CN108368111B - Crystal forms of pyrido [1,2-a ] pyrimidinone analogs, and methods and intermediates for their preparation - Google Patents

Abstract

The invention discloses pyrido [1,2-a ]]Crystal forms of pyrimidinone analogs, and methods and intermediates for their preparation. (formula I)

Description

Crystal forms of pyrido [1,2-a ] pyrimidinone analogs, and methods and intermediates for their preparation

Technical Field

The invention relates to a crystal form of a pyrido [1,2-a ] pyrimidone analog, and a preparation method and an intermediate thereof.

Background

The PI3K pathway is the most frequently mutated site in human cancer cells, and can cause cell proliferation, activation and signal amplification. PI3K and mTOR are the two most important kinases in the PI3K signaling pathway.

PI3 kinase (phosphatidylinositol 3-kinase, PI3Ks) belongs to the lipid kinase family and is capable of phosphorylating the inositol ring 3' -OH end of phosphatidylinositol. Phosphatidylinositol 3-kinase (PI 3K) is a lipid kinase composed of a regulatory subunit p85 or p101 and a catalytic subunit p110, and plays a key role in proliferation, survival, metabolism and the like of cells by activating downstream Akt and the like by catalyzing phosphorylation of phosphatidylinositol 4, 5-diphosphate (PIP 2) to phosphatidylinositol 3, 4, 5-triphosphate (PIP 3). Therefore, inhibition of phosphoinositide 3 kinase can affect PI3K pathway, thereby inhibiting proliferation and activation of cancer cells.

The tumor suppressor PTEN (phosphatase and tension homolog deleted on chromosome ten) dephosphorylates PIP3 to generate PIP2, thereby realizing negative regulation of PI3K/Akt signal channel, inhibiting cell proliferation and promoting cell apoptosis. The close relationship between PI3K and tumorigenesis is suggested by frequent occurrence of PI3K gene mutation and amplification in cancer, deletion of PTEN in cancer and the like

mTOR (mammalian target of rapamycin) is a serine/threonine protein kinase present in the cytosol, belongs to the phosphatidylinositol 3-kinase related kinase family, and plays an important role in regulating the signaling of many pathways. mTOR has been identified as a downstream target for PI 3K/Akt. Two distinct mTOR complexes, mTORC1 and mTORC2, are now found in cells. The two perform different functions, mTORC1 primarily stimulates cell growth and proliferation, while mTORC2 regulates cell survival and cytoskeleton through activation of AKT, PKC, and other kinases. Studies have shown that mTOR signaling pathways are involved in carcinogenesis, and that simultaneous inhibition of the activity of both mTOR complexes in cancer cells has a broader and more potent anticancer effect.

The PI3K-mTOR dual inhibitor can block multiple links in information transmission at the same time, and can more effectively prevent kinase information transmission, thereby overcoming or delaying the generation of drug resistance.

In the patent applications W02008163636 by Nowa company and W02008144463 by GSK company, a series of compounds having inhibitory effects on both PI3K and mTOR are reported, and these compounds have good tumor therapeutic activity. However, currently, there are no drugs having inhibitory effects on both PI3K and mTOR on the market, so that there is a need to develop multidrug drugs having inhibitory effects on both PI3K and mTOR for the treatment of cancer.

Disclosure of Invention

The invention provides a preparation method of a compound 1,

which comprises the following steps:

wherein the content of the first and second substances,

the base C is selected from pyridine, 2, 6-dimethylpyridine and Et₃N、4-DMAP、LiOH、Cs₂CO₃And K₂CO₃；

Solvent C is selected from pyridine, dichloromethane, toluene, acetonitrile, acetone, DMF and THF;

the mol ratio of the compound 7 to the compound 8 is 1: 1-3;

the mol ratio of the compound 7 to the alkali C is 1: 1-3.

In some embodiments of the invention, the preparation of compound 1 comprises the steps of,

wherein the content of the first and second substances,

the base A is selected from potassium carbonate, sodium carbonate, cesium carbonate, potassium hydroxide or sodium hydroxide;

solvent a is selected from DMF, DMSO or NMP.

In some embodiments of the invention, the molar ratio of the base A to the compound 5 is selected from 1-3: 1.

