**Synthesis, Molecular Docking, and Neuroprotective Effect of 2-Methylcinnamic Acid Amide in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)—An Induced Parkinson's Disease Model**

**Maya Chochkova 1,\*, Rusi Rusew <sup>2</sup> , Reni Kalfin <sup>3</sup> , Lyubka Tancheva <sup>3</sup> , Maria Lazarova <sup>3</sup> , Hristina Sbirkova-Dimitrova <sup>2</sup> , Andrey Popatanasov <sup>3</sup> , Krasimira Tasheva <sup>4</sup> , Boris Shivachev <sup>2</sup> , Nejc Petek <sup>5</sup> and Martin Štícha <sup>6</sup>**


**Abstract:** Parkinson's disease (PD) has emerged as the second most common form of human neurodegenerative disorders. However, due to the severe side effects of the current antiparkinsonian drugs, the design of novel and safe compounds is a hot topic amongst the medicinal chemistry community. Herein, a convenient peptide method, TBTU (O-(benzotriazole-1-yl)-N,N,N0 ,N0 -tetramethyluronium tetrafluoroborate), was used for the synthesis of the amide (*E*)-*N*-(2-methylcinnamoyl)-amantadine (CA(2-Me)-Am; 3)) derived from amantadine and 2-methylcinnamic acid. The obtained hybrid was studied for its antiparkinsonian activity in an experimental model of PD induced by MPTP. Mice (C57BL/6,male, 8 weeks old) were divided into four groups as follows: (1) the control, treated with normal saline (i.p.) for 12 consecutive days; (2) MPTP (30 mg/kg/day, i.p.), applied daily for 5 consecutive days; (3) MPTP + CA(2-Me)-Am, applied for 12 consecutive days, 5 days simultaneously with MPTP and 7 days after MPTP; (4) CA(2-Me)-Am +oleanoic acid (OA), applied daily for 12 consecutive days. Neurobehavioral parameters in all experimental groups of mice were evaluated by rotarod test and passive avoidance test. Our experimental data showed that CA(2-Me)-Am in parkinsonian mice significantly restored memory performance, while neuromuscular coordination approached the control level, indicating the ameliorating effects of the new compound. In conclusion, the newly synthesized hybrid might be a promising agent for treating motor disturbances and cognitive impairment in experimental PD.

**Keywords:** amantadine; 2-methylcinnamic acid; single crystal X-ray diffraction; Parkinson's disease

#### **1. Introduction**

Globally, the growth of the older population, comprising 7 % or more of the total population, is projected to reach about 2 billion people by 2050 [1]. Thereafter, this can be associated with increasing prevalence of age-related diseases as neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease, among others. According to the WHO (World Health Organization), amongst the neurological disorders, there is a growing concern around the disability and death caused by PD [2]. Parkinsonism is the second most common neurodegenerative disaster after Alzheimer's disease [3,4]. Currently,

**Citation:** Chochkova, M.; Rusew, R.; Kalfin, R.; Tancheva, L.; Lazarova, M.; Sbirkova-Dimitrova, H.; Popatanasov, A.; Tasheva, K.; Shivachev, B.; Petek, N.; et al. Synthesis, Molecular Docking, and Neuroprotective Effect of 2-Methylcinnamic Acid Amide in 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP)—An Induced Parkinson's Disease Model. *Crystals* **2022**, *12*, 1518. https:// doi.org/10.3390/cryst12111518

Academic Editor: Abel Moreno

Received: 8 September 2022 Accepted: 21 October 2022 Published: 26 October 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

there are no strategies that can stop the brain cell injury afflicted by PD. The multifactorial nature of this incurable pathology requires an effective multi-target concept that can hit diverse targets. However, the almost all of the central nervous system drug candidates do not efficiently penetrate the blood–brain barrier. Therefore, to solve this problem, it is wildly accepted that the addition of a lipophilic rest to main structure can modify absorption, distribution, metabolism, or excretion (ADME) properties of an entire molecule.

Accordingly, adamantane core has been known as a precise building block that can alter the lipophilicity on a lead compound, without increasing its toxicity. Indeed, there are many adamantane-based compounds that are currently used in clinical practice and also as potential therapeutics [5,6]. However, the end of the era of aminoadamantanes, including rimantadine and amantadine as antiviral, is delineated, since they have faced increasing resistance against influenza A strains [7–9]. Surprisingly, a random finding resurrected the role of amantadine in the efficacy for symptomatic alleviation in PD [10], as well as for other movement disorders [11]. Additionally, the dual effects of amantadine on parkinsonian signs and symptoms and levodopa-induced dyskinesias are due to its dopaminergic and glutamatergic properties [11]. Amantadine is the only glutamate antagonist drug that is prescribed against PD, often used to treat dyskinesia. However, the clinical use of amantadine is limited because of concerns regarding its safety and tolerability issues, as well as the duration of its antidyskinetic efficacy. Hence, the search for new agents with powerful antiparkinsonian action and good biological tolerance is an important task for chemists and biologists.

In this regard, cinnamic acid (CA) has been known as a plant 3-phenylpropenoic acid and represents one of the constituents in the common spice cinnamon. This unsaturated carboxylic acid has been obtained through phenylpropanoid pathway as a deaminated plant product of its amino acid precursor phenylalanine. Besides having a plethora of activities such as antidiabetic [12] and anti-cancer [13] effects, cinnamic acid emerges with a new function in protecting dopaminergic neurons via PPARα [14]. Moreover, an earlier result [15] reveals that cinnamic acid improves memory by suppressing the oxidative stress and cholinergic dysfunction in the brain of diabetic mice.

Inspired by the above-mentioned results for CA, in our study, we report the synthesis of a hybrid molecule consisting of methylated cinnamic acid and amantadine. Furthermore, the newly obtained derivative was examined as a potential neuroprotective agent in the MPTP experimental mouse model of PD.

#### **2. Materials and Methods**

#### *2.1. General Methods*

2-Methyl-cinnamic acid, amantadine, and other reagents were purchased from Angene Chemical, Sigma Aldrich (FOT, Bulgaria), whereas all solvents were obtained from Thermo Fisher Scientific (Bulgaria) and applied with no further purification. Thin-layer chromatography (TLC) was carried out on precoated Kieselgel 60F<sup>254</sup> plates (Merck, Germany) with detection by UV absorbance at 254 nm. A TLC plate was visualized by Ce-PMo reagent solution followed by heating. Flash chromatography of the target amide was performed on prepackaged BÜCHI FlashPure EcoFlex silica columns.

The newly amide 3 was synthesized according to the modified literature method [16]. The NMR spectra were recorded in deuterated solvents with (CH3)4Si as the internal standard on a Bruker Ascend neo NMR 600 instrument (Bruker, Billerica, MA, USA) at 600 MHz for <sup>1</sup>H nuclei and at 151 MHz for <sup>13</sup>C nuclei. A Bruker Compact QTOF-MS (Bruker Daltonics, Bremen, Germany) controlled by the Compass 1.9 Control software was used to measure the mass spectrum. The monoisotopic mass values were calculated using Data analysis software v 4.4 (Bruker Daltonics, Germany). The analysis was conducted in the positive ion mode at a scan range from m/z 50 to 1000, and nitrogen was used as nebulizer gas at a pressure of 4 psi and flow of 3 L/min for the dry gas. The capillary voltage and temperature were set at 4500 V and 220 ◦C, respectively.

#### *2.2. Synthesis of (E)- N-(2-Methylcinnamoyl)-Amantadine (3)*

2-Methylcinnamic acid (1.8 g, 11.4 mmol) was suspended in 30 mL of CH2Cl2, and then, after adding Et3N (1.6 mL, 11.4 mmol), the obtained colorless liquid was treated by solid TBTU (3.7 g, 11.4 mmol). After being stirred for ≈10 min, to the mixture, we added amantadine (2.4 g, 12.6 mmol) and Et3N (1.8 mL, 12.6 mmol), dissolved (under sonication) in 40 mL CH2Cl2. Thus, the reaction mixture was stirred at room temperature for 3 h, and then was diluted with an additional 30 mL CH2Cl2. The organic phase was washed with 5% aqueous NaHCO<sup>3</sup> (5 × 50 mL) and brine (3 × 50 mL), dried over Na2SO4, and concentrated in vacuo. Furthermore, after purification, the amide was obtained (3.3 g, 89%) as white crystals.

Compound (**3**): white crystals (CH3CN); mp 188–189 <sup>0</sup>C; <sup>1</sup>H NMR (DMSO-d6, 600 MHz) δ 7.59 (s, 1H), 7.56 (d, J = 15.6 Hz, 1H), 7.48 (d, J = 7.3 Hz, 1H), 7.27–7.20 (m, 3H), 6.58 (d, J = 15.6 Hz, 1H), 2.35 (s, 3H), 2.03 (bs, 3H), 2.00 (bs, 6H), 1.64 (bs, 6H); <sup>13</sup>C NMR (DMSO-d6, 151 MHz) δ 164.5, 137.0, 135.7, 134.4, 131.1, 129.4, 126.8, 126.3, 125.3, 51.4, 41.5, 36.5, 29.3, 19.9; HRMS m/z 318.1830 (calcd for C20H25NNaO, 318.1828).

#### *2.3. Single Crystal X-ray Diffraction (SCXRD) of (E)-N-(2-methylcinnamoyl)-amantadine (3)*

Single crystals of compound 3 were grown from 1:1 *v/v* benzene methanol solution. A crystal with suitable size and quality was selected and was mounted on a glass capillary. The diffraction peak intensities and coordinates were collected on Bruker D8 Venture diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) equipped with a PhotonII CMOS detector using micro-focus Mo*K*α radiation (λ = 0.71073 Å). Data were processed with CrysAlisPro software [17]. The structure was solved with intrinsic methods using ShelxT [18] and refined by the full-matrix least-squares method on the *F* <sup>2</sup> with ShelxL program [19]. All non-hydrogen atoms were located successfully from the Fourier map and were refined anisotropically. Hydrogen atoms were placed on calculated positions (C–Haromatic = 0.93, C–Hmethyl = 0.96 Å, and C–Hmethylenic = 0.97 Å, riding on the parent atom (*Ueq* = 1.2). The H atom near the nitrogen was located from a different Fourier map. Complete crystallographic data for the structure of the title compound reported in this paper were deposited in the CIF format at the Cambridge Crystallographic Data Center as 2205297. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, deposited on 5 September 2022 (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +441223336033; e-mail: deposit@ccdc.cam.ac.uk).

