CN118994104A

CN118994104A - Compound serving as PARP-NAMPT double-target inhibitor and application thereof

Info

Publication number: CN118994104A
Application number: CN202411047864.9A
Authority: CN
Inventors: 代霖霖; 李冬冬; 王慧; 支爽; 杨子博
Original assignee: Tianjin Institute Of Pharmaceutical Sciences Tianjin Pharmaceutical And Health Research Center
Current assignee: Tianjin Institute Of Pharmaceutical Sciences Tianjin Pharmaceutical And Health Research Center
Priority date: 2024-08-01
Filing date: 2024-08-01
Publication date: 2024-11-22
Anticipated expiration: 2044-08-01
Also published as: CN118994104B

Abstract

The invention relates to a compound serving as a PARP-NAMPT double-target inhibitor, and a preparation method and application thereof. The compound serving as the PARP-NAMPT double-target inhibitor is a compound shown in a formula (I) or pharmaceutically acceptable salt thereof. The invention also relates to a preparation method and application of the compound shown in the formula (I). The compound has stronger PARP-NAMPT enzyme inhibition activity and obvious anti-tumor activity. Thus, the compounds of the present invention are useful in the preparation of a medicament for the treatment of malignant tumors and diseases associated with differentiation and proliferation.

Description

Compound serving as PARP-NAMPT double-target inhibitor and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a compound serving as a PARP-NAMPT double-target inhibitor and application thereof.

Background

Cancer is severely threatening human life health, and with the increasing incidence and mortality worldwide, cancer is not only a major factor in human mortality, but is also a global public health problem.

The phenomenon of "synthetic death" of cancer has been recognized as a reliable strategy for anticancer therapy. Wherein targeting DNA repair defects has become an effective cancer treatment strategy. However, cancers with defective DNA repair often rely on backup DNA repair pathways, which are "deadly spots" that can be targeted to eliminate cancer cells, and are the basis for synthetic lethality. PARP is a family of proteins involved in the repair of Single Strand Breaks (SSBs) in DNA, which function by binding to sites of DNA damage. The success of Poly ADP Ribose Polymerase (PARP) inhibitors in the treatment of BRCA-deficient breast and ovarian cancers demonstrates synthetic lethality. PARP enzyme inhibitors (such as olaparib, lu Kapa, nilaparib, fluzopanib, pamphlet Mi Pani and talazapanib) have been approved for the treatment of breast and ovarian cancer in BRCA mutant patients.

Nicotinamide ribosyltransferase (nicotinamide phosphoribosyltransferase, NAMPT) catalyzes Nicotinamide (NAM) to nicotinamide mononucleotide (nicotinaminde mononucleotide, NMN), regulates the level of NAD, an essential energy substance for mammalian cells, is a rate-limiting enzyme of the NAD production pathway, and plays a vital role in cellular physiological activity. Research shows that NAMPT is closely related to the occurrence and development of tumors, and has become a very important new target in anti-tumor drug research. Tumor cells have very high NAD consumption and metabolism rate, and are more dependent on NAD than normal cells and are more susceptible to NAMPT inhibitors. NAD in tumor cells is used as an essential coenzyme to participate in the synthesis of various tumor essential substances, and NAD can obviously reduce the level of active oxygen free radicals in the environment and protect tumor cells. NAMPT plays an important role in angiogenesis and in the induction of vascular endothelial growth factor production.

Research shows that PARP inhibitor and NAMPT inhibitor have synergistic synthetic lethal effect, and the combination of the PARP inhibitor and NAMPT inhibitor expands the clinical application of the PARP inhibitor. However, such multicomponent dosage forms often suffer from the disadvantages of complex dose setting, drug-drug interactions, pharmacokinetic differences, and low patient compliance. The multi-target medicine can act on single chemical molecules of a plurality of targets in a disease network simultaneously, can generate a synergistic effect on each target, enables the total effect to be larger than the sum of single effects, achieves the optimal treatment effect, simplifies the administration scheme, improves the compliance of patients, and avoids the problems caused by medicine-medicine interaction and different absorption, distribution, metabolism and excretion processes among the components. Therefore, the design of the PARP-NAMPT double-target inhibitor is expected to become a new strategy for developing novel antitumor drugs.

Disclosure of Invention

A first object of the present invention is to provide a compound as a PARP-NAMPT dual-target inhibitor and a pharmaceutically acceptable salt thereof.

The second object of the invention is to provide an application of the compound serving as a PARP-NAMPT double-target inhibitor in preparing anti-tumor drugs. Experimental results show that the compound has strong inhibition activity on PARP-1 and NAMPT, has obvious in vitro anti-breast cancer cell proliferation activity, is stronger than that of a positive drug Olaparib, is independent of a cell BRCA mutation state, has an IC ₅₀ value of 0.09-30.05 mu M, can obviously inhibit the growth of human breast cancer cell strain MDA-MB-468 nude mice transplanted tumor, and has excellent in vivo tumor growth inhibition activity.

A third object of the present invention is to provide a method for preparing a compound as a PARP-NAMPT dual-target inhibitor as described above, and pharmaceutically acceptable salts thereof.

