Design and synthesis of amino acids-conjugated heterocycle derived ureas/thioureas as potent inhibitors of protein glycation

Article abstract of DOI:10.1134/S1068162014040128

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

Full text of DOI:10.1134/S1068162014040128

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2014, Vol. 40, No. 4, pp. 443–454. © Pleiades Publishing, Ltd., 2014.
Design and Synthesis of Amino AcidsꢀConjugated Heterocycle
Derived Ureas/Thioureas as Potent Inhibitors
of Protein Glycation1
C. S. Shantharam, D. M. Suyoga Vardhan, R. Suhas, and D. Channe Gowda2
Department of Studies in Chemistry, Manasagangotri, University of Mysore, Mysore 570006, Karnataka, India
Received October 2, 2013; in final form, January 31, 2014
Abstract—Protein glycation is believed to play an important role in the development of longꢀterm 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
positive control, Rutin, with IC50 = 41.9 M. Thus, the title compounds represent novel class of potent antiꢀ
glycating agents.
µ
M against the
µ
Keywords: amino acids, conjugation, heterocycle, urea/thiourea, antiglycation
DOI: 10.1134/S1068162014040128
2 1
INTRODUCTION
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
active (Fig. 1). Hence, there is still a great need to
design, screen, and identify more effective antiglycaꢀ
tion agents.
Glycation research poses great challenge to scienꢀ
tists. The physiological effects of protein glycation are
diverse, intricate, and complex [1]. Advanced glycaꢀ
tion endꢀproducts 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 nonꢀ
enzymatic and comprises 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 intraꢀ and intermolecular crossꢀlinking of the
proteins, to AGE’s [4]. In this regard, glycation
research remains a fertile ground of challenging invesꢀ
tigation nearly a century after its discovery and the
appreciation of its potential therapeutic importance in
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,
bifunctional 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 linkꢀ
ers is considered to play major role in medicinal chemisꢀ
try. In this respect, several reports concerning the conjuꢀ
gation approaches have been published [9–12].
Abbreviations: AGE’s, advanced glycation end products; BSA,
bovine serum albumin; EDCI, 1ꢀ(3ꢀdimethylaminopropyl)ꢀ3ꢀ
ethylꢀcarbodiimide; Het, [3ꢀ(4ꢀpiperidyl)ꢀ6ꢀfluoroꢀ1,2ꢀbenꢀ
zisoxazole]; HOBt, 1ꢀhydroxybenzotriazole; NMM,
morpholine; TCA, trichloroacetic acid.
The article is published in the original.
Corresponding author: phone: +91 821 241ꢀ96ꢀ64, eꢀmail:
dchannegowda@yahoo.co.in.
Nꢀmethyl
Many articles discuss interesting biological activiꢀ
ties of 1,2ꢀbenzisoxazole, including antidiabetic [13],
hypoglycemic [14], anticonvulsant, antitumor [15],
antimicrobial [16], antiulcer, and anticancer. Recently,
1
2
443

444
SHANTHARAM et al.
N
Cl
N
NO2
O
N
H
N
H
N
HO
O
N
CH2
O
R
O2N
O
2
O
N
O
H
LRꢀ90
BisꢀSchiff base of isatin
Metronidazole ester derivative
IC50 = 275 µM
IC50 = 243.95 µM
IC50 = 218.97 µM
O
R
O
Cl
Cl
R
H
H
O
O
N
N
N
Br
N
Cl
NH
O
O
O
O
O
Benzoquione derivative
2,4,6ꢀTrichloro hydrazone
derivative
NꢀAroylated isatin
Urea derivative of phenyl
isocyanate
IC50 = 74 µM
derivative
IC50 = 27.2 µM
IC50 = 18.01 µM
IC50 = 4.26 µM
Fig. 1. Some of the known AGE’s inhibitors.
