Novel 5-Hydroxy, 5-Substituted Benzenesulfonamide Pyrimidine-2,4,6-Triones Attenuate Lipopolysaccharide-Induced Acute Lung Injury via Inhibition of the Gelatinases, MMP-2 and MMP-9

Article abstract of DOI:10.1002/ddr.21319

(Table presented.). A novel series of ten 5-hydroxy, 5-substituted benzene sulfonamide pyrimidine-2,4,6-triones were synthesized and their structures ascertained using ¹H-NMR, ¹³C-NMR, mass and elemental analysis. These compounds wer

Full text of DOI:10.1002/ddr.21319

DRUG DEVELOPMENT RESEARCH 00 : 00–00 (2016)
Research Article
Novel 5-Hydroxy, 5-Substituted Benzenesulfonamide
Pyrimidine-2,4,6-Triones Attenuate Lipopolysaccharide-
Induced Acute Lung Injury via Inhibition of the
Gelatinases, MMP-2 and MMP-9
Wei He,1 Jie Jiang,1 Ze-Qian Yu,2 and Jia-Hua Zhou2*
1Department of Cardiothoracic Surgery, Zhongda Hospital, Medical School of Southeast
University, Nanjing, Jiangsu 210009, People’s Republic of China
2Department of Hepatobiliary Surgery, Zhongda Hospital, Medical School of Southeast
University, Nanjing, Jiangsu 210009, People’s Republic of China
Strategy, Management and Health Policy
Enabling
Preclinical Development
Toxicology, Formulation
Drug Delivery,
Clinical Development
Phases I-III
Preclinical
Research
Postmarketing
Phase IV
Technology,
Genomics,
Proteomics
Regulatory, Quality,
Manufacturing
Pharmacokinetics
ABSTRACT
A novel series of ten 5-hydroxy, 5-substituted benzene sulfonamide pyrimidine-2,4,6-
triones were synthesized and their structures ascertained using H-NMR, 13C-NMR, mass and elemental
analysis. These compounds were subsequently tested for inhibition of MMP-2 and MMP-9 where most
exhibited activity with compound 5i being the most potent against MMP-2 and MMP-9 with IC50 val-
ues of 2.35 nM and 8.24 nM, respectively. Compound 5i was further analyzed in a mouse LPS-
induced acute lung injury model where it had protective activity. Histochemical studies indicated that
5i improved the vascular integrity of the lung.
1
Key words: synthesis; sulfonamide pyrimidine-2,4,6-triones; MMP-2; MMP-9
metal enzymes that are responsible for degrading all
types of extracellular matrix proteins [Nelson et al.,
2000] overactivation and/or dysregulation of which
may be involved in various inflammatory, malignant,
and degenerative disease states. Aberrant expression
of MMP has been associated with numerous chronic
lung diseases [Vignola et al., 1988]. The bronchoal-
veolar lavage (BAL) fluid of patients with ARDS
INTRODUCTION
Acute lung injury (ALI) and acute respiratory
distress syndrome (ARDS) are two major life threaten-
ing sickness associated with acute and severe inflam-
mation of lungs which leads to respiratory failure
[Malemud, 2006]. Extensive studies on the pathogene-
sis of ALI have allowed elucidation of alterations in
lung endothelial and epithelial barriers as causal to the
disease. The characteristic of ALI include disruption
of alveolar capillary membranes which results in exces-
sive neutrophil infiltration, pulmonary edema, release
of pro-inflammatory, and cytotoxic mediators
[Grommes and Soehnlein, 2011]. Due to compromised
lung compliance in ALI, it is associated with signifi-
cant mortality (34–58%; MacCallum and Evans, 2005]
and morbidity with no effective treatment.
†Conflict of interest: The authors declare no conflict of
interest.