#### *2.4. Neurobehavioral Studies*

Mice (C57BL/6, male, 8 weeks old) were obtained from Erboj (Animal Breeding Center, Slivniza, Sofia). The animals were housed two per cage under constant laboratory conditions (25 ± 3 ◦C, 12/12 h light/dark cycle) with food and water available ad libitum. The habituation period was 5 days before the start of the experiment. The protocol of all experiments was in accordance with the requirements of the European Communities Council Directive 86/609/EEC and rules of the Bioethics Committee 30/03/2021, Institute of Neurobiology, Bulgarian Academy of Sciences.

CA(2-Me)-Am (3) was dissolved in oleanolic acid (OA). We tested five doses of amide 3 applied per os on 25 male mice C57BL/6 and found the dose of 20 mg/kg to be the most effective.

Mice were divided into four experimental groups (*n* = 8 in each group) as follows: (1) control, treated with normal saline (i.p.) for 12 consecutive days; (2) MPTP (30 mg/kg/day, i.p.) applied daily for 5 consecutive days in accordance with the work of Shin et al. [20]; (3) MPTP (30 mg/kg, i.p.) + CA(2-Me)-Am (20 mg/kg, per os) applied for 12 consecutive days, 5 days simultaneously with MPTP and 7 days after MPTP; (4) MPTP (30 mg/kg, i.p.) + OA (per os) applied daily for 12 consecutive days.

All mice training was conducted before MPTP administration.

#### 2.4.1. Rotarod Test

Mice from all experimental groups were placed on a gyratory with a fixed speed of 7 rpm/min, and the time on rotarod was determined. The observation period was 5 min. All animals were pre-trained on the rotarod apparatus before treatment in order to reach stable performance. The training consisted of one session per day over 3 consecutive days. The test was made on the 13th day, and the average time per group was calculated after the experiment and repeated four times [21].

#### 2.4.2. Passive Avoidance Test

Learning and memory performance in mice was evaluated using passive avoidance learning test [22]. Acquisition phase: during this phase, each animal was placed in the illuminated compartment. When the rodents innately entered into the dark compartment, they received a mild electrical foot shock (0.5 mA, 3 s). In this trial, the initial latency (IL) of entrance into the dark chamber of each animal was recorded, and mice with ILs > 60 s were excluded from the study. Test phase: on the 13th and 14th days, each mouse was placed in the illuminated chamber, and the entry into the dark chamber was measured as step through latency (STL). The behavioral observations were carried out between 9 a.m. and 12 a.m.

#### 2.4.3. Statistical Analysis

The results were expressed as means ± the standard error of the mean (SEM) or as percentage changes over the mean compared to the control. Statistical analyses of the data were performed by one-way analysis of variance (ANOVA) followed by Dunnett post hoc comparison test. Differences were considered significant at *p* < 0.05.

#### **3. Results and Discussion**

#### *3.1. Chemistry*

Herein, the 2-methylcinnamic acid amide (CA(2-Me)-Am; 3) was synthesized as outlined in Scheme 1. Generally, the amidation of 2-methylcinnamic acid (CA(2-Me)-OH; 1) with amantadine (Am; 2) was carried out in the presence of tertiary amine (triethylamine, Et3N) and by one of the preferred coupling reagents for in situ activation, such as TBTU [16] to amide 3. *Crystals* **2022**, *12*, × FOR PEER REVIEW 5 of 12

(2-methylcinnamoyl)-amantadine.

tures [23–27].

The structure of the newly obtained compound (3) was confirmed by the 1H NMR, 13C NMR, HRMS, and single crystal analysis powder diffraction. The title compound (CA(2-Me)-Am; 3) crystallized in the monoclinic *P*21/c space group, with one molecule in the asymmetric unit (Figure 1). The bond distances and angles (Table 1) within the ada-The structure of the newly obtained compound (3) was confirmed by the <sup>1</sup>H NMR, <sup>13</sup>C NMR, HRMS, and single crystal analysis powder diffraction. The title compound (CA(2-Me)-Am; 3) crystallized in the monoclinic *P*21/c space group, with one molecule in the asymmetric unit (Figure 1). The bond distances and angles (Table 1) within the

mantane and 2-methylcinnamic acid were comparable with those observed in other struc-

Empirical formula C20H25NO Formula weight 295.41 Temperature/K 290.00 Crystal system monoclinic Space group P21/c a/Å 14.692(2) b/Å 11.900(2) c/Å 9.9904(18) α/° 90 β/° 104.943(5) γ/° 90 Volume/Å3 1687.6(5) Z 4 ρcalcg/cm3 1.163 μ/mm-1 0.071 F(000) 640.0 Crystal size/mm3 0.3 × 0.25 × 0.2 Radiation MoKα (λ = 0.71073)

2Θ range for data collection/° 4.466 to 52.842

Reflections collected 10,910

Data/restraints/parameters 3443/0/205 Goodness-of-fit on F2 1.018 Final R indexes (I > =2σ (I)) R1 = 0.0605, wR2 = 0.1171 Final R indexes (all data) R1 = 0.1142, wR2 = 0.1405

Largest diff. peak/hole/e Å<sup>−</sup>3 0.17/−0.13

Index ranges -18 ≤ h ≤ 17, -14 ≤ k ≤ 12, -12 ≤ l ≤ 12

Independent reflections 3443 (Rint = 0.0595, Rsigma = 0.0654)

adamantane and 2-methylcinnamic acid were comparable with those observed in other structures [23–27].

**Table 1.** The most important data collection and crystallographic refinement parameters for (E)=N-(2 methylcinnamoyl)-amantadine.


The angle between the phenyl and acrylamide moieties was 29.8 ◦ , disclosing that the conjugation was not stringent, e.g., the conjugation could be disrupted. In the molecule of (*E*)–*N*-(2-methylcinnamoyl)-amantadine, one hydrogen donor (N–H) and one acceptor (carbonyl oxygen) were present. In the crystal structure, the molecules produced onedimensional chains with a graph set *C* 1 1 (4) [28,29] (Figure 2a). The three-dimensional packing of the molecules (Figure 2b) did not reveal additional weak interactions, and thus the stabilization of the crystal structure was achieved by the N1-H1 . . . O1 hydrogen bond (N1 . . . O1 of 3.065(5) Å). The angle between the phenyl and acrylamide moieties was 29.8 °, disclosing that the conjugation was not stringent, e.g., the conjugation could be disrupted. In the molecule of (*E*)–*N*-(2-methylcinnamoyl)-amantadine, one hydrogen donor (N–H) and one acceptor (carbonyl oxygen) were present. In the crystal structure, the molecules produced one-dimensional chains with a graph set ଵ ଵ(4) [28,29] (Figure 2a). The three-dimensional packing of the molecules (Figure 2b) did not reveal additional weak interactions, and thus the stabilization of the crystal structure was achieved by the N1-H1…O1 hydrogen bond (N1…O1 of 3.065(5) Å).

**Figure 1.** (**a**) ORTEP [30] view and numbering scheme of the molecule present in the asymmetric unit of (*E*)–*N*-(2-methylcinnamoyl)-amantadine; the thermal ellipsoids were drawn with 50% probability, and hydrogen atoms are shown as small spheres with arbitrary radii. (**b**) Observed angle between the mean plane of the phenyl (C1/C15/C16/C17/C18/C19) and acrylamide (C13/C12/C11/O1/N1) moieties (C13/C12/C11/O1/N1). **Figure 1.** (**a**) ORTEP [30] view and numbering scheme of the molecule present in the asymmetric unit of (*E*)–*N*-(2-methylcinnamoyl)-amantadine; the thermal ellipsoids were drawn with 50% probability, and hydrogen atoms are shown as small spheres with arbitrary radii. (**b**) Observed angle between the mean plane of the phenyl (C1/C15/C16/C17/C18/C19) and acrylamide (C13/C12/C11/O1/N1) moieties (C13/C12/C11/O1/N1).

**Figure 2.** The observed (**a**) hydrogen bonding interaction stabilizing the crystal structure of (*E*)–*N*- (2-methylcinnamoyl)-amantadine and (**b**) a view along the *b* axis of the three-dimensional packing

There was no significant change in the weight of the control mice over the 12-day period. In those treated with MPTP, we observed a 6.92% weight gain within the group. In the MPTP + CA(2-Me)-Am and MPTP + OA mice, weight reduction was recorded at

of the molecules and formation of C11(4) chains propagating along the [010] plane.

the end of the observed period at 17.41% and 15.51%, respectively (Figure 3).

*3.2. In Vivo Evaluation of Amide 3 in an Experimental Mouse Model of PD*  3.2.1. Effect of CA(2-Me)-Am (3) on the Weight of Experimental Animals

The angle between the phenyl and acrylamide moieties was 29.8 °, disclosing that the conjugation was not stringent, e.g., the conjugation could be disrupted. In the molecule of (*E*)–*N*-(2-methylcinnamoyl)-amantadine, one hydrogen donor (N–H) and one acceptor (carbonyl oxygen) were present. In the crystal structure, the molecules produced one-di-

ing of the molecules (Figure 2b) did not reveal additional weak interactions, and thus the stabilization of the crystal structure was achieved by the N1-H1…O1 hydrogen bond

**Figure 1.** (**a**) ORTEP [30] view and numbering scheme of the molecule present in the asymmetric unit of (*E*)–*N*-(2-methylcinnamoyl)-amantadine; the thermal ellipsoids were drawn with 50% probability, and hydrogen atoms are shown as small spheres with arbitrary radii. (**b**) Observed angle

ଵ(4) [28,29] (Figure 2a). The three-dimensional pack-

mensional chains with a graph set ଵ

(N1…O1 of 3.065(5) Å).

**Figure 2.** The observed (**a**) hydrogen bonding interaction stabilizing the crystal structure of (*E*)–*N*- (2-methylcinnamoyl)-amantadine and (**b**) a view along the *b* axis of the three-dimensional packing of the molecules and formation of C11(4) chains propagating along the [010] plane. **Figure 2.** The observed (**a**) hydrogen bonding interaction stabilizing the crystal structure of (*E*)–*N*-(2 methylcinnamoyl)-amantadine and (**b**) a view along the *b* axis of the three-dimensional packing of the molecules and formation of C<sup>1</sup> 1 (4) chains propagating along the [10] plane.

