Synthesis and physiochemical property evaluation of carbamate derivatives of scutellarin methyl ester

Article abstract of DOI:10.1016/j.cclet.2013.02.003

A series of carbamate prodrugs of scutellarin methyl ester (4a-4e) were prepared in the presence of bis(trichloromethyl) carbonate (BTC) with scutellarin methyl esters and l-amino acid tert-butyl ester hydrochloride as starting materials. In vitro stability and aqueous solubility of target compounds were evaluated. The results indicated that compounds 4d and 4e have higher solubility and in vitro stability than scutellarin respectively.

Full text of DOI:10.1016/j.cclet.2013.02.003

Chinese Chemical Letters 24 (2013) 338–340
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Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Synthesis and physiochemical property evaluation of carbamate derivatives of
scutellarin methyl ester
*
*
Feng-Jie Jiang, Xiao-Zhong Fu , Shan-Wu Wang, Yong Huang, Wen Zhou, Ai-Min Wang, Yong-Lin Wang
School of Pharmacy, Guiyang Medical College, Guiyang 550004, China
A R T I C L E I N F O
A B S T R A C T
Article history:
A series of carbamate prodrugs of scutellarin methyl ester (4a–4e) were prepared in the presence of
bis(trichloromethyl) carbonate (BTC) with scutellarin methyl esters and -amino acid tert-butyl ester
Received 20 November 2012
Received in revised form 14 January 2013
Accepted 23 January 2013
Available online 11 March 2013
L
hydrochloride as starting materials. In vitro stability and aqueous solubility of target compounds were
evaluated. The results indicated that compounds 4d and 4e have higher solubility and in vitro stability
than scutellarin respectively.
ß 2013 Xiao-Zhong Fu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights
reserved.
Keywords:
Scutellarin
Bis(trichloromethyl) carbonate
Physiochemical property
1. Introduction
prodrugs of scutellarin methyl ester via sub-structure combination
methods, and to preliminarily evaluate their physiochemical
properties.
In this letter, five novel carbamate prodrugs of scutellarin
methyl ester (4a–4e) were obtained, and their physiochemical
properties were evaluated. It is expected that the results of the
reported study might be able to afford some valuable information
on new scutellarin prodrug design.
Scutellarin (40,5,6-trihydroxyflavone-7-glucuronide) is a pri-
mary active ingredient in breviscapine, which is extracted from the
Chinese herb, Erigeron breviscapus [1]. It has been confirmed that
scutellarin can protect against cerebral ischemia-reperfusion
injury by cellular mechanisms of attenuating H2O2-induced
cytotoxicity, reducing intracellular accumulation of reactive
oxygen species (ROS), Ca2+ and lipid peroxidation [2]. Despite its
in vitro and in vivo bioactivity, scutellarin has little therapeutic use
given its poor absorption in the gut [3]. In the structural
modification of scutellarin, glucuronic acid carboxyl and 40-
hydroxyl moieties play important roles, because the above two
moieties can be easily modified, that makes it possible to design
and synthesize novel scutellarin prodrugs with improved bioac-
tivity and physiochemical properties by using scutellarin as the
lead compound [4–6].
2. Experimental
As shown in Scheme 1, scutellarin methyl ester was prepared
according to literature procedures [4]. L-Amino acid tert-butyl ester
hydrochlorides (1a–e) were treated with triphosgene [bis (tri-
chloromethyl) carbonate (BTC) in anhydrous CH2Cl2 at low
temperature to afford amino acid ester isocyanate (2a–e),
quantitatively. Subsequently, 2a–e were treated with scutellarin
methyl ester in anhydrous THF/DMF in the presence of Et3N to
afford tert-butyl ester carbamate of scutellarin methyl ester (3a–e).
Removal of tert-butyl protecting group in trifluoroacetic acid
solution at low temperature gave the target compounds 4a–4e as
yellow solids. Yields were in the range of 71–90%.
