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Abstract

In order to develop a series of novel compounds with antiplatelet aggregation activities, 3,15-disuccinate-12-coumarin substituted derivatives were designed and synthesized based on the natural product andrographolide. In vitro antiplatelet aggregation activities were evaluated by the turbidimetric method with thrombin, adenosine diphosphate (ADP), arachidonic acid (AA), and collagen as inducers. The biological evaluation revealed that compound 11k showed significant inhibition activity for thrombin, AA, and collagen-induced platelet aggregation at the same time and exhibited a dose-dependent behavior. Compound 11c showed the highest antiplatelet aggregation activity induced by ADP. Most of the derivatives had no significant cytotoxicity. Therefore, our work proved that 3,15-disuccinate-12-coumarin substituted andrographolide derivatives had the potential to become a novel candidate structure for antiplatelet aggregation and deserved further study.

Keywords

andrographolide Design 3,15-Disuccinate-12-coumarin substituted andrographolide derivativesx synthesis anti-platelet aggregation

Thrombus is a quite common cardiovascular disease and seriously threatens human health and life quality.^1,2 Platelet aggregation is one of the important factors leading to thrombosis. Antiaggregation drugs are often used as the top-priority drugs to treat thrombosis.^3,4 Currently, a variety of antiplatelet aggregation drugs have been successfully marketed. According to different mechanisms, there are thromboxane A₂inhibitor (aspirin),⁵ phosphodiesterase inhibitor (cilostazol),⁶ 5-hydroxytrypamine receptor antagonist (sarpogrelate),⁷ platelet membrane glycoproteins Ⅱb/Ⅲa receptor inhibitor (tirofiban),⁸ adenosine diphosphate (ADP) induction inhibitor (clopidogrel),⁹ Ca²⁺ channel antagonist (flunarizine),^10
-12 and protease active receptor-1 (PAR-1) antagonist (vorapaxar sulfate).¹³

Andrographolide 1 (Figure 1), a labdane diterpene, is abundant in the aerial parts of Andrographis paniculata Nees with up to 2.0%.¹⁴ Andrographolide possesses rich biological activities such as antiproliferation,¹⁵ antihepatotoxicity,¹⁶ antimalaria,¹⁷ anti-HIV,¹⁸ anticancer,¹⁹ antibacterium,²⁰ antithrombosis,²¹ anti-inflammatory,²² anti-influenza virus,²³ and antihyperglycemia.²⁴ In recent years, there were a few publications reporting A. paniculata extract and its active diterpenoids possessing antiplatelet aggregation activity.^25
-27 In 2011 and 2013, Lien et al and Lu et al illustrated possible mechanisms of antiplatelet aggregation of andrographolide such as regulating the extrinsic apoptotic or inhibiting endothelial nitric oxide synthase/cyclic guanosine monophosphate.^28,29 In 2017, Liu et al developed a series of novel and potent orally active PAR1 derivatives based on andrographolide and vorapaxar.³⁰ In our previous work, we had found that andrographolide and dehydroandrographolide 2 (Figure 1) had low antiplatelet aggregation activities with ADP as inducer. Their sulfonate 3 (medicinal composition of Xiyanping) and succinate 4 (Figure 1) (medicinal composition of Chuanhuning) showed better antiplatelet aggregation activities, especially the latter,^31
-33 the reason for which were that andrographolide and dehydroandrographolide possessed poor aqueous solubility, but their sulfonate and succinate possessed high aqueous solubility.^34,35

Figure 1

Figure 1 Anti-platelet aggregation compounds (Andrographolides and Coumarins).

The well-known Coumarins, easily available natural compounds, exhibit a wide range of biological activities such as acetylcholinesterase inhibitor,³⁶ antioxidant,³⁷ antitumor,³⁸ anti-inflammatory,³⁹ alpha-chymotrypsin inhibitor,⁴⁰ and anti-HIV.⁴¹ In addition, coumarin derivatives have obvious antiplatelet aggregation activities.^42

-46 Among them, warfarin 5, dicoumarol 6, and acenocoumarol 7 (Figure 1) are marketed drugs for treating thromboembolic diseases.^47,48

Based on the above, it is highly worthwhile to develop accessible and effective methods to construct novel antiplatelet aggregation compounds with andrographolide and coumarins.

Results and Discussion

Preparation of Target Compounds 11

By analyzing the structure, andrographolide had many reaction sites such as 3,19-OH, C-8 = C-17, C-12, C-15. Based on our previous work, to improved aqueous solubility, 2 molecules of carboxylates were still introduced at 3,19-OH. To improve bioactivities, coumarins tried to be introduced at C-12. As expected, we successfully designed the synthetic route to prepare the new skeleton compounds 11 with a combination of andrographolide and coumarins, shown as Scheme 1. First, andrographolide 1 underwent dehydration under pyridine at reflux. At the same time, 2 molecules of monosuccinates were introduced at 3,19-OH to give the intermediate 8.⁴⁹ Afterward, intermediate 8 degraded to the key intermediate 9 by selective oxidation of the C-12,13 olefin bond under potassium permanganate (KMnO₄).⁵⁰ Lastly, target derivatives 11 (3,15-disuccinate-12-coumarin substituted andrographolide derivatives) were synthesized from intermediate 9 reacting with 4-methylcoumarins 10 under potassium tert-butoxide (t-BuOK).⁵¹

Scheme 1

The synthetic route of derivatives 11. Reagents and conditions: (a) pyridine, succinic anhydride, reflux, 12 hours, then, 4 M hydrochloric acid, 60°C, 30 minutes; (b) potassium permangante, tetrahydrofuran, −5°C; (c) potassium tert-butoxide, 4-methylcoumarins 10, dimethyl sulfoxide , room temperature, 5 hours, then, acetic acid, 1 hour, then potassium bicarbonate.

Biological Properties

In vitro antiplatelet aggregation activities of 11a-11l were assessed using Sprague Dawley (SD) male rats arterial blood by the turbidimetric method.⁵² Thrombin, ADP, arachidonic acid (AA), and collagen were used as inducers. Aspirin was employed to be the positive control. All the results were summarized in Table 1. Those compounds with significant antiplatelet aggregation activities were chosen to investigate the relationship between activity and concentration (Figure 2).

Table 1

The IR and IC₅₀ of Target Compounds 11 In Vitro.

Entry	Compounds	Dose(μM)	Thrombin (0.1 U/mL)		ADP (5 mM)		AA (20 μM)		Collagen (1 mg/mL)
Entry	Compounds	Dose(μM)	IR (%)^a	IC₅₀ (μM)^b	IR (%)^a	IC₅₀ (μM)^b	IR (%)^a	IC₅₀ (μM)^b	IR (%)^a	IC₅₀ (μM)^b
1	Aspirin	1.7	24.32 ± 0.25	-	54.03 ± 0.15	0.27	38.33 ± 0.13	0.44	49.27 ± 0.17	0.47
2	1	1.7	23.55 ± 0.23**	-	26.64 ± 0.14*	-	21.46 ± 0.10	-	31.48 ± 0.15*	-
3	3	1.7	30.31 ± 0.16*	-	38.25 ± 0.16**	-	27.65 ± 0.12	-	36.63 ± 0.12	-
4	4	1.7	35.56 ± 0.14**	-	42.54 ± 0.14	0.71	30.48 ± 0.15*	-	41.78 ± 0.13	-
5	11a	1.7	38.71 ± 0.16**	-	41.65 ± 0.12	0.86	31.58 ± 0.16	-	38.56 ± 0.10*	-
6	11b	1.7	42.57 ± 0.22	0.72	47.71 ± 0.11	0.65	23.56 ± 0.12	-	37.55 ± 0.14	-
7	11c	1.7	46.87 ± 0.14*	0.53	53.69 ± 0.15**	0.31	35.75 ± 0.17	0.72	39.36 ± 0.17*	-
8	11d	1.7	36.67 ± 0.21*	-	42.18 ± 0.11**	0.68	20.86 ± 0.10	-	33.78 ± 0.10	-
9	11e	1.7	44.83 ± 0.09**	0.59	37.25 ± 0.18**	-	31.47 ± 0.13	-	44.57 ± 0.16	0.81
10	11f	1.7	41.14 ± 0.05*	0.67	44.67 ± 0.06*	0.75	30.15 ± 0.12	-	42.75 ± 0.14*	-
11	11g	1.7	47.25 ± 0.06	0.53	43.13 ± 0.18	0.46	37.25 ± 0.13*	0.68	49.86 ± 0.18	0.70
12	11h	1.7	42.36 ± 0.13**	0.42	38.75 ± 0.09**	0.64	35.29 ± 0.13	0.63	47.57 ± 0.15	0.67
13	11i	1.7	47.74 ± 0.21*	0.51	43.47 ± 0.14	0.73	36.66 ± 0.13	0.57	48.76 ± 0.14	0.59
14	11j	1.7	43.38 ± 0.18	0.46	40.54 ± 0.21**	0.81	33.43 ± 0.16	-	51.72 ± 0.10	0.54
15	11k	1.7	50.14 ± 0.17**	0.36	41.69 ± 0.13	0.68	46.73 ± 0.12*	0.48	56.24 ± 0.13*	0.50
16	11l	1.7	45.49 ± 0.07*	0.68	36.05 ± 0.22*	-	42.48 ± 0.13	0.56	50.55 ± 0.14*	0.69

ADP, adenosine diphosphate; AA, arachidonic acid; IC₅₀, half-maximal inhibitory concentration.

