Gram‐Scale Synthesis of an Arylsulfonamide‐Type Alkaline Phosphatase Inhibitor

Abstract

Alkaline phosphatases (ALP) are present in most living organisms. This family of metalloenzymes catalyzes transphosphorylation reactions and hydrolyzes phosphate monoesters. ALP enzymes have significant roles in several physiological processes and disease states. Inhibition of these enzymes makes it possible to prevent or treat certain diseases, and their role in normal physiology can also be examined. Several inhibitors with diverse chemical structures have been reported. One of them, known as SBI‐425, has a low IC₅₀ value (16 nM). An alternative, scalable method was established to prepare SBI‐425 in larger amounts for in vivo experiments. In three simple steps, 4‐chloroanisole was transformed to the appropriate sulfonyl chloride, which was used to acylate 3‐aminonicotinamide. Without the need for chromatography or crystallization, the desired inhibitor was obtained with >90% purity (based on UPLC‐MS analysis). The synthesis could also be carried out on a 10‐fold scale, with identical outcomes. The final product was subjected to biological testing, and it was found to be effective in inhibiting ALP activity in mouse serum.

A larger amount of a tissue‐nonspecific alkaline phosphatase inhibitor, 5‐((5‐chloro‐2‐methoxyphenyl)sulfonamido)nicotinamide, was synthesized in five steps using a parallel method without the need for chromatography or crystallization. Because a single batch was used for in vivo studies in a large population of mice, the pharmacological results were not affected by deviations in impurities.

1. Introduction

The role and properties of alkaline phosphatase (ALP) have been extensively investigated since its discovery. Numerous varieties of this homodimeric metalloenzyme have been identified [1, 2], which are widely distributed across species (from bacteria to humans) [3]. ALP is a membrane‐bound glycoprotein; there are at least four isoenzymes, tissue‐nonspecific (TNAP) and tissue‐specific isoenzymes: placental (PLAP), intestinal (IAP), and germ cell (GCAP) [1, 2]. Circulating levels of ALP within the plasma reflect the activity of these forms within different tissues, however, the exact mechanisms and regulation of the release are not clear [4]. These facts indicate that ALP plays roles in fundamental biochemical processes [5], however, its exact physiological function is largely unknown, although its role in bone metabolism [6, 7, 8] and cell growth and differentiation has been demonstrated [9, 10]. A few in vitro functions have been attributed to ALP: protein phosphatase activity, phosphotransferase activity, and hydrolysis of organic phosphomonoesters of low molecular weight. Because its substrate specificity is low, it will hydrolyze all kinds of phosphomonoesters, for example, extracellular nucleotides [11, 12, 13]. Serum activity of ALP has been used for decades to identify various disease states [14, 15, 16, 17, 18, 19, 20, 21, 22]. This is related to the ability of the enzyme to hydrolyze extracellular nucleotides, thereby regulating the levels of nucleotides and adenosine, which act as signaling molecules capable of triggering cellular responses [5, 23, 24].

In order to investigate and get an insight into the physiological role of ALP, the development of small molecules that selectively inhibit this enzyme is crucial. It is known that ALP is uncompetitively inhibited by certain amino acids (leucine, phenylalanine). Other well‐known inhibitors are theophylline [25, 26, 27] and levamisole [28, 29]. In recent years, several studies summarizing the results of small molecule ALP inhibitors have appeared in the literature [30, 31, 32]. Among them, a few arylsulfonamides were tested and found to be potent inhibitors of tissue‐nonspecific alkaline phosphatase (TNAP). The presence of a 3‐pyridyl or 3‐quinolinyl moiety on the nitrogen of the sulfonamide was found to be essential, as well as an alkoxy group in the ortho position of the aromatic ring bearing the sulfonamide group [33]. Keeping these motifs fixed, a structure–activity study was performed involving 35 analogues by an American research group in 2018 [34]. One of the synthesized derivatives, SBI‐425 (1), exhibited excellent inhibitory activity (IC₅₀ = 16 nM, selective TNAP inhibition and oral administration) (Figure 1).

