An active intermediate ZT-1-PVP and its preparation method and application

Abstract

The present invention provides an active intermediate and a preparation method and application thereof. The active intermediate includes a derivative of huperzine A and polyvinylpyrrolidone (PVP). Furthermore, the derivative of huperzine A is ZT‑1, and the active intermediate is ZT‑1‑PVP. The present invention also provides a preparation method of ZT‑1‑PVP and its application in the preparation of medicines and foods for treating Alzheimer's disease. ZT‑1‑PVP can perfectly solve the instability problem of the oxime group in the ZT‑1 molecule, so that in the process of ZT‑1 drug formulation, whether from the storage of raw materials, the intermediate links from raw materials to preparations, or the preparation processes involving tablets, capsules, dispersants, granules, and the stable 2-year storage period of the final drug product, its instability problem has been perfectly solved.

Description

Technical Field

[0001] The present invention relates to an active intermediate in the application of preparations, and in particular to an active intermediate ZT-1-PVP and a preparation method thereof, and application thereof in the treatment of Alzheimer's disease. Background Art

[0002] Huperzine A is an alkaloid extracted from the phenolic part of the Chinese herbal medicine Huperzia serrata. It is an acetylcholinesterase inhibitor (AChEI) and can be directly used in clinical treatment of Alzheimer's Disease (AD).

[0003] ZT-1 (hupervan) is a derivative of huperzine A, and its chemical name is [5R-(5α,9β,11E)-5-[[(5-chloro-2-hydroxy-3-methoxyphenyl)methylene]amino]-11-ethylidene-5,6,9,10-tetrahydro-7-methyl-5,9-bridged methylenecyclooctane[b]pyridin-2(1H)-one; or: N-(2-hydroxy-3-methoxy-5-chloro-benzylidene)-huperzine A.

[0004] When Huperzine A is used directly to treat AD, the therapeutic dose window of Huperzine A is narrow, the dosage is limited, and the side effects are large. The results of European clinical phase I and II trials conducted by the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, showed that after the derivative ZT-1 was prepared, the therapeutic dose window for treating AD was greatly expanded, and the side effects were also greatly reduced.

[0005] Phase I and II clinical trials conducted in Europe also proved that after ZT-1 is absorbed into the human body and enters the blood, it is automatically hydrolyzed into the active ingredient, huperzine A, through a non-enzymatic reaction, and exerts the pharmacological effect of an acetylcholinesterase inhibitor, which significantly increases the acetylcholine (Ach) content in the synaptic cleft within the distribution area, thereby enhancing neuronal excitation conduction, strengthening the excitatory effect of the learning and memory brain areas, and playing a role in improving cognitive function, enhancing memory retention and promoting memory retrieval.

[0006] There are oxime groups in the molecular structure of ZT-1 formed by amine-aldehyde condensation. Under normal circumstances, oxime groups are particularly easy to decompose in acidic and humid environments, which is not conducive to the preservation of ZT-1 drugs when used as raw materials. It is also not conducive to the stability of ZT-1 products during the preparation process of tablets, capsules, dispersants, granules, etc., and cannot meet the requirement that the finished ZT-1 tablets, capsules, dispersants, and granules have a stable storage period of 2 years. Summary of the invention

[0007] In view of the above-mentioned defects of the prior art, the object of the present invention is to provide an active intermediate of a huperzine A derivative that can be stably stored, and a preparation method and application thereof.

[0008] In order to achieve the above object, the technical solution of the present invention is:

[0009] An active intermediate, comprising a derivative of huperzine A and polyvinylpyrrolidone (PVP) as shown in formula (I), wherein the general molecular formula of the polyvinylpyrrolidone is (C6H9NO)n;

[0010]

[0011] Wherein, n is greater than or equal to 1.

[0012] Furthermore, the derivative of Huperzine A is ZT-1 as shown in formula (II); the active intermediate is named ZT-1-PVP;

[0013]

[0014] Furthermore, the content of ZT-1 accounts for 0.001% to 90% (w / w), 0.01% to 50% (w / w), 0.05% to 30% (w / w), 0.25% to 10% (w / w), 1% to 8% (w / w) or 2% to 6% of the total weight of ZT-1-PVP; the value of n is 143 to 5143, 186 to 2057, 215 to 1543, 286 to 1028, 358 to 514 or 396 to 486; the K value of the polyvinyl pyrrolidone is 10 to 360, 13 to 144, 15 to 108, 20 to 72, 25 to 36 or 28 to 32.

[0015] It should be noted that when the content of ZT-1 accounts for 0.001% to 90% (w / w) of the total weight of ZT-1-PVP, the polyvinyl pyrrolidone macromolecule has the function of protecting ZT-1 from molecular decomposition in acid, alkali, water and high temperature environments; when the content of ZT-1 is 0.01% to 50% (w / w) to 0.05% to 30% (w / w), this anti-decomposition protection can be effectively achieved. Under normal circumstances, the content of ZT-1 is 0.25% to 10% (w / w) to 1% to 8% (w / w), and the protective effect of polyvinyl pyrrolidone is best when it is 2% to 6% (w / w).

[0016] Regarding K value, polyvinyl pyrrolidone is divided into four grades according to its average molecular weight, which is usually expressed as K value. Different K values ​​represent the corresponding PVP average molecular weight range. K value is actually a characteristic value related to the relative viscosity of polyvinyl pyrrolidone aqueous solution, and viscosity is a physical quantity related to the molecular weight of polymer. Therefore, K value can be used to characterize the average molecular weight of polyvinyl pyrrolidone. Generally, the larger the K value, the greater the viscosity and the stronger the adhesion.

[0017] In some embodiments, the huperzine A derivative ZT-1 forms the active intermediate with polyvinyl pyrrolidone as shown in formula (I) through a non-chemical bond.

[0018] A method for preparing the active intermediate as described above, wherein the preparation steps include:

[0019] Dissolving polyvinyl pyrrolidone (PVP) in an organic solvent, and adding a pH adjuster to adjust the pH of the solution to 6.5-7.0 to prepare a polyvinyl pyrrolidone solution;

[0020] Add an appropriate amount of ZT-1 to the polyvinyl pyrrolidone (PVP) solution; stir evenly and then dry under reduced pressure to obtain ZT-1-PVP.

[0021] In some embodiments, the organic solvent is selected from one or more of dichloromethane, chloroform, methanol, ethanol, acetone, and ethyl acetate; the pH adjuster is selected from one or more of 0.1% KOH methanol solution, 0.1% ammonia methanol solution, and 0.5% HCl methanol solution.

[0022] In some embodiments, the synthesis of ZT-1 includes, but is not limited to, a "de novo" approach and a "one-pot" approach;

[0023] The steps of the "from scratch" method are:

[0024] First, 5-chloro-o-vanillin (5-Cl-o-Vanillin) is prepared, and then huperzine A and 5-chloro-o-vanillin are poured into an alcohol solvent to form a suspension solution, and an amine-aldehyde condensation reaction is performed to obtain ZT-1;

[0025] The steps of the "one-pot" synthesis method are:

[0026] o-Vanillin, huperzine A and dithionyl chloride are mixed in an appropriate molar ratio; the molar ratio of o-vanillin to huperzine A is (1.005-1.2):1, and the best is 1.05:1; the molar ratio of huperzine A to dithionyl chloride is 1:(5-20), and the best is 1:10; the best molar ratio of o-vanillin, huperzine A and dithionyl chloride is 1.05:1:10; after the raw materials are mixed in proportion, they are stirred and reacted at 10-30° C. to obtain ZT-1.

[0027] In some embodiments, the source of Huperzine A includes natural extraction and chemical synthesis;

[0028] The natural extraction steps are as follows: collecting the whole plant or the aerial part of Huperzine serrata, drying and crushing it, and then putting it into an extraction container, soaking it with a 0.5%-1% dilute HCl aqueous solution (3-4 times), collecting and combining the acid water extract, adding ammonia water or a 5%-10% NaOH aqueous solution for alkalization, and extracting it with dichloromethane or chloroform; combining the extracts, concentrating and recovering the solvent to obtain a crude extract of Huperzine serrata alkaloids; after multiple steps of solvent extraction and column chromatography separation and purification steps, finally performing silica gel or alumina column chromatography, collecting the eluate containing Huperzine A, and concentrating it to obtain crude Huperzine A crystals; and obtaining pure Huperzine A after multiple recrystallization with ethanol or other organic solvents;

[0029] The chemical synthesis steps are: firstly obtain racemic (±) huperzine A through chemical synthesis, and then obtain L-huperzine A through chemical resolution; the huperzine A obtained through chemical synthesis must meet the conditions that the content of (-) huperzine A is greater than 99% and the content of (+) huperzine A is less than 0.5% after HPLC testing, so that it can be used to prepare ZT-1-PVP active intermediate.

[0030] In some embodiments, the preparation steps of 5-chloro-o-vanillin in the "from scratch" method are:

[0031] Dissolve o-vanillin in a haloalkane solvent, cool to below 0°C, add 3 times the molar ratio of thionyl chloride dropwise while keeping the reaction solution below 0°C, continue stirring at below 0°C for 2 hours, then warm to room temperature and continue reacting for more than 3 hours; evaporate the solvent and excess unreacted thionyl chloride under vacuum, wash the resulting crude material with petroleum ether for more than 2 times, and then recrystallize with a mixed solvent of ethyl acetate / petroleum ether (1:5) for more than 1 time to obtain 5-chloro-o-vanillin with a melting point of 116-118°C;

[0032] The halogenated alkane solvent includes but is not limited to CH2Cl2;

[0033] The ZT-1 obtained by the "from scratch" method is stirred at room temperature for more than 1 day until the amine-aldehyde condensation reaction is completed, and 1 / 3 to 2 / 3 volume (preferably 1 / 2 volume) of the solvent is evaporated under reduced pressure, crystallized, and filtered; the filtered product is vacuum dried and recrystallized for more than two times to obtain ZT-1 crystals with a melting point of 202-204°C;

[0034] The ZT-1 obtained by the "one-pot" method is obtained by reducing pressure to recover excess thionyl chloride after reaching the end point of the reaction, adding ethanol for recrystallization 2 or more times, and the obtained ZT-1 crystals have a melting point of 202-204°C.

