Synthesis and pharmacological evaluation of novel limonin derivatives as anti-inflammatory and analgesic agents with high water solubility

Article abstract of DOI:10.1016/j.bmcl.2014.02.003

A novel series of water-soluble derivatives of limonin were synthesized by introducing various tertiary amines onto the C (7)-position of limonin. Ten target compounds were characterized and screened for their anti-inflammatory and analgesic activity in v

Full text of DOI:10.1016/j.bmcl.2014.02.003

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1851–1855
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and pharmacological evaluation of novel limonin
derivatives as anti-inflammatory and analgesic agents with
high water solubility
c
b
c
Yun Yang a, Xinhui Wang b, Qihua Zhu c, Guoqing Gong b, , Danmeng Luo , Aidou Jiang , Liyan Yang ,
⇑
Yungen Xu a,c,
⇑
a Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing, Jiangsu 21009, China
b Department of Pharmacology, China Pharmaceutical University, Nanjing, Jiangsu 210009, China
c Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing, Jiangsu 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of water-soluble derivatives of limonin were synthesized by introducing various tertiary
amines onto the C (7)-position of limonin. Ten target compounds were characterized and screened for
their anti-inflammatory and analgesic activity in vivo. Compound 3c exhibited the strongest analgesic
and anti-inflammatory activity among the limonin and its derivatives tested; its analgesic activity is more
potent than that of aspirin and its anti-inflammatory activity is stronger than that of naproxen.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 24 November 2013
Revised 20 January 2014
Accepted 1 February 2014
Available online 10 February 2014
Keywords:
Limonin derivatives
Solubility
Analgesic activity
Anti-inflammatory activity
Limonoids are typical secondary metabolites, and are abundant
in citrus fruit and other plants found in the Rutaceae and Meliaceae
families. Limonin (1) is known as the most abundant limonoid in
the natural environment and is isolated from navel orange juice
in which it works as bitter principle. As limonoids possess various
therapeutic effects, such as antitumor,1–4 anti-inflammatory,5,6
analgesic,6 antibacteria,7 antimalaria,7 antifeedant8 etc., the chem-
istry and pharmacology of these compounds have attracted great
interest in medicinal chemistry.
A number of literature reported that Evodiae Fructus, originates
from a variety of Evodia species (Rutaceae), has been used in clinics
as NSAIDs in the treatment of traditional Chinese medicine.9
Limonin isolated from Evodiae Fructus is a potent anti-inflamma-
tory and analgesic agent.10–12 However, these active substances
suffered from poor aqueous solubility (<0.005 mg/ml), which results
in low bioavailability and poses challenges in its formulation
development. Therefore, obtaining the water-soluble derivatives
of limonin by structural modification has become one of the key
issues before limonoids are applied in clinics.
limonin to yield five nitrogen-containing derivatives which were
treated with a solution of ether saturated with hydrogen chloride
to form hydrochloride (3a–3e). In addition, five tertiary amine
derivatives (6a–6e) of desoxylimonin (4)13 were designed and
synthesized to evaluate contributions of the oxygen bridge be-
tween C(14) and C(15) to anti-inflammatory and analgesic activity.
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
(1) Limonin
(4) Desoxylimonin
The synthetic routes for compounds 3a–3e and 6a–6e are
depicted in Schemes 1 and 2. Compound 4 was prepared from limo-
nin according to the literature method.13 Treatment of 1 or 4 with
hydroxylamine hydrochloride yielded exclusively limonin-7-oxime
(2)8 or desoxylimonin 7-oxime (5). 2 was alkylated with 1-(2-chlo-
roethyl) piperidine to give 7-[2-(piperidin-1-yl)ethoxyimino]
To increase the water solubility of limonin derivatives, various
tertiary amine moieties were introduced onto C (7)-position of
⇑
Corresponding authors. Tel.: +86 025 86185307; fax: +86 025 83271487 (G.G.);
tel.: +86 025 83322067; fax: +86 025 83271487 (Y.X.).
