Design, Synthesis, and Biological Activity Studies of Istradefylline Derivatives Based on Adenine as A2A Receptor Antagonists.

Due to its double bond, istradefylline rapidly isomerizes to Z-istradefylline when exposed to normal daylight in dilute solution. To solve the poor photostability of the istradefylline solution, a series of istradefylline derivatives (in total 17 compounds, including II-1 and II-2 series) were designed and synthesized, and their biological activity in inhibiting cAMP was evaluated. The IC50 values of compounds II-1-3, II-2-1, II-2-2, II-2-3, II-2-4, and II-2-6 were 7.71, 6.52, 6.16, 7.23, 7.96, and 9.68 μg/mL, respectively, which had the same order of activity as that of istradefylline (IC50 value was 1.94 μg/mL). The preliminary structure-activity relationship suggested that the 6-amino in adenine played an important role in binding an A2A receptor. The results of photostability experiments showed that the photostability of the target compounds of II-1 and II-2 series was improved when compared with that of istradefylline.

Introduction

Purine was one of the first confirmed natural products isolated from gallstones in 1776 by Carl Wilhelm Scheele. The term purine was first coined in 1884 by the German chemist Emile Fischel, and it was first synthesized in 1898. Purines are essential components of various molecules of living organisms and play vital roles in many biological processes.[1,2] Purine is an aromatic heterocyclic organic compound that consists of an imidazole ring and a pyrimidine ring.

Purine rings are the most common nitrogen-containing heterocyclic rings in nature. Not only are a large number of purine derivatives found in various marine organisms and plants but they also are the core structures of adenine and guanine in nucleic acids (RNA and DNA).[3] Purines and their derivatives have been widely used for the treatment of various diseases in the medical industry because of their various biological activities. For example, theophylline, diprophylline, proxyphylline, bamifylline, and other derivatives (Figure ) have the characteristics of bronchiectasis antiasthma (antiasthma drugs),[4−6] reproterol can show sympathomimetic activity (antiasthmatic), and pentoxifylline is primarily used as a vasodilator (cardiovascular drug).[7−10] Caffeine, the purine derivative with the largest industrial output, stimulates the central nervous system, causing an increase in heart activity, metabolism, respiration, and blood pressure.[11,12] In 1965, Robbins and Holmes synthesized 8-bromoguanosine, which has excellent immune-enhancing activity.[13,14]

Figure 1

Structure of purine derivatives.

In addition, several existing A2A receptor inhibitors with purine or purine analogue skeleton rings are used in Parkinson’s Disease (PD), such as istradefylline (KW-6002), a xanthine derivative, which was marketed in Japan in 2013.

In humans, A2A adenosine receptors are expressed in a huge array of organs and tissues, including the heart, lungs, liver, cardiovascular tissues, neutrophils, leukocytes, and endothelial cells, with remarkable implications in the regulation of inflammatory and immune responses. On account of these reasons, there is a growing interest in the adenosine A2A receptor (AA2AR) as a drug target. Agonists are explored as anti-inflammatory agents, and antagonists have emerged as an attractive target to treat neurodegenerative diseases (Parkinson’s disease, Huntington’s disease, and Alzheimer’s disease).[15]

Istradefylline, a novel selective AA2AR antagonist, was developed by Kyowa Japan Hakko Kogyo, which is able to improve the motor symptoms of patients with Parkinson’s disease by activating the γ-aminobutyric acid (GABA) pathway of the subthalamic nucleus. In adjunct with levodopa–carbidopa, istradefylline is able to shorten the off period and prolong the on period with better tolerance and safety. It can significantly reduce the end-of-dose phenomenon of levodopa treatment.[16−23] At present, istradefylline is applied for approval and initial clinical trials by many pharmaceutical companies based on its great drug efficacy.

Although istradefylline is applied for initial clinical trials, it has disadvantages, such as low bioavailability and poor photostability, that still exist. Its conjugated double bond structure is stable in the trans-configuration in the solid state, but it has poor photosensitivity in solution and easily converts into the cis-configuration. In general, the lower the concentration, the faster the conversion. Therefore, it is necessary to solve the poor photostability of the istradefylline solution to meet the requirements of multiple dosage forms and to find other AA2AR antagonists with better pharmacological activity.