*P < 0.05 vs aspirin. **P < 0.01 vs aspirin.

^aIRs were expressed as the means ± standard error of 6 experimental replicates (n = 6).

^b“–”, not tested.

Figure 2

Antiplatelet aggregation activities of compounds 11 at different concentrates (final concentration: 1.7, 3.4, and 6.8 µM). (a) Thrombin, (b) adenosine diphosphate (ADP), (c) arachidonic acid (AA), and (d) collagen. Data were expressed as the means ± standard error of 6 experimental replicates (n = 6).

Compared Entry 3 with Entry 4, the IR of compound 4 was obviously higher than compound 3, which revealed that succinic acid salinization could be more beneficial to improve the activities of derivatives. Therefore, in our current work, all the derivatives were succinated at 3,19-OH (Scheme 1).

For thrombin, AA, and collagen as inducers, 11c, 11g, 11i, 11k, and 11l showed prominent antiplatelet aggregation activities equivalent or superior to the positive control. The same substituents attaching to different positions would lead to different activities. Analyzing Entries 6, 9, and 13, the order of activity was 7′-CH₃ (11i) > 6′-CH₃ (11e) > 3′-CH₃ (11b). Analyzing Entries 8, 12, and 16, the order of activity was 7′-Cl (11l) > 6′-Cl (11h) > 3′-Cl (11d). Over all, the activities of 7′-substituted derivatives (11i-11l) were higher than those of 3′-substituted (11b-11d) and 6′-substituted (11e-11h) derivatives. Different substituents attaching to the same positions also resulted in different activities. When attaching to 3′ position, the order of inhibition rate was 3′-COOCH₂CH₃ (11 c) > 3′-CH₃ (11b) > 3′-Cl (11d) (for thrombin, AA, and collagen). When attaching to 6′ position, the orders of inhibition rate were 6′-OCOCH₃ (11g) > 6′-CH₃ (11e) > 6′-Cl (11h) > 6′-OCH₃ (11f) (for thrombin), 7′-OCOCH₃ (11g) > 6′-Cl (11h) > 6′-CH₃ (11e) > 6′-OCH₃ (11f) (for AA and collagen). When attaching to 7′ position, the orders of inhibition rate were 7′-OCOCH₃ (11k) > 7′-CH₃ (11i) > 7′-Cl (11l) > 7′-OCH₃ (11j) (for thrombin), 7′-OCOCH₃ (11k) > 7′-Cl (11l) > 7′-CH₃ (11i) > 7′-OCH₃ (11j) (for AA), and 7′-OCOCH₃ (11k) > 7′-OCH₃ (11j) > 7′-Cl (11l) > 7′-CH₃ (11i) (for collagen). The electron effect of substituents may had little impact on activities. When methoxyl and chloro groups were introduced into 3′, 6′, 7′ positions, antiplatelet aggregation activities would subside, especially for thrombin and AA. Interestingly, 11k strongly inhibited thrombin, AA, and collagen-induced platelet aggregation at the same time and exhibited a dose-dependent behavior (Figure 2(a, c, d)).

For ADP as inducer, only 11c displayed good antiplatelet aggregation activities and also exhibited a dose-dependent behavior (Figure 2(b)). The activities of 3′-substituted derivatives (11b-11d) were higher than the 6′-substituted (11e-11h) and 7′-substituted (11i-11l). When chloro group attaching to 3′, 6′, 7′ positions, antiplatelet aggregation activities also subsided.

To sum up, for thrombin, AA, and collagen as inducers, 11k (for thrombin, IR: 50.14%, half-maximal inhibitory concentration [IC₅₀]: 0.36 µM; for AA, IR: 46.73%, IC₅₀: 0.48 µM; for collagen, IR: 56.24%, IC₅₀: 0.50 µM) showed the best antiplatelet aggregation activity. For ADP as inducer, 11c (IR: 53.69%, IC₅₀: 0.31 µM) displayed the highest antiplatelet aggregation activity and was nearly equivalent to the positive control drug aspirin.

Cytotoxicity Assay In Vitro

Mouse fibroblast cells (L929) were selected to evaluate cell toxicity of target derivatives 11. The absorbance and survival rates were summarized in Table 2. The result revealed that most of derivatives had no significant cytotoxicity. Among them, the survival rates of 11c, 11e, 11j, and 11k were higher than aspirin at the dose of 10 and 100 µM.

Table 2

The Absorbance and Survival Rates of Target Compounds 11 In Vitro.

Compounds	Dose(μM)	Absorbance	Survival rate (%)^a	Compounds	Dose(μM)	Absorbance	Survival rate(%)^a
Blank group	-	0.202	-	11f	10 100	0.240 0.229	68.30 ± 0.06* 47.67 ± 0.07
Control group	-	0.258	-	11g	10 100	0.241 0.231	70.35 ± 0.06 51.64 ± 0.06*
Aspirin	10 100	0.242 0.232	72.02 ± 0.08 54.33 ± 0.06*	11h	10 100	0.244 0.229	74.08 ± 0.08 47.67 ± 0.06
11a	10 100	0.239 0.226	66.02 ± 0.06 43.56 ± 0.09*	11i	10 100	0.245 0.232	77.23 ± 0.10* 54.33 ± 0.06
11b	10 100	0.238 0.226	64.60 ± 0.11 43.56 ± 0.09	11j	10 100	0.247 0.233	79.69 ± 0.09* 55.87 ± 0.06
11c	10 100	0.245 0.237	77.23 ± 0.10* 62.31 ± 0.05	11k	10 100	0.245 0.236	77.23 ± 0.10** 60.30 ± 0.08
11d	10 100	0.245 0.231	77.23 ± 0.10* 51.64 ± 0.06	11l	10 100	0.241 0.227	70.35 ± 0.06* 45.37 ± 0.08
11e	10 100	0.247 0.238	79.69 ± 0.09** 64.60 ± 0.11

*P < 0.05 vs aspirin. **P < 0.01 vs aspirin.

^aSurvival rates were expressed as the means ± standard error of 6 experimental replicates (n = 6).

Conclusion

Through our efforts, 3,15-disuccinate-12-coumarin substituted derivatives 11 were designed and synthesized based on the natural product andrographolide. By preliminary biological evaluation, these synthesized compounds showed prominent antiplatelet aggregation activities in response to thrombin, ADP, AA, and collagen agonists in vitro. Compound 11k showed the best activities when thrombin, AA, and collagen were inducers. Among them, compound 11c had the highest antiplatelet aggregation activities when ADP was inducer. Most of the derivatives had no significant cytotoxicity. Therefore, our research results proved that 3,15-disuccinate-12-coumarin substituted andrographolide derivatives had the potential to become a novel candidate structure for antiplatelet aggregation. The most active compounds were to be selected to measure bleeding time and mechanisms of action in later work.

Experimental

General

Infrared spectra were determined on a Nicolet Avatar-370 spectrometer in potassium bromide (ν in cm⁻¹). Melting points were measured on a Büchi B-540 capillary melting point apparatus and uncorrected. Electrospray ionization-mass spectra (ESI-MS) were determined on a Thermo Finnigan LCQ-Advantage. High-resolution MS (HRMS) was determined on an Agilent 6210 TOF instrument. ¹H nuclear magnetic resonance (NMR) and ¹³C NMR spectra were recorded on Varian Mercury Plus-400 spectrometer (400 and 100 MHz) in dimethyl sulfoxide (DMSO)-d ₆ or D₂O, δ in parts per million, J in Hertz, using tetramethylsilane as an internal standard. Platelet aggregation rates were measured on LG-PABER Platelet aggregation apparatus (made in Beijing Shidi Scientific Instrument Co. LTD, Beijing). Column chromatography was carried out on silicagel (200-300 grading). Andrographolide 1 was provided by Chengdu Biochemical Biotechnology co. LTD (Chengdu, China). All other analytical reagents like pyridine, tetrahydrofuran (THF), methanol (MeOH), acetic acid (AcOH), DMSO, succinic anhydride, KMnO₄, t-BuOK, 4-methylcoumarins were commercially available and used directly without further purification.

