Parallel synthesis and biological evolution of quinic
acid derivatives as immuno-suppressing agents
against T-cell receptors
†
Chih-Yu Huang,aLi-Hsun Chen,bHsuan-Yu Huang,aFeng-Sheng Kao,aYun-Ta Lee,bc Manikandan Selvaraju,bChung-Ming Sun*bcand Hueih-Min Chen*a
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.
Introduction
Immuno-response to exclude the invasion of harmful outside materials is the major defense system in human and other living creatures. Immuno-suppressive treatment for over-reacting patients to control immune responses is highly demanded as the over-reaction of immune cells cannot be self-controlled and may become a big burden for the entire life. In particular, there are two signals (Signal 1 and Signal 2) produced during the conjugation of T-cells and antigen-presenting cells (APC cells such as B-cells or dendritic cells) to activate the adaptive immune responses.1–4 The specialized
formation of intercellular contact aer activation of resting T-cells by APC cells is termed as immunological synapses.5–7A more recent study using single cell force spectroscopy showed that T-cells can be activated by dendritic cells than by B-cells based on the quantity measurement of IL-2 and IFNg secreted from T-cells aer activation.8 Signal 1 is created as the T-cell
receptor (TCR) of T-cells, which strongly binds with major histocompatibility complex (MHC) of APC cells. Signal 2 is a co-stimulation signal, which occurs simultaneously with Signal 1 by two concurrent bindings: CD28 of T-cell to CD80 of APC cell (weaker binding) and CD154 of T-cell to CD40 of APC cell (stronger binding). Aer stimulation (Signal 1) and
co-stimulation (Signal 2) bindings, the strength of these immuno-responses can be estimated by IL-2 released from activated T-cells. If Signal 2 is inhibited, the total release of IL-2 would be reduced consequently.9In the current work, a mild
blocker for mainly blocking CD28 therefore can be found to only inhibit Signal 2 by directly blocking T-cells on their membrane surfaces.
Many known therapeutics, such as cyclosporine A (CSA), cyclophosphamide, tacrolimus (FK506) and azathioprine, have been developed to treat immuno-suppression. Although they are selective and potent to prevent the rejection of organ transplants and in diseases involving the immune system, they block both Signal 1 and Signal 2 to penetrate inside the cell and this causes severely toxic side effects. Under this consideration, development of mild immuno-suppressive agents that partially reduce the immune responses without rough side-effects is urgently needed.
Cynarin is a biologically active chemical constituent of Cyn-ara cardunculus.11Earlier, we reported that Cynarin in Echinacea
purpurea is able to block Signal 2 of T-cell activation specically for immuno-suppression.9,10The blocking effect between CD28
of T-cell and CD80 of B-cell was identied by a small molecule, Cynarin (Cyn), aer owing through an immobilized receptor (AFTIR). Computer simulation of this effect is displayed in Fig. 1. A key blocking factor (KBF) was indicated by a red circle and a main blocking body (quinic acid-like) was assigned. Physical contact of the immunological synapses mentioned above between CD28 of T-cells and CD80 of B-cells was a key 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 natural product Cynarin was found to effectively block the
aNational Applied Research Laboratories, National Nano Device Laboratories,
Biomedical Group, Hsinchu 300-10, Taiwan. E-mail: [email protected]
bDepartment of Applied Chemistry, National Chiao-Tung University, Hsinchu 300,
Taiwan. E-mail: [email protected]
cDepartment 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: 10.1039/c5ra06095h
Cite this: RSC Adv., 2015, 5, 50801
Received 6th April 2015 Accepted 20th May 2015 DOI: 10.1039/c5ra06095h www.rsc.org/advances
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Published on 08 June 2015. Downloaded by University of Oxford on 18/07/2016 07:26:38.
