BR102018073535A2 - PROCESS OF OBTAINING THE PHYSETINIDOL PALMITATE COMPOUND AND ITS USE AS ANALGESIC AND ANTIRREABSORTIVE AGENT - Google Patents

Abstract

the present invention, particularly if applied, inserts the area in the field of chemical, organic synthesis, and describes the process of obtaining fisetinidol palmitate (2) and its use as an analgesic and antiresorptive agent.

Description

PROCESS OF OBTAINING THE PHYSETINIDOL PALMITATE COMPOUND AND ITS USE AS ANALGESIC AND ANTIRREABSORTIVE AGENT

Field of the invention:

[1] The present invention falls within the field of chemistry, particularly applied to the area of organic synthesis, and describes the process of obtaining the compound Fismitinidol Palmitate (2) thus obtained and its use as an analgesic and antiresorptive agent.

Fundamentals of the invention:

[2] Fisetinidol (1), a flavonoid of the flavan-3ol class, is a secondary metabolite obtained from species of the genus Bauhinia, such as B. ungulata, B. pulchella, B. acuruana and B. cheilantha, belonging to the Fabaceae family, subfamily Caesalpinoideae of the order Fabales, and has antioxidant activity. The semi-synthetic fisetinidol palmitate derivative (2) of this invention was obtained from fisetinidol (1), isolated from Bauhinia pulchella. Fisetinidol palmitate (2) is a solid compound at room temperature and more chemically stable than fisetinidol (1).

[3] Several species of the genus Bauhinia are used in folk medicine to treat diseases such as diabetes, infections, pain and inflammation. Studies reveal that plants of this genus have several activities such as antitumor, antipyretic, anti-ulcer, antimalarial and antioxidant properties. These pharmacological activities are attributed to the presence of terpenes, steroids, alkaloids and mainly, flavonoids in their chemical compositions. Cytotoxic activity against cells

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2/13 carcinogenic was presented by the essential oil of Bauhinia pulchella, through the presence of constituents such as β-pinene, α-pinene, E-caryophyllene, caryophyllene oxide.

[4] Studies have shown that substances derived from the genus Bauhinia have antinociceptive, antifungal, antibacterial and anti-inflammatory action in the formalin model. Compounds of the genus Bauhinia, containing flavonoids and tannins were shown to be effective in reducing nociceptive effect in abdominal contractions induced by acetic acid in rats, attenuating both phases in the formalin model; in addition, it has anti-inflammatory activity in models of histamine and carrageenan-induced paw edema through modulation of the regulation or release of pro-inflammatory mediators, consequently reducing the nociceptive and inflammatory response.

State of the art:

The present invention is a method for isolating fisetinidol (1) and obtaining fisetinidol palmitate (2) from the natural product (1).

[5] The process of obtaining the present invention has the advantage of using a natural flavonoid structurally modified in just one reaction step, generating the bioactive compound. The use of this bioactive compound in experimental tests of bone pain and inflammation brings a new molecule with analgesic and anti-resorptive effect for the treatment of periodontitis and that does not present toxicity. Namely, periodontitis represents a serious public health problem, being responsible for the loss of teeth in

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3/13 adults. The worsening and chronicity of the clinical picture can cause leave from work and decrease social interactions, with a consequent decrease in quality of life. Despite the high prevalence, there are still no effective therapies, being necessary to search for new drugs, hence the importance of the present invention, bringing a new molecule with analgesic effect in this pain.

Brief description of the invention:

The present invention describes the process of obtaining the thus obtained fisetinidol palmitate (2) and its use as an analgesic and antiresorptive agent.