*Correspondence to: Jia-hua Zhou, Department of Hepa-
tobiliary Surgery, Zhongda Hospital, Medical School of South-
east University, China. E-mail: zhoujiahua05@hotmail.com
Received 10 May 2016; Accepted 16 June 2016
Matrix metalloproteinases (MMP) are a diverse
Published online in Wiley Online Library (wileyonlinelibrary.
family of zinc containing extracellular proteinases com). DOI: 10.1002/ddr.21319
C
V
2016 Wiley Periodicals, Inc.

2
HE ET AL.
secondary to severe trauma or septic shock has ele- 1402, 1132 (SO2), 887, 658; 1H-NMR (400MHz,
vated levels of MMP-9 in comparison with controls CDCl3-d6, TMS) d ppm: 10.62 (s, 2H, 2 3 NH),
[Ricou et al., 1996]. A similar pattern was observed 7.86–7.72 (m, 4H,4 3 CH, Ar-H), 7.70–7.06 (m, 5H,
in the lungs of newborns with ARDS, along with ele- 5 3 CH, ArAH), 3.87 (br,s, 1H, NH), 3.61 (s, 1H,
vated concentrations of MMP-2 [Cederqvist et al., OH), 3.18 (s, 2H,CH2); 13C-NMR (100MHz, CDCl3)
2001]. Patients with pneumonia also have increased d ppm:201.1, 170.2, 150.4, 142.2, 139.7, 131.9, 129.6,
MMP-9 expression [Hartog et al., 2003]. Thus, selec- 129.1, 127.3, 126.7, 116.2, 91.5, 41.2; Mass: 418.41
tive inhibition of MMP-2 and MMP-9 may represent (M 1 H)1; Elemental analysis for C18H15N3O7S: Cal-
a treatment option for ALI patients.
culated: C, 51.80; H, 3.62; N, 10.07. Found: C,
The present manuscript describes the synthesis 51.82; H, 3.61; N, 10.07.
and inhibitory activity of a series of novel 5-hydroxy, 5-
substituted benzene sulfonamide pyrimidine-2,4,6- 4-Fluoro-N-(4-(2-(5-hydroxy-2,4,6-trioxohexahydro-
trione derivatives against MMP-2 and MMP-9. Further,
to assess the beneficial effect on ALI, the most active
inhibitor, 5i was also tested in Lipopolysaccharide
(LPS)-stimulated lung injury in mice.
pyrimidin-5 yl)acetyl)phenyl)benzenesulfonamide
(5b)
Yield: 72%; m.p: 193–1958C; MW: 435.38; Rf:
0.62; FTIR (mmax; cm21 KBr): 3275 (NAH), 3059
(CAHAromatic), 2937, 2932 (CAHaliphatic), 1664
(C@O), 1408, 1135 (SO2), 883 (F), 652; 1H-NMR
(400 MHz, CDCl3-d6, TMS) d ppm: 10.58 (s, 2H, 2
3 NH), 7.98–7.72 (m, 4H, 4 3 CH, ArAH), 7.38–
7.05 (m, 4H, 4 3 CH, ArAH), 3.89 (br,s, 1H, NH),
3.63 (s, 1H, OH), 3.21 (s, 2H,CH2); 13C-NMR (100
MHz, CDCl3) d ppm: 201.1, 170.2, 166.1, 150.5,
142.2, 135.3, 130.8, 129.6, 126.7, 116.3, 115.9, 91.6,
41.2; Mass: 436.37 (M 1 H)1; Elemental analysis for
C18H14FN3O7S: Calculated: C, 49.66; H, 3.24; N,
9.65. Found: C, 49.68; H, 3.23; N, 9.64.
METHODS AND MATERIALS
Chemistry
Analytical grade chemical were used directly.
Melting points were determined in open capillary tube
melting point apparatus and are uncorrected. Silica
gel-G coated Al-plates (0.5 mm thickness; Merck)
were used to check the completion of reaction and
the plates were illuminated under UV (254 nm). FTIR
spectra were recorded on Perkin Elmer-Spectrum RX-
I spectrometer, while elemental analysis was carried
out on a Vario EL III CHNOS elemental analyzer.
