7H- azulene [1,2,3-i,j] isoquinolin-7-one compound, single crystal and use thereof

Abstract

A 7H-azulene [1,2,3-i,j] isoquinolin-7-one compound, a single crystal and a use thereof are provided. The compound has a structure as shown in a formula (I). The present invention obtains a crude extract of Acorus calamus L., then extracts with different polar solvents in sequence and separates column chromatography to obtain a novel alkaloid compound, which is expected to be developed into a novel class of drug exerting neuroprotective effects, so as to prevent and treat senile dementia, stroke and epilepsy diseases.

##STR00001##

Claims

1. A class of compounds having a mother nucleus structure of 7H-azulene [1,2,3-i,j] isoquinolin-7-one, which are the compounds as shown in a formula (I); or stereoisomer, oxynitride, pharmaceutically acceptable salt or prodrug of the compounds as shown in the formula (I): ##STR00006## wherein, each of R.sup.1, R.sup.2 and R.sup.3 is independently hydrogen, hydrazine, F, Cl, Br, I, hydroxy, amino, nitro, cyano, C.sub.1-6 alkyl, hydroxy C.sub.1-6 alkyl, amino C.sub.1-6 alkyl , C.sub.1-6 haloalkyl, C.sub.1-6 haloalkoxy, C.sub.1-6 alkoxy C.sub.1-6 alkyl, C.sub.2-6 alkenyl, alkynyl, C.sub.1-6 alkylamino, or C.sub.1-6 alkoxy; or carbon atoms attached between R.sup.1 and R.sup.2 together form an oxygen-containing C.sub.2-7 heterocycle.

2. The compounds, as recited in claim 1, wherein, each of R.sup.1, R.sup.2 and R.sup.3 is independently hydrogen, hydrazine, F, Cl, Br, I, hydroxy, amino, nitro, cyano, C.sub.1-4 alkyl, hydroxy C.sub.1-4 alkyl, amino C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, C.sub.1-4 haloalkoxy, C.sub.1-4 alkoxy C.sub.1-4 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-4 alkylamino, or C.sub.1-4 alkoxy; or carbon atoms attached between R.sup.1 and R.sup.2 together form oxygen-containing C.sub.2-5 heterocycle.

3. The compounds, as recited in claim 1, wherein, each of R.sup.1, R.sup.2 and R.sup.3 is independently hydrogen, hydrazine, F, Cl, Br, I, hydroxy, amino, nitro, cyano, methyl, ethyl, n-propyl, isopropyl, n-butyl, hydroxymethyl, 1-hydroxyethyl, methoxy, ethoxy, n-propoxy, isopropoxy or n-butoxy group, or carbon atoms attached between R.sup.1 and R.sup.2 together form oxygen-containing cyclopropane, cyclobutane, cyclopentane or cyclohexane.

4. The compounds, as recited in claim 1, which are compounds with one of following structures; or stereoisomer, oxynitride, pharmaceutically acceptable salt or prodrug of the compound with one of the following structures: ##STR00007##

5. A single crystal of a compound as shown in a formula (II), wherein the single crystal has following single crystal structure parameters: ##STR00008## TABLE-US-00008 crystal system monoclinic system space group P121/c1 cell parameters a = 7.5352(8) , = 90, b = 11.7589(10) , = 95.301(6), c = 15.6709(14) , = 90, volume 1382.6(2) .sup.3 number of molecules per 4 unit cell Z:

6. A pharmaceutical composition comprising arbitrary compounds as recited in claim 1, and pharmaceutically acceptable carrier, excipient, diluent, adjuvant, vehicle or combination thereof.

7. A method for preparing a medicament for preventing, treating, curing or ameliorating diseases caused by damaged nerve cells, using a pharmaceutical composition as recited in claim 6.

8. A method for preparing a single crystal as recited in claim 5, comprising steps of: dissolving a crude product of the formula (II) in a solvent and standing, sealing when crystals are precipitated, keeping stand to obtain the single crystal.