[6] Process of obtaining fisetinidol palmitate (2) characterized by understanding the steps of:

a) Dissolve (-) - fisetinidol (30.3 mg; 0.11 mmol) in pyridine (1.5 ml) and chloroform (2 ml) at room temperature and add palmitoyl chloride (1 ml;

3.3 mmol) and 4,4-dimethylamino pyridine (DMAP) in catalytic amounts;

b) Keep the reaction mixture obtained in step (a) under magnetic stirring at room temperature for 19 hours, with monitoring by thin layer chromatography;

c) Dilute the solution obtained in step (b) with dichloromethane (20 mL), transfer it to a separating funnel, wash successively with solution

saturated in sulfate copper (4 x 10 mL) and solution saturated in chloride sodium (1 x 10 mL), dry with sulfate from s anhydrous hate d) Remove the solvent the middle one reactional obtained in

step (c) in a rotary evaporator; and

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e) Purify the reaction product obtained in (d) by chromatography on silica gel (44.33 g), using a mixture of hexane: ethyl acetate (90:10), as eluent.

[7] The compound has a structural formula: C79H134O9 and is called fisetinidol palmitate (2) to treat bone inflammation and pain.

Brief description of the figures:

[8] Figure 1 refers to the reaction scheme of the process for obtaining fisetinidol palmitate (2).

[9] Figure 2 refers to the molecular structure of fisetinidol palmitate (2).

[10] Figure 3 refers to the experimental test that demonstrates the analgesic activity of fisetinidol palmitate.

[11] Figure 4 refers to the assessment of alveolar bone loss from fisetinidol palmitate.

Detailed description of the invention [12] The present invention is a process for obtaining the palmitate compound of fisetinidol (2) which comprises the following steps:

a) Dissolve (-) - fisetinidol (1) (30.3 mg; 0.11 mmol) in pyridine (1.5 mL) and chloroform (2mL) at room temperature and add palmitoyl chloride (1 mL; 3.3 mmol) and 4,4-dimethylamino pyridine (DMAP) in catalytic amounts;

¹³ C NMR data of fisetinidol (1) (CD3OD, 75 MHz): δ 82.96 (C-2); 68.81 (C-3); 33.10 (C-4); 131.28 (C-5); 109.47 (C-6);

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157.84 (0-7), · 103.62 (C-8); 156.13 (C-9); 112.58 (C-10);

132.24 (C-1 '); 115.11 (C-2 '); 146.22 (C-3 'and C-4'); 116.13 (C-5 '); 119.87 (C-6 '); ¹ H NMR data of fisetinidol (CD3OD, 300 MHz): δ 4.64 (d, J = 7.2 Hz, H-2); 4.00 (dt, J

= 7.6 and 5.2 Hz, H-3); 2.68 (dd, J = 15.7 and 8.0 Hz, H-3a); 2.87 (dd, J = 15.7 and 5.0 Hz, H-3b ); 6.86 (s, H-5) ; 6.34 (dd; J = 8.2 and 2.4 Hz , H- 6); 6.29 (d, J = 2.3 Hz, H-8); 6.83 (d, J = 1.9 Hz, H-2 ' ); 6.76 d, J = 8.0 Hz, H-5 '); 6.71 (dd, J = 8.2 and 1.8 Hz, H-6 ').

b) Keep the reaction mixture obtained in step (a) under magnetic stirring at room temperature for 19 hours, with monitoring by thin layer chromatography;

c) Dilute the solution obtained in step (b) with dichloromethane (20 mL), transfer it to a separating funnel, wash successively with solution

saturated in sulfate copper (4 x 10 mL) and solution saturated in chloride sodium (1 x 10 mL), dry with sulfate from s anhydrous hate ; d) Remove the solvent the middle one reactional obtained in

step (c) in a rotary evaporator; and

e) Purify the reaction product obtained in (d) by chromatography on silica gel (44.33 g), using a mixture of hexane: ethyl acetate

90:10), as an eluent.

13] In addition, the object of the present invention is fisetinidol palmitate (2) (46.0 mg; 0.0374 mmol; 35.4%).

