KR20230171296A - A method for preparing acylsulfonamide-based DNA-encoding compound - Google Patents

Abstract

The present invention relates to a DNA-encoded library, which is characterized by being linked with an acylsulfonamide functional group. The present invention also relates to a method for producing an acyl sulfonamide-based DNA-encoding library, which is characterized in that it is produced by reacting sulfonyl azide and an alkyne compound under mild conditions using a copper catalyst in a water solvent.

Description

Method for preparing acylsulfonamide-based DNA-encoding compound {A method for preparing acylsulfonamide-based DNA-encoding compound}

The present invention relates to a method for producing DNA-encoded compounds necessary for constructing a DNA-Encoded Library (DEL). The present invention also relates to DEL libraries with new functional groups.

The importance of new screening platforms for deriving key substances in new drug development has always been clear, and in small molecule drug development, screening of hundreds of thousands of compound libraries is considered the starting point in determining the success of new drug development. However, in order to build a high-throughput screening (HTS) system to screen a large number of compounds, there are many financial and spatial constraints depending on the size and type of the library.

To overcome this, DNA is combined with small molecule compounds and used as a barcode. As a result, DNA-encoded library technology that enables rapid analysis began to emerge ("DNA-Encoded Chemistry: Drug Discovery from a Few Good Reactions", Chem. Rev. 2021, 121, 12, 7155-7177). This technology has been improved over a long period of time and is currently used by many pharmaceutical companies in conjunction with HTS to derive various new drug candidates. For example, small molecule inhibitors targeting RIPK1 and sEH in clinical trials developed by GSK were selected through chemical modification of hit compounds obtained from DNA encoding library screening.

However, existing synthetic methods for constructing DNA-encoded libraries are limited. Above all, there are limits to the use of various bases because the reaction must be performed in a polar solvent, such as water, in which DNA dissolves. The use of acids in the reaction can affect the stability of the DNA, and at high temperatures, the DNA becomes depurinated and loses its role as a barcode to recognize attached small molecules. As a result, reactions performed under conventional heating conditions are not applicable to the preparation of DNA-conjugation compounds. In addition, because the negative charge of phosphate in the DNA framework has a metal chelating effect, there is a problem in the application of recently developed transition metal catalysts. For this reason, in order to expand the versatility of the current technology, the development of a reaction to construct a DNA-encoded library (DEL) that can introduce new functional groups is required.

J. AM. CHEM. SOC. 2005, 127, 2038-2039

Therefore, the problem to be solved by the present invention is to provide a new synthetic method for constructing a DNA-encoded library.

Another problem to be solved by the present invention is to provide a DNA-encoding library with a new functional group.

First, while trying to produce a DNA-encoded library, the present inventors had the idea of producing library compounds by linking DNA and candidate compounds centered on the N-acylsulfonamide moiety.

N-acylsulfonamide moiety is a moiety used in various drugs (candidates) such as Asunaprevir, Tapotoclax, Parecoxib, and GNE-0310, and has stable properties even under hydrolysis conditions in vivo, ensuring metabolic stability required for drug candidates. In that it can be said to be an attractive functional group.

N-acylsulfonamides can be easily synthesized through general organic reactions. For example, it has been reported that the desired product can be obtained when amide reacts with a strong base and sulfonyl chloride under low temperature conditions as shown in the reaction equation below.

However, the application of sulfonyl chloride is challenging in DEL because of its poor stability in aqueous solvents, and the introduction of a strong base (e.g. NaH) to promote deprotonation also limits reaction conditions in the presence of water.

To solve this problem, the present inventors discovered that N-acylsulfonamide moiety-based DEL can be effectively prepared through a reaction based on the DNA of an alkyne under conditions of using a copper reagent with water as a nucleophile. did. This method can be applied to the preparation of various types of N-acylsulfonamide compounds using commercially available and easily accessible water-stable sulfonyl azide.