9. A method for preparing a compound as shown in a formula (II), comprising steps of: washing rhizomes of Acorus calamus L. for removing impurities, drying in shade, pulverizing into coarse powder, extracting with an alcohol solvent, and concentrating an extract under a reduced pressure until the extract is viscous, so as to obtain an alcohol extract; suspending the alcohol extract in water, extracting with different polar solvents in sequence, concentrating a solvent extract under a reduced pressure; and separating residue by column chromatography to obtain the compound as shown in the formula (II).

10. The method, as recited in claim 9, wherein the alcohol solvent is a C.sub.1-4 alcohol solvent; the water is distilled water or purified water; the different polar solvents are cyclohexane, petroleum ether, chloroform, dichloromethane, ethyl acetate, n-butanol or a combination thereof.

Description

DESCRIPTION OF THE DRAWINGS

[0062] FIG. 1 HR-ESI-MS spectrum of 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-one.

[0063] FIG. 2 IR spectrum of 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-one.

[0064] FIG. 3 UV spectrum of 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-one.

[0065] FIG. 4 .sup.1H NMR spectrum of 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-one.

[0066] FIG. 5 .sup.13C NMR spectrum of 6-hydroxy-5,8-dimethoxy-7H-azulene 8 1,2,3i,j] isoquinolin-7-one.

[0067] FIG. 6 HSQC spectrum of 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-one.

[0068] FIG. 7 .sup.1H-.sup.1H COSY spectrum of 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3---i,j] isoquinolin-7-one.

[0069] FIG. 8 HMBC spectrum of 6-hydroxy-5,8-dimethoxy-7H-azulene isoquinolin-7-one.

[0070] FIG. 9 X-ray single crystal diffraction structure of 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-one.

[0071] FIG. 10 uint cell stacking diagram of 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-one.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0072] The present invention will be further illustrated with the following embodiments but is not limited thereby. Those skilled in the art can learn from the contents of the specification and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are Obvious to those skilled in the art and are considered to be included in the present invention. The method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention.

Embodiment 1

Preparation of Crude Product of 6-hydroxy-5,8-dimethoxy-7H-azulene isoquinolin-7-one

[0073] Pulverizing dried rhizomes (10 kg) of Acorus calamus L. into coarse powder, and extracting medicinal materials with 95% ethanol four times at room temperature for 24 h each time; combining extract, and then concentrating under reduced pressure at 45 C. to obtain an ethanol extract((weighs 1.1 kg).

[0074] Adding 1L distilled water into obtained ethanol extract for suspending, and extracting with petroleum ether (1 L5 times) and chloroform (1 L5 times) in sequence; concentrating under reduced pressure to obtain a petroleum ether extract (extract 1 weights 618.6 g) and a chloroform extract (extract 2 weighs 58.9 g).

[0075] Eluting the extract 2 through silica gel column chromatography (200-300 mesh, 6.0100 cm, 1.0 kg) with a gradient of petroleum ether-acetone (50:1.fwdarw.0.1), so as to obtain 6 fragments (Fr. 1-Fr. 6), eluting Fr. 4 (9.0 g) through Sephadex LH-20 column with petroleum ether-chloroform-methanol (4:5:1) (flow rate of 15 drops.Math.min.sup.1). so as to obtain crude product of 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-one (weighs 5 mg).

Embodiment 2

Preparation of Single Crystal of 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-one

[0076] Dissolving 5 mg crude product of 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-one obtained in the embodiment 1 into 2-3L ethanol, slowly volatilizing obtained solution at a room temperature, and sealing when crystals are precipitated; waiting for 7 to 10 days before filtering, adding a small amount of ethanol to wash surfaces of filter residue, and drying residue at room temperature, so as to obtain a deep to red needle crystal, weighs of the single crystal, 4.25 mg, wherein a yield is 85%.

Embodiment 3

Structure Identification of 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-one

[0077] 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-one is dark red needle crystal. mp>254 C., yellow fluorescence under TLC ultraviolet lamp (254 nm), showing brownish red by modified Dragendorff s reagent.

[0078] UV (MeOH) .sub.max(log ) 207 nm (3.82), 242 nm (3.37), 269 nm (3.219, 314 nm (3.08), 335 nm (3.06).

