RU2820938C2 - Pentacyclic compound salt and crystal thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to monohydrochloride 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4",3":4',5']thieno[2',3':4,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione of formula (I), as well as type A, B, C, D, E, F crystals with diffraction peaks at diffraction angles (2θ ± 0.2°), given in the claim, in powder X-ray diffraction obtained using CuKα as an X-ray source. Invention relates to monohydrobromide 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4",3":4',5']thieno[2',3':4,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione of formula (I), as well as its crystal with diffraction peaks at diffraction angles (2θ ± 0.2°), given in the claim, in powder X-ray diffraction obtained using CuKα as an X-ray source. Invention relates to crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4",3":4',5']thieno[2',3':4,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, represented by formula (I), with diffraction peaks at diffraction angles (2θ ± 0.2°) 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 obtained using CuKα as an X-ray source. Invention also relates to a pharmaceutical composition having an activating effect on cholinergic neurons, containing a monohydrochloride salt or a monohydrobromide of a compound of formula (I) or a crystal of a compound of formula (I) as an active ingredient and pharmaceutically acceptable additives.

EFFECT: compound 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4",3":4',5']thieno[2',3':4,5]pyrimido[1],2-a]thieno[3,2-f][1,4]diazepine-4,13-dione in the form of its monohydrochloride or monohydrobromide salt, also in crystalline form, capable of activating cholinergic neurons and/or has neuroprotective action.

12 cl, 19 dwg, 3 tbl, 9 ex

Description

Technical field

[0001] The present invention relates to pharmaceutically acceptable salts of a pentacyclic compound having cholinergic neuronal activating and/or neuroprotective effects, as well as crystals of the pentacyclic compound and pharmaceutically acceptable salts thereof. The present invention also relates to pharmaceutical compositions containing the above salts or crystals as the active substance.

State of the art

[0002] Cholinergic neurons, which release acetylcholine as a transmitter, are widely distributed in the forebrain from the basal nucleus of Meynert and the septal nucleus of the basal forebrain to the hippocampus, amygdala and cerebral cortex and are involved in the processes of modulation of memory, learning, cognition and attention (non-patent literature source 1). In addition, cholinergic neurons in the pedunculopontine tegmental nucleus and laterodorsal tegmental nucleus of the brainstem are distributed in the striatum, nucleus accumbens, substantia nigra, and thalamus, and are thought to be involved in the regulation of motivation and wakefulness (Non-Patent Literature 2-4).

[0003] In particular, the role of cholinergic neurons in the basal forebrain has been elucidated to a large extent through analysis using many animal models, such as the lesion model. In particular, a correlation between functional impairment of cholinergic neurons and impairment of memory and learning ability has been shown in animal models (non-patent literature 5-7), and cognitive performance has been shown to be improved by increasing acetylcholine with cholinesterase inhibitor use and enhancing function cholinergic neurons (non-patent literature sources 8-12).

[0004] Nerve growth factor (NGF) has been reported to demonstrate a neuroprotective effect on cholinergic neurons in an animal model of cholinergic neuron loss. (Non-Patent Literature 13-15).

[0005] Particularly in the case of Alzheimer's disease (AD), loss of cholinergic neurons is found in the early stage of AD and is one of the pathological manifestations of AD. The accumulation of senile plaques due to the deposition of beta-amyloid and neurofibrillary tangles due to tau protein aggregation are also pathological manifestations of AD, and in particular, neurofibrillary tangles are known to increase with the progression of the pathological condition and this leads to neuronal death. Neurofibrillary tangles are found in the nucleus basalis of Meynert and the entorhinal cortex from early AD. Among other things, it has been reported that loss of cholinergic neurons in the nucleus basalis of Meynert due to tau protein aggregation is detected at an earlier stage, and that there is a correlation between such loss and a decrease in cognitive function scores (Non-Patent Literatures 16 and 17). As with AD, hyperphosphorylation and abnormal accumulation of tau protein are found in genetically modified mice with the P301S mutation, which has been found in hereditary frontotemporal dementia (P301S transgenic mice expressing human tau). Consequently, neurofibrillary tangles, which are a pathological manifestation of AD, are formed (Non-Patent Literature 18), and they cause cognitive dysfunction due to synaptic impairment, neurodegeneration and neuronal loss. Based on these results, transgenic mice with the P301S mutation expressing human tau have been widely used as AD-like animal models (non-patent literature 19-22), and improvements in cognitive decline and suppression of disease pathology can be expected Alzheimer's disease by suppressing AD-like pathological changes in transgenic mice with the P301S mutation expressing human tau protein.

[0006] In addition, numerous analyzes using genetically modified mice and animal models of disorders suggest that deficits in axonal transport are one of the causes of cholinergic neuron loss (Non-Patent Literature 23-25). Including the axon of cholinergic neurons, which projects from the septal region into the hippocampus, is impaired in the hippocampal fimbria and fornix lesion model, and the impairment of retrograde transport of molecules associated with survival and function leads to neuronal loss (non-proprietary literature 26 -28). Impaired retrograde transport is also found in genetically modified mice (Non-Patent Literatures 23 and 24), and loss of cholinergic neurons due to damage to the hippocampal fimbriae and fornix reflects one aspect of the pathological condition. Thus, improvements in cognitive decline and suppression of pathological development in Alzheimer's disease can be expected by suppressing or improving the loss of cholinergic neurons in this disorder model.

[0007] Dementia with Lewy bodies (DLB) and Parkinson's disease (PD) are progressive neurodegenerative diseases in which abnormal inclusion bodies (Lewy bodies), primarily composed of alpha-synuclein, form within neurons and lead to neuronal degeneration and loss . Cognitive dysfunction develops if Lewy bodies are primarily distributed in the cerebral cortex, and parkinsonism develops if Lewy bodies are primarily distributed in the brain stem. In addition to this, psychiatric symptoms such as visual hallucination, hallucination and delusion, sleep disturbance and autonomic symptoms also develop. The diagnosis is dementia with Lewy bodies if the dementia appears before or within one year of the onset of parkinsonism, and the diagnosis is Parkinson's disease with dementia (PDD) if the parkinsonism appears before one year or more of the onset of dementia. Dementia with Lewy bodies, Parkinson's disease with dementia, and Parkinson's disease are pathologically similar diseases and are collectively called Lewy body disease (LBD), although they differ in cognitive dysfunction and the order of onset and degree of parkinsonism. In dementia with Lewy bodies and Parkinson's disease with dementia, as in the case of Alzheimer's disease, neurons of the basal nucleus of Meynert, which is the nucleus that is the source of the cholinergic nerve, degenerate and die, and it is reported that there is severe impairment of cholinergic neurons in the hippocampus and cortex brain (non-patent literature sources 29-31). Additionally, there is a correlation between the development of cholinergic neuron impairment and cognitive dysfunction (Non-Patent Literature 29), and cholinesterase inhibitors have been demonstrated to provide improvements in cognitive function. Based on these results, it was concluded that cognitive function improves due to improved cholinergic neuron function and, as with Alzheimer's disease, improvements in cognitive decline and suppression of pathological development in Lewy body dementia and Parkinson's disease can be expected with dementia by suppressing or reducing the loss of cholinergic neurons in several models of the disorder.

[0008] Therefore, based on these results, improvement in cognitive decline due to cholinergic neuron dysfunction can be expected by achieving functional activation and/or neuroprotective effect on cholinergic neurons in clinical practice.

List of cited literature

Non-patent literature

[0009] [Non-Patent Literature 1] Everitt BJ et al . "Central cholinergic systems and cognition." Annu. Rev. Psychol. 48 (1997) 649-684.

[Non-Patent Literature 2] Gulledge AT. et al . "Cholinergic inhibition of neocortical pyramidal neurons." J. Neurosci. 25 (2005) 10308-20.

[Non-Patent Literature 3] Daniel Dautan D. et al . "A major external source of cholinergic innervation of the striatum and nucleus accumbens originates in the brainstem." J. Neurosci. 34 (2014) 4509-18.

[Non-Patent Literature 4] M Steriade M. et al . "Neuronal activities in brain-stem cholinergic nuclei related to tonic activation processes in thalamocortical systems." J. Neurosci. 10 (1990) 2541-59.

[Non-Patent Literature 5] Fischer W. et al . "Progressive decline in spatial learning and integrity of brain cholinergic neurons in rats during aging." Neurobiol. Aging 13 (1992) 9-23.

[Non-Patent Literature 6] Leanza G. et al . "Selective lesioning of the basal forebrain cholinergic system by intraventricular 192 IgG-saporin: behavioral, biochemical and stereological studies in the rat." Eur. J. Neurosci. 7 (1995) 329-43.

[Non-Patent Literature 7] Leanza G. et al . "Selective immunolesioning of the basal forebrain cholinergic system disrupts short-term memory in rats." Eur. J. Neurosci. 8 (1996) 1535-44.

[Non-Patent Literature 8] Ogura H. et al . "Donepezil, a centrally acting acetylcholinesterase inhibitor, alleviates learning deficits in hypocholinergic models in rats." Methods Find Exp Clin Pharmacol. 22 (2000) 89-95.

[Non-Patent Literature 9] Spowart-Manning L. et al . "Spatial discrimination deficits by excitotoxic lesions in the Morris water escape task." Behav Brain Res. 156 (2005) 269-76.