Results and Discussion

Istradefylline uses xanthine as its pharmacophore, which has a similar structure to adenine; however, their pharmacological activities are completely different. In medical applications, adenine is involved in the synthesis of DNA and RNA, which can promote leukocytosis and increase the number of white blood cells. Based on this, it can be used for leukopenia caused by tumor radiotherapy, tumor chemotherapy, psychotropic drugs, and benzene poisoning. Adenine has also been reported as a new type of adenosine receptor antagonist.[24−27] Consequently, it can be concluded that adenine modified by substituents may also result in a pharmacologically active A2A receptor antagonist. Based on the structural properties of adenine, we study the structure–activity relationship of adenine (Figure ).

Figure 2

Structure of istradefylline and adenine.

According to literature reports,[28] a phenoxy group in the 2-position gave high AA2AR and AA2BR selectivity (nearly 400-fold), but the selectivity over AA1R was poor. The nitrogen atom in the 3-position appeared not to be important, since 3-nitrogen-free-adenine derivatives were almost as potent as the corresponding purines.

The 6-amino replaced by alkoxy or alkynyl groups greatly diminishes the affinity for the adenosine receptor subtypes, suggesting that a N–H group is required to provide a H-bond donor for a good interaction with all adenosine receptors. The relevant references have described that the amino group is a necessary element for the receptor interaction but slight modifications are allowed.[28]

The nitrogen atom in the 7-position is not required for high AA2AR affinity. Derivatives with the hydrogen atom in the 8-position substituted by a 1,2,3-triazole ring are developed as potent AA2AR antagonists. A propargyl substituent at N9 replacing the ribose moiety present in the agonistic adenosine derivatives appears to be favorable for the high AA2AR affinity.

According to the structure–activity relationship of adenine, it was found that the 6-amino plays an important role in the affinity for the A2A receptor, while the 8-position can be substituted by a benzene ring or an aromatic ring as potent AA2AR antagonists. The amino group is a vital element for the receptor interaction, and slight modifications are allowed.

According to the structure–activity relationship of adenine, two series containing a total of 17 novel compounds were designed and synthesized by maintaining the skeleton of adenine based on bioisosterism (Table ). The pharmacological activity of the compounds was screened, and the photostability of the solution was tested. Moreover, docking studies are presented in this paper as well.

Table 1

Target Compounds of Series II

Design

On the basis of keeping the skeleton of adenine unchanged, the target compounds of series II-1 were designed by modifying the side chain. On the basis of keeping the side chain unchanged, the target compounds of series II-2 were designed by modifying the adenine group (Figure ).

Figure 3

Target compounds of series II.

Synthesis

2-Deoxyadenosine was suspended in a sodium acetate and acetic acid buffer solution (pH 4.3). To this suspension, a glacial acetic acid/sodium acetate buffer solution (pH 4.3) containing bromine was added dropwise. The mixture was adjusted to pH 7 with a saturated sodium hydroxide aqueous solution at room temperature. The slurry was filtered, and 8-bromo-2′-deoxyadenosine was obtained. 8-Bromo-2′-deoxyadenosine, (E)-2-aromatic-vinyl boronic acids, and Pd(OAc)2 were added into the reaction bottle, and then the solution of acetonitrile and water with TPPTS (triphenylphosphine-3,3′,3″-trisulfonic acid trisodium salt) and Na2CO3 were also added into the reaction bottle. Protected by nitrogen, the mixture was stirred to maintain the reaction for 1.5 h at 95 °C. After the reaction, the mixture was cooled to room temperature, and the pH of the mixture was adjusted to 9 with 2 mol/L HCl. The precipitate was filtered. The filter was dried for 1 h at 120 °C and then ground in ethyl acetate. The intermediates were obtained. The intermediates were dissolved in 2 mol/L HCl. Protected by nitrogen, the mixture was stirred at the temperature of 100 °C for 2 h. After the reaction, the mixture was cooled to room temperature, and then the pH of the mixture was adjusted to 9 with a saturated NaHCO3 aqueous solution. The precipitate was filtered and ground in ethyl acetate. The target compounds II-1 were obtained. The synthetic route to target compounds II-1 is described in Scheme .