FIGURE 1.

Structure of SBI‐425 (1).

SBI‐425 (1) was later shown to inhibit ectopic calcification in TNAP‐overexpressing mice and the Abcc6‐/‐ mouse model of the calcification disease, pseudoxanthoma elasticum (PXE) [35, 36, 37, 38].

Based on biological needs, multigram amounts of SBI‐425 (1) were requested in a single batch for in vivo studies in a large population of mice. One‐batch synthesis was crucial in order to get comparable pharmacological results unaffected by the deviation of the impurities. Although a draft scheme of preparation was published [34], no relevant detailed description was presented for compound 1. Furthermore, when the only published synthesis of 1 that includes details [39] was attempted in our laboratory, we were unable to reproduce it. Therefore, we decided to develop and describe a different procedure for the synthesis of SBI‐425 (1).

2. Results and Discussion

2.1. Synthesis

To synthesize TNAP inhibitor 1, we attempted to follow the pathway in Scheme 1, however, obstacles arose already in the first step. The formation of sulfonic acid chloride 4 was attempted in a one‐pot reaction using chlorosulfonic acid (Scheme 2), as described in Scheme 1 [34, 39]. This method is similar to the transformation occurring in the synthesis of sulfonamides [40].

SCHEME 1.

Synthetic pathway of SBI‐425 (1) reported in the literature [34, 39].

SCHEME 2.

Unsuccessful synthesis of sulfonyl chloride 4 using chlorosulfonic acid.

Stirring 4‐chloroanisole (2) in 6 equivalents of chlorosulfonic acid (without solvent) at 25°C did not result in the desired chloride 4, exclusively sulfonic acid 3 was formed (Table 1, entry 1). Neither raising the temperature (up to 80°C) nor increasing the amount of acid (to 12 equivalents) nor a longer reaction time (48 h) altered the outcome (Table 1, entry 2). When 4‐chloroanisol (2) was treated with 2.1 equivalents of chlorosulfonic acid in dichloromethane, instead of the formation of the desired compound 4, sulfonic acid 3 spontaneously and continuously precipitated from the reaction mixture in 77% yield (Table 1, entry 3).

TABLE 1.

Reaction conditions used in experiments to obtain sulfonic acid 3 / sulfonyl chloride 4.

Entry	Substrate	Reagent	Equivalent	Solvent	Temp.,°C	Time, h	Yield, %
1	2	ClSO3H	6	—	0 → 25	24	3: 76, 4: 0
2	2	ClSO3H	12	—	0 → 80	48	3: 70, 4: 0
3	2	ClSO3H	2.1	CH2Cl2	0–5	1	3: 77, 4: 0
4	2	ClSO3H	1.2	CH2Cl2	0–5	1	3: 84
5	2	ClSO3H	3	CH2Cl2	0–5	1	3: 16
6	2	ClSO3H	2.1	2‐CH3‐THF	0 → 25	1	3: ‐a, 4: ‐a
7	2	ClSO3H	3.1	2‐CH3‐THF	0 → 25	72	3: ‐a, 4: ‐a
8	2	ClSO3H	5	2‐CH3‐THF	50	2	3: ‐a, 4: ‐a
9	2	SO2Cl2	1.1	CH2Cl2	0 → 25	24	3: ‐a, 4: ‐a
10	3	SOCl2	2	CH2Cl2	20	24	4: ‐a
11	3	SOCl2	2	CH2Cl2	40	4	4: ‐a
12	3	POCl3	2	CH2Cl2	20	24	4: ‐a

^a Reaction mixture was not worked up.

Since sulfonic acid 3 proved to be barely soluble in dichloromethane, the reaction was carried out in 2‐methyltetrahydrofuran in the hope that the acid 3 remaining in the solution would thus be converted into acid chloride 4. After stirring the reaction mixture at 25°C for 60 min, TLC analysis indicated the predominant presence of the starting compound (2) (Table 1, entry 6). The addition of 1 more equivalent of chlorosulfonic acid did not change the progress of the reaction, even when the reaction time was significantly extended (72 h) (Table 1, entry 7).