[0035] A drug containing the active intermediate as claimed above, wherein the drug dosage forms include but are not limited to tablets, capsules, oral dispersions, oral granules and solution preparations.

[0036] Use of the active intermediate as described above in preparing medicines or foods for treating Alzheimer's disease and / or myasthenia gravis.

[0037] Furthermore, the active intermediate plays a role in treating Alzheimer's disease and / or myasthenia gravis by inhibiting the activity of acetylcholinesterase.

[0038] A use of the above active intermediate in preparing medicines or foods for treating cholinergic nervous system dysfunction.

[0039] Beneficial effects of the present invention:

[0040] The active intermediates can perfectly solve the instability problem of the oxime group in the ZT-1 molecule, so that in the process of ZT-1 drug development, from the storage of raw materials, the intermediate links from raw materials to preparations, to the preparation processes such as tablets, capsules, dispersants, granules, and a stable 2-year storage period, the stability of ZT-1 has been perfectly solved.

[0041] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is the ultraviolet spectrum of ZT-1 in Example 3.

[0043] Figure 2 This is the circular dichroism spectrum of ZT-1 in Example 3.

[0044] Figure 3 This is the infrared spectrum of ZT-1 in Example 3.

[0045] Figure 4 is the ZT-1 in Example 3 1 H NMR ( 1 H NMR) and 13 C NMR ( 13 C NMR) data assignment diagram.

[0046] Figure 5 This is the mass spectrum of ZT-1 in Example 3.

[0047] Figure 6 This is the X-ray diffraction structure diagram of ZT-1 in Example 3.

[0048] Figure 7This is the HPLC analysis chart of the ZT-1 sample in Experiment 1 of Example 6 after being placed in a 0.5% HCl medium for 24 hours.

[0049] Figure 8 This is the HPLC analysis chart of the ZT-1 sample in Experiment 2 of Example 6 after being placed in a 0.5% NaOH medium for 1 hour.

[0050] Fig. 9 This is the HPLC analysis chart of the ZT-1-PVP sample in Experiment 1 of Example 7 after it was placed in a distilled water medium for 24 hours.

[0051] Fig.10 This is the HPLC analysis chart of the ZT-1-PVP sample in Experiment 2 of Example 7 after being placed in a 0.5% HCl medium for 24 hours.

[0052] Fig.11 This is the HPLC analysis chart of the ZT-1-PVP sample in Experiment 3 of Example 7 after being placed in rat plasma medium for 24 hours.

[0053] Fig.12 This is a process flow chart for preparing ZT-1 granules using ZT-1-PVP particles in Example 15.

[0054] Fig.13A This is the blood concentration curve of ZT-1 in the human body after administration of different doses of ZT-1 in Section 4 of Example 16.

[0055] Fig. 13B This is the blood concentration curve of the active ingredient Huperzine A in vivo after administration of different doses of ZT-1 in Section 4 of Example 16.

[0056] Fig.14A This is the average blood concentration curve of ZT-1 on the first day of the DEB-02-ZT-03 clinical trial conducted in Section 4 of Example 16 with repeated doses (from 0.5 to 1.5 mg / day, oral administration for 14 days).

[0057] Fig. 14B This is the average blood concentration curve of huperzine A on the first day of the DEB-02-ZT-03 clinical trial conducted in Section 4 of Example 16 with repeated doses (from 0.5 to 1.5 mg / day, oral administration for 14 days).

[0058] Fig. 14C This is the average blood concentration curve of ZT-1 on the 14th day of the DEB-02-ZT-03 clinical trial conducted in Section 4 of Example 16 with repeated doses (from 0.5 to 1.5 mg / day, oral administration for 14 days).

[0059] Fig.14D This is the average blood concentration curve of huperzine A on the 14th day of the DEB-02-ZT-03 clinical trial conducted in Section 4 of Example 16 with repeated doses (from 0.5 to 1.5 mg / day, oral administration for 14 days).

[0060] Fig.15 This is a graph showing the relationship between ZT-1 dose and acetylcholinesterase inhibition rate in the test of erythrocyte acetylcholinesterase activity and plasma butyrylcholinesterase activity in Section 5.1.1 of Example 16.

[0061] Fig.16 This is the concentration-effect relationship diagram between the blood concentration of huperzine A and the erythrocyte acetylcholinesterase activity in Section 5.1.3 of Example 16.

[0062] Fig.17A This is the result of the test on attention (simple reaction time) in Section 5.1.4 of Example 16, which shows the effect of ZT-1 on cognitive deficits induced by scopolamine in volunteers in the DEB-02-ZT-01 Part 2 clinical trial. Donepezil is the donepezil group and Placebo is the placebo group.

[0063] Fig. 17B This is the result graph of the situational secondary reproduction test (immediate word recall-correct recall) in Section 5.1.4 of Example 16, which shows the effect of ZT-1 on cognitive deficits induced by scopolamine in volunteers in the DEB-02-ZT-01 Part 2 clinical trial. Donepezil is the donepezil group and Placebo is the placebo group.

[0064] Fig.18 This is a graph of changes in the AD Assessment Scale Cognitive Score (ADAS-cog) values ​​in Section 3.1 of Example 17.

[0065] Fig.19 This is a graph of the response rates of each test group in Section 3.1 of Example 17 when the AD Assessment Scale Cognitive Part Score (ADAS-cog) value changes.

[0066] Fig. 20 This is a graph of changes in the Mini-Mental State Examination (MMSE) values ​​in Section 3.2 of Example 17.

[0067] Fig.21 This is a graph of the response changes corresponding to the Neuropsychiatric Inventory Questionnaire (NPI-Q) value of “serious” in Section 3.3 of Example 17.

[0068] Fig. 22This is a graph of the response changes of the Neuropsychiatric Inventory Questionnaire (NPI-Q) value corresponding to "sadness" in Section 3.3 of Example 17.

[0069] Fig.23 Schematic diagram of ZT-1-PVP, (I) is the general chemical formula of polyvinyl pyrrolidone, and (II) is the chemical structural formula of ZT-1.

[0070] Description of the drawings: Donepezil is the donepezil group, and Placebo is the placebo group. DETAILED DESCRIPTION

[0071] In order to make the technical means, creative features, objectives and effects of the invention easier to understand, the invention is further described below with reference to specific illustrations. However, the invention is not limited to the following implementation cases.

[0072] It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with this technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantive technical significance. Any structural modification, change in proportion or adjustment of size, without affecting the effects and purposes that can be achieved by the present invention, should still fall within the scope of the technical contents disclosed by the present invention.

[0073] Example 1 Preparation of ZT-1 by "De novo" method

[0074] 1. Dissolve 15.2 g (0.1 mol) of o-vanillin in 50 ml of CH2Cl2, cool to 0°C, add 35.7 g (0.3 mol) of thionyl chloride dropwise while keeping the reaction solution at 0°C, continue stirring at 0°C for 2 hours, then warm to room temperature and continue reacting for 3 hours; evaporate the solvent and excess unreacted thionyl chloride under vacuum decompression, wash the resulting crude material with petroleum ether twice, and then recrystallize twice with a mixed solvent of ethyl acetate / petroleum ether (1:5) to obtain 5-chloro-o-vanillin with a melting point of 116-118°C, with a yield of 66.5%; yield = (actual yield / theoretical yield) x 100%, the yields below are all calculated according to this formula.

[0075] 2. Pour 500 mg (207 mmol) of huperzine A and 401 mg (215 mmol) of 5-chloro-o-vanillin into 40 ml of anhydrous ethanol to form a suspension solution; stir at room temperature for 48 hours to cause an amine-aldehyde condensation reaction; after the reaction is completed, evaporate half the volume of the solvent on a rotary evaporator under reduced pressure, place and cool to crystallize, and filter to obtain a crude ZT-1 product; the vacuum-dried product is recrystallized from anhydrous ethanol twice to obtain 602 mg of ZT-1 yellow solid crystals with a melting point of 202-204°C, and the yield is 70.97%.

[0076] Example 2 Preparation of ZT-1 by "one-pot" method

[0077] 1.6469 g (10.8328 mmol) of o-vanillin, 2.5 g (10.3169 mmol) of huperzine A, and 12.28 g (103.169 mmol) of thionyl chloride were mixed in a raw material molar ratio of 1.05:1:10, stirred at 10-30°C for 12 hours, and a sample of the reaction solution was taken and analyzed by high-performance thin-layer chromatography or high-performance liquid chromatography to determine the reaction endpoint. Excess thionyl chloride was recovered under reduced pressure, and ethanol was added for recrystallization twice to obtain 3.12 g (yield 73.67%) of ZT-1 yellow solid crystals with a melting point of 202-204°C.

[0078] Example 3 Spectroscopic Data Determination of ZT-1

[0079] 1. UV spectrum of ZT-1

[0080] 2 mg ZT-1 was completely dissolved in 100 ml methanol and the UV spectrum was measured using a Shimazu UV-260 spectrophotometer. The data are shown in Table 1 and Figure 1 .

[0081] Table 1 UV spectrum absorption peak and absorbance of ZT-1

[0082] serial number Wavelength [nm] Absorbance Absorption peak 1 300.0 0.2859 Absorption peak 2 231.00 0.6970 Absorption peak 3 200.00 0.8962

[0083] 2. Circular dichroism spectrum (CD) of ZT-1

[0084] 4.813 mg ZT-1 was completely dissolved in 5 ml methanol and the circular dichroism (CD) spectrum was measured using a JASCO 500-C instrument. The data are shown in Table 2 and Figure 2 .

[0085] Table 2 Circular dichroism (CD) peak and valley wavelengths and molar ellipticity [θ] values ​​of ZT-1

[0086] Wavelength [nm] [θ] 231 71406.9 268 -1179.9 285 7867.4 319 -22692.8

[0087] 3. Infrared spectrum (IR) of ZT-1

[0088] According to the ratio of ZT-1 to potassium bromide KBr of 0.25% to 0.5%, the two were mixed and pressed into tablets, and the infrared spectrum (IR) of the ZT-1 sample was measured using a Perkin-Elmer 983 infrared spectrometer. The data are shown in Table 3 and Figure 3 .