Scheme 1

Synthesis of the Target Compounds of Series II-1

6-Chloro-4,5-diaminopyrimidine, substituted (or unsubstituted) cinnamic acid, and ammonium chloride were added to a flask and refluxed in POCl3 for 24 h. After cooling to room temperature, the mixture was poured into ice water. Then, ammonia solution (25%, pH 7–8) was used to neutralize and obtain the precipitation products. The precipitates were filtered and dried in a vacuum drier, and the intermediates were obtained. The intermediates, aniline compounds, and concentrated HCl in isopropanol were stirred under reflux for 2–3 h. After being cooled, a solid precipitate was obtained, and then the suspension was diluted with a sodium bicarbonate solution. The precipitate was filtered and dried in a desiccator at 50 °C. Target compounds II-2 were finally obtained after recrystallization in ethyl acetate. The preparation of target compounds II-2 is shown in Scheme .

Scheme 2

Synthesis of the Target Compounds of Series II-2

Biological Activity

To test the biological activity of the designed and synthesized istradefylline derivatives, two series of compounds, target compounds of series II-1 and II-2, were evaluated. ADA (adenosine deaminase) and NECA (adenosine-5′-(N-ethylcarboxamide)) were used to stimulate hADORA2A-HEK293 to construct a cAMP cell model for compound screening. This method is more reliable to observe the drug effect at the cellular level.

For 17 designed compounds, the effect of different compounds on cAMP was quantified by the enzyme-linked immunosorbent assay (ELISA), and the content of cAMP in cells was also quantified by ELISA for compound screening. The determined cAMP concentration–absorbance standard curve is shown in Figure with the linear equation y = −0.0044x + 1.2584. The cAMP content of the supernatant after the treatment in the white medium group, stimulation control group, and administration group was measured, and the cAMP release inhibition rate (%) = [1 – (administration group – blank medium group)/(stimulation control group – blank medium group)] × 100%.

Figure 4

Enzyme-linked immunosorbent assay to detect the effect of different compounds on cAMP.

For the synthesized target compounds of series II, different compounds were applied to treat A2A receptor high-expression model cells at a final concentration of 10 μg/mL. An ELISA kit was used to detect the cAMP content, and the inhibitory activity of compounds II on the A2A receptor was investigated. The activity test results of the target compounds of series II are shown in Table .

Table 2

Results of Pharmacological Activity of Target Compounds of Series IIa

number	cAMP expression inhibition rate	IC50 (μg/mL)	number	cAMP expression inhibition rate	IC50 (μg/mL)
Istradefylline	96.19	1.94	II-1–9	0.9	>100
II-1–1	5.7	>100	II-1-10	1.2	>100
II-1-2	3.1	>100	II-1–11	0.3	>100
II-1–3	48.19	7.71	II-2–1	81.2	6.52
II-1–4	2.4	>100	II-2-2	85.2	6.16
II-1–5	6.8	>100	II-2-3	64.5	7.23
II-1–6	1.88	>100	II-2–4	58.9	7.96
II-1–7	14.1	65.3	II-2–5	2.5	>100
II-1–8	6.49	>100	II-2–6	54.7	9.68

>100 means no activity.

The pharmacological activity results showed that the target compounds of series II have certain pharmacological activities. Among these compounds, six compounds, II-1-3, II-2-1, II-2-2, II-2-3, II-2-4, and II-2–6, showed an inhibition rate on cAMP expression in model cells at a concentration of 10 μg/mL of 48.19, 81.2, 85.2, 64.5, 58.9, and 54.7%, respectively. Compounds II-2-1 and II-2-2 exhibited prominent cAMP expression inhibition rates of 81.2 and 85.2%, respectively, which were similar to the activity of istradefylline (96.19%).

It can be concluded from Table that the pharmacological activity of II-1 series compounds were generally less than those of II-2 series target compounds, which indicated that the aromatic amines were helpful in improving the pharmacological activity of adenine.

From Table , the IC50 values of compounds II-1-3, II-2-1, II-2-2, II-2-3, II-2-4, and II-2-6 were 7.71, 6.52, 6.16, 7.23, 7.96, and 9.68 μg/mL, respectively. The IC50 values of these compounds had the same order of activity as that of istradefylline (IC50 value was 1.94 μg/mL).

Photostability

Due to the instability of the istradefylline solution, it is isomerized under light conditions. To study the photostability of representative target compounds with better activity in each series, the following experimental methods were used.

