CN110128501A

CN110128501A - A kind of camptothecine compounds and its preparation method and application targeting FAP enzyme

Info

Publication number: CN110128501A
Application number: CN201910425487.0A
Authority: CN
Inventors: 王英召; 张旭; 刘艳玲; 黄小根
Original assignee: Beijing Sea Source Medical Science And Technology Co Ltd
Current assignee: Beijing Sea Source Medical Science And Technology Co Ltd
Priority date: 2019-05-21
Filing date: 2019-05-21
Publication date: 2019-08-16

Abstract

The present embodiments relate to a kind of camptothecine compounds and its preparation method and application for targeting FAP enzyme.The camptothecine compounds of targeting FAP enzyme, as starting material, are substituted respectively and are modified by 10- difluoromethyl camptothecine compounds and proline analog derivative, then the two gains are reacted, and finally obtain the camptothecine compounds of targeting FAP enzyme.The preparation method step is simple, easy to operate, and the requirement to equipment is low, can be efficiently obtained above-mentioned camptothecine compounds.The camptothecine compounds of targeting FAP enzyme have preferable anti-tumor activity, have broad application prospects in the preparation of antitumor drugs.

Description

Camptothecin compound targeting FAP enzyme and preparation method and application thereof

Technical Field

The invention relates to the technical field of organic synthesis, in particular to a camptothecin compound targeting FAP enzyme and a preparation method and application thereof.

Background

Camptothecin is a natural plant alkaloid, the molecule is of a five-ring structure and contains a pyrrole [3,4-b ] quinoline ring, a conjugated pyridine ring and an α -hydroxyl six-membered internal alicyclic ring (shown in the following formula), and the camptothecin can be used for treating various malignant tumors, such as bone cancer, liver cancer, bladder cancer, leukemia and the like, in 1985, Hisang and the like find that the camptothecin and derivatives thereof use DNA topoisomerase I as a target to inhibit the synthesis of DNA in an organism to play an anti-tumor role.

Fibroblast Activation protease (FAP enzyme) is a membrane serine peptidase, one of the serine protease family members of type II, with dipeptidyl peptidase and collagenase activities, and tumor-associated fibroblasts expressing FAP in the tumor microenvironment are the earliest identified tumor stromal cell type. The FAP enzyme is activated by interaction of fibroblasts in tumor stroma and cancer cells, is the most main host cell in a tumor microenvironment, has the functions of promoting growth and invasion of tumor cells and immunosuppression, has stable genome and is not easy to resist drugs, and thus can be used as a new target for tumor immunotherapy.

FAP enzymes are generally very rare in human normal cells, but their presence in the human Tumor Microenvironment (TME) is 10-100 times greater than in plasma. The cytotoxic small molecule drug and the special polypeptide are crosslinked to form a temporary inactive prodrug, then the prodrug is delivered to tumor tissues, and the peptide bond specific peptide chain cleavage enzyme activity of FAP is utilized to release the prodrug, so that the target cells (cancer cells) are killed.

Disclosure of Invention

In a first aspect of the embodiments of the present invention, there is provided a camptothecin compound targeting FAP enzyme, which can be used as a prodrug enabling tumor FAP enzyme to be specifically degraded. It has no activity in vitro, and after entering into body, it can produce high concentration active medicine in tumor part to inhibit tumor, reduce medicine consumption and reduce toxicity.

The second aspect of the embodiments of the present invention provides a preparation method of the above-mentioned camptothecin compound targeting FAP enzyme, which has the advantages of simple steps, convenient operation, low requirement for equipment, and capability of efficiently obtaining the above-mentioned camptothecin compound targeting FAP enzyme.

The third aspect of the embodiment of the invention provides an application of the camptothecin compound targeting the FAP enzyme in preparing an anti-tumor drug.

Specifically, the first aspect of the embodiments of the present invention provides a camptothecin compound targeting FAP enzyme, wherein the compound has a structural formula shown in formula 1,

in the formula 1, R¹Selected from hydrogen, C1-C6 alkyl or C1-C6 substituted alkyl, R¹The binding site of (a) is any one of three unsubstituted sites on the benzene ring; r²Selected from hydrogen, C1-C6 alkyl or C1-C6 substituted alkyl; r³Selected from hydrogen, amino, C1-C6 alkyl or C1-C6 substituted alkyl.