[0089] Table 3 Infrared spectrum (IR) absorption peaks of ZT-1

[0090]

[0091]

[0092] 4. H NMR spectrum of ZT-1 ( 1 H NMR) and carbon nuclear magnetic resonance spectroscopy ( 13 C NMR)

[0093] ZT-1 was dissolved in deuterated chloroform (CDCl3) to prepare a solution, and trimethylsilane (TMS) was used as the internal standard to measure the molecular weight of ZT-1. 1 H NMR and 13 C NMR data of ZT-1 1 H NMR and 13 C NMR is shown in Table 4 below:

[0094] Table 4ZT-1 1 H NMR and 13 C NMR data table (0.20mol / L, in CDCl3)

[0095] Location <![CDATA[δ H ]]> <![CDATA[δ C ]]> Location <![CDATA[δ H ]]> <![CDATA[δ C ]]> 2 165.3 12 5.04q,J=6.8 114.7 3 6.32d, J=9.5 117.7 13 1.54d, J=6.8 12.5 4 7.09d, J=9.5 140.2 14 1.56s 22.7 4a 119.3 1’ 117.8 5 64.8 2’ 153.1 6 2.73d, J = 16.1 46.5 3’ 149.8 2.26d,J=16.1 4’ 6.81d,J=2.1 114.6 7 133.3 5’ 122.2 8 5.41brd,J=4.5 124.3 6’ 6.80d, J=2.1 122.1 9 3.61brs 32.8 -N=CH- 8.17s 165.2 10 2.95dd,J=4.9;16.6 35.0 <![CDATA[-OCH3]]> 3.83s 56.2 2.75d, J=16.6 >NH 13.34 10a 144.4 -OH 14.40 11 138.3

[0096] The code numbers of the positions in Table 4 are the same as Figure 4 The coded numbers in the ZT-1 molecular formula correspond to .

[0097] 5. Mass spectrometry (MS) data of ZT-1

[0098] The mass spectrum (MS) data of ZT-1 was measured using a JMSO-D300S high-resolution mass spectrometer. The mass spectrum (MS) data of ZT-1 are shown in Table 5 and Figure 5 .

[0099] Table 5 Mass Spectrum (MS) Data of ZT-1

[0100] Mass Composition Delta(mmu) <![CDATA[M + ]]> 410.1405 <![CDATA[C 23 H 23 ClN2O3]]> -0.7 M-1 409.1324 <![CDATA[C 23 H 22 ClN2O3]]> -0.5 M-15 395.1171 <![CDATA[C 22 H 20 ClN2O3]]> -0.9 M-55 355.0852 <![CDATA[C 19 H 16 ClN2O3]]> -0.3 M-157 253.1350 <![CDATA[C 16 H 17 N2O]]> -0.9 M-171 239.1183 <![CDATA[C 15 H 15 N2O]]> 0.1 M-180 226.1257 <![CDATA[C 15 H 16 NO]]> -2.5

[0101] 6. X-ray diffraction analysis of ZT-1

[0102] X-ray diffraction analysis of ZT-1 crystals was performed using a Rigaku AFC7R X-ray diffraction analyzer, graphite monochromatic Mo-Kα radiation and a 12kW rotating anode X-ray generator. The data from the X-ray diffraction analysis determined the absolute conformation of the ZT-1 molecule. The X-ray diffraction analysis data showed that: (1) the molecular structure of ZT-1 retains a complete huperzine A molecular fragment; (2) the stereo conformation of the C=N double bond is an E-type conformation; (3) the X-ray diffraction analysis data of ZT-1 is completely consistent with other spectral data of ZT-1.

[0103] The ZT-1 structure obtained by X-ray diffraction analysis is shown in Figure 6 .

[0104] Example 4 Preparation of active intermediate ZT-1-PVP, method 1

[0105] Dissolve 50g PVP (Polyvinylpyrrolidone, K28-32, with an average molecular weight of 44000-54000) in 250ml dichloromethane, and adjust the pH of the solution to 6.5-7.0 with 0.1% KOH methanol solution to prepare a dichloromethane solution of PVP (K28-32). Add 1g ZT-1 to the dichloromethane solution of PVP (K28-32); stir evenly and dry under reduced pressure to obtain 50.592g (yield, 99.20%) of active intermediate ZT-1-PVP particles.

[0106] Example 5 Preparation of active intermediate ZT-1-PVP, method 2

[0107] Dissolve 100g PVP (Polyvinylpyrrolidone, K28-32) in 300ml chloroform, adjust the pH of the solution to 6.5-7.0 with 1% ammonia methanol solution to prepare a chloroform solution of PVP (K28-32). Add 2g ZT-1 to the chloroform solution of PVP (K28-32); stir evenly and dry under reduced pressure to obtain 101.4084g (yield, 99.42%) of active intermediate ZT-1-PVP particles.

[0108] Example 6 Stability test of ZT-1 in 0.5% HCl aqueous solution and 0.5% NaOH aqueous solution

[0109] Test 1: 0.5% HCl aqueous solution medium

[0110] 5 mg ZT-1 (purity greater than 99.5%) samples were placed in four 10 ml conical flasks, and 0.5% HCl aqueous solution was added. After vigorous shaking for a while, the samples were placed at room temperature for 1 hour, 3 hours, 6 hours, and 24 hours respectively; the samples at the time points were extracted with dichloromethane, and the method was as follows: 10 ml of dichloromethane was added each time, and a total of three extractions were performed; the extracts were combined, and then anhydrous magnesium sulfate was added as a desiccant for drying; the desiccant was filtered to remove the desiccant, and the filtrate was combined and the sample was concentrated by vacuum evaporation. ZT-1 and its impurity content were tested by high performance liquid chromatography (HPLC) using the normalization method. The test conditions of HPLC were as follows: Waters 2487 (UV) high performance liquid chromatograph was used, the chromatography column was Turner Kromasil Silica 5μ250x 4.6mm, the mobile phase was: CH2Cl2:EtOAc:CH3OH 63:30:7, the flow rate was 1 ml / min, and the detection wavelength was 300 nm. The HPLC analysis results of the samples placed in 0.5% HCl medium for 24 hours are shown in Table 6 and Figure 7 .

[0111] Table 6 HPLC analysis results of ZT-1 samples placed in 0.5% HCl aqueous solution for 24 hours

[0112]

[0113] Test 2: 0.5% NaOH aqueous solution medium

[0114] The ZT-1 sample was subjected to the same procedures as in Example 6, Experiment 1, except that the solution added was a 0.5% aqueous solution of NaOH. The HPLC analysis results of the sample after the 0.5% aqueous solution of NaOH was left for 1 hour are shown in Tables 7 and Figure 8 .

[0115] Table 7 HPLC analysis results of ZT-1 samples placed in 0.5% NaOH aqueous solution for 1 hour

[0116]

[0117] The content of ZT-1 at various time points in the above-mentioned Test 1 and Test 2 is shown in Table 8.

[0118] Table 8 Content of ZT-1 in acidic aqueous solution (pH 1-2) and alkaline aqueous solution (pH 13-14)

[0119]

[0120] From the analysis of the results in Table 8, the following conclusions can be drawn: the ZT-1 sample is unstable in the 0.5% HCl aqueous solution medium. The 24-hour sampling analysis shows that 65.5027% of ZT-1 has been converted into intermediates, and the content of ZT-1 itself is only 30.0832%; it is even more unstable in the 0.5% NaOH aqueous solution medium. The 1-hour sampling analysis shows that the content of ZT-1 itself is only 23.7645%, and most of ZT-1 has been converted into huperzine A, and the content of huperzine A is as high as 75.97%.

[0121] Example 7 Stability test of active intermediate ZT-1-PVP in different media

[0122] Test 1 Stability test of active intermediate ZT-1-PVP in distilled water medium

[0123] 289 mg ZT-1-PVP particle sample (equivalent to 5 mg ZT-1) was placed in 4 10 ml conical flasks, and 2.5 ml distilled water was added. After vigorous shaking for a while, the samples were placed at room temperature for 1 hour, 3 hours, 6 hours, and 24 hours respectively; the samples at the time points were extracted with dichloromethane, and the method was as follows: 10 ml dichloromethane was added each time, and a total of three extractions were performed; the extracts were combined, and then anhydrous magnesium sulfate was added as a desiccant for drying; the desiccant was filtered to remove the desiccant, and the filtrate was combined and the sample was concentrated by vacuum evaporation. ZT-1 and its impurity content were tested by high performance liquid chromatography (HPLC) using the normalization method. The test conditions of HPLC were as follows: SHIMADZU instrument was used, the chromatography column was Alltima C18 5μ, 150x 4.6mm, the mobile phase was: CH2Cl2: EtOAc: CH3OH 63: 30: 7, the flow rate was 1 ml / min, and the detection wavelength was 300 nm. The content of ZT-1 after being placed in the distilled water medium for 1 hour, 3 hours, 6 hours, and 24 hours is shown in Table 9 below.

[0124] Table 9 ZT-1-PVP content of ZT-1 in distilled water medium for 1 hour, 3 hours, 6 hours, and 24 hours

[0125] Sampling time (hours) 0 1 3 6 24 ZT-1 content (ZT-1-PVP in distilled water) 99.70％ 99.13％ 99.76％ 99.35％ 99.10％

[0126] The HPLC analysis results of the samples taken from ZT-1-PVP in distilled water after 24 hours are shown in Tables 10 and Fig. 9 .

[0127] Table 10 HPLC analysis results of samples taken from ZT-1-PVP in distilled water medium at 24 hours

[0128]

[0129] Test 2 Stability test of active intermediate ZT-1-PVP in 0.5% HCl aqueous solution medium

[0130] The conditions and operation were the same as those in Example 7, Experiment 1, except that 2.5 ml of 0.5% HCl aqueous solution was added to the ZT-1-PVP particle sample. The content of ZT-1 after the 0.5% HCl aqueous solution sample with ZT-1-PVP was left for 1 hour, 3 hours, 6 hours, and 24 hours is shown in Table 11.

[0131] Table 11 ZT-1-PVP content in 0.5% HCl medium for 1 hour, 3 hours, 6 hours, and 24 hours

[0132] Sampling time (hours) 0 1 3 6 24 ZT-1 content (ZT-1-PVP in 0.5% HCl) 99.70％ 96.49％ 94.62％ 94.47％ 92.56％

[0133] The HPLC analysis results of ZT-1-PVP samples taken at 24 hours in 0.5% HCl aqueous solution are shown in Table 12 and Fig.10 .

