Indole-fused hydrogenated phytocyanine compounds, their preparation methods and applications

Abstract

This invention discloses indole-fused hydrogenated morpholino compounds, their preparation methods, and applications, belonging to the technical fields of fluorescent materials and antitumor compounds. Using enamine ketones and amines as reaction substrates, a series of indole-fused hydrogenated morpholino compounds with different substituents were synthesized in highly polar solvents via the Stork enamine reaction and intramolecular acylation reaction. These compounds exhibit fluorescence wavelengths in the range of 430-700 nm and show certain inhibitory activity against lung cancer A549, breast cancer MDA-MB-231, colon cancer HCT116, liver cancer HepG2, and prostate cancer DU145, providing more options for the preparation of tumor drugs.

Description

Technical Field

[0001] This invention belongs to the technical field of fluorescent materials and antitumor compounds, and particularly relates to indole-fused hydrogenated morpholine compounds, their preparation methods and applications. Background Technology

[0002] Morpholins are six-membered heterocyclic compounds containing two nitrogen atoms, and their core skeletons are widely found in natural products and pharmaceutical active molecules. As important organic molecular skeletons, morpholins have broad applications in medicinal chemistry and materials science. Over the past few decades, researchers have developed various efficient synthetic methods. However, existing strategies often rely on expensive metal catalysts or harsh reaction conditions, limiting their application in industrial production. Therefore, developing new methods for constructing morpholin rings with mild conditions, economical steps, and environmental friendliness remains of significant research value and practical importance. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention proposes indole-fused hydrogenated morpholino compounds, their preparation methods, and applications.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] One of the technical solutions of the present invention:

[0006] This invention provides an indole-fused hydrogenated oxaloline compound with the following structural formula:

[0007]

[0008] Where R is selected from H, halogen, methyl (Me), methoxy, cyano, or nitro. 1 Selected from hydroxyalkyl or alkyl.

[0009] The indole-fused hydrogenated morpholine compounds of this invention exhibit fluorescence wavelengths in the range of 430-700 nm and show good anti-proliferative effects against five cell lines: lung cancer A549, breast cancer MDA, colon cancer HCT116, liver cancer HepG2, and prostate cancer DU145, providing usable compounds for the development of anticancer drugs.

[0010] Furthermore, when R is a halogen, it includes monohalogen substitution and dihalogen substitution; the halogen is F, Cl or Br; the hydroxyalkyl group is C2-C7 hydroxyalkyl (hydroxyethyl, hydroxypropyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl, hydroxyheptyl); the alkyl group is n-butyl, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

[0011] Furthermore, the indole-fused hydrogenated porphyrin compound is selected from the following compounds:

[0012]

[0013] The second technical solution of the present invention:

[0014] This invention also provides a method for preparing the above-mentioned indole-fused hydrogenated oxaloline compounds, using enamine ketone compounds and amine compounds as reaction substrates, and synthesizing the indole-fused hydrogenated oxaloline compounds in a highly polar solvent via the Stork enamine reaction and intramolecular acylation reaction.

[0015] The structural formula of the enaminoketone compound is as follows: R is selected from H, halogen, methyl (Me), methoxy, cyano, or nitro;

[0016] The structural formula of the amine compound is as follows: R 1 Selected from hydroxyalkyl or alkyl.

[0017] Furthermore, the molar ratio of the enamine ketone compound and the amine compound is 1:(1-10), more preferably 1:(1-5), and most preferably 1:5.

[0018] Furthermore, the temperature of the overstork enamine reaction and the intramolecular acylation reaction is 45-80°C, more preferably 60-80°C, and most preferably 80°C.

[0019] Furthermore, the highly polar solvent is selected from methanol, acetonitrile, and chloroform, with methanol being the preferred highly polar solvent.

[0020] The third technical solution of the present invention:

[0021] The present invention also provides the application of the above-mentioned indole-fused hydrogenated morpholine compounds in the preparation of fluorescent materials and / or semiconductor materials.

[0022] For example, the indole-fused hydrogenated oxalool compounds of the present invention can be used to prepare organic light-emitting diodes and / or fluorescent probes.

[0023] The fourth technical solution of the present invention:

[0024] The present invention also provides the application of the above-mentioned indole-fused hydrogenated morpholine compounds in the preparation of antitumor drugs.

[0025] Furthermore, the antitumor drugs include drugs for the prevention and / or treatment of lung cancer, drugs for the prevention and / or treatment of breast cancer, drugs for the prevention and / or treatment of liver cancer, and drugs for the prevention and / or treatment of prostate cancer.

[0026] Compared with the prior art, the present invention has the following advantages and technical effects:

[0027] The present invention provides a simple method for preparing indole-fused hydrogenated boronicoline compounds under mild reaction conditions, resulting in high yields and purity of the compounds, and enabling large-scale preparation.

