Design, Synthesis and Anti-Platelet Aggregation Activity Study of Ginkgolide-1,2,3-triazole Derivatives

Ginkgolides are the major active component of Ginkgo biloba for inhibition of platelet activating factor receptor. An azide-alkyne Huisgen cycloaddition reaction was used to introduce a triazole nucleus into the target ginkgolide molecules. A series of ginkgolide-1,2,3-triazole conjugates with varied functional groups including benzyl, phenyl and heterocycle moieties was thus synthesized. Many of the designed derivatives showed potent antiplatelet aggregation activities with IC50 values of 5~21 nM.


Introduction
Ginkgo biloba, also named maidenhair tree, the only surviving species from the family Ginkgoaceae has existed for more than 180 million years, and for this reason it was called a "living fossil" by Darwin. G. biloba has been used as a traditional Chinese medicine for a long time for the treatment of lung weakness, asthma, coughing, cancer, etc [1,2]. Ginkgo has also been popular in the Western world since 1965, when a German company developed from ginkgo extracts a botanical medicine named EGB761, with various effects on central nervous system (CNS) diseases, including Alzheimer's disease, dementia, hypomnesia, etc [3,4]. The main components of ginkgo extracts are terpene trilactones (including ginkgolides and bilobalide) and flavonoids [5,6]. Since flavonoids are deemed to hardly penetrate the blood-brain barrier, it is assumed the terpene trilactones from ginkgo extracts should be the major active components for the CNS effects and cardiovascular activity [7,8]. As a natural phospholipid agonist of the platelet activating factor of platelet activating factor receptor (PAFR), platelet activating factor (PAF) regulates various physiological activities of the CNS and peripheral nervous system, including platelet aggregation, blood pressure regulation, inflammation, long-term enhancement of CNS, etc [9]. It's reported that ginkgolides could competitively inhibit the platelet-activating factor receptor (PAFR), resulting in the observed CNS protection and antithrombotic effects [10,11].
To date, 10 ginkgolides (ginkgolide A~Q) [5,6,12] and two bilobalides (bilobalide and bilobanol) [13,14] have been isolated and from G. biloba and their structures elucidated. Most natural ginkgolides displayed significant activity against PAFR, while the bilobalides didn't. In particular, ginkgolide B is the most potent PAFR antagonist discovered in nature [1]. Since the 1970s, many investigations on the structural modifications and structure-activity relationship of ginkgolides It is revealed that both ring C and ring D are essential for anti-PAFR activity [15]. Substituents at the C-7 position decrease the activity [16]. It's noteworthy that the introduction of bulky or aromatic substituents at 10-OH could help to increase activity against PAFR [17,18]. The 1,2,3-triazole moiety, also known simply as triazole, acts as an important structural fragment widely used to construct new drug molecules [19]. The triazole is an electron isostere of the amide group, which easily forms hydrogen bonds, coordination bonds, etc., helping to form a variety of non-covalent bond interactions with target proteins [19,20].
In this paper, a series of ginkgolide derivatives with 1,2,3-triazole moieties connected with various benzyl, phenyl and heterocycle moieties at the C-10 position were designed and synthetized. Their antiplatelet aggregation activities were also evaluated and several derivatives displayed more potent inhibitory effects against PAFR than the natural ginkgolide B, with IC50 values of 5~21 nM, or about 10 to 20 times higher than the natural compound.