Preparation of istradefylline reference standard: Two milligram of the compound was precisely weighed and transferred into a 25 mL volumetric flask, and then it was diluted with an acetonitrile and potassium dihydrogen phosphate solution to obtain a 0.05 mol/mL solution. After the compound was dissolved completely, it was stored in the dark, and its purity was detected by high-performance liquid chromatography (HPLC).

Preparation of samples for light exposure: The purity of the samples was determined by HPLC after 1 h of light exposure.

HPLC method: The HPLC system was equipped with a DAD detector and an Agilent 1200. A column temperature chamber, an automatic sampler, and an Agilent XDB C18 (250 × 4.6 mm2, 5 μm) were used. Acetonitrile–0.05 mol/mL potassium dihydrogen phosphate (1/5–5/1, V/V) was used as the mobile phase. The detection wavelength was 210–365 nm, and the column temperature was 30 °C. The flow rate was 1.0 mL/min. The injection volume was 20 μL.

The results of the photostability test are shown in Table S1. The experimental results prove that the photostability of the target compounds of series II-1 and II-2 was significantly better than that of istradefylline. After 1 h of light exposure, istradefylline was isomerized (about 1/3), while the designed and synthesized target compounds were rarely isomerized, which proved that they had better photostability and were significantly superior to istradefylline.

According to the analysis of the structures, the reason for the better photostability of compounds II-1 and II-2 may be the hyperconjugation effect caused by the hydrogen atom on the imino group or the amino group with the carbon–carbon double bond, which makes the double bond more stable.

Molecular Docking

To simulate and estimate the relationship between compounds and receptor biomacromolecules and optimize lead compounds, molecular docking simulation studies were carried out using Avogadro, UCSF Chimera, PyMOL, Ledock, and Discovery Studio. According to the results of the A2A receptor inhibitory activity test, compounds II-2-1 and II-2-2 with prominent cAMP expression inhibitory activity were selected for molecular docking.

Computer-aided drug design methods were adopted, and the target protein (PDB ID: 3REY) was used as the receptor. Molecular docking was carried out with representative target compounds as ligands, and the results are shown in Figures and 6. The results of molecular docking showed that the target compounds could bind well to the target site and interact with various important amino acids of receptors.

Figure 5

Molecular docking results of compound II-2-1 with adenosine A2A receptors.

Figure 6

Molecular docking results of compound II-2-2 with adenosine A2A receptors.

In compound II-2-1, there was a π–π stacking interaction between the heterocyclic core and Phe168. At the same time, the nitrogen atom on the heterocyclic ring combined with the NH2 group on the donor Asn253 to form a hydrogen bond. The heterocyclic ring had a certain hydrophobic effect with Met270. The benzene ring had a π-sigma interaction with Val84, and the terminal benzene ring had a certain π-anion force with Glu.

In compound II-2-2, there was a π–π stacking interaction between the heterocyclic core and Phe168, and the nitrogen atom on the heterocyclic ring combined with the NH2 group on the donor Asn253 to form a hydrogen bond. The heterocyclic ring had a certain hydrophobic effect with Met270. The hydroxybenzene ring had a weak hydrophobic interaction with Ala63 and Val84, and the terminal benzene ring had a certain π-anion force with Glu.

The istradefylline as a ligand was also subjected to molecular docking research, and the results of molecular docking are shown in Figure .

Figure 7

Molecular docking results of istradefylline with adenosine A2A receptors.

There was a π–π stacking interaction between the purine ring core and Phe154, and the carbonyl group on the purine combined with the NH2 groups on the donors Leu79 and Asn239 to form hydrogen bonds. The ethyl group on N1 formed a hydrophobic interaction with Leu235 and changed the rotamerization of Met163 and His236. The ethyl group on N3 combined with Val78 to give rise to an effect. The benzene ring had a certain strong hydrophobic effect on Leu253.

A comparison of the results of molecular docking of compounds II-2-1 and II-2-2 with that of istradefylline showed that the designed target compounds were reasonable, and they can bind to the target site well and interact with multiple important amino acids of receptors. The abovementioned results of SAR analysis and molecular docking study may provide a basis for the rational design of more potent adenosine A2A receptor antagonists.