[0028] The maximum fluorescence emission wavelength of the indole-fused hydrogenated borane compounds of this invention is affected by the electronic effects of substituents. When the benzene ring contains electron-withdrawing groups (-F, -Cl, -Br, -CN), the excitation and emission wavelengths of the product exhibit a slight blue shift; when it contains electron-donating groups (-Me, -OMe), the excitation and emission wavelengths of the product exhibit a red shift. Moreover, compounds with electron-donating groups exhibit weaker fluorescence and a larger Stokes shift. Extending the carbon chain causes a slight red shift in the excitation and emission wavelengths of the product; replacing the chain structure with a cyclic structure also results in a slight red shift in the excitation and emission wavelengths of the compound. In summary, the indole-fused hydrogenated borane compounds of this invention exhibit fluorescence wavelengths in the range of 430-700 nm and quantum yields in the range of 15.3%-72.9%, demonstrating good fluorescence performance. Simple structural modifications can alter their fluorescence properties, potentially leading to applications in fluorescent materials and other fields.

[0029] The indole-fused hydrogenated boronic acid compounds of this invention exhibited inhibitory effects on five cell lines: lung cancer A549, breast cancer MDA-MB-231, colon cancer HCT116, liver cancer HepG2, and prostate cancer DU145. Overall, these compounds showed stronger inhibitory effects on colon cancer HCT116 than on the other four cancer cell lines, demonstrating a certain degree of cell selectivity. Analysis of the substituents revealed that four compounds with bromine substitution at position 4, bromine substitution at position 7, and dichloro substitution at positions 4,6 and 4,7 exhibited good inhibitory effects on the proliferation of all five cancer cell lines. The remaining compounds showed relatively low cytotoxicity against these cancer cells, indicating that these four compounds have a certain degree of universality against cancer cells. This also confirms that the scaffold possesses certain antitumor activity and a structure-activity relationship. Attached Figure Description

[0030] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0031] Figure 1 The structural formula and absolute configuration of compound 3aa;

[0032] Figure 2 This is a synthetic mechanism route diagram for compound 3aa;

[0033] Figure 3 For some compounds in 10 -5Fluorescence spectrum of M in methanol solution;

[0034] Figure 4 These are the normalized fluorescence emission spectra of some compounds;

[0035] Figure 5 For the standard in methanol (10 -5 Fluorescence excitation and emission spectra in M). Detailed Implementation

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0038] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0039] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0040] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0041] Unless otherwise specified, the room temperature in this invention is 25±2℃.

[0042] All raw materials used in the embodiments of this invention were obtained through commercial purchase.

[0043] This invention uses indole-based enamine ketone 1 and amino compound 2 as substrates to synthesize indole-fused hydrogenated oxaloline compounds via site activation-intramolecular acylation reaction in a highly polar solvent using the Stork reaction. The reaction route is as follows:

[0044]

[0045] Where R is selected from H, halogen, methyl (Me), methoxy, cyano, or nitro; R 1 Selected from hydroxyalkyl or alkyl groups, and when the substituent R is halogen, it includes monohalogen substitution and dihalogen substitution.

[0046] In this embodiment of the invention, in the preparation method of indole-fused hydrogenated oxalool compounds, the reaction process is monitored by thin-layer chromatography (TLC). The developing solvent is petroleum ether and ethyl acetate in a volume ratio of 1:1. The reaction is considered complete when the starting material spot disappears. At a temperature of 45-80°C, the reaction takes 2-24 hours to complete. After the reaction is complete, the reaction is stopped, an appropriate amount of silica gel is added, and the solvent is evaporated using a rotary evaporator. Then, the reaction system is separated by column chromatography. The mobile phase used in the separation process is petroleum ether and ethyl acetate in a volume ratio of (1-2):1. The entire separation process is monitored by TLC to obtain a pure product. The pure product is obtained by evaporating the solvent from the product segment using a rotary evaporator.

[0047] The lung cancer A549, breast cancer MDA-MB-231, colon cancer HCT116, liver cancer HepG2, and prostate cancer DU145 cell lines used in the embodiments of this invention were purchased from Hunan Fenghui Biotechnology Co., Ltd.

[0048] In the embodiments of the present invention, the maximum emission wavelength and excitation wavelength of indole-fused hydrogenated morpholine compounds were determined by fluorescence spectroscopy, and the relative fluorescence quantum yield of each compound was obtained by reference method. It was found that this type of compound has good fluorescence properties, proving that it has broad prospects in the field of preparing fluorescent materials.

[0049] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0050] The technical solution of the present invention will be further illustrated by the following embodiments.

[0051] Example 1

[0052] Using compounds 1a and 2a as substrates, compound 3aa was synthesized via site activation-intramolecular acylation using a Stork reaction in different solvents. The specific steps were as follows: Substrate 1a was added to a 10 mL reaction flask, followed by 0.5 mL of the corresponding reaction solvent. The mixture was then stirred and dissolved using a magnetic stirrer. Substrate 2a was then added, and the reaction was carried out. The reaction process was monitored using thin-layer chromatography (TLC) with petroleum ether and ethyl acetate as the developing solvents in a volume ratio of 1:1. The reaction was considered complete when the starting material spot disappeared. The reaction was stopped, silica gel was added, and the solvent was evaporated using a rotary evaporator. The reaction system was then separated by column chromatography. The mobile phase used in the separation process was petroleum ether and ethyl acetate in a volume ratio of 2:1. The entire separation process was monitored using thin-layer chromatography (TLC) to obtain a pure product. The solvent in the product fraction was evaporated using a rotary evaporator to obtain the pure product. The solvent types, the molar ratio of compounds 1a and 2a, the reaction temperature, and the time are shown in Table 1. The reaction route is as follows:

[0053]

[0054] Table 1

[0055]

[0056]

[0057] Note: [b] The yield is the isolated yield of the purified product 3aa. [c] The reaction was carried out at the specified temperature for 24 h. [d] At the specified feed metering ratio

[0058] Follow up the reaction until it is complete.