Chemistry
Drugability is improved when the triazole moiety is introduced into some leads [20]. The azidealkyne Huisgen cycloaddition reaction, also known as the Huisgen 1,3-dipolar cycloaddition, has been proved to be a powerful tool in construction of triazoles [21]. In this reaction, the azide moiety reacts to a terminal alkyne group to form the triazole ring. The copper(I) catalyst improves both the reaction rate and selectivity. A series of ginkgolide-1,2,3-triazole conjugates with varied functional groups including benzyl, phenyl and heterocycle moieties was synthesized via this method.
Azides 4a-4ee were synthesized by firstly mixing the corresponding aniline, sodium nitrite and concentrated hydrochloride in ethyl acetate at 0 • C for 30 min, then adding sodium azide to the system at room temperature and stirring for another two hours (Scheme 3) [22][23][24]. The resulting products are listed in Table 1.  Azides 4a-4ee were synthesized by firstly mixing the corresponding aniline, sodium nitrite and concentrated hydrochloride in ethyl acetate at 0°C for 30 minutes, then adding sodium azide to the system at room temperature and stirring for another two hours (Scheme 3) [22][23][24]. The resulting products are listed in Table 1.  Azides 4ff-4gg were synthesized by mixing the corresponding benzyl bromide and sodium azide in DMF for two hours (Scheme 4) [22,25]. The resulting products are listed in Table 2.
Firstly, the newly synthesized 10-substituted 1,2,3-triazole-ginkgolide B derivatives (5) were tested by the method of Born [26,27]. The result was given in Figure 3. Which showed some of them exhibited considerable activity better than ginkgolide B. The best results were obtained with compounds 5a, 5n, 5p, 5ff and 5gg.
From the results shown above, we can preliminarily conclude that non-substituted benzyl (compound 5a) and phenyl (compound 5ff) 1,2,3-triazole conjugates have significantly enhanced antiplatelet aggregation activities compared to ginkgolide B (1). We can also see that the substitution at the metaor/and parapositions of the benzyl group reduce the activity. Some small steric hindrance groups (such as methyl and cyano groups) substituted at the ortho-position of the phenyl (compounds 5n and 5p) and benzyl (compound 5gg) groups maintain or slightly reduce the activity. From the results shown above, we can preliminarily conclude that non-substituted benzyl (compound 5a) and phenyl (compound 5ff) 1,2,3-triazole conjugates have significantly enhanced antiplatelet aggregation activities compared to ginkgolide B (1). We can also see that the substitution at the meta-or/and para-positions of the benzyl group reduce the activity. Some small steric hindrance groups (such as methyl and cyano groups) substituted at the ortho-position of the phenyl (compounds 5n and 5p) and benzyl (compound 5gg) groups maintain or slightly reduce the activity.
In further studies, 10-substituted 1,2,3-triazole-ginkgolide A 5' and ginkgolide C 5'' derivatives, having the same moieties as the most active ginkgolide B derivatives 5a, 5n, 5p, 5ff and 5gg, were synthesized and their antiplatelet aggregation activities tested and reported as inhibition ratios at 50 nM). The results are shown in Figure 4. Some of them exhibit better activity than not only their precursors 1' or 1'', but also ginkgolide B (1). The best results were obtained with compounds 5'a, 5'ff, 5'gg and 5''gg. The most active compounds obtained by method above, 5a, 5n, 5p, 5q, 5ff, 5gg, 5'a, 5'ff, 5'gg and 5''gg, were further examined in order to get their activity expressed as an IC50 value. The results are listed in Table 3. 1 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j k 5l 5m 5n 5o 5p 5q 5r 5s 5t 5u 5v 5w 5x 5y 5z 5aa 5bb 5cc 5dd 5ee 5ff 5gg Platelet aggregation (%) 0  In further studies, 10-substituted 1,2,3-triazole-ginkgolide A 5 and ginkgolide C 5" derivatives, having the same moieties as the most active ginkgolide B derivatives 5a, 5n, 5p, 5ff and 5gg, were synthesized and their antiplatelet aggregation activities tested and reported as inhibition ratios at 50 nM). The results are shown in Figure 4. Some of them exhibit better activity than not only their precursors 1 or 1", but also ginkgolide B (1). The best results were obtained with compounds 5 a, 5 ff, 5 gg and 5"gg.  From the results shown above, we can preliminarily conclude that non-substituted benzyl (compound 5a) and phenyl (compound 5ff) 1,2,3-triazole conjugates have significantly enhanced antiplatelet aggregation activities compared to ginkgolide B (1). We can also see that the substitution at the meta-or/and para-positions of the benzyl group reduce the activity. Some small steric hindrance groups (such as methyl and cyano groups) substituted at the ortho-position of the phenyl (compounds 5n and 5p) and benzyl (compound 5gg) groups maintain or slightly reduce the activity.
As we can see, compounds 5a, 5p, 5ff, 5gg and 5 a display promising antiplatelet aggregation activity with IC 50 values ranging from 5-21 nM. Among them compounds 5ff and 5gg were the best among the series of compounds, showing about a 20-fold increase in comparison with the natural ginkgolide B (1). In order to verify if the most active ginkgolide-1,2,3-triazole derivatives could also be considered as potential antiplatelet aggregation therapeutics, compounds 5a, 5n, 5p, 5q, 5ff, 5gg, 5 a, 5 ff and 5"gg were examined to confirm their cytotoxicity using an LDH assay [28]. The results are shown in Figure 5. As we can see, compounds 5a, 5p, 5ff, 5gg and 5'a display promising antiplatelet aggregation activity with IC50 values ranging from 5-21 nM. Among them compounds 5ff and 5gg were the best among the series of compounds, showing about a 20-fold increase in comparison with the natural ginkgolide B (1).
In order to verify if the most active ginkgolide-1,2,3-triazole derivatives could also be considered as potential antiplatelet aggregation therapeutics, compounds 5a, 5n, 5p, 5q, 5ff, 5gg, 5'a, 5'ff and 5''gg were examined to confirm their cytotoxicity using an LDH assay [28]. The results are shown in Figure 5. In addition, compounds 5a, 5p, 5ff, 5gg and 5'a were examined to confirm their toxicity on platelets using the LDH assay [29]. The results are shown in Figure 6.  In addition, compounds 5a, 5p, 5ff, 5gg and 5 a were examined to confirm their toxicity on platelets using the LDH assay [29]. The results are shown in Figure 6. As shown in results above, these most active compounds did not demonstrate toxicity towards cardiomyocytes and platelets (P > 0.05) up to 10 μM (almost two order of magnitude higher than IC50 of ginkgolide B), which suggest that they have a broad therapeutic window/safety window.