[0079] IR spectrum shows the existence of hydroxyl group (3444 cm.sup.1) and phenyl ring (1598, 1553, 1497 and 1444 cm.sup.1) functional groups.

[0080] HR-ESI-MS m/z 308.09061 [M+H].sup.+ (calcd. for C.sub.18H.sub.14NO.sub.4 308.09228), 330.07248 [M+Na].sup.+ (calcd. for C.sub.18H.sub.13NO.sub.4Na 330.07423), which is determined to have a molecular formula of C.sub.1814H.sub.13NO.sub.4 and an unsaturation degree of 13.

[0081] According to NMR data, the compound has 18 carbons, including ten quaternary carbons, six tertiary carbons, and two methyl carbons. .sup.1H NMR spectrum (see Table 1) shows two methoxy methyl signals .sub.H 4.07 (3H, s, 5-OCH.sub.3) and 4.10 (3H, s, 8-OCH.sub.3); a set of each coupled olefinic protons signals .sub.H 8.64 (1H, d, J=5.0 Hz, H-2) and 7.68 (1H, d, J=5.0 Hz, H-3), indicating there are two ortho-hydrogen protons at the 2,3 position on the isoquinoline ring; another set of each coupled olefinic proton signals .sub.H 7.29 (1H, d, J=10.0 Hz, H-9), 7.50 (1H, t, J=10.0 Hz, H-10) and 8.29 (TH, d, J=10.0 Hz, H-11). .sup.13C NMR spectrum (see Table 1) shows a ketone carbonyl carbon signal .sub.C 176.0 (C-7), three oxygenated quaternary carbon signals .sub.C 157.9 (C-5), 154.2 (C-6), 163.1 (C-8), and two oxygenated methyl carbon signals .sub.C 57.0 (5, 8-OCH.sub.3); According to the molecular formula, there is interred to be one hydroxyl group in the structure.

TABLE-US-00002 TABLE 1 NMR data of 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-one .sub.C .sub.H HMBC No. (ppm) (ppm, J in Hz) .sup.1H-.sup.1H COSY (H.fwdarw.C) 2 145.1 8.64 (1H, d, 5.0) H-3 3, 3a, 11b 3 120.4 7.68 (1H, d, 5.0) H-2 2, 3b, 4 3a 129.9 3b 122.4 4 106.7 7.15 (1H, s) 3, 3b, 5, 6 5 157.9 6 154.2 6a 115.8 6b 141.6 7 176.0 8 163.1 9 117.4 7.29 (1H, d, 10.0) H-10 7, 8, 10, 11 10 133.1 7.50 (1H, t, 10.0) H-9, H-11 8, 11a 11 125.2 8.29 (1H, d, 10.0) H-10 6b, 9, 11b 11a 144.3 11b 152.3 5-OCH.sub.3 57.0 4.07 (3H, s) 5 8-OCH.sub.3 57.0 4.10 (3H, s) 8 .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD/DMSO-d.sub.6 (1:1:0.25)) and .sup.13C NMR (125 MHz, CDCl.sub.3/CD.sub.3OD/DMSO-d.sub.6 (1:1:0.25)).

[0082] Cross-signal peaks of the .sup.1H-.sup.1H COSY spectrum indicates that the compound has two proton-bearing structural fragments, corresponding to H-9/H-10/H-11 and H-2/H-3. In HMBC spectrum, strong correlations from 5-OCH.sub.3 to C-5, 8-OCH.sub.3 to C-8, indicating that two methoxy groups are respectively attached at the C-5 and C-8 positions. HMBC correlations from H-9/H-10 to C-8 indicate that C-8 is connected to C-9; HMBC correlation from H-9 to C-7 indicates that C-7 is connected to C-8: HMBC correlation from II-10 to C-11a indicates that C-11a is connected to C-11; and HMBC correlations from H-11 to C-6b/C-11b indicates that C-11b and C-6b are connected to C-11a. Chemical shift of a C-6b position is towards a low field, which may be affected by - conjugated effect of carbony group. According to the above information, the structure is initially inferred as the formula (III). The above analysis is as shown in the formula (III).