Synthesis of Derivatives

Synthesis of intermediate 8

Andrographolide 1 (60.0 g, 171.3 mmol) was dissolved in anhydrous pyridine (200 mL) and then succinic anhydride (48.0 g, 479.6 mmol) was added. The mixture was stirred at reflux until total consumption of the starting material about 12 hours under monitoring of thin-layer chromatography (TLC). Afterward, most of the pyridine should be evaporated under reduced pressure. The mixture cooled to 60°C and was poured to 4M hydrochloric acid slowly with stirring vigorously for 30 minutes to keep pH = 5-6. Lastly, the mixture was filtered and washed with water. The residue was purified by column chromatography (dichloromethane [CH₂Cl₂]:MeOH:AcOH = 20:1:0.05) and afforded intermediate 8, milky solid, 50.2 g, yield 55.1%. ¹H NMR (400 MHz, DMSO-d ₆): δ = 7.65 (1H, s, 14 H), 6.23 (1H, dd, J = 6.6 Hz, J = 15.8 Hz, 11H), 5.10 (1H, s, 17 Ha), 4.92 (2H, s, 15H), 4.87 (1H, d, J = 15.8 Hz, 12H), 4.43 (1H, s, 17-Hb), 4.13 (1H, d, J = 11.8 Hz, 19 Ha), 3.88-3.64 (2H, m, 3H, 19-Hb), 2.83-2.52 (9H, m, 9,22,23,26,27H), 2.15-1.42 (9H, m, 1,2,5,6,7H), 1.04 (3H, s, 18 H), 0.97 (3H, s, 20 H). ¹³C NMR (100 MHz, DMSO-d ₆): δ = 174.7 (C-24), 174.5 (C-28), 173.1 (C-21), 173.0 (C-25), 170.4 (C-16), 148.8 (C-8), 138.6 (C-11), 136.1 (C-14), 131.7 (C-13), 120.2 (C-12), 109.1 (C-17), 75.1 (C-3), 70.3 (C-15), 65.9 (C-19), 60.5 (C-9), 47.7 (C-5), 40.8 (C-4), 39.5 (C-10), 36.3 (C-7), 33.6 (C-1), 29.3 (C-22), 29.2 (C-26), 29.1 (C-23), 29.0 (C-27), 24.3 (C-2), 24.0 (C-6), 21.7 (C-18), 14.3 (C-20) ppm. MS (ESI): m/z (%) =533.2 ([M + H]⁺, 100%). HRMS (ESI) calcd for C₂₈H₃₇O₁₀ [M + H]⁺: 533.2388; found 533.2396.

Synthesis of intermediate 9

Intermediate 8 (48.0 g, 90.2 mmol) was dissolved in THF (200 mL) and then KMnO₄ (21.4 g, 135.3 mmol) was added slowly for 2 hours at −5°C. Then the mixture continued to be stirred for another 5 hours until total consumption of compound 8 under the monitoring of TLC. After reaction, the mixture was filtered off and the solvent was removed under vacuum. The residue was purified by column chromatography (CH₂Cl₂:MeOH:AcOH = 40:1:0.05) and afforded intermediate 9, white solid, 25.3 g, yield 62.1%. ¹H NMR (400 MHz, DMSO-d ₆): δ = 9.72 (1H, d, J = 2.4 Hz, 11H), 5.25 (1H, s, 12Ha), 5.16 (1H, s, 12-Hb), 4.12 (1H, d, J = 11.8 Hz, 14-Ha), 3.89-3.68 (2H, m, 3H, 14-Hb), 2.85-2.57 (9H, m, 9,17,18,21,22H), 2.10–1.42 (9H, m, 1,2,5,6,7H), 1.03 (3H, s, 13H), 0.93 (3H, s, 15H). ¹³C NMR (100 MHz, DMSO-d ₆): δ = 205.2 (C-11), 174.7 (C-19), 174.5 (C-23), 173.1 (C-16), 173.0 (C-20), 145.1 (C-8), 109.1 (C-12), 75.2 (C-3), 67.6 (C-9), 65.9 (C-14), 47.5 (C-5), 40.4 (C-4), 38.5 (C-10), 36.3 (C-7), 32.9 (C-1), 29.3 (C-17), 29.2 (C-21), 29.1 (C-18), 29.0 (C-22), 23.9 (C-2), 23.7 (C-6), 21.8 (C-13), 14.3 (C-15) ppm. MS (ESI): m/z (%) =453.2 ([M + H]⁺, 100%). HRMS (ESI) calcd for C₂₃H₃₃O₉ [M + H]⁺: 453.2125; found 453.2136.

Synthesis of product 11 (11a was selected as example)

Compound 9 (2.0 g, 4.4 mmol) and t-BuOK (0.64 g, 5.7 mmol) were dissolved in dry DMSO (20 mL) and then 4-methylcoumarin (1.1 g, 6.6 mmol) was added. The mixture was stirred at room temperature for about 5 hours until total consumption of compound 9 under the monitoring of TLC. Then a mixed solution of AcOH and water (15 mL, 3:1) was added to the mixture with stirring for about 2 hours. After reaction, sodium bicarbonate solution was dropped in to neutralize the mixture. CH₂Cl₂ (3 × 50 mL) was poured into the mixture. The combined CH₂Cl₂ extract was washed with brine (3 × 30 mL) and dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was dissolved in ethanol (10 mL) and then potassium bicarbonate saturated solution (1.1 equiv.) was dropped slowly at room temperature. Afterward, the mixture produced a large number of bubbles and changed gradually from clear to milky white turbidity. Lastly, the mixture was filtered and filter cake was dried to give the following compounds.

Monopotassium 3,15-disuccinate-12-coumarin andrographolide 11a

Pale yellow solid, 1.18 g, yield 42.5%, purity 96.2%. ¹H NMR (400 MHz, D₂O): δ = 7.84-7.81 (1H, m, 5′-H), 7.65-7.45 (3H, m, 6′,7′,8′-H), 6.47 (1H, s, 3′-H), 6.19 (1H, d, J = 15.8 Hz, 12H), 5.72 (1H, m, 11H), 5.03 (1H, s, 13Ha), 4.87 (1H, s, 13-Hb), 4.13 (1H, d, J = 11.8 Hz, 15-Ha), 3.93-3.78 (2H, m, 3H, 15-Hb), 2.83-2.52 (9H, m, 9,18,19,22,23H), 2.12-1.62 (9H, m, 1,2,5,6,7H), 1.05 (3H, s, 14H), 0.97 (3H, s, 16H). ¹³C NMR (100 MHz, D₂O): δ = 174.7 (C-20), 174.6 (C-24), 173.2 (C-17), 173.1 (C-21), 162.2 (C-4′), 159.3 (C-2′), 150.1 (C-9′), 148.8 (C-8), 135.5 (C-11), 129.1 (C-5′), 128.3 (C-7'), 128.0 (C-12), 127.2 (C-10′), 125.4 (C-6′), 111.2 (C-8′), 109.1 (C-13), 104.7 (C-3′), 75.0 (C-3), 65.9 (C-15), 60.9 (C-9), 47.5 (C-5), 40.9 (C-4), 39.8 (C-10), 36.7 (C-7), 33.6 (C-1), 29.5 (C-18), 29.2 (C-22), 29.1 (C-19), 29.0 (C-23), 24.3 (C-2), 24.0 (C-6), 21.9 (C-14), 14.7 (C-16) ppm. MS (ESI): m/z (%) =633.2 ([M + H]⁺, 100%). HRMS (ESI) calcd for C₃₃H₃₈KO₁₀ [M + H]⁺: 633.2102; found 633.2116.

