RU2804280C2 - Phenyl-2-hydroxy-acetylamino-2-methyl-phenyl compounds - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to a compound of the following formula

, where R is selected from the group consisting of and ; X is CH or N; R¹ is hydrogen or fluorine; and R² is C₁-C₃ alkyl; or to its pharmaceutically acceptable salt. The invention also relates to a pharmaceutical composition having PERK inhibitor activity based on the said compound.

EFFECT: obtaining new compounds and a pharmaceutical composition based on them which can be used in medicine as effective agents for the treatment of prostate cancer.

11 cl, 4 tbl, 9 ex

Description

The present invention relates to novel phenyl-2-hydroxy-acetylamino-2-methyl-phenyl compounds, pharmaceutical compositions comprising the compounds, methods of using the compounds for the treatment of physiological disorders, as well as intermediates and methods that can be used to synthesize the compounds .

The present invention relates to the field of treatment of cancer and other diseases and disorders associated with protein kinase R (PKR)-like protein kinase endoplasmic reticulum (PERK). PERK, an eIF2 kinase involved in the unfolded protein response (UPR), regulates protein synthesis, helps cells mitigate the effects of endoplasmic reticulum stress, and is involved in tumorigenesis and cancer cell survival.

Tumor cells thrive in a hostile microenvironment caused mainly by nutrient and oxygen limitation, high metabolic demand and oxidative stress. These stresses are known to impair the ability of the endoplasmic reticulum (ER) to fold proteins, causing a cellular remediation response known as the unfolded protein response (UPR). ER stress contributes to increased tumorigenic potential of cancer cells, tumor metastasis, tumor drug resistance, and the ability of cancer cells to evade effective immune responses.

There are three major ER transmembrane sensors of the UPR: 1) inositol-requiring enzyme (IRE1α/IRE1β, encoded by ERN1 and ERN2 , respectively); 2) PKR-like ER kinase (PERK, also known as PEK, encoded by EIF2AK3 ); and 3) activating transcription factor 6α (encoded by ATF6 ). Each of these three sensors is similarly regulated through binding of the luminal ER chaperone protein GRP78 or BiP (encoded by HSPA5 ). When ER protein folding demands exceed capacity, reduced BiP binding results in activation of these ER sensor proteins, resulting in the induction of coordinated signaling pathways to increase ER folding capacity and alleviate underlying stress. Effective responses lead to cell adaptation and survival, while irreparable ER stress causes cell death and apoptosis.

PERK is a type I transmembrane serine/threonine kinase and a member of a family of kinases that phosphorylate eukaryotic translation initiation factor 2α (eIF2-α) and regulate translation initiation. Other family members include HRI (EIF2AK1), PKR (EIF2AK2), and GCN2 (EIF2AK4). Each eIF2 kinase responds to different cellular stress signals to regulate general translation and gene-specific translational control. Phosphorylation of eIF2 results in decreased initiation of overall translation due to decreased activity of the exchange factor eIF2B, reducing the amount of protein entering the ER (and thus protein folding load) and the need for ATP translation. Phosphorylation of eIF2 also increases the translation of several mRNAs in a gene-specific manner, including the transcription factor ATF4. Transcriptional targets of ATF4 include numerous genes involved in cell adaptation and survival, including several genes involved in protein folding, nutrient uptake, amino acid metabolism, redox homeostasis, and autophagy ( 4 ). Selective inhibition of the PERK branch of the UPR is predicted to profoundly affect tumor cell growth and survival. Thus, compounds that inhibit PERK are believed to be useful in the treatment of cancer.

With the current state of medical treatment, patients with pancreatic cancer often have a poor prognosis, even if the disease is detected in the early stages. Thus, there remains a significant need for new and effective treatments for pancreatic cancer. The compounds of the present invention are PERK inhibitors and are believed to be useful in the treatment of cancer, in particular pancreatic cancer.

WO 2015/136463 discloses certain pyrrolidinone derivatives that have PERK inhibitory activity and further discloses compounds useful for the treatment of cancer and diseases associated with activated unfolded protein response, including pancreatic cancer.

Accordingly, the present invention relates to a compound of formula I:

Where

R is selected from the group consisting of

And ;

X represents CH or N;

R ¹ represents hydrogen or fluorine; And

R ² represents C ₁ -C ₃ alkyl;

or a pharmaceutically acceptable salt thereof.

Furthermore, the present invention relates to a compound of formula Ia:

Where

R is selected from the group consisting of

And ;

X represents CH or N;

R ¹ represents hydrogen or fluorine; And

R ² represents C ₁ -C ₃ alkyl;

or a pharmaceutically acceptable salt thereof.

Furthermore, the present invention relates to a compound of formula I or Ia:

where R represents

.

In addition, the present invention relates to a compound of formula I or Ia: wherein X represents CH or N; R ¹ represents hydrogen or fluorine; and R ² represents methyl or isopropyl; or a pharmaceutically acceptable salt thereof.

Furthermore, the present invention relates to a compound of formula I or Ia: wherein R is

or a pharmaceutically acceptable salt thereof.

Furthermore, the present invention relates to the compound 3-amino-6-[4-[[(2R)-2-(3,5-difluorophenyl)-2-hydroxy-acetyl]amino]-2-methyl-phenyl]-N -methyl-pyrazine-2-carboxamide, which can be represented by the formula

or a pharmaceutically acceptable salt thereof.

Furthermore, the present invention relates to the compound 2-amino-5-[4-[[(2R)-2-(3,5-difluorophenyl)-2-hydroxy-acetyl]amino]-2-methyl-phenyl]-N -isopropyl-pyridine-3-carboxamide, which can be represented by the formula

or a pharmaceutically acceptable salt thereof.

Furthermore, the present invention relates to the compound (2R)-N-[4-(4-amino-7-methyl-pyrrolo[2,3-d]pyrimidin-5-yl)-3-methyl-phenyl]-2- (3-fluorophenyl)-2-hydroxy-acetamide, which can be represented by the formula

or a pharmaceutically acceptable salt thereof.

