Patent Application
20230266302
Sulfonamides are a crucial class of bioisosteres that are prevalent in a wide range of pharmaceuticals. Unfortunately, the ability to produce sulfonamides directly from heteroaryl aldehyde reagents remains limited. In particular, methods are needed for the direct installation of sulfonamide functionality on heteroarene scaffolds. This disclosure provides new methods for the production of a wide range of N-benzyl sulfonamides via a one-pot reduction of intermediate N-heteroarene-containing N-sulfonyl imines generated from commercially available aldehydes under mild conditions. Additionally, the following disclosure provides a new approach for regioselective incorporation of a sulfonamide unit to heteroarene scaffolds.
In one aspect, the present disclosure provides a method of preparing N-benzyl sulfonamides. The method comprises the steps of adding a sulfonamide and a heteroaromatic compound having a functional group such as an aldehyde, alcohol, ketone or amine to a single reaction vessel. The reaction is initiated by adding elemental iodine and an oxidizing agent such as a hypervalent iodine compound to the reaction vessel under non-acidic conditions. The resulting compound is an imine which is then reduced to the desired sulfonamide.
Also provided is a compound for treating cancer comprising a N-benzyl sulfonamide and a metabolic inhibitor.
Also provided is a method for determining the ATP levels of a cell following treatment with a metabolic inhibitor and a N-benzyl sulfonamide.
Throughout this disclosure, the terms “about”, “approximate”, and variations thereof, are used to indicate that a value includes the inherent variation or error for the device, system, the method being employed to determine the value, or the variation that exists among the study subjects.
Method for Preparing Sulfonamides from Heteroaryl Aldehydes
In one embodiment, the method disclosed reacts a heteroaromatic compound having a functional group such as an aldehyde or precursor to an aldehyde as reflected in
Alternatively, the step of converting the sulfonamide to an iminoiodinane in situ can be skipped. In this case the iminoiodinane reagent can be prepared in advance of the reaction and used as the N-substrate as reflected in
A non-limiting list of suitable hypervalent iodine reagents for use in the present method would include: phenyliodine(III) diacetate (PhI(OAc)2), phenyliodine bis(trifloroacetate) (PIFA), iodosylbenzene (PhIO), [hydroxyl(tosyloxy)iodo]benzene (HTIB, Koser reagent), PhICl2, TolIF2, diaryl iodonium salts, Togni's reagents, μ-oxobis(trifluoroacetoxyiodobenzene) (μ-oxo BTI), Dess-Martin periodinane, 2-Iodoxybenzoic acid (IBX), cyano(trifluoromethylsulfonyloxy)iodobenzene (Stang's reagent), 1-phenyl-2-(phenyl-λ3-iodaneylidene)-2-((trifluoromethyl)sulfon-yl)ethan-1-one (Shibata reagent II), iodosodilactone. In general, any hypervalent iodine compound will function in the reactions described herein provided that it serves as an oxidizing agent to form intermediate iminoiodinane in conjunction with a sulfonamide reagent.
A non-limiting list of heteroaromatic compounds suitable for use in the following method include but are not limited to those structures identified in
Reducing agents appropriate for use in the following method include but are not limited to: sodium borohydride, lithium aluminum hydride, lithium borohydride, diisobutyl aluminum hydride, sodium cyanoborohydride, sodium triacetoxyborohydride, 9-borabicyclo(3.3.1)nonane (9-BBN), and Hantzsch ester. Those skilled in the art will be familiar with other approaches for reducing the sulfonyl imine to the sulfonamide. For example, a catalytic reduction in the presence of hydrogen will perform satisfactorily.
As discussed above, the two-step method utilizes R3, a heteroaromatic compound having a functional group R2 capable of reacting with the N-sulfonamide component of the N-substrate.
