Ligand for orphan nuclear receptor Nur77 and uses thereof
10808005 ยท 2020-10-20
Assignee
Inventors
- Xiaokun Zhang (Xiamen, CN)
- Xiangzhi Lin (Xiamen, CN)
- Ying Su (Xiamen, CN)
- Zhiping Zeng (Xiamen, CN)
- Mengjie Hu (Xiamen, CN)
- Qiang Luo (Xiamen, CN)
- Yi Zhu (Xiamen, CN)
- Alitongbieke Gulimiran (Xiamen, CN)
Cpc classification
G01N2333/912
PHYSICS
C07J63/00
CHEMISTRY; METALLURGY
A61K31/56
HUMAN NECESSITIES
C07J63/008
CHEMISTRY; METALLURGY
International classification
C07J63/00
CHEMISTRY; METALLURGY
Abstract
Provided are use of a compound of Formula I as a ligand of orphan nulear receptor Nur77, and in the prevention or treatment of a orphan nulear receptor Nur77 associated disease, ##STR00001##
Claims
1. A method for treating a triple negative breast cancer, comprising administering to a subject in need thereof an effective amount of a compound selected from (1)-(2), or a tautomer, a stereoisomer or a pharmaceutically acceptable salt or ester thereof: ##STR00083## wherein, in formula (I), X represents NH, N(R), O, CH.sub.2 or halogen; wherein, when X is halogen, R.sub.1 is absent; when the bond between Y and the carbon atom attached thereto is a single bond, Y represents H, halogen, OR, SR or NRR; when the bond between Y and the carbon atom attached thereto is a double bond, Y represents O, S or NR; R.sub.1 is absent or represents H, PO(OR).sub.2, C.sub.1-6alkyl, glycosyl, C.sub.1-6alkoxycarbonyl-C.sub.1-6alkyl, 3- to 8-membered cycloalkyl-aminoacyl, aryl-C.sub.1-6alkyl or aryl, wherein the C.sub.1-6alkyl, glycosyl, C.sub.1-6alkoxycarbonyl-C.sub.1-6alkyl, 3- to 8-membered cycloalkyl-aminoacyl, aryl-C.sub.1-6alkyl and aryl are unsubstituted or substituted with one or more substituents selected from the group consisting of halogen, hydroxy, amino, C.sub.1-6alkyl, C.sub.1-6alkoxy, C.sub.1-6alkylamino and C.sub.1-6alkanoyl; R.sub.2 represents H, D, PO(OR).sub.2, CONH.sub.2, NH.sub.2, NHR, NRR, NHCOR, NRCOR, NHCOOR, NHCONHR, NHCONRR, NRCONHR, NRCONRR, OH, OR, OCONHR, OCONRR, SH, SR, SOR, SOOR, SO.sub.2NHR, nitro, halogen, glycosyl, cyano, trifluoromethyl, C.sub.1-6alkyl, 3- to 8-membered cycloalkyl, 3- to 8-membered heterocycloalkyl, aryl, C.sub.1-6alkyl-substituted aryl, 6- to 15-membered heteroaryl, alkenyl, alkynyl, sulfinyl, sulfonic acid group or sulfonate group; wherein the C.sub.1-6alkyl, 3- to 8-membered cycloalkyl, 3- to 8-membered heterocycloalkyl, aryl, C.sub.1-6alkyl-substituted aryl, 6- to 15-membered heteroaryl, alkenyl and alkynyl are unsubstituted or substituted with one or more substituents selected from the group consisting of amino, halogen, hydroxy, oxy, C.sub.1-6alkyl, C.sub.1-6alkoxy, C.sub.1-6alkylthio, C.sub.1-6alkanoyl, 3- to 8-membered cycloalkyl, 3- to 8-membered oxocycloalkyl, cyano, trifluoromethyl, C.sub.1-6alkoxycarbonyl, C.sub.1-6alkylamido, ureido group, carbamate, carboxyl and aryl; R.sub.3 and R.sub.4 each independently is absent or represents H, C.sub.1-6alkyl, C.sub.1-6alkanoyl, C.sub.1-6alkoxycarbonyl, glycosyl, aryl-C.sub.1-6alkyl or aryl, wherein the C.sub.1-6alkyl, C.sub.1-6alkanoyl, C.sub.1-6alkoxycarbonyl, glycosyl, aryl-C.sub.1-6alkyl and aryl are unsubstituted or substituted with one or more substituents selected from the group consisting of halogen, hydroxy, amino, C.sub.1-6alkyl, C.sub.1-6alkoxy, C.sub.1-6alkylamino and C.sub.1-6alkanoyl; R and R each independently is selected from H, C.sub.1-6alkyl, 3- to 8-membered cycloalkyl, aryl-C.sub.1-6alkyl or aryl, wherein the C.sub.1-6alkyl, 3- to 8-membered cycloalkyl, aryl-C.sub.1-6alkyl and aryl are unsubstituted or substituted with one or more substituents selected from the group consisting of halogen, hydroxy, amino, C.sub.1-6alkyl, C.sub.1-6alkoxy, and C.sub.1-6alkylamino; R represents C.sub.1-6alkyl or aryl; in Formula (I) represents single bond or double bond.
2. The method according to claim 1, wherein, in formula (I), the bond between Y and the carbon atom attached thereto is a double bond, and Y represents O.
3. The method according to claim 1, wherein, in formula (I), X in the compound represents NH, N(R), O, CH.sub.2 or halogen; R represents C.sub.1-6alkyl or 3- to 8-membered cycloalkyl; wherein, when X is halogen, R.sub.1 is absent.
4. The method according to claim 1, wherein, in formula (I), R.sub.1 is absent or represents hydrogen, C.sub.1-4alkyl, PO(OR).sub.2, monoglycosyl, C.sub.1-4alkoxycarbonyl-C.sub.1-4alkyl, 3- to 6-membered cycloalkyl-aminoacyl, aryl-C.sub.1-4alkyl or aryl; wherein the C.sub.1-4alkyl, monoglycosyl, C.sub.1-4alkoxycarbonyl-C.sub.1-4alkyl, 3- to 6-membered cycloalkyl-aminoacyl, aryl-C.sub.1-4alkyl and aryl are unsubstituted or substituted with one or more substituents selected from the group consisting of: halogen, hydroxy, amino, C.sub.1-4alkyl, C.sub.1-4alkoxy, C.sub.1-4alkylamino and C.sub.1-4alkanoyl; R represents C.sub.1-4alkyl.
5. The method according to claim 1, wherein, in formula (I), R.sub.2 represents H, D, OH, PO(OR).sub.2, C.sub.1-6alkyl, 9- to 15-membered fused heteroaryl or sulfonate; wherein the C.sub.1-6alkyl or 6- to 15-membered heteroaryl is unsubstituted or substituted with one or more substituents selected from the group consisting of amino, halogen, hydroxy, oxy, C.sub.1-6alkyl, C.sub.1-6alkoxy, C.sub.1-6alkanoyl, cyano, trifluoromethyl and carboxyl; R represents H, C.sub.1-6alkyl or aryl.
6. The method according to claim 1, wherein, in formula (I), the Carbon 7 and Carbon 8 of the compound are linked with a carbon-carbon double bond.
7. The method according to claim 1, wherein, in formula (I), the bond between Y and the carbon atom attached thereto in the compound is a double bond.
8. The method according to claim 1, wherein, in formula (I), the bond between Y and the carbon atom attached thereto in the compound is a single bond.
9. The method according to claim 1, wherein the compound has the following structure: ##STR00084## wherein R.sub.3 and R.sub.4 each independently represents H, C.sub.1-6alkyl or C.sub.1-6alkanoyl.
10. The method according to claim 1, wherein the compound has the following structure: ##STR00085## wherein R.sub.4 represents H, C.sub.1-6alkanoyl, C.sub.1-6alkoxycarbonyl or monoglycosyl substituted with one or more C.sub.1-6alkanoyl groups.
11. The method according to claim 1, wherein the compound is selected from the following compounds: ##STR00086## ##STR00087## ##STR00088## ##STR00089## ##STR00090## ##STR00091## ##STR00092## ##STR00093## ##STR00094## ##STR00095## ##STR00096## ##STR00097## ##STR00098## ##STR00099##
Description
DRAWINGS
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EXAMPLE 1. CHARACTERIZATION OF COMPOUND YXY101
(47) (1) Surface Plasmon Resonance (SPR)
(48) The binding of YXY101 to Nur77 was tested by surface plasmon resonance. Briefly, 50 g purified ligand binding domain of Nur77 (Nur77-LBD) protein was coupled to CM5 of Biacore; the binding of YXY101 (20 m) to Nur77-LBD was subsequently tested by Biacore T200. Unrelated compounds were used as controls. The test results were shown in
(49)
(50) (2) Binding Kinetics of YXY101 to Nur77
(51) The dissociation constant (Kd) of YXY101 to Nur77 was tested by surface plasmon resonance. Briefly, Biacore T200 instrument was used to measure the binding of YXY101 in different concentrations (0.04 M, 0.08 M, 0.16 M, 0.32 M, 0.64 M) to Nur77-LBD. The test results were shown in
(52) The results showed that the dissociation constant (Kd) of the compound YXY101 to Nur77-LBD was 292 nM.
(53) (3) Circular Dichroism Spectroscopy (CD)
(54) The binding of YXY101 to Nur77-LBD was further analyzed by circular dichroism spectroscopy (CD). Briefly, YXY101 (1 ml, 1 mg/ml) was added to the phosphate buffer (10 m, pH 7.4) of Nur77-LBD protein (1 ml, 1 mg/ml) and incubated at 4 C. for 3 h. 0.7 ml of the incubation buffer was detected with Jasco J-810 spectropolarimeter. The CD spectra was obtained from 190 nm to 260 nm. Nur77-LBD solution (i.e., no YXY101 was added) alone was used as control. The results of the detection were shown in
(55) The results showed that the compound YXY101 was able to change the CD spectrum of Nur77-LBD, indicating that the compound YXY101 was able to bind to Nur77-LBD.
(56) (4) High Performance Liquid Chromatography (HPLC) Analysis
(57) The binding of YXY101 to Nur77-LBD was further analyzed by high performance liquid chromatography (HPLC). Briefly, YXY101 (600 uL, 0.1 mg/ml) was incubated with purified Nur77-LBD protein (5 ml, 1 mg/ml). After incubation for 3 h at 4 C., the complex of YXY101 and Nur77-LBD was captured using Ni beads. The complex was then degenerated with chloroform, and YXY101 in the degenerated product was extracted. Then, HPLC spectrometer (Shimadzu L C 20A, Japan) was used to detect YXY101 in the extracted product, wherein ODS column (5 um, 4.6*250 mm) was used, and the mobile phase was 0.2% H.sub.3PO.sub.4 acetonitrile. The detection wavelength was 425 nm. In addition, the above experiment was repeated using YXY101 and RXR-LBD (ligand binding domain of retinoic X receptor ) as control. The results of the detection were shown in
(58) The results showed that the compound YXY101 was able to bind to Nur77-LBD to form a complex, but not to RXR-LBD.
(59) (5) Dual Luciferase Reporter Assay
(60) The binding of YXY101 to Nur77-LBD was further analyzed by dual luciferase reporter assay. In addition, the experiment was repeated using YXY101 and glucocorticoid receptor (GR) as control. The results were shown in
(61) The results showed that the compound YXY101 was able to inhibit the transactivation of Nur77, but had no significant effect on the transactivation of glucocorticoid receptor (GR). This indicated that the compound YXY101 was capable of binding to Nur77-LBD and inhibiting its transcriptional activity; but not to GR.
(62) (6) Molecular Simulation
(63) The docking of YXY101 to Nur77 (PDB code: 4JGV) was performed using AutoDock V4.2. The conformation of YXY101 was generated by the Lamarckian genetic algorithm. In the crystal structure of Nur77, grid center was chosen aroud the reported coordinates (12.08, 18.29, 4.233) of THPN, and the grid size was set to 40*40*40 (X, Y, Z) grid points with a spacing of 0.375 A between grid points.
(64) In the molecular docking, the standard docking protocol was applied: the number of randomly placed individuals was 150; the maximum number of energy evaluation was 2.5 million; the rate of gene mutations was 0.02; the rate of crossover was 0.8; the probability of performing local evaluation was 0.06; the lower bound on rho was 0.01; PyMOL Version 0.99 was used for molecular visualization. The results were shown in
(65) The molecular docking studies showed that YXY101 binded to the known hydrophobic grooves on the surface of Nur77 protein mainly by hydrophobic interaction.