Khan et al. have synthesized benzoxazole derivatives ates to obtain urea/thiourea derivatives, respectively
and found that they act as possible antiglycating agents (Scheme 1). All the derivatives were obtained in high
[17]. In view of the broad spectrum of the biological yields.
activity exhibited by these benzisoxazoles, the present
The evidence for the formation of title compounds
work aimed to incorporate [3ꢀ(4ꢀpiperidyl)ꢀ6ꢀfluoroꢀ
was obtained from IR, 1H NMR, mass, and elemental
1,2ꢀbenzisoxazole] to be explored as medicinally
analysis. IR spectra of the ureas and thioureas exhibꢀ
important compounds.
ited peaks at 1615–1644 cm–1 (CO), ~2040 cm–1
(CS), and ~3300 cm–1 (NH). PMR spectra showed a
A variety of activities are associated with ureas and
thioureas including antiglycation [18, 19], anti–HIV
[20], antiꢀinflammatory [21], antimicrobial [22, 23],
and antidepressant [24]. Some of the urea compounds
are multiꢀstage glycation inhibitors with the highest
postꢀAmadori activities [25].
singlet for –NH at
another –NH at ~ 8.01–8.10 for urea derivatives. On
the other hand, a singlet at ~5.50–5.90 (–NH) and
multiplet at ~8.01–8.20 (NH) was observed for thioꢀ
δ
~ 5.50–5.90 and a multiplet for
δ
δ
δ
urea derivatives. All the other peaks exactly matched the
structure. Also, % of each element (C, H, N, and S) of
the synthesized compounds was confirmed by elemenꢀ
tal analysis and the values were found to be within
0.4% of the calculated ones. Further, structures of
the compounds were confirmed by mass spectrometry
analyses. The physical, mass spectrometry, IR, and
NMR data of the synthesized compounds are reported
in Tables 1 and 2.
Taking into consideration the need for more glycaꢀ
tion inhibitors and the aforementioned biological sigꢀ
nificance of amino acids, benzisoxazole, and
urea/thiourea functions, and coupled with our
research focused on synthesis of novel potential bioacꢀ
tive agents [10–12], the aim of this investigation was to
develop some new chemotherapeutic agents by joining
in one single structure these important biologically
active scaffolds seeking an improvement in the activity.
In Vitro Antiglycation Activity
RESULTS AND DISCUSSION
The main aim of the present work was to synthesize
some analogues, which can act as possible antiglycatꢀ
ing agents. Hence, the general structure has been
designed with the parameters discussed below.
Heterocycle, [3ꢀ(4ꢀpiperidyl)ꢀ6ꢀfluoroꢀ1,2ꢀbenꢀ
zisoxazole] · HCl, was synthesized by following the
previously reported method [26]. This was further
conjugated to Bocꢀamino acids using EDCI/HOBt as
coupling agent and NMM as base. Boc group of the
(i) Formation of the hybrid analogues by conjugation of
conjugates was removed using TFA and reacted with Het to different classes of amino acids, viz., hydrophobic,
various substituted phenyl isocyanates/isothiocyanꢀ phenolic, acidic, and basic, in order to improve potency, as
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Table 2. Physical and mass data of the synthesized compounds (I–IV/1–40)
Elemental analysisa
Rf Values
Enꢀ
try
Yield M.P.
Theoretiꢀ
cal mol wt values
MS
Rfa
98:2:3 95:5:3
) 0.53 0.67
Rfb
Mol. For.