##STR00005##

Embodiment 4

Single Crystal Structure of 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-one

[0083] (1) X-Ray Single Crystal Diffraction Method for Measuring Structure

[0084] Selecting a single crystal of 0.0500.0800.150 mm.sup.3, and collecting diffraction data with a BRUKER SMART APEX 1000 CCD diffractotneter, wherein Mo K ray (=0.071073 nm) monochromated by a graphite monochromator is used at 296(2) K for collecting the diffraction data by - scanning. Using Broker's SAINTPLUS to program to restore data and use SADABS program for empirical absorption correction. Directly resolving and refining the structurewith SHEXLS-97 and SHELXL-97 programs [Sheldrick, G. M. SHELX597 and SHELXL97 University of Gottingen, Germany, 2008]. Structurally refining all non-hydrogen atoms with full matrix least squares; and anisotropically refining all non-hydrogen atoms. With theoretical hydrogenation and modification of hydrogen atom isotropic thermal parameters, crystal data and structural parameters are listed in Table 2.

TABLE-US-00003 TABLE 2 crystal data and structural parameters of 6-hydroxy-5,8-dimethoxy- 7H-azulene [1,2,3-i,j] isoquinolin-7-one Item Data Chemical formula C.sub.18H.sub.13NO.sub.4 Formula weight 307.29 Temperature 296(2) K Wavelength 0.71073 Crystal size 0.050 0.080 0.150 mm.sup.3 Crystal system monoclinic Space group P 1 21/c 1 Unit cell dimensions a = 7.5352(8) = 90 b = 11.7589(10) = 95.301(6) c = 15.6709(14) = 90 Volume 1382.6(2) .sup.3 Z 4 Density (calculated) 1.476 mg/cm.sup.3 Absorption coefficient 0.106 mm.sup.1 F(000) 640 Theta range for data collection 2.17 to 25.00 Index ranges 8 <= h <= 8, 13 <= k <= 13, 18 <= l <= 18 Reflections collected 9965 Independent reflections 2423 [R(int) = 0.0233] Coverage of independent 99.8% reflections Absorption correction multi-scan Max. and min. transmission 0.9947 and 0.9843 Structure solution technique direct methods Structure solution program SHELXS-97 (Sheldrick, 2008) Refinement method Full-matrix least-squares on F.sup.2 Refinement program SHELXL-97 (Sheldrick, 2008) Function minimized w(F.sub.o.sup.2 F.sub.c.sup.2).sup.2 Data/restraints/parameters 2423/0/213 Goodness-of-fit on F.sup.2 1.040 /.sub.max 0.001 Final R indices 1965 data; R.sub.1 = 0.0351, I > 2(I) wR.sub.2 = 0.0901 R indices (all data) R.sub.1 = 0.0465, wR.sub.2 = 0.0977 Weighting scheme w = 1/[.sup.2(F.sub.o.sup.2) + (0.0511P).sup.2 + 0.2796P] where P = (F.sub.o.sup.2 + 2F.sub.c.sup.2)/3 Largest diff. peak and hole 0.138 and 0.166 e.sup.3 R.M.S. deviation from mean 0.035 e.sup.3

[0085] (2) Single Crystal Structure of 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-One

[0086] Non-hydrogen atomic coordinates and thermal parameters of 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-one are listed in Table 3, partial bond lengths and bond angles are listed in Tables 4 and 5