#### *3.2. In Vivo Evaluation of Amide 3 in an Experimental Mouse Model of PD*  3.2.1. Effect of CA(2-Me)-Am (3) on the Weight of Experimental Animals *3.2. In Vivo Evaluation of Amide 3 in an Experimental Mouse Model of PD* 3.2.1. Effect of CA(2-Me)-Am (3) on the Weight of Experimental Animals

There was no significant change in the weight of the control mice over the 12-day period. In those treated with MPTP, we observed a 6.92% weight gain within the group. In the MPTP + CA(2-Me)-Am and MPTP + OA mice, weight reduction was recorded at the end of the observed period at 17.41% and 15.51%, respectively (Figure 3). There was no significant change in the weight of the control mice over the 12-day period. In those treated with MPTP, we observed a 6.92% weight gain within the group. In the MPTP + CA(2-Me)-Am and MPTP + OA mice, weight reduction was recorded at the end of the observed period at 17.41% and 15.51%, respectively (Figure 3). *Crystals* **2022**, *12*, × FOR PEER REVIEW 7 of 12

#### 3.2.2. Rotarod Test

**Time on rotarod (sec)**

3.2.2. Rotarod Test The studies performed demonstrated that the group of mice treated either with the MPTP toxin or with MPTP + OA spent less time on the rotating lever of the rotarod apparatus as compared to the controls, which is an indication of a motor-impairing effect. The reduction was by 21.13 % (*p* < 0.05) for the MPTP group, and by 20.99 % (*p* < 0.05) for the MPTP + OA group (Figure 4). In the MPTP + CA(2-Me)-Am group, the time that experimental animals spent on the rotary lever was comparable to that of the control group (Figure 4). The studies performed demonstrated that the group of mice treated either with the MPTP toxin or with MPTP + OA spent less time on the rotating lever of the rotarod apparatus as compared to the controls, which is an indication of a motor-impairing effect. The reduction was by 21.13 % (*p* < 0.05) for the MPTP group, and by 20.99 % (*p* < 0.05) for the MPTP + OA group (Figure 4). In the MPTP + CA(2-Me)-Am group, the time that experimental animals spent on the rotary lever was comparable to that of the control group (Figure 4).

**Figure 4.** Effect of CA(2-Me)-Am on neuromuscular coordination. The asterisks above bars indicate significant differences in number of falls per minute for each experimental group versus the control at \* *p* < 0.05. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed

The administration of the MPTP toxin caused a decrease in the step-through latency time by 31.56% (*p* < 0.01) at 1st h and by 33.45% (*p* < 0.01) at 24th h after the training of mice as compared to controls, which is evidence of memory and learning deficits. Administration of CA(2-Me)-Am increased the latent reaction time by 33.49% (*p* < 0.05) at the 1st h and by 33.84% (*p* < 0.05) at 24th h as compared to the MPTP-treated group, an indication of a memory-protective effect of the newly synthesized amantadine derivative (Figure 5).

by Dunnett's post hoc comparison test.

3.2.3. Passive Avoidance Test

**10**

**15**

**20**

**Body Weight (g)**

**25**

ure 4).

**Control**

**MPTP + OA**

**MPTP + CA(2-Me)-Am**

**MPTP**

**Figure 4.** Effect of CA(2-Me)-Am on neuromuscular coordination. The asterisks above bars indicate significant differences in number of falls per minute for each experimental group versus the control at \* *p* < 0.05. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Dunnett's post hoc comparison test. **Figure 4.** Effect of CA(2-Me)-Am on neuromuscular coordination. The asterisks above bars indicate significant differences in number of falls per minute for each experimental group versus the control at \* *p* < 0.05. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Dunnett's post hoc comparison test.

**Figure 3.** Effect of CA(2-Me)-Am on the weight of the experimental animals. Data are presented as

The studies performed demonstrated that the group of mice treated either with the MPTP toxin or with MPTP + OA spent less time on the rotating lever of the rotarod apparatus as compared to the controls, which is an indication of a motor-impairing effect. The reduction was by 21.13 % (*p* < 0.05) for the MPTP group, and by 20.99 % (*p* < 0.05) for the MPTP + OA group (Figure 4). In the MPTP + CA(2-Me)-Am group, the time that experimental animals spent on the rotary lever was comparable to that of the control group (Fig-

means with their respective standard errors (m ± S.E.M; n = 8; \* *p* < 0.05).

#### 3.2.3. Passive Avoidance Test 3.2.3. Passive Avoidance Test

**1 Day 5 Day 12 Day**

3.2.2. Rotarod Test

The administration of the MPTP toxin caused a decrease in the step-through latency time by 31.56% (*p* < 0.01) at 1st h and by 33.45% (*p* < 0.01) at 24th h after the training of mice as compared to controls, which is evidence of memory and learning deficits. Administration of CA(2-Me)-Am increased the latent reaction time by 33.49% (*p* < 0.05) at the 1st h and by 33.84% (*p* < 0.05) at 24th h as compared to the MPTP-treated group, an indication of a memory-protective effect of the newly synthesized amantadine derivative (Figure 5). The administration of the MPTP toxin caused a decrease in the step-through latency time by 31.56% (*p* < 0.01) at 1st h and by 33.45% (*p* < 0.01) at 24th h after the training of mice as compared to controls, which is evidence of memory and learning deficits. Administration of CA(2-Me)-Am increased the latent reaction time by 33.49% (*p* < 0.05) at the 1st h and by 33.84% (*p* < 0.05) at 24th h as compared to the MPTP-treated group, an indication of a memory-protective effect of the newly synthesized amantadine derivative (Figure 5). *Crystals* **2022**, *12*, × FOR PEER REVIEW 8 of 12

**Figure 5.** The effect of CA(2-Me)-Am on initial latency (IL) and step-through latency (STL) in a single-trial passive avoidance test in a mouse model of PD. Significance vs. control group: \*\**p* < 0.01; significance vs. MPTP-treated group: # *p* < 0.05. Statistical analysis was performed by one-way analysis of variance (ANOVA), followed by Dunnett's post hoc comparison test. **Figure 5.** The effect of CA(2-Me)-Am on initial latency (IL) and step-through latency (STL) in a single-trial passive avoidance test in a mouse model of PD. Significance vs. control group: \*\**p* < 0.01; significance vs. MPTP-treated group: # *p* < 0.05. Statistical analysis was performed by one-way analysis of variance (ANOVA), followed by Dunnett's post hoc comparison test.

**Detected Hydrogen Bonding Interaction** 

D…A 3.38 Å

D…A 3.09 Å

#### *3.3. Molecular Docking 3.3. Molecular Docking*

lected targets associated with PD.

The docking of (*E*)-*N*-(2-methylcinnamoyl)-amantadine (**3**) was performed against four different targets associated with PD: A2aAR (3EML) [31], COMT (1H1D) [32], MAO-B (2C65) [33], and NMDA (7SAD) [34] (Table 2). The docking of (*E*)-*N*-(2-methylcinnamoyl)-amantadine (**3**) was performed against four different targets associated with PD: A2aAR (3EML) [31], COMT (1H1D) [32], MAO-B (2C65) [33], and NMDA (7SAD) [34] (Table 2).

**Table 2.** Molecular docking score (kcal/mol) of (*E*)–*N*-(2-methylcinnamoyl)-amantadine against se-

**Molegro Virtual Docker Score** 

(*E*)–*N*-(2-methylcinnamoyl) amantadine

A2aAR (3EML) [31] -83.578 C=O…O-H Tyr271

MAO-B (2C65) [33] -119.889 C=O…N-H Gly58

NMAD, *N*-methyl-*D*-aspartate receptor; COMT, catechol-*O*-methyltransferase; A2aAR, A2A aden-

The docking approach involved predicting the conformation and orientation of ligands within a targeted binding site. Initially, the reference drug memantine present in 7SAD [34] was employed to adjust the docking parameters. The structures of the target enzymes were obtained from the Protein Data Bank (PDB), the coordinates of the small molecules were generated from the crystal structure of the (*E*)–*N*-(2-methylcinnamoyl) amantadine, and positioning in the active site and docking were conducted using Molegro Virtual Docker (MVD2019.7.0.0-2019-03-18-1B win32). Details regarding the docking validation are provided in Figures S4 and S5. The UCSF Chimera [35] and Ligplot+ 2.2.5 [36] were used for visualization and interactions detection. On the basis of the scores obtained from the docking results, the interaction of (*E*)–*N*-(2-methylcinnamoyl)-amantadine with MAO-B was most favorable (score of –119.889, Figure 6). The second possibility not to be excluded is the interaction with COMT (–111.957). However, while for MAO-B, a hydro-

NMAD (7SAD) [34] -80.240 No COMT (1H1D) [32] -111.957 No

osine receptor; MAO-B, monoamine oxidase B.


**Table 2.** Molecular docking score (kcal/mol) of (*E*)–*N*-(2-methylcinnamoyl)-amantadine against selected targets associated with PD.

NMAD, *N*-methyl-*D*-aspartate receptor; COMT, catechol-*O*-methyltransferase; A2aAR, A2A adenosine receptor; MAO-B, monoamine oxidase B.

The docking approach involved predicting the conformation and orientation of ligands within a targeted binding site. Initially, the reference drug memantine present in 7SAD [34] was employed to adjust the docking parameters. The structures of the target enzymes were obtained from the Protein Data Bank (PDB), the coordinates of the small molecules were generated from the crystal structure of the (*E*)–*N*-(2-methylcinnamoyl)-amantadine, and positioning in the active site and docking were conducted using Molegro Virtual Docker (MVD2019.7.0.0-2019-03-18-1B win32). Details regarding the docking validation are provided in Figures S4 and S5. The UCSF Chimera [35] and Ligplot+ 2.2.5 [36] were used for visualization and interactions detection. On the basis of the scores obtained from the docking results, the interaction of (*E*)–*N*-(2-methylcinnamoyl)-amantadine with MAO-B was most favorable (score of –119.889, Figure 6). The second possibility not to be excluded is the interaction with COMT (–111.957). However, while for MAO-B, a hydrogen bonding interaction was detected for COMT, no hydrogen bonding interaction "enzyme . . . ligand" was identified (Figure 7). Interestingly, the active sites of COMT and MAO-B shared a large amount of analogical AA and thus it may be possible to design a ligand that will interact with both enzymes.