The present invention relates to a method of treating cancer in a patient in need of such treatment, comprising administering to the patient an effective amount of a compound of formula I or Ia, or a pharmaceutically acceptable salt thereof. The present invention also provides a method of inhibiting PERK activity resulting in antitumor activity in a patient in need of such treatment, comprising administering to the patient an effective amount of a compound of Formula I or Ia, or a pharmaceutically acceptable salt thereof.

The present invention also provides a method of treating pancreatic cancer in a patient in need of such treatment, comprising administering to the patient an effective amount of a compound of formula I or Ia, or a pharmaceutically acceptable salt thereof.

Furthermore, the present invention relates to a compound of formula I or Ia, or a pharmaceutically acceptable salt thereof, for use in therapy, in particular for the treatment of pancreatic cancer. Furthermore, the present invention relates to a compound of formula I or Ia or a pharmaceutically acceptable salt thereof for use in the treatment of pancreatic cancer. In a further embodiment, the present invention relates to the use of a compound of the invention or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of pancreatic cancer.

The invention further relates to a pharmaceutical composition containing a compound of the invention or a pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable carriers, diluents or excipients. In a further embodiment, the composition further comprises one or more other therapeutic agents. The invention also relates to a method for preparing a pharmaceutical composition, comprising mixing a compound of formula I or Ia or a pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable carriers, diluents or excipients. The present invention also covers new intermediates and methods for the synthesis of compounds of formula I and Ia.

As used herein, the terms “treating” or “treating” include limiting, slowing, stopping or reversing the progression or severity of an existing symptom or disorder.

As used herein, the term “effective amount” refers to an amount or dose of a compound of the invention, or a pharmaceutically acceptable salt thereof, that, when administered single or multiple doses to a patient, provides the desired effect in the patient being diagnosed or treated.

The effective amount can be readily determined by one skilled in the art using known techniques and by observing results obtained under similar circumstances. When determining the effective amount for a patient, a number of factors are considered, including, but not limited to: the patient's gender; its size, age and general health; a specific disease or disorder; the extent or extent or severity of the disease or disorder; individual patient response; the specific compound administered; method of administration; characteristics of the bioavailability of the administered drug; selected dosing regimen; use of concomitant medications; and other relevant circumstances.

The compounds of the present invention are generally effective over a wide range of dosages. For example, dosages per day typically range from about 0.1 to about 50 mg/kg body weight. In some cases, dosage levels below the lower end of the above range may be more than adequate, while in other cases even higher doses may be used with acceptable side effects, and therefore the above dosage range is in no way intended to limit the scope of the invention. It is understood that the amount of compound actually administered will be determined by the physician taking into account the relevant circumstances, including the condition being treated, the route of administration chosen, the actual compound or compounds administered, the age, weight and response of the individual patient, and the severity of the patient's symptoms.

The compounds of the present invention are preferably prepared in the form of pharmaceutical compositions administered by any route that makes the compound bioavailable, including oral, intravenous and transdermal routes of administration. Most preferably, such compositions are for oral administration . Such pharmaceutical compositions and methods for their preparation are well known in the art (see, for example, Remington: The Science and Practice of Pharmacy, LV Allen, Editor, ^22nd Edition, Pharmaceutical Press, 2012).

It is understood that the compounds of formula I may exist as stereoisomers. Embodiments of the present invention include all enantiomers, diastereomers, and mixtures thereof. The specific enantiomer of a compound of formula I is represented by a compound of formula Ia

where R and R ¹ are as previously defined.

One skilled in the art will also understand that the Cahn-Ingold-Prelog designation (R) or (S) for all chiral centers will vary depending on the substitution pattern of the particular compound. The individual enantiomers or diastereomers can be prepared starting from chiral reagents or using stereoselective or stereospecific synthetic methods. Alternatively, individual enantiomers or diastereomers can be isolated from mixtures by standard chiral chromatography or crystallization techniques at any convenient point in the synthesis of the compounds of the invention. See, for example, J. Jacques, et al ., “ Enantiomers, Racemates, and Resolutions ,” John Wiley and Sons, Inc., 1981, and E. L. Eliel and S. H. Wilen, “ Stereochemistry of Organic Compounds ,” Wiley-Interscience, 1994. The individual enantiomers of the compounds of the invention are a preferred embodiment of the invention.

A pharmaceutically acceptable salt of the compounds of the invention may be formed, for example, by reacting a suitable free base of the compound of the invention and a suitable pharmaceutically acceptable acid in a suitable solvent under standard conditions well known in the art. The formation of such salts is well known and appreciated in the art. See, for example, Gould, P.L., “Salt selection for basic drugs,” International Journal of Pharmaceutics , 33 : 201-217 (1986); Bastin, R. J., et al . “Salt Selection and Optimization Procedures for Pharmaceutical New Chemical Entities,” Organic Process Research and Development , 4 : 427–435 (2000); and Berge, S. M., et al ., “Pharmaceutical Salts,” Journal of Pharmaceutical Sciences , 66 : 1-19, (1977).