[0059] Table 1 shows that, using indolyl enamine ketone 1a and ethanolamine 2a as the starting substrates, groups 1-11 were selected for solvent selection at a 1:1 ratio of 1a:2a (0.05 mmol / 0.05 mmol), a reaction temperature of 28 °C, and a solvent volume of 0.5 mL. The selected solvents included low-polarity toluene, dichloroethane, dichloromethane, chloroform, and tert-butanol; moderately polarity diethyl ether, ethyl acetate, tetrahydrofuran, and dioxane; and highly polarity methanol and acetonitrile. It can be seen that the reaction yield was very low (7%-8%) when using dichloroethane, toluene, and tert-butanol as solvents, while the reaction yield increased (21%-22%) when using tetrahydrofuran and diethyl ether as solvents. Overall, the reaction yield was optimal (33%) when the reaction occurred in highly polar methanol (group 4). Therefore, methanol was determined to be the optimal solvent for this reaction.

[0060] To further improve the reaction yield, temperature was selected. The reaction accelerated slightly at 45℃ (Group 12); the yield increase was minimal at 60℃ (Group 13); however, a significant increase in yield occurred at 80℃, reaching 72% within the same timeframe (Group 14). Next, the feed ratio was investigated (Groups 15-17). Increasing the amount of substrate 2a accelerated the reaction rate and increased the yield. At a feed ratio of 1:3 (molar ratio, the same below), the yield increased to 84%; with further increases in feed ratio, the template reaction reached complete, with a yield of 99% (Groups 16-17). Notably, at a feed ratio of 1:10, the reaction was only 3 hours faster than at a feed ratio of 1:5. Considering environmental protection, economic efficiency, and sustainable development, minimizing unnecessary losses, and improving raw material utilization, a molar ratio of 1:5 was ultimately selected as the optimal feed ratio for this reaction.

[0061] Through a series of condition screenings for this reaction, the 16th group was finally determined as the optimal reaction conditions, namely, using indole-based enamine ketone compound 1a and ethanolamine 2a as the starting substrates, with a dosage ratio of indole-based enamine ketone 1a:ethanolamine 2a of 1:5 (0.05 mmol / 0.25 mmol), 0.5 mL of methanol as the solvent, and carrying out the reaction at 80 °C. The subsequent synthesis of indole-fused hydrogenated boronic acid compounds was carried out under these reaction conditions.

[0062] Example 2

[0063] Based on the optimal conditions in group 16 of Example 1, using compound 1 (0.2 mmol, 1.0 eq) and compound 2a (1.0 mmol, 5.0 eq) as substrates (i.e., a molar ratio of 1:5), indole-fused hydrogenated oxaloline compound 3 was synthesized in methanol (2.0 mL) via a site activation-intramolecular acylation reaction using the Stork reaction. The synthetic route is as follows:

[0064]

[0065] Where R is F, Cl, Br, methyl, methoxy, cyano, or nitro; reaction time, reaction products, and yield are shown in Table 2.

[0066] Table 2

[0067]

[0068]

[0069] Note: Yield refers to the separation yield of the major product 3 (including 3ba-3ua) after purification.

[0070] As shown in Table 2, the reaction exhibited good functional group tolerance, yielding a series of indole-containing fused-ring hydrogenated boronicoline compounds 3aa-3ua, with excellent yields (48%-94%).