Using 5 equivalents of chlorosulfonic acid and heating the reaction mixture also resulted in incomplete conversion (only sulfonic acid 3 was detected by TLC, the presence of the desired sulfonyl chloride 4 could not be indicated (Table 1, entry 8)). An experiment was carried out using sulfuryl chloride (1.1 equivalents) instead of chlorosulfonic acid, which also did not provide sulfonyl chloride 4 (Table 1, entry 9).

After several unsuccessful one‐step attempts, we decided to look for an alternative route. Sulfonic acid 3 was synthesized in dichloromethane using 1.2 equivalents of chlorosulfonic acid. Compound 3 was obtained in a yield of 84%, and it could be used without purification based on NMR analysis (Table 1, entry 4). The use of larger amounts of chlorosulfonic acid should be avoided, because the yield is significantly reduced. When 3 equivalents of chlorosulfonic acid were used under the same conditions, only a 16% yield could be reached (Table 1, entry 5). Chlorosulfonic acid dissolves the expected sulfonic acid, which may account for the reduced precipitation of the product. In addition, tar formation was observed.

When sulfonic acid 3 was treated with thionyl chloride (Table 1, entries 10 and 11) or phosphorus oxychloride (Table 1, entry 12), the reactions did not result in the formation of sulfonyl chloride 4, no trace of 4 was detected.

Although reduced nucleophilicity of the OH group of sulfonic acid 3 initially appeared to be a plausible explanation for the failed acid chloride generation, closer examination suggests otherwise. A more likely explanation is the formation of an intramolecular hydrogen bond between the acidic proton of the sulfonic acid and the oxygen atom of the ortho‐methoxy group, resulting in a stabilized six‐membered ring. Disruption of this interaction by salt formation rendered the sulfonic acid amenable to conversion into the corresponding acid chloride.

Salt formation was carried out in methanol with an equimolar amount of potassium hydroxide (Scheme 3). After the solutions of the base and acid 3 (both in methanol) were mixed, potassium salt 5 immediately precipitated (in a yield of 83%), which was dried and used directly in the next step. Salt 5 was suspended in dry toluene and was treated with excess thionyl chloride in the presence of a catalytic amount of DMF. After 3 h at 70°C, the volatiles were removed, the residue was dissolved in toluene, and the cloudy solution was filtered through Celite. Concentration of the clear solution gave the desired sulfonyl chloride 4 in excellent yield (95%). Based on NMR analysis, no purification was necessary. A similar method using sulfonic acid salt to obtain sulfonyl chloride has already been described [41]. Despite its multistep nature, this approach provides rapid access to sulfonyl chloride 4 and avoids the decomposition of large amounts of chlorosulfonic acid.

SCHEME 3.

Synthesis of sulfonyl chloride 4 via salt formation.

After that, the resulting acid chloride 4 could be reacted with the commercially available nicotinic acid derivative 6 (Scheme 4). When methyl 5‐aminonicotinate (6) was acylated with sulfonyl chloride 4 in dichloromethane or in 2‐methyl‐THF in the presence of triethylamine, intermediate 7 could not be isolated due to the formation of unidentified side products. Surprisingly, when the reaction was carried out in pyridine without additional base, compound 7 partially precipitated from the reaction mixture after 2 h. After pouring the mixture into water and filtration of the precipitate, sulfonamide 7 was isolated with excellent yield (89%). Based on its NMR and HRMS spectra, no further purification was necessary.

SCHEME 4.

Sulfonamide formation with 3‐aminonicotinic acid methyl ester (6).

