Synthesis and biological evaluations of novel bendazac lysine analogues as potent anticataract agents

Article abstract of DOI:10.1016/j.bmcl.2010.02.061

Novel bendazac analogues and their salts have been designed and prepared. The resulting compounds (13c-d, 15c, 17c) showed very good aqueous solubility (>100 mg/mL). An in vitro assay showed that most of the resulting compounds had potent protective activ

Full text of DOI:10.1016/j.bmcl.2010.02.061

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2115–2118
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluations of novel bendazac lysine analogues
as potent anticataract agents
c
c
c
a,
Hong Shen a, Shaohua Gou a,b, , Jianping Shen , Yanqin Zhu , Yindi Zhang , Xuetai Chen
*
*
a School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, PR China
b Pharmaceutical Research Center and School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China
c Institute of Clinical Pharmacology, Nanjing Medical University, Nanjing 210029, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel bendazac analogues and their salts have been designed and prepared. The resulting compounds
(13c–d, 15c, 17c) showed very good aqueous solubility (>100 mg/mL). An in vitro assay showed that most
of the resulting compounds had potent protective activity against the oxidative damage. Particularly,
compound 13d was also able to enhance the WSP and T-AOC level in the H2O2/FeCl3-induced oxidative
damage model, indicating the resulting compound may protect the lens through an antioxidant pathway.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 17 November 2009
Revised 12 February 2010
Accepted 13 February 2010
Available online 19 February 2010
Keywords:
Bendazac lysine analogues
Anticataract agents
Aqueous solubility
Cataract, often causing severe loss of vision and eventually
blindness, is a multifactorial disease process, and is induced by var-
ious toxic factors, environmental stressors, and gene mutations.1
Surgery, with lens extraction and intraocular lens implantation, is
the only currently available treatment, but the high operation cost
limits its applications in developing countries.2 In addition, poster-
ior capsule opacification (PCO) hinders the ultimate success of cat-
aract surgery as well. It is of necessary need to develop novel
agents preventing or delaying the appearance of PCO in patients
submitted to cataract surgery.3 So cataract prevention or medical
treatment that could slow the progression of cataract is an impor-
tant task. Among the few drugs currently available for the medical
treatment of cataract, bendazac lysine (BDL) (Fig. 1) is a topical
non-steroidal anti-inflammatory drug.4,5
Bendazac is one of agents that have been introduced for the
treatment of cataracts. It can protect patients from the loss of vi-
sion, thus delaying the need for surgical intervention.6 Bendazac
and its main metabolite, the 5-hydroxy derivative, provide antiox-
idant effects as scavengers of oxygen-derived free radicals. Hence
both of them endowed with protein-stabilizing properties.7–12 It
was shown that bendazac protected lens crystallins against dena-
turation, glycation, and carbamylation.13–16 It is generally thought
that bendazac stabilizes proteins by binding to specific sites, but a
direct antioxidant activity was also demonstrated.17–21 The most
common bendazac-related adverse reactions were laxative activity
and other gastrointestinal disturbances; furthermore a few cases of
hepatotoxicity have been reported.22–24
In this paper a series of novel bendazac analogues have been de-
signed, synthesized and evaluated for their anticataract activity.
Among these analogues, a malonic acid group was connected to
the bendazac skeleton by replacing the original acetic acid func-
tional group. The presence of this dicarboxylic group is expected
to increase the polarity of the resulting compound for the activity.
Furthermore, different salts of the dicarboxylic acids with inor-
ganic bases (NaOH, KOH) and amino acids (L-histidine, L-lysine)
have also been prepared. The conversion from the free acid to
the corresponding salts will probably give a great increase of the
aqueous solubility and decrease the irritation of bendazac. The
benzyl substituted indazolone, which is believed to be essential
for the activity of bendazac, is maintained in the backbone of the
analogues. Another structural modification is that different substi-
tuent groups have been introduced to the benzyl ring. Through this
OH
O
O
COOH
NH
2
N
.
N
H N
2
* Corresponding authors. Tel./fax: +86 25 83272381 (S.G.).