EXAMPLE 2. YXY101 INHIBITS THE BIOLOGICAL EFFECT OF TNF
(66) HepG2 cells were treated with different concentrations of YXY101 (0 M, 0.25 M, 0.5 M, 1 M, 2 M or 4 M) for 1 hour, and then exposed to TNF (0 ng/mL or 20 ng/mL) for 30 minutes. IB and phosphorylated IKK/ in the cells were then detected by WB. The results were shown in
(67) The results of
(68) HepG2 cells were treated with YXY101 (0 or 1 M) for 1 hour, and then exposed to TNF (20 ng/mL) for 30 minutes. The p65 subunit of NF-B in the cells was then detected by immunofluorescence staining method. Untreated cells were used as controls. The results were shown in
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(70) The NF-B reporter gene was transfected into HEK-293T cells and then treated with YXY101 (0 or 1 M) and TNF (20 ng/mL). NF-B activity in the cells was then analyzed. Untreated cells were used as controls. The results were shown in
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(72) A variety of cancer cell lines (LO2, SMMC-7721, QGY-7703, HeLa, H460) were treated with different concentrations of YXY101 (0 M, 1 M or 4 M) for 1 hour, and then exposed to TNF (0 ng/mL or 20 ng/mL) for 30 minutes. IB and phosphorylated IKK/ in the cells were then detected by WB. The results were shown in
(73) The results showed that TNF was able to induce phosphorylation of IKK/ and degradation of IB in various cell lines, whereas YXY101 was able to inhibit TNF-induced phosphorylation of IKK/ and degradation of IB.
(74) In addition, HepG2 cells were also applied in the experiment. Briefly, HepG2 cells were treated with various compounds (YXY101, XS0284, and XS0287) in specified concentrations for a specified period of time, and then exposed to TNF (0 ng/mL or 20 ng/mL) for 30 minutes. IB in the cells was then detected by WB. The results were shown in
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(76) The results of
EXAMPLE 3. Nur77 MEDIATES THE INHIBITORY EFFECT OF YXY101 ON BIOLOGICAL EFFECTS OF TNF
(77) SiRNA, Nur77 SiRNA or RXR SiRNA as controls were transfected into HepG2 cells. Subsequently, the HepG2 cells were treated with different concentrations of YXY101 (0 M, 1 M or 4 M) for 1 hour, and then exposed to TNF (0 ng/mL or 20 ng/mL) for 30 minutes. Nur77, RXR and IB in the cells were then detected by WB. The results were shown in
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(79) The above results were also confirmed by using MEF cells and Nur77/MEF cells (i.e., MEF cells not expressing Nur77). Briefly, MEF cells and Nur77/MEF cells were treated with different concentrations of YXY101 (0 M or 1 M) for 1 hour, and then exposed to TNF (0 ng/mL or 20 ng/mL) for 30 minutes. Subsequently, IB in the cells was detected by WB. The results were shown in
(80) The results showed that YXY101 was able to inhibit TNF-induced degradation of IB in the MEF cells expressing Nur77; however, in Nur77/MEF cells, YXY101 lost the ability to inhibit IB degradation. These results indicated that the inhibitory effect of YXY101 on the biological effects of TNF was mediated by Nur77.
(81) In addition, MEF cells and Nur77/MEF cells were treated with YXY101 (0 or 1 M) for 1 hour, and then exposed to TNF (20 ng/mL) for 30 minutes. Subsequently, the p65 subunit of NF-B in the cells was detected by immunofluorescence staining. Untreated cells were used as controls. The results were shown in
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EXAMPLE 4. YXY101 HAS SIGNIFICANT ANTI-TUMOR ACTIVITY, AND IS PARTICULARLY SENSITIVE TO TRIPLE-NEGATIVE BREAST CANCER, THE BIOLOGICAL FUNCTION IS DEPENDENT ON Nur77
(84) Different breast cancer cells (MDA-MB-231; MDA-MB-468; BT549; SKBR3; T47D and MCF-7) were treated with different concentrations of YXY101 (1 M, 1.3 M, 1.6 M, 1.9 M, 2.2 M, 2.5 M, 2.8 M, 3.1 M, 3.4 M, 3.7 M, 4.0 M) for 72 h, and the proliferation ratio of the breast cancer cells were determined. Curves of the cancer cell proliferation ratio in relation to the concentrations of YXY101 were plotted, and the IC50 of YXY101 was determined. The results were shown in
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(87) Further, MDA-MB-231 and MCF-7 cells were treated with different concentrations of YXY101 (0 M, 0.25 M, 0.5 M, 1 M, 2 M, or 4 M), followed by detection of parp and ER in the cells by WB. The results were shown in
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(89) Further, MDA-MB-231 was treated with different concentrations of YXY101 (0 M, 2 M, or 4 M) for different period of time (12 h or 24 h), followed by detection of parp and p-mTOR in the cells by WB. The results were shown in
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(91) In addition, MDA-MB-231 cells were also treated with YXY101 in combination with TNF for different period of time (1 h, 6 h or 12 h), followed by detection of parp in the cells by WB. The results were shown in
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EXAMPLE 5. INHIBITORY EFFECT OF YXY101 ON THE PROLIFERATION OF BREAST CANCER TUMORS
(93) In this section, we tested the anti-tumor effect of YXY101 alone or in combination with TNF on triple-negative breast cancers via nude mice xenograft experiments.
(94) 1) Laboratory Animals and Reagents
(95) MDA-MB-231 cells; BALB/c (nu/nu) nude mice, weighed 18-20 g, female, raised in SPF animal house, fodder, drinking water, animal cages, litter were all autoclaved, the litter was changed every two days, and strictly aseptic operations were performed. Under sterile conditions, the cells in the logarithmic growth phase for inoculation were collected and washed with serum-free medium, the number of viable cells was counted under inverted microscope and the survival rate >95%, the cell concentration was adjusted to 110.sup.6/ml, and the tumor cells were re-suspended in PBS to prepare the cell suspension. Each nude mouse was inoculated on right subaxillary with 0.2 ml of the above cell suspension, and the tumor growth condition was observed regularly.
(96) 2) Grouping and Administration
(97) The drug-administered groups: when the diameter of the transplanted tumor of nude mice reached about 0.5 cm, the nude mice without hemorrhage, necrosis and infection were selected for experiment. The nude mice were weighed, the tumor diameter of which was measured, and then grouped, 6 nude mice in each group; the mice of the experimental groups were administered with TNF (12010.sup.4 U/kg), YXY101 (2 mg/kg), or the combination thereof, and the mice of the control group were given the same amount of normal saline. TNF was administered by intratumoral injection every other day; YXY101 was administered intragastrically daily. The nude mice were sacrificed 6 hours after the last administration, and the tumor weight was measured.
(98) During the treatment, the food intake and body weight of the nude mice were not significantly reduced, the activity was normal, and no symptoms such as loose hair and diarrhea appeared. At the end of treatment, no death occurred in each group of nude mice. After the nude mice were sacrificed, the autopsy showed tumors had clear boundaries, uneven surface, tough texture, significant local expansion of blood vessels, and necrosis occurred in the central area of some tumors. No metastasis was observed in all groups of nude mice, and no obvious changes in appearance in the heart, liver, spleen, lung, kidney and other organs was observed in the treatment groups of nude mice.
(99) 3) Observation Index
(100) Drawing of Tumor Growth Curves:
(101) The formula for calculating the tumor volume (TV) is: V=abc; wherein a, b and c represent length, width and height, respectively. The tumor volume was calculated based on the measurement results, and the tumor growth curve was plotted with time as abscissa and tumor volume as ordinate.
(102) The anti-tumor activity evaluation index was the relative tumor proliferation rate T/C (%): wherein T represents the RTV of the treatment group; C represents the RTV of the negative control group. The therapeutic effect evaluation criteria were: T/C %>40% for ineffective; T/C %40% and p<0.05 for effective.
(103) The experimental results were shown in
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EXAMPLE 6. INHIBITORY EFFECT OF YXY101 ON PROLIFERATION AND METASTASIS OF BREAST CANCER
(105) 1) Laboratory Animals and Reagents
(106) MMTV-PYVT transgenic mice of breast cancer, 9-week-old, female, were raised in SPF animal house, fodder, drinking water, animal cages, litter were all autoclaved, the litter was changed every two days, strictly aseptic operations were performed.
(107) 2) Grouping and Administration
(108) Female MMTV-PYVT transgenic mice of breast cancer were housed under the conditions of temperature 231 C., humidity: 40-60%, natural light, freely drinking of water, and freely access to chow diet. Thirty-six mice with tumor began to grow in chest were selected and randomly divided into three groups, and each group was subdivided into the control group and the YXY101 group.
(109) The administration was performed as follows:
(110) 11 Wk time point
(111) Control group: 9-week-old mice were given normal saline at 7:00 m every day before the fodder was given;
(112) YXY101 group: 9-week-old mice were intragastrically administered once at a dose of 2 mg/kg at 7:00 m every day.
(113) 13 k time point
(114) Control group: 11-week-old mice were given normal saline at 7:00 m every day before the fodder was given;
(115) YXY101 group: 11-week-old mice were intragastrically administered once at a dose of 2 mg/kg at 7:00 m every day.
(116) 17 k time point
(117) Control group: 15-week-old mice were given normal saline at 7:00 m every day before the fodder was given;
(118) YXY101 group: 15-week-old mice were intragastrically administered once at a dose of 2 mg/kg at 7:00 m every day.
(119) After continuous administration for two weeks according to the above method, each animal was bled from the eye, and the supernatant was taken to determine the serum inflammation index; tumor tissue having clear boundary, uneven surface, and tough texture was routinely treated and embedded for immunostaining.
(120) The experimental results were shown in
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(123) The experimental results in
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(125) In summary, we creatively screened drug targeting at Nur77 by using SPR technology, thereby obtaining the drug molecule YXY101 that specifically binds to Nur77, it showed significant anti-tumor activity, especially sensitive to triple-negative breast cancer, and this therapeutic activity for breast cancer was dependent on Nur77. The compound was a very promising active molecule for the treatment of triple negative breast cancer, and the screening method was an effective way to develop drug molecule that specifically binds to Nur77 and for the target treatment of triple negative breast cancer.
EXAMPLE 7. INFLAMMATION CAN INDUCE TUMORIGENESIS TO A CERTAIN EXTENT, AND THIS PROCESS IS Nur77 RELEVANT
(126) In the previous study, we found that the development of breast cancer, especially triple-negative breast cancer, was also accompanied by inflammatory response and high activation of mTOR signaling pathway. Chronic inflammation was also an important basis for the development of breast cancer, and many non-steroidal anti-inflammatory drugs effective to other tumors had also been used for the prevention and treatment of breast cancer.
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(128) In combination with Example 6 and Example 7, Nur77 was not only important for the development of breast cancer, but also can be used as a target for the treatment of breast cancer. Therefore, Nur77 was a valuable biochemical indicator for the detection of development of breast cancer, and can be used to evaluate breast cancer progression and treatment strategies.
EXAMPLE 8. YXY101 WAS ABLE TO SIGNIFICANTLY INHIBIT mTOR ACTIVITY, WHICH IN TURN INHIBITS TUMORS, AND THIS PROCESS RELIES ON Nur77
(129) In this section, the internal environment of tumor was experimentally simulated, that was, TNF, a cytokine that caused acute inflammation, was used to simulate tumorigenesis. This method could provide an experimental basis for screening drugs inhibiting tumors. By this method, Nur77-dependent compounds that inhibited the mTOR signaling pathway were screened to find a drug effective in the targeting treatment of triple-negative breast cancer.
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(131) In conclusion, the inhibition of mTOR signaling pathway can be used as an effective indicator for screening anticancer drugs. By this method, compounds screened having potent activity of inhibiting mTOR signaling pathway via Nur77 have potential anticancer activity.