[Non-Patent Literature 10] Bruce AP. et al. "Choline acetyltransferase activity and cognitive domain score of Alzheimer's patients." Neurobiol. Aging. 21 (2000) 11-17

[Non-Patent Literature 11] Rogers SL. et al . "The efficacy and safety of donepezil in patients with Alzheimer's disease: results of a US Multicentre, Randomized, Double-Blind, Placebo-Controlled Trial. The Donepezil Study Group." Dementia. 7 (1996) 293-303

[Non-Patent Literature 12] Mori E. et al . "Donepezil for dementia with Lewy bodies: a randomized, placebo-controlled trial." Ann Neurol. 72 (2012) 41-52

[Non-Patent Literature 13] Mufson EJ. et al . "Human cholinergic basal forebrain: chemoanatomy and neurologic dysfunction." J. Chem. Neuroanat. 26 (2003) 233-242

[Non-Patent Literature 14] Mufson EJ. et al . "Cholinergic system during the progression of Alzheimer's disease: therapeutic implications." Expert. Rev. Neurother. 8 (2008) 1703-1718

[Non-Patent Literature 15] Schliebs R. et al . "The significance of the cholinergic system in the brain during aging and in Alzheimer's disease." J. Neural. Transm 113 (2006) 1625-1644

[Non-Patent Literature 16] Vana L et al . "Progression of tau pathology in cholinergic Basal forebrain neurons in mild cognitive impairment and Alzheimer's disease." Am J Pathol. 179 (2011) 2533-2550.

[Non-Patent Literature 17] Gómez-Isla T et al . "Neuronal loss correlates with but exceeds neurofibrillary tangles in Alzheimer's disease." Ann Neurol. 41 (1997) 17-24.

[Non-Patent Literature 18] Lee VM et al . "Neurodegenerative tauopathies." Annu. Rev. Neurosci. 24 (2001) 1121-1159.

[Non-Patent Literature 19] Allen B et al . "Abundant tau filaments and nonapoptotic neurodegeneration in transgenic mice expressing human P301S tau protein." J. Neurosci. 22 (2002) 9340-9351.

[Non-Patent Literature 20] Xu H et al . "Memory deficits correlate with tau and spine pathology in P301S MAPT transgenic mice." Neuropathol. Appl. Neurobiol. 40 (2014) 833-43.

[Non-Patent Literature 21] Yoshiyama Y et al . "Synapse loss and microglial activation precede tangles in a P301S tauopathy mouse model." Neuron. 53 (2007) 337-351.

[Non-Patent Literature 22] Hoffmann NA et al . "Impaired plasticity of cortical dendritic spines in P301S tau transgenic mice." Acta Neuropathol Commun. 1 (2013) 82.

[Non-Patent Literature 23] Salehi A et al . "Increased App Expression in a Mouse Model of Down's Syndrome Disrupts NGF Transport and Causes Cholinergic Neuron Degeneration" Neuron 51 (2006) 29-42.

[Non-Patent Literature 24] Onishi T et al . "Early-onset cognitive deficits and axonal transport dysfunction in P301S mutant tau transgenic mice" Neuroscience Research 80 (2014) 76-85.

[Non-Patent Literature 25] Xu W et al . "Amyloid precursor protein-mediated endocytic pathway disruption induces axonal dysfunction and neurodegeneration" J. Clin. Invest. 126 (2016) 1815-33.

[Non-Patent Literature 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 [ ³ H]acetylcholine synthesis, [ ³ H]acetylcholine release and choline acetyltransferase activity" Neuroscience 42 (1991) 639-49.

[Non-Patent Literature 27] Gilmor ML et al . "Coordinate expression of the vesicular acetylcholine transporter and choline acetyltransferase following septohippocampal pathway lesions" J. Neurochem. 71 (1998) 2411-20.

[Non-Patent Literature 28] Gu H et al. "Recombinant human NGF-loaded microspheres promote survival of basal forebrain cholinergic neurons and improve memory impairments of spatial learning in the rat model of Alzheimer's disease with fimbria-fornix lesion" Neurosci. Lett. 453 (2009) 204-9.

[Non-Patent Literature 29] Shimada, H. et al ., "Mapping of brain acetylcholinesterase alterations in Lewy body disease by PET" Neurology, vol.73, pp. 273-278, 2009.

[Non-Patent Literature 30] Tiraboschi, P. et al ., "Cholinergic dysfunction in diseases with Lewy bodies" Neurology 54 (2000) 407-411.

[Non-Patent Literature 31] Perry, EK et. al ., "Neocortical cholinergic activities differentiate Lewy body dementia from classical Alzheimer's disease", NeuroReport, vol.5, pp.747-749 (1994).

Brief description of the invention

Technical problem

[0010] The inventors of the present invention have discovered that the 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]thieno[3,2-f][1,4]diazepine-4,13-dione, hereinafter referred to as “compound (I )") is capable of activating cholinergic neurons and/or has a neuroprotective effect. Therefore, there is a possibility of using compound (I) as a means for improving cognitive ability reduced as a result of dysfunction of cholinergic neurons.

[0011] The physical properties of compounds used as pharmaceuticals, and their salts, and crystals thereof generally greatly influence the bioavailability of the drug, the purity of the drug, and the composition of the pharmaceutical. Therefore, it is an object of the present invention to provide pharmaceutically acceptable salts of compound (I), which can potentially be used as a drug substance for pharmaceuticals, and crystals thereof.

The solution of the problem

[0012] As a result of careful studies of compound (I) in view of the above-mentioned circumstances, the inventors of the present invention discovered salts of compound (I) and crystals thereof, thereby completing the present invention.

[0013] Thus, the present invention relates to the following items <1>-<35>.

<1> Monohydrochloride or monohydrobromide 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4, 5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione represented by formula (I):

.

<1> Crystal 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4,5] pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione or 5,10-dimethyl-5,6,9,10,11,12-monohydrochloride or monohydrobromide hexahydropyrido[4'',3'':4',5']thieno[2',3':4,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine -4,13-diones.

<3> Crystal 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4,5] pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, having diffraction peaks at diffraction angles (2θ±0.2°) 9.0°, 11, 1° and 23.6° in powder X-ray diffraction using CuKα as the X-ray source.

<3.1> Crystal 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4,5] pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, having diffraction peaks at diffraction angles (2θ±0.2°) 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 CuKα in as a source of X-ray radiation.

<3.2> Crystal 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4,5] pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, the powder X-ray diffraction pattern of which is shown in Figure 1 by powder X-ray diffraction using CuKα as the X-ray source .

<4> Type A crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4) monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, having diffraction peaks at diffraction angles (2θ±0.2°) 11.6° , 20.8° and 25.7° in powder X-ray diffraction using CuKα as the X-ray source.

<4.1> Type A crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4) monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, having diffraction peaks at diffraction angles (2θ±0.2°) 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 with using CuKα as an X-ray source.

<4.2> Type A crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4) monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, the powder X-ray diffraction pattern of which is shown in Figure 2 with powder X-ray diffraction using CuKα as X-ray source.

<4.3> Type A crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4) monohydrochloride ,5]pyrimido[1,2-a]thieno[4,2-f][1,4]diazepine-4,13-dione, characterized by peaks at chemical shifts (δ±0.5 ppm) 164, 0 ppm, 129.6 ppm and 36.5 ppm in ¹³ C solid-state NMR spectrum with glycine (176.03 ppm) as external standard.

<5>Type B crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4'' monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, having diffraction peaks at diffraction angles (2θ±0.2°) 9.7° , 10.1° and 17.9° in powder X-ray diffraction using CuKα as the X-ray source.

<5.1> Type B crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4) monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, having diffraction peaks at diffraction angles (2θ±0.2°) 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 with using CuKα as an X-ray source.

<5.2> Type B crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4) monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, the powder X-ray diffraction pattern of which is shown in Figure 3 with powder X-ray diffraction using CuKα as X-ray source.

<5.3> Type B crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4) monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, characterized by peaks at chemical shifts (δ±0.5 ppm) 160, 1 ppm, 133.4 ppm and 130.7 ppm in ¹³ C solid-state NMR spectrum with glycine (176.03 ppm) as external standard.

<6>Type C crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4) monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, having diffraction peaks at diffraction angles (2θ±0.2°) 6.0° , 7.7° and 16.9° in powder X-ray diffraction using CuKα as the X-ray source.

<6.1> Type C crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4) monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, having diffraction peaks at diffraction angles (2θ±0.2°) 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 with using CuKα as an X-ray source.

<6.2> Type C crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4) monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione having a powder X-ray diffraction pattern as shown in Figure 4, with powder X-ray diffraction using CuKα as an X-ray source.

<6.3> Type C crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4) monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, characterized by peaks at chemical shifts (δ±0.5 ppm) 159, 6 ppm, 127.6 ppm and 38.9 ppm in ¹³ C solid-state NMR spectrum with glycine (176.03 ppm) as external standard.

<7> Type D crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4) monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, having diffraction peaks at diffraction angles (2θ±0.2°) 6.6° , 14.6° and 26.4° in powder X-ray diffraction using CuKα as the X-ray source.

<7.1> Type D crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4) monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, having diffraction peaks at diffraction angles (2θ±0.2°) 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 with using CuKα as an X-ray source.

<7.2> Type D crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4) monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, the powder X-ray diffraction pattern of which is shown in Figure 5 with powder X-ray diffraction using CuKα as X-ray source.

<8> Type E crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4) monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, having diffraction peaks at diffraction angles (2θ±0.2°) 6.4° , 11.3° and 27.3° in powder X-ray diffraction using CuKα as the X-ray source.

<8.1> Type E crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4) monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, having diffraction peaks at diffraction angles (2θ±0.2°) 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 with using CuKα as an X-ray source.

<8.2> Type E crystal of the monohydrochloride of compound (I) having a powder x-ray diffraction pattern as shown in Figure 6, powder x-ray diffraction using CuKα as the x-ray source.

<9> Type F crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4) monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, having diffraction peaks at diffraction angles (2θ±0.2°) 7.3° , 9.3° and 10.7° in powder X-ray diffraction using CuKα as the X-ray source.

<9.1> Type F crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4) monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, having diffraction peaks at diffraction angles (2θ±0.2°) 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 with using CuKα as an X-ray source.

<9.2> Type F crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4) monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione having a powder X-ray diffraction pattern as shown in Figure 7, with powder X-ray diffraction using CuKα as an X-ray source.

