CRYSTAL FORM OF SGLT INHIBITOR AND APPLICATION THEREOF

Abstract

Crystal form A of a compound represented by formula (I) and an application thereof in preparing a drug for treating an SGLT1/SGLT2-related disease.

##STR00001##

Claims

1. Crystal form A of a compound represented by formula (I), wherein an X-ray powder diffraction pattern of the crystal form A has characteristic diffraction peaks at the following 2θ angles: 5.09±0.20°, 9.35±0.20°, and 13.24±0.20° ##STR00005##

2. The crystal form A according to claim 1, wherein the X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 5.09±0.20°, 9.35±0.20°, 13.24±0.20°, 14.35±0.20°, 15.86±0.20°, 17.04±0.20°, 18.04±0.20°, and 19.16±0.20°.

3. The crystal form A according to claim 2, wherein the X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 5.09±0.20°, 7.13±0.20°, 9.35±0.20°, 11.98±0.20°, 13.24±0.20°, 14.35±0.20°, 15.86±0.20°, 17.04±0.20°, 18.04±0.20°, and 19.16±0.20°.

4. The crystal form A according to claim 3, wherein the X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 3.33°, 4.84°, 5.09°, 7.13°, 9.35°, 10.17°, 11.98°, 13.24°, 14.35°, 14.62°, 15.38°, 15.86°, 17.04°, 18.04°, 19.16°, 19.95°, 20.26°, 20.70°, 21.71°, 22.52°, 24.43°, 26.11° and 28.41°.

5. The crystal form A according to claim 4, wherein the XRPD pattern of the crystal form A is as shown in FIG. 1.

6. The crystal form A according to claim 1, wherein a differential scanning calorimetry curve of the crystal form A has an endothermic peak starting at 134.7±3° C.

7. The crystal form A according to claim 6, wherein the DSC curve is as shown in FIG. 2.

8. The crystal form A according to claim 1, wherein a thermal gravimetric analysis curve of the crystal form A has a weight loss of 0.97% at 110±3° C.

9. The crystal form A according to claim 8, wherein the TGA curve is as shown in FIG. 3.

10. A preparation method for a crystal form A of the compound represented by formula (I), comprising an anti-solvent addition method, a slow cooling method, a room temperature suspension stirring method, a gas-liquid permeation method, a slow volatilization method, or a temperature cycling method.

11. The preparation method according to claim 10, wherein the anti-solvent addition method comprises: 1) adding the compound represented by formula (I) into a solvent to form a saturated solution; and 2) adding an anti-solvent to the solution, wherein the solvent is isopropanol, dichloromethane, tetrahydrofuran, acetone or ethyl acetate; and the anti-solvent is H.sub.2O or n-pentane.

12. The preparation method according to claim 10, wherein the room temperature suspension stirring method comprises: 1) preparing a saturated solution at 50° C. with the compound of formula (I) in a solvent; and 2) cooling the solution from 50° C. to 5° C. at a rate of 0.1° C. per minute; and wherein the solvent is a mixture of toluene and n-pentane at a volume ratio of 2:1, ethyl acetate and n-pentane at a volume ratio of 4:1, water and 1,4-dioxane at a volume ratio of 9:1, methyl isobutyl ketone and n-pentane at a volume ratio of 3:1, trichloromethane and 1-n-pentanol at a volume ratio of 1:1 or N, N-dimethylformamide and H.sub.2O at a volume ratio of 1:1.

13. The preparation method according to claim 10, wherein the slow cooling method comprises: 1) adding a compound of formula (I) to a solvent to form a suspension; and 2) carrying out crystal transformation by magnetically stirring the suspension; and wherein the solvent is 2-butanol, H.sub.2O, a mixture of isopropanol and H.sub.2O at a volume ratio of 98:2 to 85:15, a mixture of n-pentane and acetone at a volume ratio of 9:1, a mixture of n-pentane:ethanol at a volume ratio of 19:1, a mixture of anisole and n-pentane at a volume ratio of 4:1, a mixture of dimethyl sulfoxide and H.sub.2O at a volume ratio of 3:1, a mixture of methyl isobutyl ketone and n-butanol H at a volume ratio of 2:1, a mixture of dichloromethane and n-pentane at a volume ratio of 1:1, a mixture of 2-butanone and n-pentane at a volume ratio of 3:1, a mixture of toluene and n-heptane at a volume ratio of 3:1, a mixture of xylene and n-butanol at a volume ratio of 1:1, a mixture of ethyl acetate and n-heptane at a volume ratio of 4:1 or a mixture of methanol and H.sub.2O at a volume ratio of 4:1.