[0134] Table 12 HPLC analysis results of ZT-1-PVP samples taken at 24 hours in 0.5% HCl medium

[0135]

[0136] Experiment 3: Stability test of active intermediate ZT-1-PVP in rat plasma medium

[0137] Except that the solution added to the ZT-1-PVP particle sample was 2.5 ml of rat plasma, the other conditions and operations were the same as those of Experiment 1 in Example 7. The content of ZT-1 in rat plasma after 1 hour, 3 hours, 6 hours, and 24 hours is shown in Table 13.

[0138] Table 13 ZT-1 content of ZT-1-PVP in rat plasma after 1 hour, 3 hours, 6 hours and 24 hours

[0139] Sampling time (hours) 0 1 3 6 24 ZT-1 content (ZT-1-PVP placed in rat plasma) 99.70％ 99.04％ 98.95％ 98.94％ 97.61％

[0140] The results of HPLC analysis of ZT-1-PVP in rat plasma at 24 hours are shown in Tables 14 and Fig.11 .

[0141] Table 14 HPLC analysis results of ZT-1-PVP samples taken at 24 hours in rat plasma environment

[0142]

[0143] Example 8 Study on the long-term stability of active intermediate ZT-1-PVP

[0144] The ZT-1-PVP dried granule sample (containing 5 g ZT-1, 95 g PVP with a K value of 30) was stored at room temperature, and the content of ZT-1 was determined by HPLC at 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 24 months, 27 months, and 30 months, respectively. The content of ZT-1 and its impurity content were tested by high performance liquid chromatography (HPLC) by normalization method. The HPLC test conditions are as follows: Waters 2487 (UV) instrument was used, the chromatography column was Turner Kromasil Silica 5μ250x 4.6mm, the mobile phase was: CH2Cl2: EtOAc: CH3OH 63:30:7, the flow rate was 1 ml / min, and the detection wavelength was 300 nm. The HPLC analysis results of the ZT-1-PVP dried granule sample stored for 30 months at room temperature are shown in Table 15.

[0145] Table 15 Analysis of ZT-1 content during long-term storage of active ZT-1-PVP dried granules at room temperature

[0146]

[0147] Example 9 Preparation of ZT-1 Solution Formulation Using Active Intermediate ZT-1-PVP Particles

[0148] Prescription: 275mg ZT-1-PVP granules (containing 5mg ZT-1), distilled water

[0149] Preparation method: Weigh 275 mg of ZT-1-PVP particles, add an appropriate amount of distilled water, stir at room temperature to dissolve, and then add distilled water to quantify to 25 ml. The hydrophilicity of ZT-1-PVP makes ZT-1-PVP dissolve in water to obtain a uniformly translucent ZT-1 aqueous solution with a concentration of 0.5 mg / ml.

[0150] Application: Use active ZT-1-PVP particles to prepare ZT-1 solution preparations for animal testing and animal and human pharmacology studies.

[0151] Example 10 Preparation of ZT-1 Tablets Using Active Intermediate ZT-1-PVP Granules

[0152] Prescription: ZT-1-PVP granules (ZT-1 content 5.28% w / w) 60.0g, lactose 900.0g, microcrystalline cellulose 100.0g, carboxymethyl cellulose 30.0g, magnesium stearate 10.0g;

[0153] Preparation method: Weigh 60.0g of active ZT-1-PVP particles and dissolve them in 300ml of anhydrous ethanol; add 100.0g of microcrystalline cellulose, 900.0g of lactose, and 30.0g of carboxymethyl cellulose, stir well, evaporate the ethanol at 80°C in a vacuum, and set aside; weigh 10.0g of magnesium stearate, mix it with the medicinal material obtained in the above drying step, pass it through a 60-mesh sieve 3 times, and directly compress it into tablets to make 10,000 tablets, each containing 316.8μg ZT-1.

[0154] Application: The ZT-1 tablets prepared from the ZT-1-PVP particles of this example are used for tablet preparation process research before Phase I clinical trials.

[0155] Example 11 Preparation of ZT-1 Phase I Clinical Trial Tablet Samples Using Active Intermediate ZT-1-PVP Granules

[0156] Prescription: ZT-1-PVP granules (ZT-1 content 5.28% w / w) 473.485 g, lactose 4326.515 g, microcrystalline cellulose 500.0 g, carboxymethyl cellulose 150.0 g, magnesium stearate 50.0 g;

[0157] Preparation method: Weigh 473.485g active ZT-1-PVP particles and dissolve them in 1500ml anhydrous ethanol; add 500.0g microcrystalline cellulose, 4326.515g lactose, and 150.0g carboxymethyl cellulose, stir well, evaporate the ethanol at 80℃ under vacuum, and set aside; weigh 50.0g magnesium stearate and mix it with the drug material obtained in the above drying step, pass through a 60-mesh sieve 3 times, and directly compress it into tablets to make 50,000 tablets, each containing 500.00μg ZT-1. This is the batch 1 sample of the Phase I clinical trial.

[0158] The prescription and preparation method of this example were repeated for two batches to obtain batch 2 and batch 3 samples of phase I clinical trial.

[0159] Application: The ZT-1 tablets of Batch 1, Batch 2 and Batch 3 prepared using active ZT-1-PVP particles were subjected to Phase I clinical trials on humans. See Example 16 for details.

[0160] Example 12 Preparation of ZT-1 Phase II Clinical Trial Tablet Samples Using Active Intermediate ZT-1-PVP Granules

[0161] Prescription: ZT-1-PVP granules (ZT-1 content 5.28% w / w) 946.97g, lactose 8653.03g, microcrystalline cellulose 1000.0g, carboxymethyl cellulose 300.0g, magnesium stearate 100.0g;

[0162] Preparation method: Weigh 946.97g active ZT-1-PVP particles and dissolve them in 3000ml anhydrous ethanol; add 1000.0g microcrystalline cellulose, 8653.03g lactose, and 300.0g carboxymethyl cellulose, stir well, evaporate the ethanol at 80℃ under vacuum, and set aside; weigh 100.0g magnesium stearate and mix it with the drug material obtained in the above drying step, pass through a 60-mesh sieve 3 times, and directly compress it into tablets to make 100,000 tablets, each containing 500.00μg ZT-1. This is the batch 4 sample of the Phase II clinical trial.

[0163] The prescription and preparation method of this example were repeated for two batches to obtain batch 5 and batch 6 samples of the Phase II clinical trial.

[0164] Application: The ZT-1 tablets of Batch 4, Batch 5 and Batch 6 prepared using active ZT-1-PVP particles were subjected to Phase II clinical trials on humans. See Example 17 for details.

[0165] In the future, once ZT-1 tablets obtain production approval in China or overseas, they will be produced according to the above prescription and method for use by AD patients.

[0166] Example 13 Preparation of ZT-1 capsules using active intermediate ZT-1-PVP particles

[0167] Prescription: 100g active ZT-1-PVP granules (ZT-1 content 5.28% w / w), 520g starch, 240g lactose, 240g microcrystalline cellulose, 50g gum arabic, 50g dextrin;

[0168] Preparation of core material solution: Weigh 100 g of ZT-1-PVP particles, dissolve in 200 ml of water, add 520 g of starch, 240 g of lactose, and 240 g of microcrystalline cellulose, stir evenly, and prepare a core material solution.

[0169] Preparation of wall material solution: weigh 50g of gum arabic and 50g of dextrin, add 1000ml of water to make them swell and disperse, and finally add 4000ml of water, stir evenly to make a wall material solution.

[0170] Mixing: Add the core material solution to the wall material solution, stir and mix to make a mixed solution.

[0171] Drying: Use a spray dryer to spray dry the mixed solution.

[0172] Loading into capsules: Select No. 5 capsules and load qualified drug particles that have passed the moisture test (less than 9.0%), disintegration test (should be completely disintegrated within 30 minutes), and dissolution (release) test (in accordance with the standards of the 2020 edition of the Chinese Pharmacopoeia; the content of ZT-1 is determined by HPLC, and the ultraviolet detection wavelength used is 300nm) into capsules.

[0173] Final product testing: Conduct visual inspection, filling volume difference measurement (±7.5%) and other verification work.

[0174] Example 14 Preparation of ZT-1 powder using active intermediate ZT-1-PVP particles

[0175] Prescription: ZT-1-PVP granules (ZT-1 content 5.28%) 100g

[0176] Preparation method: Take the above samples, weigh and bag them directly.

[0177] Example 15 Preparation of ZT-1 Granules Using Active Intermediate ZT-1-PVP Granules

[0178] Prescription: ZT-1-PVP granules (ZT-1 content 5.28%) 6g, dextrin 1000.0g, powdered sugar 980.0g, tartaric acid 10g, 50% ethanol (volume fraction) appropriate amount

[0179] Preparation method: The preparation process of ZT-1 granules is shown in Fig.12 .

[0180] 1000g dextrin and 980g sugar powder were crushed and sieved (100 mesh) respectively; then the two powders were added to the mixing tank in equal amounts and stirred to obtain a mixed powder. 6g ZT-1-PVP particles (containing 0.5g ZT-1) and 10g tartaric acid were stirred and dissolved in an appropriate amount of 50% alcohol (the amount of alcohol was determined by the appropriate softness of the prepared soft material), and the solution was poured into the mixed powder to make a soft material with appropriate softness. The soft material was squeezed and sieved to obtain wet granules, vacuum dried at 80℃, and the drying time was set according to the moisture content below 6%. The dried product was sieved through a 10-mesh sieve and an 80-mesh sieve, and the whole granules were packaged into 1000 small packages of finished products, that is, granules containing 316.8μg ZT-1 per package were obtained.

[0181] Example 16: Three batches of ZT-1 tablets (batch 1, batch 2 and batch 3 samples in Example 11) were prepared using the active intermediate ZT-1-PVP and a Phase I clinical trial was conducted.