E-mail addresses: gonggq@hotmail.com (G. Gong), xyg64@126.com (Y. Xu).
http://dx.doi.org/10.1016/j.bmcl.2014.02.003
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

1852
Y. Yang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1851–1855
O
O
O
CH2CH2
N
N
3a: R=
O
O
O
O
O
O
O
O
O
O
a
b
O
R
3b
: R= CH2CH2
O
O
O
O
O
O
O
N
O
N
3c
3d
: R=-CH2CH2N(CH2CH3)2
: R=-CH2CH2CH2N(CH3)2
3e: R=-CH2CH2N(CH3)2
O
OH
O
HCl
1
2
Scheme 1. Reagents and conditions: (a) hydroxylamine hydrochloride, pyridine, anhydrous ethanol, reflux; (b) (i) corresponding alkyl chloride hydrochloride, NaOH, TBAB
(Tetrabutylammonium Bromide), anhydrous THF, 80 °C; (ii) hydrogen chloride, anhydrous ether, anhydrous dichlormethane.
O
O
O
O
O
O
O
O
O
O
O
O
a
b
O
O
O
O
O
O
O
O
OH
N
O
5
1
4
CH2CH2
6b: R= CH2CH2
N
N
6a
: R=
O
O
O
O
O
R
c
O
6c
: R=-CH2CH2N(CH2CH3)2
6d: R=-CH2CH2CH2N(CH3)2
6e
O
O
N
HCl
: R=-CH2CH2N(CH3)2
Scheme 2. Reagents and conditions: (a) hydroiodic acid, acetic acid; (b) hydroxylamine hydrochloride, pyridine, anhydrous ethanol, acetonitrile, reflux; (c) (i) corresponding
alkyl chloride hydrochloride, NaOH, TBAB, anhydrous THF, 80 sC; (ii) hydrogen chloride, anhydrous ether, anhydrous dichlormethane.
Table 1
pKa, logD7.4, solubility and analgesic activity by acetic acid-induced writhing response in mice of indicated compound
Entry
pKa
logD7.4
Intrinsic solubility (mg/mL)
Acetic acid induced writhing test
Number of writhingsa
Dose (mg/kg)
Inhibition rate (%)
b
Control
Aspirin
1
4
3a
3b
3c
3d
3e
6a
6b
6c
6d
—
—
—
—
—
—
—
—
—
—
—
44.0 6.7⁄⁄⁄
—
200
70
70
70
70
70
70
70
70
70
70
70
70
12.4 2.4
71.82
45.91
24.61
70.45
57.95
80.00
64.09
68.64
44.32
50.00
33.18
60.71
30.00
<0.005c
<0.005c
25.3
4.5
23.8 2.8⁄⁄⁄
33.17 5.5⁄⁄⁄
13.0 3.6⁄⁄⁄
18.5 3.0⁄⁄⁄,
8.87
6.11
9.17
9.28
8.78
8.52
6.84
9.49
9.39
8.88
0.44
2.45
0.26
0.37
0.59
0.81
2.52
0.57
0.19
0.32
#
14.5
9.6
17.8
19.8
2.1
1.9
9.4
28.9
8.8 2.7⁄⁄⁄
15.8 6.2
13.8 2.2⁄⁄⁄
24.5 5.9⁄⁄
22.0 4.8⁄⁄⁄
29.4 2.0
17.3 6.2⁄⁄⁄
30.8 1.7
6e
a
Statistical analysis was performed using one-way ANOVA (⁄p <0.05, ⁄⁄p <0.01, ⁄⁄⁄p <0.001 as compared with the respective control; #p <0.05 as compared with 1). The
results are expressed as mean SEM (n = 8).
b
—: Not determined.
c
Equilibrium solubility values measured by the classical saturation shake flask method.
limonin which was treated with a solution of ether saturated with
hydrogen chloride to form hydrochloride 3a. Accordingly, com-
pounds 3b–3e or 6a–6e were obtained respectively by treating 2
or 5 with corresponding alkyl chloride in different yields. Com-
pounds 3a–3e and 6a–6e were characterized by IR, 1H/13C NMR
and HRMS spectroscopy.14,15 The absence of a broad singlet peak
at d ꢀ 7.70 ppm (C@NOH) and the emergence of a new triplet peak
around d ꢀ 4.20 ppm which is attributed to the proton of O–CH2 in
the 1H NMR spectrum provided good support for the formation of
oxime ether derivatives.