E-mail addresses: sgou@seu.edu.cn (S. Gou), xtchen@nju.edu.cn (X. Chen).
Figure 1. Bendazac lysine (BDL).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.061

2116
H. Shen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2115–2118
The synthetic route of new compounds is described in Scheme
R
O
1. Indazolone was prepared by diazotization of anthranilic acid, fol-
lowed by reduction to the hydrazine and ring closure,25 then
indazolone reacting with (un)substituted benzyl chlorine formed
a series of N-substituted indazolone.26 Followed by substitution
with diethyl 2-bromomalonate and then hydrolyzation, com-
pounds 13–17 were obtained (Scheme 1). These diacids were re-
O
i
ii
OH
NH
NH
N
N
N
2
H
OH
1
2
3-7
acted with NaOH, KOH, L-lysine, and L-histidine, respectively, to
generate their corresponding salts (Scheme 2). However, attempts
to prepare their amine salts like those of triethylamine and tribu-
tylamine with the resulting diacids failed, only unstable com-
pounds, which could hardly be isolated and purified, were
obtained.
R
R
iv
iii
N
N
N
N
O
O
The aqueous solubility of compounds 13–17 and their salts has
been measured, which is given in Table 1.
OH
OH
O
O
O
13: R=H
14: R=p-CH
15: R=m-F
16: R=m-Cl
17: R=p-Cl
3
O
It was found that the solubility of the corresponding amino acid
salts was quite good (all over 50 mg/mL), while that of the corre-
sponding sodium or potassium salts was rather low. Among all
these compounds, 17c showed the best solubility, unfortunately,
obvious moisture absorption was also found. Moreover, 15 ex-
pressed better solubility than other diacids and bendazac. In order
to study the antioxidant activity of the resulting compounds, 13a–
d, 15a, 15c, and 16c were selected as representative samples to
perform further in vitro evaluations.
O
O
13-17
8-12
Scheme 1. Preparation of the study compounds (13–17). Reagents and conditions:
(i) HCl and NaNO2; (ii) NaOH, substituted benzyl chloride, 70 °C, 2 h; (iii)
BrCH(COOEt)2, potassium carbonate and TEBA, reflux, 4 h; (iv) KOH, reflux, 2 h.
way the structure–activity relationship of the benzyl moiety can be
studied. In order to investigate the anticataract activity of the ana-
logues, some compounds (13a–d, 15a, 15c, and 16c) have been
evaluated in vitro.
The ability of compounds to retard H2O2/FeCl3-induced cell
damage in isolated lenses cultured in Dulbecco’s Modified Eagle
Medium (DMEM) was tested, using the recently modified Lou’s
R
N
N
i
O
13a-d, 14a-d, 15a-d, 16a-d, 17a-d
OH
OH
O
O
13-17
R
R
N
N
13b: R=H
14b: R=p-CH3
15b: R=m-F
16b: R=m-Cl
13a: R=H
N
N
O
O
14a: R=p-CH3
15a: R=m-F
16a: R=m-Cl
17a: R=p-Cl
OK
ONa
O
O
OK
ONa
17b: R=p-Cl
O
O
R
R
OH
O
H
N
NH2
N
N
N
N
.
N
O
2
.
O
2
OH
OH
OH
OH
H2N
O
O
COOH
H2N
O
O
13d: R=H
13c: R=H
14d: R=p-CH3
15d: R=m-F
16d: R=m-Cl
17d: R=p-Cl
14c: R=p-CH3
15c: R=m-F
16c: R=m-Cl
17c: R=p-Cl
Scheme 2. Preparation of the study compounds (13a–d, 14a–d, 15a–d, 16a–d, and 17a–d). Reagents and conditions: (i) corresponding base or amino acid, H2O, rt, 2 h.

H. Shen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2115–2118
2117
Table 1
13b > 13c > > 16c, and for rat lenses is 13d > 13a > 15a > 15c >
16c > BDL > 13c > > 13b.
The aqueous solubility of compounds
The activities of the resulting compounds and salts are related
to the nature of the cation group. It has been shown that the salts
with histidine have higher activity than those with lysine, sodium
or potassium cations. For example, the activity order is
13d > 13a > 13b > 13c.