The amino group referred to in the examples of the present invention is-NH-which comprises both a nitrogen atom and two hydrogens₂Also includes-NH₂In which one or both hydrogen atoms are replaced by alkyl, alkoxy, acyl, alkoxycarbonyl or aryloxycarbonyl radicals, e.g., -N (CH)₃)₂， -N(CH₂CH₃)₂，-N(CH₂CH₂CH₃)₂，-NHCH₂CH₃，-NHOCH₂CH₃， -NHCOCH₃，-NHCOOCH₃-NHCbz, etc. The C1-C6 alkyl group may be a straight chain or branched chain alkyl group, including but not limited to methyl, ethyl, n-propyl, isopropyl, t-butyl, and the like. The C1-C6 substituted alkyl refers to a C1-C6 alkyl group in which at least one hydrogen atom is substituted with halogen, amino, hydroxyl, alkoxy, aryl or heterocycle. Such heterocycles include, but are not limited to, furan, thiophene, piperidine, piperazine, and derivatives thereof.

Furthermore, the structural formula of the camptothecin compound targeting the FAP enzyme is shown as a formula 2,

in the formula 2, R¹Selected from hydrogen, C1-C4 alkyl or C1-C4 substituted alkyl; r²Selected from hydrogen, C1-C4 alkyl or C1-C4 substituted alkyl; r³Selected from hydrogen, amino, C1-C4 alkyl or C1-C4 substituted alkyl.

The C1-C4 alkyl group may be a straight chain or branched chain alkyl group, including but not limited to methyl, ethyl, n-propyl, isopropyl, t-butyl, and the like. The C1-C4 substituted alkyl refers to a C1-C4 alkyl group in which at least one hydrogen atom is substituted with halogen, amino, hydroxyl, alkoxy, aryl or heterocycle. Such heterocycles include, but are not limited to, furan, thiophene, piperidine, piperazine, and derivatives thereof.

Further, in formula 2, R¹Selected from hydrogen orR⁴、R⁵Are respectively selected from hydrogen, C1-C6 alkyl, C1-C6 substituted alkyl or aryl; r²Selected from hydrogen, ethyl orR³is-NHR⁶，R⁶Selected from hydrogen, benzyloxycarbonyl or acetyl.

A second aspect of the embodiments of the present invention provides a preparation method of a camptothecin compound targeting FAP enzyme, the preparation method including:

reacting a 10-difluoromethylcamptothecin compound with Boc-glycine to obtain a glycine ester intermediate, and reacting the glycine ester intermediate with hydrochloric acid to obtain a substituted 10-difluoromethylcamptothecin intermediate;

reacting the proline derivative with aminoacetic ester to obtain a proline intermediate, and reacting the proline intermediate with an inorganic alkali reagent to obtain a substituted proline compound; and

reacting the substituted 10-difluoromethyl camptothecin intermediate with a substituted proline compound to obtain the camptothecin compound targeting the FAP enzyme; wherein,

the structural formula of the camptothecin compound targeting FAP enzyme is shown as a formula 1,

the structural formula of the 10-difluoromethylcamptothecin compound is shown as formula 3; the structural formula of the glycine ester intermediate is shown as a formula 4; the structural formula of the substituted 10-difluoromethyl camptothecin intermediate is shown as a formula 5,

the structural formula of the proline derivative is shown as a formula 6; the structural formula of the proline intermediate is shown as a formula 7; the structural formula of the substituted proline compound is shown as a formula 8,

wherein R is¹Selected from hydrogen, C1-C6 alkyl or C1-C6 substituted alkyl, R¹The binding site of (a) is any one of three unsubstituted sites on the benzene ring; r²Selected from hydrogen, C1-C6 alkyl or C1-C6 substituted alkyl; r³Selected from hydrogen, amino, C1-C6 alkyl or C1-C6 substituted alkyl.

