d, and 17f establish H-bond with Thr200.
Moreover, the nitro group 17d is in a good position to form an H-bond with Asp132 (Figure S4B).
For what concerns CA XII, all compounds except 8f and 9e establish H-bonds with Lys67. Other recurrent H-
bonds exist with Thr91 and/or Gln92 and varied groups of compounds 8f, 9e, 17f and 18f (Figure S5A).
Moreover, compounds 9e, 17d, 17c and 18f establish H-bonds by the carbonyl or the pyrazoline nitrogen
with Thr200. Finally, compounds 9e, 17c, 17d bearing an H-bond acceptor group in para to the non-
sulfamate ring establish interaction with Ser132 (Figure S5B).
3. Conclusions
Four new series of aromatic sulfamates were synthesized by a sulfamoylation reaction of the corresponding
phenolic precursors. The reported derivatives bearing 3,5-diarylpyrazoline moieties as spacers between
the benzenesulfamate fragment which binds the zinc ion from the active site, and the tail of the inhibitor
were investigated for the inhibition of four human hCAs, namely the cytosolic hCA I and II (off target
isoforms) and the trans-membrane, tumor-associated hCA IX and XII enzymes (anticancer drug targets). The
pyrazoline sulfamates did not effectively inhibited hCA I, whereas many low nanomolar inhibitors were
evidenced against hCA II (KIs in the range of 0.42–90.1 nM), IX (KIs in the range of 0.72–63.6 nM), and XII
(KIs in the range of 0.88–85.2 nM). The best substitution fragments at the pyrazoline ring included for CA II
a 4-sulfamic group on the 3-aryl and halogens on the 5-aryl or a methoxy group on the 3-aryl and a 4-
sulfamate group on the 5-aryl; for CA IX and CA XII the sulfamic group on the 3- or 4-position of the 5-aryl
and an electronwithdrawing group on the 4-postion of the 3-aryl ring. Docking analyses suggested that the
selectivity and potency showed by some compounds may be related to the number of hydrogen bonds
between the sulfamate compounds and the CA isoforms.
4. Experimental section
4.1. General methods
All commercially available solvents and reagents were used without further purification. NMR spectra were
recorded on an Inova 500 spectrometer (Varian, Palo Alto, CA, USA). The chemical
shifts (δ) are reported in part per million downfield from tetramethylsilane (TMS), which was used as
internal standard. The spectra were recorded in hexadeuteriodimethylsulphoxide (DMSO-d6). Infrared
spectra were recorded on a Vector 22 spectrometer (Bruker, Bremen, Germany) in Nujol mulls. The main
bands are given in cm-1. Positive-ion electrospray ionization (ESI) mass spectra were recorded on a double-

focusing MAT 95 instrument (Finnigan, Waltham, MA, USA) with BE geometry. Melting points (mp) were
determined with a SMP1 Melting Point apparatus (Stuart Scientific, Stone, UK) and are uncorrected. All
1
products reported showed H NMR spectra in agreement with the assigned structures. The purity of the
tested
compounds
was
determined by combustion elemental analyses conducted by the Microanalytical Laboratory of the
Chemistry Department of the University of Ferrara with a MT-5 CHN recorder elemental analyser
(Yanagimoto, Kyoto, Japan) and the values found were within 0.4% of theoretical values. Pyrazolines 7a
[51], 7b [52], 7c [53], 7d [44], 7i [52], 7l [54], 15a [55], 15b [55], 15f [55], 16a [43], and sulfamates 8a, 8d,
8e, 8g [44] have been prepared as previously described.
4.1.1. General procedure for the preparation of 1-acetyl-3,5-diaryl- 4,5-dihydro-1H-pyrazoles 6, 7, 15 and
16.
To a solution of chalcone derivative 4, 5, 13, 14 (1 mmol) in acetic acid (3 mL) hydrazine hydrate (0.3 mL, 6
mmol) was added. The mixture was refluxed under stirring for 3 h, and then poured onto crushed ice. The
precipitate was filtered off, washed with cold water, and crystallized from methanol to give the titled
pyrazolines.
4.1.1.1. 1-(5-(4-fluorophenyl)-3-(3-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (7f)
Following the general procedure, the title compound was prepared starting from chalcone 5f. Yield 79%.
M.p. 218-219 °C. 1H NMR (DMSO-d6) 2.33 (s, 3H, CH3), 2.99 (dd, J = 14.0, 3.5 Hz, 1H, CH), 3.93 (dd, J = 14.0,
3.0 Hz,1H, CH), 5.73 (dd, J = 13.5, 3.5 Hz, 1H, CH), 6.85 (d, J = 6.5 Hz, 1H, Ar), 7.05 (d, J = 7.0 Hz, 1H, Ar),
7.16 (m, 1H, Ar), 7.21 (d, J = 8.0 Hz, 2H, Ar), 7.29 (d, J = 8.5 Hz, 2H, Ar), 7.47 (s, 1H, Ar), 9.63 (s, 1H, OH). IR
(Nujol) 3272, 1635, 1575 cm-1. m/z 299 (m+H)+. Anal. calcd. for C17H15FN2O2 (298.31) C, 64.45; H, 5.07; N,
9.39. Found C, 64.49; H, 5.11; N, 9.36.
4.1.1.2. 1-(5-(2-Chlorophenyl)-3-(3-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (7g)
Following the general procedure, the title compound was prepared starting from chalcone 5g. Yield 84%.
M.p. 212-214 °C. 1H NMR (DMSO-d6) 2.36 (s, 3H, CH3), 3.01 (dd, J = 14.0, 3.0 Hz, 1H, CH), 3.94 (dd, J = 14.5,
3.5 Hz,1H, CH), 5.76 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.87 (d, J = 7.5 Hz, 1H, Ar), 7.07 (d, J = 7.0 Hz, 1H, Ar),
7.16 (m, 1H, Ar), 7.23 (d, J = 7.5 Hz, 2H, Ar), 7.31 (d, J = 8.0 Hz, 2H, Ar), 7.49 (s, 1H, Ar), 9.64 (s, 1H, OH). IR
(Nujol) 3231, 1643, 1573 cm-1. m/z 315 (m+H)+. Anal. calcd. for C17H15ClN2O2 (314.77) C, 64.87; H, 4.80; N,
8.90. Found C, 64.94; H, 4.78; N, 8.86.
4.1.1.3. 1-(5-(3-Chlorophenyl)-3-(3-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (7h)
Following the general procedure, the title compound was prepared starting from chalcone 5h. Yield 92%.
M.p. 127-128 °C. 1H NMR (DMSO-d6) 2.31 (s, 3H, CH3), 3.32 (dd, J = 3.0, 14.0 Hz, 1H, CH), 3.84 (dd, J = 14.5,
3.5 Hz, 1H, CH), 5.54 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.87 (d, J =7.5 Hz, 1H, Ar), 7.18 (d, J = 7.0 Hz, 1H, Ar), 7.23
(m, 1H, Ar), 7.27 (d, J = 7.5 Hz, 2H, Ar), 7.31 (d, J = 8.0 Hz, 2H, Ar), 7.37 (s, 1H, Ar), 9.66 (s, 1H, OH). IR (Nujol)
3172, 1640, 1574 cm-1. m/z 315 (m+H)+. Anal. calcd. for C17H15ClN2O2 (314.77) C, 64.87; H, 4.80; N, 8.90.
Found C, 64.83; H, 4.81; N, 8.93.
4.1.1.4. 1-(5-(2,4-Dichlorophenyl)-3-(3-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (7j)
Following the general procedure, the title compound was prepared starting from chalcone 5j. Yield 92%.
