TWI323657B - Catalyzed reaction for forming indole-based compounds and their application in anticancer agents - Google Patents

Description

1323657 IX. Description of the invention: • [Technical field to which the invention pertains] The present invention relates to a method for forming a compound having n structures by catalytic reaction, particularly relating to a catalytic formation of a ruthenium by Lewis acid. 'Methods of structural compounds and their use in anticancer drugs. [Prior Art] The cancer cells that have been identified are transformed by the mutation of multiple cancer-related genes (such as tumor suppressor genes, proto-oncogenes, etc.) in the body, and they are jumped at the molecular level. The regulation mechanism of death and growth, cancer cells are not easy to age, die and have the ability to proliferate rapidly. In past studies, it has been found that receptors and message molecules that stimulate growth at midnight, such as erb_B, her-2 and Ki-ras, C-myC, and anti-apoptotic molecules, such as Bc1_Xl, are found in cancer cells. There are a lot of performance or excessive activation, these molecules we call. Onc〇genes. On the other hand, some important regulatory molecules in the cell cycle, such as P53, pl6 and pRb, may be removed from the entire ** sequence of the gene bank or severely on the DMA sequence promoter. Thiolation may also result in mutations that cause these molecules to fail to perform their normal functions, thus making the cell cycle unmanageable, prompting the proliferation and proliferation of cancer cells. Therefore, we often call these molecules tumor suppressor genes. (tumor suppressor genes). Of course, the formation of cancer cells is not the only one, "the scorpion's scorpion molecule is out of regulation. Many known and unknown molecules can participate in it. It is also due to the complexity of cancer cells themselves. It adds to our difficulty in cancer treatment. Xianli's research suggests that the role of chemotherapy is to cause necrosis of tumor cells, but recent literature reports that many anticancer agents are causing cell physiology confusion. Planned death of cells (programmed ceU de_, the so-called planned death almost all tissue cells.. When cells age, damage, lose function, most cells will eliminate these useless cells through suicidal behavior, such death mode It is different from general necrosis, called apoptosis. It is because the cell death does not cause inflammation like necrosis, and the apoptotic cells are quickly phagocytosed by neighboring cells, so no Adjacent cell necrosis or immune system imbalance caused by cell death. It is first isolated in mammals. The apoptotic molecule is the bd 2 gene' when the overexpression of bcl-2gene inhibits 叩叩[0, some cancers rely on bcl-2 and related genes to prevent cell death. Prognosis of prostate cancer and colorectal cancer (prognosis) is also related to the performance of bcl_2. In addition, p53 gene has also been extensively studied. When DNA is damaged, P53 is expressed in a large amount, leaving the cells in the G1/S phase until the DNA repairs, and then enters the normal cycle. When DNA damage is too severe, P53 will cause cells to enter apoptosis. In many cancers, defects in p53 gene can be detected. The p53 gene is the most widely mutated gene in cancer cells in the literature. Derivatives are known for their biological activity, for example:

Page 7/44 page 1323657 Cell induction of apoptosis (apoptosis), organic chemists have long been continually striving to synthesize different kinds of compounds with a ruthenium structure, including one-degree σ 朵 朵, /3-β 卜 朵 石 肖 肖 化合物 化合物 化合物 化合物 化合物 化合物 化合物 化合物 化合物 化合物 化合物 化合物 化合物 化合物 化合物 化合物 化合物 。 。 。. Until now, only a handful of literature has reported studies related to the above compounds. Case 1 is that chemists such as Keer have observed that under the conditions of rolling, in addition to the main product, another secondary product with a yield of only 6% is also isolated. Product triterpene cyclohexane (Harrington, P.; Keer, MA

Can. J. Chem. 1998, 76, 1256.); Case 2 is that chemists such as Shi later reported that the same product can be obtained if a metal triflate is used as a catalyst. (Shi, μ.; Cui, s._c; u, Q-丄Tetrahedron 2004, 60, 6679). However, both of the above synthetic methods have their disadvantages, such as harsh reaction conditions [13 kbar high pressure], long reaction time (1-3 days), and the necessity to use expensive tri-gas methanesulfonic acid metal. The salt is a catalyst. In view of this, it is still necessary to develop low-cost, low-toxicity, non-contaminating and (four) capacitive (four) system to understand the structure of the compound and adopt a new catalytic strategy to improve the yield and quickly produce the desired product. At present, the industry has paid considerable attention to the development direction. SUMMARY OF THE INVENTION In order to meet the requirements of the industry, the present invention provides a novel method for catalyzing a reaction to form a compound having a structure and an application thereof for an anticancer drug. The purpose of the 8th/to 44 page 1323657 is to use Lewis acid as a catalyst to provide easy operation and enthalpy (four) process to form a compound having a lion. The catalyst used in the present invention has the advantages of high reaction efficiency and reaction. The latter mixture is easy to handle and the reaction can be carried out under mild or moderate conditions such as room temperature, which produces a medium to very reduced rate of mono-product. Accordingly, the present invention can meet economic benefits and industrial applicability. Another object of the present invention is to provide a pharmaceutical composition for treating cancer in a patient in need of cancer treatment, which comprises a compound having a three-and-one structure (3 also offering h(d), its enantiomer, diastereomer, medicine An acceptable salt or any combination thereof, for a cancer cell in which the tumor suppressor gene p53 is mutated. According to the above-mentioned purposes, the present invention discloses a method for forming a compound having a structure, which comprises a catalytic reaction, And the above-mentioned reaction is carried out by catalyzing the α, a point-unsaturated ketone to react with a derivative or a derivative thereof, or a catalyzed α, a-unsaturated aldehyde and an anthracene. The ruthenium or a derivative thereof is reacted. The above Lewis acid comprises one of the following groups: a metal halide, a halogen, an inorganic ammonium salt and an organic acid (salt). On the other hand, the present invention also discloses three The use of a compound of 3-indole based on an anticancer drug. [Embodiment] The direction of the invention in this invention is to form a catalytic reaction to form a 0-introduction frame. Compound of page In order to be able to fully understand the present invention, detailed process steps or constituent structures will be set forth in the following description. Apparently, the present invention is not limited to the specific details familiar to those skilled in the art. The well-known composition or process steps are not specifically described in the details to avoid causing the invention to be unnecessarily closed. The present invention will be described in detail as follows, but in addition to this fine bribe, this radiant feeding The method of the present invention is not limited, and the scope of the patent after the gamma is subject to the standard. The first embodiment of the present invention discloses a method for forming a compound of the structure of the (4) structure, which includes - a room temperature catalytic reaction where the room temperature is less than or equal to 3 ° C ' and the catalytic reaction described above is based on at least one Lewis acid to catalyze the alpha, point-unsaturated ketones and hydrazine or derivatives thereof The reaction is carried out. The above-mentioned Lewis acid comprises the following groups: metal halides, halogens, inorganic coatings and organic % acids (salts), wherein the metal halides described above comprise one of the following groups: gasification (InCl3), gasification brocade (CeCl3) and chlorinated sulphate (A1C13). Secondly, the above inorganic ammonium salt comprises cerium ammonium nitrate (CAN). Further, 'the above organic sulfonic acid (salt) The class contains one of the following groups: alkylsulfonic acid (salt) [eg: dodecylsulfonic acid (salt)], aryl. sulfonic acid (salt) [eg: quinone basic acid (4- Methylbenzenesulfonic acid)], aryl sulfonic acid (salt) [eg: dodecyl benzene sulfonic acid (salt)], divinyl benzene cross-linking acidification page 10 / total 44 page 1323657 polystyrene and perfluoro Cross-acid resin (Nafion). The above-mentioned divinylbenzene cross-linking acidified polystyrene-thipropone is an acidic ion exchange resin, which has been widely used in industrial catalysts. The products that are commercialized are Amberlyst>15, Amberlyst XN- 1005, Amberlyst XN-1010, Amberlyst XN-1011, Amberlyst XN-1008, Amberlite 200...etc. In the present embodiment, the above a, 々-Bab and ketones comprise one of the following groups:

