阅读说明：本技术 五环化合物的盐及其晶体 (Salt of pentacyclic compound and crystal thereof ) 是由 吉田贤史 大桥芳章 星川环 佐藤信明 栉田郁雄 于 2020-03-03 设计创作，主要内容包括：由式(I)所表示的化合物的盐或其晶体具有用作药物的原料药的可能性。(The salt of the compound represented by the formula (I) or the crystal thereof has a possibility of being used as a drug substance of a medicament.)

1.5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5' represented by formula (I) ']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivatives-monohydrochloride or monohydrobromide salts of 4, 13-diketones:

2.5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5' represented by formula (I) ']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivatives-4, 13-dione or 5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [4",3":4',5' represented by formula (I) ']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivatives-crystals of the monohydrochloride or monohydrobromide salt of 4, 13-dione:

3.5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5' represented by formula (I) ']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivatives-crystals of 4, 13-dione:

in powder X-ray diffraction using CuK α as an X-ray source, diffraction peaks were found at diffraction angles (2 θ ± 0.2 °) of 9.0 °, 11.1 ° and 23.6 °.

4.5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5' represented by formula (I) ']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivatives-form a crystals of 4, 13-dione monohydrochloride:

in powder X-ray diffraction using CuK α as an X-ray source, diffraction peaks were found at diffraction angles (2 θ ± 0.2 °) of 11.6 °, 20.8 ° and 25.7 °.

5.5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5' represented by formula (I) ']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivatives-form B crystals of 4, 13-dione monohydrochloride:

in powder X-ray diffraction using CuK α as an X-ray source, diffraction peaks were found at diffraction angles (2 θ ± 0.2 °) of 9.7 °, 10.1 ° and 17.9 °.

6.5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5' represented by formula (I) ']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivatives-form C crystals of 4, 13-dione monohydrochloride:

in powder X-ray diffraction using CuK α as an X-ray source, diffraction peaks were found at diffraction angles (2 θ ± 0.2 °) of 6.0 °, 7.7 ° and 16.9 °.

7.5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5' represented by formula (I) ']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivatives-form D crystals of 4, 13-dione monohydrochloride:

in powder X-ray diffraction using CuK α as an X-ray source, diffraction peaks were found at diffraction angles (2 θ ± 0.2 °) of 6.6 °, 14.6 ° and 26.4 °.

8.5, 10-dimethyl-5, 6,9,10,11, 12-hexahydro represented by formula (I)Pyrido [4",3":4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivatives-form E crystals of 4, 13-dione monohydrochloride:

in powder X-ray diffraction using CuK α as an X-ray source, diffraction peaks were found at diffraction angles (2 θ ± 0.2 °) of 6.4 °, 11.3 ° and 27.3 °.

9.5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5' represented by formula (I) ']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivatives-form F crystals of 4, 13-dione monohydrochloride:

in powder X-ray diffraction using CuK α as an X-ray source, diffraction peaks were found at diffraction angles (2 θ ± 0.2 °) of 7.3 °, 9.3 ° and 10.7 °.

10. 5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5' represented by formula (I) ']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivatives-crystals of 4, 13-dione monohydrobromide salt:

in powder X-ray diffraction using CuK α as an X-ray source, diffraction peaks were found at diffraction angles (2 θ ± 0.2 °) of 7.8 °, 24.5 ° and 25.2 °.

11. 5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5' represented by formula (I) ']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivatives-form a crystals of 4, 13-dione monohydrochloride:

it is measured at 587cm in Raman spectrum^-1Has Raman shift peak (+/-2 cm)^-1)。

12. 5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5' represented by formula (I) ']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivatives-form a crystals of 4, 13-dione monohydrochloride:

it is measured at 587cm in Raman spectrum^-1、1428cm^-1And 1493cm^-1Has Raman shift peak (+/-2 cm)^-1)。

13. 5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5' represented by formula (I) ']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivatives-form a crystals of 4, 13-dione monohydrochloride:

it is measured at 587cm in Raman spectrum^-1、763cm^-1、1428cm^-1、1493cm^-1And 1688cm^-1Has Raman shift peak (+/-2 cm)^-1)。

14.5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5' represented by formula (I) ']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivatives-form a crystals of 4, 13-dione monohydrochloride:

it is at 409cm in Raman spectrometry^-1、587cm^-1、763cm^-1、976cm^-1、1428cm^-1、1493cm^-1And 1688cm^-1Has Raman shift peak (+/-2 cm)^-1)。

15. A pharmaceutical composition comprising the salt of claim 1 or the crystal of any one of claims 2 to 14.

Technical Field

The present invention relates to a pharmaceutically acceptable salt of a pentacyclic compound having a cholinergic neuron activating effect and/or a neuroprotective effect and a crystal of the pentacyclic compound and the pharmaceutically acceptable salt thereof. The present invention also relates to a pharmaceutical composition comprising the above salt or crystal as an active ingredient.

Background

Cholinergic neurons that release acetylcholine as a transmission substance project widely in the forebrain from the basal nuclei of Meynert (Meynert) and the septal nuclei of the basal forebrain to the hippocampus, amygdala, and cerebral cortex, and are involved in the regulation of memory, learning, cognition, and attention (non-patent document 1). Furthermore, it is considered that cholinergic neurons of the foot bridge nucleus and dorsolateral tegmental nucleus of the brain stem project toward the striatum, nucleus accumbens, substantia nigra or thalamus and participate in the regulation of motivation or wakefulness (non-patent documents 2 to 4).

In particular, the role of cholinergic neurons in the basal forebrain has been more elucidated by analysis using a number of animal models, such as injury models. In particular, the correlation between dysfunction of cholinergic neurons and decline in memory and learning has been shown in animal models (non-patent documents 5 to 7), and it has been shown that cognitive performance is improved by increasing the amount of acetylcholine and enhancing the function of cholinergic neurons using cholinesterase inhibitors (non-patent documents 8 to 12).

It has been reported that Nerve Growth Factor (NGF) shows a neuroprotective effect on cholinergic neurons in an animal model showing a deletion of cholinergic neurons (non-patent documents 13 to 15).

In particular, in Alzheimer's Disease (AD), a loss of cholinergic neurons is found from an early stage of AD, and is one of the pathological features of AD. Accumulation of senile plaques caused by deposition of amyloid β and neurofibrillary tangles caused by tau aggregation are also pathological features of AD, and in particular neurofibrillary tangles are known to increase with progression of the disease state and cause neuronal death. Neurofibrillary tangles are found in the basal nucleus of menatt and the entorhinal cortex from early stages of AD. Among them, it has been reported that tau aggregation is found early to cause the deletion of cholinergic neurons in the basal nucleus of menatet, and that there is a correlation between the loss and decrease of cognitive function score (non-patent documents 16 and 17). Like AD, hyperphosphorylation and abnormal accumulation of tau protein was found in genetically modified mice bearing the P301S mutation, which P301S mutation has been found in familial frontotemporal dementia (human tau P301S transgenic mice). Therefore, neurofibrillary tangles (pathological features of AD) are formed (non-patent document 18) and cause cognitive dysfunction through synaptic damage, neurodegeneration, and loss of neurons. Based on these findings, human τ P301S transgenic mice are widely used as AD-like animal models (non-patent documents 19 to 22), and improvement of cognitive decline in alzheimer's disease and inhibition of progression of the disease state can be expected by inhibiting AD-like lesions in human τ P301S transgenic mice.

Furthermore, multiple analyses using genetically modified mice and animal models of disorders have shown that axonal transport defects are one of the causes of cholinergic neuron deletion (non-patent documents 23-25). Among them, axons of cholinergic neurons projecting from septal regions to the hippocampus are damaged in a fornix hippocampal umbrella injury model, and neuronal loss is caused by impaired reverse transport of molecules involved in survival and function (non-patent documents 26 to 28). Reverse trafficking injury was also found in genetically modified mice (non-patent documents 23 and 24), and deletion of cholinergic neurons caused by fornix hippocampal injury reflects one aspect of the disease state. Thus, improvement of cognitive decline and inhibition of progression of the disease state in alzheimer's disease can be expected by inhibiting or ameliorating the loss of cholinergic neurons in this model of disorder.

Dementia with lewy bodies (DLB) and Parkinson's Disease (PD) are progressive neurodegenerative disorders in which abnormal inclusion bodies (lewy bodies), composed mainly of alpha synuclein, appear in neurons and cause degeneration and loss of neurons. Progression of cognitive dysfunction results if lewy bodies are predominantly distributed in the cerebral cortex and parkinson's disease if they are predominantly distributed in the brainstem. In addition to this, mental symptoms such as hallucinations, hallucinations and delusions, sleep disturbances and the development of autonomic symptoms may also result. Dementia with lewy bodies is diagnosed if the dementia occurs before or within one year after the onset of parkinson's disease, and dementia with parkinson's disease (PDD) is diagnosed if parkinson's disease occurs one or more years before the onset of dementia. Dementia with lewy bodies, dementia with parkinson's disease and parkinson's disease are pathologically identical diseases and are collectively called Lewy Body Disease (LBD), although these differ in the order and extent of appearance of cognitive dysfunction and parkinson's disease. In dementia with lewy bodies and dementia with parkinson's disease, neurons of the basal nucleus of menait (nucleus of cholinergic nerve origin) degenerate and delete, similar to alzheimer's disease, and it has been reported that severe cholinergic neuron disorders occur in the hippocampus and cortex (non-patent documents 29 to 31). Furthermore, there is a correlation between the progression of cholinergic neuronal disorders and cognitive dysfunction (non-patent document 29), and it has been demonstrated that cholinesterase inhibitors can improve cognitive functions. Based on these findings, cognitive function is improved by improving the function of cholinergic neurons, and improvement of cognitive decline and inhibition of disease state progression in dementia with lewy bodies and dementia with parkinson's disease can be expected by inhibiting or improving the absence of cholinergic neurons in several disorder models, similar to alzheimer's disease.

Thus, based on these findings, improvement in the decline in cognitive performance caused by cholinergic neuronal dysfunction can be expected by achieving functional activation and/or neuroprotective effects on cholinergic neurons in clinical practice.

List of citations

Non-patent document

[ non-patent document 1] Evertt BJ et al, "Central Cholinergic systems and verification", "Annu. Rev. Psychol.48(1997)649- > 684.

[ non-patent document 2] Gulledge AT.et al, "Choline scientific inhibition of neoorganic pyramids neurones," J.Neurosci.25(2005)10308-20.

