WO2021009768A1

WO2021009768A1 - An oral pharmaceutical composition for alpha- amylase inhibition

Info

Publication number: WO2021009768A1
Application number: PCT/IN2020/050479
Authority: WO
Inventors: Gopal Lal Khatik; Zakari Umar YA'U; Bashir Usman; Khurana NAVNEET; Jeena Gupta; Manish Vyas
Original assignee: Lovely Professional University
Priority date: 2019-07-12
Filing date: 2020-05-28
Publication date: 2021-01-21

Abstract

The present invention relates to an oral pharmaceutical composition of synthesized amino acid derivative which is inhibitor of alpha-amylase. In particular, the invention refers to the use of the composition for the treatment of diabetes mellitus type II. The said composition is in the form of a liquid, gel, pill, capsule or tablet.

Description

Title of Invention

AN ORAL PHARMACEUTICAL COMPOSITION FOR ALPHA- AMYLASE INHIBITION

TECHNICAL FIELD

[01] The present invention relates to an oral pharmaceutical composition of synthesized amino acid derivative which is inhibitor of alpha- amylase. In particular, the invention refers to the use of the composition for a remarkable effect on the treatment by administering the drug to the patient with diabetes mellitus type II.

BACKGROUND

[02] Diabetes mellitus is a group of metabolic disorder which is associated with elevated blood sugar level. Worldwide, it is one of the major health concern and related dead. Although diabetes mellitus incidence rate is lower in the developing countries while in contrast to the developed countries, which is widely believed to be related to the adapted lifestyles. Current treatment includes administration of some oral hypoglycemic agents, injections as well as inhalable. Focus nowadays is more on the production of 1 hypoglycemic agent which will balance the sugar level instead of that which will decrease it to an unprecedented fatal level.

[03] European patent application number EP0543076A2, disclosed deobesitogen as partially purified alpha-amylase inhibitor obtained from wheat. The said compound is obtained by subjecting a concentrate of the aqueous extract from wheat flour to column chromatography to remove sugar and a part of inorganic matters from the a-amylase inhibitor and then drying.

[04] Japanese patent application number JPH0812584A, disclosed alpha-amylase inhibitor which inhibits porcine pancreas-derived alpha- amylase, human salivary origin alpha-amylase, barley derived alpha-amylase but does not inhibit the alpha-amylase from Aspergillus microorganisms. The said alpha- amylase inhibitor is obtained from a microorganism. [05] Chinese patent application number CN106798742A, disclosed a new application of tetrahydroisoquinoline alkaloid as alpha-amylase inhibitors. The said compounds are synthesized in four step reaction with an overall yield of 28%.

[06] The prior art search showed most of the alpha-amylase inhibitors are isolated from the natural products. The isolated compounds are of variable purity. There is a need for alpha-amylase inhibitors with potent activity and which can be synthesized easily in high purity.

SUMMARY

[07] The present invention relates to an oral pharmaceutical composition of synthesized amino acid derivative which is inhibitor of alpha- amylase.

[08] In one of the embodiments of the invention the alpha-amylase inhibitors were designed by molecular docking approach

[09] In another embodiment of the invention the biological activity of most active compound was evaluated in in-vivo model.

[0010] In yet another embodiment of the invention the biochemical parameters were evaluated after oral administration of active compound in rats.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The foregoing detailed description of embodiments is well understood when read in conjunction with the attached drawings.

[0012] Figure 1 illustrates interactions of the compound Zu06 within the binding site of 4gqr protein.

[0013] Figure 2 illustrates a scheme for the synthesis of compound Zu06 and Zu08.

[0014] Figure 3 illustrates NMR spectra of Zu06 and Zu08. [0015] Figure 4 illustrates a flow chart which describes procedure used for alpha-amylase inhibition testing.

[0016] Figure 5 illustrates effect of treatments on body weight of rats.

[0017] Figure 6 illustrates results of starch tolerance test.

[0018] Figure 7 illustrates effect of treatment on plasma glucose level.

