WO2011113175A1

WO2011113175A1 - Cytarabine prodrug derivatives and use for resisting cancer or tumor thereof

Info

Publication number: WO2011113175A1
Application number: PCT/CN2010/000321
Authority: WO
Inventors: 高峰; 徐峻
Original assignee: Gao Feng; Xu Jun
Priority date: 2010-03-15
Filing date: 2010-03-15
Publication date: 2011-09-22

Abstract

The cytarabine prodrug derivatives of general formulas (I), (II) and (III), their synthetic routes, preparations and preparative methods thereof, and use for resisting cancer or tumor. The derivatives are prepared by chemical modification on N4, O5 positions of cytarabine, which can avoid losing efficacy and inducing toxicity caused by N4-amino group metabolism, and make O5-hydroxy group be easily phosphorylated and activated. The derivatives can increase bioavailability, solubility and liposolubility, and reduce multidrug resistance.

Description

Cytarabine prodrug derivative and its use in anticancer and antitumor

The invention relates to the technical field of medicine, in particular to a novel cytarabine prodrug derivative and a synthetic route thereof, and the invention also relates to a cytarabine prodrug derivative preparation, a preparation method thereof and a cytarabine The use of drug derivatives and their preparations in anti-cancer and anti-tumor.

Background technique

Cancer is currently the most important disease that threatens human health. The existing methods of treating cancer include: surgical resection, radiotherapy, chemotherapy or the combination of these methods. Chemotherapy has gained widespread use and has been used in the treatment of many different types of cancer. However, most of the anticancer drugs used in chemotherapy are limited to delaying the deterioration of cancer and prolonging the life of the patient. Although the pathogenesis of various types of cancer varies, they are actually a large group of syndromes with common characteristics. In addition to the strong metabolism and constant differentiation of cancer cells, the physiological difference from normal cells is not large. This is a huge challenge for developing drugs that selectively clear cancer cells without killing normal cells. Another major challenge in the development of anticancer drugs is cancer cell resistance, which is the drug resistance caused by a period of chemotherapy. The used chemotherapy drugs, even if the dose is increased, will no longer play a role in cancer cells. Metastasis of tumor cells often also prevents treatment with chemotherapy. So far, no anticancer drug has been able to cure all cancers. Finding new, highly effective, highly selective, low-toxic, non-resistant, and urgently needed new anticancer drugs remains challenging. Most chemotherapy and anticancer drugs have serious side effects, which can lead to the inability of chemotherapy to continue. Therefore, existing drugs are greatly limited in the treatment of different types of tumors. Therefore, finding new and effective anticancer drugs with high efficiency and low toxicity is still urgently needed to maintain human health.

Cytarabine is an analog of cytidine nucleoside, an inhibitor of DNA polymerase. It can block DNA synthesis, can also be incorporated into DNA, interfere with DNA replication, and can also block the reduction of cytosine nucleotides into Deoxycytosine (Sylvester, RK, Fisher, AJ, and Lobell, Μ·, Drug Intelligence & Clinical Pharmacy: Vol. 21, No. 2, pp. 177-180 (1987); Boyer et al., Novel Cytarabine Monophospate Prodrugs, United States Patent Application Publication, Pub. No. : US 2007/0037774 A 1, (Feb. 15, 2007); Colon-Cesario, M" Wang, J" Ramos, X., Garcia, HG, Davila , JJ, Laguna, J., Rosado, C, and Pena de Ortiz, SJ Neurosci., 26(20): 5524 -5533 (2006)).

Currently, cytarabine is mainly used for the treatment of acute leukemia. It is the best for acute myeloid leukemia. It is also effective for acute monocytic leukemia and acute lymphoblastic leukemia. It has certain curative effect on malignant lymphoma, lung cancer, digestive tract cancer, head and neck cancer. It has viral keratitis and epidemic. Conjunctivitis and the like also have a certain effect, however, it is ineffective for most solid tumors. The activity of cytarabine is not very high. In order to improve the efficacy, cytarabine is generally associated with other drugs, such as: methoxy daunorubicin, all-trans retinoic acid combined with arsenic trioxide, pirarubicin, topotecan - Etoposide-cyclophosphamide, fludarabine, etc. are used in combination. Cytarabine has side effects such as myelosuppression and digestive tract reaction. A few patients may have side effects such as abnormal liver function, fever, and rash (Bolwell, BJ, Cassileth, PA, Gale, RP Leukemia. 2(5): 253-60 ( 1988); Kimby, E., Nygren, P., Glimelius, B. Acta Oncol. 40(2-3): 231-22 (2001); Stamatopoulos, K. Leukemia Research, Volume 22, Issue 8 , pp 759 - 761, (2003); Burnett, AK, Milligan, D., Prentice, AG, Goldstone, AH, McMullin, MF, Hills, RK, Wheatley, K. Cancer. 109(6): 1007-10 (2007)).

Cytarabine is an antimetabolite, which is phosphorylated by deoxycytidine in cells, converted to active cytarabine, and further converted to the corresponding diphosphate and cytarabine. effect. Cytarabine inhibits DNA polymerase by interfering with the deoxycytidine triphosphate required for DNA synthesis, interfering with nucleotide incorporation into DNA, and inhibiting nucleotide reductase, preventing nucleotides from being converted into Deoxynucleotide, but has no significant effect on the synthesis of RNA and protein. It belongs to the cell cycle-specific drug acting on S phase, and is most sensitive to the role of cells in S proliferative phase, and has a G1/S and S/G2 transition period. Also has a role. Immediately after intravenous injection, it disappears from the blood, 40% can pass the blood-brain barrier, and the drug is mainly metabolized in the liver to inactive glucoside, 70% ~ 90% through the kidney. In order to develop new anticancer drugs that are effective against solid tumors such as liver cancer, new drugs for liver targeting must be sought. Obviously, Anti-hepatitis drugs can be an excellent reference. For example, miftine and adefovir (PMEA) have been approved as anti-viral therapeutics for hepatitis B (Starrett, et al., "Synthesis, oral bioavailability determination, and in vitro evaluation of prodrugs of the antiviral agent 9 - [2 - (pho sphonomethoxy)ethy 1] adenine (PMEA)," J Med Chem., 37(12): 1857-64 (1994); Shaw, et al""Pharmacokinextics and Metabolism of Selected Prodrugs of PMEA in Rats," Drug Metabolism Dis., 25(3): 362-366 (1997); Wacher, VJ, et al., Advanced Drug Delivery Reviews 46:89-102 (2001); Wacher, et al., "Active Secretion And Enterocytic Drug Metabolism Barriers to Drug Absorption," Adv. Drug Del. Rev., 46:89-102 (2001); Murono, et al., "Prevention and inhibition of nasopharyngeal carcinoma growth by antiviral phosphonated nucleoside analogs," Cancer Res , 61(21): 7875-7 (2001). In 2003, scientists from Metabasis Therapeutics, Inc. K. Raja Reddy, Mark D. Erion, Michael C. Matelich, Joseph J. Kopcho proposed the use of cyclic nucleoside nucleosides. As an anti-liver cancer treatment drug Prodrug (United States Patent 7,214,668;), when the compound enters the liver, is catalyzed by the CYP 3A4 metabolite of the liver to catalyze dissociation into acyclic nucleoside derivatives to have anticancer activity. 2007, Metabasis Therapeutics, Inc. Novel Cytarabine Monophospate Prodrugs, United States Patent Application Publication, Pub. No. : US 2007/0037774 A 1, Boyer et al., Feb . 15, 2007; Phosphonic acid based prodrugs of PMEA and its analogues, United States Patent 7,214,668, Reddy, et al. May 8, 2007). The core of these patent applications is to attach the ring to the -OCH2-ribose position on the ribose ring of cytarabine, the O5 position, and the amino group on the cytosine cytosine ring is not modified.