Further, the above-mentioned cockroach or its derivative contains one of the following groups:

Wherein X is a halogen, and R4, R5 and R6 are independently selected from one of the group consisting of a hydrogen atom and a linear alkyl group (e.g., methyl, ethyl). A second embodiment of the present invention discloses a method for forming a compound having a ruthenium structure, which comprises a room temperature catalytic reaction, wherein the room temperature system is lower than or equal to 3 〇C, and the above catalytic reaction system Catalyzing α, a point-unsaturated aldehyde (a,/5-unsaturated aldehyde) with "anthraquinone or a derivative thereof by at least one Lewis acid, wherein the above Lewis acid, hydrazine or its derivative The selection of the object is the same as that of the first embodiment. Further, the above α,/5-unsaturated mix comprises one of the following groups: Page 11 of 44 r S ··. 1323657

Page 12 of 44 S 1 1323657 Table 1. Reaction data of a , cer cerjum ammonium nitrate (CAN) ' a, do not saturate steroid 1 and 吲哚 2a (Icqmv) 2 ( 4equiv) 3 (cqtfiv)_T?nie{h)_ 4a (%) 54 {%) 0.1 20 12 0.1 20 0.3 6 0.3 13 0,5 2 0.5 4 0.3 1 0.3 1/6 03 72 〇j 4 2823202028282-28282828 4aa {—) 5aa (— 4m (14) 5aa(S; 4 曲(一) 5δό{9: 4aa(8) 5as(9: 41⁄2 () Safi (« 4sa (a) 5oa (8; (99) Sbii 4κά (90) 5έϋ {*» 4ea(60) In f-Sea mXMR yields, — - To observe the catalytic effect of diammonium hexahydrate (CAN), use dimercaptopurine at room temperature (〇]\ /130) / water (volume ratio 5 / 1) as a solvent 'we found 1 equivalent of 2-cyclohexen-1-one la and 4 equivalents (eqUiv)吲哚2a was carried out under conditions of diammonium hexahydrate using different catalytic ruthenium (the results are as indicated by reaction i and the table). The reaction was first carried out without adding a catalyst, although the mixing system was stirred for 20 hours. There is no flaw in the result, 4_plus The product 4aa of the reaction was also produced without first performing the 1>4_addition reaction and then continuing the addition of the reaction product 5aa [Experiment i (entry).] In contrast, 0.1 equivalent of six was added to the reaction system. After the diammonium nitrate decoration (CAN), the result was significantly improved after 12 hours of reaction, and 14% of the 5aa product of the same was obtained (Experiment 2); it is pleasing that after the same reaction was carried out for 2 hours 93% of the single-product 5 pumping can be obtained in a step-by-step manner (Experiment 3). In order to obtain better results, we have also tried to increase the enthalpy of the diammonium hexahydrate hexadium hexahydrate (CAN) to 0.3 equivalents. As predicted, the yield of 4aa can be reduced to 8% after 6 hours, and then increased to 92% (Experiment 4). Fortunately, '99% of the single reaction can be obtained after 13 hours of the same reaction: product 5aa (Experiment 5). Although the addition of hexazachoic acid to the 〇5 § can significantly accelerate the reaction', but after 2 hours of reaction, the yield of the products 4aa and 5aa is reduced to only 11〇. /. and 77% (Experiment 6), and only 85% of 5aa was obtained after 4 hours of reaction (Experiment 7). A reasonable explanation may be because a slight excess (CAN), although there will be a clearer _catalytic side, makes the reaction too sturdy, affecting the stability of the reaction system and causing some of the starting materials or products in the reaction process. The medium is broken and the yield is lowered. According to the results of the results, we can observe that the hexa-Nitrate II (CAN) ritual has a highly efficient catalytic effect, and the catalytic effect is 0.3 eq. In addition to la, other starting materials such as lb-e are also tested in similar cases, and the experimental results are shown in Run 8-11. Based on the data in Table 1, we observed some interesting phenomena, especially regarding the differences between the reactants la and lb-e. For example, only the reactant la may be subjected to a 1,4-addition reaction to produce 4 aa, and then 4 aa may be further reacted with the called bud 2a to obtain 5 aa. However, the reactant lb-e can only undergo a 丄4_ addition reaction to produce a high yield of 4ba-4ea, but does not proceed with other reactions such as a 1,2-addition reaction. Regarding the fact that ia and ib produce different products respectively, the 14th page/total 44 pages of 1323657, we speculate that it is mainly because of the "torsion tension factor, (torsi〇nal strain effect) caused by this factor during the reaction to the product Or the intermediate product plays a very important role, for example: when the first equivalent of 2a is first subjected to a 1,4-addition reaction and added to or after ib, 3-indolylcydohexanone 4aa or 3-indolylcyclopentanone 4ba. When the 12-addition reaction is continued, the mixed orbital of the carbon atom of the carbonyl group in 4aa is converted from sp2 to sp3 orbital and produces a hexagonal product 5aa. As a result of this change, 5aa has a staggered or chair-like configuration that can significantly reduce the torsional tension within the compound and increase the stability of 5 aa; the opposite case if a five-ring is formed The product 5ba, however, exhibits an overlapping configuration that causes the compound to rise, which in turn makes the pentacyclic product 5ba less stable.