[0182] 1. Introduction

[0183] So far, the following three Phase I clinical trials of ZT-1 have been conducted on humans:

[0184] DEB-02-ZT-01Part 1 [1] and Part 2 [2,3] ;

[0185] DEB-02-ZT-03;

[0186] DEB-02-ZT-03;

[0187] Since ZT-1 is a prodrug that is hydrolyzed into huperzine A in vivo, we have summarized all the clinical data on huperzine A in this section 3. These studies on huperzine A were mainly conducted in China, with the aim of evaluating the therapeutic effect of huperzine A on senile memory impairment and dementia (see Table 17).

[0188] 2. Phase I clinical study of ZT-1

[0189] Phase I clinical study of ZT-1 was conducted in Utrecht, the Netherlands, Europe (see Table 16).

[0190] Table 16 Phase I clinical study of ZT-1

[0191]

[0192]

[0193] 3. Clinical research on Huperzine A

[0194] Huperzine A, the active metabolite of ZT-1, has been clinically tested in healthy volunteers and elderly patients with memory impairment or dementia at a daily oral dose of 50 μg to 1500 μg. [4-11] A double-blind placebo-controlled trial of 0.2 mg of huperzine A daily in patients with AD [12,13] The results showed that Huperzine A can effectively improve the cognitive ability of AD patients and has good tolerance. The most common adverse reactions are nausea, vomiting and diarrhea related to cholinergic nerve properties, and the intensity of these side effects is mild. No serious adverse reactions occurred during the study. The relevant studies are summarized in Table 17.

[0195] Table 17 Clinical studies of Huperzine A

[0196]

[0197]

[0198]

[0199] 4. Pharmacokinetics and metabolism of ZT-1 in humans

[0200] In the clinical study of DEB-02-ZT-01, Part 1, a total of 24 people (13 men and 11 women) participated in the trial. The pharmacokinetic study of ZT-1 was conducted under the conditions of single oral administration of 1mg to 3mg on an empty stomach and 1.5mg after meals. The concentrations of ZT-1 and huperzine A in human plasma and human urine were determined by LC / MS / MS, respectively.

[14] .

[0201] After administration of different doses of ZT-1, the pharmacokinetic curves of ZT-1 and Huperzine A are shown in Fig.13A -B; Pharmacokinetic parameters in human blood are shown in Table 18.

[0202] Table 18 Pharmacokinetic parameters of single oral dose of ZT-1

[0203]

[0204] like Fig.13A As shown in Figure 2, after oral administration of ZT-1 on an empty stomach, it is quickly absorbed and converted into the active metabolite huperzine A. The highest blood concentration of ZT-1 (C max ) is generally 1.25 to 2.5 hours after administration (t max ) appeared, and the highest plasma concentration of the active metabolite huperzine A (C max ) generally 2 to 3 hours after administration (t max For both compounds, absorption lag times of about 0.25 to 0.5 hours were generally observed.

[0205] As shown in Table 18 and Fig.13A -B, the plasma concentration of ZT-1 decreased monophasically with time in most subjects after administration and absorption; a second phase of elimination was observed in a few subjects. The plasma concentration of huperzine A decreased monophasically with time in all subjects. ZT-1 could still be quantitatively detected in the plasma 36 hours after ZT-1 administration; 48 to 72 hours after ZT-1 administration, the plasma concentration of huperzine A could still be quantitatively measured. The average terminal half-life (t Ω ) appears to be independent of dose, ZT-1t Ω 3 to 8 hours, Huperzine A Ω The mean retention time (MRT) of ZT-1 is about 7 hours, while the mean retention time (MRT) of Huperzine A is about 27 hours. In the 1mg to 3mg dose range, the average C max About 4.5nM~43nM; the average C maxThe drug exposure is not proportional to the ZT-1 dose. In the 1mg to 3mg ZT-1 dose range, there is a higher-dose-proportional systemic gain of ZT-1. In the 1mg to 2mg ZT-1 dose range, the systemic gain of huperzine A is proportional to the dose, but at 3mg ZT-1, there is a higher-dose-proportional systemic gain of huperzine A. Because the t Ω The MRT was quite synchronized at all dose levels, so the reason for the deviation from dose proportionality is unlikely to be a nonlinear pharmacokinetic relationship formed by the limitation of systemic metabolic capacity. The most likely reason is from nonlinear absorption / first-pass metabolism or distribution.

[0206] There were no statistically significant differences in the terminal half-lives of ZT-1 and huperzine A; however, at some dose levels, the C values ​​of ZT-1 and huperzine A in female subjects were significantly different. max The data and AUC were statistically higher than those of male subjects; the reason for this can be attributed to the different body structures and weights of men and women.

[0207] Since ZT-1 cannot be detected in urine, only Huperzine A in urine was measured. The excretion ratio of Huperzine A in urine remained consistent at all ZT-1 dose levels, with approximately 20% of the administered ZT-1 excreted in the form of Huperzine A in urine within 30 hours on a molar basis; further, approximately 33% of the administered ZT-1 was excreted in the form of Huperzine A in urine within 120 hours as time went by (Huperzine A was no longer detectable in urine after 120 hours).

[0208] As shown in Table 18 and Fig.13A -B, after taking the drug after a meal, the highest blood concentration of ZT-1 and huperzine A (C max ) appears (t max ) is delayed, t of ZT-1 max Delayed 2.5 to 4 hours; t of Huperzine A max Delayed 1.25 to 4 hours. Average C of ZT-1 max and AUC decreased by approximately 10% to 50% (not significant), whereas the average C max The AUC and the 1.5 mg dose of ZT-1 increased statistically by about 50% to 80%. The terminal half-life and MRT values ​​were similar when the doses were taken before and after meals. Therefore, we believe that food increases the systemic exposure of huperzine A, but this conclusion needs more research data to confirm.

[0209] Subsequently, a repeated-dose (from 0.5 to 1.5 mg / day, oral administration for 14 days) DEB-02-ZT-03 clinical trial was conducted to obtain further relevant pharmacokinetic data. Fig.14A -D is the average pharmacokinetic curve of ZT-1 and huperzine A at different dosages. Fig.14A It is the average blood drug concentration curve of ZT-1 on day 1; Fig. 14B This is the average blood concentration curve of Huperzine A in vivo on day 1; Fig. 14C This is the mean blood concentration curve of ZT-1 on day 14; Fig.14D This is the average blood concentration curve of huperzine A on the 14th day.

[0210] Careful analysis of the average blood concentration curves of ZT-1 and huperzine A for each day of the 14-day continuous dosing process revealed the following patterns of change: after repeated single-dose administration for 3-5 days, the body was able to reach a stable blood concentration; compared with the blood concentration after a single dose, the blood concentration increased by 2 times; within the range of 1mg to 1.5mg, the drug exposure and the dose showed a good positive proportional relationship. At all dose levels, the terminal half-life (t Ω ) are very similar, with the former being 5 hours and the latter being 20 hours; these data are consistent with the data obtained from the previous single-dose clinical trial of DEB-02-ZT-01.

[0211] 5. Safety and effectiveness of ZT-1

[0212] 5.1. Effectiveness and Pharmacodynamics

[0213] In the DEB-02-ZT-01 Part 1 clinical study, the pharmacodynamic evaluation criteria were changes in erythrocyte acetylcholinesterase activity, changes in plasma butyrylcholinesterase activity, changes in pupil diameter, and salivary flow.

[0214] In the DEB-02-ZT-01 Part 1 clinical study, the effect of ZT-1 in reversing scopolamine-induced cognitive impairment was measured using a task selection from the computerized CDR (cognitive drug research) cognitive assessment system.

[0215] 5.1.1 Erythrocyte acetylcholinesterase activity and plasma butyrylcholinesterase activity

[0216] Spectrophotometric determination of erythrocyte acetylcholinesterase activity and plasma butyrylcholinesterase activity

[15] The sample before administration of each subject (a total of 24 subjects, including 13 males and 11 females) was used as a reference (100% activity), and the activity value of the sample after administration was measured to calculate the percentage of activity decrease.

[0217] like Fig.15 As shown, the inhibitory activity of acetylcholinesterase can be observed in samples of all dose levels of ZT-1 administration, while the inhibitory activity of acetylcholinesterase in the placebo samples only changed slightly (-6±22.4% to 15±13.7%). The average inhibition rate of acetylcholinesterase was 33±10.1% to 70±11.9% 1-4 hours after administration of ZT-1 in the dose range of 1 to 3 mg; the inhibitory activity of acetylcholinesterase can still be observed 30 hours after administration. The inhibitory activity of acetylcholinesterase after postprandial administration of 1.5 mg ZT-1 was slightly higher than that after fasting administration, and the average maximum inhibition peak was delayed in the case of postprandial administration.

[0218] No inhibitory activity of plasma butyrylcholinesterase was observed when the ZT-1 dose reached 3 mg, which further supports the conclusion that ZT-1 is highly selective in in vitro experiments.

[0219] 5.1.2 Pupil Detection and Saliva Flow

[0220] 5.1.2.1 Pupil diameter

[0221] The Compact Integrated Pupillograph / E instrument was used to measure pupil diameter; the effectiveness of the drug was defined as a change of more than 0.5 mm in pupil diameter before and after administration. The time points for pupil measurement were: immediately before administration, 2 hours after administration, and 4 hours after administration for all ZT-1 doses; and an additional time point of 6 hours after administration for the three doses and routes of administration: 1.5 mg post-meal administration, 2 mg pre-meal administration, and 3 mg pre-meal administration. In addition, new delayed pupil test time points can be added as long as the drug continues to work.

[0222] A small miotic effect (mean pupil constriction less than 0.5 mm) was observed at 10 hours after administration of the 2.0 mg dose and 6 hours after administration of the 3.0 mg dose; the values ​​measured at 4 hours after administration of the 1.0 mg and 1.5 mg fasting doses were similar to those measured with placebo. The 1.5 mg dose was administered after a meal, and its pupil constriction effect was maximal at 12 hours after administration, with a mean maximum constriction of 0.61 ± 0.60 mm.

[0223] 5.1.2.2 Saliva flow

[0224] Dental rollers were placed on the right side of the mouth between the gums and cheeks for 180 seconds, and salivary flow was expressed in mg / min. Measurements were made immediately before and within 30 hours after dosing. An increase in salivary flow was observed at all doses tested, proportional to the dose; placebo controls showed only minor changes. Of note, there was high variability within the subjects.

