KR20170105033A - Synthetic ligidine analogs as antitumor agents, salts, solvates and prodrugs thereof, and methods for preparing the same - Google Patents

Abstract

상기 화학식(F)에서, R, R₁ 및 R₂는 수소, 아릴, 융합된 아릴, 헤테로아릴, 포화된 카보사이클릭, 부분 포화된 카보사이클릭, 포화된 헤테로사이클릭, 부분 포화된 헤테로사이클릭, C_1-10 알킬, 할로알킬, 알케닐, 알키닐, 아릴알킬, 아릴알케닐, 아릴알키닐, 헤테로아릴알킬, 헤테로아릴알케닐, 알케닐알킬, 알키닐알킬, 카보사이클로알킬, 헤테로사이클로알킬, 하이드록시알킬, 아미노알킬, 카복시알킬, 니트로알킬, 시아노알킬, 아세트아미도알킬, 및 아실옥시알킬로부터 선택된다. 또한, 이의 제조 방법이 제공된다.There is provided a compound having the formula:

In the above formula (F), R, R ₁ and R ₂ are selected from the group consisting of hydrogen, aryl, fused aryl, heteroaryl, saturated carbocyclic, partially saturated carbocyclic, saturated heterocyclic, partially saturated heterocyclic click, C _1-10 alkyl, haloalkyl, alkenyl, alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroaryl alkenyl, alkyl, alkenyl, alkynyl, alkyl, cycloalkyl carbonyl, heteroaryl Cycloalkyl, hydroxyalkyl, aminoalkyl, carboxyalkyl, nitroalkyl, cyanoalkyl, acetamidoalkyl, and acyloxyalkyl. Also provided is a method of making the same.

Description

Synthetic ligidine analogs as antitumor agents, salts, solvates and prodrugs thereof, and methods for preparing the same

This application is a continuation-in-part of U.S. Patent Application Serial No. 13 / 678,987, filed November 16,

The present invention relates to synthetic ligand analogues, their salts, solvates and prodrugs as anticancer agents, and methods of making the same.

Cancer is the second leading cause of death in the United States. Cancer is a class of more than 100 diseases characterized by unregulated abnormal cell growth and proliferation. Anticancer drugs are one of many tools used to combat cancer. They may be used alone or in combination with other therapies to combat the disease. The incidence of cancer is expected to increase only in the future as the average life span continues to increase, and accordingly, the development of new anti-cancer drugs is the highest priority in the drug development community. Of particular importance are numerous novel anticancer drugs derived from natural sources. Of all the novel anticancer drugs developed between 1981 and 2006, only about 20% were fully synthetic, and thus the novel natural compounds were found to be an excellent source for forming stronger and more selective agents than those currently used in hospitals As well.

Plant-derived natural product as an anticancer source

The plant has a long history of use in the treatment of cancer, as the course's Hippocrates of Cos dates back as long as 4 centuries BC, using oils obtained from narcissus Narcissus poeticus L. to treat uterine tumors. Several plant-derived compounds are among the best anticancer drugs used clinically; These include vinca alkaloids, vinblastine (Fig. 1) and vincristine, and taxane taxanes (Fig. 1) and their semisynthetic analogues docetaxel. Vinca alkaloids are one of the first plant-derived anticancer drugs to advance clinical use and were first isolated in the 1950s from Madagascar periwinkle plants.

Many other plant-derived compounds have been used as inducers in the development of more potent and less toxic anticancer drugs. Among these is a tree that is used extensively in the grapes and grape pilrum pilrok toxin (podophyllotoxin) (Fig. 2) wherein the mitotic (antimitotic) isolated from (Podophyllum) in the plant, and a typical hanyakje kamto teka And camptothecin (Fig. 2), a topoisomerase inhibitor isolated from camptotheca acuminate . Although grape candelotoxin and camptothecin failed clinical trials because of their severe toxicity and poor water solubility, both acted as inducers in the development of several clinically used anticancer drugs. These include three semisynthetic derivatives of podophyllotoxin teniposide, etoposide (Fig. 2), and etoposide phosphate, as well as the camptothecin analog topotecan (Fig. 2 ), And irinotecan, all of which are currently clinically used for the treatment of a variety of cancers.

Marine-derived natural products as a source for anticancer drugs

In addition to plant-derived anticancer drugs, a relatively new resource for the development of new anticancer drugs is marine natural compounds. These include natural products separated from aquatic fungi, cyanobacteria, sponges, and tunicates. Cyanobacteria are abundant sources for new bioactive agents to obtain about 300 new alkaloids, which are likely to be produced as toxins to save predators. Marine alkaloids derived from cyanobacteria are well characterized and exhibit various mechanisms of action against a variety of cancer cell lines, including, but not limited to, histone deacetylase (HDAC) inhibition, And cell cycle arrest at G ₁ targeting tubulin. Fungi generally produce a large number of secondary metabolites that exhibit promising high anticancer potential, but the mode of action (MOA) of many of these alkaloids is not fully understood.

Marine sponges and picans have proven to produce some of the most interesting and useful marine-derived alkaloids to date. A number of identified compounds have reached clinical trials, many of which have well-defined MOA similar to those of the plant-derived natural product taxol and camptothecin. These include hemiasterlin and its synthetic analog E7974, which exhibit potent anti-tubulin activity, and makaluvamine, which inhibits topoisomerase II (TOP II). Circulating fluid has been known for a long time as a major source of compounds for the treatment of cancer. Among them, lamellarin and related pyrrole-derived alkaloids have been shown to have promising biological activities such as cytotoxicity, reversal of multidrug resistance (MDR), HIV-1 integrase inhibition, antibiotic activity, It is remarkable as a group of marine alkaloids with aldose reductase inhibition, immunomodulation, antioxidant activity, and the like. In view of their attractive new structure, interesting biological properties, and the difficulty of obtaining large quantities from natural sources, marine pyrrole-derived alkaloids have attracted considerable attention to organic and pharmaceutical chemists. Currently, several marine natural products are in clinical trials, and a drug, trabectedin, has recently been approved in Europe as the first marine-derived anticancer drug.