Cytarabine is generally not used to treat liver cancer because its N4 amino group (see Figure 1) is metabolically deactivated and causes toxicity when its nucleoside skeleton structure enters the liver; on the other hand, its glycocalyx structure The O5 hydroxyl group (see Figure 1) must be activated by phosphorylation, and this activation process is too slow in the liver. Summary of the invention

The present invention aims to overcome the deficiencies of the above prior art and provide an efficient, low toxicity, non-resistance, a cytarabine prodrug derivative capable of being rapidly activated, and a synthetic route of a cytarabine prodrug derivative and a preparation method of a cytarabine prodrug derivative preparation, and the present invention also provides an arsenic cell Experimental data on the use of glycoside prodrug derivatives and their formulations in anticancer and antitumor applications.

The cytarabine prodrug derivative of the present invention, characterized in that the cytarabine prodrug derivative has any one of the following general formulae (I), (II) and (III) compound of:

Wherein W represents any one of C=O, S(O)O and C(O)O;

Wherein Y represents (CH ₂ ) n or H ₂ C-0-CH ₂ , wherein n=2~6;

X represents any one of OH, O-P OXOR^ and phosphorus Stt, and the monument Stt is any one of a monophosphoric acid group, a diphosphate group and a triphosphate group;

1. R ² is a C _1S saturated or unsaturated aliphatic group containing one or more unsaturated groups, the unsaturated group comprising a cis or trans isomer; A represents O or S or CH ₂ or no group;

P = l-5;

R ³ is an amino group, an alkyl-substituted amino group, an aryl-substituted amino group, a heterocyclic-substituted amino group, NHOH, NHOR ⁴ and any of the following groups:

Wherein R ⁴ is a halogen atom, an amino group, a nitro group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkylthio group, a carboxyl group, a hydroxyl group, a cyano group, a trifluoromethyl group, a benzyl group, a phenyl group, an aromatic group, Any one of an acyl group, a carbonyl group, a substituted amino group, a yellow acid group, an amide group, a yellow amide group, an amino acid, a carbocyclic group and a heterocyclic group.

Wherein R ¹ may be any one of H, Cw ₈ alkyl, C ₁₈ cycloalkyl, benzyl, phenyl and aromatic ring; R ² AH, dC ₁₈ alkyl, C _3-18 cycloalkyl Any one of C _2-18 alkenyl, C _2-18 alkynyl, cycloalkenyl, benzyl, phenyl, aryl, cyclic amino, carbocyclyl, heterocyclyl, anilino and substituted anilino . Each of these groups may be further substituted, and may contain a hetero atom therein.

The cytarabine prodrug derivative of the formula (I) of the present invention includes a representative formula having the following structural formula

The cytarabine prodrug derivative of the formula (III) of the present invention includes a representative compound having the following structural formula:

For the sake of clarity, but not limitation of the invention, all technical terms used in the present invention are the same as those commonly used and understood by those skilled in the art of the invention. The patent applications or published applications and other papers cited in the present invention are the original references and are not modified.

As used herein, "a" or "an" or "a" or "an"

As used herein, "alkyl" means various saturated straight-chain, side-chain or cyclic hydrocarbon groups, particularly including small alkyl groups containing ten or ten carbons or less. For example, mercapto, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, isohexyl, heptyl, octyl and decyl groups, etc. Some typical examples in this definition.

The "base group" used in the present invention has the same definition as the above "alkyl group", but it must have at least one carbon-carbon double bond (C=C), so the alkenyl group used in the present invention includes two to ten carbon atoms. a linear, branched or cyclic hydrocarbon group having at least one carbon-carbon double bond, such as a vinyl group, a propenyl group, a butenyl group, a pentenyl group, and the like.

The "block group" used in the present invention is the above alkyl group or a sulfhydryl group and contains at least one carbon-carbon triple bond. Therefore, the alkynyl group includes a linear chain having two to ten carbon atoms and containing at least one carbon-carbon triple bond. A branched or cyclic hydrocarbon or alkynyl group such as ethynyl, propynyl, butynyl and pentynyl.

"Saturated" in the present invention means that the group does not contain an unsaturated bond, such as a carbon-carbon double bond or a carbon-carbon triple bond; and "unsaturated" means that the group contains one or more carbon-carbon doubles. Key or carbon-carbon triple bond.

The "cycloalkyl group" used in the present invention is a cyclic hydrocarbon group and preferably a cycloalkyl group having three to eight carbons. Thus cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane and cyclooctane are all under this definition typical example. A cycloalkyl group contains one or two carbon-carbon double bonds to form a "cycloalkenyl group". The cycloalkyl group may also carry an alkyl group, an alkenyl group, an alkynyl group, and other groups.

The "aromatic group" used in the present invention is a cyclic conjugated aromatic system and may contain one or more non-carbon atoms (other than carbon other nitrogen such as nitrogen) such as phenyl, naphthyl and pyridine in the ring. Base.

The "heterocyclic group" which is also commonly used in the present invention means a cyclic group and a compound in which any plurality of atoms are constituted by a covalent bond, and which contains at least one non-carbon atom. Specifically, a heterocyclic group includes a five- and six-membered ring system containing a nitrogen, sulfur or oxygen non-carbon atom such as pyrazole, pyrrole, pyridine or pyrimidine.

The "alkoxy group" in the present invention means an alkyloxy group formed by linking an oxygen atom to a linear or branched alkyl group. Examples of such alkoxy groups include a decyloxy group, an ethoxy group, a propoxy group or an isopropoxy group and the like.

Similarly, "alkylthio" refers to an alkylthio group formed by linking a sulfur atom to a straight or branched alkyl group. Examples of such an alkylthio group include a methylthio group, an ethylthio group, a propylthio group or an isopropylthio group.

The "halogen atom group" in the present invention is fluorine, chlorine, bromine or iodine.

The "amino acid" in the present invention means a substituted natural and non-natural amino acid, a pure L- or D-configuration or a racemic mixture, and a group derived from an amino group.

It is particularly worthy to further clarify that the various substituents defined above also include groups which are further substituted, wherein these new substituents may also contain other groups. For example, a ruthenium atom on an alkyl or aromatic group is substituted by an amino group, a halogen or other group to become a new group belonging to each of the above definitions.

The "acid riding" or "calate" used in the present invention is that the highest oxidation state of the pentavalent phosphorus atom is bonded to four oxygen atoms, one oxygen atom is connected to the phosphorus atom by a double bond, and the two oxygen atoms are single bonds. It is connected to a phosphorus atom, and these two oxygen atoms may be a hydrogen atom, a negative charge or various alkyl groups, aromatic groups and the like as defined above, such as -P(=O)(O-) ₂ , -P( O) (OR) ₂ . Another oxygen atom on the phosphorus atom is attached to the derivative of the present invention.

The "prodrug" in the present invention means a compound which cleaves or increases the biological action formed by a certain structural unit in the body after the cytarabine prodrug derivative of the present invention is used in the body.

The organic solvents mentioned in the present invention include: benzotriazole hexafluorophosphate-1 benzyl-oxytripyrrole Phosphorus (PyBOP), 4-dimethylaminopyridine (DMAP), hydrazine, hydrazine-dimercaptocarboxamide (DMF), dimethyl sulfoxide (DMSO), polyethylene glycol (PEG) and tetrahydrofuran (THF) ).

Another technical problem solved by the present invention is to provide a simple and effective synthetic route for a plurality of cytarabine prodrug derivatives of the formula (I), (II) or (III), the technical disclosures of which are as follows :

1. A synthetic route for cytarabine prodrug derivatives, characterized by:

(1) The acid anhydride compound is mixed with a fatty alcohol, heated under reflux or stirred at room temperature for 3-5 hours, and the first intermediate product obtained by cooling (B) is directly used in the next reaction without further purification;

(2) Acytosolic, first intermediate (B), PyBOP and DMAP are dissolved in DMF, stirred at room temperature ~60 ° C for 7 to 24 hours, and the reaction solution is purified by column chromatography and ridge ridge to obtain the general formula. (I) The arabinose 3 glucosides organism.