In some embodiments of the invention, the preparation of compound 1 comprises the steps of:

the base B is selected from potassium carbonate, sodium carbonate, barium hydroxide, potassium phosphate, cesium carbonate, potassium fluoride, cesium fluoride, sodium hydroxide, potassium tert-butoxide, sodium tert-butoxide, potassium acetate or sodium acetate;

the solvent b is selected from 1, 4-dioxane, DMSO, THF, 1, 4-dioxane/water or THF/water;

in the solvent b, the volume ratio of 1, 4-dioxane or THF to water is selected from 3-6: 1;

the catalyst is selected from Pd (dppf) Cl₂Or Pd (PPh)₃)₄。

In some embodiments of the present invention, the volume ratio of 1, 4-dioxane or THF to water in the solvent b is selected from 5: 1.

In some embodiments of the invention, the molar ratio of the base B to the compound 6 is selected from 1-3: 1.

In some embodiments of the invention, the preparation of compound 1 comprises the steps of:

the present invention also provides compounds of the formula:

the invention provides a crystal form I of a compound 1, and an XRPD pattern of the crystal form I is shown in a figure 1.

In some embodiments of the present invention, the XRPD pattern analysis data of the crystalline form I of compound 1 above is as shown in table-1:

table-1 form I XRPD pattern analysis data for compound 1

The invention provides a compound 1 in a crystal form II, wherein an XRPD pattern is shown in figure 4.

In some embodiments of the invention, the XRPD pattern data for form II of compound 1 above is shown in table-2.

TABLE-2 Compound II form XRPD pattern analysis data

The invention provides a form III crystal form of compound 1, wherein an XRPD pattern is shown as figure 7.

In some embodiments of the present invention, the crystalline form III profile data of compound 1 above is shown in table-3.

TABLE-3 XRPD pattern data for form III of Compound 1

The invention provides a compound 1 in a crystal form IV, wherein an XRPD pattern is shown in figure 10.

In some embodiments of the invention, the XRPD pattern data for form IV of compound 1 above is shown in table-4.

TABLE-4 XRPD pattern data for form IV of Compound 1

No.	2θ	d(A)	I％	No.	2θ	d(A)	I％
								1	5.246	16.8322	47.8	15	16.681	5.3102	29.6
2	6.074	14.5392	7.9	16	18.371	4.8255	8.5
								3	6.723	13.1361	47.2	17	19.894	4.4593	16.0
4	7.708	11.4600	100.0	18	20.818	4.2634	5.5
								5	10.308	8.5742	9.1	19	22.712	3.9119	9.0
6	11.098	7.9658	37.7	20	23.934	3.7149	10.1
								7	12.182	7.2595	5.5	21	24.389	3.6467	8.5
8	12.656	6.9887	18.4	22	24.804	3.5866	15.0
								9	12.836	6.8911	24.5	23	26.026	3.4208	5.5
10	13.501	6.5530	34.3	24	26.817	3.3217	12.3
								11	14.355	6.1651	30.7	25	28.965	3.0800	6.6
12	15.356	5.7655	5.8	26	29.513	3.0242	6.4
								13	15.729	5.6293	6.1	27	29.992	2.9769	5.3
14	16.145	5.4854	7.5	28	32.587	2.7456	4.9

The invention provides a crystal form V of a compound 1, and an XRPD pattern of the crystal form V is shown in figure 13.

In some embodiments of the invention, the XRPD resolution data for form V of compound 1 above is shown in table-5.

TABLE-5 XRPD pattern analysis data for form V of Compound 1

The invention provides a compound 1 crystal form VI, wherein an XRPD pattern is shown as figure 16.

In some embodiments of the present invention, XRPD pattern analysis data for form VI of compound 1 above is shown in table-6.

Table-6 XRPD pattern analysis data for compound 1 form VI

No.	2θ	d(A)	I％	No.	2θ	d(A)	I％
								1	8.735	10.1143	7.3	20	25.663	3.4684	8.9
2	10.470	8.4419	13.7	21	26.412	3.3717	22.9
								3	12.424	7.1185	100.0	22	27.159	3.2807	4.7
4	13.786	6.4183	50.6	23	27.617	3.2273	11.7
								5	15.166	5.8372	1.7	24	27.970	3.1873	15.0
6	16.883	5.2471	10.9	25	30.714	2.9086	8.8
								7	18.006	4.9222	1.3	26	31.291	2.8562	2.4
8	18.711	4.7385	4.1	27	31.781	2.8133	1.8
								9	19.386	4.5749	5.7	28	32.389	2.7618	10.4
10	19.604	4.5247	2.5	29	33.730	2.6551	2.3
								11	20.572	4.3138	11.7	30	34.838	2.5731	2.1
12	20.986	4.2296	2.9	31	35.198	2.5476	1.6
								13	21.402	4.1484	11.4	32	35.603	2.5195	2.9
14	22.425	3.9614	13.2	33	35.902	2.4992	5.2
								15	23.297	3.8150	2.7	34	37.087	2.4221	3.2
16	24.123	3.6863	79.2	35	37.641	2.3877	1.2
								17	24.596	3.6164	11.8	36	38.231	2.3522	2.2
18	25.013	3.5570	3.5	37	38.760	2.3213	1.6
								19	25.306	3.5165	5.2	38	39.747	2.2659	2.7

The invention also aims to provide application of the stable properties of the I crystal form, the II crystal form, the III crystal form, the IV crystal form, the V crystal form and the VI crystal form of the compound 1 in preparing medicaments for treating diseases related to mTOR/P13K receptors.