As a powerful new anti-cancer drug Rigidine

Although intensive studies have been done on marine natural products, there are still a number of very potentially useful medicines that have not been studied sufficiently. Among these are the picans Eudistoma cf. Found in Okinawa and New Guinea. B, C, D (Figure 3) and E separated from the rigida . Rigidins have been studied less as natural products due to their lower availability from natural sources, but they have a variety of interesting biological properties. They have been reported to exhibit cytotoxicity against cancer cell lines, and it has been found that lrigidine A also has calmodulin antagonistic activity.

The ligdin introduces a pyrrolo [2,3-d] pyrimidine ring system which is considered to be a "privileged medicinal scaffold" (PMS). PMS is a molecular skeleton that can act as a ligand for a variety of apparent targets. The pyrrolo [2,3-d] pyrimidine ring system is similar to the purine skeleton and, in addition to the ligand, it also contains several other natural products such as the nucleoside anticancer antibiotics tubercidin, (toyocamycin), sangivamycin (sangivamycin) is a common motif. Thus, the ligand is an unexamined class of marine alkaloids with a high potential for multiobjective activity.

Conventionally, the inventors have discovered a three-component chemical reaction that results in efficient synthesis of marine alkaloids (see FIG. 4). Although these natural products have been reported to have weak antiproliferative and calmodulin-antagonistic activity, their biological properties have rarely been studied due to insufficient amounts of available materials from separation and lack of efficient chemical synthesis. The inventors have developed an efficient synthetic approach that allows them to produce sufficient amounts of material for biological research.

The evaluation of these synthesized alkaloids against several cancer cell lines showed very weak antiproliferative activity (IC ₅₀ values of> 100 μM). The current discovery addresses the consequence that alteration of these reported synthetic methods to novel four-component reactions results in the preparation of ligand analogs having a hypoxanthine-like skeleton that is not conventionally known See three representative possible compounds in Figure 5).

In addition, the evaluation of these compounds against several cancer cell lines showed a remarkable and unexpectedly strong antiproliferative effect at very low concentrations.

Novel hypoxanthine-type analogues of marine alkaloids are described herein.

Accordingly, one aspect of the present application includes a compound of formula I:

Wherein R ₁ and R ₂ are selected from the group consisting of hydrogen, aryl, fused aryl, heteroaryl, saturated carbocyclic, partially saturated carbocyclic, saturated and partially saturated heterocyclic, C alkyl, Alkenyl, alkynyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, carbocycloalkyl, heterocycloalkyl, hydroxyalkyl, aminoalkyl, carboxyalkyl, nitroalkyl, cyanoalkyl, ≪ / RTI > acetamidoalkyl, and acyloxyalkyl.

In each alkyl, aryl, cycloalkyl or heteroaryl, one or more available hydrogens may be substituted or unsubstituted with fluorine, and the resulting compound, or a pharmaceutically acceptable salt, solvate or prodrug thereof, And is within the scope of the application.

In another embodiment, the compounds of formula I include isotopic labels (also known as isotope tracers or markers). Thus, the present application also includes compounds of formula I wherein one or more atomic isotope labels are substituted with, for example, ² H, ³ H, ¹³ C, ¹⁵ N, ¹²³ I, ¹⁸ F.

In another aspect of the present application, there is provided a pharmaceutical composition comprising one or more compounds of formula I as defined above, and / or a pharmaceutically acceptable salt, solvate and / or prodrug thereof, and a pharmaceutically acceptable carrier, Among other things, it includes pharmaceutical compositions comprising nanoparticles, liposomes, microbubbles, hydrocolloid beads.

In a further aspect of the present application, there is provided a medicament comprising one or more compounds of formula I for treating cancer, and / or a pharmaceutically acceptable salt, solvate and / or prodrug thereof, and a pharmaceutically acceptable carrier Composition.

In yet another further aspect of the present application, there is provided a method of treating cancer comprising administering to said mammal an effective amount of at least one compound of formula I as defined above, and / or a pharmaceutically acceptable salt, solvate and / or prodrug thereof, Comprising administering to a subject in need thereof.

A further aspect of the present application is a process for the preparation of a compound of formula I which comprises reacting a cyanoacetamide or a synthetic equivalent thereof, including triethyl orthoformate or its synthetic equivalent, as well as a suitable base and solvent , I.e. in the presence of them, reacting a compound of formula (II) with a compound of formula (III): < EMI ID =

In Formula II, R ₂ is selected from the group consisting of hydrogen, aryl, fused aryl, heteroaryl, saturated carbocyclic, partially saturated carbocyclic, saturated heterocyclic, partially saturated heterocyclic, Alkenyl, alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, carbocycloalkyl, heterocycloalkyl, hydroxyalkyl, aminoalkyl, carboxyalkyl, nitroalkyl, cyano Alkyl, acetamidoalkyl, and acyloxyalkyl,

In Formula III above, R ₁ is selected from the group consisting of hydrogen, aryl, fused aryl, heteroaryl, saturated carbocyclic, partially saturated carbocyclic, saturated heterocyclic, partially saturated heterocyclic, Alkenyl, alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, carbocycloalkyl, heterocycloalkyl, hydroxyalkyl, aminoalkyl, carboxyalkyl, nitroalkyl, cyano Alkyl, acetamidoalkyl, and acyloxyalkyl. Examples of suitable solvents include, among others, ethanol, iso-propanol, butanol, acetonitrile, DMF, examples of suitable bases include K ₂ CO ₃ , Na ₂ CO ₃ , Cs ₂ CO ₃ , DBU, NaOH, KOH.

Other features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating the specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present application will become apparent to those skilled in the art from the detailed description.

The present application will now be described in more detail with reference to the accompanying drawings.
Figure 1 shows the structure of paclitaxel and vinblastine.
2 shows a representative structure of a plant-derived anticancer agent and a clinically used derivative thereof.
Fig. 3 shows the structures of the ligands A, B, C and D. Fig.
Figure 4 shows the previously developed synthetic route to natural ligand.
Figure 5 shows an illustrative embodiment of the four-component reaction of the present application resulting in the synthesis of hypoxanthine-type ligandin analogs.
Figure 6 is a graph showing cell cycle analysis using HeLa cells treated with Rig8 for 24 hours; The error bar represents the standard deviation between trials.
Figure 7 shows the induction of apoptosis in RIG8.
Figure 8 shows the structure of the ligands A, B, C, D and their synthetic analogs based on 7-deaza hypochactin (A) and the novel C2-substituted-7-deaza hypoxanthine (B) Respectively.
Figure 9 shows 22 cell images against U373 hybrids and SKMEL melanoma cells showing cell death at a MTT colorimetric assay-associated GI ₅₀ value of 0.3 [mu] M.