(%)
(°C)
% C
% H
% N
% S
(I
85
84
89
87
91
89
82
85
83
77
89
79
87
95–96 C26H30FN3O4
110 C33H34FN3O5
Gum C29H34FN3O6
Gum C31H39FN4O6
83 C28H27FN4O3
467.53
642.54
539.60
582.66
486.54
502.60
532.63
532.63
532.63
581.50
565.43
581.50
565.43
520.59
660.17
677.62
707.64
468.5
66.79
6.47
8.99
(8.91)
6.54
(6.51)
7.79
(7.75)
9.62
(9.58)
11.52
–
–
(M + 1) (66.73) (6.45)
643.5 61.68 5.33
(M + 1) (61.64) (5.29)
540.6 64.55 6.35
(M + 1) (64.50) (6.31)
583.6 63.90 6.75
(M + 1) (63.94) (6.77)
486.5 69.12 5.59
(
II) 0.57 0.76
III) 0.58 0.71
IV) 0.55 0.70
(
–
(
–
(
1
) 0.51 0.65
) 0.47 0.69
) 0.47 0.70
) 0.46 0.65
) 0.45 0.61
) 0.50 0.62
) 0.49 0.70
) 0.47 0.69
) 0.44 0.69
–
(M + 1) (69.08) (5.53) (11.50)
503.6 66.91 5.41 11.15
(2
130 C28H27FN4O2S
60 C29H29FN4O3S
78 C29H29FN4O3S
90 C29H29FN4O3S
70 C28H26BrFN4O2S
100 C28H26BrFN4O3
80 C28H26BrFN4O2S
108 C28H26BrFN4O3
6.38
(M + 1) (66.88) (5.45) (11.09) (6.33)
533.6 65.39 5.49 10.52 6.02
(M + 1) (65.35) (5.46) (10.57) (6.05)
533.6 65.39 5.49 10.52 6.02
(M + 1) (65.33) (5.52) (10.55) (6.07)
533.6 65.39 5.49 10.52 6.02
(M + 1) (65.36) (5.42) (10.56) (6.09)
583.5
(M + 2) (57.81) (4.56)
567.0 59.48 4.63
(M + 2) (59.46) (4.59)
583.5 57.83 4.51
(M + 2) (57.79) (4.46)
567.0 59.48 4.63
(M + 2) (59.52) (4.56)
521.5 64.60 5.03
(3
(4
(5
(6
57.83
4.51
9.63
(9.60)
9.91
(9.58)
9.63
(9.58)
9.91
(9.58)
10.76
5.51
(5.47)
–
(7
(8
5.51
(5.46)
–
(9
(
10) 0.47 0.66
11) 0.51 0.65
12) 0.54 0.70
13) 0.50 0.72
14) 0.46 0.65
15) 0.53 0.74
16) 0.51 0.65
17) 0.46 0.62
18) 0.47 0.68
19) 0.46 0.65
20) 0.46 0.63
21) 0.50 0.67
80 112–115 C28H26F2N4O2S
6.16
(M + 1) (64.64) (5.07) (10.71) (6.09)
(
88
86
83
79–84 C35H31Cl2FN4O4
83–87 C35H31Cl2FN4O3S
77 C36H33Cl2FN4O4S
661.1
(M + 1) (63.59) (4.68)
678.6 62.04 4.61
(M + 1) (62.01) (4.66)
706.9
(M+)
706.9
(M+)
706.9
(M+)
758.5
(M + 2) (55.52) (4.04)
742.4 56.77 4.08
(M + 2) (56.71) (4.04)
758.5 55.57 4.00
(M + 2) (55.57) (4.06)
742.4 56.77 4.08
(M + 2) (56.73) (4.03)
696.6 60.43 4.35
(M + 1) (60.41) (4.31)
559.6 66.65 5.59
63.54
4.72
8.47
(8.43)
8.27
(8.21)
7.92
–
(
4.73
(4.78)
4.53
(
61.10
(61.14) (4.76)
61.10 4.70
(61.15) (4.76)
61.10 4.70
(61.06) (4.74)
55.57 4.00
4.70
(7.91)
(4.53)
(
86
84
105 C36H33Cl2FN4O4S
84 C36H33Cl2FN4O4S
707.64
707.64
7.92
(7.94)
7.92
4.53
(4.49)
4.53
(
(7.88)
(4.49)
(
81
75
78
84
79
68 C35H30BrCl2FN4O3S 756.51
83–87 C35H30BrCl2FN4O4 740.45
112 C35H30BrCl2FN4O3S 756.51
7.41
(7.46)
7.57
(7.53)
7.41
(7.36)
7.57
(7.52)
8.05
4.24
(4.28)
–
(
(
4.24
(4.21)
–
(
100 C35H30BrCl2FN4O4
78–82 C35H30Cl2FN4O3S
740.45
695.61
558.60
(
4.61
(4.66)
–
(8.01)
10.03
(
85 128–132 C31H31FN4O5
(M + 1) (66.61) (5.54) (10.08)
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Theoretiꢀ MS
Table 2. (Contd.)
Elemental analysisa
Rf Values
Enꢀ
try
Yield M.P.
Mol. For.
