New saccharin derivatives as tyrosinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.03.033

A newly series of 6-(phenylurenyl/thiourenyl) saccharin (6a-y) derivatives were synthesized and their inhibitory effects on the diphenolase activity of banana tyrosinase were evaluated. A 70-fold purification of the enzyme with 6.85% yield was achieved by using a Sepharose 4B-l-tyrosine-p-amino benzoic acid affinity column. The result showed that all the synthesized compounds inhibited the tyrosinase enzyme activity. Among the compounds synthesized, 6-(3-iodophenylthiourenyl) saccharin (6s) was found to be most active one (K_i = 3.95 μM) and the inhibition kinetics analyzed by Lineweaver-Burk double reciprocal plots revealed that compound 6s was a competitive inhibitor. Structure-activity relationships study showed that generally, most of the 6-(phenylthiourenyl) saccharin derivatives (6m-y) exhibited higher inhibitory activity than 6-(phenylurenyl) saccharin derivatives (6a-l). An electron-withdrawing group at 3-position of phenylurenyl-ring increased in activity and the halogen series at 3-position of phenylthiourenyl-ring showed a qualitative relationship for higher inhibitory activity with increasing size and polarizability. We also calculated HOMO-LUMO energy levels and dipole moments of some selected the synthesized compounds (6a, 6h, 6m and 6s) using Gaussian software.

Full text of DOI:10.1016/j.bmc.2012.03.033

Bioorganic & Medicinal Chemistry 20 (2012) 2811–2821
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New saccharin derivatives as tyrosinase inhibitors
Nahit Gençer a, , Dudu Demir a, Fatih Sonmez b, Mustafa Kucukislamoglu b
⇑
a Department of Chemistry, Faculty of Art and Sciences, Balikesir University, Balikesir 10145, Turkey
b Department of Chemistry, Faculty of Art and Sciences, Sakarya University, Sakarya 54140, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
A newly series of 6-(phenylurenyl/thiourenyl) saccharin (6a–y) derivatives were synthesized and their
Received 28 November 2011
Revised 13 March 2012
Accepted 15 March 2012
Available online 23 March 2012
inhibitory effects on the diphenolase activity of banana tyrosinase were evaluated. A 70-fold purification
of the enzyme with 6.85% yield was achieved by using a Sepharose 4B-L-tyrosine-p-amino benzoic acid
affinity column. The result showed that all the synthesized compounds inhibited the tyrosinase enzyme
activity. Among the compounds synthesized, 6-(3-iodophenylthiourenyl) saccharin (6s) was found to be
most active one (Ki = 3.95 lM) and the inhibition kinetics analyzed by Lineweaver–Burk double recipro-
Keywords:
Saccharin
Urea
Thiourea
Tyrosinase inhibitors
cal plots revealed that compound 6s was a competitive inhibitor. Structure–activity relationships study
showed that generally, most of the 6-(phenylthiourenyl) saccharin derivatives (6m–y) exhibited higher
inhibitory activity than 6-(phenylurenyl) saccharin derivatives (6a–l). An electron-withdrawing group
at 3-position of phenylurenyl-ring increased in activity and the halogen series at 3-position of phenyl-
thiourenyl-ring showed a qualitative relationship for higher inhibitory activity with increasing size
and polarizability. We also calculated HOMO–LUMO energy levels and dipole moments of some selected
the synthesized compounds (6a, 6h, 6m and 6s) using Gaussian software.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
bioactive molecules.13 It was also employed in the synthesis of phe-
nolic ether prodrugs, with the intention of increasing their oral bio-
Saccharin, 1,2-benzisothiazole-3-one-1,1-dioxide, is
a
well-
availability.14 Being a weak acid, saccharin readily forms salts with
various basic active pharmaceutical ingredients (API) thus resulting
in highly soluble saccharinates, which may be used above all in
pediatric medication, on account of saccharin being a potent sweet-
ener able to mask the bitter taste of many drugs.15 The use of sac-
charin acting as a co-crystal former was also reported. Behaving
both like a hydrogen bond donor and acceptor, it forms highly sol-
uble co-crystals with certain APIs.16 In addition, certain substituted
saccharin derivatives have been used as intermediates in the prep-
aration of the 4-hydroxy-1,2-benzothiazine 1,1-dioxide (oxicam)
ring system, a ring-expanded saccharin, derivatives of which pos-
sess significant anti-inflammatory properties.17
known heterocyclic compound and has been used as a sweetener
in the form of its sodium salt since 1885. Yet it is also a heterocycle
of pharmaceutical importance, being a key structural element of
certain CNS-active drugs.1 Saccharin compounds have been inten-
sively investigated mainly due to its suspected cancerogenic
nature.2 Many biological activities have been attributed to this
group, such as inhibitors of serine proteases,3 cathepsin G and pro-
teinase 3,4
a1a and a
1c adrenergic receptor antagonists,5 human
mast cell tryptase inhibitors,6 analgesics,7 5-HT1a receptors,8 alde-
hyde dehydrogenase inhibitors,9 anti-anxiety and antibacterial.10
Recently, as an orally active bioavailable human leukocyte elastase
(HLE) inhibitor, [6-methoxy-4-isopropyl-3-oxo-1,2-benzisothiaz-
ole-2(3H)-yl]methyl-2,6-dichloro-3-[2-(4-morpholinyl)ethoxy]be
nzoate S,S-dioxide (WIN) series,4 saccharin-based inhibitors, were
found to have potent HLE inhibitory activity. Additionally, sacchari-
nate complexes with amine derivatives have been useful as antidote
for metal poisoning11 and DNA-alerting ability.12 Its importance has
increased over the years and it can be viewed as a privileged scaffold
in the field of medicinal chemistry. This particular heterocycle can
either be a substituent of a larger compound that assumes the role
of a framework, or it can play the role of the pharmacophore of
Since N,N0-disubstituted ureas and thioureas are linked to a
series of biological activities including antiglycating,18 MCH-R1
antagonists,19 P2Y1 receptor antagonists,20 heparanase inhibitors,21
anti-HIV,22 cytostatic and antioxidant,23 and proliferation inhibi-
tors24 properties, 6-aminosaccharin was reacted with isocyanates
and isothiocyanates to get product unsymmetrically substituted
urea and thiourea derivatives. In addition, this groups have re-
ported25 that aromatic sulfonamides or bis-sulfonamides possess-
ing among others the unsubstituted urea or thiourea moieties
attached to the aromatic ring, not only behave as very potent inhib-
itors against three isozymes, human CA I, human CA II and bovine
isozyme CA, but possess an unexpectedly high affinity for the slow
isozyme human CA I.