TABLE-US-00004 TABLE 3 atomic coordinates and thermal parameters (.sup.2) of 6-hydroxy-5,8- dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-one x/a y/b z/c U(eq) O4 0.54332(17) 0.36641(9) 0.03189(7) 0.0499(3) O1 0.88401(17) 0.01371(10) 0.68249(7) 0.0518(3) O2 0.78729(16) 0.14489(9) 0.79346(6) 0.0461(3) O3 0.66800(16) 0.27925(9) 0.89912(7) 0.0468(3) N1 0.77066(18) 0.85096(11) 0.10287(8) 0.0449(4) C18 0.4696(3) 0.42065(15) 0.10268(11) 0.0525(5) C12 0.5949(2) 0.25617(13) 0.04246(9) 0.0374(4) C13 0.6633(2) 0.20968(12) 0.96562(9) 0.0339(3) C14 0.71886(18) 0.09709(12) 0.96788(9) 0.0314(3) C16 0.79460(18) 0.01647(12) 0.90842(9) 0.0314(3) C1 0.82203(19) 0.04581(12) 0.82173(9) 0.0351(3) C2 0.8853(2) 0.96352(13) 0.76058(10) 0.0387(4) C17 0.9179(4) 0.94719(18) 0.60936(12) 0.0780(7) C15 0.70583(18) 0.03724(12) 0.04503(9) 0.0334(3) C10 0.64227(19) 0.08017(13) 0.11938(9) 0.0369(4) C11 0.5851(2) 0.19540(13) 0.11632(9) 0.0397(4) C9 0.6447(2) 0.00142(15) 0.18679(10) 0.0459(4) C8 0.7068(2) 0.89220(15) 0.17502(11) 0.0488(4) C7 0.76780(19) 0.92678(13) 0.03904(9) 0.0359(4) C6 0.82580(19) 0.91211(12) 0.95254(9) 0.0347(3) C5 0.8997(2) 0.80954(13) 0.92658(10) 0.0413(4) C4 0.9524(2) 0.78497(13) 0.84766(11) 0.0452(4) C3 0.9412(2) 0.85373(13) 0.77388(11) 0.0447(4) Note: structure number sequence in Table 3 is shown in FIG. 9.

TABLE-US-00005 TABLE 4 bond lengths of 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-one chemical bond bond length O4C12 1.3590(18) O1C2 1.3578(18) O2C1 1.2652(18) O3H6 0.99(2) N1C8 1.359(2) C18H1 0.96 C12C11 1.368(2) C13C14 1.388(2) C14C16 1.480(2) C16C1 1.435(2) C2C3 1.368(2) C17H13 0.96 C15C7 1.386(2) C10C9 1.404(2) C11H7 0.93 C9H12 0.93 C7C6 1.472(2) C5C4 1.364(2) C4C3 1.407(2) C3H8 0.93 O4C18 1.4354(18) O1C17 1.430(2) O3C13 1.3279(17) N1C7 1.3387(19) C18H5 0.96 C18H4 0.96 C12C13 1.459(2) C14C15 1.4100(19) C16C6 1.417(2) C1C2 1.417(2) C17H3 0.96 C17H2 0.96 C15C10 1.395(2) C10C11 1.421(2) C9C8 1.385(2) C8H11 0.93 C6C5 1.405(2) C5H10 0.93 C4H9 0.93 Note: structure number sequence in Table 3 is shown in FIG. 9.

TABLE-US-00006 TABLE 5 bond angles of 6-hydroxy-5,8-dimethoxy-7Z7-azulene [1,2,3-i,j] isoquinolin-7-one chemical bond bond angle C12O4C18 117.15(12) C13O3H6 106.5(11) O4C18H5 109.5 H5C18H1 109.5 H5C18H4 109.5 O4C12C11 124.35(14) C11C12C13 123.59(14) O3C13C12 116.99(13) C13C14C15 116.95(13) C15C14C16 106.35(12) C6C16C14 107.67(12) O2C1C16 120.64(13) C16C1C2 122.82(13) O1C2C1 108.99(13) O1C17H3 109.5 H3C17H13 109.5 H3C17H2 109.5 C7C15C10 122.65(13) C10C15C14 126.31(14) C15C10C11 116.32(13) C12C11C10 119.11(14) C10C11H7 120.4 C8C9H12 120.2 N1C8C9 126.28(14) C9C8H11 116.9 N1C7C6 128.63(14) C5C6C16 130.88(14) C16C6C7 107.32(12) C4C5H10 116.5 C5C4C3 128.53(15) C3C4H9 115.7 C2C3H8 114.3 C2O1C17 119.85(14) C7N1C8 113.68(14) O4C18H1 109.5 O4C18H4 109.5 H1C18H4 109.5 O4C12C13 112.06(12) O3C13C14 125.30(13) C14C13C12 117.71(12) C13C14C16 136.69(13) C6C16C1 129.64(13) C1C16C14 122.66(13) O2C1C2 116.51(13) O1C2C3 121.59(14) C3C2C1 129.41(14) O1C17H13 109.5 O1C17H2 109.5 H13C17H2 109.5 C7C15C14 111.04(13) C15C10C9 114.10(15) C9C10C11 129.58(15) C12C11H7 120.4 C8C9C10 119.52(15) C10C9H12 120.2 N1C8H11 116.9 N1C7C15 123.76(14) C15C7C6 107.61(12) C5C6C7 121.79(14) C4C5C6 126.93(15) C6C5H10 116.5 C5C4H9 115.7 C2C3C4 131.43(15) C4C3H8 114.3 Note: structure number sequence in Table 3 is shown in FIG. 9.