In vitro inhibition of PERK enzymatic activity (isolated)

Recombinant human EIF2AK2 (PKR) catalytic domain (amino acids 252–551), EIF2AK3 (PERK) catalytic domain (amino acids 536–1116), GFP-eIF2α substrate, and terbium-labeled phospho-eIF2α antibody (Invitrogen, Carlsbad, CA) were prepared. The HIS-SUMO-GCN2 catalytic domain (amino acids 584-1019) was expressed and purified from E. coli . TR-FRET kinase assays were performed in the absence or presence of inhibitors in a reaction buffer consisting of 50 mM HEPES, pH 7.5, 10 mM MgCl ₂ , 1.0 mM EGTA and 0.01% Brij-35 and 100-200 nM GFP-eIF2α substrate. PKR assays contain 14 ng/mL enzyme and 2.5 μM ATP ( Km , approximately ~2.5 μM), PERK assays contain 62.5 ng/mL enzyme and 1.5 μM ATP ( Km , approximately ~1.5 μM ), and GCN2 assays contain 3 nM enzyme and 90 μM ATP ( Km , approximately ∼200 μM). The test compound was added, the reaction was initiated by adding enzyme and incubated at room temperature for 45 minutes. The reaction was stopped by adding EDTA to a final concentration of 10 mM, terbium-labeled phospho-eIF2α antibody was added at a final concentration of 2 nM and incubated for 90 minutes. The resulting fluorescence was monitored using an EnVison® Multilabel reader (PerkinElmer, Waltham, MA). TR-FRET ratios and resulting IC ₅₀ values were determined using a 4-parameter nonlinear logistic equation as shown (A+((BA)/(1+((C/x) ^∧ D)))), where Y=% specific inhibition , A=bottom of the curve, B=top of the curve, C=absolute IC ₅₀ (concentration causing 50% inhibition) and D=Hill's slope.

The compounds of Examples 1, 5 and 9 were tested essentially as described above and showed the IC ₅₀ values shown in Table 1. These data demonstrate that the compounds of Examples 1, 5 and 9 inhibit the activity of the isolated PERK enzyme in vitro .

Table 1

Example No. Enzyme IC ₅₀ (µM) PERK GCN2 PKR Example 1 0.0022±0.0012
(N=3) 18.1±1.5
(N=2) >20
(N=1) Example 5 0.0020±0.0005
(Ν=3) 10.8±2.1
(N=4) >20
(N=1) Example 9 0.0024±0.0010
(N=4) 16.4±2.9
(N=4) Not determined

In vitro inhibition of PERK enzyme activity (whole cell)

GripTite™ 293 cells (Invitrogen, Carlsbad, CA) expressing GFP-eIF2α were seeded at 10,000 cells per well in 384-well plates and allowed to attach overnight. Cells were pretreated with test compounds for 1 hour. Tunicamycin (1 μM) was added to induce PERK activity, and the plates were incubated at 37°C for 2 h. The culture medium was removed and the cells were lysed in a buffer consisting of 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 5 mM NaF, protease inhibitors (Sigma, St. Louis, MO ), phosphatase inhibitors (Sigma, St. Louis, MO), and 2 nM terbium-labeled anti-phospho-eIF2 antibody (Invitrogen, Carlsbad, CA). Cell lysates were incubated for 2 hours in the dark at room temperature and fluorescence was monitored with an EnVison® Multilabel reader (PerkinElmer, Waltham, MA). TR-FRET ratios and resulting IC ₅₀ values were determined from the corresponding inhibition curves using uninduced (100% inhibition) and induced (0% inhibition) wells as controls.

The compounds of Examples 1, 5 and 9 were tested substantially as described above and showed the IC ₅₀ values shown in Table 2. These data demonstrate that the compounds of Examples 1, 5 and 9 inhibit whole cell PERK enzyme activity in vitro .

table 2

Example No. Cell IC ₅₀ (µM) Example 1 0.054±0.060
(N=9) Example 5 0.117±0.102
(N=14) Example 9 0.028±0.011
(N=12)

In vivo Inhibition of pancreatic cancer (xenograft mouse model)

Female nude mice (Harlan Laboratories) were implanted subcutaneously with 5×10 ⁶ BxPC-3 cells with Matrigel in the right flank, and tumor growth was monitored using a caliper. Compound administration was started when tumors reached ~250 mm ³ and mice were administered twice daily by oral gavage (8 animals per group) for 28 days. The compounds are prepared in 10% gum acacia containing 0.05% antifoam or 20% captisol in 25 mM NaPO ₄ buffer, pH 2, for 30 compounds of Examples 5 and 9, respectively. Control animals were administered only the carrier gum acacia. Tumor volumes were estimated using the formula l × w ² × (π/6), where l represents the larger measured diameter and w represents the smaller perpendicular diameter. The percentage delta T/C was calculated using the formula 100×[(TT ₀ )/(CC ₀ )], and the percentage regression using the formula 100×[(TT ₀ )/T ₀ ], where T and C represent the average tumor volumes in the treated and control groups, respectively. T ₀ and C ₀ represent the corresponding initial mean tumor volumes. Convert percent delta T/C to percent delta tumor growth inhibition (TGI) using the equation 100 - percent delta T/C. For statistical analysis, convert tumor volume data to log10 scale to equalize variance across time and treatment groups. Analyze log ₁₀ volume data using two-way repeated measures ANOVA (spatial statistical power correlation model) across time and treatment using mixed procedures in the SAS software package (version 9.3). Comparison of the treatment group with the control group at each time point.

The compounds of Examples 5 and 9 were tested essentially as described above and showed the tumor growth inhibition values shown in Tables 3 and 4, respectively. These data demonstrate that the compounds of Examples 5 and 9 inhibit pancreatic tumor growth in vivo .