Only one additional step was required to complete the synthesis, the transformation of the ester group to an amide, as reported in the literature [34, 39]. However, conversion of compound 7 into SBI‐425 (1) was not successful in 25% (w/w) aqueous ammonia or 3 M ethanolic ammonia (Scheme 5). In the former case, the amide formation was observed, but the hydrolysis of the ester 7 into the appropriate carboxylic acid proceeded faster. In the latter case, there was only low conversion (ca. 10% was estimated based on LC‐MS measurement), even in a sealed vessel in a microwave reactor at 100°C.

SCHEME 5.

Attempted transformation of ester 7 into SBI‐425 (1).

The unsuccessful amide formation suggested that the conversion of the ester group should be performed before the acylation of the amino group. When methyl 5‐aminonicotinate (6) was treated with aqueous ammonia at room temperature (Scheme 6), it was observed that the amide formation proceeded faster than the hydrolysis of the ester group. According to UPLC‐MS analysis, the crude product, which was obtained after concentration of the reaction mixture, contained the desired amide 8 in 95% purity (25°C, 24 h). A small sample was purified by thin‐layer chromatography to prove the structure of amide 8, although compound 8 was used successfully further without purification.

SCHEME 6.

Formation of 5‐aminonicotinamide (8).

Then, crude 5‐aminonicotinamide 8 was acylated with sulfonyl chloride 4 in pyridine (Scheme 7). After completion of the reaction (3 h), the mixture was concentrated. The residue was treated with ethyl acetate, which resulted in formation of a precipitate, that was washed with methanol. This method gave SBI‐425 (1) in 67% yield and >90% purity (based on UPLC‐MS analysis).

SCHEME 7.

Synthesis of SBI‐425 (1) from sulfonyl chloride 4 and amide 8.

Scaling up the original synthesis 10‐fold resulted in similar yields (Table 2). The only major deviation concerned compound 8, which was purified during the first synthesis but used as crude in the later experiment. The purification was performed by thin‐layer chromatography without any optimization, which may have led to greater material loss. When the purification of compound 8 is omitted on the smaller scale, the overall yields of the two syntheses are comparable (40% and 43%, respectively).

TABLE 2.

Comparison of the yields of the smaller and larger‐scale syntheses.

	Smaller scale	Larger scale
Compound	Yield, %	Yield, %
3	77	84
5	83	81
4	94	97
8	23a	100b
1	67	65
Overall	9 (40c)	43

^a After preparative thin‐layer chromatography.

^b Crude product with 91% purity.

^c Without the purification of compound 8.

2.2. In Vitro Inhibition Test of Compound 1

SBI‐425 (1) is a well‐characterized TNAP inhibitor [34, 42]. TNAP is widely expressed throughout the body and has a well‐established role in mineralization processes; however, it is also implicated in several pathways in which its physiological function is not yet fully understood, such as inflammatory processes, lipid metabolism, and adipocyte differentiation. Accordingly, SBI‐425 (1) can be used as a valuable tool to investigate these physiological pathways and has been shown to be effective and safe in in vivo mouse studies [35, 37]. However, long‐term oral administration of SBI‐425 (1) presents practical challenges. Due to its poor water solubility, administration via oral gavage in DMSO is poorly tolerated over extended periods. An alternative approach is administration via incorporation into the regular chow; however, this method requires relatively large amounts of the compound. Therefore, the development of a large‐scale synthesis enables broader availability of this valuable tool for long‐term in vivo studies.

The effect of the SBI‐425 (1) inhibitor on ALP activity was evaluated in three mouse serum samples (no. 1941, 1942, and 1943) (Figure 2). The assay measures total ALP activity, as 4‐methylumbelliferyl phosphate is a general substrate for ALP and is not specific to TNAP. Residual ALP activity in the serum samples was determined at the 30 min time point and expressed as a percentage of untreated controls. For serum sample no. 1941, residual activities of 8.59%, 2.68%, and 2.07% were observed at 4, 20, and 40 µM SBI‐425 (1), respectively. For serum sample no. 1942, the corresponding values were 10.24%, 4.22%, and 2.41%, while for serum sample No. 1943 they were 9.03%, 2.61%, and 2.34%. In all cases, SBI‐425 (1) was highly effective even at the lowest tested concentration (4 µM). These results indicate a strong, concentration‐dependent inhibition of total serum ALP activity by SBI‐425 (1) across all samples.