Explanation of Specific Embodiments

While most terms used in this application will be readily apparent to those skilled in the art, applicants will further emphasize the definition of the term solvate, prodrug, and cancer. The term "solvate" as used herein refers to a compound of the present application, or a pharmaceutically acceptable salt thereof, wherein molecules of a suitable solvent are introduced in the crystal lattice. The compounds of the present application also generally comprise a prodrug, which is a functional derivative of a compound of the present application which is a conceptually derived compound and which is readily convertible in vivo. Prodrugs of the compounds of the present application may be conventional esters formed with available hydroxyl or amino groups. Finally, the term "cancer" as used herein refers to a class of diseases or disorders characterized by uncontrolled cell division and the ability of cells to invade other tissues.

Example

The following non-limiting examples are illustrative of the present application.

The compounds of formula I (1H- pyrrolo [2,3-d] pyrimidin -4 (7H) - one) General Procedure for the Preparation of: 2.5 ml of EtOH and RC (OEt) of 2.5 ml ₃ (R = H or a solution of CH ₃₎ N- (2- oxo-2-substituted) methane sulfonamide (0.676 mmol), selected or appropriate aldehyde (0.879 mmol) and cyanoacetamide (0.072 g, 0.879 mmol) in a mixture of Anhydrous granulated K ₂ CO ₃ (0.052 g, 0.372 mmol) was added in one portion. The mixture was purged with nitrogen for 5 min and refluxed under a nitrogen atmosphere for 24 h. The formation of pyrrole was controlled by TLC. After increasing the reaction temperature to 150 < 0 > C, the reaction mixture was heated again for 10 hours with simultaneous evaporation of ethanol. The mixture was then cooled to room temperature and the precipitate formed was collected by filtration and washed with EtOH (2 mL) and diethyl ether on a filter. The mother liquor was evaporated and the residue was subjected to column chromatography with MeOH / CH ₂ Cl ₂ = 1 / 40-1/20 gradient.

Example 1. 6- Benzoyl -5- (3,5 -dibromophenyl ) -lH -pyrrolo [2,3-d] pyrimidin- 4 (7H) -one (Rig8): 77%;

_{C 19 H 12 Br 2 N 3} 0 2 HRMS m / z (ESI) for (M + H ⁺⁾ calcd 471.9296, found 471.9301.

Example 2. 6- Benzoyl -5- (5- bromopyridin- 3 - yl) -lH -pyrrolo [2,3-d] pyrimidin- 4 (7H) -one (Rig9): 88%;

_{C 18 H 11 BrN 4 Na0 2} (M + Na +) HRMS m / z (ESI) calcd for 416.9963, found 416.9965.

Example 3. 5- (3,5-dibromophenyl) -6- (4-methoxybenzoyl) -lH- pyrrolo [2,3- d] pyrimidin- 4 (7H) 10): 60%;

HRMS m / z (ESI) calcd for C ₂₀ H ₁₃ Br ₂ N ₃ NaO ₃ (M + Na ⁺ ) 523.9221, found 523.9221.

Example 4. 6- (4- (benzyloxy) benzoyl) -5- (3,5-dibromo-phenyl) -1H- pyrrolo [2,3-d] pyrimidin -4 (7H) - one ( Rig 11): 61%;

HRMS m / z (ESI) calcd for C ₂₆ H ₁₇ Br ₂ N ₃ NaO ₃ (M + Na ⁺ ) 599.9534, found 599.9532.

Example 5 5- (3,5-dibromo-phenyl) -6- (4-fluoro-benzoyl) -1H- pyrrolo [2,3-d] blood limiter Dean -4 (7H) - one (Rig13 ): 56%;

_{C 19 H 11 Br 2 FN 3} O 2 HRMS m / z (ESI) for (M + H ⁺⁾ calcd 489.9202, found 489.9182.

Example 6. 6- (4-bromo-benzoyl) -5- (3,5-dibromo-phenyl) -1H- pyrrolo [2,3-d] pyrimidin -4 (7H) - one (Rig14) : 54%;

_{C 19 H 11 Br 3 N 3} O 2 HRMS m / z (ESI) for (M + H ⁺⁾ calcd 549.8401, found 549.8395.

Example 7. 7- (4 -methoxybenzoyl ) -5- (3,4,5 -trimethoxyphenyl ) -lH- pyrrolo [2,3- d] pyrimidin- 4 (7H) Rig 19 ): 56%;

_{C 23 H 22 N 3 O 6} HRMS m / z (ESI) for (M + H ⁺⁾ calcd 436.1509, found 436.1501.

Example 8. 6- Benzoyl -5- (6- bromopyridin-2 -yl) -1H -pyrrolo [2,3-d] pyrimidin- 4 (7H) -one (Rig30): 60%;

_{C 18 H 12 BrN 4 0 2} HRMS m / z (ESI) for (M + H ⁺⁾ calcd 395.0144, found 395.0125.

Example 9. 6-benzoyl-5- (3-iodo-4,5-dimethoxy-phenyl) -1H- pyrrolo [2,3-d] pyrimidin -4 (7H) - one (Rig31): 37 %;

_{C 21 H 17 IN 3 0 4} (M + H +) HRMS m / z (ESI) calcd for 503.0297, found 503.0280.

Example 10 6-benzoyl-5- (3-Bromo-4,5-dimethoxy-phenyl) -1H- pyrrolo [2,3-d] pyrimidin -4 (7H) - one (Rig32): 51 %;

_{C 21 H 17 BrN 3 0 4} HRMS m / z (ESI) calcd for (M ⁺ H +) 456.0382, found 456.0370.

Example 11. 6- Benzoyl -5- (3- bromophenyl ) -lH -pyrrolo [2,3-d] pyrimidin- 4 (7H) -one (Rig 33): 54%;

_{C 19 H 13 BrN 3 0 2} HRMS m / z (ESI) for (M + H ⁺⁾ calcd 394.0191, found 394.0183.

Example 12. 6- Benzoyl -5- (3 -chlorophenyl ) -lH -pyrrolo [2,3-d] pyrimidin- 4 (7H) -one (Rig 34): 61%;

_{C 19 H 13 ClN 3 0 2} HRMS m / z (ESI) for (M + H ⁺⁾ calcd 350.0696, found 350.0694.