Rfa
98:2:3 95:5:3
Rfb
(%)
(°C)
cal mol wt values
% C
% H
5.44
% N
% S
(
22) 0.43 0.61
23) 0.45 0.62
24) 0.47 0.64
25) 0.46 0.62
26) 0.45 0.66
27) 0.47 0.64
28) 0.46 0.63
29) 0.45 0.65
30) 0.45 0.61
31) 0.51 0.65
32) 0.47 0.66
33) 0.45 0.61
34) 0.45 0.60
35) 0.49 0.65
36) 0.46 0.64
37) 0.46 0.62
91
79
91 C31H31FN4O4S
90 C32H33FN4O5S
574.67
604.69
575.6
64.79
9.75
5.58
(M + 1) (64.73) (5.47)
(9.73)
(5.52)
(
604.1
(M+)
604.1
(M+)
604.1
(M+)
655.5
63.56
(63.52) (5.56)
5.50
9.27
(9.23)
5.30
(5.36)
(
81
84
83 C32H33FN4O5S
96 C32H33FN4O5S
604.69
604.69
63.56 5.50
(63.51) (5.46)
63.56 5.50
(63.59) (5.45)
56.97 4.63
9.27
(9.21)
5.30
(5.34)
(
9.27
(9.30)
5.30
(5.35)
(
83
75
79
91
79
86
76
81
131 C31H30BrFN4O4S
123 C31H30BrFN4O5
Gum C31H30BrFN4O4S
156 C31H30BrFN4O5
118 C31H30F2N4O4S
150 C33H36FN5O5
653.56
637.50
653.56
637.50
592.66
601.67
617.73
647.76
647.76
647.76
696.63
680.56
696.63
680.56
635.72
8.57
(8.53)
4.91
(4.94)
(M + 2) (56.91) (4.60)
639.5 58.41 4.74
(M + 2) (58.45) (4.70)
655.5 56.97 4.63
(M + 2) (56.94) (4.68)
639.5 58.41 4.74
(M + 2) (58.40) (4.71)
593.6 62.82 5.10
(M + 1) (62.87) (5.14)
602.6 65.88 6.03
(
8.79
(8.73)
–
(
8.57
(8.51)
4.91
(4.95)
(
8.79
(8.75)
–
(
9.45
(9.49)
5.41
(5.36)
(
11.64
–
(M + 1) (65.86) (6.01) (11.62)
(
107 C33H36FN5O4S
90–93 C34H38FN5O5S
618.7 64.16 5.87 11.34
5.19
(M + 1) (64.12) (5.91) (11.39) (5.15)
648.7 63.04 5.91 10.81 4.95
(M + 1) (63.01) (5.95) (10.84) (4.97)
648.7 63.04 5.91 10.81 4.95
(M + 1) (63.08) (5.96) (10.85) (4.99)
648.7 63.04 5.91 10.81 4.95
(M + 1) (63.06) (5.88) (10.76) (4.91)
698.6 56.90 5.06 10.05 4.60
(M + 2) (56.91) (5.04) (10.09) (4.64)
(
(
79 128–130 C34H38FN5O5S
(
84
81
79
83
86
78
134 C34H38FN5O5S
108 C33H35BrFN5O4S
89 C33H35BrFN5O5
(
(
682.5
(M + 2) (58.26) (5.22) (10.27)
698.6 56.90 5.06 10.05
58.24
5.18
10.29
–
(38) 0.50 0.66
82–85 C33H35BrFN5O4S
86–88 C33H35BrFN5O5
Gum C33H35F2N5O4S
4.60
(M + 2) (56.94) (5.09) (10.08) (4.57)
(39) 0.46 0.61
682.5
(M + 2) (58.20) (5.22) (10.25)
636.7 62.35 5.55 11.02
58.24
5.18
10.29
–
(40) 0.43 0.59
5.04
(M + 1) (62.31) (5.51) (11.07) (5.08)
a
= Values given in the parentheses are the calculated for elemental analysis. The values are within 0.4% of the calculated ones.
well as biological properties. Conversion of Nꢀterminal less ents (electron donating and electron withdrawing),
stable amide bond into more resistant urea/thiourea bond.
in order to evaluate their effects on the biological acꢀ
(ii) Structural variability of R', different substituꢀ tivity of the compounds.