[ non-patent document 3] Daniel Dautan D.et al, "A major external source of the strain and nucleus accumulations orientations in the branched," J.Neurosci.34(2014)4509-18.

[ non-patent document 4] M Steriade M.et al, "neurological activities in branched-linear genetic nucleic acid related to a genetic activation process in genetic systems," J.Neurosci.10(1990)2541-59.

[ non-patent document 5] Fischer W.et al, "Progressive cycle in specific learning and integration of for ebral cyclic nerves in rates reducing formation," neurobiol. aging 13(1992)9-23.

[ non-patent document 6] Leanza G.et al, "Selective release of the basic for ebral and veterinary systems by intragenic 192 IgG-saponin: behavioural, biochemical and scientific students in the said rate," Eur.J.neurosci.7(1995)329-43.

[ non-patent document 7] Leanza G.et al, "Selective immunization of the basal for ebral and nutritional system disorders short-term memory in rates," Eur.J.Neurosci.8(1996)1535-44.

[ non-patent document 8] Ogura H.et al, "Donepezil, a Central acting acetylcholinesterase inhibitor, alleviates leirning details in hypothiochloric models in rates," Methods Find exp.Clin.Pharmacol.22(2000)89-95.

[ non-patent document 9] Spot-manufacturing L.et. "Spatial characterization strategies by ablation strategies in the Morris water area task." Behav. brain Res.156(2005)269-76.

[ non-patent document 10] Bruce AP.et al, "Choline acetyl transferase activity and cognitive domain score of Alzheimer's patents," neurobiol.aging.21(2000)11-17.

[ non-patent document 11] Rogers SL. et al, "The efficiency and safety of therapy in tissues with Alzheimer's disease: stresses of US Multicentre, Randomized, Double-Blind, Placebo-Controlled Trial. The therapy Study group," Dementia 7(1996) 293-.

[ non-patent document 12] Mori E.et al, "Donepzil for the division with Lewy bodies: a randomized, placebo-controlled tertiary," Ann. neuron.72 (2012)41-52.

[ non-patent document 13] Mufson EJ.et al, "Human choleretic basic for ebrain: chemoanatomi and neurologic kinetic therapy" "J.chem.neuroanat.26 (2003)233-

[ non-patent document 14] Mufson EJ.et al, "Choline system duringhe progress of Alzheimer's disease" thermal imaging "expert. Rev. Neurother.8(2008)1703-1718.

[ non-patent document 15] Schliebs R.et al, "The design of The catalytic system in The catalytic reduction and in Alzheimer's disease" -J.Neural.Transm 113(2006) 1625-.

[ non-patent document 16] Vana L et al, "progress of tau pathology in cholinergic basic for ebrain nerves in fine cognitive interference and Alzheimer's disease" am.J.Pathol.179(2011) 2533-.

[ non-patent document 17] Gomez-Isla T et al, "neural low coefficients with butyl exceeds of neural columns in Alzheimer's disease," Ann. neuron.41 (1997)17-24.

[ non-patent document 18] Lee VM et al, "neuroactive tauopathies," Annu. Rev. Neurosci.24(2001)1121- "1159.

[ non-patent document 19] Allen B et al, "Absundant tau filters and nonproptopic neurogenetic in transforming human P301S tau protein", "J.Neurosci.22 (2002) 9340-" 9351.

[ non-patent document 20] Xu H et al, "Memory devices corolate with tau and thread pathology in P301S MAPT transcriptional microorganism," Neuropathol.appl.Neurobiol.40(2014)833-43.

[ non-patent document 21] Yoshiyama Y et al, "Synapse loss and microbial activation precursors in a P301S tauopathophathy mouse model," Neuron 53(2007)337-351.

[ non-patent document 22] Hoffmann NA et al, "affected plastics of scientific classes in P301S tau transgenic. Acta Neuropathol Commun.1(2013)82.

[ non-patent document 23] Salehi A et al, "incorporated App Expression in a Mouse Model of Down's synthetic dispersions NGF Transport and catalysts Choline nerve regeneration" nerve 51(2006)29-42.

[ non-patent document 24] Onishi T et al, "Early-on set cognitive configurations and axnal transport gravity function inP301S mutant tau transport" Neuroscience Research 80(2014)76-85.

[ non-patent document 25] Xu W et al, "analog pre-cursor protein-mediated end path displacement indexes axonality document 126(2016)1815-33.

[ non-patent document 26]Lapchak PA et al.“Effect of recombinant human nerve growth factor on presynaptic cholinergic function in rat hippocampal slices following partial septohippocampal lesions:measures of[³H]acetylcholine synthesis,[³H]acetylcholine release and choline acetyltransferase activity”Neuroscience 42(1991)639-49.

[ non-patent document 27] Gilmor ML et al, "Coordinate expression of the vesicular acrylic transporter and cholene acetic transfer enzyme following a partially halogenated carboxylic path loss" J.Neurochem.71(1998)2411-20.

[ non-patent document 28] Gu H et al, "Recombinant human NGF-loaded microspheres promoter overview of basic for ebral branched peptides and reactive media interactions of specific depletion in the rate model of Alzheimer's disease with complex-variant implementation" neurosci. Lett 453(2009)204-9.

[ non-patent document 29] Shimada, H.et al, "Mapping of blain acetyl cholinesterase orientations in Lewy body disease by PET," Neurology, vol.73, pp.273-278,2009.

[ non-patent document 30] Tiraboshi, P.et al, "Choline kinetic functions in diseases with Lewy bodies" Neurology 54(2000)407- "411.

[ non-patent document 31] Perry, E.K. et al, "novel chlorine microorganisms free body differentiation from structural Alzheimer's disease", NeuroReport, vol.5, pp.747-749(1994).

Disclosure of Invention

Technical problem

The inventors have found that a compound represented by the following formula (I) (5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [4",3":4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivatives-4, 13-dione, hereinafter also referred to as "Compound (I)") has a cholinergic neuron activating effect and/or a neuroprotective effect. Therefore, compound (I) has a potential use as an agent for improving cognitive impairment caused by dysfunction of cholinergic neurons.

In general, physical properties of a compound used as a drug and a salt thereof and crystals thereof have a great influence on bioavailability of the drug, purity of a raw material drug, formulation of a pharmaceutical preparation, and the like. Therefore, an object of the present invention is to provide a pharmaceutically acceptable salt of compound (I) and crystals thereof, which have the possibility of being used as a drug substance of a medicament.

Solution to the problem

In view of the above, the present inventors have conducted intensive studies on the compound (I), and as a result, have found a salt of the compound (I) and crystals thereof, thereby completing the present invention.

Namely, the present invention relates to the following <1> to <35 >.

<1>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5' represented by formula (I) ']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivatives-monohydrochloride or monohydrobromide salts of 4, 13-diketones:

<2>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivatives-4, 13-dione or 5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [4",3":4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivatives-crystals of the monohydrochloride or monohydrobromide salt of 4, 13-dione.

<3>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesThe crystals of the (E) -4, 13-dione have diffraction peaks at diffraction angles (2 θ ± 0.2 °) of 9.0 °, 11.1 ° and 23.6 ° in powder X-ray diffraction using CuK α as an X-ray source.

<3.1>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesThe crystal of (E) -4, 13-dione has diffraction peaks at diffraction angles (2 θ ± 0.2 °) of 9.0 °, 11.1 °, 14.5 °, 18.1 °, 20.0 °, 21.9 °, 23.6 °, 24.4 °, 24.9 ° and 28.5 ° in powder X-ray diffraction using CuK α as an X-ray source.

<3.2>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesUse of crystals of (E) -4, 13-dione in powder X-ray diffraction Using CuK alpha as X-ray SourceHaving the powder X-ray diffraction pattern of figure 1.

<4>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesThe type a crystal of the (E) -4, 13-dione monohydrochloride has diffraction peaks at diffraction angles (2 θ ± 0.2 °) of 11.6 °, 20.8 ° and 25.7 ° in powder X-ray diffraction using CuK α as an X-ray source.

<4.1>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesThe form A crystal of the (E) -4, 13-dione monohydrochloride has diffraction peaks at diffraction angles (2 theta + -0.2 DEG) of 6.1 DEG, 7.8 DEG, 11.6 DEG, 16.2 DEG, 19.9 DEG, 20.8 DEG, 25.2 DEG, 25.7 DEG, 26.9 DEG and 29.9 DEG in powder X-ray diffraction using CuK alpha as an X-ray source.

<4.2>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesThe form a crystals of the (e) -4, 13-dione monohydrochloride have the powder X-ray diffraction pattern of fig. 2 in powder X-ray diffraction using CuK α as the X-ray source.

<4.3>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesUse of form A crystals of (E) -4, 13-dione monohydrochloride with glycine as external reference (176.03ppm)¹³In the C solid state NMR spectrum, peaks were observed at chemical shifts (. delta. +. 0.5ppm) of 164.0ppm, 129.6ppm and 36.5 ppm.

<5>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesThe B-type crystal of the (E) -4, 13-dione monohydrochloride has diffraction peaks at diffraction angles (2 θ ± 0.2 °) of 9.7 °, 10.1 ° and 17.9 ° in powder X-ray diffraction using CuK α as an X-ray source.

<5.1>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesThe form B crystal of the (E) -4, 13-dione monohydrochloride has diffraction peaks at diffraction angles (2. theta. + -0.2 DEG) of 6.3 DEG, 9.7 DEG, 10.1 DEG, 17.9 DEG, 19.0 DEG, 19.4 DEG, 23.4 DEG, 26.3 DEG, 27.3 DEG and 32.0 DEG in powder X-ray diffraction using CuK alpha as an X-ray source.

<5.2>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesThe form B crystals of the (e) -4, 13-dione monohydrochloride have the powder X-ray diffraction pattern of fig. 3 in powder X-ray diffraction using CuK α as the X-ray source.

<5.3>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesUse of crystals of form B of (E) -4, 13-dione monohydrochloride with glycine as external reference (176.03ppm)¹³In the C solid state NMR spectrum, peaks were observed at chemical shifts (. delta. +. 0.5ppm) of 160.1ppm, 133.4ppm and 130.7 ppm.

<6>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [ 2]2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesThe C-type crystal of the (E) -4, 13-dione monohydrochloride has diffraction peaks at diffraction angles (2 θ ± 0.2 °) of 6.0 °, 7.7 ° and 16.9 ° in powder X-ray diffraction using CuK α as an X-ray source.

