Synthesis of 4H-chromene derivatives

Abstract

Substituted 4H-chromene derivatives are a new class of compounds that bind to Bcl-2 protein and induce apoptosis in tumor cells. The present invention is directed to an efficient synthetic method for the preparation of these compounds from salicylaldehyde derivatives and alkyl cyanoacetates under solid phase.

Description

[0001] This application claims priority under 35 U.S.C. §119 based upon U.S.

[0002] Provisional Patent Application No. 60/265,766 filed on Feb. 1, 2001.

[0003] 1. FIELD OF THE INVENTION

[0004] The present invention relates to the fields of organic chemistry and molecular biology and, more particularly, is directed to a method for synthesizing 4H-chromene derivatives.

[0005] 2. BACKGROUND OF THE INVENTION

[0006] Bcl-2 and a family of related proteins regulate apoptosis or programmed cell death and are implicated in a number of human diseases such as cancer.

[0007] Specifically, Bcl-2 can contribute to neoplastic cell expansion by preventing normal cell turnover caused by physiological cell death mechanisms. High levels of Bcl-2 gene expression are found in a wide variety of human cancers and can lead to tumor cell resistance to conventional chemotherapy and radiotherapy.

[0008] Synthetic peptides that bind to a functional surface pocket of Bcl-2 have in vitro activity for inducing apoptosis in cell-free systems and in HeLa cells.

[0009] Furthermore, Bcl-2 binding peptides containing a fatty acid as a cell permeable moiety can induce apoptosis in vitro and have in vivo activity in slowing human myeloid leukemia growth in severe combined immunodeficient mice. These studies suggest that peptides or other small molecules targeted to the Bcl-2 surface pocket could have important clinical applications.

[0010] The organic compound HA14-1 (FIG. 1), a 4H-chromene derivative, exhibits binding activity for the surface pocket of Bcl-2 protein (IC50 =9 μM) and induces apoptosis of tumor cells. The discovery of this Bcl-2 binding compound provides a promising lead compound for the development of potential anti-cancer agents and prompted the chemical synthesis of a series of HA14-1 analogs in order to study its structure-activity relationship and increase its potency.

[0011] While there are currently two methods for the preparation of 4H-chromene derivatives, both methods have limitations. The first method involves the cyclization of salicylaldehyde derivatives with alkyl cyanoacetates in the presence of ammonium acetate at 5-10° C., which produces analogs of HA14-1.

[0012] The reaction temperature (5-10° C.) is crucial for obtaining the desired products. If the temperature is just slightly raised to 15° C., the reaction will fail to give the desired product. In another procedure, aluminum oxide (Al2O3) is used as the catalyst instead of ammonium acetate. However, this procedure is further limited by low yields.

[0013] The present invention provides a procedure for the preparation of 4H-chromene derivatives that overcomes the limitations of the current methodology. In one embodiment of the present invention, molecular sieve, more particularly, molecular sieve 3Å, is used as the catalyst. This novel catalyst as disclosed in the present invention, allows the reaction to take place under milder conditions, about 15-300° C., and gives higher yields of 4H-chromene derivatives, about 86%.

DEFINITIONS

[0014] In the present invention, “4H-chromene derivatives”, “HA14-1 derivatives” and “HA14-1 analogs” are used interchangeably. They include molecules of the formula: 1

[0015] Within the scope of the present invention, but not being limited to, R. and R2 are hydrogen, CH3, CH2CH3, CH2CH=CH2, CH2Br, CF3, NH2, OH, OCH3, CN, NO2, Cl, Br, F, COOH or COOCH3; and, R3 is hydrogen, CH3, CH2CH3, CH2CH2CH3, CH2CH2CH2CH3, C(CH3)3, CH2Ph or CH2CH2OCH3.

[0016] “structure-activity relationship” as used herein, means the relationship between the structure of a peptide or a molecule and its ability to bind to the functional surface pocket of Bcl-2, thus inhibiting the biological activity of Bcl-2 and inducing apoptosis in cancer cells.

BRIEF DESCRIPTION OF THE FIGURES

[0017] FIG. 1 illustrates the chemical structure of HA14-1.

[0018] FIG. 2 illustrates the synthesis of 4H-chromene derivatives.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention provides a method for preparing 4H-chromene derivatives. In the new method as disclosed herein, a salicylaldehyde derivative, an alkyl cyanoacetate, and a suitable molecular sieve were combined to produce a 4H-chromene derivative, as shown in FIG. 2. In one embodiment of the present invention molecular sieve 3Å was used as the catalyst to produce the 4H-chromene derivatives. In yet another embodiment of the present invention, the process is carried out at a temperature of about 15-30° C., for about 8-20 hours.

[0020] In still another embodiment of the present invention, the process is carried out at room temperature for 14 hours.

