NSAID conjugates with carnosine and amino acids

Article abstract of DOI:10.1055/s-0033-1339920

Benzotriazole-mediated syntheses of novel bioconjugates of nonsteroidal anti-inflammatory drugs with carnosine and with amino acids were prepared in yields of 50-97% as potential drug candidates. Georg Thieme Verlag Stuttgart . New York.

Full text of DOI:10.1055/s-0033-1339920

PAPER
▌3369
paper
NSAID Conjugates with Carnosine and Amino Acids
Benzotriazole-Mediated Syntheses of Novel Bioconjugates
Sandhyamayee Sahu,a Siva S. Panda,a Abdullah M. Asiri,b,c Alan R. Katritzky*a,b
a
Center for Heterocyclic Compounds, Department of Chemistry, University of Florida, Gainesville, FL 32611-7200, USA
Fax +1(352)3929199; E-mail: katritzky@chem.ufl.edu
b
Chemistry Department, King Abdulaziz University, Jeddah 21589, Saudi Arabia
c
Center of Excellence for Advanced Materials Research (CEAMR), King Abdulaziz University, Jeddah 21589, Saudi Arabia
Received: 06.09.2013; Accepted: 22.09.2013
COOH
Abstract: Benzotriazole-mediated syntheses of novel bioconju-
gates of nonsteroidal anti-inflammatory drugs with carnosine and
with amino acids were prepared in yields of 50–97% as potential
drug candidates.
H
N
COOH
ibuprofen (1 + 1')
COOH
mefenamic acid (2)
Key words: NSAIDs, carnosine, amino acid, prodrug, conjugates,
coupling, benzotriazole methodology
Cl
H
N
COOH
Nonsteroidal anti-inflammatory drugs (NSAIDs) includ-
ing ibuprofen (1 + 1′), mefenamic acid (2), diclofenac (3),
naproxen (4), indomethacin (5) (Figure 1), and many oth-
ers are highly effective analgesic, antipyretic, and anti-in-
flammatory agents. It is generally believed that they act by
inhibiting prostaglandin synthesis, particularly in in-
flamed tissue.1,2 They do not interfere with the actual dis-
ease process, but rather offer symptom relief, even when
administered chronically. However, drugs of this type are
responsible for substantial morbidity and mortality as a re-
sult of complications associated with gastroduodenal dis-
orders, such as perforation and bleeding,3–5 The NSAIDs
often cause dyspepsia and adverse upper gastrointestinal
(GI) effects, and frequently require GI co-medications and
diagnostic procedures.
Cl
MeO
naproxen (4)
diclofenac (3)
COOH
MeO
N
O
Cl
indomethacin (5)
Figure 1 Nonsteroidal anti-inflammatory drugs 1–5
lori associated gastritis,16 duodenal ulcers induced by
mepirizole,13 and colitis induced by 2,4,6-trinitrobenzene-
sulfonic acid.17
NSAIDs affect several biological processes such as in-
flammation (COX inhibition), angiogenesis, cell differen-
tiation, and apoptosis (induction independent of COX
inhibition), which in turn potentially influence carcino-
genesis.6 However, NSAIDs are not currently recom-
mended for routine use as cancer preventive agents due to
their chronic safety issues.
–
HN
N
N
N
O
O
Zn2+
O–
OH
H2N
N
H2N
N
H
H
O
O
To improve the GI tolerance of NSAIDs by temporarily
masking the free carboxylic group, numerous ester and
amide prodrugs of NSAIDs have been designed and eval-
uated.7–9 but it has not yet been possible to avoid the sys-
temic side effects of the parent drugs by this approach.
carnosine (6)
polaprezinc (7)
Figure 2 Carnosine (6) and polaprezinc (7)
Facino et al. reported recently that L-carnosine, an en-
dogenous dipeptide present at millimolar concentrations
in tissues such as skeletal muscle, selectively quenches
unsaturated aldehydes both in vitro and in vivo.18,19 De-
spite this promising finding, the therapeutic use of L-car-
nosine is limited because it is rapidly hydrolyzed in
human plasma by carnosinase.20
Carnosine (6) and its derivatives such as polaprezinc (PZ,
7) (Figure 2), a chelate of zinc and L-carnosine, are widely
used as an anti-ulcer drugs.10 Depending on dose, PZ can
prevent gastric mucosal lesions and mucosal cell damages
induced by ethanol,11 monochloramine,12 histamine,13
HCl-aspirin,13 indomethacin,13 water-immersion stress,13
burn shock,14 ischemia reperfusion,14,15 Helicobactor py-
Use of NSAIDs can cause major gastrointestinal and hep-
atotoxicity side effects whereas carnosine is used for the
treatment of gastrointestinal and hepatotoxicity. Amino
acids have frequently been used as carriers for drugs be-
cause of their ability to transplant into mammalian tis-
SYNTHESIS 2013, 45, 3369–3374
Advanced online publication: 04.11.2013
0
0
3
9
-
7
8
8
1
1
4
3
7
-
2
1
0
X
DOI: 10.1055/s-0033-1339920; Art ID: SS-2013-M0615-OP
© Georg Thieme Verlag Stuttgart · New York

3370
S. Sahu et al.
PAPER
Synthesis of NSAID Benzotriazolides 9a + 9a′, 9b–e
Table 1 Preparation of NSAID Benzotriazolides 9a–e
Using previously reported methods (Scheme 1, Table 1),22
the carboxylic groups of NSAIDs were activated with 1H-
benzotriazole (8) in yields of 64–78%. Compound 9a,b
and 9d,e were prepared by treating NSAIDs 1,2,4, and 5
with 1H-benzotriazole in the presence of SOCl2 in THF at
room temperature, whereas compound 9c was prepared by
treating diclofenac (3) with 1H-benzotriazole in the pres-
ence of DCC as coupling agent in dichloromethane at
room temperature.
