These novel compounds are useful as active ingredients of medicaments or for the preparation of medicaments, respectively (in particular medicaments for the treatment of conditions associated with deficits in perception, concentration, learning or memory). These disorders may be associated, for example, with alzheimer's disease, schizophrenia, and other diseases. The novel compounds are also suitable, for example, for the preparation of medicaments and/or for the treatment of such diseases (in particular for cognitive disorders associated with such diseases). These compounds of the invention exhibit PDE9 inhibitory properties.
Background
Inhibition against phosphodiesterase 9A (PDE9A) is one of the relevant concepts for the current search for new effective ways of treating cognitive disorders caused by central nervous system disorders like alzheimer's disease, schizophrenia and other diseases or by any other neurodegenerative process of the brain. By the present invention, novel compounds that follow this concept are proposed.
Phosphodiesterase 9A is a member of a large family of phosphodiesterases. These enzymes regulate the content of cyclic nucleotides 5'-3' cyclic adenosine monophosphate (cAMP) and 5'-3' cyclic guanosine monophosphate (cGMP). These cyclic nucleotides (cAMP and cGMP) are important second messengers and therefore play a central role in the cell signaling cascade. In particular it reactivates protein kinases, respectively, but not exclusively. The cAMP-activated protein kinase is referred to as protein kinase a (pka) and the cGMP-activated protein kinase is referred to as protein kinase g (pkg). Activated PKA and PKG in turn can phosphorylate a number of cellular effector proteins (e.g., ion channels, G-protein coupled receptors, structural proteins, transcription factors). The second messengers cAMP and cGMP can in this way control a variety of physiological processes in a variety of organs. However, these cyclic nucleotides may also act directly on effector molecules. Thus, it is known that: for example, cGMP acts directly on ion channels and thus can affect cellular ion concentrations (see: Wei et al, prog. neurobiol.,1998,56, pages 37 to 64). These Phosphodiesterases (PDEs) are the control mechanism for cAMP and cGMP activity and thus in turn control the corresponding physiological processes. PDEs can hydrolyze these cyclic monophosphates to the inactive monophosphates AMP and GMP. Currently, 11 PDE families have been defined based on sequence homology of these corresponding genes. Each PDE gene within a family is distinguished alphabetically (e.g., PDE1A and PDE 1B). If different splice variants also occur in the gene, a further numbering indication (PDE1A1) is added after these letters.
Human PDE9A was cloned and sequenced in 1998. The amino acid identity with other PDEs is not more than 34% (PDE8A), but not less than 28% (PDE 5A). PDE9A has a Michaelis-Menten constant (Km) of 170nM, indicating a high affinity for cGMP. In addition, PDE9A is selective for cGMP (Km =230 μ M for cAMP). PDE9A lacks the cGMP binding domain, indicating that the enzyme activity is not regulated by cGMP. Western blot analysis showed that PDE9A is expressed in humans, particularly in the testis, brain, small intestine, skeletal muscle, heart, lung, thymus and spleen. The highest expression occurs in the brain, small intestine, kidney, prostate, colon and spleen (Fisher et al, J.biol.chem.,1998,273(25), pages 15559 to 15564; Wang et al, Gene,2003,314, pages 15 to 27). The gene for human PDE9A is located on chromosome 21q22.3 and comprises 21 exons. 4 alternative splice variants of PDE9A have been identified (Guipponi et al, hum. Genet.,1998,103, pages 386 to 392). Conventional PDE inhibitors do not inhibit human PDE 9A. Thus, IBMX, dipyridamole (dipyridamole), SKF94120, rolipram (rolipram) and vinpocetine (vinpocetin) still had no inhibitory effect on the isolated enzyme at concentrations up to 100 μ M. IC of Zaprast (zaprinast) has been demonstrated50At 35 μ M (Fisher et al, J.biol.chem.,1998,273(25), pages 15559 to 15564).
Murine PDE9A was cloned and sequenced in 1998 by Soderling et al (j.biol.chem.,1998,273(19), pages 15553 to 15558). It resembles the human form, has high affinity for cGMP and a Km of 70 nM. Particularly, the high expression is shown in the kidney, the brain, the lung and the liver of the mouse. IBMX concentrations below 200 μ M also failed to inhibit murine PDE 9A; IC of zaprinast5029 μ M (Soderling et al, J.biol.chem.,1998,273(19), pages 15553 to 15558). PDE9A has been found to be strongly expressed in some regions of the rat brain. These regions include the olfactory bulb, hippocampus, cortex, basal ganglia and basal forebrain (Andreeva et al, j. neurosci.,2001,21(22), pages 9068 to 9076). In particular, the hippocampus, cortex and basal forebrain play important roles in learning and memory. As already mentioned above, PDE9A is characterized by a particularly high affinity for cGMPAnd a force. Thus, in contrast to PDE2A (Km = 10. mu.M; Martins et al, J.biol.chem.,1982,257, pp. 1973 to 1979), PDE5A (Km = 4. mu.M; Francis et al, J.biol.chem.,1980,255, pp. 620 to 626), PDE6A (Km = 17. mu.M; Gillespie and Beavo, J.biol.chem.,1988,263(17), pp. 8133 to 8141) and PDE11A (Km = 0.52. mu.M; Fawcett et al, Proc.Nat.Acad.Sci.,2000,97(7), pp. 3702 to 3707), PDE9A is active even at low physiological concentrations. In contrast to PDE2A (Murashima et al, Biochemistry,1990,29, pages 5285 to 5292), the catalytic activity of PDE9A cannot be increased by cGMP because it does not have a GAF domain (PDE activity is increased ectopically via the cGMP binding domain) (Beavo et al, Current Opinion in Cell Biology,2000,12, pages 174 to 179). Thus PDE9A inhibitors may increase baseline cGMP concentrations.
This summary is intended to demonstrate that PDE9A is involved in specific physiological processes in a characteristic and unique manner, which can characteristically distinguish the role of PDE9A with any other PDE family member.
WO2004/099210 discloses 6-arylmethyl-substituted pyrazolopyrimidinones as PDE9 inhibitors.
WO2004/099211 discloses 6-cyclylmethyl-and 6-alkylmethyl-substituted pyrazolopyrimidines and their use for improving cognition, concentration, etc.
DE10238722 discloses the use of PDE 9A-inhibitors for improving cognition, attention focusing.
WO2004/018474 discloses phenyl-substituted pyrazolopyrimidines and their use for improving perception, concentration learning and/or memory.
WO2004/026876 discloses alkyl substituted pyrazolopyrimidines and their use for improving perception, attention-focusing learning ability and/or memory performance.
WO2004/096811 discloses heterocyclic bicyclic rings as PDE9 inhibitors for the treatment of diabetes (including type 1 and type 2 diabetes), hyperglycemia, dyslipidemia, impaired glucose tolerance, metabolic syndrome and/or cardiovascular diseases.
WO2009068617 discloses PDE9 inhibiting compounds derived from pyrazolopyrimidinones having a substituted phenylmethyl-or pyridyl-methyl group in the 6-position.
WO2010112437 discloses PDE9 inhibiting compounds derived from pyrazolopyrimidinones having a phenyl or heteroaryl substituted arylmethyl-or heteroaryl-methyl group in the 6-position.
WO2009/121919 discloses PDE9 inhibitors derived from pyrazolopyrimidinones having a non-aromatic heterocyclic group, in particular tetrahydropyranyl, in the 1-position.
WO2010/026214 discloses PDE9 inhibitors derived from pyrazolopyrimidinones having a cycloalkyl or cycloalkenyl group (especially 4, 4-difluorocyclohexyl) in the 1-position.
Some of the prior art is directed to chemical nucleoside derivatives. Examples thereof can be found in WO2002/057425, which discloses nucleoside derivatives as inhibitors of RNA-dependent RNA viral polymerase, or in WO2001/060315, which discloses nucleoside derivatives for the treatment of hepatitis C infections, or in EP679657, which discloses compounds as ribonucleoside analogues, or in US2002058635, which discloses purine and carbohydrate rings (pentose rings) of purine L-nucleoside compounds, which are both modified, functionalized or both modified and functionalized. Thus, the carbohydrate ring must, for example, exhibit at least one esterified hydroxyl group.
WO2005/051944 discloses oxetane-containing nucleosides for the treatment of nucleoside analogue related disorders, such as disorders associated with cell proliferation and infection.
WO2006/084281 discloses inhibitors of E1 activating enzymes having a sulfonamide moiety.
Pyrazolopyrimidinones disclosed in WO1998/40384 are PDE1, 2 and 5 inhibitors and are useful for the treatment of cardiovascular and cerebrovascular disorders and disorders of the urogenital system.
Pyrazolopyrimidines as described in CH396924, CH396925, CH396926, CH396927, DE1147234, DE1149013 have coronary artery dilating effect and can be used for the treatment of myocardial blood flow disorders.
Pyrazolopyrimidines as described in US3732225 have anti-inflammatory and hypoglycemic effects.
The styrylpyrazolopyrimidinones described in DE2408906 are useful as antimicrobial agents, and also as anti-inflammatory agents for the treatment of, for example, edema.
Object of the Invention
Changes in the substitution pattern of pyrazolopyrimidinones can produce desired changes in relation to biological activity, altering their affinity for different target enzymes, respectively.
It is therefore an object of the present invention to provide compounds as described herein (particularly in the claims) which are effective in modulating PDE9A and for the purpose of drug development, particularly for the treatment of diseases or conditions affected by PDE9A modulation.
It is another object of the present invention to provide compounds for the preparation of medicaments for the treatment of central nervous system disorders.