Table 3

Tumor Volume Summary Values Day after implantation Control media Example 5
30 mg/kg po
2 times/day p-value T/C ^b (%) TGI ^c (%) Average (mm ³ ) SE ^a Average (mm ³ ) SEa 21 108.3 4.6 123.9 13.3 N.A. N.A. N.A. 24 115.8 7.9 132.8 11.6 N.A. N.A. N.A. 32 153.4 13.5 143.4 10.4 N.A. N.A. N.A. 35 163.9 10.7 174.6 17.2 N.A. N.A. N.A. 39 180.5 13.4 183.8 19.0 N.A. N.A. N.A. 47 206.4 19.2 213.7 20.7 N.A. N.A. N.A. ^52d 252.2 39.6 252.0 21.6 N.A. N.A. N.A. 60 337.0 52.9 311.8 26.7 0.667 71.5 28.5 67 498.1 78.2 387.8 33.2 0.182 55.8 44.2 72 602.3 94.6 435.0 37.3 0.084 52.7 47.3 74 720.0 113.0 487.5 41.8 0.039* 50.7 49.3 76 762.5 119.7 528.8 45.3 0.052 54.5 45.5 79 971.5 152.5 593.2 50.8 0.010* 47.7 52.3

a) Standard error of the geometric mean tumor volume

b) Calculated using 100×[(TT ₀ )/(CC ₀ )], where T and C represent the mean tumor volumes in the treatment and control groups, respectively, T ₀ and C ₀ represent the corresponding baseline mean tumor volumes.

c) TCI is tumor growth inhibition calculated using 100 - %T/C

d) Day of randomization and start of treatment

*Significant, p < 0.05

Table 4

Tumor Volume Summary Values Day after implantation Control media Example 9
30 mg/kg po
2 times/day p-value T/C ^b (%) TGI ^c (%) average (mm ³ ) SE ^a average (mm ³ ) SE ^a 21 108.3 4.6 111.9 7.5 N.A. N.A. N.A. 24 115.8 7.9 134.3 8.4 N.A. N.A. N.A. 32 153.4 13.5 153.7 12.4 N.A. N.A. N.A. 35 163.9 10.7 162.6 14.6 N.A. N.A. N.A. 39 180.5 13.4 167.1 12.8 N.A. N.A. N.A. 47 206.4 19.2 196.4 15.5 N.A. N.A. N.A. ^52d 252.2 39.6 244.2 28.4 N.A. N.A. N.A. 60 337.0 52.9 284.8 33.2 0.367 40.8 59.2 67 498.1 78.2 317.2 36.9 0.018* 27.4 72.6 72 602.3 94.6 380.5 44.3 0.016* 37.3 62.7 74 720.0 113.0 418.3 48.7 0.005* 36.0 64.0 76 762.5 119.7 480.4 55.9 0.015* 45.1 54.9 79 971.5 152.5 541.6 63.1 0.002* 40.5 59.5

a) Standard error of the geometric mean tumor volume

b) Calculated using 100×[(TT ₀ )/(CC ₀ )], where T and C represent the mean tumor volumes in the treatment and control groups, respectively, T ₀ and C ₀ represent the corresponding baseline mean tumor volumes.

c) TCI is tumor growth inhibition calculated using 100 - %T/C

d) Day of randomization and start of treatment

*Significant, p < 0.05

The compounds of the present invention or their salts can be prepared by various methods known to one skilled in the art, some of which are illustrated in the diagrams, preparations and examples below. One skilled in the art will recognize that the specific synthetic steps for each of the described methods can be combined in different ways or in combination with steps from different schemes to obtain the compounds of the invention or salts thereof. The products of each step in the schemes below can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, grinding and crystallization. In the diagrams below, all substituents, unless otherwise noted, are as previously defined. Reagents and starting materials are readily available to one skilled in the art. Without limiting the scope of the invention, the following schemes, preparations and examples are provided to further illustrate the invention. In addition, one skilled in the art will appreciate that compounds of formula Ia can be prepared using a starting material with an appropriate stereochemical configuration that can be prepared by one skilled in the art. For example, the schemes below use starting materials with a configuration substantially corresponding to Formula Ia.

Typically, a compound of formula I can be prepared from a compound of formula III (Scheme 1). More specifically, a compound of formula III is reacted with a compound of formula II and a suitable coupling reagent such as HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3 -oxide hexafluorophosphate) in the presence of a suitable amine base such as N,N-diisopropylethylamine or trimethylamine. A compound of formula I can be separated into its isomers using chiral chromatography.

Accordingly, a compound of formula Ia can be prepared from a compound of formula IIa. More specifically, a compound of formula III is reacted with a compound of formula IIa and a suitable coupling reagent such as HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3 -oxide hexafluorophosphate) in the presence of a suitable amine base such as N,N-diisopropylethylamine or trimethylamine. The compound of formula IIa can be prepared from the compound of formula II with a lipolytic enzyme such as Lipase PS Amano SD. Additional information regarding this optical resolution method can be found in Mendiola, J. et al, Org. Process Res. Dev . 2012, 16, 1312−1316.

Scheme 1

Typically, a compound of formula III can be prepared from a compound of formula IV. The compound of formula III can be prepared by treating the compound of formula R-Br with 3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline in the presence of a base such as K ₂ CO ₃ , and a palladium catalyst such as Pd(dppf) ₂ Cl ₂ .

Scheme 2

R-Br is or

The compound of formula R-Br, represented by a compound of formula VI or VII, can be prepared by methods known in the field of chemistry, as well as by methods described in the following Preparations and Examples.

Receipt 1

Synthesis of 4-chloro-7-methyl-pyrrolo[2,3-d]pyrimidine.

Add Cs ₂ CO ₃ (845 g, 2.60 mol) at 15°C to a solution of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (200 g, 1.29 mol) in N-methyl-2 -pyrrolidone (1.20 l). Heat to 23°C, add MeI (202 g, 1.43 mol) dropwise over 30 minutes, and stir for 4 hours. After this time, pour into ice water (2.00 L) and stir for 30 min. Filtered, then suspended the substance in H ₂ O (1.00 L). Filter and dry to give the title compound (180 g, 81%). ES/MS m/z ( ³⁵ Cl) 168.0 (M+H).

Receipt 2

Synthesis of 5-bromo-4-chloro-7-methyl-pyrrolo[2,3-d]pyrimidine.

Add N-bromosuccinimide (418 g, 2.35 mol) in portions over 20 min at 15°C to a solution of 4-chloro-7-methyl-pyrrolo[2,3-d]pyrimidine (355 g, 2.12 mol) in dichloromethane (3.19 L), and stirred at 23°C for 3 hours. After this time, filtered, washed with H ₂ O (5.32 L) and dried to give the title compound (448 g, 86 %) as a white solid. ES/MS m/z ( ³⁵ Cl, ⁷⁹ Br) 245.9 (M+H).