Entry Product 9
Yield Mp
(%)
(°C)
75–7723
N
N
N
1
63
O
9a + 9a′
N
N
N
O
SOCl2, THF
O
H
or
O
N
N
DCC, CH2Cl2
2
78
111–112
N
+
R
N
N
R
OH
20 °C, 3 h
N
H
1 + 1', 2–5
N
8
9a + 9a' + 9b–e
9b
O
Scheme 1 Synthesis of NSAID benzotriazolides 9a–e
Cl
N
N
H
N
Synthesis of NSAID Conjugates 10a + 10a′, 10b–e with
Carnosine
N
3
4
66
67
128–130
Cl
NSAID-carnosine conjugates 10a + 10a′, 10b–e were ob-
tained by coupling 9a + 9a′ and 9b–e, respectively, with
carnosine (6) in aqueous acetonitrile in the presence of so-
dium hydroxide at –5 to 20 °C in 55–72% yields (Scheme
2, Table 2).
9c
N
183–18523
N
N
O
MeO
HN
N
9d
N
N
N
O
O
R
+
OH
H2N
N
O
N
MeO
H
9a + 9a' + 9b–e
O
N
6
N
N
5
76
79–81
MeCN–H2O (3:1), NaOH
–5 °C to 20 °C
O
HN
N
Cl
O
O
9e
OH
R
N
N
H
O
H
10a + 10a' + 10b–e
sue.21 In the present work, we have synthesized novel
NSAID conjugates with carnosine as well as with amino Scheme 2 Synthesis of NSAID conjugates 10a–e with carnosine
acids as potential anti-inflammatory agents.
Table 2 Preparation of NSAID Conjugates 10a + 10a′, 10b–e with Carnosine
Entry
Reactant 9
Product 10
Yield (%)
Mp (°C)
1
2
3
4
5
9a + 9a′
9b
DL-ibuprofen-carnosine-OH (10a + 10a′)
mefenamic-carnosine-OH (10b)
diclofenac-carnosine-OH (10c)
naproxen-carnosine-OH (10d)
57
72
55
56
57
175–177
108–110
193–195
230–233
163–165
9c
9d
9e
indomethacin-carnosine-OH (10e)
Synthesis 2013, 45, 3369–3374
© Georg Thieme Verlag Stuttgart · New York

PAPER
Benzotriazole-Mediated Syntheses of Novel Bioconjugates
3371
Table 3 Preparation of NSAID Conjugates 12a–i with Amino Acids
Reactant 9
Product 12
Yield (%)
Mp (°C)
9a + 9a′
9a + 9a′
9b
DL-ibufrofen-L-His-OH (12a + 12a′)
DL-ibufrofen-L-Thr-OH (12b + 12b′)
mefenamic Acid-L-Ala-OH (12c)
mefenamic Acid-DL-Ala-OH (12c + 12c′)
mefenamic Acid-L-Trp-OH (12d)
naproxen-L-His-OH (12e)
57
63
97
55
79
65
67
50
76
54
61
50
76–77
206–207
54–55
9b
55–56
9b
115–116
111–112
145–146
172–173
151–153
125–126
93–94
9d
9d
naproxen-L-Thr-OH (12f)
9d
naproxen-DL-Thr-OH (12f + 12f′)
naproxen-L-Ala-OH (12g)
9d
9e
indomethacin-L-His-OH (12h)
indomethacin-L-Thr-OH (12i)
indomethacin-DL-Thr-OH (12i + 12i′)
9e
9e
92–93
Commercial reagents were purchased from Sigma-Aldrich and
were used without purification. Solvents were purified by distilla-
tion. Melting points were determined on a capillary point apparatus
equipped with a digital thermometer. NMR spectra were recorded
in CDCl3 or CD3OD on Mercury or Gemini NMR spectrometers op-
erating at 300 MHz for 1H (with TMS as an internal standard) and
75 MHz for 13C. Elemental analyses were performed on a Carlo
Erba-EA1108 instrument. Analytical TLC was performed on E.
Merck silica gel 60 F254 plates and visualized by UV and KMnO4
staining. Yields refer to chromatographically and spectroscopically
pure compounds. Mass spectrometry was done with electrospray
ionization (ESI).
Synthesis of NSAID Conjugates 12a–i with Amino
Acids
NSAID amino acid conjugates were obtained by coupling
9a + 9a′ and 9b–e, respectively, with amino acids 11 in
aqueous acetonitrile in the presence of triethylamine at
25 °C in 53–97% yields (Scheme 3, Table 3). Histidine,
threonine, tryptophan, and alanine were used because
these amino acids help directly or indirectly in the treat-
ment of inflammation.23–25 The chirality of starting mate-
rials was preserved in the products, as we can see
duplication of peaks in 13C NMR spectra in the case of ra-
cemic mixtures only.
Benzotriazole Derivatives of NSAIDs 9a + 9a′, 9b–e; General
Procedure
SOCl2 (2.64 mmol) was added to a solution of 1H-benzotriazole (8;
1.03 g, 8.7 mmol) in anhydrous CH2Cl2 (60 mL) at r.t. and the reac-
tion mixture was stirred for 20 min. Ibuprofen (1 + 1′; 453 mg, 2.2
mmol), mefenamic acid (2; 530 mg, 2.2 mmol), naproxen (4; 506
mg, 2.2 mmol), or indomethacin (5; 787 mg, 2.2 mmol) was added
and the mixture was stirred for 3 h at r.t. The white precipitate
formed was filtered off and the filtrate was concentrated under re-
duced pressure. Each residue was diluted with EtOAc (50 mL) and
each solution was washed with aq 4 M HCl (3 × 15 mL) and dried
(MgSO4). Removal of the solvent under reduced pressure gave the
desired products 9a + 9a′, 9b, 9d, and 9e. For the preparation of
compound 9c, diclofenac (3; 651 mg, 2.2 mmol) was treated with
1H-benzotriazole (8; 1.03 g, 8.7 mmol) in the presence of DCC (453
mmol, 2.2 mmol) in anhydrous CH2Cl2 (60 mL) at r.t. and the reac-
tion mixture was stirred for 3 h. The white precipitate formed was
filtered off through Celite and the filtrate was concentrated under
reduced pressure. The residue was diluted with EtOAc (50 mL) and
the solution was washed with aq 10% Na2CO3 (3 × 15 mL) and
dried (MgSO4). Removal of the solvent under reduced pressure
gave the desired product.