It is another object of the present invention to provide compounds that exhibit advantageous safety profiles.
It is another object of the present invention to provide compounds with advantageous selectivity profiles, which advantageously inhibit PDE9A compared to other PDE family members and other pharmacological targets and thereby provide advantages.
It is another object to provide a medicament which can be used not only for the treatment but also for the prevention or amelioration of the corresponding disease or condition.
The invention further provides a pharmaceutical composition comprising a compound as described herein (particularly in the claims) and a pharmaceutically acceptable carrier.
The invention further provides a method of treating any condition as described herein in a mammal (preferably a human being) in need of such treatment, comprising administering to the mammal a therapeutically effective amount of a compound as described herein (particularly in the claims).
The invention further provides a compound as described herein, particularly in the claims, for use in a method of treatment of the human or animal body by therapy.
Detailed Description
Embodiment 1 of the present invention:
the compounds of the invention are characterized by the following general formula (I):
wherein the compound is selected from:
and salts thereof (preferably pharmaceutically acceptable salts thereof).
R in the above compound group1And D are defined.
With respect to all exemplified compounds: pyridine (II)Cycloalkyl in the 6-position of the pyrazolopyrimidinone group relative to the pyrazolopyrimidinone group and the substituent R1May be in the cis or trans configuration.
In this aspect, the compounds of the invention may have the following configuration:
wherein R is1And D is as defined above.
These stereochemically defined embodiments are a further aspect of the present invention.
Table 1 below may provide a summary of the above listed compounds of the invention, wherein R has the same definition1、R2The compounds of m, n and D are classified as a group of family of compounds, which are groups of compounds having the same general chemical structure if stereochemistry is not considered. Members of these compound families are exemplified in the section of the exemplary embodiments.
Table 1:
and salts thereof (preferably pharmaceutically acceptable salts thereof, solvates thereof, and solvates of the foregoing salts).
Within this group of compounds, compounds that are in the trans configuration relative to the substituent at the cyclobutyl group may be preferred over compounds having the cis configuration. One of the possible trans-configuration compounds may exhibit a therapeutic advantage. The more effective the compound is, the more preferred the compound is. Another criterion that may distinguish preferred compounds according to the invention is the balance between therapeutic efficacy and safety, such as e.g. selectivity relative to other PDE family members, e.g. PDE 1C.
According to the experimental part, the absolute stereochemistry of a compound having a lower therapeutic effect than its enantiomer is R, R as shown by single crystal X-ray structural analysis of a pair of trans-configured compounds. The absolute stereochemistry of compounds with higher therapeutic efficacy is therefore S, S.
For this compound, the S, S configuration is represented by the structure of the following formula (IId):
it is also speculated that such compounds exhibiting the same absolute stereochemistry may be more active compounds of the present invention than other members of the same family of compounds. According to the invention, these more active compounds are preferred over less active compounds in this same family of compounds. This family of compounds is a group of compounds that differ only in their stereochemical properties by chemical structure.
These different stereoisomers are individual embodiments according to the present invention.
Other embodiments of the invention:
embodiment 2 of the present invention pertains to compounds according to embodiment 1 of the present invention, which exhibit stereochemistry as shown in formula (IIa) below
Embodiment 3 of the present invention pertains to a compound according to embodiment 1 of the present invention, wherein the compound exhibits stereochemistry as shown in formula (IIb) below
Embodiment 4 of the present invention pertains to compounds according to embodiment 1 of the present invention, wherein the compounds exhibit stereochemistry as shown in formula (IIc) below
Embodiment 5 of the present invention pertains to a compound according to embodiment 1 of the present invention, wherein the compound exhibits stereochemistry as shown in formula (IId) below
Terms and definitions
Terms not explicitly defined herein shall have the meaning that would be given to them by a person skilled in the art in light of the disclosure and the context. Examples include the 1 or 2 letter code for a particular substituent or atom, e.g., H for hydrogen, N for nitrogen, C for carbon, O for oxygen, S for sulfur, and the like.
As used in this specification, unless indicated to the contrary, the following terms have the meanings indicated, the customary usage being given below.
Unless otherwise indicated below, the conventional definitions of the designated terms and conventional stable valencies are assumed and apply to all formulae and groups.
In general, if a term is expressly defined in a given context, then these specific definitions shall prevail over the more general definitions outlined in this paragraph.
Generally, a chemical structure or compound refers to all "tautomers and isomeric forms and mixtures," whether individual geometric or optical isomers or racemic or non-racemic mixtures of isomers, unless the specific stereochemistry or isomeric form is specifically indicated in the compound name or structure. With a particular definition taking precedence.
The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings (or possibly animals) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
"pharmaceutically acceptable salts" of these compounds according to the invention are also subject matter of the present invention. The term "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts (preferably addition salts) thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues/moieties (e.g., amino functional groups) of the compounds of the invention; the acidic residues/moieties in the compounds of the invention may form salts with bases or organic bases. Such pharmaceutically acceptable salts include, for example, the conventional non-toxic salts or the quaternary ammonium salts formed from the parent compound and a non-toxic inorganic or organic acid. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like; and salts prepared with organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfonic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid and the like.
The physiologically acceptable salts with bases, which are also organic amines having 1 to 16C atoms, such as, for example, ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine (procaine), dibenzylamine, N-methylmorpholine, dehydroabietylamine, arginine, lysine, ethylenediamine, methylpiperidine and the like, can also include salts with conventional bases, such as, for example, alkali metal salts (e.g., sodium salts and potassium salts), alkaline earth metal salts (e.g., calcium salts and magnesium salts), and ammonium salts.
The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, which has basic or acidic properties, by conventional chemical methods. In general, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of the two; nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are generally preferred.
"prodrugs" are intended to mean compounds which are designed to release the biologically active compounds according to the present invention in vivo when these prodrugs are administered to a mammalian patient. Prodrugs of compounds according to the present invention are prepared by modifying functional groups present in the compounds of the present invention in such a way that the modifications are reconverted back to the original functional groups under physiological conditions. It is understood that prodrugs of the compounds according to the invention are also subject matter of the present invention.
"metabolites" are to be understood as derivatives of the compounds according to the invention which are formed in vivo. Active metabolites are those metabolites that cause pharmacological effects. It is understood that metabolites, in particular active metabolites, of the compounds according to the invention are also subject matter of the present invention.
Some of these compounds may form "solvates". For the purposes of the present invention, the term "solvates" refers to the complex forms of these compounds which are coordinated in the solid or liquid state with solvent molecules. Hydrates are a particular form of solvate in which coordination occurs with water. According to the present invention, the term is preferably applied to solid solvates, such as amorphous or more preferably crystalline solvates.
The 'skeleton': the backbone of the compounds according to the invention is represented by the following core structure. The numbering of the positions of the ring member atoms is indicated in bold:
it is obvious to the person skilled in the art that the framework can be described by its tautomeric "enol" form.
In the context of the present invention, both structural representations of the skeleton should be regarded as subject matter of the present invention, even if only one of these two representations is suggested. Without intending to be limited or restricted, it is believed that the equilibrium between tautomeric forms of most compounds under ambient conditions and pharmaceutical compositions comprising these compounds under relevant conditions is biased toward the presentation of the pyrazolopyrimidin-4-one. Thus, all embodiments are represented by pyrazolo pyrimidin-4-one derivatives or, more precisely, pyrazolo [3,4-d ] pyrimidin-4-one derivatives.
As used herein, expressions such as "prevention", "prophylactic treatment" or "prophylactic treatment" are to be understood as synonyms and have the meaning of reducing the risk of developing the condition described hereinbefore, especially for high risk patients in the condition or corresponding prior medical history. Thus, the expression "prevention of disease" as used herein means the management and care of an individual at risk of developing disease prior to the clinical onset of disease. The purpose of prevention is to combat the development of a disease, condition, or disorder, and includes administration of the active compounds to prevent or delay the onset of symptoms or complications, and to prevent or delay the development of the associated disease, condition, or disorder. The success of the prophylactic treatment is reflected by a statistically reduced incidence of the condition in a population of patients at risk of the condition compared to an equivalent population of patients who have not been prophylactically treated.
The term "treatment" or "therapy" preferably means the therapeutic treatment of a patient (e.g., preferably a human) who has developed one or more of the conditions in acute or chronic form, including symptomatic treatment, to alleviate the symptoms of a particular indication, or for treatment, to reverse or partially reverse the condition, or to delay the progression of the indication as much as possible, depending on the condition and its severity. Thus, the expression "treatment of a disease" as used herein means the management and care of a patient who has developed a disease, condition or disorder. The purpose of the treatment is to combat the disease, condition, disorder or symptoms thereof. Treatment includes administration of the active compound to eliminate or control the disease, condition, or disorder, as well as to alleviate symptoms or complications associated with the disease, condition, or disorder.
The following reaction schemes generally illustrate how the compounds of the present invention are prepared by way of example. The abbreviated substituents, if not defined in the context of these reaction formulae, may be as defined in the embodiments of formula (I):
reaction formula 1: in the first step, 2-ethoxymethylene-malononitrile is condensed with monosubstituted hydrazine by heating in a suitable solvent such as ethanol in the presence of a base (e.g., triethylamine) to form the corresponding 5-amino-1H-pyrazole-4-carbonitrile. In a second step, these compounds are converted to the corresponding amides by, for example, treating the ethanol solution with ammonia (25% aqueous solution) and hydrogen peroxide (35% aqueous solution). In a third step, a 4-oxo-4, 5-dihydro-1H-pyrazolo [3,4-d ] pyrimidin-6-yl substituted nitrile is synthesized using a dinitrile under basic conditions (e.g., sodium hydride in ethanol) with heating. The nitrile function is further converted to a heteroaryl substituent as described in equation 2 to yield pyrazolo [3,4-d ] pyrimidin-4-one as the final product. [ see, e.g., A.Miyashita et al, Heterocycles1990,31,1309ff ].