Example 13. 6- Benzoyl -5-phenyl-lH -pyrrolo [2,3-d] pyrimidin- 4 (7H) -one ( Rig 35 ): 71%;

_{C 19 H 14 N 3 0 2} (M + H +) HRMS m / z (ESI) calcd 316.1086, found on 31 6.1087.

Example 14. 6- Benzoyl -5- (3- iodophenyl ) -lH -pyrrolo [2,3-d] pyrimidin- 4 (7H) -one (Rig 36): 68%;

_{C 19 H 13 IN 3 0 2} HRMS m / z (ESI) calcd for (M ⁺ H +) 443.0086, found 443.0080.

Example 15. 6- Benzoyl -5- (2,6- dichlorophenyl ) -lH -pyrrolo [2,3-d] pyrimidin- 4 (7H) -one (Rig 40): 22%;

C ₁₉ H ₁₂ Cl ₂ N ₃ O ₂ HRMS m / z (ESI) calculated for (M + H ⁺ ) 384.0307, found 384.0299

Example 16. 6- Benzoyl -5- (pyridin-3-yl) -lH -pyrrolo [2,3-d] pyrimidin- 4 (7H) -one (Rig41): 61%;

_{C 18 H 13 N 4 0 2} (M + H +) HRMS m / z (ESI) calcd for 317.1039, found 317.1038.

Example 17. 6- Benzoyl -5- (3 -hydroxyphenyl ) -lH -pyrrolo [2,3-d] pyrimidin- 4 (7H) -one (Rig42): 26%;

_{C 19 H 14 N 3 O 3} HRMS m / z (ESI) for (M + H ⁺⁾ calcd 332.1035, found 332.1032

Example 18 3- (6- Benzoyl -4-oxo-4,7 -dihydro- lH -pyrrolo [2,3-d] pyrimidin- 5 - yl) benzonitrile (Rig43): 47%;

_{C 20 H 13 N 4 0 2} (M + H +) HRMS m / z (ESI) calcd for 341.1039, found 341.1041.

Example 19. 6- (4 -Hydroxybenzoyl ) -5-phenyl-lH -pyrrolo [2,3-d] pyrimidin- 4 (7H) -one (Rig 45): 88%;

_{C 19 H 13 KN 3 O 3} HRMS m / z (ESI) for (M + H ⁺⁾ calcd 370.0594, found 370.0595.

Antitumor activity

(a) Cell culture.

Human T-cell leukemia cell line Jurkat (ATCC TIB-152, E6-1 clone) was treated with 10% FBS (Invitrogen), 100 mg / L penicillin G, 100 mg / L streptomycin, 1.0 mM sodium (Invitrogen) supplemented with 0.5 g / L pyruvate, 1.5 g / L sodium bicarbonate, and 4.5 g / L glucose (all from Sigma). Human cervical cancer cell line Hela (ATCC S3) was cultured in DMEM (Invitrogen) supplemented with 10% FBS, 100 mg / L penicillin G, and 100 mg / L streptomycin. MCF-7 (human mammary carcinoma) cells were treated with 1.0 mM sodium pyruvate, 1% GlutaMax ™ -1 (Invitrogen), 100 μg / mL penicillin, 100 g / mL streptomycin, and 10% FBS Were cultured in supplemented DMEM. Human uterine sarcoma MES-SA and MES-SA / Dx5 cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 mg / L penicillin G and 100 mg / L streptomycin. All cells were incubated (incubation) at 37 ℃ in a humid atmosphere of 5% C0 ₂ is present. Other cell lines were maintained in a manner similar to that previously described.

(b) MTT assay.

 In order to evaluate the antiproliferative properties of the compounds of formula I, mitochondrial dehydrogenase activity was measured using MTT assay. Cell lines were prepared by trypsinization and seeding 4 x 10 cells per well with microplates. All compounds were reconstituted in dimethyl sulfoxide (DMSO) at a concentration of 100 mM or 25 mM before treatment. Cells were grown for 24 hours before treatment at concentrations ranging from 0.004 to 100 [mu] M and incubated in 200 [mu] L of media for 48 hours. 20 μL of MTT reagent in serum-free medium (5 mg / mL) was added to each well and incubated for an additional 2 hours. The medium was removed and the formazan crystals produced were redissolved in 100 μL of DMSO and the optical density (OD) was measured at a wavelength of 490 nm using a Thermomax Molecular Device plate reader. The experiment was performed four times and repeated at least twice for each compound per cell strain. Cells treated with 0.1% DMSO were used as a negative control whereas phenyl arsine oxide (PAO) was used as a positive death control.

(c) Cell cycle test

To evaluate the effect of analogues on the cell cycle, cell cycle analysis was performed. The initial density of the trypsinized HeLa cells and 70-80% fusion, 10% FBS, 2 × 10 ⁵ per well of cells in 6 well plates in the 100 mg / L of penicillin G and 100 mg / L streptomycin, supplemented DMEM Lt; / RTI > The cells were allowed to attach overnight and before treatment with the compound of formula I at concentrations ranging from 0.1 to 10 [mu] M. After 24 hours, the cells were washed three times with DMEM, trypsinized, pelleted and resuspended in 200 μL of culture medium containing 10 μM (2 μL / mL) of vybrant orange dye . The sample in the labeling solution was transferred to a Falcon tube and incubated in a water bath for 30 minutes at 37 < 0 > C. The samples were then analyzed using a Becton Dickinson FACscan flow cytometer with CellQuest software. Using a 488 nm laser with parameters that can be identified in Appendix B; 10,000 cases were collected for analysis. Cells treated with 0.1% DMSO were used as a negative control and cells treated with colchicine were used as a positive control.

(d) Cell apoptosis assay.

2 x 10 joule cart cells / mL were plated in 24 well plates, treated with the desired compound, and incubated for the required time (h).

Cells were centrifuged at 400 G for 1 min. The supernatant was discarded and the cells were resuspended in HHB (3 μL CaCl ₂ (1.5 M) per mL HHB, 2 μL per mL HHB (10 mg / mL) propidium iodide (Sigma) and 20 mL per 1 mL HHB V-FITC / propidium iodide solution in 100 μL of Annexin-V-FITC (Southern Biotech, Birmingham, AL). The sample in the labeling solution was transferred to a Falcon tube and incubated in a water bath at 37 占 폚 for 20 minutes. The samples were then analyzed using a Becton Dickinson FACscan flow cytometer with CellQuest software. The results were tabulated as% of Annexin-V-FITC positive apoptotic cells.