<6.1>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesThe C-type crystal of the (e) -4, 13-dione monohydrochloride has diffraction peaks at diffraction angles (2 θ ± 0.2 °) of 6.0 °, 7.7 °, 9.7 °, 11.4 °, 15.8 °, 16.9 °, 18.1 °, 23.2 °, 25.4 ° and 27.6 ° in powder X-ray diffraction using CuK α as an X-ray source.

<6.2>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesThe form C crystals of the (e) -4, 13-dione monohydrochloride have the powder X-ray diffraction pattern of fig. 4 in powder X-ray diffraction using CuK α as the X-ray source.

<6.3>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesUse of the C-form crystals of (E) -4, 13-dione monohydrochloride with glycine as external reference (176.03ppm)¹³In the C solid state NMR spectrum, peaks were observed at chemical shifts (. delta. +. 0.5ppm) of 159.6ppm, 127.6ppm and 38.9 ppm.

<7>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesThe D-form crystal of the (E) -4, 13-dione monohydrochloride has diffraction peaks at diffraction angles (2 theta + -0.2 DEG) of 6.6 DEG, 14.6 DEG and 26.4 DEG in powder X-ray diffraction using CuK alpha as an X-ray source.

<7.1>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesThe form D crystal of the (E) -4, 13-dione monohydrochloride has diffraction peaks at diffraction angles (2. theta. + -0.2 DEG) of 6.6 DEG, 14.6 DEG, 16.1 DEG, 20.5 DEG, 21.0 DEG, 23.0 DEG, 24.5 DEG, 26.4 DEG, 28.0 DEG and 32.5 DEG in powder X-ray diffraction using CuK alpha as an X-ray source.

<7.2>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesForm D crystals of the (E) -4, 13-dione monohydrochloride having the powder X-ray diffraction pattern of FIG. 5 in powder X-ray diffraction using CuK α as the X-ray source.

<8>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesThe E-type crystal of the (E) -4, 13-dione monohydrochloride has diffraction peaks at diffraction angles (2 θ ± 0.2 °) of 6.4 °, 11.3 ° and 27.3 ° in powder X-ray diffraction using CuK α as an X-ray source.

<8.1>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivatives-4, 13-diketonesForm E crystals of the monohydrochloride have diffraction peaks at diffraction angles (2 θ ± 0.2 °) of 6.4 °, 11.3 °, 15.7 °, 18.0 °, 19.2 °, 22.8 °, 24.6 °, 25.4 °, 26.0 ° and 27.3 ° in powder X-ray diffraction using CuK α as the X-ray source.

<8.2> type E crystals of the monohydrochloride salt of compound (I) having the powder X-ray diffraction pattern of FIG. 6 in powder X-ray diffraction using CuK α as an X-ray source.

<9>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesThe form F crystal of the (E) -4, 13-dione monohydrochloride has diffraction peaks at diffraction angles (2. theta. + -0.2 DEG) of 7.3 DEG, 9.3 DEG and 10.7 DEG in powder X-ray diffraction using CuK alpha as an X-ray source.

<9.1>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesThe form F crystal of the (E) -4, 13-dione monohydrochloride has diffraction peaks at diffraction angles (2 theta + -0.2 DEG) of 5.9 DEG, 7.3 DEG, 9.3 DEG, 10.7 DEG, 13.8 DEG, 15.6 DEG, 16.4 DEG, 18.7 DEG, 25.1 DEG and 26.8 DEG in powder X-ray diffraction using CuK alpha as an X-ray source.

<9.2>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesForm F crystals of the (E) -4, 13-dione monohydrochloride having the powder X-ray diffraction pattern of FIG. 7 in powder X-ray diffraction using CuK α as the X-ray source.

<10>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesThe crystals of the (E) -4, 13-dione monohydrobromide salt had diffraction peaks at diffraction angles (2 θ ± 0.2 °) of 7.8 °, 24.5 ° and 25.2 ° in powder X-ray diffraction using CuK α as an X-ray source.

<10.1>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesThe crystals of (E) -4, 13-dione monohydrobromide have diffraction peaks at diffraction angles (2 θ ± 0.2 °) of 6.0 °, 7.8 °, 10.0 °, 11.7 °, 17.8 °, 20.8 °, 23.5 °, 24.5 °, 25.2 ° and 27.3 ° in powder X-ray diffraction using CuK α as an X-ray source.

<10.2>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesThe crystals of the (E) -4, 13-dione monohydrobromide salt have a powder X-ray diffraction pattern of FIG. 8 in powder X-ray diffraction using CuK α as the X-ray source.

<11>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesForm A crystals of the monohydrochloride salt of (E) -4, 13-dione, which, in Raman spectroscopy, is at 587cm^-1Has Raman shift peak (+/-2 cm)^-1)。

<12>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesMono-of (E) -4, 13-dioneForm A crystals of the hydrochloride salt at 587cm in Raman spectroscopy^-1、1428cm^-1And 1493cm^-1Has Raman shift peak (+/-2 cm)^-1)。

<13>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesForm A crystals of the monohydrochloride salt of (E) -4, 13-dione, which, in Raman spectroscopy, is at 587cm^-1、763cm^-1、1428cm^-1、1493cm^-1And 1688cm^-1Has Raman shift peak (+/-2 cm)^-1)。

<14>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesForm A crystals of the monohydrochloride salt of (E) -4, 13-dione, which, in Raman spectroscopy, is at 409cm^-1、587cm^-1、763cm^-1、976cm^-1、1428cm^-1、1493cm^-1And 1688cm^-1Has Raman shift peak (+/-2 cm)^-1)。

<15>5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4', 3': 4',5']Thieno [2',3':4,5]Pyrimido [1,2-a ] s]Thieno [3,2-f][1,4]Diaza derivativesForm A crystals of the monohydrochloride salt of a 4, 13-dione having a spectrum of FIG. 19 in Raman spectroscopy.

<16> a pharmaceutical composition comprising the salt according to <1> or the crystal according to any one of <2> to <15 >.

<17> the pharmaceutical composition according to <16>, which is a cholinergic neuron activator.

<18> the pharmaceutical composition according to <16>, which is a cholinergic neuron protective agent.

<19> the pharmaceutical composition according to <16> for the treatment of cognitive dysfunction.

<20> a therapeutic agent for cognitive dysfunction, which comprises the salt according to <1> or the crystal according to any one of <2> to <15 >.

<21> a method for treating cognitive dysfunction, the method comprising administering to a patient the salt according to <1> or the crystal according to any one of <2> to <15 >.

<22> the salt according to <1> or the crystal according to any one of <2> to <15> for use in treating cognitive dysfunction.

<23> use of the salt according to <1> or the crystal according to any one of <2> to <15> for manufacturing a therapeutic agent for cognitive dysfunction.

<24> a therapeutic agent for alzheimer's disease comprising the salt according to <1> or the crystal according to any one of <2> to <15 >.

<25> a method for treating alzheimer's disease, which comprises administering the salt according to <1> or the crystal according to any one of <2> to <15> to a patient.

<26> the salt according to <1> or the crystal according to any one of <2> to <15> for use in treating alzheimer's disease.

<27> use of the salt according to <1> or the crystal according to any one of <2> to <15> for the manufacture of a therapeutic agent for alzheimer's disease.

<28> a therapeutic agent for dementia with lewy bodies, which comprises the salt according to <1> or the crystal according to any one of <2> to <15 >.

<29> a method for treating dementia with Lewy bodies, which comprises administering the salt according to <1> or the crystal according to any one of <2> to <15> to a patient.

<30> the salt according to <1> or the crystal according to any one of <2> to <15> for use in treating dementia with lewy bodies.

<31> use of the salt according to <1> or the crystal according to any one of <2> to <15> for manufacturing a therapeutic agent for dementia with lewy bodies.

<32> a therapeutic agent for dementia in Parkinson's disease, comprising the salt according to <1> or the crystal according to any one of <2> to <15 >.

<33> a method for treating dementia in parkinson's disease, which comprises administering to a patient the salt according to <1> or the crystal according to any one of <2> to <15 >.

<34> the salt according to <1> or the crystal according to any one of <2> to <15> for use in treating parkinson's disease dementia.

<35> use of the salt according to <1> or the crystal according to any one of <2> to <15> for manufacturing a therapeutic agent for Parkinson's disease dementia.

Advantageous effects of the invention

According to the present invention, a salt of compound (I) and crystals thereof having good physical properties, which are expected to have the possibility of being used as a drug substance of a drug, can be provided.

Drawings

Fig. 1 is a powder X-ray diffraction pattern of the crystals of compound (I) obtained in example 1. The abscissa represents the diffraction angle (2 θ), and the ordinate represents the peak intensity.

Fig. 2 is a powder X-ray diffraction pattern of form a crystals of the monohydrochloride salt of compound (I) obtained in example 2. The abscissa represents the diffraction angle (2 θ), and the ordinate represents the peak intensity.

Fig. 3 is a powder X-ray diffraction pattern of form B crystals of the monohydrochloride salt of compound (I) obtained in example 4. The abscissa represents the diffraction angle (2 θ), and the ordinate represents the peak intensity.

Fig. 4 is a powder X-ray diffraction pattern of form C crystals of the monohydrochloride salt of compound (I) obtained in example 3. The abscissa represents the diffraction angle (2 θ), and the ordinate represents the peak intensity.

Fig. 5 is a powder X-ray diffraction pattern of form D crystals of the monohydrochloride salt of compound (I) obtained in example 5. The abscissa represents the diffraction angle (2 θ), and the ordinate represents the peak intensity.

Fig. 6 is a powder X-ray diffraction pattern of form E crystals of the monohydrochloride salt of compound (I) obtained in example 6. The abscissa represents the diffraction angle (2 θ), and the ordinate represents the peak intensity.

Fig. 7 is a powder X-ray diffraction pattern of form F crystals of the monohydrochloride salt of compound (I) obtained in example 7. The abscissa represents the diffraction angle (2 θ), and the ordinate represents the peak intensity.

FIG. 8 is a powder X-ray diffraction pattern of crystals of Compound (I) monohydrobromide salt obtained in example 8. The abscissa represents the diffraction angle (2 θ), and the ordinate represents the peak intensity.

FIG. 9 is a crystal form A of the monohydrochloride salt of Compound (I) obtained in example 2¹³C solid state NMR spectrum. The abscissa represents chemical shift (δ), and the ordinate represents peak intensity.

FIG. 10 is a B-form crystal of the monohydrochloride salt of Compound (I) obtained in example 4¹³C solid state NMR spectrum. The abscissa represents chemical shift (δ), and the ordinate represents peak intensity.