1H-NMR spectra were recorded on a Bruker Avance
II 400 NMR Spectrometer and 13C-NMR spectra on
a Bruker Avance II 100 NMR spectrometer in
DMSO-d6 using TMS as internal standard. Mass spec-
3-Fluoro-N-(4-(2-(5-hydroxy-2,4,6-trioxohexahydro-
pyrimidin-5-yl)acetyl)phenyl)benzenesulfonamide
(5c)
Yield: 63%; m.p: 208–2098C; MW: 435.38; Rf:
tra were obtained using a VG-AUTOSPEC spectrome- 0.54; FTIR (mmax; cm21 KBr): 3279 (NAH), 3064
ter. For induction of ALI, LPS (Escherichia coli, (CAHAromatic), 2942, 2939 (CAHaliphatic), 1667
serotype 0111:B4) was used along with dimethyl sulf- (C@O), 1412, 1139 (SO2), 889 (F), 659; 1H-NMR
oxide (DMSO), and other reagents, were procured (400 MHz, CDCl3-d6, TMS) d ppm: 10.62 (s, 2H, 2
from Sigma–Aldrich (USA). The final volume of 3 NH), 7.89–7.72 (m, 3H,3 3 CH, ArAH), 7.70–
DMSO in the reaction mixture was less than 0.5%.
7.08 (m, 5H, 5 3 CH, ArAH), 3.85 (br, s, 1H, NH),
Synthesis of compound 3 was performed as pre- 3.67 (s, 1H, OH), 3.24 (s, 2H,CH2); 13C-NMR (100
viously reported [Karimi Zarchi and Aslani, 2012; MHz, CDCl3) d ppm: 201.2, 170.2, 161.7, 150.4,
Xiang et al., 2012 ].
142.1, 141.3, 130.6, 129.6, 126.7, 122.9, 118.7, 116.2,
Synthesis of compound 5(a–j): The relevant 113.9, 91.4, 41.2; Mass: 436.42 (M 1 H)1; Elemental
methyl ketone (3) and 0.5 mmol of alloxan monohy- analysis for C18H14FN3O7S: Calculated: C, 49.66; H,
drate (0.08 g) were suspended in 5 mL of glacial ace- 3.24; N, 9.65. Found: C, 49.65; H, 3.25; N, 9.65.
tic acid and refluxed at 1158C for 3 h. The resulting
mixture was precipitated, cooled, and re-crystallized N-(4-(2-(5-Hydroxy-2,4,6-trioxohexahydropyrimidin-
from ethanol to afford pure products.
5-yl)acetyl)phenyl)23-methylbenzenesulfonamide
(5d)
N-(4-(2-(5-Hydroxy-2,4,6-trioxohexahydropyrimidin-
Yield: 68%; m.p: 212–2138C; MW: 431.42; Rf:
0.49; FTIR (mmax; cm21 KBr): 3282 (NAH), 3068
5-yl)acetyl)phenyl)benzenesulfonamide (5a)
Yield: 67%; m.p: 204–2058C; MW: 417.39; Rf: (CAHAromatic), 2946, 2935 (CAHaliphatic), 1673
1
0.52; FTIR (mmax; cm21 KBr): 3272 (NAH), 3052 (C@O), 1417, 1142 (SO2), 881, 664; H-NMR (400
(CAHAromatic), 2935 (CAHaliphatic), 1672 (C@O), MHz, CDCl3-d6, TMS) d ppm: 10.64 (s, 2H, 2 3
Drug Dev. Res.

BENZENESULFONAMIDE PYRIMIDINE-2,4,6-TRIONES AS MMP2 AND MMP9 INHIBITORS
3
NH), 7.77–7.72 (m, 3H,3 3 CH, ArAH), 7.67–7.06 3.61 (s, 1H, OH), 3.22 (s, 2H,CH2); 13C-NMR (100
(m, 5H, 5 3 CH, ArAH), 3.87 (br, s, 1H, NH), 3.69 MHz, CDCl3) d ppm: 201.3, 170.3, 150.5, 145.9,
(s, 1H, OH), 3.23 (s, 2H,CH2), 2.28 (s, 3H, CH3); 142.2, 129.7, 128.2, 126.7, 124.2, 116.2, 91.5, 41.2;
13C-NMR (100 MHz, CDCl3) d ppm: 201.3, 170.2, Mass: 463.40 (M 1 H)1; Elemental analysis for
150.3, 142.2, 139.7, 138.8, 132.2, 129.7, 128.9, 126.7, C18H14N4O9S: Calculated: C, 46.76; H, 3.05; N,
124.3, 116.2, 91.5, 41.2, 21.3; Mass: 432.48 12.12. Found: C, 46.78; H, 3.04; N, 12.13.