⇑
Corresponding author. Tel.: +90 266 612 1278; fax: +90 266 612 1215.
E-mail address: ngencer@balikesir.edu.tr (N. Gençer).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.033

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N. Gençer et al. / Bioorg. Med. Chem. 20 (2012) 2811–2821
Tyrosinase (monophenol or o-diphenol, oxygen oxidoreductase,
2.2. Biological evaluation of synthesized compounds for
tyrosinase inhibitory activity
EC 1.14.18.1), also known as polyphenol oxidase (PPO), is a copper-
containing monooxygenase that is widely distributed in microor-
ganisms, animals, and plants.26 Tyrosinase could catalyze two dis-
tinct reactions involving molecular oxygen in the hydroxylation of
monophenols to o-diphenols (monophenolase) and in the oxida-
tion of o-diphenols to o-quinones (diphenolase).27 Due to the high
reactivity, quinines could polymerize spontaneously to form high
molecular weight brown pigments (melanins) or react with amino
acids and proteins to enhance brown color of the pigment pro-
duced.28,29 Previous reports confirmed that tyrosinase not only
was involved in melanising in animals, but also was one of the
main causes of most fruits and vegetables quality loss during post
harvest handling and processing, leading to faster degradation and
shorter shelf life.30 Recently, investigation demonstrated that var-
ious dermatological disorders, such as age spots and freckle, were
caused by the accumulation of an excessive level of epidermal pig-
mentation.31,32 Tyrosinase has also been linked to Parkinson’s and
other neurodegenerative diseases.33 In insects, tyrosinase is un-
iquely associated with three different biochemical processes,
including sclerotization of cuticle, defensive encapsulation and
melanisation of foreign organism, and wound healing.34 These pro-
cesses provide potential targets for developing safer and effective
tyrosinase inhibitors as insecticides and ultimately for insect con-
trol. Thus, the development of safe and effective tyrosinase inhib-
itors is of great concern in the medical, agricultural, and cosmetic
industries. However, only a few such as kojic acid, arbutin, tropo-
lone, and 1-phenyl-2-thiourea are used as therapeutic agents and
cosmetic products.32,35
For evaluating the tyrosinase inhibitory activity, all the synthe-
sized compounds were subjected to tyrosinase inhibition assay
with catechol as substrate. The result showed that all the synthe-
sized compounds (6a–y) inhibited the tyrosinase enzyme activity.
The inhibition constants of 6a–y analogues against PPO were
summarized in Table 1. We have determined the Ki values of
3.95–40.18
lM for the inhibition of banana PPO. Among the com-
pounds synthesized, 6s (Ki = 3.95
l
M) was found to be the most ac-
tive one and the inhibition kinetics analyzed by Lineweaver–Burk
double reciprocal plots revealed that compound 6s was a compet-
itive inhibitor (Fig. 1). It was reported that DTT and sodium meta-
bisulfite were the most effective inhibitors of banana root PPO
activity, followed by
concentrations to achieve 50% inhibition were 63, 65, 322 and
437
M respectively.37 Acidulants, such as citric acid can inhibit
L-cysteine and L-ascorbic acid. The minimum
l
PPO activity by reducing pH and/or chelating Cu in a food prod-
uct.38,39 Ascorbic acid can also be considered as an effective com-
pound at higher concentrations. The mechanism of ascorbic acid
inhibition involves the reduction of quinones generated by
PPO.40,41 It was reported that Ki value of ascorbic acid was found
to be 530 l
M.42 Our results are better than this study.
Reducing agents, antioxidants, and enzymatic inhibitors pre-
vent browning chemically reducing the o-quinones. The effect of
these reducing agents can be considered temporary because these
compounds are oxidized irreversibly by reaction with pigment
intermediates, endogenous enzymes and metals such as copper.
In this study series of 23 new 6-(phenylurenyl/thiourenyl) sac-
charin (6a–y) derivatives were synthesized and evaluated effect on
tyrosinase, for same molecules, such as urea and thioure deriva-
tives including aromatic sulfonamides or heterocyclic groups, have
widely biological activities and pharmacologic properties.4,8,25
Additionally, we presented structure–activity relationship analyses
and molecular energy calculations of some compounds.
Among sulfur containing agents, L-cystein is an effective compound
to prevent enzymatic browning. Direct inhibition of polyphenol
oxidase by cystein through the formation of stable complexes with
copper has also been proposed.43 Halim and Montgomery (1978)
showed in a series of publications that cystein can inhibit enzy-
matic browning of pear juice concentrate more effectively than
sulfite.44 Kahn (1985) used cystein to inhibit browning of cut or
pureed avocados and bananas.45 As the concentration of inhibitors
raised, the residual enzyme activity drastically decreased. All
inhibitors manifested a similar relationship between the enzyme
activity and enzyme concentration. From the progress curve ob-
tained, all compounds (6a–y) showing solid lines below the lines
of enzyme activity have indicative of enzyme inhibition and vice
versa. The result may be related to the structure of tyrosinase con-
tained a type-3 copper center with a coupled dinuclear copper ac-
tive site in the catalytic core. The purification procedures are
summarized in Table 2. As seen in Table 2, finally, PPO was purified
up to 70-fold.
2. Results and discussion
2.1. Chemistry
The synthetic procedures employed to obtain the target com-
pounds 6a–y are depicted in Scheme 1. The 6-aminosaccharin (5)
was synthesized from 4-nitrotoluene (1) in four steps by known
procedures.36 Compound 5 was reacted with phenylisocyanate or
phenylisothiocyanate derivatives to get product 6-(phenylurenyl/
thiourenyl) saccharin compounds (6a–y) in high yields.
All new compounds were characterized by 1H NMR, 13C NMR, IR
and MS. In the infrared spectra of compounds 6a–y, it was possible
to observe the absorptions between 3581 and 3030 cmꢀ1 relating
to NH stretch for urea derivatives, between 3099 and 2980 cmꢀ1
relating to NH stretch for thiourea derivatives, about 3300 cmꢀ1
relating to NH stretch for saccharin, absorptions in about
1725 cmꢀ1 from saccharin carbonyl moiety stretch and absorp-
tions between 1654 and 1710 cmꢀ1 from urea and thiourea car-
bonyl moiety stretching, absorptions between 1330 and 1120 cm
2.3. Structure–activity relationship (SAR)
Some notions about SAR emerged from these studies: For the
synthesized urea derivatives; (a) when electron-donating groups
(methoxy, methyl) at phenyl-ring compared with electron-with-
drawing groups (nitro, halogen), electron-withdrawing groups at
3-position of phenyl-ring increased the tyrosinase inhibitory activ-
ity (Table 1, for inhibitory activity, 6e, 6h and 6k > 6b, 6c and 6d).