M.p. 162-163 °C. 1H NMR (DMSO-d6) 2.34 (s, 3H, CH3), 3.02 (dd, J = 4.0, 14.0 Hz, 1H, Ch), 3.90 (dd, J = 13.5,
3.5 Hz, 1H, CH), 5.71 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.87 (d, J =7.5 Hz, 1H, Ar), 7.08 (d, J = 7.0 Hz, 1H, Ar), 7.22
(m, 2H, Ar), 7.66 (m, 2H, Ar), 7.72 (s, 1H, Ar), 9.65 (s, 1H, OH). IR (Nujol) 3264, 1644, 1574 cm-1. m/z 349
(m+H)+. Anal. calcd. for C17H14Cl2N2O2 (349.21) C, 58.47; H, 4.04; N, 8.02. Found C, 58.42; H, 4.02; N, 8.05.
4.1.1.5. 1-(5-(2,5-Dichlorophenyl)-3-(3-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (7k)
Following the general procedure, the title compound was prepared starting from chalcone 5k. Yield 91%.
M.p. 231-233 °C. 1H NMR (DMSO-d6) 2.35 (s, 3H, CH3), 3.25 (dd, J = 4.0, 14.0 Hz, 1H, CH), 4.07 (dd, J = 13.5,
3.5 Hz, 1H, CH), 5.68 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.93 (d, J = 7.5 Hz, 1H, Ar), 7.35 (d, J = 7.0 Hz, 1H, Ar),
7.56 (m, 2H, Ar), 7.73 (m, 2H, Ar), 8.06 (s, 1H, Ar), 10.1 (s, 1H, OH). IR (Nujol) 3320, 1636, 1571 cm-1. m/z
349 (m+H)+. Anal. calcd. for C17H14Cl2N2O2 (349.21) C, 58.47; H, 4.04; N, 8.02. Found C, 58.41; H, 4.05; N,
8.06.
4.1.1.6. 1-(3-(3-Hydroxyphenyl)-5-(naphthalen-1-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (7m)
Following the general procedure, the title compound was prepared starting from chalcone 5m. Yield 71%.
M.p. 180-181 °C. 1H NMR (DMSO-d6) 2.44 (s, 3H, CH3), 2.98 (dd, J = 14.0, 4.0 Hz, 1H, CH), 4.22 (dd, J = 13.5,

4.0 Hz, 1H, CH), 6.17 (dd, J = 13.0, 3.5 Hz, 1H, CH), 6.62 (d, J = 8.0 Hz, 2H, Ar), 7.08 (d, J = 8.5 Hz, 2H, Ar),
7.62 (m, 3H, Ar), 7.70 (m, 2H, Ar), 7.92 (m, 2H, Ar), 8.15 (s, 1H, Ar), 9.42 (s, 1H, OH). IR (Nujol) 3212, 1643
cm-1. m/z 331 (m+H)+. Anal. calcd. for C21H18N2O4 (330.38) C, 76.34; H, 5.49; N, 8.48. Found C, 76.41; H,
5.50; N, 8.45.
4.1.1.7. 1-(5-(4-Hydroxyphenyl)-3-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (15d)
Following the general procedure, the title compound was prepared starting from chalcone 13d. Yield 41%.
1
M.p. 130-131 °C. H NMR (DMSO-d6) 2.29 (s, 3H, CH3), 3.11 (dd, J = 13.0, 3.0 Hz, 1H, CH), 3.88 (dd, J = 14,
4.0 Hz, 1H, CH), 5.61 (dd, J = 13.0, 3.5 Hz, 1H, CH), 6.77 (d, J = 8.5 Hz, 2H, Ar), 7.14 (d, J = 8.0 Hz, 2H, Ar),
8.22 (d, J = 7.0 Hz, 2H, Ar), 8.30 (d, J = 7.5 Hz, 2H, Ar), 9.66 (s, 1H, OH). IR (Nujol) 3259, 1702, 1606 cm-1. m/z
326 (m+H)+. Anal. calcd. for C17H15N3O4 (325.32) C, 62.76; H, 4.65; N, 12.92. Found C, 62.70; H, 4.66; N,
12.96.
4.1.1.8. 1-(3-(2,4-Dichlorophenyl)-5-(4-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (15g)
Following the general procedure, the title compound was prepared starting from chalcone 13g. Yield 62%.
1
M.p. 127-128°C. H NMR (DMSO-d6) 2.28 (s, 3H, CH3), 3.13 (dd, J = 14.0, 4.0 Hz, 1H, CH), 3.81 (s, 2H, NH2),
3.86 (dd, J = 13.5, 3.5 Hz, 1H, CH), 5.44 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.73 (d, J = 8.5 Hz, 2H, Ar), 7.02 (d, J =
8.0 Hz, 2H, Ar), 7.61 (d, J = 7.5 Hz, 1H, Ar), 7.80 (d, J = 8.0 Hz, 1H, Ar), 7.81 (s, 1H, Ar), 9.35 (s, 1H, OH). IR
(Nujol) 3318, 1645, 1587 cm-1. m/z 349 (m+H)+. Anal. calcd. for C17H14Cl2N2O2 (349.21) C, 58.47; H, 4.04; N,
8.02. Found C, 58.43; H, 4.05; N, 8.06.
4.1.1.9. 1-(3-(3,4-Dichlorophenyl)-5-(4-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (15h)
Following the general procedure, the title compound was prepared starting from chalcone 13h. Yield 84%.
M.p. 122-123°C. 1H NMR (DMSO-d6) 2.31 (s, 3H, CH3), 3.09 (dd, J = 14.0, 4.5 Hz, 1H), 3.88 (dd, J = 13.5, 3.5
Hz, CH), 5.52 (dd, J = 13.0, 3.0 Hz, 1H), 6.73 (d, J = 8.0 Hz, 2H, Ar), 7.01 (d, J = 8.0 Hz, 2H, Ar), 7.76 (d, J = 7.5
Hz, 1H, Ar), 7.89 (d, J = 8.0 Hz, 1H, Ar), 7.94 (s, 1H, Ar), 9.33 (s, 1H, OH). IR (Nujol) 3250, 1646, 1595 cm-1.
m/z 349 (m+H)+. Anal. calcd. for C17H14Cl2N2O2 (349.21) C, 58.47; H, 4.04; N, 8.02. Found C, 58.52; H, 4.03;
N, 7.99.
4.1.1.10. 1-(5-(3-Hydroxyphenyl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (16b)
Following the general procedure, the title compound was prepared starting from chalcone 14b. Yield 83%.
1
M.p. 98-100 °C. H NMR (DMSO-d6) 2.32 (s, 3H, CH3), 2.35 (s, 3H, CH3), 2.80 (dd, J = 13.0, 3.5 Hz, 1H, CH),
3.41 (dd, J =13.5, 3.5 Hz, 1H, CH), 4.73 (dd, J = 14.5, 3.0 Hz, 1H, CH), 6.65 (d, J = 8.5 Hz, 1H, Ar), 6.79 (d, J =
8.5 Hz, 1H, Ar), 7.20 (m, 1H, Ar), 7.52 (d, J = 7.0 Hz, 2H, Ar), 7.68 (d, J = 7.0 Hz, 2H, Ar), 9.32 (s, 1H, OH). IR
(Nujol) 3328, 1595 cm-1. m/z 295 (m+H)+. Anal. calcd. for C18H18N2O2 (294.35) C, 73.45; H, 6.16; N, 9.52.
Found C, 73.51; H, 6.18; N, 9.47.
4.1.1.11. 1-(5-(3-hydroxyphenyl)-3-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (16d)
Following the general procedure, the title compound was prepared starting from chalcone 14d. Yield 71%.