FIG. 11 is a crystal form C of the monohydrochloride salt of Compound (I) obtained in example 3¹³C solid state NMR spectrum. The abscissa represents chemical shift (δ), and the ordinate represents peak intensity.

FIG. 12 is a graph of thermal analysis TG-DTA of form A crystals of the monohydrochloride salt of Compound (I) obtained in example 2. The abscissa represents temperature, the left ordinate represents weight change of TG, and the right ordinate represents heat flow of DTA.

FIG. 13 is a graph of thermal analysis TG-DTA of form B crystals of the monohydrochloride salt of Compound (I) obtained in example 4. The abscissa represents temperature, the left ordinate represents weight change of TG, and the right ordinate represents heat flow of DTA.

FIG. 14 is a graph of thermal analysis TG-DTA of the C-form crystal of the monohydrochloride salt of Compound (I) obtained in example 3. The abscissa represents temperature, the left ordinate represents weight change of TG, and the right ordinate represents heat flow of DTA.

FIG. 15 is a graph of thermal analysis TG-DTA of the D form crystal of the monohydrochloride salt of Compound (I) obtained in example 5. The abscissa represents temperature, the left ordinate represents weight change of TG, and the right ordinate represents heat flow of DTA.

FIG. 16 is a graph of thermal analysis TG-DTA of the form E crystal of the monohydrochloride salt of Compound (I) obtained in example 6. The abscissa represents temperature, the left ordinate represents weight change of TG, and the right ordinate represents heat flow of DTA.

FIG. 17 is a graph of thermal analysis TG-DTA of form F crystals of the monohydrochloride salt of Compound (I) obtained in example 7. The abscissa represents temperature, the left ordinate represents weight change of TG, and the right ordinate represents heat flow of DTA.

FIG. 18 is a graph of thermal analysis TG-DTA of the crystals of Compound (I) monohydrobromide obtained in example 8. The abscissa represents temperature, the left ordinate represents weight change of TG, and the right ordinate represents heat flow of DTA.

Fig. 19 shows a raman spectrum of form a crystals of the monohydrochloride salt of compound (I) obtained in example 2.

Detailed Description

The salt of the compound (I) of the present invention, crystals thereof, and a production method thereof are described in detail below.

As used herein, "salt" refers to a chemical substance consisting of compound (I) as a basic component, and a certain number of equivalents of acid relative to compound (I).

As used herein, examples of the "salt" include salts of inorganic acids, salts of organic acids, and salts of acidic amino acids, etc., wherein pharmaceutically acceptable salts are preferred.

Examples of the salts of inorganic acids include hydrochloride, hydrobromide, sulfate, nitrate and phosphate; and suitable examples of the salt of an organic acid include salts of organic carboxylic acids (e.g., acetic acid, succinic acid, fumaric acid, maleic acid, tartaric acid, malic acid, citric acid, lactic acid, stearic acid, and benzoic acid) and salts of organic sulfonic acids (e.g., methanesulfonic acid (methanesulfonic acid), ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid); among them, hydrochloric acid, hydrobromic acid, and phosphoric acid are preferable.

Examples of salts of acidic amino acids include aspartate and glutamate.

The salts of the present invention may also be anhydrates or hydrates or solvates. As used herein, hydrate or solvate refers to a solid formed by compound (I) or a salt thereof together with water molecules or solvent molecules, and the solid may also be a crystal: and examples of the solvent of the solvate may include ketone-based solvents such as acetone, 2-butanone, cyclohexanone; ester-based solvents such as methyl acetate and ethyl acetate; ether-based solvents such as 1, 2-dimethoxyethane, t-butyl methyl ether; alcohol-based solvents such as methanol, ethanol, 1-propanol, isopropanol; polar solvents, such as N-methyl-2-pyrrolidone, N-dimethylformamide, dimethylsulfoxide. The amount of the water molecule or solvent molecule of the compound (I) or a salt thereof is not particularly limited, and may be, for example, 1 molecule or 2 molecules.

As used herein, "crystal" refers to a crystal of an anhydrate or hydrate of compound (I) or a salt thereof.

As used herein, preferred examples of the crystals of compound (I) and hydrochloride and hydrobromide salts of compound (I) include:

a crystal of compound (I) having diffraction peaks at diffraction angles (2 θ ± 0.2 °) of 9.0 °, 11.1 ° and 23.6 ° in powder X-ray diffraction;

a crystal of compound (I) having diffraction peaks at diffraction angles (2 θ ± 0.2 °) of 9.0 °, 11.1 °, 14.5 °, 18.1 °, 20.0 °, 21.9 °, 23.6 °, 24.4 °, 24.9 ° and 28.5 ° in powder X-ray diffraction;

form a crystals of the monohydrochloride salt of compound (I) having diffraction peaks at diffraction angles (2 θ ± 0.2 °) of 11.6 °, 20.8 ° and 25.7 ° in powder X-ray diffraction;

form a crystal of the monohydrochloride salt of compound (I) having diffraction peaks at diffraction angles (2 θ ± 0.2 °) of 6.1 °, 7.8 °, 11.6 °, 16.2 °, 19.9 °, 20.8 °, 25.2 °, 25.7 °, 26.9 ° and 29.9 ° in powder X-ray diffraction;

in that¹³Form A crystals of the monohydrochloride salt of compound (I) having peaks at chemical shifts (delta. + -. 0.5ppm) of 164.0ppm, 129.6ppm and 36.5ppm in the C solid state NMR spectrum;

in Raman spectroscopy, at 587cm^-1Has Raman shift peak (+/-2 cm)^-1) Form A crystals of the monohydrochloride salt of compound (I) of (1);

in Raman spectroscopy, at 587cm^-1、1428cm^-1And 1493cm^-1Has Raman shift peak (+/-2 cm)^-1) Form A crystals of the monohydrochloride salt of compound (I) of (1);

in Raman spectroscopy, at 587cm^-1、763cm^-1、1428cm^-1、1493cm^-1And 1688cm^-1Has Raman shift peak (+/-2 cm)^-1) Form A crystals of the monohydrochloride salt of compound (I) of (1);

in Raman spectroscopy, at 409cm^-1、587cm^-1、763cm^-1、976cm^-1、1428cm^-1、1493cm^-1And 1688cm^-1Has Raman shift peak (+/-2 cm)^-1) Form A crystals of the monohydrochloride salt of compound (I) of (1);

form a crystals of the monohydrochloride salt of compound (I) having substantially the spectrum shown in fig. 19 in raman spectroscopic measurement;

form B crystals of the monohydrochloride salt of compound (I) having diffraction peaks at diffraction angles (2 θ. + -. 0.2 ℃) of 9.7 °, 10.1 ° and 17.9 ° in powder X-ray diffraction;

form B crystals of the monohydrochloride salt of compound (I) having diffraction peaks at diffraction angles (2 θ. + -. 0.2 °) of 6.3 °, 9.7 °, 10.1 °, 17.9 °, 19.0 °, 19.4 °, 23.4 °, 26.3 °, 27.3 ° and 32.0 ° in powder X-ray diffraction;

in that¹³Form B crystals of the monohydrochloride salt of compound (I) having peaks at chemical shifts (delta. + -. 0.5ppm) of 160.1ppm, 133.4ppm and 130.7ppm in the C solid state NMR spectrum;

form C crystals of the monohydrochloride salt of compound (I) having diffraction peaks at diffraction angles (2 θ ± 0.2 °) of 6.0 °, 7.7 ° and 16.9 ° in powder X-ray diffraction;

form C crystals of the monohydrochloride salt of compound (I) having diffraction peaks at diffraction angles (2 θ ± 0.2 °) of 6.0 °, 7.7 °, 9.7 °, 11.4 °, 15.8 °, 16.9 °, 18.1 °, 23.2 °, 25.4 ° and 27.6 ° in powder X-ray diffraction;

in that¹³C-type crystals of the monohydrochloride salt of compound (I) having peaks at chemical shifts (delta. + -. 0.5ppm) of 159.6ppm, 127.6ppm and 38.9ppm in a C solid state NMR spectrum;

form D crystals of the monohydrochloride salt of compound (I) having diffraction peaks at diffraction angles (2 θ ± 0.2 °) of 6.6 °, 14.6 ° and 26.4 ° in powder X-ray diffraction;

form D crystals of the monohydrochloride salt of compound (I) having diffraction peaks at diffraction angles (2 θ. + -. 0.2 °) of 6.6 °, 14.6 °, 16.1 °, 20.5 °, 21.0 °, 23.0 °, 24.5 °, 26.4 °, 28.0 ° and 32.5 ° in powder X-ray diffraction;

form E crystals of the monohydrochloride salt of compound (I) having diffraction peaks at diffraction angles (2 θ ± 0.2 °) of 6.4 °, 11.3 ° and 27.3 ° in powder X-ray diffraction;

form E crystals of the monohydrochloride salt of compound (I) having diffraction peaks at diffraction angles (2 θ ± 0.2 °) of 6.4 °, 11.3 °, 15.7 °, 18.0 °, 19.2 °, 22.8 °, 24.6 °, 25.4 °, 26.0 ° and 27.3 ° in powder X-ray diffraction;

form F crystals of the monohydrochloride salt of compound (I) having diffraction peaks at diffraction angles (2 θ ± 0.2 °) of 7.3 °, 9.3 ° and 10.7 ° in powder X-ray diffraction;

form F crystals of the monohydrochloride salt of compound (I) having diffraction peaks at diffraction angles (2 θ ± 0.2 °) of 5.9 °, 7.3 °, 9.3 °, 10.7 °, 13.8 °, 15.6 °, 16.4 °, 18.7 °, 25.1 ° and 26.8 ° in powder X-ray diffraction;

crystals of compound (I) monohydrobromide salt having diffraction peaks at diffraction angles (2 θ ± 0.2 °) of 7.8 °, 24.5 ° and 25.2 ° in powder X-ray diffraction; and

crystals of the monohydrobromide of compound (I) having diffraction peaks at diffraction angles (2 θ ± 0.2 °) of 6.0 °, 7.8 °, 10.0 °, 11.7 °, 17.8 °, 20.8 °, 23.5 °, 24.5 °, 25.2 ° and 27.3 ° in powder X-ray diffraction, and the like.

A diffraction peak in the powder X-ray diffraction,¹³Chemical shift in C solid state NMR spectrum and raman shift peak in raman spectroscopic measurement are unique to each of the crystal of the compound (I), the a-F type crystal of the compound (I) monohydrochloride salt, and the crystal of the compound (I) monohydrobromide salt, and they are characteristic peaks of the crystal.