Therefore, the present invention relates to a method for producing a DNA-encoded library compound, which involves reacting a compound represented by the following formula 1, which is linked to a DNA chain, with a sulfonyl azide compound represented by the formula 2 below. A method for producing an acylsulfonamide-based DNA-encoding compound represented by the following formula (3) is provided.

[Formula 1]

[Formula 2]

[Formula 3]

The present invention also provides a method for producing a DNA-encoded library compound, by reacting a compound represented by the following formula 4, which is linked to a DNA chain, with an alkyne compound represented by the formula 5, A method for producing an acylsulfonamide-based DNA-encoding compound represented by Formula 6 is provided.

[Formula 4]

[Formula 5]

[Formula 6]

In one aspect of the present invention, the compound of Formula 1 or Formula 4 is obtained by reacting Formula 7 below with 4-ethynylbenzoic acid or 4-(azidosulfonyl)benzoic acid, respectively.

[Formula 7]

The present inventors performed a three-component reaction using tosyl azide, diisopropylamine (DIPEA), and an alkyne combined with double-stranded DNA commonly used in split-pool DEL synthesis, and the results are summarized in Table 1 below. indicated.

(In Table 1 above, unless otherwise specified, the reaction conditions were starting material (20 nmol in buffer), Cu reagent (60 equiv), tosyl azide (80 equiv), and DIPEA (80 equiv). The yield was HPLC-MS was analyzed.)

As shown in Table 1 above, under ambient conditions in DMF, reaction with CuSO ₄ ·H ₂ O, a well-known reagent for click chemistry, in the presence of DNA-conjugated molecules did not give the desired product. Therefore, various copper reagents were used in the experiment, including CuI, CuBr·SMe ₂ , and CuCl, with CuI providing the best results. It was found that increasing the equivalent weight of DIPEA and tosyl azide compared to the starting material also affected the reaction efficiency. Additionally, alternative polar aprotic solvents such as DMSO and MeCN (entries 6 and 8) did not provide good conversion. Improving the solubility of the DNA-conjugated starting material using water as a solvent resulted in low conversion rates due to the low solubility of the organic reagents, while the reactivity was low in carbonate and MOPS (3-( N -morpholino)propanesulfonic acid) buffered solutions. As shown in entries 12 and 13, when the reaction time was shortened, the reaction conversion rate also improved.

Therefore, in a preferred aspect of the present invention, the method according to the present invention is characterized by using a copper compound as a catalyst, and more preferably by using CuI as a catalyst. In one preferred embodiment of the invention, the process according to the invention is carried out in the presence of water, more preferably in a borate buffer.

The substrate range of the reaction under the conditions of one preferred embodiment of the present invention was investigated and the results are summarized in Table 2 below.

(In Table 2 above, the reaction conditions were starting material (20 nmol in borate buffer), CuI (40 equiv), sulfonly azide derivative (40 equiv), and DIPEA (60 equiv). The yield was analyzed by HPLC-MS. )

First, we evaluated the utility of the developed reaction by initially using aryl sulfonyl azides containing various electron-withdrawing and electron-donating groups in the reaction (Table 2, entries 1-9). In the case of derivatives with electron-donating groups such as methyl or methoxy groups, the desired product was obtained in high yield (87% and 73%, respectively). Additionally, bromide- and chloride-substituted substrates, which can increase the diversity of library composition through further functionalization via Suzuki cross-coupling reaction, were also efficiently converted to the corresponding N-acylsulfonamides in yields of 78% and 80%, respectively. Compounds containing nitro, acyl, and cyano groups that are common in pharmacological intermediates or that may enhance their pharmacological activity have also been investigated and found to provide the desired products in high yields (entries 5–7). Derivatives containing fluoride, such as those containing trifluoromethyl and difluoromethoxy groups, also showed high reactivity. Additionally, the scope of this method has been further expanded to ring systems including hetero, double and fused structures, as well as 2-chlorothiophene and N-methyl imidazole substituted DNA conjugated N-acylsulfonamides, with 78% and 60% oxidation, respectively. Obtained in % yield. More specifically, the bicyclic sulfonyl azide successfully participated in the reaction to provide the desired product with acceptable conversion (entries 12-16), demonstrating that it is amenable to starting materials with various substituents of the present invention. . Cross-linked camphorsulfone azide was also converted to the desired compound in high yield (entry 17), and a substrate containing a typical pyrazole structure was also successfully converted to the desired product under optimized conditions (entries 19 and 20).