Parkinson's disease has high social significance, resulting from a progressive loss of nigrostriatal dopaminergic neurons. The decrease in striatal dopaminergic innervation due to this loss is responsible for motor disturbances characteristic of the disease, such as akinesia, muscular rigidity, and tremor, and cognitive function impairment later appears. Amantadine is an agent that raises the concentration of dopamine in the synaptic cleft in PD. Currently, levodopa is considered to be the gold standard for symptomatic treatment of PD. However, long-term treatment with levodopa is complicated by motor fluctuations and dyskinesia. Everything stated above requires the search for compounds that can replace levodopa and improve the efficacy of amantadine in the treatment of PD. In this line of thinking, we performed investigations of a newly obtained amantadine derivative CA(2- Me)-Am (**3**) on neuromuscular coordination, learning, and memory in an experimental mouse model of PD. The obtained data showed that amide 3 restored neuromuscular coordination and memory performance of parkinsonian animals to the control level, giving an indication of its beneficial protective effects.

ligand that will interact with both enzymes.

**Figure 6.** Visualization of the docking studies and molecular interaction of (*E*)-*N*-(2-methylcinnamoyl)-amantadine with (**a**) COMT and (**b**) MAO-B. The hydrogen bonding interaction *N*-H…O=C is shown in as red dashed line with the A…D distance of 3.096 Å. **Figure 6.** Visualization of the docking studies and molecular interaction of (*E*)-*N*-(2 methylcinnamoyl)-amantadine with (**a**) COMT and (**b**) MAO-B. The hydrogen bonding interaction *N*-H . . . O=C is shown in as red dashed line with the A . . . D distance of 3.096 Å.

gen bonding interaction was detected for COMT, no hydrogen bonding interaction "enzyme…ligand" was identified (Figure 7). Interestingly, the active sites of COMT and MAO-B shared a large amount of analogical AA and thus it may be possible to design a

*Crystals* **2022**, *12*, × FOR PEER REVIEW 10 of 12

**Figure 7.** Observed interactions after docking of (*E*)–*N*-(2-methylcinnamoyl)-amantadine into the active site of two putative PD targets (**a**) MAO-B (2C65) and (**b**) COMT (1H1D). The hydrogen bonding interactions are shown in green; the hydrophobic contacts are shown as for enzymes and with for ligands; the similar residues for both enzymes are shown as . **Figure 7.** Observed interactions after docking of (*E*)–*N*-(2-methylcinnamoyl)-amantadine into the active site of two putative PD targets (**a**) MAO-B (2C65) and (**b**) COMT (1H1D). The hydrogen bonding interactions are shown in green; the hydrophobic contacts are shown as **Figure 7.** Observed interactions after docking of (*E*)**–***N*-(2-methylcinnamoyl)-amantadine into the active site of two putative PD targets (**a**) MAO-B (2C65) and (**b**) COMT (1H1D). The hydrogen bonding interactions are shown in green; the hydrophobic contacts are shown as for for enzymes **Figure 7.** Observed interactions after docking of (*E*)**–***N*-(2-methylcinnamoyl)-amantadine into the active site of two putative PD targets (**a**) MAO-B (2C65) and (**b**) COMT (1H1D). The hydrogen bonding interactions are shown in green; the hydrophobic contacts are shown as for **Figure 7.** Observed interactions after docking of (*E*)**–***N*-(2-methylcinnamoyl)-amantadine into the active site of two putative PD targets (**a**) MAO-B (2C65) and (**b**) COMT (1H1D). The hydrogen bonding interactions are shown in green; the hydrophobic contacts are shown as for

.

enzymes and with for ligands; the similar residues for both enzymes are shown as . and with enzymes and with for ligands; the similar residues for both enzymes are shown as . for ligands; the similar residues for both enzymes are shown as enzymes and with for ligands; the similar residues for both enzymes are shown as .

Parkinson's disease has high social significance, resulting from a progressive loss of nigrostriatal dopaminergic neurons. The decrease in striatal dopaminergic innervation due to this loss is responsible for motor disturbances characteristic of the disease, such as akinesia, muscular rigidity, and tremor, and cognitive function impairment later appears. Amantadine is an agent that raises the concentration of dopamine in the synaptic cleft in Parkinson's disease has high social significance, resulting from a progressive loss of nigrostriatal dopaminergic neurons. The decrease in striatal dopaminergic innervation due to this loss is responsible for motor disturbances characteristic of the disease, such as akinesia, muscular rigidity, and tremor, and cognitive function impairment later appears. Parkinson's disease has high social significance, resulting from a progressive loss of nigrostriatal dopaminergic neurons. The decrease in striatal dopaminergic innervation due to this loss is responsible for motor disturbances characteristic of the disease, such as akinesia, muscular rigidity, and tremor, and cognitive function impairment later appears. Parkinson's disease has high social significance, resulting from a progressive loss of nigrostriatal dopaminergic neurons. The decrease in striatal dopaminergic innervation due to this loss is responsible for motor disturbances characteristic of the disease, such as akinesia, muscular rigidity, and tremor, and cognitive function impairment later appears. Moreover, the molecular docking study investigations showed that from the considered four targets, (*E*)–*N*-(2-methylcinnamoyl)-amantadine interacted preferably with MAO-B, followed by COMT. The detected hydrogen bonding interaction could be used for development of modified potential antiviral drug candidates.

PD. Currently, levodopa is considered to be the gold standard for symptomatic treatment of PD. However, long-term treatment with levodopa is complicated by motor fluctuations and dyskinesia. Everything stated above requires the search for compounds that can re-Amantadine is an agent that raises the concentration of dopamine in the synaptic cleft in PD. Currently, levodopa is considered to be the gold standard for symptomatic treatment of PD. However, long-term treatment with levodopa is complicated by motor fluctuations Amantadine is an agent that raises the concentration of dopamine in the synaptic cleft in PD. Currently, levodopa is considered to be the gold standard for symptomatic treatment of PD. However, long-term treatment with levodopa is complicated by motor fluctuations Amantadine is an agent that raises the concentration of dopamine in the synaptic cleft in PD. Currently, levodopa is considered to be the gold standard for symptomatic treatment of PD. However, long-term treatment with levodopa is complicated by motor fluctuations In conclusion, our results demonstrated ameliorating effects of the newly synthesized compound CA(2-Me)-Am in an experimental model of Parkinson's disease, which deserves further investigations.

place levodopa and improve the efficacy of amantadine in the treatment of PD. In this line of thinking, we performed investigations of a newly obtained amantadine derivative CA(2-Me)-Am **(3**) on neuromuscular coordination, learning, and memory in an experiand dyskinesia. Everything stated above requires the search for compounds that can replace levodopa and improve the efficacy of amantadine in the treatment of PD. In this line of thinking, we performed investigations of a newly obtained amantadine derivative CA(2-Me)-Am **(3**) on neuromuscular coordination, learning, and memory in an and dyskinesia. Everything stated above requires the search for compounds that can replace levodopa and improve the efficacy of amantadine in the treatment of PD. In this line of thinking, we performed investigations of a newly obtained amantadine derivative CA(2-Me)-Am **(3**) on neuromuscular coordination, learning, and memory in an and dyskinesia. Everything stated above requires the search for compounds that can replace levodopa and improve the efficacy of amantadine in the treatment of PD. In this line of thinking, we performed investigations of a newly obtained amantadine derivative CA(2-Me)-Am **(3**) on neuromuscular coordination, learning, and memory in an **Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/cryst12111518/s1, NMR and MS spectra of compound 3 are provided.

mental mouse model of PD. The obtained data showed that amide 3 restored neuromuscular coordination and memory performance of parkinsonian animals to the control level, giving an indication of its beneficial protective effects. Moreover, the molecular docking study investigations showed that from the considexperimental mouse model of PD. The obtained data showed that amide 3 restored neuromuscular coordination and memory performance of parkinsonian animals to the control level, giving an indication of its beneficial protective effects. Moreover, the molecular docking study investigations showed that from the experimental mouse model of PD. The obtained data showed that amide 3 restored neuromuscular coordination and memory performance of parkinsonian animals to the control level, giving an indication of its beneficial protective effects. Moreover, the molecular docking study investigations showed that from the experimental mouse model of PD. The obtained data showed that amide 3 restored neuromuscular coordination and memory performance of parkinsonian animals to the control level, giving an indication of its beneficial protective effects. Moreover, the molecular docking study investigations showed that from the **Author Contributions:** Synthesis, supervision, writing and manuscript conceptualization, M.C.; NMR spectroscopy studies, N.P. and M.Š.; study, M.Š.; single crystal X-ray diffraction experiments, R.R. and H.S.-D.; docking studies, writing, B.S.; neurobehavioral studies, writing, R.K., L.T., M.L., A.P. and K.T. All authors have read and agreed to the published version of the manuscript.

ered four targets, (*E*)–*N*-(2-methylcinnamoyl)-amantadine interacted preferably with MAO-B, followed by COMT. The detected hydrogen bonding interaction could be used for development of modified potential antiviral drug candidates. considered four targets, (*E*)**–***N*-(2-methylcinnamoyl)-amantadine interacted preferably with MAO-B, followed by COMT. The detected hydrogen bonding interaction could be considered four targets, (*E*)**–***N*-(2-methylcinnamoyl)-amantadine interacted preferably with MAO-B, followed by COMT. The detected hydrogen bonding interaction could be considered four targets, (*E*)**–***N*-(2-methylcinnamoyl)-amantadine interacted preferably with MAO-B, followed by COMT. The detected hydrogen bonding interaction could be **Funding:** This work was funded by the Bulgarian National Science Fund (BNSF), grant number KP-06-Russia/7-2019.

In conclusion, our results demonstrated ameliorating effects of the newly synthesized compound CA(2-Me)-Am in an experimental model of Parkinson's disease, which deserves further investigations. used for development of modified potential antiviral drug candidates. In conclusion, our results demonstrated ameliorating effects of the newly synthesized compound CA(2-Me)-Am in an experimental model of Parkinson's disease, which used for development of modified potential antiviral drug candidates. In conclusion, our results demonstrated ameliorating effects of the newly synthesized compound CA(2-Me)-Am in an experimental model of Parkinson's disease, which used for development of modified potential antiviral drug candidates. In conclusion, our results demonstrated ameliorating effects of the newly synthesized compound CA(2-Me)-Am in an experimental model of Parkinson's disease, which **Institutional Review Board Statement:** The animal study protocol was approved by the Commission of Bioethics (CBE) at the Institute of Neurobiology, Bulgarian Academy of Sciences— BAS/CBE/018/2020.