EXAMPLE 9. INDUCTION OF APOPTOSIS OF YXY101 DERIVATIVES ON TRIPLE-NEGATIVE BREAST CANCER CELLS
(132) Different breast cancer cells (MDA-MB-231 and MCF-7) were treated with different concentrations of YXY101 derivative XS0503 (0.16 M, 0.31 M, 0.625 M, 1.25 M, 2.5 M, 5 M, 10.0 M) for 24 h, and the proliferation ratio of breast cancer cells were measured. Curves of the cancer cell proliferation ratio in relation to the concentrations of XS0503 were plotted, and the IC50 of YXY101 was determined. The results were shown in
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(134) Further, MDA-MB-231 was treated with different YXY101 derivatives, XS0077, XS0335, XS0419, XS0474, or XS0488 (0.16 M, 0.31 M, 0.625 M, 1.25 M, 2.5 M, 5 M, 10.0 M), followed by measurement of MDA-MB-231 proliferation ratio. The results in
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(136) Further, YXY101 (4 M) or different YXY101 derivatives (4 M), XS0394, XS0395, XS0419, XS0420, XS0421, XS0284, XS0335, XS0488, XS0491, XS0492, XS0418, XS0502, XS0503, XS0506, XS0507, XS0508, or XS0077, and TNF (20 ng/ml) were used to treat MDA-MB-231 cells and PARP in the cells were subjected to immunoblotting analysis. The results
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EXAMPLE 10. CHARACTERIZATION OF OTHER COMPOUNDS BY SURFACE PLASMON RESONANCE (SPR)
(138) According to the methods described in Examples 1 and 2, the binding of various YXY101 derivatives (XS0418, XS0419, XS0474, XS0394, XS0492, XS0491, XS0488) to Nur77-LBD was detected by SPR using Biacore T200 instrument. The results were shown in
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EXAMPLE 11. XS0284 IS MORE EFFECTIVE IN TREATING HYPERLIPEMIA INDUCED BREAST CANCER THAN YXY101
(140) In order to prove that YXY101 derivatives can also inhibit breast cancer, we selected XS0284 showing strong inhibitory effect on P-mTOR, P-p70S6K and PS6 for confirmation. The results of
(141) These results indicated that a series of YXY101 derivatives (such as XS0284) can also inhibit tumor development, and they were even superior to YXY101 in term of tumor inhibition.
EXAMPLE 12. THE TUMOR INHIBITION EFFECTS OF YXY101 AND ITS DERIVATIVES IS DEPENDENT ON Nur77
(142) In order to prove the anti-tumor effect of YXY101 and its derivatives is relied on Nur77, we selected YXY101 and its derivative XS0284, which showed significant inhibitory effect on P-mTOR, P-p70S6K and PS6 for confirmation.
(143) The above results indicated that YXY101 and its derivatives rely on Nur77 to exert their tumor suppressing effect.
EXAMPLE 13. THE ACUTE TOXIC-SIDE EFFECTS OF XS0284 ARE LOWER THAN YXY101
(144) In this example, acute toxicity model mice were established by single intragastric injection of 200 mg/kg of YXY101 or its derivative XS0284 or by single intraperitoneal injection of 20 mg/kg of YXY101 or its derivative XS0284. In this example, after intragastric administration or intraperitoneal injection of YXY101 or XS0284, the mice were observed for food intake, drinking of water, spontaneous activity, mental state, movement of the limbs, bowel quality, hair gloss, etc., and any possible toxic reactions and time points of oneset as well as offset thereof were recorded in details. The tissue morphologies of the heart, liver, small intestine, white fat and kidney were observed by Histopathological method.
(145) The experimental results were shown in
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(147) These results indicated that a series of derivatives of YXY101, such as XS0284, had less acute toxicity to animals than YXY101. It further suggested that this series of compounds had stronger targeting and specificity than YXY101, and this series of YXY101 derivatives was possibly to be developed as a safer anticancer drug.
(148) Preparation of Compound XS0077
(149) ##STR00046##
(150) Compound YXY101 (50 mg, 0.11 mmol) was dissolved in 2 mL of DMF with stirring, followed by an addition of sodium hydrogencarbonate (56 mg, 0.66 mmol) and methylene chloride (42 L, 0.66 mmol), then subjected to an reaction with stirring at room temperature for 12 hours. The reaction was quenched with 1 mol/L HCl (1 mL), then the resulting mixture was added with 9 mL of purified water, and extracted with ethyl acetate three times (5 mL each time). The organic layer was collected, dried over anhydrous sodium sulfate, and then subjected to vacuum evaporation to remove organic solvent ethyl acetate, thereby obtaining an orange-red solid of mixed crude product. The orange-red solid product was obtained by column chromatography, n-hexane and ethyl acetate (hexane/ethyl acetate=10:1) was used as eluent, and the column was packed with 300-400 mesh silica gel.
(151) .sup.1H NMR (600 MHz, DMSO-d6) ppm 0.44 (s, 3H), 0.91 (d, J=14.31 Hz, 1H), 1.07 (s, 3H), 1.12 (s, 3H), 1.21 (s, 3H), 1.30-1.35 (m, 1H), 1.38 (s, 3H), 1.41-1.46 (m, 1H), 1.50-1.59 (m, 3H), 1.61-1.72 (m, 4H), 1.78-1.86 (m, 1H), 1.95 (td, J=13.98, 3.76 Hz, 1H), 2.06 (d, J=14.12 Hz, 1H), 2.09 (s, 3H), 2.17-2.22 (m, 1H), 2.31 (d), J=15.77 Hz, 1H), 3.48 (s, 3H), 6.35 (d, J=7.15 Hz, 1H), 6.39 (d, J=1.28 Hz, 1H), 7.07 (dd, J=7.15, 1.10 Hz, 1H), 8.72 (s, 1H).
(152) .sup.13C NMR (151 MHz, DMSO-d6) ppm 10.10, 17.96, 21.41, 28.08, 29.19, 29.42, 30.12, 30.34, 31.34, 32.23, 32.91, 34.39, 36.02, 37.83, 38.8, 39.83, 41.99, 43.64, 44.48, 51.44, 117.26, 118.05, 120.18, 126.89, 133.13, 146.42, 162.94, 167.80, 177.93, 177.96.
EXAMPLE 15. PREPARATION OF COMPOUND XS0284
(153) ##STR00047##
(154) Compound YXY101 (50 mg, 0.11 mmol) was dissolved in methanol (2.5 mL) with stirring under nitrogen atmosphere. Followed by an addition of an aqueous solution of sodium hydrogen sulfite (14 mg, 0.13 mmol, dissolved in 1 mL of water) and allowed to react at room temperature for 3 hours. After the reaction, the reaction system was concentrated by a rotary evaporator to give colorless crystals. The colorless crystals were recrystallized from methanol/water, and the obtained product was dried in vacuo to afford compound XS0284 (56.6 mg) as white solid.
(155) .sup.1H NMR (600 MHz, DMSO-d6) ppm 0.59 (s, 3H) 0.86 (d, J=12.10 Hz, 1H) 1.05 (s, 3H) 1.09 (s, 3H) 1.18 (s, 3H) 1.43-1.60 (m, 6H) 1.62 (s, 3H) 1.78-1.85 (m, 1H) 1.93-2.04 (m, 3H) 2.21 (s, 3H) 2.32 (d, J=15.22 Hz, 1H) 4.48 (d, J=6.24 Hz, 1H) 5.81 (d, J=6.60 Hz, 1H) 6.58 (s, 1H) 7.61 (br.s., 1H) 8.81 (br.s., 1H).
(156) .sup.13C NMR (151 MHz, DMSO-d6) ppm 13.57, 18.46, 21.64, 29.04, 29.98, 30.47, 30.60, 30.64, 31.94, 32.92, 34.80, 35.11, 36.90, 36.96, 37.90, 38.07, 43.95, 44.39, 60.19, 108.96, 118.87, 123.26, 124.39, 140.89, 141.93, 144.02, 150.07, 180.00.
EXAMPLE 16. PREPARATION OF COMPOUND XS0285
(157) ##STR00048##
(158) Compound YXY101 (50 mg, 0.11 mmol) was dissolved in N,N-dimethylformamide (2 mL) with stirring. Sodium hydrogencarbonate (50.4 mg, 0.6 mmol) was added, then benzyl bromide (0.17 mg, 20 L) was added, and the reaction was carried out under stirring at room temperature for 24 hours. The reaction was stopped, and the reaction mixture was added with deionized water (15 mL), and extracted with ethyl acetate for three times. The combined ethyl acetate layer was washed with saturated aqueous solution of NaCl three times, dried over anhydrous Na.sub.2SO.sub.4, concentrated by a rotary evaporator to give a crude product (dark red-brown oily matter). The crude product was separated and purified by a rapid column chromatography (ethyl acetate:n-hexane) and dried in vacuo to afford compound XS-0285 (44 mg) as red solid.
(159) .sup.1H NMR (600 MHz, CHLOROFORM-d) ppm 0.50 (s, 3H) 0.97 (d, J=13.75 Hz, 1H) 1.09 (s, 3H) 1.21 (s, 3H) 1.22-1.25 (m, 3H) 1.25-1.28 (m, 1H) 1.41 (s, 3H) 1.47-1.58 (m, 3H) 1.58-1.72 (m, 5H) 1.87 (d, J=6.05 Hz, 1H) 1.99-2.11 (m, 3H) 2.21 (d, J=1.65 Hz, 3H) 2.24 (d, J=14.12 Hz, 1H) 2.44 (d, J=15.77 Hz, 1H) 4.93 (d, J=12.29 Hz, 1H) 5.02 (d, J=12.47 Hz, 1H) 6.32 (d, J=7.15 Hz, 1H) 6.49 (s, 1H) 7.01 (d, J=6.97 Hz, 1H) 7.27-7.30 (m, 2H) 7.30-7.36 (m, 3H).
(160) .sup.13C NMR (151 MHz, CHLOROFORM-d) ppm 10.29, 18.53, 21.58, 28.61, 29.53, 29.90, 30.55, 30.76, 31.57, 32.76, 33.27, 34.69, 36.35, 38.25, 39.43, 40.44, 42.93, 44.25, 45.03, 66.32, 117.36, 118.12, 119.62, 127.38, 128.24, 128.32, 128.64, 134.24, 135.68, 146.06, 164.79, 170.27, 177.95, 178.36.
EXAMPLE 17. PREPARATION OF COMPOUND XS0335
(161) ##STR00049##
(162) Compound YXY101 (50 mg, 0.11 mmol) was dissolved in dichloromethane (1 mL) with stirring. Palladium carbon (5 mg) was added, then dichloromethane (1 mL) was added, and hydrogen was continuously introduced, and reacted at room temperature for 24 hrs. The reaction was stopped. The reaction mixture was added with deionized water (15 mL), extracted with ethyl acetate three times. The ethyl acetate layers were combined, then washed three times with saturated NaCl, dried over anhydrous Na.sub.2SO.sub.4, concentrated by a rotary evaporator to give a crude product (colorless oily matter). The crude product was separated and purified by a rapid column chromatography (ethyl acetate:n-hexane) and dried in vacuo to afford compound XS-0335 (46 mg) as white solid.
(163) .sup.1H NMR (600 MHz, METHANOL-d.sub.4) ppm 0.93-1.00 (m, 2H) 1.03 (s, 3H) 1.11 (s, 3H) 1.19 (t, J=3.48 Hz, 4H) 1.26 (s, 3H) 1.41-1.45 (m, 6H) 1.47-1.55 (m, 2H) 1.57 (d, J=8.25 Hz, 1H) 1.59-1.68 (m, 2H) 1.83-1.98 (m, 3H) 2.06-2.11 (m, 4H) 2.11-2.20 (m, 4H) 2.43 (d, J=15.77 Hz, 1H) 2.68 (d, J=14.67 Hz, 1H) 6.67 (s, 1H).
(164) .sup.13C NMR (151 MHz, METHANOL-d.sub.4) ppm 11.94, 18.51, 20.54, 26.82, 28.66, 30.74, 31.15, 31.57, 31.61, 32.02, 32.09, 32.32, 33.63, 34.65, 37.09, 37.56, 38.53, 39.45, 39.85, 41.69, 58.12, 58.48, 112.24, 121.74, 129.36, 141.47, 142.39, 144.19, 182.95.