Definitions and description:

as used herein, the following terms and phrases are intended to have the following meanings unless otherwise indicated. A particular phrase or term should not be considered as ambiguous or unclear without special definition, but rather construed in a generic sense. When a trade name appears herein, it is intended to refer to its corresponding commodity or its active ingredient.

The intermediate compounds of the present invention may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other chemical synthetic methods, and equivalents thereof well known to those skilled in the art, with preferred embodiments including, but not limited to, the examples of the present invention.

The chemical reactions of the embodiments of the present invention are carried out in a suitable solvent that is compatible with the chemical changes of the present invention and the reagents and materials required therefor. In order to obtain the compounds of the present invention, it is sometimes necessary for a person skilled in the art to modify or select the synthesis steps or reaction schemes based on the existing embodiments.

An important consideration in any synthetic route planning in the art is the selection of suitable protecting groups for reactive functional groups, such as amino groups in the present invention. Greene and Wuts (protective groups In Organic Synthesis, Wiley and Sons, 1991) are the authorities of this area for trained practitioners. All references cited herein are incorporated herein in their entirety.

The present invention will be specifically described below by way of examples, which are not intended to limit the present invention in any way.

The proton NMR data were recorded on a Bruker Avance III 400(400MHz) spectrometer with chemical shifts expressed in terms of (ppm) at tetramethylsilane low field. L C/MS or Shimadzu MS contained a DAD: SPD-M20A (L C) and Shimadzu Micromass2020 detector.Mass spectrometer was equipped with an electrospray ion source (ESI) operating in a positive or negative mode.

The invention employs the following abbreviations: DCM represents dichloromethane; PE represents petroleum ether; EA represents ethyl acetate;DMF represents N, N-dimethylformamide; DMAC for N, N-dimethylacetamide; DMSO represents dimethyl sulfoxide; EtOAc for ethyl acetate; THF represents tetrahydrofuran; EtOH stands for ethanol; MeOH represents methanol; NMP stands for N-methylpyrrolidone; 2-METHF represents 2-methyltetrahydrofuran; i-PrOH represents 2-propanol; HCl (g) represents hydrogen chloride gas; HOAc represents acetic acid; TFA represents trifluoroacetic acid; DIPEA stands for diisopropylethylamine; DIEA stands for diisopropylethylamine; NMM stands for N-methylmorpholine; et (Et)₃N represents triethylamine; pd (PPh)₃)₄Represents palladium tetratriphenylphosphine; pd (dppf) Cl₂Represents 1, 1' -bis (diphenylphosphino) ferrocene palladium chloride; pd (PPh)₃)₂Cl₂Represents dichlorobis (triphenylphosphine) palladium (II); pd (OAc)₂Represents palladium acetate.

The compound is made by hand or

The software names, and the commercial compounds are under the supplier catalog name.

The powder X-ray diffraction (XRPD) method of the invention

The instrument model is as follows: bruker D8 advanced X-ray diffractometer

And (3) testing conditions are as follows: the detailed XRPD parameters are as follows:

x-ray generator Cu, k α,

tube voltage: 40kV, tube current: 40mA.

Scattering slit: 0.60mm

Detector slit: 10.50mm

Backscatter slit: 7.10mm

Scanning range: 4-40deg

Step length: 0.02deg

Rate: 0.1S

Sample pan rotation speed: 15rpm

Differential thermal analysis (DSC) method of the present invention

The instrument model is as follows: TA Q2000 differential scanning calorimeter

And (3) testing conditions are as follows: a sample (0.5-1 mg) is placed in a DSC aluminum pot for testing, and the method comprises the following steps: the room temperature is 300 ℃, and the heating rate is 10 ℃/min.

The present invention is a Thermal Gravimetric Analysis (TGA) method

The instrument model is as follows: TA Q5000IR thermogravimetric analyzer

And (3) testing conditions are as follows: a sample (2-5 mg) is placed in a TGA platinum pan for testing, and the method comprises the following steps: the room temperature is 300 ℃, and the heating rate is 10 ℃/min.