The reaction takes place over a period of about 8 to about 48 hours with stirring or mixing at a temperature of about 20° C. to about 60° C. under an atmosphere that is inert to the reactants. Typically, the reaction takes place over a period of about 18 hours to 26 hours with stirring or mixing at a temperature of about 40° C. to about 60° C. under argon gas. Other gases suitable for use in this method would include: nitrogen or air. Following the reaction period, the reaction products are typically cooled to room temperature, e.g. a temperature of about 17° C. to about 22° C., followed by removal of the any remaining liquid component. The remaining solid material is dissolved in a mixture of methanol and dichloromethane and cooled to a room temperature, e.g. about 17° C. to about 22° C. Alternative solvents for use in this step include, but are not limited to: ethanol, isopropyl alcohol, chloroform, ethyl acetate, diethyl ether, acetonitrile, tetrahydrofuran, and dichloroethane. The amount of solvent used in this step is not critical. In general, that amount sufficient to completely dissolve the reaction products will be used. The first step of the reaction process is complete when the intermediate imine product is formed. The presence of the sulfonyl imine product can be confirmed by nuclear magnetic resonance (1H NMR, 13C NMR) and mass spectrometry.
Following dissolution of the initial reaction products in solvent, the conversion of the sulfonyl imine intermediate to the sulfonamide occurs by a reduction reaction. In most instances, the reduction of the sulfonyl imine intermediate can take place in the same reaction vessel as the first step. Thus, the preparation of the sulfonamide on heteroaromatic scaffold can be referred to as a “one pot” method. The reduction of imine to sulfonamide is brought about by stepwise addition of a reducing agent, such as sodium borohydride, to the reaction vessel while the reaction vessel is at a temperature less than 40° C. Approximately, 5 equivalents of sodium borohydride are added over a period of 1-5 minutes followed by 10 minutes of stirring. Then a second portion of 5 equivalents of sodium borohydride is added over a period of 1-5 minutes followed by an additional 10 minutes of stirring. The desired temperature during the addition of the reducing agent is that temperature which prevents potential decomposition of the product. After approximately 10 minutes to about 45 minutes the solution is allowed to warm to room temperature or a temperature of about 20° C. with continued stirring or mixing. Any remaining reducing agent is neutralized by addition of water. A solvent extraction process is used to isolate the resulting sulfonamide which is subsequently dried by treatment with sodium sulfate under a vacuum. Final purification of the sulfonamide can be performed by flash chromatography or any other convenient method such as recrystallization. Typical flash chromatography will use a blend of hexanes and ethyl acetate as the eluent. Advantageously, the reaction pathways of the present method preclude the formation of byproducts such as would occur under benzylic amidation via C—H activation.
Without intending to be bound by theory and simply to provide a better understanding of the reactions described herein,
The following examples demonstrate the use of a variety of heteroaromatic compounds as the R3 scaffold material for production of N-benzyl-sulfonamides and a variety of N-substrates with functional groups R1. The final products are depicted as compounds 1-30 in
In addition to the foregoing N-benzyl sulfonamides, the following compounds have also been generated using the methods discussed above. Each of the foregoing and following compounds are depicted in
Many of the resulting N-benzyl sulfonamides produced by the foregoing method will have pharmacological properties. In particular, many of the compounds will be effective anti-cancer compounds. To determine which compounds will be effective as anti-cancer compounds a new method is needed.
In addition to providing the novel method for preparation of N-benzyl sulfonamides, the present disclosure also provides a method for determining the effectiveness of the resulting N-benzyl sulfonamides as pharmacological compounds. Depending on the location of the sulfonamide functionality on the scaffold and the type of sulfonamide functionality, the resulting N-benzyl sulfonamides should have effectiveness in treatment of cancer. The following methods were developed to assess the in vitro effectiveness of the resulting compounds when combined with a metabolic inhibitor.
The N-benzyl sulfonamide library of compounds of
The cytotoxicity tests were carried out in the following manner. Living cells are known to convert resazurin to the fluorescent compound resorufin. Test systems which rely upon this reaction are commercially available. One such test is known at the Cell Titer Blue Cell Viability test assay from Promega. Cell cultures for each of the identified cancer lines were obtained from ATCC and maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and pen/strep. As known to those skilled in the art, DMEM typically includes the components identified below.