To further evaluate the substrate range of the method according to the invention, DNA-conjugated sulfone azides were tested using various terminal aryl alkynes under optimal reaction conditions, and the results are summarized in Table 3 below.

As shown in Table 3 above, most aryl alkynes gave the desired product at moderate conversion. More specifically, aryl alkynes bearing various electron donating groups such as methyl, methoxy and tert-butyl groups at para-, meta- or ortho-positions were efficiently converted to the corresponding acylsulfonamides with moderate efficiency (Table 3, Items 1-5). Additionally, arenes containing electron-withdrawing groups were also resistant under optimized conditions (entries 6–11). Additionally, heteroarenes such as thiophene and pyridine substituted alkynes and disubstituted aryl alkyne derivatives have been found to be reactive under reaction conditions optimized to provide the products (entries 12-15).

From the results in Tables 2 and 3 above, it is confirmed that the production method of the present invention can be used for various starting materials.

Another aspect of the present invention also provides DNA-Encoded Library (DEL) compounds represented by Formula 3 and DEL consisting of these compounds.

The present invention provides an efficient synthetic method providing bioactive N-acylsulfonamides from corresponding DNA conjugated alkynes based on a copper-mediated three-component reaction. The synthetic method of the present invention can be performed under mild conditions and is applicable to a wide range of substrates. It showed high reactivity even when it contained a heterocyclic functional group that could exhibit drug activity. Additionally, the method of the present invention can be applied to sequential protocols in the presence of DNA splicing to provide the possibility of library construction. Established reaction ranges along with proven DNA compatibility are useful for the preparation of N-acylsulfonamides containing DNA encoding libraries to aid in the discovery of hit compounds against target proteins in the pharmaceutical industry.

Figure 1 shows the results of PCR amplification of compound 2a according to one aspect of the present invention. PCR amplification was evaluated using gel electrophoresis (E-Gel EX 4% agarose).
Figure 2 shows the Sanger sequencing results of the PCR amplification product (top: HP-primer, bottom: 2a-primer). The sequence in the red box in Figure 2 is the sequence present during the three-component reaction.

Hereinafter, to aid understanding of the present invention, it will be described in detail through examples. However, the embodiments according to the present invention may be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the present invention are provided to more completely explain the present invention to those with average knowledge in the field to which the present invention pertains.

General Procedure I: Amide Coupling Reaction with Headpiece

Headpiece solution (2mM in water; 120nmol, 60μL) with 60μL pH 8.5 buffer solution (250mM), 40 equivalents of ethynyl-benzoic acid (200mM in DMA, 24μL) and 40 equivalents of DMT-MM (200mM in water, 24μL). was added. The mixture was then vortexed. The reaction was performed at room temperature at 850 rpm for 15 hours. Once the reaction was complete, the following “EtOH precipitation protocol” and lyophilization were performed.

In this experiment, The structure of Chemical Formula 1a below was used.

[Formula 1a]

* Ethanol precipitation and lyophilization procedure

10% of the total volume of 5M NaCl solution was added to the DNA aqueous solution, and then cold ethanol (2.5 times the amount of ethanol stored at -20°C) was added. The mixture was stored in a -80°C freezer for more than 1 hour. The sample was centrifuged at 4°C in a microcentrifuge at 10000 rpm for approximately 30 minutes. The supernatant was removed, the pellet (precipitate) was cooled in liquid nitrogen, and then placed in a freeze dryer. After freeze-drying, the dried pellet was recovered.