14. The preparation method according to claim 10, wherein the gas-liquid permeation method comprises: 1) preparing a clear solution by adding the compound of formula (I) to anisole at room temperature; and 2) placing the clear solution in an atmosphere of n-pentane to allow well contacting of the n-pentane with the solution to induce crystallization.

15. The preparation method according to claim 10, wherein the slow volatilization method comprises: 1) dissolving the compound of formula (I) in a solvent to form a clear solution; and 2) sealing the clear solution with a sealing film, and making holes in the sealing film to allow slow volatilization and crystallization; and wherein the solvent is ethanol or a mixture of ethanol and H.sub.2O at a volume ratio of 4:1.

16. The preparation method according to claim 10, wherein the temperature cycling method comprises: 1) dissolving the compound of formula (I) in a solvent to form a turbid solution of 50° C.; and 2) carrying out crystal transformation by circulating the turbid solution at a speed of 0.1° C./minute according to a procedure of 50° C. to 5° C.; and wherein the solvent is n-pentane, H.sub.2O, a mixture of acetone and H.sub.2O at a volume ratio of 4:1, a mixture of 2-butanone and n-heptane at a volume ratio of 3:1, a mixture of isopropyl acetate and n-butanol at a volume ratio of 1:1 or a mixture of xylene and n-heptane at a volume ratio of 4:1.

17. A preparation method for crystal form A of a compound represented by formula (I), comprising: 1) adding acetone to the compound of formula (I) to dissolve; and 2) adding n-heptane, stirring at room temperature for 3-4 hours, and filtering.

18. A method for treating an SGLT1/SGLT2 related disease, comprising administering the crystal form A according to claim 1 to a subject in need.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0078] FIG. 1 is an XRPD pattern using Cu-Kα radiation of the crystal form A of a compound represented by formula (I);

[0079] FIG. 2 is a DSC curve of the crystal form A of a compound represented by formula (I);

[0080] FIG. 3 is a TGA curve of the crystal form A of a compound represented by formula (I);

[0081] FIG. 4 is a DVS curve of the crystal form A of a compound represented by formula (I).

DETAILED DESCRIPTION

[0082] In order to better understand the content of the present disclosure, the present disclosure is further described in detail below by referring to the examples, which are not intended to adversely limit the present disclosure.

Example 1: Preparation of a Compound Represented by Formula (I)

[0083] ##STR00004##

[0084] Step 1: Synthesis of Compound C

[0085] Tetrahydrofuran (3.2 L) and compound A (400.12 g, 1.70 mol, 1 eq) are sequentially added to a pre-dried three-necked flask (10 L). After replacing with nitrogen, an internal temperature thereof is cooled to less than −60° C. Then n-butyllithium (2.5 M, 712 mL, 1.05 eq) is slowly added dropwise thereto, stirred for 0.5 hours, then Compound B (250.00 g, 1.86 mol, 1.1 eq) in tetrahydrofuran (250 mL) is added dropwise, and the resulted solution is slowly warmed to 0° C. and stirred for 0.5 hours. After the reaction is completed, the reaction solution is slowly poured into water (3 L) at 0-5° C. for quenching, then stirred for 10 minutes, and allowed to separate into layers by standing, wherein an organic phase is reserved, and an aqueous phase is extracted with ethyl acetate (2 L×2). Organic phases are merged, washed with saturated sodium chloride solution (1 L), and concentrated under reduced pressure to obtain a crude compound C which will be directly used for the next reaction.