Reaction formula 2: the 4-oxo-4, 5-dihydro-1H-pyrazolo [3,4-d ] pyrimidin-6-yl-substituted nitrile is mixed with methanol and treated with acetyl chloride or with a saturated solution of ethanol containing hydrochloric acid. The intermediate is treated in a second step with a methanolic solution containing ammonia to form the corresponding amide. With 1,1,3, 3-tetraalkoxypropane to give pyrazolo [3,4-d ] pyrimidin-4-one substituted with pyrimidin-2-yl as the final product.
Other alternative methods for preparing pyrazolo [3,4-d ] pyrimidin-4-ones are known in the art and may also be used in the synthesis of these compounds of the invention (see, e.g., P.Schmidt et al, Helvetica Chimica acta1962,189,1620 ff.).
Reaction formula 3: the monosubstituted hydrazine derivative used in step 1 of reaction formula 1 may be prepared as follows: after reductive amination of tert-butyl hydrazinecarboxylate with a ketone, a deprotection step is carried out as shown in scheme 3, wherein D is cyclopentyl or cyclohexyl as defined by general formula (I) [ see, for example, J.W.Timberlake et al, "Chemistry of Hydrazo-, Azo-and Azoxy Groups"; patai, s., eds.; 1975, chapter 4; hung et al, Journal of Organic Chemistry1981,46, pages 5413 to 5414 ].
Reaction formula 4: as depicted in equation 1, in a first step, 2-ethoxymethylene-malononitrile is condensed with monosubstituted hydrazine in the presence of a base (e.g., triethylamine) by heating in a suitable solvent such as ethanol to form the corresponding 5-amino-1H-pyrazole-4-carbonitrile. In a second step, these compounds are converted into the corresponding amides by, for example, treating an ethanol solution with ammonia (25% aqueous solution) and hydrogen peroxide (35% aqueous solution). In a third step, under basic conditions (e.g., sodium hydride in ethanol), with a catalyst such as sodium chloride1And R2Heating substituted cyclobutyl or cyclopentyl carboxylate to obtain pyrazolo [3,4-d]Pyrimidin-4-one as the final product. [ see, e.g., A.Miyashita et al, Heterocycles1990,31,1309ff]. In the experimental part (examples 29 to 34), R is described in more detail1Is a process of preparing pyridine group.
Other information may also be found in the following:
WO2004/099210 (in particular, page 9 last paragraph through page 14 line 8, the disclosure of which is incorporated by reference),
for more information on the general preparation of compounds wherein D is tetrahydropyranyl, see WO2009/121919 (in particular, pages 120 to 125 and the experimental part thereof, the disclosure of which is incorporated by reference),
for more information on D as 4, 4-difluorocyclohexyl, see WO2010/026214 (in particular, pages 59 to 63 and the experimental part thereof, the disclosure of which is incorporated by reference),
and in the experimental part of this specification (exemplary embodiments). The latter relates in particular to the preparation of the following two building blocks:
method of treatment
The present invention relates to compounds that are believed to be effective in treating disease. The compounds according to the invention are potent and selective inhibitors of phosphodiesterase 9A and can be used for the development of medicaments. These agents are preferably used for the treatment of diseases where the effect of treating, preventing or ameliorating a disease state is produced by the inhibition of PDE 9A. Preferably, these drugs are useful for improving perception, concentration, cognition, learning or memory, such as those symptoms that occur particularly in conditions/diseases/syndromes, such as: mild cognitive impairment, age-related learning and memory impairment, age-related memory loss, vascular dementia, craniocerebral trauma, stroke, dementia occurring after stroke (post-stroke dementia), post-traumatic dementia, general attention-concentration impairment, attention-concentration impairment in children with learning and memory problems, alzheimer's disease, dementia with lewy bodies, dementia with accompanying frontal lobe degeneration (including Pick's syndrome), parkinson's disease, progressive nuclear palsy, dementia with accompanying corticobasal degeneration, Amyotrophic Lateral Sclerosis (ALS), Huntington's disease, multiple sclerosis, thalassemia, creutzfeldt-jakob dementia, HIV dementia, epilepsy, temporal lobe epilepsy, schizophrenia (with dementia), Korsakoff's psychoses (Korsakoff's psychoses) or cognitive disorders associated with depression or bipolar disorder.
Another aspect of the invention relates to the treatment of diseases that can be treated by modulation of PDE9A, in particular diseases involving sleep disorders (e.g. insomnia or lethargy), bipolar disorder, metabolic syndrome, obesity, diabetes (including type 1 or type 2 diabetes), hyperglycemia, dyslipidemia, impaired glucose tolerance, or diseases of the testis, brain, small intestine, skeletal muscle, heart, lung, thymus, or spleen.
The medical aspects of the invention can thus be summarized as: compounds of formula (I) or (II) as defined herein, in particular classes of compounds as defined herein, are considered to be useful as medicaments.
Such a medicament is preferably used in a method of treatment of a central nervous system disorder.
In another use, the medicament is for use in a method of treatment or prophylaxis, preferably a method of treatment of a central nervous system disorder treatable by inhibition of PDE 9.
In another use, the medicament is for use in a method of treatment or prophylaxis, preferably for use in a method of treatment of a disease treatable by inhibition of PDE9 (particularly PDE 9A).
In a most preferred alternative application, the medicament is for use in a method of treatment or prevention (preferably a method of treatment), for the treatment, alleviation and/or prevention of cognitive disorders associated with perception, concentration, cognition, learning or memory, preferably for cognitive disorders associated with a disease or condition as described in this paragraph.
In another use, the medicament is for use in a method of treatment or prophylaxis (preferably a method of treatment) for treating or ameliorating or preventing a cognitive disorder associated with: age-related learning and memory disorders, age-related memory loss, vascular dementia, craniocerebral trauma, stroke, dementia resulting from stroke (post-stroke dementia), post-traumatic dementia, general attention-concentration disorder, attention-concentration disorder in children with learning and memory problems, Alzheimer's disease, dementia with Lewy bodies, dementia with accompanying frontal lobe degeneration (including pick's syndrome), Parkinson's disease, progressive nuclear palsy, dementia with corticobasal degeneration, Amyotrophic Lateral Sclerosis (ALS), huntington's disease, multiple sclerosis, thalamic degeneration, creutzfeldt-jakob dementia, HIV dementia, epilepsy, temporal lobe epilepsy, schizophrenia (with dementia), korsakoff's psychosis or cognitive disorders associated with depression or bipolar disorder.
In another use, the medicament is for use in a method of treatment or prophylaxis, preferably for use in a method of treatment of alzheimer's disease, schizophrenia or cognitive disorders associated with alzheimer's disease or with schizophrenia.
In another use, the medicament is for use in a method of treatment or prophylaxis, preferably for use in a method of treatment of a sleep disorder, a bipolar disorder, a metabolic syndrome, obesity, diabetes, hyperglycemia, dyslipidemia, impaired glucose tolerance, or a disease of the testis, brain, small intestine, skeletal muscle, heart, lung, thymus, or spleen.
In another aspect of the invention, the invention relates to a method of treating or preventing a condition or disease selected from the group consisting of the conditions or diseases listed above, wherein the method comprises administering to a human in need thereof a therapeutically effective amount of a compound according to the invention.
Another aspect of the present invention relates to a compound of the present invention for use as a medicament in a method of treatment or prophylaxis, preferably a method of treatment. If indicated, the method of treatment or the medicament is preferably for the treatment of a condition or disease selected from the group of conditions or diseases as described above under the heading "method of treatment".
Pharmaceutical composition
A medicament for administration (which is also a subject of the present invention), comprising:
a therapeutically effective amount of a compound according to the invention as pharmaceutically active ingredient and
-a drug carrier.
By "therapeutically effective amount" is meant an amount of a compound of formula (I) which, if the medicament is administered over a suitable course of treatment for a patient's condition, will be sufficient to effectively treat, prevent or slow the progression of the corresponding disease, or to improve the condition of a patient suffering from such a disease. Wherein the "therapeutically effective amount" in monotherapy may be different from the "therapeutically effective amount" in combination therapy with another drug.
The dosage range of a compound of formula (I) suitable for daily administration may generally be from 0.1 to 5000mg, preferably from 0.1 to 1000mg, preferably from 2 to 500mg, more preferably from 5 to 250mg, most preferably from 10 to 100 mg. Dosage units (e.g. tablets) may preferably comprise from 2 to 250mg, particularly preferably from 10 to 100mg, of a compound according to the invention.
The actual pharmaceutically effective amount or therapeutic dose depends on factors known to those skilled in the art, such as the age, weight, sex or other condition of the patient, the route of administration, the severity of the disease, and the like.