FIGURE 2.

The effect of SBI‐425 (1) on mouse sera ALP activity (1941, 1942, and 1943: sample numbers).

3. Conclusion

Although the synthetic scheme for the target compound 1 had been reported previously [34], no details regarding reaction conditions, work‐up procedures, or purification were disclosed. In the absence of sufficient experimental information, the reported route could not be reproduced; therefore, an alternative, versatile, and scalable synthetic pathway was developed. Direct conversion of sulfonic acid 3 to the corresponding acid chloride 4 was unsuccessful; however, compound 4 could be efficiently prepared via the potassium salt of sulfonic acid 3.

Inhibitor 1 was successfully synthesized by acylation of 5‐aminonicotinamide (8) with compound 4 in pyridine. When the ester analogue 6 was used instead of amide 8, difficulties were encountered during the amide formation step. In the developed synthetic route, no crystallization or chromatographic purification was required for any intermediate. The effectiveness of ALP inhibitor 1 was confirmed in vitro, with strong inhibition observed even at a concentration of 4 µM. The larger‐scale batch of TNAP inhibitor 1 was successfully applied in mouse experiments, the results of which have recently been published [43].

4. Experimental Section

4.1. General

All reagents and solvents were purchased from Merck. For reaction monitoring and analysis, a Shimadzu LC‐40D XR UPLC‐MS system was used, equipped with a SIL‐40C XR autosampler, an SPD‐M40 photodiode array detector, an RF‐20A XS fluorescent detector and an LCMS‐2020 DUIS mass spectrometer operated in alternating negative and positive modes. An Ascentis Express C18, 2 μm UHPLC column (L × I.D. 5 cm × 2.1 mm) was used at 40°C provided by a CTO‐40s column oven. Gradient elution was applied with 0.1% v/v TFA in CH₃CN. Melting point was determined using a Stuart SMP10 apparatus. NMR spectra were recorded on a Bruker DRX‐500 spectrometer operating at 500 MHz (¹H) or 125 MHz (¹³C). HRMS measurements were carried out on a high‐resolution Q‐Exactive Focus hybrid quadrupole‐orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) used with a heated electrospray ionization source. Samples were dissolved in an acetonitrile‐water 1:1 (v/v) mixture containing 0.1% (v/v) formic acid. Flow injection analysis was performed using a 50 μL 1/min eluent flow provided by a Thermo Scientific UPLC. Under the applied conditions, the compounds form protonated molecules, [M + H]⁺ in positive ionization ESI. Mouse serum samples originate from the Institute of Molecular Life Sciences, RCNS, HUN‐REN, Budapest.

4.2. 5‐Chloro 2‐methoxybenzenesulfonic acid (3)

4.2.1. Smaller Scale

4‐Chloroanisole (30 mmol, 3.67 ml) was dissolved in dichloromethane (20 ml), the solution was cooled to 0°C, and chlorosulfonic acid (63 mmol, 4.19 ml) was added dropwise. The mixture was stirred at 0°C for 1 h, then the mixture was left to warm to 25°C while it was stirred (30 min). Then the precipitate was filtered and was washed with hexane. To the filtrate, hexane was added (70 ml), and the precipitate formed was filtered and washed with dichloromethane three times.

Yield: 5.13 g (77%); white solid; Mp 106°C–110°C.

¹H NMR (300 MHz, CD₃OD) δ [ppm]: 7.81 (d, J = 2.8 Hz, 1H, ArH, H‐6), 7.42 (dd, J = 8.8, 2.7 Hz, 1H, ArH, H‐4), 7.09 (d, J = 8.8 Hz, 1H, ArH, H‐3), 3.91 (s, 3H, OCH₃).