General Experimental Procedures
All solvents and reagents of analytical grade were obtained from commercial sources. Flash chromatography was performed using silica gel (200-300 mesh, Qingdao Marine Chemical Group Co., Qingdao, China). All reactions were monitored by TLC on silica gel plates (Merck, Darmstadt, Germany). NMR spectra were recorded in CDCl3 or DMSO at 400 or 600 MHz for 1 H-NMR and 125 or 150 MHz for 13 C-NMR on an Ascent 400 or 600 spectrometer (Bruker, Fallanden, Switzerland). The solvent signal was used as an internal standard. ESI-MS were recorded on an 1200/MSD mass spectrometer (Agilent, Santa Clara, CA, USA). HREIMS were recorded on a LTQ Orbitrap XL mass spectrometer (Thermo, Bremen, Germany).

General Procedures for the Preparation 10-O-propargylated Ginkgolides
Propargyl bromide (2.4 mmol) were slowly added to a mixture of ginkgolide (1, 1' or 1'', 2.0 mmol) and K2CO3 (4.0 mmol) in acetonitrile (15 mL  As shown in results above, these most active compounds did not demonstrate toxicity towards cardiomyocytes and platelets (p > 0.05) up to 10 µM (almost two order of magnitude higher than IC 50 of ginkgolide B), which suggest that they have a broad therapeutic window/safety window.

General Experimental Procedures
All solvents and reagents of analytical grade were obtained from commercial sources. Flash chromatography was performed using silica gel (200-300 mesh, Qingdao Marine Chemical Group Co., Qingdao, China). All reactions were monitored by TLC on silica gel plates (Merck, Darmstadt, Germany). NMR spectra were recorded in CDCl 3 or DMSO at 400 or 600 MHz for 1 H-NMR and 125 or 150 MHz for 13 C-NMR on an Ascent 400 or 600 spectrometer (Bruker, Fallanden, Switzerland). The solvent signal was used as an internal standard. ESI-MS were recorded on an 1200/MSD mass spectrometer (Agilent, Santa Clara, CA, USA). HREIMS were recorded on a LTQ Orbitrap XL mass spectrometer (Thermo, Bremen, Germany).

General Procedures for the Preparation 10-O-propargylated Ginkgolides
Propargyl bromide (2.4 mmol) were slowly added to a mixture of ginkgolide (1, 1 or 1", 2.0 mmol) and K 2 CO 3 (4.0 mmol) in acetonitrile (15 mL). The reaction mixture was refluxed for 24 h under an argon atmosphere and then was extracted with EtOAc three times. The combined organic phases were dried over anhydrous MgSO 4 , filtered, and concentrated in vacuo. The residue was purified by chromatography (SiO 2 , petroleum ether (PE)/EtOAc stepwise elution, 1:1 to EtOAc).

Conclusions
In summary, a series of ginkgolide-1,2,3-triazole conjugates were synthesized through a copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of the corresponding 10-O-propargylated ginkgolides with benzyl, phenyl and heterocyclic azides. Five of them (compounds 5a, 5p, 5ff, 5gg and 5 a) displayed promising antiplatelet aggregation activities with IC 50 values ranging from 5-21 nM. Compounds 5ff and 5gg, having a benzyl group attached at the triazole nucleus were the best among the series of compounds. The most active compounds may be regarded as safe towards normal cells and platelets at therapeutic concentrations.