<10> Crystal of monohydrobromide 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4.5 ]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, having diffraction peaks at diffraction angles (2θ±0.2°) 7.8°, 24 .5° and 25.2° in powder X-ray diffraction using CuKα as the X-ray source.

<10.1> Crystal of monohydrobromide 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4.5 ]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, having diffraction peaks at diffraction angles (2θ±0.2°) 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 CuKα as a source of X-ray radiation.

<10.2> Crystal of monohydrobromide 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4.5 ]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione having a powder X-ray diffraction pattern as shown in Figure 8 by powder X-ray diffraction using CuKα in as a source of X-ray radiation.

<11> Type A crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4) monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, characterized by a Raman shift peak (± 2 cm ^-1 ) at 587 cm ^-1 obtained using Raman spectroscopy.

<12> Type A crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4] monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, characterized by Raman shift peaks (± 2 cm ^-1 ) at 587 cm ^-1 , 1428 cm ^-1 and 1493 cm ^-1 obtained using Raman spectroscopy.

<13> Type A crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4) monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, characterized by Raman shift peaks (2 cm ^-1 ) at 587 cm ^-1 , 763 cm ^-1 , 1428 cm ^-1 , 1493 cm ^-1 and 1688 cm ^-1 obtained using Raman spectroscopy.

<14> Type A crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4) monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, characterized by Raman shift peaks (± 2 cm ^-1 ) at 409 cm ^-1 , 587 cm ^-1 , 763 cm ^-1 , 976 cm ^-1 , 1428 cm ^-1 , 1493 cm ^-1 and 1688 cm ^-1 obtained using Raman spectroscopy.

<15> Type A crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4) monohydrochloride ,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione, characterized by the spectrum shown in Figure 19 and obtained by Raman spectroscopy.

<16> A pharmaceutical composition containing a salt according to <1> or a crystal according to any of <2> - <15>.

<17> Pharmaceutical composition according to <16>, which is an agent that activates cholinergic neurons.

<18> Pharmaceutical composition according to <16>, which is an agent that prevents damage to cholinergic neurons.

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

<20> A therapeutic agent for the treatment of cognitive dysfunction, containing a salt according to <1> or a crystal according to any of <2> - <15>.

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

<22> Salt according to <1> or crystal according to any of <2> - <15> for use in the treatment of cognitive dysfunction.

<23> Use of a salt according to <1> or a crystal according to any of <2> to <15> in the production of a therapeutic agent for the treatment of cognitive dysfunction.

<24> A therapeutic agent for the treatment of Alzheimer's disease containing a salt according to <1> or a crystal according to any one of <2> to <15>.

<25> A method of treating Alzheimer's disease, comprising administering to a patient a salt according to <1> or a crystal according to any one of <2> to <15>.

<26> A salt according to <1> or a crystal according to any of <2> to <15> for use in the treatment of Alzheimer's disease.

<27> Use of a salt according to <1> or a crystal according to any of <2> to <15> in the production of a therapeutic agent for treating Alzheimer's disease.

<28> A therapeutic agent for the treatment of dementia with Lewy bodies, containing a salt according to <1> or a crystal according to any of <2> - <15>.

<29> A method of treating dementia with Lewy bodies, comprising administering to the patient a salt according to <1> or a crystal according to any of <2> - <15>.

<30> Salt according to <1> or crystal according to any of <2> - <15> for use in the treatment of dementia with Lewy bodies.

<31> Use of a salt according to <1> or a crystal according to any of <2> to <15> in the production of a therapeutic agent for the treatment of dementia with Lewy bodies.

<32> A therapeutic agent for the treatment of Parkinson's disease with dementia, containing a salt according to <1> or a crystal according to any of <2> - <15>.

<33> A method of treating Parkinson's disease with dementia, comprising administering to a patient a salt according to <1> or a crystal according to any one of <2> - <15>.

<34> Salt according to <1> or crystal according to any of <2> - <15> for use in the treatment of Parkinson's disease with dementia.

<35> Use of a salt according to <1> or a crystal according to any of <2> to <15> in the production of a therapeutic agent for the treatment of Parkinson's disease with dementia.

Advantageous effects of the present invention

[0014] According to the present invention, it is possible to obtain salts of compound (I) and crystals thereof, which are expected to be useful as drug substances of pharmaceuticals and to have good physical properties.

Brief description of graphic materials

[0015]

Figure 1 shows a powder X-ray diffraction pattern of a crystal of compound (I) obtained in Example 1. The x-axis is the diffraction angle (2θ), and the ordinate is the intensity of the peaks.

Figure 2 shows the powder X-ray diffraction pattern of a type A crystal of the monohydrochloride of compound (I) obtained in Example 2. The x-axis is the diffraction angle (2θ), and the ordinate is the peak intensity.

Figure 3 shows the powder X-ray diffraction pattern of a type B crystal of the monohydrochloride of compound (I) obtained in Example 4. The x-axis represents the diffraction angle (2θ), and the ordinate represents the intensity of the peaks.

Figure 4 shows the X-ray powder diffraction pattern of a type C crystal of the monohydrochloride of compound (I) obtained in Example 3. The x-axis represents the diffraction angle (2θ), and the ordinate represents the intensity of the peaks.

Figure 5 shows the powder X-ray diffraction pattern of a type D crystal of the monohydrochloride of compound (I) obtained in Example 5. The x-axis represents the diffraction angle (2θ), and the ordinate represents the intensity of the peaks.

Figure 6 shows the powder X-ray diffraction pattern of a type E crystal of the monohydrochloride of compound (I) obtained in Example 6. The x-axis represents the diffraction angle (2θ), and the ordinate represents the intensity of the peaks.

Figure 7 shows the powder X-ray diffraction pattern of a type F crystal of the monohydrochloride of compound (I) obtained in Example 7. The x-axis represents the diffraction angle (2θ), and the ordinate represents the intensity of the peaks.

Figure 8 shows the powder X-ray diffraction pattern of the monohydrobromide crystal of compound (I) obtained in Example 8. The x-axis is the diffraction angle (2θ), and the ordinate is the peak intensity.

Figure 9 shows the ¹³ C solid-state NMR spectrum of a type A solid crystal of the monohydrochloride of compound (I) obtained in Example 2. The x-axis represents the chemical shift (δ), and the ordinate represents the peak intensity.

Figure 10 shows the ¹³ C solid-state NMR spectrum of a type B solid crystal of the monohydrochloride of compound (I) obtained in Example 4. The x-axis represents the chemical shift (δ), and the ordinate represents the peak intensity.

Figure 11 shows ^{the 13} C solid-state NMR spectrum of a type C solid crystal of the monohydrochloride of compound (I) obtained in Example 3. The x-axis represents the chemical shift (δ), and the ordinate represents the peak intensity.

Figure 12 shows the TG-DTA thermal analysis curves of a type A crystal of the monohydrochloride of compound (I) obtained in Example 2. The x-axis shows the temperature, the left y-axis shows the weight change under TG, and the right y-axis shows the heat flow during DTA. .

Figure 13 shows the TG-DTA thermal analysis curves of the type B crystal of the monohydrochloride of compound (I) obtained in Example 4. The x-axis shows the temperature, the left y-axis shows the weight change during TG, and the right y-axis shows the heat flow during DTA. .

Figure 14 shows the TG-DTA thermal analysis curves of the Type C crystal of the monohydrochloride of compound (I) obtained in Example 3. The x-axis shows the temperature, the left y-axis shows the weight change during TG, and the right y-axis shows the heat flow during DTA. .

Figure 15 shows the TG-DTA thermal analysis curves of a type D crystal of the monohydrochloride of compound (I) obtained in Example 5. The x-axis shows the temperature, the left y-axis shows the weight change during TG, and the right y-axis shows the heat flow during DTA. .

Figure 16 shows the TG-DTA thermal analysis curves of the type E crystal of the monohydrochloride of compound (I) obtained in Example 6. The x-axis shows the temperature, the left y-axis shows the weight change under TG, and the right y-axis shows the heat flow during DTA. .

Figure 17 shows the TG-DTA thermal analysis curves of the type F crystal of the monohydrochloride of compound (I) obtained in Example 7. The x-axis shows the temperature, the left y-axis shows the weight change under TG, and the right y-axis shows the heat flow during DTA. .

Figure 18 shows the TG-DTA thermal analysis curves of the monohydrobromide crystal of compound (I) obtained in Example 8. The x-axis shows the temperature, the left y-axis shows the weight change during TG, and the right y-axis shows the heat flow during DTA.

Figure 19 shows the Raman spectrum of type A crystals of the monohydrochloride of compound (I) obtained in Example 2.

Description of Embodiments

[0016] Next, the salts of compound (I) of the present invention, their crystals and the method for their preparation will be described in detail.

[0017] As used herein, the term “salt” refers to a chemical substance consisting of compound (I) as a main component and a certain amount of acid equivalents relative to compound (I).

[0018] Examples of the “salt” used herein include inorganic acid salts, organic acid salts and acidic amino acid salts, and among these, pharmaceutically acceptable salts are preferred.

[0019] Examples of inorganic acid salts include salts of hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid; and examples of organic acid salts include salts of organic carboxylic acids such as acetic acid, succinic acid, fumaric acid, maleic acid, tartaric acid, malic acid, citric acid, lactic acid, stearic acid and benzoic acid, as well as salts of organic sulfonic acids, such as methanesulfonic acid (mesylate), ethanesulfonic acid, benzenesulfonic acid and p-toluenesulfonic acid (tosylate); and among them, hydrochloric acid, hydrobromic acid and phosphoric acid are preferred.

[0020] Examples of acidic amino acid salts include salts of aspartic acid and glutamic acid.