[0071] This invention involves substituting different positions of the indole benzene ring with electron-withdrawing groups of varying properties. Substituting different halogen atoms (-F, -Cl, -Br) at the 4-position of the indole benzene ring had minimal overall impact on the reaction (3ba, 3ca, 3da), with yields of 89%, 91%, and 78%, respectively. Introducing an electron-donating group (-Me) at the 4-position prolonged the reaction time to 13 hours, but the yield remained at 90%. Subsequently, different electron-withdrawing groups (-F, -Cl, -Br, -CN) and electron-donating groups (-CH3, -OCH3) were substituted at the 5-position of the indole. Introducing halogen atoms (-F, -Cl, -Br) maintained good yields (70%-94%); introducing the electron-withdrawing group -CN, possibly due to its deactivation effect on the indole benzene ring, accelerated the reaction rate, achieving complete reaction in 4 hours, while also maintaining a good yield (91%). When the substituent was -Me, 3ia yielded excellent results (90%), but the reaction rate slowed down (13 h). When the 5-position was substituted with an electron-donating group -OMe, the yield decreased (62%). Subsequently, different electron-donating groups (3la-3na) were introduced at the 6-position, and the yields were well maintained (77%-94%). When substitutions were made at the 7-position of the indole benzene ring (-Cl, -Br, -Me), the steric hindrance at the 7-position likely restricted the formation of intermediates, resulting in a slower reaction and decreased yields of 55%, 48%, and 62%, respectively. However, when an electron-withdrawing group -NO2 was introduced at the 7-position, the steric hindrance did not affect the reaction rate. Instead, -NO2 likely reduced the electron cloud density on the benzene ring through an inductive effect, delocalizing some electrons from the pyrrole ring to the benzene ring, thereby increasing the reactivity of the carbonyl carbon at the 2-position of the indole and accelerating the reaction rate. The reaction was completed in 3 h, and the yield was well maintained (85%). Finally, halogen atom di-substitution at different positions on the indole benzene ring was performed (3sa, 3ta, 3ua). Similarly, the increased steric hindrance from di-substitution was considered, potentially affecting the yield of these substituted products. With bis-Cl substitution, the yields of 4-6 and 4-7 di-substitution were better than those of 5-7 di-substitution. The 4-6 di-substituted compound (3sa) achieved a 90% yield, the 4-7 di-substituted compound (3ua) an 89% yield, and the 5-7 di-substituted compound only 72% yield. When the 7-position of indole was substituted with a nitrogen atom to form a 7-N heteroindole, the reaction time was shortened and the yield increased. This is likely because the nitrogen atom has a higher electronegativity, resulting in a higher electron cloud density around it, while the electron cloud density in other parts of the indole ring decreases, thus increasing the nucleophilic reactivity.

[0072] When the indole ring is replaced with a pyrrole ring, the product obtained is (Denoteed as 3wa), the specific reaction process is as follows: at 80℃, pyrrole enamine ketone (0.2 mmol, 1.0 eq) and compound 2 (1.0 mmol, 5.0 eq) are used as substrates. The reaction is carried out in methanol (2.0 mL) by site activation-intramolecular acylation via the Stork reaction. The reaction is complete in 11 h. The yield of this reaction is significantly reduced to only 11%. This may be because, compared to indole, only the lone pair of electrons of the N ring is conjugated with the ring, lacking the conjugation of a benzene ring. This makes the electron density of the pyrrole ring higher than that of indole, resulting in lower nucleophilic reactivity and thus a lower yield.

[0073] Substitution with different substituents at different positions on the indole benzene ring revealed that the electron-donating groups of different properties had some influence on the reaction results, but generally maintained the yield well. In summary, steric hindrance had a relatively greater impact on the reaction yield, with substitution at the 7-position resulting in a lower yield. Secondly, the introduction of the electron-donating group -Me prolonged the reaction time, but the yield remained good. Thirdly, the introduction of electron-withdrawing groups -CN and -NO2 had a dominant inductive effect, which accelerated the reaction and also yielded excellent yields.

[0074] Example 3

[0075] Based on the optimal conditions in group 16 of Example 1, using compound 1a (0.2 mmol, 1.0 eq) and compound 2 (1.0 mmol, 5.0 eq) as substrates (i.e., a molar ratio of 1:5), indole-fused hydrogenated oxaloline compound 3 was synthesized in methanol (2.0 mL) via a site activation-intramolecular acylation reaction using the Stork reaction. The synthetic route is as follows:

[0076]

[0077] Where R is C2-C7 hydroxyalkyl, n-butyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; reaction time, reaction products, and yield are shown in Table 3.

[0078] Table 3

[0079]

[0080]

[0081] Note: Yield refers to the separation yield of the main product 3 (including 3ab-3al) after purification.

[0082] Table 3 shows that, although the yield of the target compound (3ab-3af) decreased slightly (77%-88%) with increasing C atom number, it remained relatively high. When the substrate was changed to tert-butylamine, the reaction slowed down and the yield decreased, indicating that increasing steric hindrance worsened the reaction. In this invention, the chain amine was replaced with a cyclic amine, and the yield of the compound (3ai-3al) (53%-87%) increased with increasing ring size. The amino group acts as a nucleophile, attacking the protonated carbonyl carbon to form a tetrahedral intermediate. Since smaller cyclic amine rings have greater ring strain, electrons become more crowded, and the rigid conformation restricts adjacent sites, reducing the reactivity of the α-C of the carbonyl group. Through substrate expansion of amine compound 2, it was found that when ethanolamine was replaced with a long-chain amine or a cyclic amine structure, except for the tert-butylamine compound, the other compounds maintained high yields, demonstrating the good functional group tolerance of this reaction.

[0083] Example 4: Scale-up reaction

[0084] To investigate the potential of the reaction of this invention in industrial production, compound 1a was taken ( 835 mg (2.8 mmol) was placed in a 100 mL round-bottom flask, dissolved in 28 mL of methanol, and then compound 2a was added. 840 μL (14.0 mmol) was added and reacted at 80 °C. The reaction was monitored by TLC until complete. The solvent was evaporated and the product 3aa was purified by column chromatography. The yield was 98% (809 mg, 2.74 mmol). Even after scale-up, the product maintained an excellent yield, demonstrating the potential for industrial-scale production.