[0225] According to the dose order of ZT-1 1.0mg, 1.5mg (administered on an empty stomach), 1.5mg (administered after a meal), 2.0mg, and 3.0mg, the saliva flow rate increased after administration compared with that before administration, with the maximum values ​​being 98±179.3%, 138±142.5%, 136±192.8%, 169±274.9%, and 212±229.5%, respectively; the mean change (±SD) values ​​before and after administration in the placebo group ranged from -16±61.3% to 16±128.7%; except for the 1.0mg ZT-1 fasting dose group, in which the maximum mean increase in saliva flow rate occurred 12 hours after administration, and the 1.5mg ZT-1 postprandial dose, in which the maximum mean increase in saliva flow rate occurred 4 hours after administration, the maximum mean increase in saliva flow rate for the fasting administration route in the other dose groups occurred 2 hours after administration. In the 3 mg ZT-1 group, saliva flow was still increasing at a mean increase of 106±168.1% (±SD) at 30 hours after administration.

[0226] 5.1.3 Pharmacokinetic-pharmacodynamic relationship

[0227] For each pharmacodynamic measure, the mean response for each dose level was plotted versus time to reveal the relationship between ZT-1 dose and pupil diameter, salivary flow, and acetylcholinesterase inhibitory activity.

[0228] Furthermore, the pharmacodynamic measurements at each time point were plotted against the ZT-1 and huperzine A concentrations at that time point and were also extrapolated to all dose levels.

[0229] Linear regression showed that there was a small but statistically significant decrease in pupillary volume and an increase in salivary flow from baseline corresponding to increasing ZT-1 concentrations and increasing huperzine A concentrations. The pharmacokinetic and pharmacodynamic results suggest that the sigmoid Emax model may be appropriate to describe the concentration-effect relationship between huperzine A plasma concentrations and erythrocyte acetylcholinesterase activity. The estimated EC50 was 5.9 nM (see Fig.16 )

[0230] 5.1.4 Cognitive Ability Assessment

[0231] In the DEB-02-ZT-01 Part 2 clinical trial, the effect of ZT-1 on improving cognitive ability was preliminarily evaluated; the main purpose of this clinical trial was to assess whether ZT-1 has the effect of antagonizing cognitive impairment induced by scopolamine in healthy elderly volunteers. This is a 4-way, repeated test, placebo and positive control, randomized, double-blind, crossover clinical trial. Donepezil, the most commonly used cholinesterase inhibitor in clinical practice, was selected as the positive internal standard. The CDR (Cognitive Drug Research) evaluation system was used to evaluate cognitive function.

[0232] On each medication day (Day 1, Day 15, Day 29, Day 43), each volunteer was subcutaneously injected with 0.5 mg of scopolamine; before and 45 minutes after the injection of scopolamine, a series of CDR tests including attention, working memory, and scene replay were used to identify cognitive impairment caused by scopolamine. Subsequently, the volunteers were given placebo, 1 mg ZT-1, 1.5 mg ZT-1, or 10 mg Donepezil in a double-blind mode; then the CDR series of tests were also performed at four time points of 0.5 hours, 2 hours, 4.5 hours, and 6 hours after administration to determine the extent to which the therapeutic drug reversed cognitive impairment; 10 volunteers participated in this clinical study and obtained complete data.

[0233] The CDR test battery is highly sensitive to the cognitive impairment induced by scopolamine, and the initial impairments obtained in the tests are basically the same in most subjects. Fig.17A -B, ZT-1 was able to reduce impairment in the following three main areas, namely attention, working memory, and context re-recall:

[0234] Fig.17A The data show a test of attention: Simple Reaction Time. The first two time points reflect the cognitive impairment caused by scopolamine before the injection of the study drug; the following four time points reflect the study drug preventing further cognitive impairment or promoting the recovery of cognitive function. The Simple Reaction Time test scores show that both the 1 mg dose of ZT-1 and the 1.5 mg dose of ZT-1 showed significant therapeutic effects compared with placebo, and the low dose of 1 mg ZT-1 was statistically more effective than the high dose of 1.5 mg ZT-1. Compared with placebo, 10 mg donepezil also showed a good therapeutic effect. Therefore, ZT-1 can reduce cognitive impairment induced by scopolamine, and the efficacy of the 1.5 mg dose of ZT-1 is equivalent to that of 10 mg donepezil.

[0235] The positive treatment effect of ZT-1 was also evident in the scenario re-creation test. Fig. 17B The immediate word recall test showed that ZT-1 and donepezil reversed the cognitive impairment induced by scopolamine injection 0.75 hours earlier.

[0236] As Fig.17A and Fig. 17B As shown in the results, 1mg ZT-1, 1.5mg ZT-1 and donepezil can all reduce the cognitive impairment caused by scopolamine, and the degree of reduction is very significant. Overall, by testing the subjects' performance in attention, working memory, scene re-reproduction, hand-eye coordination, etc., we can confirm that ZT-1 has the ability to reduce the cognitive impairment induced by scopolamine. This finding suggests that ZT-1 can be an effective symptomatic treatment for cognitive deficits in patients with AD.

[0237] 5.2 Safety and Tolerability

[0238] The safety and tolerability evaluation criteria of the three Phase I clinical studies of ZT-1 summarized in this chapter are: incidence of adverse reactions, changes in physical signs relative to baseline (blood pressure, heart rate, body temperature), changes in hematological indicators, changes in blood biochemistry and urine analysis indicators, coagulation status (prothrombin time), and 12-lead ECG (including RR, QT, and QTcB intervals).

[0239] 5.2.1. DEB-02-ZT-01, Part 1 Phase I Clinical Study

[0240] For subjects who took the fasting 1 mg, fasting 1.5 mg, 2 mg (Cohort 1), and 3 mg ZT-1 doses, safety assessments were conducted from before dosing to 30 hours after dosing; for subjects who took the 2 mg (Cohort 2) and 1.5 mg postprandial ZT-1 doses, safety assessments were conducted from before dosing to 144 hours after dosing; for all other subjects participating in this phase of the study, safety assessments were conducted from before dosing to follow-up after the last dose (the follow-up time was scheduled for at least 7 days after the last dose). The research results are shown in Table 19.

[0241] 5.2.1.1 Adverse reactions

[0242] Table 19 Adverse reactions considered possibly related to the study drug in DEB-02-ZT-01, Part 1 Phase I clinical trial

[0243]

[0244]

[0245]

[0246] Most of the common adverse reactions, including headache, nausea, vomiting, dizziness, sweating, abnormal bowel sounds, pale face, excessive salivation, loose stools, flatulence, diarrhea, abdominal pain, drowsiness, muscle cramps, restlessness, and feeling cold, were considered to be possibly related to the study drug ZT-1. Table 19 summarizes all adverse reactions that may be related to the study drug.

[0247] A total of 108 adverse reactions possibly related to the study drug occurred in 18 of the 24 subjects. Except for a few adverse reactions that lasted a little longer in the 3mg ZT-1 dose, most adverse reactions occurred within a few hours after dosing and disappeared within 24 hours in all subjects.

[0248] The incidence of adverse reactions of low-dose ZT-1 1 mg and 1.5 mg on an empty stomach was similar to that of the placebo group, but the incidence of adverse reactions of high-dose ZT-1 2 mg and 3 mg was significantly increased; the incidence of adverse reactions of 1.5 mg ZT-1 after meal administration was higher than that of 1.5 mg ZT-1 after fasting administration.

[0249] In the 3mg ZT-1 group of 8 volunteers, 7 of them had a total of 50 adverse reactions that may be related to the study drug. The subjects who took 3mg ZT-1 had higher incidences of adverse reactions not only in the nervous system and gastrointestinal system, but also in other aspects, including restlessness, fatigue, feeling cold and chills, sweating, pale skin, double vision, blurred vision, decreased visual acuity, etc.

[0250] Most of the adverse reactions were mild (79 mild adverse reactions, only mild symptoms, not affecting the normal function of body organs), and a few were moderate (28 moderate adverse reactions, the symptoms were severe enough to make the subjects feel uncomfortable and have affected the normal function of body organs). These moderate adverse reactions occurred mainly in the 2mg and 3mg high-dose groups of ZT-1. There was a special severe nausea adverse reaction in the 1.5mg post-meal administration group, which lasted about 5 hours and recovered naturally.

[0251] 5.2.1.2 Serious adverse reactions, deaths, and adverse reactions leading to withdrawal from clinical trials

[0252] No serious adverse reactions occurred in all ZT-1 subjects; there were no adverse reactions related to the trial drug ZT-1 that caused the subjects to withdraw from the trial; and no deaths occurred during the trial.

[0253] 5.2.1.3 Other safety parameters

[0254] 5.2.1.3.1. Safety Assessment of Clinical Laboratory Measurement Data

[0255] Comparison of the test results of hematological and biochemical indices of the subjects before and after administration showed that there was no trend change in hematological, biochemical indices and PT values ​​related to ZT-1 administration; urine analysis before and after administration showed that no changes in urine indicators occurred.

[0256] During the clinical study, blood biochemistry tests showed one adverse reaction that may be related to ZT-1: a female subject who took a 1.5 mg dose of ZT-1 after a meal following the placebo test had an increase in transaminase (the value was less than 2.5 times the reference upper limit).

[0257] 5.2.1.3.2 Physical signs

[0258] The measurement data showed that the subjects' average diastolic and systolic blood pressure did not change with the administration time and dosage (dosage below 3 mg).

[0259] When 1mg, 1.5mg, and 2mg ZT-1 were administered on an empty stomach, many subjects had an increase in diastolic blood pressure (increase greater than 20mmHg) compared to placebo; at the same time, heart rate increased (increase greater than 15bpm) compared to placebo at this dose. No increase in diastolic blood pressure or increased heart rate was observed when 1.5mg ZT-1 was administered after a meal or 3mg on an empty stomach.

[0260] The changes in the average heart rate and average body temperature before and after administration of each dose group were compared with those of the placebo group, and the situations of the dose group and the placebo group were similar.

[0261] 5.2.1.3.3 ECG

[0262] Comparison of ECG data before and after administration of each dose of ZT-1 showed no significant changes in RR interval, QT interval, and QTc interval.