The increase in electronegativities of these groups changed propor-
tionally the effect of compounds on enzyme inhibition (for electro-
negativity, NO2 > F > Cl, for inhibitory activity, 6h > 6k > 6e), (b)
when the electron-withdrawing groups are at meta-position of
compounds exhibited higher inhibitory activity than at para-posi-
tion (compare 6e with 6f, 6h with 6i, 6k with 6l). For the synthe-
sized thiourea derivatives; (c) the halogen series at 3-position of
phenyl-ring showed a qualitative relationship for higher inhibitory
activity with increasing size and polarizability (for size and
ꢀ1
relating to saccharin SO2 stretch. The 1H NMR spectra for all
the synthesized urea and thiourea compounds show signals be-
tween 8.88 and 10.93 ppm relating to hydrogens attached to the
nitrogen. The signals for aromatic hydrogens are between 6.62
and 8.25 ppm. In this same regions are the saccharin NH protons.
Through the 13C NMR data, a sign can be seen 161–162 ppm,
relating to saccharin carbonyl. This is followed by the sign
152.0–153.0 ppm for urea carbonyl and 180.0–182.0 ppm for thio-
urea carbonyl.

N. Gençer et al. / Bioorg. Med. Chem. 20 (2012) 2811–2821
2813
O
O
CH3
CH3
O
S
CH3
O
S
NH
S
NH
S
O
O
NH2
Cl
NH3(aq) , ether
40 oC , 2h
ClSO3H
CrO3/H2SO4
rt, 24h
Pd-C. EtOH
2h, reflux
O
O
O
O
60 oC , 20h
NO2
1
NO2
NO2
NH2
NO2
2
3
4
5
6a
O
6b
6c
6d
6e
6f
6g
6h
N
C
X
O
R
X
R
O
O
O
O
O
O
O
X=O,S
DMF, TEA,
H
3-OCH3
4-OCH3
4-CH3
6l
3-Cl
4-Cl
6n
3,4-di-Cl 3-NO2
12 h, rt
6i
6j
6k
O
6m
S
6o
6p
S
NH
S
O
O
X
R
O
O
O
S
S
4-NO2
2-F
3-F
4-F
H
4-CH3
6v
4-OCH3
2-F
H
N
NH
6q
S
6r
S
6s
S
6t
6u
S
6y
R
X
6a-y
X
R
S
S
S
3-F
4-F
3-I
3-Cl
2,4-di-Cl 3,5-di-Cl 4-NO2
Scheme 1. Synthesis of 6-(phenylurenyl/thiourenyl)saccharin derivatives.
Table 1
Inhibitory effect of saccharin derivatives on banana tyrosinase activities
Compd
X
R
Ki (l
M)
Inhibition type
Compd
X
R
Ki (lM)
Inhibition type
5
—
O
O
O
O
O
O
O
O
O
O
O
—
H
13.62
13.40
14.08
16.55
16.21
5.85
40.18
14.95
16.87
21.96
35.07
12.13
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
6l
O
S
S
S
S
S
S
S
S
S
S
S
4-F
H
4-CH3
4-OCH3
2-F
3-F
4-F
3-I
3-Cl
15.61
10.66
9.96
9.81
4.23
11.10
14.80
3.95
28.30
10.09
13.74
19.53
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
6a
6b
6c
6d
6e
6f
6g
6h
6i
6m
6n
6o
6p
6q
6r
6s
6t
6u
6v
6y
3-OCH3
4-OCH3
4-CH3
3-Cl
4-Cl
3,4-di-Cl
3-NO2
4-NO2
2-F
2,4-di-Cl
3,5-di-Cl
4-NO2
6j
6k
3-F
polarizability, I > Cl > F, for inhibitory activity, 6s > 6t > 6q), (d)
there was an inverse relationship between the inhibitory activity
and the distance between the substituent at the phenyl ring and
the sulfur atom at the thiourea group. The inhibitory activity was
increased by substituent closer to sulfur atom (6p (R: 2-F) > 6q
(R: 3-F) > 6r (R: 4-F) and 6u (R: 2,4-di-Cl) > 6t (R: 3-Cl) > 6v (R:
3,5-di-Cl)).
compatible with inhibition effect of 6m (Ki = 10.66
(Ki = 13.40 M). EHOMO–LUMO of the compound 6h, showed the
highest inhibitory activity (Ki = 16.87 M) among the synthesized
urea derivatives, is 3.557 eV. EHOMO–LUMO of the compound 6s,
showed the highest inhibitory activity (Ki = 3.95 M) among the
synthesized all compounds, is 3.460 eV. Our result showed a clear
relation between HOMO–LUMO energy differences and the po-
tency of these compounds as tyrosinase inhibitors.
lM) and 6a
l
D
l
D
l
Additionally, the calculated dipole moment of these compounds
(6a: 3.1473 Debye, 6h: 4.502 Debye, 6m: 3.8624 Debye, 6s: 4.6391
Debye) showed a linear relationship for higher inhibitory activity
with increasing dipole moment (for inhibitory activity,
6s > 6h > 6m > 6a). This relation probably to be explained that
the rise in the dipole moment of the molecule increases enzyme
inhibition activity for possibility of binding to copper ions in the
active cite of enzyme.
2.4. Quantum chemical calculations
In order to understand the experimental results obtained,
molecular calculations were performed using Gaussian soft-
ware,46a,b some selected the synthesized compounds (6a, 6h, 6m
and 6s) (Fig. 2).