N
R2
N
N
R1
OH
H2N
+
O
O
9a + 9a' + 9b–e
11
MeCN–H2O, Et3N
20 °C, 4 h
O
R2
OH
R1
N
H
O
12a+12a', 12b + 12b', 12c + 12c', 12d–e, 12f + 12f', 12g–h, 12i + 12i
Scheme 3 Synthesis of NSAID conjugates 12a–l with amino acids
In summary, benzotriazole-activated nonsteroidal anti-
inflammatory drugs are sufficiently reactive coupling re-
agents to form amides under mild conditions, which offer
an efficient preparation of chirally pure nonsteroidal anti-
inflammatory drug-conjugates with carnosine and amino
acids in good yields. The chirality of starting materials
was preserved in the products, as evidenced by NMR
spectra.
1-(1H-Benzo[d][1,2,3]triazol-1-yl)-2-(4-isobutylphenyl)propan-
1-one (Ibuprofen-Bt, 9a + 9a′)
Yield: 0.47 g (63%); white microcrystals; mp 75–77 °C.
1H NMR (CDCl3): δ = 8.27 (d, J = 8.1 Hz, 1 H), 8.06 (d, J = 8.1 Hz,
1 H), 7.60 (t, J = 7.5 Hz, 1 H), 7.48–7.38 (m, 3 H), 7.08 (d, J = 7.8
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2013, 45, 3369–3374

3372
S. Sahu et al.
PAPER
Hz, 2 H), 5.39–5.37 (m, 1 H), 2.39 (d, J = 7.2 Hz, 2 H), 1.81–1.72
(m, 4 H), 0.84 (d, J = 6.3 Hz, 6 H).
13C NMR (CDCl3): δ = 173.8, 146.4, 141.3, 136.6, 131.6, 130.5,
triazole 9 (1 equiv) in MeCN (3 mL) at –5 °C and the mixture was
stirred for 12 h at r.t. After completion of the reaction, MeCN was
evaporated and the mixture was poured over crushed ice followed
by the addition of citric acid (1 mL) maintaining the pH at 4–5. The
precipitate obtained was then dried and washed with Et2O (5 mL)
and then with EtOAc (5 mL) to obtain the product in pure form.
129.8, 127.8, 126.3, 120.3, 114.8, 45.2, 44.6, 30.3, 22.6, 18.9.
Anal. Calcd for C19H21N3O: C, 74.24; H, 6.89; N, 13.67. Found: C,
73.96; H, 6.97; N, 13.70.
{3-[2-(4-Isobutylphenyl)propanamido]propanoyl}-L-histidine
(Ibuprofen-carnosine, 10a + 10a′)
(1H-Benzo[d][1,2,3]triazol-1-yl){2-[(2,3-dimethylphenyl)ami-
no]phenyl}methanone (Mefenamic acid-Bt, 9b)
Yield: 0.15 g (57%); white microcrystals; mp 175–177 °C.
Yield: 0.55 g (78%); yellow solid; mp 111–112 °C.
1H NMR (DMSO-d6): δ = 8.13 (d, J = 7.8 Hz, 1 H), 7.94 (br s, 1 H),
7.58 (s, 1 H), 7.18 (d, J = 8.1 Hz, 2 H), 7.05 (d, J = 7.8 Hz, 1 H),
6.81 (s, 1 H), 4.40–4.39 (m, 1 H), 3.52–3.50 (m, 2 H), 3.21–3.12 (m,
2 H), 2.89–2.81 (m, 2 H), 2.38 (d, J = 7.2 Hz, 2 H), 2.24–2.20 (m, 2
H), 1.81–1.76 (m, 1 H), 1.27 (d, J = 7.2 Hz, 3 H), 0.84 (d, J = 6.6
Hz, 6 H).
13C NMR (DMSO-d6): δ = 176.2, 172.9, 170.3, 139.5, 139.2, 134.5,
132.6, 128.7, 126.9, 116.8, 71.7, 52.1, 44.3, 35.4, 35.2, 29.7, 28.4,
22.2, 18.6.
1H NMR (CDCl3): δ = 9.06 (s, 1 H), 8.29 (d, J = 9.0 Hz, 1 H), 8.18
(d, J = 9.0 Hz, 1 H), 8.09 (d, J = 8.1 Hz, 1 H), 7.69 (t, J = 8.4 Hz, 1
H), 7.54 (t, J = 7.6 Hz, 1 H), 7.38 (t, J = 7.9 Hz, 1 H), 7.21 (d, J =
8.7 Hz, 1 H), 7.14 (t, J = 7.8 Hz, 1 H), 7.06 (d, J = 6.3 Hz, 1 H), 6.89
(d, J = 8.7 Hz, 1 H), 6.81 (t, J = 7.9 Hz, 1 H), 2.35 (s, 3 H), 2.24 (s,
3 H).
13C NMR (CDCl3): δ = 167.8, 151.0, 146.0, 138.6, 138.3, 135.6,
134.9, 132.9, 132.4, 130.1, 127.4, 126.4, 126.1, 123.1, 120.4, 116.7,
114.8, 114.7, 112.6, 20.9, 14.3.
Anal. Calcd for C22H30N4O4: C, 63.75; H, 7.29; N, 13.52. Found: C,
63.48; H, 7.57; N, 13.24.
Anal. Calcd for C21H18N4O: C, 73.67; H, 5.30; N, 16.36. Found: C,
73.27; H, 5.40; N, 16.36.