In order to achieve the first object, the present invention adopts the following technical scheme:

A compound serving as a PARP-NAMPT double-target inhibitor can target PARP and NAMPT simultaneously and play a double role in inhibiting PARP and NAMPT. The compound is characterized by having a structure shown in the following general formula (I), and optical isomer, diastereoisomer and racemate mixture thereof, and pharmaceutically acceptable salt thereof;

Wherein:

X is an oxygen atom or a sulfur atom;

by "pharmaceutically acceptable salt" is meant the therapeutically effective and non-toxic salt form of the compound of formula (I). Many such salts are known in the art. Cationic salts formed on any acidic group (e.g., carboxyl) or anionic salts formed on any basic group (e.g., amino), many of which are known in the art, such as cationic salts including salts of alkali metals (e.g., sodium and potassium) and alkaline earth metals (magnesium and calcium) and organic salts (e.g., ammonium salts). The anionic salts may also be conveniently obtained by treating the basic forms (I) and (II) with the corresponding acids, such acids including mineral acids such as sulfuric acid, nitric acid, phosphoric acid, hydrochloric acid and the like; or organic acids such as acetic acid, propionic acid, glycolic acid, 2-hydroxypropionic acid, 2-oxopropionic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, 2-hydroxy-1, 2, 3-malonic acid, ethanesulfonic acid, benzenesulfonic acid, cyclohexylsulfinic acid, 2-hydroxybenzoic acid, 4-amino-2-hydroxybenzoic acid, and the like. In addition, the skilled artisan can take one salt and another salt depending on factors such as solubility, stability, ease of formulation, and the like. Determination and optimization of these salts is within the experience of the skilled artisan.

In the above-mentioned compound as PARP-NAMPT double-target inhibitor and its pharmaceutically acceptable salt, the pharmaceutically acceptable salt contains no crystal water or contains one or more crystal water.

As used herein, "optical isomers," "enantiomers," "diastereomers," "racemates," and the like define all possible stereoisomeric forms of the compounds or physiological derivatives of the present invention. Unless otherwise indicated, the chemical designation of the compounds of the invention includes mixtures of all possible stereochemical forms, including all diastereomers and enantiomers of the basic structural molecule, as well as individual isomeric forms of the compounds of the invention which are substantially pure, i.e., wherein less than 10%, preferably less than 5%, particularly less than 2%, most preferably less than 1% of the other isomers are present.

The compounds of formula (I) may also exist in other protected forms or derivatives, which are obvious to a person skilled in the art and are intended to be included within the scope of the present invention.

As a preferred embodiment of the present invention, the compound as a PARP-NAMPT dual-target inhibitor is preferably:

compound A1:1- (4- ((4- (2-fluoro-5- ((4-oxo-3, 4-dihydro-phthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) sulfonyl) phenyl) -3- (pyridin-3-ylmethyl) urea;

Compound A2:1- (4- ((5- (2-fluoro-5- ((4-oxo-3, 4-dihydro-phthalazin-1-yl) methyl) benzoyl) hexahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) sulfonyl) phenyl) -3- (pyridin-3-ylmethyl) urea;

Compound A3: 2-fluoro-5- ((4-oxo-3, 4-dihydro-phthalazin-1-yl) methyl) -N- (1- ((4- (3- (pyridin-3-ylmethyl) ureido) phenyl) sulfonyl) piperidin-4-yl) benzamide;

compound A4: n- (1- (2-fluoro-5- ((4-oxo-3, 4-dihydro-phthalazin-1-yl) methyl) benzoyl) piperidin-4-yl) -4- (3- (pyridin-3-ylmethyl) ureido) benzenesulfonamide;

Compound A5:1- (4- ((7- (2-fluoro-5- ((4-oxo-3, 4-dihydro-phthalazin-1-yl) methyl) benzoyl) -2, 7-diazaspiro [4.4] nonan-2-yl) sulfonyl) phenyl) -3- (pyridin-3-ylmethyl) urea;

Compound A6:1- (4- ((6- (2-fluoro-5- ((4-oxo-3, 4-dihydro-phthalazin-1-yl) methyl) benzoyl) -2, 6-diazaspiro [3.3] heptan-2-yl) sulfonyl) phenyl) -3- (pyridin-3-ylmethyl) urea;

compound A7:1- (4- ((9- (2-fluoro-5- ((4-oxo-3, 4-dihydro-phthalazin-1-yl) methyl) benzoyl) -3, 9-diazaspiro [5.5] undec-3-yl) sulfonyl) phenyl) -3- (pyridin-3-ylmethyl) urea;

Compound A8: 2-fluoro-N- (2- ((N-methyl-4- (3- (pyridin-3-ylmethyl) ureido) phenyl) sulfamido) ethyl) -5- ((4-oxo-3, 4-dihydro-phthalazin-1-yl) methyl) benzamide;

Compound A9:1- (4- ((4- (2-fluoro-5- ((4-oxo-3, 4-dihydro-phthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) sulfonyl) phenyl) -3- (pyridin-3-ylmethyl) thiourea;

Compound a10: n- (4- ((4- (2-fluoro-5- ((4-oxo-3, 4-dihydro-phthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) sulfonyl) phenyl) -1, 3-dihydro-2H-pyrrole [3,4-c ] pyridine-2-carboxamide;

Compound a11:1- (4- ((4- (2-fluoro-5- ((4-oxo-3, 4-dihydro-phthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) sulfonyl) phenyl) -3- ((4-fluoropyridin-3-yl) methyl) urea;

compound a12: n- (4- ((4- (2-fluoro-5- ((4-oxo-3, 4-dihydro-phthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) sulphonyl) phenyl) thieno [2,3-c ] pyridine-2-carboxamide.

Their structural formulae and nuclear magnetic mass spectrometry data are shown in table 1 below:

TABLE 1 structural formulas and Nuclear magnetic Mass Spectrometry data for preferred Compounds of the invention

。

The invention has the advantages that:

1. Through enzyme inhibition activity and in-vitro anti-tumor cell proliferation activity experiments, the preferred compound provided by the invention not only has strong inhibition activity on PARP-1 and NAMPT, but also has obvious in-vitro anti-breast cancer cell proliferation activity, and the preferred compound has excellent in-vivo tumor growth inhibition activity.

2. Compared with the prior art, the invention opens up a new way for deeply researching and developing the antitumor drug with a new structure type and provides a new strategy.

Detailed Description

The invention is described below by means of specific embodiments. The technical means used in the present invention are methods well known to those skilled in the art unless specifically stated. Further, the embodiments should be construed as illustrative, and not limiting the scope of the invention, which is defined solely by the claims. Various changes or modifications to the materials ingredients and amounts used in these embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The raw materials and reagents used in the invention are all commercially available.