M.p. 139-140 °C. 1H NMR (DMSO-d6) 2.19 (s, 3H, CH3), 3.15 (dd, J = 3.0, 14.0 Hz, 1H, CH), 3.98 (dd, J =14.5,
3.5 Hz, 1H, CH), 5.63 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.89 (d, J = 7.5 Hz, 1H, Ar), 7.12 (d, J = 7.0 Hz, 1H, Ar),
7.21 (m, 1H, Ar), 7.54 (d, J = 8.5 Hz, 2H, Ar), 7.69 (d, J = 8.0 Hz, 2H, Ar), 9.33 (s, 1H, OH). IR (Nujol) 3347,
1604 cm-1. m/z 326 (m+H)+. Anal. calcd. for C17H15N3O4 (325.32) C, 62.76; H, 4.65; N, 12.92. Found C, 62.70;
H, 4.66; N, 12.89.
4.1.1.12. 1-(3-(4-Chlorophenyl)-5-(3-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (16e)
Following the general procedure, the title compound was prepared starting from chalcone 14e. Yield 67%.
M.p. 109-110 °C. 1H NMR (DMSO-d6) 2.22 (s, 3H, CH3), 3.13 (dd, J = 14.0, 3.0 Hz, 1H), 3.96 (dd, J = 14.5, 3.5
Hz, CH), 5.01 (dd, J = 13.5, 3.0 Hz, 1H), 6.81 (d, J = 7.5 Hz, 1H, Ar), 6.98 (d, J = 7.0 Hz, 1H, Ar), 7.22 (m, 1H,
Ar), 7.46 (d, J = 7.5 Hz, 2H, Ar), 7.58 (d, J = 8.0 Hz, 2H, Ar), 9.36 (s, 1H, OH). IR (Nujol) 3322, 1652, 1591 cm-1.
m/z 315 (m+H)+. Anal. calcd. for C17H15ClN2O4 (314.77) C, 64.87; H, 4.80, N, 8.90. Found C, 64.82; H, 4.79; N,
8.95.
4.1.1.13. 1-(3-(2,4-Dichlorophenyl)-5-(3-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (16g)
Following the general procedure, the title compound was prepared starting from chalcone 14g. Yield 51%.
M.p. 124-125 °C. 1H NMR (DMSO-d6) 2.31 (s, 3H, CH3), 3.17 (dd, J = 14.0, 4.0 Hz, 1H, CH), 3.97 (dd, J =13.5,
3.5 Hz,1H, CH), 5.03 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.67 (d, J = 7.5 Hz, 1H, Ar), 6.91 (d, J = 7.0 Hz, 1H, Ar), 7.11
(m, 1H, Ar), 7.19 (d, J = 7.5 Hz, 1H, Ar), 7.53 (d, J = 8.0 Hz, 1H, Ar), 7.79 (s, 1H, Ar), 9.12 (s, 1H, OH). IR (Nujol)

3330, 1672, 1585 cm-1. m/z 349 (m+H)+. Anal. calcd. for C17H14Cl2N2O2 (349.21) C, 58.47; H, 4.04; N, 8.02.
Found C, 58.52; H, 4.05; N, 7.98.
4.1.1.14. 1-(3-(3,4-Dichlorophenyl)-5-(3-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (16h)
Following the general procedure, the title compound was prepared starting from chalcone 14h. Yield 73%.
M.p. 114-116 °C. 1H NMR (DMSO-d6) 2.33 (s, 3H, CH3), 3.18 (dd, J = 14.0, 4.0 Hz, 1H, CH), 4.01 (dd, J = 13.5,
3.5 Hz, 1H, CH), 5.13 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.52 (d, J = 7.5 Hz, 1H, Ar), 6.81 (d, J = 7.0 Hz, 1H, Ar),
7.31 (m, 1H, Ar), 7.44 (d, J = 7.5 Hz, 1H, Ar), 7.58 (d, J = 8.0 Hz, 1H, Ar), 7.89 (s, 1H, Ar), 9.12 (s, 1H, OH). IR
(Nujol) 3407, 1669, 1590 cm-1. m/z 349 (m+H)+. Anal. calcd. for C17H14Cl2N2O2 (349.21) C, 58.47; H, 4.04; N,
8.02. Found C, 58.42; H, 4.02; N, 8.05.
4.1.2. General procedure for the preparation of 1-acetyl-3,5-diaryl-4,5-dihydro-1H-pyrazole sulfamates 8a-
m, 9a-m, 17a-h, 18a-h. To an ice-cooled stirred solution of pyrazolines 6, 7, 15, 16 (1 mmol) in anhydrous
DMA (10 mL), freshly prepared sulfamoyl chloride (0.81 g, 7 mmol) in DMA (5 mL) was added dropwise in
30 min. The obtained mixture was stirred at room temperature overnight, then water (30 mL) was added.
The mixture was stirred for additional 2 h, then the formed precipitate was filtered off and dried.
4.1.2.1 4-(1-Acetyl-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl sulfamate (8b)
Following the general procedure, the title compound was prepared starting from pyrazoline 6b. Yield 22%.
M.p. 140-142 °C. 1H NMR (DMSO-d6) 2.15 (s, 3H, CH3), 3.11 (dd, J = 14.0, 4-0 Hz, 1H, CH), 3.36 (dd, J = 13.0,
3.5 Hz, 1H, CH), 3.61 (s, 3H, CH3), 4.22 (s, 2H, NH2), 5.47 (dd, J = 13.5, 4.0 Hz, 1H, CH), 6.83 (d, J = 8.0, 2H,
Ar), 7.09 (d, J = 8.5 Hz, 2H, Ar), 7.62 (d, J = 7.0 Hz, 2H, Ar), 7.87 (d, J = 7.5 Hz, 2H, Ar). IR (Nujol) 3155, 1629,
1516 cm-1. m/z 374 (m+H)+. Anal. calcd. for C18H19N3O4S (373.43) C, 57.89; H, 5.13; N, 11.25. Found C, 57.93;
H, 5.15; N, 11.21.
4.1.2.2. 4-(1-Acetyl-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl sulfamate (8c)
Following the general procedure, the title compound was prepared starting from pyrazoline 6c. Yield 61%.
M.p. 119-120 °C. 1H NMR (DMSO-d6) 2.15 (s, 3H, CH3), 3.09 (dd, J = 14.0, 4.0 Hz, 1H, CH), 3.68 (dd, J = 13.0,
3.5 Hz, 1H, CH), 3.72 (s, 3H, OCH3), 3.87 (s, 2H, NH2), 5.44 (dd, J = 13.5, 4.0 Hz, 1H, CH), 6.59 (d, J = 8.0 Hz,
2H, Ar), 6.84 (d, J = 8.5 Hz, 2H, Ar), 7.66 (d, J = 7.0 Hz, 2H, Ar), 7.85 (d, J = 7.5 Hz, 2H, Ar). IR (Nujol) 3198,
1611 cm-1. m/z 390 (m+H)+. Anal. calcd. for C18H19N3O5S (389.43) C, 55.52; H, 4.92; N, 10.79. Found C, 55.48;
H, 4.93; N, 10.83.
4.1.2.3. 4-(1-Acetyl-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl sulfamate (8f)
Following the general procedure, the title compound was prepared starting from pyrazoline 6f. Yield 95%.
M.p. 134-135°C. 1H NMR (DMSO-d6) 2.33 (s, 3H, CH3), 3.23 (dd, J = 13.0, 5.0, Hz, 1H, CH), 3.92 (dd, J = 12.0,
4.5 Hz, 1H, CH), 4.16 (s, 2H, NH2), 5.66 (dd, J = 13.0, 5.0 Hz, 1H, CH), 7.37 (d, J = 8.5 Hz, 2H, Ar), 7.43 (d, J =
7.5 Hz, 2H, Ar), 7.71 (d, J = 8.0 Hz, 2H, Ar), 7.87 (d, J = 9.0 Hz, 2H, Ar). IR (Nujol) 3265, 1652, 1513 cm-1. m/z
378 (m+H)+. Anal. calcd. for C17H16FN3O4S (377.39) C, 54.10; H, 4.27; N, 11.37. Found C, 54.15; H, 4.26; N,
11.34.