(3-{2-[(2,3-Dimethylphenyl)amino]benzamido}propanoyl)-L-
histidine (Mefenamic Acid-carnosine, 10b)
1-(1H-Benzo[d][1,2,3]triazol-1-yl)-2-{2-[(2,6-dichlorophe-
nyl)amino]phenyl}ethan-1-one (Diclofenac-Bt, 9c)
Yield: 0.44 g (66%); white microcrystals; mp 128–130 °C.
1H NMR (CDCl3): δ = 8.23 (d, J = 8.1 Hz, 1 H), 8.06 (d, J = 8.4 Hz,
1 H), 7.58 (t, J = 7.6 Hz, 1 H), 7.46–7.41 (m, 1 H), 7.30–7.26 (m, 2
H), 7.10–7.05 (m, 1 H), 6.97–6.89 (m, 3 H), 6.53 (d, J = 8.1 Hz, 1
H), 4.84 (s, 2 H).
13C NMR (CDCl3): δ = 171.0, 146.5, 143.1, 137.9, 131.5, 131.4,
130.8, 129.8, 129.1, 128.6, 126.6, 124.4, 123.2, 122.6, 120.5, 118.9,
114.8, 38.9.
Yield: 0.19 g (72%); white microcrystals; mp 108–110 °C.
1H NMR (DMSO-d6): δ = 9.58 (s, 1 H), 8.59–8.52 (m, 1 H), 8.26 (d,
J = 7.2 Hz, 1 H), 7.91 (d, J = 7.5 Hz, 1 H), 7.83 (s, 1 H), 7.61 (d, J =
7.5 Hz, 1 H), 7.42 (s, 1 H), 7.23 (t, J = 7.6 Hz, 1 H), 7.11–7.04 (m,
2 H), 6.93–6.90 (m, 2 H), 6.83 (d, J = 8.1 Hz, 1 H), 6.72 (t, J = 7.0
Hz, 1 H), 4.48–4.45 (m, 1 H), 3.50–3.37 (m, 2 H), 3.10–2.86 (m, 2
H), 2.46–2.39 (m, 2 H), 2.26 (s, 3 H), 2.10 (s, 3 H).
13C NMR (DMSO-d6): δ = 176.2, 173.1, 171.4, 170.4, 169.1, 146.1,
139.3, 137.7, 134.5, 132.7, 131.9, 129.3, 128.7, 125.8, 125.4, 125.0,
119.6, 117.0, 116.8, 113.9, 59.8, 44.0, 40.3, 35.1, 28.5, 20.3.
(S)-1-(1H-Benzo[d][1,2,3]triazol-1-yl)-2-(6-methoxynaphtha-
len-2-yl)propan-1-one (Naproxen-Bt, 9d)
Yield: 0.48 g (67%); white microcrystals; mp 183–185 °C.
HRMS: m/z calcd for C24H27N5O4 [M + H]+ 450.2136; found:
450.2150.
1H NMR (CDCl3): δ = 8.25 (d, J = 9 Hz, 1 H), 8.02 (d, J = 9 Hz, 1
H), 7.86 (s, 1 H), 7.67 (d, J = 6.0 Hz, 2 H), 7.61–7.55 (m, 2 H), 7.41
(t, J = 9.0 Hz, 1 H), 7.10–7.03 (m, 2 H), 5.55–5.49 (m, 1 H), 3.85 (s,
3 H), 1.80 (d, J = 9.0 Hz, 3 H).
13C NMR (CDCl3): δ = 173.7, 157.9, 146.4, 134.5, 134.1, 131.5,
130.5, 129.5, 129.1, 127.6, 127.1, 126.6, 126.3, 120.3, 119.3, 114.7,
105.7, 55.5, 44.9, 18.8.
[3-(2-{2-[(2,6-Dichlorophenyl)amino]phenyl}acetamido)pro-
panoyl]-L-histidine (Diclofenac-carnosine, 10c)
Yield: 0.14 g (55%); white microcrystals; mp 193–195 °C.
1H NMR (DMSO-d6): δ = 8.43 (d, J = 9.9 Hz, 2 H), 8.20 (d, J = 5.7
Hz, 1 H), 7.67 (s, 2 H), 7.50 (d, J = 10.5 Hz, 2 H), 7.43 (s, 1 H),
7.20–7.10 (m, 2 H), 7.01 (s, 1 H), 6.85 (s, 2 H), 6.28 (d, J = 10.5 Hz,
1 H), 4.44–4.42 (m, 1 H), 3.56 (s, 2 H), 3.26–3.20 (m, 2 H), 3.04–
2.83 (m, 2 H), 2.30–2.28 (m, 2 H).
13C NMR (DMSO-d6): δ = 173.0, 171.6, 170.1, 142.9, 137.2, 134.6,
133.1, 130.4, 129.3, 129.1, 127.1, 125.5, 125.3, 124.9, 120.6, 116.7,
115.9, 114.8, 52.2, 43.9, 35.4, 35.0, 28.8.
Anal. Calcd for C20H17N3O2: C, 72.49; H, 5.17; N, 12.68. Found: C,
72.05; H, 5.08; N, 12.76.
1-(1H-Benzo[d][1,2,3]triazol-1-yl)-2-[1-(4-chlorobenzoyl)-5-
methoxy-2-methyl-1H-indol-3-yl]ethan-1-one (Indomethacin-
Bt, 9e)
HRMS: m/z calcd for C23H23Cl2N5O4 [M + Na]+: 504.1187; found:
504.1199.
Yield: 0.49 g (76%); yellow microcrystals; mp 79–81 °C.
1H NMR (CDCl3): δ = 8.27 (d, J = 8.1 Hz, 1 H), 8.14 (d, J = 7.8 Hz,
1 H), 7.69–7.61 (m, 3 H), 7.56–7.54 (m, 3 H), 7.14 (d, J = 2.4 Hz, 1
H), 6.87 (d, J = 9.0 Hz, 1 H), 6.67 (d, J = 9.0 Hz, 1 H), 4.79 (s, 2 H),
3.84 (s, 3 H), 2.53 (s, 3 H).