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Table 3. Antiglycation activity of the compounds
F
X
O
HN
HN
O
N
R'
N
R
R =
O
C
C6H4
O
CH2
O
C
O
R =
CH2 CH2
O
R =
NH
CH2
4
R =
C6H5
CH2
Cl
Cl
R'
X
IC50
IC50
IC50
IC50
Comp
Comp
Comp
Comp
H
H
O
(
(
(
(
(
(
(
(
(
1
2
3
4
5
6
7
8
9
)
)
)
)
)
)
)
)
)
)
)
75.6 0.5
70.3 0.3
3.6 0.2
4.8 0.5
3.0 0.4
6.4 0.3
9.3 0.5
6.5 0.2
9.2 0.4
23 0.6
(
(
(
(
(
(
(
(
(
(
11
12
13
14
15
16
17
18
19
20
)
)
)
)
)
)
)
)
)
)
80.2 0.2
74.4 0.3
3.0 0.3
4.4 0.4
1.9 0.2
4.8 0.7
8.8 0.3
6.5 0.6
8.2 0.2
15.4 0.4
(
(
(
(
(
(
(
(
(
(
21
22
23
24
25
26
27
28
29
30
)
)
)
)
)
)
)
)
)
)
64.1 0.8
55.2 0.2
2.9 0.3
4.8 0.5
2.5 0.3
5.2 0.2
8.2 0.6
6.5 0.2
8.0 0.5
15.1 0.5
370.2 0.8
Het
(31
(32
(33
(34
(35
(36
(37
(38
(39
(40
(IV
)
81.8 0.8
74.1 0.7
3.8 0.3
4.5 0.4
3.0 0.4
6.1 0.3
9.1 0.2
7.0 0.4
7.4 0.8
20.6 0.7
365.8 0.9
>500
S
S
S
S
S
O
S
O
S
)
)
)
)
)
)
)
)
)
)
2ꢀOCH3
3ꢀOCH3
4ꢀOCH3
2ꢀBr
3ꢀBr
3ꢀBr
4ꢀBr
2ꢀF
(10
(
–
I
425.4 0.6
Rutin
(
II)
390.7 0.4
41.9 0.7
(III)
–
IC
( M) is a measure of the effectiveness of a compound at its half maximal inhibitory concentration. Values are means of three meaꢀ
µ
50
surements; SD ranges are <5% of the mean in all cases.
Earlier work from our research group revealed that that compounds containing F, Br, and OMe as the subꢀ
stituents exhibited high activity. In this direction, we
projected our current work by incorporating represenꢀ
tative 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.
compounds containing simple amino acids like Gly
and Pro having different substituents on the aromatic
ring of urea/thiourea analogues possess promising
antiglycation activity [27]. The IC50 values were in the
range of 3.6–76.1
μM. Further, it was demonstrated
Antiglycation activity of the synthesized comꢀ
pounds (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
Dose response curve
3
15 25 35
14
80
70
60
50
40
30
20
10
showed IC50 value of 41.9 M. The results revealed
μ
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 repꢀ
resentative amino acid from each class. Among the
amino acid analogues, compounds containing Tyr
0
1
2 3
Concentration,
4
5
µ
M
derivative exhibited the most potent activity (11
On the other hand, its parent analogue Phe ( )–(10
presented moderate activity. Further, those comꢀ
–20).
1
)
Fig. 2. Experimental doseꢀresponse curves of the most
active compounds of the series.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
No. 4
2014

DESIGN AND SYNTHESIS OF AMINO ACIDSꢀCONJUGATED HETEROCYCLE
453
pounds containing Glu (21)–(30) exhibited higher
General Procedure for the Conjugation of Het
with BocꢀXaa, where Xaa = Phe ( ), Tyr(2,6ꢀCl2ꢀBzl)
II), Glu(OBzl) (III), and Lys(Z) (IV
I
activity than Lys (31)–(40). Hence, the order of activꢀ
ity based on the amino acids was found to be Tyr > Glu >
Lys > Phe.
(
)
NMM (1.1 mL, 0.01 mol) was added to BocꢀXaa
(0.01 mol) and HOBt (1.53 g, 0.01 mol) dissolved in
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.