General procedure II: Synthesis of Sulfonyl Azides

To a solution of NaN ₃ (1.5 equiv) in water (2M) was added dropwise a solution of sulfonyl chloride (1.0 equiv) in acetone (1 mL) at 0°C. The reaction mixture was warmed to room temperature and stirred for 4-12 hours. Acetone was removed under reduced pressure and the reaction mixture was extracted with EtOAc. The combined organic layers were dried over MgSO ₄ and the solvent was removed under reduced pressure. The product was used without further purification. (Yield 24%~99%)

General procedure III: Three-component Reaction on DNA

Each DNA-tagged ethinylbenzene derivative (20 nmol, 40 μL, 1.0 eq.) was added to 40 μL pH 8.5 buffered solution (250 mM), followed by each sulfonyl azide derivative (1200 nmol, 30 μL). , 60 eq.), CuI (1200 nmol, 60 eq.), and DIPEA (1600 nmol, 32 μL, 80 eq.) were added. The mixture was vortexed. The reaction was performed at room temperature at 850 rpm for 3 hours. After the reaction, the scavenger sodium diethyldithiocarbamate (2 equivalents of Cu, 48 μL, 100 mM in water) was added. Upon addition of the scavenger, a brown precipitate was observed. Samples were vortexed and centrifuged (3 min, 4 °C, 10000 rpm) and the supernatant was separated. Once this process was completed, the “EtOH precipitation protocol” and lyophilization were performed.

The DNA-Encoded libraries prepared through the methods of General procedures I to III above are summarized in Table 4 below.

General procedure IV: Amide Coupling Reaction with Headpiece

The headpiece solution (2mM in water; 120nmol, 60μL) was added with 60μL pH 8.5 borate buffer solution (250mM), 40 equivalents of 4-(azidosulfonyl)benzoic acid (200mM in DMA, 24μL) and 40 equivalents of DMT-MM (24μL in water). 200mM, 24μL) was added. The mixture was then vortexed. The reaction was performed at room temperature at 850 rpm for 15 hours. When the reaction was completed, the “EtOH precipitation protocol” and lyophilization process were performed.

General procedure V: Three-component Reaction on DNA

Each DNA-tagged sulfonyl azide derivative (20 nmol, 40 μL, 1.0 eq.) in 40 μL pH 8.5 buffer solution (250 mM) and each ethynylbenzene derivative (1200 nmol, 30 μL, 60 eq.). .), CuI (1200 nmol, 60 eq.) and DIPEA (1600 nmol, 32 μL, 80 eq.) were added. The mixture was then vortexed. The reaction was performed at room temperature at 850 rpm for 3 hours. After the reaction, the scavenger sodium diethyldithiocarbamate (2 equivalents of Cu, 48 μL 100 mM in water) was added. Upon addition of the scavenger, a brown precipitate was observed. Samples were vortexed and centrifuged (3 min, 4 °C, 10000 rpm) and the supernatant was separated. Once this process was completed, the “EtOH precipitation protocol” and lyophilization were performed.

The DNA-Encoded libraries prepared through the methods of General procedures IV and V are summarized in Table 5 below.

Synthesis Example 1: Synthetic Application of on-DNA Three-component Reactions

Step 1: Preparation of the F-moc Protection Conjugate

Fmoc-Gly-OH (200 mM DMF solution; 2000 nmol, 10 μL, 40 equiv), DMT-MM (200 mM aqueous solution, 2000 nmol, 10 μL, 40 equiv) in HP-NH ₂ (2 mM in water; 50 nmol, 25 μL, 1.0 equiv). ), and pH 8.5 buffer solution (250 mM, 25 μL) were mixed and vortexed. Afterwards, the reaction was performed at room temperature at 850 rpm for 15 hours. 5M NaCl solution (10% by volume) and cold ethanol (2.5 times by volume, ethanol stored at -20°C) were added. Afterwards, the mixture was stored in a -80°C freezer for more than 1 hour. Samples were centrifuged at 4°C for approximately 30 minutes in a microcentrifuge at 15000 rpm. The supernatant was removed, the pellet (precipitate) was cooled with liquid nitrogen, and then placed in a freeze dryer. After freeze-drying, the dried pellet was recovered.