The yield of the purified fisetinidol palmitate product (2) was 35.4% (46.0 mg). ¹³ C NMR data of fisetinidol palmitate (2) (CDCl3, 75 MHz): δ 77.66 (C-2); 68.83 (CPetition 870180151694, of 11/14/2018, page 15/33

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3); 130.12 (C-5); 114.59 (C-6); 153.79 (C-7); 109.89 (C-

8); 150.32 (C-9); 116.44 (C-10); 136.60 (C-1 '); 121.66 (C-

2'); 142.11 ( C-3 '); 142.22 (C-4 '); 123.62 C-5 '); 124.22 (C-6 ' ); 173.0 8 (C = O); 172.23 (C = O); 170.88 (C = O); 170.84 (C = O) ; 34.42; 34.24; 34.07; 31.95; 29.73; 29.69; 29.63; 29.52 ; 29.48; 29.39; 29.34; 29, 29; 29.24; 29.20; 29.19; 29.13 ; 29.01; 28.29; 24.99; 24.95; 24.92; 24.81; 22.71; 14.12 ; Dice NMR ¹ H from fisetinidol palmitate (2) (CDCl 3, 300 MHz): δ 5, 22 (d, J = 5.6 Hz, H-2) ; 5.34 (dd, J = 10, 8 and 5.5, H-3); 3 , 06 (dd , J = 16 , 4 and 4.3 Hz, H- 4 eq.); 2.86 (s, H-5) dd, J = 16.4 and 6.0 Hz, H-4 ax. ; 7.31 (s, H- 5); 6 , 70 (dd, J = 8.2 and 1.8 Hz, H-6) ; 6.74 ( sl, H-8 ); 7.28

(d, J = 5.1 Hz, H-2 '); 7.20 (d, J = 6.9 Hz, H-5 '); 7.06 (d,

J = 8.2 Hz, H-6 '); 2.59-2.54 (m, 10H), 2.28 (t, J = 7.4 Hz, 3H), 1.81-1.73 (m), 1.65-1.30 (m) , 0.93 (t, J = 6.7 Hz).

[14] The compound N- [4- (4'-O-acetyl-a-L-raminosyloxy) benzyl] 2- (pyridinyl-4-carbonyl) hydrazine-1-carbothioamide (MC-D7) has application as an analgesic.

Example of embodiment

Evaluation of the analgesic activity of Fisetinidol Palmitate [15] Analgesic activity and the reduction of alveolar bone loss of fisetinidol palmitate were evaluated in a model of formalin-induced inflammatory hypernociception in rats.

[16] Male Wistar rats with a body mass between 170 to 220 g from the Central Animal Hospital of the Campus do Pici and the Animal Hospital of the UFC - Campus of Sobral were used.

[17] The animals were kept in plastic boxes with free access to water and food at an average temperature of 22

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7/13 ± 2 ° C obeying 12h light-dark cycles. The experiment was submitted to and approved by the Ethics Committee for the Use of Animals (CEUA - UFC Campus Sobral) (Protocol number 12/15). Every effort has been made to minimize the suffering of the animals as well as the number of animals used in accordance with the guidelines of the Society

Brazilian Science in Laboratory Animals (SBCAL / COBEA) and international standards (Guide for care and use of laboratory animals - NIH publication 85-23, revised 1985).

[18] Experimental periodontitis (PE) was induced as previously described (BEZERRA et al., 2002).

Briefly, the animals were anesthetized with ketamine and xylazine (90:10 mg / kg, i.p.), and a sterile nylon suture thread (3.0 Nylpoint, Ceará, Brazil) was placed around the left second maxillary molar. To allow the wire to pass, a guide was used through the mesial and distal interproximal spaces of the animal's left 2nd molar. The experimental groups were composed of n = 6. At 11 days after periodontitis induction, the jaws were removed for morphometric analysis. For the quantification of bone resorption, the two hemiarches were assembled in blocks of beeswax and photographed in high resolution. The digitized images were analyzed with ImageJ® software for the quantification of alveolar bone loss (LIMA et al., 2004).