DMF (10 mL/g of compound) and cooled to
EDCI (1.91 g, 0.01 mol) was added under stirring
while maintaining the temperature at . The reacꢀ
0°C.
0°C
tion mixture was stirred for an additional 10 min and a
preꢀcooled solution of [3ꢀ(4ꢀpiperidinyl)ꢀ6ꢀfluoroꢀ
To summarize, a series of new urea/thiourea derivꢀ
atives of amino acid–Het conjugates were synthesized
and evaluated concerning their antiglycation activity.
Maximum activity was observed for thiourea derivaꢀ
tives possessing electron donating substituent in the
aromatic ring. The –OMe group, particularly at the
para position, was found to have potent activity
1,2ꢀbenzisoxazole]
⋅ HCl (2.57 g, 0.01 mol) and
NMM (1.1 mL, 0.01 mol) in DMF (20 mL) was added
slowly. After 20 min, pH of the solution was adjusted to
8 by the addition of NMM and the reaction mixture
was stirred overnight at room temperature. DMF was
removed under reduced pressure and the residue was
poured into about 200 mL iceꢀcold 90% saturated
KHCO3 solution and stirred for 30 min. The precipiꢀ
tated product was taken into CHCl3 and washed
(
1.9 M). Presence of Tyr derivative was found to be
μ
critical to enhance the activity. Attending the strucꢀ
tural variation points in the general backbone, we have
made an attempt to improve/modulate antiglycation
activity through the choice of amino acid and the isocyꢀ
anate/isothiocyanate reactant. The results presented in
this work encourage us to continue in this line.
sequentially with 5% NaHCO3 solution (
water ( 20 mL), 0.1 N cold HCl solution (
and finally brine (2 20 mL). The CHCl3 layer was dried
2
×
20 mL),
2
×
2
×
20 mL),
×
over anhydrous Na2SO4 and the solvent was removed
under reduced pressure. The products thus obtained were
recrystallized from ether/petroleum ether, filtered, and
dried to get desired conjugates (I)–(IV).
EXPERIMENTAL
Bocꢀamino acids, EDCI, HOBt, and TFA were
purchased from Advanced Chem. Tech. (Louisville,
Kentucky, USA). NMM and substituted phenyl isocyꢀ
anates/isothiocyanates were purchased from Sigmaꢀ
Aldrich Co., USA. All solvents and reagents used for
the synthesis were of analytical grade. Silica gel for
TLC was purchased from Sisco Research Laboratories
Pvt. Ltd. (Mumbai, India). Progress of the reaction
was monitored by TLC using silica gelꢀcoated glass
plates with the solvent system comprising chloroꢀ
General Procedure for the Synthesis of Ureido and
Thioureido Derivatives (1)–(40)
BocꢀXaaꢀHet (0.3 g) conjugates (I)–(IV) were
stirred with 3.0 mL of TFA for 45 min at room temperꢀ
ature. After completion of the reaction monitored by
TLC, the reaction mixture was concentrated at high
vacuum to get TFA · HꢀXaaꢀHet which was then tritꢀ
urated with dry ether, filtered, washed with ether, and
dried under vacuum (yield: 100%).
form/methanol/acetic acid in the ratio of 98 : 2 :
3
Raf and 95 : 5 : 3 (Rbf
)
)
; the compounds on TLC plates
A solution of TFA · HꢀXaaꢀHet (0.5 mmol) in
DMF (10 mL/g of compound) was cooled to 0°C and
0.5 mmol NMM was added. Phenyl isocyanates/
isothiocyanates (0.5 mmol) were added dropꢀwise to
the mixture while maintaining the temperature at 0°C.
(
were detected by iodine vapors. Melting point was
determined on Superfit (India) melting point apparaꢀ
tus and are uncorrected. FTꢀIR was performed with a
1
Jasco spectrometer. H NMR spectra were obtained
The reaction mixture was stirred for 8 h, slowly warmꢀ
ing to room temperature. DMF was evaporated under
high vacuum and the residue was poured into about 20 mL
iceꢀcold 90% saturated KHCO3 solution and stirred
for 15 min. The precipitated compound was extracted
with ethyl acetate and washed with 5% NaHCO3 soluꢀ
on a VARIAN 400 MHz instrument using DMSOꢀd6
and the chemical shifts are reported as parts per milꢀ
lion ( ppm) using TMS as an internal standard. Mass
δ
spectrometry analysis was performed on an Agilent
LCꢀMS equipment. Elemental analysis was perꢀ
formed by using VARIO EL III CHNS Elementar.