The physiochemical properties including pKa (ionization
constants), logD7.4 (partition coefficient at pH 7.4) and aqueous
solubility of ten target compounds were summarized in Table 1.
The pKa, logD7.4 and aqueous solubility were determined accord-
ing to the method of Avdeef and Tsinman16 on a Gemini Profiler
instrument (pION) by the ‘goldstandard’ Av-deef-Bucher potentio-
metric titration method.17
All target compounds (3a–3e and 6a–6e) were evaluated in vivo
for their analgesic and anti-inflammatory activity. The analgesic ef-
fect was evaluated using acetic acid-induced writhing tests18,19

Y. Yang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1851–1855
1853
the method reported in previous literature23 with minor modifica-
tions. The limonin, desoxylimonin and their derivatives (100 mg/
kg, i.g.) with naproxen (150 mg/kg, i.g.) as positive controls were
suspended in 0.5% CMC-Na. The vehicle control group treated with
xylene was given 0.5% CMC-Na with the same method. 90 min
Table 2
The inflammatory activity by xylene-induced ear swelling test in mice of indicated
compounds
Entry
Xylene-induced ear swelling test
Dose (mg/kg)
Swollen extent (%)a
Inhibition rate (%)
after administration of the corresponding drugs, 25 ll of xylene
b
Control
Naproxen
1
4
—
132.8 16.3
84.1 6.4⁄⁄⁄
89.3 14.1⁄⁄
111.7 9.9
—
was applied to anterior and posterior surfaces of right ear lobe. Left
ear was considered as control. 30 min after xylene application, the
mice were sacrificed. Circular sections on both ears were taken
using a cork borer with a diameter of 8 mm and weighed. Degree
of swelling caused by the xylene was measured based on the
weight of left ear without stimulus. The results are summarized
in Table 2 and are expressed as swollen extent.
As shown in Table 1, the aqueous solubility of ten target
compounds was superior to that of 1 and 4. There is a significant
negative correlation between the pKa and logD7.4 of the ten tested
compounds. Overall, compound 3 series with an oxygen bridge be-
tween C(14) and C(15) were more water-soluble than compound 6
series (except compound 6e) with a double bond. Piperidino-
ethoxy analogue 3a showed the most significant increase in
water-solubility, whereas morpholinyl ethoxy analogue 3b showed
poor water-solubility among compounds 3a–3e.
150
100
100
100
100
100
100
100
100
100
100
100
100
36.65
32.74
15.89
54.77
53.01
75.46
46.08
45.46
25.36
27.61
18.43
26.35
24.55
#
3a
3b
3c
3d
3e
6a
6b
6c
6d
6e
60.1 16.0⁄⁄⁄,
62.4 9.4⁄⁄⁄
32.6 8.2⁄⁄⁄,
###
71.6 11.2⁄⁄⁄
72.4 13.1⁄⁄⁄
99.1 6.1⁄
96.1 3.2⁄
108.2 6.3
97.8 5.1⁄
100.2 7.4⁄
Statistical analysis was performed using one-way ANOVA (⁄p <0.05, ⁄⁄p <0.01,
a
⁄⁄⁄p <0.001 as compared with the respective control; #p <0.05, ##p <0.01, ###p
<0.001, as compared with 1). The results are expressed as mean SEM (n = 8).
b
—: Not determined.