(M 1 H)1; Elemental analysis for C19H17N3O7S: Cal-
culated: C, 52.90; H, 3.97; N, 9.74. Found: C, 52.92; N-(4-(2-(5-Hydroxy-2,4,6-trioxohexahydropyrimidin-
H, 3.98; N, 9.74.
5-yl)acetyl)phenyl)22-(trifluoromethyl) benzenesul-
fonamide (5h)
N-(4-(2-(5-Hydroxy-2,4,6-trioxohexahydropyrimidin-
5-yl)acetyl)phenyl)24-methylbenzenesulfonamide
(5e)
Yield: 62%; m.p: 241–2428C; MW: 485.39; Rf:
0.79; FTIR (mmax; cm21 KBr): 3293 (NAH), 3069
(CAHAromatic), 2958, 2952 (CAHaliphatic), 1691
1
Yield: 71%; m.p: 221–2238C; MW: 431.42; Rf: (C@O), 1429, 1155 (SO2), 782 (CF3), 682; H-NMR
0.58; FTIR (mmax; cm21 KBr): 3284 (NAH), 3066 (400 MHz, CDCl3-d6, TMS) d ppm: 10.59 (s, 2H, 2
(CAHAromatic), 2942, 2939 (CAHaliphatic), 1678 3 NH), 7.90–7.72 (m, 4H,4 3 CH, ArAH), 7.62–
1
(C@O), 1415, 1149 (SO2), 881, 669; H-NMR (400 7.06 (m, 4H, 4 3 CH, ArAH), 3.85 (br, s, 1H, NH),
MHz, CDCl3-d6, TMS) d ppm: 10.62 (s, 2H, 2 3 3.63 (s, 1H, OH), 3.24 (s, 2H,CH2); 13C-NMR (100
NH), 7.74–7.72 (m, 4H,4 3 CH, ArAH), 7.40–7.06 MHz, CDCl3) d ppm: 201.2, 170.3, 150.4, 142.2,
(m, 4H, 4 3 CH, ArAH), 3.89 (br,s, 1H, NH), 3.64 135.2, 132.2, 130.1, 129.6, 128.2, 127.1, 126.7, 125.4,
(s, 1H, OH), 3.21 (s, 2H,CH2), 2.31 (s, 3H, CH3); 116.2, 120.2, 91.4, 41.2; Mass: 486.42 (M 1 H)1; Ele-
13C-NMR (100 MHz, CDCl3) d ppm: 201.2, 170.4, mental analysis for C19H14F3N3O7S: Calculated: C,
150.4, 142.3, 137.8, 136.9, 129.7, 129.4, 128.3, 126.8, 47.01; H, 2.91; N, 8.66. Found: C, 47.03; H, 2.90; N,
116.3,91.4, 41.2, 21.3; Mass: 432.45 (M 1 H)1; Ele- 8.68.
mental analysis for C19H17N3O7S: Calculated: C,
52.90; H, 3.97; N, 9.74. Found: C, 52.91; H, 3.96; N, N-(4-(2-(5-Hydroxy-2,4,6-trioxohexahydropyrimidin-
9.72.
5-yl)acetyl)phenyl)24-(trifluoromethy) benzenesulfo-
namide (5i)
N-(4-(2-(5-Hydroxy-2,4,6-trioxohexahydropyrimidin-
5-yl)acetyl)phenyl)23-nitrobenzenesulfonamide (5f)
Yield: 73%; m.p: 245–2468C; MW: 485.39; Rf:
0.72; FTIR (mmax; cm21 KBr): 3295 (NAH), 3064
Yield: 74%; m.p: 218–2198C; MW: 462.39; Rf: (CAHAromatic), 2962, 2958 (CAHaliphatic), 1682
1
0.64; FTIR (mmax; cm21 KBr): 3283 (NAH), 3068 (C@O), 1431, 1152 (SO2), 786 (CF3), 689; H-NMR
(CAHAromatic), 2949, 2941 (CAHaliphatic), 1681 (400 MHz, CDCl3-d6, TMS) d ppm: 10.63 (s, 2H, 2
1
(C@O), 1423, 1152 (SO2), 878 (NO2), 671; H-NMR 3 NH), 7.90–7.79 (m, 4H,4 3 CH, ArAH), 7.72–
(400 MHz, CDCl3-d6, TMS) d ppm: 10.65 (s, 2H, 2 7.05 (m, 4H, 4 3 CH, ArAH), 3.87 (br,s, 1H, NH),
3 NH), 8.54–8.02 (m, 4H,4 3 CH, ArAH), 7.72– 3.61 (s, 1H, OH), 3.21 (s, 2H,CH2); 13C-NMR (100
7.06 (m, 4H, 4 3 CH, ArAH), 3.87 (br, s, 1H, NH), MHz, CDCl3) d ppm: 202.1, 170.2, 150.4, 143.1,
3.62 (s, 1H, OH), 3.24 (s, 2H,CH2); 13C-NMR (100 142.2, 134.3, 129.7, 128.8, 127.2, 126.8, 124.1, 116.2,
MHz, CDCl3) d ppm: 201.2, 170.3, 150.4, 148.2, 91.4, 41.2; Mass: 486.38 (M 1 H)1; Elemental analy-
142.2, 140.3, 133.5, 129.8, 129.6, 127.3, 126.8, 123.1, sis for C19H14F3N3O7S: Calculated: C, 47.01; H,
116.2, 91.2, 41.2; Mass: 463.42 (M 1 H)1; Elemental 2.91; N, 8.66. Found: C, 47.02; H, 2.92; N, 8.66.