EXAMPLE 18. PREPARATION OF COMPOUNDS XS0366, XS0434-XS0438, XS0440, XS0441, XS0443, XS0463 AND XS0464
(165) ##STR00050##
(166) Taking the preparation of XS0366 for example, compound YXY101 (100 mg, 0.22 mmol) was dissolved in dichloromethane (4 mL) under stirring. Indole (52 mg, 0.44 mmol) was added, followed by an addition of aluminum trichloride hexahydrate (5.3 mg, 0.022 mmol), and the reaction was conducted under stirring at room temperature for 5 hours. The reaction was stopped, and the reaction system was added with deionized water (15 mL), then extracted three times with ethyl acetate. The combined ethyl acetate layer was washed with saturated NaCl three times, dried over anhydrous Na.sub.2SO.sub.4, and concentrated by a rotary evaporator to obtain a crude product (brown oily matter). The crude product was separated and purified by rapid column chromatography (ethyl acetate:n-hexane), dried in vacuo to afford compound XS0366 (122.2 mg) as purple red solid.
(167) .sup.1H NMR (600 MHz, CHLOROFORM-d) ppm 0.73 (br. s., 3H) 0.87-0.90 (m, 1H) 0.95-1.00 (m, 3H) 1.01 (br. s., 3H) 1.14 (br. s., 3H) 1.19 (t, J=7.06 Hz, 1H) 1.25-1.27 (m, 1H) 1.34 (br. s., 3H) 1.42-1.57 (m, 4H) 1.57-1.76 (m, 4H) 1.90 (s, 3H) 1.99-2.07 (m, 2H) 2.10-2.17 (m, 1H) 2.40 (d, J=15.04 Hz, 1H) 4.90 (d, J=5.69 Hz, 1H) 6.21 (d, J=6.24 Hz, 1H) 6.23 (br. s., 1H) 6.79 (br. s., 1H) 7.11 (t, J=7.43 Hz, 1H) 7.16 (t, J=7.43 Hz, 1H) 7.28 (d, J=7.89 Hz, 1H) 7.75 (d, J=7.70 Hz, 1H) 7.88 (br. s., 1H).
(168) .sup.13C NMR (151 MHz, CHLOROFORM-d) ppm 11.53, 18.84, 21.93, 28.89, 29.63, 29.75, 30.40, 30.54, 30.72, 31.55, 32.84, 34.62, 35.50, 36.74, 36.94, 37.76, 40.35, 43.62, 44.28, 108.93, 111.30, 119.09, 119.28, 120.23, 121.55, 121.58, 121.67, 127.10, 127.84, 136.49, 139.92, 142.17, 142.83, 147.48, 184.30.
(169) According to the above method, the following compounds were also synthesized in the present invention:
(170) ##STR00051## ##STR00052## ##STR00053## ##STR00054##
EXAMPLE 19. PREPARATION OF COMPOUND XS0395
(171) ##STR00055##
(172) Compound YXY101 (50 mg, 0.11 mmol) was weighed in a 25 ml reaction flask, and 4 ml of acetone was added thereto and dissolved under stirring, and then a drop of concentrated hydrochloric acid was added as catalyst, and the reaction was carried out at room temperature for 12 hours. The reaction was stopped, and the reaction mixture was directly concentrated to remove solvent. The residue was purified by silica gel column chromatography with ethyl acetate:n-hexane=4:1, affording a white solid in a yield of 51%.
(173) .sup.1H NMR (600 MHz, DMSO-d6) ppm 0.62 (s, 3H), 0.82-1.86 (m, 1H), 1.04 (s, 3H), 1.09 (s, 3H), 1.15 (s, 3H), 1.25-1.38 (m, 4H), 1.39 (s, 3H), 1.42-1.50 (m, 2H), 1.55-1.71 (m, 4H), 1.77 (td, J=13.9, 6.1 Hz, 1H), 1.93-2.02 (m, 2H), 2.03 (s, 3H), 2.09 (s, 3H), 2.27-2.36 (m, 2H), 2.67 (dd, J=16.2, 2.7 Hz, 1H), 3.71-3.78 (m, 1H), 5.72 (d, J=6.4 Hz, 1H), 6.63 (s, 1H), 7.91 (s, 1H), 8.94 (s, 1H), 12.06 (br.s., 1H).
(174) .sup.13C NMR (151 MHz, DMSO-d6) ppm 11.64, 18.43, 22.63, 29.01, 29.90, 30.36, 30.47, 30.62, 30.77, 31.44, 31.87, 32.88, 32.92, 34.94, 35.60, 36.79, 36.85, 36.98, 37.74, 39.87, 43.74, 44.36, 51.72, 109.06, 120.00, 122.04, 126.53, 140.46, 141.62, 143.85, 149.91, 179.96.208.01.
EXAMPLE 20. PREPARATION OF COMPOUND XS0419
(175) ##STR00056##
(176) Compound YXY101 (50 mg, 0.11 mmol) was dissolved under stirring in 2 mL of deuterated methanol, then sodium borohydride (44 mg, 1.1 mmol) was added, the reaction was carried out at room temperature for 30 min. The reaction was quenched with 1 mol/L HCl (1 mL), and then 9 mL of pure water was added, and extracted with dichloromethane (5 mL each time) three times. The organic layers were combined, dried over anhydrous sodium sulfate, and then the organic solvent dichloromethane was rapidly removed by vacuum distillation to obtain compound XS0419 (50.1 mg) as a white solid.
(177) .sup.1H NMR (600 MHz, DMSO-d.sub.6) ppm 0.66 (s, 3H) 0.85 (d, J=13.9 Hz, 1H) 1.05 (s, 3H) 1.11 (s, 3H) 1.17 (s, 3H) 1.22 (s, 3H) 1.29 (ddd, J=13.7, 4.4 Hz, 1H) 1.36-1.41 (m, 1H) 1.43-1.51 (m, 3H) 1.53-1.59 (m, 1H) 1.59-1.63 (m, 1H) 1.63-1.69 (m, 1H) 1.79 (ddd, J=13.8, 6.5 Hz, 1H) 1.86 (ddd, J=13.8, 5.0 Hz, 1H) 1.94-2.00 (m, 2H) 2.01 (s, 3H) 2.04 (d, J=13.6 Hz, 1H) 2.34 (d, J=15.6 Hz, 1H) 2.91 (dd, J=20.0, 1.5 Hz, 1H) 3.18 (dd, J=20.5, 6.2 Hz, 1H) 5.72 (dd, J=6.1, 1.8 Hz, 1H) 6.61 (s, 1H) 7.82 (s, 1H) 8.80 (s, 1H) 12.05 (br. s., 1H).
(178) .sup.13C NMR (151 MHz, DMSO-d.sub.6) ppm 11.53, 18.10, 22.70, 27.30, 28.45, 29.46, 29.77, 30.08, 30.19, 31.39, 32.44, 34.06, 34.09, 34.40, 36.07, 36.56, 37.14, 39.44, 43.25, 43.83, 108.17, 117.67, 120.10, 123.10, 139.35, 140.56, 143.11, 149.22, 179.51.
EXAMPLE 21. PREPARATION OF COMPOUND XS0462
(179) ##STR00057##
(180) First, Celastrol (135.2 mg, 0.3 mmol) was dissolved in 2 mL of DMF under stirring, followed by an addition of sodium hydrogencarbonate (138.6 mg, 1.65 mmol) and ethyl bromide (234 L, 0.15 mmol), the reaction was carried out under stirring at room temperature for 12 hours. The reaction was quenched with 1 mol/L HCl (1 mL), 9 mL of pure water was added. The resulting mixture was extracted three times with ethyl acetate (15 mL each time). The organic layers were combined, dried over anhydrous sodium sulfate, and then the organic solvent ethyl acetate was removed by vacuum evaporation to afford a crude product as orange-red mixture solid. The crude product was separated by column chromatography with n-hexane and ethyl acetate (hexane/ethyl acetate=10:1) as eluent, using 300-400 mesh silica gel packed column, affording an orange-red solid product.
(181) .sup.1H NMR (600 MHz, DMSO-d.sub.6) ppm 0.47 (s, 3H), 0.91 (d, J=14.1 Hz, 1H), 1.07 (s, 3H), 1.11 (s, 3H), 1.12-1.15 (m, 3H), 1.21 (s, 3H), 1.31-1.36 (m, 1H), 1.38 (s, 3H), 1.41-1.46 (m, 1H), 1.52-1.58 (m, 3H), 1.61-1.71 (m, 4H), 1.78-1.87 (m, 1H), 1.90-1.99 (m, 1H), 2.03-2.08 (m, 1H), 2.09 (s, 3H), 2.21 (d, J=11.2 Hz, 1H), 2.34 (d, J=15.6 Hz, 1H), 3.91 (m, 2H), 6.35 (d, J=7.2 Hz, 1H), 6.39 (s, 1H), 7.05-7.10 (m, 1H), 8.73 (s, 1H).
(182) .sup.13C NMR (151 MHz, DMSO-d.sub.6) ppm 10.53, 14.28, 18.50, 21.84, 28.55, 29.57, 29.75, 30.58, 31.76, 32.76, 33.32, 34.78, 36.43, 38.20, 39.27, 40.13, 40.44, 42.43, 44.04, 44.93, 60.33, 117.74, 118.51, 120.56, 127.29, 133.67, 146.86, 163.40, 168.31, 177.83, 178.41.
EXAMPLE 22. PREPARATION OF COMPOUND XS0474
(183) ##STR00058##
(184) Compound YXY101 (50 mg, 0.11 mmol) was dissolved under stirring in 2 mL of deuterated methanol, then sodium borodeuteride (48.4 mg, 1.1 mmol) was added, the reaction was carried out at room temperature for 30 min. The reaction was quenched with 1 mol/L HCl (1 mL), and then 9 mL of pure water was added. The resulting mixture was extracted three times with dichloromethane (5 mL each time). The organic layers were combined, dried over anhydrous sodium sulfate, and then the organic solvent dichloromethane was rapidly removed by vacuum distillation to obtain compound XS0474 (50.2 mg) as a white solid.
(185) .sup.1H NMR (600 MHz, DMSO-d.sub.6) ppm 0.66 (s, 3H) 0.85 (d, J=12.84 Hz, 1H) 1.04 (s, 3H) 1.10 (s, 3H) 1.17 (s, 3H) 1.22 (s, 3H) 1.29 (td, J=13.66, 4.40 Hz, 1H) 1.35-1.41 (m, 1H) 1.43-1.52 (m, 3H) 1.53-1.58 (m, 1H) 1.58-1.63 (m, 1H) 1.63-1.69 (m, 1H) 1.79 (td, J=13.66, 6.60 Hz, 1H) 1.86 (td, J=13.71, 5.04 Hz, 1H) 1.94-1.99 (m, 2H) 2.01 (s, 3H) 2.04 (d, J=12.10 Hz, 1H) 2.34 (d, J=15.59 Hz, 1H) 3.16 (d, J=6.05 Hz, 1H) 5.71 (d, J=6.24 Hz, 1H) 6.61 (s, 1H) 7.82 (s, 1H) 8.83 (s, 1H) 12.03 (br. s., 1H).
(186) .sup.13C NMR (151 MHz, DMSO-d.sub.6) ppm 11.60, 18.15, 22.74, 26.98, 28.52, 29.51, 29.83, 30.14, 30.25, 31.45, 32.50, 34.15, 34.21, 34.46, 36.14, 36.62, 37.21, 39.49, 43.31, 43.89, 108.23, 117.68, 120.20, 123.11, 139.45, 140.60, 143.18, 149.32, 179.59.
EXAMPLE 23. PREPARATION OF COMPOUND XS0503
(187) ##STR00059##
(188) First, pristimerin (250 mg, 0.54 mmol) was dissolved in 20 mL of tetrahydrofuran, followed by an addition of LiAlH.sub.4 (1.2 mL, 1.1 mmol), and the reaction was carried out under stirring at room temperature for 2 h. The reaction was quenched with 10 mL of deionized water, and then acidified with 1 mol/L HCl (5 mL). The resulting mixture was performed three times with ethyl acetate (15 mL each time). The organic layers were combined, dried over anhydrous sodium sulfate, and the organic solvent ethyl acetate was removed by vacuum distillation to afford a crude product as orange-yellow mixture solid. The crude product was separated by chromatography column with n-hexane and ethyl acetate (hexane/ethyl acetate=2:1) as eluent, using 300-400 mesh silica gel packed column, affording an orange-yellow solid product.