[0019] Figure 8 illustrates effect of treatment on plasma total cholesterol.

[0020] Figure 9 illustrates effect of treatment on GSH, TBARS and CAT activity.

[0021] Figure 10 illustrates histology of pancreas on 29^th day of treatment.

[0022] Data is represented as mean±SD, n =7. *, **, *** represents p <

0.05, p < 0.01, p < 0.001, respectively, when compared with vehicle control group; #, ##, ### represents p < 0.05, p < 0.01, p < 0.001, respectively, when compared with negative control group; and ^L, ^LL, ^LLL represents p < 0.05, p < 0.01, p < 0.001, respectively, when compared with positive control.

DETAILED DESCRIPTION

[0023] The invention as such will now be discussed in detail.

[0024] In one of the embodiments of the invention the alpha-amylase inhibitors were designed by molecular docking approach.

[0025] The aryl amido derivatives of (S)-amino acids similar to an antidiabetic drug nateglinide were designed by molecular docking approach. The sixteen designed compounds were drawn first in 2-dimension and further in 3 -dimension using ChemDraw and geometric repulsion energy was minimized by using molecular mechanics method. All geometry minimized structures were then transformed into readable protein data bank (pdb) format using AutoDockTools (ADT). Autodock-Vina 1.5.6 was used for further molecular docking purposes. [0026] The target alpha-amylase protein (PDB: 4gqr) was prepared for docking using the standard protocol in ADT. In short, all water molecules, co factors, and co-crystallized ligands were removed from the protein structure and structure converted into pdbqt format. The grid box was centered over the co-crystallized ligand having grid size 26 A⁰, 24 A⁰ and 32 A⁰ with center at 48.266, 30.220, and -56.950 in x, y, and z directions. The result of molecular docking of sixteen designed ligands is summarized in Table 1.

O CO₂H

Ri X A

FI ^R

Table 1: Binding affinities of sixteen designed ligands.

[0027] Referring now to Figure 1, which illustrates the interaction of best- docked compound Zu06 with 4gqr. The compound Zu06 showed one hydrogen bond interaction with Argl95 residue and hydroxyl of carboxylic acid of Zu06. It was also observed that the naphthalene part of the ligand is buried in the hydrophobic region of the binding site with amino acid resuidues like Asp300, Thrl63, Trp59 etc..

[0028] In another embodiment of the invention the best-docked compound

Zu06 along with the second best-docked compound Zu08 were synthesized.

[0029] In the first step of synthesis methyl ester of amino acid was synthesized. The acidic group in amino acid was esterified using methanol and sulfuric acid. Firstly amino acid was suspended in a mixture of methanol and sulfuric acid at 0-8 °C afterward the suspension was refluxed at 70 °C for 12- 24 hours. The completion of the reaction was checked using thin layer chromatography (TLC).

[0030] In the second step of synthesis the amino acid methyl ester was coupled with aromatic acid. The coupling reaction was done using the protocol of Montalbetti el al. 2005. In short, naphthoic acid (1.1 equivalent), DCC (2.1 equivalent), HOBt (0.1 equivalent), and Et3N (1.1 equivalent) were dissolved in DMF at room temperature, then mixed with the amino acid methyl ester obtained in the first step. The completion of the reaction was checked using thin layer chromatography (TLC).

[0031] In the third step of synthesis the final compound was synthesized.

Firstly, the ester of the amino acid peptide was hydrolyzed to its corresponding amino acid by the addition of 1 M lithium hydroxide monohydrate in THF/H₂0 (1 : 1) at room temperature. The completion of reaction was checked using thin layer chromatography (TLC). The work-up of the reaction was done by extracting with ethyl acetate, acidifying the aqueous layer to pH 2, then extracting again with ethyl acetate. The ethyl acetate extract obtained after the acidification step was dried with anhydrous sodium sulfate and concentrated to give the final product.

[0032] Referring now to Figure 2, which illustrates a scheme for the synthesis of Zu06 and Zu08.