**Supplementary Materials:** The following supporting information can be downloaded at: deserves further investigations. deserves further investigations. deserves further investigations. **Informed Consent Statement:** Not applicable.

www.mdpi.com/xxx/s1, NMR and MS spectra of compound 3 are provided.

www.mdpi.com/xxx/s1, NMR and MS spectra of compound 3 are provided.

www.mdpi.com/xxx/s1, NMR and MS spectra of compound 3 are provided.

www.mdpi.com/xxx/s1, NMR and MS spectra of compound 3 are provided.

**Supplementary Materials:** The following supporting information can be downloaded at:

**Supplementary Materials:** The following supporting information can be downloaded at:

**Supplementary Materials:** The following supporting information can be downloaded at:

**Data Availability Statement:** Crystallographic data for the structure of the title compound *(E*)- *N*-(2-methylcinnamoyl)-amantadine (CA(2-Me)-Am; 3)) was deposited in the CIF format with the Cambridge Crystallographic Data Center as 2205297. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, deposited on 05 September 2022 (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +441223336033; e-mail: deposit@ccdc.cam.ac.uk).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


*Article* 

#### *Article* **Studies on the Crystal Forms of Istradefylline: Structure, Solubility, and Dissolution Profile Studies on the Crystal Forms of Istradefylline: Structure, Solubility, and Dissolution Profile**

**Yiyun Wang 1,2, Youwei Xu 2,3, Zhonghui Zheng <sup>2</sup> , Min Xue 1,\*, Zihui Meng 1,\*, Zhibin Xu <sup>1</sup> , Jiarong Li <sup>1</sup> and Qing Lin <sup>4</sup> Yiyun Wang 1,2, Youwei Xu 2,3, Zhonghui Zheng 2, Min Xue 1,\*, Zihui Meng 1,\*, Zhibin Xu 1, Jiarong Li 1 and Qing Lin 4**


**Abstract:** Istradefylline as a selective adenosine A2A-receptor antagonist is clinically used to treat Parkinson's disease and improve dyskinesia in its early stages. However, its crystal form, as an important factor in the efficacy of the drug, is rarely studied. Herein, three kinds of crystal forms of istradefylline prepared from ethanol (form I), methanol (form II), and acetonitrile (form III) are reported by use of a crystal engineering strategy. These three crystal forms were characterized and made into tablets for dissolution testing. Both the solubility and the dissolution rates were also determined. The dissolution rate of form I and form III is significantly higher than form II at pH 1.2 (87.1%, 58.2%, and 87.7% for form I, form II, and form III, respectively), pH 4.5 (88.1%, 58.9%, and 87.1% for form I, form II, and form III, respectively) and pH 6.8 (87.5%, 58.2%, and 86.0% for form I, form II, and form III, respectively) at 60 min. Considering the prepared solution and the proper dissolution profile, form I is anticipated to possess promising absorption for bioavailability. **Abstract:** Istradefylline as a selective adenosine A2A-receptor antagonist is clinically used to treat Parkinson's disease and improve dyskinesia in its early stages. However, its crystal form, as an important factor in the efficacy of the drug, is rarely studied. Herein, three kinds of crystal forms of istradefylline prepared from ethanol (form I), methanol (form II), and acetonitrile (form III) are reported by use of a crystal engineering strategy. These three crystal forms were characterized and made into tablets for dissolution testing. Both the solubility and the dissolution rates were also determined. The dissolution rate of form I and form III is significantly higher than form II at pH 1.2 (87.1%, 58.2%, and 87.7% for form I, form II, and form III, respectively), pH 4.5 (88.1%, 58.9%, and 87.1% for form I, form II, and form III, respectively) and pH 6.8 (87.5%, 58.2%, and 86.0% for form I, form II, and form III, respectively) at 60 min. Considering the prepared solution and the proper dissolution profile, form I is anticipated to possess promising absorption for bioavailability.

**Keywords:** Parkinson's disease; istradefylline; solubility; crystal form; dissolution **Keywords:** Parkinson's disease; istradefylline; solubility; crystal form; dissolution

#### **1. Introduction 1. Introduction**

The adenosine A2A-receptor is closely related to Parkinson's disease (PD). A suitable antagonist could enhance the function of dopamine on D2 receptor neurons and result in some anti-Parkinson's effect [1–6]. Istradefylline (Figure 1) (KW-6002, (*E*)-8-(3,4 dimethoxystyryl)-1,3-diethyl-7-methyl-dihydro-1*H*-purine-2,6-dione) is the first approved adenosine A2A-receptor antagonist that can improve the motor function of PD patients through its neuronal activity [7,8]. Moreover, it has also received extensive attention in pharmacology. Istradefylline was reported as a promising drug for movement disorders treatment [9]. In addition, Shin-ichi Uchida reported that istradefylline enhances the anti-parkinsonian activity of low doses of dopamine agonists [10–13]. The adenosine A2A-receptor is closely related to Parkinson's disease (PD). A suitable antagonist could enhance the function of dopamine on D2 receptor neurons and result in some anti-Parkinson's effect [1–6]. Istradefylline (Figure 1) (KW-6002, (*E*)-8-(3,4-dimethoxystyryl)-1,3-diethyl-7-methyl-dihydro-1*H*-purine-2,6-dione) is the first approved adenosine A2A-receptor antagonist that can improve the motor function of PD patients through its neuronal activity [7,8]. Moreover, it has also received extensive attention in pharmacology. Istradefylline was reported as a promising drug for movement disorders treatment [9]. In addition, Shin-ichi Uchida reported that istradefylline enhances the antiparkinsonian activity of low doses of dopamine agonists [10–13].

**Figure 1.** Chemical structure of istradefylline. **Figure 1.** Chemical structure of istradefylline.

It is known that the originality of the pharmacological activity of a drug has an important influence on the effective absorption and utilization of the drug in the body.

**Citation:** Wang, Y.; Xu, Y.; Zheng, Z.; Xue, M.; Meng, Z.; Xu, Z.; Li, J.; Lin, Q. Studies on the Crystal Forms of Istradefylline: Structure, Solubility, and Dissolution Profile. *Crystals* **2022**, *12*, 917. https://doi.org/10.3390/ cryst12070917 Zheng, Z.; Xue, M.; Meng, Z.; Xu, Z.; Li, J.; Lin, Q. Studies on the Crystal Forms of Istradefylline: Structure, Solubility, and Dissolution Profile. *Crystals* **2022**, *12*, x. https://doi.org/10.3390/xxxxx

**Citation:** Wang, Y.; Xu, Y.;

Academic Editors: Abel Moreno and Brahim Benyahia Academic Editors: Abel Moreno and Brahim Benyahia

Received: 15 April 2022 Accepted: 26 June 2022 Published: 28 June 2022 Received: 15 April 2022 Accepted: 26 June 2022 Published: 28 June 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. tral with regard to jurisdictional claims in published maps and institutional affiliations.

**Publisher's Note:** MDPI stays neu-

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

It is remarkable that the solubility and dissolution of a drug in oral tablets significantly affects its absorption and metabolism in the body. The particle size and crystal form of a drug also affect pharmacological efficacy, due to their ability to alter the physicochemical properties of solubility, dissolution, and dosage forms. During the crystallization of a drug, different crystal structures can be formed, as the packing of molecules in space change at different temperatures, solutions, and pressures [14–17]. Generally, the appearance, melting point, dissolution, and other aspects of the same drug are significantly different in diverse crystal forms, which correspondingly affect clinical efficacy [18–22]. However, there are few reports on the solubility, crystal form, and dissolution of istradefylline.

Drug crystallization form depends on many factors, such as solvent, temperature and cooling rate, stirring speed and time, water content in solvent, and impurities in product. Based on descriptions in the literature [23–27], five crystal forms of istradefylline from ethanol/THF/isopropanol/n-propanol, methanol, acetonitrile, dichloromethane, and DMF/H2O have been reported, while the melting points, acceleration tests, and longterm stability studies of three forms have been described; namely, the melting points of form I, form II, and form III were reported as 191.93 ◦C, 191.14 ◦C, and 191.14 ◦C by DSC analysis, respectively. However, the particle size, physical properties, and single crystal data of crystal forms were not reported in these patents, except for PXRD. Generally, dichloromethane and DMF were excluded in the manufacturing process due to their harmful impact on the quality of the medicine of the solution.

In this paper, we primarily discuss the dissolution rate of istradefylline in consideration of its adsorption in pharmacokinetics. In order to avoid the influence of other factors, the solubility, crystal form, particle size, and physical and chemical properties of istradefylline were also studied. The solubility and crystal form of istradefylline in seven single solvents and five mixed solvents were studied in a temperature range from 293.15 K to 333.15 K. Based on the solubility and powder diffraction data, three different crystal forms of istradefylline were obtained from ethanol (form I), methanol (form II), and acetonitrile (form III). These were consistent with the three crystal forms reported by patent NO. CN104744464A [23]. In addition, all of them were characterized by solubility, HPLC analysis, TGA and FT-IR [28,29]. In order to keep the particle size and specific surface area roughly uniform, each of the istradefylline forms was ground in a mortar for five minutes before the tablet preparation. Then, the dissolution rate of istradefylline was investigated according to the "Guidelines for determination and comparison of dissolution curves of common oral solid preparations" of Chinese pharmacopoeia. In this study, the dissolution rates of form I and form III were significantly higher than that of form II. These results have guiding significance for istradefylline tablet production.

#### **2. Materials and Methods**

#### *2.1. Materials*

Istradefylline with a purity of 99.5% was provided by Shandong Xinhua pharmaceutical Co., Ltd., Zibo, China. Acetonitrile, methanol, ethanol, ethyl acetate, n-propanol, isopropanol, and n-butanol were purchased from J.T. Baker Co., Ltd. without further purification (analytical pure), Shanghai, China. Hydrochloric acid, sodium hydroxide, potassium dihydrogen phosphate, sodium acetate trihydrate, and acetic acid were purchased from Sinopharm Group Chemical Reagent Co., Ltd. without further purification (analytical pure), Shanghai, China. Lactose (Lactose Anhydrous, NF DTHV) was provided by Kerry Inc.-Rothschild, Shanghai, China. Microcrystalline cellulose (MCC, Microcrystalline Cellulose, VIVAPUR®, PH 102) was provided by J. Rettenmaier & Sohne GmbH + Co. KG, Germany. Crospovidone (PVPP, Kollidon®, CL-F) was provided by BASF SE. Magnesium stearate (LIGAMED®, MF-2-V) was provided by Peter Greven Nederland CV. Sodium laurylsulfonate (SDS) was purchased from J&K Chemicals, Beijing, China. Purified water (18.25 MΩ·cm−<sup>1</sup> ) was obtained from a Millipore Mili-Q Plus water system. All saturated solutions prepared for HPLC detection were filtered through 0.22 µm filter membrane before usage.