[0085] To determine the configuration of the product, compound 3aa was then cultured in single crystals, and the absolute configuration of compound 3aa was obtained after X-ray diffraction analysis. Figure 1 Based on the absolute configuration of compound 3aa, the absolute configurations of other compounds can be deduced. The synthetic mechanism route of compound 3aa is shown below. Figure 2 As shown, the reaction mechanism is as follows: the lone pair electrons of primary amine 2 first attack the carbonyl group of enamine ketone 1, undergoing nucleophilic addition to give hemiketamine intermediate A. Then, one molecule of water is eliminated to give enamine B. Enamine tautomerism forms an imine, and the resulting intermediate C is a secondary ketimine with a more stable structure. Intermediate C has both imine and enamine structures. The N atom of the enamine and the β-carbon atom of the enamine are both nucleophilic sites. The β-carbon atom of the enamine (with the inductive effect of the electron-withdrawing functional group imine on the β-carbon atom of the enamine, the nucleophilic ability is stronger) nucleophilically attacks the 2-ester carbonyl group of indole, which then undergoes an intramolecular cyclization reaction to give compound 3aa.

[0086] The crystal structure data of the synthesized compounds are as follows:

[0087] Compound 3aa, yellow solid, 57.7 mg, 98% yield, mp 176.2-177.7 °C. 1 H NMR (400MHz, DMSO-d6) δ13.30(s,1H),7.88(d,J=8.2Hz,1H),7.76(d,J=8.0Hz,1H),7.35-7.28(m,1H),7.25-7.18(m,1H),7.00(s,1H) ,5.18(t,J=4.9Hz,1H),3.68(q,J=4.9Hz,2H),3.59(q,J=5.2Hz,2H),2.83(t,J=6.1Hz,2H),2.80-2.72(m,2H),1.94(p,J=6.2Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ173.83,172.68,151.57,133.13,130.98,125.36,123.30,122 .17,122.04,111.61,99.04,95.69,59.66,46.46,29.97,27.13,20.83.HRMS(ESI)m / z calcdfor C 17 H 18 N3O2[M+H] + =296.1399,found=296.1403.

[0088] Compound 3ba, yellow solid, 56.6 mg, 89% yield, mp 214.6-215.2℃. 1 H NMR (400MHz, DMSO-d6) δ13.30(s,1H),7.72(d,J=8.4Hz,1H),7.28(q,J=7.9Hz,1H),7.01(d,J=12.4Hz,2H),5.18(t,J=4.8 Hz,1H),3.70(q,J=5.1Hz,2H),3.62(q,J=5.4Hz,2H),2.86(t,J=6.4Hz,2H),2.77(t,J=6.2Hz,2H),1.95(p,J=6.2Hz,2H). 13C NMR (101MHz, DMSO-d6) δ173.42, 173.08, 156.50 (d, J = 247.0Hz), 152.56, 135.37 (d, J = 11.0Hz), 131.44, 123.64 (d, J = 7.6 Hz), 114.87 (d, J = 23.5Hz), 108.24 (d, J = 3.5Hz), 106.10 (d, J = 18.2Hz), 99.23, 90.94, 59.59, 46.56, 29.92, 27.17, 20.72. 19 F NMR(376MHz,DMSO-d6)δ-121.69(dd,J=10.9,5.4Hz).HRMS(ESI)m / z calcd for C 17 H 17 N3O2F[M+H] + =314.1305, found=314.1309.

[0089] Compound 3ca, yellow solid, 60.0 mg, 91% yield, mp 237.6-238.7 °C. 1 H NMR(400MHz,DMSO-d6)δ13.31(s,1H),7.86(s,1H),7.31(s,2H),6.96(s,1H), 5.18(s,1H),3.70(s,2H),3.63(s,2H),2.87(s,2H),2.78(s,2H),1.96(s,2H). 13 C NMR(101MHz,DMSO-d6)δ173.27,173.17,152.72,133.70,131.78,126.04,123.91,123 .78,121.52,110.87,99.32,93.20,59.59,46.59,29.92,27.18,20.70.HRMS(ESI)m / z calcd for C 17 H 17 N3O2Cl[M+H] + =330.1009, found=330.1013.

[0090] Compound 3da, yellow solid, 59.2 mg, 78% yield, mp 231.8-232.6 °C. 1H NMR (400MHz, DMSO-d6) δ13.30(s,1H),7.90(d,J=8.3Hz,1H),7.46(d,J=7.3Hz,1H),7.23(t,J=7.8Hz,1H),6.88(s,1H),5.18( t,J=4.8Hz,1H),3.70(q,J=4.9Hz,2H),3.61(q,J=5.3Hz,2H),2.85(t,J=5.3Hz,2H),2.80-2.72(m,2H),1.95(p,J=6.2Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ173.24,173.14,152.71,133.34,131.73,125.74,124.71,124 .09,115.22,111.34,99.31,94.86,59.60,46.60,29.92,27.17,20.70.HRMS(ESI)m / z calcd for C 17 H 17 N3O2Br[M+H] + =374.0504, found=374.0508.