13C NMR (CDCl3): δ = 169.4, 165.4, 156.3, 146.5, 139.6, 137.1,
133.9, 131.4, 130.8, 129.3, 126.6, 120.5, 115.2, 114.7, 112.2, 111.1,
101.5, 55.9, 31.7, 13.9.
{3-[(S)-2-(6-Methoxynaphthalen-2-yl)propanamido]propano-
yl}-L-histidine (Naproxen-carnosine, 10d)
Yield: 0.15 g (56%); white microcrystals; mp 230–233 °C.
1H NMR (DMSO-d6): δ = 8.13 (d, J = 7.5 Hz, 1 H), 8.02 (t, J = 5.7
Hz, 1 H), 7.78–7.68 (m, 2 H), 7.60 (s, 1 H), 7.41 (d, J = 8.6 Hz, 1
H), 7.26–7.23 (m, 1 H), 7.12 (d, J = 8.8 Hz, 1 H), 6.80 (s, 1 H), 4.46–
4.33 (m, 1 H), 3.85 (s, 3 H), 3.70–3.63 (m, 1 H), 3.25–3.13 (m, 2 H),
2.93–2.77 (m, 2 H), 2.30–2.17 (m, 2 H), 1.38 (d, J = 6.9 Hz, 3 H).
HRMS: m/z calcd for C25H19ClN4O3 [M + H]+: 459.1218; found:
13C NMR (TFA-d4): δ = 182.0, 176.8, 174.1, 133.4, 133.0, 129.4,
128.8, 128.1, 126.4, 125.5, 119.9, 118.2, 117.6, 116.1, 112.3, 108.6,
55.2, 52.0, 45.8, 36.9, 34.1, 26.0, 15.7.
459.1201.
NSAID-Carnosine Conjugates 10a + 10a′, 10b–e; General Pro-
cedure
To a solution of carnosine (6; 1 equiv) in H2O (1 mL) and NaOH
(1.5 equiv) was added a solution of the respective NSAID benzo-
Anal. Calcd for C23H26N4O5: C, 63.00; H, 5.98; N, 12.78. Found: C,
62.62; H, 5.98; N, 12.49.
Synthesis 2013, 45, 3369–3374
© Georg Thieme Verlag Stuttgart · New York

PAPER
Benzotriazole-Mediated Syntheses of Novel Bioconjugates
3373
(3-{2-[1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]acetamido}propanoyl)-L-histidine (Indomethacin-carno-
sine, 10e)
1H NMR (DMSO-d6): δ = 9.01 (br s, 1 H), 7.47(d, J = 8.1 Hz, 1 H),
7.21 (t, J = 7.9 Hz, 1 H), 7.14 (d, J = 7.8 Hz, 1 H), 7.06 (t, J = 7.6
Hz, 1 H), 6.95 (d, J = 6.9 Hz, 1 H), 6.87 (d, J = 8.1 Hz, 1 H), 6.75
(d, J = 6.9 Hz, 1 H), 6.69 (t, J = 7.5 Hz, 1 H), 4.82–4.75 (m, 1 H),
2.31 (s, 3 H), 2.17 (s, 3 H), 1.57 (d, J = 7.2 Hz, 3 H).
13C NMR (DMSO-d6): δ = 177.7, 169.7, 147.7, 139.4, 138.3, 133.0,
131.4, 127.9, 126.1, 126.0, 121.6, 117.0, 115.8, 115.1, 48.5, 20.9,
18.4, 14.1.
Yield: 0.14 g (57%); yellow microcrystals; mp 163–165 °C.
1H NMR (DMSO-d6): δ = 8.19 (d, J = 7.8 Hz, 1 H), 8.09 (s, 1 H),
7.82 (s, 1 H), 7.42–7.60 (m, 4 H), 7.10 (s, 1 H), 6.94–6.88 (m, 2 H),
6.69 (d, J = 8.4 Hz, 1 H), 4.45–4.39 (m, 1 H), 3.75 (s, 3 H), 3.48 (s,
2 H), 3.25–3.17 (m, 2 H), 3.00–2.80 (m, 2 H), 2.30–2.24 (m, 2 H),
2.21 (s, 3 H).
13C NMR (DMSO-d6): δ = 172.9, 171.3, 170.3, 169.3, 155.5, 137.5,
135.1, 134.5, 134.2, 132.6, 131.1, 130.2, 129.0, 116.7, 114.5, 114.3,
11.3, 101.8, 55.4, 52.0, 43.6, 35.4, 35.1, 31.1, 28.4, 13.4.
Anal. Calcd for C18H20N2O3: C, 69.21; H, 6.45; N, 8.97. Found: C,
69.57; H, 6.55; N, 8.86.
{2-[(2,3-Dimethylphenyl)amino]benzoyl}-L-tryptophan (Mefe-
namic Acid-L-tryptophan, 12d)
Yield: 0.20 g (79%); orange solid; mp 115–116 °C.
HRMS: m/z calcd for C28H28 ClN5O6 [M + H]+: 566.1728; found:
566.1800.
1H NMR (300 MHz, CDCl3): δ = 8.97 (br s, 1 H), 8.10 (s, 1 H), 7.58
(d, J = 7.5 Hz, 1 H), 7.24 (d, J = 8.4 Hz, 1 H), 7.17–7.02 (m, 6 H),
6.99–6.91 (m, 2 H), 6.82 (d, J = 8.7 Hz, 1 H), 6.71 (d, J = 6.9 Hz, 1
H), 6.51 (t, J = 7.5 Hz, 1 H), 5.09–5.05 (m, 1 H), 3.50–3.30 (m, 2
H), 2.27 (s, 3 H), 2.11 (s, 3 H).
13C NMR (CDCl3): δ = 176.4, 169.9, 147.5,139.5, 138.3, 136.3,
132.9, 131.3, 128.1, 127.8, 126.1, 126.1, 123.4, 122.4, 121.5, 120.0,
118.7, 117.1, 116.0, 115.1, 111.6, 109.7, 53.7, 27.4, 20.8, 14.1.