The result of acetic acid-induced writhing test (Table 1)
revealed that compounds 3a–3e, 6b and 6d possessed more potent
analgesic activity than that of 1. Compound 3c exhibited the most
potent analgesic activity and higher inhibition rate of pain thresh-
old compared with aspirin (200 mg/kg, i.g.). As shown in Figures 1
and 2, all the tested compounds exhibited moderate analgesic
apacity, and their analgesic effects were more potent than that of
1 in the tail-immersion test; however, compounds 4 displayed no
analgesic activities at the administered dose. The tail-immersion
latencies of compounds 3a–3e showed a significant increase. The
maximal anti-nociceptive response was obtained between 30 and
90 min, while compounds 6a–6e reached maximal anti-nocicep-
tive responses after 60 min of gavage administration. Significant
analgesic effects were observed at 60 min for 3a and 6d, and
90 min for 3c.
and tail-immersion tests20–22 in mice. In the acetic acid-induced
writhing test, limonin and desoxylimonin (70 mg/kg, i.g.14), and
aspirin (200 mg/kg, i.g.), used as positive control, and compounds
3 and 6 (70 mg/kg, i.g.) were suspended in 0.5% CMC-Na.14 The
model group was given 0.5% CMC-Na using the same method.
One hour after gavage administration of the corresponding drug,
mice were injected intraperitoneally with 0.1 ml/10 g body weight
of 0.7% acetic acid solution in saline. The number of writhes was
recorded 15 min after the chemical stimulus. The results are sum-
marized in Table 1. In the tail-immersion test, all the compounds
were administrated in the same way used in the writhing test,
using a same single dosage (100 mg/kg, i.g.). Tail-curl latency time
was measured before administration and 30, 60, 90 and 120 min
afterward.
Ear swelling induced by xylene in mice was used to evaluate the
anti-inflammatory activities of the target compounds according to
The results revealed that the inhibition rates on mouse ear
swollen (Table 2) of compounds 3a, 3b and 3c were higher than
Figure 1. Antinociceptive effect of compounds 3a–3e (100 mg/kg), 1 (100 mg/kg), 4 (100 mg/kg) and Aspirin (100 mg/kg) in the tail-immersion test in mice. Statistical
analysis was performed using one-way ANOVA (⁄p <0.05, ⁄⁄p <0.01, ⁄⁄⁄p <0.001 as compared with the respective control; #p <0.05 as as compared with 1). The results are
expressed as mean SEM (n = 8).

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Y. Yang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1851–1855
Figure 2. Antinociceptive effect of compounds 6a–6e (100 mg/kg), 1 (100 mg/kg), 4 (100 mg/kg) and Aspirin (100 mg/kg) in the tail-immersion test in mice. Statistical
analysis was performed using one-way ANOVA (⁄p <0.05, ⁄⁄p <0.01, ⁄⁄⁄p <0.001 as compared with the respective control; #p <0.05 as as compared with 1). The results are
expressed as mean SEM (n = 8).
those of 1 and naproxen. The inhibition rates of compounds 3d and
3e were equivalent to that of naproxen. However, compounds 6a–
6e displayed weaker anti-inflammatory activities at the adminis-
tered dose.
References and notes
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As a whole, compounds 4 and 6a–6e exhibited less analgesic
and anti-inflammatory activities than those of compounds 1 and
3a–3e. This indicated that the oxygen bridge between C(14) and
C(15) played an important role in analgesic and anti-inflammatory
activity. Among all compounds, 3c exhibited the highest analgesic
activity, the best activity in acetic acid-induced writhing test, and
remarkable activity in tail-immersion test with high aqueous solu-
bility (14.5 mg/ml). Compound 3c also showed the best anti-
inflammatory activity in mouse ear swelling test, even stronger
than that of naproxen at the administered dosage.