(189) .sup.1H NMR (600 MHz, DMSO-d.sub.6) ppm 0.76 (s, 3H) 0.83-0.87 (m, 1H) 0.89 (s, 3H) 1.11 (s, 3H) 1.21 (s, 3H) 1.23-1.25 (m, 1H) 1.26 (s, 3H) 1.27-1.34 (m, 2H) 1.48 (dd, J=6.7, 3.9 Hz, 1H) 1.50-1.54 (m, 1H) 1.55-1.59 (m, 1H) 1.60 (d, J=5.0 Hz, 1H) 1.64 (dd, J=9.2, 4.6 Hz, 2H) 1.65-1.69 (m, 2H) 1.69-1.72 (m, 1H) 1.91 (d, J=5.3 Hz, 1H) 1.93-1.97 (m, 1H) 2.02 (s, 3H) 2.92 (d, J=19.4 Hz, 1H) 2.96 (dd, J=10.3, 4.8 Hz, 1H) 3.15-3.20 (m, 1H) 3.21 (t, J=6.0 Hz, 1H) 4.44 (t, J=5.0 Hz, 1H) 5.73 (dd, J=6.1, 1.7 Hz, 1H) 6.61 (s, 1H) 7.80 (s, 1H) 8.78 (s, 1H).
(190) .sup.13C NMR (151 MHz, CHLOROFORM-d) ppm 11.49, 19.35, 25.65, 27.80, 28.10, 28.75, 29.36, 30.31, 30.45, 30.57, 32.27, 32.89, 33.58, 34.24, 36.55, 36.82, 36.95, 37.68, 42.91, 43.15, 71.82, 108.42, 118.15, 120.42, 125.27, 139.96, 141.09, 141.87, 151.04.
EXAMPLE 24. PREPARATION OF COMPOUND XS0508
(191) ##STR00060##
(192) XS0077 (50 mg, 0.11 mmol) was weighed and placed in a 50 ml round bottom bottle and added with 3 ml of methanol for dissolution, the air was replaced with nitrogen for protection, then sodium bisulfite solution (28 mg NaHSO.sub.3-1 ml H.sub.2O, 0.26 mmol) was added, and the reaction was carried out at room temperature for 3 h under protection of nitrogen. The reaction was stopped, the solvent was removed via concentration by distillation under reduced pressure. The residual solid was added with pyridine for dissolution, then filtered and concentrated in vacuo to give a white solid in a yield of 90%.
(193) .sup.1H NMR (600 MHz, DMSO-d6) ppm 0.57 (s, 3H) 1.07 (s, 3H) 1.12-1.14 (m, 1H) 1.14-1.16 (m, 1H) 1.17 (s, 3H) 1.21 (s, 3H) 1.22-1.23 (m, 2H) 1.24-1.28 (m, 4H) 1.28-1.32 (m, 2H) 1.38 (s, 3H) 1.40-1.46 (m, 2H) 1.47-1.52 (m, 2H) 1.57 (d, J=6.79 Hz, 1H) 1.59-1.63 (m, 4H) 1.64-1.72 (m, 6H) 1.75-1.83 (m, 4H) 1.92-1.96 (m, 1H) 1.97-2.01 (m, 1H) 2.03-2.07 (m, 1H) 2.09 (s, 3H) 2.18 (d, J=10.82 Hz, 2H) 2.73 (d, J=15.04 Hz, 1H) 3.40-3.49 (m, 1H) 3.80-3.89 (m, 1H) 6.36 (d, J=7.34 Hz, 1H) 6.40 (d, J=0.92 Hz, 1H) 7.07 (dd, J=6.97, 0.92 Hz, 1H) 7.73 (d, J=8.07 Hz, 1H) 8.71 (s, 1H).
(194) .sup.13C NMR (151 MHz, DMSO-d6) ppm 10.08, 18.32, 21.83, 24.68, 24.75, 25.13, 25.29, 25.63, 25.67, 28.38, 28.59, 29.91, 30.13, 31.02, 31.08, 31.39, 31.82, 31.91, 31.93, 32.36, 33.10, 36.02, 36.13, 37.67, 38.99, 42.16, 42.81, 44.45, 44.49, 50.05, 54.77, 117.23, 117.93, 120.04, 126.75, 133.25, 146.43, 153.97, 163.09, 168.74, 175.44, 177.83.
EXAMPLE 25. PREPARATION OF COMPOUND XS0536
(195) ##STR00061##
(196) XS0077 (150 mg, 0.32 mmol) was weighed and placed in a heavy wall pressure vessel, added with potassium carbonate (220 mg, 1.6 mmol), and 4 ml of acetone with stirring to dissolve the sample at room temperature, then added with 100 L of dimethyl sulfate solution; the reaction mixture was transferred to 70 C. oil bath and heated for 8 h. The reaction was quenched with 1 mol/L HCl, followed by an addition of water, then extracted with ethyl acetate three times, dried over anhydrous sodium sulfate, and the solvent was concentrated by a rotary evaporator. The residue was purified by silica gel column chromatography with ethyl acetate:n-hexane=1:20 to afford a pure product as white solid, yield 21%.
(197) .sup.1H NMR (600 MHz, CHLOROFORM-d) ppm 0.61 (s, 3H) 0.94 (d, J=13.75 Hz, 1H) 1.08 (s, 3H) 1.17 (s, 3H) 1.22 (s, 3H) 1.34 (s, 3H) 1.40 (dd, J=14.12, 4.22 Hz, 1H) 1.44 (dd, J=14.40, 2.84 Hz, 1H) 1.51-1.57 (m, 2H) 1.57-1.63 (m, 1H) 1.63-1.69 (m, 2H) 1.73 (d, J=11.37 Hz, 1H) 1.85 (td, J=13.71, 6.33 Hz, 1H) 2.01-2.07 (m, 1H) 2.07-2.13 (m, 2H) 2.17 (s, 3H) 2.18-2.23 (m, 1H) 2.44 (d, J=15.59 Hz, 1H) 3.02 (d, J=19.99 Hz, 1H) 3.28 (dd, J=20.00, 5.87 Hz, 1H) 3.54 (s, 3H) 3.77 (s, 3H) 3.87 (s, 3H) 5.77 (d, J=4.58 Hz, 1H) 6.78 (s, 1H).
(198) .sup.13C NMR (151 MHz, CHLOROFORM-d) ppm 11.77, 18.28, 22.73, 27.85, 28.85, 29.87, 30.29, 30.50, 30.85, 31.55, 32.88, 34.16, 34.51, 34.79, 36.83, 37.15, 37.52, 40.45, 43.69, 44.33, 51.47, 55.88, 60.30, 106.30, 117.63, 125.49, 127.80, 144.62, 144.80, 149.13, 150.89, 179.05.
EXAMPLE 26. PREPARATION OF COMPOUND XS0286
(199) ##STR00062##
(200) Compound YXY101 (50 mg, 0.11 mmol) was dissolved in N,N-dimethylformamide (2 mL) under stirring. Catalysts EDCl (85.4 mg, 0.55 mmol) and HOBT (74.3 mg, 0.55 mmol) were added and stirred for dissolution; p-tert-butylaniline (49.2 mg, 0.33 mmol) was added, and the reaction was carried out under stirring at room temperature for 36 hours. The reaction was stopped, and the reaction system was added with deionized water (15 mL) and extracted three times with ethyl acetate. The combined ethyl acetate layer was washed with saturated NaCl three times, dried over anhydrous Na.sub.2SO.sub.4, concentrated by a rotary evaporator to obtain a crude product (dark-red oily matter). The crude product was purified by rapid column chromatography (ethyl acetate:n-hexane), dried in vacuo to afford compound XS0286 (10 mg) as dark-red solid.
(201) .sup.1H NMR (600 MHz, CHLOROFORM-d) ppm 0.64 (s, 3H) 1.08 (d, J=13.20 Hz, 1H) 1.15 (s, 3H) 1.24-1.27 (m, 6H) 1.40-1.42 (m, 4H) 1.48-1.57 (m, 3H) 1.63 (m, 5H) 1.65-1.79 (m, 5H) 1.85 (d, J=11.92 Hz, 1H) 1.91 (d, J=6.24 Hz, 1H) 1.98-2.06 (m, 3H) 2.08 (d, J=12.10 Hz, 2H) 2.18 (d, J=1.65 Hz, 3H) 2.54 (d, J=15.77 Hz, 1H) 6.29 (d, J=6.79 Hz, 1H) 6.45 (s, 1H) 6.92-7.00 (m, 2H) 7.30-7.34 (m, 2H) 7.34-7.37 (m, 2H) 7.37-7.41 (m, 1H).
(202) .sup.13C NMR (151 MHz, CHLOROFORM-d) ppm 10.21, 18.49, 21.75, 28.58, 29.63, 29.69, 30.41, 30.83, 31.33, 33.24, 33.73, 34.35, 34.98, 36.32, 38.06, 39.38, 41.10, 42.96, 44.47, 45.11, 76.81, 77.02, 77.23, 116.99, 117.95, 119.53, 119.81, 125.89, 127.38, 133.95, 135.11, 146.01, 147.26, 164.68, 170.02, 175.83, 178.38.
EXAMPLE 27. PREPARATION OF COMPOUND XS0287
(203) ##STR00063##
(204) Compound YXY101 (50 mg, 0.11 mmol) was dissolved in N,N-dimethylformamide (2 mL) under stirring. Catalysts EDCl (85.4 mg, 0.55 mmol) and HOBT (74.3 mg, 0.55 mmol) were added and stirred for dissolution; p-methoxyaniline (40.6 mg, 0.33 mmol) was added, and the reaction was carried out under stirring at room temperature for 36 hours. The reaction was stopped. The reaction system was added with deionized water (15 mL) and extracted three times with ethyl acetate. The combined ethyl acetate layer was washed with saturated NaCl three times, dried over anhydrous Na.sub.2SO.sub.4, concentrated by a rotary evaporator to obtain a crude product (dark-red oily matter). The crude product was purified by rapid column chromatography (ethyl acetate:n-hexane), dried in vacuo to afford compound XS0287 (20 mg) as dark-red solid.
(205) .sup.1H NMR (600 MHz, CHLOROFORM-d) ppm 0.53 (s, 3H) 1.05 (d, J=13.94 Hz, 1H) 1.13 (s, 3H) 1.21 (s, 3H) 1.24 (s, 3H) 1.26 (s, 3H) 1.35 (s, 3H) 1.48-1.54 (m, 2H) 1.58 (d, J=7.34 Hz, 2H) 1.64-1.72 (m, 2H) 1.78-1.89 (m, 3H) 2.03-2.11 (m, 2H) 2.18 (s, 3H) 2.55 (d, J=15.77 Hz, 1H) 3.79 (s, 3H) 6.25 (d, J=7.15 Hz, 1H) 6.34 (s, 1H) 6.91 (m, J=8.99 Hz, 2H) 6.96 (d, J=7.15 Hz, 1H) 7.40 (m, J=8.80 Hz, 2H) 7.48 (s, 1H).
(206) .sup.13C NMR (151 MHz, CHLOROFORM-d) ppm 10.23, 18.30, 21.75, 28.46, 29.39, 29.70, 30.30, 30.79, 31.52, 32.86, 33.81, 34.89, 36.21, 37.99, 39.28, 40.96, 42.96, 44.43, 45.12, 55.48, 114.21, 117.18, 117.82, 119.38, 121.99, 127.34, 130.98, 134.06, 146.06, 156.41, 164.80, 170.23, 175.88, 178.42.
EXAMPLE 28. PREPARATION OF COMPOUND XS0394
(207) ##STR00064##
(208) Compound YXY101 (50 mg, 0.11 mmol) and triethylamine (37 L) were dissolved in re-distilled tetrahydrofuran (3.0 mL, 0.26 mmol) under stirring, then cooled to 30 C. Ethyl chloroformate (22 L, 0.23 mmol) was added dropwise to the reaction liquor 10 minutes later. After being conducted at 30 C. for 12 hours under stirring, the reaction was stopped. The insoluble material was filtered off by a Buchner funnel with a fritted glass disc and washed with tetrahydrofuran to give a yellow filtrate. The yellow filtrate was added with 10 mL of pure water, and extracted with ethyl acetate three times (15 mL each time). The organic layer was combined, washed three times with saturated brine (30 mL each time), and dried over anhydrous sodium sulfate. The organic solvent was removed by distillation under reduced pressure to give a crude product as oily mixture. The product as yellow solid was obtained by column chromatography separation with n-hexane and ethyl acetate (hexane/ethyl acetate=10:1) as eluent, and using 300-400 mesh silica gel packed column.