EXAMPLE 1 [0033] Synthesis of amino acid methyl ester hydrochloride: 3 g of L- proline was transferred into 100 mL RBF, 30 mL of methanol was added into the flask and placed on a magnetic stirrer at room temperature till proline was dissolved. 1.5 equiv (2.08 ml) of sulfuric acid was added into the reaction mixture dropwise with shaking and then refluxed at 70 °C for about 24 hours, the product was concentrated on a rotary evaporator and weight of the product was calculated for the next step. The reaction completion was checked in TLC using methanol: distilled water in a ratio of 1: 1 and then dipped in Ninhydrin solution then heated to observe the spots.

[0034] (S)-methyl pyrrolidine-2 -carboxylate hydrochloride: White crystalline solid; 64% yield; M.P= 73 °C, IR (cm ¹) = 1087.89 (C-O), 1745.64 (C=0), 3001 (C-H), 3328.28 (N-H); Mass (m/z) = 165.06.

EXAMPLE 2

[0035] Synthesis of N-peptide of organic acid with amino acid methyl ester: Naphthoic acid 3.43 g (1 equiv), HoBt 0.31 g (0.1 equiv), Et₃N 5.83 mL (2.1 equiv), DCC 4.51 g (1.1 equiv), DMF 30 mL and THF 30 mL were transferred in to a 250 mL RBF and stirred on magnetic stirrer for 2 hours, 1- proline methyl ester 4.5 g was added in to the RBF and stirred for about 48 hours, the product was concentrated on rotary evaporator. 20 mL of NaHCCf solution in water was added into the RBF and then transferred in to separating funnel and extracted with 25 mL of ethyl acetate four times, the combined ethyl acetate layer was filtered and the filtrate was concentrated. The product was subjected to column chromatography to separate the two spots using silica gel G as the stationary phase and ethyl acetate: n-hexane 50:50 ratio. The IR spectra of separated compounds were taken to select the desired product. Completion of the reaction was checked in ethyl acetate: n-hexane in the ratio of 1: 1.

[0036] (S)-methyl l-(l-naphthoyl)pyrrolidine-2 -carboxylate: white crystalline solid, M.P=94 °C, 52% yield, IR (cm^-1) = 1087.89 (C-O), 1745.64 (C=0), 3328.28 (N-H); Mass (m/z) = 283.13. [0037] (.V) -methyl 2-amino-3-(4-hydroxyphenyl)propanoate hydrochloride: white crystalline solid, 63% yield, M.P. = 146 °C, IR(cm‘) = 1056.06 (C-O), 2087.05 (C=0), 2900 (CH), 3336.0 (N-H).

EXAMPLE 3

[0038] Hydrolysis of the amino acid peptide: (S)-methyl 1-(1- naphthoyl)pyrrolidine-2-carboxylate was taken 2.1 g and dissolved in THF (25 mL) was mixed with Lithium hydroxide monohydrate 0.35 g (1 equiv) was dissolved in 25 mL distilled water and the two solutions were mixed in RBF, and then stirred at room temperature for about 5 hours. Water (20 mL) was added and extracted with 20 mL of ethyl acetate two times, the aqueous layer was acidified with HC1 to pH = 2 and then extracted with ethyl acetate 25 mL portions 4 times. The ethyl acetate layer was concentrated and dried product was collected from the RBF. The final product was sent for IR analysis, proton NMR and ¹³C NMR, and mass analysis for the product characterization.

[0039] (S)-l-(l-naphthoyl)pyrrolidine-2 -carboxylic acid (Zu08): White crystalline solid, Yield = 64%; M.P. = 81 °C; IR (cm ¹) = 3424 (OH), 2933 (CH), 1695 (C=C), 1269 (C-N); 1H-NMR (400 MHz, DMSO) d: 0.83-0.93 (m, 1H), 1.10-1.24 (m, 3H), 1.30-1.32 (m, 1H), 1.91 (s, 1H), 3.02 (d, 1H, J=4.5 Hz), 7.58-7.63 (m, 3H), 7.49-8.01 (m, 1H), 8.14 (d, 2H, J=6.6 Hz ), 8.86 (d, 1H, J= 7.9 Hz), 13.08 (bs, 1H, C02H); 13C-NMR (100 MHz, DMSO): 21.10, 45.27, 124.88, 125.45, 126.18, 127.56, 127.67, 128.60, 129.25, 129.84, 130.48, 132.92, 133.42, 134.88, 168.62, 172.00; MS-ESI (m/z): 270.23 (M+l); Anal. Calcd for C16H15N03: C, 71.36; H, 5.61; N, 5.20; Found: C, 71.38; H, 5.59; N, 5.23.