Examples

Preparation of the compound: 1- (4- ((4- (2-fluoro-5- ((4-oxo-3, 4-dihydro-phthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) sulphonyl) phenyl) -3- (pyridin-3-ylmethyl) urea (A1)

The synthetic route is as follows:

Reagents and conditions: (a) Triethylamine, dichloromethane, room temperature for 2 hours, yield 75%; (b) Hydrogen, palladium on carbon, dichloromethane/methanol, room temperature, overnight, 92% yield; (c) Triphosgene, triethylamine, dichloromethane, room temperature, 2 hours; (c) Triethylamine, dichloromethane, room temperature overnight, yield 85%; (d) Trifluoroacetic acid, dichloromethane, room temperature, 2 hours, yield 97%; (e) 1H-benzotriazol-1-yl-oxy-tripyrrolidinylphosphonium hexafluorophosphate, N, N-diisopropylethylamine, N, N-dimethylformamide, at room temperature for 2 hours, in 65% yield.

(One) preparation of intermediate 2a:4- (4-Nitrophenyl) sulfonyl) piperazine-1-carboxylic acid tert-butyl ester

P-Nitrophenesulfonyl chloride 1 (2.2 g,10.0 mmol) (commercially available) and 1-t-butoxycarbonyl-piperazine (2.2 g,12.0 mmol) (commercially available) were dissolved in 50 mL dichloromethane, and triethylamine (3.0 g,30.0 mmol) was added dropwise thereto and then stirred at room temperature for 2 hours. After the reaction is finished, distilled water is added to terminate the reaction, dichloromethane is used for extraction, the organic phase is dried and concentrated, and white solid 3.4 g is obtained through column chromatography separation, the yield is obtained 92%.¹H-NMR (600 MHz, DMSO-d₆) δ: 8.43 (d, J = 8.9 Hz, 2H), 7.99 (d, J = 8.9 Hz, 2H), 3.43-3.35 (m, 4H), 2.97-2.89 (m, 4H), 1.32 (s, 9H).

(II) preparation of intermediate 3a:4- (4-Aminophenyl) sulfonyl) piperazine-1-carboxylic acid tert-butyl ester

Intermediate 2a (2 g,5.4 mmol) was dissolved in 25mL of a mixed solution of dichloromethane/methanol (2/1), and palladium on carbon (57 mg, 0.5 mmol) was added and stirred under hydrogen overnight. After the reaction was completed, the reaction solution was filtered, and the solvent was distilled off under reduced pressure to give 1.9 g as a white solid in yield 95%）.¹H-NMR (600 MHz, DMSO-d₆) δ: 7.32 (d, J = 8.6 Hz, 2H), 6.64 (d, J = 8.6 Hz, 2H), 6.09 (s, 2H), 3.39-3.34 (m, 4H), 2.75-2.67 (m, 4H), 1.33 (s, 9H).

(III) preparation of intermediate 4a:4- ((4- (3- (pyridin-4-ylmethyl) ureido) phenyl) sulfonyl) piperazine-1-carboxylic acid tert-butyl ester

Triphosgene (131 mg,0.4 mmol) was dissolved in 5mL dichloromethane and stirred at 0 ℃, then intermediate 3a (341 mg,1.0 mmol) and triethylamine (146 mg,1.4 mmol) were dissolved in 5mL dichloromethane, after which the solution was added to the triphosgene solution and stirred at ambient temperature for 2h. 3-methylaminopyridine (120 mg,1.11 mmol) and triethylamine (146 mg,1.4 mmol) were dissolved in 5mL dichloromethane and the system was added slowly and reacted overnight at room temperature. After the reaction was completed, the solvent was removed by filtration and distillation under reduced pressure, and the crude product was purified by column chromatography to give 559 mg as a white solid in yield 85%.¹H-NMR (600 MHz, DMSO-d⁶) δ: 9.41 (s, 1H), 8.49 (d, J = 6.1 Hz, 2H), 7.64(d, J = 9.0 Hz, 2H), 7.57 (d, J = 9.0 Hz, 2H), 7.28 (d, J = 6.1 Hz, 2H), 7.01 (t, J = 5.9 Hz, 1H), 4.33 (d, J = 6.2 Hz, 2H), 3.41-3.34 (m, 4H), 2.82-2.75 (m, 4H), 1.32 (s, 9H).

(IV) preparation of intermediate 5a:1- (4- (piperazin-1-ylsulfonyl) phenyl) -3- (pyridin-4-ylmethyl) urea

Intermediate 4a (475 mg,1.00 mmol) was dissolved in a 10 mL trifluoroacetic acid/dichloromethane (1/1) mixed solvent and stirred at room temperature for 2h. After the reaction, the solution is concentrated by reduced pressure distillation and purified by column chromatography to obtain a white solid 364 mg with yield 97%.¹HNMR (600 MHz, DMSO-d⁶) δ: 9.67 (s, 1H), 8.72 (s, 1H), 8.59 (d, J = 6.2 Hz, 2H), 7.69 (d, J = 8.9Hz, 2H), 7.63 (d, J = 8.9 Hz, 2H), 7.46 (d, J = 5.9 Hz, 2H), 7.32 (t, J = 5.9 Hz, 1H), 4.41 (d, J = 6.0 Hz, 2H), 3.19-3.15 (m, 4H), 3.08-3.01 (m, 4H).

