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EHOOPrÁHH^ECRAa XHMH3, 2014, moM 40, № 4, c. 479-490
DESIGN AND SYNTHESIS OF AMINO ACIDS-CONJUGATED HETEROCYCLE DERIVED UREAS/THIOUREAS AS POTENT INHIBITORS OF PROTEIN GLYCATION
© 2014 C. S. Shantharam, D. M. Suyoga Vardhan, R. Suhas, and D. Channe Gowda#
Department of Studies in Chemistry, Manasagangotri, University of Mysore, Mysore 570006, Karnataka, India
Received October 2, 2013; in final form January 31, 2014
Protein glycation is believed to play an important role in the development of long-term disorders associated with diabetic complications. In view of the wide occurrence of advanced glycation end products (AGE's) and the oxidative stress derived from them in a variety of diabetic complications, it would be of great interest to identify and develop AGE inhibitors. In this study, synthesis and in vitro antiglycation activity of a small library of forty urea/thiourea derivatives of Phe/Tyr/Glu/Lys—benzisoxazole hybrids are reported. Structures of the compounds were confirmed by IR, NMR, mass spectrometry, and elemental analysis. Most of the title compounds exhibited promising activity. Best antiglycation activity was found for Tyr analogue with methoxy group as a substituent particularly at the para position with IC50 value of 1.9 p.M against the positive control, Rutin, with IC50 = 41.9 p.M. Thus, the title compounds represent novel class of potent antiglycating agents.
Keywords: amino acids, conjugation, heterocycle, urea/thiourea, antiglycation
DOI: 10.7868/S0132342314040125
INTRODUCTION
Glycation research poses great challenge to scientists. The physiological effects of protein glycation are diverse, intricate, and complex [1]. Advanced glycation end-products are involved in the pathogenesis of diabetes and neurological diseases like Alzheimer's disease. They also contribute to the development of atherosclerosis and joint diseases and cause aging of many tissues [2, 3]. The Maillard reaction is non-enzymatic and comprises of two stages: the early stage consists of reaction between the carbonyl group of a reducing sugar and primary amino group of a protein and produces the corresponding Schiff base, which undergoes an Amadori rearrangement. Then, complex oxidation, dehydration, and condensation reactions lead, via intra- and intermolecular cross-linking of the proteins, to AGE's [4]. In this regard, glycation research remains a fertile ground of challenging investigation nearly a century after its discovery and the appreciation of its potential therapeutic importance in human health. On the other hand, several classes of organic compounds have been developed as inhibitors of protein glycation, but are found to be moderately
Abbreviations: AGE's, advanced glycation end products; BSA, bovine serum albumin; EDCI, 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide; Het, [3-(4-piperidyl)-6-fluoro-1,2-ben-zisoxazole]; HOBt, 1-hydroxybenzotriazole; NMM, N-methyl morpholine; TCA, trichloroacetic acid.
#Author for correspondence (tel.: +91 821 241-96-64, e-mail: dchannegowda@yahoo.co.in).
active (Fig. 1). Hence, there is still a great need to design, screen, and identify more effective antiglycation agents.
In the field of biomedical research, conjugation of amino acids to small bioactive molecules is found promising and successful approach employed for developing new leads with enhanced potency because of their low toxicity, biocompatibility, likeliness, and favored interaction of amino acid residues with the biological system. Moreover, other studies have shown that many amino acids are competitive inhibitors of protein glycation [5, 6]. While in most cases amino acids are directly conjugated to the parent drugs, bifunc-tional linkers have been used to further increase the structural diversity and expand the types of parent drugs that could be linked to amino acids [7, 8]. Thus, the rationale for the introduction of amino acids as linkers is considered to play major role in medicinal chemistry. In this respect, several reports concerning the conjugation approaches have been published [9—12].
Many articles discuss interesting biological activities of 1,2-benzisoxazole, including antidiabetic [13], hypoglycemic [14], anticonvulsant, antitumor [15], antimicrobial [16], antiulcer, and anticancer. Recently, Khan et al. have synthesized benzoxazole derivatives and found that they act as possible antiglycating agents [17]. In view of the broad spectrum of the biological activity exhibited by these benzisoxazoles, the present work aimed to incorporate [3-(4-piperidyl)-6-
O
LR-90 IC50 = 275 ^M
N
\
N °2N
O
Bis-Schiff base of isatin Metronidazole ester derivative IC50 = 243.95 ^M IC50 = 218.97 ^M
O
O
IC50 = 74 ^M
H H
^ Br
Y
O
Benzoquione derivative 2,4,6-Trichloro hydrazone #-Aroylated isatin Urea derivative of phenyl
derivative IC50 = 27.2 ^M
derivative IC50 = 18.01 ^M
isocyanate IC50 = 4.26 ^M
Fig. 1. Some of the known AGE's inhibitors.
fluoro-1,2-benzisoxazole] to be explored as medicinally important compounds.