[0091] Compound 3ea, yellow solid, 55.9 mg, 90% yield, mp 199.5-201.0℃. 1 H NMR (400MHz, DMSO-d6) δ13.30(s,1H),7.69(d,J=8.3Hz,1H),7.20(t,J=7.7Hz,1H),7.00(d,J=6.2Hz,2H),5.15(s,1H),3.68( d,J=5.2Hz,2H),3.61(q,J=4.9,4.3Hz,2H),2.86(t,J=6.2Hz,2H),2.76(t,J=6.2Hz,2H),2.56(s,3H),1.95(p,J=6.1Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ173.87,172.66,151.61,132.98,131.09,130.53,125.56,123.52 ,121.79,109.22,99.06,94.38,59.72,46.46,30.01,27.15,20.85,18.79.HRMS(ESI)m / z calcd for C 18 H 20 N3O2[M+H] +=310.1556,found=310.1556.

[0092] Compound 3fa, yellow solid, 58.7 mg, 94% yield, mp 260.1-262.4℃. 1 H NMR (400MHz, DMSO-d6) δ13.34(s,1H),7.88(dd,J=8.8,4.6Hz,1H),7.50(dd,J=9.9,2.5Hz,1H),7.16(td,J=9.2,2.6Hz,1H) ,6.97(s,1H),5.17(s,1H),3.69(s,2H),3.65-3.58(m,2H),2.88(t,J=5.5Hz,2H),2.81-2.72(m,2H),1.95(p,J=6.3Hz,2H). 13 C NMR (101MHz, DMSO-d6) δ173.34, 173.05, 158.63 (d, J = 235.1Hz), 151.98, 132.30, 129.99, 125.36 (d, J = 11.0Hz), 113.05 (d ,J=10.0Hz),112.26(d,J=27.2Hz),106.00(d,J=23.4Hz),99.14,95.48(d,J=5.4Hz),59.61,46.54,29.90,27.18,20.80. 19 F NMR(376MHz,DMSO-d6)δ-121.19(td,J=9.8,4.5Hz).HRMS(ESI)m / z calcd for C 17 H 17 N3O2F[M+H] + =314.1305, found=314.1308.

[0093] Compound 3ga, yellow solid, 60.0 mg, 91% yield, mp 252.4-253.8℃. 1 H NMR (400MHz, DMSO-d6) δ13.31 (s, 1H), 7.84 (d, J = 21.0Hz, 2H), 7.29 (s, 1H), 6.96 (s ,1H),5.17(s,1H),3.66(d,J=17.9Hz,4H),2.89(s,2H),2.77(s,2H),1.96(s,2H). 13C NMR(101MHz,DMSO-d6)δ173.44,173.15,152.33,132.14,131.45,126.51,126.12,123 .38,121.13,113.30,99.22,95.18,59.60,46.58,29.94,27.21,20.76.HRMS(ESI)m / z calcd for C 17 H 17 N3O2Cl[M+H] + =330.1009,found=330.1011.

[0094] Compound 3ha, yellow solid, 52.8 mg, 70% yield, mp 235.9-237.4 °C. 1 H NMR (400MHz, DMSO-d6) δ13.31(s,1H),7.97(s,1H),7.83(d,J=8.7Hz,1H),7.40(d,J=8.3Hz,1H),6.96(s,1H), 5.17(s,1H),3.66(dt,J=16.8,5.3Hz,4H),2.90(t,J=6.2Hz,2H),2.77(t,J=6.2Hz,2H),1.96(p,J=6.3Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ173.44,173.14,152.36,131.95,131.63,126.81,125.82,124.2 6,114.59,113.65,99.24,95.08,59.60,46.57,29.94,27.21,20.75.HRMS(ESI)m / zcalcd for C 17 H 17 N3O2Br[M+H] + =374.0504,found=374.0504.

[0095] Compound 3ia, yellow solid, 57.4 mg, 93% yield, mp 225.1-226.2 °C. 1H NMR (400MHz, DMSO-d6) δ13.24(s,1H),7.69(d,J=8.5Hz,1H),7.43(s,1H),7.07(d,J=8.5Hz,1H),6.82(s,1H),5.10(s,1H),3 .61(t,J=5.1Hz,2H),3.53(q,J=5.4Hz,2H),2.79(t,J=6.2Hz,2H),2.68(t,J=6.2Hz,2H),2.35(s,3H),1.87(p,J=6.1Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ173.80,172.67,151.30,131.74,130.94,130.85,125.59,125.35 ,121.25,111.34,99.02,95.12,59.68,46.44,29.96,27.15,21.78,20.86.HRMS(ESI)m / z calcd for C 18 H 20 N3O2[M+H] + =310.1556,found=310.1557.

[0096] Compound 3ja, yellow solid, 40.1 mg, 62% yield, mp 214.5-215.7℃. 1 H NMR (400MHz, DMSO-d6) δ13.35(s,1H),7.76(d,J=9.0Hz,1H),7.18(s,1H),6.95(d,J=8.5Hz,1H),6.89(s,1H),5.15(s, 1H),3.80(s,3H),3.68(s,2H),3.61(t,J=5.3Hz,2H),2.85(t,J=6.1Hz,2H),2.75(t,J=6.1Hz,2H),1.98-1.89(m,2H). 13 C NMR(101MHz,DMSO-d6)δ173.45,172.74,155.50,151.29,131.14,128.69,125.81,115.06 ,112.52,101.83,99.00,95.19,59.67,55.70,46.45,29.91,27.13,20.88.HRMS(ESI)m / z calcd for C 18 H 20 N3O3[M+H] + =326.1505, found=326.1509.