analysis for C18H14N4O9S: Calculated: C, 46.76; H,
3.05; N, 12.12. Found: C, 46.77; H, 3.06; N, 12.11.
N-(4-(2-(5-Hydroxy-2,4,6-trioxohexahydropyrimidin-
5-yl)acetyl)phenyl)24-methoxybenzenesulfonamide
(5j)
N-(4-(2-(5-Hydroxy-2,4,6-trioxohexahydropyrimidin-
5-yl)acetyl)phenyl)24-nitrobenzenesulfonamide (5g)
Yield: 76%; m.p: 194–1958C; MW: 447.42; Rf:
Yield: 67%; m.p: 226–2288C; MW: 462.39; Rf: 0.63; FTIR (mmax; cm21 KBr): 3291 (NAH), 3062
0.69; FTIR (mmax; cm21 KBr): 3287 (NAH), 3072 (CAHAromatic),
2959,2952 (CAHal1iphatic),
1676
(CAHAromatic), 2952, 2947 (CAHaliphatic), 1685 (C@O), 1435, 1158 (SO2), 794, 678; H-NMR (400
1
(C@O), 1427, 1157 (SO2), 875 (NO2), 678; H-NMR MHz, CDCl3-d6, TMS) d ppm: 10.61 (s, 2H, 2 3
(400 MHz, CDCl3-d6, TMS) d ppm: 10.61 (s, 2H, 2 NH), 7.72–7.64 (m, 4H,4 3 CH, ArAH), 7.12–7.06
3 NH), 8.39–8.12 (m, 4H,4 3 CH, ArAH), 7.72– (m, 4H, 4 3 CH, ArAH), 3.89 (br, s, 1H, NH), 3.82
7.05 (m, 4H, 4 3 CH, ArAH), 3.86 (br,s, 1H, NH), (s, 3H, OCH3), 3.64 (s, 1H, OH), 3.24 (s, 2H,CH2);
Drug Dev. Res.

4
HE ET AL.
13C-NMR (100 MHz, CDCl3) d ppm: 201.2, 170.2, operation group received vehicle ip for 30 min fol-
150.3, 142.2, 132.3, 129.6, 126.8, 126.2, 116.2, 114.2, lowed by intratracheal (i.t.) instillation of 50 mL
91.4, 55.9, 41.3; Mass: 448.44 (M 1 H)1; Elemental
saline. The four treatment groups were injected with
vehicle, 10, 100, 1000 mg/kg of compound 5i i.p. for
30 min respectively, followed by 100 mg/50 mL of
LPS i.t. After 6 h, the mice were sacrificed and sam-
ples collected.
analysis for C19H17N3O8S: Calculated: C, 51.00; H,
3.83; N, 9.39. Found: C, 51.02; H, 3.83; N, 9.40.