[0087] FIG. 9 illustrates a single crystal diffraction structure of 6-hydroxy-5,8-dimethoxy-7H-azulene isoquinolin-[1,2,3-i,j] isoquinolin-7-one. FIG. 10 is a cell stacking diagram thereof.

[0088] The single crystal of 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-one belongs to a monoclinic system and the space group is P 1 21/c 1. Unit cell parameters are: a=7.5352(8) , b=11,7589(10) , c=15.6709(14) , =90, =95.301(6), =90; Z=4, V=1382.6(2) 3, D.sub.c=1.476 mg/cm.sup.3, F(000)=640, =0.09 mm.sup.1, 1965 observable points [I>2(I)], observable point refinement final deviation factor R1=0.0351, wR2=0.0901.

[0089] In the crystal structure of 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-one, the intermolecular structure is maintained stable by - interaction.

Embodiment 5

Protective Effect of 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-One on Damaged Nerve Cells

[0090] Sample: 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,,j] isoquinolin-7-one.

[0091] Cell line: PC12 cells (rat adrenal medullary pheochromocytoma clone), highly differentiated, purchased from Changsha Yingrun Biotechnology Co., Ltd.

[0092] Reagents: DMFM high sugar medium, D-Hanks (Solarbio, no calcium and magnesium, no phenol red), 100 penicillin-streptomycin solution (Beyotime), fetal bovine serum (FBS) (gibco), trypsin (Beijing Suo Labao), tetramethylazo salt method (Sigma, USA), amyloid protein (A.sub.25-35) (Sigma, USA), sterile 96-well plate (NEST).

[0093] Instruments: HFsafe-1500 ultra-clean workbench, FIF151UV CO.sub.2 incubator (Shanghai Li shen Scientific instrument Co., Ltd.); XSP-15C inverted microscope (Shanghai Changfang Optical Instrument Co., Ltd.); Multiskan MK3 microplate reader (USA Thermo); ultrapure water preparation instrument (Milli-Q, USA).

[0094] Cell culture: culturing PC12 cells cultured in TC-treated flasks, wherein a DEME high-sugar medium (pH 7.2) was adopted, containing 10% fetal bovine serum, 100 U.Math.mL.sup.1 penicillin, 100 g.Math.mL.sup.1 streptomycin; incubating at 37 C. in 5% CO.sub.2 cell culture incubator; when cell adherence is up to 90%, digesting with 0.25% trypsin, then add perfusate and pipetting to a single cell suspension; passing or inoculating to an appropriate culture plate, and keeping incubating for 48 h as experimental cells.