[0097] Compound 3 kDa, yellow solid, 58.2 mg, 91% yield, mp 267.6-268.2 °C. 1 H NMR (400MHz, DMSO-d6) δ13.27(s,1H),8.35(s,1H),8.01(d,J=8.6Hz,1H),7.59(d,J=8.6Hz,1H),7. 12(s,1H),5.19(s,1H),3.75-3.56(m,4H),2.94-2.86(m,2H),2.83-2.75(m,2H),2.01-1.91(m,2H). 13 C NMR(101MHz,DMSO-d6)δ173.37,173.31,153.45,133.83,133.05,128.61,124.92,124.68 ,120.64,113.02,104.15,99.31,96.58,59.54,46.65,29.97,27.25,20.64.HRMS(ESI)m / z calcd for C 18 H 17 N4O2[M+H] + =321.1352,found=321.1353.

[0098] Compound 3la, yellow solid, 59.4 mg, 94% yield, mp 204.9-206.5 °C. 1 H NMR (400MHz, DMSO-d6) δ13.25(s,1H),7.79(dd,J=8.8,5.4Hz,1H),7.57(dd,J=9.7,2.3Hz,1H),7.10(td,J=9.3,2.4Hz,1H),7.02(s,1H ),5.17(t,J=4.5Hz,1H),3.69(q,J=4.5Hz,2H),3.60(q,J=5.2Hz,2H),2.85(t,J=6.1Hz,2H),2.78-2.72(m,2H),1.94(p,J=6.3Hz,2H). 13CNMR(101MHz,DMSO-d6)δ173.71,172.71,159.94(d,J=238.9Hz),152.12,132.72(d,J=12.5Hz),131.93(d,J=3.6Hz),123 .92(d,J=10.1Hz),122.17,111.43(d,J=25.4Hz),98.85,97.10(d,J=26.3Hz),96.05,59.64,46.47,29.95,27.14,20.75. 19 F NMR(376MHz,DMSO-d6)δ-117.68(td,J=9.7,5.4Hz).HRMS(ESI)m / z calcd for C 17 H 17 N3O2F[M+H] + =314.1305, found=314.1309.

[0099] Compound 3ma, yellow solid, 58.9 mg, 89% yield, mp 249.6-250.9℃. 1 H NMR(400MHz,DMSO-d6)δ13.27(s,1H),8.09-7.57(m,2H),7.22(s,1H),7.01(s,1 H),5.17(s,1H),3.65(d,J=22.3Hz,4H),2.87(s,2H),2.76(s,2H),1.95(s,2H). 13 C NMR(101MHz,DMSO-d6)δ173.56,172.94,152.37,133.06,131.90,127.96,123.91,123 .83,122.54,110.99,99.11,95.90,59.60,46.53,29.93,27.16,20.72.HRMS(ESI)m / z calcd for C 17 H 17 N3O2Cl[M+H] + =330.1009, found=330.1013.

[0100] Compound 3na, yellow solid, 57.7 mg, 77% yield, mp 272.8-274.3 °C. 1H NMR (400MHz, DMSO-d6) δ13.28(s,1H),8.00(s,1H),7.72(d,J=8.6Hz,1H),7.33(d,J=8.5Hz,1H),7.00(s,1H), 5.17(s,1H),3.68(s,2H),3.66-3.58(m,2H),2.88(t,J=5.8Hz,2H),2.80-2.71(m,2H),1.95(p,J=6.2Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ173.54,173.00,152.43,133.48,131.72,125.03,124.13 ,115.99,114.02,99.17,95.91,59.61,46.55,29.94,27.19,20.73.HRMS(ESI)m / z calcd for C 17 H 17 N3O2Br[M+H] + =374.0504,found=374.0504.

[0101] Compound 3oa, yellow solid, 36.3 mg, 55% yield, mp 255.2-256.3℃. 1 H NMR (400MHz, DMSO-d6) δ13.26(s,1H),7.72(d,J=8.1Hz,1H),7.33(d,J=7.4Hz,1H),7.16(t,J=7 .8Hz,1H),7.09(s,1H),5.17(s,1H),3.75-3.57(m,4H),2.88(s,2H),2.75(s,2H),1.96(s,2H). 13 CNMR(101MHz,DMSO-d6)δ173.16,172.89,151.26,132.30,128.16,128.11,124.94,12 2.29,121.21,118.03,98.83,96.77,59.60,46.55,30.20,27.19,20.81.HRMS(ESI)m / z calcdfor C 17 H 17 N3O2Cl[M+H] + =330.1009,found=330.1012.

