Parallel synthesis and biological evolution of quinic acid derivatives as immuno-suppressing agents against T-cell receptors

Article abstract of DOI:10.1039/c5ra06095h

A simple protocol for the synthesis of quinic acid derivatives was established and their biological evolution against T-cells is studied. Results showed that one of the derivatives, Cyn-1324, has low toxicity on T-cells and a high effect on reducing Signal 2 of T-cell immune responses. In vitro binding measurements of atomic force spectroscopy further indicated that the blocking effect of Cyn-1324 between CD28 and CD80 was about 31 ± 4%. In vivo animal tests also confirmed that Cyn-1324 can reduce the allergic responses from ovalbumin-induced mice with little toxicity. Based on these observations, Cyn-1324 can be a mild immuno-suppressive candidate for future drug development.

Full text of DOI:10.1039/c5ra06095h

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PAPER
Parallel synthesis and biological evolution of quinic
acid derivatives as immuno-suppressing agents
against T-cell receptors†
Cite this: RSC Adv., 2015, 5, 50801
a
b
a
a
bc
Chih-Yu Huang, Li-Hsun Chen, Hsuan-Yu Huang, Feng-Sheng Kao, Yun-Ta Lee,
Manikandan Selvaraju, Chung-Ming Sun* and Hueih-Min Chen*a
b
bc
A simple protocol for the synthesis of quinic acid derivatives was established and their biological evolution
against T-cells is studied. Results showed that one of the derivatives, Cyn-1324, has low toxicity on T-cells
and a high effect on reducing Signal 2 of T-cell immune responses. In vitro binding measurements of atomic
force spectroscopy further indicated that the blocking effect of Cyn-1324 between CD28 and CD80 was
about 31 ꢀ 4%. In vivo animal tests also confirmed that Cyn-1324 can reduce the allergic responses from
ovalbumin-induced mice with little toxicity. Based on these observations, Cyn-1324 can be a mild
immuno-suppressive candidate for future drug development.
Received 6th April 2015
Accepted 20th May 2015
DOI: 10.1039/c5ra06095h
www.rsc.org/advances
co-stimulation (Signal 2) bindings, the strength of these
immuno-responses can be estimated by IL-2 released from
Introduction
Immuno-response to exclude the invasion of harmful outside activated T-cells. If Signal 2 is inhibited, the total release of IL-2
9
materials is the major defense system in human and other would be reduced consequently. In the current work, a mild
living creatures. Immuno-suppressive treatment for over- blocker for mainly blocking CD28 therefore can be found to
reacting patients to control immune responses is highly only inhibit Signal 2 by directly blocking T-cells on their
demanded as the over-reaction of immune cells cannot be self- membrane surfaces.
controlled and may become a big burden for the entire life. In
Many known therapeutics, such as cyclosporine A (CSA),
particular, there are two signals (Signal 1 and Signal 2) cyclophosphamide, tacrolimus (FK506) and azathioprine, have
produced during the conjugation of T-cells and antigen- been developed to treat immuno-suppression. Although they
presenting cells (APC cells such as B-cells or dendritic cells) to are selective and potent to prevent the rejection of organ
1
–4
activate the adaptive immune responses.
formation of intercellular contact aer activation of resting block both Signal 1 and Signal 2 to penetrate inside the cell and
The specialized transplants and in diseases involving the immune system, they
5–7
T-cells by APC cells is termed as immunological synapses.
A
this causes severely toxic side effects. Under this consideration,
more recent study using single cell force spectroscopy showed development of mild immuno-suppressive agents that partially
that T-cells can be activated by dendritic cells than by B-cells reduce the immune responses without rough side-effects is
based on the quantity measurement of IL-2 and IFNg secreted urgently needed.