(III) preparing a target product A1:1- (4- ((4- (2-fluoro-5- ((4-oxo-3, 4-dihydro-phthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) sulphonyl) phenyl) -3- (pyridin-3-ylmethyl) urea

5- [ (3, 4-Dihydro-4-oxo-1-phthalazinyl) methyl ] -2-fluorobenzoic acid (1.49 g,5 mmol) (commercially available), intermediate 5a (2.06 g,5.5 mmol), 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (3.12 g,6 mmol) was dissolved in 20 mL N, N-dimethylformamide and N, N-diisopropylethylamine (1.94 g,15 mmol) was added and stirred at room temperature for 2H. After the reaction is finished, distilled water 50 mL is added, the mixture is filtered, and a filter cake is purified by column chromatography to obtain white solid 2.13 g, and the yield is obtained 65%.¹H NMR (600 MHz, DMSO-d₆) δ 8.52 – 8.45 (m, 2H), 8.19 (s, 2H), 7.94 – 7.88 (m, 1H), 7.80 – 7.66 (m, 5H), 7.60 – 7.50 (m, 3H), 7.44 – 7.31 (m, 3H), 4.43 – 4.39 (m, 2H), 4.25 (d, J = 0.9 Hz, 2H), 3.31 (dd, J = 10.9, 5.6 Hz, 2H), 3.29 – 3.20 (m, 3H), 3.18 (s, 4H). ¹³C NMR (150 MHz, DMSO-d₆) δ 166.06, 166.02, 159.51, 157.74, 156.82, 156.08, 148.67, 148.45, 142.83, 140.15, 136.27, 135.40, 133.99, 133.28, 132.03, 131.89, 131.87, 131.56, 131.52, 130.48, 130.43, 129.05, 128.52, 128.18, 125.91, 125.51, 124.38, 124.29, 122.91, 119.63, 115.65, 115.50, 48.43, 45.88, 44.17, 38.72. ESI-MS: m/z [M+H]⁺: 656.21.

Examples

The compound A2 was obtained in 52% yield by substituting tert-butyl hexahydropyrrolo [3,4-c ] pyrrole-2 (1H) -carboxylate (commercially available) for 1-tert-butoxycarbonyl-piperazine used in step (one) of example 1, otherwise as in example 1.

Examples

The compound A3 was obtained in 55% yield by substituting 4-amino-1-t-butoxycarbonyl piperidine (commercially available) for 1-t-butoxycarbonyl-piperazine used in step (one) of example 1 in the same manner as in example 1.

Examples

The compound A4 was obtained in 49% yield by substituting 1-t-butoxycarbonyl-piperazine used in step (one) of example 1 with 1-t-butoxycarbonyl-4-aminopiperidine (commercially available) in the same manner as in example 1.

Examples

The compound A5 was obtained in a yield of 51% by substituting 2-Boc-2, 7-diaza-spiro [4.4] nonane (commercially available) for 1-t-butoxycarbonyl-piperazine used in step (one) of example 1, otherwise.

Examples

The compound A6 was obtained in 46% yield by substituting tert-butyl 2, 6-diazaspiro [3.3] heptane-2-carboxylate (commercially available) for 1-t-butoxycarbonyl-piperazine used in step (one) of example 1, otherwise.

Examples

The compound A7 was obtained in 38% yield by substituting tert-butyl 3, 9-diazaspiro [5.5] undecane-3-carboxylate (commercially available) for 1-t-butoxycarbonyl-piperazine used in step (one) of example 1, otherwise as in example 1.

Examples

The compound A8 was obtained in 40% yield by substituting tert-butyl 2- (methylamino) ethylcarbamate (commercially available) with 1-t-butoxycarbonyl-piperazine used in step (one) of example 1, otherwise as in example 1.

Examples

Preparation of the compound: 1- (4- ((4- (2-fluoro-5- ((4-oxo-3, 4-dihydro-phthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) sulphonyl) phenyl) -3- (pyridin-3-ylmethyl) thiourea (A9)

The synthetic route is as follows:

Reagents and conditions: (a) 1,1' -thiocarbonyldiimidazole, acetonitrile, 60 ℃ for 4 hours at a yield of 70%; (b) Trifluoroacetic acid, dichloromethane, room temperature, 2 hours, yield 95%; (c) 1H-benzotriazol-1-yl-oxy-tripyrrolidinylphosphonium hexafluorophosphate, N, N-diisopropylethylamine, N, N-dimethylformamide, at room temperature for 2 hours, in 55% yield.

(One) preparation of intermediate 6:4- ((4- (3- (pyridin-4-ylmethyl) thiourea) phenyl) sulfonyl) piperazine-1-carboxylic acid tert-butyl ester

1,1' -Thiocarbonyldiimidazole (2.67 g,15 mmol) and intermediate 3a (3.41 g,10 mmol) were dissolved in 100 mL anhydrous acetonitrile, and after stirring at room temperature for 2 hours, 3-methylaminopyridine (2.16 g,20 mmol) was added thereto, and the reaction mixture was heated to 60℃and reacted for another 4 hours. After the reaction is finished, the reaction solution is concentrated, and the white solid 3.44 g is obtained by column chromatography separation, the yield 70%.¹H NMR (600 MHz, DMSO-d₆) δ 12.12 (s, 1H), 10.71 (t, J = 5.9 Hz, 1H), 8.78 (p, J= 1.2 Hz, 1H), 8.50 (dt, J = 3.8, 1.7 Hz, 1H), 7.91 (dtt, J = 7.9, 1.9, 1.0 Hz, 1H), 7.77 – 7.71 (m, 2H), 7.55 – 7.49 (m, 2H), 7.33 (dd, J = 7.9, 4.3 Hz, 1H), 4.72 – 4.68 (m, 2H), 3.15 (d, J = 5.6 Hz, 2H), 3.09 – 2.96 (m, 4H), 1.39 (s, 5H).¹³C NMR (150 MHz, DMSO-d₆) δ 176.23, 154.85, 148.67, 148.45, 140.65, 135.84, 135.40, 133.99, 128.99, 122.91, 120.41, 79.30, 48.43, 46.91, 45.84, 28.27.