The acid anhydride compound is any one of succinic anhydride, glutaric anhydride, adipic anhydride, and diglycolic anhydride; the fatty alcohol is methanol, ethanol, n-octanol, lauryl alcohol, n-nonanol, and tetradecyl Any of alcohol, n-hexadecanol and stearyl alcohol. (Refer to synthetic route 1~5, 8, 9, 11, 12, 16-23)

2. A synthetic route for cytarabine prodrug derivatives characterized by:

(1) 2,6-dimercaptoaminobenzene or cycloalkylbenzimidazole, an acid anhydride compound and DMAP are dissolved in tetrahydrofuran, and the reaction is carried out at a temperature of 35 to 50 ° C for 3 to 5 hours to precipitate a solid, and the second intermediate product is obtained. (C) used directly in the next reaction without purification;

(2) Dissolving cytarabine, second intermediate product (C), PyBOP and DMAP in DMF, stirring at room temperature for 12-24 hours, pouring the reaction solution into water, precipitating the solid or purifying the reaction solution by column chromatography. A cytarabine prodrug derivative of the formula (II) is obtained.

The acid anhydride compound is any one of succinic anhydride, glutaric anhydride, adipic anhydride, and diglycolic anhydride. (Refer to synthetic route 14, 15)

3. A synthetic route for cytarabine prodrug derivatives characterized by:

(1) The acid anhydride compound is mixed with a fatty alcohol, heated under reflux or stirred at room temperature, and reacted for 3 to 5 hours, and the first intermediate product (B) obtained by cooling is directly used in the next reaction without further purification;

(2) The first intermediate product (B), SOCl ₂ and 1-2 drops of DMF are dissolved in dichloromethane, heated to reflux After 3 to 4 hours, the solvent and excess SOCl ₂ were removed by rotary evaporation to give a third intermediate (D), which was used in the next step without further purification;

(3) cytarabine hydrochloride and the third intermediate product (D) are dissolved in DMF, stirred at room temperature for 2 to 4 days, and the oil obtained by distilling off DMF under reduced pressure is stirred and solidified with diethyl ether to obtain a semi-solid product. The NaHCO ₃ aqueous solution is neutralized, filtered, and the obtained solid is washed with water until neutral, and then recrystallized from ethyl acetate, filtered, or the obtained solid is dissolved in methanol, and the insoluble matter is removed by filtration, and then purified by column chromatography to obtain the formula (III). A cytarabine derivative.

The acid anhydride compound is any one of succinic anhydride, glutaric acid, adipic anhydride, and diethylene glycol; the fatty alcohol is decyl alcohol, ethanol, n-octanol, n-nonanol, lauryl alcohol, Any one of tetradecanol, n-hexadecanol and stearyl alcohol. (Refer to synthetic route 6, 7, 10, 13)

A method for preparing a cytarabine prodrug derivative preparation, characterized in that:

(1) dissolving a cytarabine prodrug derivative having a structural formula of any one of the formula (I), (II) or (III) into water, physiological saline, a cyclodextrin aqueous solution, a water-soluble organic solvent a preparation solution prepared by combining a solvent of any one or more of a nonionic surfactant, a water-soluble lipid, a fatty acid, a fatty acid ester, and a phospholipid;

(2) The preparation solution is further diluted with physiological saline or glucose injection to prepare a cytarabine prodrug derivative preparation.

The organic solvent is any one of ethanol, propylene glycol, glycerin, glyceride, polyethylene glycol (PEG), hydrazine, fluorenyl-dimercapto amide (DMF) and dimercapto sulfoxide (DMSO) or A variety of combinations of solvents.

The cytarabine prodrug derivative preparation of the present invention is characterized in that it is a product prepared by the above-described preparation method of the cytarabine prodrug derivative.

Use of cytarabine prodrug derivatives for anti-cancer and anti-tumor. Such cancers include, but are not limited to, leukemia, solid tumors, lung cancer, colon cancer, liver cancer, central nervous system tumors, ovarian cancer, and kidney cancer.

The use of the cytarabine prodrug derivative preparation of the invention for anticancer and antitumor. The cancer includes, but is not limited to, cancer including leukemia, solid tumor, lung cancer, colon cancer, liver cancer, central nervous system tumor, Ovarian cancer and kidney cancer. The cytarabine prodrug derivative preparation may also be used in combination with other chemotherapeutic drugs in anti-cancer and anti-tumor, the other chemotherapeutic drugs including alkylating agents, plant alkaloids, antibacterial anti-tumor amides, platinum Drugs, anti-metabolites and other known anti-cancer drugs. In the combination process, at least one of the representative cytarabine prodrug derivatives having the above-listed enumeration is employed.

The pharmaceutical preparation is prepared by using the cytarabine prodrug derivative of the present invention as a component, and can be administered orally or parenterally. The parenteral administration referred to herein means subcutaneous, intravascular, intraarterial, ^! Within a few flesh, in the atria, in the synovium, intrathoracic injection or drip.

The present invention contemplates the design of cytarabine prodrugs using new design techniques. Cytarabine (see Figure 1) cannot be used to treat liver cancer. On the one hand, when its nucleoside skeleton structure enters the liver, its N4 amino group is metabolically deactivated and causes toxicity. On the other hand, its glycoside structure The O5 hydroxyl group on it must be activated by phosphorylation, and this activation process is too slow in the liver.

The cytarabine prodrug derivative of the present invention is designed by chemically modifying the position of N4, 05 to design a novel prodrug derivative, thereby avoiding metabolic failure of N4 amino group and causing toxicity; It is easily activated by phosphorylation. The main beneficial effects are: increased bioavailability, reduced multi-drug resistance (multi-targeted design techniques), increased solubility, and increased ester solubility. The invention provides a synthetic route of a cytarabine prodrug derivative, a cytarabine prodrug derivative preparation and a preparation method thereof, and proves the cytarabine prodrug derivative of the present invention through a large amount of experimental data. Use in anti-cancer and anti-tumor.

DRAWINGS

Figure 1 is a schematic diagram showing the structure of cytarabine and the position of modification of N4, 05.

Fig. 2 is a view showing the structure of a representative cytarabine prodrug derivative of the formula -I of the present invention.

Fig. 3 is a view showing the structure of a representative cytarabine prodrug derivative of the formula -II of the present invention.

Fig. 4 is a view showing the structure of a representative cytarabine prodrug derivative of the formula -III of the present invention. Fig. 5 is a graph showing the drug concentration-inhibition rate of the cytosine prodrug derivative of the present invention inhibiting the BEL-7402 liver cancer cell line. detailed description

The synthetic routes of some representative cytarabine prodrug derivatives of the present invention are listed below. Other similar cytarabine prodrug derivatives of the patents of the invention are synthesized by the same or similar methods.

1

Synthesis of the first intermediate product B1 (Scheme 1): Succinic anhydride (25 g, 250 mmol) was dissolved in methanol (50 ml), and heated under reflux for 3 hours. The obtained first intermediate B 1 was used in the next reaction without further purification.

Synthesis of cytarabine prodrug derivative 1 (Scheme 1): cytarabine (1.0 g, 4.1 mmol), first intermediate Β 1 (0.6 g, 4,5 mmol), PyBOP ( 2.35 g, 4.5 Methyl) and DMAP (0.055 g, 0.41 mmol) were dissolved in DMF (20 ml) and stirred at room temperature for 24 hours. The reaction solution was purified by column chromatography (silica gel, solvent: dichloromethane / decane = 9/1) to give cytidine prodrug derivative 1 ( 16.9 mg ) , LC (UV 254 nm ) purity > 95%. LC-MS m/z 358 [M + H] dec. (M. C ₁₄ H ₁₉ N ₃ 0 ₈ , molecular weight 357 ). Synthetic route 2:

Synthesis of the first intermediate product B 2 (Scheme 2): Glutaric anhydride (28.5 g, 250 mmol) was dissolved in ethanol (100 ml), and heated under reflux for 5 hours. The obtained first intermediate product B2 was used in the next step without further purification.