[0040] (.Y)-2-( 1 -naphthamido)-3-(4-hydroxyphcnyl)propanoic acid

(Zu06): white crystalline solid, 48% yield, M.P.= 249 °C; IR (cm ¹) = 1041.60 (C-O), 1631.83 (C=0), 2983.01 (C-H), 3426.66 (N-H); Ή-NMR: 2.51-2.53 (m, 1H), 2.51-2.91 (m, 1H), 4.31-4.41 (m, 1H), 5.80 (bs, 2H, NH, OH), 6.66- 6.67 (m, 2H), 6.68-6.71 (m, 2H), 7.01-7.04 (m, 2H), 7.12 (d, J =8Hz, 1H), 7.45-7.46 (m, 2H), 7.47-7.49 (m, 2H), 7.81-7.91 (m, 2H).

[0041] Referring now to Figure 4, which shows the 'H-NMR of Zu06 and

Zu08 and ¹³C-NMR ofZu08.

[0042] In yet another embodiment of the invention the synthesized compounds were evaluated for alpha-amylase inhibition activity.

[0043] The flow diagram of the method used for the alpha-amylase inhibition measurement is shown in Figure 4. The result of alpha-amylase inhibition activity is shown in Table 2. The results in the table are average of the triplicate run. Both of the synthesized compounds Zu06 and Zu08 showed good inhibition of alpha-amylase enzyme. The IC₅₀ of both ligands was 90.41 pg/mL, and 49.27 pg/mL, respectively. The observed activity was compared with alpha-amylase inhibition activity of acarbose (IC₅₀ = 82.04 pg/mL).

Table 2: :% inhibition of alpha amylase by acarbose

[0044] In yet another embodiment of the invention the solution of test compound (Zu08) was prepared by dissolving 441 mg compound in 6.34 mL ethanol which was diluted further with distilled water to 100 mL. The solution was sonicated for 10 minutes at 35 °C and used orally in a dose of 5 mg/kg or 10 mg/kg and otherwise stored in refrigerator at 10 °C.

[0045] In yet another embodiment of the invention the biological activity was also evaluated in in-vivo model. The body weights of animals were recorded on 1^st day, 15^th day, 22^nd day and 29^th day (Table 3). No significant difference in body weight of rats among different groups was observed on day 1. On the 15^th day after 2 weeks of dietary modification, significant increase in body weight was observed in rats fed with high fat diet (HFD) (group 3, 4, 5 and 6), as compared to rats fed with NPD (control and test compound per se group), but no significant difference was observed between positive control, ZU08 low dose and high dose treated groups in comparison to negative control group. On 22^nd day after one week of streptozotocin (STZ) injection, no significant difference was observed between all the groups in comparison to the vehicle control which signifies loss of body weight in the groups injected with STZ probably due to development of diabetes. On 29^th day; no significant difference in body weight was observed in ZU08 per se treated group as compared with vehicle control group while no significant difference of bodyweight in negative control, ZU08 low and high dose treated group in comparison to the vehicle control, also a significant decrease in body weight was observed in positive control in comparison with vehicle control. However, body weight was significantly decreased in positive control and ZU08 high dose groups in comparison to the negative control group while there is a significant difference of body weight in ZU08 low and high dose treated groups when compared with positive control group. The effect of different treatments on weight of rats was shown in Table 3 and Figure 5.