[0095] Determination of viability of PC12 cells by A.sub.25-35 with different concentrations: the cultured PC12 cells were seeded at 110.sup.4 cells/well in a 96-well plate, and the cells were grown to a logarithmic growth phase and replaced with serum-free medium for subsequent use. The experiment was divided into 8 groups, 4 wells in each group. The blank control group: 200 L of serum-free medium to each well. The A.sub.25-35 model group: each group was added with serum-free medium for preparing 200 L A.sub.25-35 solution with final concentration of 5, 10, 20, 30, 40, 50 and 60 mol/L; the cells were cultured in an incubator containing 5% CO.sub.2 at 37 C. for 24 h, and the cell viability was measured with MTT by: adding 10 L of an MTT solution (prepared by PBS at a concentration of 5 mg.Math.mL.sup.1) into the wells; after incubating for 4 h in the cell culture incubator, taking out and quickly flipping the 96-well plate; vertically flapping to remove the solution in the wells, so avoid cross-interference; adding 100 L of dimethyl sulfoxide (DMSO), measuring optical density (OD) of each well under 490 nm with enzyme-linked immunosorbent assay, and calculating cell viability, so as to determine concentration required for A.sub.25-35 induced PC12 cells oxidative stress response model.

Cell viability/%=(drug group OD-blank control group OD)/(control groupblank group OD)100%

[0096] Protective effect of 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-one on PC12 cells damaged by A.sub.25-35: cell seeding in the 96-well plate was the same as above, and the experiment was divided into 5 groups, 4 holes in each group. Blank control group: 200 L of serum-free medium to each well; A.sub.25-35 model group: each group was added with serum-free medium for preparing 200 L A.sub.25-35 solution with final concentration of 30 mol/L; drug treatment group: each group was added with serum-free medium for preparing drugs with final concentration of 0.5, 1.0 and 5.0 g.Math.mL.sup.1, and then A.sub.25-35 was added to a final concentration of 30 mol.Math.L.sup.1; wherein final volume of each well was 2004 L; culture and determination methods are the same as above.

[0097] Statistical methods: data were represented with xs, t test was used for comparison between groups, P<0.05 was considered as significant difference, and P<0.01 was extremely significant difference, which was statistically significant.

[0098] Experimental Results:

[0099] (1) Determination of viability of PC12 cells by A.sub.25-35 with different concentrations: after A.sub.25-35 with different concentrations and PC12 cells were incubated together for 24 h, the cell viability decreased significantly, wherein in the range of 10-40 mol.Math.L.sup.1, A.sub.25-35 had a significant dose-related association with PC12 cell damage. 5 mol.Math.L.sup.1 A.sub.25-35 had very little cell damage, only a small number of cells became round. As the concentration increased, the cells aggregated and fell off, which are diffused, and the cell viability also decreased linearly. When the concentration was as high as 40-60 mol.Math.L.sup.1, the degree of damage was similar, and the change was not significant. In this experiment, A.sub.25-35 with a concentration of 30 mol.Math.L.sup.1 was selected as the damage concentration of the damaged nerve cell model.

[0100] (2) Protective Effect of 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-one on PC12 cells damaged by A.sub.25-35:

TABLE-US-00007 TABLE 6 protective effect of 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-one on PC12 cells damaged by A.sub.25-35 (x s, n = 4) concentration sample (ug .Math. mL.sup.1) cell viability (%) blank control 100.00 0.97 model 51.90 1.88* 6-hydroxy-5,8- 0.5 85.80 1.60** dimethoxy-7H- 1.0 102.80 1.80** azulene [1,2,3-i,j] 5.0 77.40 3.80** isoquinolin-7-one *P < 0.01 compared with blank control group; **P < 0.01 compared with model group

[0101] From the analysis of Table 6, it was found that compared with blank control group, the cell viability was significantly decreased after adding 30 mol.Math.L.sup.1 A.sub.25-35 injury factor (P<0.01). Compared to model group, 6-hydroxy-5,8-dimethoxy-7H-azulene [1,2,3-i,j] isoquinolin-7-one with three different concentrations all significantly increased cell viability (P<0.01), wherein viability of PC12 damaged cells was >95% at a concentration of 1.0 g.Math.mL.sup.1. The compound of 7H-azulene [1,2,3-i,j] soquinolin-7-one can be used for preparing a drug for diseases caused by damaged nerve cells, especially for the treatment of senile dementia, stroke and epilepsy.

[0102] The above description is only the preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes can be made to the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and scope of the present invention are intended to be included within the scope of the present invention.