The technical effects are as follows:

the compound 1 provided by the invention has stable properties of crystal forms I, II, III, IV, V and VI, good solubility and hygroscopicity, and good pharmaceutical prospect.

The process for synthesizing the compound 1 and the intermediate thereof has the advantages of cheap and easily-obtained raw materials, and overcomes the defects of large toxicity of used reagents, harsh reaction conditions, difficult separation and purification, difficult industrialization and the like.

Drawings

FIG. 1 is an XRPD spectrum of Cu-K α radiation of form I.

Figure 2 is a DSC profile of form I.

Figure 3 is a TGA profile of form I.

FIG. 4 is an XRPD spectrum of Cu-K α radiation of form II.

Figure 5 is a DSC profile of form II.

Fig. 6 is a TGA profile of form II.

Figure 7 is an XRPD spectrum of Cu-K α radiation of form III.

Figure 8 is a DSC profile of form III.

Figure 9 is a TGA profile of crystalline form III.

Figure 10 is an XRPD spectrum of Cu-K α radiation of form IV.

Figure 11 is a DSC profile of form IV.

Figure 12 is a TGA profile of form IV.

FIG. 13 is an XRPD spectrum of Cu-K α radiation of form V.

Figure 14 is a DSC profile of form V.

Fig. 15 is a TGA profile of form V.

Figure 16 is an XRPD spectrum of Cu-K α radiation of form VI.

Figure 17 is a DSC profile of form VI.

Fig. 18 is a TGA profile of crystalline form VI.

Detailed Description

For better understanding of the present invention, the following description is given with reference to specific examples, but the present invention is not limited to the specific embodiments.

Reference example 1 preparation of Compound 5

Preparation of methyl 2- (benzyloxy) acetate (2)

Dichloromethane (640 ml) was added to a 3.0 l three necked round bottom flask, methanol (149.70 g, 187.13 ml) was added, pyridine (236.09 ml, 2.92 mol) was added, the mixture was cooled to 0 ℃ with an ice water bath, 2-benzyloxycarbonyl chloride (227.00 g, 1.17 mol) was added dropwise to the round bottom flask under nitrogen protection, the temperature was controlled at 0-10 ℃ and dropwise added after the addition was completed, the ice water bath was removed and the reaction solution was stirred at 20 ℃ for 1.5 hours, sampling was performed and the T L C (petroleum ether/ethyl acetate 5/1) showed complete reaction, water (1000 ml) was added to the round bottom flask, stirring was carried out for 10 minutes, the organic layer was separated and collected, the organic layer was washed with 1.0 mol/l dilute hydrochloric acid (600 ml 2), the organic layer was separated and collected, the organic layer was washed with 20% sodium carbonate solution (400 ml), the organic layer was separated and collected, the organic layer was dried with anhydrous sodium sulfate (80 g), filtered under reduced pressure, the filtrate was concentrated to give a colorless product purity of 5912%).¹H NMR(400MHz，CHLOROFORM-d)ppm 7.41-7.28(m，5H)，4.64(s，2H)，4.12(s，2H)，3.77(s，3H)；LCMS(ESI)m/z：181(M+1).

Preparation of methyl 2- (benzyloxy) -3- (dimethylamino) acrylate (3)

Methyl 2- (benzyloxy) acetate (207 g, 1.11 mol) was charged into a 3 l round bottom flask, T-butoxy bis (dimethylamino) methane (233 g, 1.34 mol) was added, the reaction temperature was controlled at 90-100 ℃ for 16 hours, and a sample was taken to check that T L C (PE/EA ═ 5/1) showed completion of the reaction, the reaction was cooled to 60 ℃, and the reaction was concentrated with an oil pump to give a yellow oily product (275 g, crude) which was used directly in the next reaction.

¹H NMR(400MHz，CHLOROFORM-d)ppm 7.44-7.30(m，5H)，6.87(s，1H)，4.72(s，2H)，3.73(s，3H)，2.98(s，6H).

Preparation of 3- (benzyloxy) -7-bromo-4H-pyrido [1,2-a ] pyrimidin-4-one (4)

Methyl 2- (benzyloxy) -3- (dimethylamino) acrylate (130 g, 552.53 mmol) was charged to a 3 l round bottom flask, acetic acid (1.25 l) was added, and 2-amino-5-bromopyridine (100.6 g, 552.53 mmol) was added. The temperature of the reaction solution was controlled at 120 ℃ and 130 ℃ and the reaction was stirred for 16 hours. Sampling and detecting, and completing the reaction. The reaction mixture was cooled to 60 ℃ and concentrated, the solvent was evaporated, ethyl acetate (500 ml) was added, stirring was carried out for 10min, filtration was carried out, ethyl acetate (1 l) was added to the cake, stirring was carried out for 10min, and filtration was carried out, and the cake was spin-dried to give a compound (132.5 g, purity: 92%, yield: 66.62%) as a yellow solid.