Animals
The present study was performed according to
the recommendations in the Guide for the Care and
Use of Laboratory Animals and approved by the
Institutional Committee on the Ethics of Animal
Experiments of the Medical School of Southeast Uni-
versity. Sodium phenobarbital was used as anesthetic
prior to all surgeries. For the study, specific
pathogen-free male BALB/c mice (16–20 g; 6–
8 weeks) were maintained under specific pathogen-
free conditions in the animal center facilities of the
Institute. Mice were kept in a temperature-controlled
room (12-h dark and light cycles) and provided ad
libitum access to food and water. They were accli-
mated for at least for 7 days to the surrounding envi-
ronment prior to experimentation.
Bronchoalveolar lavage fluid collection
BAL was performed as described previously
[Kuo et al. 2011; Li et al. 2012]. Briefly, after eutha-
nasia, the trachea was exposed and intubated with a
tracheal cannula. Collection was performed after
repeated washing of airways and lungs with 1 mL of
cold saline three times. Then respective BALFs were
pooled and collected on ice. These were then centri-
fuged at 500 3 g for 5 min at 48C. The resulting
cell-free supernatant was stored at 2208C for mea-
surement of protein concentration using the Bio-Rad
protein assay. Gemsa stain was used for the determi-
nation of total leukocyte content by counting the cells
in the pellet.
For induction of ALI, LPS (Escherichia coli,
serotype 0111:B4) was used along with DMSO, and
other reagents were procured from Sigma–Aldrich
(USA). The final volume of DMSO in the reaction
mixture was less than 0.5%.
RESULTS AND DISCUSSION
Chemistry
The synthesis of the target compounds was
achieved in two-step reaction as shown in Figure 1.
The reaction was started with synthesis of N-(4-
acetyl-N-(P-substituted) phenyl benzenesulfonamide,
3 reacting compound 1 with relevant aromatic sul-
phonamides (2) in the presence of pyridine under
vigorous reflux conditions. Compound 3 was also
used as starting substrate to react with compound 4
Murine model of LPS-induced lung inflammation
LPS was used to induce ALI in mice as
described in previous studies [Kuo et al. 2011; Li
et al. 2012]. Forty mice were taken and randomly
divided into five groups, including a sham operation
group and four treatment groups. The sham to furnish compounds 5(a–j). The structure of the
Fig. 1. Reagents and condition: (a) Reflux for 4 h; (b) Reflux, 3 h.
Drug Dev. Res.

BENZENESULFONAMIDE PYRIMIDINE-2,4,6-TRIONES AS MMP2 AND MMP9 INHIBITORS
5
pattern of the trifluoromethyl as in compound 5h
leads to a slight loss of activity. The least activity was
associated by compound possessing no phenyl substit-
uent 5a. More surprisingly, introduction of a fluorine
substituent, compound 5b, led to marked improve-
ment of potency against both MMP-2 and MMP-9.
TABLE 1. MMP Inhibition Study of Compound 5(a–j)
IC50* (in nM)
Compound
MMP-2
MMP-9
5a
5b
5c
5d
5e
5f
5g
5h
980 6 0.13
14.81 6 0.66
21.01 6 0.73
49.43 6 0.36
61.23 6 0.34
39.21 6 0.83
28.34 6 0.71
5.31 6 0.63
2.35 6 0.21
643.62 6 0.15
26.5 6 0.2
887.03 6 0.63
29.11 6 0.41 Changing the pattern of substitution from ortho- to
28.32 6 0.53
63.26 6 0.11
79.46 6 0.68
41.16 6 0.60
36.04 6 0.39
15.76 6 0.15
8.24 6 0.29
443.45 6 0.24
7.3 6 0.62
para- (compound 5c), resulted in a marginal loss in
activity. Activity further declined on introducing elec-
tron donating methyl substituents, compound 5d and
5e against both MMPs. Replacing methyl with an
electron withdrawing group, e.g., nitro in compound
5f, increased activity against MMP-2 and MMP-9,
i.e., 39.21 6 0.83 and 41.16 6 0.60 nM, respectively,
while a modest increase in activity was observed in
the isomer, para-NO2, compound 5g.
5i
5j
CGS27023A
*The mean of three experiments 6 standard deviation.