[0021] The salt of the present invention may be in anhydride, hydrated or solvated form. Hydrate or solvate in the context of the present description refers to a solid substance that is formed by compound (I) or a salt thereof and water molecules or solvent molecules; this solid may be a crystal; examples of the solvent in the solvate include ketone-based solvents such as acetone, 2-butanone and cyclohexanone; ester solvents such as methyl acetate and ethyl acetate; ether solvents such as 1,2-dimethoxyethane and tert-butyl methyl ether; alcohol-based solvents such as methanol, ethanol, 1-propanol and isopropanol; polar solvents such as N-methyl-2-pyrrolidone, N,N-dimethylformamide and dimethyl sulfoxide. The number of water molecules or solvent molecules per compound (I) or salt thereof is not particularly limited, and, for example, may be one molecule or two molecules.

[0022] As used herein, the term “crystal” refers to a crystal of the anhydride or hydrated form of compound (I) or a salt thereof.

[0023] In the context of the present description, preferred examples of crystals of compound (I) and hydrochloride and hydrobromide of compound (I) include:

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

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

a type A crystal of the monohydrochloride of compound (I), characterized by diffraction peaks at diffraction angles (2θ±0.2°) of 11.6°, 20.8° and 25.7° in powder x-ray diffraction examination;

type A crystal of the monohydrochloride of compound (I), characterized by diffraction peaks at diffraction angles (2θ±0.2°) 6.1°, 7.8°, 11.6°, 16.2°, 19.9°, 20.8° , 25.2°, 25.7°, 26.9° and 29.9° in X-ray powder diffraction study;

type A crystal of the monohydrochloride of compound (I), characterized by peaks at chemical shifts (δ±0.5 ppm) 164.0 ppm, 129.6 ppm. and 36.5 ppm in ¹³ C solid-state NMR spectrum;

a type A crystal of the monohydrochloride of compound (I), characterized by a Raman shift peak (± 2 cm ^-1 ) at 587 cm ^-1 obtained by Raman spectroscopy;

a type A crystal of the monohydrochloride of compound (I), characterized by Raman shift peaks (± 2 cm ^-1 ) at 587 cm ^-1 , 1428 cm ^-1 and 1493 cm ^-1 obtained by Raman spectroscopy;

type A crystal of the monohydrochloride of compound (I), characterized by Raman shift peaks (± 2 cm ^-1 ) at 587 cm ^-1 , 763 cm ^-1 , 1428 cm ^-1 , 1493 cm ^-1 and 1688 cm ^-1 obtained by spectroscopy Raman scattering;

type A crystal of the monohydrochloride of compound (I), characterized by Raman shift peaks (± 2 cm ^-1 ) at 409 cm ^-1 , 587 cm ^-1 , 763 cm ^-1 , 976 cm ^-1 , 1428 cm ^-1 , 1493 cm ^-1 and 1688 cm ^-1 obtained using Raman spectroscopy;

a type A crystal of the monohydrochloride of compound (I), essentially characterized by the spectrum shown in Figure 19 and obtained by Raman spectroscopy;

a type B crystal of the monohydrochloride of compound (I), characterized by diffraction peaks at diffraction angles (2θ±0.2°) of 9.7°, 10.1° and 17.9° in powder X-ray diffraction examination;

type B crystal of the monohydrochloride of compound (I), characterized by diffraction peaks at diffraction angles (2θ±0.2°) 6.3°, 9.7°, 10.1°, 17.9°, 19.0°, 19, 4°, 23.4°, 26.3°, 27.3° and 32.0° in X-ray powder diffraction study;

type B crystal of the monohydrochloride of compound (I), characterized by peaks at chemical shifts (δ±0.5 ppm) 160.1 ppm, 133.4 ppm. and 130.7 ppm in ¹³ C solid-state NMR spectrum;

a type C crystal of the monohydrochloride of compound (I), characterized by diffraction peaks at diffraction angles (2θ±0.2°) of 6.0°, 7.7° and 16.9° in powder x-ray diffraction examination;

type C crystal of the monohydrochloride of compound (I), characterized by diffraction peaks at diffraction angles (2θ±0.2°) 6.0°, 7.7°, 9.7°, 11.4°, 15.8°, 16, 9°, 18.1°, 23.2°, 25.4° and 27.6° in X-ray powder diffraction study;

type C crystal of the monohydrochloride of compound (I), characterized by peaks at chemical shifts (δ±0.5 ppm) 159.6 ppm, 127.6 ppm. and 38.9 ppm in ¹³ C solid-state NMR spectrum;

a type D crystal of the monohydrochloride of compound (I), characterized by diffraction peaks at diffraction angles (2θ±0.2°) of 6.6°, 14.6° and 26.4° in powder X-ray diffraction examination;

type D crystal of the monohydrochloride of compound (I), characterized by diffraction peaks at diffraction angles (2θ±0.2°) 6.6°, 14.6°, 16.1°, 20.5°, 21.0°, 23, 0°, 24.5°, 26.4°, 28.0° and 32.5° in X-ray powder diffraction study;

a type E crystal of the monohydrochloride of compound (I), characterized by diffraction peaks at diffraction angles (2θ±0.2°) of 6.4°, 11.3° and 27.3° in a powder X-ray diffraction study;

type E crystal of the monohydrochloride of compound (I), characterized by diffraction peaks at diffraction angles (2θ±0.2°) 6.4°, 11.3°, 15.7°, 18.0°, 19.2°, 22, 8°, 24.6°, 25.4°, 26.0° and 27.3° in X-ray powder diffraction study;

a type F crystal of the monohydrochloride of compound (I), characterized by diffraction peaks at diffraction angles (2θ±0.2°) of 7.3°, 9.3° and 10.7° in powder X-ray diffraction examination;

type F crystal of the monohydrochloride of compound (I), characterized by diffraction peaks at diffraction angles (2θ±0.2°) 5.9°, 7.3°, 9.3°, 10.7°, 13.8°, 15, 6°, 16.4°, 18.7°, 25.1° and 26.8° in X-ray powder diffraction study;

a monohydrobromide crystal of compound (I) characterized by diffraction peaks at diffraction angles (2θ±0.2°) of 7.8°, 24.5° and 25.2° in X-ray powder diffraction examination; And

monohydrobromide crystal of compound (I), characterized by diffraction peaks at diffraction angles (2θ±0.2°) 6.0°, 7.8°, 10.0°, 11.7°, 17.8°, 20.8° , 23.5°, 24.5°, 25.2° and 27.3° in X-ray powder diffraction study;

[0024] The powder X-ray diffraction peaks, chemical shifts in ^{the 13} C solid-state NMR spectrum, and Raman shift peaks obtained by Raman spectroscopy described above are unique to each of the crystal of compound (I), crystal types A to F monohydrochloride compound (I) and the monohydrobromide crystal of compound (I), and they are characteristic peaks for the crystals.

[0025] Typically, the diffraction angles (2θ) of powder x-ray diffraction may contain errors within ±0.2°, so it should be appreciated that the diffraction angle values described above include numerical values within approximately ±0.2°. Thus, not only crystals whose diffraction angles of peaks in powder x-ray diffraction are exactly the same, but also crystals whose diffraction angles of peaks are the same within an error of approximately ±0.2° are the same in a particular compound or its salt and are included in the present invention .

[0026] In this context, for example, the expression “having a diffraction peak at a diffraction angle (2θ±0.2°) of 9.0°” means “having a diffraction peak at a diffraction angle (2θ) from 8.8° to 9.2 °", and the same applies to the case of other diffraction angles.

[0027] In general, even when the crystalline form is the same, the peak intensity or half-width of diffraction angles (2θ) in powder X-ray diffraction is different for each measurement depending on the different measurement conditions and the difference in the size and shape of the powder crystal particles used as a sample for measurements, and it is not always possible to obtain a constant peak intensity or half-width. Therefore, when comparing powder X-ray diffraction patterns, if a difference in peak intensity or half-width is observed at the same diffraction angle (2θ), it does not mean that the difference is caused by a difference in crystalline forms. Therefore, it means that a crystal having a powder X-ray diffraction pattern that differs in this manner from the diffraction peaks characteristic of a particular crystal of the present invention has the same crystalline form as the crystal of the present invention. Moreover, in this context, the expression “characterized by the powder X-ray diffraction pattern shown in Figure 1” means that the entire crystal exhibiting the powder X-ray diffraction pattern shown in Figure 1 is the same crystal as the crystal of the present invention, not only in the case where the X-ray powder diffraction pattern having characteristic diffraction peaks coincides with the X-ray powder diffraction pattern shown in figure 1 within an error of ±0.2°, but also in the case of the powder diffraction pattern having different peak intensity or half-width , but at the same time having characteristic diffraction angles that correspond to the powder X-ray diffraction pattern shown in figure 1, within an error of ±0.2°.

[0028] As used herein, the expression “chemical shifts (δ±0.5 ppm) 164.0 ppm, 129.6 ppm and 36.5 ppm.” means “characterized by peaks substantially equivalent to the chemical shifts (δ±0.5 ppm) 164.0 ppm, 129.6 ppm. and 36.5 ppm if the measurements of ^{the 13} C solid-state NMR spectrum are obtained under normal measurement conditions or under conditions substantially the same as those described herein.”

[0029] In general, when determining whether "substantially equivalent peaks" are "characterized" or not, it must be taken into account that the chemical shift δ in a ¹³ C solid-state NMR spectrum may contain an error of ±0.5 ppm, so the values chemical shifts described above include numerical values ranging from about ±0.5 ppm. Thus, not only crystals whose chemical shifts match perfectly in the ¹³ C solid-state NMR spectra, but also crystals whose chemical shifts match within an error of approximately ±0.5 ppm are included in the present invention. Therefore, in this context, for example, “characterized by a peak at a chemical shift (δ±0.5 ppm) 164.0 ppm.” means the presence of a peak at a chemical shift (δ) in the range from 163.5 to 164.5 ppm, and the same applies to the case of other chemical shifts in the ¹³ C solid-state NMR spectrum.