Monopotassium 3,15-disuccinate-12-(3′-methylcoumarin) andrographolide 11b

White solid, 1.16 g, yield 40.9%, purity 95.7%. ¹H NMR (400 MHz, D₂O): δ = 7.86-7.83 (1H, m, 5′-H), 7.65–7.42 (3H, m, 6′,7′,8′-H), 6.15 (1H, d, J = 15.8 Hz, 12H), 5.75 (1H, m, 11H), 5.04 (1H, s, 13Ha), 4.87 (1H, s, 13-Hb), 4.11 (1H, d, J = 11.8 Hz, 15-Ha), 3.92-3.79 (2H, m, 3H, 15-Hb), 2.83-2.55 (9H, m, 9,18,19,22,23H), 2.43 (3H, s, 3′-CH₃), 2.12-1.68 (9H, m, 1,2,5,6,7 H), 1.01 (3H, s, 14H), 0.90 (3H, s, 16H). ¹³C NMR (100 MHz, D₂O): δ = 174.7 (C-20), 174.4 (C-24), 173.4 (C-17), 173.1 (C-21), 162.4 (C-4′), 159.4 (C-2′), 150.2 (C-9′), 148.7 (C-9), 135.4 (C-11), 129.2 (C-5′), 128.2 (C-7′), 128.0 (C-12), 127.3 (C-10), 125.4 (C-6′), 120.2 (C-3′), 111.1 (C-8′), 109.2 (C-13), 75.1 (C-3), 65.8 (C-15), 60.9 (C-9), 47.6 (C-5), 40.8 (C-4), 39.8 (C-10), 36.9 (C-7), 33.5 (C-1), 29.4 (C-18), 29.2 (C-22), 29.0 (C-19), 28.8 (C-23), 24.3 (C-2), 24.0 (C-6), 21.4 (C-14), 14.7 (C-16), 10.9 (3′-CH₃) ppm. MS (ESI): m/z (%) =647.2 ([M + H]⁺, 100%). HRMS (ESI) calcd for C₃₄H₄₀KO₁₀ [M + H]⁺: 647.2259; found 647.2273.

Monopotassium 3,15-disuccinate-12-(3′-ethoxyformylcoumarin) andrographolide 11c,

Pale yellow solid, 1.04 g, yield 33.5%, purity 96.4%. ¹H NMR (400 MHz, D₂O): δ = 7.85-7.82 (1H, m, 5′-H), 7.66–7.48 (3H, m, 6′,7′,8′-H), 6.19 (1H, d, J = 15.8 Hz, 12H), 5.73 (1H, m, 11H), 5.04 (1H, s, 13Ha), 4.88 (1H, s, 13-Hb), 4.21 (2H, q, J = 8.2 Hz, 3′-COOCH ₂CH₃), 4.14 (1H, d, J = 11.8 Hz, 15-Ha), 3.91-3.88 (2H, m, 3H, 15-Hb), 2.83-2.56 (9H, m, 9,18,19,22,23H), 2.12-1.62 (9H, m, 1,2,5,6,7 H), 1.29 (3H, t, J = 8.2 Hz, 3′-COOCH₂ CH ₃), 1.03 (3H, s, 14 H), 0.97 (3H, s, 16 H). ¹³C NMR (100 MHz, D₂O): δ = 174.7 (C-20), 174.6 (C-24), 173.4 (C-17), 173.1 (C-21), 165.0 (C-3′-COOCH₂CH₃), 159.2 (C-2), 150.1 (C-9′), 148.6 (C-8), 144 (C-4′) 135.5 (C-11), 129.2 (C-5′), 128.3 (C-7′), 128.0 (C-12), 127.1 (C-10′), 125.4 (C-6′), 115.1 (C-3′), 111.2 (C-8′), 109.2 (C-13), 75.1 (C-3), 65.9 (C-15), 61.4 (C-3′-COOCH ₂CH₃), 60.8 (C-9), 47.5 (C-5), 40.8 (C-4), 39.9 (C-10), 36.8 (C-7), 33.4 (C-1), 29.5 (C-18), 29.3 (C-22), 29.1 (C-19), 29.0 (C-23), 24.2 (C-2), 24.0 (C-6), 21.8 (C-14), 14.7 (C-16), 14.2 (C-3′-COOCH₂ CH ₃) ppm. MS (ESI): m/z (%) =705.2 ([M + H]⁺, 100%). HRMS (ESI) calcd for C₃₆H₄₂KO₁₂ [M + H]⁺: 705.2313; found 705.2330.

Monopotassium 3,15-disuccinate-12-(3′-chlorocoumarin) andrographolide 11d

Pale yellow solid, 1.33 g, yield 45.5%, purity 96.2%. ¹H NMR (400 MHz, D₂O): δ = 7.86-7.82 (1H, m, 5′-H), 7.65-7.42 (3H, m, 6′,7′,8′-H), 6.17 (1H, d, J = 15.8 Hz, 12H), 5.79 (1H, m, 11H), 5.04 (1H, s, 13Ha), 4.88 (1H, s, 13-Hb), 4.13 (1H, d, J = 11.8 Hz, 15-Ha), 3.93-3.84 (2H, m, 3H, 15-Hb), 2.80-2.52 (9H, m, 9,18,19,22,23H), 2.12-1.68 (9H, m, 1,2,5,6,7H), 1.05 (3H, s, 14H), 0.96 (3H, s, 16H). ¹³C NMR (100 MHz, D₂O): δ = 174.6 (C-20), 174.5 (C-24), 173.2 (C-17), 173.1 (C-21), 159.7 (C-2′), 153.4 (C-4′), 150.1 (C-9′), 148.6 (C-8), 135.5 (C-11), 129.2 (C-5′), 128.3 (C-7′), 128.0 (C-12), 127.3 (C-10′), 125.4 (C-6′), 116.7 (C-3′), 111.1 (C-8′), 109.2 (C-13), 75.1(C-3), 65.9 (C-15), 60.8 (C-9), 47.5 (C-5), 40.8 (C-4), 39.7 (C-10), 36.7 (C-7), 33.7 (C-1), 29.4 (C-18), 29.2 (C-22), 29.1 (C-19), 29.0 (C-23), 24.2 (C-2), 24.0 (C-6), 21.7 (C-14), 14.9 (C-16) ppm. MS (ESI): m/z (%) = 667.2 ([M + H]⁺, C₃₃H₃₇ ³⁵ClKO₁₀, 100%), 669.2 ([M + H]⁺, C₃₃H₃₇ ³⁷ClKO₁₀, 33%). HRMS (ESI) calcd for C₃₃H₃₇ ³⁵ClKO₁₀ [M + H]⁺: 667.1712, found 667.1734; for C₃₃H₃₇ ³⁷ClKO₁₀ [M + H]⁺: 669.1683, found 669.1698.

Monopotassium 3,15-disuccinate-12-(6′-methylcoumarin) andrographolide 11e

white solid, 1.14 g, yield 40.2%, purity 95.3%. ¹H NMR (400 MHz, D₂O): δ = 7.59-7.56 (1H, m, 5′-H), 7.31–7.26 (2H, m, 7′,8′-H), 6.46 (1H, s, 3′-H), 6.19 (1H, d, J = 15.8 Hz, 12H), 5.73 (1H, m, 11H), 5.03 (1H, s, 13Ha), 4.86 (1H, s, 13-Hb), 4.14 (1H, d, J = 11.8 Hz, 15-Ha), 3.93-3.83 (2H, m, 3H, 15-Hb), 2.83-2.53 (9H, m, 9,18,19,22,23H), 2.34 (3H, s, 6′-CH₃), 2.12-1.65 (9H, m, 1,2,5,6,7H), 1.02 (3H, s, 14H), 0.98 (3H, s, 16H). ¹³C NMR (100 MHz, D₂O): δ = 174.7 (C-20), 174.6 (C-24), 173.2 (C-17), 173.1 (C-21), 162.2 (C-4′), 159.3 (C-2′), 148.8 (C-8), 147.2 (C-9′), 135.6 (C-11), 135.4 (C-6′), 132.3 (C-7′), 128.0 (C-12), 127.2 (C-10′), 127.1 (C-5′), 116.9 (C-8′), 109.1 (C-13), 104.7(C-3′), 75.0 (C-3), 65.9 (C-15), 60.7 (C-9), 47.5 (C-5), 40.9 (C-4), 39.8 (C-10), 36.9 (C-7), 33.4 (C-1), 29.5 (C-18), 29.2 (C-22), 29.1 (C-19), 29.0 (C-23), 24.3 (C-2), 24.1 (C-6), 21.9 (C-14), 21.6 (6′-CH₃), 14.5 (C-16) ppm. MS (ESI): m/z (%) =647.2 ([M + H]⁺, 100%). HRMS (ESI) calcd for C₃₄H₄₀KO₁₀ [M + H]⁺: 647.2259; found 647.2273.