As known to those skilled in the art, pen/strep is a combination of penicillin and streptomycin used to prevent bacterial and fungal contamination of mammalian cell cultures. The pen/strep solution contains 5,000 Units of Penicillin G (sodium salt) which acts as the active base, and 5,000 micrograms of Streptomycin (sulfate) (base per milliliter), formulated in 0.85% saline.
The test method provides for incubating the cell cultures at 37° C. Additionally, the cell cultures are kept under an atmosphere containing 5% CO2. After allowing for proliferation of the cells, the cells were distributed across a plurality of test cells containing from 100 μL DMEM plus 10% FBS and allowed to attach to the surface of the cells. Typically, the time for attachment will require about 12 hours to about 18 hours. Following attachment, the cells were treated with either a solvent control or the N-benzyl sulfonamide compound of interest dissolved in a suitable solvent such as but not limited to DMSO. Typically, about 18 hours to about 36 hours are required to determine the effect of the N-benzyl sulfonamide compound of interest on cell viability. Following treatment of the cells with the N-benzyl sulfonamide or control, the toxicity of the compound to the cells will be determined by addition of 10 μl of CellTiter-Blue reagent, i.e. resazurin. Typically, the resazurin is added between about 18 hours to about 36 hours after treating the cells with the N-benzyl sulfonamides or the control. The cells are allowed to consume and convert the resazurin to resorufin for about one to four hours. Subsequently, the fluorescence of resazurin is measured by excitation at 560 nm and recording the emission at 590 nm within an instrument configured to measure fluorescence intensity. Two commercially available systems are the BioTek Cytation 5 plate reader and the Promega Glomax Multi+detection system. For compounds exhibiting cytotoxicity, the half maximal inhibitory concentration (IC50) values were determined using non-linear regression analysis in Graph-Prism software. The method for determining IC50 values is well known in the art and will not be discussed further. As known to those skilled in the art, IC50 is a quantitative measure that indicates how much of a particular inhibitory substance (e.g. drug) is needed to inhibit, in vitro, a given biological process or biological component by 50%. Therefore, each N-benzyl sulfonamide will be tested over a range of concentrations. Typically, the concentration ranges will be: 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM. The control in this method is normally dimethyl sulfonamide (DMSO) or other solvent suitable for dissolving the compounds to be tested.
Method for Determining ATP Levels Following Treatment with Two Component Compositions
The conventional cytotoxicity screening method described above can identify compounds that on their own reduce cell viability; however, the above method will miss biologically active compounds that do not cause cell death. The above method does not provide any information about a compound's biological targets or mechanism of action. Therefore, one aspect of the present invention includes a screening method suitable for identifying compounds which directly inhibit ATP metabolism by select pathways. Additionally, the following method permits identification of the pathway inhibited. Further, the rapid testing method does not require cell death during the exposure of the cells to the compound of interest.
The improved method for identifying such compounds measures ATP levels as a function of light emitted by any ATP dependent luciferase or modified luciferases, i.e. luciferase derivatives. The method uses a conventional luminescent assay to determine the number of viable cells in a culture. The improvement provided by the present method results from pre-treating cells used in the assays with a metabolic inhibitor prior to treatment with the compound of interest. Luminescent assays for determining cytotoxicity are well known in the art. Assays, kits and methods for measuring ATP levels are disclosed by U.S. Pat. Nos. 7,741,067 and 7,083,911, incorporated herein by reference. Commercially available assays, marketed as the CellTiter-Glo® and CellTiter-Blue® from Promega Corporation, are particularly suited for carrying out the method described below; however, other similar luminescent or fluorescent assays will perform equally well in the described method.
The commercially available assays are configured for the purposes of determining cell viability. In normal usage, the test determines ATP levels in untreated cells, i.e. the control. Corresponding cells are treated with an agent of interest that is suspected of reducing cell viability. In the common practice, resulting data is presented as a comparison of the ATP levels in the treated and untreated cells with the decrease in ATP levels indicative of the effectiveness of the agent. The data may be presented as a direct comparison of the assay output levels or as a percentage using the untreated control cells ATP level as 100%.