[0030] Typically, Raman shift peaks (cm ^-1 ) obtained using Raman spectroscopy may have an error of ±2 cm ^-1 , so the peak values described above should be considered to include numerical values within approximately ±2 cm ^-1 . Thus, not only are crystals whose Raman shift peaks obtained by Raman spectroscopy perfectly matched, but also crystals whose Raman shift peaks matched to within about ±2 cm ^-1 are the same in a particular compound or its salts and are included in the present invention.

[0031] As used herein, for example, the expression “characterized by a Raman shift peak (±2 cm ^-1 ) at 587 cm ^-1 obtained by Raman spectroscopy” means “characterized by a Raman shift peak at 585 cm ^-1 to 589 cm ^-1 obtained by Raman spectroscopy,” and the same applies to the case of other Raman shifts.

[0032] In general, even when the crystalline form is the same, the peak intensities or half-widths of the Raman shift obtained by Raman spectroscopy measurements differ in each measurement depending on different measurement conditions and variations in the size and shape of the crystal particles of the powder used as sample for measurement, and the same intensity or half-width of the peak is not always obtained. Therefore, when comparing Raman spectroscopy measurements, if a difference in peak intensity or half-width of the same Raman shift peak (cm ^-1 ) is observed, it does not mean that the difference is caused by a difference in crystal forms. Thus, this means that a crystal exhibiting a Raman spectrum so different from the Raman shift peaks characteristic of a particular crystal of the present invention has the same crystalline form as the crystal of the present invention. In addition, in the context of the present description, the expression "characterized by the spectrum shown in Figure 19 obtained by Raman spectroscopy" means that the entire crystal exhibiting the Raman spectrum shown in Figure 19 is the same crystal as the crystal of the present invention , not only in the case where the Raman spectrum with characteristic peaks of the Raman shift (cm ^-1 ) corresponds to the Raman spectrum shown in figure 19, within an error of ±2 cm ^-1 , but also in the case of the Raman spectrum having different intensity or half-width of peaks in the presence of characteristic peaks of the Raman shift, which coincide within an error of ±2 cm ^-1 .

[0033] The following describes methods for preparing salts of compound (I), crystals and the like, which represent one embodiment of the present invention.

[0034] Method for preparing compound (I)

Compound (I) can be prepared by methods well known to those skilled in the art. For example, compound (I) can be synthesized by the method described in the comparative example described below.

[0035] Method for producing salt of compound (I)

The salts of compound (I) according to the present invention can be prepared by methods for preparing conventional salts. In particular, the salts can be prepared, for example, by suspending or dissolving compound (I) in a solvent with heating if necessary, and then adding an acid to the resulting suspension or solution, followed by stirring or keeping the mixture at room temperature or by cooling for a period of several minutes to several days. Using these preparation methods, salts of compound (I) can be obtained in crystalline or amorphous forms. Moreover, in addition to these production methods, the amorphous form can be obtained by performing an operation such as lyophilization, if necessary. Examples of the solvent used herein include alcohol-based solvents such as ethanol, 1-propanol and isopropanol; acetonitrile; ketone-based solvents such as acetone and 2-butanone; ester solvents such as ethyl acetate; saturated hydrocarbon solvents such as hexane and heptane; ether solvents such as tert-butyl methyl ether and water. These solvents can be used alone or in combination of two or more of them.

[0036] Method for producing a crystal of compound (I) or a salt thereof

A crystal of compound (I) or a salt thereof can be obtained by the above method for preparing compound (I) or a method for producing a salt thereof, or can be obtained by heating and dissolving compound (I) or a salt thereof in a solvent, and then cooling the resulting solution while stirring to effect crystallization .

[0037] The compound (I) or a salt thereof used for crystallization may be in any form, that is, solvated or hydrated or anhydride, amorphous or crystalline form (including a form consisting of a plurality of polymorphic crystals), or a mixture thereof.

[0038] Examples of the solvent used for crystallization include alcohol-based solvents such as methanol, ethanol, isopropanol and 1-propanol; acetonitrile; amide-based solvents such as N,N-dimethylformamide; ester solvents such as ethyl acetate; saturated hydrocarbon solvents such as hexane and heptane; ketone-based solvents such as acetone and 2-butanone; ether solvents such as tert-butyl methyl ether and water. These solvents can be used alone or in combination of two or more of them.

[0039] The amount of solvent used can be selected accordingly, provided that the amount capable of dissolving compound (I) or its salt when heated, or the amount that allows the suspension to be stirred, is the lower limit, and the amount at which the yield of the crystal is not significantly reduced , is the upper limit.

[0040] During crystallization, a seed crystal (eg, a crystal of compound (I) or a salt thereof to be prepared) may or may not be introduced. The temperature at which the seed crystal is introduced is not particularly limited, but is preferably from 0 to 80°C.

[0041] The temperature for heating and dissolving the compound (I) or its salt can be suitably selected depending on the solvent so that the compound (I) or its salt can be dissolved at this temperature, but this temperature is preferably in the range of 50°C to the initial boiling point of the reflux solvent is more preferably from 55 to 80°C.

[0042] Since rapid cooling can produce crystals of different characters (polymorphism), cooling during crystallization is preferably carried out with appropriate regulation of the cooling rate, taking into account the effect on the quality and grade of crystals, preferably cooling at a rate of, for example, from 5 to 40° C/hour. More preferably, cooling is carried out at a rate of, for example, 5 to 25° C./hour.

[0043] The final crystallization temperature can be suitably selected depending on yield, quality, or the like. crystals and preferably it is from -25 to 30°C.

[0044] The desired crystal can be obtained by separating the crystals obtained by crystallization using a conventional filtration procedure, washing the filtered crystals with a solvent if necessary, and then drying. As a solvent for washing the crystal, solvents 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 can be used alone or in combination of two or more of them.

[0045] The crystals separated by the filtration procedure can be dried, suitably exposed to air or a nitrogen stream, or by heating.

[0046] The drying time can be suitably selected as the time required for the residual solvent content to fall below a predetermined value depending on the amount to be produced, the drying apparatus, the drying temperature and the like. Drying can also be carried out in a stream of air or at reduced pressure. The degree of pressure reduction can be suitably selected depending on the quantity to be produced, the drying device, the drying temperature and the like. The resulting crystal can also be left in the air after drying if necessary.

[0047] Crystals of compound (I) and salts of compound (I) obtained by the method described above are capable of activating cholinergic neurons and/or have neuroprotective effects, as evidenced by the activity data in the pharmacological test examples described below, and there is the possibility of their use in as a means to improve cognitive ability reduced as a result of dysfunction of cholinergic neurons.

[0048] [Pharmaceutical composition]

Another embodiment of the present invention is a pharmaceutical composition containing crystals of compound (I) and pharmaceutically acceptable additives. The pharmaceutical composition can be prepared by mixing pharmaceutically acceptable additives with crystals of compound (I). The pharmaceutical composition of the present invention can be prepared according to a known method, for example, the method described in the General Rules for Preparations in Japanese Pharmacopoeia 17th Edition.

The pharmaceutical composition according to the present embodiment can be administered to a patient in a suitable manner depending on its dosage form.

[0049] The dose of compounds (I) according to the present invention depends on the severity of symptoms, age, gender, body weight, dosage form, type of salt, specific type of disease and other conditions; however, in general, the daily dose for an adult, if administered orally, is from about 30 μg to 10 g, preferably from 100 μg to 5 g, and more preferably from 100 μg to 1 g; the daily dose for an adult in the case of injection is from about 30 μg to 1 g, preferably from 100 μg to 500 mg, and more preferably from 100 μg to 300 mg; and wherein the above dose is administered one or more times.

Examples

[0050] Crystals of the compound (I) of the present invention can be obtained, for example, using the methods described in the following examples, and the effect of the compounds can be confirmed using the methods described in the following test examples. However, these are just examples, and the present invention is in no way limited to the following specific examples and can be modified without departing from the scope of the present invention.

[0051] Powder X-ray diffraction of the crystals obtained in the following examples was carried out by fixing the obtained crystal on the stage of an X-ray powder diffraction device, and measurements were carried out under any of the following conditions.

(Conditions when skipping)

X-ray source: CuKα

Voltage: 45 kV

Current: 40mA

Optical system: focusing mirror

Soller gap: 0.02°

Detector: X'Celerator (semiconductor detector)

Scan range: 5° - 35°

Step size: 0.017°

Scan step time: 600 sec

Sample holder: Kapton film

(Conditions when reflected)

X-ray source: CuKα

Voltage: 50 kV

Current: 300mA

Slit: 0.5mm deflection slit, diffusion slit open, light receiving slit open

Detector: scintillation counter

Scanning speed: 5°/min

Scan step: 0.02°

Scan range: 5° - 35°

Sample holder: aluminum holder

[0052] The sample was accurately weighed in an aluminum sample cell, and thermal analysis was performed under the following conditions.

(Measurement conditions)

Atmosphere: in a stream of nitrogen gas (100 ml/min)

Control: empty aluminum sample cuvette

Temperature rise rate: 10°C/min

Scan step: 1 sec

Temperature measurement range: from room temperature to 320°C

[0053] A ¹³ C solid state NMR spectrum of the crystal was measured by placing approximately 300 mg of the solid sample in a sample tube under the following conditions.

(Measurement conditions)

Device used: Avance 400 MHz (manufactured by BRUKER) with 7 mm CPMAS sensor (manufactured by BRUKER)

Measured cores: ¹³ C (resonant frequency 100.6248425 MHz)

Measuring temperature: room temperature

Pulse mode: CPTOSS measurement

Rotation speed: 5000Hz

Pulse repetition time: 3 sec

Contact time: 1ms

Total number of times: 5120 times

Reference material: glycine (external standard: 176.03 ppm)

[0054] The Raman spectrum of the crystals was measured by placing the sample on the stage of a Raman microspectroscope under the following measurement conditions.