Example 2 CHO /CHO /

H3c 6a

Page 15 / a total of 44 pages 1323657 Table 2 · Yu 6; 6 cerium ammonium nitrate (CAN) catalyzed, α, no unsaturated aldehydes 6 and 吲哚 2a reaction data ^ Xty___ t 3 ( Egoiv)_Time (b ot mio)____(%)__81 (%) &€bers.«ic al It it I- n αα°1α°· 10 min lb 5 Ml? 15 min 8汹{TM») 8iw ( 29) 8ca(66) ^ (SO) 8ca(99) 7昶(99) m ($4) 7ca 02) 7da (20) 7ea (-) Page 16 of 44 ^ S ) 1 NMR yidds. Further Studies have shown that acids with similar structures to ketones [eg, a, cold-unsaturated aldehyde 6 (6) can also be reacted with 2a, and the experimental results are as indicated in Reaction 2 and Table 2. For example, 2-crotonaldehyde 6a and 2a can produce 99% of a single product 7aa after reacting at room temperature for 1 hour using 0.1 equivalent of diammonium hexahydrate (CAN) as a catalyst (experiment) 1, identified and analyzed by NMR), the mixture was not found to have the formation of bis(indolylmethane) 8aa (identified and analyzed by GCMS). However, when the starting material used 3-methyl-2-butyraldehyde (d-methylcrotonaldehyde) 6b, 64% of 7ba and 29% of 8ba (perspective 2) were produced. On the other hand, the reaction result of 6c or 6d is similar to that of 6a or 6b, which can produce 32% of 7ca and 66% of 8ca (experiment 3) and 20% of 7da and 80% of 8da (experiment 4), respectively. It is quite different that when starting with 6e, 99% of the single product can be obtained from 8 (Experiment 5) ° 1323657 According to the results of Table 1 and Table 2, we find that in different cases, α, The unsaturated ketone 1 may produce only a single product 4 or 5 and may also produce products 4 and 5 at the same time (may be abbreviated as product 4 or / and 5); in the same situation, the α /3-unsaturated aldehyde 6 may also produce only a single Product 7 or 8 or both products 7 and 8. The reaction mechanism for producing the product 7 and the reaction mechanism for the product 5 are similar. First, the reactant 6 is subjected to the 1,4·addition reaction, and then the 1>2_addition reaction is continued to produce the final product 7. However, the reaction mechanism of products 8 and 4 is not the same, wherein product 4 is produced via a 1,4-addition reaction route, and product 8 is produced via an ι,2-addition reaction. The above different experimental results may be due to the fact that the aldehyde and the ketone have different steric hindrances from each other, and since the steric hindrance of the aldehyde group is much smaller than that of the ketone group, the aldehyde is more susceptible to the 12-addition reaction than the ketone. The above assumptions can also be carried out by reacting with a mixture of 1 equivalent of diammonium hexahydrate (CAN) to catalyze the progress of the reaction, while ketones require at least 0.3 equivalents to catalyze a similar reaction. In addition, when there are different steric obstacles inside the molecular structure, the products 7 or / and . 8 can be obtained and the ratio between them is very different. For example, when using 6a, only 7 aa can be obtained. 1), while using 6b-d can produce 7 and 8 (entries 2-4) at the same time; but if you use 6e, you only get 8ea (Experiment 5) 〇 Example 3 Page / Total 44 Page 1323657 〇

N h la k4 2a: R4=H 2b: R4=CH3

Et2〇

4aa

Reaction 3

NH 4aa

h 9 reaction 4

Et20, rt

Table 3. Reaction data of a, point-unsaturated _ class 1 and |^2a/2b catalyzed by iodine

Entry t (I tqoiv)_2(4eqaiv)_h (egaiv)_^Tlme(h)

12545AV7S9W 18la3al83aJa,b,ew3e is 28·29'28^®28·28^28 0.15 IS 03 1 0,1 2 0.5 1 I Ϊ 5/12 <5.3 VI 03 m 0.3 72 W 5 4ask {28) 4aa 4aa (*««·) 4ab 4bM9Z) 4c» (91) Object (4) 4c« ip) 5sjtf{49> 细满$m(n) 5ίΐβ<99) (62) S»h(99) S>a {«&lt ;-) 5ca (««) 5da(^) Sp». atmR yields. We also tried to use iodine as a catalyst, and the reaction procedure was similar to that of the sampler. When 1 equivalent of α, stone-unsaturated ketone 1 and 4 equivalents of ruthenium 2a are reacted in a solution of 1 ml of diethyl ether (Ethyl ether; Et2〇) in a dish as a catalyst, it is possible to simultaneously produce product 4 And 5, or only produce a single page 18 / a total of 44 pages