The compounds according to the invention can be administered by oral, parenteral (intravenous, intramuscular, etc.), intranasal, sublingual, inhalation, intrathecal, topical or rectal routes. Suitable formulations for administration of a compound according to the invention include, for example, patches, tablets, capsules, pills, pellets, dragees, powders, lozenges, suppositories, liquid preparations (e.g., solutions, suspensions, emulsions, drops, syrups, elixirs) or gaseous preparations (e.g., aerosols, sprays, etc.). The amount of pharmaceutically active compound should be in the range of 0.05 to 90% by weight, preferably 0.1 to 50% by weight of the total composition. Suitable tablets can be obtained, for example, by mixing the active substance with the following known excipients: such as inert diluents (e.g., calcium carbonate, calcium phosphate or lactose), disintegrating agents (e.g., corn starch or alginic acid), binding agents (e.g., starch or gelatin), lubricating agents (e.g., magnesium stearate or talc), and/or delayed release agents (e.g., carboxymethylcellulose, cellulose acetate phthalate, or polyvinyl acetate). These tablets may also comprise several layers.
Coated tablets may thus be prepared by coating cores prepared analogously to tablets with substances customarily used for coating tablets, for example collidone or shellac, gum arabic, talc, titanium dioxide or sugar. The core may also be composed of a number of layers in order to achieve delayed release or to prevent incompatibility. Similarly, the tablet coating may be composed of a number of layers to achieve delayed release, and excipients as described above for the tablets may be used.
A syrup or elixir containing an active substance according to the invention or a combination thereof may additionally contain a sweetening agent such as saccharin, cyclamate, glycerol or sucrose and a flavour enhancer such as a flavouring agent such as vanillin (vanillin) or orange extract. It may also contain suspension adjuvants or thickeners (such as sodium carboxymethylcellulose), wetting agents (such as, for example, the condensation products of fatty alcohols with ethylene oxide) or preservatives (such as p-hydroxybenzoates).
Solutions can be prepared in a customary manner, for example by adding isotonic agents, preservatives such as p-hydroxybenzoates or stabilizers such as alkali metal salts of ethylenediamine tetraacetic acid, optionally using emulsifiers and/or dispersants, while if water is to be used as diluent, for example, organic solvents can optionally be used as solubilizers or cosolvents and the solutions can be transferred into injection bottles or ampoules or infusion bottles.
Capsules comprising one or more active substances or combinations of active substances can be prepared, for example, by mixing these active substances with inert carriers, such as lactose or sorbitol, and encapsulating them in gelatin capsules.
Suitable suppositories may be prepared, for example, by mixing with carriers provided for this purpose, such as neutral fats or polyethylene glycols or derivatives thereof.
Excipients which may be used include, for example, water, pharmaceutically acceptable organic solvents such as paraffins (e.g. petroleum fractions), vegetable oils (e.g. arachis oil or sesame oil), mono-or polyhydric alcohols (e.g. ethanol or glycerol); carriers such as, for example, natural mineral powders (e.g. kaolin, clay, talc, chalk), synthetic mineral powders (e.g. highly dispersed silicic acid and silicates), sugars (e.g. sucrose, lactose and glucose), emulsifiers (e.g. lignin, spent sulfite liquor, methylcellulose, starch and polyvinylpyrrolidone) and lubricants (e.g. magnesium stearate, talc, stearic acid and sodium lauryl sulfate).
For oral use, these tablets may contain, in addition to the indicated carrier, additives such as sodium citrate, calcium carbonate and dicalcium phosphate, and various other substances such as starch (preferably potato starch), gelatin and the like. Lubricants such as magnesium stearate, sodium lauryl sulfate and talc may also be used to prepare these tablets. For aqueous suspensions, these actives may be combined with various flavor enhancers or colorants in addition to the excipients described above.
The dosage of the compounds according to the invention naturally depends highly on the method of administration and the condition to be treated.
In combination with other active substances
In another aspect, the invention relates to a combination therapy wherein a compound according to the invention is co-administered together with another active compound. The invention therefore also relates to pharmaceutical preparations which provide such a combination of pharmaceutically active ingredients, one of which is a compound of the invention. These combinations may be fixed dose combinations (the pharmaceutically active ingredients used for combination being components of the same pharmaceutical formulation) or free dose combinations (the pharmaceutically active ingredients being in separate pharmaceutical formulations).
Thus, a further aspect of the present invention relates to the combination of each compound of the present invention (preferably at least one compound according to the invention) with another active compound selected from the group consisting of: inhibitors of beta-secretase; gamma-secretase inhibitors; a gamma-secretase modulator; amyloid aggregation inhibitors such as azamailed (alzhemed); substances which act directly or indirectly to protect nerves and/or to improve the condition of the disease; antioxidants, such as vitamin E, ginkgo biloba (ginko biloba) or bilobalide; anti-inflammatory substances, e.g. Cox inhibitors, additionally or exclusively havingAn NSAID of reduced profile; HMG-CoA reductase inhibitors such as statins; acetylcholinesterase inhibitors such as donepezil, rivastigmine (rivastigmine), tacrine, galantamine; NMDA receptor antagonists, such as memantine; AMPA receptor agonists; AMPA receptor positive modulators, AMPkines, glycine transporter 1 inhibitors; monoamine receptor reuptake inhibitors; substances that regulate neurotransmitter concentration or release; induced growth hormone fractionSecreted substances such as ibutemoram mesylate (ibutamoren mesylate) and capromorelin (capromorelin); a CB-1 receptor antagonist or inverse agonist; antibiotics such as minocycline or rifampin; PDE1, PDE2, PDE4, PDE5 and/or PDE10 inhibitors, GABAA receptor inverse agonists; GABAA α 5 receptor inverse agonist; GABAA receptor antagonists; a nicotinic receptor agonist or partial agonist or positive modulator; an α 4 β 2 nicotinic receptor agonist or partial agonist or positive modulator; an alpha 7 nicotinic receptor agonist or partial agonist; histamine receptor H3 antagonists; a 5-HT4 receptor agonist or partial agonist; 5-HT6 receptor antagonists; alpha 2-adrenoceptor antagonists, calcium antagonists; a muscarinic receptor M1 agonist or partial agonist or positive modulator; muscarinic receptor M2 antagonists; muscarinic receptor M4 antagonists; a metabotropic glutamate receptor 5 positive ectopic modulator; metabotropic glutamate receptor 2 antagonists; a metabotropic glutamate receptor 2/3 agonist; metabotropic glutamate receptor 2 positive ectopic modulators and other substances that modulate the receptor or enzyme in a manner that increases the therapeutic efficacy and/or safety of the compounds according to the present invention and/or reduces unwanted side effects.
The invention further relates to pharmaceutical compositions comprising one or more, preferably one, active substances. At least one active substance is selected from the compounds according to the invention and/or the corresponding salts thereof. Preferably, the composition comprises only one of the active compounds. If more than one active compound is present, the other may be selected from the aforementioned combination partners, such as azametide, vitamin E, bilobalide, donepezil, rivastigmine, tacrine, galantamine, memantine, ibumoram mesylate, carmorelin, minocycline and/or rifampin. Optionally, the composition comprises other ingredients such as inert carriers and/or diluents.
The compounds according to the invention may also be used in combination with immunotherapy (e.g. active immunization with A.beta.or a part thereof or passive immunization with humanized anti-A.beta.antibodies or antibody fragments) for the treatment of the above-mentioned diseases and conditions.
The compounds according to the invention can also be combined with entacapone (Dimebon).
The compounds according to the invention may also be combined with antidepressants such as amitriptyline, imipramine hydrochloride (TOFANIL), imipramine maleate (SURMOTIL), lofepramine, desipramine (NORPRAMIN), doxepin (SINEQUAN, ZONALON), trimipramine (SURONTIL).
Alternatively, the compounds according to the invention may also be combined with 5-hydroxytryptamine (5-HT) reuptake inhibitors such as alapropyl ester, citalopram (CELEXA, CIPRAMIL), escitalopram (LEXAPRO, CIPRALEX), clomipramine (ANAFRANIL), duloxetine (CYMBALTA), femoxetine (MALELIL), fenfluramine (PONDIMIN), norfenfluramine, fluoxetine (PROZAC), fluvoxamine (LUVOX), indapadine, milnacipran (IXEL), paroxetine (PAXIL, SEROXAT), sertraline (ZOLOFT, LUSTRAL), trazodone (DESYREL, MOLIPAAX), venlafaxine (EFXOR), zimelidine (NORMB, ZELMID), bifaxed, norfalcaricin (IST), praseofcine and tesofenmene.
The combination according to the invention may be provided simultaneously in a single and identical dosage form, i.e. in the form of a combined preparation, e.g. the two components may be incorporated in one tablet, e.g. in different layers of the tablet. The combination may also be provided separately in free combination, i.e. the compound of the invention is provided in one dosage form and one or more of the above-mentioned combination partners are provided in another dosage form. The two dosage forms may be the same dosage form, e.g. co-administered in two tablets, one containing a therapeutically effective amount of a compound of the invention and the other containing a therapeutically effective amount of a combination partner as described above. If desired, different administration forms can also be combined. Any suitable type of administration form may be provided.
The combination of a compound according to the invention or a physiologically acceptable salt thereof and another active substance can be used simultaneously or at staggered time points, but in particular close in time. If administered simultaneously, the two active substances are provided to the patient together; if administration is carried out at staggered time points, the two active substances are provided to the patient sequentially within a time period of less than or equal to 12 hours, in particular less than or equal to 6 hours.
The dosage or administration form is not limited, and any suitable dosage form may be used in the context of the present invention. For example, these dosage forms may be selected from solid preparations such as patches, tablets, capsules, pills, dragees, powders, troches, suppositories, liquid preparations such as solutions, suspensions, emulsions, drops, syrups, elixirs, or gaseous preparations such as aerosols, sprays and the like.
These dosage forms are preferably formulated in dosage units, each dosage unit being adapted to provide a single dose of each active ingredient. These ingredients are selected accordingly according to the route of administration and the dosage form.