(209) .sup.1H NMR (600 MHz, DMSO-d.sub.6) ppm 1.05-1.07 (m, 1H) 1.08 (s, 3H) 1.09-1.12 (m, 1H) 1.18 (s, 3H) 1.23 (s, 3H) 1.27 (t, J=7.06 Hz, 3H) 1.28-1.31 (m, 1H) 1.41 (s, 3H) 1.43-1.50 (m, 4H) 1.53 (dd, J=12.10, 5.69 Hz, 1H) 1.59 (d, J=7.89 Hz, 1H) 1.65 (d, J=10.64 Hz, 2H) 1.70-1.78 (m, 2H) 1.79-1.85 (m, 1H) 1.90 (td, J=13.98, 3.39 Hz, 1 H) 1.95-2.01 (m, 1H) 2.01-2.06 (m, 1H) 2.15 (s, 3H) 2.26 (d, J=15.22 Hz, 1H) 4.21 (q, J=1.00 Hz, 2H) 6.30 (d, J=7.15 Hz, 1H) 6.40 (d, J=0.73 Hz, 1H) 7.29 (dd, J=7.52, 0.73 Hz, 1H) 12.07 (br. s, 1H).
(210) .sup.13C NMR (151 MHz, CHLOROFORM-d) ppm 11.17, 14.09, 19.00, 21.96, 28.60, 29.35, 29.36, 30.47, 30.67, 31.49, 32.67, 33.38, 34.53, 36.20, 38.24, 39.13, 40.16, 42.88, 44.05, 45.34, 65.05, 117.76, 117.78, 122.70, 126.08, 133.37, 142.54, 152.62, 163.30, 172.96, 183.86, 183.90.
EXAMPLE 29. PREPARATION OF COMPOUND XS0418
(211) ##STR00065##
(212) First, compound YXY101 (50 mg, 0.11 mmol) was stirred and dissolved in 2 mL of tetrahydrofuran, followed by an addition of potassium carbonate (15.2 mg, 0.11 mmol). Ethyl bromoacetate (18.37 mg, 0.11 mmol) was dissolved in 1 mL of tetrahydrofuran, and then added dropwise to the reaction solution. The reaction was carried out under stirring at room temperature for 4 hours, then quenched with 1 mol/L HCl (1 mL), 9 mL of pure water was added. The resulting mixture was extracted three times with ethyl acetate (15 mL each time). The collected organic layer was dried over anhydrous sodium sulfate, and the organic solvent ethyl acetate was removed by distillation in vacuo to afford a crude product as orange-red mixture solid. The product as orange-red solid was obtained by column chromatography separation with n-hexane and ethyl acetate (hexane/ethyl acetate=10:1) as eluent, using 300-400 mesh silica gel packed column.
(213) .sup.1H NMR (600 MHz, CHLOROFORM-d) ppm 0.53 (s, 3H) 0.99 (d, J=14.31 Hz, 1H) 1.11 (s, 3H) 1.25 (t, J=7.20 Hz, 3H) 1.27 (s, 3H) 1.28 (s, 3H) 1.39-1.44 (m, 1H) 1.45 (s, 3H) 1.51 (dd, J=14.95, 4.13 Hz, 1H) 1.54-1.58 (m, 1H) 1.60 (d, J=7.89 Hz, 1H) 1.63-1.67 (m, 1H) 1.67-1.71 (m, 1H) 1.75 (dd, J=15.96, 8.07 Hz, 1H) 1.80-1.82 (m, 1H) 1.83-1.85 (m, 1H) 1.85-1.92 (m, 1H) 2.06 (td, J=14.12, 3.85 Hz, 1H) 2.13-2.18 (m, 1H) 2.21 (s, 3H) 2.25 (d, J=14.31 Hz, 1H) 2.48 (d, J=15.96 Hz, 1H) 4.19 (q, J=7.15 Hz, 2H) 4.41 (d, J=15.77 Hz, 1H) 4.54 (d, J=15.96 Hz, 1H) 6.35 (d, J=7.15 Hz, 1H) 6.53 (d, J=1.10 Hz, 1H) 7.02 (dd, J=7.06, 1.19 Hz, 1H)
(214) .sup.13C NMR (151 MHz, CHLOROFORM-d) ppm 10.23, 14.04, 18.57, 21.61, 28.59, 29.57, 29.74, 30.47, 30.63, 31.55, 32.56, 33.50, 34.67, 36.31, 38.24, 39.39, 40.40, 42.91, 44.19, 45.01, 60.52, 61.28, 117.15, 118.13, 119.52, 127.37, 134.09, 145.98, 164.74, 167.70, 170.03, 177.53, 178.31
EXAMPLE 30. PREPARATION OF COMPOUNDS XS0421, XS0457, XS0473 AND XS0493
(215) ##STR00066##
(216) Taking the synthesis of XS0421 as an example: Compound YXY101 (50 mg, 0.11 mmol) was weighed in a 50 ml sealed tube, then dimethyl phosphite (123 mg, 1.1 mmol) and 2.7 mg of aluminum chloride hexahydrate (0.1 eq) were added, and 2 mL of DCM was added for dissolution. The tube was sealed, and the reaction was performed at room temperature for 6 h. The reaction was stopped and quenched with saturated NaCl. The resulting mixture was extracted with ethyl acetate three times, the organic phases were combined and dried with anhydrous Na.sub.2SO.sub.4, and concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography with ethyl acetate:n-hexane=1:2 to gave a white solid, yield 55%.
(217) .sup.1H NMR (600 MHz, DMSO-d.sub.6) ppm 0.61 (3H, s) 0.86 (1H, d, J=12.84 Hz) 1.06 (3H, s) 1.10 (3H, s) 1.19 (3H, s) 1.22-1.27 (1H, m) 1.27-1.32 (1H, m) 1.40 (1H, d, J=4.95 Hz) 1.43 (1H, d, J=5.32 Hz) 1.49 (1H, d, J=8.07 Hz) 1.51-1.55 (1H, m) 1.57 (3H, s) 1.59-1.60 (1H, m) 1.60-1.64 (1H, m) 1.76-1.85 (1H, m) 1.93-1.97 (1H, m) 1.99 (1H, d, J=2.20 Hz) 2.00-2.02 (1H, m) 2.02-2.06 (1H, m) 2.13 (3H, s) 2.32 (1H, d, J=15.59 Hz) 3.49 (3H, d, J=8.80 Hz) 3.51 (3H, d, J=8.99 Hz) 4.19 (1H, dd, J=23.66, 6.24 Hz) 5.64 (1H, dd, J=6.33, 3.21 Hz) 6.66 (1H, s) 7.97 (1H, s) 9.04 (1H, br. s.) 12.04 (1H, s).
(218) .sup.13C NMR (151 MHz, DMSO-d.sub.6) ppm 12.60, 14.00 (1 C, s) 18.04, 21.58 (1 C, d, J=6.60 Hz) 22.11, 28.78, 29.48, 30.02 (1 C, d, J=13.20 Hz) 30.18, 31.00, 31.45, 32.42, 33.49, 33.57, 34.57, 36.42, 37.43, 37.89, 38.79, 43.88, 52.31 (1 C, d, J=6.60 Hz) 52.81 (1 C, d, J=6.60 Hz) 109.32, 114.64 (1 C, d, J=12.10 Hz) 117.90 (1 C, d, J=7.70 Hz) 121.35 (1 C, d, J=4.40 Hz) 140.50 (1 C, d, J=6.60 Hz) 141.05 (1 C, d, J=3.30 Hz) 144.08 (1 C, d, J=3.30 Hz) 150.62 (1 C, d, J=12.10 Hz) 179.51.
(219) According to the above preparation method, the following compounds were also synthesized in the present invention:
(220) ##STR00067##
EXAMPLE 31. PREPARATION OF COMPOUNDS XS0439, XS0442, XS0444-XS0449, XS0478-XS0480, XS0487 AND XS0490
(221) ##STR00068## ##STR00069## ##STR00070## ##STR00071## ##STR00072##
(222) The preparation method is exemplified by XS0439: Compound YXY101 (100 mg, 0.22 mmol) was dissolved in dichloromethane (4 mL) under stirring. 7-Methoxy-substituted indole (65.3 mg, 0.44 mmol) was added, then aluminum trichloride hexahydrate (5.3 mg, 0.022 mmol). The reaction was carried out under stirring at room temperature for 5 hours. The reaction was stopped, the reaction mixture was added with deionized water (15 mL) and extracted three times with ethyl acetate. The ethyl acetate layers were combined, washed with saturated NaCL three times, dried over anhydrous Na.sub.2SO.sub.4, concentrated by a rotary evaporator to obtain a crude product (brown oily matter). The crude product was separated and purified by rapid column chromatography (ethyl acetate:n-hexane=1:4), dried in vacuo to afford a product as purple red solid.
(223) .sup.1H-NMR (DMSO-d.sub.6) ppm 0.71 (s, 3H), 0.86 (d, J=9.9 Hz, 1H), 0.96 (s, 3H), 1.01 (s, 3H), 1.10 (s, 3H), 1.22-1.29 (m, 2H), 1.32 (s, 3H), 1.34-1.40 (m, 2H), 1.45 (d, J=8.1 Hz, 1H), 1.50-1.61 (m, 3H), 1.62-1.75 (m, 2H), 1.79 (s, 3H), 1.96-2.08 (m, 3H), 2.34 (d, J=15.4 Hz, 1H), 3.88 (s, 3H), 4.79 (d, J=5.9 Hz, 1H), 6.12 (d, J=6.2 Hz, 1H), 6.18 (s, 1H), 6.62 (d, J=7.7 Hz, 1H), 6.73 (s, 1H), 6.90 (t, J=7.9 Hz, 1H), 7.22 (d, J=7.9 Hz, 1H), 7.88 (br. s., 1H), 8.99 (br. s., 1H), 10.69 (s, 1H), 12.04 (br. s., 1H).
(224) .sup.13C-NMR (DMSO-d.sub.6) ppm 11.9, 18.5, 22.3, 29.1, 29.9, 30.4, 30.5, 30.6, 31.8, 32.9, 34.9, 35.4, 35.5, 35.6, 36.8, 36.9, 37.8, 43.5, 44.3, 55.4, 101.8, 108.8, 112.2, 119.2, 119.8, 121.1, 121.6, 122.6, 126.3, 126.7, 128.5, 140.8, 141.4, 144.0, 146.6, 147.0, 180.0.
EXAMPLE 32. PREPARATION OF COMPOUNDS XS0486, XS0491 AND XS0492
(225) ##STR00073##
(226) Taking the preparation of compound XS0486 as an example: First, Compound YXY101 (50 mg, 0.11 mmol) was dissolved in dioxane (600 L), triethylamine (150 L, 0.33 mmol) was added, and then dioxane (100 L) was added to wash the residue on the wall of the reaction bottle, and the reaction solution was cooled to 0 C. Dimethyl phosphite (110 mg, 1.1 mmol) was dissolved in 50 L of carbon tetrachloride, then the dissolved dimethyl phosphite was slowly added dropwise into the reaction solution dissolved with Celastrol, the reaction was carried out under stirring at 0 C. for 12 h, then 10 mL of ice-cold deionized water was added, and 10 mL of saturated ammonium chloride solution was added for quenching the reaction. The resulting mixture was extracted with ethyl acetate three times (15 mL each time), and the organic layer was collected and dried over anhydrous sodium sulfate. The organic solvent ethyl acetate was then removed by distillation under reduced pressure to give a crude product as red mixture solid. The crude product was separated by rapid column chromatography with 300-400 mesh silica gel packed column using n-hexane and ethyl acetate (hexane/ethyl acetate=1:1) as eluent to give a red solid product.