Compound 13d, which showed potent activity on both rabbit
and rat lenses, was selected for further test. Different concentra-
tions of 13d (5, 10, and 15 mM) were tested for its protective effect
on rabbit lenses.
As shown in Table 3, both BDL and 13d have shown protective
activities against oxidation compared with the control group. A
clear dose-dependent manner of 13d has been observed, the higher
concentration is, the better activity shows. It is also worthwhile to
note that the activity of 13d is higher than BDL at the same concen-
tration (10 mM).
Furthermore, the levels of water-solvent protein (WSP), total-
antioxidant capacity (T-AOC) and malondialdehyde (MDA, a mar-
ker of lipid peroxidation) in lens homogenate of each group were
also detected with the reagent kit individually.
As shown in Table 4, the levels of WSP and T-AOC of BDL and
13d group were, as expected, much higher than that of the control
group, while the level of MDA was obviously lower. Both BDL and
13d showed positive protective activities. It was observed that at
the equal molar dose 13d showed a better activity than BDL as
judging from all the three different investigations. Besides, a clear
dose-dependent manner of the WSP-protective activity of 13d was
also found. Altogether, the biological results indicate that the
resulting compounds, especially 13d, are potent antioxidants and
can effectively protect lens from the oxidative damage.
In conclusion, a series of novel bendazac analogues, which are
characterized by replacing the original acetic acid group with the
malonic acid group as well as introducing different substituent
groups onto the benzyl ring, have been synthesized,28,29 and their
salts30 are evaluated for the antioxidant activity in isolated
lenses. The aqueous solubility of the compounds has been
measured, and it was found most of them possessed quite good
solubility, especially 15 showed better solubility (2.0 mg/mL)
than bendazac (<1.0 mg/mL). The pharmacological results indi-
cated that most compounds exhibited better activities than BDL
in protecting lens against the oxidative damage. In particular,
Compounds
Solubility (mg/mL)
Bendazac
BDL
13
13a
13b
13c
13d
14
14a
14b
14c
14d
15
15a
15b
15c
15d
16
16a
16b
16c
16d
17
17a
17b
17c
17d
<1.0
93.8
<1.0
3.0
2.2
123.2
180.0
<1.0
<1.0
<1.0
57.4
71.0
2.0
3.2
2.4
134.4
56.6
<1.0
1.2
<1.0
195.2
115.2
<1.0
<1.0
<1.0
312.0
51.0
protocol.27 Transparent cultured lenses were randomly divided
into control group and different sample groups. Control group
lenses were incubated in DMEM supplemented with FeCl3
(0.02%) and hydrogen peroxide (2% for rabbit lenses and 0.5% for
rat lenses) which induced cataract formation. For the sample
groups, tested compounds and BDL were added into culture media
of transparent isolated lenses, respectively. FeCl3 (0.02%) and
hydrogen peroxide (2% for rabbit lenses and 0.5% for rat lenses)
were added into culture media posterior to an hour. After 24 h,
all lenses were photographed and the degree of opacification was
graded as follows:
À, when there was absence of opacification;
+, when there was a slight degree of opacification;
++, when there was a middle degree of opacification;
+++, when there was extensive mature nuclear opacity.
compound 13d, bis(L-histidine) 2-(1-benzyl-1H-indazol-3-yloxy)
malonate, was found to be able to enhance WSP and T-AOC level
in the H2O2/FeCl3-induced oxidative impairment model, indicat-
ing the resulting compound may protect the lenses through a
antioxidant pathway.
As seen in Table 2, the order of activities of protecting isolated
rabbit lenses against oxidation is 15a > BDL > 13d > 13a >
Table 2
Ability of compounds on protecting lens against oxidative damage in vitro
Group*
Percentage of different degree of opacificationa (%)
Percentage of different degree of opacificationb (%)
À
+
++
+++
À
+
++
+++
Control
BDL
13a
13b
13c
13d
15a
15c
0
0
0
0
0
0
0
–
0
30
75
60
30
30
65
80
–
40
25
40
40
35
35
20
–
30
0
0
30
35
0
0
–
65
0
0
0
0
0
0
0
0
0
20
55
80
0
15
85
60
55
55
45
40
20
50
70
15
35
45
30
35
5
0
50
15
0
5
0
15
16c
0
35
*
The concentration of tested sample is 0.5 mM.
a
Tested by isolated rabbit lenses.
b
Tested by isolated rat lenses.