(Measurement conditions)

Instrument used: RENISHAW Raman microscope with Via Reflex technology

Laser wavelength: 785 nm

Diffraction grating: 1200 lines/mm

Goal: 50 times

Scan mode: continuous

Exposure time: 5 sec

Total number of times: 5 times

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

Error:±2cm ^-1

[0055] The compounds described with reference to documents, etc., were prepared in accordance with such documents, etc.

[0056] Moreover, abbreviations used in the present description are well known and understandable to one skilled in the art. In the present description, the following abbreviations apply.

DMSO: dimethyl sulfoxide

IPA: isopropanol

n-: normal

TEA: triethylamine

THF: tetrahydrofuran

¹ H-NMR: Proton Nuclear Magnetic Resonance Spectrometry

MS: mass spectrometry

[0057] The term “room temperature” in the following examples and comparative examples generally refers to the range from about 10°C to about 35°C. % refers to percentage mass fraction unless otherwise noted.

[0058] Chemical shifts in proton nuclear magnetic resonance spectra are indicated in units of δ (ppm) relative to tetramethylsilane, and coupling constants are recorded in hertz (Hz). Cleavage structures are indicated by the following abbreviations:

s: singlet; d: doublet; t: triplet; q: quartet; m: multiplet; br.s: wide singlet.

[0059] In the reactions using a microwave reactor, the comparative examples used Initiator(TM) or Initiator+(TM) from Biotage.

[0060] Regarding chromatography, the silica gel used was Silica Gel60, manufactured by Merck (70-230 mesh ASTM), or PSQ60B, manufactured by Fuji Silysia Chemical Ltd., or a prepacked column {column: Hi-Flash(TM) Column (silica gel ), manufactured by YAMAZEN, size: one of S (16x60 mm), M (20x75 mm), L (26x100 mm), 2L (26x150 mm) and 3L (46x130 mm); or Biotage(TM) SNAP Ultra Silica Cartridge, manufactured by Biotage, size: one of 10g, 25g and 50g}.

The NH silica gel used was CHROMATOREX NH-DM2035, manufactured by Fuji Silysia Chemical Ltd., or a prepacked column {column: Hi-Flash (TM) Column (amino-modified silica gel), manufactured by YAMAZEN, size: one of S (16× 60 mm), M (20×75 mm), L (26×100 mm), 2L (26×150 mm) and 3L (46×130 mm); or Presep (TM) (Luer Lock) NH2(HC), manufactured by Wako Pure Chemical Industries, Ltd., size: one of M type (14 g/25 ml), L type (34 g/70 ml), 2L type ( 50 g/100 ml) and type 3L (110 g/200 ml)}.

The neutral alumina used was Alumina 90, Active Neutral, 70-230 mesh, Merck, E6NXX.

[0061] The names of the compounds shown below, with the exception of commonly used reagents, were used as those shown in "E-Notebook" version 12 (PerkinElmer).

[0062] Comparative Example 1

Synthesis of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4,5]pyrimido[1 ,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione (hereinafter referred to as “compound (I)”)

[0063]

(1) Synthesis of ethyl 2-amino-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylate

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

¹ H-NMR (400 MHz, CDCl ₃ ) δ (ppm): 1.33 (t, J=7.0 Hz, 3H), 2.44 (s, 3H), 2.62-2, 70 (m, 2H), 2.79-2.88 (m, 2H), 3.37 (t, J=2.0 Hz, 2H), 4.26 (q, J=7.3 Hz, 2H ), 5.97 (br. s, 2H).

MS (electrospray ionization (ESI)) mass/charge: 241 [M+H] ⁺

[0064]

(2) Synthesis of 4-methyl-3,4-dihydro-1H-thieno[2,3-e][1,4]diazepine-2,5-dione

1H,2H,4H-Thieno[2,3-d][1,3]oxazine-2,4-dione (CAS No. 103979-54-0) (600 mg, 3.55 mmol) was added to the sarcosine solution (790 mg, 8.87 mmol) 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 layer was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The resulting solid was dried to give the title compound (430 mg).

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

MS (electrospray ionization (ESI)) mass/charge: 197 [M+H] ⁺

[0065]

(3) Synthesis of compound (I)

Phosphorus oxychloride (1.43 mL, 15.3 mmol) was added at room temperature to a mixture of 4-methyl-3,4-dihydro-1H-thieno[2,3-e][1,4]diazepine-2,5- dione (1.00 g, 5.10 mmol) obtained in step (2), ethyl-2-amino-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3- carboxylate (1.84 g, 7.64 mmol) obtained in step (1), and 1,4-dioxane (30 ml). The reaction mixture was stirred at room temperature for 5 minutes and then stirred at 90°C for 2 hours. Sodium ethoxide (20% ethanol solution, 21.7 mL, 56.1 mmol) was added over 5 minutes to the reaction mixture cooled to room temperature. The reaction mixture was stirred at room temperature for 1.5 hours. Ethyl acetate, saturated aqueous sodium hydrogen carbonate and water were successively added to the reaction mixture, and the organic layer was separated. The aqueous layer was extracted with ethyl acetate. The combined organic layer was 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 resulting solid was triturated with ethanol and the precipitates were collected by filtration. The resulting solid was washed with ethanol and dried under reduced pressure to obtain the title compound (712 mg).

¹ H-NMR (400 MHz, CDCl ₃ ) 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=14.8 Hz, 1H), 4.57 (d, J=14.8 Hz, 1H), 7 .35 (d, J=6.2 Hz, 1H), 7.39 (d, J=5.9 Hz, 1H).

MS (electrospray ionization (ESI)) mass/charge: 373 [M+H] ⁺

[0066] Example 1

Preparation of a crystal of compound (I)

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

X-ray powder diffraction peak (reflection method, 2θ±0.2°): 9.0°, 11.1°, 14.5°, 18.1°, 20.0°, 21.9°, 23.6°, 24 ,4°, 24.9°, 28.5°

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

[0067] Example 2

Preparation of type A crystal of compound (I) monohydrochloride

101 mg of compound (I) was added to a screw cap test tube. 0.2 ml of 1.5 M hydrochloric acid was added here and dissolved. 1.8 ml of IPA was added here, irradiated with ultrasonic waves, and then stirred with a stirrer at 40°C for 24 hours. After stirring at room temperature for an additional 1 hour, the sample was collected by filtration (0.2 μm), washed with 0.5 ml IPA/water (9/1 v/v) and air dried under a stream of nitrogen. The residue was dried at 70°C for about 1 hour to obtain the title crystal (103 mg).

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

¹³ C-NMR (100 MHz, solid state) δ (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 peaks (cm ^-1 ): 409, 587, 763, 976, 1428, 1493, 1688

The powder X-ray diffraction pattern of type A crystal of monohydrochloride of compound (I) prepared by the above method is shown in Figure 2, ¹³ C solid state NMR spectrum is shown in Figure 9, TG-DTA thermal analysis curves are shown in Figure 12, and Raman spectrum is shown in Figure 19.

[0068] Example 3

Preparation of type C crystal of monohydrochloride of compound (I)

1020 mg of compound (I) was added to a test tube with a screw cap. 1.5 equivalents (353 μl) of hydrochloric acid was dissolved in 20 ml of methanol and this solution was added to the sample. The sample was stirred with a stirrer at room temperature for 2 days. The sample was collected on a filter by filtration (0.2 μm). The resulting solid was dried under reduced pressure for about 2 hours, then dried at 70°C for 1 hour to obtain the title crystal (1048 mg).

X-ray powder diffraction peak (transmission method, 2θ±0.2°): 6.0°, 7.7°, 9.7°, 11.4°, 15.8°, 16.9°, 18.1°, 23 ,2°, 25.4°, 27.6°

¹³ C-NMR (100 MHz, solid state) δ (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 X-ray powder diffraction pattern of the C-type crystal of the monohydrochloride of compound (I) obtained by the above method is shown in Figure 4, ^{the 13} C solid-state NMR spectrum is shown in Figure 11, and the TG-DTA thermal analysis curves are shown in Figure 14.

[0069] Example 4

Preparation of type B crystal of monohydrochloride of compound (I).

303 mg of the hydrochloride crystals obtained in Example 3 were added to a platinum crucible and heated at 160°C for 15 minutes to obtain the title crystal (293 mg).

X-ray powder diffraction peak (transmission method, 2θ±0.2°): 6.3°, 9.7°, 10.1°, 17.9°, 19.0°, 19.4°, 23.4°, 26 ,3°, 27.3°, 32.0°

¹³ C-NMR (100 MHz, solid state) δ (ppm): 162.0, 160.1, 153.8, 151.1, 133.4, 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 type B crystal of compound (I) monohydrochloride obtained by the above method is shown in Figure 3, ¹³ C solid-state NMR spectrum is shown in Figure 10, and TG-DTA thermal analysis curves are shown in Figure 13.

[0070] Example 5

Preparation of type D crystal of monohydrochloride of compound (I)

227 mg of a mixture of hydrochloride crystals obtained in examples 2 and 3 and 8 ml of ethanol were added to a test tube with a screw cap. The mixture was stirred with a stirrer at 65°C. After about 1 hour, the mixture was irradiated with ultrasonic waves and stirred at the same temperature for one day. The sample was collected on a filter by filtration (0.2 µm) to obtain the title crystal (203 mg).

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

The powder X-ray diffraction pattern of type D crystal of compound (I) monohydrochloride obtained by the above method is shown in Figure 5, and the TG-DTA thermal analysis curves are shown in Figure 15.

[0071] Example 6

Preparation of type E crystal of monohydrochloride of compound (I)

108 mg of hydrochloride crystals obtained in example 2 and 5 ml of acetonitrile were added to a test tube with a screw cap. The mixture was stirred with a stirrer at 60°C for 24 hours, and the sample was collected on a filter by filtration (0.2 μm). The resulting solid and 5 ml of acetonitrile were again added to a screw cap test tube and stirred with a stirrer at 60°C for 24 hours. The sample was collected on the filter by filtration (0.2 μm) under a stream of nitrogen to obtain the title crystal (89.7 mg).