(227) .sup.1H NMR (600 MHz, DMSO-d.sub.6) ppm 0.60 (s, 3H) 0.96 (d, J=13.57 Hz, 1H) 1.08 (s, 3H) 1.23 (s, 3H) 1.24 (s, 3H) 1.38 (s, 3H) 1.41-1.48 (m, 2H) 1.55-1.60 (m, 3H) 1.61-1.67 (m, 2H) 1.69-1.74 (m, 1H) 1.78 (dd, J=16.14, 7.89 Hz, 1H) 1.81-1.88 (m, 1H) 1.95 (td, J=14.12, 3.85 Hz, 1H) 2.02 (d, J=13.94 Hz, 1H) 2.09 (s, 3H) 2.21-2.24 (m, 1H) 2.27 (d, J=15.96 Hz, 1H) 3.74 (dd, J=11.55, 5.14 Hz, 6H) 6.36 (d, J=7.15 Hz, 1H) 6.39 (d, J=1.28 Hz, 1H) 7.08 (dd, J=6.97, 1.10 Hz, 1H) 8.75 (s, 1H).
(228) .sup.13C NMR (151 MHz, DMSO-d.sub.6) ppm 10.07, 18.53, 21.43, 28.03, 29.09, 29.24, 29.71, 30.07, 31.21, 31.46, 32.76, 33.92, 35.85, 37.71, 38.78, 41.23 (d, J=5.50 Hz,) 41.97, 43.37, 44.45, 55.15 (dd, J=17.61, 5.50 Hz, 2 C) 117.24, 118.13, 120.12, 126.89, 133.12, 146.44, 162.82, 167.43, 172.21 (d, J=11.00 Hz) 177.98.
EXAMPLE 33. PREPARATION OF COMPOUND XS0488
(229) ##STR00074##
(230) First, compound XS0077 (80 mg, 0.17 mmol) was dissolved in tetrahydrofuran (4 mL), triethylamine (370 L, 2.5 mmol) was added, then 4-DMAP (24.7 mg, 0.22 mmol) was added, stirred evenly, and n-butyryl chloride (113 L, 1.1 mmol) was added. The reaction was carried out under stirring at room temperature for 30 min, then quenched with 10 mL of saturated ammonium chloride solution. The resulting mixture was extracted with ethyl acetate three times (15 mL each time). The organic layer was collected and dried over anhydrous sodium sulfate, the organic solvent ethyl acetate was removed by distillation under reduced pressure to give a crude product as red mixture solid. Separation was performed by column chromatography with 300-400 mesh silica gel packed column, using n-hexane and ethyl acetate (hexane/ethyl acetate=4:1) as eluent to give a product as bright yellow solid.
(231) .sup.1H NMR (600 MHz, DMSO-d.sub.6) ppm 0.46-0.51 (m, 3H) 0.91 (d, J=1.00 Hz, 1H) 0.95-1.01 (m, 3H) 1.08 (s, 3H) 1.12 (s, 3H) 1.23 (s, 3H) 1.36 (ddd, J=13.80, 4.20 Hz, 1H) 1.42 (s, 3H) 1.44-1.48 (m, 1H) 1.54-1.60 (m, 3H) 1.63 (s, 2H) 1.66-1.68 (m, 2H) 1.70 (d, J=9.17 Hz, 2H) 1.83 (dd, J=13.57, 7.70 Hz, 1H) 1.93-1.99 (m, 1H) 2.06 (d, J=13.94 Hz, 1H) 2.10 (s, 3H) 2.22 (d, J=7.34 Hz, 1H) 2.32 (d, J=15.59 Hz, 1H) 2.55 (t, J=7.15 Hz, 2H) 3.49 (s, 3H) 6.38 (s, 1H) 6.41 (d, J=7.15 Hz, 1H) 7.31 (d, J=6.97 Hz, 1H).
(232) .sup.13C NMR (151 MHz, DMSO-d.sub.6) ppm 13.41, 18.05, 21.59, 28.06, 29.11, 29.42, 30.11, 30.16, 30.32, 31.32, 32.21, 32.31, 33.06, 34.36, 34.91, 36.00, 37.90, 38.77, 39.83, 42.22, 43.64, 44.85, 51.48, 117.99, 122.13, 125.21, 133.52, 136.69, 142.26, 162.81, 170.65, 171.21, 176.21, 177.91.
EXAMPLE 34. PREPARATION OF COMPOUND XS0506
(233) ##STR00075##
(234) Compound YXY101 (50 mg, 0.11 mmol) was weighed in a 50 ml round bottom flask, dicyclohexylcarbodiimide (23 mg, 0.11 mmol) and glucose (24 mg, 0.11 mmol) were added, then 2 ml of DCM was added for dissolution. The reaction was carried out at room temperature for 12 h, quenched with a large amount of water, extracted with ethyl acetate three times. The organic phases were combined and dried over anhydrous Na.sub.2SO.sub.4, and concentrated under reduced pressure to remove solvent. Separation and purification were performed by silica gel column with ethyl acetate:n-hexane=1:4 to afford an orange-red solid, yield 42%.
(235) .sup.1H NMR (600 MHz, DMSO-d.sub.6) ppm 0.57 (s, 3H) 1.07 (s, 3H) 1.12-1.14 (m, 1H) 1.14-1.16 (m, 1H) 1.17 (s, 3H) 1.21 (s, 3H) 1.22-1.23 (m, 2H) 1.24-1.28 (m, 4H) 1.28-1.32 (m, 2H) 1.38 (s, 3H) 1.40-1.46 (m, 2H) 1.47-1.52 (m, 2H) 1.57 (d, J=6.79 Hz, 1H) 1.59-1.63 (m, 4H) 1.64-1.72 (m, 6H) 1.75-1.83 (m, 4H) 1.92-1.96 (m, 1H) 1.97-2.01 (m, 1H) 2.03-2.07 (m, 1H) 2.09 (s, 3H) 2.18 (d, J=10.82 Hz, 2H) 2.73 (d, J=15.04 Hz, 1H) 3.40-3.49 (m, 1H) 3.80-3.89 (m, 1H) 6.36 (d, J=7.34 Hz, 1H) 6.40 (d, J=0.92 Hz, 1H) 7.07 (dd, J=6.97, 0.92 Hz, 1H) 7.73 (d, J=8.07 Hz, 1H) 8.71 (s, 1H).
(236) .sup.13C NMR (151 MHz, DMSO-d.sub.6) ppm 10.08, 18.32, 21.83, 24.68, 24.75, 25.13, 25.29, 25.63, 25.67, 28.38, 28.59, 29.91, 30.13, 31.02, 31.08, 31.39, 31.82, 31.91, 31.93, 32.36, 33.10, 36.02, 36.13, 37.67, 38.99, 42.16, 42.81, 44.45, 44.49, 50.05, 54.77, 117.23, 117.93, 120.04, 126.75, 133.25, 146.43, 153.97, 163.09, 168.74, 175.44, 177.83.
EXAMPLE 35. PREPARATION OF COMPOUND XS0507
(237) ##STR00076##
(238) Compound YXY101 (50 mg, 0.11 mmol) was weighed in a 50 ml round bottom flask, added with 2 ml of DCM for dissolution, then transferred to 78 C. and stirred. DAST (150 ul, 10 eq) was added and reacted at 78 C. for 1 h. The reaction solution was directly poured into a large amount of ice to quench the reaction. The aqueous phase was extracted three times with DCM. The organic phases were combined and dried over anhydrous Na.sub.2SO.sub.4. The organic phase was concentrated under reduced pressure to remove solvent. Separation and purification were performed by column chromatography with ethyl acetate:n-hexane=1:4 system to gave an orange-red solid, yield 58%.
(239) .sup.1H NMR (600 MHz, DMSO-d.sub.6) ppm 0.56 (s, 3H) 0.98 (d, J=11.92 Hz, 1H) 1.08 (s, 3H) 1.22 (s, 3H) 1.29 (s, 3H) 1.38 (s, 3H) 1.43-1.47 (m, 1H) 1.48-1.52 (m, 1H) 1.55-1.59 (m, 2H) 1.60-1.64 (m, 1H) 1.67 (d, J=4.03 Hz, 1H) 1.71 (d, J=3.85 Hz, 1H) 1.80 (dd, J=16.41, 8.16 Hz, 1H) 1.83-1.87 (m, 1H) 1.88-1.92 (m, 1H) 1.96 (d, J=17.61 Hz, 1H) 1.95-1.95 (m, 1H) 2.09 (s, 3H) 2.20 (d, J=1.83 Hz, 2H) 6.35 (d, J=7.34 Hz, 1H) 6.38 (d, J=1.28 Hz, 1H) 7.06 (dd, J=6.97, 1.28 Hz, 1H) 8.73 (s, 1H).
(240) .sup.13C NMR (151 MHz, DMSO-d.sub.6) ppm 10.07, 18.85, 21.41, 27.89, 29.26, 29.33, 29.46, 29.94, 31.16, 32.71, 33.79, 35.78, 37.87, 38.74, 40.04, 40.22, 41.89, 43.26, 44.33, 117.21, 118.14, 120.15, 126.99, 132.88, 146.41, 162.78, 167.39 (d, J=418.15 Hz, 1 C), 166.71, 177.97.
EXAMPLE 36. PREPARATION OF COMPOUND XS0509
(241) ##STR00077##
(242) Compound YXY101 (50 mg, 0.11 mmol) was weighed in a 25 ml heavy wall pressure vessel, added with tetrabutylammonium bromide (TBAB, 17.5 mg, 0.05 mmol), and 2 ml of dichloromethane (DCM) was added for dissolution, and then 5% NaOH (180 l) was add dropwise. The reaction was carried out at room temperature for 30 min, then transferred to 50 C. oil bath, and added dropwise with dichloromethane solution of 2,3,4,6-tetraacetoxy--D-glucopyranose bromide (57 mg-1 ml, 0.138 mmol), and reacted at 50 C. for 12 h. The reaction was stopped, and a large amount of water and saturated brine were added. The resulting mixture was extracted with DCM for 3 times. The organic phases were combined and dried over anhydrous Na.sub.2SO.sub.4. The organic phase was concentrated under reduced pressure, and purified by silica gel column chromatography with ethyl acetate:n-hexane=4:1 system to obtain a yellow solid, yield 25%.
(243) .sup.1H NMR (600 MHz, DMSO-d.sub.6) ppm 0.51 (s, 3H) 0.95 (d, J=13.57 Hz, 1H) 1.08 (d, J=6.24 Hz, 6H) 1.22 (s, 3H) 1.23 (br. s., 1H) 1.38 (s, 3H) 1.41 (d, J=4.22 Hz, 1H) 1.46 (d, J=11.92 Hz, 1H) 1.56 (d, J=7.52 Hz, 2H) 1.58 (s, 3H) 1.60-1.63 (m, 1H) 1.64 (d, J=4.95 Hz, 2H) 1.66-1.69 (m, 1H) 1.81-1.87 (m, 1H) 1.91 (s, 3H) 1.94 (d, J=3.67 Hz, 1H) 1.97 (s, 3H) 2.00-2.04 (m, 1H) 2.08 (s, 3H) 2.12 (s, 3H) 2.19 (d, J=8.80 Hz, 1H) 2.31 (d, J=15.59 Hz, 1H) 3.85-3.90 (m, 1H) 3.92-3.96 (m, 1H) 4.30 (dd, J=8.16, 4.13 Hz, 1H) 5.05 (dd, J=10.09, 8.44 Hz, 1H) 5.23 (d, J=3.48 Hz, 1H) 5.29 (dd, J=10.45, 3.67 Hz, 1H) 5.83 (d, J=8.25 Hz, 1H) 6.36 (d, J=7.15 Hz, 1H) 6.37 (d, J=0.92 Hz, 1H) 7.06 (d, J=6.97 Hz, 1H) 8.71 (s, 1H).
(244) .sup.13C NMR (151 MHz, DMSO-d.sub.6) ppm 10.06, 18.63, 19.97, 20.31, 20.42, 21.57, 28.11, 28.59, 29.22, 29.97, 30.07, 31.31, 32.62, 32.84, 34.57, 35.93, 37.76, 38.52, 40.05, 40.24, 41.97, 43.46, 44.54, 61.76, 67.35, 67.85, 69.78, 71.21, 91.64, 117.29, 118.08, 120.11, 126.93, 133.07, 146.36, 162.86, 167.78, 169.24, 169.41, 169.57, 169.97, 176.51, 177.93.