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H. Shen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2115–2118
12. Guglielmotti, A.; de Joannon, A. C.; Cazzolla, N.; Marchetti, M.; Soldo, L.;
Table 3
Cavallo, G.; Pinza, M. Pharm. Res. 1995, 32, 369.
Antioxidant activity of different concentrations of 13d
13. Silvestrini, B.; Catanese, B.; Barillari, G.; Iorio, E.; Valeri, P. Int. J. Tissue React.
1983, 5, 217.
Group
Percentage of different degree of opacificationa (%)
14. Lewis, B. S.; Harding, J. J. Exp. Eye Res. 1988, 47, 217.
15. Marques, C.; Ramalho, J. S.; Pereira, P.; Mota, M. C. Doc. Ophthalmol. 1995, 90,
395.
16. Lewis, B. S.; Rixon, K. C.; Harding, J. J. Exp. Eye Res. 1986, 43, 973.
17. Musci, G.; Silvestrini, B. Drugs Exp. Clin. Res. 1987, 13, 289.
18. Schmut, O.; Hofmann, H. Wien. Klin. Wochenschr. 1985, 97, 819.
19. Mira, M. L.; Azevedo, M. S.; Manso, C. Free Radical Res. 1987, 4, 125.
20. Woollard, A. C. S.; Wolff, S. P.; Bascal, Z. A. Free Radical Biol. Med. 1990, 9,
299.
À
+
++
+++
Control
0
30
65
70
10
0
40
15
0
0
0
30
0
0
0
0
BDL 10 mM
13d 5 mM
13d 10 mM
13d 15 mM
20
30
90
100
a
Tested by isolated rabbit lenses.
21. Crabbe, M. J. C.; Hoe, S. T. Enzyme 1991, 45, 188.
22. Balfour, J. A.; Clissold, S. P. Drugs 1990, 39, 575.
23. Prieto de Paula, J. M.; Rodríguez Rodríguez, E.; Villamandos Nicás, V.; Sanz de la
Fuente, H.; Prada Mínguez, A.; del Portillo Rubí, A. Rev. Clin. Esp. 1995, 195, 387.
24. Laporte, J. R.; Capellà, D. Med. Clin. (Barc) 1995, 104, 396.
25. Baiocchi, L.; Corsi, G.; Palazzo, G. Synthesis 1978, 9, 633.
26. Gordon, D. W. Synlett 1998, 1065.
Table 4
Levels of WSP, T-AOC, and MDA in lens homogenate of each group
Group
WSP mg/mL
T-AOC u/mg protein MDA nmol/mg protein
27. Wang, G.-M.; Raghavachari, N.; Lou, M. F. Exp. Eye Res. 1997, 64, 693.
28. Preparation of compounds 8–12. 32 g (0.134 mol) diethyl 2-bromomalonate was
added into a solution of 3–7 (0.054 mol), 18 g (0.131 mol) potassium carbonate
and 1 g TEBA in 150 mL of 1,2-dimethoxyethane, followed refluxing for 4 h.
After cooled to room temperature, the precipitate was filtered and the volatiles
were evaporated in vacuo. The crude product was column chromatographed
Control
BDL 5 mM
13d 5 mM
7.40 0.73
9.34 1.65
0.04 0.05
0.17 0.05*
0.46 0.27
0.22 0.10
0.04 0.02*
0.03 0.02*
0.08 0.04*
12.99 1.95* 0.21 0.12*
13d 10 mM 16.78 5.96* 0.64 0.03*
13d 15 mM 19.04 1.03* 0.34 0.16*
(n-hexane/ethyl acetate = 3:1). Data for 8. Yield: 59%, colorless oil. IR (
m
, cmÀ1):
*
Compared with control group, p <0.01.