Conclusions

Although istradefylline is a novel selective adenosine A2A receptor antagonist, its poor photostability affects the preparation safety, efficacy, and bioavailability of the drug. To solve the poor photostability of the istradefylline solution, 17 compounds of two series based on adenine were designed and synthesized by maintaining the skeleton of adenine based on bioisosterism. After obtaining the compounds, we stimulated hADORA2A-HEK293 with ADA and NECA to construct a cAMP cell model and tested the inhibitory activity of cAMP expression on all of the synthesized target compounds. The test results showed that the target compounds of series II-2 showed good cell cAMP expression inhibitory activity. Among the target compounds, cAMP expression inhibition rates of compounds II-2-1 and II-2-2 reached 81.2 and 85.2%, respectively, which were equivalent to the activity of istradefylline. In addition, the IC50 values of compounds II-1-3, II-2-1, II-2-2, II-2-3, II-2-4, and II-2-6 were 7.71, 6.52, 6.16, 7.23, 7.96, and 9.68 μg/mL, respectively, which had the same order of activity as that of istradefylline (IC50 value was 1.94 μg/mL). This suggested that the side chains had a lower effect, and the 6-amino in adenine played an important role in binding the A2A receptor. In addition, the photostability of representative target compounds with good activity in each series was tested, and the target compounds of series II-1 and II-2 were rarely isomerized, which proved that the designed and synthesized target compounds had better photosensitivity and were significantly superior to istradefylline. This suggested that compounds with adenine attached to trans-double bonds were more stable than compounds with xanthine attached to trans-double bonds. The experimental results provided an idea for the future development of novel A2A receptor antagonists.

Experimental Section

Instruments and Reagents of Synthesis

Dioxane, sodium hydroxide (NaOH), methylene chloride (CH2Cl2), chloroform, toluene, methanol, hydrochloric acid, potassium carbonate, dimethylformamide (DMF), petroleum ether, ethyl acetate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, acetone, TEBA, anhydrous sodium sulfate, sodium methoxide, sodium acetate, sodium thiosulfate, and acetonitrile were of analytical grade, and all were purchased from Sinopharm Group Chemical Reagent Co., Ltd. The remaining materials were provided by Shandong Xinhua Pharmaceutical Co., Ltd. The instruments used in this work were as follows: a circulating water vacuum pump (SHB-III, Zhengzhou Great Wall Science, Industry and Trade Co., Ltd.), an electric blast drying oven (GZX-9240MBE, Shanghai Boxun Industrial Co., Ltd.), an electronic balance scale (PB3002-S, METTLER TOLEDO), a constant-temperature magnetic stirrer (DF-101S, Zhengzhou Great Wall Industry and Trade Co., Ltd.), a three-purpose UV instrument (ZF-2, Shanghai Anting Electronic Instrument Factory), and a high-performance liquid chromatograph (Agilent-1200, United States Agilent Technologies). A Bruker nuclear magnetic resonance instrument and a Bruker mass spectrometer were also used.

Experimental Materials and Instruments

HEK293 cells stably transfected with adenosine A2A (hADORA2A) were purchased from Shanghai Saiye. Dulbecco’s modified Eagle’s medium (DMEM)/high sugar medium (BI), penicillin–streptomycin solution, pancreatin cell digest (Sigma, USA), fetal bovine serum (Gibco, USA), dimethyl sulfoxide (DMSO) (Biosharp, China), a cAMP Elisa kit (R & D, USA), NECA (Sigma, USA), and ADA (Sigma, USA) were used.

A Synergy 2 multifunctional microplate reader (Bio-Tek, USA), a cell incubator (Thermo, USA), and a high-speed refrigerated centrifuge (Thermo, USA) were also employed.

Enzyme-Linked Immunosorbent Assay to Detect the Content of cAMP in Cells

HEK293 cells (hADORA2A-HEK293) were stably transfected with adenosine A2A (hADORA2A) in the logarithmic growth phase, digested with 0.25% trypsin for about 3 min, inoculated into 6 cm culture dishes at 1 × 105 cells per well, and cultivated for 24 h. After that 80 μL of buffer was added to it, and then these cells were incubated at 37 °C for 45 min. NECA (500 nm) and different compounds were added (final concentration 10 μg/mL), and these cells were stimulated for 30 min. The final values of the IC50 were 1.1, 3.3, 10, 30, and 90 μg/mL.