8
from T-cells aer activation. Signal 1 is created as the T-cell
Cynarin is a biologically active chemical constituent of Cyn-
11
receptor (TCR) of T-cells, which strongly binds with major ara cardunculus. Earlier, we reported that Cynarin in Echinacea
histocompatibility complex (MHC) of APC cells. Signal 2 is a purpurea is able to block Signal 2 of T-cell activation specically
co-stimulation signal, which occurs simultaneously with Signal for immuno-suppression. The blocking effect between CD28
by two concurrent bindings: CD28 of T-cell to CD80 of APC cell of T-cell and CD80 of B-cell was identied by a small molecule,
9,10
1
(
(
weaker binding) and CD154 of T-cell to CD40 of APC cell Cynarin (Cyn), aer owing through an immobilized receptor
stronger binding). Aer stimulation (Signal 1) and (AFTIR). Computer simulation of this effect is displayed in
Fig. 1. A key blocking factor (KBF) was indicated by a red circle
a
and a main blocking body (quinic acid-like) was assigned.
National Applied Research Laboratories, National Nano Device Laboratories,
Biomedical Group, Hsinchu 300-10, Taiwan. E-mail: hmchen@narlabls.org.tw
b
Physical contact of the immunological synapses mentioned
Department of Applied Chemistry, National Chiao-Tung University, Hsinchu 300, above between CD28 of T-cells and CD80 of B-cells was a key
Taiwan. E-mail: cmsun@mail.nctu.edu.tw
point that we applied to establish a novel drug screening
method to obtain an immuno-suppressive compound. The
reason is that this structural contact between CD28 and CD80
can be properly blocked by suitable molecules. Accordingly, a
c
Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, 100,
Shih-Chuan 1st Road, Kaohsiung 807-08, Taiwan
†
Electronic supplementary information (ESI) available. CCDC 1058469. For ESI
and crystallographic data in CIF or other electronic format see DOI:
0.1039/c5ra06095h
natural product Cynarin was found to effectively block the
1
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Fig. 1 Computer simulation for blocking between CD28/T-cell and CD80/B-cell by Cynarin.
binding between CD28 and CD80: i.e., the immunological using a strong acid catalyst. In addition, the cytotoxicity of
10
synapses of Signal 2 can be shut down by Cyn. This blocking quinic acid may be reduced if its C-1 carboxyl group is modied
effect induced by Cyn on T-cells has been further conrmed by to other functional groups.
1
2
using atomic force spectroscopy. However, we found that the
Lactonization and ketalization of quinic acid 1 was carried
out in the presence of a catalytic amount of sulfuric acid in
9
toxicity of Cyn on T-cells is observed. Our computer simulation
showed that the main blocking interactions between Cynarin acetone to provide the acetal lactone intermediates 2 in a single
and CD28 came from the quinic acid-like structure. Two step. Under reux conditions, the reaction took 2 h to complete
symmetrical side chains (di-caffeoyl group) may not be essential conversion with a yield of 85%. Alternatively, the use of
ꢁ
for the effect of “blocking”. Hence, as a part of our ongoing microwave irradiation in a closed vessel system at 100
C
research to nd better immuno-suppressive agents, we synthe- dramatically reduced the reaction time to only 5 min with a
sized a series of Cynarin derivatives by using quinic acid (QA) as maximum yield of 91%. The next transformation is the ami-
a novel scaffold targeted on KBF structure. Many natural nolysis of lactone intermediates 2 with various amines. The
product (NP) drugs and NP-derived compounds have been reaction was accomplished under microwave irradiation at
1
3,14
ꢁ
found and applied in clinical trials.
screening method is a powerful technique to nd potential
target drugs from a signicant number of NP or NP-like conversion under the conventional reuxing conditions.
Structure-based virtual 100 C for 5 min to obtain various quinic acid derivatives.
Such aminolysis of lactones requires 18 h to complete
15–22
compound libraries.