4.1.2.4. 4-(1-Acetyl-5-(3-chlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl sulfamate (8h)
Following the general procedure, the title compound was prepared starting from pyrazoline 6h. Yield 91%.
M.p. 118-120 °C. 1H NMR (DMSO-d6) 2.28 (s, 3H, CH3), 3.13 (dd, J = 14.0, 3.0 Hz, 1H, CH), 3.76 (dd, J = 14.5,
3.5 Hz, 1H, CH), 3.82 (s, 2H, NH2), 5.51 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.82 (d, J = 7.5Hz, 1H, Ar), 6.86 (d, J =
7.0 Hz, 1H, Ar), 7.32 (m, 1H, Ar), 7.46 (s, 1H, Ar), 7.62 (d, J = 7.5 Hz, 2H, Ar), 7.88 (d, J = 8.0 Hz, 2H, Ar). IR
(Nujol) 3222, 1610 cm-1. m/z 394 (m+H)+. Anal. calcd for C17H16ClN3O4S (393.84) C, 51.84; H, 4.09; N, 10.67.
Found C, 51.89; H, 4.07; N, 10.63.
4.1.2.5. 4-(1-Acetyl-5-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl sulfamate (8i)
Following the general procedure, the title compound was prepared starting from pyrazoline 6i. Yield 31%.
M.p. 123-124°C. 1H NMR (DMSO-d6) 2.28 (s, 3H, CH3), 3.15 (dd, J = 14.0, 3.5 Hz, 1H, CH), 3.82 (dd, J = 13.0,
3.5 Hz, 1H, CH), 4.67 (s, 2H, NH2), 5.55 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.85 (d, J = 8.0 Hz, 2H, Ar), 7.21 (d, J =
8.0 Hz, 2H, Ar), 7.63 (d, J = 7.5 Hz, 2H, Ar), 7.88 (d, J = 8.0 Hz, 2H, Ar). IR (Nujol) 3329, 1634, 1609 cm-1. m/z
394 (m+H)+. Anal. calcd. for C17H16ClN3O4S (393.84) C, 51.84; H, 4.09; N, 10.67. Found C, 51.79; H, 4.10; N,
10.70.
4.1.2.6. 4-(1-Acetyl-5-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl sulfamate (8j)
Following the general procedure, the title compound was prepared starting from pyrazoline 6j. Yield 49%.
M.p. 183-184 °C. 1H NMR (DMSO-d6) 2.32 (s, 3H, CH3), 3.14 (dd, J = 14.0, 4.0 Hz, 1H, CH), 3.88 (s, 2H, NH2),
3.95 (dd, J = 13.5, 3.5 Hz, 1H, CH), 5.68 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.83 (d, J = 8.5Hz, 2H, Ar), 7.07 (d, J =

8.0 Hz, 2H, Ar), 7.62 (d, J = 7.5 Hz, 1H, Ar), 7.85 (d, J = 8.0 Hz, 1H, Ar), 8.09 (s, 1H, Ar). IR (Nujol) 3296, 1630,
1599 cm-1 m/z 428 (m+H)+. Anal. calcd. for C17H15Cl2N3O4S (428.29) C, 47.67; H, 3.53; N, 9.81. Found C,
47.71; H, 3.52; N, 9.84.
4.1.2.7. 4-(1-Acetyl-5-(2,5-dichlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl sulfamate (8k)
Following the general procedure, the title compound was prepared starting from pyrazoline 6k. Yield 50%.
M.p. 174–176°C. 1H NMR (DMSO-d6) 2.31 (dd, J = 16.0, 4.5 Hz, 1H, CH), 2.35 (s, 3H, CH3), 3.94 (m, 1H, CH),
5.74 (dd, J = 12.5, 4.5 Hz, 1H, CH), 7.11 (m, 1H, Ar), 7.36 (m, 3H, Ar), 7.66 (s, 1H, Ar), 7.85 (m, 2H, Ar), 8.09
(s, 2H, NH2). IR (Nujol) 3308, 1637, 1603 cm-1. m/z 428 (m+H)+. Anal. calcd. for C17H15Cl2N3O4S (428.29) C,
47.67; H, 3.53; N, 9.81. Found C, 47.62; H, 3.54; N, 9.78.
4.1.2.8. 4-(1-Acetyl-5-(2,6-dichlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl sulfamate (8l)
Following the general procedure, the title compound was prepared starting from pyrazoline 6l. Yield 75%.
M.p. 138-140°C. 1H NMR (DMSO-d6) 2.24 (s, 3H, CH3), 3.16 (dd, J = 14.0, 4.0 Hz, 1H, CH), 3.78 (dd, J = 13.0,
3.5 Hz, 1H, CH), 3.88 (s, 2H, NH2), 6.05 (dd, J = 13.5, 4.0 Hz, 1H, CH), 6.85 (d, J = 8.0 Hz, 2H, Ar), 7.31 (m, 2H,
Ar), 7.39 (m, 2H, Ar), 7.88 (d, J = 7.5 Hz, 2H, Ar). IR (Nujol) 3387, 1638, 1602 cm-1. m/z 428 (m+H)+. Anal.
calcd. for C17H15Cl2N3O4S (428.29) C, 47.67; H, 3.53; N, 9.81. Found C, 47.70; H, 3.54; N, 9.83.
4.1.2.9. 4-(1-Acetyl-5-(naphthalen-1-yl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl sulfamate (8m)
Following the general procedure, the title compound was prepared starting from pyrazoline 6m. Yield 71%.
M.p. 116-118 °C. 1H NMR (DMSO-d6) 2.39 (s, 3H, CH3), 3.10 (dd, J = 14.0, 4.0 Hz, 1H, CH), 3.44 (s, 2H, NH2),
4.13 (dd, J = 13.5, 4.0 Hz, 1H, CH), 6.30 (dd, J = 13.0, 3.5 Hz, 1H, CH), 7.14 (d, J = 8.0 Hz, 2H, Ar), 7.32 (d, J =
8.5 Hz, 2H, Ar), 7.44 (m, 3H, Ar), 7.62 (m, 2H, Ar), 7.87 (m, 2H, Ar). IR (Nujol) 3285, 1646 cm-1. m/z 410
(m+H)+. Anal. calcd. for C21H19N3O4S (409.46) C, 61.60; H, 4.68; N, 10.26. Found C, 61.55; H, 4.69; N, 10.29.
4.1.2.10. 3-(1-Acetyl-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl sulfamate (9a)
Following the general procedure, the title compound was prepared starting from pyrazoline 7a. Yield 28%.
M.p. 103-105 °C. 1H NMR (DMSO-d6) 2.33 (s, 3H, CH3), 3.08 (dd, J = 13.0, 3.5 Hz, 1H, CH), 3.88 (dd, J = 14.5,
3.5 Hz, 1H, CH), 5.54 (s, 2H, NH2), 5.57 (dd, J = 14.5, 3.0 Hz, 1H, CH), 6.88 (d, J = 8.0 Hz, 1H, Ar), 6.83 (d, J =
8.5 Hz, 1H, Ar), 7.26 (m, 1H, Ar), 7.55 (m, 3H, Ar), 7.88 (m, 2H, Ar), 8.05 (s, 1H, Ar). IR (Nujol) 3261, 1615 cm-
1. m/z 360 (m+H)+. Anal. calcd. for C17H17N3O4S (359.40) C, 56.81; H, 4.77; N, 11.69. Found C, 56.86; H, 4.75;
N, 11.72.
4.1.2.11. 3-(1-Acetyl-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl sulfamate (9b)
Following the general procedure, the title compound was prepared starting from pyrazoline 7b. Yield 88%.