(e) Results

MTT  black: Anti-proliferation  activation

The compounds of formula (I) synthesized against antiproliferative activity against cancer cell lines of one panel, including human Hela cervix and MCF-7 breast adenocarcinoma, were evaluated. Many of the analogues exhibited the efficacy of nM, and the properties and results of clinically used anticancer agents are shown in Table 1. In addition, the selected compounds exhibited similar levels of activity against colon and head and neck cancer cell lines as well as cells derived from tumor metastasis (Table 1A).

Table 1. Anti-proliferative properties of the compounds of formula (I)

^a Concentration required to reduce the viability of cancer cells by 50% after 48 h of treatment with the indicated compound for DMSO control ± SD from two independent experiments, each of which was carried out repeatedly eight times, as determined by MTT assay.

Table 1A. The antiproliferative properties of the compound of formula (1)

^a Concentration required to reduce cell viability to 50% after 48 h with the indicated compound for DMSO control as measured by MTT assay. ^b ND = not measured.

Importantly, glioma, melanoma, non-small-cell lung, and many other types of cancer are known to be associated with a gloomy prognosis.

Thus, the increased incidence of glioma and melanoma has not been improved by improved therapeutic options over the years. Therefore, a new type of drug is urgently needed to combat cancer that rarely responds to current therapies. Thus, the human U737 hybrids (from GBM, from astrocyte), human Hs683 malignant brain glioma, human A549 non-small-cell-lung cancer (NSCLC), human SKMEL-28 And the most potent compounds Rig8, Rig9 and Rig35 against the mouse B16F10 melanoma cell line were tested and the results are shown in Table 2. Thus, the discovery that the compounds of formula I exhibit potent activity against cell lines exhibiting a cancer with a dark prognosis is highly encouraging.

Table 2. Anti-proliferative properties of the most potent compounds of formula I against cancer cell lines showing cancer with a dark prognosis

^a Concentration required to reduce the viability of cancer cells by 50% after 48 h of treatment with the indicated compound for DMSO control ± SD from two independent experiments, each of which was carried out repeatedly eight times, as determined by MTT assay.

Taxol and vinblastine-like drugs have been very successful in treating tumors early, but this is common when the tumor recurs and does not respond to these drugs due to overexpression of p-gp. The cells of such tumors are referred to as multiple drug resistance (MDR). Thus, compounds of formula I were evaluated for antiproliferative activity in MDR cancer cell lines. This was done using MES-SA / Dx5, an endometrial sarcoma cell overexpressing p-gp. The parent drug-reactive MES-SA cell line was used as a control.

Data for Rig8 and Rig35 as well as control Taxol and Vinblastin are shown in Table 3. As can be seen, Taxus and Vinblastin were approximately 1000-fold less potent for MDR cells than for parental MES-SA uterine sarcoma cells, although there was only a small change in the sensitivity of the parent and MDR cell lines using Rig8 and Rig35. These results suggest that the compounds of formula I are not sensitive to the action of p-gp and thus have significant clinical potential.

Table 3. Anti-proliferative effect of selected compounds of formula I against MDR cells

Cell cycle test

In a further anti-cancer assay of compounds of formula I, cell cycle assays were carried out using cell permeable DNA binding dye Vibri- orte orange. In a normal population, there are (2n) G ₀ / G ₁ cells in which there is a set of paired chromosomes per cell, followed by S or synthetic steps in which DNA is synthesized in the preparation for splicing (variable amounts of DNA are present during this step ) And finally the cell was distributed among the three major stages of the cell cycle of the (2n) G ₂ / M stage in which the chromosomes of each cell are now cloned. Such DNA content can be measured using fluorescent DNA selective probes, such as vibran-orange, that produce a signal proportional to the DNA mass per cell. This dye interacts with double-stranded DNA (dsDNA) in living cells, becomes fluorescent upon binding, and can thus be used to track DAN content as cells progress through various stages of the cell cycle using flow cytometry . Cell cycle analysis was performed on HeLa cells using Rig8 at various concentrations above and below the respective GI of ₅₀ . Since colchicine is known as an antimitotic drug, it was used as a positive control whereas Hela cells treated with 0.1% DMSO were used as a negative control. Figure 6 shows the results of this analysis using Rig 8 for Hela cells.

As can be seen in FIG. 6 and Table 4, Rig8 caused a dose-dependent cell cycle arrest at the G ₂ / M stage using Hela cell lines.

Table 4. Flow cytometric analysis of Rig8 using Hela cells

^a % relative DNA content after 24 h treatment of Hela cells with the indicated compound from two independent experiments, each performed 3 times ± SD

Apoptosis  black

A number of currently used chemotherapeutic agents are strongly associated with their ability to induce apoptosis in cancer cells, and accordingly, a number of major screens for novel anti-cancer directing agents have now been shown to inhibit the ability of compounds with antiproliferative activity It is not based on confirmation, but rather on the basis of their apoptosis inducing properties. Thus, compounds of formula I were tested for their ability to induce apoptosis in jurkat cells by flow cytometry annexin-V / propidium iodide assay, and after 48 hours of treatment with Rig8 at different concentrations, The proportion of apoptotic cells is shown in Fig.

As discussed specifically above, novel analogs of marine alkaloids were prepared using a new synthetic method developed for the construction of pyrrolo [2,3-d] pyrimidine ring systems. The synthesized compounds exhibited antiproliferative efficacy of sub-muM to nM against drug resistant cells, such as, for example, hybrids, melanoma and non-small cell lung cancer. The above-mentioned compounds include a compound (A, Fig. 8) in which position C2 is present unsubstituted. Thus, a further series of C2-substituted-deaza hypoxanthines (B, FIG. 8) were prepared and these compounds were determined to be evaluated for anticancer activity. In addition, such compounds have also been found to have potent antiproliferative activity against a number of cancer cell lines.

result

(a) a C2-substituted -7- Deaza hypophoxin  Synthetic and SAR

In order to develop a synthetic approach to C2-substituted 7-deazahypoxanthin B, the previously developed multicomponent reaction (MCR) of sulfonamidoacetophenone and aldehyde and cyanoacetamide was used to synthesize pyrrole 1 was prepared (Table 5). Thereafter, pyrimidine ring closure was followed using a reaction of 1 with various aromatic and aliphatic esters catalyzed by sodium ethoxide in ethanol reported for the associated purine system. Desirable 7-deaza hypoxanthine 2-17 was obtained in varying but acceptable yields (Table 5), indicating that in most cases these products are expected after acidification of the reaction mixture and no further purification is required do.