We report herein the preliminary Furthermore, the aminolysis was performed with various
results which indicate that one of the NP-derived compounds, amines with different electronic natures to give a variety of
Cyn-1324, has low toxicity and more chemical stability as quinic acid analogues, as shown in Table 1. All the amines gave
compared to that of Cynarin. In vivo animal tests further satisfactory yields under similar reaction conditions. The
conrmed that Cyn-1324 has an immuno-suppressive effect on structure of compound 3k is also conrmed by X-ray crystal-
28
ovalbumin-induced allergic mice and therefore potentially is lography, as shown in Fig. 2.
qualied for development as a drug candidate in the future.
Biology
To investigate the potential biological applications of quinic
acid analogues obtained by this synthetic protocol, preliminary
tests of the analogues for cytotoxicity were performed. The
Results and discussion
Chemistry
Synthesis of quinic acid (QA) derivatives. The synthesis of results demonstrated that Cyn-1324 (3k) could effectively
quinic acid derivatives was accomplished in a straightforward inhibit the proliferation of T-cell receptors. The efficacy is
way by two steps: lactonisation/ketalisation followed by ami- comparable with that of Cynarin, which served as a control
nolysis, as shown in Scheme 1. Earlier, modications at C-1/C-5 study.
hydroxyl groups and formation of macrocycles between
The percentage cell survival and efficacy of Cyn and Cyn-1324
23–27
carboxylate and C-3 hydroxyl group are reported.
Stable against T-cells were investigated. The relationship between cell
quinic acid derivatives can be synthesized by converting cis C-3/ survival (T-cells) vs. concentration of Cyn and Cyn-1324 is
C-4 hydroxyl groups into a ketal via a reaction with a ketone shown in Fig. 3. The results implied that Cyn-1324 has low
Scheme 1 Synthesis of quinic acid analogues 3.
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Table 1 Synthesis of quinic acid analogues 3
Table 1 (Contd.)
Entry
R
1
R
2
(C]O)
H
2
NR
3
Isolated yield
76%
Entry
R
1
R
2
(C]O)
H
2
NR
3
Isolated yield
88%
3
a
3
3
p
q
3
3
3
3
b
c
81%
86%
93%
91%
89%
d
e
3
3
r
80%
82%
s
3
f
88%
3
3
g
90%
87%
3t
75%
h
3
3
i
j
68%
92%
3
3
3
k
l
93%
72%
85%
Fig. 2 ORTEP representation of compound 3k (Cyn-1324).
toxicity on T-cells up to 1000 mM when compared to that of Cyn.
At high concentration (1000 mM), Cyn became very toxic
whereas the cell survival rate was less than 5%. Similar results
were observed with the efficacy test against T-cells and are
shown in Fig. 4. For the efficacy test (blocking CD28 on T-cells
with the results of reducing IL-2 release), both Cyn and
Cyn-1324 reduced IL-2 production. The reduction rates were
similar at higher concentration for both compounds. To iden-
tify the blocking ability of Cyn-1324 on CD28 of T-cell, a real
binding measurement was completed by using atomic force
spectroscopy (AFM). Comparison of the unbinding force
distribution between CD28 and CD80 without and with the
m
3
3
n
o
84%
86%
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CD80); be ¼ blocking effect; bf ¼ binding force). These experi-
ments were also done by investigating the blocking effect
between CD154 and CD40, and less than 5% was observed.
The immuno-suppressive effect on mice was assessed by
using ovalbumin (OVA) as an immunization inducer and
cyclosporine A (CSA) as a reference drug. For their efficacy
investigation (testing quantity change of IgG and IgE), mice
were divided into four groups (5 mice per group): OVA/Cyn-1324
1 2 3
(n group); OVA/CSA (n group); OVA only (n group) and PBS
buffer only (n group). Blood samples were collected and both
4
Fig. 3 Cytotoxicity tests on T-cells.
quantities of IgG/IgE were measured. Results showed that IgG
was reduced about 30% for the n group (OVA/Cyn-1324) and
1
45% for the n
2
group (OVA/CSA) as compared with the n
3
group
(OVA only) at day 14.