#### *2.2. HPLC analysis*

The qualitative and quantitative determinations of istradefylline were performed on a Shimadzu HPLC system (Kyoto, Japan) comprising of two LC-20AT pumps, one SPD-20 UV detector, and a SIL-10A auto-sampler. The liquid chromatographic condition was optimized on an Agilent ZORBOX C18 chromatographic column (150 mm × 4.6 mm, 5 µm) with acetonitrile and water (60/40, *v*/*v*) as the stationary and mobile phase, respectively. The flow rate was confirmed as 1.0 mL·min−<sup>1</sup> , while the UV-determined wavelength was 355 nm, and the sample injection volume was 20 µL.

#### *2.3. Solubility of Istradefylline in Diverse Organic Solvents and Solvent Mixtures with Water*

A certain amount of istradefylline powder was placed into a glass vial with 10 mL of acetonitrile, methanol, ethanol, ethyl acetate, n-propanol, isopropanol, n-butanol, methanol/water (30/70, *v*/*v*), ethanol/water (24/76, *v*/*v*), ethanol/water (55/45, *v*/*v*), acetonitrile/water (25/75, *v*/*v*), and acetonitrile/water (58/42, *v*/*v*), respectively. Then, the vials were incubated in a thermostat water bath for 12 h with magnetic stirring at 293.15 K, 303.15 K, 313.15 K, 323.15 K, and 333.15 K, each measured by a thermometer inside each glass vial. The temperature fluctuation of the thermostat water bath was controlled within ±0.5 K with temperature uncertainty of ±0.1 K. Then, all solutions were left to stand for a further 12 h at the corresponding temperature until the dissolution equilibrium was obtained. Then, 2 mL of supernatant from each vial was withdrawn by a syringe with a 0.22 µm filter membrane for HPLC analysis. All of the experiments were carried out three times simultaneously to obtain data averages (Table S1).

#### *2.4. Preparation of Single Crystal*

First, 1 g istradefylline was added into each of three 100 mL single-mouth flasks with 20 mL ethanol, 50 mL methanol, or 20 mL acetonitrile, respectively. Then, the mixture was stirred and heated at 78 ◦C, 64.5 ◦C and 81.0 ◦C, respectively, until completely dissolved, followed by being cooled down to room temperature. Stirring continued for 2 h for crystallization. Consequently, forms I, II, and III of istradefylline were obtained by filtration.

#### *2.5. X-ray Diffraction*

Single-crystal X-ray diffraction data were collected using a Bruker apex2 X-ray diffractometer equipped with a Mercury CCD detector with graphite monochromated Mo-K α radiation (λ = 0.71073 Å) at 296 K. The structures were solved by direct methods and refined by full-matrix least-squares on F2 values (SHELXL-97). Non-hydrogen atoms were refined anisotropically. Hydrogen atoms were fixed at calculated positions and refined using a riding mode. Powder X-ray diffraction (PXRD) patterns of samples were collected on a Bruker D8 Focus X-ray diffractometer with Cu Kα radiation (λ = 1.54 Å) at a scanning rate of 0.02◦ s −1 from 5◦ to 50◦ in 2θ.

#### *2.6. Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA)*

The DSC and TGA was determined by TGA/DSC1/1100LF(Mettler Toledo, Switzerland). The temperature range was 25~1100 ◦C; temperature accuracy was ±0.3 ◦C; calorimetric accuracy was ±1%; balance sensitivity was 0.1 µg; heating rate was 0.1~100 K/min.

The experiment was performed under N<sup>2</sup> atmosphere at 1 atm with a heating rate of 10 ◦C/min in a temperature range of 30~400 ◦C.

#### *2.7. Fourier-Transform Infrared Spectral Analysis (FT-IR)*

FT-IR analysis was collected in a range of 3600–1600 cm−<sup>1</sup> using KBr pellets and a Thermo iD7 ATR infrared spectrometer (Thermo Fisher Technology (China) Co., Ltd., Shanghai, China).

#### *2.8. Particle Size and Specific Surface Area Analysis (BET)*

The particle sizes of forms I/II/III were determined by a Malvern 2000 laser particle size analyzer (Malvern, England). The specific surface areas (N<sup>2</sup> adsorption) of forms I/II/III were detected by the specific surface-area analyzer BK200B (Beijing Jingwei Gaobo Science and Technology Co., Ltd., Beijing, China.).

The samples were mixed thoroughly and evenly (loose clumps were gently pressed with a spoon to completely disperse) and flatly laid on the sample table of the Scirocco 2000 dry sampler. Vibration injection speed was 30~80%; relaxation-dispersed air pressure was 2.5 bar; cost of shading was 1~5%; measuring time was 10 s; background time was 10 s.

Specific surface-area analysis was performed under N<sup>2</sup> adsorption with adsorption temperature of 77.35 K on a BK200B using the static capacity method, while the temperature was controlled at 40 ◦C for 360 min.

#### *2.9. Dissolution Study*

The instruments used for tablets included a circulating water vacuum pump (SHB-III, Zhengzhou Great Wall Science, Industry and Trade Co., Ltd., Zhengzhou, China), an electrothermal blast drying box (GZX-9240MBE, Shanghai Boxun Industrial Co., Ltd., Shanghai, China), an electronic balance (PB3002-S, METTLER TOLEDO), a constant-temperature magnetic stirrer (DF-101S, Zhengzhou Great Wall Industry and Trade Co., Ltd.), an ultravioletabsorption spectrophotometer (UV1800, Shimadzu, Kyoto, Japan), a dissolution tester (SNTR-8400AT, Shimadzu), a single-stamping-sheet machine (YP-1, HangZhou XuZhong Food Machinery Co., Ltd.), and a high-efficiency coating machine (JCB/K-3/5/10, Wenzhou Jianpai Pharmaceutical Machinery Co., Ltd., Wenzhou, China, nozzle diameter of 1 mm).

In order to prepare the istradefylline tablets, a prescribed amount of istradefylline was weighed and ground in a mortar for 5 min. Lactose, microcrystalline cellulose PH102, and PVPP were weighed and mixed with istradefylline by hand for 3 min. Then, magnesium stearate (MS) was weighed and added into the above mixture and mixed again by hand for 3 min. A single-stamping-sheet machine (mold circular concave Φ7.1 mm, tablet weight 140 mg, tablet hardness −5 kp) was used for tableting. The coating liquid prepared by Opadry 03K19229 (solid content: 8%) was coated on a high-efficiency coating machine. The inlet air temperature was 70 ◦C, the atomization gas pressure was 0.3 MPa, the rotation speed was 8 rpm, and the spraying speed was 7 rpm. Coating-weight gain was controlled at about 3%. The batch size was 1000 pieces. The tablet speed was 1000 tablets/h, and the coating batch was 800 tablets/batch. The spray speed was 7.0 g/min. The prescription ingredients are shown in Table 1 in detail.


**Table 1.** Prescription ingredients list.

According to the dissolution and release determination method (Chinese Pharmacopoeia 2020 Edition, general rule of the fourth part 0931, second method), six tablets of istradefylline were put into a beaker filled with 900 mL buffer solution of pH 1.2, pH 4.5, and pH 6.8, while the rotation speed was fixed at 75 r/min. Then, 10 mL samples were

taken out at 5 min, 10 min, 15 min, 30 min, 45 min, and 60 min and filtrated with 0.22 µm filter membrane. Then, 2 mL filtrate was diluted to 10 mL with a diluent (acetonitrilewater (50:50)), and the test sample was obtained. Next, 25 mg istradefylline was precisely weighed and dissolved into 25 mL acetonitrile in a volumetric flask. Then, 1 mL istradefylline solution was diluted to 5 mL with a diluent (acetonitrile-water (50:50)), and the solution was mixed. Next, 2 mL solution was precisely measured and transferred into a 10 mL volumetric flask. Then, 2 mL dissolution medium was added, and the solution was diluted to scale with a diluent (acetonitrile-water (50:50)), upon which the reference solution was obtained.

The same amount of diluent (acetonitrile-water (50:50)) was added to the two cuvettes, and then they were placed in channel 1 and channel 2 of UV-Vis spectrophotometer, respectively. After the instrument was zeroed, the reference solution and sample solution were placed in channel 2 and measured at a wavelength of 362 nm. Each group of samples was repeatedly tested six times, and the RSD of all samples at each time point was less than 10%, which proved that each sample had good uniformity.

Computational Formula:

$$\text{Dissolution} = \frac{\text{Atest} \times \text{C reference} \times 5 \times 900}{\text{A reference} \times 20} \times 100\%$$

$$\text{Cumulative dissociation} = \text{A}\_{\text{n}} + \frac{(\text{A}\_{\text{n}} - 1 + \dots \dots + \text{A}\_{\text{l}}) \times 10}{900}$$

where Atest is UV absorbance of sample, Areference is UV absorbance of reference substance, Creference is concentration of reference substance.