The dosage of the combination partner described above is suitably from 1/5, which is the lowest generally recommended dose, to 1/1, which is the generally recommended dose.
Depending on the nature of the formulation, these dosage forms are administered to the patient daily, for example 1,2, 3 or 4 times. If a delayed or sustained release formulation or other pharmaceutical formulation, it may be administered in a different manner (e.g., 1 time per week or month, etc.). The compounds of the present invention are preferably administered three or less times daily, more preferably once or twice daily.
Examples
Pharmaceutical composition
The examples are illustrative of possible pharmaceutical formulations and are not intended to be limiting:
the term "active substance" denotes one or more compounds according to the invention (including salts thereof). The term "active" may also include additional actives if a combination of the foregoing with one or more other actives.
Example A
Tablet comprising 100mg of active substance
Example B
Tablet comprising 150mg of active substance
Example C
Hard gelatin capsules containing 150mg of active substance
Example D
Example E
Composition (A): ampoules containing 10mg of active substance
Active substance 10.0mg
Proper amount of 0.01N hydrochloric acid
Double distilled Water to 2.0mL
Example F
Composition (A): ampoules containing 50mg of active substance
Active substance 50.0mg
Proper amount of 0.01N hydrochloric acid
Double distilled Water to 10.0mL
All of the above formulations can be prepared following standard procedures.
Biological assay
The in vitro effect of the compounds of the invention can be shown by the following bioassay.
PDE9a2 assay protocol:
PDE9A2 enzyme activity was measured using the Scintillation Proximity Assay (SPA), generally according to the protocol of the manufacturer (GEHealthcare, form Amersham Biosciences, product number: TRKQ 7100).
As an enzyme source, a lysate of SF9 cells expressing human PDE9A2 (1% Triton X-100 in PBS supplemented with protease inhibitors, centrifuged at 13,000rpm for 30 minutes to remove cell debris) was used. The total amount of protein included in the assay varied with the infection and production efficacy of SF9 cells and ranged from 0.1 to 100 ng.
In general, the detection conditions are as follows:
total volume detected: 40 microliter
Protein amounts of 0.1 to 50ng
Substrate concentration (cGMP): 20 nM; about 1mCi/l
Culture time: 60min at room temperature
Final DMSO concentration: 0.2 to 1%
The assay was performed in 384-well format. The test reagents and enzymes and substrates are diluted with detection buffer. The assay buffer contained 50mM Tris, 8.3mM MgCl21.7mM EGTA, 0.1% BSA, 0.05% Tween 20; the pH of the assay buffer was adjusted to 7.5. Specific inhibition by use of an excess of PDE9An agent (e.g. a compound according to WO2004/099210 or WO2004/099211, such as one of the enantiomers of example 37, e.g. 1- (2-chlorophenyl) -6- [ (2R) -3,3, 3-trifluoro-2-methyl-propyl]-1, 5-dihydro-4H-pyrazolo [3,4-d]Pyrimidin-4-one).
Reference documents:
Wunder F,Tersteegen A,Rebmann A,Erb C,Fahrig T,Hendrix M.Characterization of the first potent and selective PDE9inhibitor using a cGMPreporter cell line.Molecular Pharmacology.2005Dec;68(6):1775-81.
van der Staay FJ,Rutten K,L,Devry J,Erb C,Heckroth H,Karthaus D,Tersteegen A,van Kampen M,Blokland A,Prickaerts J,ReymannKG,UH,Hendrix M.The novel selective PDE9inhibitor BAY73-6691improves learning and memory in rodents.Neuropharmacology.2008Oct;55(5):908-18.
PDE1C assay protocol:
the assay was performed similarly to the PDE9A2 assay, with the following differences: PDE1C was substituted for PDE9A2, and the detection buffer additionally contained 50nM Calmodulin (Calmodulin), 3mMCaCl2. By using the same inhibitor as above (1- (2-chlorophenyl) -6- [ (2R) -3,3, 3-trifluoro-2-methyl-propyl)]-1, 5-dihydro-4H-pyrazolo [3,4-d]Pyrimidin-4-one).
IC50The determination of (1):
IC50the positive control can be set to 100 and the negative control to 0 using GraphPadPrism or other suitable software for calculation. To calculate IC50Dilutions of the test compound (substrate) were selected and tested according to the protocol described above.
Data of
The following IC for PDE9A2 inhibition50Value [ nM)]Indicating that compounds according to the invention inhibit PDE9 (in particular PDE9a 2). This demonstrates that these compounds provide useful pharmacological properties. These examples are not intended to be limiting.
The table also provides selectivity values (selectivities) showing that the compound is selective for PDE9A in preference to PDE 1C. Selectivity is (IC inhibiting PDE 1C)50[nM]) /(IC inhibiting PDE9A250[nM]) The ratio of (a) to (b).
Example numbers refer to the final examples summarized in the exemplary embodiments section and defined in the above compound family table (table 2).
All data can be measured according to the procedures described herein. Enantiomer 1 or enantiomer 2 of this definition is related to the elution order of the enantiomers in chiral SFC and chiral HPLC.
Table 2:
trans racemic mixture
In vivo effects:
it is believed that a positive in vitro potency result for these compounds of the invention may be converted to a positive in vivo potency.
The in vivo activity of the compounds of the invention can be measured in a new target recognition test according to the program of Prickaerts et al (neuroscience2002,113, pages 351 to 361), in a social recognition test or in a T-maze spontaneous alternation test according to the program described by van der Staay et al (Neuropharmacology2008,55, pages 908 to 918). Additional information on biological tests can also be found in both citations.
In addition to being inhibitory against the target PDE9, the compounds according to the invention may provide other advantageous pharmacokinetic properties.
For example, compounds according to the present invention may exhibit one or more advantages in the areas of safety, balanced metabolism, lower risk of causing drug-drug interactions, and/or balanced clearance.
The compounds may also exhibit one or more additional or alternative advantages in the areas of bioavailability, high absorption scores, blood-brain transport properties, favorable (e.g., high average) residence times (mrt), favorable exposure to compartment effector regions, and the like.
Chemical process
The compounds presented below and their preparation, some of which are the subject of the present invention, some of which need to be further described. Compounds 23 to 34 are the subject of the present invention.
Abbreviations:
LC-MS method:
method 1
MS equipment type is Waters Micromass ZQ; HPLC equipment types Waters Alliance2695, Waters2996 diode array detector; column Varian Microsorb100C18, 30X 4.6mm, 3.0 μm; eluent A is water and 0.13% TFA, eluent B is ACN; gradient 0.0min5% B → 0.18min5% B → 2.0min98% B → 2.2min98% B → 2.3min5% B → 2.5min5% B; the flow rate is 3.5 mL/min; and UV detection is 210-380 nm.
Method 2
MS equipment type is Waters Micromass ZQ; HPLC equipment types Waters Alliance2695, Waters2996 diode array detector; column Varian Microsorb100C18, 30X 4.6mm, 3.0 μm; eluent A is water and 0.13% TFA, eluent B is MeOH; gradient 0.0min5% B → 0.35min5% B → 3.95min100% B → 4.45min100% B → 4.55min5% B → 4.9min5% B; the flow rate is 2.4 mL/min; and UV detection is 210-380 nm.
Method 3
MS equipment type is Waters Micromass ZQ; HPLC equipment types Waters Alliance2695, Waters2996 diode array detector; column Varian Microsorb C18, 20X 4.6mm, 5.0 μm; eluent A is water and 0.15% TFA, eluent B is MeOH; gradient 0.0min5% B → 0.25min5% B → 1.90min100% B → 2.05min100% B → 2.15min5% B → 2.25min5% B; the flow rate is 5.2 mL/min; and UV detection is 210-400 nm.
Method 1E hydro
Instrument LC/MS ThermoFinnigan. HPLC Surveyor DAD, MSQ quadrupole; column Synergi Hydro-RP80A, 4um, 4.60 × 100 mm; eluent A is 90 percent of water, 10 percent of acetonitrile and 10mM of ammonium formate; eluent B = ACN90% +10% H2O+NH4COOH10 mM; gradient A (100) for 1.5min, then within 10min to B (100) for 1.5 min; the flow rate is 1.2mL/min, and the UV detection is 254 nm; APCI is the ion source.
Chiral SFC method:
method 4
SFC device type Berger "Analytix"; a column of Daicel IC, 250 mm. times.4.6 mm, 5.0 μm; eluent CO225% MeOH/0.2% DEA (isocratic); the flow rate is 4.0mL/min and 10 min; the temperature is 40 ℃; and UV detection is 210/220/254 nm.
Method 5
SFC device type Berger "Analytix"; the column is Daicel ADH, 250mm multiplied by 4.6mm, 5.0 μm; eluent CO225% MeOH/0.2% DEA (isocratic); the flow rate is 4.0mL/min and 10 min; the temperature is 40 ℃; UV detection was 210/220/254 nm.
Chiral HPLC method:
method 6
HPLC equipment type Agilent 1100; a column, Daicel chiralcel OJ-H, 250mm × 4.6mm, 5.0 μm; eluent hexane/EtOH 80: 20; the flow rate is 1mL/min, and the temperature is 25 ℃; UV detection variable (200 to 500 nm).
Method 6.1:
HPLC equipment type Agilent 1100; column Daicel chiralcel OJ-H, 250mm × 4.6mm, 5.0 μm; eluent hexane/EtOH 85:15, flow rate 1mL/min, temperature 25 ℃; UV detection variable (200 to 500 nm).