Oppositely, about 23% (n
1 2
group) and 12% (n
group)
reduction of IgE was observed, as shown in Fig. 6. This implied
that Cyn-1324 might not be a better candidate to reduce IgG, but
gives a stronger reduction in IgE as compared with cyclosporine
A. There are four types of hypersensitivity reactions: (a) type I
(
anaphylactic or immediate-type) reaction; (b) type II (cytotoxic)
reaction; (c) type III (immune complex) reaction; (c) type IV
cell-mediated or delayed-type) reaction. Two main factors of
(
immune responses, IgE and IgG, are related to type I and type
IV, respectively. The results of over-reacting behavior will
increase the production of both IgE and IgG. Suppressing type I
symptoms (reducing IgE production) is done by blocking T-cells
from binding with APC cells.
However, the reducing strategy should be only “mild” since
one would expect to bring the immune responses back to
normal, but not completely suppress them. Our current results
showed that Cyn-1324 could reduce IgE to a certain extent (23%
on average) when compared with 12% (on average) using
cyclosporine A. This means that Cyn-1324 could be a potential
candidate for the treatment of anaphylactic immune disease
Fig. 4 Efficacy tests on T-cells.
addition of Cyn-1324 is shown in Fig. 5a and b, respectively. A
larger part distribution of higher unbinding forces was
observed without interruption by Cyn-1324 whereas a larger
part distribution of lower unbinding forces was observed with
the addition of Cyn-1324. The average unbinding force of CD28/
CD80 was about 41.9 (ꢀ5.3) pN. However, the average
unbinding force was reduced to about 29.1 (ꢀ3.3) pN aer block
by Cyn-1324. Thus the “blocking effect” was observed to be
about 31 ꢀ 4% as compared with that Cynarin of 25 ꢀ 7%
(type I), and is better than cyclosporine A. More experiments will
be carried out to investigate its pharmacokinetics in animals to
classify the efficacy with time-release so that immediate
(be
¼
bf (CD28/CD80)-bf(CD28/Cyn-1324/CD80)/bf(CD28/
Fig. 5 In vitro blocking effect is tested by AFM. (a) Unbinding forces distribution diagram between CD28 and CD80 (41.9 pN on average). (b)
Unbinding forces distribution diagram between CD28 and CD80 after addition of Cyn-1324 (29.1 pN on average). The distribution of higher
4
ꢃ1
unbinding forces is reduced with the addition of Cyn-1324. The loading rate and contact time of AFM were 1.44 ꢂ 10 pN s and 0.5 s,
respectively.
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Fig. 6 Animal model test of the immuno-suppressive effects induced by Cyn-1324 and CSA. Both IgG and IgE were used to test Cyn-1324-
treated and CSA-treated ovalbumin (OVA)-sensitized mice. Mice were sensitized by intraperitoneal injections for the n (PBS buffer only), n
OVA), n (OVA + Cyn-1324) and n (OVA + CSA) groups. Blood was obtained from the tail vein. Serum samples were analyzed by mouse-IgG and
1
2
(
3
4
IgE ELISA test. (a) IgG reduction; (b) IgE reduction. Results shown here were taken at day 14. Beyond day 14, similar results were obtained.