Generally, differentiation between HOMO and LUMO reflects
the intensity of electron affinity, and lower differentiation suggests
higher electron affinity.47 The explaining to different effect of the
synthesized urea derivatives and thiourea derivatives on tyrosi-
nase enzyme, we performed quantum chemical calculations of 6a
and 6m structures to calculate HOMO and LUMO energy levels
3. Conclusions
In conclusion, series of new 12 urea and 11 thiourea saccharin
derivatives were prepared from 6-aminosaccharin and their activ-
ities as tyrosinase inhibitors were examined. Most of phenylthio-
and dipole moment. HOMO–LUMO energy differences (
LUMO) of 6m (3.727 eV) are lower than 6a (4.045 eV). These results
DEHOMO–

2814
N. Gençer et al. / Bioorg. Med. Chem. 20 (2012) 2811–2821
400
350
300
250
500
450
400
350
300
250
200
150
100
50
control
200
150
100
50
control
0.03 mM [6d]
0.10 mM [6d]
0.03 mM [5]
0.10 mM [5]
0
0
-100
0
100
200
300
400
-100
0
100
200
300
400
1/[S] (M-1)
1/[S] (M-1)
500
450
400
350
300
250
200
150
100
50
350
300
250
200
150
100
50
control
control
0.01 mM [6e]
0.03 mM [6e]
0.03 mM [6a]
0.10 mM [6a]
0
0
-100
-100
-100
0
100
200
300
400
-100
0
100
200
300
400
1/[S] (M-1)
1/[S] (M-1)
400
350
300
250
200
150
100
50
500
450
400
350
300
250
200
150
100
50
control
control
0.03 mM [6b]
0.10 mM [6b]
0.10 mM [6f]
0.20 mM [6f]
0
0
-100
0
100
200
300
400
0
100
200
300
400
1/[S] (M-1)
1/[S] (M-1)
450
400
350
300
250
200
150
100
50
400
350
300
250
200
150
100
50
control
control
0.03 mM [6c]
0.10 mM [6c]
0.03 mM [6g]
0.10 mM [6g]
0
0
-100
0
100
200
300
400
0
100
200
300
400
1/[S] (M-1)
1/[S] (M-1)
Figure 1. Ki graphics of saccharin derivatives on BPPO.

N. Gençer et al. / Bioorg. Med. Chem. 20 (2012) 2811–2821
2815
350
300
250
200
150
100
50
400
350
300
250
200
150
control
0.03 mM [6l]
0.10 mM [6l]
control
100
0.03 mM [6h]
0.08 mM [6h]
50
0
0
-100
0
100
100
100
200
300
400
-100
0
100
200
300
400
1/[S] (M-1)
1/[S] (M-1)
350
300
250
200
150
100
50
800
700
600
500
400
300
200
100
0
control
control
0.03 mM [6m]
0.10 mM [6m]
0.06 mM [6i]
0.10 mM [6i]
0
-100
0
200
300
400
-100
0
100
200
300
400
1/[S] (M-1)
1/[S] (M-1)
500
450
400
350
300
250
200
150
100
50
800
700
600
500
400
300
200
100
0
control
0.10 mM [6j]
0.20 mM [6j]
control
0.03 mM [6n]
0.10 mM [6n]
0
-100
0
100
200
300
400
-100
0
200
300
400
1/[S] (M-1)
1/[S] (M-1)
600
500
400
300
200
100
0
800
700
600
500
400
300
200
100
0
control
control
0.03 mM [6k]
0.10 mM [6k]
0.03 mM [6o]
0.10 mM [6o]
-100
0
100
200
300
400
-100
0
100
200
300
400
1/[S] (M-1)
1/[S] (M-1)
Fig. 1 (continued)

2816
N. Gençer et al. / Bioorg. Med. Chem. 20 (2012) 2811–2821
250
900
800
700
600
500
400
300
200
100
0
200
control
150
100
50
0.01 mM [6p]
0.05 m M [6p]
control
0.06 mM [6t]
0.10 mM [6t]
0
-100
0
100
200
300
400
-100
0
100
200
300
400
1/[S] (M-1)
1/[S] (M-1)
300
250
200
150
100
50
700
600
500
400
300
200
100
0
control
control
0.03 mM [6q]
0.10 mM [6q]
0.02 mM [6u]
0.04 mM [6u]
0
-100
0
100
200
300
400
-100
0
100
200
300
400
1/[S] (M-1)
1/[S] (M-1)
450
400
350
300
250
200
150
100
50
500
450
400
350
300
250
200
150
100
50
control
control
0.03 mM [6r]
0.10 mM [6r]
0.03 mM [6v]
0.10 mM [6v]
0
0
-100
0
100
200
300
400
-100
0
100
200
300
400
1/[S] (M-1)
1/[S] (M-1)
250
200
150
100
50
450
400
350
300
250
200
150
100
50
control
0.06 mM [6y]
0.10 mM [6y]
control
0.01 mM [6s]
0.05 mM [6s]
0
0
-100
0
100
200
300
400
-100
0
100
200
300
400
1/[S] (M-1)
1/[S] (M-1)
Fig. 1 (continued)

N. Gençer et al. / Bioorg. Med. Chem. 20 (2012) 2811–2821
2817
Table 2
Summary of the purification of tyrosinase from banana
Purification step
Volume
(mL)
Total
activity
Activity (U/
mLdak)
Total protein
(mg)
Specific activity (U/mg
protein)
Overall yield
(%)
Purification
fold
Extract
Ammonium sulfate
precipitation
25
10
502,500
125,250
20,100
12,525
0.205
0.045
24,51,219
27,83,333
—
24.93
—
1.13
Affinity chromatography
2
34,400
17,200
0.00020
1720,00,000
6.85
70.17
6a
6h
6m
6s
Etot.= -1403.438689 a.u.;
Dipole moment (μ)=
3.1473 Debye
Etot.=-1726.387575 a.u.; Etot.=-960.69788616 a.u.;
Etot.=-1607.936297a.u.;
Dipole moment (μ)=
4.502 Debye
Dipole moment (μ)= Dipol
moment
(μ)=
3.8624 Debye
4.6391 Debye
EHOMO = -6.285 eV;
ELUMO = -2.240 eV;
ΔEHOMO-LUMO= 4.045 eV
EHOMO = -6.159 eV;
ELUMO = -2.432 eV;
EHOMO
ELUMO
=
=
-6.561 eV;
-3.101 eV;
EHOMO = -6.077 eV;
ELUMO = -2.520 eV;
ΔEHOMO-LUMO= 3.557 eV
ΔEHOMO-LUMO= 3.727 eV ΔEHOMO-LUMO= 3.460 eV
Figure 2. The calculated HOMO and LUMO for compounds 6a, 6h, 6m and 6s using HF method with 6-31G basis set.
urenylsaccharin derivatives (6m–y) inhibited the tyrosinase en-
4. Experimental
4.1. General
zyme activity more than phenylurenylsaccharin derivatives (6a–
l). The present study revealed that activity could also be influ-
enced by the type, electronegativity and position of substituents
on the phenyl ring. Among the compounds synthesized, com-
pound 6s showed the highest inhibitory activity. Additionally,
there was a clear relation between HOMO–LUMO energy differ-
ences, dipole moment and the potency of these compounds as
tyrosinase inhibitors. Kinetic study revealed that compound 6s
acted as a noncompetitive inhibitor of banana tyrosinase with
Melting points were taken on a Barnstead Electrothermal 9200.