[19] To assess nociceptive behavior, saline (50pL) or formalin 0.5% (50pL) was applied in the region

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8/13 of the animal's upper left lip. The procedure took place between 6:00 am and 4:00 pm in a quiet room maintained at 23 ± 2 ° C (Rosland, 1991). The rats were placed individually for 10 minutes in a mirrored wooden chamber (30 x 30 x 30 cm) with glass on the front to minimize stress. After anesthesia with isoflurane (3%, 30s), each animal received 50 pL of formalin (0.5%), dissolved in sterile saline, or 50 pL of saline (0.9%) in the upper left lip. The injection was performed with a 30 gauge needle connected to a 50pL Hamilton syringe according to the method described previously (Clavelou et al., 1995). After the rats regained consciousness, approximately 30 seconds, they returned to the test chamber for an observation period of 45 minutes. A trained examiner assessed nociceptive responses by the total accumulated number of seconds the animal used rubbing the orofacial region asymmetrically with the ipsilateral front or rear and the number of heads raised. All tests used the double-blind study in which the person who injected the solutions will not be the same person who assessed behavioral responses (Luccarini et al., 2006).

[20] A first group of animals went through the procedure described above and received 0.9% saline solution (1 mL / 200 g of weight) orally for 11 days and received an injection of 0.9% saline solution (50 pL) on the upper left lip, corresponding to the saline group used as a control, represented in white in the graph in figure 3.

[21] A second group of animals went through the procedure described above and received 0.9% saline (1 mL / 200 g

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9/13 weight) orally for 11 days and received an injection of 0.5% formalin (50 pL) in the upper left lip, shown in white in the second column in the graph in figure 3, and was named as Saline + Formalin Group 0.5%.

[22] A third group of animals went through the procedure described above, underwent PD and received 0.9% saline solution (1 mL / 200 g of weight) orally for 11 days and 0.5% formalin injection ( 50 pL) on the upper left lip, represented in white in the third column in the graph in figure 3, and was named as Group DP (Periodontal Disease + Formalin 0.5%).

[23] Two other groups of animals were submitted to PD and each group received a different dose of fisetinidol palmitate for 11 days. After that period, they receive the injection of Formalin in the manner described above. Fisetinidol palmitate was administered for 11 days at doses of 0.01 or 0.1 mg / kg orally, in two groups of animals, respectively, represented in black columns in the graph in figure 3.

[24] The groups described above underwent evaluation of alveolar bone loss. The doses of fisetinidol palmitate (0.01 or 0.1 mg / kg, orally), in two groups, respectively, are represented in the black columns in the graph in figure 4.

Subchronic toxicity test [25] A safety test of fisetinidol palmitate was also performed to evaluate and measure whether the compound had any toxicity, local or systemic, as well as morbidity and mortality.

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10/13 [26] Subchronic toxicity (repeated doses), which consisted of administering a dose of 0.01 or 0.1 mg / kg fisetinidol palmitate, (orally), according to the animal's weight, for 14 days consecutive hours at the same time. The observation of the same behavioral parameters of morbidity and mortality was performed, however, for 15 minutes followed daily, subsequent to the administration of fisetinidol palmitate or control substance. Aiming to observe if there were behavioral changes that could indicate morbidity and also mortality, namely: piloerection, aggression, drowsiness, abdominal contortion, licking, somersaults, agitation, sexual drive, weight loss and death (VAL et al., 2014).

[33] On the 15th day, the animals were anesthetized with ketamine + xilasine and then the animals were euthanized. In order to assess changes not detectable by the naked eye or by laboratory means in those animals that did not show behavioral changes, after euthanasia, the heart, liver, spleen and kidney were removed for microscopic analysis by a pathologist, performed in optical microscope coupled to a digital image capture system. The increases used were: 100x, for general morphological aspects and 400x, for detailed analysis of histological events, both cellular and in the extracellular matrix (MEC), with blind analysis of experimental groups (test drugs), according to ISO 10993-11 .