In general, the diffraction angle (2 θ) in powder X-ray diffraction may contain errors in the range of. + -. 0.2 °, and it is understood that the values of the diffraction angle also include values in the range of about. + -. 0.2 °. Therefore, in some compounds or salts thereof, not only crystals in which the diffraction angles of peaks in powder X-ray diffraction are completely uniform, but also crystals in which the diffraction angles of peaks are uniform within an error of about. + -. 0.2 ℃ are the same and are included in the present invention.

As used herein, for example, "having a diffraction peak at a diffraction angle (2 θ ± 0.2 °) of 9.0 ° means" having a diffraction peak at a diffraction angle (2 θ) of 8.8 ° to 9.2 °, and the same applies to the case of other diffraction angles.

In general, even if the crystal form is the same, the peak intensity or half-peak width of the diffraction angle (2 θ) differs for each measurement of powder X-ray diffraction, depending on different measurement conditions and variations in the size and form of powder crystal particles used as a measurement sample, and does not always exhibit a constant peak intensity or half-peak width. Therefore, in comparison of powder X-ray diffraction patterns, even if there is a difference in peak intensity or half-value width thereof at the same diffraction angle (2 θ), it does not mean that the difference comes from differences in crystal form. Therefore, it means that a crystal (having a powder X-ray diffraction pattern) having such a difference in diffraction peak characteristics with respect to the specific crystal of the present invention has the same crystal form as the crystal of the present invention. In addition, as used herein, "the powder X-ray diffraction pattern having fig. 1" means that all the crystals showing the powder X-ray diffraction pattern shown in fig. 1 are the same crystals as the crystals of the present invention, not only in the case where the powder X-ray diffraction pattern having a characteristic diffraction peak matches the powder X-ray diffraction pattern shown in fig. 1 within an error range of ± 0.2 °, but also in the case of powder X-ray diffraction patterns having different peak intensities or half-peak widths (within an error range of ± 0.2 °, having a characteristic diffraction angle matching the powder X-ray diffraction pattern shown in fig. 1).

As used herein, "chemical shifts (. delta. +. 0.5ppm) of 164.0ppm, 129.6ppm and 36.5 ppm" means "carried out under the usual measurement conditions or under the substantially same conditions as in the present specification¹³C solid-state NMR spectrum measurement showed peaks substantially equivalent to chemical shifts (. delta. +. 0.5ppm) of 164.0ppm, 129.6ppm and 36.5ppm, respectively.

In general, when determining whether or not "substantially equivalent peaks" are present,¹³chemical shift δ in the C solid state NMR spectrum may have an error in the range of ± 0.5ppm, and it is understood that the above-mentioned chemical shift value also includes values in the range of ± 0.5ppm or so. Therefore, not only do¹³Crystals whose chemical shifts are completely consistent in the C solid state NMR spectrum are included in the present invention, and crystals whose chemical shifts are consistent within an error of about. + -. 0.5ppm are also included in the present invention. Thus, as used herein, for example, "having a peak at a chemical shift (δ ± 0.5ppm) of 164.0 ppm" means having a peak in the range of chemical shifts (δ)163.5ppm to 164.5ppm, and applies equally to¹³Chemical shift in C solid state NMR spectrum.

Generally, the Raman shift peak (cm) in Raman spectroscopy^-1) May be within + -2 cm^-1Contains errors, it is understood that the peak values are also included in the range of about. + -. 2cm^-1A numerical value within the range of (1). Therefore, in some compounds or salts thereof, not only crystals having raman shift peaks completely identical in raman spectroscopic measurement, but also raman shift peaks at about ± 2cm^-1The same applies to consistent crystals within the error of (1), and is included in the present invention.

As used herein, for example, "in Raman Spectroscopy, at 587cm^-1Has Raman shift peak (+/-2 cm)^-1) "means" in Raman spectroscopy, at 585cm^-1～589cm^-1With Raman shift peak ", other Raman shift cases are also the sameThe same is true.

In general, even if the crystal form is the same, the peak intensity or half-peak width of raman shift in raman spectroscopic measurement differs for each measurement depending on different measurement conditions and variations in the size and form of the powder crystal particles used as a measurement sample, and does not always exhibit a constant peak intensity or half-peak width. Therefore, in comparison of Raman spectroscopic measurements, even at the same Raman shift peak (cm)^-1) There is a difference in their peak intensities or half widths, which does not mean that the difference is from the difference in crystal form. Therefore, this means that a crystal (having a raman spectrum) having such a difference with respect to the raman shift peak characteristics of some of the crystals of the present invention has the same crystal form as the crystal of the present invention. Further, as used herein, "in raman spectroscopic measurement, with the spectrum of fig. 19" means that all of the crystals showing the raman spectrum shown in fig. 19 are the same crystals as those of the present invention, not only in having characteristic raman shift peaks (cm)^-1) In the Raman spectrum of (A) and the Raman spectrum shown in FIG. 19 are within. + -. 2cm^-1But also in the case of raman spectra with different peak intensities or half-value widths (although with a peak intensity of ± 2 cm)^-1Characteristic raman shift peaks that match within the error range of (a).

Hereinafter, a method for producing a salt, crystal or the like of the compound (I) as one embodiment of the present invention will be described.

Process for producing Compound (I)

Compound (I) may also be a compound produced by methods well known to those skilled in the art. For example, the compound (I) can be synthesized by the methods described in the reference examples described below.

Process for producing salt of compound (I)

The salt of compound (I) according to the present invention can be obtained by a method generally used for producing salts. Specifically, a salt can be produced by, for example, suspending or dissolving the compound (I) in a solvent while heating as needed, then adding an acid to the resulting suspension or solution, and then stirring or allowing the mixture to stand at room temperature, or cooling for several minutes to several days. By using these production methods, the salt of compound (I) can be obtained in a crystalline or amorphous form. Further, according to need, in addition to these production methods, an amorphous body can be obtained by performing an operation such as freeze drying. Examples of the solvent used herein include alcohol-based agents such as ethanol, 1-propanol, isopropanol; acetonitrile; ketone-based solvents such as acetone and 2-butanone; ester-based solvents such as ethyl acetate; saturated hydrocarbon-based solvents such as hexane and heptane; ether-based solvents such as t-butyl methyl ether and water. These solvents may be used alone or in combination of two or more.

Process for producing crystals of Compound (I) or salt thereof

The crystals of compound (I) or a salt thereof can be prepared by the above-mentioned method for producing compound (I), or a method for preparing a salt thereof, or can also be prepared by dissolving compound (I) or a salt thereof in a solvent by heating and crystallizing it by cooling under stirring.

The compound (I) or a salt thereof used for crystallization may be in any form, i.e., may be a solvate or hydrate or an anhydrate, may be in an amorphous or crystalline form (including one composed of multiple polymorphs), or a mixture thereof.

Examples of the solvent used in the crystallization include alcohol-based solvents such as methanol, ethanol, isopropanol, and 1-propanol; acetonitrile; amide-based solvents such as N, N-dimethylformamide; ester-based solvents such as ethyl acetate; saturated hydrocarbon-based solvents such as hexane and heptane; ketone-based solvents such as acetone and 2-butanone; ether-based solvents such as t-butyl methyl ether and water. These solvents may be used alone or in combination of two or more.

The amount of the solvent to be used may be appropriately selected, provided that the amount capable of dissolving the compound (I) or a salt thereof by heating or the amount capable of stirring the suspension is a lower limit and the yield of crystals is not significantly reduced is an upper limit.

In the crystallization, a seed crystal (for example, a crystal of the desired compound (I) or a salt thereof) may be added or not added. The temperature at which the seed crystal is added is not particularly limited, and is preferably 0 ℃ to 80 ℃.

The temperature at which the compound (I) or a salt thereof is heated and dissolved may be appropriately selected depending on the solvent so that the compound (I) or a salt thereof can be dissolved at the temperature, but the temperature is preferably in the range of 50 ℃ to the temperature at which the reflux of the crystalline solvent is started, more preferably 55 ℃ to 80 ℃.

Since the quenching causes crystals of different forms (polymorphs), cooling at the time of crystallization should be performed by appropriately controlling a cooling rate, for example, 5 to 40 c/hr, in consideration of influence on the quality and grade of crystals. More preferably, the cooling is performed at a cooling rate of, for example, 5 to 25 ℃/hour.

The final crystallization temperature may be appropriately selected according to the yield, quality, etc. of the crystals, and it is preferably-25 ℃ to 30 ℃.

The crystals obtained by crystallization can be isolated by ordinary filtration procedures, and the filtered crystals are washed with a solvent if necessary, and then dried to obtain the objective crystals. As the solvent for washing the crystals, those similar to those used for crystallization can be used. Preferably, examples thereof include ethanol, acetone, 2-butanone, ethyl acetate, diethyl ether, tert-butyl methyl ether and hexane. These solvents may be used alone or in combination of two or more.

The crystals separated by the filtration procedure can be dried by suitably placing under the atmosphere or under a stream of nitrogen, or by heating.

The drying time may be appropriately selected depending on the production amount, the drying apparatus, the drying temperature, and the like until the amount of the residual solvent is less than a specific amount. Drying can also be carried out under ventilation or under reduced pressure. The degree of the pressure reduction can be appropriately selected depending on the production amount, the drying apparatus, the drying temperature, and the like. After drying, the crystals obtained can also be put in the atmosphere as required.

The crystals of compound (I) and the salt of compound (I) obtained by the production method described above have a cholinergic neuron-activating effect and/or a neuroprotective effect (as shown by activity data in pharmacological test examples described below), and are likely to be used as an agent for improving cognitive function deterioration caused by dysfunction of cholinergic neurons.

[ pharmaceutical composition ]

Another embodiment of the present invention is a pharmaceutical composition comprising a crystal of compound (I) and a pharmaceutically acceptable additive. The pharmaceutical composition can be produced by mixing pharmaceutically acceptable additives with the crystals of compound (I). The pharmaceutical composition according to the present invention can be produced by a known method, for example, a method described in General Rules for Preparations of Japanese Pharmacopoeia, Seventeenth Edition.

The pharmaceutical composition according to this embodiment may be appropriately administered to a patient depending on its dosage form.