Receipt 3

Synthesis of 5-bromo-7-methyl-pyrrolo[2,3-d]pyrimidin-4-amine.

Stir a suspension of 5-bromo-4-chloro-7-methyl-pyrrolo[2,3-d]pyrimidine (454 g 1.84 mol) in ammonia (30% in H ₂ O, 3.63 L) at 120°C in a Hastelloy™ pressure vessel for 18 hours. Cool to 20°C, filter, wash with H ₂ O (1.80 L) and methanol (900 ml), and dry to give the title compound (351 g, 82% ) as a white solid. ES/MS m/z ( ⁷⁹ Br) 227.2 (M+H).

Receipt 4

Synthesis of 3-amino-6-bromo-pyrazine-2-carboxylic acid.

Add 3-aminopyrazine-2-carboxylic acid (50.0 g, 369.4 mmol) to a solution of N-bromosuccinimide (61.2 g, 377.3 mmol) and dimethylformamide (236.3 g, 3.2 mol) at 0°C. After 1 hour at room temperature, an orange solid formed. The solid residue was washed with ethyl acetate (500 ml) and discarded. Dry the organic phase over sodium sulfate, filter and concentrate under reduced pressure to give the title compound as a white solid (32.0 g, 146.7 mmol, 41%). ES/MS m/z ( ⁷⁹ Br/ ⁸¹ Br) 217.1/219.0 (M+H).

Receipt 5

Synthesis of 3-amino-6-bromo-N-methyl-pyrazine-2-carboxamide.

A solution of 3-amino-6-bromo-pyrazine-2-carboxylic acid (214 g, 983 mmol) in dimethylformamide (1.07 L) was treated with methylamine hydrochloride (79.7 g, 1.18 mol) and N, N-diisopropylethylamine (445 g, 3.44 mol) at 23°C. To the resulting suspension, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium hexafluorophosphate 3-oxide (449 g, 1.18 mol) was added over 30 minutes. After 30 minutes, add H ₂ O (4.29 L) over 2 hours. Stir at 23°C for 30 minutes and then 1 hour at 10°C. Filter, wash the solid with H ₂ O (2 x 428 ml), and dry to give the title compound (227 g, 82%). ES/MS m/z ( ⁷⁹ Br) 231.0 (M+H).

Receipt 6

Synthesis of 2-amino-5-bromo-N-isopropyl-pyridine-3-carboxamide.

Add propan-2-amine (42.5 g, 0.719 mol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (127 g, 0.664 mol) and hydroxybenzotriazole (89.7 g, 0.660 mol) to the suspension of 2- amino-5-bromo-pyridine-3-carboxylic acid (120 g, 0.553 mol) in tetrahydrofuran (1.2 L) at 12°C. Stir the mixture at 23°C overnight. Ethyl acetate (250 ml) and saturated aqueous NaHCO ₃ (250 ml) were added, the phases were separated, and the aqueous layer was extracted with ethyl acetate (2×150 ml). The combined organic phases were washed with H ₂ O (300 ml) and saturated aqueous NaCl (300 ml), and concentrated under reduced pressure to give the title compound (125 g, 88%). ES/MS m/z ( ⁷⁹ Br) 258.0 (M+H).

Receipt 7

Isolation of (2R)-2-(3,5-difluorophenyl)-2-hydroxy-acetic acid.

Maintain PS Amano lipase (see Mendiola, J. et al, Org. Process Res. Dev. 2012, 16, 1312-1316) in diatomaceous earth before use by mixing 200 g diatomaceous earth and 200 g PS Amano SD lipase. H ₂ O was added to coat the solid and the mixture was stirred. Remove H ₂ O in an oven at 4 mbar and 40°C for 16 hours. Control H ₂ O below 1% by Karl Fischer titration to determine water.

Added supported lipase PS amano SD (250 g) and vinyl acetate (312 ml; 3.36 mol) to a suspension of racemic 2-(3,5-difluorophenyl)-2-hydroxyacetic acid (125 g, 664 mmol) in methyl tert-butyl ether (2.50 L) and the mixture was stirred at 26°C for 72 hours. After this time, filtered, washed the solid with methyl t-butyl ether (1.50 L) and concentrated the combined filtrates under reduced pressure. Suspend the residue in dichloromethane (160 ml) at 23°C for 4 hours. Filter, wash the solid with petroleum ether (150 ml), and dry to give the title compound (47.0 g, 36%). ^1H NMR (d ₆ -DMSO) δ 5.11 (s, 1H), 6.20 (brs, 1H), 7.11-7.21 (m, 3H), 12.8 (brs, 1H) The absolute configuration was determined using vibrational circular dichroism (see Freedman TB et al, Chirality , 2003 Nov., 15(9), 743-758). Chiral HPLC: Rt=7.39 min (UV); Column: Chiralpak® AD 4.6x150 mm 5 µm; 5% EtOH in n-hexane (0.05% TFA) isocratic; Flow rate: 1.5 ml/min, ee >98%.

Receipt 8

Isolation of (2R)-2-(3-fluorophenyl)-2-hydroxy-acetic acid.

Maintain PS Amano SD lipase (see Mendiola, J. et al, Org. Process Res. Dev . 2012, 16, 1312-1316) in diatomaceous earth before use by mixing 100 g diatomaceous earth and 100 g PS Amano SD lipase. H ₂ O was added to coat the solid and the mixture was stirred. Remove H ₂ O in an oven at 4 mbar and 40°C for 16 hours. Control H ₂ O below 1% by Karl Fischer titration to determine water.