An active transporting mechanism has been applied in
designing flavonoids L-amino acid prodrugs such as quercetin–
carbamate conjugates [7–9]. In these compounds, carbamate
structural fragment are crucial for enhanced water solubility and
cell permeability. In addition, studies indicated that esterification
of glucuronic acid carboxyl of scutellarin is a critical factor for
improving its pharmacokinetic properties [5]. Based upon above
results, we designed and synthesized a series of novel carbamate
In the process of synthesizing target compounds we initially
focused our attention on ‘one-pot two-step’ strategy, in which L-
amino acid tert-butyl ester can be firstly converted to correspond-
ing activated urethanes by using bis(4-nitrophenyl) carbonate
(NPC), followed by alcoholysis with scutellarin methyl ester to
obtain carbamates derivatives [8,10]. However the results were
disappointing, as the reaction mixture was very complicated and
very little tert-butyl ester carbamates of scutellarin methyl ester
* Corresponding authors.
E-mail addresses: xiaozhong_fu@sohu.com (X.-Z. Fu), ylwang_gmc@163.com
(Y.-L. Wang).
1001-8417/$ – see front matter ß 2013 Xiao-Zhong Fu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2013.02.003

[
F.-J. Jiang et al. / Chinese Chemical Letters 24 (2013) 338–340
339
OH
O
O
HO
OH
O
OH
O
OHO
b
O
O
OH
O
O
HO
O
HO
OH
H N
N
2
O
O
NH
O
O
O
O
O
C
O
a
R
O
R
R
O
HO
OHO
2a-2e
3a-3e
1a-1e
R
-CH
-CH(CH )
-CH Ph
-CH CH COOBu -CH CH COOH
-CH COOBu
R
1
-CH
-CH(CH )
3 2
-CH Ph
2
OH
O
O
HO
O
a
b
c
d
e
OH
3
3
O
NH
3 2
O
O
OH
c
O
2
O
R
1
HO
t
2 2 2 2
OH O
4a-4e
t
-CH COOH
2
2
Scheme 1. The synthesis of compounds 4a–4e. Reagents and conditions: (a) bis(trichloromethyl) carbonate (BTC), pyr, DCM, ꢀ10 8C, 2 h, 1 mol/L HCl, yield 90–95%; (b)
anhydrous THF/DMF, Et3N, 50 8C, yield 40–55%; and (c) TFA, 0 8C, 5–6 h, yield 71–90%.
were obtained. Moreover, auxiliary reagent NPC and its degrada-
tion products were very difficult to remove from the reaction
mixture.
666.5. HRMS: calcd. for C32H30NO15 [M+H]+ 668.1615, found
668.1596. 4d: 1H NMR (400 MHz, DMSO-d6):
13.06 (brs, 1H),
d
10.46 (brs, 1H), 8.22 (d, 1H, J = 8.0 Hz), 7.99 (d, 2H, J = 8.4 Hz), 7.13
(s, 1H), 6.97 (d, 2H, J = 8.8 Hz), 6.90 (s, 1H), 5.53 (d, 1H, J = 5.6 Hz),
5.37–5.36 (m, 2H), 4.23 (d, 1H, J = 9.6 Hz), 4.08–4.02 (m, 1H), 3.67
(s, 3H), 2.42–2.36 (m, 2H), 2.08–2.02 (m, 1H), 1.89–1.82 (m, 1H);
ESI-MS (m/z) [M+H]+:650.3, [MꢀH]ꢀ: 648.3. HRMS calcd. for
2.1. The synthetic method and spectra data of the target compounds
The synthetic method: Under N2 atmosphere L-amino acid tert-
butyl ester hydrochloride (2.5 mmol) was treated with pyridine
(0.8 mL) in anhydrous CH2Cl2 (50 mL) at ꢀ10 8C for 15 min, then
3 mL CH2Cl2 solution which contained bis(trichloromethyl)
carbonate (BTC) (3.8 mmol) was added to the mixture. The
reaction mixture was stirred at that temperature for 2 h. Then
the mixture was washed twice with icecold 0.1 mol/L HCl and
saturated NaCl aqueous solution respectively, then dried over
Mg2SO4, filtered, and concentrated in vacuo to obtained amino acid
ester isocyanate (2a–e). Under N2 atmosphere scutellarin methyl
ester (1.37 mmol) was dissolved in 15 mL anhydrous DMF, then
10 mL anhydrous THF solution which contained compounds 2a–e
(2.05 mmol) and Et3N (0.2 mmol) were added to the mixture. The
reaction mixture was stirred at 50 8C for 10 h. After that the solvent
was evaporated in vacuo, the residue was purified by using routine
flash column chromatography on silica gel (eluent: CHCl3/
MeOH = 15:1–20:1 v/v). The obtained intermediates were further
deprotected by using trifluoroacetic acid at 0 8C – r.t. to obtain the
target compounds (4a–4e).