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binding between CD28 and CD80: i.e., the immunological synapses of Signal 2 can be shut down by Cyn.10This blocking
effect induced by Cyn on T-cells has been further conrmed by using atomic force spectroscopy.12However, we found that the
toxicity of Cyn on T-cells is observed. Our computer simulation9
showed that the main blocking interactions between Cynarin and CD28 came from the quinic acid-like structure. Two symmetrical side chains (di-caffeoyl group) may not be essential for the effect of “blocking”. Hence, as a part of our ongoing research tond better immuno-suppressive agents, we synthe-sized a series of Cynarin derivatives by using quinic acid (QA) as a novel scaffold targeted on KBF structure. Many natural product (NP) drugs and NP-derived compounds have been found and applied in clinical trials.13,14Structure-based virtual
screening method is a powerful technique to nd potential target drugs from a signicant number of NP or NP-like compound libraries.15–22 We report herein the preliminary results which indicate that one of the NP-derived compounds, Cyn-1324, has low toxicity and more chemical stability as compared to that of Cynarin. In vivo animal tests further conrmed that Cyn-1324 has an immuno-suppressive effect on ovalbumin-induced allergic mice and therefore potentially is qualied for development as a drug candidate in the future.
Results and discussion
Chemistry
Synthesis of quinic acid (QA) derivatives. The synthesis of quinic acid derivatives was accomplished in a straightforward way by two steps: lactonisation/ketalisation followed by ami-nolysis, as shown in Scheme 1. Earlier, modications at C-1/C-5 hydroxyl groups and formation of macrocycles between carboxylate and C-3 hydroxyl group are reported.23–27 Stable quinic acid derivatives can be synthesized by converting cis C-3/ C-4 hydroxyl groups into a ketal via a reaction with a ketone
using a strong acid catalyst. In addition, the cytotoxicity of quinic acid may be reduced if its C-1 carboxyl group is modied to other functional groups.
Lactonization and ketalization of quinic acid 1 was carried out in the presence of a catalytic amount of sulfuric acid in acetone to provide the acetal lactone intermediates 2 in a single step. Under reux conditions, the reaction took 2 h to complete conversion with a yield of 85%. Alternatively, the use of microwave irradiation in a closed vessel system at 100 C dramatically reduced the reaction time to only 5 min with a maximum yield of 91%. The next transformation is the ami-nolysis of lactone intermediates 2 with various amines. The reaction was accomplished under microwave irradiation at 100C for 5 min to obtain various quinic acid derivatives.
Such aminolysis of lactones requires 18 h to complete conversion under the conventional reuxing conditions. Furthermore, the aminolysis was performed with various amines with different electronic natures to give a variety of quinic acid analogues, as shown in Table 1. All the amines gave satisfactory yields under similar reaction conditions. The structure of compound 3k is also conrmed by X-ray crystal-lography,28as shown in Fig. 2.
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 demonstrated that Cyn-1324 (3k) could effectively inhibit the proliferation of T-cell receptors. The efficacy is comparable with that of Cynarin, which served as a control study.
The percentage cell survival and efficacy of Cyn and Cyn-1324 against T-cells were investigated. The relationship between cell survival (T-cells) vs. concentration of Cyn and Cyn-1324 is shown in Fig. 3. The results implied that Cyn-1324 has low
Fig. 1 Computer simulation for blocking between CD28/T-cell and CD80/B-cell by Cynarin.
Scheme 1 Synthesis of quinic acid analogues 3.
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
Table 1 Synthesis of quinic acid analogues 3
Entry R1R2(C]O) H2NR3 Isolated yield
3a 76% 3b 81% 3c 86% 3d 93% 3e 91% 3f 88% 3g 90% 3h 87% 3i 68% 3j 92% 3k 93% 3l 72% 3m 85% 3n 84% 3o 86% Table 1 (Contd. )
Entry R1R2(C]O) H2NR3 Isolated yield
3p 88%
3q 89%
3r 80%
3s 82%
3t 75%
Fig. 2 ORTEP representation of compound 3k (Cyn-1324).
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 aer block by Cyn-1324. Thus the “blocking effect” was observed to be about 31 4% as compared with that Cynarin of 25 7% (be ¼ bf (CD28/CD80)-bf(CD28/Cyn-1324/CD80)/bf(CD28/
CD80); be¼ 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 (n1group); OVA/CSA (n2group); OVA only (n3group) and PBS
buffer only (n4group). Blood samples were collected and both
quantities of IgG/IgE were measured. Results showed that IgG was reduced about 30% for the n1group (OVA/Cyn-1324) and
45% for the n2group (OVA/CSA) as compared with the n3group
(OVA only) at day 14.