Supported lipase PS amano SD (200 g) and vinyl acetate (269 ml; 2.90 mol) were added to a suspension of racemic 2-(3-fluorophenyl)-2-hydroxyacetic acid (96 g, 560 mmol) in methyl tert-butyl ether ( 2.00 L), and the mixture was stirred at 26°C for 90 hours. After this time, filtered, washed the solid with methyl tert-butyl ether (1.50 L) and concentrated the combined filtrates under reduced pressure. Suspended the residue. in dichloromethane (160 ml) at 23°C for 4 hours. Filter, wash the solid with petroleum ether (150 ml), and dry to give the title compound (31.0 g, 32%). ¹ H NMR (d ₆ -DMSO) δ 5.07 (s, 1H), 6.17 (brs, 1H), 7.12 (m, 1H), 7.23 (m, 1H), 7.39 (m, 1H ), 12.8 (brs, 1H). [α] _D ²⁰ = -119° (C=2.83, acetone). Chiral HPLC: Rt=10.22 min (UV); Column: Chiralpak® AD 4.6x150 mm 5 µm; 5% EtOH in n-hexane (0.05% TFA) isocratic; Flow rate: 1.5 ml/min, ee >98%.

Receipt 9

Synthesis of 3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline.

Heat a suspension of tricyclohexylphosphine (59.85 g, 213 mmol) in 1,4-dioxane (2.98 L) at 95°C for 10 min until a solution is obtained. Then, 4-bromo-3-methylaniline (752 g, 2.67 mol), bis(pinacolato)diborone (745.17 g, 2.93 mol), potassium acetate (524 g, 5.34 mol), and palladium(II) acetate (23.96 g, 107 mmol), and continued to heat the mixture at 95°C for 4 hours. After this time, cooled to 23°C, diluted with methyl tert-butyl ether (2.5 L ), filtered through kieselguhr, and washed the solid with methyl tert-butyl ether (1 L). The filtrates were combined, washed with H₂O (1.5 L) and saturated aqueous NaCl (1.2 L), and concentrated under reduced pressure to give the title compound (593 g, 95%). To obtain an analytical sample, suspend with hexane (1.6 ml/g) at 40°C for 2 hours, then cool to 23°C, filter and wash the solid with hexane (2 x 0.5 ml/g). ES/MSm/z 234.1 (M+H).

Receipt 10

Synthesis of 2-amino-5-(4-amino-2-methyl-phenyl)-N-isopropyl-pyridine-3-carboxamide.

Add 3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (93.6 g, 0.401 mol), K ₂ CO ₃ (119 g, 0.860 mol ) and Pd(dppf) ₂ Cl ₂ (10.6 g, 140 mmol) to a solution of 2-amino-5-bromo-N-isopropyl-pyridine-3-carboxamide (74.0 g, 0.287 mol) in dioxane (888 ml) and H ₂ O (296 ml), and heated the mixture to 55°C overnight. Cool to 23°C, add ethyl acetate (150 ml), filter the resulting suspension through kieselguhr, and wash the solid with ethyl acetate (50 ml). Wash the combined filtrates with H ₂ O (30 ml) and saturated aqueous NaCl (300 ml), and concentrate under reduced pressure to give the title compound (78.0 g, 96%). ES/MS m/z 285.1 (M+H).

Receipt eleven

Synthesis of 3-amino-6-(4-amino-2-methylphenyl)-N-methylpyrazine-2-carboxamide.

Add 3-amino-6-bromo-N-methylpyrazine-2-carboxamide (99.1 g, 429 mmol), Na ₂ CO ₃ (2 M in H ₂ O, 500 ml, 1.00 mol), and 1 ,1'-bis(diphenylphosphino)ferrocene)palladium(II) chloride (19 g, 22.8 mmol) to a solution of 3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane) -2-yl)aniline (122 g, 450 mmol) in 1,4-dioxane (3.00 L), and the mixture was heated to 85°C for 32 hours. Cooled to 30°C, added ethyl acetate (4.00 l), filtered through a pad of silica gel, and washed the solid with ethyl acetate (3×1.00 l). The combined filtrates were washed with H ₂ O (2 x 1.50 L) and concentrated under reduced pressure. Purify the residue by chromatography (eluent: petroleum ether/ethyl acetate 5:1-1:1) to give the title compound (80 g, 72%) as a yellow solid. ES/MS m/z 258.1 (M+H).

Receipt 12

Synthesis of 5-(4-amino-2-methyl-phenyl)-7-methyl-pyrrolo[2,3-d]pyrimidin-4-amine.

Add Pd(II) acetate (635 mg, 2.83 mmol), cataCXium A™ (2.03 g, 5.65 mmol) and saturated aqueous NaHCO ₃ (186 mL, 188 mmol) to the 5-bromo-7- methyl-pyrrolo[2,3-d]pyrimidin-4-amine (21.4 g, 94.3 mmol) and 3-methyl-4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)aniline (28.6 g, 123 mmol) in 2-methyl-tetrahydrofuran (214 ml) at 23°C, and the mixture was stirred in a sealed tube at 100°C for 3 hours. Cooled to 23° C, filtered through a pad of kieselguhr, and washed the solid with H ₂ O (50 ml) and ethyl acetate (100 ml). The organic layer was separated, washed with saturated aqueous NaCl (50 ml) and concentrated under reduced pressure. The residue was purified by chromatography (eluent: 0-100% hexane/acetone) to give the title compound (12.1 g, 51%) as a yellow solid. ES/MS m/z 254.1 (M+H).

Example 1

Synthesis of 2-amino-5-[4-[[(2R)-2-(3,5-difluorophenyl)-2-hydroxy-acetyl]amino]-2-methyl-phenyl]-N-isopropyl-pyridine-3- carboxamide.