Step 2: F-moc Deprotection.

F-moc protected DNA-conjugate compound (25 μL, 50 mmol, 2 mM) was added with 5 μL 10% piperidine in pH 8.5 buffered solution (25 μL, 250 mM). The mixture was vortexed. Afterwards, it was reacted at room temperature for 2 hours. 5M NaCl solution (10% by volume) and cold ethanol (2.5 times by volume, ethanol storage -20°C) were added. The mixture was then stored in a -80°C freezer for at least 30 minutes. The reaction was centrifuged at 4°C for about 30 minutes in a microcentrifuge at 10000 rpm. The supernatant was removed, the pellet (precipitate) was cooled in liquid nitrogen, and then placed in a lyophilizer overnight. After freeze-drying, the dried pellet was recovered.

Step 3: Amide Coupling.

To the deprotected compound (25 μL, 50 mmol), pH 8.5 buffer solution (25 μL), 40 equivalents of 4-ethynyl-benzoic acid (10 μL, 200 mM DMA solution) and 40 equivalents of DMT-MM (10 μL, 200 mM aqueous solution) were added. and vortexed the mixture. The reaction was performed at room temperature at 850 rpm for 15 hours. 5M NaCl solution (10% by volume) and cold ethanol (2.5 times by volume, ethanol stored at -20°C) were added. The mixture was stored in a -80°C freezer for more than 30 minutes. Samples were centrifuged at 4°C for approximately 30 minutes in a microcentrifuge at 10000 rpm. The supernatant was removed, the pellet (precipitate) was cooled with liquid nitrogen, and then placed in a freeze dryer. After freeze-drying, the dried pellet was recovered.

Step 4: Three-component Reaction

The prepared alkyne derivative (0.5 mM aqueous solution, 50 nmol, 100 μL, 1.0 eq) was mixed with pH 9.5 buffer solution (100 μL 250 mM), TsN3 (40 mM DMF solution, 3000 nmol, 75 μL, 60 eq), and CuI (3000 μL). nmol, 60 eq) and DIPEA (50 mM in DMF solution; 4000 nmol, 80 μL, 80 eq) were added and the mixture was vortexed. The reaction was performed at room temperature at 850 rpm for 3 hours. After the reaction, the scavenger sodium diethyldithiocarbamate (2 eq. of Cu, 90 μL, 100 mM in water) was added. Upon addition of the scavenger, a brown precipitate was observed. The samples were vortexed and centrifuged (3 min, 4 °C, 10000 rpm) and the supernatant was separated. 5M NaCl solution (10% by volume) and cold ethanol (2.5 times by volume, ethanol stored at -20°C) were added. The mixture was stored in a -80°C freezer for more than 1 hour. Samples were centrifuged at 4°C for approximately 30 minutes in a microcentrifuge at 10000 rpm. The supernatant was removed, the pellet (precipitate) was cooled with liquid nitrogen, and then placed in a freeze dryer. After freeze-drying, the dried pellet was recovered.

DNA Damage Evaluation

[Ligation]

The substrate containing the headpiece (HP) of Formula 1a was dissolved in 100mM phosphate buffer (pH 8.0). 5 μL of 100 μM HP or 5 μL of 100 μM compound 2a and 8 μL of 100 μM primer were mixed in the presence of ligase (total volume: 20 μl). This ligation solution was stored at 16°C for 20 hours. DNA ligation was confirmed by resolving the DNA solution on a 4% agarose gel. To purify DNA from the ligation solution, 3.75 μL of 4M NaCl and 45 μL of 100% EtOH were added to 15 μL of the ligation solution, and this solution was maintained at -20 °C for 12 hours. After centrifugation at 14,000 rpm for 5 minutes, the DNA pellet was dissolved in water.