The dose of compound (I) according to the present invention varies depending on the severity of symptoms, age, sex, body weight, dosage form, type of salt, specific type of disease and other conditions; however, in general, the daily oral dose for an adult is about 30 μ g to 10g, preferably 100 μ g to 5g, and more preferably 100 μ g to 1 g; the dose administered by injection per day for an adult is about 30 μ g to 1g, preferably 100 μ g to 500mg, and more preferably 100 μ g to 300 mg; and the above dose is administered one or more times.

Examples of the invention

The crystals of the compound (I) of the present invention can be produced by, for example, the methods described in the following examples, and the effects of the compound can be confirmed by the methods described in the following test examples. However, these are merely examples, and the present invention is not limited to the following specific examples in any way and can be modified within a range not departing from the scope of the present invention.

Powder X-ray crystal diffraction of the crystals produced in the following examples the obtained crystals were loaded on a sample stage of a powder X-ray diffraction apparatus, and measurement was performed under any of the following conditions.

(conditions of Transmission)

X line source: CuKa

Voltage: 45kV

Current: 40mA

An optical system: focusing mirror

A soller slit: 0.02 degree

A detector: x' Celerator (semiconductor detector)

Scanning range: 5 to 35 DEG

Step length: 0.017 degree

Scanning step time: 600sec

A sample holder: kapton film

(reflection Condition)

X line source: CuKa

Voltage: 50kV

Current: 300mA

Slit: 0.5mm divergence slit, scattering slit opening, receiving slit opening

A detector: scintillation counter

Scanning speed: 5 °/min

Sampling interval: 0.02 degree

Scanning range: 5 to 35 DEG

A sample holder: aluminum retainer

The samples were precisely weighed in an aluminum sample pan and subjected to thermal analysis under the following conditions.

(measurement conditions)

Atmosphere: under nitrogen flow (100mL/min)

Comparison: empty aluminium sample dish

The heating rate is as follows: 10 ℃/min

Sampling interval: 1sec

Measurement temperature range: room temperature to 320 DEG C

The measurement of crystals was carried out by enclosing about 300mg of a solid sample in a sample tube under the following conditions¹³C solid state NMR spectrum.

(measurement conditions)

The apparatus used was: avance 400MHz (manufactured by Bruker) 7mm-CPMAS Probe (manufactured by Bruker)

Nuclear measurement:¹³c (resonance frequency 100.6248425MHz)

Measuring the temperature: at room temperature

Pulse mode: CPTOSS measurement

Rotating speed: 5000Hz

Pulse repetition time: 3sec

Contact time: 1msec

And (4) accumulating times: 5120 times

Reference materials: glycine (external reference: 176.03ppm)

The raman spectrum of the crystal was measured by placing the sample on the sample stage of a raman microspectrometer under the following measurement conditions.

(measurement conditions)

The apparatus used was: RENISHAW Raman microscope inVia Reflex

Laser wavelength: 785nm

Diffraction grating: 1200 lines/mm

An objective lens: 50 times of

Scanning mode: in succession

Exposure time 5sec

And (4) accumulating times: 5 times (twice)

Measurement range: 400-1800cm^-1(Raman shift)

Error: plus or minus 2cm^-1

The compounds described by the literature names and the like are indicated to be produced according to these literatures and the like.

Further, the abbreviations used in this specification are well known and common to those skilled in the art. In the present specification, the following abbreviations are used.

DMSO, DMSO: dimethyl sulfoxide

IPA: isopropanol (I-propanol)

n-: is just

TEA: triethylamine

THF: tetrahydrofuran (THF)

¹H-NMR: proton nuclear magnetic resonance spectroscopy

MS: mass spectrometry

The term "room temperature" in the following examples and production examples generally means from about 10 ℃ to about 35 ℃. Unless otherwise indicated,% means weight percent.

Chemical shifts in proton nmr spectra are expressed in δ units (ppm) relative to tetramethylsilane, and coupling constants are reported in hertz (Hz). Split form dial is as follows:

s: singlet, d: doublet, t: triplet, q: quartet, m: multiple seams, br.s: a wide single peak.

In the reaction using the microwave reactor of the reference example, Initiator (TM) or Initiator + (TM) manufactured by the company of betaizil was used.

For the chromatography, as the silica gel, silica gel 60(70-230 mesh ASTM) produced by Merck corporation (Merck) or PSQ60B produced by Fuji Silysia Chemical Ltd) was used, or a pre-packed column { column: hi-flash (tm) column (silica gel) produced by YAMAZEN corporation, size: one of S (16x60mm), M (20x75mm), L (26x100mm), 2L (26x150mm), and 3L (46x130 mm); or using a Biotage (TM) SNAP Ultra Silica cartridge manufactured by the Bytaizil company, size: one of 10g, 25g, and 50g }.

As the NH silica gel, CHROMATOREX NH-DM2035 produced by Fuji Silysia Chemical Ltd, or a pre-packed column { column: hi-flash (TM) column (Amino) from Yamazen, Inc., size: one of S (16x60mm), M (20x75mm), L (26x100mm), 2L (26x150mm), and 3L (46x130 mm); or Presep (TM) (luer Lock) NH2(HC) manufactured by Wako Pure Chemical Industries, Ltd, size: one of M type (14g/25mL), L type (34g/70mL), 2L type (50g/100mL), and 3L type (110g/200 mL).

As neutral alumina, alumina 90 neutral active, 70-230 mesh, Merck, E6NXX, was used.

As the names of the compounds shown below, those shown in "E-Notebook [ E-handbook ]" 12 th edition (PerkinElmer)) were used in addition to the commonly used reagents.

Reference example 1

5, 10-dimethyl-5, 6,9,10,11, 12-hexahydropyrido [ 4]",3":4',5']Thieno [2',3':4,5]Pyrimidine as one kind of food Pyrido [1,2-a ]]Thieno [3,2-f][1,4]Diaza derivatives Synthesis of (E) -4, 13-dione (hereinafter referred to as Compound (I))

(1) Ethyl 2-amino-6-methyl-4, 5,6, 7-tetrahydrothieno [2,3-c ]]Synthesis of pyridine-3-carboxylic acid esters

TEA (61.6mL, 442mmol) was added to a mixture of 1-methyl-4-piperidone (CAS No. 1445-73-4) (51.5mL, 442mmol), ethyl cyanoacetate (CAS No. 105-56-6) (47.2mL, 442mmol), sulfur (CAS No.7704-34-9) (14.2g, 442mmol) and ethanol (800mL) at room temperature. The reaction mixture was stirred at 40 ℃ for 15 hours, and then concentrated under reduced pressure. The residue was purified by column chromatography (NH silica gel, ethyl acetate). The concentrated residue obtained was triturated with ethyl acetate. The precipitate was collected by filtration, washed with ethyl acetate, and dried under reduced pressure to give the title compound (58.4 g).

¹H-NMR(400MHz,CDCl₃)δ(ppm):1.33(t,J＝7.0Hz,3H),2.44(s,3H),2.62-2.70(m,2H),2.79-2.88(m,2H),3.37(t,J＝2.0Hz,2H),4.26(q,J＝7.3Hz,2H),5.97(br.s,2H)。

MS(ESI)m/z：241[M+H]⁺

(2) 4-methyl-3, 4-dihydro-1H-thieno [2,3-e][1,4]Diaza derivatives Synthesis of (E) -2, 5-diketones

1H,2H, 4H-thieno [2,3-d ] [1,3] oxazine-2, 4-dione (CAS number 103979-54-0) (600mg, 3.55mmol) was added to a solution of sarcosine (790mg, 8.87mmol) in water (12 mL). The reaction mixture was heated at reflux for 1.5 hours. The reaction mixture was cooled to room temperature. Chloroform was added to the reaction mixture, and the organic layer was separated. The aqueous layer was extracted with chloroform (twice) and ethyl acetate (3 times). The combined organic layers were dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The obtained solid was dried to give the title compound (430 mg).

¹H-NMR(400MHz,CDCl₃)δ(ppm)：3.23(s,3H),3.99(s,2H),6.90(d,J＝5.9Hz,1H),7.29(d,J＝5.7Hz,1H),8.39(br.s,1H)。

MS(ESI)m/z：197[M+H]⁺

(3) Synthesis of Compound (I)

Phosphorus oxychloride (1.43mL, 15.3mmol) was added to the 4-methyl-3, 4-dihydro-1H-thieno [2,3-e ] obtained in step (2) at room temperature][1,4]Diaza derivatives-2, 5-dione (1.00g, 5.10mmol), ethyl 2-amino-6-methyl-4, 5,6, 7-tetrahydrothieno [2, 3-c) obtained in step (1)]Pyridine-3-carboxylate (1.84g, 7.64mmol), and 1, 4-dioxane (30 mL). The reaction mixture was stirred at room temperature for 5 minutes and at 90 ℃ for 2 hours. Sodium ethoxide (20% solution in ethanol, 21.7mL, 56.1mmol) was added to the reaction mixture cooled to room temperature over 5 minutes. The reaction mixture was stirred at room temperature for 1.5 hours. Ethyl acetate, a saturated aqueous sodium bicarbonate solution, and water were sequentially added to the reaction mixture, and the organic layer was separated. The aqueous layer was extracted with ethyl acetate. The combined organic layers were dried over anhydrous magnesium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 20% -50% methanol/ethyl acetate). The obtained solid was triturated with ethanol and the precipitate was collected by filtration. The obtained solid was washed with ethanol and dried under reduced pressure to give the title compound (712 mg).

¹H-NMR(400MHz,CDCl₃)δ(ppm)：2.52(s,3H),2.71-2.87(m,2H),3.05-3.30(m,5H),3.59-3.75(m,2H),4.23(d,J＝14.8Hz,1H),4.57(d,J＝14.8Hz,1H),7.35(d,J＝6.2Hz,1H),7.39(d,J＝5.9Hz,1H)。

MS(ESI)m/z：373[M+H]⁺

Example 1

Preparation of crystals of Compound (I)

To 1.5L of 0.3M hydrochloric acid was added 152.08g of compound (I), and to the solution was added 450ml of ethyl acetate, followed by stirring for 5 minutes. The aqueous layer was separated and washed with 450ml of ethyl acetate, and insoluble matter was removed by filtration. To the filtrate was added 100ml of a 1N aqueous solution of sodium hydroxide in a water bath at 20 ℃ and the mixture was stirred for 15 minutes. To the mixture was added 350ml of a 1N aqueous sodium hydroxide solution, and the resulting suspension was stirred for 2 hours 30 minutes. The resulting crystals were collected by filtration, washed with 300ml, 450ml, 300ml of water, 300ml, 350ml and 300ml of ethanol in this order, and dried under reduced pressure to obtain 141.7g of the titled crystals.