Table 5. Synthesis of C2-substituted -7-deaza hypoxanthin 2-17 and antiproliferative activity

^a MTT assay, the concentration required to reduce the viability of cancer cells to 50% after 48 h of treatment with the indicated compound for DMSO control < RTI ID = 0.0 > SD < / RTI > from two independent experiments, . ^b Synthesized using the method described in ref. 13 (ref. ^c CO (OEt) Obtained as a by-product of the reaction of ₂ and 1.

The obtained compounds were evaluated for antiproliferative activity using MCF-7 cells as a model for human cervical adenocarcinoma as a model for Hela cell and breast adenocarcinoma. Cells were treated with each compound for 48 h and cell viability was assessed using the MTT method (Table 5). The results demonstrated that most of the C2-substituted 7-deaza-hypoxanthine showed antiproliferative activity when compared to the ligandin-related 7-deaattactin 2 containing carbonyl groups at C2. The activity was found to be doubled to one-place μM for C2-aryl (5,6), C2-OEt (8) and branched C2-alkyl (9). The linear C2-alkyl derivative 10-12 showed efficacy of less than or equal to μM, and C2-pentynyl compounds 13 and 17 as well as C2-methyl were nM.

(b) C2- methyl -7- Deaza hypophoxin MCR - based synthesis and SAR

The potent activity of C2-methyl analog 13 led to further studies of C2-methyl compounds by altering the aldehyde-derived C7 aromatic group. In order to facilitate the synthesis of such compounds, a new four-component cyclic condensation of various aldehydes with methylsulfonamido acetophenone, cyanoacetamide and triethylorthosacetate was developed (Table 6). In this reaction, the entire pyrrolo [2,3-d] pyrimidine skeleton is assembled in one step, and its success depends on the optimized temperature range, wherein the reaction is first maintained at 90 [ (Similar to 1 in Table 5) and then maintained at 150 [deg.] C to bring the fourth component orthoacetate to the process, resulting in the closure of the pyrimidine moiety of the molecule.

Table 6. MCR-based synthesis and antiproliferative activity of C2-methyl-7-deaza hypoxanthine 13 and 18-23

^a Concentration required to reduce the viability of the cells to 50% after 48 h of treatment with the indicated compound for DMSO control ± SD from two independent experiments, each of which was repeated four times, as measured by MTT assay.

The anti-proliferative effects of compounds 13 and 18-23, in addition to the Hela and MCF-7 used previously, have been shown to be effective in the treatment of cancers, such as U-87 human oligodendrocytes and A549 human non- ) ≪ / RTI > carcinoma cells in vitro. The newly synthesized compound 18-23 was found to be inferior to the original C7-phenyl analog 13 but retained efficacies of less than < RTI ID = 0.0 > M < / RTI > except for the pyridine-containing variant 21,

(c) Quantitative video microscope

The bad prognosis of schizophrenia, melanoma, and NSCLC patients is generally attributed to these malignant drug resistant properties, and accordingly, 7-deaza hypoxanthine 13 and 18-23 have been shown to counteract U87 human melanoma and A549 NSCLC carcinoma cell lines It was encouraging to be active. Since the tubulin-targeting agent is generally less effective against cancer cells that are resistant to induction of apoptosis, compound 22 can be used against U373 human hybrids and SKMEL human melanoma cells, which have been reported to exhibit apoptotic resistance And further evaluated. MTT-based GI ₅₀ values of 0.3 μM obtained in both of these cell lines indicate that these compounds retain similar levels of activity (compared to GI ₅₀ values in Table 6) in these cell types. Computer-assisted phase-contrast microscopy (quantitative video microscopy) was used to confirm cell death with the principle mechanism of action associated with the in vitro growth inhibition of these compounds in these cell lines.

As shown in the following cell image (FIG. 9), 22 inhibited cancer cell proliferation by inducing apoptosis when actually assayed at its MTT-based GI ₅₀ values in U373 and SKMEL melanoma cells.

Example

(a) Synthetic chemistry

All reagents, solvents and catalysts were purchased from commercial sources (Acros Organics and Sigma-Aldrich) and used without purification. All reactions were performed in open-air or oven-dried flasks under nitrogen and applied by TLC pre-coated (250 μm) silica gel 60 F254 glass-backed plates (EMD Chemicals Inc.). Visualization was achieved by UV light. Flash column chromatography was performed on silica gel (32-63 [mu] m, 60 A pore size). ^{The 1} H and ¹³ C NMR spectra were recorded on a Bruker 400 spectrometer. Chemical shifts (δ) were reported in ppm relative to the TMS internal standard. The abbreviations are: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) . HRMS analysis was performed using Waters Synapt G2 LCMS. The purity of> 95% of the compound synthesized by UPLC / MS analysis was confirmed.

Example 1: 2-Amino-5-benzoyl-4-phenyl -1H- pyrrole-3-carboxamide (1): N- methyl sulfonamido acetophenone ¹³ (0.084 g, 0.4 mmol) in EtOH (3 mL), To the stirred solution of benzaldehyde (0.055 g, 0.52 mmol) and cyanoacetamide (0.044 g, 0.52 mmol) was added anhydrous granulated K ₂ CO ₃ (0.028 g, 0.2 mmol). The mixture was then refluxed for 14 h. The reaction mixture was then cooled to room temperature and concentrated. The crude product was purified by flash chromatography on silica gel using CH ₂ Cl ₂ / AcOEt (2: 1) as eluent to give 74 mg of desired pyrrole 1 (61% yield).

Example 2: 2-Amino-5-benzoyl-4-phenyl-1H-pyrrole-3-carboxamide (1): 61%;

_{C 18 H 16 N 3 0 2} (M + H) HRMS m / z (ESI) calcd for 306.1243, found 306.1248.