¹³C NMR (75 MHz, CD₃OD) δ [ppm]: 155.63 (ArC, C‐2), 133.68 (ArC, C‐1), 131.34 (ArC, C‐4), 128.07 (ArC, C‐6), 124.04 (ArC, C‐5), 113.34 (ArC, C‐3), 55.15 (OCH₃).

4.2.2. Larger Scale

4‐Chloroanisole (60 mmol, 7.35 ml) was dissolved in dichloromethane (50 ml), the solution was cooled to 0°C, and chlorosulfonic acid (72 mmol, 4.79 ml) was added dropwise. The mixture was stirred at 0°C for 1 h, then the mixture was left to warm to 25°C while it was stirred (30 min). Then the precipitate was filtered and was washed with hexane.

Yield: 12.80 g (84%).

4.3. 5‐Chloro 2‐methoxybenzenesulfonic acid potassium salt (5)

4.3.1. Smaller Scale

5‐Chloro 2‐methoxybenzenesulfonic acid (5 mmol, 1.11 g) was dissolved in methanol (10 ml), and KOH (5 mmol, 281 mg) in methanol (5 ml) was added. Precipitate was formed immediately. The suspension was stirred for 20 min, then the precipitate was filtered and dried in a desiccator.

Yield: 1.08 g (83%); white solid; Mp 94°C–97°C.

4.3.2. Larger Scale

5‐Chloro 2‐methoxybenzenesulfonic acid (57.5 mmol, 12.80 g) was dissolved in methanol (35 ml), and KOH (57.5 mmol, 3.23 g) in methanol (25 ml) was added. Precipitate was formed immediately. The suspension was stirred for 20 min, then the precipitate was filtered, and dried in a desiccator.

Yield: 12.10 g (81%).

4.4. 5‐Chloro 2‐methoxybenzenesulfonyl chloride (4)

4.4.1. Smaller Scale

5‐Chloro 2‐methoxybenzenesulfonic acid potassium salt (4.2 mmol, 1.08 g) was suspended in dry toluene (10 ml), and DMF (0.6 mmol, 50 μl) and thionyl chloride (10.4 mmol, 0.75 ml) were added. The mixture was stirred at 70°C for 3 h, then it was concentrated. Toluene was added to the residue, and the mixture was concentrated again. The residue was dissolved in toluene and filtered through Celite. The filtrate was concentrated; the residue was dissolved in dichloromethane and concentrated again.

Yield: 0.95 g (94%).; white solid; Mp 103°C–104°C.

¹H NMR (300 MHz, CDCl₃) δ [ppm]: 7.96 (d, J = 2.7 Hz, 1H, ArH, H‐6), 7.65 (dd, J = 8.9, 2.6 Hz, 1H, ArH, H‐4), 7.10 (d, J = 8.9 Hz, 1H, ArH, H‐3), 4.08 (s, 3H, OCH₃).

¹³C NMR (75 MHz, CDCl₃) δ [ppm]: 155.90 (ArC, C‐2), 136.87 (ArC, C‐4), 132.55 (ArC, C‐1), 129.35 (ArC, C‐6), 125.52 (ArC, C‐5), 114.59 (ArC, C‐3), 56.99 (OCH₃).

4.4.2. Larger Scale

5‐Chloro 2‐methoxybenzenesulfonic acid potassium salt (46.5 mmol, 12.12 g) was suspended in dry toluene (100 ml), and DMF (4 mmol, 0.31 ml) and thionyl chloride (116 mmol, 8.40 ml) were added. The mixture was stirred at 70°C for 3 h, then concentrated. Toluene was added to the residue, and the mixture was concentrated again. The residue was dissolved in toluene and filtered through Celite. The filtrate was concentrated; the residue was dissolved in dichloromethane and concentrated again.

Yield: 10.80 g (97%).