Method 7
HPLC equipment type Agilent 1100; the column is Chiralpak AD-H, 250mm multiplied by 4.6mm, 5.0 mu m; eluent hexane/isopropanol 80: 20; the flow rate is 1mL/min, and the temperature is 25 ℃; UV detection variable (200 to 500 nm).
HPLC equipment type Agilent 1100; the column is Chiralpak AD-H, 250mm multiplied by 4.6mm, 5.0 mu m; eluent hexane/isopropanol 80: 20; the flow rate is 1mL/min, and the temperature is 25 ℃; UV detection variable (200 to 500 nm).
Microwave heating:
·CEM instruments, equipped with 10 and 35mL containers;
· Biotage Initiator60。
general description relating to structural features
Compounds having one or more chiral centers the formulae depicted in the experimental section below do not necessarily show all the stereochemical possibilities of these compounds, but only one of them is shown. However, in these cases, terms like "trans-racemic mixture" or "cis-racemic mixture" are added alongside the indicated structural formula to indicate that there are other stereochemical possibilities.
An example is as follows. The structural formula is shown as
The appended term "trans-racemic mixture" indicates that there is also a second stereochemical possibility:
thus, the compound produced should be
This principle also applies to the other formulae shown.
Starting compounds:
example 1A (trans-racemic mixture)
2.00g (13.9mmol) of trans-cyclobutane-1, 2-dicarboxylic acid are mixed with 16mL of EtOH at 0 ℃ and 2.21mL (30.5mmol) of thionyl chloride are slowly added. The mixture was allowed to warm to room temperature and stirred for 1 h. The solvent was removed under reduced pressure and the product was then filtered through an activated basic alumina packing. 2.71g (98%) of product are obtained.
HPLC-MS (method 1) Rt=1.34min
MS(ESI pos):m/z=201(M+H)+
The following examples were synthesized by a similar procedure to example 1A using the corresponding diacids as starting materials.
Example 2A (racemic mixture)
8.00g (89.7mmol) of 2-aminopropionic acid are mixed with 88.0mL (0.93mol) of acetic anhydride and 88.0mL of pyridine. The reaction mixture was stirred at 100 ℃ for 135 min. The solvent was removed under reduced pressure. Toluene was added to the residue and the solvent was removed under reduced pressure, then 204mL (816mmol) of HCl (4M in water) was added and the mixture was refluxed for 3 h. The solvent was removed under reduced pressure. 1-Butanol (20mL) was added to the residue, followed by removal of the solvent under reduced pressure. 11.6g of the title compound are obtained as the hydrochloride salt.
MS(ESI pos):m/z=88(M+H)+
Example 3A (trans-racemic mixture)
1.00g (4.09mmol) of 5-amino-1- (4, 4-difluoro-cyclohexyl) -1H-pyrazole-4-carboxylic acid amide (see PCT patent application WO2010/026214, example 8A) were mixed with 15mL of anhydrous EtOH, 2.46g (12.3mmol) of example 1A and 0.66g (16.4mmol) of sodium hydride (60% suspension in mineral oil) were added. The reaction mixture was heated to 140 ℃ in a microwave oven for 30 min. The mixture was allowed to cool to room temperature and then sodium hydroxide solution (4M aqueous solution) was added. The solvent was removed under reduced pressure. The residue was purified by preparative HPLC (eluent A: water +0.13% TFA, eluent B: MeOH). 0.70g (49%) of product are obtained.
HPLC-MS (method 1) Rt=1.24min
MS(ESI pos):m/z=353(M+H)+
The following examples were synthesized in a similar manner to the preparation of example 3A, using the corresponding amides and esters as starting materials (see PCT patent publications WO2010/026214, WO2009/121919 and WO 2004/09921).
Example 4A (trans-racemic mixture)
0.200g (0.568mmol) of example 3A were mixed with 0.157mL (1.14mmol) of triethylamine and 5mL of DMMF. 0.237g (0.624mmol) of HATU was added to the mixture, and the reaction mixture was stirred at room temperature for 10 min. 0.042g (0.568mmol) of acetohydrazide are added to the mixture and the reaction mixture is stirred at room temperature for 1 h. The mixture was purified by preparative HPLC (eluent A: water +0.13% TFA, eluent B: MeOH). 30mg of product are obtained.
HPLC-MS (method 1) Rt=1.03min
MS(ESI pos):m/z=409(M+H)+
Example 5A (trans-racemic mixture)
0.150g (0.426mmol) of example 3A was mixed with 2mL of THF. The mixture was cooled to 0 ℃ and 0.036 was addedmL (0.426mmol) of oxalyl chloride and 1 drop of DMF. The reaction mixture was stirred at 0 ℃ for 1 h. 2mL of ACN and 0.426mL (0.851mmol) of trimethylsilyldiazomethane (2M in hexane) were added to the reaction mixture. The mixture was stirred for 2h, then 0.213mL HCl (4M in two) was added slowlyIn an alkane). The reaction was stirred for 3 h. Ethyl acetate and saturated aqueous sodium bicarbonate were added to the mixture. The organic layer was washed with water and brine, and then dried over sodium sulfate. The solvent was partially evaporated until the volume reached about 2 mL. These mixtures were used in the next step without further purification.
HPLC-MS (method 1) Rt=1.40min
MS(ESI pos):m/z=385/387(Cl)
The following examples were synthesized in a similar manner to example 5A using the corresponding acids as starting materials.
Example 6A (Trans-mixture of stereoisomers)
0.200g (0.628mmol) of example 3B are mixed with 1mL of DMF. 0.261mL (1.89mmol) of triethylamine and 0.222g (0.691mmol) of TBTU were added. The reaction mixture was stirred at room temperature for 10 min. Then 0.078g (0.628mmol) of example 2A was added and the mixture was stirred at room temperature for 1 h. The mixture was purified by preparative HPLC (eluent A: water +0.13% TFA, eluent B: MeOH). 190mg of product are obtained.
HPLC-MS (method 3) Rt=1.03min
MS(ESI pos):m/z=388(M+H)+
Example 7A (trans-racemic mixture)
0.200g (0.628mmol) of example 3B are mixed with 1mL of DMF. 0.174mL (1.26mmol) of triethylamine and 0.222g (0.691mmol) of TBTU were added. The reaction mixture was stirred at room temperature for 10 min. Then 0.066g (0.628mmol)2, 2-dimethoxy-ethylamine was added and the mixture was stirred at room temperature for 1 h. HCl (2M aqueous) was then added and the mixture was purified by preparative HPLC (eluent A: water +0.13% TFA, eluent B: MeOH). The residue was mixed with 5mL acetone and 1mL HCl (2M aq) and stirred under nitrogen overnight. The mixture was then extracted with DCM. The organic layer was evaporated and purified by preparative HPLC (eluent A: water +0.13% TFA, eluent B: MeOH). 170mg of product are obtained.
HPLC-MS (method 3) Rt=1.01min
MS(ESI pos):m/z=360(M+H)+
Example 8A (Trans-mixture of stereoisomers)
0.200g (0.568mmol) of example 3A was mixed with 1.0mL of DMF. 0.432mL (2.84mmol) of DIPEA and 0.200g (0.624mmol) of TBTU were added. The reaction mixture was stirred at room temperature for 10 minutes. Then 0.140g (1.14mmol) of example 2A are added and the mixture is stirred at room temperature for 2 h. The mixture was purified by preparative HPLC (eluent A: water +0.13% TFA, eluent B: MeOH). 70mg (29%) of product are obtained.
HPLC-MS (method 1) Rt=1.23min
MS(ESI pos):m/z=422(M+H)+
Synthesis of the following example Using the corresponding nucleophile as starting Material, by a similar procedure as in example 8A
For example.
Example 9A (trans-racemic mixture)
0.182g (0.430mmol) of Dess-Martin (Dess-Martin) periodinane was mixed with 2.5mL of DCM. At room temperature, 0.160g (0.391mmol) of example 8D in 2.5mL of DCM was added. The reaction mixture was stirred at room temperature for 30min, followed by 30min at 30 ℃.10 mL of sodium thiosulfate solution (10% aqueous solution) and 10mL of saturated sodium bicarbonate solution were added to the mixture and the mixture was stirred for 20 min. The organic layer was separated and the aqueous layer was extracted with DCM. The organic layer was washed with saturated sodium bicarbonate solution, dried and evaporated. 93mg (58%) of product are obtained.
HPLC-MS (method 1) Rt=1.18min
MS(ESI pos):m/z=408(M+H)+
The following examples were synthesized in analogy to the procedure for example 9A, using the corresponding alcohols as starting materials.
Example 10A (Trans-mixture of stereoisomers)
0.450g of example 3C was mixed with 3.5mL of DMF and 0.273g (2.21mmol) of example 2A. 1.00mL (6.64mmol) of DIPEA and 0.390g (1.22mmol) of TBTU were added and the mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent A: water +0.13% TFA, eluent B: MeOH). 360mg (83%) of product are obtained.
HPLC-MS (method 1) Rt=0.85min
MS(ESI pos):m/z=395(M+H)+
Example 11A (trans-racemic mixture)
300mg (1.23mmol) of 5-amino-1- (4, 4-difluoro-cyclohexyl) -1H-pyrazole-4-carboxylic acid amide (cf. WO2010/026214, example 8A) were mixed under nitrogen with 4mL of anhydrous EtOH, 326mg (3.07mmol) of trans-cyclobutane-1, 2-dicarbonitrile and 0.197g (4.91mmol) of sodium hydride (60% suspension in mineral oil). The reaction mixture was heated to 140 ℃ in a microwave oven for 45 min. The solvent was removed under reduced pressure. The residue was purified by preparative HPLC (eluent A: water +0.13% TFA, eluent B: MeOH). 210mg (51%) of the title compound are obtained.