Methods and Results

[0021] Synthesis of HA14-1

[0022] To a suspension of the 5-bromosalicylaldehyde (0.010 mol) in dry ethyl alcohol (30 ml) was added ethyl cyanoacetate (0.022 mol) and 3.0 grams of molecular sieve 3Å powder (Aldrich Chemical Company). The resulting mixture was stirred at room temperature overnight (14 h). Most of the 5-bromosalicylaldehyde disappeared within the first 2 hours as determined by thin layer chromatography (TLC). The molecular sieve was then filtered off and washed 3 times with tetrahydrofuran. The filtrates were combined and the solvent was removed under vacuum. The residue solidified when incubated at-24° for 2 hours. The desired product was obtained by crystallization in 85% ethanol as a single diastereoisomeric pair. The yield was 86%.

[0023] Catalytic activity of other catalysts

[0024] Other types of molecular sieves, such as molecular sieves 4Å and 5Å, as well as Al2O3, were also compared using this procedure. All solid catalysts tested catalyzed the reactions, with type-3Å giving the best yields. (Table 1). Table 1. Comparison of various solid catalysts

Yield of 3 Å, %
Molecular sieve 3 Å    86.1
Molecular sieve 4 Å    56.5
Molecular sieve 5 Å    50.2
Alumnium Oxide (Al2O3) 62.9

[0025] Preparation of other 4H-chromene derivatives

[0026] The new method of using molecular sieve 3Å, as disclosed herein, also was applied to the preparation of several 4H-chromene derivatives. The yields are shown in Table 2.

[0027] Table 2. Preparation of 4H-chromene derivatives using molecular sieve 3Å

	R1	R2	R3	Yield* %
3 Å	6-Br	H	—CH2CH3	86.1
(HA14-1)
3b	6-Br	H	—C(CH3)3	82.5
3c	6-Br	H	—CH2Ph	60.0
3d	6-Br	H	—CH2CH2OCH3	74.6
3e	6-Cl	H	—CH2CH3	70.7
3f	6-Cl	H	—C(CH3)3	74.8
3g	6-Cl	H	—CH2CH2OCH3	72.9
3h	6-NO2	H	—C(CH3)3	84.0
3i	H	8-CH2CH═CH2	—CH2CH3	78.1
3j	H	8-CH2CH═CH2	—CH2CH2CH2CH3	85.3
3k	5-Br	8-OCH3	—CH2CH3	45.7
3l	6-NO2	8-CH2Br	—CH2CH2CH2CH3	51.3
*Isolated yield, without optimization

Discussion

[0028] The present invention relates to a procedure for the synthesis of 4H-chromene derivatives. This method features mild reaction conditions, high 15 yields, and facile manipulation. This novel methodology presented herein has been used to synthesize a series of HA14-1 analogs for investigating the structure-activity relationship of the analogs. The present invention thus provides a novel method for the optimization of synthesizing lead compounds for use in the development of novel therapeutic agents that will induce apoptosis.

[0029] While this invention has been described with a reference to specific embodiments, it will obvious to those of ordinary skill in the art that variations in these methods and compositions may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the claims.

Claims

1. A method for preparing a 4H-chromene derivative comprising the steps a) combining a salicylaldehyde derivative, an alkyl cyanoacetate and a suitable molecular sieve to form said 4H-chromene derivative; and b) isolating said 4H-chromene derivative.

2. The method of claim 1, wherein said salicylaldehyde derivative has a formula of:

3. The method of claim 2, wherein said suitable molecular sieve is molecular sieve 3Å.

4. The method of claim 3, wherein the step a) of combining said salicylaldehyde derivative and said alkyl cyanoacetate is carried out under a suitable temperature for a sufficient period of time.

5. The method of claim 4, wherein said suitable temperature is about 15-30° C.

6. The method of claim 5, wherein said sufficient period of time is about 8-20 hours.

7. The method of claim 1, wherein said 4H-chromene derivative is HA14-1, said salicylaldehyde derivative is 5-bromosalicylaldehyde, and said alkyl cyanoacetate is ethyl cyanoacetate.

8. A method for preparing a 4H-chromene derivative of a formula: from a salicylaldehyde derivative of a formula:

9. The method of claim 8, wherein in said molecular sieve is molecular sieve 3Å.

10. The method of claim 9, wherein said first solvent is dry ethyl alcohol.

11. The method of claim 10, wherein said second solvent is tetrahydrofuran.

12. The method of claim 11, wherein the molar ratio of salicylaldehyde derivative : alkyl cyanoacetate is about 1:2.

13.The method of claim 12, wherein the reaction time of step d) is 14 hours.

14. The method of claim 8, wherein said 4H-chromene derivative is HA14-1, said salicylaldehyde derivative is 5-bromosalicylaidehyde, and said alkyl cyanoacetate is ethyl cyanoacetate.