NSAID-Amino Acid Conjugates 12a–i; General Procedure
To a solution of the amino acid 11 (1 equiv) in H2O (1 mL) and Et3N
(1.5 equiv) was added a solution of the respective NSAID benzotri-
azole 9 (1 equiv) in MeCN (3 mL) at 20 °C and the mixture was
stirred for 4 h at r.t. After completion of the reaction, MeCN was
evaporated and the mixture was poured over crushed ice followed
by the addition of citric acid (1 mL) maintaining the pH at 4–5. The
precipitate obtained was then dried and washed with Et2O (5 mL)
and then with EtOAc (5 mL) to afford the product in pure form.
HRMS: m/z calcd for C26H25 N3O3 [M + H]+: 428.1969; found:
428.1963.
[2-(4-Isobutylphenyl)propanoyl]-L-histidine (Ibuprofen-L-his-
tidine, 12a + 12a′)
[(S)-2-(6-Methoxynaphthalen-2-yl)propanoyl]-L-histidine
(Naproxen-L-histidine, 12e)
Yield: 0.14 g (65%); white solid; mp 111–112 °C.
Yield: 0.13 g (57%); white microcrystals; mp 76–77 °C.
1H NMR (DMSO-d6): δ = 8.21 (s, 1 H), 7.69 (d, J = 15 Hz, 2 H),
7.18–7.04 (m, 5 H), 6.84 (s, 1 H), 7.75 (s, 1 H), 4.41 (m, 1 H), 3.61
(s, 1 H), 2.94–2.84 (m, 1 H), 2.58–2.55 (m, 1 H), 2.39 (s, 2 H), 1.79
(br s, 1 H), 1.26 (d, J = 15.3 Hz, 3 H), 0.84 (br s, 6 H).
13C NMR (DMSO-d6): δ = 173.2, 139.0, 134.4, 132.8, 128.6, 127.0,
126.9, 116.5, 52.0, 44.25, 29.6, 29.3, 22.2, 18.8, 18.2.
1H NMR (DMSO-d6): δ = 8.28 (d, J = 7.2 Hz, 1 H), 7.83–7.63 (m,
3 H),7.63 (s, 1 H), 7.43 (d, J = 8.7 Hz, 1 H), 7.27 (s, 1 H), 7.14 (d,
J = 9.3 Hz, 1 H), 6.83 (s, 1 H), 4.50–4.37 (m, 1 H), 3.87–3.72 (m, 4
H), 3.02–2.78 (m, 2 H), 1.35 (d, J = 6.9 Hz, 3 H).
13C NMR (DMSO-d6): δ = 173.2, 173.0, 156.9, 137.1, 134.6, 133.1,
129.1, 128.3, 126.7, 126.5, 125.4, 118.5, 116.9, 105.7, 55.2, 52.2,
44.7, 28.9, 18.7.
HRMS: m/z calcd for C19H25N3O3 [M – H]–: 342.1823; found:
342.1839.
HRMS: m/z calcd for C20H21N3O4 [M + H]+: 368.1605; found:
368.1602.
[2-(4-Isobutylphenyl)propanoyl]-L-threonine (DL-Ibuprofen-L-
threonine, 12b + 12b′)
Yield: 0.13 g (63%); white microcrystals; mp 206–207 °C.
1H NMR (DMSO-d6): δ = 7.20 (d, J = 7.2 Hz, 3 H), 7.06 (d, J = 7.5
Hz, 2 H), 3.85 (br s, 1 H), 3.71 (t, J = 7.3 Hz, 2 H), 2.39 (d, J = 6.9
Hz, 2 H), 1.81–1.77 (m, 2 H), 1.28 (d, J = 6.9 Hz, 3 H), 0.83 (d, J =
6.9 Hz, 6 H), 0.66 (d, J = 6.9 Hz, 2 H).
[(S)-2-(6-Methoxynaphthalen-2-yl)propanoyl]-L-threonine
(Naproxen-L-threonine, 12f)
Yield: 0.13 g (67%); white solid; mp 145–146 °C.
1H NMR (DMSO-d6): δ = 7.87 (d, J = 8.7 Hz, 1 H), 7.79–7.72 (m,
3 H), 7.48 (d, J = 8.7 Hz, 1 H), 7.27 (s, 1 H), 7.13 (d, J = 8.7 Hz, 1
H), 4.26–4.22 (m, 1 H), 4.16–4.11 (m, 1 H), 4.04–3.97 (m, 1 H),
3.86 (s, 3 H), 1.43 (d, J = 7.2 Hz, 3 H), 1.08 (d, J = 6.3 Hz, 2 H).
13C NMR (DMSO-d6): δ = 174.5, 172.6, 157.5, 137.7, 133.6, 129.6,
128.9, 127.3, 127.0, 126.0, 119.0, 106.2, 67.0, 58.1, 55.7, 44.9,
20.9, 19.4.
13C NMR (DMSO-d6): δ = 172.8, 172.2, 139.9, 139.0, 128.6, 127.2,
66.4, 57.4, 44.7, 44.3, 22.2, 20.4, 20.1, 19.0, 18.3.
Anal. Calcd for C17H25NO4: C, 66.43; H, 8.20; N, 4.56. Found: C,
66.79; H, 8.57; N, 4.29.
HRMS: m/z calcd for C18H21NO5 [M + Na]+: 354.1312; found:
354.1218.
{2-[(2,3-Dimethylphenyl)amino]benzoyl}-L-alanine (Mefenam-
ic Acid-L-alanine, 12c)
Yield: 0.18 g (97%); yellow solid; mp 54–55 °C.
1H NMR (CD3OD): δ = 7.51 (d, J = 7.5 Hz, 1 H), 7.07 (t, J = 7.5 Hz,
1 H), 6.95–6.87 (m, 2 H), 6.79 (d, J = 6.0 Hz, 1 H), 6.67–6.57 (m, 2
H), 4.48–4.44 (m, 1 H), 2.17 (s, 3 H), 2.02 (s, 3 H), 1.37 (d, J = 7.2
Hz, 3 H).