8. Ruberto, G.; Renda, A.; Tringali, C.; Napoli, E. M.; Simmonds, M. S. J. Agric. Food
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In summary, a series of novel limonin oxime ether derivatives
3a–3e and desoxylimonin oxime ether derivatives 6a–6e were syn-
thesized and evaluated for their analgesic and anti-inflammatory
activity and water-solubility. The investigation of analgesic and
anti-inflammatory effects and solubility indicated that oximation
of limonin and subsequent introduction of tertiary amino group
by etherification of limonin oxime improved water solubility and
bioactivity. In general, compounds 1 and 3a–3e exhibited more po-
tent analgesic and anti-inflammatory efficacies than those of com-
pounds 4 and 6a–6e, suggesting that oxygen bridge between C(14)
and C(15) in limonin derivatives is important for improvement of
analgesic and anti-inflammatory activities. In particular, Com-
pound 3c exhibited the greatest analgesic and anti-inflammatory
activity among all the compounds tested; its analgesic activity is
more potent than that of aspirin and its anti-inflammatory activity
is stronger than that of naproxen. It has been identified as a prom-
ising analgesic and anti-inflammatory candidate compound with
high water-solubility.
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14. General procedure: To a solution of compounds 2 or 5 (1 mmol) in anhydrous
THF (25 ml), NaOH (10 mmol) was added, then the reaction mixture was
stirred for 30 min at room temperature. Alkyl chloride hydrochloride (3 mmol)
and TBAB (catalytic amount) were added, and the mixture was heated to reflux
and monitored by thin layer chromatography (TLC). After completion of the
reaction, the solvent was evaporated and the crude reaction mixture was
diluted with water (50 ml) and acidified to pH 6 with HCl (1 M). The aqueous
layer was extracted twice with dichlormethane and the combined organic
layer was washed with brine, dried over MgSO4, and evaporated to dryness.
The crude product was purified by column chromatography (MeOH/CH2Cl2
1:50). The purified product thus obtained was dissolved in anhydrous
dichlormethane (3 ml) and treated with a saturated solution of dry hydrogen
chloride in diethyl ether. The solid separated out was filtered, washed with
diethyl ether, and dried, to give 3 or 6. Symbols: CMC-Na: Carboxymethyl
Cellulose Sodium; i.g.: Intragastric Administration.
15. Physical and spectral data of selected compound. Compound 3c: Yield 43%, white
solid, mp 78 °C (decomposition); IR (KBr,
m
cmÀ1): 3434, 2948, 2648, 1740,
1626, 1464 1023, 810; 1H NMR (CDCl3, 500 MHz), d (ppm): 12.21 (s, 1H), 7.45
(d, J = 1.6 Hz, 2H), 6.39 (s, 1H), 5.55 (s, 1H), 4.73 (d, J = 12.4 Hz, 1H), 4.66 (br s,
1H), 4.38 (d, J = 11.4 Hz, 2H), 4.01 (br s, 1H), 3.82 (s, 1H), 3.49 (dt, J1 = 57.4,
J2 = 28.7 Hz, 2H), 3.27 (br s, 5H), 2.97 (d, J = 16.1 Hz, 1H), 2.71 (d, J = 16.4 Hz,
1H), 2.43 (t, J = 17.0 Hz, 1H), 2.14–1.74 (m, 6H), 1.47 (br s, 6H), 1.35 (s, 3H),
1.29 (s, 3H), 1.21 (s, 3H), 1.04 (s, 3H); 13C NMR (CDCl3, 300 MHz), d (ppm):
169.34, 167.76, 161.73, 143.27, 140.95, 119.92, 109.58, 80.38, 79.13, 78.57,
Acknowledgments
The Project was supported by the National Undergraduate
Training Programs for Innovation and Entrepreneurship of China.

Y. Yang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1851–1855
1855
67.39, 65.82, 64.67, 60.48, 54.33, 49.86, 49.74, 47.38, 46.84, 46.30, 46.12, 38.14,
35.77, 32.93, 30.26, 21.53, 21.44, 20.43, 19.58, 17.79, 8.36, 8.34; HR-ESIMS m/z
585.3165 [M+H]+ (calcd for C32H45N2O8, 585.3170).
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