HPLC-MS (method 3) Rt=1.19min
MS(ESI pos):m/z=334(M+H)+
Example 11B (trans-racemic mixture)
0.457g (19.6mmol) of sodium hydride (60% suspension in mineral oil) is added to a solution of 0.8g (3.805mmol) of 5-amino-1- (tetrahydro-pyran-4-yl) -1-H-pyrazole-4-carboxylic acid amide (see PCT patent application WO2010/026214) in 8mL of anhydrous EtOH at room temperature under nitrogen. After stirring for 1h, 1.2g (11.42mmol) trans-cyclobutane-1, 2-dicarbonitrile are added and the reaction mixture is heated to 140 ℃ in a microwave oven for 45 min. The solvent was removed under reduced pressure. The residue was dissolved in DCM, water was added and the phases were separated. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. The crude product was purified by flash chromatography (Cy/EtOAc from 80/20 to 100%) to afford the title compound as a yellow solid. (0.64g, 55%)
HPLC-MS (method 1Eh) Rt=6.21min
MS(APCI):m/z=300(M+H)+
Example 11C (trans-racemic mixture)
0.47g (11.74mmol) of sodium hydride (60% suspension in mineral oil) is added to a solution of 0.85g (3.91mmol) of 5-amino-1- (4-methyl-pyridin-3-yl) -1H-pyrazole-4-carboxylic acid amide (see PCT patent application WO2004/09921) in10 mL of anhydrous EtOH at room temperature under nitrogen. After stirring for 1h, 1.28g (11.74mmol) trans-cyclobutane-1, 2-dicarbonitrile is added and the reaction mixture is heated to 140 ℃ in a microwave oven for 45 min. The reaction mixture was then loaded onto an SCX column (cartridge), the ammonia fractions were collected and evaporated, and the residue was purified by flash chromatography (DCM/MeOH90:10) to give the title compound as a white solid. (0.63g, 52%).
HPLC-MS (method 1Eh) Rt=5.92min
MS(APCI pos):m/z=307(M+H)+
Example 12A (trans-racemic mixture)
190mg (0.570mmol) of example 11A were mixed with 0.281mL of toluene and 0.093mL (2.30mmol) of anhydrous MeOH. 0.103mL (1.45mmol) of acetyl chloride was slowly added at 0 ℃. The mixture was stirred at room temperature for 12 h. The solvent was removed under reduced pressure. 0.5mL MeOH was added to the residue. Then 0.407mL (2.85mmol) ammonia (7M in MeOH) was added at 0 ℃ and the mixture was allowed to warm to room temperature. After 30min, the reaction mixture was treated with water, followed by addition of TFA to adjust the pH to pH = 1. The mixture was purified by preparative HPLC (eluent A: water +0.13% TFA, eluent B: MeOH) to yield 110mg (42%) of the product as the trifluoroacetate salt.
HPLC-MS (method 3) Rt=1.04min
MS(ESI pos):m/z=351(M+H)+
Example 12B (trans-racemic mixture)
Acetyl chloride (2.27mL, 30.82mmol) was slowly added to anhydrous EtOH (5mL) and anhydrous CHCl which had been cooled at 0 deg.C3(5mL) and the mixture was stirred for 20 minutes (0 deg.C). Example 11B (0.410g, 1.027mmol) in anhydrous CHCl was added dropwise3(5mL) and the mixture was stirred at room temperature overnight. The solvent was evaporated under reduced pressure, the residue was dissolved in anhydrous EtOH (5mL), and 6.4mL of 7.0M ammonia in MeOH (30.82mmol) was added. The mixture was stirred at room temperature for 12 h. The solvent was removed under reduced pressure. The final product is obtained in the form of the hydrochloride,and used in the next step without further purification. (0.37g, content 50% by HPLC-MS).
HPLC-MS (method 1Eh) Rt=5.38min
MS(APCI pos):m/z=317(M+H)+
Example 12C (trans-racemic mixture)
Acetyl chloride (4.38mL, 61.7mmol) was slowly added to anhydrous EtOH (4mL) and anhydrous CHCl which had been cooled at 0 deg.C3(10mL) and the mixture was stirred for 20 minutes (0 deg.C). Example 11C (0.63g,2.057mmol) in anhydrous CHCl was added dropwise3(5mL) and the mixture was stirred at room temperature overnight. The solvent was evaporated under reduced pressure and the residue was dissolved in dry MeOH (10mL) and 10.3mL of 7.0M ammonia in MeOH (72mmol) was added. The mixture was stirred at room temperature for 12 h. The solvent was removed under reduced pressure. The final product was obtained as the hydrochloride salt, which was used in the next step without further purification. (0.85g, 84% by 1H-NMR).
HPLC-MS (method 1Eh) Rt=5.15min
MS(APCI pos):m/z=324(M+H)+
Example 13A (trans-racemic mixture)
Propargylamine (1.4mL, 20.4mmol) was added to a solution of 1.6g (10.24mmol) of methyl 2-acetyl-cyclobutanecarboxylate (prepared as described in j.med.chem,25,109,1982) in anhydrous EtOH (12mL), followed by 0.122g (0.307mmol) of gold sodium trichloride. The reaction mixture was heated to 140 ℃ in a microwave oven for 45 minutes, the solid was filtered off and the organic phase was evaporated. The crude product was purified by flash chromatography (Cy/EtOAc70:30) to afford the title compound as a yellow-green oil. (0.18g, 9.2%).
HPLC-MS (method 1Eh) Rt=0.87min
MS(APCI pos):m/z=192(M+H)+
Exemplary embodiments
Example 1 (trans-racemic mixture)
22.0mg (0.306mmol) of propan-2-one oxime are mixed with 2mL of anhydrous THF, and 0.471mL (1.22mmol) of n-butyllithium (2.6mol/L in toluene) are carefully added to the mixture. The reaction mixture was stirred at room temperature for 30 minutes. 0.110g (0.278mmol) of example 8B in 1mL of anhydrous THF was carefully added over a period of 10 minutes. After 30 minutes, the reaction mixture was added to 0.28mLH2SO4With 4mL of a THF/water mixture (4: 1). The mixture was refluxed for 1.5 h. Saturated aqueous sodium bicarbonate was added and extracted with ethyl acetate. The organic layer was dried and the solvent was evaporated. The residue was purified by preparative HPLC (eluent A: water +0.13% TFA, eluent B: MeOH). 8mg (8%) of product were obtained.
HPLC-MS (method 1) Rt=1.40min
MS(ESI pos):m/z=390(M+H)+
Example 2 (trans-racemic mixture)
0.190g of example 6A was mixed with 3mL of DME and 0.273g (1.14mmol) of Burgis reagent. The reaction mixture was heated to 130 ℃ in a microwave oven for 1 h. The solvent was evaporated and the residue was purified by preparative HPLC (eluent A: water +0.13% TFA, eluent B: MeOH). 70mg (55%) of product are obtained.
HPLC-MS (method 1) Rt=1.11min
MS(ESI pos):m/z=370(M+H)+
The following examples were synthesized in analogy to the procedure of example 2, using the corresponding amides as starting materials.
Example 9 (trans-racemic mixture)
0.062g (0.832mmol) thioacetamide in 2mL EtOH was added dropwise to the solution of example 5A synthesized starting from 0.426mmol of the above-mentioned example 3A. The reaction mixture was stirred overnight. The mixture was purified by preparative HPLC (eluent A: water +0.13% TFA, eluent B: MeOH). 62mg of the title compound were obtained.
HPLC-MS (method 1) Rt=1.37min
MS(ESI pos):m/z=406(M+H)+
The following examples were synthesized in analogy to the procedure of example 9, using the corresponding starting materials.
Example 13 (trans-racemic mixture)
100mg (0.215mmol) of example 12A are mixed with 1.00mL (6.07mmol) of 1,1,3, 3-tetramethoxypropane. The reaction mixture was heated to 175 ℃ for 1h using a microwave oven. The reaction mixture was treated with DCM/MeOH and 1 drop triethylamine. The solvent was removed under reduced pressure. The mixture was purified by preparative HPLC (eluent A: water +0.13% TFA, eluent B: MeOH) to yield 45mg (54%) of the title compound.
HPLC-MS (method 3) Rt=1.36min
MS(ESI pos):m/z=387(M+H)+
Enantiomers of the title compound were separated by HPLC using a chiral stationary phase.
Enantiomer separation method:
HPLC equipment type is Berger Minigram; the column is Daicel IC, 5.0 μm, 250mm × 10 mm; method eluent CO230% MeOH/0.2% DEA (isocratic); the flow rate is 10mL/min, and the temperature is 40 ℃; the pressure is 100 bar; UV detection at 210nm
The following examples were synthesized by a similar procedure to example 13, using the corresponding dialdehyde diacetal as the starting material.
Example 17 (trans-racemic mixture)
176mg (0.431mmol) of example 4A are mixed at room temperature with 3mL of THF and 122mg (0.302mmol) of Lawson's reagent. The mixture was then stirred at 60 ℃ for 6 h. The reaction mixture was treated with water and then diluted with DCM. The mixture was filtered through basic alumina and eluted with DCM and EtOH. The solvent was removed under reduced pressure. The residue was purified by preparative HPLC (eluent A: water +0.13% TFA, eluent B: MeOH). 45mg (26%) of product are obtained.