Table 3: Effect of different treatments on body weight of rats

[0046] In yet another embodiment of the invention the Starch Tolerance

Test (STT) was conducted. STT was conducted on 22^nd day of the study, PGL was evaluated at 0, 30, 60, and 120 minutes after given respective treatments and starch administration, change in PGL (delta) was calculated by substituting PGL at 0 minute from PGL of 30, 60, and 120 minutes of each animal, the mean delta values of different groups were compared as shown in Table 4 and Figure 6. Delta PGL at 30 minutes; significant difference of PGL was observed in ZU08 per se group, as it prevents rise of PGL even after starch administration as compared to control group, and there is significant difference of delta PGL in negative control, positive control, and ZU08 treatment groups as compared to control group. But there was significant reduction of PGL in both positive and ZU08 treated groups in comparison to the negative control.

Table 4: Results of Starch Tolerance test

[0047] Delta PGL at 60 minutes; similarly significant difference in PGL was observed in ZU08 per se group, ZU08 low dose and negative control in comparison to the control group while no significant fall of PGL in positive control, but significant decrease of PGL was seen in ZU08 high dose (p < 0.05) when compared with the control group, and significant reduction of PGL in both positive and ZU08 treated groups in comparison to the negative control while no significant difference in PGL between ZU08 high dose treated group and positive control but highly significant between ZU08 low dose and positive control.

[0048] Delta PGL at 120 minutes; there was a significant difference PGL in ZU08 per se group, positive control and ZU08 high dose group in comparison to the control , but highly significant difference in negative control group while no any significant difference in ZU08 low dose. The PGL of positive control and ZU08 treated groups were significantly different in comparison to the negative control. Moreover; no significant difference between positive and ZU08 high dose treated group, and ZU08 low dose PGL was significantly different in comparison to the positive control.

[0049] In yet another embodiment of the invention the Plasma glucose

(PGL) was evaluated. The plasma glucose levels were compared on 22^nd day and 29^th day of study. On 22^nd day; there is highly significant increase of PGL in all the HFD+STZ treated groups in comparison to the control which confirm the type 2 diabetic condition, but no significant difference of PGL between positive control and ZU08 treated groups in comparison to the negative control. On 29^th day; still no any significant difference between control and ZU08 per se group. However, significant difference of PGL was observed in all HFD+STZ (diabetic) groups, but the PGL of positive control and ZU08 treated groups was significantly reduced in comparison to the negative control, but no significant difference was observed between ZU08 high dose and low dose treated groups as compared with positive control. The effects of respective treatments were shown in Table 5 and Figure 7.

Table 5: Effects of different treatment on plasma glucose level.

[0050] In yet another embodiment of the invention the Total cholesterol was evaluated. The plasma total cholesterol levels were evaluated on 22^nd and 29^th day (Table 6) of study and then compared. On the 22^nd day; the results shows that there is no significant difference in PTC level of ZU08 per se group but PTC significantly increased in negative control, positive control and ZU08 treated groups in comparison to the control group, however no significant difference was observed in positive control and ZU08 treated groups when compared with negative control and likewise no significant difference in ZU08 treated groups in comparison to positive control. On 29^th day; similarly no significant difference was seen between control and ZU08 per se group, and significantly different in negative control, positive control, and ZU08 treated groups in comparison to the control. But PTC was decrease in positive control and ZU08 treated groups as compared to negative control while no significant decrease between positive and ZU08 low dose treated group but highly significant decrease was observed in ZU08 high dose in comparison to positive control as shown in Figure 8 and Table 6.

Table 6: Effects of different treatment on plasma total cholesterol

[0051] In yet another embodiment of the invention the Estimation of reduced glutathione (GSH) was carried out. GSH level in pancreas was estimated on 29^th day, the equation of calibration curve in Figure 9A was used for the calculation of GSH level in the samples. No significant difference was observed between control and ZU08 per se group, but GSH level was significantly decreased in negative control (p < 0.001) in comparison to the control, the level was significantly different in positive control (p < 0.05) and ZU08 low dose (p < 0.01) while no significant difference in ZU08 high dose treated group as compared to control group. However; positive control and ZU08 treated groups showed a significant difference in comparison to the negative control as shown in Figure 9A and Table 7.