[PCR Procedure]

The ligated DNA was used in PCR experiments according to the following instructions. PCR was performed using 1 μL of forward primer, 1 μL of reverse primer, 1 μL of ligated DNA, 1 μL of pfu polymerase, 1 μL of dNTP, 10 μL of 5 × polymerase buffer, and 35 μL of water.

Forward primer sequence: ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT NNN NTG ACT CCC AAA TCG ATG TG

Reverse primer sequence: TGG AGT TCA GAC GTG TGC TCT TCC GAT CTG CAG GTG AAG CTT GTC GG

PCR was performed according to the following method:

- 5 minutes at 95°C; [30 seconds at 92°C; 15 seconds at 60°C; 15 seconds at 72°C]; 5 minutes at 72℃

PCR amplification was evaluated through gel electrophoresis, and the results are shown in Figure 1. As shown in Figure 1, it was confirmed that Compound 2a was also ligated in the same manner as the headpiece of Formula 1a. Afterwards, Sanger sequencing described later was performed on the PCR product, and through this, it was confirmed whether there was any abnormality in the headpiece part of compound 2a .

[Sanger Sequencing procedure]

PCR products were submitted to GENEWIZ for Sanger sequencing using primers (5'->3', AGT TCA GAC GTG TGC TCT TCC). These primers (capable of sequencing in the “reverse” direction) were chosen to obtain high-quality data for the barcode region that was present during the three-component reaction.

The results are shown in Figure 2. When comparing the sequencing results of the HP-primer and the sequencing results of the compound 2a-primer, it was confirmed that the DNA was not damaged and remained stable when manufacturing the DNA-Encoded library of the present invention.

Through Synthesis Example 1 and its evaluation, the applicability of the developed three-component reaction method was confirmed. Step 1 shows that commercially available and easily accessible building blocks such as amino acids, aryl alkynes and sulfonyl azides can potentially be applied using the currently developed method. In step 1, amino acids can be smoothly introduced using a simple amide coupling method. After deprotection of the Fmoc group and introduction of the aryl alkyne derivative in steps 2 and 3, the desired N-acylsulfonamide derivative was obtained by complete conversion through step 4. Afterwards, DNA reactivity was evaluated to demonstrate the applicability of the newly developed method. After ligation, the corresponding conjugated molecule was successfully obtained, and the sequence of the desired oligo region was confirmed through Sanger sequencing.

Claims (8)

In the method of producing a DNA-encoded library compound,
To prepare an acylsulfonamide-based DNA-encoding compound represented by Formula 3 by reacting a compound represented by Formula 1 below, which is linked to a DNA chain, with a sulfonyl azide compound represented by Formula 2 below. method.
[Formula 1]

[Formula 2]

[Formula 3]
The method of claim 1, wherein the compound of Formula 1 is obtained by reacting Formula 7 with 4-ethynylbenzoic acid.
[Formula 7]
In the method of producing a DNA-encoded library compound,
A method of producing an acylsulfonamide-based DNA-encoding compound represented by Formula 6 by reacting a compound represented by Formula 4 below, which is linked to a DNA chain, with an alkyne compound represented by Formula 5 below.
[Formula 4]

[Formula 5]

[Formula 6]
The method of claim 3, wherein the compound of Chemical Formula 4 is obtained by reacting Chemical Formula 7 with 4-(azidosulfonyl)benzoic acid.
[Formula 7]
The method of producing an acylsulfonamide-based DNA-encoding compound according to any one of claims 1 to 4, wherein the method uses a copper compound as a catalyst. The method of claim 5, wherein the copper compound is CuI. The method of producing an acylsulfonamide-based DNA-encoding compound according to any one of claims 1 to 4, wherein the method is performed in the presence of water. The method of claim 8, wherein the water is a borate buffer solution.