M.p. 117-118 °C. 1H NMR (DMSO-d6) 2.17 (s, 3H, CH3), 2.41 (s, 3H, CH3), 3.17 (dd, J = 13.0, 3.5 Hz, 1H, CH),
3.89 (dd, J = 13.5, 3.5 Hz, 1H, CH), 5.21 (s, 2H, NH2), 5.33 (dd, J = 14.5, 3.0 Hz, 1H, CH), 6.70 (d, J = 8.5 Hz,
1H, Ar), 6.88 (d, J = 8.5 Hz, 1H, Ar), 7.12 (m, 1H, Ar), 7.41 (d, J = 7.0 Hz, 2H, Ar), 7.84 (d, J = 7.0 Hz, 2H, Ar),
8.09 (s, 1H, Ar). IR (Nujol) 3246, 1614 cm-1. m/z 374 (m+H)+. Anal. calcd. for C18H19N3O4S (373.43) C, 57.89;
H, 5.13; N, 11.25. Found C, 57.93; H, 5.11; N, 11.22.
4.1.2.12. 3-(1-Acetyl-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl sulfamate (9c)
Following the general procedure, the title compound was prepared starting from pyrazoline 7c. Yield 42%.
M.p. 116-118°C. 1H NMR (DMSO-d6) 2.28 (s, 3H, CH3), 3.12 (dd, J = 13.0, 3.5 Hz, 1H, CH), 3.72 (s, 3H, OCH3),
3.79 (dd, J = 13.5, 3.5 Hz, 1H, CH), 3.90 (s, 2H, NH2), 5.49 (dd, J = 14.5, 3.0 Hz, 1H, CH), 6.88 (d, J = 8.5 Hz,
1H, Ar), 7.04 (d, J = 8.5 Hz, 1H, Ar), 7.12 (m, 1H, Ar), 7.27 (d, J = 7.0 Hz, 2H, Ar), 7.71 (d, J = 7.0 Hz, 2H, Ar),
8.06 (s, 1H, Ar). IR (Nujol) 3246, 1614 cm-1. m/z 390 (m+H)+. Anal. calcd. for C18H19N3O5S (389.43) C, 55.52;
H, 4.92; N, 10.79. Found C, 55.59; H, 4.91; N, 10.77.
4.1.2.13. 3-(1-Acetyl-5-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl sulfamate (9d)
Following the general procedure, the title compound was prepared starting from pyrazoline 7d. Yield 47%.
M.p. 203-205°C. 1H NMR (DMSO-d6) 2.32 (s, 3H, CH3), 3.11 (dd, J = 13.0, 3.5 Hz, 1H, CH), 3.17 (dd, J = 13.5,
3.5 Hz, 1H, CH), 3.87 (s, 2H, NH2), 5.68 (dd, J = 14.5, 3.0 Hz, 1H, CH), 6.89 (d, J = 8.5 Hz, 1H, Ar), 7.17 (d, J =
8.5 Hz, 1H, Ar), 7.25 (m, 1H, Ar), 7.49 (d, J = 7.0 Hz, 2H, Ar), 7.57 (d, J = 7.0 Hz, 2H, Ar), 8.20 (s, 1H, Ar). IR
(Nujol) 3239, 1634 cm-1. m/z 405 (m+H)+. Anal. calcd. for C17H16N4O6S (404.40) C, 50.49; H, 3.99; N, 13.85.
Found C, 50.56; H, 4.01; N, 13.81.
4.1.2.14. 3-(1-Acetyl-5-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl sulfamate (9e)

Following the general procedure, the title compound was prepared starting from pyrazoline 7e. Yield 40%.
M.p. 114-115 °C. 1H NMR (DMSO-d6) 2.34 (s, 3H, CH3), 3.04 (dd, J = 14.0, 3.0 Hz, 1H, CH), 3.59 (dd, J = 14.5,
3.5 Hz, 1H, CH), 3.98 (s, 2H, NH2), 5.69 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.88 (d, J = 7.5 Hz, 1H, Ar), 7.17 (d, J =
7.0 Hz, 1H, Ar), 7.28 (m, 1H, Ar), 7.45 (d, J = 8.5 Hz, 2H, Ar), 7.88 (d, J = 8.9 Hz, 2H, Ar), 7.98 (s, 1H, Ar). IR
(Nujol) 3209, 1642, 1584 cm-1. m/z 428 (m+H)+. Anal. calcd. for C18H16F3N3O4S (427.40) C, 50.58; H, 3.77; N,
9.83. Found C, 50.64; H, 3.76; N, 9.80.
4.1.2.15. 3-(1-Acetyl-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl sulfamate (9f)
Following the general procedure, the title compound was prepared starting from pyrazoline 7f. Yield 27%.
M.p. 94-95 °C. 1H NMR (DMSO-d6) 2.29 (s, 3H, CH3), 3.12 (dd, J = 14.0, 3.0 Hz, 1H, CH), 3.74 (dd, J = 14.5, 3.5
Hz, 1H, CH), 3.88 (s, 2H, NH2), 5.54 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.87 (d, J = 7.5 Hz, 1H, Ar), 7.15 (d, J = 7.9
Hz, 1H, Ar), 7.22 (m, 1H, Ar), 7.39 (d, J = 8.5 Hz, 2H, Ar), 7.55 (d, J = 8.0 Hz, 2H, Ar), 8.06 (s, 1H, Ar). IR (Nujol)
3264, 1607 cm-1. m/z 378 (m+H)+. Anal. calcd. for C17H16FN3O4S (377.39) C, 54.10; H, 4.27; N, 11.13. Found
C, 54.04; H, 4.28; N, 11.17.
4.1.2.16. 3-(1-Acetyl-5-(2-chlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl sulfamate (9g)
Following the general procedure, the title compound was prepared starting from pyrazoline 7g. Yield 54%.
M.p. 121-123 °C. 1H NMR (DMSO-d6) 2.35 (s, 3H, CH3), 2.96 (dd, J = 14.0, 3.0 Hz, 1H, CH), 3.93 (dd, J = 14.5,
3.5 Hz, 1H, CH), 4.88 (s, 2H, NH2), 5.77 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.87 (d, J = 7.5 Hz, 1H, Ar), 7.08 (d, J =
7.0 Hz, 1H, Ar), 7.22 (m, 1H, Ar), 7.38 (d, J = 7.5 Hz, 2H, Ar), 7.72 (d, J = 8.0 Hz, 2H, Ar), 8.08 (s, 1H, Ar). IR
(Nujol) 3237, 1642, 1574 cm-1. m/z 394 (m+H)+. Anal. calcd. for C17H16ClN3O4S (393.84) C, 51.84; H, 4.09; N,
10.67. Found C, 51.79; H, 4.11; N, 10.69.
4.1.2.17. 3-(1-Acetyl-5-(3-chlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl sulfamate (9h)
Following the general procedure, the title compound was prepared starting from pyrazoline 7h. Yield 44%.
M.p. 96-98 °C. 1H NMR (DMSO-d6) 2.34 (s, 3H, CH3), 3.14 (dd, J = 3.0, 14.0 Hz, 1H, CH), 3.64 (dd, J = 14.5, 3.5
Hz, 1H, CH), 3.82 (s, 2H, NH2), 5.58 (dd, J = 13.5, 3.0 Hz, 1H, CH), 7.16 (d, J = 7.5 Hz, 1H, Ar), 7.27 (d, J = 7.0
Hz, 1H, Ar), 7.39 (s, 1H, Ar), 7.55 (d, J = 7.5 Hz, 2H, Ar), 7.72 (d, J = 8.0 Hz, 2H, Ar), 8.05 (s, 1H, Ar). IR (Nujol)
3262, 1614, 1574 cm-1. m/z 394 (m+H)+. Anal. calcd. for C17H16ClN3O4S (393.84) C, 51.84; H, 4.09; N, 10.67.
Found C, 51.88; H, 4.08; N, 10.71.