(II) preparation of intermediate 7:

Intermediate 6 (4.91 g,10 mmol) was dissolved in a mixed solvent of 100mL trifluoroacetic acid/dichloromethane (1/1) and stirred at room temperature for 2h. After the reaction is finished, the solution is concentrated by reduced pressure distillation and purified by column chromatography to obtain white solid 3.72 g with yield 95%.¹H NMR (600 MHz, DMSO-d₆) δ 12.12 (s, 1H), 10.71 (t, J = 5.9 Hz, 1H), 8.80 – 8.76 (m, 1H), 8.50 (dt, J = 3.8, 1.7 Hz, 1H), 7.94 – 7.88 (m, 1H), 7.76 – 7.71 (m, 2H), 7.55 – 7.49 (m, 2H), 7.33 (dd, J = 7.9, 4.2 Hz, 1H), 4.70 (dd, J = 6.1, 1.1 Hz, 2H), 3.00 (d, J = 10.0 Hz, 2H), 2.82 – 2.72 (m, 5H).¹³C NMR (150 MHz, DMSO-d₆) δ 176.23, 148.67, 148.45, 140.65, 135.84, 135.40, 133.99, 128.99, 122.91, 120.41, 46.91, 43.31, 42.60.

(III) preparing a target product A9:1- (4- ((4- (2-fluoro-5- ((4-oxo-3, 4-dihydro-phthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) sulphonyl) phenyl) -3- (pyridin-3-ylmethyl) thiourea

5- [ (3, 4-Dihydro-4-oxo-1-phthalazinyl) methyl ] -2-fluorobenzoic acid (1.49 g,5 mmol) (commercially available), intermediate 7 (2.15 g,5.5 mmol), 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (3.12 g,6 mmol) was dissolved in 20 mL N, N-dimethylformamide and N, N-diisopropylethylamine (1.94 g,15 mmol) was added and stirred at room temperature for 2H. After the reaction is finished, distilled water 50mL is added, the mixture is filtered, and a filter cake is purified by column chromatography to obtain white solid 1.85 g, and the yield is increased 55%.¹H NMR (600 MHz, DMSO-d₆) δ 12.12 (s, 1H), 11.40 (s, 1H), 10.71 (t, J= 5.9 Hz, 1H), 8.80 – 8.76 (m, 1H), 8.52 – 8.45 (m, 2H), 8.19 (dd, J = 3.8, 2.5 Hz, 1H), 7.94 – 7.88 (m, 1H), 7.77 – 7.66 (m, 2H), 7.60 – 7.54 (m, 1H), 7.54 – 7.49 (m, 2H), 7.38 (dd, J = 10.1, 8.3 Hz, 1H), 7.33 (dd, J = 7.9, 4.2 Hz, 1H), 4.70 (dd, J = 6.0, 1.0 Hz, 2H), 4.25 (d, J = 2.0 Hz, 1H), 4.25 (s, 2H), 3.30 (dt, J = 11.0, 5.7 Hz, 2H), 3.29 – 3.20 (m, 2H), 3.18 (d, J = 11.5 Hz, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 176.23, 166.06, 166.02, 159.51, 157.74, 156.08, 148.67, 148.45, 142.83, 140.65, 135.84, 135.40, 133.99, 133.28, 132.03, 131.89, 131.87, 131.56, 131.52, 130.48, 130.43, 128.99, 128.52, 128.18, 125.91, 125.51, 124.38, 124.29, 122.91, 120.41, 115.65, 115.50, 48.43, 46.91, 45.88, 38.72. ESI-MS: m/z [M+H]⁺: 672.18.

Example 10:

The compound A10 was obtained in a yield of 51% by substituting 2, 3-dihydro-1H-pyrrolo [3,4-c ] pyridine (commercially available) for 3-aminomethylpyridine (commercially available) used in step (III) of example 1, otherwise by example 1.

Example 11:

The compound A11 was obtained in 56% yield by substituting 4-fluoropyridine-3-methylamine (commercially available) for 3-aminomethylpyridine used in step (III) of example 1 in the same manner as in example 1.

Example 12:

Preparation of the compound: n- (4- ((4- (2-fluoro-5- ((4-oxo-3, 4-dihydro-phthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) sulphonyl) phenyl) thieno [2,3-c ] pyridine-2-carboxamide (A12)

The synthetic route is as follows:

reagents and conditions: (a) 1H-benzotriazol-1-yloxy tripyrrolidinylphosphonium hexafluorophosphate, N, N-diisopropylethylamine, N, N-dimethylformamide, overnight at room temperature, yield 52%; (b) Trifluoroacetic acid, dichloromethane, room temperature, 2 hours, yield 95%; (c) 1H-benzotriazol-1-yl-oxy-tripyrrolidinylphosphonium hexafluorophosphate, N, N-diisopropylethylamine, N, N-dimethylformamide, at room temperature for 2 hours, yield 57%.

(One) preparation of intermediate 8c:4- ((4- (thiophene [2,3-c ] pyridine-2-carboxamide) phenyl) sulfonyl) piperazine-1-carboxylic acid tert-butyl ester

Thieno [2,3-c ] pyridine-2-carboxylic acid (0.89 g,5 mmol) (commercially available), intermediate 3a (1.87 g,5.5 mmol), 1H-benzotriazole-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (3.12 g,6 mmol) was dissolved in 20mL of N, N-dimethylformamide, followed by addition of N, N-diisopropylethylamine (1.94 g,15 mmol) and stirred at room temperature overnight. After the reaction is finished, distilled water 50mL is added, the mixture is filtered, and a filter cake is purified by column chromatography to obtain white solid 1.31: 1.31 g, and the yield is increased 52%.¹H NMR (600 MHz, DMSO-d₆) δ 10.33 (s, 1H), 9.53 (dd, J = 3.7, 1.3 Hz, 1H), 9.15 (dd, J = 4.0, 2.1 Hz, 1H), 8.59 (d, J = 1.5 Hz, 1H), 8.12 (d, J = 2.2 Hz, 1H), 7.99 – 7.93 (m, 2H), 7.80 – 7.74 (m, 2H), 3.15 (d, J = 5.6 Hz, 2H), 3.09 – 2.96 (m, 3H), 1.39 (s, 5H).¹³C NMR (150 MHz, DMSO-d₆) δ 158.57, 154.85, 152.54, 144.79, 143.61, 139.49, 136.46, 134.31, 132.04, 128.95, 121.21, 120.14, 118.23, 79.30, 48.43, 45.84, 28.27.