Monopotassium 3,15-disuccinate-12-(6′-methoxylcoumarin) andrographolide 11f

Pale yellow solid, 1.22 g, yield 41.8%, purity 96.0%. ¹H NMR (400 MHz, D₂O): δ = 7.79-7.76 (1H, m, 8′-H), 7.20-7.16 (1H, m, 7′-H), 6.90-6.86 (1H, m, 5′-H), 6.47 (1H, s, 3′-H), 6.17 (1H, d, J = 15.8 Hz, 12H), 5.71 (1H, m, 11H), 5.03 (1H, s, 13Ha), 4.88 (1H, s, 13-Hb), 4.16 (1H, d, J = 11.8 Hz, 15-Ha), 3.90-3.78 (5H, m, 3H, 15-Hb, 4′-CH₃), 2.83-2.56 (9H, m, 9,18,19,22,23H), 2.12-1.65 (9H, m, 1,2,5,6,7H), 1.04 (3H, s, 14H), 0.98 (3H, s, 16H). ¹³C NMR (100 MHz, D₂O): δ = 174.6 (C-20), 174.5 (C-24), 173.5 (C-17), 173.1 (C-21), 162.2 (C-4′), 159.3 (C-2′), 157.4 (C-6′), 148.8 (C-8), 142.4 (C-9′), 135.5 (C-11), 128.2 (C-10′), 128.1 (C-12), 124.2 (C-8′), 117.8 (C-7′), 110.9 (C-5′), 109.1 (C-13), 104.7 (C-3′), 75.0 (C-3), 65.8 (C-15), 60.9 (C-9), 55.9 (6′-OCH₃), 47.5 (C-5), 40.9 (C-4), 39.8 (C-10), 36.9 (C-7), 33.5 (C-1), 29.5 (C-18), 29.2 (C-22), 29.1 (C-19), 29.0 (C-23), 24.3 (C-2), 24.1 (C-6), 21.8 (C-14), 14.5 (C-16) ppm. MS (ESI): m/z (%) = 663.2 ([M + H]⁺, 100%). HRMS (ESI) calcd for C₃₄H₄₀KO₁₁ [M + H]⁺: 663.2208; found 663.2219.

Monopotassium 3,15-disuccinate-12-(6′-methanoylcoumarin) andrographolide 11g

Pale yellow solid, 1.02 g, yield 33.6%, purity 96.3%. ¹H NMR (400 MHz, D₂O): δ = 7.87-7.84 (1H, m, 8′-H), 7.17-7.12 (2H, m, 5′,7′-H), 6.47 (1H, s, 3′-H), 6.19 (1H, d, J = 15.8 Hz, 12H), 5.72 (1H, m, 11H), 5.03 (1H, s, 13Ha), 4.87 (1H, s, 13-Hb), 4.13 (1H, d, J = 11.8 Hz, 15-Ha), 3.93-3.78 (2H, m, 3H, 15-Hb), 2.83-2.52 (9H, m, 9,18,19,22,23H), 2.28 (3H, s, 6′-OCOCH₃), 2.12-1.62 (9H, m, 1,2,5,6,7H), 1.05 (3H, s, 14H), 0.97 (3H, s, 16H). ¹³C NMR (100 MHz, D₂O): δ = 174.7 (C-20), 174.6 (C-24), 173.4 (C-17), 173.1 (C-21), 169.0 (6′-OCOCH₃), 162.2 (C-4′), 159.3 (C-2′), 148.8 (C-8), 148.0 (C-6′), 146.9 (C-9′), 135.5 (C-11), 128.0 (C-12), 127.6 (C-10′), 123.9 (C-8′), 121.3 (C-7′), 118.1 (C-5′), 109.1 (C-13), 104.7 (C-3′), 75.1 (C-3), 65.8 (C-15), 60.9 (C-9), 47.4 (C-5), 40.7 (C-4), 39.6(C-10), 36.8 (C-7), 33.5 (C-1), 29.5 (C-18), 29.3 (C-22), 29.1 (C-19), 29.0 (C-23), 24.2 (C-2), 24.0 (C-6), 21.7 (C-14), 20.3 (6′-OCOCH ₃), 14.6 (C-16) ppm. MS (ESI): m/z (%) =691.2 ([M + H]⁺, 100%). HRMS (ESI) calcd for C₃₅H₄₀KO₁₂ [M + H]⁺: 691.2157; found 691.2172.

Monopotassium 3,15-disuccinate-12-(6′-chlorocoumarin) andrographolide 11h

pale yellow solid, 1.16 g, yield 39.5%, purity 96.4%. ¹H NMR (400 MHz, D₂O): δ = 8.04-8.01 (1H, m, 5′-H), 7.48-7.36 (2H, m, 7′,8′-H), 6.46 (1H, s, 3′-H), 6.18 (1H, d, J = 15.8 Hz, 12H), 5.75 (1H, m, 11H), 5.05 (1H, s, 13Ha), 4.87 (1H, s, 13-Hb), 4.17 (1H, d, J = 11.8 Hz, 15-Ha), 3.90-3.88 (2H, m, 3H, 15-Hb), 2.83-2.57 (9H, m, 9,18,19,22,23H), 2.14-1.65 (9H, m, 1,2,5,6,7H), 1.04 (3H, s, 14H), 0.93 (3H, s, 16H). ¹³C NMR (100 MHz, D₂O): δ = 174.7 (C-20), 174.6 (C-24), 173.2 (C-17), 173.1 (C-21), 162.2 (C-4′), 159.3 (C-2′), 148.8 (C-8), 148.2(C-9′), 135.5 (C-11), 131.0 (C-6′), 129.3(C-7′), 128.6 (C-10′), 128.0 (C-12), 126.8 (C-5′), 122.9 (C-8′), 109.1 (C-13), 104.7 (C-3′), 75.1 (C-3), 65.9 (C-15), 60.8 (C-9), 47.5 (C-5), 40.8 (C-4), 39.8 (C-10), 36.8 (C-7), 33.4 (C-1), 29.5 (C-18), 29.4 (C-22), 29.2 (C-19), 29.1 (C-23), 24.3 (C-2), 24.0 (C-6), 21.9 (C-14), 14.8 (C-16) ppm. MS (ESI): m/z (%) = 667.2 ([M + H]⁺, C₃₃H₃₇ ³⁵ClKO₁₀, 100%), 669.2 ([M + H]⁺, C₃₃H₃₇ ³⁷ClKO₁₀, 33%). HRMS (ESI) calcd for C₃₃H₃₇ ³⁵ClKO₁₀ [M + H]⁺: 667.1712, found 667.1736; for C₃₃H₃₇ ³⁷ClKO₁₀ [M + H]⁺: 669.1683, found 669.1699.

Monopotassium 3,15-disuccinate-12-(7'-methylcoumarin) andrographolide 11i

Yellow solid, 1.07 g, yield 37.6%, purity 95.5%. ¹H NMR (400 MHz, D₂O): δ = 7.74-7.71 (1H, m, 5′-H), 7.03-7.00 (2H, m, 6′,8′-H), 6.48 (1H, s, 3′-H), 6.18 (1H, d, J = 15.8 Hz, 12H), 5.73 (1H, m, 11H), 5.03 (1H, s, 13Ha), 4.87 (1H, s, 13-Hb), 4.14 (1H, d, J = 11.8 Hz, 15-Ha), 3.92-3.78 (2H, m, 3H, 15-Hb), 2.83-2.61 (9H, m, 9,18,19,22,23H), 2.34 (3H, s, 7′-CH₃), 2.12-1.62 (9H, m, 1,2,5,6,7H), 1.03 (3H, s, 14H), 0.98 (3H, s, 16H). ¹³C NMR (100 MHz, D₂O): δ = 174.7 (C-20), 174.6 (C-24), 173.2 (C-17), 173.1 (C-21), 162.1 (C-4′), 159.3 (C-2′), 150.0 (C-9′), 148.8 (C-8), 143.1 (C-7′), 135.5 (C-11), 126.7 (C-5'), 128.0 (C-12), 124.2 (C-10′), 125.7 (C-6′), 117.2 (C-8′), 109.2 (C-13), 104.7 (C-3′), 75.0 (C-3), 65.9 (C-15), 60.8 (C-9), 47.5 (C-5), 40.8 (C-4), 39.8 (C-10), 36.6 (C-7), 33.4 (C-1), 29.5 (C-18), 29.3 (C-22), 29.1 (C-19), 29.0 (C-23), 24.3 (C-2), 24.0 (C-6), 21.9(C-14), 21.2 (7′-CH₃), 14.7 (C-16) ppm. MS (ESI): m/z (%) =647.2 ([M + H]⁺, 100%). HRMS (ESI) calcd for C₃₄H₄₀KO₁₀ [M + H]⁺: 647.2259; found 647.2276.