¹H NMR (400MHz, CH L OROFORM-d) d 9.13(d, J1.7 Hz, 1H), 8.05(s, 1H), 7.56(dd, J2.0, 9.5Hz, 1H), 7.49-7.41(M, 3H), 7.40-7.27(M, 3H), 5.29(s, 2H); L (esi) M/z 333 (isotope M +1).

Preparation of 7-bromo-3-hydroxy-4H-pyrido [1,2-a ] pyrimidin-4-one (5)

Trifluoroacetic acid (1 l) was added to a 3 l round bottom flask, 3- (benzyloxy) -7-bromo-4H-pyrido [1,2-a ] pyrimidin-4-one (260 g, 785.12 mmol) was added, the reaction solution was allowed to warm at 90-100 ℃ and stirred for 2 hours, sampling was performed, L CMS showed substantial completion of the reaction, the reaction solution was cooled to 60 ℃, concentrated, the solvent was evaporated, ethyl acetate (1 l) was added, stirring was performed for 30 minutes, then, ethyl acetate (1 l) was added to the filter cake, stirring was performed for 30 minutes, filtering was performed, and the filter cake was dried under reduced pressure at 40 ℃ for 60 hours to obtain a yellow solid compound (167 g, content 95.75%, purity 100%, yield 88.25%).

¹H NMR (400MHz, DMSO-d6) d 9.92(br, 1H), 8.87(d, J1.6 Hz, 1H), 8.05(s, 1H), 7.71(dd, J2.0, 9.6Hz, 1H), 7.50(d, J9.6 Hz, 1H); MS m/z: 240.9(M +1), 242.9 (isotope M +1).

EXAMPLE 1 preparation of Compound 1

Preparation of 7-bromo-3- (2-methoxyethoxy) -4H-pyrido [1,2-a ] pyrimidin-4-one (6)

7-bromo-3-hydroxy-4H-pyrido [1,2-a ] pyrimidin-4-one (140 g, 0.58 mol), 2-bromoethyl methyl ether (97 g, 0.70 mol), potassium carbonate (241 g, 1.74 mol), and N, N-dimethylformamide (0.241 k g of 1.4 l was stirred at 95 ± 5 ℃ for 3.5 to 4.5 hours, the reaction solution was rotary-dried under reduced pressure using an oil pump at 50 ± 5 ℃, a dichloromethane and methanol mixture (1.6 l, V/V ═ 15: 1) was added to the residue, stirred at 20 ± 5 ℃ for 1.5 ± 0.5 hours, filtered, and the filter cake was rinsed with a dichloromethane and methanol mixture (0.64 l, V/V ═ 15: 1). the filtrate was collected and concentrated under reduced pressure to give compound 3(180 g, yield: 100%, purity: 99.95%).

1H NMR(400MHz，CDCl₃) 3.42(s, 3H), 3.76(t, J ═ 4.8Hz, 2H), 4.31(t, J ═ 4.8Hz, 2H), 7.44(d, J ═ 9.6Hz, 1H), 7.54(dd, J ═ 9.6Hz, 2Hz, 1H), 8.52(s, 1H), 9.08(d, J ═ 1.6Hz, 1H), L cms (esi) M/z 301 (isotope M +1).

Preparation of 7- (5-amino-6-methoxypyridin-3-yl) -3- (2-methoxyethoxy) -4H-pyrido [1,2-a ] pyrimidin-4-one (7)