(II) preparation of intermediate 9c: n- (4- (piperazine-1-ylsulfonyl) phenyl) thiophene [2,3-c ] pyridine-2-carboxamide

Intermediate 8c (5.02 g,10 mmol) was dissolved in a mixed solvent of 100 mL trifluoroacetic acid/dichloromethane (1/1) and stirred at room temperature for 2h. After the reaction is finished, the solution is concentrated by reduced pressure distillation and purified by column chromatography to obtain white solid 3.82 g with yield 95%.¹H NMR (600 MHz, DMSO-d₆) δ 10.33 (s, 1H), 9.53 (dd, J = 3.8, 1.3 Hz, 1H), 9.15 (dd, J = 4.0, 2.1 Hz, 1H), 8.59 (d, J = 1.5 Hz, 1H), 8.12 (d, J= 2.2 Hz, 1H), 7.99 – 7.93 (m, 2H), 7.80 – 7.74 (m, 2H), 3.00 (d, J = 10.0 Hz, 2H), 2.82 – 2.72 (m, 5H).¹³C NMR (150 MHz, Common NMR Solvents) δ 158.57, 152.54, 144.79, 143.61, 139.49, 136.46, 134.31, 132.04, 128.95, 121.21, 120.14, 118.23, 43.31, 42.60.

(III) preparing a target product A12: n- (4- ((4- (2-fluoro-5- ((4-oxo-3, 4-dihydro-phthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) sulphonyl) phenyl) thieno [2,3-c ] pyridine-2-carboxamide

5- [ (3, 4-Dihydro-4-oxo-1-phthalazinyl) methyl ] -2-fluorobenzoic acid (1.49 g,5 mmol) (commercially available), intermediate 9c (2.21 g,5.5 mmol), 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (3.12 g,6 mmol) was dissolved in 20 mL N, N-dimethylformamide and N, N-diisopropylethylamine (1.94 g,15 mmol) was added and stirred at room temperature for 2H. After the reaction is finished, distilled water 50 mL is added, the mixture is filtered, and a filter cake is purified by column chromatography to obtain white solid 1.94: 1.94 g, and the yield is increased 57%.¹H NMR (600 MHz, DMSO-d₆) δ 11.40 (s, 1H), 10.33 (s, 1H), 9.53 (dd, J= 3.7, 1.3 Hz, 1H), 9.15 (dd, J = 4.0, 2.1 Hz, 1H), 8.59 (d, J = 1.5 Hz, 1H), 8.49 – 8.45 (m, 1H), 8.19 (dd, J = 3.5, 2.3 Hz, 1H), 8.12 (d, J = 2.1 Hz, 1H), 7.98 – 7.93 (m, 2H), 7.80 – 7.71 (m, 4H), 7.71 – 7.66 (m, 1H), 7.60 – 7.54 (m, 1H), 7.38 (dd, J = 10.1, 8.2 Hz, 1H), 4.25 (s, 1H), 3.30 (dt, J = 11.0, 5.7 Hz, 2H), 3.27 – 3.20 (m, 2H), 3.18 (d, J = 11.5 Hz, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 166.06, 166.02, 159.51, 158.57, 157.74, 156.08, 152.54, 144.79, 143.61, 142.83, 139.49, 136.46, 134.31, 133.28, 132.04, 131.89, 131.87, 131.56, 131.52, 130.48, 130.43, 128.95, 128.52, 128.18, 125.91, 125.51, 124.38, 124.29, 121.21, 120.14, 118.23, 115.65, 115.50, 48.43, 45.88, 38.72. ESI-MS: m/z [M+H]⁺: 683.16.

Example 13:

test for inhibition of PARP-1 enzyme Activity in vitro of target Compounds

1. Experimental materials

PARP1 colorimetric assay kit (cat# 80580, BPS Bioscience); PBS (Cat#21600-010, gibco); tween-20 (cat#p9416, sigma); ELISA stop solution (Cat#C1058, solarbio), positive drug Olaparib (olaharib).

2. Measurement method

(1) Diluting 10 XPARP buffer solution with deionized water to obtain 1 XPARP buffer solution, diluting the composite stock solution to 1 mu M with DMSO for later use, and diluting 1 mu M compound solution to 10 nM with 1 XPARP buffer solution. The tray was washed 3 times with 200 μl/well PBST, 200 μl/well blocking buffer was added, and incubation was performed at room temperature of 90 min. The tray was washed 3 more times with 200 μl/well PBST.

(2) Preparing a main mixed solution: n holes× (2.5 μl 10×parp buffer+2.5 μl 10×parp detection liquid+5 μl activated dna+15 μl distilled water), 25 μl was added per hole.

(3) The uniform compound was diluted by adding 5 μl/well and the same volume of 1×parp buffer containing 10% DMSO was added to the carrier control well and blank well. PARP1 was thawed on ice and diluted to 1.0 ng/ul with 1 x PARP buffer. 20 [ mu ] L of diluted PARP enzyme is added into a non-blank hole. 20 μl/well 1×parp buffer was added to the blank wells. The reaction was carried out at room temperature for 60 minutes. The trays were washed 3 times with 200 μl/well PBST.