In most instances, following the treatment period, the commercially available assays use a lysis buffer to break the cells apart and release ATP. The releasing agent also contains an enzyme which catalyzes a light emitting reaction. Typically, the enzyme is luciferase and its substrate D-luciferin. In the presence of ATP, luciferase catalyzes a reaction that emits light (see
In the present method, the method of using the available assays has been modified to measure the short-term effect of compounds of interest on ATP levels in the cells. The present method does not result in cell death and does not measure cell viability. In the modified method, the control and test cells are initially treated with the metabolic inhibitor for a period of about thirty minutes to about four hours. Typically, the treatment of the cells with the metabolic inhibitor is for one hour prior to adding the compound to be screened for anti-cancer properties. However, simultaneous treatment with the metabolic inhibitor and the compound being screened should provide satisfactory results. Thus, the modified luminescent assays have been adapted to screen for compounds that directly inhibit ATP metabolism. ATP synthesis in cells occurs over multiple biochemical pathways. These pathways are very responsive to metabolic inhibitors and/or changes in available nutrients. By incorporating a metabolic inhibitor which is known to impact certain pathways, the disclosed method provides for measurement of a compound's direct effects on the remaining metabolic pathways available for ATP synthesis in the cell.
Because cancers exhibit dysregulation of metabolic pathways, compounds identified in the disclosed method are potential anti-cancer therapeutics. In this screening methodology, cells used in the assays are pretreated with a metabolic inhibitor such as 2-deoxyglucose (2-DG) (inhibits ATP production via glycolysis) or rotenone (inhibits ATP production by mitochondria). As a consequence of the pre-treatment with a metabolic inhibitor, the cell must utilize the remaining uninhibited pathways to maintain cellular ATP levels.
Following treatment of the control and test cells with the metabolic inhibitor, the method calls for addition of the compound to be screened for anti-cancer properties to the cells. Following addition of the compound of interest, the assay is allowed to continue for about one hour to about four hours or depending on the luminescing agent up to eighteen hours. However, approximately 60 minutes will be sufficient to determine the ATP inhibiting effect of the compound to be screened on the cells. Following completion of the selected time period, the ATP level within the cell is determined. ATP levels can be determined by luminescence according to standard measuring procedures, i.e. the luminescence level of the assay from the living cells treated with the two component composition is compared to the luminescence level of the assay from the living cells without the two component composition, i.e., the control experiment. Thus, the ATP levels are determined without killing the cells during the incubation of the cells in the presence of the compound of interest. As a result, the measured reduction in ATP level corresponds directly to the inhibition of the metabolic pathways uninhibited by the metabolic blocker.
Thus, the method provides the ability to screen compounds for the ability to specifically target the uninhibited pathway being used to generate ATP. Furthermore, because cells pre-treated with a metabolic inhibitor targeting a first known pathway are forced to use an alternative known pathway that is not inhibited to maintain ATP levels, the screening method provides immediate mechanistic information about the active compound mechanism of action. These results are provided in a relatively short time period of about ninety minutes to about five hours.
A wide variety of ATP luminescing detection reagents are available commercially. So long as the reagent produces a luminescence in the presence of ATP, the reagent will be suitable for use in the present method. Suitable reagents include but are not limited to any ATP dependent luciferase such as but not limited to firefly luciferase, other modified luciferase based reagents, i.e. luciferase derivatives. According to the rapid method for determining the anti-cancer activity of a compound, a sample of living cells is distributed across a number of testing wells. Typically, 96-well plates are used; however, the number of wells is not critical to the current method. When using a 96-well plate the number of living cells will commonly be about 20,000. The sample wells contain a cell growth medium to promote cell health and growth and an additive to prevent bacterial contamination of the wells. One common example of the cell growth medium is DMEM with 10% FBS as described above. One example of the additive to prevent bacterial contamination is a solution of penicillin G and streptomycin referred to commonly as Pen-Strep. The Pen-Strep solution typically contains 5000 units of penicillin G and 5000 micrograms of streptomycin. Thus, the rapid determination of anti-cancer compounds can be carried out using the following method.