IR spectra were measured on a SHIMADZU Prestige-21 (200 VCE)
spectrometer. 1H and 13C NMR spectra were measured on spec-
trometer at VARIAN Infinity Plus 300 and at 75 Hz, respectively.
1H and 13C chemical shifts are referenced to the internal deuterated
solvent. Mass spectra were obtained using MICROMASS Quattro
LC–MS-MS spectrometer. Solvents were dried following standard
Ki value of 3.95 lM.

2818
N. Gençer et al. / Bioorg. Med. Chem. 20 (2012) 2811–2821
methods. All chemicals were purchased from Merck, Alfa Easer,
Sigma–Aldrich and Fluka.
temperature for 12 h and then poured into cold 1 M HCl. The pre-
cipitate was filtered and washed with cold water. The crude prod-
ucts were recrystallizated from ethanol over 99% purity.
Compounds 6a–y were obtained with 69–94% yields.
4.2. Synthesis procedures and spectral data
4.2.1. 2-Methyl-5-nitrobenzenesulfonylchloride (2)
4.2.6. 6-(Phenylurenyl) saccharin (6a)
A solution of 4-nitrotoluene (13.7 g, 0.1 mol) in chlorosulfonic
acid (27 mL, 0.4 mol) was stirred at 60 °C for 20 h. The solution
was cooled and poured into crash ice carefully. The precipitated
product was filtered and washed with abundant cold water than
dried in the vacuum-oven at 40 °C. The product was provided a
brown solid in 96% yield. 1H NMR (CDCl3, 300 MHz) d/ppm: 2.93
(3H, s), 7.69 (1H, d, J = 8.2 Hz), 8.48 (1H, d, J = 8.4 Hz), 8.92 (1H,
s); 13C NMR (CDCl3, 75 MHz) d/ppm: 20.9, 124.4, 129.4, 135.0,
143.8, 145.6, 146.3.
White powder, 87% yield, mp 180.1–180.3 °C; IR: 3549, 3300,
3207, 1710, 1684, 1593, 1548, 1346, 1226, 1172, 1138 cmꢀ1 1H
;
NMR (DMSO-d6, 300 MHz) d/ppm: 7.01 (1H, t, J = 7.3 Hz), 7.30
(2H, t, J = 7.8 Hz), 7.40 (1H, br, NH), 7.47 (2H, d, J = 8.3 Hz), 7.71
(1H, d, J = 8.3 Hz), 7.89 (1H, d, J = 8.5 Hz), 8.27 (1H, s), 9.02 (1H, s,
NH), 9.58 (1H, s, NH); 13C NMR (DMSO-d6, 75 MHz) d/ppm:
108.9, 119.5, 120.5, 123.4, 123.5, 126.6, 129.6, 139.5, 141.7,
147.1, 152.8, 161.5; LC–MS (m/z): 318.09 [MH+].
4.2.7. 6-(3-Methoxyphenylurenyl) saccharin (6b)
4.2.2. 2-Methyl-5-nitrobenzenesulfonamide (3)
Yellowish powder, 79% yield, mp 218.2–218.4 °C; IR: 3581,
To a solution of 2-methyl-5-nitrobenzenesulfonylchloride (10 g,
0.43 mol) in diethyl ether (200 mL) was added aqueous ammonia
solution (35% solution, 40 mL) dropwise over 20 min keeping the
temperature below 10 °C, then the mixture was stirred at 40 °C
for 2 h and diethyl ether was evaporated. After the precipitated
product was filtered and washed with abundant cold water, it
was dried in the vacuum-oven at 40 °C. The product was provided
a fawn-colored solid in 88% yield. 1H NMR (CDCl3, 300 MHz) d/
ppm: 2.79 (3H, s), 7.22 (2H, s, NH2), 7.52 (1H, d, J = 8.5 Hz), 8.24
(1H, d, J = 8.2 Hz), 8.80 (1H, s); 13C NMR (CDCl3, 75 MHz) d/ppm:
20.6, 122.9, 126.0, 133.5, 143.4, 144.6, 145.7.
3352, 3053, 1741, 1714, 1595, 1537, 1321, 1284, 1205, 1144 cm
;
ꢀ1
1H NMR (DMSO-d6, 300 MHz) d/ppm: 3.75 (3H, s), 6.62 (1H,
d, J = 7.7 Hz), 6.99 (1H, t, J = 7.9 Hz), 7.19–7.25 (3H, m), 7.73
(1H, d, J = 8.5 Hz), 7.90 (1H, d, J = 8.5 Hz), 8.28 (1H, s), 9.06 (1H,
s, NH), 9.60 (1H, s, NH); 13C NMR (DMSO-d6, 75 MHz) d/ppm:
55.7, 105.2, 108.8, 108.9, 111.7, 120.7, 123.5, 126.6, 130.4,
140.8, 141.8, 147.0, 152.7, 160.4, 161.6; LC–MS (m/z): 348.40
[MH+].
4.2.8. 6-(4-Methoxyphenylurenyl) saccharin (6c)
Yellowish powder, 76% yield, mp 251.7–252.0 °C; IR: 3500,
3344, 3030, 1731, 1708, 1597, 1527, 1348, 1255, 1207, 1134 cm
ꢀ1
4.2.3. 6-Nitrosaccharin (4)
;
1H NMR (DMSO-d6, 300 MHz) d/ppm: 3.73 (3H, s), 6.91 (2H,
To solution chromium trioxide (20.4 g, 0.204 mol) in water
(150 mL) was added concentrated sulfuric acid (190 mL) dropwise
in the ice bath. 2-Methyl-5-nitrobenzenesulfonamide (10 g,
46.3 mmol) was added and the mixture was stirred at room tem-
perature for 24 h. The mixture was filtered and the residue washed
with water, dissolved in sodium bicarbonate solution and filtered
under vacuum. The filtrate was acidified with concentrated HCl.