Powder X-ray diffraction peak (reflection method, 2 θ ± 0.2 °): 9.0 °, 11.1 °, 14.5 °, 18.1 °, 20.0 °, 21.9 °, 23.6 °, 24.4 °, 24.9 °, 28.5 °

A powder X-ray diffraction pattern of the crystal of the compound (I) obtained by the above-described method is shown in fig. 1.

Example 2

Preparation of form A crystals of Compound (I) monohydrochloride

To a screw tube was added 101mg of compound (I). 0.2mL of 1.5M hydrochloric acid was added thereto and dissolved. IPA (1.8 mL) was added, and the mixture was irradiated with ultrasonic waves and then stirred at 40 ℃ for one day with a stirrer. After further stirring at room temperature for 1 hour, the sample was collected by filtration using a filter (0.2 μm), rinsed with 0.5mL of IPA/water (9/1, v/v), and air-dried under a stream of nitrogen. The residue was dried at 70 ℃ for about 1 hour to give the title crystals (103 mg).

Powder X-ray diffraction peak (transmission method, 2 θ ± 0.2 °): 6.1 °, 7.8 °, 11.6 °, 16.2 °, 19.9 °, 20.8 °, 25.2 °, 25.7 °, 26.9 °, 29.9 °

¹³C-NMR (100MHz, solid state) delta (ppm) 164.0,162.5,160.5,153.9,151.6,150.7,133.6,131.1,129.6,128.4,126.9,125.2,123.7,121.3,120.3,119.5,53.7,52.0,50.9,44.7,36.5,22.6

Raman shift peak (cm)^-1): 409. 587, 763, 976, 1428, 1493, 1688 the powder X-ray diffraction pattern of the a-form crystal of the monohydrochloride of compound (I) obtained by the above method is shown in fig. 2, and¹³the C-solid state NMR spectrum is shown in FIG. 9, the thermal analysis TG-DTA is shown in FIG. 12, and the Raman spectrum is shown in FIG. 19.

Example 3

Preparation of form C crystals of Compound (I) monohydrochloride

1020mg of compound (I) are added to a screw tube. 1.5 equivalents of hydrochloric acid (353 μ L) were dissolved in 20mL of methanol and added to the sample. The sample was stirred with a stirrer at room temperature for 2 days. Samples were collected by filtration through a filter (0.2 μm). After drying the obtained solid under reduced pressure for about 2 hours, it was then dried at 70 ℃ for 1 hour to give the title crystals (1048 mg).

Powder X-ray diffraction peak (transmission method, 2 θ ± 0.2 °): 6.0 °, 7.7 °, 9.7 °, 11.4 °, 15.8 °, 16.9 °, 18.1 °, 23.2 °, 25.4 °, 27.6 °

¹³C-NMR (100MHz, solid state) delta (ppm) 162.5,160.5,159.6,153.8,151.1,134.1,131.6,128.4,127.6,125.6,120.0,54.0,52.6,50.9,44.3,43.5,38.9,32.3,22.4

The powder X-ray diffraction pattern of the C-form crystal of the monohydrochloride of Compound (I) obtained by the above-mentioned method is shown in FIG. 4, in which¹³The C-solid state NMR spectrum is shown in FIG. 11, and the thermal analysis TG-DTA is shown in FIG. 14.

Example 4

Preparation of form B crystals of Compound (I) monohydrochloride

The hydrochloride crystal 303mg obtained in example 3 was added to a platinum crucible, and heated at 160 ℃ for 15 minutes to give the title crystal (293 mg).

Powder X-ray diffraction peak (transmission method, 2 θ ± 0.2 °): 6.3 °, 9.7 °, 10.1 °, 17.9 °, 19.0 °, 19.4 °, 23.4 °, 26.3 °, 27.3 °, 32.0 °

¹³162.0,160.1,153.8,151.1,133 in C-NMR (100MHz, solid state) delta (ppm).4,130.7,128.3,126.9,125.6,120.3,51.2,43.6,32.3,22.3

The powder X-ray diffraction pattern of the B-form crystal of the monohydrochloride of Compound (I) obtained by the above-mentioned method is shown in FIG. 3, in which¹³The C-solid state NMR spectrum is shown in FIG. 10, and the thermal analysis TG-DTA is shown in FIG. 13.

Example 5

Preparation of form D crystals of Compound (I) monohydrochloride

227mg of the mixture of hydrochloride crystals obtained in examples 2 and 3, respectively, and 8mL of ethanol were added to a screw test tube. The mixture was stirred with a stirrer at 65 ℃. After about 1 hour, the mixture was irradiated with ultrasonic waves and stirred at the same temperature for one day. Samples were collected by filtration through a filter (0.2 μm) to give the title crystals (203 mg).

Powder X-ray diffraction peak (transmission method, 2 θ ± 0.2 °): 6.6 °, 14.6 °, 16.1 °, 20.5 °, 21.0 °, 23.0 °, 24.5 °, 26.4 °, 28.0 °, 32.5 °

A powder X-ray diffraction pattern of the D-form crystal of the monohydrochloride salt of compound (I) obtained by the above method is shown in fig. 5, and a thermal analysis TG-DTA pattern is shown in fig. 15.

Example 6

Preparation of form E crystals of the monohydrochloride salt of Compound (I)

108mg of the hydrochloride crystals obtained in example 2 and 5mL of acetonitrile were added to a screw test tube. The mixture was stirred with a stirrer at 60 ℃ for one day, and the sample was collected by filtration through a filter (0.2 μm). The obtained solid and 5mL of acetonitrile were again added to a screw test tube and stirred with a stirrer at 60 ℃ for one day. The sample was collected by filtration through a filter (0.2 μm) under a nitrogen stream to give the title crystals (89.7 mg).

Powder X-ray diffraction peak (transmission method, 2 θ ± 0.2 °): 6.4 °, 11.3 °, 15.7 °, 18.0 °, 19.2 °, 22.8 °, 24.6 °, 25.4 °, 26.0 °, 27.3 °

A powder X-ray diffraction pattern of the E-type crystal of the monohydrochloride salt of compound (I) obtained by the above-described method is shown in fig. 6, and a thermal analysis TG-DTA is shown in fig. 16.

Example 7

Preparation of form F crystals of the monohydrochloride salt of Compound (I)

101mg of the hydrochloride crystals obtained in example 2 and 5mL of ethanol were added to a screw-top test tube. The mixture was stirred with a stirrer at 60 ℃ for one day. Samples were collected by filtration through a filter (0.2 μm). The solid obtained and 5mL of ethanol were again added to a screw test tube and stirred with a stirrer at 60 ℃ for 4 hours. Samples were collected by filtration through a filter (0.2 μm) to give the title crystals (75.0 mg).

Powder X-ray diffraction peak (transmission method, 2 θ ± 0.2 °): 5.9 °, 7.3 °, 9.3 °, 10.7 °, 13.8 °, 15.6 °, 16.4 °, 18.7 °, 25.1 °, 26.8 °

A powder X-ray diffraction pattern of the F-form crystal of the monohydrochloride salt of compound (I) obtained by the above-described method is shown in fig. 7, and a thermal analysis TG-DTA is shown in fig. 17.

Example 8

Preparation of crystals of Compound (I) monohydrobromide

To a screw tube, 933mg of compound (I) was added. 1.5 equivalents (434. mu.L) of hydrobromic acid were dissolved in 20mL of methanol and added to the sample. The mixture was stirred with a stirrer at room temperature for 3 days. The sample was collected by filtration through a filter (0.2 μm) and dried at 60 ℃ for 1 hour to give the title crystals (1111 mg).

Powder X-ray diffraction peak (transmission method, 2 θ ± 0.2 °): 6.0 °, 7.8 °, 10.0 °, 11.7 °, 17.8 °, 20.8 °, 23.5 °, 24.5 °, 25.2 °, 27.3 °

A powder X-ray diffraction pattern of the compound (I) monohydrobromide salt obtained by the above-described method is shown in fig. 8, and a thermal analysis TG-DTA is shown in fig. 18.

< pharmacological test examples >

Measurement of acetylcholine (ACh) Release in rat Primary septal neuron culture systems in the Presence of NGF

(1) Rat primary compartment neuron culture

Compartments were isolated from 18-day old Sprague-Dawley (Sprague-Dawley, SD) rats (Charles River Laboratories Japan, Inc.) and cultured. Specifically, under isoflurane anesthesia, fetuses were aseptically removed from pregnant rats. Brains were extracted from each foetus and immersed in ice-cooled L-15 medium (11415-. Septal regions were dissected from the extracted brains under a stereomicroscope. The dissected compartment was subjected to enzymatic treatment in an enzyme solution containing 0.25% trypsin (15050-. In this case, the enzyme reaction was terminated by the addition of inactivated horse serum (26050-. The enzyme-treated solution was centrifuged at 1000rpm for 3 minutes, and the supernatant was removed. An amount of 10mL of the culture medium was added to the obtained cell pellet. The media used were Du's modified eagle's medium (044-. The cells of the cell pellet to which the medium was added were redispersed by gentle pipetting, and then centrifuged again at 1000rpm for 3 minutes, and the supernatant was removed. An amount of 10mL of the culture medium was added to the obtained cell mass, and the cell dispersion was filtered through a 40- μm nylon mesh (cell filtration net) to remove the cell mass, thereby obtaining a neuronal cell suspension. Neuronal cell suspensions were diluted in culture medium and 10% inactivated bovine serum (26140-079, zemer feishell scientific) and 10% inactivated horse serum were added. Thereafter, 100. mu.L/well of the suspension was seeded in a 96-well plate (354461, CORNING Corning) precoated with poly-D-lysine to an initial culture density of 1.4X10⁵Individual cell/cm². The inoculated cells were incubated at 37 ℃ in an incubator with 5% CO₂After 2 days of culture in 95% air, 120. mu.L of fresh medium was used instead of the wholeMedium, and then cells were cultured for 5 days.

(2) Addition of Compounds

On day 7 of culture, compounds were added in the following manner. The solution of test compound in DMSO was diluted with the culture medium so that the concentration was 10-fold higher than the final concentration. NGF was prepared at 0.3ng/mL (450-01, PeProteck, INC.). Each of the two solutions was added in an amount of 15 μ L/well, and the mixture was mixed well. The final DMSO concentration was 0.1% or less. In addition, only DMSO and NGF were added to the control group.