[0086] Step 2: Synthesis of Compound D

[0087] Compound C (493.04 g, 1.70 mol, 1 eq), acetic acid (2 L) and concentrated sulfuric acid (16.56 g, 0.17 mol, 0.1 eq) are sequentially added to a pre-dried three-necked flask (5 L), heated to 100° C. and stirred for 1 hour. After the reaction is completed, the solvent is evaporated under reduced pressure. The residue is dissolved in ethyl acetate (3 L), neutralized with 0.5M NaOH, and then the organic phase is washed successively with saturated NaHCO.sub.3 solution (1 L) and saturated NaCl solution (0.5 L), followed by reduced pressure concentration to obtain a crude product. Methanol (5740 mL) is added to the crude product, heated to 40° C. to allow well dispersion of the system, then cooled to room temperature, stirred for 4 hours, and filtered. The filtrate is concentrated under reduced pressure to obtain a crude product, into which methanol (1150 mL) is added and heating is performed to dissolve. The resulted solution is cooled to room temperature, stirred for 2 hours, and filtered by suction to obtain a compound D. .sup.1H NMR (400 MHz, CHCl.sub.3-d) δ ppm 7.43-7.49 (m, 2H), 7.21-7.27 (m, 2H), 5.88-5.95 (m, 1H), 2.64-2.77 (m, 4H), 2.12-2.26 (m, 2H).

[0088] Step 3: Synthesis of Compound E

[0089] To a pre-dried 50 L reaction kettle, 1,4-dioxane (22 L) is added, then compound D (2200.00 g, 4.02 moL, 1 eq), bis(pinacolato)diboron (2147.20 g, 4.22 moL, 1.05 eq), potassium acetate (1185.80 g, 5.73 moL, 1.5 eq) and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium (58.80 g, 0.03 moL, 0.01 eq) are successively added thereto, heated under nitrogen protection to 100° C. and reacted for 5 hours. The resulted reaction solution is concentrated under reduced pressure and then dissolved in ethyl acetate (22 L), water (22 L) is added thereto to perform washing, and the aqueous phase is extracted with ethyl acetate (11 L). Organic phases are merged and concentrated under reduced pressure to obtain a crude product. N-heptane (3.5 L) is added to the crude product, stirred at room temperature for 1 hour, filtered, and the filter cake is collected and dried under vacuum for 16 hours to give a compound E. .sup.1H NMR (400 MHz, CHCl.sub.3-d) δ ppm 7.78 (d, J=8.3 Hz, 2H), 7.38 (d, J=8.1 Hz, 2H), 5.94-6.00 (m, 1H), 2.67-2.79 (m, 4H), 2.19 (tt, J=13.7, 6.6 Hz, 2H), 1.36 (s, 12H).

[0090] Step 4: Synthesis of Compound G

[0091] To a pre-dried 50 L reaction kettle, 1,4-dioxane (12 L) and water (3 L) are added, then compound E (1493.00 g, 2.96 mol, 1.0 eq), compound F (1139.45 g, 3.55 mol, 1.2 eq), potassium carbonate (614.78 g, 4.44 mol, 1.5 eq) and tetrakis(triphenylphosphine)palladium (171.43 g, 0.14 mol, 0.05 eq) are successively added thereto, and stirred under nitrogen protection at 50° C. for 16 hours. After the reaction is cooled, ethyl acetate (7.5 L) and water (7.5 L) are added thereto and stirred, followed by standing to separate into layers. The separated aqueous phase is extracted with ethyl acetate (7.5 L). Organic phases are merged and concentrated under reduced pressure to obtain a crude product. Ethanol (15 L) is added to the crude product, heated to 80° C. and stirred until the solution is clear. Then the solution is cooled to 30° C. and stirred for 2 hours at this temperature, filtered, and the filter cake is collected. The filter cake is subjected to palladium removal to give a compound G. .sup.1H NMR (400 MHz, CHCl.sub.3-d) δ ppm 7.29 (s, 1H), 7.26 (s, 1H), 7.17-7.22 (m, 2H), 7.01-7.06 (m, 3H), 5.88 (br s, 1H), 5.29-5.36 (m, 1H), 5.18-5.24 (m, 1H), 5.12 (t, J=9.7 Hz, 1H), 4.52 (d, J=9.8 Hz, 1H), 4.39 (d, J=9.8 Hz, 1H), 3.93-4.05 (m, 2H), 2.65-2.76 (m, 4H), 2.58 (q, J=7.5 Hz, 2H), 2.13-2.24 (m, 5H), 2.10 (s, 3H), 2.01 (s, 3H), 1.73 (s, 3H), 1.55 (s, 3H), 1.13 (t, J=7.5 Hz, 3H).