Further, in the step of obtaining the glycine ester intermediate by reacting the 10-difluoromethylcamptothecin compound with Boc-glycine, DCM (dichloromethane), EDCI (1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride) and DMAP (4-dimethylaminopyridine) are added, and the reaction is carried out at room temperature for 15-20 hours.

Further, in the step of reacting the glycine ester intermediate with hydrochloric acid to obtain the substituted 10-difluoromethyl camptothecin intermediate, an ethyl acetate solution is also added, and the reaction is carried out at room temperature for 2-3 hours.

Further, in the step of reacting the proline derivative with aminoacetate to obtain a proline intermediate, DIEA (N, N-diisopropylethylamine) and HATU (2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate) are added for reaction at room temperature for 8-12 min; then Gly-OMe.HCl (glycine methyl ester hydrochloride) was added and the reaction was carried out at room temperature for 3.5 to 4.5 hours.

Furthermore, in the step of reacting the proline intermediate with an inorganic base reagent to obtain the substituted proline compound, THF (tetrahydrofuran) and water are added for reaction at room temperature for 2.5-3.5 hours.

Further, in the step of reacting the substituted 10-difluoromethyl camptothecin intermediate with the substituted proline compound to obtain the camptothecin compound targeting the FAP enzyme, DCM, DMAP and DIEA are added, and the system is cooled to 0 ℃; EDCI was then added to the system in 4 portions and allowed to react at room temperature for 16-18 hours.

In some embodiments, the FAP enzyme-targeting camptothecin compounds of the invention have the structural formula:

any one of the above.

The third aspect of the embodiment of the invention provides an application of any one of the camptothecin compounds targeting FAP enzyme in preparing an anti-tumor drug.

The embodiment of the invention has the beneficial effects that:

the embodiment of the invention provides a camptothecin compound targeting FAP enzyme, and a preparation method and application thereof. The preparation method has simple steps, convenient operation and low requirement on equipment, and can efficiently obtain the camptothecin compound. The camptothecin compound targeting FAP enzyme has better anti-tumor activity and wide application prospect in preparing anti-tumor drugs.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

Fig. 1 is a photograph of groups of tumor entities according to an embodiment of the present invention.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

Preparation of Compounds 1-4

(1) Taking 10-difluoromethyl camptothecin compound 1b as a starting material. Adding compound 1b (200mg,0.47mmol), Boc-glycine (165mg,0.94mmol), DCM (5mL) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) (448mg,2.34mmol) into a 50mL three-necked flask, stirring the mixture at room temperature for 15-20 hours, detecting the completion of the reaction of the raw materials by TLC, stopping the reaction, adding 20mL of DCM, washing the mixture with 20mL of sodium chloride for 2 times, adding anhydrous sodium sulfate to the organic phase, drying, filtering, concentrating the filtrate under reduced pressure to obtain a crude product, eluting the crude product by column chromatography, purifying (hexane: EA ═ 1:1-2), and concentrating to obtain 0.21 g of a white solid with yield: 78 percent.

(2) Adding 1-2(0.25g) and 10mL of saturated HCl/EA solution into a three-necked bottle, reacting at room temperature for 2-3 hours, detecting that the raw materials completely react by TLC, concentrating the reaction solution, adding 6mL of isopropanol into the obtained pale yellow oily matter, heating to completely dissolve the pale yellow oily matter, naturally cooling while stirring, separating out yellow solid after about 30 minutes, continuously stirring for 2-3 hours, filtering, leaching the filter cake with 2mL of isopropanol, drying at 40-50 ℃ after pumping to obtain 0.21 g of yellow solid, and obtaining the yield: 95 percent.

ESI-MS：484.46[M+H]

¹H NMR(400MHz,CDCl₃)δ8.56(s,1H),8.45(b,3H),8.15(d,J＝12Hz, 1H),8.01(d,J＝12Hz,1H),7.33(s,1H),7.34(t,J＝56Hz,1H),5.62(s, 2H),5.51(m,2H),4.35(d,J＝20Hz,1H),3.35(d,J＝20Hz,1H),3.25(m, 2H),2.18(m,2H),1.30(m,3H),0.90(m,3H).