(3) ACh Release measurement

One day after compound addition, ACh release was measured by HPLC in the following manner. After the medium was eliminated, warm buffer was added to the wells at 100 μ L/well, and the buffer was removed immediately. Thereafter, 120. mu.L/well of buffer supplemented with 10. mu.M choline, 10. mu.M physostigmine, and 6mM KCl was added. The buffer was prepared by adding 125mM NaCl, 25mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid, 1.2mM KH₂PO₄、1.2mM MgSO₄、2.2mM CaCl₂(2H₂O) and 10mM glucose to sterile water, and the final pH of the solution was set to 7.4. The buffer-supplemented 96-well plates were incubated at 37 ℃ in 5% CO₂After 40 min incubation at-95% air, 80. mu.L of buffer was collected. The internal standard solution IPHC (5X 10)^-7M) was added to the collected buffer in an amount of 6 μ L, and the buffer was transferred to a tube for HPLC measurement and HPLC measurement was performed. The results were expressed as the percentage of ACh concentration in the control buffer (% of control) by the effect of each compound, and the concentration of the compound of reference example 1 showing an increase of 20% from ACh concentration in the control buffer was 0.1 μ M.

Measurement of Choline acetyltransferase (ChAT) mRNA expression levels in rat compartments

(1) Compound administration

In this study, SD male rats (Charles River Laboratories Japan, Inc.) weighing about 250g to 350g were used. The compound was dissolved in 0.01mol/L hydrochloric acid and administered orally.

(2) Sampling

Whole brain tissue was collected under pentobarbital anesthesia 24 hours after compound administration. The medial septal nuclei were isolated from whole brain on ice and frozen with liquid nitrogen, and then stored at-80 ℃.

(3) Measurement of ChAT mRNA expression levels

For RNA purification, RNeasy Plus mini kit (# 74136: QIAGEN) was used in this study. RNA purification was performed by the method described in the kit. After RNA purification, total RNA concentration was measured by using a QIAxpert instrument (qiagen). cDNA was synthesized using SuperScript (R) VILO (TM) cDNA synthesis kit (# 11754: Seimer Feishol science). The synthesis of cDNA was performed by the method described in the kit. The synthesized cDNA was diluted 4-fold with RNase-free water, and the diluted cDNA solution was used as a sample. Taqman Universal PCR premix (# 4304437: Saimer Fielder technologies), Taqman (R) gene expression assay, INVENTRIED (# 4331182: Saimer Fielder technologies), RNase-free water, and the cDNA solutions were each mixed in amounts of 10. mu.l, 1. mu.l, 4. mu.l, and 5. mu.l, and the resulting mixture was used as a measurement sample solution. Quantitative polymerase chain reaction (qPCR) was performed by the fluorescent probe method using ABI PRISM (R)7900HT (seimer feishell scientific). Analysis was performed by SDS 2.4 (Saimer Feishell science). The result was calculated as 56.4% at 10mg/kg by percentage of the amount of ChAT mRNA expression level in the compound administration group of reference example 1 increased from the amount of ChAT mRNA expression level in the vehicle administration group.

Measurement of acetylcholine (ACh) in rat cerebrospinal fluid (CSF)

(1) Background of the invention

The correlation between increases and decreases in neurotransmitters in the brain and increases and decreases in neurotransmitters in cerebrospinal fluid (CSF) was revealed by studies in rodents, and was also observed in humans (Lowe S et al Psychopharmacology 219(2012) 959-. Thus, changes in acetylcholine in the CSF are measured to determine changes in acetylcholine in the brain caused by the test compound.

(2) Compound administration

In this study, Fischer344 male rats (Charles River Laboratories Japan, Inc.) weighing about 150g to 250g were used. The test compound was orally administered to rats at 10mg/kg once daily for three days. The carrier used was 0.01mol/L hydrochloric acid.

(3) Sampling

CSF was collected from the cisterna magna in tubes containing AchE inhibitors 24 hours after administration of vehicle and test compound under pentobarbital anesthesia. The collected CSF was centrifuged at 3500x g at 4 ℃ for 10 minutes, and the supernatant was collected. The collected supernatant was frozen with liquid nitrogen and then stored at-80 ℃.

(4) Ach measurement by LC-MS

To 10. mu.L of CSF was added 50. mu.L of acetylcholine-d 9 chloride (ACh-d9) at a final concentration of 0.34nmol/L as an internal standard. The mixture was pipetted and centrifuged at 1500x g for 10 min at 4 ℃. The supernatants were collected and subjected to LC/MS (NexeraX2(MS), TSQ Altis (HPLC)), and Ach was detected as a precursor ion at m/z 146.050 and as a product ion at m/z 87.071, and ACh-d9 was detected as a precursor ion at m/z155.088 and as a product ion at m/z 87.000 as an internal standard. The result was calculated to be 156.8% by calculating the percentage (% control) of the ACh concentration in CSF in the compound-administered group of reference example 1 increased in the CSF compared to the vehicle-administered group.

Evaluation of human tau P301S transgenic mice

(1) Compound administration

In this study, human τ P301S transgenic mice were orally administered with test compounds once daily for three months from four months to seven months of age. The carrier used was 0.01mol/L hydrochloric acid.

(2) Sampling

On the first day of administration (four months of age) and the second day of the last administration, mice of the vehicle administration group and the test compound administration group were anesthetized under pentobarbital (50mg/kg, intraperitoneal administration) and perfused with PBS. After perfusion, forebrains containing the medial compartment were collected and fixed with 4% paraformaldehyde.

(3) Preparation of frozen brain-coronal sections

The collected forebrains containing the medial compartment were immersed in 4% paraformaldehyde and shaken overnight. The infusion solution was replaced with a 7.5% sucrose solution. It was immersed in a 7.5% sucrose solution and shaken overnight, and the immersion solution was replaced with a 15% sucrose solution, and it was immersed and shaken overnight. The infusion solution was replaced with a 30% sucrose solution and immersed and shaken overnight. Coronal frozen sections of the brain with a thickness of 30 μm were prepared from the forebrain containing the medial compartment by using a microtome (Leica, SM 2000R).

(4) Immunohistochemistry for Choline acetyltransferase (ChAT) Positive cells

Prepared brain coronary cryosections were stained with DAB (DAB peroxidase substrate kit (Vector, SK-4100)) using ChAT antibody (Santa Cruz, SC-20672) as the primary antibody. Images of sections containing The medial compartment, such as "The mouse Brain in stereotaxic coordinates", were taken by means of an integrated fluorescence microscope (Keyence, BZ-X710)]"(COMPACT THIRD EDITION THIRD EDITION of contract)],Keith B.J.Franklin&George Paxinos) and ChAT positive cells around the long axis of the medial compartment were counted by BZ analysis software (keyence corporation). Results are shown as the percentage of the number of ChAT positive cells in vehicle administration group and test compound administration group relative to the number of ChAT positive cells at the time of initial administration (four months of age). Data are presented as mean ± SEM. Analysis of initial application by unpaired t-testAnd the differences between the vehicle-treated group and the compound-treated group were also analyzed by unpaired t-test (significance:^#)。P<a value of 0.05 is considered statistically significant. Statistical analysis was performed using GraphPad Prism version 7.02. The results are shown in Table 1.

[ Table 1]

Neuroprotective and restorative effects of cholinergic neurons using a rat model of fornix hippocampal umbrella injury

(1) Preparation of rat model of fornix hippocampal umbrella injury

In this study, male Sprague-Dawley rats (Charles River Laboratories Japan, Inc.) weighing about 250g to 350g were used. In three drugs: rats were anesthetized with a combination of midazolam (2mg/kg, subcutaneously), medetomidine hydrochloride (0.15mg/kg, subcutaneously), and butorphanol tartrate (2.5mg/kg, subcutaneously) and fixed with a brain stereotactic apparatus (naishige co., Ltd.). The skull was exposed and a 5mm wide hole was drilled in the skull from the midline 2mm posterior to bregma. A razor having a width of 4mm was inserted into bregma at a depth of 5.5mm to cut fornix the hippocampal umbrella. After hemostasis, the scalp was sutured. After surgery, the rats were brought back into cages and allowed to recover from anesthesia. In the sham group, a 5mm wide hole was drilled in the skull from the midline 2mm posterior to bregma, but without penetrating the razor.

(2) Compound administration

The test compounds were orally administered to rats once daily for five to nine days after surgery (example 1: 10mg/kg) or seven to fourteen days after surgery (example 3: 3 mg/kg). The carrier used was 0.01mol/L hydrochloric acid. In the sham group, the vehicle was orally administered once a day similarly to the test compound administration group.

(3) Sampling

Rats were anesthetized under pentobarbital and perfused with ice-cold PBS through the heart. After perfusion, the forebrain containing the medial compartment was collected and submerged in 4% paraformaldehyde and shaken overnight. The infusion solution was replaced with a 7.5% sucrose solution. It was immersed in a 7.5% sucrose solution and shaken overnight, and the immersion solution was replaced with a 15% sucrose solution, and it was immersed and shaken overnight. The infusion solution was replaced with a 30% sucrose solution and immersed and shaken overnight. Coronal frozen sections of the brain with a thickness of 30 μm were prepared from the forebrain containing the medial compartment by using a microtome (Leica, SM 2000R).

(4) Immunization of choline acetyltransferase (ChAT) positive cells and vesicular acetylcholine transporter (VAChT) Histochemistry

The prepared brain coronary frozen sections were stained with DAB (DAB peroxidase substrate kit (vkk, SK-4100)) using ChAT antibody (santa cruz, SC-20672) or VAChT antibody (Merck Millipore, ABN100) as primary antibody. Images of sections containing The medial compartment or hippocampus, such as "The mouse Brain in stereotaxic coordinates", were taken by means of an integrated fluorescence microscope (Ginshi, BZ-X710)]"(COMPACT THIRD EDITION THIRD EDITION of contract)],Keith B.J.Franklin&George Paxinos), and the Optical Density (OD) in ChAT positive cells in the medial compartment or hippocampus VAChT was measured by BZ analysis software (kirnshi). The results are shown as the percentage of the number of ChAT positive cells or OD in hippocampus VAChT in the medial compartment in vehicle administration group and test compound administration group relative to the number of ChAT positive cells or OD in hippocampus VAChT in the medial compartment in sham operation group. Data are presented as mean ± SEM. Differences between vehicle-treated and compound-treated groups were analyzed by unpaired t-test (significance:^#)。P<a value of 0.05 is considered statistically significant. Statistical analysis was performed using GraphPad Prism version 7.02. The results are shown in tables 2 and 3.

[ Table 2]

[ Table 3]