[0092] Step 5: Synthesis of Compound of Formula (I)

[0093] Anhydrous methanol (8.5 L) is added to a pre-dried 50 L reaction kettle, then compound G (855.83 g, 1.42 moL, 1 eq) and 25% sodium methoxide in methanol solution (171 mL) are successively added thereto. The resulted reaction solution is stirred at room temperature for 16 hours. After the reaction is completed, standing and filtering are performed. Water (8.5 L) is added into the filtrate, heated to 40° C. and stirred for 2 hours, and then stirred at room temperature for 16 hours. Then filtration is carried out and a solid crude product is collected. Anhydrous dichloromethane (11 L) is added to the crude product, followed by standing to allow separating into layers, and the organic phase is collected and concentrated to obtain the compound of formula (I). .sup.1H NMR (400 MHz, CH.sub.3OH-d.sub.4) δ ppm 7.32 (d, J=8.3 Hz, 2H), 7.23-7.27 (m, 1H), 7.17-7.22 (m, 2H), 7.12 (d, J=8.3 Hz, 2H), 5.92 (br s, 1H), 4.40 (d, J=9.5 Hz, 1H), 4.15 (d, J=9.0 Hz, 1H), 4.03 (s, 2H), 3.36-3.51 (m, 3H), 2.58-2.74 (m, 6H), 2.10-2.24 (m, 5H), 1.11 (t, J=7.5 Hz, 3H).

Example 2: Preparation of Crystal Form A of Compound Represented by Formula (I)

[0094]

TABLE-US-00003 TABLE 2 Chinese and English names of solvents English English Reagents name Reagents name Methanol MeOH N,N-dimethylformamide DMF Ethanol EtOH Anisole Anisole Isopropanol IPA n-heptane n-Heptane Acetone Acetone Toluene Toluene Methyl isobutyl MIBK Dichloromethane DCM ketone Ethyl acetate EtOAc Dimethyl sulfoxide DMSO Isopropyl acetate IPAc Water H.sub.2O Tetrahydrofuran THF 2-Butanone MEK 1,4-Dioxane 1,4-dioxane 2-Butanol 2-BuOH n-hexane n-Hexane Chloroform CHCl.sub.3 n-butanol 1-BuOH Xylene Xylene 1-pentanol 1-pentanol n-pentane n-Pentane

[0095] Method 1: Anti-Solvent Addition Method

[0096] Nearly saturated solutions in different solvents are prepared respectively with approximately 15 mg of the compound of formula (I), and a corresponding anti-solvent is added into each solution. The obtained solids are separated and dried to perform XRPD tests. The experimental results are as follows:

TABLE-US-00004 Experiment No. Solvents Anti-solvents Crystal form of solid 1 IPA H.sub.2O Crystal form A 2 DCM N-Heptane Crystal form A 3 THF N-Heptane Crystal form A 4 Acetone N-Pentane Crystal form A 5 EtOAc N-Pentane Crystal form A

[0097] Method 2: Slow Cooling Method

[0098] Nearly saturated solutions of 50° C. in different solvents are prepared respectively with approximately 15 mg of the compound of formula (I), cooled from 50° C. to 5° C. at a rate of 0.1° C. per minute, and resulted solids are collected to preform XRPD tests. The experimental results are as follows:

TABLE-US-00005 Experiment No. Solvents (v/v) Crystal form of solids 1 Toluene/n-Heptane, 2:1 Crystal form A 2 EtOAc/n-Pentane, 4:1 Crystal form A 3 H.sub.2O/1,4-Dioxane, 9:1 Crystal form A.sup.# 4 MIBK/n-Pentane, 3:1 Crystal form A 5 CHCl.sub.3/1-Pentanol, 1:1 Crystal form A Note: for .sup.#samples, a large amount of solids of crystal form A are generated when equilibrated at 50° C.