Treated with a mixture of (2R)-2-(3,5-difluorophenyl)-2-hydroxyacetic acid (29.0 g, 0.154 mol), 2-amino-5-(4-amino-2-methyl-phenyl)-N- isopropyl-pyridine-3-carboxamide (43.83 g, 0.154 mol), and N,N-diisopropylethylamine (39.8 g, 0.308 mol) in tetrahydrofuran (960 ml), (1-[bis(dimethylamino)methylene]- 1H-1,2,3-triazolo[4,5-b]pyridinium hexafluorophosphate 3-oxide) (87.9 g, 0.231 mol) at 0°C for 30 min, and then heated to 20°C and stirred for 2 hours. Ethyl acetate (50 ml) was added and the mixture was filtered. The filtrate was concentrated under reduced pressure and the residue was purified by chromatography (eluent: 2:1 petroleum ether/ethyl acetate) and then by supercritical liquid chromatography, SFC (Column: Chiralpak® IC 30×250 mm 5 µm (Daicel); MeOH/ CO ₂ = 30:70 isocratic; Flow rate: 80 g/min; Back pressure: 100 bar; Column temperature: 40°C) to obtain the title compound (27.5 g, 39%) as a white solid. ES/MS m/z 455.2 (M+H).

Example 2

Synthesis of 2-amino-5-[4-[[2-(3,5-difluorophenyl)-2-hydroxy-acetyl]amino]-2-methyl-phenyl]-N-isopropyl-pyridine-3-carboxamide.

Add 2-amino-5-(4-amino-2-methyl-phenyl)-N-isopropyl-pyridine-3-carboxamide (1000.5 mg, 3.5 mmol) to the 2-(3,5-difluorophenyl) solution -2-hydroxy-acetic acid (793 mg, 4.2 mmol), (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium hexafluorophosphate 3-oxide) (1.7 g, 4.6 mmol), N,N-diisopropylethylamine (909.5 mg, 7.0 mmol) in tetrahydrofuran (7.9 g, 93.6 mol). After 2 hours at room temperature, 3 ml of ethyl acetate was added and the reaction mixture was stirred for 10 minutes. The solid was filtered off and the organic phase was reduced under reduced pressure. Wash the residue with saturated aqueous NaHCO ₃ (10 ml) and extract with DCM (2×10 ml). The organic phase was dried over sodium sulfate, filtered and concentrated under reduced pressure.

The residue was purified by HPLC, Rt (retention time) = 2.036 minutes (UV), LC column: XTerra MS C18 (2.1 x 50 mm, 3.5 µm; H ₂ O:acetonitrile; 0.25 min gradient at 5%B; 5%B to 100%B in 3 min; hold 0.25 min at 100%B; Column temperature: 50°C; Flow rate 1.1 ml/min to obtain the title compound as a mixture isomer 1 and isomer 2 as a white solid (0.97 g, 60%) ES/MS (m/z): 455.4 (M+H).

Example 3 and 4

Separation of 2-amino-5-[4-[[2-(3,5-difluorophenyl)-2-hydroxy-acetyl]amino]-2-methyl-phenyl]-N-isopropyl-pyridine-3-carboxamide into isomer 1 and isomer 2.

A mixture of isomer 1 and isomer 2 was separated using Chiralcel® OD-H (4.6 x 100 mm, 5 µm), 20% MeOH-DMEA (0.2%) in CO ₂ ), 2.5 ml/min, 100 bar outlet pressure, 35°C temperature to obtain individual isomer 1 and isomer 2 as a white solid.

Example 3: 2-amino-5-[4-[[2-(3,5-difluorophenyl)-2-hydroxy-acetyl]amino]-2-methyl-phenyl]-N-isopropyl-pyridine-3-carboxamide isomer 1. Rt (retention time) = 1.131 minutes (430 mg, ee > 98%), ES/MSm/z 455.4 (M+H).

Example 4: 2-amino-5-[4-[[2-(3,5-difluorophenyl)-2-hydroxy-acetyl]amino]-2-methyl-phenyl]-N-isopropyl-pyridine-3-carboxamide isomer 2. Rt (retention time) = 1.823 minutes (404 mg, ee > 98%), ES/MSm/z 455.4 (M+H).

Example 5

Synthesis of 3-amino-6-[4-[[(2R)-2-(3,5-difluorophenyl)-2-hydroxy-acetyl]amino]-2-methyl-phenyl]-N-methyl-pyrazin-2- carboxamide.

N, N- (15.3 ml 87.5 mmol) and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium hexafluorophosphate 3-oxide (33, 2 g, 87.5 mmol) to a solution of 3-amino-6-(4-amino-2-methylphenyl)-N-methylpyrazin-2-carboxamide (18.0 g, 70.0 mmol) and (2R)-2 -(3,5-difluorophenyl)-2-hydroxy-acetic acid (13.2 g, 70.0 mmol) in tetrahydrofuran (90.0 ml), and the mixture was stirred at 23°C for 5 hours. After this time, concentrated the mixture under reduced pressure, suspended the residue in ethyl acetate (100 ml) for 15 minutes, filtered, and washed the solid with ethyl acetate (2 x 25 ml). The combined filtrates were concentrated under reduced pressure and the residue was purified by chromatography (eluent: hexane/acetone 2:1, then hexane/ethanol 4:1). Dissolve the substance in methanol (115 ml), add silica-thiol resin (0.4 g/g), and the resulting suspension is stirred at 23°C for 8 hours. After this time, filter, and wash the solid with methanol ( 2×12 ml). The combined filtrates were concentrated under reduced pressure. Purified with SFC (Column: Chiralpak® IC 4.6 x 100 mm 5 µm; 35% methanol (0.2% N, N-dimethylethylamine) in CO ₂ isocratic; Flow rate: 2.5 ml/min; Back pressure : 100 Bar; Column Temp: 40°C) to give the title compound (19.7 g, 62%). ES/MS m/z 428.1 (M+H).

Example 6

Synthesis of 3-amino-6-[4-[[2-(3,5-difluorophenyl)-2-hydroxy-acetyl]amino]-2-methyl-phenyl]-N-methyl-pyrazine-2-carboxamide.