From the structure–activity relationship studies
(SAR), the inhibitory potency of the compounds
described is greatly influenced by minor structural
variations. Compounds containing electron withdraw-
ing groups (5b, 5c, 5f, 5g, 5h, and 5i) exhibit greater
inhibition than their analogs with electron donating
substituent (5d, 5e, and 5j) although no substitution
render any compound inactive. Apart from this, the
synthesized compounds were confirmed on their
physicochemical characteristics and spectroscopic
(IR, 1H-NMR,13C-NMR, and mass spectral data)
investigation. The strong intensity bands at 3430,
3139 and 2543 cm21 were attributable to 2NH and
C@N, respectively confirming the formation of a cya-
noacetylhydrazono derivative. An upward field singlet
1
in H-NMR spectrum at 3.1 ppm was observed for
CH2 group and downfield singlet appearing at 9.2
ppm was due to addition of NH group. Moreo-
ver,13C-NMR displayed a signal at 31.5 ppm for CH2
and another signal at 125.7 ppm assigned to CAN.
Pharmacological Activity
MMP inhibition studies
As a result of the attractive drug like properties
of barbiturates several 5,5-disubstituted pyrimidine-
2,4,6-triones, exhibiting high inhibition over several
MMP subtypes have been discovered [Foley et al.,
2001; Blagg et al., 2005; Reiter et al., 2006;Wang
et al., 2011]. The presence of a sulfonamide group
also contributes to MMP inhibition. To search for
novel MMP inhibitors, these two pharmacophores
was linked together to generate novel hybrid conju-
gates. Their design was based on the assumption
that, the appropriate balance between hydrophilic/
lipophilic properties was efficiently achieved on
substituting a 5-OH group because of its hydrogen
bonding properties. As shown in Table 1, most of the
compounds exhibit potent inhibitory activity against
both the target MMPs as compared to CGS27023A, a
reference standard [Kasaoka et al., 2008]. Among
the newly synthesized derivatives, compound 5i was
the most potent analog against both MMP-2 and
MMP-9, with IC50 values of 2.35 6 0.21 and 8.24 6
0.29 nM, respectively. Changing the substitution
Fig. 2. Effect of compound 5i on LPS-induced protein accumula-
tion and leukocytes migration in BALF. A: Protein contents quantifi-
cation in cell-free BALF exemplified pulmonary permeability. B:
Leukocyte count in BALF. Values are expressed as mean 6 S.D.
Drug Dev. Res.

6
HE ET AL.
Fig. 3. Compound 5i improves LPS induced lung injury (original magnification 3100). Healthy lung (control) and LPS induced lung injury;
Treatment with compound 5i at concentrations of (A) 25, (B) 50, and (C) 100 mg/kg. [Color figure can be viewed in the online issue, which
is available at wileyonlinelibrary.com.]
CONCLUSIONS
position of substitution has much impact on activity
with para-substituted compounds being more active
than their meta analogs.
In the present paper a novel series of potent
MMP-2 and MMP-9 inhibitors were synthesized
among which compound 5i was the most potent ana-
log against MMP-2 and MMP-9, with IC50 values of
2.35 6 0.21 and 8.24 6 0.29 nM. Compound 5i was
also active in vivo in reducing LPS-induced ALI.
Effect of compound 5i on LPS-induced ALI in mice
Infection caused by Gram-negative pathogens is
responsible for ALI progression with LPS, an endo-
toxin constituting major portion of outer membrane
of Gram-negative bacteria serving as a determinant
pathological factor in ALI [Chen et al., 2010]. In the
present study, administration of LPS i.t. leads to
acute lung inflammation in mice via the recruitment
of macrophages and neutrophils leading to a loss of
vascular integrity resulting in protein leakage and leu-
kocyte infiltration.
The effect of compound 5g was evaluated for
effects on vascular permeability and leukocyte migra-
tion in ALI mice measuring the total BALF protein
concentration to assess the integrity of the vascular
membrane barrier. IB i.t. administration of LPS, pro-
tein concentration of BALF was increased nearly
fourfold in comparison to control mice. In mice pre-
treated with compound 5i at 100 and 1000 lg/kg for
30 min the BALF protein concentration was reduced
(Fig. 2A). LPS administration for 6 h led to an
approximate fivefold increase in lung leukocyte infil-
tration (Fig. 2B), With 30 min of 5i pretreatment,
LPS-induced leukocyte migration was inhibited in a
dose-dependent manner with an approximate IC50
value of 1000 lg/kg (Fig. 1b).
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