Monopotassium 3,15-disuccinate-12-(7'-methoxylcoumarin) andrographolide 11j

Pale yellow solid, 1.07 g, yield 36.9%, purity 96.3%. ¹H NMR (400 MHz, D₂O): δ = 7.74-7.71 (1H, m, 5′-H), 6.96-6.92 (2H, m, 6′,8′-H), 6.45 (1H, s, 3′-H), 6.19 (1H, d, J = 15.8 Hz, 12H), 5.72 (1H, m, 11H), 5.02 (1H, s, 13-Ha), 4.86 (1H, s, 13-Hb), 4.12 (1H, d, J = 11.8 Hz, 15-Ha), 3.93-3.81 (5H, m, 3H, 15-Hb, 7′-OCH₃), 2.83-2.64 (9H, m, 9,18,19,22,23H), 2.13-1.67 (9H, m, 1,2,5,6,7H), 1.04 (3H, s, 14H), 0.96 (3H, s, 16H). ¹³C NMR (100 MHz, D₂O): δ = 174.7 (C-20), 174.6 (C-24), 173.2 (C-17), 173.1 (C-21), 162.1 (C-4′), 160.3 (C-7′), 159.3 (C-2′), 151.1 (C-9′), 148.8 (C-8), 135.5 (C-11), 130.8 (C-5′), 128.0 (C-12), 119.2 (C-10′), 111.2 (C-6′), 109.1 (C-13), 104.7 (C-3′), 98.2 (C-8′), 75.1 (C-3), 65.7 (C-15), 60.9 (C-9), 55.8 (7′-OCH₃), 47.6 (C-5), 40.9 (C-4), 39.8 (C-10), 36.7 (C-7), 33.4 (C-1), 29.4 (C-18), 29.2 (C-22), 29.1 (C-19), 29.0 (C-23), 24.1 (C-2), 24.0 (C-6), 21.6 (C-14), 14.7 (C-16) ppm. MS (ESI): m/z (%) =663.2 ([M + H]⁺, 100%). HRMS (ESI) calcd for C₃₄H₄₀KO₁₁ [M + H]⁺: 663.2208; found 663.2219.

Monopotassium 3,15-disuccinate-12-(7'-methanoylcoumarin) andrographolide 11k

Pale yellow solid, 1.19 g, yield 39.2%, purity 95.2%. ¹H NMR (400 MHz, D₂O): δ = 7.83–7.80 (1H, m, 5′-H), 7.41-7.38 (1H, m, 8′-H), 7.28-7.25 (1H, m, 6′-H), 6.46 (1H, s, 3′-H), 6.18 (1H, d, J = 15.8 Hz, 12H), 5.71 (1H, m, 11H), 5.03 (1H, s, 13Ha), 4.86 (1H, s, 13-Hb), 4.12 (1H, d, J = 11.8 Hz, 15-Ha), 3.93–3.78 (2H, m, 3H, 15-Hb), 2.83-2.52 (9H, m, 9,18,19,22,23H), 2.28 (3H, s, 7′-OCOCH₃), 2.11-1.61 (9H, m, 1,2,5,6,7H), 1.03 (3H, s, 14H), 0.96 (3H, s, 16H). ¹³C NMR (100 MHz, D₂O): δ = 174.7 (C-20), 174.6 (C-24), 173.2 (C-17), 173.1 (C-21), 169.1 (7′-OCOCH₃), 162.4 (C-4′), 159.4 (C-2′), 152.7 (C-7′), 150.5 (C-9′), 148.7 (C-8), 135.5 (C-11), 130.1 (C-5′), 128.0 (C-12), 124.0 (C-10′), 118.4 (C-6′), 114.9 (C-8′), 109.1 (C-13), 104.8 (C-3′), 75.1 (C-3), 65.9 (C-15), 60.8 (C-9), 47.5 (C-5), 40.9 (C-4), 39.6 (C-10), 36.7 (C-7), 33.5 (C-1), 29.4 (C-18), 29.2 (C-22), 29.1 (C-19), 29.0 (C-23), 24.2 (C-2), 24.0 (C-6), 21.7 (C-14), 20.3 (7′-OCOCH ₃), 14.5 (C-16) ppm. MS (ESI): m/z (%) =691.2 ([M + H]⁺, 100%). HRMS (ESI) calcd for C₃₅H₄₀KO₁₂ [M + H]⁺: 691.2157; found 691.2174.

Monopotassium 3,15-disuccinate-12-(7'-chlorocoumarin) andrographolide 11l

Yellow solid, 1.19 g, yield 40.5%, purity 96.2%. ¹H NMR (400 MHz, D₂O): δ = 7.80-7.78 (1H, m, 5′-H), 7.25-7.20 (2H, m, 6′,8′-H), 6.48 (1H, s, 3′-H), 6.17 (1H, d, J = 15.8 Hz, 12H), 5.72 (1H, m, 11H), 5.02 (1H, s, 13-Ha), 4.88 (1H, s, 13-Hb), 4.12 (1H, d, J = 11.8 Hz, 15-Ha), 3.94-3.79 (2H, m, 3H, 15-Hb), 2.83-2.62 (9H, m, 9,18,19,22,23H), 2.12-1.79 (9H, m, 1,2,5,6,7H), 1.05 (3H, s, 14H), 0.94 (3H, s, 16H). ¹³C NMR (100 MHz, D₂O): δ = 174.7 (C-20), 174.6 (C-24), 173.2 (C-17), 173.1(C-21), 162.4 (C-4′), 159.6 (C-2′), 151.6 (C-9′), 148.7 (C-8), 135.6 (C-11), 135.1 (C-7′), 128.2 (C-5′), 128.0 (C-12), 125.5 (C-6'), 125.2 (C-10′), 119.6 (C-8′), 109.1 (C-13), 104.7 (C-3′), 75.0 (C-3), 65.9 (C-15), 60.9 (C-9), 47.2 (C-5), 40.8 (C-4), 39.8 (C-10), 36.8 (C-7), 33.4 (C-1), 29.5 (C-18), 29.3 (C-22), 29.1 (C-19), 29.0 (C-23), 24.3 (C-2), 24.0 (C-6), 21.8 (C-14), 14.5 (C-16) ppm. MS (ESI): m/z (%) =667.2 ([M + H]⁺, C₃₃H₃₇ ³⁵ClKO₁₀, 100%), 669.2 ([M + H]⁺, C₃₃H₃₇ ³⁷ClKO₁₀, 33%). HRMS (ESI) calcd for C₃₃H₃₇ ³⁵ClKO₁₀ [M + H]⁺: 667.1712, found 667.1738; for C₃₃H₃₇ ³⁷ClKO₁₀ [M + H]⁺: 669.1683, found 669.1601.

Biological Assays In Vitro

Fresh arterial blood was taken from groin of SD male rats with 3.8% sodium citrate as anticoagulant (9:1 by volume). Then, whole blood samples were centrifuged at 1000 rpm/min for 10 minutes at room temperature to give the platelet-rich plasma (PRP). The residue continued to be centrifuged at 3000 rpm/min for 10 minutes to prepare platelet-poor plasma (PPP). PPP was employed to be the blank control. Thrombin, ADP, AA, collagen were employed as inducers. Aspirin was selected to be the positive control. Normal saline was selected as the negative control. The sample groups (5 µL) were prepared with target compounds dissolved in normal saline (1.7 μmol/L, 3.4μmol/L, 6.8μmol/L) and then was added into PRP-2 (200 µL) and incubated for 2 minutes, as well as positive control and negative control. Afterward, adding 20 µL thrombin (0.1 U/mL), ADP (5 mM/L), AA (20μM), collagen (1 mg/mL), respectively, induced platelet aggregation. IR could be calculated by the formula IR = [1 − (SG/NC)] × 100% (SG and NC represented the platelet aggregation rates of sample group and negative control, respectively)

Cytotoxicity Assay In Vitro

Cell toxicity of target derivatives 11 were evaluated on mouse fibroblast cells (L929) by Cell Counting Kit-8 (CCK-8) assays.⁵³ First, test compounds were dissolved and diluted into 10 µM/L and 100 µM/L with DMSO, respectively. Cells were added on 96-well microplates (1 × 10⁴ cell/well) and then cultivated at 37°C in a humidified atmosphere of carbondioxide (5%) for 24 hours. Second, test compound was added into the cells and continued to be incubated at 37°C for 48 hours. Third, the medium was removed and replaced with fresh complete medium of RPMI-1640 (100 µL). CCK-8 solution (10 µL/well) was added to the microplates. Lastly, the absorbance of test solution was measured at 450 nm on Bio-Tek Flx800 fluorescence microplate reader. According to the formula to calculated relative survival rate: relative survival rate (%) = {[Abs(test cells) − Abs(blank cells)]/[Abs(controlled cells) − Abs(blank cells)]} × 100%.