General Procedure for Synthesis of Deaza Hypoxanthin 3-17. The selected ethyl ester (1.28 mmol) and pyrrole 1 (50 mg, 0.16 mmol) were added to a solution of EtONa in EtOH prepared by dissolving sodium metal (30 mg, 1.3 mmol) in EtOH (2 ml). The mixture was then refluxed overnight for 10 h. The reaction mixture was then diluted with H ₂ O and neutralized with 1M HCl. The formed precipitate was collected by filtration and dried under vacuum overnight. In most cases, the product deaza hypoxanthine was> 95% pure, but they could be further purified using column chromatography (5% MeOH in CHCl ₃ ).

Selected characterization data for the compounds of Table 5:

Example 3: 6-Benzoyl-2-phenyl-5-phenyl-lH-pyrrolo [2,3-d] pyrimidin-4 (7H) -one (3): 41%;

HRMS m / z (ESI) calcd for C ₂₅ H ₂₀ DN ₄ O ₂ (M + NH ₄ ) 410.1727, found 410.1722.

Example 4: 6-benzoyl-2- (2-ethoxycarbonylethyl) -5-phenyl-lH-pyrrolo [2,3-d] pyrimidin-4 (7H) -one (10): 60%;

HRMS m / z (ESI) calcd for C ₂₄ H ₂₁ N ₃ NaO ₄ (M + Na) 438.1430, found 438.1431.

Example 5: 6-Benzoyl-2-butyl-5-phenyl-lH-pyrrolo [2,3-d] pyrimidin-4 (7H) -one (11): 42%;

HRMS m / z (ESI) calcd for C ₂₃ H ₂₁ N ₃ NaO ₂ (M + Na) 394.1531, found 394.1528.

Example 6: 6-Benzoyl-2-methyl-5-phenyl-lH-pyrrolo [2,3-d] pyrimidin-4 (7H) -one (13): 49%;

C ₂₀ H ₁₅ N ₃ NaO ₂ Calculated HRMS m / z (ESI) for (M + Na) 352.1062, found 352.1068.

Example 7: 6-Benzoyl-2- (difluoromethyl) -5-phenyl-lH-pyrrolo [2,3-d] pyrimidin-4 (7H) -one (15): 47%;

_{C 20 H 14 F 2 N 3} O 2 Calcd HRMS m / z (ESI) for (M + H) 366.1054, Found 366.1054.

Example 8: 6-Benzoyl-5-phenyl-2- (trifluoromethyl) -lH-pyrrolo [2,3-d] pyrimidin-4 (7H) -one (16): 66%;

_{C 20 H 12 F 3 N 3} Na0 2 Calculated HRMS m / z (ESI) for (M + Na) 406.0779, found 406.0777.

General procedure for the synthesis of deaza hypoxanthine 13 and 18-23. EtOH (2.5 mL) and MeC (OEt) ₃ (2.5 mL), N- (2- oxo-2-aryl-ethyl) -methanesulfonamide (0.676 mmol), the selected aldehyde (0.879 mmol), cyanoacetamide in a mixture of ( to _{0.072 g, K 2 C0 3 (} 0.052g, 0.372 mmol) of anhydrous granular at a time to a solution of Chemistry 0.879 mmol) was added. The mixture was purged for 5 minutes with N _2, and heated under a nitrogen atmosphere for 24 hours at 90 ℃. The formation of the intermediate pyrrole was monitored by TLC. After the reaction temperature was increased to 150 ° C, the reaction mixture was heated for 3-6 h. The mixture was cooled to room temperature and the precipitate formed was collected by filtration and washed with EtOH (2 mL) and Et ₂ O (2 mL) to provide the desired 7-deaza hypoxanthine 13 and 17-23. An additional amount of product was obtained by evaporation of the mother liquor and purification of the residue by column chromatography with MeOH / CH ₂ Cl ₂ = 1 / 40-1/20 gradient.

Characterization data for the compounds of Table 2

Example 9: 6-benzoyl-5- (3-chlorophenyl) -2-methyl- lH- pyrrolo [2,3- d] pyrimidin-4 (7H)

C ₂₀ H ₁₅ ClN ₃ O ₂ Calcd HRMS m / z (ESI) for (M + H) 364.0853, Found 364.0851.

Example 10: 6-benzoyl-5- (3-bromophenyl) -2-methyl- lH- pyrrolo [2,3- d] pyrimidin-

HRMS m / z (ESI) calcd for C ₂₀ H ₁₄ BrN ₃ NaO ₂ (M + Na) 430.0167, found 430.0168.

Example 11: 6-Benzoyl-5- (3-fluorophenyl) -2-methyl-lH-pyrrolo [2,3-d] pyrimidin-4 (7H) -one (19): 43%;

C ₂₀ H ₁₄ FKN ₃ O ₂ Calcd HRMS m / z (ESI) for (M + K) 386.0707, found 386.0708.

Example 12: 6-Benzoyl-2-methyl-5- (pyridin-3-yl) -lH- pyrrolo [2,3-d] pyrimidin-4 (7H) -one (20): 65%;

C ₁₉ H ₁₄ N ₄ Na0 ₂ Calculated HRMS m / z (ESI) for (M + Na) 353.1014, found 353.1019.

Example 13: 6-benzoyl-5- (5-bromopyridin-3- yl) -2-methyl- lH- pyrrolo [2,3- d] pyrimidin- ;

_{C 19 H 14 BrN 4 0 2} (M + H) HRMS m / z (ESI) calcd for 409.0300, found 409.0287.

Example 14: 6-Benzoyl-5- (3,5-dibromophenyl) -2-methyl-lH-pyrrolo [2,3-d] pyrimidin-4 (7H) -one (22): 58%;

_{C 20 H 14 Br 2 N 3} 0 2 HRMS m / z (ESI) for (M + H ⁺⁾ calcd 485.9453, found 485.9438.

Example 15: 6-Benzoyl-2- (pent-4-ynyl) -5-phenyl-lH-pyrrolo [2,3-d] pyrimidin-4 (7H) -one (23): 72%;

HRMS m / z (ESI) calculated for C ₂₄ H ₁₉ N ₃ NaO ₂ (M + Na) 404.1375, found 404.1374.