C
28H28NO17 [M+H]+ 650.1357, found 650.1352. 4e: 1H NMR
(400 MHz, DMSO-d6): d13.05(brs, 1H), 10.45 (brs, 1H), 8.30 (d, 1H,
J = 8.4 Hz), 7.97 (d, 2H, J = 8.8 Hz), 7.10 (s, 1H), 6.96 (d, 2H,
J = 8.8 Hz), 6.90 (s, 1H), 5.35 (d, 1H, J = 7.2 Hz), 4.36 (m, 1H), 4.22
(d, 1H, J = 9.6 Hz), 3.67 (s, 3H), 3.37–3.26 (m, 3H), 2.79–2.74 (m,
1H), 2.66–2.62 (m, 1H); ESI-MS (m/z) [M+H]+: 636.3, [MꢀH]ꢀ:
634.3. HRMS: calcd. for C27H26NO17 [M + H]+ 636.1201, found
636.1181.
2.2. UPLC–MS/MS analysis conditions for identification of target
compounds 4a–4e
LC analyses were performed on a Waters ACQUITY UPLC
instrument coupled to a binary pump, an autosampler, and a
column compartment. The samples were separated on a BEH Cl8
column (2.1 mm ꢁ 50 mm, 1.7
m
m), guarded by an Waters Van
m). The mobile
Guard BEH C18 column (2.1 mm ꢁ 5 mm, 1.7
m
phase consisted of acetonitrile containing 0.1% formic acid (A)
and water containing 0.1% formic acid (B). The elution gradient
was as follows: 10% A (0ꢀ2 min), 90% A (2ꢀ3 min), 10% A
(3 min). The mobile phase flow rate was 0.35 mL/min, and the
column temperature was set at 45 8C. The injection volume was
Spectra data of compounds 4a–4e: 4a: 1H NMR (400 MHz,
DMSO-d6): d 13.06 (brs, 1H), 10.46 (brs, 1H), 8.23 (d, 1H, J = 7.6 Hz),
7.99 (d, 2H, J = 8.8 Hz), 7.13 (s, 1H), 6.97 (d, 2H, J = 9.2 Hz), 6.92 (s,
1H), 5.37 (d, 1H, J = 7.2 Hz), 4.23 (d, 1H, J = 9.6 Hz), 3.67 (s, 3H),
3.53–3.34 (m, 3H), 1.36 (d, 3H, J = 7.6 Hz). ESI-MS (m/z) [M+H]+:
592.3, [MꢀH]ꢀ: 590.3. HRMS: calcd. for C26H26NO15 [M+H]+:
1
m
L.