X-ray powder diffraction peak (transmission method, 2θ±0.2°): 6.4°, 11.3°, 15.7°, 18.0°, 19.2°, 22.8°, 24.6°, 25 ,4°, 26.0°, 27.3°

The X-ray powder diffraction pattern of the E type crystal of compound (I) monohydrochloride obtained by the above method is shown in Figure 6, and the TG-DTA thermal analysis curves are shown in Figure 16.

[0072] Example 7

Preparation of type F crystal of monohydrochloride of compound (I)

101 mg of hydrochloride crystals obtained in example 2 and 5 ml of ethanol were added to a test tube with a screw cap. The mixture was stirred with a stirrer at 60°C for 24 hours. The sample was collected on a filter by filtration (0.2 μm). The resulting solid and 5 ml of ethanol were again added to the screw cap test tube and stirred with a stirrer at 60°C for 4 hours. The sample was collected on a filter by filtration (0.2 μm) to obtain the title crystal (75.0 mg).

X-ray powder diffraction peak (transmission method, 2θ±0.2°): 5.9°, 7.3°, 9.3°, 10.7°, 13.8°, 15.6°, 16.4°, 18 ,7°, 25.1°, 26.8°

The powder X-ray diffraction pattern of the F type crystal of the monohydrochloride of compound (I) obtained by the above method is shown in Figure 7, and the TG-DTA thermal analysis curves are shown in Figure 17.

[0073] Example 8

Preparation of the monohydrobromide crystal of compound (I)

933 mg of compound (I) was added to a screw cap test tube. 1.5 equivalents (434 μl) of hydrobromic acid was dissolved in 20 ml of methanol and this solution was added to the sample. The mixture was stirred with a stirrer at room temperature for 3 days. The sample was collected on a filter by filtration (0.2 μm) and dried at 60°C for 1 hour to obtain the title crystal (1111 mg).

X-ray powder diffraction peak (transmission method, 2θ±0.2°): 6.0°, 7.8°, 10.0°, 11.7°, 17.8°, 20.8°, 23.5°, 24 ,5°, 25.2°, 27.3°

The powder X-ray diffraction pattern of the monohydrobromide of compound (I) prepared by the above method is shown in Figure 8, and the TG-DTA thermal analysis curves are shown in Figure 18.

[0074] <Examples of pharmacological studies>

Measurement of acetylcholine (ACh) release in a primary culture system of rat septal neurons in the presence of NGF

(1) Primary culture of rat septal neurons

The septal region was isolated from Sprague-Dawley (SD) rats (Charles River Laboratories Japan, Inc.) at 18 days of gestational age and cultured. In particular, fetuses were removed under sterile conditions from pregnant rats under isoflurane anesthesia. The brain was removed from each fetus and immersed in ice-cold L-15 medium (11415-064, Thermo Fisher Scientific). The septal region was excised from the extracted brain under a stereoscopic microscope. The excised septal area was enzymatically digested in an enzyme solution containing 0.25% trypsin (15050-065, Thermo Fisher Scientific) and 0.01% DNase (D5025-150KU, Sigma) at 37°C for 30 minutes, thereby ensuring cell dispersion. In this case, the enzymatic reaction was stopped by adding inactivated horse serum (26050-088, Thermo Fisher Scientific). The enzyme-treated solution was centrifuged at 1000 rpm. for 3 minutes and the supernatant was removed. Medium in an amount of 10 ml was added to the resulting cell mass. The medium used was Dulbecco's modified Eagle's medium (044-29765, WAKO), supplemented with N2 supplement (17502-048, Thermo Fisher Scientific), 1 mM sodium pyruvate (11360-070, Thermo Fisher Scientific), and penicillin-streptomycin (15140 -1221, Thermo Fisher Scientific). The cells in the cell mass to which the medium was added were redispersed by gentle pipetting and then centrifuged again at 1000 rpm for 3 minutes and the supernatant was removed. The medium in an amount of 10 ml was added to the resulting cell mass and the cell dispersion was filtered through a 40 μm nylon mesh (Cell Strainer) to remove the cell mass, thereby obtaining a suspension of neuronal cells. The neuronal cell suspension was diluted with medium and 10% inactivated bovine serum (26140-079, Thermo Fisher Scientific) and supplemented with 10% inactivated horse serum. Thereafter, 100 μl/well of the suspension was seeded into a 96-well plate (354461, CORNING) pre-coated with poly-D-lysine, so that the initial culture density was 1.4 x 10 ⁵ cells/cm ² . After the seeded cells were cultured at 5% CO ₂ -95% air in a 37°C incubator for 2 days, the entire medium was replaced with 120 μl of fresh medium and the cells were then cultured for 5 days.

(2) Adding a connection

On the 7th day of cultivation, the compound was added as follows. A solution of the test compound in DMSO was diluted with media so that the concentration was 10 times higher than the final concentration. NGF (450-01, PEPRO TECH, INC.) was prepared at a concentration of 0.3 ng/mL. Each of these two solutions was added in an amount of 15 μl/well and the mixture was mixed well. The final DMSO concentration was 0.1% or less. Moreover, only DMSO and NGF were added to the control group.

(3) Measurement of ACh release

One day after addition of the compound, the amount of ACh released was measured by HPLC as follows. Warmed buffer was added at 100 μl/well per well after the medium was removed, and the buffer was immediately removed. After this, a buffer was added to which 10 μM choline, 10 μM physostigmine and 6 mM KCl were added at 120 μL/well. The buffer was prepared by adding 125 mM NaCl, 25 mM 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid, 1.2 mM _{KH2PO4 ,} 1.2 mM _MgSO4 , 2.2 mM _CaCl2 ( _2H2O ) and 10 mM glucose to sterilized water and the final pH of the solution was adjusted to 7.4. After incubating the buffer-added 96-well plate in 5% CO ₂ -95% air in an incubator at 37°C for 40 minutes, 80 μl of buffer was withdrawn. IPHC internal standard solution (5×10 ^-7 M) was added in an amount of 6 μl to the collected buffer and the buffer was transferred to an HPLC tube and analyzed by HPLC. The results are presented as the effect of each compound as a percentage (% of control) of the ACh concentration in the control group buffer, and the compound concentration in Comparative Example 1 showing a 20% increase over the ACh concentration in the control group buffer was 0.1 μM.

[0075] Measurement of choline acetyltransferase (ChAT) mRNA expression levels in the septal region of rats

(1) Connection introduction

Male SD rats (Charles River Laboratories Japan, Inc.) with a body weight of about 250 to 350 g were used in this study. The compound was dissolved in 0.01 mol/L hydrochloric acid and administered orally.

(2) Sampling

24 hours after compound administration, all brain tissue was collected under pentobarbital anesthesia. The medial septum was isolated from the whole brain on ice and frozen with liquid nitrogen and then stored at -80°C.

(3) Measurement of ChAT mRNA expression levels

The RNeasy Plus Mini kit (#74136: QIAGEN) was used for RNA purification in this study. RNA purification was performed using the method described in the kit. After RNA purification, total RNA concentration was measured using a QIAxpert instrument (QIAGEN). cDNA was synthesized using the SuperScript(R)VILO(TM) cDNA Synthesis Kit (#11754: Thermo Fisher Scientific). cDNA synthesis was performed using the method described in the kit. The synthesized cDNA was diluted 4 times with RNase-free water, and the diluted cDNA solution was used as a sample. Taqman Universal PCR Master Mix (#4304437: Thermo Fisher Scientific), Taqman Gene Expression Assay Kits (R), INVENTORIED (#4331182: Thermo Fisher Scientific), RNase-free water and cDNA solution were mixed in 10 µl , 1 μL, 4 μL, and 5 μL, respectively, and the resulting mixture was used as the sample solution to be measured. Quantitative polymerase chain reaction (qPCR) was performed using an ABI PRISM(R) 7900HT (Thermo Fisher Scientific) using a fluorescent probe method. Analysis was performed using SDS 2.4 (Thermo Fisher Scientific). The results were calculated by the percentage increase in the ChAT mRNA expression levels in the compound administration group of Comparative Example 1 compared with the ChAT mRNA expression levels in the vehicle administration group to 56.4% at 10 mg/kg.

[0076] Measuring the concentration of acetylcholine (ACh) in the cerebrospinal fluid (CSF) of rats

(1) Prerequisites

A correlation between increases and decreases in brain neurotransmitters and cerebrospinal fluid (CSF) neurotransmitters has been found in rodent studies, and a correlation has also been observed in humans (Lowe S et al . Psychopharmacology 219 (2012) 959-970). Accordingly, changes in CSF acetylcholine concentration were measured to determine changes in brain acetylcholine concentration with test compounds.

(2) Connection introduction

Male Fischer 344 rats (Charles River Laboratories Japan, Inc.) with a body weight of approximately 150 to 250 g were used in this study. The test compounds were administered orally to the rats once daily at 10 mg/kg for three days. The carrier medium used was 0.01 mol/L hydrochloric acid solution.

(3) Sampling

24 hours after administration of vehicle medium and test compounds, CSF was collected from the medullary cistern into a tube containing AChE inhibitors under pentobarbital anesthesia. The collected CSF was centrifuged at 3500 x g at 4°C for 10 minutes and the supernatant was collected. The collected supernatant was frozen using liquid nitrogen and then stored at -80°C.

(4) Measurement of Ach concentration using LC-MS

50 μL of acetylcholine-d9-chloride (ACh-d9) was added to 10 μL of CSF at a final concentration of 0.34 nmol/L as an internal standard. The mixture was pipetted and centrifuged at 1500 x g at 4°C for 10 minutes. The supernatant was collected and examined by LC/MS (NexeraX2 (MS), TSQ Altis (HPLC)), identifying Ach as corresponding to a precursor ion peak at m/c of 146.050 and a product ion peak at m/c , equal to 87.071, the concentration of ACh-d9 was determined as an internal standard by the precursor ion peak at a mass/charge value of 155.088 and the product ion peak at a mass/charge value of 87.000. The results were calculated by the percentage (% of control) of the ACh concentration in the cerebrospinal fluid (CSF) in the Comparative Example 1 administration group increased compared with the cerebrospinal fluid ACh concentration in the vehicle administration group to 156.8%.