Synthesis of cytarabine prodrug derivative 2 (Scheme 2): cytarabine (1.0 g, 4.1 mmol), first intermediate Β2 (0.79 g, 4.9 mmol), PyBOP (2.35 g, 4.5 mmol), And DMAP (0.055 g, 0.41 mmol) was dissolved in DMF (10 ml) and stirred at room temperature for 12 hours. The reaction solution was purified by column chromatography (silica limb, developing solvent: dichloromethane/methanol = 9/1) to obtain cytarabine prodrug derivative 2 (46.9 mg), LC (UV 254 nm) purity. >95%. LC-MS m/z 386 [M + H] + (Molecular formula C ₁₆ H ₂₃ N ₃ 0 ₈ , molecular weight 385 ).

Synthesis of the first intermediate product B3 (Route 3): glutaric anhydride (11. 4 g, 100 mmol) dissolved in furfuryl alcohol

(50 ml), heated to reflux and allowed to react for 3 hours. The obtained first intermediate product B3 was used in the next reaction without further purification.

Synthesis of cytarabine prodrug derivative 3 (Scheme 3): cytarabine (1.0 g, 4.1 mmol), first intermediate B3 (0.72 g, 4.9 mmol), PyBOP (2.35 g, 4,5 mmol) And DMAP (0.055 g, 0.41 mmol) dissolved in DMF (10 ml) and stirred at room temperature for hr. Reaction chromatography through column chromatography Purification (silica gel, developing solvent: dichloromethane / methanol = 9/1) afforded cytidine prodrug derivative 3 (78.3 mg), LC (UV 254 nm) purity 91%. LC-MS m/z 372 [M + H] ⁺ (Molecular formula C ₁₅ H ₂₁ N ₃ O ₈ , molecular weight 371 ).

Synthesis of the first intermediate product B 4 (Scheme 4): Glutaric anhydride (3.4 g, 30 mmol) was dissolved in lauryl alcohol (10 ml, 45 mmol) at a controlled temperature of 30. C, reaction for 5 hours. The first intermediate product B4 obtained was used in the next step without further purification.

Synthesis of cytarabine prodrug derivative 4 (Scheme 4): cytarabine (1.0 g, 4.1 mmol), first intermediate Β 4 ( 1.36 g, 4.5 mmol), PyBOP (2.35 g, 4.5 mmol) And DMAP (0.055 g, 0.41 mmol) was dissolved in DMF (10 ml) and stirred at room temperature for 12 hours. The reaction solution was purified by column chromatography (silica gel, developing solvent: methylene chloride / decyl alcohol = 9/1) to obtain cytarabine prodrug derivative 4 (169.3 mg), LC (UV 254 nm) The purity is 98%. LC-MS m/z 564 [M + H] dec. (M.s. C ₂₆ H ₄₃ N ₃ 0 ₈ , molecular weight 525 ) ; 1H NMR (600 MHz, DMSO-d ₆ ) δ 10.84 (s, 1H) , 8.06 (d , 1H) , 7.20 ( d, 1H) , 6.05 ( d, 1H) , 5.49 ( d, 2H ) , 5.07 (t, 1H) , 4.06 (m, 1H) , 3.99 (t, 2H ) , 3.92 ( m, 1H) , 3.82 ( m, 1H) , 3.61 (m, 2H) , 2.43 (t, 2H ) , 2.32 ( t, 2H ) , 1.79 (m, 2H) , 1.55 (m, 2H) , 1.24 ( m, 18H ) , 0.85 (t, 3H ). Synthetic route 5:

Synthesis of the first intermediate product B 5 (Scheme 5): Succinic anhydride (3.0 g, 30 mmol) was dissolved in lauryl alcohol ( 8.37 g, 45 mmol), stirred at room temperature for 5 hours. The obtained first intermediate Β5 was used in the next reaction without further purification.

Synthesis of cytarabine prodrug derivative 5 (Scheme 5): cytarabine (1 g, 4.1 mmol), first intermediate Β5 (1.3 g, 4.5 mmol), PyBOP ( 2.35 g, 4.5 mmol) and DMAP (0.055 g, 0.41 mmol) was dissolved in DMF (10 mL). The reaction solution was purified by column chromatography (silica gel,ield: methylene chloride/methanol = 9/1) to afford cytidine prodrug derivative 5 ( 16.3 mg). The purity of LC (UV 254 nm) was 98%. LC-MS m/z 512 [M + H] ⁺ (Molecular formula C ₂₅ H ₄₁ N ₃ 0 ₈ , molecular weight 511 ).

Synthesis of the first intermediate B6 (Scheme 4): Glutaric anhydride (3.4 g, 30 mmol) was dissolved in lauryl alcohol (10 ml, 45 mmol) at a controlled temperature of 30. C, reaction for 5 hours. The first intermediate product B6 obtained was used in the next step without further purification.

Synthesis of the third intermediate D6 (Scheme 6): First intermediate B6 (4.0 g, 13.3 mmol), SOCl ₂ (3.17 g, 26.6 mmol) and 1 drop of DMF in dichloromethane (20 ml), heated Reflux for 3 hours. The solvent and excess SOCl ₂ were removed by rotary evaporation under reduced pressure to give a third intermediate product D6. The obtained product was used in the next reaction without further purification.

Synthesis of cytarabine prodrug derivative 6 (Scheme 6): cytarabine hydrochloride (0.7 g, 2.5 mmol) and the third intermediate D6 (1.04 g, 3.26 mmol) were dissolved in DMF (6 ml). Stir at room temperature for 2 days. DMF was distilled off under reduced pressure. The oil was stirred and solidified with 12.5 ml of ft for three times. The resulting semi-solid products with pro 5 ml, 1 mol / NaHC0 ₃ solution and the L. Filtration - the obtained solid was washed with water until neutral, recrystallized from 60 ml of ethyl acetate and filtered to give cytidine prodrug derivative 6 (89.4 mg) with LC (UV 254 nm) purity >95%. LC-MS m/z 564 [M + H] dec. (M. C ₂₆ H ₄₃ N ₃ O ₈ , molecular weight 525); 1HNMR (600 MHz, DMSO-^) 57.46 (d, 1H), 7.10 (brs, 1H) , 7.01 (brs, 1H) , 6.08 ( d, 1H) , 5.65 ( d, 1H) , 5.57 ( m, 2H ) , 4.29 (dd, 1H) , 4.19 ( dd, 1H ) , 3.98 ( m, 3H ) , 3.90 (m, 1H) , 3.88 ( s, 1H) , 2.38 (t, 2H) , 2.32 (t, 2H ) , 1.78 ( m, 2H ) , 1.55 ( m, 2H ) , 1.24 (m, 18H) , 0.85 (t, 3H).

Synthesis of the first intermediate product B 7 (Scheme 2): Glutaric anhydride (28.5 g, 250 mmol) was dissolved in ethanol (100 ml), and heated under reflux for 5 hours. The obtained first intermediate B7 was used in the next reaction without further purification.

Synthesis of the third intermediate D7 (Scheme 7): first intermediate B7 (6.0 g, 37.5 mmol), SOCl ₂ (8.9 g, 75 mmol), and 1 drop of DMF in dichloromethane (40 ml). Heat to reflux for 3 hours. The solvent and excess SOCl ₂ were removed by rotary evaporation to give a third intermediate D.

Synthesis of cytarabine prodrug derivative 7 (Scheme 7): cytarabine hydrochloride (1.4 g, 5 mmol) and third intermediate D7 (1.0 g, 5.6 mmol) dissolved in DMF (12 ml) Stir at room temperature for 2 days. DMF was distilled off. The oil was stirred and solidified with 25 ml of diethyl ether for three times. The obtained semisolid crude product was neutralized with 10 ml of a 1 mol/L aqueous NaHCO ₃ solution. The mixture was extracted three times with ethyl acetate. It was filtered, concentrated, and precipitated to precipitate crystals. Filtration gave cytarabine prodrug derivative 7 (25.3 mg) with LC (UV 254 nm) purity >95%. LC-MS m/z 386 [M + H] ⁺ (Molecular formula C ₁₆ H ₂₃ N ₃ 0 ₈ , molecular weight 385 ). Synthetic route 8:

Synthesis of the first intermediate product B8 (Scheme 8): Diethylene glycol anhydride (0.58 g, 5 mmol) was dissolved in ethanol (20 ml), and the temperature was controlled at 40 C for 5 hours. The first intermediate product B8 obtained was used in the next step without further purification.