13C NMR (CD3OD): δ = 176.5, 171.9, 148.2, 141.0, 139.2, 133.4,
132.0, 129.9, 127.0, 126.9, 122.3, 118.7, 118.2, 115.6, 20.8, 17.7,
14.1.
[(S)-2-(6-Methoxynaphthalen-2-yl)propanoyl)-DL-threonine
(Naproxen-DL-threonine, 12f + 12f′)
Yield: 0.10 g (50%); white solid; mp 172–173 °C.
1H NMR (DMSO-d6): δ = 7.89 (d, J = 8.6 Hz, 1 H), 7.79–7.73 (m,
3 H), 7.49 (d, J = 8.7 Hz, 1 H), 7.20 (s, 1 H), 7.14 (d, J = 8.7 Hz, 1
H), 4.27–4.19 (m, 1 H), 4.16–4.08 (m, 1 H), 4.05–3.98 (m, 1 H),
3.86 (s, 3 H), 1.45–1.38 (m, 3 H), 1.08 (d, J = 6.0 Hz, 2 H), 0.84 (d,
J = 6.3 Hz, 1 H).
13C NMR (DMSO-d6): δ = 174.6, 172.7, 157.6, 137.8, 133.7, 129.7,
129.0, 127.4, 127.1, 126.1, 119.1, 106.3, 67.1, 58.2, 55.8, 45.1,
21.1, 20.8, 19.5, 19.0.
Anal. Calcd for C18H20N2O3: C, 69.21; H, 6.45; N, 8.97. Found: C,
68.99; H, 6.64; N, 8.84.
{2-[(2,3-Dimethylphenyl)amino]benzoyl}-DL-alanine (Mefe-
namic Acid-DL-alanine, 12c + 12c′)
Yield: 0.10 g (55%); yellow solid; mp 55–56 °C.
Anal. Calcd for C18H21NO5: C, 65.24; H, 6.39; N, 4.23. Found: C,
65.36; H, 6.64; N, 3.99.
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2013, 45, 3369–3374

3374
S. Sahu et al.
PAPER
[(S)-2-(6-Methoxynaphthalen-2-yl)propanoyl)-L-alanine
(Naproxen-L-alanine, 12g)
Yield: 0.14 g (76%); white solid; mp 151–153 °C.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synthesis.SnoIufproi
m
tgioSrantnugIifoop
r
itmnatr
1H NMR (DMSO-d6): δ = 8.34 (d, J = 7.2 Hz, 1 H), 7.78–7.72 (m,
3 H), 7.46 (d, J = 7.8 Hz, 1 H), 7.26 (s, 1 H), 7.13 (d, J = 7.2 Hz, 1
H), 4.25–4.20 (m, 1 H), 3.86–3.60 (m, 4 H), 1.39 (d, J = 7.2 Hz, 3
H), 1.28 (d, J = 6.9 Hz, 3 H).
13C NMR (DMSO-d6): δ = 174.1, 173.1, 156.9, 137.2, 133.1, 129.1,
128.3, 126.0, 126.4, 125.3, 118.5, 105.6, 55.1, 47.5, 44.4, 18.7,
17.3.
References
(1) Ferreira, S. H.; Vane, J. R. Annu. Rev. Pharmacol. 1974, 14,
57.
(2) Vane, J. R. Nat. New Biol. 1971, 231, 232.
(3) Ng, S. C.; Chan, F. K. L. Curr. Opin. Gastroenterol. 2010,
26, 611.
(4) Weber, J. C. P. Advances in Inflammation Research; Vol. 6;
Chap. 1; Raven Press: New York, 1984.
Anal. Calcd for C17H19NO4: C, 67.76; H, 6.36; N, 4.65. Found: C,
68.10; H, 6.48; N, 4.37.
(5) Straube, S.; Tramer, M. R.; Moore, R. A.; Derry, S.;
McQuay, H. J. BMC Gastroenterol. 2009, 9, 41.
(6) Shiff, S. J.; Shivaprasad, P.; Santini, D. L. Curr. Opin.
Pharmacol. 2003, 3, 352.
(7) Wallace, J. L.; Reuter, B.; Cicala, C.; McKnigh, W.;
Grisham, M.; Cirino, G. Eur. J. Pharmacol. 1994, 257, 249.
(8) Tammara, V. K.; Narurkar, M. M.; Crider, A. M.; Khan, M.
A. Pharm. Sci. 1994, 83, 644.
(9) Mahfouz, N. M.; Omar, F. A.; Aboul-Fadl, T. Eur. J. Med.
Chem. 1999, 34, 551.
(10) Matsukura, T.; Tanaka, H. Biochemistry 2000, 65, 817.
(11) Arakawa, T.; Satoh, H.; Nakamura, A.; Nebiki, H.; Fukuda,
T.; Sakuma, H.; Nakamura, H.; Ishikawa, M.; Seiki, M.;
Kobayashi, K. Dig. Dis. Sci. 1990, 35, 559.
{2-[1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]acetyl}-L-histidine (Indomethacin-L-histidine, 12h)
Yield: 0.12 g (54%); yellow solid; mp 125–126 °C.
1H NMR (DMSO-d6): δ = 8.44 (s, 1 H), 8.04 (s, 1 H), 7.71–7.65 (br
s, 4 H), 7.07 (s, 1 H), 6.94 (s, 2 H), 7.68 (s, 1 H), 3.74 (s, 3 H), 3.54
(s, 2 H), 3.00–2.95 (m, 2 H), 2.16 (s, 3 H).
13C NMR (DMSO-d6): δ = 175.8, 172.9, 171.5, 169.6, 167.9, 155.6,
137.7, 135.2,134.5,134.3, 132.4, 131.2, 130.9, 129.1, 116.7, 114.5,
114.2, 111.5, 101.8, 55.5, 52.2, 43.6, 30.8, 13.4.