Reacting 7-bromo-3- (2-methoxyethoxy) -4H-pyrido [1,2-a ]]Pyrimidin-4-one (175 g, 0.55 mol), 2-methoxy-5- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-3-amine (158 g, 0.6 mol), potassium carbonate (227 g, 1.64 mol), 1, 4-dioxane (1.7 l) and water (350 ml) were added to a round bottom flask, pd (dppf) Cl was added₂(16.4 g, 0.016 mol). The mixture is stirred for 4-4.5 hours at 90-95 ℃ under the protection of nitrogen. After the reaction, the reaction solution was concentrated under reduced pressure at an external temperature of 50. + -. 5 ℃. To the residue was added water to R1 and stirred for 0.5-1 hour, filtered, the filter cake rinsed with 0.35 kg of water, spin-dried under reduced pressure, dichloromethane (1.164 kg) was added to the filter cake at 20 ℃, stirred for 0.5-1 hour, filtered and the filter cake spin-dried under reduced pressure at 60 ℃. Methylene chloride (3.99 kg) and methanol (1.185 kg) were added to the filter cake to dissolve it. To the solution was added TMT solution (1.16 g of thiocyanuric acid, 4.5 g of sodium chloride, 114.55 g of ethanol, 14.65 g of ammonia water, 161 g of water) at 20 ℃ and stirred for 18. + -. 0.5 hours. Filtering, and concentrating the filtrate at 45-50 deg.C under reduced pressure. 0.875 kg of water was added to the crude product and stirred for 16. + -. 0.5 hours. Filtering, and spin-drying the filter cake at 45-50 deg.C under reduced pressure. It was dissolved with dichloromethane (4.655 kg) and methanol (1.38 kg). To the solution was added TMT solution (1.16 g of thiocyanuric acid, 4.5 g of sodium chloride, 114.55 g of ethanol, 14.65 g of ammonia water, 161 g of water) at 20 ℃ and stirred for 16. + -. 0.5 hours. Filtering, and concentrating the filtrate at 45-50 deg.C under reduced pressure. 0.875 kg of water was added to the crude product and stirred for 2. + -. 0.5 hours. Filtration and spin-drying of the filter cake at 45-50 ℃ under reduced pressure gave the product (174 g, purity: 96.96%, yield: 84.59%).

¹H NMR(400MHz，DMSO-d6)d＝8.91(d，J＝1.6Hz，1H)，8.21(s，1H)，8.04(dd，J＝9.6Hz，2Hz，1H)，7.79(d，J＝2Hz，1H)，7.67(d，J＝9.6Hz，1H)，7.26(d，J＝2.4Hz，1H)，5.25(s，2H)，4.22-4.24(m，2H)，3.92(s，3H)，3.66-3.69(m，2H)，3.32(s，3H)；LCMS(ESI)m/z：343(M+1).。

Preparation of N- (2-methoxy-5- (3- (2-methoxyethoxy) -4-oxo-4H-pyrido [1,2-a ] pyrimidin-7-yl) pyridin-3-yl) -2, 4-dimethylthiazole-5-sulfonamide (1)

Adding 7- (5-amino-6-methoxypyridin-3-yl) -3- (2-methoxyethoxy) -4H-pyrido [1,2-a ] pyrimidin-4-one (170g, 454.86mmol) to R1 in a 3L round-bottomed flask, adding pyridine (1.7L) to R1, cooling R1 to 0-10 deg.C, adding SM4(142.91g, 659.54mmol) dropwise to R1, controlling the temperature of R1 to 10-20 deg.C, stirring for 20-21 hours, sampling to detect that the starting material is substantially reacted, pouring the reaction solution into 5L% water, adding 3L% ethyl acetate, stirring for 40 minutes, filtering the mixture with celite (200g), rinsing the filter cake with ethyl acetate (700m L), collecting the filtrate, standing for layering, separating, extracting the aqueous phase with ethyl acetate (2L ≦ 4), combining the organic phases, rinsing with saturated sodium acetate (L%), rinsing the filter cake with ethyl acetate (700m L%), concentrating the filtrate under reduced pressure, adding the filtrate with sodium sulfate, filtering the filtrate, concentrating the aqueous phase with sodium sulfate, concentrating the filtrate under reduced pressure, adding the filtrate at room temperature to obtain a filtrate (1.9%) and concentrating the residue, adding the residue after cooling, adding sodium sulfate, filtering the residue, concentrating the residue at room temperature to obtain a filtrate (90% purity, filtering the residue, concentrating the residue at room temperature, filtering the residue at room temperature to obtain a concentration under 0% of 5% of the residue, filtering temperature, filtering the filtrate, filtering the residue, filtering the.

¹H NMR(400MHz，CHLOROFORM-d)d＝9.09(s，1H)，8.21(s，1H)，8.17(d，J＝2.4Hz，1H)，8.01(d，J＝2.4Hz，1H)，7.74-7.67(m，2H)，7.29(br，1H)，4.35(t，J＝4.8Hz，2H)，3.97(s，3H)，3.80(t，J＝4.8Hz，2H)，3.45(s，3H)，2.64(s，3H)，2.56(s，3H)；LCMS(ESI)m/z：518(M+1).