The results or 5' results are shown in Reactions 3 and Table 3. When eight of the catalysts in 0.15 were used, the crusting produced 4 aa and 5 aa simultaneously (Experiment 1). When the star of 9 was raised to 0.3 or 0.5 equivalent and reacted for 2 hours or 1 hour, it was improved to obtain 93% or 99% of the single product 5aa (Experiments 3 and 4). However, when the amount of iodine was increased to 1 equivalent, only 62% of 5 aa was produced (Experiment 5). The above results show that the use of 〇·3 equivalents of iodine has been able to catalyze and iE reactions as well. Similarly, if a reaction is carried out using la-d and 2a or 2b, respectively, different kinds of products 4 can be obtained (Experiments 6-10). Compared with the data of Catalyst CAN in Table 1, most of the starting materials except Id can produce a medium to very high yield of product 4 under the conditions of iodine as a catalyst. For Id, only 49% of 4da is produced by iodine catalysis, and 62% of yield is obtained by CAN catalysis. The only difference is that CAN is a catalyst that requires dimethyl hydrazine (DMSO)/H2 hydrazine solvent and iodine. An ether solvent is required for the catalyst. The reaction mechanism for the products 4aa and 5aa is as follows. First, a 1,4-addition reaction is carried out to produce 4aa, and then 4aa is further subjected to a 1,2-addition reaction with 2a to produce 5aa. In order to prove the above-mentioned reaction mechanism, 1 equivalent of 4 aa and 3 equivalents of 2a and 3 equivalents of a mixture were mixed and reacted in 1 ml of diethyl ether, and after 1 hour, 87% of 5 aa was obtained (reaction 4) ). The above results may explain why the same reaction produces products 4 and 5 simultaneously in a shorter reaction time, but after a longer period of time, only product 19 is obtained on page 19 of 44 pages 1323657. Example 4 R3vCHO τ R^R2 6

2a: R4 = H 2b: R4 = CH, 9

R4 reaction 5 J 4. Reaction of residual and surface 6 and ah 2 under the catalysis of ruthenium (10)ine

Batry 1 2 No 4 You 5 ik 6 <ie 7 % 9 Kx 10 ϋ K* 12 ^-M<ocyi4, r^^h, r3^. h m ? H. 63⁄4 213⁄42123^3⁄43⁄423283⁄43⁄43⁄43⁄4

Izicqupf) HR 棘h4m ^b^tnd^jEsahane was afeto general B 99% <rf M^ind«l>l^ictlMiusc »ca m$ gfiflaafed. 1* I 4J .3> I n I at I 1· II ϋ ϋ ϋ ο ϋ 0 ΰ ϋ 0 ϋ ΰ o

Time 15 min ΗΙκβϊβ lOtiBn 3h 30 nan 3mk I0n»n iOnan Cover 0 spot ia iQmn f is cut 9, "^ 明^^9) 2)7, for eeee("("{2i<79i ee τ ΛΓ 7 ? 7 ? 7 7 ?· ? 7 ? · According to the results of Examples 1 to 3, we also tried to react the starting materials 6 and 2 and use the α ΐ _ _ _ side, the experimental results such as the reaction 5 and the table As shown in Figure 4. When the dish is a catalyst, the starting materials (6) and ❿ only produce the single-products 7aa_cb and 7fa_gb, and no other products such as 8 are produced (entries 1_6 and 9-12 in Table 4). The results in Table 4 found that 'the use of hexahydrate bismuth (CAN) or angstrom as catalyst will result in slightly different experimental results on page 20 of 44 1323657. The possible reason is because diammonium hexahydrate (CAN) is It is a strong Lewis acid, so it has a high activity; but iodine is a weak Lewis acid, so the activity is mild. Because the Lewis acidity of the two is poor, the effect on the reaction is not the same. For example In the same reaction, when using iodine as a catalyst, the starting materials 6a e and 6f_g are first subjected to hydrazine, 4 addition reaction, and then continue to proceed. 1>2•Addition reaction singularity-product I has a particularly pronounced effect on starting materials 6b and 6c (entries 3_6); but if starting materials 6b and 6c are sieved with diammonium dichloride (CAN) As a catalyst, the mixtures 7 and 8 are produced (she ies 3-4 in Table 2). However, for the starting material Mβ, °, sda and 8ea produced by the 1,2-addition reaction are the main products ( Experiment 7) and the only product (Experiment 8), the results were similar whether using ruthenium or hexamethylene diammonium (CAN) as a catalyst. There were no special results above, except for the difference in Lewis acidity. In addition, it can be explained by the different steric obstacle factors of the starting material itself.

Reaction 6 Page 21 of 44 '«: S ) Table 5 uses, under the conditions of his catalyst, the reaction data of α, 々 unsaturated ketones 1 £1 and 0 noise extraction 2 & time solvent yield % ~~ ~---- (4aa/5aa) — EtOH 23/4(SM 83) EtOH 12/2(SM 72) EtOH 10/90 丄day ·*—— acetone/H2O 0/99 1 day ~ — Et2〇0 /85 EtOH 19/lliS.M. 261 Iday Ether 5/91 — —2dayS EtOH 7/24{SM 72) catalyst(mmole) _InCl3(0.1)_

CeCl3(0.1) AIC13(0.1) Dodecylbenzene sulfonic acid __Acid (0.1)_ 4-methylbenzenesulfonic acid(Ol) p-toluenesulfonic acid Amberlyst-15 (O.lg) TCT(0.05) ' NBS( Ol) 'Based on the results of Examples 1 through 4, we also tried to use other catalysts to catalyze the reaction starting materials la and 2a. The experimental results are shown in Reaction 6 and Table 5. When 0.1 equivalent of metal halide is used as the catalyst, the amount of product 4aa catalyzed by indium sulfide (InCl3) and gasification sieve (CeCl3) is more, but chlorinated sulphide (AICI3) is the main product of catalytic formation 5aa. And its yield is as high as 9〇% (entries I·3 in Table 5). Secondly, the catalytic effect of organic acid reflux (salt) is very different. When 0.1 equivalent of dodecylbenzene sulfonic acid is used, 99% of mono-product 5aa can be obtained (experiment 4), while 0.1 equivalent is used. For the 4-methylbenzenesulfonic acid, 85% of the single product 5aa can be obtained (Experiment 5); relatively ' 〇.lg acidic ion exchange resin page 22 / 44 pages 1323657