Oppositely, about 23% (n1 group) and 12% (n2 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 (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
Fig. 3 Cytotoxicity tests on T-cells.
Fig. 4 Efficacy tests on T-cells.
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
unbinding forces is reduced with the addition of Cyn-1324. The loading rate and contact time of AFM were 1.44 104pN s1and 0.5 s,
respectively.
responses of type I disease can be dose-controlled. Possible mechanisms of type I immune response blocked by Cyn-1324 is shown in Fig. 7. In the future, a clinically curing strategy (sup-pressing the allergic reaction) is that the extra production of IgE from B-cells due to the incoming allergen particles will be partially ceased by taking Cyn-1324, which will block the 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/ 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 benets 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.
Conclusion
In conclusion, we have synthesized a series of quinic acid derivatives for their potential application as immuno-suppres-sive agents against T-cell receptors. Among these compounds,
we found that compound 3k (Cyn 1324) shows similar efficacy with lower toxicity as compared to Cynarin. The mechanism of action of Cyn-1324 on the immune system is identied. Because of the easy synthesis and low toxicity of Cyn 1324, it may be further developed to a possible candidate to eliminate allergic type I disease in the future.
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)
To a solution of compound 1 (0.1 g, 0.52 mmol) in acetone (10 mL) was added conc. H2SO4 (2 drops) and the reaction
mixture was allowed to stir under reux for 2 hours. Aer the completion of the reaction, the solvent was evaporated, diluted with ethyl acetate (15 mL), washed with water (2 30 mL) fol-lowed by brine solution (20 mL). The obtained crude compound 2a (0.1 g, 91%) was pure enough to proceed to the next step. To a solution of compound 2a (0.1 g, 0.46 mmol) in dichloromethane (5 mL) was added triethylamine (0.07 g, 0.7 mmol) followed by 2-phenylethylamine (0.083 g, 0.7 mmol) and the reaction
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-Cyn-1324-treated ovalbumin (OVA)-sensitized mice. Mice were sensitized by intraperitoneal injections for the n1(PBS buffer only), n2
(OVA), n3(OVA + Cyn-1324) and n4(OVA + CSA) groups. Blood was obtained from the tail vein. Serum samples were analyzed by mouse-IgG and
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.
mixture was subjected to microwave irradiation for 5 minutes at 60C. Aer the completion of the reaction, the solvent was evap-orated, diluted with ethyl acetate (25 mL), and then washed with water (2 50 mL) and brine solution (30 mL). The crude product was puried by ash chromatography using 5% methanol/ dichloromethane to afford the pure product 3a (0.2 g, 76%). Spectral data (3aR,5R,7R,7aS)-5,7-dihydroxy-2,2-dimethyl-N-(2-phenylethyl)-hexahydro-1,3-benzodioxole-5-carboxamide (3a). 1H NMR (300 MHz, CDCl3) d 7.33–7.28 (m, 5H), 7.04 (s, 1H), 4.86 (s, 1H), 4.51 (m, 1H), 4.12 (m, 1H), 3.81 (m, 1H), 3.52 (dd, J¼ 9.2, 6.8 Hz, 2H), 3.39 (s, 1H), 2.90–2.73 (m, 1H), 2.81 (m, 1H), 2.37 (m, 1H), 2.05–1.88 (m, 2H), 1.48 (s, 3H), 1.33 (s, 3H);13C NMR (75 MHz, CDCl3) d 176.6, 138.5, 128.8, 128.7, 126.7, 108.7, 76.1, 72.9, 72.1, 65.9, 40.5, 37.0, 35.6, 34.4, 27.1, 24.4; MS (EI) 335.2; HRMS (EI) calcd for C18H25NO5335.1733; found 335.1729; IR (cm1, neat)
3359, 1644.
Abbreviations
APC Antigen presenting cell
Cyn Cynarin
KBF Key blocking factor CSA Cyclosporine A
MHC Histocompatibility complex ORTEP Oak ridge thermal ellipsoid plot
OVA Ovalbumin
TCR T-cell receptor
QA Quinic acid
Acknowledgements
The authors thank the National Health Research Institutes (NHRI-EX104-10431N1) of Taiwan for thenancial 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.
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