(b) Cell culture

Human cancer cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), the European Collection of Cell Culture (ECACC, Salisbury, UK) and Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) . Human cervical adenocarcinoma cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). Human breast carcinoma MCF-7 cells were cultured in RPMI supplemented with 10% FBS. U87 and U373 cells were cultured in DMEM culture medium (Lonza cord 12-136F, Verbier, Belgium) while SKMEL-28 and A549 cells were cultured in 10% heat-inactivated FBS (Lonza, FBS South America Code DE14-801 F ) Supplemented with RPMI culture medium (Lonza; code 12-115F). Cell culture medium was supplemented with 4 mM glutamine (Lonza cord BE 17-605E), 100 μg / ml gentamycin (Lonza code 17-5182), and penicillin-streptomycin (200 units / ml and 200 μg / ml) -602E). All cell lines were cultured in T25 flasks, maintained, and grown at 37 ° C, 95% humidity, 5% CO ₂ .

(c) Anti-proliferation  characteristic

MTT assay was used to evaluate the antiproliferative properties of the synthesized compounds. Cell lines were evaluated by trypsin treatment of each cell line and seeding 4 x 10 ³ cells per well in a 95-well plate. All compounds were dissolved in DMSO at a concentration of 100 mM or 50 mM before cell treatment. Cells were grown for 24 h and then treated with compounds at concentrations ranging from 0.04 to 100 [mu] M and incubated in 200 [mu] l of media for 48 h. 20 μL of MTT reagent in serum-free medium (5 mg / mL) was added to each well and incubated for an additional 2 h. The medium was removed and the formazan crystals produced were redissolved in 200 [mu] L of DMSO. A ₄₉₀ was measured using a Thermomax Molecular Device plate reader. The experiment was performed 4 times and repeated at least twice for each compound per cell line. Cells treated with 0.1% DMSO were used as controls and 1 [mu] M phenyl arsenic oxide (PAO) was used as a positive death control.

d) Quantitative video microscope

The effect of 22 on the viability of human U373 hybrids and SKMEL melanoma cells was characterized in vitro using a computer-assisted phase contrast video microscope as described elsewhere.

The present invention, of course, is by no means limited to the specific disclosure of the specification and drawings, as well as any variations within the scope of the appended claims.

Claims (12)

Compounds having the formula:

Wherein R, R ₁ and R ₂ are selected from the group consisting of hydrogen, aryl, fused aryl, heteroaryl, saturated carbocyclic, partially saturated carbocyclic, saturated heterocyclic, partially saturated heterocyclic, C _{1 -10} alkyl, haloalkyl, alkenyl, alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroaryl alkenyl, alkyl, alkenyl, alkynyl, alkyl, a carbonyl-cycloalkyl, heterocycloalkyl, hydroxy Hydroxyalkyl, aminoalkyl, carboxyalkyl, nitroalkyl, cyanoalkyl, acetamidoalkyl, and acyloxyalkyl. Comprising administering an effective amount of one or more compounds of formula < RTI ID = 0.0 > (I). ≪ / RTI > Comprising administering an effective amount of at least one compound of the formula of claim 1. A method of treating breast cancer, comprising administering an effective amount of at least one compound of formula (I). A method of treating brain cancer comprising administering an effective amount of at least one compound of the formula of claim 1. A method of treating a melanoma, comprising administering an effective amount of at least one compound of formula (I). Comprising administering to a patient in need thereof an effective amount of a compound of formula < RTI ID = 0.0 > 1 < / RTI > A method of treating head and neck cancer, comprising administering an effective amount of at least one compound of formula (I). A method of treating tumor metastasis comprising administering an effective amount of at least one compound of formula (I). A pharmaceutical composition comprising a compound of formula (I) as defined above, as well as a pharmaceutically acceptable salt and / or solvate thereof, and a pharmaceutically acceptable carrier. A process for preparing a compound of formula (I) wherein R = H or R = Me, comprising reacting cyanoacetamide or a synthetic equivalent thereof and triethylorthoformate or triethylorthosacetate, or a synthetic equivalent thereof As well as in the presence of a suitable base and a solvent, with a compound of formula (III): < EMI ID =

In Formula II, R ₂ is hydrogen, aryl, fused aryl, heteroaryl, saturated carbocyclic, partially saturated carbocyclic, saturated hetero cyclic, part cyclic, saturated heterocyclic, C _1-10 alkyl, Alkyl, alkenyl, alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, carbocycloalkyl, heterocycloalkyl, hydroxyalkyl, aminoalkyl, carboxyalkyl, nitroalkyl , Cyanoalkyl, acetamidoalkyl, and acyloxyalkyl,
In Formula III, R ₁ is hydrogen, aryl, fused aryl, heteroaryl, saturated carbocyclic, partially saturated carbocyclic, saturated hetero cyclic, part cyclic, saturated heterocyclic, C _1-10 alkyl, Alkyl, alkenyl, alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, carbocycloalkyl, heterocycloalkyl, hydroxyalkyl, aminoalkyl, carboxyalkyl, nitroalkyl , Cyanoalkyl, acetamidoalkyl, and acyloxyalkyl. A process for the preparation of a compound of formula (R ≠ H and R ≠ Me)
Comprising the step of forming a suitable base and in the presence of ethyl carboxylate to a compound of formula 1 (RC0 ₂ Et) or methyl (RC0 ₂ Me) and the ester compound of the formula by reacting one or more of claim 1 in a solvent:

Wherein R ₁ and R ₂ are independently selected from the group consisting of hydrogen, aryl, fused aryl, heteroaryl, saturated carbocyclic, partially saturated carbocyclic, saturated heterocyclic, partially saturated heterocyclic, C _{1 -10} alkyl, haloalkyl, alkenyl, alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroaryl alkenyl, carbonyl cycloalkyl, heterocycloalkyl, hydroxyalkyl, aminoalkyl, carboxyalkyl , Nitroalkyl, cyanoalkyl, acetamidoalkyl, and acyloxyalkyl,
In the formula RC0 ₂ Et or RC0 ₂ Me, R is aryl, fused aryl, heteroaryl, saturated carbocyclic, partially saturated carbocyclic, cyclic, saturated heterocyclic, partially cyclic, saturated heteroaryl, C _{1 -10} alkyl, haloalkyl, alkenyl, alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroaryl alkenyl, alkyl, alkenyl, alkynyl, alkyl, a carbonyl-cycloalkyl, heterocycloalkyl, hydroxy Hydroxyalkyl, aminoalkyl, carboxyalkyl, nitroalkyl, cyanoalkyl, acetamidoalkyl, and acyloxyalkyl.

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