Synthesis of cytarabine prodrug derivative 8 (Scheme 8): cytarabine (1 g, 4.1 mmol), first intermediate Β8 (0.81 g, 5.0 mmol) PyBOP ( 2.35 g, 4.5 mmol) and DMAP (0.055 g, 0.41 mmol) dissolved in DMF (10 ml). The reaction solution was purified by column chromatography (silica gel, solvent: methylene chloride / methanol = 15/1) to give cytidine prodrug derivative 8 (191.5 mg), LC (UV 254 nm) purity >90% LC-MS m/z 388 [M + H] ⁺ (Molecular formula C ₁₅ H ₂₁ N ₃ O ₉ , molecular weight 387 ) Synthetic Route 9:

Synthesis of the first intermediate product B9 (Scheme 9): Diethylene glycol anhydride (1.776 g, 11 mmol) was dissolved in lauryl alcohol (2.42 g, 13 mmol) at a controlled temperature of 40 C for 4 hours. The first intermediate product B9 obtained was used in the next step without further purification.

Synthesis of cytarabine prodrug derivative 9 (Scheme 9): cytarabine (1 g 4.1 mmol), first intermediate B 9 (1.51 g, 5.0 mmol), PyBOP (2.35 g, 4.5 mmol), And DMAP (0.055 g, 0.41 mmol) was dissolved in DMF (10 ml) and stirred at room temperature for 12 hours. Reaction solution through the column Purification by chromatography (silica gel, developing solvent: dichloromethane / decyl alcohol = 15/1) gave cytarabine prodrug derivative 9 (90.1 mg) with LC (UV 254 nm) purity >90%. LC-MS m/z 528 [M+H] + (M. C ₂₅ H ₄₁ N ₃ O ₉ , molecular weight 527). Synthetic route 10:

Synthesis of the first intermediate product BIO (Scheme 10): Diethylene glycol anhydride (1.776 g, ll mmol) was dissolved in lauryl alcohol (2.42 g, 13 mmol) and the temperature was controlled 40. C, reaction for 4 hours. The first intermediate product B10 obtained was used in the next step without further purification.

Synthesis of the third intermediate D10 (Scheme 10): First intermediate B10 (1.81 g, 6.0 mmol), SOCl ₂ (1.43 g, 12 mmol) and 1 drop of DMF dissolved in dichloromethane (20 ml), heated Reflux for 4 hours. The solvent and excess SOCl ₂ were removed by rotary evaporation to give the third intermediate D10, which was used directly in the next step without further purification.

Synthesis of cytarabine prodrug derivative 10 (Scheme 10): cytarabine hydrochloride (1.4 g, 5 mmol) and third intermediate D10 (1.92 g, 6.0 mmol) are dissolved in DMF (12 ml) Stir at room temperature for 2 days. DMF was distilled off. The oil was stirred and solidified with 25 ml of acetic acid for three times. The obtained semisolid crude product was neutralized with 10 ml of a 1 mol/L aqueous solution of NaHC0 ₃ . Filtration, the obtained solid was washed with EtOAc EtOAc (EtOAc) (EtOAc) Cytarabine prodrug derivative 10 (118.0 mg), LC (UV 254 nm) purity > 90%. LC-MS mz 528 [M + H] ⁺ (Molecular formula C ₂₅ H ₄₁ N ₃ O ₉ , molecular weight 527). Synthetic route 11:

11

Synthesis of the first intermediate product B11 (Scheme 11): Adipic acid ( 1.276 g, 11 mmol) was dissolved in methanol (2.42 g, 13 mmol) at a controlled temperature of 40. C, reaction for 4 hours. The first intermediate product B11 (monodecyl adipate) was used in the next step without further purification.

Synthesis of cytarabine prodrug derivative 11 (Scheme 11): cytarabine (1 g, 4.1 mmol), first intermediate Bl 1 (monodecyl adipate) (0.72 g, 4.1 mmol), PyBOP (2.35 g, 4.5 mmol) and DMAP (0.055 g, 0.41 mmol) were dissolved in DMF (10 ml) and stirred at room temperature for 12 hours. The reaction solution was purified by column chromatography (silica gel,ield: methylene chloride/methanol = 10/1) to give cytidine prodrug derivative 11 (58.6 mg), LC (UV 254 nm) purity >95% . LC-MS 々386 [M + H] ⁺ (Molecular formula C ₁₆ H ₂₃ N ₃ O ₈ , molecular weight 385 ).

12 Synthesis of the first intermediate product B12 (Scheme 12): Adipic anhydride ( 1.276 g, 11 mmol) was dissolved in ethanol (2.42 g, 13 mmol) at a temperature of 40. C, reaction for 4 hours. The obtained first intermediate product B12 (monoethyl adipate) was used in the next step without further purification.

Synthesis of cytarabine prodrug derivative 12 (Scheme 12): cytarabine (1 g, 4.1 mmol), first intermediate Β12 (monoethyl adipate) (0.8 g, 4.1 mmol), PyBOP (2.35 g, 4.5 mmol) and DMAP (0.055 g, 0.41 mmol) were dissolved in DMF (10 ml). Counter The solution was purified by column chromatography (silica gel, developing solvent: dichloromethane / decyl alcohol = 10/1) to obtain cytarabine prodrug derivative 12 (47.9 mg), LC (UV 254 nm) purity> 95%. LC-MS m/z 400 [M + H] ⁺ (M. C ₁₇ H ₂₅ N ₃ O ₈ , molecular weight 399 ).

Ol) was dissolved in methanol (2.42 g, 13 mmol) and the temperature was controlled at 40 °. C, reaction for 4 hours. The obtained first intermediate product B13 (monomethyl succinate) was used in the next step without further purification.

Synthesis of the third intermediate D13 (Scheme 13): first intermediate B13 (726 mg, 5.5 mmol), SOCl ₂ (1.31 g, 11 mmol) and 1 drop of DMF dissolved in dichloromethane (10 ml), heated Reflux for 3 hours. The solvent and excess SOCl ₂ were removed by rotary evaporation under reduced pressure to give a third intermediate product D13 which was used for the next step without further purification.

Synthesis of cytarabine prodrug derivative 13 (Scheme 13): cytarabine hydrochloride (1.4 g, 5 mmol) and third intermediate D13 (820 mg, 5.5 mmol) dissolved in DMF (10 ml) Stir at room temperature for 2 days. DMF was distilled off. The oil is mixed with a boat and solidified. The obtained semisolid crude product was neutralized with an aqueous NaHCO ₃ solution. Spin dry, solid dissolved in methanol, filtered to remove insolubles. Then purified by column chromatography (silica gel, developing solvent: dichloromethane / decyl alcohol = 4/1) to obtain cytarabine prodrug derivative 13 (128.4 mg), LC (UV 254 nm) purity > 90 %. LC-MS m/z 358 [M + H] ⁺ (Molecular formula C ₁₄ H ₁₉ N ₃ O ₈ , molecular weight 357). Synthetic route 14:

Synthesis of the second intermediate C14 (Scheme 14): 2,6-Dimethylaminobenzene (1.21 g, 10 mmol) in the above synthetic route was dissolved in 10 ml of THF, then glutaric anhydride (1.14 g, 10 mmol) was added. The mixture was reacted with DMAP (0.12 g, 1 mmol) at 50 ° C for 5 hours, and the filtrate was evaporated to dryness.