#### **3. Results and Discussion**

#### *3.1. Solubility of Istradefylline*

A perfect chromatogram of istradefylline as a symmetrical sharp peak was obtained, as shown in Figure 2. The relationship between the chromatogram peak area and concentration expressed as calibration curve is graphically displayed in Figure 2. The linear fitting equation was Y = 4.42X + 7.06 with a concentration range of 0.001 mg·mL−<sup>1</sup> to 0.1 mg·mL−<sup>1</sup> , while the linear dependence was 0.9999. The linearity was used to calculate the istradefylline concentration in the supernatant of each vial in the experiment by HPLC detection. *Crystals* **2022**, *12*, x FOR PEER REVIEW 6 of 13

**Figure 2.** HPLC chromatogram of istradefylline. Insets show the linear relationship between the **Figure 2.** HPLC chromatogram of istradefylline. Insets show the linear relationship between the chromatogram peak area (Y) and the concentration (X) of istradefylline in acetonitrile.

chromatogram peak area (Y) and the concentration (X) of istradefylline in acetonitrile. In this study, the solubility data of istradefylline in common organic solvents in the In this study, the solubility data of istradefylline in common organic solvents in the range of 293.15 K to 333.15 K were determined by an established HPLC method with

=

classified in these solvents by powder X-ray diffraction method.

range of 293.15 K to 333.15 K were determined by an established HPLC method with mil-

solute in the solution. The mass of the solute in the sample solution can be calculated according to Equation (1), while the concentration of istradefylline in saturated solution was estimated by the liquid chromatographic method according to the calibration curve,

where *m* is the mass of istradefylline in saturated solution, *c* is the corresponding concentration, and *v* is the volume after diluted. The mole fraction of the solute can be readily

where *x* is the mole fraction of the solute istradefylline, *m*<sup>1</sup> is the mass of the solute calculated by Equation (1), *M*<sup>1</sup> is the molecular weight of solute, *m*0 is the mass of the solution, and *M*<sup>2</sup> is the molecular weight of solvent. The precise solubility of this compound in seven single-solvents and five mixed-solvents in the range of 293.15 K to 333.15 K is recorded in Table S1. Furthermore, the temperature influence on the solubility of istradefylline was also studied. Solubility increased at an exponential rate with rising temperature in all solvents, as shown in Figure 3. Generally, the solubility of chemicals is an endothermic process, so increasing the temperature is beneficial for increasing the solubility of drugs. Besides the solubility data, which were useful in the quality control and process improvement that followed, three kinds of crystalline forms were also determined and

ଵ/ଵ ଵ/ଵ + ሺ − ଵሻଶ

=∙ (1)

(2)

calculated as follows:

milligram-grade usage. The solubility of istradefylline was expressed by mole fraction of the solute in the solution. The mass of the solute in the sample solution can be calculated according to Equation (1), while the concentration of istradefylline in saturated solution was estimated by the liquid chromatographic method according to the calibration curve,

$$
\mathfrak{m} = \mathfrak{c} \cdot \mathfrak{v} \tag{1}
$$

where *m* is the mass of istradefylline in saturated solution, *c* is the corresponding concentration, and *v* is the volume after diluted. The mole fraction of the solute can be readily calculated as follows:

$$\infty = \frac{m\_1/M\_1}{m\_1/M\_1 + (m\_0 - m\_1)M\_2} \tag{2}$$

where *x* is the mole fraction of the solute istradefylline, *m*<sup>1</sup> is the mass of the solute calculated by Equation (1), *M*<sup>1</sup> is the molecular weight of solute, *m*<sup>0</sup> is the mass of the solution, and *M*<sup>2</sup> is the molecular weight of solvent. The precise solubility of this compound in seven single-solvents and five mixed-solvents in the range of 293.15 K to 333.15 K is recorded in Table S1. Furthermore, the temperature influence on the solubility of istradefylline was also studied. Solubility increased at an exponential rate with rising temperature in all solvents, as shown in Figure 3. Generally, the solubility of chemicals is an endothermic process, so increasing the temperature is beneficial for increasing the solubility of drugs. Besides the solubility data, which were useful in the quality control and process improvement that followed, three kinds of crystalline forms were also determined and classified in these solvents by powder X-ray diffraction method. *Crystals* **2022**, *12*, x FOR PEER REVIEW 7 of 13

**Figure 3.** Temperature dependence of mole fraction of istradefylline in several solvents. **Figure 3.** Temperature dependence of mole fraction of istradefylline in several solvents.

It is known that the solubility of istradefylline is very low in aqueous media in the pH range from 1.0 to 12.0, so the solubility of istradefylline in aqueous solutions of different pH was tested. The test results were shown in Table 2. From the test results, it can be seen that the solubility of istradefylline decreased when the pH increased, since istrade-It is known that the solubility of istradefylline is very low in aqueous media in the pH range from 1.0 to 12.0, so the solubility of istradefylline in aqueous solutions of different pH was tested. The test results were shown in Table 2. From the test results, it can be seen that the solubility of istradefylline decreased when the pH increased, since istradefylline is a weakly alkaline drug that has greater solubility in acidic solutions.

fylline is a weakly alkaline drug that has greater solubility in acidic solutions.

2 0.39 3 0.32 4 0.31 7 0.27 8 0.18 10 0.11 12 0.10

**pH Solubility of Istradefylline (µg/mL)** 

The istradefylline crystalline solids from the saturated solutions were characterized by X-ray powder diffraction (Figure 4). Three forms can be clearly distinguished from the significant differences present among their diffraction patterns. Form I could be obtained in a wide variety of solvent systems, including ethyl acetate, n-propanol, isopropanol, nbutanol, ethanol, ethanol/water (w = 0.1828), and ethanol/water (w = 0.4886). Its powder diffraction pattern is characterized by peaks at 2θ = 6.98°, 11.02°, 13.98°, 15.68°. Form II crystallized in methanol and methanol/water (w = 0.2553). Its characteristic diffraction peaks can be found at 2θ = 8.68°, 11.86° and 12.12°. Form III, obtained in acetonitrile, acetonitrile/water (w = 0.2105), and acetonitrile/water (w = 0.5127), shows characteristic diffraction peaks at 2θ = 9.74°, 10.24°, 12.38° and 25.07°. These results were consistent with

*3.2. Powder X-ray Diffraction* 

**Table 2.** Solubility of istradefylline in different pH at 293.15 K.

the three forms disclosed by patent number CN104744464A.


**Table 2.** Solubility of istradefylline in different pH at 293.15 K.

#### *3.2. Powder X-ray Diffraction*

The istradefylline crystalline solids from the saturated solutions were characterized by X-ray powder diffraction (Figure 4). Three forms can be clearly distinguished from the significant differences present among their diffraction patterns. Form I could be obtained in a wide variety of solvent systems, including ethyl acetate, n-propanol, isopropanol, nbutanol, ethanol, ethanol/water (w = 0.1828), and ethanol/water (w = 0.4886). Its powder diffraction pattern is characterized by peaks at 2θ = 6.98◦ , 11.02◦ , 13.98◦ , 15.68◦ . Form II crystallized in methanol and methanol/water (w = 0.2553). Its characteristic diffraction peaks can be found at 2θ = 8.68◦ , 11.86◦ and 12.12◦ . Form III, obtained in acetonitrile, acetonitrile/water (w = 0.2105), and acetonitrile/water (w = 0.5127), shows characteristic diffraction peaks at 2θ = 9.74◦ , 10.24◦ , 12.38◦ and 25.07◦ . These results were consistent with the three forms disclosed by patent number CN104744464A. *Crystals* **2022**, *12*, x FOR PEER REVIEW 8 of 13

**Figure 4.** Comparison of powder X-ray diffraction (after grinding) and crystallographic data of istradefylline. (**a**) Form I made in ethanol; (**b**) Form II made in methanol; (**c**) Form III made in acetonitrile. **Figure 4.** Comparison of powder X-ray diffraction (after grinding) and crystallographic data of istradefylline. (**a**) Form I made in ethanol; (**b**) Form II made in methanol; (**c**) Form III made in acetonitrile.

For further study, three forms were obtained using ethanol, methanol, and acetoni-

crystal structure and crystallography data are shown in Figures S1–S3 and Table 3.

**Compounds Form I Form II Form III**  Chemical formula C20H24N4O4 C20H26N4O5 C22H29N5O5 Formula weight 384.43 402.45 443.50 Crystal system monoclinic monoclinic monoclinic Space group *P*21 *P*21*/c P*21/*m* a/Å 13.6762(17) 4.5436(5) 9.430(9) b/Å 4.7483(7) 23.776(2) 7.129(7) c/Å 16.464(2) 18.6282(17) 17.587(18) α/° 90 90 90 β/° 112.39(4) 95.065(7) 103.514(10) γ/° 90 90 90 vol/Å3 988.5(2) 2004.6(3) 1149.5(19) Z 2 4 2 ρcalcg/cm3 1.292 1.334 1.281

**Table 3.** Crystallographic data and structure refinement parameters.

*3.3. Single-Crystal X-Ray Diffraction* 

#### *3.3. Single-Crystal X-ray Diffraction*

For further study, three forms were obtained using ethanol, methanol, and acetonitrile as the crystallization solvents, respectively (CCDC number: 2043873-2043875). Their crystal structure and crystallography data are shown in Figures S1–S3 and Table 3.


**Table 3.** Crystallographic data and structure refinement parameters.

According to the crystallographic data and structure refinement parameters, three forms can be effectively distinguished. From the single-crystal structure, it can be seen that form I was pure crystal, form II was monohydrate crystal, and form III was monohydrate of acetonitrile solvent complex. The three forms were all monoclinic systems with different space groups, with form I, II and III exhibiting *P*21, *P*21/*c* and *P*21/*m*, respectively. The molecular packing and intermolecular interactions of the three forms were different (Figure 5). Form I has strong intermolecular hydrogen bonds between oxygen atoms and the nearby hydrogen on the other side of the molecule (C=O . . . H, 2.37 Å and 3.16 in form I), without π-π stacking. Form II and form III have hydrogen bonds between oxygen atoms and the nearby hydrogens of water molecules (O5=O2 . . . H, 2.878 Å in form II and O5A=O3 . . . H, 2.955 Å in form III). Although abundant hydrogen bonds were constructed through the interactions between water molecules and the crystal of form II and form III, their bond energies of about 15~30 kJ·mol−<sup>1</sup> were much lower than those of general chemical bonds. This suggests that these hydrogen bonds are fragile, and the water molecules are transferred during the drying process of istradefylline. Otherwise, form II was a head-to-head π-π interaction, and form III was a head-to-tail π-π interaction. The π-π stacking interaction of form II, at 3.55 Å, was stronger than that of form III, at 3.54 Å, between two molecules. It is well-known that π-π stacking is detrimental to the solubility of compounds. Different π-π stacking forms also have an effect on solubility, which may lead to the weak solubility and dissolution of the crystalline form II of istradefylline.

**Figure 5.** Single-crystal molecular structure of istradefylline in (**a**) ethanol (form I), (**b**) methanol (form II) and (**c**) acetonitrile (form III), respectively, and π-π stacking and hydrogen-bond lengths. **Figure 5.** Single-crystal molecular structure of istradefylline in (**a**) ethanol (form I), (**b**) methanol (form II) and (**c**) acetonitrile (form III), respectively, and π-π stacking and hydrogen-bond lengths.