(3) A100 mL single neck flask was charged with compound 2-1(Cbz-Gly-Pro-OH) (3g,9.8 mmol), DMF (50mL), DIEA (3.8g,29.4mmol) and 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU) (4.85g,12.7mmol), stirred at room temperature for 10min, added Gly-OMe.HCl (1.48g,11.75mmol), stirred at room temperature for 4h, the system was poured into 300mL water, DCM (200mL x 3) was extracted, the organic phases were combined, washed with aqueous HCl (0.1M,200mL), NaHCO was added₃Washed with aqueous solution (0.1M,200mL) and water (200 mL. times.3)Wash, saturated NaCl (100mL) wash, Na₂SO₄Drying, spin drying, chromatography on 200-mesh 300-mesh silica gel column (Hex/EA is 1:1-0:1), collecting the product, and concentrating to obtain 2.9g of white solid with the yield of 78%.

(4) In a 100mL single neck flask was added compound 2-2(2.9g,7.7mmol), THF (25mL), LiOH. H2O (1.45g,34.6mmol), water (25mL) and MeOH (5 mL). After stirring at room temperature for 3h, the organic solvent was removed by concentration under reduced pressure, the remaining aqueous phase was adjusted to pH3 with HCl (2M), and the aqueous phase was purified with chloroform: isopropanol (1:3, 100mL x 3) extraction, combined organic phases, washed with saturated aqueous NaCl (50mL), Na₂SO₄Drying; concentration under reduced pressure gave 2.5g of a white solid in a yield of 90%.

(5) A50 mL single-necked flask was charged with compounds 2-3(380mg,1.05mmol), 1-3(300 mg,0.7mmol), DCM (10mL), DMAP (9mg,0.07mmol) and DIEA (180mg, 1.41mmol), the system was cooled to 0 deg.C, EDCI (540mg,2.81mmol) was added to the system in 4 portions; stirring was carried out at room temperature for 17 hours, TLC detection reaction was essentially complete, stirring was stopped, 100mL DCM was added for dilution, water (60mL x 2) was used for washing, saturated NaCl (60mL x 2) was used for washing, anhydrous Na₂SO₄Drying, spin-drying, purifying with 200-mesh 300-mesh silica gel column chromatography (Hex/EA: 2:1-0:1), collecting the product, concentrating, stirring with Hex/EA (10/1,20mL) for 20min, filtering, and drying to obtain 310mg of light yellow solid with a yield of 57%.

ESI-MS：772.27[M+H]

Example 2

In addition to the above methods, compounds 1-4 can be utilized to obtain compounds 1-5.

Preparation of Compounds 1-5

Compound 1-4(0.3g) and HBr (35% in AcOH,4mL) were added to a single vial, reacted at room temperature for 2 hours, the system was spun dry, dissolved in 100mL aqueous HCl (0.1M), the aqueous phase was washed with methyl tert-butyl ether (50 mL. times.3), and the aqueous phase was washed with NaHCO₃The pH was adjusted to about 8, extracted with DCM (60 mL. times.4), the DCMs were combined and washed with saturated NaCl (50mL) and anhydrous Na₂SO₄Drying, concentrating the organic phase under reduced pressure to about 30mL, adding 0.3mL HCl (4M in EA) to generate solid, standing for 20min, removing supernatant, adding 30mL DCM, stirring, standing for 20min, removing supernatant, and drying the solid under negative pressure by an oil pump to obtain 70mg of yellow solid, and spin-drying the supernatant to obtain 160mg of yellow solid with a yield of 88%.

ESI-MS：638.23[M+H]

Example 3

In addition to the above methods, compounds 1-5 can be utilized to obtain compounds 1-6.

Preparation of Compounds 1-6

To a single-necked flask, compound 1-5(160mg,025mmol), CHCl was added₃(10mL), DIEA (162mg,1.25mmol) and Ac₂O (77mg,0.75mmol), reaction at room temperature for 1 hour, spin-drying the system, adding 30mL DCM, washing the organic phase with citric acid (10%, 30mL), NaHCO₃Washed (1%, 30mL), saturated NaCl (30mL), Na₂SO₄Drying, spin drying, chromatography on 200-300 mesh silica gel column (DCM/MeOH 50:1-15:1), collection of the product and concentration to 150 mg of light yellow solid, 93% yield.