[0077] Evaluation in a transgenic mouse with the P301S mutation expressing human tau protein

(1) Connection introduction

In this study, test compounds were administered orally once daily for three months to human tau-expressing P301S transgenic mice aged four to seven months. The carrier medium used was 0.01 mol/L hydrochloric acid solution.

(2) Sampling

On the first day of administration (at four months of age) and the day after the last administration, mice in the vehicle group and the test compound group were anesthetized with pentobarbital (50 mg/kg, i.p.) and perfused with PBS. After perfusion, the forebrain, including the medial septal region, was collected and fixed with 4% paraformaldehyde.

(3) Obtaining a coronal section of frozen brain

The collected forebrain, including the medial septal region, was immersed in 4% paraformaldehyde and shaken overnight. The dipping solution was replaced with a 7.5% sucrose solution. The resulting solution was immersed in a 7.5% sucrose solution and shaken overnight, and the immersion solution was replaced with a 15% sucrose solution, and the resulting solution was immersed in it and shaken overnight. The dipping solution was replaced with a 30% sucrose solution and the resulting solution was dipped and shaken overnight. Coronal 30-μm-thick frozen brain sections were obtained from the forebrain, including the medial septal region, using a microtome (Leica, SM2000R).

(4) Immunohistochemical study of cells positive for choline acetyltransferase (ChAT)

The resulting frozen brain coronal sections were stained with DAB (DAB PEROXIDASE SUBSTRATE KIT (Vector, SK-4100)) using anti-ChAT antibody (Santa Cruz, SC-20672) as the primary antibody. The slice image, including the medial region of the septum, as shown in "The mouse Brain in stereotaxic coordinates" (COMPACT THIRD EDITION, Keith BJ Franklin & George Paxinos), was obtained using a universal fluorescence microscope (KEYENCE, BZ-X710) and ChAT- positive cells near the main axis of the medial septal region were counted using BZ analysis software (KEYENCE). Results are presented as the percentage of ChAT-positive cells in the vehicle group and test compound group relative to the number of ChAT-positive cells at the time of initial administration (at four months of age). Data are given as mean ±SEM. Differences between the initial administration group and the vehicle-treated group (significant: *) were analyzed using an unpaired t test, as well as differences between the vehicle-treated group and the compound-treated group (significant: ^# ). analyzed using unpaired t test. A P value <0.05 was considered statistically significant. Statistical analyzes were performed using GraphPad Prism version 7.02. The results are presented in Table 1.

[0078] [Table 1]

Treatment group Ratio (%) of the number of ChAT-positive cells compared to that at initial administration Group at time of initial introduction 100.0±4.5 Group with introduction of carrier medium 83.0±5.8* Comparative Example 1 Compound Compound Introduction Group
(dose: 5 mg/kg) 105.3±4.3 ^#

[0079] Neuroprotective and restorative effects on cholinergic neurons in a rat model of hippocampal fimbria and fornix lesions

(1) Development of a rat model with lesions of the hippocampal fimbria and cerebral fornix

Male Sprague-Dawley rats (Charles River Laboratories Japan, Inc.) with a body weight of approximately 250 to 350 g were used in this study. The rats were anesthetized using a combination of three drugs: midazolam (2 mg/kg s.c.), medetomidine hydrochloride ( 0.15 mg/kg s.c.) and butorphanol tartrate (2.5 mg/kg s.c.), and fixed using a stereotactic brain apparatus (Narishige Co., Ltd.). The skull was exposed and a 5 mm wide hole was drilled into the skull from the midline 2 mm behind the bregma. A 4 mm wide blade was inserted into the bregma to a depth of 5.5 mm, cutting the fimbriae of the hippocampus and the cerebral fornix. After the bleeding stopped, the scalp was sutured. After surgery, the rat was returned to its cage and allowed to recover from anesthesia. In the sham group, a 5 mm wide hole was drilled into the skull from the midline 2 mm behind the bregma, but without inserting a blade.

(2) Connection introduction

The test compounds were administered to rats orally once daily from five days to nine days after surgery (Example 1: 10 mg/kg) or from seven days to fourteen days after surgery (Example 3: 3 mg/kg). The carrier medium used was 0.01 mol/L hydrochloric acid solution. In the sham group, the vehicle was administered orally once daily, similar to the test compound group.

(3) Sampling

Rats were anesthetized with pentobarbital and perfused transcardially with ice-cold PBS. After perfusion, the forebrain, including the medial septal region, was collected and immersed in 4% paraformaldehyde and shaken overnight. The dipping solution was replaced with a 7.5% sucrose solution. The resulting solution was immersed in a 7.5% sucrose solution and shaken overnight, and the immersion solution was replaced with a 15% sucrose solution, and the resulting solution was immersed in it and shaken overnight. The dipping solution was replaced with a 30% sucrose solution and the resulting solution was dipped and shaken overnight. Coronal 30-μm-thick frozen brain sections were obtained from the forebrain, including the medial septal region, using a microtome (Leica, SM2000R).

(4) Immunohistochemical study of cells positive for choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT)

The resulting frozen brain coronal sections were stained with DAB (DAB PEROXIDASE SUBSTRATE KIT (Vector, SK-4100)) using anti-ChAT antibody (Santa Cruz, SC-20672) or anti-VAChT antibody (Merck Millipore, ABN100) as primary antibody. The slice, including the medial septal region or hippocampus, as shown in “The mouse Brain in stereotaxic coordinates” (COMPACT THIRD EDITION, Keith BJ Franklin & George Paxinos) was imaged using a universal fluorescence microscope (KEYENCE, BZ-X710) and the number of ChAT-positive cells in the medial septal region or optical density (OD) of hippocampal VAChT was measured using BZ analysis software (KEYENCE). Results are presented as the percentage of the number of medial septal ChAT-positive cells or hippocampal OD VAChT in the vehicle group and test compound group relative to the number of medial septal ChAT-positive cells or hippocampal VAChT OD in the sham group. Data are presented as mean ±SEM. Differences between the vehicle-treated group and the compound-treated group (significant: ^# ) were analyzed using an unpaired t test. A P value <0.05 was considered statistically significant. Statistical analyzes were performed using GraphPad Prism version 7.02. The results are presented in Tables 2 and 3.

[0080] [Table 2]

Compound Number of ChAT-positive cells (%) at initial injection Number of ChAT-positive cells (%) in the group with the introduction of carrier medium Number of ChAT-positive cells (%) in the test compound group Comparative example 1 57.0±7.5 38.4±5.0 74.1±9.3 ^#

[0081] [Table 3]

Compound Hippocampal VAChT OD (%) at initial administration Hippocampal VAChT OD (%) in the vehicle group Hippocampal VAChT OD (%) in test compound group Comparative example 1 51.7±13.1 19.5±6.4 66.1±14.2 ^#

Claims (31)

1. Monohydrochloride or monohydrobromide 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4,5 ]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione represented by formula (I): . 2. Crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4,5]pyrimido [1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione represented by formula (I): , with diffraction peaks at diffraction angles (2θ±0.2°) 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 obtained using CuKα as the X-ray source. 3. Type A crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4, monohydrochloride 5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione represented by formula (I): , with diffraction peaks at diffraction angles (2θ±0.2°) 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 obtained using CuKα as the X-ray source. 4. Type B crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4, monohydrochloride 5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione represented by formula (I): , with diffraction peaks at diffraction angles (2θ±0.2°) 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 obtained using CuKα as the X-ray source. 5. Type C crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4, monohydrochloride 5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione represented by formula (I): , with diffraction peaks at diffraction angles (2θ±0.2°) 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 obtained using CuKα as the X-ray source. 6. Type D crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4, monohydrochloride 5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione represented by formula (I): , with diffraction peaks at diffraction angles (2θ±0.2°) 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 obtained using CuKα as the X-ray source. 7. Type E crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4, monohydrochloride 5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione represented by formula (I): , with diffraction peaks at diffraction angles (2θ±0.2°) 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 obtained using CuKα as the X-ray source. 8. Type F crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4, monohydrochloride 5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione represented by formula (I): , with diffraction peaks at diffraction angles (2θ±0.2°) 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 obtained using CuKα as the X-ray source. 9. Crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4,5] monohydrobromide pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione represented by formula (I): , with diffraction peaks at diffraction angles (2θ±0.2°) 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 obtained using CuKα as the X-ray source. 10. Type A crystal of 5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4, monohydrochloride 5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione represented by formula (I): , characterized by peaks at chemical shifts (δ±0.5 ppm) 164.0 ppm, 129.6 ppm. and 36.5 ppm in ¹³ C solid-state NMR spectrum with glycine (176.03 ppm) as external standard. 11. Crystal according to claim 10, characterized by peaks at chemical shifts (δ±0.5 ppm) 164.0 ppm, 162.5 ppm, 160.5 ppm, 153, 9 ppm, 151.6 ppm, 150.7 ppm, 133.6 ppm, 131.1 ppm, 129.6 ppm, 128.4 m .d., 126.9 ppm, 125.2 ppm, 123.7 ppm, 121.3 ppm, 120.3 ppm, 119.5 ppm ., 53.7 ppm, 52.0 ppm, 50.9 ppm, 44.7 ppm, 36.5 ppm and 22.6 ppm in ¹³ C solid-state NMR spectrum with glycine (176.03 ppm) as external standard. 12. Pharmaceutical composition having an activating effect on cholinergic neurons, containing a salt according to claim 1 or a crystal according to any of claims. 2-11 as an active ingredient and pharmaceutically acceptable additives.