Example 2 preparation of form I

Compound 1, about 50mg, was added to 0.3m L methanol (acetonitrile or acetone) to form a suspension, the suspension sample was shaken for 2 days (protected from light) on a homoeothermic homogenizer (40 ℃), the residual solid was centrifuged and dried overnight in a vacuum oven at 40 ℃ to give compound 1, form I.

Example 3 preparation of crystalline form II

The preparation process of the crystal form II is the same as that of the crystal form I, and only the solvent methanol is changed into 0.3m L ethanol (0.35m L isopropanol or ethyl acetate).

Example 4 preparation of crystalline form III:

and adding about 50mg of the compound 1 into 0.35m L ethanol-water mixed solvent (ethanol-water 3: 1, v: v) to form suspension, placing a suspension sample on a homoeothermic homogenizer (at 40 ℃), shaking (keeping out of the sun) for 1 day to obtain a solution, centrifuging, taking supernate, placing the supernate in a ventilation cabinet for natural volatilization, and drying the residual solid in a vacuum drying box at 40 ℃ overnight to obtain the II crystal form of the compound 1.

Example 5 preparation of form IV

The preparation process of the crystal form IV is the same as that of the crystal form I, and only the solvent methanol is changed into a mixed solvent of 0.35m L acetone-water (acetone-water 1: 2, v: v).

Example 6 preparation of form V

Taking about 50mg of compound 1, adding 0.15m L Tetrahydrofuran (THF) to obtain a small amount of solid residues, adding 0.15m L THF continuously to obtain a solution, centrifuging, taking the supernatant, placing the supernatant in a fume hood for natural volatilization, and drying the residual solid in a vacuum drying oven at 40 ℃ overnight to obtain the V crystal form of compound 1.

Example 7 preparation of form VI

Compound 1(170 g) was dissolved in anhydrous dichloromethane (2.66 kg), the solution was filtered through silica gel (170g, 100-. The filtrates were combined and concentrated to 145 g at 45-50 ℃ under reduced pressure. To the resulting solid was added absolute ethanol (711 g), and the mixture was stirred at 80-85 ℃ for 12-14 hours. The mixture was cooled to 20. + -. 10 ℃ and filtered. The filter cake was rinsed with absolute ethanol (237 g) and dried under vacuum at 40-45 ℃ to constant weight to give N- (2-methoxy-5- (3- (2-methoxyethoxy) -4-oxo-4H-pyrido [1,2-a ] pyrimidin-7-yl) pyridin-3-yl) -2, 4-dimethylthiazole-5-sulfonamide (133 g, purity: 98.94%, yield: 78.2%) as form VI.

Solid stability test of VI crystal form under conditions of high temperature, high humidity and strong illumination

About 5mg of the crystal form VI sample is weighed, placed at the bottom of a glass sample bottle and spread into a thin layer. The sample is sealed with aluminum foil paper, and the aluminum foil paper is pricked with small holes to ensure that the sample can be fully contacted with ambient air, and is placed in a constant temperature and humidity box under the humidity condition of 40 ℃/75 percent. And taking a 25mgVI crystal form sample, and lofting according to the method for detecting the crystal form of the sample. The samples placed under the above conditions were sampled and tested at months 1,2 and 3, and the test results were compared with the initial test results at month 0, and the test results are shown in the following table-7:

solid stability test of form VI of Table-7

Time point (moon)	Appearance of the product	Crystal form	Content (%)	Total impurities (%)
					0	Yellow powder	VI crystal form	99.8	0.18
1	Yellow powder	VI crystal form	99.6	0.19
					2	Yellow powder	VI crystal form	100.3	0.17
3	Yellow powder	VI crystal form	99.4	0.17

And (4) experimental conclusion: the crystal form of the invention has good stability and is easy to prepare.

Experimental examples in vitro cell Activity assay

Experimental procedures and methods:

1. MCF-7 cells were plated at 2.5 × 10 per well⁴The density of individuals was plated in 96-well plates (the culture medium used was the whole culture medium containing 10% FBS).

2. The following day the wells were drained of medium and a concentration (primary screening) or series of concentrations (IC) were added₅₀Test) was dissolved in a serum-free culture solution, and cells were cultured in a 96-well plate for 2 hours.

3. Insulin was dissolved in serum-free medium, and cells were added and cultured for 30 minutes at a final insulin concentration of 10. mu.g/ml.

4. When waiting for the reaction, the lysate was prepared as follows:

a) the enhancement liquid (Enhancer Solution) needs to be taken out of the refrigerator and melted in advance.

b) The enhancing Solution (Enhancer Solution) was diluted 10-fold with 5 Xlysis Buffer (L sys Buffer) to prepare a concentrated lysate.

c) And diluting the concentrated lysate by 5 times with double distilled water to prepare lysate.