Statistical Analysis

Results were presented as the means ± standard error. Data were analyzed with one-way analysis of variance (SPSS software) to evaluate statistical significance of the differences. The level of significance was considered at P < 0.05 and P < 0.01.

Footnotes

Acknowledgements

We are grateful to the Natural Science Foundation of Jiangxi Province (No. 20171BAB215075), Traditional Chinese Medicine Research Key Projects of Health and Family Planning Commission of Jiangxi Province (No. 2017Z018 and No. 2018A314) for financial support.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Xue Li

References

Lüscher

. Pulmonary hypertension, gender issues, and quality of care. Eur Heart J. 2016;37(1):1-3.doi:10.1093/eurheartj/ehv699

De Luca

Savonitto

van’t Hof

AWJ.

Suryapranata

. Platelet GP IIb-IIIa receptor antagonists in primary angioplasty: back to the future. Drugs. 2015;75(11):1229-1253.doi:10.1007/s40265-015-0425-7

AS.

Mozaffarian

Roger

et al. Executive summary: heart disease and stroke statistics--2014 update: a report from the American Heart Association. Circulation. 2014;129(3):399-410.doi:10.1161/01.cir.0000442015.53336.12

Langer

HF.

Gawaz

. Platelet-vessel wall interactions in atherosclerotic disease. Thromb Haemost. 2008;99(3):480-486.doi:10.1160/TH07-11-0685

Berrettini

De Cunto

Parise

Grasselli

Nenci

. “In vitro” and “ex vivo” effects of picotamide, a combined thromboxane A2-synthase inhibitor and -receptor antagonist, on human platelets. Eur J Clin Pharmacol. 1990;39(5):495-500.doi:10.1007/BF00280943

1963845

Hiatt

WR.

Money

SR.

Brass

. Long-term safety of cilostazol in patients with peripheral artery disease: the Castle study (cilostazol: a study in long-term effects. J Vasc Surg. 2008;47(2):330-336.doi:10.1016/j.jvs.2007.10.009

Kajiwara

Soejima

Miyamoto

Ogawa

. Effects of additional treatment of sarpogrelate to aspirin therapy on platelet aggregation and plasma plasminogen activator inhibitor activity in patients with stable effort angina. Thromb Res. 2011;128(6):547-551.doi:10.1016/j.thromres.2011.06.001

Namazi

MH.

Safi

Vakili

Saadat

Karimi

Bagheri

. Comparison between intracoronary abciximab and intravenous eptifibatide administration during primary percutaneous coronary intervention of acute ST-segment elevation myocardial infarction. J Tehran Heart Cent. 2013;8(3):132-139.

Ray

. Clopidogrel resistance: the way forward. Indian Heart J. 2014;66(5):530-534.doi:10.1016/j.ihj.2014.08.012

10.

Piccini

Nuti

Paoletti

et al. Possible involvement of dopaminergic mechanisms in the antimigraine action of flunarizine. Cephalalgia. 1990;10(1):3-8.doi:10.1046/j.1468-2982.1990.1001003.x

11.

Belforte

JE.

Magariños-Azcone

Armando

Buño

Pazo

. Pharmacological involvement of the calcium channel blocker flunarizine in dopamine transmission at the striatum. Parkinsonism Relat Disord. 2001;8(1):33-40.doi:10.1016/S1353-8020(01)00006-2

12.

Cortelli

Santucci

Righetti

et al. Neuroendocrinological evidence of an anti-dopaminergic effect of flunarizine. Acta Neurol Scand. 1988;77(4):289-292.doi:10.1111/j.1600-0404.1988.tb05912.x

13.

Whellan

DJ.

Tricoci

Chen

et al. Vorapaxar in acute coronary syndrome patients undergoing coronary artery bypass graft surgery: subgroup analysis from the tracer trial (thrombin receptor antagonist for clinical event reduction in acute coronary syndrome. J Am Coll Cardiol. 2014;63(11):1048-1057.doi:10.1016/j.jacc.2013.10.048

14.

Tan

WSD.

Liao

Zhou

Wong

WSF

. Is there a future for andrographolide to be an anti-inflammatory drug? Deciphering its major mechanisms of action. Biochem Pharmacol. 2017;139:71-81.doi:10.1016/j.bcp.2017.03.024

15.

Peng

Sun

et al. Sar studies of 3,14,19-derivatives of andrographolide on anti-proliferative activity to cancer cells and toxicity to zebrafish: an in vitro and in vivo study. RSC Adv. 2015;5(29):22510-22526.doi:10.1039/C5RA00090D

16.

Chen

Y-B.

Huang

X-Y

et al. Synthesis, structure-activity relationships and biological evaluation of dehydroandrographolide and andrographolide derivatives as novel anti-hepatitis B virus agents. Bioorg Med Chem Lett. 2014;24(10):2353-2359.doi:10.1016/j.bmcl.2014.03.060

17.

Mishra

Dash

AP.

Swain

BK.

Dey

. Anti-malarial activities of Andrographis paniculata and Hedyotis corymbosa extracts and their combination with curcumin. Malar J. 2009;8:26.doi:10.1186/1475-2875-8-26

18.

Tang

Liu

Wang

et al. Synthesis and biological evaluation of andrographolide derivatives as potent anti-HIV agents. Arch Pharm. 2012;345(8):647-656.doi:10.1002/ardp.201200008

19.

Kandanur

SGS.

Golakoti

NR.

Nanduri

. Synthesis and in vitro cytotoxicity of novel C-12 substituted-14-deoxy-andrographolide derivatives as potent anti-cancer agents. Bioorg Med Chem Lett. 2015;25(24):5781-5786.doi:10.1016/j.bmcl.2015.10.053

20.

Wang

Zhang

et al. Design, synthesis and antibacterial activity of novel andrographolide derivatives. Bioorg Med Chem. 2010;18(12):4269-4274.doi:10.1016/j.bmc.2010.04.094

21.

WJ.

Lin

KH.

Hsu

MJ.

Chou

DS.

Hsiao

Sheu

. Suppression of NF-κB signaling by andrographolide with a novel mechanism in human platelets: regulatory roles of the p38 MAPK-hydroxyl radical-ERK2 cascade. Biochem Pharmacol. 2012;84(7):914-924.doi:10.1016/j.bcp.2012.06.030

22.

Lim

JCW.

Chan

TK.

DSW.

Sagineedu

SR.

Stanslas

Wong

WSF

. Andrographolide and its analogues: versatile bioactive molecules for combating inflammation and cancer. Clin Exp Pharmacol Physiol. 2012;39(3):300-310.doi:10.1111/j.1440-1681.2011.05633.x

23.

Yuan

Zhang

Sun

et al. The semi-synthesis of novel andrographolide analogues and anti-influenza virus activity evaluation of their derivatives. Bioorg Med Chem Lett. 2016;26(3):769-773.doi:10.1016/j.bmcl.2015.12.100

24.

Husen

Pihie

AHL.

Nallappan

. Screening for antihyperglycaemic activity in several local herbs of malaysia. J Ethnopharmacol. 2004;95(2-3):205-208.doi:10.1016/j.jep.2004.07.004

25.

Sirikarin

Palo

Chotewuttakorn

Chandranipapongse

Limsuvan

Akarasereenont

. The effects of Andrographis paniculata on platelet activity in healthy thai volunteers. Evid Based Complement Alternat Med. 2018;2018:1-9.doi:10.1155/2018/2458281

26.

Singh

IN.

Mondal

SC.

Singh

Garg

. Platelet-activating factor (PAF)-antagonists of natural origin. Fitoterapia. 2013;84:180-201.doi:10.1016/j.fitote.2012.11.002

27.

Thisoda

Rangkadilok

Pholphana

Worasuttayangkurn

Ruchirawat

Satayavivad

. Inhibitory effect of Andrographis paniculata extract and its active diterpenoids on platelet aggregation. Eur J Pharmacol. 2006;553(1-3):39-45.doi:10.1016/j.ejphar.2006.09.052

28.

Lien

L-M.

C-C.

Hsu

W-H

et al. Mechanisms of andrographolide-induced platelet apoptosis in human platelets: regulatory roles of the extrinsic apoptotic pathway. Phytother Res. 2013;27(11):1671-1677.doi:10.1002/ptr.4911

29.