A variety of activities are associated with ureas and thioureas including antiglycation [18, 19], anti-HIV [20], anti-inflammatory [21], antimicrobial [22, 23], and antidepressant [24]. Some of the urea compounds are multi-stage glycation inhibitors with the highest post-Amadori activities [25].
Taking into consideration the need for more glyca-tion inhibitors and the aforementioned biological significance of amino acids, benzisoxazole, and urea/thiourea functions, and coupled with our research focused on synthesis of novel potential bioactive agents [10—12], the aim of this investigation was to develop some new che-motherapeutic agents by joining in one single structure these important biologically active scaffolds seeking an improvement in the activity.
RESULTS AND DISCUSSION
Heterocycle, [3-(4-piperidyl)-6-fluoro-1,2-ben-zisoxazole] • HCl, was synthesized by following the previously reported method [26]. This was further conjugated to Boc-amino acids using EDCI/HOBt as coupling agent and NMM as base. Boc group of the conjugates was removed using TFA and reacted with various substituted phenyl isocyanates/isothiocyanates to obtain urea/thiourea derivatives, respectively (Scheme). All the derivatives were obtained in high yields.
The evidence for the formation of title compounds was obtained from IR, 1H NMR, mass spectrometry, and elemental analysis. IR spectra of the ureas and thioureas exhibited peaks at 1615—1644 cm-1 (CO),
~2040 cm-1 (CS), and ~3300 cm-1 (NH). PMR spectra showed a singlet for -NH at 6 ~ 5.50-5.90 and a multiplet for another -NH at 6 ~ 8.01-8.10 for urea derivatives. On the other hand, a singlet at 6 ~ 5.50-5.90 (-NH) and multiplet at 6 ~ 8.01-8.20 (NH) was observed for thiourea derivatives. All the other peaks were exactly matching the structure. Also, % of each element (C, H, N, and S) of the synthesized compounds was confirmed by elemental analysis and the values were found to be within ±0.4% of the calculated ones. Further, structures of the compounds were confirmed by mass spec-trometry analyses. The physical, mass spectrometry, IR, and NMR data of the synthesized compounds are reported in Tables 1 and 2.
In Vitro Antiglycation Activity
The main aim of the present work was to synthesize some analogues, which can act as possible antiglycat-ing agents. Hence, the general structure has been designed with the parameters discussed below.
(i) Formation of the hybrid analogues by conjugation of Het to different classes of amino acids, viz., hydrophobic, phenolic, acidic, and basic, in order to improve potency, as well as biological properties. Conversion of ^-terminal less stable amide bond into more resistant urea/thiourea bond.
(ii) Structural variability of R', different substitu-ents (electron donating and electron withdrawing), in order to evaluate their effects on the biological activity of the compounds.
Dose response curve - 15 -25—35 -3 - 14
0 1 2 3 4 5
Concentration,
Fig. 2. Experimental dose-response curves of the most active compounds of the series.
Earlier work from our research group revealed that compounds containing simple amino acids like Gly and Pro having different substituents on the aromatic ring of urea/thiourea analogues possess promising an-tiglycation activity [27]. The IC50 values were in the range of 3.6—76.1 ^M. Further, it was demonstrated that compounds containing F, Br, and OMe as the sub-stituents exhibited high activity. In this direction, we projected our current work by incorporating representative amino acids from different classes, like Phe, Tyr, Glu, and Lys. A small library of forty title compounds were synthesized by varying different parameters as anchored above.
Antiglycation activity of the synthesized compounds (I—IV/1—40) was evaluated by the method of Nakagawa et al. [28] and the results are presented in Table 3 and Fig. 2. Rutin was used as a standard and showed IC50 value of 41.9 ^M. The results revealed that most of the synthesized compounds showed excellent inhibition activity. However, no activity was detected in case of amino acids or Het tested alone. This clearly indicated that conjugation plays a central role in enhancing the biological activity [10, 11].
As discussed earlier, the present work involved representative amino acid from each class. Among the amino acid analogues, compounds containing Tyr derivative exhibited the most potent activity (11—20). On the other hand, its parent analogue Phe (1)—(10) presented moderate activity. Further, those compounds containing Glu (21)—(30) exhibited higher activity than Lys (31)—(40). Hence, the order of activity based on the amino acids was found to be Tyr > Glu > Lys > Phe.
The nature of R' substituent (isocyanates or isothiocyanates) was also found to be crucial for the antiglycation activity. The most active compounds were those with aromatic rings possessing an electron donating —OCH3 substituent.
To summarize, a series of new urea/
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