(4) Streptavidin-HRP was diluted 1:50 with blocking buffer, 50 μl of diluted streptavidin-HRP was added per well and incubated for 30min at room temperature. The tray was washed 3 times with 200 μl/well PBST, 100 μl/well HRP colorimetric substrate 20min was added at room temperature, 100 μl/well 2M sulfuric acid was added, and OD 450 nm was read on a microplate reader.

The experimental results (table 2) show that these compounds all show good PARP1 inhibitory activity, wherein compounds A1, A2 and A9 show comparable inhibitory activity to the positive control olaparib.

TABLE 2 inhibition of PARP-1 Activity of target Compounds

Example 14:

Test for in vitro inhibition of NAMPT enzyme Activity by target Compounds

1. Experimental materials

Test solution: bovine serum albumin (BSA, kingmorn) +Tris-HCl (pH 7.5) +MgCl ₂ (Inactive), ATP solution (100 mM, inactive), PRPP solution (40 mM, source leaf), DTT solution (2 mM, inactive); compound gradient dilution solution; NAM solution (0.2. Mu.M, manufactured); 20% acetophenone 2M KOH 88% formic acid; positive drug FK866.

2. Measurement method

(1) A freshly prepared test solution, 20. Mu.L, and 0.5. Mu.L of each concentration gradient compound solution, was added to a 96-well plate and allowed to stand at room temperature for 5min.

(2) Adding 4.5 mu L of NAM solution into the 96-well plate, and adding double distilled water into the blank control group; after mixing, the mixture was reacted at 37℃to give 15 min.

(3) After the reaction, the reaction was terminated by heating at 95℃for 1 min and rapidly cooling on ice.

(4) To the terminated enzyme reaction solution, 10. Mu.L of 20% acetophenone and 2M KOH were added, followed by rapid centrifugation, and then, the mixture was mixed with a vortex mixer and reacted at 0℃for 10 minutes.

(5) A further 45. Mu.L of 88% formic acid was added and the reaction was carried out at 37℃with 10 min.

(6) After cooling, 85. Mu.L of the reaction system was transferred to a black flat bottom 96 Kong Yingguang plate.

(7) NAMPT protein catalyzes the conversion of nicotinamide NAM to product nicotinamide mononucleotide NMN; in the detection reaction, NMN is converted to a fluorescent derivative by a two-step chemical reaction, and a maximum fluorescence signal is detected at excitation wavelength 382 nm and emission wavelength 445 nm. The fluorescence values at excitation light 382 nm and emission light 445 nm were measured with a microplate reader, and the inhibitory activity of the compounds on NAMPT was calculated with GRAPHPAD PRISM software.

Experimental results show that most of the compounds of the invention show good NAMPT inhibition activity, and the results are shown in a table 3, wherein the difference of the connectors has a significant effect on the enzyme inhibition activity of the compounds, and the activity of the compounds except the compounds A4, A7 and A8 is far stronger than that of positive control medicine FK866.

TABLE 3 inhibition of NAMPT by target compounds

Example 15:

In vitro antitumor Activity test of target Compounds (MTT method)

1. Experimental materials

MTT, PRMI1640 medium, fetal bovine serum, 96-well plate, CO ₂ constant temperature incubator, BIO-TEK Uquant multifunctional enzyme label instrument, human breast cancer cells (MDA-MB-231), human breast cancer cells (MDA-MB-436) and human breast cancer cells (MDA-MB-468), positive control Olaparib.

2. Experimental method

(1) Cells were inoculated, single cell suspensions were prepared with 10% fetal bovine serum in culture, and 5000 cells per well were inoculated into 96-well plates, 100 μl per well volume, and cultured overnight.

(2) Preparing a solution of a compound to be tested, and diluting a DMSO stock solution of the compound to be tested into 5 concentrations to be tested by using a culture solution in a sterile table, wherein the adjacent concentrations are diluted by two times.

(3) Solutions of different concentrations of the compound were added to 96-well plates that had been incubated overnight, 100 μl each, and 3 multiplex wells for each concentration. Since the periphery has an edge effect and is easily contaminated, no cells, no compound, and 100. Mu.L of the culture medium was added as a blank. A further 100% well, i.e.100. Mu.L of cells and compound-free medium, was placed and incubated in a 37℃incubator for 48 hours.

(4) Staining, adding 10 mu LMTT solution (5 mg/mL, prepared by PBS) into a 96-well plate for staining, centrifuging at 2500rps for 10 minutes after incubation for 4 hours, then sucking out the culture solution from the well by a discharge gun, adding 150 mu LDMSO, vibrating for 5-10 minutes on a vibrating plate to fully dissolve formazan, and measuring the OD value of each well at 570nm by an enzyme-labeling instrument.

Inhibition (%) = (100% mean OD of wells-mean OD of wells of compound)/(100% mean OD of wells-mean OD of wells of blanks) ×100%. Based on the inhibition values of the respective concentrations, linear regression was performed to calculate the concentration of the drug inhibiting cell growth by 50%, i.e., IC ₅₀.

The experimental results (table 4) show that these target compounds have significant anti-tumor cell proliferation activity, all stronger than the positive drug Olaparib, and independent of the cellular BRCA mutant status, with IC ₅₀ values between 0.09-30.05 μm, with compounds A1, A9 and a10 being the most active potential compounds.

TABLE 4 results of in vitro anti-tumor cell proliferation Activity of target Compounds

Example 16: preferred compounds have in vivo antitumor effects

According to the experimental results, a model of human breast cancer cell strain MDA-MB-468 nude mice transplantation tumor is selected to test the in-vivo anti-tumor activity of the compounds A1 and A9, the administration dosage is 50 mg/kg, and the intraperitoneal injection is carried out twice daily for 14 days. The results show (table 5) that both compounds A1 and A9 showed excellent in vivo inhibition activity, with tumor inhibition rates of 78.95% and 73.68%, respectively, significantly better than the positive Olaparib control group (30.53%) at the same dose. Furthermore, no significant changes in mice death and body weight were found during dosing, indicating that compounds A1 and A9 were less toxic in vivo and of further research value.