Synthesis of cytarabine prodrug derivative 14 (Scheme 14): cytarabine ( 2.43 g, 10 mmol), second intermediate C14 (2.35 g, 10 mmol), PyBOP (5.7 g, 11 mmol) It was dissolved in DMF (10 ml) with DMAP (0.12 g, 1 mmol) and stirred at room temperature for 24 hours. The reaction solution was purified by column chromatography (silica gel, solvent: methylene chloride / methanol = 10/1) to give cytidine prodrug derivative 14 (139.4 mg), LC (UV 254 nm) purity >95 %). LC-MS m/z 461 [M + H] + (Molecular formula C ₂₂ H ₂₈ N ₄ O ₇ , molecular weight 460).

Synthesis of second intermediate C15 (Scheme 15): cycloalkylbenzimidazole (0.53 g, 2.1 mmol), succinic anhydride (0.25 g, 2.5 ml) and DMAP (0.2 g, 1.6 mmol) in Route 15 above. ) Dissolved in tetrahydrofuran (20 ml) at a controlled temperature of 35. C, reaction for 3 hours. After pouring into water, a solid was precipitated, and the second intermediate C15 of the obtained solid was filtered and used directly to the next reaction without further purification. Synthesis of cytarabine prodrug derivative 15 (Scheme 15): cytarabine (0.39 g, 1.6 mmol), second intermediate C15 (0.47 g, 1.3 mmol), PyBOP (0.84 g, 1.5 mmol) and DMAP (0.055 g, 0.41 mmol) was dissolved in DMF (10 ml) and stirred at room temperature over the weekend. The reaction solution was poured into water to precipitate a solid to obtain a cytarabine prodrug derivative 15 (104.8 mg), and the purity of LC (UV 254 nm) was >99%. LC-MS w3⁄4 599 [M + Na]+ (M. C ₂₅ H ₂₉ ClN ₆ O _s , molecular weight 576). Synthetic road 16

Synthesis of the first intermediate product B16 (Scheme 16): n-nonanol (4 g, 26 mmol) was dissolved in 20 ml of tetrahydrofuran, then glutaric anhydride (2.5 g, 22 mmol) was added, and the reaction was stirred at 60 ° C for 4 hours. . The first intermediate product B16 obtained was used in the next step without further purification.

Synthesis of cytarabine prodrug derivative 16 (Scheme 16): cytarabine (1 g, 4.1 mmol), first intermediate B16 ( 1.36 g, 5 mmol), PyBOP ( 2.35 g, 4.5 mmol), And DMAP (0.055 g, 0.45 mmol) was dissolved in DMF (10 ml) and stirred at room temperature for 12 hours. The reaction solution was poured into water to precipitate a solid, and the solid was purified by column chromatography (silica gel, developing solvent: dichloromethane / decyl alcohol = 15/1) to obtain cytarabine prodrug derivative 16 (70.2 mg). ). LC (UV 254 nm) purity 97%. LC-MS m/z 495 [M + H] ⁺ (M.p. C ₂₄ H ₃₉ N ₃ O ₈ , molecular weight 497 ). Synthetic route 17

HO

Synthesis of the first intermediate product B17 (Scheme 17): glutaric anhydride (2.28 g, 20 mmol) was mixed with n-tetradecyl alcohol (4.28 g, 20 mmol), heated and melted, reacted for 4 hours, and cooled to give a white solid. The first intermediate B17 was used directly in the next reaction without further purification.

Synthesis of cytarabine prodrug derivative 17 (Scheme 17): cytarabine (1 g, 4.1 mmol), first intermediate B17 ( 1.48 g, 4.5 mmol), PyBOP (2.35 g, 4.5 mmol), And DMAP (0.055 g, 0.45 mmol) was dissolved in DMF (10 ml) and stirred at 40 °C for 12 hours. The reaction solution was poured into water to precipitate a solid, and the solid was purified by column chromatography (silica gel, developing solvent: dichloromethane / methanol = 15/1) to obtain cytarabine prodrug derivative 17 (31.6 mg). . LC (UV 254 nm) purity 95%. LC-MS m/z 554 [M + H] ⁺ (Molecular formula C ₂₈ H ₄₇ N ₃ O _s , molecular weight 553 ) „ Synthesis Route 18:

Synthesis of the first intermediate product B18 (Scheme 18): Glutaric anhydride (0.51 g, 4.5 mmol) was mixed with n-hexadecanol (1.2 g, 4.9 mmol), heated and melted, reacted for 4 hours, and cooled to give a white solid. of The first intermediate B 18 was used directly in the next reaction without further purification.

Synthesis of cytarabine prodrug derivative 18 (Scheme 18): cytarabine (1 g, 4.1 mmol), first intermediate B18 (1.6 g, 4.5 mmol), PyBOP ( 2.35 g, 4.5 mmol), It was dissolved in DMF (10 ml) with DMAP (0.055 g, 0.45 mmol) and stirred at 60 ° C for 12 hours. The reaction solution is poured into water to precipitate a solid, and the solid is purified by column chromatography (silica gel, developing solvent: dichlorodecane/decyl alcohol = 15/1) to obtain cytarabine prodrug 4 staining organism 18 (166.7) Mg). The purity of LC (UV 254 nm) was 97%. LC-MS m/z 564 [M+Na] ⁺ (M. C ₃₀ H ₅₁ N ₃ O ₈ , molecular weight 581).

Synthesis of the first intermediate product B19 (Scheme 19): glutaric anhydride (2.24 g, 19.6 mmol) was mixed with n-octadecyl alcohol (5.4 g, 20 mmol), heated and melted, reacted for 4 hours, and cooled to give a white solid. The first intermediate B 19 was used directly in the next reaction without further purification.

Synthesis of cytarabine prodrug derivative 19 (Scheme 19): cytarabine (1 g, 4.1 mmol), first intermediate B19 (1.73 g, 4.9 mmol), PyBOP ( 2.35 g, 4.5 mmol), And DMAP (0.055 g, 0.45 mmol) was dissolved in DMF (10 ml) and stirred at 50 ° C for 12 hours. The reaction solution was poured into water to precipitate a solid, and the solid was purified by column chromatography (silica gel, developing solvent: dichloromethane/decyl alcohol = 15/1) to obtain cytarabine prodrug derivative 19 (149.6 mg). ). LC (UV 254 nm) purity 99%. LC-MS m/z 632 [M+Na (M.p. C ₃₂ H ₅₅ N ₃ O ₈ , molecular weight 609 ). Synthetic route 20:

Synthesis of the first intermediate product B20 (Scheme 20): n-nonanol (0.77 g, 4.9 mmol) was dissolved in 20 ml of tetrahydrofuran, then diglycolic anhydride (0.52 g, 4.5 mmol) was added, and the reaction was stirred under reflux for 4 hours. The first intermediate product B20 obtained was used in the next step without further purification.

Synthesis of cytarabine prodrug derivative 20 (Scheme 20): cytarabine (lg, 4.1 mmol), first intermediate B20 (1.23 g, 4.5 mmol), PyBOP ( 2.35 g, 4.5 mmol), and DMAP (0.055 g, 0.45 mmol) was dissolved in DMF (10 ml) and stirred at 40 °C for 12 h. The reaction solution was poured into water to precipitate a solid, and the solid was purified by column chromatography (silica gel, solvent: methylene chloride / decyl alcohol = 15/1) to obtain cytarabine prodrug derivative 20 (31.3 mg). LC (UV 254 nm) purity 97%. LC-MS m/z 500 [M + H] ⁺ (M. C ₂₃ H ₃₇ N ₃ O ₉ , molecular weight 499 ). Synthetic route 21:

_A O

Synthesis of the first intermediate product B21 (Scheme 21): Diethylene glycol anhydride (0.52 g, 4.5 mmol) was mixed with tetradecyl alcohol (1.05 g, 4.9 mmol), heated and thawed, reacted for 4 hours, and cooled to give a white solid. The first intermediate B 21 was used directly in the next reaction without further purification.