EXAMPLE 37. PREPARATION OF COMPOUND XS0514, XS0515
(245) ##STR00078##
(246) Taking the synthesis of XS0514 as an example: XS0419 (50 mg, 0.11 mmol) was weighed in a 50 ml round bottom flask, added with 2 ml DCM for dissolution, then transferred to 78 C. and stirred, followed by an addition of diethylaminosulfurtrifluoride (DAST, 150 ul, 10 eq), and reacted at 78 C. for 1 h. The reaction solution was poured into a large amount of ice to quench the reaction. The aqueous phase was extracted three times with DCM. The organic phase was combined, washed with saturated NaHCO.sub.3, dried with anhydrous Na.sub.2SO.sub.4, and concentrated under reduced pressure. Separation and purification were performed by silica gel column chromatography with ethyl acetate:n-hexane=1:8 system to give an orange-red solid, yield 58%.
(247) .sup.1H NMR (600 MHz, DMSO-d.sub.6) ppm 0.61 (s, 3H) 0.95 (d, J=14.49 Hz, 1H) 1.06 (s, 3H) 1.18 (s, 3H) 1.23 (s, 3H) 1.28 (s, 3H) 1.41 (d, J=14.86 Hz, 2H) 1.47 (dd, J=13.85, 4.31 Hz, 2H) 1.54 (d, J=7.70 Hz, 1H) 1.58-1.63 (m, 2H) 1.74-1.84 (m, 2H) 1.88 (d, J=6.05 Hz, 1H) 1.94 (d, J=10.64 Hz, 1H) 1.98 (d, J=10.09 Hz, 1H) 2.01 (s, 3H) 2.07 (d, J=17.97 Hz, 1H) 2.24 (d, J=15.96 Hz, 1H) 2.89-2.94 (m, 1H) 3.16-3.21 (m, 1H) 5.74 (d, J=4.77 Hz, 1H) 6.61 (s, 1H) 7.81 (br. s., 1H) 8.78 (br. s., 1H).
(248) .sup.13C NMR (151 MHz, DMSO-d.sub.6) ppm 11.56, 18.66, 22.57, 27.30, 28.27, 29.21, 29.54, 29.98, 30.02, 30.08, 31.18, 33.69, 33.99, 34.13, 36.05, 36.27, 37.14, 40.25, 43.12, 43.42, 108.21, 118.07, 120.13, 123.02, 139.23, 140.61, 143.17, 148.76, 167.64 (d, J=380.73 Hz, 1 C).
EXAMPLE 38. PREPARATION OF COMPOUND XS0516
(249) ##STR00079##
(250) XS0077 (50 mg, 0.11 mmol) was weighed in a 25 ml heavy wall pressure vessel, added with tetrabutylammonium bromide (TBAB, 17.5 mg, 0.05 mmol), and 2 ml of dichloromethane (DCM) was added for dissolution, then 5% NaOH (180 L) was added dropwise. The mixture were reacted at room temperature for 30 min, subsequently transferred to 50 C. oil bath, added dropwise with 2,3,4,6-tetraacetoxy--D-glucopyranose bromide-dichloromethane solution (57 mg-1 ml, 0.138 mmol) and reacted at 50 C. for 12 h. The reaction was stopped, a large amount of water and saturated brine were added, and the mixture was extracted with DCM for 3 times. The organic phases were combined, dried over anhydrous Na.sub.2SO.sub.4 concentrated under reduced pressure, and purified by silica gel column chromatography with ethyl acetate:n-hexane=4:1 to afford a yellow solid, yield 25%.
(251) .sup.1H NMR (600 MHz, CHLOROFORM-d) ppm 0.53 (s, 3H) 0.97 (d, J=14.31 Hz, 1H) 1.10 (s, 3H) 1.18 (s, 3H) 1.26 (s, 3H) 1.38 (td, J=14.03, 4.58 Hz, 1H) 1.47 (s, 3H) 1.50 (dd, J=15.59, 4.58 Hz, 1H) 1.53-1.56 (m, 1H) 1.58 (d, J=8.25 Hz, 1H) 1.62-1.65 (m, 2H) 1.66 (d, J=5.32 Hz, 1H) 1.67-1.72 (m, 2H) 1.76-1.80 (m, 1H) 1.82-1.87 (m, 1H) 1.87-1.91 (m, 1H) 1.98 (s, 3H) 2.01 (s, 3H) 2.10-2.14 (m, 1H) 2.15 (s, 3H) 2.18 (s, 3H) 2.23 (s, 3H) 2.42 (d, J=15.77 Hz, 1H) 3.55 (s, 3H) 3.86 (t, J=7.34 Hz, 1H) 4.06 (dd, J=11.10, 7.61 Hz, 1H) 4.16 (dd, J=11.10, 6.14 Hz, 1H) 5.11 (dd, J=10.36, 3.58 Hz, 1H) 5.31 (d, J=7.89 Hz, 1H) 5.38-5.43 (m, 2H) 6.31 (d, J=7.15 Hz, 1H) 6.39 (d, J=1.10 Hz, 1H) 7.02 (dd, J=7.06, 1.01 Hz, 1H).
(252) .sup.13C NMR (151 MHz, CHLOROFORM-d) ppm 11.54, 18.33, 20.61, 20.70, 21.02, 21.86, 28.57, 29.57, 29.85, 30.52, 30.82, 31.55, 32.65, 33.67, 34.70, 36.34, 38.20, 39.24, 40.37, 42.30, 44.27, 45.07, 51.54, 60.81, 66.90, 69.09, 70.49, 70.84, 100.37, 117.93, 123.33, 126.92, 134.32, 134.96, 145.71, 162.50, 170.07, 170.33, 170.35, 170.37, 170.61, 178.68, 179.14.
EXAMPLE 39. PREPARATION OF COMPOUND XS0534
(253) ##STR00080##
(254) XS0077 (150 mg, 0.32 mmol) was weighed and dissolved in 4 mL of acetone with stirring, anhydrous potassium carbonate (180 mg, 1.3 mmol) and dimethyl sulfate (95 L, 0.96 mmol) were added under stirring, the mixture was reacted under oil bath at 70 C. for 12 h. The reaction was quenched with 1 mol/L HCl, then adjusted to pH=7, and extracted with ethyl acetate three times. The organic phases were combined, dried over Na.sub.2SO.sub.4, concentrated to remove solvent under reduced pressure, and separated and purified by silica gel column chromatography with ethyl acetate:n-hexane=1:10 system to afford a white solid, yield 24.3%.
(255) .sup.1H NMR (600 MHz, DMSO-d.sub.6) ppm 0.51 (s, 3H) 0.87-0.92 (m, 1H) 1.06 (s, 3H) 1.12 (s, 3H) 1.18 (s, 3H) 1.27 (s, 3H) 1.32-1.37 (m, 1H) 1.37-1.42 (m, 1H) 1.47-1.52 (m, 3H) 1.55-1.61 (m, 2H) 1.65 (dd, J=15.68, 8.16 Hz, 1H) 1.80 (td, J=13.43, 7.24 Hz, 1H) 1.84-1.90 (m, 1H) 1.97 (td, J=13.75, 4.03 Hz, 1H) 2.02 (s, 3H) 2.06 (d, J=13.57 Hz, 1H) 2.12-2.18 (m, 1H) 2.33 (d, J=15.77 Hz, 1H) 2.95 (dd, J=20.17, 1.40 Hz, 1H) 3.21 (dd, J=20.72, 6.24 Hz, 1H) 3.46-3.50 (m, 3H) 3.77 (s, 3H) 5.72 (dd, J=6.42, 1.28 Hz, 1H) 6.72 (s, 1H) 8.10 (s, 1H).
(256) .sup.13C NMR (151 MHz, DMSO-d.sub.6) ppm 11.35, 17.69, 22.41, 27.31, 28.41, 29.42, 29.90, 30.13, 30.25, 31.36, 32.37, 34.01, 34.21, 34.36, 36.41, 36.49, 37.11, 39.85, 43.23, 43.80, 51.37, 55.82, 105.29, 117.48, 119.79, 124.58, 139.03, 141.52, 145.83, 148.73, 178.04.
EXAMPLE 41. PREPARATION OF COMPOUND XS0420
(257) ##STR00081##
(258) Compound YXY101 (50 mg, 0.11 mmol) was weighed in a 50 ml round bottom flask, PTSA (100 mg, excess) and 2 mL of toluene were added, and the mixture was stirred at room temperature for 6 h quenched by saturated NaHCO.sub.3 solution, and extracted with ethyl acetate three times. The organic phases were combined, dried with anhydrous Na.sub.2SO.sub.4, concentrated under reduced pressure to remove solvent, and separated and purified by silica gel column chromatography with ethyl acetate:n-hexane=1:4 system to afford an off-white solid in 70% yield.
(259) .sup.1H NMR (600 MHz, DMSO-d.sub.6) ppm 1.03 (s, 3H) 1.06 (s, 3H) 1.07-1.11 (m, 1H) 1.15 (s, 3H) 1.18 (s, 3H) 1.21-1.24 (m, 1H) 1.28-1.32 (m, 1H) 1.37 (td, J=13.75, 5.32 Hz, 1H) 1.46 (dd, J=8.62, 4.03 Hz, 2H) 1.48-1.50 (m, 2H) 1.51-1.55 (m, 1H) 1.58 (dd, J=12.93, 1.93 Hz, 1H) 1.83-1.90 (m, 3H) 2.33 (t, J=1.00 Hz, 1H) 2.36 (s, 3H) 2.38 (s, 3H) 2.80-2.92 (m, 2H) 2.80-2.92 (m, 2H) 7.06 (d, J=8.62 Hz, 1H) 7.18 (s, 1H) 7.49 (d, J=8.44 Hz, 1H) 8.48 (br. s., 1H) 9.92 (s, 1H).
(260) .sup.13C NMR (151 MHz, DMSO-d.sub.6) ppm 10.96, 19.45, 21.73, 21.95, 23.51, 24.03, 25.45, 30.89, 30.96, 34.59, 35.08, 36.41, 37.98, 38.30, 40.71, 41.70, 43.50, 99.01, 102.91, 115.74, 120.73, 126.07, 127.17, 127.30, 129.03, 132.36, 143.60, 146.12, 175.68.
EXAMPLE 42. PREPARATION OF COMPOUND XS0502
(261) ##STR00082##
(262) Compound XS0077 (50 mg, 0.11 mmol) was weighed in a 50 ml round bottom flask, SeO.sub.2 (200 mg, excess) and 2 mL of dioxane were added, and the mixture was stirred at 55 C. for 12 hours. The reaction was quenched by the addition of deionized water, extracted with ethyl acetate for 3 times. The organic phases were dried over Na.sub.2SO.sub.4, concentrated to remove solvent under reduced pressure, and separated and purified by silica gel column chromatography with ethyl acetate:n-hexane=1:10 system to give an off-white solid, yield 16.8%.
(263) .sup.1H NMR (600 MHz, DMSO-d.sub.6) ppm 0.75 (s, 3H) 0.93-0.97 (m, 1H) 1.10 (s, 3H) 1.21 (s, 3H) 1.22 (s, 3H) 1.40 (td, J=14.08, 4.13 Hz, 1H) 1.49 (dt, J=14.53, 4.65 Hz, 1H) 1.71-1.79 (m, 2H) 1.79-1.82 (m, 1H) 1.82-1.88 (m, 1H) 2.07-2.10 (m, 1H) 2.10-2.12 (m, 1H) 2.13 (s, 3H) 2.29 (d, J=14.31 Hz, 1H) 2.46 (t, J=4.77 Hz, 1H) 2.54 (s, 3H) 3.50 (s, 3H) 6.20 (d, J=9.54 Hz, 1H) 6.45 (d, J=9.54 Hz, 1H) 7.11 (s, 1H) 9.44 (s, 1H).
(264) .sup.13C NMR (151 MHz, DMSO-d.sub.6) ppm 11.88, 17.45, 19.70, 21.88, 26.78, 29.04, 30.34, 30.42, 30.47, 31.02, 34.19, 36.47, 40.05, 40.43, 41.84, 46.15, 51.58, 122.97, 125.72, 125.78, 129.24, 136.20, 139.22, 142.11, 142.43, 144.61, 146.30, 176.92, 178.25, 181.38.
(265) Although the embodiments of the present invention have been described in detail, according to the disclosed teaching, various modifications and alternations can be made to the details of the embodiments of the present invention, which are within the scope of the present invention. The scope of the invention is defined by the appended claims and any equivalents thereof.