[0263] 5.2.1.4 Safety conclusions obtained from DEB-02-ZT-01, Part 1 Phase I clinical trial

[0264] Based on the first human trial of ZT-1, we have drawn the following conclusions on the safety and tolerability of ZT-1:

[0265] Single doses up to 1.5 mg of ZT-1 were administered orally on an empty stomach and ZT-1 showed good safety and tolerability in healthy elderly male and female subjects.

[0266] A single 3 mg oral dose of ZT-1 was not well tolerated by most elderly male and female subjects, mainly due to adverse reactions to the nervous system and gastrointestinal system. Common adverse reactions after a single 3 mg oral dose of ZT-1 were lightheadedness, vomiting, nausea, restlessness, headache, and drowsiness, which were mild to moderate in intensity. Some subjects were not able to tolerate a 2 mg dose of ZT-1 well, mainly due to adverse reactions to the nervous system and gastrointestinal system.

[0267] When a single oral dose of ZT-1 was as high as 3 mg, there were no significant changes in the hematology, blood biochemistry, and urine analysis data of healthy elderly male and female subjects; there were no changes in the coagulation status and 12-lead ECG electrocardiogram.

[0268] When 1.5 mg ZT-1 was administered to healthy elderly male and female volunteers after meals (high-fat, high-energy diet) compared with fasting, the incidence of drug-related adverse reactions, especially those of the nervous system and gastrointestinal system, was significantly higher when administered after meals; the reason for this is probably the greater exposure of huperzine A when the stomach is full (see Section 4. Pharmacokinetics and metabolism of ZT-1 in humans)

[0269] 5.2.2 DEB-02-ZT-01, Part 2 Phase I Clinical Trial

[0270] Safety evaluation was carried out throughout the four study cycles, from before dosing to 144 hours after dosing.

[0271] 5.2.2.1 Adverse reactions and other safety parameters

[0272] As shown in Table 20, of the 205 adverse reactions, none was severe; 180 were mild and 25 were moderate. After scopolamine injection to induce cognitive impairment, 116 adverse reactions occurred; 89 adverse reactions occurred after medication (placebo, 1 mg ZT-1, 1.5 mg ZT-1, 10 mg donepezil); Table 20 summarizes the adverse reactions that occurred in each period. The incidence of adverse reactions during the active drug phase was higher than that during the placebo phase. In all groups, including the placebo group, the most common adverse reactions were of the nervous system and gastrointestinal system.

[0273] Table 20 Summary of adverse reaction rates in DEB-02-ZT-01, Part 2 Phase I clinical trial

[0274]

[0275] 5.2.2.2 Physical signs

[0276] Throughout the observation period in this clinical trial, no drug-related changes in heart rate and body temperature were observed. There was no trend in the change of mean systolic and diastolic blood pressures related to time and dose level from before to after administration. Some changes occurred, and the intensity of changes was the same between the placebo group, 1mg ZT-1 group, 1.5mg ZT-1 group, and 10mg donepezil group, but these changes did not have a trend.

[0277] 5.2.2.3 Electrocardiogram (ECG)

[0278] Four subjects had obvious abnormal ECG on 1 or 2 occasions; two subjects had abnormal ECG that may be related to ZT-1 and donepezil administration: sinus bradycardia, first-degree AV block, and junctional bradycardia. All of these abnormal ECGs can return to normal ECG states under natural conditions. Compared with placebo, the average RR, QT, and QTcB intervals of subjects in the 1mg ZT-1 group, 1.5mg ZT-1 group, and 10mg donepezil group were prolonged before and 2 hours after administration. Further analysis, using the average RR, QT, and QTcB intervals before administration and the values ​​of the worst subject after administration for dispersion plotting, revealed that the RR interval was indeed prolonged; but the QTcB average interval value was dispersed, and the changes in the four groups including the placebo were similar, so it cannot be determined that the QTcB interval was prolonged after medication. The heart rate of the active drug group was slightly reduced after administration compared with the placebo group (maximum about 4 bpm), but this slight reduction in heart rate could not be confirmed to be related to ZT-1 administration.

[0279] 5.2.2.4. Safety Assessment of Clinical Laboratory Measurement Data

[0280] Comparison of the test results of hematological and biochemical indices of the subjects before and after administration showed that there was no trend change in hematological, biochemical indices and PT values ​​related to ZT-1 administration; urine analysis before and after administration showed that no abnormal changes occurred in any urine test indicators.

[0281] 5.2.3 DEB-02-ZT-03 Phase I Clinical Trial

[0282] Table 21 Incidence of adverse reactions in DEB-02-ZT-03 Phase I clinical trial

[0283]

[0284] Safety assessments were performed regularly from the day before dosing to 6 days after the last dose, and at a follow-up visit scheduled 7-14 days after dosing.

[0285] Table 21 summarizes the incidence of adverse reactions by system organ class; Table 22 summarizes the onset and duration of adverse reactions.

[0286] Table 22 Onset and duration of adverse reactions in DEB-02-ZT-03 Phase I clinical trial

[0287]

[0288] In general, a higher incidence of adverse reactions to the gastrointestinal system occurred at the 1.5 mg ZT-1 dose level; a number of upper respiratory tract infections and infections occurred at the 0.5 mg ZT-1 dose level, which was attributed to the infectious environment in the hospital ward (in winter, the subjects were confined to a small indoor area of ​​the air-conditioned ward for up to 12 days).

[0289] In general, all adverse reactions that occurred were mild or moderate in intensity.

[0290] Therefore, the preliminary safety results of the DEB-02-ZT-03 Phase I clinical trial, which was conducted within a dosage range of 0.5 mg to 1.5 mg / day, with oral administration once daily for 14 days, showed that ZT-1 has good safety and tolerability.

[0291] 5.2.4 DEB-02-ZT-104 Phase I Clinical Trial

[0292] This study was conducted on 6 healthy elderly volunteers (3 males and 3 females). The subjects were randomly given 1 mg ZT-1 orally in the morning. The order of food and medication was as follows:

[0293] A) Fast overnight the night before taking the medication, and take 1 mg ZT-1 orally on an empty stomach in the morning and have breakfast two hours later;

[0294] B) Fast overnight the night before taking the medication, and take 1 mg ZT-1 orally on an empty stomach in the morning and have breakfast half an hour later;

[0295] C) Fast overnight on the night before taking the medication, and take 1 mg of ZT-1 orally half an hour after breakfast in the morning.

[0296] The subjects' breakfast consisted of 50% carbohydrates, 30% fat, and 20% protein, with a calorie content of approximately 600; the safety evaluation work was carried out from before dosing to 60 hours after dosing, and follow-up at least 7 days after the last dose.

[0297] A total of 17 adverse reactions occurred, all of which were mild in intensity; the incidence of adverse reactions and the percentage of subjects experiencing adverse reactions were similar under the three different food conditions. The most common adverse reactions by organ class were as follows: vascular adverse reactions (5 cases in 2 subjects), gastrointestinal adverse reactions (3 cases in 2 subjects), and infectious and contagious adverse reactions (2 cases in 2 subjects); these adverse reactions and some other adverse reactions were similarly distributed under the three different food conditions. After careful study, 8 (out of 17) adverse reactions were found to be possibly related to ZT-1: flushing (n=4), nausea (n=1), diarrhea (n=1), sweating (n=1), and muscle fatigue (n=1). Other safety parameters (hematology, blood biochemistry, urinalysis, coagulation status, physical signs, ECG) measured after oral administration of ZT-1 under the three different food conditions or at different meal times did not show any special differences.

[0298] 5.2.5 Comparison of safety results between DEB-02-ZT-01 Part 1 and DEB-02-ZT-104 Phase I clinical trials

[0299] Initial safety results showed that ZT-1 showed good safety and tolerability when administered orally at a single dose of 1 mg 0.5 hours before standard breakfast, 2 hours before standard breakfast, and 0.5 hours after standard breakfast. Comparison of safety results of DEB-02-ZT-104 and DEB-02-ZT-01 Part 1I clinical trials showed that the incidence of adverse reactions in the former was lower than that in the latter (see Table 23).

[0300] The increase in adverse reactions in the DEB-02-ZT-01 Part 1I clinical trial almost all occurred at doses greater than 1 mg ZT-1. In addition, the breakfast standard in the DEB-02-ZT-01 Part 1I clinical trial was the American breakfast standard, while the DEB-02-ZT-104 Phase I clinical trial used a standard breakfast, which may have led to an increase in the incidence of adverse reactions in the former.

[0301] Table 23 Summary and comparison of adverse reactions in DEB-02-ZT-01 Part 1 and DEB-02-ZT-104 Phase I clinical trials

[0302]

[0303]

[0304] Example 17: Three batches of ZT-1 tablets (batch 4, batch 5 and batch 6 samples in Example 12) were prepared using the active intermediate ZT-1-PVP and a Phase II clinical trial was conducted.

[0305] 1. Test methods and grouping

[0306] A multicenter, randomized, double-blind, blank and positive control method was used to treat mild and moderate Alzheimer's disease (AD) patients with ZT-1 tablets.

[0307] 186 patients (107 females + 79 males) were enrolled in the safety trial. After intention-to-treat analysis (ITT), 177 patients completed the efficacy trial and were divided into the following groups (Table 24):

[0308] Table 24 Enrollment and grouping

[0309]

[0310] 2. Safety and tolerability experiments of ZT-1

[0311] As shown in Table 25, ZT-1 was well tolerated at both dose levels, and the adverse reaction reporting rates were similar among the groups: 34 cases (74%) in the placebo group; 33 cases (66%) in the 1.5 mg ZT-1 group; 32 cases (74%) in the 2 mg ZT-1 group and 35 cases (75%) in the donepezil group. Most adverse reactions were mild or moderate. The most clinically significant gastrointestinal adverse reactions were significantly lower in the 1.5 mg ZT-1 group (36%) than in the donepezil group (43%).

[0312] Table 25 Main adverse reactions

[0313]

[0314] * Note: Number of patients (number of adverse reactions) percentage

[0315] 3. Efficacy study of ZT-1

[0316] 3.1. AD Assessment Scale-Cognitive Score (ADAS-Cog)

[0317] like Fig.18 As shown, after AD patients took the drug continuously for 12 weeks, a follow-up visit was conducted at the 14th week (after 2 weeks of drug withdrawal).