Synthesis of cytarabine prodrug derivative 21 (Scheme 21): cytarabine (1 g, 4.1 mmol), first Intermediate B21 ( 1.48 g, 4.5 mmol), PyBOP ( 2.35 g, 4.5 mmol), and DMAP (0.055 g, 0.45 mmol) were dissolved in DMF (10 ml) and stirred at 50 ° C for 12 hours. The reaction solution was poured into water to precipitate a solid, and the solid was purified by column chromatography (silica gel, solvent: methylene chloride / decyl alcohol = 15/1) to obtain cytarabine prodrug derivative 21 (101.2 mg). LC (UV 254 nm) purity 95%. LC-MS m/z 556 [M + H] ⁺ (M. C ₂₇ H ₄₅ N ₃ O ₉ , molecular weight 555 ).

Synthesis of the first intermediate product B22 (Scheme 22): Diethylene glycol anhydride (0.52 g, 4.5 mmol) was mixed with cetyl alcohol (1.19 g, 4.9 mmol), heated and melted, reacted for 4 hours, and cooled to give a white solid. The first intermediate B22 was used directly in the next reaction without further purification.

Synthesis of cytarabine prodrug derivative 22 (Scheme 22): cytarabine (lg, 4.1 mmol), first intermediate B22 (1.6 g, 4.5 mmol), PyBOP ( 2.35 g, 4.5 mmol), and DMAP (0.055 g, 0.45 mmol) was dissolved in DMF (10 ml) and stirred at 60 ° C for 12 h. The reaction solution was poured into water to precipitate a solid, and the solid was purified by column chromatography (silica gel, developing solvent: dichloromethane / decyl alcohol = 15/1) to obtain cytarabine prodrug 4 contaminated organism 22 (62.9 mg) ). LC (UV 254 nm) purity 95%. LC-MS m/z 564 [M + Na] ⁺ (M. C ₂₉ H ₄₉ N ₃ 0 ₉ , molecular weight 583). Synthetic route 23:

Synthesis of the first intermediate product B23 (Scheme 23): Diethylene glycol anhydride (0.52 g, 4.5 mmol) was mixed with octadecyl alcohol (1.33 g, 4.9 mmol), heated and melted, reacted for 4 hours, and cooled to give a white solid. The first intermediate B 23 was used directly in the next reaction without further purification.

Synthesis of cytarabine prodrug derivative 23 (Scheme 23): cytarabine (lg, 4.1 mmol), first intermediate B23 (1.74 g, 4.5 mmol), PyBOP ( 2.35 g 4.5 mmol), and DMAP (0.055 g, 0.45 mmol) was dissolved in DMF (10 ml) and stirred at room temperature for 12 h. The reaction solution was poured into water to precipitate a solid, and the solid was purified by column chromatography (silica gel, developing solvent: dichloromethane / decyl alcohol = 15/1) to obtain cytarabine prodrug 4 staining organism 23 (43.2 mg) . LC (UV 254 nm) purity 95% LC-MS m/z 612 [M + H] ⁺ (Molecular formula C ₃₁ H ₅₁ N ₃ O ₉ , molecular weight 611 ). Biological activity:

The cytarabine prodrug derivatives encompassed by the present invention have a function of inhibiting tumor cell proliferation and an antitumor effect. Tumor cell cancers include, but are not limited to, blood cancer, intestinal cancer, skin cancer, breast cancer, lung cancer, liver cancer, ovarian cancer, gastric cancer, uterine cancer, brain tumor, pigmentoma, prostate cancer, and the like. Synthetic drug tumor cytotoxicity

Inhibitory effect of cytarabine prodrug derivatives and preparations thereof on tumor cells of the present invention

Test material

1) Cell line:

HL-60 cell line, suspension growth, using RPMI 1640 containing 10% fetal bovine serum (Hyclone) The cell culture medium was cultured, and the initial cell concentration was maintained at about 3*10 ⁵ /ml, and passaged 1:3 once every three days. Passage (5* 10 ⁵ /ml) one day before the experiment, the cell concentration during the experiment is between 7.5~10*10 ⁵ /ml.

The BEL-7402 cell line and the HT-29 cell line were adherently grown and cultured in D-MEM cell culture medium of 10% fetal bovine serum (Hyclone). The initial cell concentration of the conventional culture was about 3*10 ⁵ /ml, 2 ~3 days 1: 3 pass once. On the day before the experiment, 1: 2 passage, the cell concentration during the experiment was between 5~10*10 ⁵ /ml.

2) Dissolution and dilution of the drug: According to the weight and molecular weight of the cytarabine prodrug derivative provided, first add DMSO 100~200 μΐ, then add NS, so that the concentration of the drug obtained after dilution is 5 mM (note the end of DMSO) The concentration does not exceed 10%).

3) D-MEM or RPMI 1640 Cell Culture Medium, Gibco

4) Fetal bovine serum, Hyclone

5) Fine digestive juice, 0.25 % Trypsin + 0.02 % EDTA

6) PBS phosphate buffer

7) MTT solution, MTT dry powder (Sigma), fully dissolved in PBS to prepare 5 mg/ml, 0.22 μπι microfiltration filter-membrane filtration, and stored at -20 °C

8) 10% acidification SDS, 0.01N HC1

9) Centrifuge tubes, straws, etc. (BD company), 96-well plate (Costar)

2. Steps:

1) Cell seeding: The cells were cultured 24 hours after passage and grew well. The cells were routinely harvested, and the cell concentration was adjusted to 2 x ^{10 5} /ml (adherent cells) ~ 3 x ^{10 5} /ml (suspended cells) with fresh medium. The adherent cells were inoculated at 100 μΐ/well, cultured in a 37 ° C, 5% CO ₂ incubator for 24 h, and the old culture solution was discarded, and fresh culture medium was added at 95 μM/well. Suspension cells were inoculated directly to 95 μΐ/well.

2) Drug treatment: There are 6 concentration gradients for each drug, 3 duplicate wells for each concentration, and 5 duplicate wells for the drug blank control group. Ara-C control was performed simultaneously for each test. The concentrations of HT-29 and BEL-7402 cells were 5, 2.5, 1.25, 0.625, 0.3125, 0.16 mM, 5 μl per well, and the final concentrations were 0.25, 0.125, 0.0625, 0.03125, 0.016, 0.008 mM, respectively. The group was added with 5 μΐ of normal saline; the concentration of HL-60 cells added to the drug was 5×l (T ³ , ^{^{^{2.5xl0- 3, 1.25xl0- 3, 0.625xl0- 3}}} , 0.3125x10- 3, 0.16x10- 3 mM, corresponding to a final concentration followed ^{^{2.5χ10 -4, 1.25χ10 -4, 6 ·}} 25χ10 -5, 3 · 125χ10 ^-5 , 1·6χ10 ^-5 , 8xlO" ⁶ mM, 5 μΐ saline was added to the control group.

3) Cell culture and testing: After adding the drug, 37. C, 5% C0 ₂ incubator for 72h, then add MTT 10 μl per well, continue to culture for 4 h, add 100 μΐ 10% SDS (containing 0.01 N HC1 ) per well, and use Bio-rad 680 after 24 h. The absorbance (A) of each well was measured by an ELISA reader with a detection wavelength of 570 nm and a reference wavelength of 630 nm.

4) Calculation: First, average the absorbance of each replicate well (remove the data from over-disparity), calculate the inhibition rate (IR) for each drug concentration of each cell, IR(%)=(1- A _n /A ₀ ) l00 %, A _n is the average absorbance of the experimental well, A. The mean absorbance of the drug blank control wells. Using the EXCEL software, plot the drug concentration effect curve and select a reasonable calculation method to calculate the drug concentration (IC ₅₀ ) for 50% cell survival.

Figure 5 is a graph showing the drug concentration-inhibition rate of cytosine prodrug derivative inhibiting BEL-7402 liver cancer cell line, wherein JF001, JF017, JF019, JF020 and JF033 are respectively according to the above synthetic routes 1, 17, 19, The cytarabine derivative obtained by synthesizing 20 and 33.