TABLE 5 therapeutic efficacy of preferred Compounds on human breast cancer MDA-MB-468 nude mice transplantable tumors

Claims

1. A compound as a PARP-NAMPT dual-target inhibitor, characterized in that it has a structure represented by the following general formula (I), as well as its optical isomers, diastereoisomers and racemic mixtures, and pharmaceutically acceptable salts thereof; ;

in:

X is an oxygen atom or a sulfur atom;

_R1 is any one of the following structures:

;

_R2 is any one of the following structures:

.

2. The typical compounds as PARP-NAMPT dual-target inhibitor compounds according to claim 1 are as follows:

Compound A1: 1-(4-((4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)sulfonyl)phenyl)-3-(pyridin-3-ylmethyl)urea;

Compound A2: 1-(4-((5-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)sulfonyl)phenyl)-3-(pyridin-3-ylmethyl)urea;

Compound A3: 2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)-N-(1-((4-(3-(pyridin-3-ylmethyl)ureido)phenyl)sulfonyl)piperidin-4-yl)benzamide;

Compound A4: N-(1-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperidin-4-yl)-4-(3-(pyridin-3-ylmethyl)ureido)benzenesulfonamide;

Compound A5: 1-(4-((7-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)-2,7-diazaspiro[4.4]nonan-2-yl)sulfonyl)phenyl)-3-(pyridin-3-ylmethyl)urea;

Compound A6: 1-(4-((6-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)-2,6-diazaspiro[3.3]heptane-2-yl)sulfonyl)phenyl)-3-(pyridin-3-ylmethyl)urea;

Compound A7: 1-(4-((9-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)-3,9-diazaspiro[5.5]undec-3-yl)sulfonyl)phenyl)-3-(pyridin-3-ylmethyl)urea;

Compound A8: 2-fluoro-N-(2-((N-methyl-4-(3-(pyridin-3-ylmethyl)ureido)phenyl)sulfonamido)ethyl)-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamide;

Compound A9: 1-(4-((4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)sulfonyl)phenyl)-3-(pyridin-3-ylmethyl)thiourea;

Compound A10: N-(4-((4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)sulfonyl)phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide;

Compound A11: 1-(4-((4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)sulfonyl)phenyl)-3-((4-fluoropyridin-3-yl)methyl)urea;

Compound A12: N-(4-((4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)sulfonyl)phenyl)thieno[2,3-c]pyridine-2-carboxamide.

3. The method for preparing the compound as a PARP-NAMPT dual-target inhibitor according to claim 1, characterized in that:

(1) Synthesis of Compounds A1-A8

Using p-nitrobenzenesulfonyl chloride and a mono-tert-butyloxycarbonyl protected diamino intermediate as starting materials, a substitution reaction is carried out under alkaline conditions to obtain intermediates 2a-2h; then, in the presence of hydrogen and palladium carbon, the nitro group is reduced to obtain intermediates 3a-3h; intermediates 3a-3h are reacted with triphosgene and 3-aminomethylpyridine under alkaline conditions to obtain urea intermediates 4a-4h; then, deprotection is carried out under acidic conditions to obtain key intermediates 5a-5h; finally, intermediates 5a-5h are subjected to an amide condensation reaction with 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid to obtain target compounds A1-A8 ;

Reagents and conditions: (a) triethylamine, dichloromethane, room temperature, 2 hours; (b) hydrogen, palladium carbon, dichloromethane/methanol, room temperature, overnight; (c) triphosgene, triethylamine, dichloromethane, room temperature, 2 hours; triethylamine, dichloromethane, room temperature, overnight; (d) trifluoroacetic acid, dichloromethane, room temperature, 2 hours; (e) 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, room temperature, 2 hours;

(2) Synthesis method of compound A9

Intermediate 3a reacts with 1,1'-thiocarbonyldiimidazole and 3-aminomethylpyridine under alkaline conditions to obtain thiourea intermediate 6; then deprotection is performed under acidic conditions to obtain key intermediate 7; finally, intermediate 7 undergoes amide condensation reaction with 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid to obtain the target compound A9. ;

Reagents and conditions: (a) 1,1'-thiocarbonyldiimidazole, acetonitrile, 60°C, 4 hours; (b) trifluoroacetic acid, dichloromethane, room temperature, 2 hours; (c) 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, room temperature, 2 hours;

(3) Synthesis of Compounds A10 and A11

Intermediate 3a is reacted with triphosgene and 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine or 4-fluoropyridine-3-methylamine under alkaline conditions to obtain urea intermediate 8a or 8b; then deprotected under acidic conditions to obtain key intermediate 9a or 9b; finally, intermediate 9a or 9b is subjected to amide condensation reaction with 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid to obtain target compound A10 or A11 ;

Reagents and conditions: (a) triphosgene, triethylamine, dichloromethane, room temperature; triethylamine, dichloromethane, room temperature, overnight; (b) trifluoroacetic acid, dichloromethane, room temperature, 2 hours; (c) 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, room temperature, 2 hours;

Synthesis method of compound A12

Intermediate 3a and thieno[2,3-c]pyridine-2-carboxylic acid are subjected to an amide condensation reaction to obtain intermediate 8c; then, the intermediate 9c is deprotected under acidic conditions to obtain the key intermediate 9c; finally, intermediate 9c is subjected to an amide condensation reaction with 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid to obtain the target compound A12 ;

Reagents and conditions: (a) 1H-Benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, room temperature overnight; (b) trifluoroacetic acid, dichloromethane, room temperature, 2 hours; (c) 1H-Benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, room temperature, 2 hours.

4. Use of the compound as a PARP-NAMPT dual-target inhibitor according to claim 1 in the preparation of anti-tumor drugs; the tumor is breast cancer.