4.1.2.18. 3-(1-Acetyl-5-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl sulfamate (9i)
Following the general procedure, the title compound was prepared starting from pyrazoline 7i. Yield 72%.
M.p. 108-110 °C. 1H NMR (DMSO-d6) 2.29 (s, 3H, CH3), 2.95 (dd, J = 14.0, 3.0, Hz, 1H, CH), 3.80 (dd, J = 14.5,
3.5 Hz, 1H, CH), 3.91 (s, 2H, NH2), 5.54 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.88 (d, J = 7.5 Hz, 1H, Ar), 7.16 (d, J =
7.0 Hz, 1H, Ar), 7.39 (m, 1H, Ar), 7.55 (d, J = 7.5 Hz, 2H, Ar), 7.73 (d, J = 8.0 Hz, 2H, Ar), 8.06 (s, 1H, Ar). IR
(Nujol) 3265, 1615 cm-1. m/z 394 (m+H)+. Anal. calcd. for C17H16ClN3O4S (393.84) C, 51.84; H, 4.09; N, 10.67.
Found C, 51.79; H, 4.08; N, 10.70.
4.1.2.19. 3-(1-Acetyl-5-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl sulfamate (9j)
Following the general procedure, the title compound was prepared starting from pyrazoline 7j. Yield 22%.
M.p. 105-107 °C. 1H NMR (DMSO-d6) 2.33 (s, 3H, CH3), 3.14 (dd, J = 14.0, 4.0, Hz, 1H, CH), 3.88 (dd, J = 13.5,
3.5 Hz, 1H, CH), 4.01 (s, 2H, NH2), 5.78 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.87 (d, J = 7.5 Hz, 1H, Ar), 7.11 (d, J =
7.0 Hz, 1H, Ar), 7.38 (m, 2H, Ar), 7.67 (m, 2H, Ar), 8.05 (s, 1H, Ar). IR (Nujol) 3220, 1640, 1584 cm-1. m/z 428
(m+H)+. Anal. calcd. for C17H15Cl2N3O4S (428.29) C, 47.67; H, 3.53; N, 9.81. Found C, 47.71; H, 3.52; N, 9.78.
4.1.2.20. 3-(1-Acetyl-5-(2,5-dichlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl sulfamate (9k)
Following the general procedure, the title compound was prepared starting from pyrazoline 7k. Yield 88%.
M.p. 160-161 °C. 1H NMR (DMSO-d6) 2.37 (s, 3H, CH3), 3.18 (dd, J = 14.0, 4.0 Hz, 1H, CH), 3.97 (dd, J = 13.5,
3.5 Hz, 1H, CH), 4.57 (s, 2H, NH2), 5.77 (dd, J = 13.5, 3.0 Hz, 1H, CH), 7.11 (d, J = 7.5 Hz, 1H, Ar), 7.55 (d, J =
7.0 Hz, 1H, Ar), 7.69 (m, 2H, Ar), 7.73 (m, 2H, Ar), 8.06 (s, 1H, Ar). IR (Nujol) 3190, 1647, 1572 cm-1. m/z 428
(m+H)+. Anal. calcd. for C17H15Cl2N3O4S (428.29) C, 47.67; H, 3.53; N, 9.81. Found C, 47.62; H, 3.54; N, 9.77.
4.1.2.21. 3-(1-Acetyl-5-(2,6-dichlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl sulfamate (9l)
Following the general procedure, the title compound was prepared starting from pyrazoline 7l. Yield 41%.
M.p. 129-130 °C. 1H NMR (DMSO-d6) 2.25 (s, 3H, CH3), 3.22 (dd, J = 14.0, 4.0 Hz, 1H, CH), 3.72 (dd, J = 13.5,
3.5 Hz, 1H, CH), 3.88 (s, 2H, NH2), 6.07 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.14 (d, J = 7.5 Hz, 1H, Ar), 6.89 (d, J =
7.0 Hz, 1H, Ar), 7.28 (m, 1H, Ar), 7.33 (d, J = 7.5 Hz, 1H, Ar), 7.71 (d, J = 8.0 Hz, 1H, Ar), 8.09 (s, 1H, Ar). IR
(Nujol) 3230, 1642, 1576 cm-1. m/z 428 (m+H)+. Anal. calcd. for C17H15Cl2N3O4S (428.29) C, 47.67; H, 3.53; N,
9.81. Found C, 47.72; H, 3.54; N, 9.78.

4.1.2.22. 3-(1-Acetyl-5-(naphthalen-1-yl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl sulfamate (9m)
Following the general procedure, the title compound was prepared starting from pyrazoline 7m. Yield 79%.
M.p. 118-120 °C. 1H NMR (DMSO-d6) 2.42 (s, 3H, CH3), 2.96 (dd, J = 14.0, 4.0 Hz, 1H, CH), 3.04 (s, 2H, NH2),
4.12 (dd, J = 13.5, 4.0 Hz Hz, 1H, CH), 6.27 (dd, J = 13.5, 3.5 Hz, 1H, CH), 6.87 (d, J = 8.0 Hz, 2H, Ar), 7.22 (d, J
= 8.5 Hz, 2H, Ar), 7.45 (m, 3H, Ar), 7.64 (m, 2H, Ar), 7.87 (m, 2H, Ar), 8.16 (s, 1H, Ar). IR (Nujol) 3213, 1640
cm-1. m/z 410 (m+H)+. Anal. calcd. for C21H19N3O4S (409.46) C, 61.60; H, 4.68; N, 10.26. Found C, 61.67; H,
4.69; N, 10.23.
4.1.2.23. 4-(1-Acetyl-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl)phenyl sulfamate (17a)
Following the general procedure, the title compound was prepared starting from pyrazoline 15a. Yield 19%.
M.p. 124-125 °C. 1H NMR (DMSO-d6) 2.29 (s, 3H, CH3), 3.13 (dd, J = 14.0, 4.0 Hz, 1H, CH), 3.87 (dd, J = 13.5,
4.0 Hz, 1H, CH), 4.62 (s, 2H, NH2), 5.61 (dd, J = 13.5, 3.5 Hz, 1H, CH), 7.07 (d, J = 8.0 Hz, 2H, Ar), 7.23 (d, J =
8.5 Hz, 2H, Ar), 7.48 (m, 3H, Ar), 7.98 (m, 2H, Ar). IR (Nujol) 3196, 1596 cm-1. m/z 360 (m+H)+. Anal. calcd.
for C17H17N3O4S (359.40) C, 56.81; H, 4.77; N, 11.69. Found C, 56.76; H, 4.76; N, 11.73.
4.1.2.24. 4-(1-acetyl-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl sulfamate (17b)
Following the general procedure, the title compound was prepared starting from pyrazoline 15b. Yield 47%.
M.p. 126-127 °C. 1H NMR (DMSO-d6) 2.27 (s, 3H, CH3), 2.38 (s, 3H, CH3), 3.14 (dd, J = 14.0, 3.5 Hz, 1H, CH),
3.87 (dd, J =13.5, 3.5 Hz, 1H, CH), 4.23 (s, 2H, NH2), 5.57 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.69 (d, J = 8.0 Hz, 2H,
Ar), 6.98 (d, J = 8.0 Hz, 2H, Ar), 7.37 (d, J = 7.5 Hz, 2H, Ar), 7.90 (d, J = 7.0 Hz, 2H, Ar). IR (Nujol) 3346, 1637,
1607 cm-1. m/z 374 (m+H)+. Anal. calcd. for C18H19N3O4S (373.43) C, 57.89; H, 5.13; N, 11.25. Found C, 57.96;
H, 5.11; N, 11.22.
4.1.2.25. 4-(1-Acetyl-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl sulfamate (17c)
Following the general procedure, the title compound was prepared starting from pyrazoline 15c. Yield 30%.