[0099] Method 3: Room Temperature Suspension Stirring Method

[0100] Suspensions are prepared respectively with approximately 15 mg of the compound of formula (I) in different solvents. The suspensions are magnetically stirred to allow crystal transformation, then solids are collected by centrifugation and XRPD tests are performed. The experimental results are as follows:

TABLE-US-00006 Experiment No. Solvents (v/v) Crystal form of Solids 1 2-BuOH Crystal form A 2 n-Pentane/Acetone, 9:1 Crystal form A 3 n-Heptane/EtOH, 19:1 Crystal form A 4 Anisole/n-Pentane, 4:1 Crystal form A 6 MIBK/1-BuOH, 2:1 Crystal form A 7 DCM/n-Pentane, 1:1 Crystal form A 8 MEK/n-Pentane, 3:1 Crystal form A 9 IPA/H.sub.2O (98:2) Crystal form A 10 IPA/H.sub.2O (95:5) Crystal form A 11 IPA/H.sub.2O (92:8) Crystal form A 12 IPA/H.sub.2O (85:15) Crystal form A 13 H.sub.2O Crystal form A 14 Toluene/n-Heptane, 3:1 Crystal form A 15 Xylene/1-BuOH, 1:1 Crystal form A 16 EtOAc/n-Heptane, 4:1 Crystal form A 17 MeOH/H.sub.2O, 4:1 Crystal form A

[0101] Method 4: Gas-Liquid Permeation Method

[0102] A clear solution is prepared respectively with approximately 15 mg of the compound of formula (I) in a solvent at room temperature. The clear solution is placed in an atmosphere of an anti-solvent, so that the anti-solvent vapor and the solution are full contacted with to induce crystallization. The obtained solid is collected and XRPD test is carried out, and the experimental results are as follows:

TABLE-US-00007 Experiment No. Solvent Anti-solvent Crystal form of solid 1 Anisole n-Pentane Crystal form A

[0103] Method 5: Slow Volatilization Method

[0104] Clear solutions are prepared respectively with approximately 15 mg of the compound of formula (I) in different solvents. Each of the clear solutions is sealed with a sealing film, and holes are made in the sealing film to allow slow volatilization and crystallization. The resulted solids are collected and XRPD tests are performed. The experimental results are as follows:

TABLE-US-00008 Experiment No. Solvents Crystal form of solids 1 EtOH Crystal form A 2 EtOH/H.sub.2O, 4:1 Crystal form A

[0105] Method 6: Temperature Cycling Method

[0106] Suspensions are prepared respectively with approximately 15 mg of the compound of formula (I) in different solvents. The suspensions are circulated at a speed of 0.1° C./minute according to a procedure of 50° C. to 5° C. to allow crystal transformation. The resulting solids are collected and XRPD tests are performed. The experimental results are as follows:

TABLE-US-00009 Experiment No. Solvents Crystal form of solids 1 n-Heptane Crystal form A 2 H.sub.2O Crystal form A 3 Acetone/H.sub.2O, 4:1 Crystal form A 4 MEK/n-Heptane, 3:1 Crystal form A 5 IPAc/1-BuOH, 1:1 Crystal form A 6 Xylene/n-Hexane, 4:1 Crystal form A

[0107] Method 7:

[0108] 371 g of the compound of formula (I) is dissolved in acetone (530 mL), and n-heptane (4770 mL) is added thereto. The resulted solution is stirred at room temperature for 3 hours, then filtered and a filter cake is collected, obtaining the crystal form A of formula (I). .sup.1H NMR (400 MHz, CH.sub.3OH-d.sub.4) δ ppm 7.32 (d, J=8.3 Hz, 2H), 7.23-7.27 (m, 1H), 7.17-7.22 (m, 2H), 7.12 (d, J=8.3 Hz, 2H), 5.92 (br s, 1H), 4.40 (d, J=9.5 Hz, 1H), 4.15 (d, J=9.0 Hz, 1H), 4.03 (s, 2H), 3.36-3.51 (m, 3H), 2.58-2.74 (m, 6H), 2.10-2.24 (m, 5H), 1.11 (t, J=7.5 Hz, 3H).

Example 3: Hygroscopicity Study of Crystal Form a of Compound of Formula (I)

[0109] Experimental Material:

[0110] SMS DVS Advantage Dynamic Vapor Sorption Intrinsic

[0111] Experimental Method:

[0112] 10-30 mg of the crystal form A of compound of formula (I) is placed in a DVS sample tray for testing.

[0113] Experimental Results:

[0114] The DVS pattern of the crystal form A of compound of formula (I) is shown in FIG. 4. ΔW=0.1483%.