Amberlyst-15 produced only 19% of the product 4aa and 11% of the product 5aa (Experiment 6). In addition, the use of the catalyst 2,4,6-trichloro-1,3,5-trichloro-l (3,5-triazine; TCT) is quite good, can produce 5% Product 4aa and 91% of product 5aa (Experiment 7). As for the catalyst N-Bromosuccinimide (NBS), it produced 7% of the product 4aa and 24% of the product 5aa (Experiment 8). Example 6

Table 6 Reaction data of unsaturated ketones 1 with various anthraquinones and their derivatives under the catalysis of 0.3 equivalents of iodine

Indole and its deritives Time (h) solvent yield % 2a: 1-Hi0.3) 2 Et2〇4aa/5aa=0/93 2b: l-CH^To^P 5/12 Et2〇4ab/5ab=0/99 2c: 5-F(0.3) 2 Et2〇4ac/5ac=0/93 2d: 5-BriO.3) 2 Et2〇4ad/5ad=0/98 2e: 7-Eti0.15, 1 Et20 4ae/5ae= 0/37 We use 1 equivalent of α,β-unsaturated ketone 1 and 4 equivalents of hydrazine and its derivatives 2a-2e in 1 ml of diethyl ether (EGO) solution at 0.15 or ο. When the equivalent iodine (i〇dine) was reacted as a catalyst, 'only a single product 5 was produced, and the results are shown in Table 6. In the above-described examples of the present invention, it is presumed that the present invention produces a different product of page 23 of 44 pages*·S; the reason may be that the starting materials or reactants are due to the standing obstacles or/and catalysts of the yangzilin The difference between each other's Louise acidity. The catalyst used in the present invention has the advantage that the reaction scale is high, the mixed solution after the reaction is easy to handle, and the reaction can be carried out under mild or moderate conditions such as room temperature. In addition, the above-mentioned catalysts are not only cheap, secret, but also have a low impact on the ring, which is in line with the world trend of environmental protection. The cells have proliferation, differentiation and death. During the growth balance of normal tissues, 'cell proliferation, differentiation, and social coordination, and co-regulation' play an important role in the cell-to-decay and renewal, and the constant number of cells. Long _ scholars focus on tumor proliferation activity and differentiation characteristics _ research, and due to experimental methods and means, research on tumor cell death is relatively _. However, the more rounds of data show that the cell __ job has a close _. Wei, tumor is not only a disease of abnormal proliferation and differentiation, but also a disease of strong abnormality. At present, the tumors and the deaths of the Tian D D have become the focus of everyone's attention in the field of life sciences. As described in the prior art, cell death (ap〇pt〇sis) is also known as the Cellular Cell Society ((e) face eeU death). According to the book, Wyllie and Currie: Autonomous and orderly death controlled by genes that maintain the stability of the intracellular environment. It is different from cell necrosis (ne_is). Cell death is not a passive process, but an active process; it involves - series of bases - initiation, expression and regulation, not under pathological conditions from page 24 / A total of 44 pages of physical damage, but a voluntary process to take the initiative to better adapt to the living environment. The prominent change in apoptosis is the cleavage of chromosomal DNA catabolism catalyzed by endogenous endonuclease, forming a chromosomal DNA fragment of an integer multiple of about 180-200 bp, ie, chromoplast DNA. Fragmentation (DNA fragmentation). Cells that have undergone cell death, the cell membrane shrinkage, dents, chromatin becomes dense, and finally breaks into pieces; then the cell membrane divides the cytoplasm and surrounds the cytoplasmic fragment to form multiple membranes. The structurally intact/packet-shaped body is called; the dying body (ap〇pt〇ticb〇cJy). In the process of cell death, the cell is in a state of contraction, and the cytoskeletal protein is destroyed by protease. However, the structure and function of major organelles such as mitochondria and lysosome are often maintained in the late stage of apoptosis. The endoplasmic reticulum also has the function of synthesizing proteins in the early stage, and then expands into a vesicular form, which is in contact with the cell membrane to form cells. Bubbles. The cell membrane remains intact and the cell contents are not spilled, so it does not cause an inflammatory response. Therefore, apoptosis is a very special, natural cellular process that is mainly controlled by a variety of genetic genes, such as ?1*〇_叩(^〇1^:1)53,6,

Bad, Bak' and anti-apoptotic gene: Bcl-2, Bcl-xL, Bcl-w, etc., cells will know: follow the procedures set by themselves until the cells are swallowed, in order to keep the cells and tissues constant. Apoptosis has four important extrinsic features: (〇 cell cytoplasmic shrinkage, (2) chromosome condensation, (3) pNA fragmentation, (4) apoptotic bodies. The characteristic of sputum is that the cell membrane does not rupture, cells The contents will not flow out on page 25/44 pages, so there will be no inflammatory reaction. During the process of apoptosis, the double-stranded DNA in the cells will be cleaved by the endonuclease (caSpase), which will form approximately as ^ #大小, and then progress to cleavage to a nucleosome of about 185 bp, and finally formed; the dying body is phagocytized by phagocytic cells. Apoptosis has many important functions in animal development, such as morphological changes, removal is not required Construct, control the number of cells, remove abnormal or de-functioning or harmful cells, and produce differentiated cells, etc. Experimental analysis methods for detecting cell death, including (1) electrophoretic separation technology: for apoptosis, '·· After the DNA is taken, the dna ladders ladder pattern can be observed by the electrophoretic separation technique to understand the degree of DNA fragmentation. (2) Flow cytometry technique. Analysis of the proportion of each cell cycle (cell cyde), if the cell Death In the case of death, the flow cytometry will be in the normal cell cycle including 〇1 (Gap h interval 1), S (Synthesis, DNA synthesis phase), G2 (Gap 2, interval 2), and Μ (mit〇) In addition to Sls, mitosis, cells in the sub-G1 pre-sub (sub Gi) are detected, which may indicate apoptosis of the cells. A second embodiment of the present invention discloses a pharmaceutical composition for treating cancer, which comprises A compound having the following general formula:

Its enantiomers, diastereomers, pharmaceutically acceptable _ or any group thereof s 26 pages / total 44 pages 1323657 "where ' Ri, R2, R3 and R4 are among the following groups - A nitrogen atom, a silk, a substituted type silk, an aromatic group, a substituted type base, or a ring of any of ri, r2, R3 and R4. In a preferred embodiment of the present embodiment, the above compound comprises one of the following groups (hereinafter referred to as 3-ind〇ie drug):