1
M.p. 177-178 °C. H NMR (DMSO-d6) 2.31 (s, 3H, CH3), 3.11 (dd, J = 4.5, 17.5 Hz, 1H, CH), 3.84 (m, 4H, CH
and OCH3), 5.56 (d, J = 4.5, 11.5 Hz, 1H, CH), 7.02 (d, J = 8.5 Hz, 2H, Ar), 7.23 (d, J = 9.0 Hz, 2H, Ar), 7.27 (d, J
= 8.5 Hz, 2H, Ar), 7.73 (d, J = 9.0 Hz, 2H, Ar), 7.98 (s, 2H, NH2). IR (Nujol) 3321, 3167, 1637, 1568 cm-1. m/z
390 (M + H)+. Anal. calcd. for C18H19N3O5S (389.10) C, 55.52; H, 4.92; N, 10.79. Found C, 55.58; H, 4.90; N,
10.76.
4.1.2.26. 4-(1-Acetyl-3-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl sulfamate (17d)
Following the general procedure, the title compound was prepared starting from pyrazoline 15d. Yield 60%.
M.p. 134-135 °C. 1H NMR (DMSO-d6) 2.31 (s, 3H, CH3), 2.95 (s, 2H, NH2), 3.13 (dd, J = 13.0, 3.0 Hz, 1H, CH),
3.86 (dd, J = 14.0 , 4.0 Hz, 1H, CH), 5.57 (dd, J = 13.0, 3.5 Hz, 1H, CH), 6.72 (d, J = 8.5 Hz, 2H, Ar), 7.19 (d, J =
8.0 Hz, 2H, Ar), 8.13 (d, J = 7.0 Hz, 2H, Ar), 8.33 (d, J = 7.5 Hz, 2H, Ar). IR (Nujol) 3203, 1643, 1605 cm-1. m/z
405 (M + H)+. Anal. calcd. for C17H16N4O6S (404.40) C, 50.49, H, 3.99, N, 13.85. Found C, 50.53, H, 4.01; N
13.81.
4.1.2.27. 4-(1-Acetyl-3-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl sulfamate (17f)
Following the general procedure, the title compound was prepared starting from pyrazoline 15f. Yield 61%.
M.p. 118-120 °C. 1H NMR (DMSO-d6) 2.27 (s, 3H, CH3), 3.14 (dd, J = 14.0, 3.5 Hz, 1H, CH), 3.83 (dd, J = 13.5,
3.5 Hz, 1H, CH), 4.47 (s, 2H, NH2), 5.57 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.71 (d, J = 8.0 Hz, 2H, Ar), 7.09 (d, J =
8.0 Hz, 2H, Ar), 7.54 (d, J = 7.5 Hz, 2H, Ar), 7.93 (d, J = 8.0 Hz, 2H, Ar). IR (Nujol) 3187, 1640, 1592 cm-1. m/z
394 (m+H)+. Anal. calcd. for C17H16ClN3O4S (393.84) C, 51.84; H, 4.09; N, 9.00. Found C, 51.79; H, 4.08; N,
9.04.
4.1.2.28. 4-(1-Acetyl-3-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl sulfamate (17g)
Following the general procedure, the title compound was prepared starting from pyrazoline 15g. Yield 77%.
M.p. 114-115 °C. 1H NMR (DMSO-d6) 2.26 (s, 3H, CH3), 3.17 (dd, J = 14.0, 4.0 Hz, 1H, CH), 3.76 (s, 2H, NH2),
3.89 (dd, J = 13.5, 3.5 Hz, 1H, CH), 5.45 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.71 (d, J = 8.5 Hz, 2H, Ar), 7.02 (d, J =
8.0 Hz, 2H, Ar), 7.62 (d, J = 7.5 Hz, 1H, Ar), 7.78 (d, J = 8.0 Hz, 1H, Ar), 7.82 (s, 1H, Ar). IR (Nujol) 3271, 1646,
1587 cm-1. m/z 428 (m+H)+. Anal. calcd. for C17H15Cl2N3O4S (428.29) C, 47.67; H, 3.53; N, 9.81. Found C,
47.63; H, 3.52; N, 9.84.
4.1.2.29. 4-(1-acetyl-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl sulfamate (17h)
Following the general procedure, the title compound was prepared starting from pyrazoline 15h. Yield 66%
M.p. 123-125 °C. 1H NMR (DMSO-d6) 2.29 (s, 3H, CH3), 3.14 (dd, J = 14.0, 4.5 Hz, 1H), CH, 3.81 (dd, J = 13.5,
3.5 Hz, 1H, CH), 4.21 (s, 2H, NH2), 5.47 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.69 (d, J = 8.0 Hz, 2H, Ar), 7.08 (d, J =
8.0 Hz, 2H, Ar), 7.73 (d, J = 7.5 Hz, 1H, Ar), 7.81 (d, J = 8.0 Hz, 1H, Ar), 7.98 (s, 1H, Ar). IR (Nujol) 3250, 1645,

1594 cm-1. m/z 428 (m+H)+. Anal. calcd. for C17H15Cl2N3O4S (428.29) C, 47.67; H, 3.53; N, 9.81. Found C,
47.73; H, 3.54; N, 9.78.
4.1.2.30. 3-(1-Acetyl-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl)phenyl sulfamate (18a)
Following the general procedure, the title compound was prepared starting from pyrazoline 16a. Yield 51%.
M.p. 94-95 °C. 1H NMR (DMSO-d6) 2.22 (s, 3H, CH3), 3.11 (dd, J = 13.0, 3.5 Hz, 1H, CH), 3.87 (dd, J =14.5, 3.5
Hz, 1H, CH), 5.01 (s, 2H, NH2), 5.12 (dd, J = 14.5, 3.0 Hz, 1H, CH), 6.71 (d, J = 8.0 Hz, 1H, Ar), 6.83 (d, J = 8.5
Hz, 1H, Ar), 7.26 (m, 1H, Ar), 7.55 (m, 3H, Ar), 7.88 (m, 2H, Ar). IR (Nujol) 3200, 1598 cm-1. m/z 360 (m+H)+.
Anal. calcd. for C17H17N3O4S (359.40) C, 56.81; H, 4.77; N, 11.69. Found C, 56.86; H, 4.76; N, 11.65.
4.1.2.31. 3-(1-Acetyl-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl sulfamate (18b)
Following the general procedure, the title compound was prepared starting from pyrazoline 16b. Yield 88%.
M.p. 108-110 °C. 1H NMR (DMSO-d6) 2.17 (s, 3H, CH3), 2.41 (s, 3H, CH3), 3.17 (dd, J = 13.0, 3.5 Hz, 1H, CH),
3.89 (dd, J = 13.5, 3.5 Hz, 1H, CH), 5.21 (s, 2H, NH2), 5.33 (dd, J = 14.5, 3.0 Hz, 1H, CH), 6.70 (d, J = 8.5 Hz,
1H, Ar), 6.88 (d, J = 8.5 Hz, 1H, Ar), 7.12 (m, 1H, Ar), 7.41 (d, J = 7.0 Hz, 2H, Ar), 7.84 (d, J = 7.0 Hz, 2H, Ar). IR
(Nujol) 3250, 1609 cm-1. m/z 374 (m+H)+. Anal. calcd. for C18H19N3O4S (373.43) C, 57.89; H, 5.13; N, 11.25;
Found C, 57.84; H, 5.15; N, 11.29.
4.1.2.32. 3-(1-Acetyl-3-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl sulfamate (18d)
Following the general procedure, the title compound was prepared starting from pyrazoline 16d. Yield 47%.