Bovine serum albumin (BSA) was purchased from
Research Organics, Cleveland, while other chemicals
used for inhibition studies were purchased from Sigma
Aldrich.
tion (
2
×
10 mL), water (
2
×
10 ml), and 0.1 N HCl
(
2
×
10 mL) followed by brine. The organic layer was
dried over anhydrous Na2SO4 and the residue was tritꢀ
urated with hexane, filtered, and dried.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
No. 4
2014

454
SHANTHARAM et al.
6. Sensi, M., De Rossi, M.G., Celi, F.S., Cristina, A.,
Antiglycation Assay
Rosati, C., Prrett, D., Anreani, D., and Di Mario, U.,
Sodium phosphate buffer (pH 7.4) was prepared by
Diabetologia, 1993, vol. 36, pp. 797–801.
mixing Na2HPO4 and NaH2PO4 (67 mM) containing
sodium azide (3 mM); phosphate buffer saline (PBS)
was prepared by mixing NaCl (137 mM) + Na2HPO4
(8.1 mM) + KCl (2.68 mM) + KH2PO4 (1.47 mM) and
pH 10 was adjusted with NaOH (0.25 mM), while BSA
(10 mg/mL) and anhydrous glucose (50 mg/mL) solutions
were prepared in sodium phosphate buffer. Eppendorf
tubes (Tarsons, India) were used for incubation.
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,
,
Bovine serum albumin (10 mg/mL) and anhydrous
glucose (50 mg/mL) were prepared in sodium phosꢀ
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compounds was found to have no effect on the reacꢀ
11. Suhas, R., Chandrashekar, S., and Gowda, D.C., Eur.
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12. Suresha, G.P., Suhas, R., Kapfo, W., and Gowda, D.C.,
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13. Adams, A.D., Yuen, W., Hu, Z., Santini, C.,
tion at <2% (v/v). Glycated control contained 20
BSA + 20 glucose + 20 sodium phosphate
buffer, blank control contained 20 BSA and 40
sodium phosphate buffer, while the test contained 20
BSA+ 20 glucose + 20 compound ranging
μL
Jones, A.B.,
MacNaul,
K.L.,
Berger,
J.P.,
μL
μL
Doebber, T.W., and Moller, D.E., Bioorg. Med. Chem.
μL
μL
Lett., 2003, vol. 13, pp. 931–935.
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μL
μL
μL
from 0.5–500 μg/mL concentration. All the incubaꢀ
tion tubes containing the mixtures were sealed and
incubated at 37
(100%) of TCA was added into each tube and centriꢀ
fuged (15000 rpm) for 4 min at . After centrifugaꢀ
tion, the pellets were rewashed with 60 10% TCA.
°C
for 7 days. After incubation,
6
μL
4°C
μL
16. Suhas, R., Chandrashekar, S., and Gowda, D.C., Eur.
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The supernatant containing glucose, inhibitor, and an
interfering substance was removed and the pellet conꢀ
taining advanced glycated BSA end product were disꢀ
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solved in 60 L phosphate buffer solution (PBS) and
μ
transferred into 96ꢀwell ELISA plates (Tarsons,
India). Evaluation of fluorescence spectrum (excitaꢀ
tion 370 nm) and change in fluorescence intensity 19. Mendez, J.D., Xie, J., and GarciaꢀPerez, E., WASJ
,
2007, vol. 2, pp. 090–098.
(excitation 370 nm to emission 440 nm), based on
AGEs were monitored using the RFꢀ1500 (Shimadzu,
Japan) spectrofluorimeter. Percent inhibition was calꢀ
culated using the formula given below and eventually
IC50 of the compounds was found out by plotting conꢀ
centration vs percent inhibition.
20. Venkatachalam, T.K., Maob, C., and Uckun, F.M.,
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% Inhibition = [1 – (Fluorescence of sample/
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,
Fluorescence of glycated sample)] × 100.
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RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
No. 4
2014