In the present embodiment, the cancer is selected from one of the following groups or any combination thereof. Lung cancer, esophageal cancer, ovarian cancer, breast cancer, lymphoma, glandular cancer, colorectal cancer, head and neck cancer, and bladder cancer. The above-mentioned compounds having a 3-indole based structure are particularly suitable for cancer cells in which the tumor suppressor gene p53 is mutated, for example, lung cancer. Clinically, lung cancer is divided into two categories, namely, page 27/44 pages ) S) 1323657 Small Cell Lung Cancers (SCLCs) and Non-Small Cell Lung Cancers (NSCLCs), most of them Non-small cell lung cancer can be divided into adenocarcinoma lung cancer, squam〇us cell lung cancer and large cell • hmgcancer. Among them, lung adenocarcinoma is the most common in lung cancer patients, and lung cancer is often the case in non-smokers, while lung squamous cell carcinoma is common in smoking patients. Example 7 Materials and Methods (Example of triterpene cyclohexane) Cell lines The five lung cancer cell lines used in this experiment have p53 genotypes as follows: H1299 (p53 null type) &gt ; CL1-1 (p53 mutant type) > H1435 (p53 mutant), H1437 (p53 mutant), A549 (p53 wild-type); in addition, the normal lung cell line MR90 is used as a control group; two esophageal cancer cell lines, φ is KYSE170 and KYSE510 respectively. Among them, H1299, CL1-:L, H1437, A549 and IMR90 were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum, while H1435, KYSE170 and KYSE510 were cultured in containing 1% fetal bovine serum. rpmI-1640 medium. Drug treatment The drug solvent is dimethy sulphoxide (DMSO). The cells were cultured in a 37 ° C, 5% C 〇 2 incubator on a dedicated culture page; a quantitative cell (approximately 3 xi 〇 5) using a 6-well culture dish Dish culture for 12~16hr. The next day was at 37 with a different concentration of the anticancer drug 3-indole. (: culture for 24 hr. And count the survival rate of the cells after drug treatment, and understand why the anti-cancer drug 3-indole is the concentration (IC50) required for tumor cells to die by 50%. Covered cell viability test (MTT assay) • The cells are cultured in a special medium of 37. 〇, 5% c〇2 incubator; the cells in the assay (about 3x10) are incubated for 12 to 16 hr in a cucuture dish of a 6-well culture dish. The medium with the concentration of 3_ω〇ΐ6 was cultured in 3rc for 24 hr, the medium was removed, and the medium containing MTT [3- (4 5 dmethyi thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] 1 ml / well was added and placed in the incubator. 1 hour; remove the medium containing MTT, add DMSO60_well and place it on the horizontal shaker for 5 minutes; • Take 20 〇Wwe11 in a 96-well culture plate, measure the absorbance at 570 nm; and obtain the absorbance Convert to the number of cells to know the survival rate of the cells. DNA fragmentation analysis (DNAladfWage@ scrape the cells from the plate about 2 (Κ) μ1, add _ pn> tease κ, and 2_ AL buffer, mix for 15 seconds Dissipate the cells and place them on the crucible heater 10 'After the addition of 99% ethanol 200μ1; ⑽ which moves the mixture shame page 29/44 Total

S 1323657 in the column (centrifuge 8000 rpm, 1 minute, remove the lower layer of waste; add 500μ1 QIAGEN Kit AW1 buffer centrifuge 8000 rpm, 1 minute, remove the lower layer of waste; add 500μ1 QIAGEN Kit AW2 buffer centrifuge 14000 rpm , 3 minutes; then move the upper column to a new 1.5 ml centrifuge tube (eppendorf), add 200 | ilAEbuffer to room temperature for 1 minute, then centrifuge 8000 for '1 minute; finally collect the collected DNA to run electrophoresis, observe The case of DNA ladder. Detection of Cell Cycle Quantitative cells (approximately 2 x 106) were fixed at 70% alcohol with fixed cells at _2 〇. After standing in the refrigerator for 24 hours, centrifuge at 900 rpm for 5 minutes at low speed, aspirate the supernatant, leave about 50 ul of solution, spread the cell mass evenly, and add 5 mi of phosphate-based saline (PBS). The cells were washed three times with pBs. Add 1.0 ml propidium iodide (PI) / Triton X-100 staining solution, φ spread the cell mass evenly' and gently shake and mix for 30 minutes in the dark room at room temperature (final concentration is Triton-X 0.1%, RNase a 〇.2 mg/ml, & PI20 pg/ml). The sample was mixed before the machine was placed' and the sample was filtered through a 35-μιη nylon mesh. Estimate the DNA distribution of DNA cells in about 10,000 cells by flow cytometry (FACScan flow cytometry, BD, MountainView, CA> to detect the fluorescence intensity of PI in cells) to define various stages of the cell cycle and utilize M 〇fltLT Ver2.0 software calculates the proportion of cells in each cycle and analyzes the mapping. If the cells affect the original cell cycle due to drug treatment, the DNA detected by the machine is divided into 30 pages/44 pages of 1323657. It will be different from the samples of untreated drugs, including G1, S, G2 and Μ. G1 stands for “Gap 1” (interval 1), and S stands for “Synthesis', (DNA synthesis), the stage of DNA replication. G2 stands for "Gap 2" (interval 2), Μ stands for "mitosis", which is the stage of nuclear fission (chromosomal segregation) and mitochondrial (cytoplasmic division). If cells are detected in the early G1 phase, the cells may The phenomenon of DNA fragmentation that occurs in apoptosis occurs. Western Blot: Add appropriate amount of RIPA [50 mM Tris ρΗ8·0, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1%) Triton X-100, 5mM After phenylmethylsulphonyl fluoride (PMSF), lOmg/ml leupeptin, 20 mM sodium phosphate pH7.0], all cells were removed using a cell scraper and centrifuged at 4 ° C for 30 minutes. Take the supernatant 4μ1 for protein quantification, and store the remaining protein in _2〇.〇. 蛋白蛋白贝疋篁. Bio-Rad DC (Detergent- Compatible) Protein