HRMS: m/z calcd for C25H23ClN4O5 [M + Na]+: 517.1249; found:
517.1263.
[1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3-car-
bonyl]-L-threonine (Indomethacin-L-threonine, 12i)
Yield: 0.12 g (61%); white solid; mp 93–94 °C.
(12) Kato, S.; Nishiwaki, H.; Konaka, A.; Takeuchi, K. Dig. Dis.
Sci. 1997, 42, 2156.
(13) Seiki, M.; Ueki, S.; Tanaka, Y.; Soeda, M.; Hori, Y.; Aita,
H.; Yoneta, T.; Morita, H.; Tagashira, E.; Okabe, S. Folia
Pharmacol. Jpn. 1990, 95, 257.
(14) Yoshikawa, T.; Naito, Y.; Tanigawa, T.; Yoneta, T.; Kondo,
M. Biochim. Biophys. Acta 1991, 1115, 15.
(15) Yoshikawa, T.; Naito, Y.; Tanigawa, T.; Yoneta, T.; Yasuda,
M.; Ueda, S.; Oyamada, H.; Kondo, M. Free Radical Res.
Commun. 1991, 14, 289.
(16) Ishihara, R.; Iishi, H.; Sakai, N.; Yano, H.; Uedo, N.;
Narahara, H.; Iseki, K.; Mikuni, T.; Ishiguro, S.; Tatsuta, M.
Helicobacter 2002, 7, 384.
1H NMR (DMSO-d6): δ = 7.87 (s, 2 H), 7.66–7.57 (br s, 2 H), 7.49–
7.42 (m, 3 H), 7.03–6.89 (m, 2 H), 6.68–6.57 (m, 1 H), 4.41 (s, 1 H),
4.30 (s, 1 H), 3.69 (s, 1 H), 3.62 (s, 1 H), 3.26 (s, 1 H), 2.89–2.73
(m, 2 H), 1.24 (s, 1 H), 1.10 (s, 2 H).
13C NMR (DMSO-d6): δ = 167.4, 165.4, 164.1, 160.3, 148.1, 130.5,
127.5, 127.2, 126.1, 122.8, 122.5, 120.6, 117.5, 106.4, 105.2, 103.6,
103.0, 92.8, 92.6, 64.6, 58.7, 49.7, 46.6, 34.4, 22.6, 11.12.
HRMS: m/z calcd for C23H23ClN2O6 [M – H]–: 457.1245; found:
457.1169.
[1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3-car-
bonyl]-DL-threonine (Indomethacin-DL-threonine, 12i + 12i′)
Yield: 0.10 g (50%); white solid; mp 92–93 °C.
1H NMR (DMSO-d6): δ = 12.52 (br s, 1 H), 8.09 (d, J = 8.7 Hz, 1
H), 7.69–7.60 (m, 4 H), 7.21 (s, 1 H), 6.95 (d, J = 9.3 Hz, 1 H), 6.69
(d, J = 8.4 Hz, 1 H), 5.01 (s, 1 H), 4.25–4.14 (m, 2 H), 3.78 (s, 3 H),
3.70–3.60 (m, 2 H), 2.24 (s, 3 H), 1.04 (d, J = 6.0 Hz, 3 H).
(17) Yoshikawa, T.; Yamaguchi, T.; Yoshida, N.; Yamamoto,
H.; Kitazumi, S.; Takahashi, S.; Naito, Y.; Kondo, M.
Digestion 1997, 58, 464.
(18) Aldini, G.; Carini, M.; Beretta, G.; Bradamante, S.; Facino,
R. M. Biochem. Biophys. Res. Commun. 2002, 298, 699.
(19) Orioli, M.; Aldini, G.; Benfatto, M. C.; Facino, R. M.;
Carini, M. Anal. Chem. 2007, 79, 9174.
13C NMR (DMSO-d6): δ = 172.1, 171.2, 169.9, 167.8, 155.5, 137.5,
135.0, 134.2, 131.1, 130.8, 130.1, 129.0, 114.6, 114.4, 111.5, 101.8,
66.2, 57.6, 55.4, 30.7, 20.5, 13.4.
(20) Vistoli, G.; Pedretti, A.; Cattaneo, M.; Aldini, G.; Testa, B.
J. Med. Chem. 2006, 49, 3269.
(21) Rennert, R.; Neundorf, I.; Beck-Sickinger, A. G. In Nucleic
Acid and Peptide Aptamers. Methods in Molecular Biology;
Vol. 535; Humana: New York, 2009, Chap. 22, 389.
(22) Katritzky, A. R.; Jishkariani, D.; Narindoshvili, T. Chem.
Biol. Drug Des. 2009, 73, 618.
Anal. Calcd for C23H23ClN2O6: C, 60.20; H, 5.05; N, 6.10. Found:
C, 60.06; H, 5.01; N, 5.70.
(23) Hasegawa, S.; Ichiyama, T.; Sonaka, I.; Ohsaki, A.; Okada,
S.; Wakiguchi, H.; Kudo, K.; Kittaka, S.; Hara, M.;
Furukawa, S. Clin. Exp. Immunol. 2012, 167, 269.
(24) Krause, D.; Suh, H.-S.; Tarassishin, L.; Cui, Q. L.;
Durafourt, B. A.; Choi, N.; Bauman, A.; Cosenza-Nashat,
M.; Antel, J. P.; Zhao, M.-L.; Lee, S. C. Am. J. Pathol. 2011,
179, 1360 .
Acknowledgment
We thank the University of Florida and the Kenan Foundation for
financial support. This paper was also funded in part by generous
support from King Abdulaziz University, under grant No. (D-
006/431). The authors, therefore, acknowledge the technical and fi-
nancial support of KAU. We also thank Dr. C. D. Hall for useful
suggestions and English checking.
(25) Davis, R. H.; Fisher, J. S.; McGowan, L. J. Endocrinol.
1968, 41, 603.
Synthesis 2013, 45, 3369–3374
© Georg Thieme Verlag Stuttgart · New York