After the precipitated product was filtered and washed with abun-
dant cold water, it was dried in the vacuum-oven at 40 °C. The
product was obtained a white powder in 72% yield. Mp 205.6–
205.8 °C, (lit.36 205–207 °C); IR: 3103, 1720, 1608, 1541, 1354,
d, J = 9.0 Hz), 7.35 (1H, br, NH), 7.40 (2H, d, J = 9.1 Hz), 7.72 (1H,
d, J = 8.5 Hz), 7.90 (1H, d, J = 8.5 Hz), 8.29 (1H, s), 8.88 (1H, s,
NH), 9.58 (1H, s, NH); 13C NMR (DMSO-d6, 75 MHz) d/ppm: 55.8,
103.5, 108.7, 114.7, 121.4, 127.0, 132.4, 141.7, 142.7, 147.3,
152.9, 156.2, 161.6; LC–MS (m/z): 348.36 [MH+].
4.2.9. 6-(4-Methylphenylurenyl) saccharin (6d)
Yellow solid, 82% yield, mp 240.1–240.2 °C; IR: 3523, 3331,
3041, 1758, 1709, 1595, 1537, 1338, 1226, 1132 cmꢀ1 1H NMR
;
(DMSO-d6, 300 MHz) d/ppm: 2.23 (3H, s), 7.10 (2H, d, J = 8.2 Hz),
7.35 (2H, d, J = 8.5 Hz), 7.63 (1H, br, NH), 7.66 (1H, d, J = 8.5 Hz),
7.82 (1H, d, J = 8.5 Hz), 8.20 (1H, s), 8.90 (1H, s, NH), 9.49 (1H, s,
NH); 13C NMR (DMSO-d6, 75 MHz) d/ppm: 21.1, 108.8, 119.5,
123.1, 126.2, 129.3, 129.9, 132.2, 137.0, 142.6, 146.6, 152.8,
162.6; LC–MS (m/z): 332.34 [MH+].
1340, 1184, 1128 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d/ppm:
;
8.02 (1H, d, J = 8.5 Hz), 8.56 (1H, d, J = 8.2 Hz), 8.71 (1H, s), 10.2
(1H, br, NH); 13C NMR (DMSO-d6, 75 MHz) d/ppm: 117.2, 126.4,
129.8, 135.2, 143.0, 151.7, 162.2.
4.2.4. 6-Aminosaccharin (5)
4.2.10. 6-(3-Chlorophenylurenyl) saccharin (6e)
To a solution of 6-nitrosaccharin (1.14 g, 5 mmol) and cyclohex-
ene (2.54 mL, 25 mmol) in ethanol (50 mL) was added 10% Pd-C
(2.65 g) in the ice bath. The mixture was refluxed for 2 h with stir-
ring, than cooled and filtered. The filtrate was evaporated to dry-
ness. The crude product was washed with chloroform and dried
under vacuum. The product was provided a yellowish solid in
87% yield. Mp 262.4–262.6 °C, (lit.36 264–265 °C); IR: 3473, 3377,
White solid, 90% yield, mp 234.6–234.8 °C; IR: 3533, 3284,
3057, 1722, 1683, 1581, 1545, 1477, 1284, 1222, 1125 cmꢀ1 1H
;
NMR (DMSO-d6, 300 MHz) d/ppm: 7.06 (1H, dd, J = 2.1, 2.3 Hz),
7.26–7.32 (3H, m), 7.68 (1H, s), 7.72 (1H, d, J = 8.5 Hz), 7.89 (1H,
d, J = 8.5 Hz), 8.24 (1H, s), 9.22 (1H, s, NH), 9.66 (1H, s, NH); 13C
NMR (DMSO-d6, 75 MHz) d/ppm: 109.2, 117.9, 118.9, 120.8,
123.0, 123.8, 126.6, 131.2, 133.9, 141.1, 141.7, 146.8, 152.7,
161.5; LC–MS (m/z): 352.04 [MH+].
3244, 1708, 1598, 1285, 1163, 1107 cmꢀ1
;
1H NMR
(CDCl3+DMSO-d6, 300 MHz) d/ppm: 6.80 (2H, s, NH2), 6.91 (1H, d,
J = 8.5 Hz), 6.97 (1H, s), 7.60 (1H, d, J = 8.2 Hz), 9.01 (1H, br, NH);
13C NMR (CDCl3+DMSO-d6, 75 MHz) d/ppm: 103.4, 113.4, 118.4,
126.9, 142.8, 156.1, 161.8; LC–MS (m/z): 199.24 [MH+].
4.2.11. 6-(4-Chlorophenylurenyl) saccharin (6f)
White solid, 88% yield, mp 272.7–272.8 °C; IR: 3558, 3334,
3047, 1724, 1698, 1599, 1541, 1489, 1284, 1209, 1157 cmꢀ1 1H
;
NMR (DMSO-d6, 300 MHz) d/ppm: 7.35 (1H, br, NH), 7.38 (2H, d,
J = 8.5 Hz), 7.53 (2H, d, J = 8.2 Hz), 7.76 (1H, d, J = 8.5 Hz), 7.92
(1H, d, J = 8.2 Hz), 8.28 (1H, s), 9.21 (1H, s, NH), 9.68 (1H, s, NH);
13C NMR (DMSO-d6, 75 MHz) d/ppm: 109.1, 120.6, 121.0, 123.7,
126.6, 127.0, 129.4, 138.5, 141.6, 146.9, 152.7, 161.4; LC–MS (m/
z): 352.05 [MH+].
4.2.5. Synthesis of 6-(phenylurenyl/thiourenyl) saccharin
derivatives (6a–y)
To a solution of 6-aminosaccharin (1 mmol) and triethyl amine
(1 mL) in dry DMF was added phenylisocyanate or phenylisothio-
cyanate derivatives (1 mmol). The mixture was stirred at room

N. Gençer et al. / Bioorg. Med. Chem. 20 (2012) 2811–2821
2819
4.2.12. 6-(3,4-Dichlorophenylurenyl)saccharin (6g)
125.9, 126.0, 127.8, 129.4, 139.4, 140.6, 147.1, 161.3, 180.1; LC–
White solid, 91% yield, mp >350 °C; IR: 3522, 3346, 3072, 1740,
MS (m/z): 334.30 [MH+].
1710, 1585, 1529, 1473, 1336, 1298, 1199, 1169, 1126 cmꢀ1 1H
;
NMR (DMSO-d6, 300 MHz) d/ppm: 7.35 (1H, d, J = 9.0 Hz), 7.52
(1H, d, J = 8.8 Hz), 7.70 (1H, d, J = 8.8 Hz), 7.80 (1H, br, NH), 7.84
(1H, s), 7.87 (1H, d, J = 8.5 Hz), 8.21 (1H, s), 9.30 (1H, s, NH), 9.69
(1H, s, NH); 13C NMR (DMSO-d6, 75 MHz) d/ppm: 109.3, 119.6,
120.7, 121.1, 123.8, 124.8, 126.5, 131.3, 131.8, 139.7, 141.7,
146.5, 152.7, 161.7; LC–MS (m/z): 388.03 [MH+].