[27] Figure 5 shows the animals' survival rate. The group 0.01 or 0.1 mg / kg fisetinidol palmitate and Saline (both) did not present mortality in the animals. THE

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11/13 fisetinidol palmitate did not present any death, which means this derivative can be considered safe

for use in experiments and possibly in humans. [28] For whether use of palmitate from fisetinidol in repeated could cause changes in behavior what may be related s at the rate of mortality, at

behavioral changes during the 14 days were evaluated. Table 1 shows the behavioral changes. The 0.01 or 0.1 mg / kg group of fisetinidol palmitate showed no behavioral changes, similarly to the Saline control group.

[29] Histopathological analysis of four organs was performed to confirm these data, namely: heart, spleen, liver and kidney.

[30] Figure 6 shows that in Organs animal organs of the groups treated with Saline and doses 0.01 or 0.1 mg / kg of fisetinidol palmitate did not show signs of toxicity or mortality. The organs had their general morphology preserved. Column 1: stomachs of the Saline groups and doses of 0.01 or 0.1 mg / kg of fisetinidol palmitate. The parameters evaluated, such as edema, cell loss, hemorrhagic lesion and inflammatory infiltrate, did not change in the saline group. The analysis of the gastric mucosa of the animals treated with doses of 0.01 or 0.1 mg / kg of fisetinidol palmitate showed a slight loss of cells, however the other parameters analyzed did not show significant changes when compared to the untreated animal (DP + FORMALIN ), which presented significant cell loss (black circle) and edema (blue circle). Column 2: heart of the saline groups and 0.01 or 0.1 mg / kg palmitate

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12/13 fisetinidol. In the saline group, cardiomyocytes showed morphological aspects within the normal range. The group treated with 0.1 mg / kg of fisetinidol palmitate showed few hemorrhagic areas. Regarding the groups treated with 0.1 or 0.01, parameters such as presence of congested vessels and cell swelling were within normal limits. Column 3: liver from the saline groups and doses 0.01 or 0.1 mg / kg of fisetinidol palmitate. The group treated with 0.1 mg / kg of fisetinidol palmitate showed few congested vessels (blue arrows, if any). However, in relation to the presence of hemorrhagic areas and cell swelling, the groups treated with doses of 0.01 or 0.1 mg / kg of fisetinidol palmitate were similar to the saline group and showed no morphological changes. Column 4: kidney from the Saline groups and doses 0.01 or 0.1 mg / kg of fisetinidol palmitate. The group treated with 0.01 mg / kg of fisetinidol palmitate showed some congested vessels. The group treated with the dose of 0.1 mg / kg of fisetinidol palmitate was within the normal morphological pattern.

[31] Table 1 refers to behavioral changes in animals during the safety test.

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Changes Behavioral Saline (N ° of animals that presented changes) 0.01 mg / kg of fisetinidol palmitate (No. animals that presented changes) 0.1 mg / kg of fisetinidol palmitate (No. animals that showed changes) Piloerection - - - Aggressiveness - - - Somnolence - - - Contortion Abdominal - - - Licking - - - Somersaults - - - Agitation - - - Sexual drive - - Weight loss - - Death - -

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Claims (7)