W-J.

Lee

J-J.

Chou

D-S

et al. A novel role of andrographolide, an NF-kappa B inhibitor, on inhibition of platelet activation: the pivotal mechanisms of endothelial nitric oxide synthase/cyclic GMP. J Mol Med. 2011;89(12):1261-1273.doi:10.1007/s00109-011-0800-0

30.

Liu

Sun

Zhao

et al. Discovery of potent orally active protease-activated receptor 1 (PAR1) antagonists based on andrographolide. J Med Chem. 2017;60(16):7166-7185.doi:10.1021/acs.jmedchem.7b00951

31.

Zhao

S-Y.

Wei

Yin

X-M

. The platelet aggregation activity of xiyanping induced by ADP. Pract Clin Res Chin Med. 2016;16:81-83.

32.

Yin

X-M.

Zhao

S-Y.

Wang

X-Q

. The platelet aggregation activity of chuanhuning induced by ADP. Pharm Clin Chin Materia Medica. 2017;5:48-50.

33.

Duan

S-L.

Wei

Yin

X-M

. The platelet aggregation activity of andrographolide and its sulfonate. Pract Clin Res Chin Med. 2016;16:83-85.

34.

Tang

Wang

et al. Design, synthesis, and biological evaluation of andrographolide derivatives as potent hepatoprotective agents. Chem Biol Drug Des. 2014;83(3):324-333.doi:10.1111/cbdd.12246

35.

Chen

Luo

Hai

Qian

Design

. Design, synthesis, and pharmacological evaluation of the aqueous prodrugs of desmethyl anethole trithione with hepatoprotective activity. Eur J Med Chem. 2010;45(7):3005-3010.doi:10.1016/j.ejmech.2010.03.029

36.

Catto

Pisani

Leonetti

et al. Design, synthesis and biological evaluation of coumarin alkylamines as potent and selective dual binding site inhibitors of acetylcholinesterase. Bioorg Med Chem. 2013;21(1):146-152.doi:10.1016/j.bmc.2012.10.045

http://www.ncbi.nlm.nih.gov/pubmed/23199476

37.

Pintus

Matos

MJ.

Vilar

et al. New insights into highly potent tyrosinase inhibitors based on 3-heteroarylcoumarins: Anti-melanogenesis and antioxidant activities, and computational molecular modeling studies. Bioorg Med Chem. 2017;25(5):1687-1695.doi:10.1016/j.bmc.2017.01.037

38.

Albratty

El-Sharkawy

KA.

Alam

. Original research paper. Synthesis and antitumor activity of some novel thiophene, pyrimidine, coumarin, pyrazole and pyridine derivatives. Acta Pharm. 2017;67(1):15-33.doi:10.1515/acph-2017-0004

39.

Fylaktakidou

KC.

Hadjipavlou-Litina

DJ.

Litinas

KE.

Nicolaides

. Natural and synthetic coumarin derivatives with anti-inflammatory/ antioxidant activities. Curr Pharm Des. 2004;10(30):3813-3833.doi:10.2174/1381612043382710

40.

Pochet

Doucet

Schynts

et al. Esters and amides of 6-(chloromethyl)-2-oxo-2H-1-benzopyran-3-carboxylic acid as inhibitors of alpha-chymotrypsin: significance of the “aromatic” nature of the novel ester-type coumarin for strong inhibitory activity. J Med Chem. 1996;39(13):2579-2585.doi:10.1021/jm960090b

8691456

41.

Sarikaya-Bayram

Ö.

Palmer

JM.

Keller

Braus

GH.

Bayram

. One Juliet and four Romeos: VeA and its methyltransferases. Front Microbiol. 2015;6:1.doi:10.3389/fmicb.2015.00001

42.

Cristina

Jorge

Carolina

Lucinda

Francisco

Melchor

. Anti-platelet activity of flavonoid and coumarin drugs. Vascular Pharmacol. 2016;87:139-149.

43.

Chen

K-S.

C-C.

Chang

F-R

et al. Bioactive coumarins from the leaves of Murraya omphalocarpa. Planta Med. 2003;69(7):654-657.doi:10.1055/s-2003-41112

44.

Vink

Niessen

HWM

et al. Total burden of intraplaque hemorrhage in coronary arteries relates to the use of coumarin-type anticoagulants but not platelet aggregation inhibitors. Virchows Arch. 2014;465(6):723-729.doi:10.1007/s00428-014-1654-y

45.

Jain

Surin

WR.

Misra

et al. Antithrombotic activity of a newly synthesized coumarin derivative 3-(5-hydroxy-2,2-dimethyl-chroman-6-yl)-N-{2-[3-(5-hydroxy-2,2-dimethyl-chroman-6-yl)-propionylamino]-ethyl}-propionamide. Chem Biol Drug Des. 2013;81(4):499-508.doi:10.1111/cbdd.12000

46.

Kathuria

Priya

Chand

et al. Substrate specificity of acetoxy derivatives of coumarins and quinolones towards calreticulin mediated transacetylation: investigations on antiplatelet function. Bioorg Med Chem. 2012;20(4):1624-1638.doi:10.1016/j.bmc.2011.11.016

47.

Deitelzweig

Jaff

. Medical management of venous thromboembolic disease. Tech Vasc Interv Radiol. 2004;7(2):63-67.doi:10.1053/j.tvir.2004.02.003

48.

Wang

et al. Synthesis and biological evaluation of a novel class of coumarin derivatives. Bioorg Med Chem Lett. 2014;24(22):5274-5278.doi:10.1016/j.bmcl.2014.09.051

49.

Luo

X-F.

Yin

H-B

et al. Efficient acylation and one-pot synthesis of dehydroandrographolide succinate on a large scale assisted with microwave radiation. Synth Commun. 2009;39(19):3444-3452.doi:10.1080/00397910902777987

50.

Nanduri

Nyavanandi

VK.

Thunuguntla

SSR

et al. Novel routes for the generation of structurally diverse labdane diterpenes from andrographolide. Tetrahedron Lett. 2004;45(25):4883-4886.doi:10.1016/j.tetlet.2004.04.142

51.

Tasior

Voloshchuk

Poronik

YM.

Rowicki

Gryko

. Corroles bearing diverse coumarin units — synthesis and optical properties. J Porphyr Phthalocyanines. 2011;15(09n10):1011-1023.doi:10.1142/S1088424611003926

52.

Born

. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature. 1962;194:927-929.doi:10.1038/194927b0

53.

Kim

BJ.

Jung

. Calpeptin suppresses tumor necrosis factor-alpha-induced death and accumulation of p53 in L929 mouse sarcoma cells. Apoptosis. 2002;7(2):115-121.doi:10.1023/A:1014354229159

Synthesis of 3,15-Disuccinate-12-Coumarin Substituted Andrographolide Derivatives and Their Antiplatelet Aggregation Activities In Vitro

Abstract

Keywords

Results and Discussion

Preparation of Target Compounds 11

Biological Properties

Cytotoxicity Assay In Vitro

Conclusion

Experimental

General

Synthesis of Derivatives

Synthesis of intermediate 8

Synthesis of intermediate 9

Synthesis of product 11 (11a was selected as example)

Monopotassium 3,15-disuccinate-12-coumarin andrographolide 11a

Monopotassium 3,15-disuccinate-12-(3′-methylcoumarin) andrographolide 11b

Monopotassium 3,15-disuccinate-12-(3′-ethoxyformylcoumarin) andrographolide 11c,

Monopotassium 3,15-disuccinate-12-(3′-chlorocoumarin) andrographolide 11d

Monopotassium 3,15-disuccinate-12-(6′-methylcoumarin) andrographolide 11e

Monopotassium 3,15-disuccinate-12-(6′-methoxylcoumarin) andrographolide 11f

Monopotassium 3,15-disuccinate-12-(6′-methanoylcoumarin) andrographolide 11g

Monopotassium 3,15-disuccinate-12-(6′-chlorocoumarin) andrographolide 11h

Monopotassium 3,15-disuccinate-12-(7'-methylcoumarin) andrographolide 11i

Monopotassium 3,15-disuccinate-12-(7'-methoxylcoumarin) andrographolide 11j

Monopotassium 3,15-disuccinate-12-(7'-methanoylcoumarin) andrographolide 11k

Monopotassium 3,15-disuccinate-12-(7'-chlorocoumarin) andrographolide 11l

Biological Assays In Vitro

Cytotoxicity Assay In Vitro

Statistical Analysis

Footnotes

Acknowledgements

Declaration of Conflicting Interests

Funding

ORCID iD

References