[0318] Compared with the Donepezil group, the 1.5 mg dose of ZT-1 group was superior to the Donepezil group in terms of the traditional AD rating scale cognitive score at week 12. After a total of 12 weeks of continuous administration, the 1.5 mg ZT-1 group had the best overall efficacy in terms of ADAS-Cog. There was a clear trend of improvement in all treatment groups, such as in the improvement of cognitive scores: the 1.5 mg ZT-1 group was 2.6, the 2 mg ZT-1 group was 1.5, the Donepezil group was 2.5, and the placebo group was 1.1. Compared with the baseline values, the AD cognitive assessment scores of the 1.5 mg ZT-1 group (p=0.007) and the Donepezil group (p=0.011) were statistically significant.

[0319] like Fig.19 As shown in Figure 2, the response rate in the 1.5 mg ZT-1 group reached 38.3%, and the 2 mg ZT-1 group and donepezil group reached 27.5% and 29.6%, respectively, which were all higher than the 19.6% in the placebo group (p = 0.047). (See Fig.19 ).

[0320] 3.2 Mini-Mental State Examination (MMSE)

[0321] Test results: Fig. 20 Compared with baseline, the increase in MMSE in each group was: 2.0 in the ZT-1 1.5 mg group, 1.5 in the ZT-1 2 mg group, 0.61 in the Donepezil group, and 0.85 in the Placebo group. The ZT-1 1.5 mg group showed a statistically significant increase in MMSE compared with the Donepezil group (p=0.022). The increase in MMSE was most significant in the ZT-1 1.5 mg (p=0.001) and ZT-1 2 mg (p=0.034) groups.

[0322] 3.3 Neuropsychiatric Inventory Questionnaire (NPI-Q):

[0323] NPI-Q values ​​show that Fig.21 ) and "sadness" ( Fig. 22 )’s response changes.

[0324] Compared with the Donepezil group, the ZT-1 1.5 mg group had a higher value of "serious" response change (p=0.018). Similarly, compared with the Donepezil group, the ZT-1 1.5 mg group had a higher value of "sad" response change (p=0.012). This shows that ZT-1 has the effect of improving the negative emotions of AD patients.

[0325] 3.4 Others

[0326] There were no significant differences in the CDR and IADL scores of the AD rating scale between the treatment groups and the placebo group.

[0327] 4. Conclusion

[0328] Phase II clinical study results show that for patients with mild to moderate Alzheimer's disease (AD), a daily dose of 2 mg of ZT-1 is safe and effective, and a dose of 1.5 mg is even better, not only because it is more effective than the 2 mg dose, but also because it has better tolerability than donepezil, especially for adverse reactions caused by the gastrointestinal system. In addition, ZT-1 has the effect of shortening the QTc (B) interval, which donepezil does not have. Therefore, a dose of 1.5 mg of ZT-1 is very likely to replace various acetylcholinesterase inhibitors currently on the market for the clinical treatment of Alzheimer's disease (AD).

[0329] In short, the results of the Phase II clinical study fully prove that ZT-1 tablets are a potent and highly selective acetylcholinesterase inhibitor that can effectively improve memory impairment; the drug has a long duration of action, good oral absorption, and very few peripheral side effects.

[0330] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

[0331] References

[0332] 1.Jolanda van de Logt: A partially double-blind and randomized, placebo-controlled single ascending oral doses study of safety, tolerability, pharmacodynamics and pharmacokinetics of ZT-1 administered under fastening and fed conditions in healthy elderly volunteer, Study DEB-02-ZT-01,1 stpart.Clinical Study Report,May 2003.

[0333] 2.Jolanda van de Logt:A partially double-blind and randomised,placebo-controlled single ascending oral doses study of safety,tolerability,pharmacodynamics and pharmacokinetics of ZT-1administered under fastening andfed conditions in healthy elderly volunteer,Study DEB-02-ZT-01,2 nd part.Clinical Study Report,April 2003.

[0334] 3.Keith Wesnes:Report on the outcome of cognitive assessment inprotocolno.DEB-02-ZT-01:A double-blind and randomised,placebo and positivecontrolled,cross-over study(part 2)to assess the effectiveness of ZT-1inreversing scopolamine-induced cognitive-impairment in healthy elderlyvolunteer,Study DEB-02-ZT-01,2 nd part.Clinical Study Report,March 2003.

[0335] 4.Li Shu-Lan et al.,Huperzine A capsules vs tablets in treatingsenile amnesia:adouble-blinded study.Chin J New Drugs Clin Remedies 19:34-35,2000.

[0336] 5.Liu Fu-Gen et al.,Double blind control treatment of Huperzine A andplacebo in 28patients with Alzheimer’s disease.J Pharmacoepidemiol 4:196-98,195.

[0337] 6.Zhang RW et al.,Drug evaluation of huperzine A in the treatment ofsenile memory disorders.Zhongguo Yao Li Xue Bao May;12(3):250-2,1991.

[0338] 7.Zhang,Z et al.,Clinical efficacy and safety of huperzine Alpha intreatment of mild to moderate Alzheimerís disease,a placebo-controlled,double-blind,randomized trial.Zhonghua Yi Xue Za Zhi 82(14):941-4,2002.

[0339] 8.Zhang Shi-Lu,Therapeutic effects of Huperzine A on the aged withmemory impairment.New Drugs and Clinical Remedies 5:260-62,1986.

[0340] 9.Xu Si-Sun et al.,Huperzine A in capsules and tablets for treatingpatients with Alzeimerís disease.Acta Pharmacologica Sinica 20:486-90,1999.

[0341] 10.Ma Yong-Xing et al.,Double blind trial of huperzine A(HUP)oncognitive deterioration in 314cases of benign senescent forgetfulness,vascular dementia and Alzheimeís disease Ann NY Acad Sci 854:506-7,1998.

[0342] 11.Qian BC et al.,Pharmacokinetics of tablet huperzine A in sixvolunteers.ActaPharmacol Sin 16:396-98,1995

[0343] 12.Xu Si-Sun,Efficacy of tablet huperzine A on memory and cognitionin patients withbenign senescent forgetfullnes.Chin J Pharmacol Ther 2:1-4,1997.

[0344] 13.Xu Si-Sun,Efficacy of tablet huperzine A on memory,cognition andbehavior inAlzeimerís disease.Acta Pharmacol Sinica.16:391-95,1995.

[0345] 14.Benech H.,Validation of the LC / MS / MS assay for the simultaneousquantification ofZT-1 and huperzine A in rat,dog and human plasma and ofhuperzine A in human urine.Study Report No 238135.Laboratoire díEtude du des Gif surYvette,France,2003.

[0346] 15.Ellman G.L.,Courtney K.,Andres V.and Feartherstone R.,Biochen.Pharmacol.7:88,1961.

Claims

1. An active intermediate, characterized in that , consisting of a derivative of huperzine A and polyvinylpyrrolidone as shown in formula (I); ； The derivative of Huperzine A is ZT-1 as shown in formula (II); the active intermediate is named ZT-1-PVP; ； The content of ZT-1 accounts for 5.28% to 8.00% (w / w) of the total weight of ZT-1-PVP; the n value of the polyvinyl pyrrolidone is 396 to 486; the K value of the polyvinyl pyrrolidone is 28 to 32; the derivative of huperzine A and the polyvinyl pyrrolidone shown in formula (I) form the active intermediate through a non-chemical bond.

2. A method for preparing ZT-1, characterized in that: Using the "one pot" method, the preparation steps are: o-Vanillin, huperzine A and dithionyl chloride were mixed in a molar ratio of 1.05:1:10; after the raw materials were mixed in proportion, they were stirred and reacted at 10-30°C to obtain ZT-1.

3. A method for preparing the active intermediate according to claim 1, characterized in that: The preparation steps include: Dissolving polyvinyl pyrrolidone in an organic solvent, and adding a pH adjuster to adjust the pH of the solution to 6.5-7.0 to prepare a polyvinyl pyrrolidone solution; Add an appropriate amount of ZT-1 to the polyvinyl pyrrolidone solution; stir evenly and then dry under reduced pressure to obtain ZT-1-PVP; The organic solvent is selected from one or more of dichloromethane, chloroform, methanol, ethanol, acetone, and ethyl acetate; the pH regulator is selected from one or more of 0.1% KOH methanol solution, 0.1% ammonia methanol solution, and 0.5% HCl methanol solution; The synthesis of ZT-1 adopts a "one-pot" method; The steps of the "one pot" method are: o-Vanillin, huperzine A and dithionyl chloride are mixed in an appropriate molar ratio; the molar ratio of o-Vanillin to huperzine A is (1.005-1.2):1; the molar ratio of huperzine A to dithionyl chloride is 1:(5-20); after the raw materials are mixed in proportion, they are stirred and reacted at 10-30°C to obtain ZT-1.

4. The preparation method according to claim 3, wherein the source of Huperzine A is natural extraction or chemical synthesis; The natural extraction steps are as follows: collecting the whole plant or the aerial part of Huperzine serrata, drying and crushing it, soaking it with a 0.5% to 1% dilute HCl aqueous solution, collecting the acid water extract, alkalizing it, and extracting it with dichloromethane or chloroform; the extract is concentrated and the solvent is recovered to obtain a crude extract of Huperzine serrata alkaloids; after separation and purification steps, chromatography is performed, the eluate containing Huperzine A is collected, and the crude Huperzine A crystals are concentrated; and the pure Huperzine A is obtained after recrystallization with an organic solvent; The chemical synthesis steps are: first obtaining racemic (±) huperzine A, and then obtaining (-) huperzine A through chemical separation; the huperzine A obtained by chemical synthesis must meet the conditions that the content of (-) huperzine A is greater than 99% and the content of (+) huperzine A is less than 0.5%.

5. The preparation method according to claim 3, characterized in that: The "one-pot" method also includes recovering excess thionyl chloride under reduced pressure after reaching the end point of the reaction, adding ethanol for recrystallization, and obtaining ZT-1 crystals with a melting point of 202-204°C.

6. A drug containing the active intermediate according to claim 1, characterized in that: The pharmaceutical dosage forms include tablets, capsules, oral dispersions, oral granules and solution preparations.

7. Use of the active intermediate according to claim 1 in the preparation of a medicine for treating Alzheimer's disease and / or myasthenia gravis.

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