Fig. 7 Inhibition of type I hypersensitivity reaction by Cyn-1324.
responses of type I disease can be dose-controlled. Possible we found that compound 3k (Cyn 1324) shows similar efficacy
mechanisms of type I immune response blocked by Cyn-1324 is with lower toxicity as compared to Cynarin. The mechanism of
shown in Fig. 7. In the future, a clinically curing strategy (sup- action of Cyn-1324 on the immune system is identied. Because
pressing the allergic reaction) is that the extra production of IgE of the easy synthesis and low toxicity of Cyn 1324, it may be
from B-cells due to the incoming allergen particles will be further developed to a possible candidate to eliminate allergic
partially ceased by taking Cyn-1324, which will block the type I disease in the future.
attachment of T-cells to B-cells. Without activation of B-cells,
IgE is not produced. Future experiments such as pharmacoki-
netics and ADME (adsorption/distribution/metabolism/
Experimental section
General procedure for the synthesis of (3aR,5R,7R,7aS)-5,7-
dihydroxy-2,2-dimethyl-N-(2-phenylethyl)hexahydro-1,3-
benzodioxole-5-carboxamide (3a)
excretion) will be done to further support the above argu-
ments. Therefore, the mechanism of action (MoA) of Cyn-1324
on the immune system will be understood. This compound
has major valuable benets including the low cost preparation
and facile synthetic strategy with high yield. Its low toxicity may
further lead to it being a potential drug candidate to cure
allergic type I disease in the future.
To a solution of compound 1 (0.1 g, 0.52 mmol) in acetone
2 4
(10 mL) was added conc. H SO (2 drops) and the reaction
mixture was allowed to stir under reux for 2 hours. Aer the
completion of the reaction, the solvent was evaporated, diluted
with ethyl acetate (15 mL), washed with water (2 ꢂ 30 mL) fol-
lowed by brine solution (20 mL). The obtained crude compound
Conclusion
2a (0.1 g, 91%) was pure enough to proceed to the next step. To a
In conclusion, we have synthesized a series of quinic acid solution of compound 2a (0.1 g, 0.46 mmol) in dichloromethane
derivatives for their potential application as immuno-suppres- (5 mL) was added triethylamine (0.07 g, 0.7 mmol) followed by
sive agents against T-cell receptors. Among these compounds, 2-phenylethylamine (0.083 g, 0.7 mmol) and the reaction
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45185.
Spectral data
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300 MHz, CDCl ) d 7.33–7.28 (m, 5H), 7.04 (s, 1H), 4.86 (s, 1H),
.51 (m, 1H), 4.12 (m, 1H), 3.81 (m, 1H), 3.52 (dd, J ¼ 9.2, 6.8 Hz,
H), 3.39 (s, 1H), 2.90–2.73 (m, 1H), 2.81 (m, 1H), 2.37 (m, 1H),
H
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(
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4
2
2
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1
3
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.05–1.88 (m, 2H), 1.48 (s, 3H), 1.33 (s, 3H); C NMR (75 MHz,
) d 176.6, 138.5, 128.8, 128.7, 126.7, 108.7, 76.1, 72.9, 72.1,
5.9, 40.5, 37.0, 35.6, 34.4, 27.1, 24.4; MS (EI) 335.2; HRMS (EI)
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CDCl
6
3
1
1
1
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ꢃ1
calcd for C18
359, 1644.
5
H25NO 335.1733; found 335.1729; IR (cm , neat)
3
Abbreviations
2010, 49, 2860.
1
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APC
Cyn
KBF
CSA
MHC
ORTEP
OVA
TCR
QA
Antigen presenting cell
Cynarin
Key blocking factor
Cyclosporine A
Histocompatibility complex
Oak ridge thermal ellipsoid plot
Ovalbumin
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BMC Pharmacol. Toxicol., 2013, 14, 3.
T-cell receptor
Quinic acid
1
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Acknowledgements
2
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L. T. Leung, E. A. Gullen, P. Chiu, Y. C. Cheng and
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The authors thank the National Health Research Institutes
(NHRI-EX104-10431N1) of Taiwan for the nancial assistance
and for the biochemistry experiments and animal model tests
done in the National Nano Device Laboratories. The research
grant is particularly supported by “Aim for the Top University
Plan” of the National Chiao Tung University and Ministry of
Education, Taiwan. The contribution of Wei-Jen Wu to perform
experiments for CD 40 and CD154 is well-recognized.
2
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