Example 4

Preparation of Compound 4-1

(1) 10-hydroxycamptothecin (2g,5.49mmol), methanol 60mL and water 50mL are added to a three-necked flask, followed by FeSO₄.7H₂O (1.52g,5.49mmol), the system is turbid, the temperature is reduced to 0 ℃, and concentrated H is dripped₂SO₄(26mL), dropwise adding 4.8mL of hydrogen peroxide at 0 ℃, stirring for 10 minutes at 0 ℃, removing the ice bath, heating to 30 ℃, reacting for 16 hours, cooling to 0 ℃, dropwise adding 150mL of water, completing the addition in about 10 minutes, separating out solids in the dropwise adding process, stirring for 1 hour at 0 ℃, filtering, washing a filter cake with 20mL of 0 ℃ water, and drying to obtain 1.38 g of a light yellow solid product, wherein the yield is as follows: and 69 percent.

(2) 3-1(3.8g), 70mL of hydrobromic acid (48% aqueous solution) and 1.2mL of concentrated sulfuric acid were added to a three-necked flask, stirred in an oil bath at 100 ℃ overnight, and subjected to LCMS to complete reaction of the starting materials, silica gel column chromatography (DCM: MeOH ═ 30-10:1) was performed to elute the starting materials, and the product was concentrated to obtain 3g of a red solid with a yield of 68%.

(3) 3-2(3g, 6.6mmol), DMF (120mL) and N-methylpiperazine (2.2mL, 3eq.) were added to a single vial, stirred at room temperature overnight, TLC showed the starting material was reacted, TEA (3.6mL) and PhN (SO)₂CF3)₂(4.7g, 2eq), reaction at 60 ℃ for 4h, TLC showed complete reaction of starting material. Cooling the system to room temperature, pouring into 600mL of water, extracting with EA (300 mLx 3), combining EA phases, and washing the EA phases with water (300mL of x 2); the EA phase was back-extracted with HCl (1M, 200mL x 3), the aqueous phases were combined and washed with NaHCO₃Adjusting pH to about 8, extracting with EA (200mL x 3), combining EA phases, and adding saturated NaCl (2)00mL) washing, Na₂SO₄And (5) drying. The solvent was spun dry and the crude product was stirred with 50mL EA/hex (1/3) for 20min, filtered, and the filter cake was washed with 10mL EA/hex (1/3) and dried to give 2.1g of a pale yellow solid 3-4 with 53% yield.

(4) Adding 3-4(1g), Pd (PPh3)4(0.19g,0.33mmol), phosphine ligand DPPF (0.18g,0.66mmol), KBr (0.39g,0.66mmol), toluene (5mL) and anhydrous dioxane (10mL) into a pressure-resistant bottle, protecting with N2, adding [ (SIPr) Ag (CF2H)](1.26g,4.6mmol), stirring in a 70 ℃ oil bath for 16 hours, cooling the system to room temperature, pouring into 100mL of water, EA (100mL x 3) extracting, combining the EA phases, back-extracting the EA phase with HCl (1M,100mL x 3), combining the aqueous phases, and NaHCO₃Adjusting the pH value to about 8; extracting with EA (100mL x 3), combining EA phases, washing the EA phases with saturated aqueous NaCl (100mL), and drying with anhydrous Na2SO 4; the mixture was concentrated to give 0.5g of a red solid, the solid was dissolved in DCM, purified by 200-300 mesh silica gel column chromatography (DCM: MeOH: 20-10:1), the product was collected and concentrated to give a dark yellow solid, the concentrate was stirred with 10mL (DCM/MeCN: 1/10) for 0.5 hour, filtered and dried to give 0.49 g of a pale yellow solid with 59% yield.