Add 3-amino-6-(4-amino-2-methyl-phenyl)-N-methyl-pyrazine-2-carboxamide (800.0 mg, 3.2 mmol) to the 2-(3,5-difluorophenyl) solution -2-hydroxy-acetic acid (701.9 mg, 3.4 mmol), (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) (1.7 g, 4.6 mmol), N,N-diisopropylethylamine (803.2 mg, 6.3 mmol) in tetrahydrofuran (7.9 g, 93.6 mol). After 2 hours at room temperature, 3 ml of ethyl acetate was added and the reaction mixture was stirred for 10 minutes. The solid was filtered off and the organic phase was reduced under reduced pressure. The residue was washed with saturated aqueous NaHCO ₃ (10 ml) and extracted with dichloromethane (2×10 ml). The organic phase was dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography eluting with ethyl acetate:hexane (30:70) to give the title compound as a mixture of isomer 1 and isomer 2 as a brown solid (0.72 g, 1.6 mmol). ES/MS (m/z): 428.3 (M+H).

Examples 7 and 8

Separation of 3-amino-6-[4-[[2-(3,5-difluorophenyl)-2-hydroxy-acetyl]amino]-2-methyl-phenyl]-N-methyl-pyrazine-2-carboxamide into isomer 1 and isomer 2.

A mixture of isomer 1 and isomer 2 was separated using Chiralpak® OD (4.6 x 50 mm, 5 µm), 20% MeOH-DMEA (0.2%) in CO ₂ ), 5 ml/min, 100 bar outlet pressure , 35°C temperature to obtain individual isomer 1 and isomer 2.

Example 7 3-amino-6-[4-[[2-(3,5-difluorophenyl)-2-hydroxy-acetyl]amino]-2-methyl-phenyl]-N-methyl-pyrazine-2-carboxamide isomer 1. Rt (retention time) = 1.610 minutes (258 mg, ee > 98%), ES/MS m/z 428.3 (M+H).

Example 8 3-amino-6-[4-[[2-(3,5-difluorophenyl)-2-hydroxy-acetyl]amino]-2-methyl-phenyl]-N-methyl-pyrazine-2-carboxamide isomer 2. Rt (retention time) = 2.410 minutes (278 mg, ee > 98%), ES/MS m/z 428.3 (M+H).

Example 9

Synthesis of (2R)-N-[4-(4-amino-7-methyl-pyrrolo[2,3-d]pyrimidin-5-yl)-3-methyl-phenyl]-2-(3-fluorophenyl)-2 -hydroxy-acetamide.

A solution of 5-(4-amino-2-methyl-phenyl)-7-methyl-pyrrolo[2,3-d]pyrimidin-4-amine (15.5 g, 44.1 mmol) and (2R)-2 was treated -(3-fluorophenyl)-2-hydroxy-acetic acid (8.25 g, 48.5 mmol) in tetrahydrofuran (56 ml) N,N-diisopropylethylamine (9.22 ml, 52.9 mmol) and 1-[ bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide with hexafluorophosphate (20.1 g, 52.9 mmol) at 23°C for 3.5 hours. After this time, the mixture was concentrated under reduced pressure, and suspended in ethyl acetate (100 ml) for 15 minutes. Filter, wash the solid with ethyl acetate (2×15 ml), and concentrate the combined filtrates under reduced pressure. The residue was purified by chromatography (eluent: dichloromethane/methanol 0-10%) and then by SFC (column size: 5 μm, 2x25 cm; stationary phase: 2-ethylpyridine; mobile phase: CO ₂ (A)/methanol -N,N-dimethylethylamine (0.2%) (B); mobile phase composition (i.e. A/B ratio): isocratic 72/25 A/B; Flow rate: 65 ml/min; loading: 70 mg/4 .35 min) to obtain the title compound (11.7 g, 65%). ES/MS m/z 406.1 (M+H).

Claims (36)

1. Compound formula , where R is selected from the group consisting of And ; X represents CH or N; R ¹ represents hydrogen or fluorine; And R ² represents C ₁ -C ₃ alkyl; or a pharmaceutically acceptable salt thereof. 2. Compound according to claim 1 of the formula , where R is selected from the group consisting of And ; X represents CH or N; R ¹ represents hydrogen or fluorine; And R ² represents C ₁ -C ₃ alkyl; or a pharmaceutically acceptable salt thereof. 3. A compound or salt according to claim 1 or 2, wherein R is . 4. Compound or salt according to claim 1 or 2, where X represents CH or N; R ¹ represents hydrogen or fluorine; And R ² represents methyl or isopropyl. 5. A compound or salt according to claim 1 or 2, wherein R is . 6. A compound according to claim 1 or 2, which is 3-amino-6-[4-[[(2R)-2-(3,5-difluorophenyl)-2-hydroxy-acetyl]amino]-2-methyl -phenyl]-N-methyl-pyrazine-2-carboxamide, which is represented by the formula , or a pharmaceutically acceptable salt thereof. 7. A compound according to claim 1 or 2, which is 2-amino-5-[4-[[(2R)-2-(3,5-difluorophenyl)-2-hydroxy-acetyl]amino]-2-methyl -phenyl]-N-isopropyl-pyridine-3-carboxamide, which is represented by the formula , or a pharmaceutically acceptable salt thereof. 8. A compound according to claim 1 or 2, which is (2R)-N-[4-(4-amino-7-methyl-pyrrolo[2,3-d]pyrimidin-5-yl)-3-methyl- phenyl]-2-(3-fluorophenyl)-2-hydroxy-acetamide, which is represented by the formula , or a pharmaceutically acceptable salt thereof. 9. A pharmaceutical composition having protein kinase R (PKR)-like endoplasmic reticulum protein kinase (PERK) inhibitor activity, containing an effective amount of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8 with one or more pharmaceutically acceptable carriers, diluents or excipients . 10. A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8 for use in the manufacture of a medicament for the treatment of pancreatic cancer. 11. A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8 for use in the treatment of pancreatic cancer.