CLAIMS [1] Process of obtaining fisetinidol palmitate (2) characterized by understanding the steps of: a) Dissolve (-) - fisetinidol (30.3 mg; 0.11 mmol) in pyridine (1.5 ml) and chloroform (2 ml) at room temperature and add palmitoyl chloride (1 ml; 3.3 mmol) and 4,4-dimethylamino pyridine (DMAP) in catalytic amounts; b) Keep the reaction mixture obtained in step (a) under magnetic stirring at room temperature for 19 hours, with monitoring by thin layer chromatography; c) Dilute the solution obtained in step (b) with dichloromethane (20 mL), transfer it to a separating funnel, wash successively with solution saturated in sulfate copper (4 x 10 mL) and solution saturated in chloride sodium (1 x 10 mL), dry with sulfate from s anhydrous hate d) Remove the solvent the middle one reactional obtained in step (c) in a rotary evaporator; and e) Purify the reaction product obtained in (d) by chromatography on silica gel (44.33 g), using a mixture of hexane: ethyl acetate (90:10), as eluent. 2] Process, according to claim 1, characterized by the fact that fisetinidol (1) presents ¹³ C NMR data (CD3OD, 75 MHz): δ 82.96 (C-2); 68.81 (C3); 33.10 (C-4); 131.28 (C-5); 109.47 (C-6); 157.84 (C-7); 103.62 (C-8); 156.13 (C-9); 112.58 (C-10); 132.24 (C-1 '); 115.11 (C-2 '); 146.22 (C-3 'and C-4'); 116.13 (C-5 '); 119.87 Petition 870180151694, of 11/14/2018, p. 24/33 2/3 (C-6 ') and ¹ H NMR (CD3OD, 300 MHz): δ 4.64 (d, J = 7.2 Hz, H-2); 4.00 (dt, J = 7.6 and 5.2 Hz, H-3); 2.68 (dd, J = 15.7 and 8.0 Hz, H-3a); 2.87 (dd, J = 15.7 and 5.0 Hz, H-3b); 6.86 (s, H-5); 6.34 (dd; J = 8.2 and 2.4 Hz, H-6); 6.29 (d, J = 2.3 Hz, H-8); 6.83 (d, J = 1.9 Hz, H-2 '); 6.76 (d, J = 8.0 Hz, H-5 '); 6.71 (dd, J = 8.2 and 1.8 Hz, H-6 '). [3] Compound characterized by being obtained according to the process defined in claims 1 and 2 and having the structural formula C ₇ gH ₁₃₄ O9 [4] Compound according to claim 3 characterized by so-called fisetinidol palmitate (2). [5] Compound according to claim 3 or 4, characterized by spectroscopic data of NMR ¹³ C) (CDCl3, 75 MHz): δ 77.66 (C-2); 68.83 (C-3); 130.12 (C- 5); 114.59 (C-6); 153.79 (C-7); 109.89 (C-8); 150.32 (C- 9) 116.44 (C-10); 136.60 (C-1 '); 121.66 (C-2 '); 142.11 (C- 3 '); 142.22 (C- 4 '); 123.62 (C- 5 '); 124.22 (C- 6 '); 173.08 (C = O); 172.23 (C = O); 170.88 (C = O); 170.84 (C = O); 34.42; 34.24; 34.07; 31.95; 29.73; 29.69; 29.63; 29.52; 29.48; 29.39; 29.34; 29.29; 29.24; 29.20; 29.19; 29.13; 29.01; 28.29; 24.99; 24.95; 24.92; 24.81; 22.71; 14.12 and ¹ H NMR (CDCl3, 300 MHz): δ 5.22 (d, J = 5.6 Hz, H-2); 5.34 (dd, J = 10.8 and 5.5, H-3); 3.06 (dd, J = 16.4 and 4.3 Hz, H-4 eq.); 2.86 (s, H-5) dd, J = 16.4 and 6.0 Hz, H-4 ax.); 7.31 (s, H5); 6.70 (dd, J = 8.2 and 1.8 Hz, H-6); 6.74 (bs, H-8); 7.28 (d, J = 5.1 Hz, H-2 '); 7.20 (d, J = 6.9 Hz, H-5 '); 7.06 (d, J = 8.2 Hz, H-6 '); 2.59-2.54 (m, 10H), 2.28 (t, J = 7.4 Hz, 3H), 1.81-1.73 (m), 1.65-1.30 (m) , 0.93 (t, J = 6.7 Hz). [6] Use of the compound as defined in claims 3 to 5, characterized in that it is for treating pain. [7] A compound characterized by the structural formula ^C 79 ^H 142 ^O 9 Petition 870180151694, of 11/14/2018, p. 25/33 3/3 [8] Compound according to claim 7, characterized for being to treat pain.