Gof 1.017 1.036 1.052

According to the crystallographic data and structure refinement parameters, three forms can be effectively distinguished. From the single-crystal structure, it can be seen that form I was pure crystal, form II was monohydrate crystal, and form III was monohydrate of acetonitrile solvent complex. The three forms were all monoclinic systems with different space groups, with form I, II and III exhibiting *P*21, *P*21/*c* and *P*21/*m*, respectively. The molecular packing and intermolecular interactions of the three forms were different (Figure 5). Form I has strong intermolecular hydrogen bonds between oxygen atoms and the nearby hydrogen on the other side of the molecule (C=O…H, 2.37 Å and 3.16 in form I), without π-π stacking. Form II and form III have hydrogen bonds between oxygen atoms and the nearby hydrogens of water molecules (O5=O2…H, 2.878 Å in form II and O5A=O3…H, 2.955 Å in form III). Although abundant hydrogen bonds were constructed through the interactions between water molecules and the crystal of form II and form III, their bond energies of about 15~30 kJ·mol−1 were much lower than those of general chemical bonds. This suggests that these hydrogen bonds are fragile, and the water molecules are transferred during the drying process of istradefylline. Otherwise, form II was a head-to-head π-π interaction, and form III was a head-to-tail π-π interaction. The π-π stacking interaction of form II, at 3.55 Å, was stronger than that of form III, at 3.54 Å, between two molecules. It is well-known that π-π stacking is detrimental to the solubility of compounds. Different π-π stacking forms also have an effect on solubility, which may lead to the weak solubility and dissolution of the crystalline form II of istradefylline.

R1 = 0.0888, wR2 = 0.2892

R1 = 0.083, wR2 = 0.27

<sup>R</sup>R1= 0.0552,

wR2 = 0.1107

#### *3.4. TGA 3.4. TGA*

Figure S5 shows the TGA curves for form I, form II, and form III at a heating rate of 10 °C/min under N2 atmosphere. As shown in Figure S5a, the weight loss of form I was 99.98% from 191.66 to 198.08 °C, corresponding to the degradation of istradefylline molecules. In Figure S5b, the weight loss of form II was in two stages: the first weight loss of 3.23% from 27.56 °C to 52.45 °C corresponds to the release of one H2O molecule (calc. 4.48%), while the second weight loss occurred at or above 377.72 °C, corresponding to the Figure S5 shows the TGA curves for form I, form II, and form III at a heating rate of 10 ◦C/min under N<sup>2</sup> atmosphere. As shown in Figure S5a, the weight loss of form I was 99.98% from 191.66 to 198.08 ◦C, corresponding to the degradation of istradefylline molecules. In Figure S5b, the weight loss of form II was in two stages: the first weight loss of 3.23% from 27.56 ◦C to 52.45 ◦C corresponds to the release of one H2O molecule (calc. 4.48%), while the second weight loss occurred at or above 377.72 ◦C, corresponding to the degradation of istradefylline. In Figure S5c, the thermal decomposition process of cocrystal III was in two stages: the first weight loss of 15.62% from 91.16 ◦C to 121.02 ◦C corresponds to the release of one H2O molecule and one CH3CN molecule (calc. 13.32%), while the second weight loss occurred at or above 258.93 ◦C, corresponding to the degradation of istradefylline.

#### *3.5. FT-IR Analysis*

In Figure S6a, the absorbance peaks at 2966 cm−<sup>1</sup> , 2935 cm−<sup>1</sup> , and 2832 cm−<sup>1</sup> are ascribed to the presence of the methyl or methylene group. In Figure S6b, the absorbance peaks at 3482 cm−<sup>1</sup> , 2977 cm−<sup>1</sup> , 2935 cm−<sup>1</sup> , and 2841 cm−<sup>1</sup> are ascribed to the presence of the hydroxy, methyl, or methylene group. In Figure S6c, the absorbance peaks at 2979 cm−<sup>1</sup> , 2932 cm−<sup>1</sup> , 2839 cm−<sup>1</sup> , and 2217 cm−<sup>1</sup> are ascribed to the presence of the methyl or methylene group, the absorbance peaks at 3465 cm−<sup>1</sup> , 3380 cm−<sup>1</sup> and 3028 cm−<sup>1</sup> indicate the presence of water molecules, and the absorbance peak at 2217 cm−<sup>1</sup> indicates the presence of acetonitrile molecules.

#### *3.6. Particle Size and BET Analysis*

The particle size of form I, II and III was determined with a Malvern 2000 laser particlesize analyzer, and the median particle sizes were 5.6 µm, 5.2 µm, and 7.1 µm, respectively. The specific surface areas of the three forms were 5.08 m2/g, 5.51 m2/g and 5.18 m2/g, respectively. The specific surface areas of single points were 0.20000 at P/Po is 4.67 m2/g, 4.74 m2/g, and 4.72 m2/g, respectively. As can be seen from the above data, the surface area was basically the same.

#### *3.7. Dissolution Curve Test*

The dissolution of istradefylline in three crystal forms was studied in a buffer solution in which pH value was fixed at 1.2, 4.5, and 6.8, corresponding to pH of digestive solutions such as gastric juice and intestinal juice. All experiments were repeated six times, as shown in Figure 6. The dissolution rates of the three crystal forms at pH 1.2 at 5 min were 72.2%, 28.3%, and 74.7%, respectively. At 60 min, the dissolution rates of form I, II, and III reached 87.1%, 58.2%, and 87.7% respectively. The dissolution rates of the three crystal forms at pH 4.5 at 5 min were 68.3%, 29.4%, and 73.2%, respectively. At 60 min, the dissolution of form I reached 88.1%, the dissolution of form II reached 58.9%, and the dissolution of form III reached 87.1%. The dissolution rates of the three crystal forms at pH 6.8 at 5 min were 69.2%, 30.3%, and 70.7%, respectively. At 60 min, the dissolution of form I reached 87.5%, the dissolution of form II reached 58.2%, and the dissolution of form III reached 86.0%. From the dissolution profile of the three crystal forms of istradefylline, it can be inferred that the dissolution of istradefylline form I, refined in ethanol, and form III, refined in acetonitrile, show good dissolution performance, while that of form II, refined in methanol, was significantly lower. Through single-crystal studies, it was found that form II has π-π stacking, and the π-π stacking interaction of form II is stronger than that of form III between two molecules. The π-π stacking leads to weak solubility and dissolution in istradefylline. These results indicate that the solvent has a direct effect on the dissolution. *Crystals* **2022**, *12*, x FOR PEER REVIEW 11 of 13

**Figure 6.** Dissolution curve of three crystal forms of istradefylline. (**a**) pH 1.2 (0.3% SDS), (**b**) pH 4.5 **Figure 6.** Dissolution curve of three crystal forms of istradefylline.

#### **4. Conclusions**

cokinetics.

**5. Patents** 

From II and The IR of From III.

06-25.

(0.3% SDS), (**c**) pH 6.8 (0.3% SDS).

**4. Conclusions**  In this paper, the solubility of istradefylline in 12 kinds of solvents such as ethanol, methanol, and acetonitrile was studied, and three kinds of crystal forms were sequentially obtained in these solvents and proved by X-ray powder diffraction. Their single-crystal diffraction structure and data were also confirmed by single-crystal diffraction. Furthermore, the dissolution test was performed with tablets prepared from the three crystal forms of istradefylline, and it was found that the dissolution rates of the three crystal forms were different. Compared with form II, the dissolution rates of form I and form III In this paper, the solubility of istradefylline in 12 kinds of solvents such as ethanol, methanol, and acetonitrile was studied, and three kinds of crystal forms were sequentially obtained in these solvents and proved by X-ray powder diffraction. Their single-crystal diffraction structure and data were also confirmed by single-crystal diffraction. Furthermore, the dissolution test was performed with tablets prepared from the three crystal forms of istradefylline, and it was found that the dissolution rates of the three crystal forms were different. Compared with form II, the dissolution rates of form I and form III were superior. Additionally, the solubility and X-ray powder diffraction data, as well as the dissolution

were superior. Additionally, the solubility and X-ray powder diffraction data, as well as the dissolution rates, indicated that the packing of molecules and crystal forms have no-

timizing the crystallization process of istradefylline and improving absorption in pharma-

Preparation method and application of istradefylline crystal, CN113024558A, 2021-

**Supplementary Materials:** The following supporting information can be downloaded at: www.mdpi.com/xxx/s1. Table S1. Solubility of Istradefylline in Twelve Different Solvents from 293.15 K to 333.15 K. Table S2. Bond Lengths for Istradefylline. Table S3. Stability data of Istradefylline (Form I of istradefylline). Figures S1–S3. ORTEP view with labeling scheme for Istradefylline. Figure S4. The surface area of three crystal forms. Figure S5. (a) The TGA curves of From I, (b) The TGA curves of From II and (c) The TGA curves of From III. Figure S6. The IR of From I, The IR of

**Author Contributions:** Conceptualization, Y.W. and M.X.; methodology, Y.W.; software, M.X.; validation, M.X.; formal analysis, Z.Z., Z.M., Z.X., J.L. and Q.L.; investigation, Y.W.; resources, Y.W.; rates, indicated that the packing of molecules and crystal forms have notable influence on solubility and dissolution. This study offers additional insight into optimizing the crystallization process of istradefylline and improving absorption in pharmacokinetics.

#### **5. Patents**

Preparation method and application of istradefylline crystal, CN113024558A, 2021-06-25.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/cryst12070917/s1. Table S1. Solubility of Istradefylline in Twelve Different Solvents from 293.15 K to 333.15 K. Table S2. Bond Lengths for Istradefylline. Table S3. Stability data of Istradefylline (Form I of istradefylline). Figures S1–S3. ORTEP view with labeling scheme for Istradefylline. Figure S4. The surface area of three crystal forms. Figure S5. (a) The TGA curves of From I, (b) The TGA curves of From II and (c) The TGA curves of From III. Figure S6. The IR of From I, The IR of From II and The IR of From III.

**Author Contributions:** Conceptualization, Y.W. and M.X.; methodology, Y.W.; software, M.X.; validation, M.X.; formal analysis, Z.Z., Z.M., Z.X., J.L. and Q.L.; investigation, Y.W.; resources, Y.W.; data curation, Y.X.; writing—original draft preparation, Y.W. and M.X.; writing—review and editing, Y.W. and M.X. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors wish to thank the Suzhou Jingyun Pharmaceutical Technology Co., Ltd., Suzhou ReadCrystal Biotechnology Co., Ltd. and the instrumental analysis center of Beijing Institute of Technology for their powder X-ray diffraction analysis, single crystal diffraction and so on.

**Conflicts of Interest:** The authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this paper.

#### **References**