Mass spectrometry: All the mass experiments were carried out
592.1302, found 592.1290. 4b: 1H NMR (400 MHz, DMSO-d6):
d
using a Waters ACQUITY TQD (triple quadrupole mass spectrome-
ter) equipped with a Z-spray ESI source and connected to the
Waters ACQUITY UPLC system. The acquisition parameters were:
collision gas, argon (Ar); nebulizing and drying gas, nitrogen (N2);
source temperature, 120 8C; desolvation temperature, 350 8C; cone
gas flow, 50 L/h; desolvation gas flow, 650 L/h; collision gas flow,
0.16 mL/min; capillary voltage, 3.0 kV; Multi Reaction Monitor
(MRM) mode was used, positive ions mode (ESI+) the confirmation
ion pairs are (m/z): 592.0 ! 287 (4a), 620.1 ! 287 (4b),
668.1 ! 287 (4c), 650.1 ! 287 (4d), 636.1 ! 287 (4e) respective-
ly, cone voltage was 40 V, 35 V, 40 V, 35 V, and 35 V respectively.
Collision voltage was 40 V, 30 V, 35 V, 30 V, and 30 V respectively.
13.06 (brs, 1H), 10.46 (brs, 1H), 8.05 (d, 1H, J = 8.0 Hz), 7.95 (d, 2H,
J = 8.8 Hz), 7.18 (s, 1H), 6.97 (d, 2H, J = 8.8 Hz), 6.91 (s, 1H), 5.29 (d,
1H, J = 7.6 Hz), 4.36 (d, 1H, J = 9.2 Hz), 3.93–3.90 (m, 1H), 3.67 (s,
3H), 3.37–3.29 (m, 3H), 2.15–2.10 (m, 1H), 0.97–0.95 (m, 6H). ESI-
MS (m/z) [M+H]+: 620.3, [MꢀH]ꢀ: 618.4. HRMS calcd. for
C
28H30NO15 [M+H]+ 620.1615, found 620.1623. 4c: 1H NMR
(400 MHz, DMSO-d6):
d 13.01 (brs, 1H), 10.46 (brs, 1H), 8.24 (d,
1H, J = 8.4 Hz), 7.98 (d, 2H, J = 8.8 Hz), 7.32–7.24 (m, 5H), 7.12 (s,
1H), 6.97 (d, 2H, J = 8.8 Hz), 6.90 (s, 1H), 5.37 (d, 1H, J = 7.2 Hz), 4.23
(d, 1H, J = 9.2 Hz), 3.67 (s, 3H), 3.36–3.26 (m, 3H), 3.17–3.10 (m,
1H), 2.96–2.91 (m, 1H); ESI-MS (m/z) [M+H]+: 668.3, [MꢀH]ꢀ:

340
F.-J. Jiang et al. / Chinese Chemical Letters 24 (2013) 338–340
_
Table 1
The physiochemical properties of carbamate prodrugs of scutellarin methyl ester
and reference compounds.
Compounds
Stability in PBS (t1/2 h)
Stability
Aqueous
solubility
in plasma
(t1/2 min)
(mg/mL)
pH 2.0
pH 7.4
Scutellarin
–
>72
>72
>72
>72
>72
–
3
5
15.24
4a
4b
4c
4d
4e
2.5
5.0
9.0
4.0
4.0
365.80
22.54
4.73
5
5
30
30
123.10
426.51
Fig. 2. Stability of carbamate prodrugs of scutellarin methyl ester in plasma
(37 ꢂ 1 8C).
[(Fig._1)TD$FIG]
aqueous solubility of target compounds would be increased upon
40-carbamic acid esterification and 7-methyl esterification of
scutellarin.
4. Conclusion
In summary, a new series of highly water soluble scutellarin
methyl ester carbamate derivatives were synthesized. Their
absorption and other pharmacokinetical properties, and further
Fig. 1. Stability of carbamate prodrugs of scutellarin methyl ester in PBS at pH 7.4
and pH 2.0 (37 ꢂ 1 8C).
modification of scutellarin methyl ester with other L-amino acid
ester isocyanates are still under investigation in our laboratory.
3. Results and discussion
Acknowledgments
The hydrolysis product of carbamate–scutellarin methyl ester
conjugates were carbamate–scutellarin and scutellarin after
hydrolyzed in PBS (pH 7.4 and pH 2.0) and plasma, which were
confirmed by UPLC–MS/MS. The target compounds 4a–4e were
evaluated for their physiochemical properties such as chemical,
plasma stability and aqueous solubility according to Ref. [5]. The
obtained results were compared with those of scutellarin, and the
results are summarized in Table 1, Figs. 1 and 2.