4.5. Methyl 5‐((5‐chloro‐2‐methoxyphenyl)sulfonamido) nicotinate (7)

4.5.1. Smaller Scale

Methyl‐5‐aminonicotinate (1 mmol, 152 mg) was dissolved in dry pyridine (5 ml), and 5‐chloro 2‐methoxybenzenesulfonyl chloride (1 mmol, 241 mg) was added to the solution in small portions. The mixture was stirred for 2 h at 25°C. Then the mixture was poured into 100 ml water, and the precipitate was filtered, washed with water and a small amount of 2‐propanol (2 ml), and then dried.

Yield: 317 mg (89%); white solid; Mp 238°C–239°C (dec.).

¹H NMR (500 MHz, DMSO‐d ₆) δ [ppm]: 10.81 (s, 1H, SO₂NH), 8.74 (d, J = 1.9 Hz, 1H, H‐1), 8.54 (d, J = 2.6 Hz, 1H, H‐3), 8.01 (dd, J = 2.6 Hz, 1.9 Hz, 1H, H‐2), 7.77 (d, J = 2.7 Hz, 1H, H‐4), 7.68 (dd, J = 9.0, 2.7 Hz, 1H, H‐5), 7.24 (d, J = 9.0 Hz, 1H, H‐6), 3.87 (s, 3H, COOCH₃), 3.82 (s, 3H, ArOCH₃).

¹³C NMR (126 MHz, DMSO) δ [ppm]: 164.05 (COOCH₃), 154.59 (C‐8), 145.53 (C‐2), 145.19 (C‐6), 135.62 (C‐10), 134.14, 129.60 (C‐12), 127.90, 126.98 (C‐4), 126.20, 123.37, 115.16 (C‐9), 57.09 (ArOCH₃), 53.09 (COOCH₃).

4.5.2. Larger Scale

Methyl‐5‐aminonicotinate (5 mmol, 1.2050 g) was dissolved in dry pyridine (25 ml), and 5‐chloro 2‐methoxybenzenesulfonyl chloride (5 mmol, 761 mg) was added to the solution in small portions. The mixture was stirred for 3 h at 25°C. After 1 h, the mixture turned into a dense suspension. The mixture was poured into 200 ml water, and the precipitate was filtered, washed with water and a small amount of methanol (5 ml), then dried.

Yield: 1.70 g (95%).

(ESI+) m/z calcd. 357.0306; HRMS found: 357.0299 [M + H]⁺.

4.6. 5‐((5‐Chloro‐2‐methoxyphenyl)sulfonamido)nicotinamide (1)

4.6.1. Smaller Scale

5‐Aminonicotinamide (1.5 mmol, 206 mg) was dissolved in dry pyridine (7 ml), and 5‐chloro 2‐methoxybenzenesulfonyl chloride (1.5 mmol, 362 mg) was added to the solution in small portions. The mixture was stirred for 3 h at 25°C, then 50 ml toluene was added to the mixture. The organic solution was decanted, then filtered, and the organic solutions were concentrated. The precipitate was washed with methanol, EtOAc, and hexane. The combined brown, oily residue from the evaporation was treated with methanol, then filtered. The precipitate was washed with methanol, EtOAc, and hexane.

Yield: 344 mg (67%);white solid; Mp 248°C–253°C (dec.).

¹H NMR (500 MHz, DMSO‐d ₆) δ [ppm]: 10.64 (s, 1H, SO₂NH), 8.71 (d, J = 1.9 Hz, 1H, H‐2), 8.42 (d, J = 2.6 Hz, 1H, H‐6), 8.15 (s, 1H, CONH₂), 7.92 (dd, J = 2.6, 1.9 Hz, 1H, H‐4), 7.74 (d, J = 2.7 Hz, 1H, H‐12), 7.67 (dd, J = 8.9, 2.7 Hz, 1H, H‐10), 7.63 (s, 1H, CONH₂), 7.24 (d, J = 8.9 Hz, 1H, H‐9), 3.84 (s, 3H, ArOCH₃).