ESI-MS：511.21[M+H]

(5) To a single-necked flask, compound 2-3(142mg,0.392mmol), 3-5(100mg, 0.196mmol), DCM (3mL), DMAP (9mg,0.07mmol) and DIEA (51mg,0.392 mmol) were added, the system was cooled to 0 deg.C, EDCI (540mg,2.81mmol) was added in 3 portions (3 min intervals); after stirring at room temperature for 18 hours, TLC detection of a small amount of starting material remained and stopping the reaction, 100mL DCM dilution was added, water (100 mL. times.2) washing, saturated NaCl (100 mL. times.2) washing, Na₂SO₄Drying, spin-drying, performing 200-mesh silica gel column chromatography (Hex/EA is 2:1-0:1), collecting product, and concentrating to obtain 60mg pale yellow solid 2-1 with yield：36％。

ESI-MS：856.34[M+H]

Activity assay

And (3) harvesting cells in a logarithmic growth phase by adopting a cold light detection method, counting the cells by adopting a platelet counter, and detecting the cell viability by using a trypan blue exclusion method to ensure that the cell viability is over 90 percent. The cell lines, medium and cell inoculum size selected are shown in Table 1.

TABLE 1 cell lines, media and cell inoculum used in the assay

Serial number	Cell lines	Cell type	Cell number/well	Culture medium
					1	A2780	Ovarian cancer	6000	RPMI1640+10％FBS

Mu.l of each cell suspension was added to a 96-well plate, and the cells in the 96-well plate were cultured overnight at 37 ℃ under 5% CO2 and 95% humidity. 10 times of drug solution is prepared, the highest concentration is 10 mu M, 9 concentrations are diluted by 3.16 times, 10 mu l of drug solution is added into each hole of a 96-hole plate inoculated with cells, and three multiple holes are arranged for each drug concentration. The cells in the dosed 96-well plate were cultured for 72 hours under the conditions of 37 ℃ and 5% CO2 at 95% humidity, and then CTG reagent (Promega, Cat # G7572) for determining the activity of the luminescent cells was added for analysis. The CTG reagents were thawed and the cell plates were equilibrated to room temperature for 30min, an equal volume of CTG solution was added to each well, the cells were lysed by shaking for 5min on an orbital shaker, the cell plates were placed at room temperature for 20min to stabilize the luminescence signal, and the luminescence values were read using a SpectraMax (MD, 2104-0010A) multi-labeled microplate detector. Data were analyzed using GraphPad Prism 5.0 software, and dose-response curves were derived by fitting the data using non-linear S-curve regression, from which IC50 values were calculated, as shown in table 2.

TABLE 2 antitumor Activity test results

Serial number	Drug name/code	IC50(μM)
			1	1b	0.00052
2	1-4	＞10
			3	Paclitaxel	0.0055
4	Cis-platinum	1.8150

In vivo Activity Studies

Experimental methods

30 male BALB/c nude mice were inoculated with 5X 10⁶SW620 colorectal cancer cells at a concentration of 100ul PBS 8 days later, were randomly divided into 5 groups of 6 cells each. Tail vein administration, 1 time every 3 days, 5 times of continuous administration, observation of tumor volume and body weight change, 3 days after the last 1 time administration, sacrifice of mice, tumor stripping and weighing, and photographing for retention of symptoms. And (5) sorting the measured data and carrying out statistical analysis. The results are shown in tables 3,4 and 5, and in FIG. 1.

TABLE 3 mean tumor volume (unit: mm) at different times for each group³)

P <0.05, # p <0.001, compared to blank

TABLE 4 tumor weight comparisons of groups

Drug name/code	Concentration of	Tumor weight (g)	Inhibition ratio (%)
				Blank space		1.458±0.078	/
1-4	2mg/kg	0.840±0.114*	42
				1-4	10mg/kg	0.336±0.037#	77
1-4	50mg/kg	0.027±0.009#@	98
				Iritean	80mg/kg	0.329±0.043#	77

P <0.05, # p <0.001, compared to blank

In comparison with Iritean, @ p <0.001

See fig. 1, where fig. 1 shows a photograph of groups of tumor entities, where a comparison of tumor size and weight can be seen.