4.2.19. 6-(4-Methylphenylthiourenyl) saccharin (6n)
Yellow powder, 88% yield, mp 193.2–193.3 °C; IR: 3352, 3086,
3037, 1737, 1703, 1593, 1523, 1506, 1327, 1247, 1172, 1139,
1124 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d/ppm: 2.30 (3H, s),
;
7.19 (2H, d, J = 7.9 Hz), 7.25 (1H, br, NH), 7.37 (2H, d, J = 7.7 Hz),
7.90 (1H, d, J = 8.5 Hz), 7.94 (1H, d, J = 8.5 Hz), 8.46 (1H, s),
10.33 (1H, s, NH), 10.43 (1H, s, NH); 13C NMR (DMSO-d6,
75 MHz) d/ppm: 21.2, 113.6, 122.2, 124.8, 125.9, 127.9, 129.9,
135.3, 136.7, 140.6, 147.2, 161.4, 180.1; LC–MS (m/z): 348.36
[MH+].
4.2.13. 6-(3-Nitrophenylurenyl) saccharin (6h)
Yellow powder, 94% yield, mp 346.2–346.5 °C; IR: 3518, 3354,
3217, 1741, 1693, 1597, 1537, 1481, 1346, 1282, 1228, 1128 cm
ꢀ1
;
1H NMR (DMSO-d6, 300 MHz) d/ppm: 7.59 (1H, t, J = 7.9 Hz),
7.77–7.93 (5H, m), 8.27 (1H, s), 8.54 (1H, s), 9.56 (1H, s, NH),
9.75 (1H, s, NH); 13C NMR (DMSO-d6, 75 MHz) d/ppm: 109.4,
113.4, 117.8, 120.9, 124.0, 125.6, 126.6, 130.9, 140.9, 141.6,
146.7, 148.8, 152.8, 161.4; LC–MS (m/z): 363.13 [MH+].
4.2.20. 6-(4-Methoxyphenylthiourenyl) saccharin (6o)
Yellow solid, 86% yield, mp 186.1–186.3 °C; IR: 3354, 3093,
3041, 1737, 1703, 1527, 1504, 1327, 1242, 1172, 1138, 1126 cm
ꢀ1
;
1H NMR (DMSO-d6, 300 MHz) d/ppm: 3.78 (3H, s), 6.94–7.01
(3H, m), 7.36 (2H, d, J = 8.5 Hz), 7.88 (1H, d, J = 8.5 Hz), 7.94 (1H,
d, J = 8.5 Hz), 8.44 (1H, s), 10.22 (1H, s, NH), 10.35 (1H, s, NH);
13C NMR (DMSO-d6, 75 MHz) d/ppm: 55.9, 113.6, 114.5, 124.3,
125.3, 126.9, 127.6, 132.0, 142.0, 146.2, 157.6, 163.6, 180.3; LC–
MS (m/z): 364.40 [MH+].
4.2.14. 6-(4-Nitrophenylurenyl) saccharin (6i)
Yellow powder, 92% yield, mp 306.4–306.7 °C; IR: 3387, 3309,
3122, 1751, 1722, 1595, 1537, 1492, 1332, 1284, 1242, 1149,
1111 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d/ppm: 7.66–7.74 (4H,
;
m), 7.88 (1H, d, J = 8.5 Hz), 8.15 (2H, d, J = 8.7 Hz), 8.25 (1H, s),
9.75 (1H, s, NH), 9.81 (1H, s, NH); 13C NMR (DMSO-d6, 75 MHz)
d/ppm: 109.4, 118.7, 120.9, 123.9, 125.7, 126.6, 141.4, 142.2,
146.2, 146.4, 152.4, 161.2; LC–MS (m/z): 363.11 [MH+].
4.2.21. 6-(2-Fluorophenylthiourenyl) saccharin (6p)
White powder, 69% yield, mp 157.5–157.8 °C; IR: 3365, 3080,
2930, 1718, 1696, 1597, 1531, 1490, 1294, 1259, 1232, 1163,
1138, 1116 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d/ppm: 7.21–
;
4.2.15. 6-(2-Fluorophenylurenyl) saccharin (6j)
7.33 (4H, m), 7.58 (1H, t, J = 7.9 Hz), 7.82 (1H, d, J = 8.5 Hz), 7.85
(1H, d, J = 8.5 Hz), 8.31 (1H, s), 10.07 (1H, s, NH), 10.65 (1H, s,
NH); 13C NMR (DMSO-d6, 75 MHz) d/ppm: 113.6, 116.8, 123.3,
125.2, 125.8, 127.1, 127.9, 128.9, 129.4, 141.2, 146.6, 158.8,
162.0, 181.4; LC–MS (m/z): 352.34 [MH+].
White powder, 75% yield, mp 211.1–211.3 °C; IR: 3595, 3269,
3062, 1726, 1698, 1633, 1587, 1541, 1487, 1247, 1224, 1147 cm
ꢀ1
;
1H NMR (DMSO-d6, 300 MHz) d/ppm: 7.05–7.31 (4H, m), 7.70
(1H, d, J = 8.5 Hz), 7.92 (1H, d, J = 8.5 Hz), 8.11 (1H, t, J = 8.2 Hz),
8.33 (1H, s), 8.89 (1H, s, NH), 9.99 (1H, s, NH); 13C NMR (DMSO-
d6, 75 MHz) d/ppm: 108.8, 115.7, 116.0, 120.7, 121.9, 123.6,
124.3, 125.3, 126.7, 127.4, 141.7, 146.8, 152.6, 161.3; LC–MS (m/
z): 336.32 [MH+].
4.2.22. 6-(3-Fluorophenylthiourenyl) saccharin (6q)
White powder, 77% yield, mp 182.0–182.1 °C; IR: 3325, 3087,
3020, 1737, 1693, 1597, 1512, 1485, 1332, 1230, 1174, 1149,
1126 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d/ppm: 7.03 (1H, t,
;
4.2.16. 6-(3-Fluorophenylurenyl) saccharin (6k)
J = 8.2 Hz), 7.33 (1H, d, J = 8.2 Hz), 7.39–7.47 (2H, m), 7.58 (1H, d,
J = 10.1 Hz), 7.94 (1H, d, J = 8.5 Hz), 8.00 (1H, d, J = 8.5 Hz), 8.51
(1H, s), 10.60 (1H, s, NH), 10.70 (1H, s, NH); 13C NMR (DMSO-d6,
75 MHz) d/ppm: 111.3, 112.5, 113.9120.1, 122.6, 126.0, 128.2,
131.0, 140.6, 141.3, 146.8, 161.4, 164.1, 180.1; LC–MS (m/z):
352.30 [MH+].