Table 1 reports the POC values for a variety of compounds of interest. The number in the far left column of Table 1 corresponds to the compound number of compounds depicted in
As reported in Table 1 below, one group of assays included only the compound of interest. Another group of assays included the compound of interest in combination with 2-deoxyglucose and a third group of assays included the compound of interest with rotenone. Other metabolic inhibitors suitable for use in the disclosed method include but are not limited to: 2-deoxyglucose, rotenone, Lonidamine, 3-bromopyruvate, imatinib, oxythiamine, and 6-aminonicotinamide Glutaminase Inhibitor 968, 6-Diazo-5-oxo-L-norleucine, Amytal, Antimycin A, Sodium Azide, Cyanides, oligomycin, FCCP, Phloretin, Quercetin, 3BP, 3PO, DCA, NHI-1 and Oxamic acid, Fisetin, myricetin, apigenin, genistein, cyanidin, daidzein, hesperetin, naringenin, and catechin.
In Table 1 below, the cell lines tested are reported across the top row of the table. The POC values are reported for each compound of interest and each combination of interest in combination with 2DG or rotenone. A POC value of less than 50 reflects the likely inhibition of the cell line by the compound of interest or the combination of the compound of interest with the indicated metabolic inhibitor. Compound 2 in particular demonstrated reduction in luminescence, corresponding to reduced ATP activity by the cells, across many of the cell lines. When combined with 2-DG compound, 2 showed effectiveness against each cell line and a remarkable value for BxPC3 the pancreatic cancer cell line. Compound 2 would also be expected to have effectiveness against other pancreatic cancer cell lines.
While the resulting N-benzyl sulfonamides provided by the method discussed above have shown some effectiveness in vitro against select cancer cell lines, further toxicity against cancer cells would be desired. To that end, the present disclosure also provides a two-component composition which has shown a synergistic effect against cancer cells in vitro.
The two-component composition consists of a N-benzyl sulfonamide and a metabolic inhibitor. In one embodiment, the metabolic inhibitor is 2-deoxyglucose (2-DG). In another embodiment, the metabolic inhibitor is rotenone. Other metabolic inhibitors suitable for use in the two-component composition are: Lonidamine, 3-bromopyruvate, imatinib, oxythiamine, and 6-aminonicotinamide Glutaminase Inhibitor 968, 6-Diazo-5-oxo-L-norleucine, Amytal, Antimycin A, Sodium Azide, Cyanides, oligomycin, FCCP, Phloretin, Quercetin, 3BP, 3PO, DCA, NHI-1 and Oxamic acid, Fisetin, myricetin, apigenin, genistein, cyanidin, daidzein, hesperetin, naringenin, and catechin. While subsequent in vivo testing may determine a narrower range for the ratio of the N-benzyl sulfonamide to the metabolic inhibitor, the current ratio that has demonstrated effectiveness against cancer lines, as identified in the table below, is in the range of about 1:50 to about 1:1500. Thus, the metabolic inhibitor may comprise from about 75% by weight to about 99.99% by weight of the composition containing both N-benzyl sulfonamide to the metabolic inhibitor where the N-benzyl sulfonamide has the structure set forth in
The Table of
Table 1 below provides the results of testing 30 different N-benzyl sulfonamides, as depicted in
While the two-component composition of N-benzyl sulfonamide with rotenone showed effectiveness against several cell lines, the combination of 100 μM N-benzyl sulfonamide compound number 2 as identified in
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Other embodiments of the present invention will be apparent to one skilled in the art. As such, the foregoing description merely enables and describes the general uses and methods of the present invention. Accordingly, the following claims define the true scope of the present invention.
The present application claims priority to U.S. Provisional Application No. 63/056,211 filed on Jul. 24, 2020, which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/043012 | 7/23/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63056211 | Jul 2020 | US |