[0115] Experimental Conclusion:

[0116] The crystal form A of compound of formula (0.1) has a weight gain of 0.1483% caused by hygroscopicity at 25° C. and 80% RH, indicating it has almost no hygroscopicity.

Example 4: Stability Test of Crystal Form A of Compound of Formula (I)

[0117] 12 parts of crystal form A of compound of formula (I), approximately 5 mg per part, are weighed out. Each part is placed at the bottom of a HPLC vial and spread into a thin layer. Samples placed in a constant temperature and constant humidity box at 60° C./75% RH and samples placed in a 92.5% RH dryer are sealed with a sealing film on the bottle mouth, and some small holes are made in the sealing film to ensure that the sample can be brought into full contact with the ambient air. Samples placed at 60° C., or under lights or shading conditions are tightly capped (wherein the samples under shading conditions are packaged with a tin foil paper). The test results are shown in Table 3 below:

TABLE-US-00010 TABLE 3 Solid stability test results of Compound A crystal form of Formula (I) Purity Conditions Test time (area %) Crystal form 0 day — 96.48 Crystal form A 60° C. 5 days 96.38 Crystal form A 10 days 96.41 Crystal form A 92.5% RH 5 days 96.41 Crystal form A 10 days 96.47 Crystal form A Visible illuminance reaches 96.29 Crystal form A lights.sup.# 1.2E+06 Lux .Math. hrs Shading Test at the same time with 96.44 Crystal form A lights the visible lights group Visible illuminance reaches 200 95.75 Crystal form A lights + W .Math. hrs/m.sup.2 Ultraviolet lights.sup.# Shading Test at the same time with 96.43 Crystal form A lights the visible lights + ultraviolet lights group 60° C./ 1 months 96.31 Crystal form A 75% RH 2 months 96.39 Crystal form A 3 months 96.37 Crystal form A Note: .sup.#indicates compliance with ICH requirements. Conclusion: The crystal forms A of compound of formula (I) has good stability.

Example 5: Pharmacodynamic Study of Crystal Form A of Compound of Formula (I) in Rats OGTT Model

[0118] Experimental Material:

[0119] SD rats, male, aged 7-8 weeks, purchased from Zhejiang Weitonglihua Experimental Animal Technology Co. Ltd. are maintained in a special pathogen-free environment, with 4 rats in a single ventilation cage. Beddings and water in all cages are sterilized prior to use. All animals can freely obtain a standard certified commercial laboratory diet.

[0120] Experimental Method:

[0121] After the animals are adapted to the environment for one week, they are fasted for 16 hours in a metabolic cage, and randomly divided into different groups according to fasting blood glucose and body weight. The animals are administered with a drug or solvent (2 mL/kg), followed by immediately administration of 50% glucose solution (2 g/kg, 4 mL/kg), 2 hours after the administration of glucose solution, the feed is recovered, and blood samples are collected at 0 min, 15 min, 30 min, 45 min, 60 min, and 120 min for blood glucose testing. All values are expressed as average values. Statistical analysis is carried out using Graphpad Prism 6 double factor variance analysis and evaluated with Tukey's multiple comparison tests. P values less than 0.05 are considered to have statistical significance.

[0122] Experimental Results: See Table 4

TABLE-US-00011 TABLE 4 Experimental results for rat sugar tolerance Positive compound Crystal form A of Vehicle Sotagliflozin compound of formula (I) Compounds control group (3 mg/Kg) (1 mg/kg) (3 mg/kg) (10 mg/kg) OGTT Blood 1167.8 ± 1046.7 ± 1065.6 ± 977.1 ± 881.4 ± glucose levels 30.24 33.17* 34.95 36.91*** 14.75**** AUC.sub.0-2 hr (mol/L × min) Notes: 1. Data is expressed as mean ± standard error. 2. Statistical analysis is performed using two-Way ANOVA, comparing with the control group, *denotes p < 0.05, **denotes p < 0.001, ***denotes p < 0.001, ****denotes p < 0.0001.

[0123] Experimental Conclusion:

[0124] The crystal form A of compound of formula (I) can effectively and significantly reduce the blood glucose level, AUC.sub.0-2hr, in 2 hours of animals.