2981 m, 1768s, 1748s, 1619 m, 1235s, 744 m. 1H NMR (CDCl3/TMS), d (ppm):
1.29–1.33 (t, 6H, CH2CH3, J = 7.1 Hz), 4.29–4.33 (q, 4H, CH2CH3, J = 7.1 Hz), 5.34
(s, CH2C6H5), 5.77 (s, CH(COOH)2), 7.08–7.83 (m, 9H, 2Ar). EI-MS: m/z
[M]+ = 382.
Acknowledgments
29. Preparation of compounds 13–17. 16 mmol 8–12 was added to an aqueous
solution of KOH (1.8 g/35 mL). After refluxing for 2 h, the pH was adjusted to 2
with diluted hydrochloric acid, and the resulting precipitate was filtered. Data
The authors would like to thank Zhejiang Sharp Eyes Pharmacy
Corporation for the financial support to this research. We are grate-
ful to Dr. Fang Lei for his kind help in the preparation of this Letter.
for 13. Yield: 93%, white solid, mp: 182–186 °C (decomposed). IR (m
, cmÀ1):
3033 m, 2934 m, 1741s, 1620 m, 1257s, 746 m, 711 m. 1H NMR (D2O/TMS), d
(ppm): 5.30 (s, CH2C6H5), 5.36 (s, CH(COOH)2), 7.00–7.73 (m, 9H, 2Ar). ESI-MS:
m/z [MÀH]À = 325.
30. Data for 13a. Yield: 75%, white solid, mp: >280 °C. IR (
m
,
cmÀ1): 2983w,
d (ppm): 5.21(s,
References and notes
1643s, 1334 m, 741 m, 723 m. 1H NMR (D2O/TMS),
CH(COOÀ)2), 5.33 (s, CH2C6H5), 7.03–7.79 (m, 9H, 2Ar). Data for 13b. Yield:
1. Shinohara, T.; White, H.; Mulhern, M. L.; Maisel, H. Med. Hypotheses 2007, 69, 669.
2. Toh, T.; Morton, J.; Coxon, J.; Elder, M. J. Clin. Exp. Ophthalmol. 2007, 35, 664.
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2004, 36, 145.
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86%, white solid, mp: >280 °C. IR (m
, cmÀ1): 2978w, 1639s, 1328 m, 740 m,
721 m. 1H NMR (D2O/TMS), d(ppm): 5.23 (s, CH(COOÀ)2), 5.35 (s, CH2C6H5),
6.97–7.78 (m, 9H, 2Ar). Data for 13c. Yield: 86%, white solid, mp: 204–
206 °C. IR (
m
, cmÀ1): 3031s, 2941s, 1615s, 1325 m, 741 m. 1H NMR (D2O/
TMS), (ppm): 1.30–1.43 (m, 2H2NC2H4CH2CH2 of lysine), 1.60–1.63 (m,
d
2H2NC2H4CH2CH2 of lysine), 1.78–1.82 (m, 2H2NCH2CH2C2H4 of lysine),
2.90–2.93 (m, 2H2NCH2CH2C2H4 of lysine), 3.65 (m, 2CH(NH2)COOH of
lysine), 5.24 (s, CH(COOÀ)2), 5.36 (s, CH2C6H5), 7.09–7.81 (m, 9H, 2Ar). Data
for 13d. Yield: 92%, white solid, mp: 212–214 °C. IR (m,
cmÀ1): 3128 m,
3026 m, 1618s, 1329 m, 1259w, 744 m. 1H NMR (D2O/TMS), d (ppm): 3.10–
3.24 (m, 2CH2 of histidine), 3.89–3.94 (m, 2CHNH2COOH of histidine), 5.28
(s, CH(COOÀ)2), 5.34 (s, CH2C6H5), 7.05–7.80 (m, 11H, 9H of 2Ar and 2H of 2
imidazole), 8.27 (s, 2H of 2 imidazole).
9. Jaenicke, R. Naturwissenschaften 1994, 81, 423.
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