TABLE 5 mean body weights (unit: g) of the groups at different times

P <0.05 vs. blank

The three doses of the compound 1-42 mg/kg, 10mg/kg and 50mg/kg and irinotecan 80mg/kg show the antitumor activity in tumor-bearing mice. The antitumor activity of the compounds 1-4 is positively correlated with the dosage, and is enhanced with the increase of the dosage. After the administration is finished, three doses show significant anti-tumor effect, and have statistically significant difference (p is less than 0.05 or 0.001), and the tumor inhibition rate is up to 93% at 50mg/kg dose. Irinotecan 80mg/kg tumor inhibition was 63%, which was between the tumor inhibition of compounds 1-410 mg/kg and 50 mg/kg. Tumor weight inhibition rates were similar.

The compound 1-42 mg/kg and 10mg/kg have little influence on the body weight of tumor-bearing mice. Compound 1-450 mg/kg reduced the rate of body weight gain in tumor-bearing mice compared to the blank control, with a statistically significant difference (p <0.05), and no significant difference in body weight change compared to irinotecan 80 mg/kg.

The data show that the effect of the compound 1-450 mg/kg is better than that of irinotecan 80mg/kg, and the weight change is equivalent to that of irinotecan. Compound 1-410 mg/kg acts at an equivalent effect to irinotecan 80mg/kg, but weight gain is much greater than irinotecan. Therefore, the compounds 1 to 4 have obvious advantages in reducing toxicity and improving effect, and have potential for developing new drugs.

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A camptothecin compound targeting FAP enzyme is characterized in that the structural formula of the compound is shown as formula 1,

in the formula 1, R¹Selected from hydrogen, C1-C6 alkyl or C1-C6 substituted alkyl, R¹The binding site of (a) is any one of three unsubstituted sites on the benzene ring; r²Selected from hydrogen, C1-C6 alkyl or C1-C6 substituted alkyl；R³Selected from hydrogen, amino, C1-C6 alkyl or C1-C6 substituted alkyl.

2. The camptothecin compound targeting FAP enzyme according to claim 1, wherein the camptothecin compound targeting FAP enzyme has a structural formula shown in formula 2,

3. The camptothecin compound targeting FAP enzyme according to claim 2, wherein R in formula 2 is¹Selected from hydrogen orR⁴、R⁵Are respectively selected from hydrogen, C1-C6 alkyl, C1-C6 substituted alkyl or aryl; r²Selected from hydrogen, ethyl orR³is-NHR⁶，R⁶Selected from hydrogen, benzyloxycarbonyl or acetyl.

4. A preparation method of a camptothecin compound targeting FAP enzyme is characterized by comprising the following steps:

5. The method according to claim 4, wherein in the step of reacting the 10-difluoromethylcamptothecin compound with Boc-glycine to obtain the glycine ester intermediate, DCM, EDCI and DMAP are further added, and the reaction is carried out at room temperature for 15-20 hours.

6. The method according to claim 5, wherein the step of reacting the glycine ester intermediate with hydrochloric acid to obtain the substituted 10-difluoromethylcamptothecin intermediate is carried out by adding ethyl acetate solution and reacting at room temperature for 2-3 hours.

7. The method of claim 6, wherein in the step of reacting the proline derivative with aminoacetate to obtain the proline intermediate, DIEA and HATU are further added and reacted at room temperature for 8-12 min; then adding Gly-OMe.HCl, and reacting for 3.5-4.5 hours at room temperature.

8. The method according to claim 7, wherein the step of reacting the proline intermediate with an inorganic base reagent to obtain the substituted proline compound further comprises adding THF and water and reacting at room temperature for 2.5 to 3.5 hours.

9. The preparation method of claim 8, wherein in the step of reacting the substituted 10-difluoromethylcamptothecin intermediate with the substituted proline compound to obtain the FAP enzyme-targeted camptothecin compound, DCM, DMAP and DIEA are further added, and the system is cooled to 0 ℃; EDCI was then added to the system in 4 portions and allowed to react at room temperature for 16-18 hours.

10. Use of the camptothecin compounds targeting the FAP enzyme according to any one of claims 1-3 for the preparation of an anti-tumor medicament.