Assay method, take 5μΐ stan (jard or sample solution to % well culture tray), add 25μΐ reaction reagent A (alkaline copper tartrate solution), then add 200μ1 reagent b (Folin reagent) for 15 minutes, measured at 630nm Absorbance value. The standard curve was obtained by using bovine serum albumin standard as 标准5, io, 1.5, 2. 〇mg/ml as the standard protein, and the linear equation was obtained after regression, and the protein concentration in the sample was converted ( Mg/ml) ° Page 31 of 44 < S ) 1323657 Electrophoresis: Take 30pg of protein into sample buffer (3 times sample buffer 'includes 350mM Tris-HCl pH 6.8, 12% SDS, 0.02% bromophenol blue , 35% glycerol, 30% mercaptoethnol) After boiling for 5 minutes, 'SDS-PAGE (4% stacking gel and 10% or 15% resolving gel) was firstly applied at a voltage of 80 volts for 1 minute, then the voltage was adjusted to 130 volts. The electrophoresis was carried out for 70 minutes. Immunochromatography: The protein on SDS-PAGE was spun onto the PVDF membrane, and then the membrane was removed and immersed in blocking solution (10% skim milk in PBS-T) for 1 hour at room temperature with PBS_T (ph 〇sphate_based saline-0.5% Tween-20) Wash 2 times for 10 minutes each time. The primary antibody Bcl-2 (Cell Signaling Technology, Beverly, MA 01915, USA) was added separately, and after 2 hours at room temperature, it was washed 3 times with PBS-T for 10 minutes each time, and the secondary antibody HRP-goat anti was added. -rabbit IgG, after 1 hour at room temperature, was washed 3 times with PBS-T for 10 minutes each time. Finally, the reagent was applied to the membrane using an ECL Chemiluminescent Western System (Amersham, Arlington Height, IL, USA) reagent group, and after 5 minutes from the light, it was developed by x-ray filming in a dark room. In situ tumor model animals were counted by cell counter to calculate the number of cells transferred. Take 5 X 1 6 cells, rinse with 1× PBS, and rinse twice with Hanks' Balanced Salt Solution (HBSS) to obtain 100 μl HBSS. Cell suspension is injected into the four-year-old Nude mice (small

Page 32 of 44: S mouse) The back of the nude mouse (ICR-Foxnl nude mice, National Animal Experimental Center). About 1 to 14 days after the injection of tumor cells, when the tumor grows to a size of 50 mm3, the novel anticancer drug 3-indole or the clinical chemotherapy drug Taxol is injected subcutaneously into the abdomen of nude mice, and injected once every two days (〇, 2, 4). , 6, 8 day), each dose was 0.2 mg, a total of 5 injections (total dose of 50 mg / Kg). In 30 days, (1) observe the tumor size change and record, measure the longest side of the tumor a and the shortest side b 'tumor volume is set to (axb2)/2; (2) take the blood of the injected drug Perform biochemical and blood analysis; (3) take liver and kidney for slice observation. Example 8 results (taking triterpene cyclohexane as an example) Cytotoxicity test: °Indole compounds 3-indole, the results of the toxicity test on each type of tumor cells, whether in lung cancer cell lines :H1299 (p53 null type), CL1-1 (p53 mutant type), H1435 (p53 mutant), H1437 (P53 mutant), A549 (P53 wild-type) cells and two esophageal cancer cell lines 'KYSE170 and KYSE510, respectively They all have a very high toxicity, and can inhibit the growth of cancer under low dose treatment. However, in the normal lung cell IMR90, there is no obvious killing effect under the same amount of treatment (refer to the first - _ Show), shows that _ compounds have the potential to become a novel anti-cancer drug. Page 33 / Total 44 pages 1323657 Potential test: In animal experiments and body-metabolism identification analysis, my wall lung cancer cell A549 was injected into the nude mouse (ICR_FGXni) f subcutaneous, until the cell mass formed into a tumor up to 5 (W at the beginning of the injection of 3 also her, and the traditional chemotherapeutic drug (four) as the positive control group '3, indGle solvent DMS 〇 as the control group. Refer to the second figure (eight) shown in the 'comparison with the control group results, in the injection 31_6 After that, the tumor formed by lung cancer "ww I A549" did have a growth of 3 (10)%%%. On the other hand, referring to the second figure (8), the results of blood and biochemical tests were analyzed. The doctors and the laboratory staff determined that the blood biochemical values presented after injection of her were in the normal range of 0 [Glmamic oxaiacetic transaminase (GOT), glutamic pyvuvic transaminase (GPT)]; the results of tissue section staining were confirmed by the pathologist. After 3-mdole injection, there is no damage to the relevant organs of the mouse [as shown in Figure 2 (C)].__Terrace cycle and sputum cell death identification: _ To understand 3-indole inhibition of tumor cells Growing The mechanism of action was observed by flow cytometry (FLOW cytometry), DNA ladder assay and Western blotting to observe the distribution of cells in the cell cycle and the identification of programmed cell death. Referring to the third figure, through the experiment of FLOW cytometry, it can be seen that the effect of deuterated addition on cell cycle has different dose effects; lung cancer cells (A549, H1299,