HPLC-MS (method 3) Rt=1.37min
MS(ESI pos):m/z=407(M+H)+
Enantiomers of the title compound were separated by HPLC using a chiral stationary phase.
Enantiomer separation method:
HPLC equipment type is Berger Minigram; the column is Daicel ADH, 5.0 μm, 250mm × 10 mm; method eluent CO230% MeOH/0.2% DEA (isocratic); the flow rate is 10mL/min, and the temperature is 40 ℃; the pressure is 100 bar; UV detection at 210nm
The single crystal of example 19 was prepared by recrystallization from ethyl acetate and subjected to X-ray crystal analysis. The absolute configuration of example 19 was judged from the data to be (R, R).
Experiment data collection and arrangement-data were collected on a Saturn944CCD mounted on an AFC11K goniometer, irradiating CuK α from the RU200 rotating anode and the RIGAKU VARIMAX optics, at a temperature of 100K.
Summary of data Collection statistics
() The value in refers to the last resolution shell.
Fine statistics:
p2 from example 191Medium final structure factor calculation
Total number of l.s. parameters =255
GooF=S=1.154
Weighted value =1/[ sigma ^2(Fo ^2) + (0.0421 ^ P) ^2+0.38 ^ P ] where P = (max (Fo ^2,0) +2 ^ Fc ^2)/3
For 2207Fo >4sig (Fo), R1=0.0695, and for all 2334 data, R1 is 0.0829, wR2=0.1646,
the Flack x parameter =0.09 (3).
Example 20 (trans-racemic mixture)
0.060g of example 10A was mixed with 4mL of anhydrous bisAlkane and 0.074g (0.180mmol) Lawson's reagent. The reaction mixture was heated to 120 ℃ in a microwave oven for 1 h. The mixture was filtered over basic alumina and eluted with DCM and MeOH. The solvent was removed under reduced pressure. The residue was purified by preparative HPLC (eluent A: water +0.13% TFA, eluent B: MeOH). 22mg of product was obtained in the form of a salt with TFA.
HPLC-MS (method 1) Rt=0.94min
MS(ESI pos):m/z=393(M+H)+
Example 21 (trans-racemic mixture)
0.190g (0.519mmol) of example 8E was mixed with 1.38mL (8.31mmol) of triethoxymethane. The mixture was stirred at 150 ℃ for 1.5 h. The reaction mixture was cooled to room temperature and purified by preparative HPLC (eluent A: water +0.13% TFA, eluent B: MeOH). 90mg (46%) of product are obtained.
HPLC-MS (method 1) Rt=1.19min
MS(ESI pos):m/z=377(M+H)+
Example 22 (trans-racemic mixture)
13mg (0.10mmol) of CuCl226mL (0.22mmol) of nitrous acidTert-butyl ester was mixed with ACN. 22mg (0.05mmol) of the mixture of example 12 in ACN are added carefully at 0 ℃. The mixture was stirred at 25 ℃ for 1 h. 9mg (0.07mmol) of CuCl was added thereto2And 13mL (0.11mmol) of tert-butyl nitrite and stirring for a further 20 minutes. The solvent was removed under reduced pressure. The residue was dissolved in DCM and then extracted with HCl and water. The mixture was purified by preparative HPLC (eluent A: water +0.13% TFA, eluent B: MeOH) to yield 2.1mg (9%) of the product.
HPLC-MS (method 3) Rt=1.46min
MS(ESI pos):m/z=426/428(Cl)(M+H)+
Example 23 (trans-racemic mixture)
180mg (0.26mmol, content 50% by HPLC-MS) of example 12b are mixed with 1.00mL (6.07mmol) of 1,1,3, 3-tetramethoxypropane. The reaction mixture was heated to 175 ℃ for 1h using a microwave oven. The reaction mixture was treated with DCM and washed with water. The organic layer was dried over sodium sulfate and then evaporated under reduced pressure. The crude product was purified by flash chromatography (Cy/EtOAc80/20 to AcOEt/MeOH96/4) followed by a second flash chromatography (DCM100% to DCM/EtOH96/4) to afford the title compound as an off-white solid. (0.034 g).
HPLC-MS (method 1Eh) Rt=6.57min
MS(APCI pos):m/z=353(M+H)+
Enantiomers of the title compound were separated by HPLC using a chiral stationary phase.
Enantiomer separation method:
semi-preparative conditions:
HPLC semi-preparative systems Waters600 pumps; a column, Daicel chiralcel OJ-H, 250mm × 20mm, 5.0 μm; eluent hexane/EtOH 80: 20; the flow rate is 15mL/min, and the temperature is 25 ℃; UV detection at 254nm
Conditions of analysis
HPLC equipment type Agilent 1100; method 6; a column, Daicel chiralcel OJ-H, 250mm × 4.6mm, 5.0 μm; eluent hexane/EtOH 80: 20; the flow rate is 1mL/min, and the temperature is 25 ℃; UV detection at 254nm
Example 26 (trans-racemic mixture)
140mg (content: 84%, 0.33mmol) of example 12C were mixed with 1.4mL1,1,3, 3-tetramethoxypropane and 1.4mL NMP. The reaction mixture was heated to 175 ℃ for 1h using a microwave oven. The reaction mixture was then diluted with MeOH and loaded onto an SCX column. The ammonia fractions were collected and the residue was purified by flash chromatography (Cy/EtOAc from 90/10 to 100%) to give the title compound as a white solid (30 mg).
HPLC-MS (method 1Eh) Rt=6.72min
MS(APCI pos):m/z=370(M+H)+
Enantiomers of the title compound were separated by HPLC using a chiral stationary phase.
Enantiomer separation method:
semi-preparative conditions:
HPLC semi-preparative systems Waters600 pumps; a column, Daicel chiralcel OJ-H, 250mm × 20mm, 5.0 μm; eluent hexane/EtOH 80: 20; the flow rate is 15mL/min, and the temperature is 25 ℃; UV detection at 230nm
Conditions of analysis
HPLC equipment type Agilent 1100; method 6; a column, Daicel chiralcel OJ-H, 250mm × 4.6mm, 5.0 μm; eluent hexane/EtOH 80: 20; the flow rate is 1mL/min, and the temperature is 25 ℃; UV detection at 254nm
Example 29 (trans-racemic mixture)
0.066g (1.66mmol) of sodium hydride (60% suspension in mineral oil) was added to a suspension of 0.132g (0.63mmol) of 5-amino-1- (tetrahydropyran-4-yl) -1-H-pyrazole-4-carboxylic acid amide (see PCT patent application WO2010/026214) in anhydrous EtOH (1.5mL) at room temperature under nitrogen. After 10min, 0.181mg (0.945mmol) of example 13A was added and the reaction mixture was then heated to 140 ℃ in a microwave oven (power 100W) for 40 min. The reaction mixture was then diluted with DCM, water was added, the organics were separated and dried over sodium sulfate. The organics were evaporated under reduced pressure and the crude product was purified by flash chromatography (DCM/IPA98:2) to afford the title compound as a white solid. (54mg, 32%).
HPLC-MS (method 1Eh) Rt=8.01min
MS(APCI pos):m/z=352(M+H)+
Enantiomers of the title compound were separated by HPLC using a chiral stationary phase.
Enantiomer separation method:
semi-preparative conditions:
HPLC semi-preparative systems Waters600 pumps; a column, Daicel chiralcel OJ-H, 250mm × 20mm, 5.0 μm; eluent hexane/EtOH 85: 15; the flow rate is 15mL/min, and the temperature is 25 ℃; UV detection at 254nm
Conditions of analysis
HPLC equipment type Agilent 1100; method 6.1; a column, Daicel chiralcel OJ-H, 250mm × 4.6mm, 5.0 μm; eluent hexane/EtOH 85: 15; the flow rate is 1mL/min, and the temperature is 25 ℃; UV detection at 254nm
Example 32 (trans-racemic mixture)
0.066g (1.66mmol) of sodium hydride (60% suspension in mineral oil) was added to a suspension of 0.135g (0.553mmol) of 5-amino-1- (4, 4-difluoro-cyclohexyl) -1-H-pyrazole-4-carboxylic acid amide (see PCT patent application WO2010/026214) in anhydrous EtOH (1.5mL) at room temperature under nitrogen. After 10min, 0.161mg (0.837mmol) of example 13A are added and the reaction mixture is heated to 140 ℃ in a microwave oven (power 100W) for 40 min. The reaction mixture was then diluted with DCM, water was added, the organics were separated and then dried over sodium sulfate. The organics were evaporated under reduced pressure and the crude product was purified by flash chromatography (Cy/EA from 50:50 to 10:90) to afford the title compound as a white solid. (54mg, 25%).
HPLC-MS (method 1Eh) Rt=9.63min
MS(APCI pos):m/z=386(M+H)+
Enantiomers of the title compound were separated by HPLC using a chiral stationary phase.
Enantiomer separation method:
semi-preparative conditions:
HPLC semi-preparative systems Waters600 pumps; column, Daicel chiralpak AD-H, 250mm × 20mm, 5.0 μm; eluent hexane/isopropanol 80: 20; the flow rate is 10mL/min, and the temperature is 25 ℃; UV detection at 260nm
Conditions of analysis
HPLC equipment type Agilent 1100; method 7; a column, Daicel chiralcel AD-H, 250mm × 4.6mm, 5.0 μm; eluent hexane/isopropanol 80: 20; the flow rate is 1mL/min, and the temperature is 25 ℃; UV detection is 260 nm.