Table 7: Effect of different treatments on GSH, TBARS, and CAT activity of rat pancreas

[0052] In yet another embodiment of the invention the Estimation of thiobarbituric acid reactive substances (TBARS) was carried out. TBARS in rats’ pancreas was estimated on 29^th day of the study, the equation of calibration curve in Figure 9B was used in the calculation of TBARS. No significant difference of TBARS level was observed in ZU08 per se and ZU08 high dose treated groups in comparison to the control group, but significantly increased in negative control, positive control and ZU08 low dose treated groups in comparison to the control. However; there significant difference was observed between positive and ZU08 treated groups in comparison to the negative control group, but no significant difference between positive and ZU08 low dose while significant difference (p < 0.01) in ZU08 high dose was observed in comparison to the positive control group. Effects of respective treatments were shown in Table 7 and Figure 9B.

[0053] In yet another embodiment of the invention the Estimation of catalase (CAT) activity was carried out. CAT activity in rats’ pancreas was evaluated on 29^th day. No significant difference in CAT activity was observed in all ZU08 treated groups and positive control group in comparison to the control group, but significant decreased was seen in negative control group as compared with control group. But significant difference was observed in positive control and ZU08 treated groups in comparison to the negative control group, but no significant difference between positive and ZU08 treated groups. Effects of respective treatments on CAT activity were shown in Table 7 and Figure 9C.

[0054] In yet another embodiment of the invention the Histopathology of pancreas was studied. Histopathology of pancreas was done at Gargi diagnostic Lab Jalandhar. All panels were stained with hematoxylin (H) & eosin (E) and magnified at 40X.

[0055] Referring now to Figure 10. Histology of pancreas was showed in

Figure 10, it represent the control rat with vehicle group 1 (A), Test compound ZU08 (10 mg) per se group 2 (B), Test compound ZU08 (10 mg) per se group 3 (F), were showing normal appearance of the islet of Langerhans (IL) located in the exocrine tissue (ET). Figure IOC is the pancreas of STZ- HFD-induced diabetic rat group 3, showing marked degeneration of the IL with swelling of acinar cells and vacuoles formation. D and E are the pancreas of positive control (acarbose-10 mg/kg) group 4 and test compound ZU08 (5 mg) group 5 respectively showing less swelling and degeneration in comparison to negative control rat.

Claims

CLAIMS:

1. An oral pharmaceutical composition, comprising:

pharmaceutically effective amount of:

or a pharmaceutically acceptable salt thereof; and

at least one pharmaceutically acceptable carrier, adjuvant, or vehicle for treating a subject suffering from diseases and/or disorders associated with aberrant activity of alpha-amylase.

2. The oral pharmaceutical composition of claim 1 wherein:

pharmaceutically effective amount of:

or a pharmaceutically acceptable salt thereof is dissolved in water and ethanol mixture.

3. The oral pharmaceutical composition of claim 1 wherein the oral pharmaceutical composition is in the form of a liquid, gel, pill, capsule or tablet.

4. The oral pharmaceutical composition of claim 1 wherein diseases and/or disorders associated with aberrant activity of alpha-amylase comprises but not limited to diabetes.

5. An oral pharmaceutical composition, comprising:

pharmaceutically effective amount of:

or a pharmaceutically acceptable salt thereof; and at least one pharmaceutically acceptable carrier, adjuvant, or vehicle for treating a subject suffering from diseases and/or disorders associated with aberrant activity of alpha-amylase.

The oral pharmaceutical composition of claim 5 wherein:

pharmaceutically effective amount of:

7. The oral pharmaceutical composition of claim 5 wherein the oral pharmaceutical composition is in the form of a liquid, gel, pill, capsule or tablet.

8. The oral pharmaceutical composition of claim 5 wherein diseases and/or disorders associated with aberrant activity of alpha-amylase comprises but not limited to diabetes.