The results showed that scutellarin methyl ester carbamate
derivatives (4a–e) are more stable (t1/2 >72 h) in acidic condition
(pH 2) than in neutral condition (pH 7.4) (t1/2 2.5–9.0 h). The target
compounds also showed instability under the action of some
enzymes in plasma with half-life (t1/2) less than 30 min. The results
indicated that compounds 4d and 4e with a free carboxyl group in
their carbamate moiety are significantly more stable (t1/2 30 min)
than scutellarin and compounds 4a–4c (t1/2 3–5 min) in plasma.
This occurrence would be attributed to the increasing steric
hindrance of carbamate strand and the formation of intermolecu-
lar hydrogen bonds, which caused a decrease in the rate of
hydrolysis. Thus, scutellarin methyl ester carbamate derivatives
with desired rates of hydrolysis can be obtained by the use of
appropriate amino acid ester isocyanates. Except for compound 4c,
This work was supported by the grants from the National
Natural Science Foundation of China (Nos. 81260473, 81060359),
projects of Guizhou Science and Technology Department (Nos.
2012-3013, 2010-0519).
References
[1] W.D. Zhang, W.S. Chen, Y.H. Wang, Studies on the flavone glycosides from the
extract of Erigeron breviscapus, Chin. Trad. Herbal Drugs 31 (2000) 565–566.
[2] H. Hong, G.Q. Liu, Protection against hydrogen peroxide-induced cytotoxicity in
PC12 cells by scutellarin, Life Sci. 74 (2004) 2959–2973.
[3] X. Chen, L. Cui, X. Duan, Pharmacokinetics and metabolism of the flavonoid
scutellarin in humans after a single oral administration, Drug Metab. Dispos.
34 (2006) 1345–1352.
[4] J. Lu, C. Cheng, X. Zhao, PEG-scutellarin prodrugs: synthesis, water solubility and
protective effect on cerebral ischemia/reperfusion injury, Eur. J. Med. Chem. 45
(2010) 1731–1738.
[5] F. Cao, J.X. Guo, Q.N. Ping, Prodrugs of scutellarin: ethyl, benzyl and N,N-diethyl-
glycolamide ester synthesis, physicochemical properties, intestinal metabolism
and oral bioavailability in the rats, Eur. J. Pharm. Sci. 29 (2006) 385–393.
[6] Q. Zhou, X. Jiang, K. Li, Synthesis, characterization and in situ intestinal absorption
of different molecular weight scutellarin-PEG conjugates, Pharmazie 61 (2006)
660–663.
[7] M.K. Kim, K.S. Park, W.S. Yeo, In vitro solubility, stability and permeability of novel
quercetin–amino acid conjugates, Bioorg. Med. Chem. 17 (2009) 1164–1171.
[8] M. Ninomiya, K. Tanaka, Y. Tsuchida, Increased bioavailability of tricin-amino acid
derivatives via a prodrug approach, J. Med. Chem. 54 (2011) 1529–1536.
[9] P.J. Mulholland, D.R. Ferry, D. Anderson, Pre-clinical and clinical study of QC12, a
water-soluble, pro-drug of quercetin, Ann. Oncol. 12 (2001) 245–248.
[10] K.B. Simonsen, B.K. Ayida, D. Vourloumis, Novel paromamine derivatives explor-
ing shallow-groove recognition of ribosomal-decoding-site RNA, Chembiochem 3
(2002) 1223–1228.
the aqueous solubilities of target compounds (22.54–426.51
mL) were significantly higher than that of scutellarin (15.24
m
m
g/
g/
mL), especially the solubilities of compound 4d (123.10
and 4e (426.51 g/mL), which were 8 and 28 times higher than
that of scutellarin respectively. This finding suggested that
mg/mL)
m