5. The well was blotted clean of culture medium and rinsed once quickly with PBS.

6. Add 150. mu.l of freshly prepared lysate to each well and shake for 10 minutes at room temperature.

7. After confirming that all cells had shed, the lysate was transferred to a 1.5 ml tube along with the cell debris.

8. Vortex several times to mix the lysate and cells thoroughly, and then centrifuge the mixture at 12000g for 10 minutes at 4 ℃.

9. The number of E L ISA-one microplate strips required was counted, the extra microplate strips were removed from the frame and placed back in the storage bag and sealed, and each well was rinsed with 200 microliters of double distilled water to remove the preservative before using the microplate strips.

10. To each well 50 microliters of antibody mix was added. (the antibody mixture is prepared by mixing the medium antibody reagent and the enzyme-labeled antibody reagent in equal proportion, and care should be taken not to vortex when preparing the antibody mixture)

11. To each well of the E L ISA-One microplate, 25 microliters of cell lysate was added, the microplate covered with an adhesive sealing membrane, and incubated on a microplate shaker at room temperature for 1 hour.

12. Each well was washed 3 times with 150 μ l of 1X wash buffer. After the last wash, the wells were drained of wash buffer. If desired, the 1 Xwash buffer can be allowed to remain in the microplate for a maximum of 30 minutes to allow time for the substrate mixture to be prepared.

13. The substrate mixture should be prepared at any time. To each well 100. mu.l of substrate mixture was added, and the microplate was sealed with tinfoil and incubated on a microplate shaker for 10 minutes at room temperature.

14. Add 10. mu.l of stop solution to each well and mix gently (5-10 seconds) on a microplate shaker.

15. The corresponding E L ISA-One filter set is assembled, and the fluorescence signal intensity is read.

The results are shown in Table 1:

TABLE 1 in vitro cell Activity test results

Note: a is less than or equal to 50 nM.

And (4) conclusion: the compound 1 has obvious effect of inhibiting mTOR/PI 3K.

Claims (12)

1. A process for the preparation of a compound 1,

which comprises the following steps:

wherein the content of the first and second substances,

the base C is selected from pyridine, 2, 6-dimethylpyridine and Et₃N、4-DMAP、LiOH、Cs₂CO₃And K₂CO₃；

Solvent c is selected from pyridine, dichloromethane, toluene, acetonitrile, acetone, DMF and THF;

the mol ratio of the compound 7 to the compound 8 is 1: 1-3;

the molar ratio of the compound 7 to the base C is 1: 1-3.

2. The production method according to claim 1, comprising the step of,

3. the production method according to claim 1, comprising the step of,

wherein the content of the first and second substances,

the base A is selected from potassium carbonate, sodium carbonate, cesium carbonate, potassium hydroxide or sodium hydroxide;

the solvent a is selected from DMF, DMSO or NMP;

the base B is selected from potassium carbonate, sodium carbonate, barium hydroxide, potassium phosphate, cesium carbonate, potassium fluoride, cesium fluoride, sodium hydroxide, potassium tert-butoxide, sodium tert-butoxide, potassium acetate or sodium acetate;

the solvent b is selected from 1, 4-dioxane, DMSO, THF, 1, 4-dioxane/water or THF/water;

in the solvent b, the volume ratio of 1, 4-dioxane or THF to water is 3-6: 1;

the catalyst is selected from Pd (dppf) Cl₂Or Pd (PPh)₃)₄。

4. The production method according to claim 2 or 3, wherein the volume ratio of 1, 4-dioxane or THF to water in the solvent b is selected from 5: 1.

5. Compound 7 of the following formula as an intermediate for preparing compound 1:

6. the XRPD pattern of the crystal form I of the compound 1 is shown in figure 1:

7. the XRPD pattern of the crystal form II of the compound 1 is shown in figure 4:

8. form iii of compound 1 having an XRPD pattern as shown in figure 7:

9. the compound 1 in the IV crystal form has an XRPD pattern shown in figure 10:

10. the XRPD pattern of form v of compound 1 is shown in figure 13:

11. the compound 1 in the VI crystal form has an XRPD pattern shown in figure 16:

12. the application of the crystal forms I, II, III, IV, V and VI of the compound 1 in the preparation of medicaments for treating mTOR/PI3K receptor-related diseases; wherein said form i is according to claim 6, said form ii is according to claim 7, said form iii is according to claim 8, said form iv is according to claim 9, said form v is according to claim 10, and said form vi is according to claim 11;

Images