Table 1-2 lists representative cytarabine prodrug derivatives that inhibit the biological activity of different tumor cells. Among them, JF004, JF006, JF007, JF009, JF010, JF013, JF014 and JF021 are cytarabine derivatives synthesized according to the above synthetic routes 4, 6, 7, 9, 10, 13, 14 and 21, respectively.

0.06 18.62 19.46 20.01 inhibition rate of intestinal cancer is higher than cytarabine, other

JF010 is similar

Table 2. Tumor cell activity of representative cytarabine prodrug derivatives

Claims

Claim

A cytarabine prodrug derivative, characterized in that the cytarabine prodrug derivative has any one of the following general formulae (I), (II) and (III); Compound:

Formula -III wherein w represents any one of c=o, S(O)O and C(O)O;

Wherein Y represents (CH ₂ ) n or H ₂ CO-CH ₂ , wherein n=2~6;

X represents any one of OH, O-P OXOR^ and phosphorus §tt, and the phosphorus §tt is any one of a monophosphoric acid group, a diphosphate group and a triphosphate group;

R ¹ , R ² are _ _1S saturated or unsaturated aliphatic groups containing one or more unsaturated bonds, the unsaturated bonds including cis or trans isomers ; A represents O or S or CH ₂ or no group;

The cytarabine prodrug derivative according to claim 1, wherein R ¹ is H, C _1-18 alkyl, C _3-18 cycloalkyl, benzyl, phenyl and aromatic rings Any one of the groups; ² is 11, dC ₁₈ alkyl, C _3-18 cycloalkyl, C _2-18 alkenyl, C ₂ _-18 alkynyl, cycloalkenyl, benzyl, phenyl, aryl Any one of a cyclic amino group, a carbocyclic group, a heterocyclic group, an anilino group and a substituted anilino group.

The cytarabine prodrug derivative according to claim 1 or 2, wherein the cytarabine prodrug derivative is an arsenic cell when X = OH in the formula (I) Glycoside prodrug derivatives.

The cytarabine prodrug derivative according to claim 1 or 2, wherein the cytarabine prodrug derivative is an arsenic cell when X = OH in the formula (II) Glycoside prodrug derivatives.

The cytarabine prodrug derivative according to claim 1 or 2, wherein the cytarabine prodrug derivative is a cytarabine prodrug derivative of the formula (III).

The cytarabine prodrug derivative according to claim 3, wherein the cytarabine prodrug derivative of the formula (I) comprises a representative compound having the following structural formula:

The cytarabine prodrug derivative according to claim 4, wherein the formula (II) Arabin

8,

In: the cytarabine of the general formula (III):

9. A synthetic route for a cytarabine prodrug derivative characterized by:

(1) The acid anhydride compound is mixed with a fatty alcohol, heated under reflux or stirred at room temperature, and reacted for 3 to 5 hours, and the first intermediate product (B) obtained by cooling is directly used for the next reaction without further purification;

(2) Acytosolic, first intermediate (B), PyBOP and DMAP are dissolved in DMF, stirred at room temperature ~60 ° C for 7 to 24 hours, and the reaction solution is purified by column chromatography to obtain the general formula (I). A cytarabine derivative.

The synthetic route of a cytarabine prodrug derivative according to claim 9, wherein the acid anhydride compound is any one of succinic anhydride, glutaric anhydride, adipic anhydride, and diglycolic anhydride. The fatty alcohol is any one of methanol, ethanol, n-octanol, lauryl alcohol, n-decyl alcohol, n-tetradecyl alcohol, n-hexadecanol and stearyl alcohol.

11. A synthetic route for a cytarabine prodrug derivative characterized by:

(1) 2,6-diamidinoaminobenzene or cycloalkylbenzimidazole, an acid anhydride compound and DMAP are dissolved in tetrahydrofuran, and the reaction is carried out at a temperature of 35 to 50 ° C for 3 to 5 hours to precipitate a solid, and the second intermediate product is obtained. (C) used directly in the next reaction without purification;

(2) Dissolving cyanidin, second intermediate (C), PyBOP and DMAP in DMF, stirring at room temperature for 12-24 hours, pouring the reaction solution into water, precipitating the solid or purifying the reaction solution by column chromatography. A cytarabine prodrug derivative of the formula (Π) is obtained.

The synthetic route of the cytarabine prodrug derivative according to claim 11, wherein the acid anhydride compound is any one of succinic anhydride, glutaric anhydride, adipic anhydride, and diglycolic anhydride. Kind.

13. A synthetic route for a cytarabine prodrug derivative characterized by:

(2) The first intermediate product (B), SOCl ₂ and 1-2 drops of DMF are dissolved in dichloromethane, heated under reflux for 3 to 4 hours, and the solvent and excess SOCl ₂ are removed by rotary evaporation to obtain a third intermediate product (D). ), used directly in the next reaction without further purification;

(3) cytarabine hydrochloride and the third intermediate product (D) are dissolved in DMF, stirred at room temperature for 2 to 4 days, and the oil obtained by distilling off DMF under reduced pressure is stirred and solidified with diethyl ether to obtain a semi-solid product. The NaHCO ₃ aqueous solution is neutralized, filtered, and the obtained solid is washed with water until neutral, and then recrystallized from ethyl acetate, filtered, or the obtained solid is dissolved in methanol, and the insoluble matter is removed by filtration, and then purified by column chromatography to obtain the formula ( III) cytarabine derivative.

The synthetic route of the cytarabine prodrug derivative according to claim 13, wherein the acid anhydride compound is any one of succinic anhydride, glutaric anhydride, adipic anhydride, and diglycolic anhydride. The fatty alcohol is any one of decyl alcohol, ethanol, n-octanol, n-nonanol, lauryl alcohol, n-tetradecyl alcohol, n-hexadecanol and stearyl alcohol.

15. A method for preparing a cytarabine prodrug derivative preparation, characterized in that:

(1) A cytarabine having a structural formula of any one of the formula (I), (II) or (III) The drug derivative is dissolved in any one or more of water, physiological saline, aqueous cyclodextrin solution, water-soluble organic solvent, nonionic surfactant, water-soluble lipid, fatty acid, fatty acid ester, and phospholipid a combination of solvents to prepare a formulation solution;

A method of preparing a cytarabine prodrug derivative preparation according to claim 15, wherein: said organic solvent is ethanol, propylene glycol, glycerin, glycerin, polyethylene glycol, N, N- A combination solvent of any one or more of dimethyl phthalamide and dimethyl sulfoxide.

A cytarabine prodrug derivative preparation, which comprises the preparation of the preparation method of the cytarabine prodrug derivative preparation of claim 15.

18. Use of a cytarabine prodrug derivative of claim 1 for anti-cancer and anti-tumor.

The use of a cytarabine prodrug derivative according to claim 18 for anticancer and antitumor, characterized in that the cancer comprises leukemia, solid tumor, lung cancer, colon cancer, liver cancer, central nervous system tumor, ovarian cancer and Kidney cancer.

20. Use of a cytarabine prodrug derivative formulation of claim 17 for anti-cancer and anti-tumor.

The use of a cytarabine prodrug derivative preparation according to claim 20 for anticancer and antitumor, characterized in that the cancer comprises leukemia, solid tumor, lung cancer, colon cancer, liver cancer, central nervous system tumor, ovarian cancer And kidney cancer.

The use of the cytarabine prodrug derivative preparation according to claim 20 for anticancer and antitumor, characterized in that: the cytarabine prodrug derivative preparation is used in combination with other chemotherapeutic drugs in anticancer resistance In tumors, the other chemotherapeutic drugs include alkylating agents, plant alkaloids, antibacterial antitumor sulfonamides, platinum drugs, antimetabolites, and other known anticancer drugs.

The use of a cytarabine prodrug derivative preparation according to claim 22 for anticancer and antitumor, characterized in that, in said combination administration, at least one of claims 6, 7, and 8 is used. Representative cytarabine prodrug derivatives are listed.