Page 34 of 44, ·: S 1323657 H1437, H1435, CL1-1) After treatment with 1〇μ]νΐ for 24 hours, the cell cycle can be arrested in G1 phase, and the treatment of 30 μΜ dose can further lead to Increased cell cycle sub-G1 'sub-Gl increase indicates cell death. Referring to the fourth figure, the results of DNAladderassay show that the death mechanism of lung cancer cells (A549, H1299, H1435, H1437, CL1-1) is through the apoptosis of the cells, so there will be regular DNA fragmentation. situation. It is currently known that a continuous reaction of apoptosis carries out a number of apoptosis-related molecules involved, for example, Bcl-2 (B-cell leukemia/lymphoma). Referring to the fifth figure, the analysis by Western medullary analysis _ confirms that 3_indQle riding cells are related to the Bd_2 family performance. The present invention was developed from phytoehemieal Wei (4) indGle, and developed a novel chemical substance 3_ind〇le which induces cell death, whether in P53 wild-type A549 cells, p53 mutant H1437, H1435 and CL1-1 • cells and Net 111111 has extremely high toxicity in H1299 cells, and has obvious tumor growth inhibition effect in animal experiments; further in vitro (in: ν_cell mode message transmission path analysis, 3-indole Drugs can induce cell death (ap〇pt〇sis) through the Bcl-2 message transmission pathway, and have a growth inhibitory effect on cancer cells of various P53 variants, indicating that the 3-indole compound has potential Becoming a novel anti-cancer drug can improve the cure rate for different forms of cancer patients. Obviously, the bribes on the ship's actual customs towel can have many differences on page 35/44 pages"S) 1323657 It is therefore necessary to make a solution within the scope of the appended claims. In addition to the detailed description above, the present invention can be widely practiced in other embodiments. The above is only the preferred embodiment of the present invention, and is not intended to limit the application of the present invention; the other is not included in the spirit of the disclosure; Please be within the scope of the patent. Φ [Simplified description of the drawings] The first figure shows the cell survival rate of the various cancer cells after 24 hours of treatment of the 3_indQle drug according to the third embodiment of the present invention, and it can be found that the IC5 can be achieved by using about 10-indole drug. The poisoning effect of cockroaches; The second® is the inhibitory effect of the 3_indQle drug on the in situ tumor model animal test according to the third remuneration of the present invention. (A) Take 5 nymphs and 6 lung cancer cells • A549 is injected into the back of the nude mice, and the cell mass is formed into a tumor of 5〇mm3. When the injection is 3 marriages*, the traditional chemotherapy drug Taxol is used as the positive control group. : 3_ώ—The solvent DMS0 is used as the control group. The operating conditions were once every two days (〇' 2, 4, 6, 8 day), each dose being 〇 2 mg. (B) Take the blood of the mice after the injection of the drug for blood and biochemical tests for normal liver/kidney function. The liver and kidney of the mouse after injection of the drug were taken to observe the cell type; the third cell line according to the third example of the present invention, the cell cycle distribution of the various lung cancer cells treated with or without 3-mdole drug for 24 hours Fig., Page 36 of 44 1323657 where 'G1 represents cells with 2 sets of chromosomes (2n), G2/M means cells with 4 sets of chromosomes (4N), S between G1 and G2/M, representing DNA synthesis In the period, the number of chromosomes is between 2N and 4N, sub-Gl represents less than 2 sets of cell chromosomes, and DNA fragmentation occurs in cells, and apoptosis may occur. In addition, the solid arrow represents 8111)-〇1?6 increased spit intensity, the open arrow represents (}2/]^ egg spit strength increase; the fourth figure is based on the third example of the present invention, using DNA electrophoresis to detect DNA breakage The treatment of various lung cancer cell lines by 30 //M 3-indole drugs: (A) 24 hours; (B) 48, ϊ, hr; and the fifth figure according to the third example of the present invention, The Western blast method was used to observe the expression of GAPDH and Bcl-2 in Α549 and Η1437 cancer cells by 30 "Μ 3-indole drugs. Page 37 of 44

Claims (1)

Amendment IX. Scope of Application: 丨 Announcement 1. A pharmaceutical composition for treating cancer in a patient in need of cancer treatment, comprising a compound having the following general formula: Η Η a compound, a diastereomer, a pharmaceutically acceptable salt or a combination thereof, wherein W, Y, Rule 3 and R4 are one of the following groups: a gas atom, a (tetra) group, a C1 to C8 substitution type. Silk, C6~c-C18 substituted aryl C6 2. As stated in the __ i item, it is intended to form a ring. Contains one of the following groups: ^ music group 0, of which the above compounds 苐38 pages/total 44 pages In the first aspect of the medical compound, the tumor suppressor gene p53 in the cancer cell described in 1 is mutated. 1Reward of the composition, one of the above-mentioned cancer, 歹' groups or any combination thereof · Lung cancer, esophageal cancer, ie, nest cancer, breast cancer leaching (10), pancreatic, closed bowel cancer, facial Cancer and bladder cancer. 5. The pharmaceutical composition according to claim i, wherein the cancer system is a non-small cell lung cancer (Non-Small Cell Umg Caneeq 〇6. The pharmaceutical composition as described in the patent application item i, The cancer cell death described above occurs via a cell death program (ap〇pt〇sis). 7. The pharmaceutical composition according to claim 1, wherein the secretory cancer cell is inhibited from dividing. Page 39 of 44 1323657 No-Complementary XI. Schema ❹ 0 3-indole Concentration first picture page 40/44 pages 1323657 (gl) 乐ϋ^-Ι墚1200 • solvent 3-indole 1000 mi taxel 800 600 400 y 200 0 ^ Ο 14 Α549 (A) No. 41 Page / Total 44 Pages 1323657 Biochemical values set normal range 3 -ii 1 «1 π ic ! Mean + St' ! av : i 1 Mfan ^ SK - Curry 1 1 \ 1 c'aii 4 mg / dL Ol-OJ 0.15 ± 0.05 0.18 :! : 0.04 0_15 ± 0.05 serum albumin mg / dL 1.6-1.8 1.5 ± 0.1 1.7 ± 0 · 1 1.7 ± 0.1 GOT Enzyme U / dL 】 53-235 252.3 ± 0.3 217.5 ± 37 139 ± 32 GPT Enzyme U / dL 23.1 - 97.9 38 ±2.9 40.3 + 0.3 38.5 :1: 4.3 (B) _Control group ~ Liver tissue section Kidney warm tissue section: Carrying vines _: face tissue section of experimental tissue section 11 <1: ( C) Second Tuwan 42 pages / book. 44 pages 1323657 3-indole concentration 1323657 Fourth picture GAPDH family Bel-2 family 3 indole ( Λ〇[ί VI ) 3 iiulolt* ( // M ) <_ 4 Η 12 24 ^^0 mmm (1 4 8 12 24 at, *, · \5-l^ Cell mm hi fifth picture laugh 44 hundred ' total 44 1