¹³C NMR (126 MHz, DMSO) δ [ppm]: 165.50 (CONH₂), 155.25 (C‐8), 144.24 (C‐2), 143.96 (C‐6), 135.51 (C‐10), 134.75, 130.63, 129.60 (C‐12), 127.96, 126.73 (C‐4), 124.29, 116.05 (C‐9), 57.08 (ArOCH₃).

4.6.2. Larger Scale

5‐Aminonicotinamide (7.6 mmol, 1.042 g) was dissolved in dry pyridine (30 ml), and 5‐chloro 2‐methoxybenzenesulfonyl chloride (7.6 mmol, 1.832 g) was added to the solution in small portions. The mixture was stirred for 3 h at 25°C, then it was concentrated. The residue was treated with EtOAc and decanted, then the precipitate was treated with methanol. It was filtered and washed with EtOAc and hexane. The washing liquids were combined and evaporated, and the residue was treated with methanol. The precipitate was filtered and washed with EtOAc and hexane. The washing liquids were concentrated again, and the residue was treated with methanol. The precipitate was washed with EtOAc and hexane.

Yield: 1.69 g (65%).

(ESI+) m/z calcd. 342.0310; HRMS found: 342.0306 [M + H]⁺.

4.7. 5‐Aminonicotinamide (8)

4.7.1. Smaller Scale

Methyl‐5‐aminonicotinate (1.6 mmol, 250 mg) was dissolved in methanol (3 ml), and 25% ammonia solution (3 ml) was added. The mixture was stirred at 25°C for 24 h, then it was evaporated. Crude product was purified by preparative TLC (eluent dichloromethane:methanol 100:5).

Yield: 104 mg (23%); white solid; Mp 178°C–180°C.

¹H NMR (300 MHz, CD₃OD δ [ppm]: 8.24 (d, J = 1.9 Hz, 1H, H‐2), 8.09 (d, J = 2.7 Hz, 1H, H‐6), 7.52–7.45 (dd, J = 2.7, 1.9 Hz, 1H, H‐4).

¹³C NMR (75 MHz, CD₃OD δ [ppm]: 169.34 (C═O), 145.08 (C‐5), 138.19 (C‐2), 135.62 (C‐6), 130.35 (C‐3), 119.84 (C‐4).

4.7.2. Larger Scale

Methyl‐5‐aminonicotinate (6.6 mmol, 1.00 g) was dissolved in methanol (5 ml), and 25% ammonia solution (10 ml) was added. The mixture was stirred at 25°C for 24 h, then it was evaporated. Crude product was used without purification. Based on LC‐MS investigation, its purity was 91%.

4.8. Inhibition Tests

ALP inhibitor 1 was tested in vitro using the ALP Assay Kit (fluorometric) (ab83371) based on the manufacturer's instructions. ALP cleaves the phosphate group of the nonfluorescent 4‐methylumbelliferyl phosphate disodium salt (MUP), resulting in a fluorescent signal (Ex/Em = 360 nm/440 nm). The inhibition of the reaction results in a decrease of the signal. Serum samples of 3‐month‐old male CD1 mice were used in an in vitro assay in the presence of MUP fluorescent substrate (77 µM). Serum samples were preincubated with inhibitor 1 in a final concentration of 0, 4, 20, and 40 µM. SBI‐425 (1) was dissolved in DMSO (4 mM, 1.367 mg/ml), and the stock solution was kept at −80°C. The final concentration of DMSO in the reaction volume was 1% in the case of all samples. The reaction was incubated at 30°C for 95 min. Average fluorescence was detected and calculated every 5 min with a Perkin Elmer Enspire Multimode Plate Reader. Each test was run in triplicate. MUP background fluorescence was subtracted from the measured values at each time point.

Supporting Information

Supplementary Information available: [NMR, HRMS and UPLC‐MS spectra of compounds; RFU values measured after 30 min]. Additional supporting information can be found online in the Supporting Information section.

Funding

This work was supported by Nemzeti Kutatási Fejlesztési és Innovációs Hivatal (OTKA 127957, R01AR072695).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supplementary Material

Data Availability Statement

Data available in article supplementary material.