White powder, 75% yield, mp 223.2–223.5 °C; IR: 3572, 3338,
3097, 1728, 1696, 1604, 1546, 1494, 1288, 1207, 1149, 1118 cm
ꢀ1
;
1H NMR (DMSO-d6, 300 MHz) d/ppm: 7.13–7.20 (3H, m), 7.50
(1H, d, J = 8.2 Hz), 7.66 (1H, s), 7.73 (1H, d, J = 8.5 Hz), 7.91 (1H,
d, J = 8.5 Hz), 8.27 (1H, s), 9.09 (1H, s, NH), 9.62 (1H, s, NH); 13C
NMR (DMSO-d6, 75 MHz) d/ppm: 109.0, 115.9, 116.2, 117.9,
121.6, 121.8, 122.2, 123.1, 126.6, 135.7, 141.7, 147.3, 153.0,
161.3; LC–MS (m/z): 336.32 [MH+].
4.2.23. 6-(4-Fluorophenylthiourenyl) saccharin (6r)
White powder, 90% yield, mp 202.5–202.7 °C; IR: 3331, 3097,
2980, 1718, 1694, 1597, 1539, 1496, 1330, 1288, 1165, 1143,
4.2.17. 6-(4-Fluorophenylurenyl) saccharin (6l)
1105 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d/ppm: 7.23 (2H, t,
;
White powder, 75% yield, mp 237.7–237.9 °C; IR: 3572, 3338,
J = 8.8 Hz), 7.47–7.53 (3H, m), 7.94 (1H, d, J = 8.5 Hz), 7.97 (1H, d,
J = 8.5 Hz), 8.44 (1H, s), 10.35 (1H, s, NH), 10.50 (1H, s, NH); 13C
NMR (DMSO-d6, 75 MHz) d/ppm: 113.8, 116.2, 122.5, 126.0,
127.3, 128.1, 135.7, 140.7, 146.9, 158.6, 161.5, 180.6; LC–MS (m/
z): 352.34 [MH+].
3097, 1728, 1696, 1604, 1546, 1494, 1288, 1207, 1149, 1118 cm
ꢀ1
;
1H NMR (DMSO-d6, 300 MHz) d/ppm: 7.13–7.20 (3H, m), 7.50
(2H, dd, J = 4.1, 5.0 Hz), 7.73 (1H, d, J = 8.5 Hz), 7.91 (1H, d,
J = 8.5 Hz), 8.27 (1H, s), 9.09 (1H, s, NH), 9.62 (1H, s, NH); 13C
NMR (DMSO-d6, 75 MHz) d/ppm: 109.0, 116.0, 116.3, 121.4,
121.5, 123.6, 126.6, 135.9, 141.9, 147.1, 152.9, 161.5; LC–MS (m/
z): 336.32 [MH+].
4.2.24. 6-(3-Iodophenylthiourenyl) saccharin (6s)
White powder, 86% yield, mp 175.5–175.7 °C; IR: 3313, 3092,
3045, 1736, 1695, 1577, 1506, 1467, 1330, 1232, 1172, 1145,
4.2.18. 6-(Phenylthiourenyl) saccharin (6m)
1124 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d/ppm: 7.18 (1H, d,
;
White powder, 81% yield, mp 171.9–172.1 °C; IR: 3356, 3091,
J = 8.2 Hz), 7.49 (1H, d, J = 8.2 Hz), 7.55 (1H, d, J = 7.9 Hz),
7.89–7.94 (3H, m), 7.97 (1H, d, J = 8.5 Hz), 8.39 (1H, s), 10.42
(1H, s, NH), 10.59 (1H, s, NH); 13C NMR (DMSO-d6, 75 MHz) d/
ppm: 94.8, 114.0, 122.5, 124.0, 126.1, 128.2, 131.3, 132.7,
134.3, 140.7, 141.0, 146.9, 161.2, 180.3; LC–MS (m/z): 460.56
[MH+].
3049, 1735, 1705, 1533, 1494, 1323, 1240, 1170, 1136, 1124 cm
ꢀ1
;
1H NMR (DMSO-d6, 300 MHz) d/ppm: 7.20 (1H, t, J = 6.9 Hz),
7.32–7.45 (3H, m), 7.50 (2H, d, J = 7.3 Hz), 7.90 (1H, d, J = 8.5 Hz),
7.96 (1H, d, J = 8.5 Hz), 8.47 (1H, s), 10.47 (1H, s, NH), 10.62 (1H,
s, NH); 13C NMR (DMSO-d6, 75 MHz) d/ppm: 113.5, 122.2, 124.6,

2820
N. Gençer et al. / Bioorg. Med. Chem. 20 (2012) 2811–2821
4.2.25. 6-(3-Chlorophenylthiourenyl) saccharin (6t)
constant, Ki, was deduced from the points of interception of the
White solid, 92% yield, mp 189.3–189.5 °C; IR: 3348, 3088,
3008, 1739, 1687, 1595, 1548, 1487, 1348, 1307, 1251, 1176,
plots.
1151, 1118 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d/ppm: 7.39 (1H,
;
Acknowledgments
br, NH), 7.44 (1H, d, J = 8.5 Hz), 7.53 (1H, d, J = 8.8 Hz), 7.60 (1H,
t, J = 7.6 Hz), 7.72 (1H, s), 7.92 (1H, d, J = 8.5 Hz), 7.96 (1H, d,
J = 8.5 Hz), 8.42 (1H, s), 10.45 (1H, s, NH), 10.56 (1H, s, NH); 13C
NMR (DMSO-d6, 75 MHz) d/ppm: 113.9, 122.5, 126.0, 126.3,
128.1, 128.4, 129.3, 129.8, 131.5, 138.4, 140.7, 146.9, 161.4,
180.3; LC–MS (m/z): 368.36 [MH+].
This work was supported by Sakarya University Scientific
Research Project (Project No. 2011–50-02-020). We are thankful
to Dr. Davut AVCI for quantum chemical calculation using Gaussian
Software.
References and notes
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All purification steps were carried out at 25 °C. The extraction
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