M.p. 174-175 °C. 1H NMR (DMSO-d6) 2.23 (s, 3H, CH3), 3.04 (dd, J = 14.0, 3.0 Hz, 1H, CH), 3.88 (dd, J = 14.5,
3.5 Hz, 1H, CH), 4.58 (s, 2H, NH2), 5.44 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.79 (d, J = 7.5 Hz, 1H, Ar), 7.09 (d, J =
7.0 Hz, 1H, Ar), 7.23 (m, 1H, Ar), 7.47 (d, J = 8.5 Hz, 2H, Ar), 7.90 (d, J = 8.5 Hz, 2H, Ar). IR (Nujol) 3252, 1650,
1605 cm-1. m/z 405 (m+H)+. Anal. calcd. for C17H16N4O6S (404.08) C, 50.49; H, 3.99; N, 13.85. Found C,
50.554; H, 4.01; N, 13.81.
4.1.2.38. 4-(1-Acetyl-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl sulfamate (18e)
Following the general procedure, the title compound was prepared starting from pyrazoline 16e. Yield 40%.
M.p. 190-191 °C. 1H NMR (DMSO-d6) 2.32 (s, 3H, CH3), 3.18 (dd, J = 18.0, 4.0 Hz, 1H, CH), 3.88 (dd, J = 13.5,
16.5 Hz, 1H, CH), 5.59 (m, 1H, CH), 7.11 (s, 1H, Ar), 7.16 (t, J = 7.5 Hz, 2H, Ar), 7.32 (t, J = 7.5 Hz, 1H, Ar), 7.42
(t, J = 7.5 Hz, 2H, Ar), 7.85 (m, 2H, Ar), 7.99 (s, 2H, NH2). IR (Nujol) 3325, 1638, 1608 cm-1. m/z 378 (m+H)+.
Anal. calcd. for C17H16FN3O4S (377.39) C, 54.10; H, 4.27; N, 11.13. Found C, 54.05; H, 4.28; N, 11.17.
4.1.2.39. 3-(1-Acetyl-3-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl sulfamate (18f)
Following the general procedure, the title compound was prepared starting from pyrazoline 16f. Yield 69%.
M.p. 93-95 °C. 1H NMR (DMSO-d6) 2.18 (s, 3H, CH3), 3.08 (dd, J = 14.0, 3.0 Hz, 1H, CH), 4.01 (dd, J = 14.5, 3.5
Hz, 1H, CH), 4.69 (s, 2H, NH2), 5.08 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.71 (d, J = 7.5 Hz, 1H, Ar), 6.78 (d, J = 7.0
Hz, 1H, Ar), 7.22 (m, 1H, Ar), 7.36 (d, J = 7.5 Hz, 2H, Ar), 7.68 (d, J = 8.0 Hz, 2H, Ar), 7.88 (s, 1H, Ar). IR (Nujol)
3222, 1673, 1589 cm-1. m/z 394 (m+H)+. Anal. calcd. for C17H16ClN3O4S (393.84) C, 51.84; H, 4.09; N, 10.67.
Found C, 51.80; H, 4.11; N, 10.70.
4.1.2.40. 3-(1-Acetyl-3-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl sulfamate (18g)
Following the general procedure, the title compound was prepared starting from pyrazoline 16g. Yield 77%.
M.p. 124-125 °C. 1H NMR (DMSO-d6) 2.29 (s, 3H, CH3), 3.18 (dd, J = 14.0, 4.0 Hz, 1H, CH), 3.96 (dd, J = 13.5,
3.5 Hz, 1H, CH), 4.57 (s, 2H, NH2), 5.03 (dd, J = 13.5, 3.0 Hz, 1H, CH), 6.64 (d, J = 7.5 Hz, 1H, Ar), 6.88 (d, J
=7.5 Hz, 1H, Ar), 7.13 (m, 1H, Ar), 7.19 (d, J = 7.5 Hz, 1H, Ar), 7.53 (d, J = 8.0 Hz, 1H, Ar), 7.77 (s, 1H, Ar). IR
(Nujol) 3238, 1684, 1583 cm-1. m/z 428 (m+H)+. Anal. calcd. for C17H15Cl2N3O4S (428.29) C, 47.67; H, 3.53; N,
9.81. Found C, 47.71; H, 3.54; N, 9.79.
4.1.2.41. 3-(1-Acetyl-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl sulfamate(18h)
Following the general procedure, the title compound was prepared starting from pyrazoline 16h. Yield 70%.
M.p. 108-110 °C. 1H NMR (DMSO-d6) 2.23 (s, 3H, CH3), 3.22 (dd, J = 14.0, 4.0 Hz, 1H, CH), 4.01 (dd, J =13.5,
3.5 Hz, 1H, CH), 5.13 (dd, J = 13.5, 3.0 Hz, 1H, CH), 5.36 (s, 2H, NH2), 6.46 (d, J = 7.0 Hz, 1H, Ar), 6.72 (d, J
=7.0 Hz, 1H, Ar), 7.28 (m, 1H, Ar), 7.49 (d, J = 7.5 Hz, 1H, Ar), 7.75 (d, J = 8.0 Hz, 1H, Ar), 7.95 (s, 1H, Ar). IR
(Nujol) 3241, 1679, 1584 cm-1. m/z 428 (m+H)+. Anal. calcd. for C17H15Cl2N3O4S (428.29) C, 47.67; H, 3.53; N,
9.81. Found C, 47.62; H, 3.52; N, 9.85.
4.2. Carbonic anhydrase inhibition
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO2
hydration activity [50]. Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at

the absorbance maximum of 557 nm, with 20 mMHepes (pH 7.5) as buffer, and 20 mM Na2SO4 (for
maintaining constant the ionic strength), following the initial rates of the CA-catalyzed CO2 hydration
reaction for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination
of the kinetic parameters and inhibition constants. For each inhibitor at least six traces of the initial 5–10%
of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined
in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM)
were prepared in 10% DMSO aqueous solution and dilutions up to 0.01 nM were done thereafter with the
assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature
prior to assay, in order to allow for the formation of the E-I complex. The inhibition constants were
obtained by non-linear least-squares methods using PRISM 3 and the Cheng–Prusoff equation, as reported
earlier [56-58], and represent the mean from at least three different determinations. All CA isofoms were
recombinant ones obtained in-house as reported earlier [59-62].
4.3. Molecular modeling
Molecular Docking studies were carried out to try to understand the binding mechanism of our compounds
using rDock [63].
The four crystal structures of CAI (PDBID: 3w6h), CAII (PDBID: 4g0c), CAIX (PDBID: 3iai), CAXII (PDBID: 1jd0)
were retrieved from RCSB Protein Data Bank web page (http://www.rcsb.org/). The preparation of the
proteins was performed using HTMD (High Throughput MD) tool [64], in order to add hydrogens, ionize
side chain of amino acids at physiological pH using propKa, deleting water molecules and co-crystallized
small molecules (AAZ included).
3D ligands were prepared using an in-house python script developed using RDKit toolkit [65] and minimized
using MMFF94 forcefield.
Considering the high conservation of the binding mode of the sulfamide moiety of co-crystallized
compounds a tethered docking was executed [63] to enforce the partial binding modes of the sulfamates
group. The ‘dock_solv’ rDock protocol was executed, this protocol allows a full docking search but using the
desolvation scoring function, the docking grid was centered on the co-crystalized ligand and the radius
settled at 5.0 Å. For each ligand, the number of possible poses was set to 10 and the one with the best
score was used to evaluate its binding interactions.
Protocol validation was carried out with re-docking procedure, AAZ 3D structure was prepared and docked
on the four crystals as the other ligands, then the resulting poses were compared with the crystallographic
and RMSD have been calculated (Table 2).
Table 2. RMSD and SCORE of the best pose of AAZ redocked on the four CA crystals
CAI
1.54
-21.28
CAII
1.62
-23.67
CAIX
0.76
-23.90
CAXII
0.67
-25.75
RMSD
SCORE
Acknowledgements
This study was supported in part by FIR grants from the University of Cagliari (to V.O. and G.B.).
Conflict of interest
The authors declare no conflict of interest.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in the online version,
at………………………………….

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