[0102] Compound 3pa, yellow solid, 36 mg, 48% yield, mp 245.7-246.5℃. 1H NMR (400MHz, DMSO-d6) δ13.24(s,1H),7.77(d,J=8.0Hz,1H),7.51(d,J=7.4Hz,1H),7.15-7.03(m,2H) ,5.17(s,1H),3.73-3.57(m,4H),2.87(t,J=5.7Hz,2H),2.76(t,J=6.2Hz,2H),1.96(p,J=6.3Hz,2H). 13 CNMR(101MHz,DMSO-d6)δ173.17,172.87,150.83,132.31,129.07,128.43,128.26,12 2.77,121.73,104.70,98.91,96.63,59.61,46.54,30.19,27.19,20.81.HRMS(ESI)m / z calcd for C 17 H 17 N3O2Br[M+H] + =374.0504, found=374.0506.

[0103] Compound 3qa, yellow solid, 38.2 mg, 62% yield, mp 190.7-191.9℃. 1 H NMR (400MHz, DMSO-d6) δ13.27 (s, 1H), 7.55 (d, J = 7.2Hz, 1H), 7.20-6.90 (m, 3H), 5. 17(s,1H),3.76-3.54(m,4H),2.89(s,3H),2.84(s,2H),2.72(s,2H),1.94(s,2H). 13 C NMR(101MHz,DMSO-d6)δ173.65,172.44,150.51,131.76,131.09,125.89,125.25,124.26 ,121.76,119.68,98.60,96.36,59.68,46.43,30.23,27.13,20.93,20.08.HRMS(ESI)m / z calcd for C 18 H 20 N3O2[M+H] + =310.1556,found=310.1559.

[0104] Compound 3ra, yellow solid, 57.4 mg, 85% yield, mp 275.3-276.6 °C. 1H NMR (400MHz, DMSO-d6) δ13.26(s,1H),8.07(d,J=8.0Hz,1H),7.73(d,J=7.4Hz,1H),7.33(t,J=7.8Hz,1H),7.23(s,1H) ,5.19(t,J=4.7Hz,1H),3.67(dp,J=15.7,5.4Hz,4H),2.90(t,J=6.2Hz,2H),2.70-2.64(m,2H),1.94(p,J=6.2Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ173.42,173.05,152.76,137.82,133.00,128.10,126.60,121 .43,121.01,118.39,98.94,97.10,59.52,46.66,29.95,27.24,20.63.HRMS(ESI)m / z calcd for C 17 H 17 N4O4[M+H] + =341.1250,found=341.1246.

[0105] Compound 3sa, yellow solid, 66.2 mg, 90% yield, mp 265.4-266.6℃. 1 H NMR (400MHz, DMSO-d6) δ13.27(s,1H),7.82(dd,J=1.7,0.9Hz,1H),7.38(d,J=1.8Hz,1H),6.92(d,J=0.9Hz,1H),5. 19(t,J=4.8Hz,1H),3.67(dp,J=14.9,4.6Hz,4H),2.89(t,J=6.1Hz,2H),2.80-2.72(m,2H),1.96(p,J=6.0Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ173.37,172.98,153.40,133.09,132.57,127.52,127.02,122.6 8,121.68,110.45,99.40,93.41,59.54,46.66,29.88,27.21,20.62.HRMS(ESI)m / zcalcd for C 17 H 16 N3O2Cl2[M+H] + =364.0620, found=364.0623.

[0106] Compound 3ta, yellow solid, 52.9 mg, 72% yield, mp 270.6-271.9℃. 1 H NMR(400MHz,DMSO-d6)δ13.27(s,1H),7.81(s,1H),7.37(s,1H),7.05(s,1H) ,5.18(s,1H),3.67(d,J=16.0Hz,4H),2.90(s,2H),2.75(s,2H),1.96(s,2H). 13 C NMR(101MHz,DMSO-d6)δ173.26,172.75,151.95,133.48,128.41,126.74,125.78,124 .21,120.25,119.15,99.05,96.37,59.55,46.65,30.14,27.24,20.74.HRMS(ESI)m / z calcd for C 17 H 16 N3O2Cl2[M+H] + =364.0620,found=364.0620.

[0107] Compound 3ua, yellow solid, 66.0 mg, 89% yield, mp 243.2-244.3℃. 1 H NMR (400MHz, DMSO-d6) δ13.18(s,1H),7.82(d,J=1.9Hz,1H),7.67(d,J=8.7Hz,1H),7.29(dd,J=8.7,1.9Hz,1H) ,3.70(t,J=5.0Hz,2H),3.63-3.58(m,2H),2.85(t,J=6.2Hz,2H),2.71(t,J=6.3Hz,2H),1.93(p,J=6.4Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ173.05,172.66,152.83,131.04,129.25,125.46,123.36,121.8 0,120.40,111.22,99.42,99.40,59.53,46.57,29.79,27.18,20.52.HRMS(ESI)m / zcalcd for C 17 H 16 N3O2Cl2[M+H] + =364.0620,found=364.0622.

[0108] Compound 3wa, white solid, 5.5 mg, 11% yield, mp 160.1-162.0℃. 1 H NMR (500MHz, CDCl3) δ13.26(s,1H),7.35(dd,J=2.6,1.5Hz,1H),6.91-6.87(m,1H),6.43(dd,J=4.2,2.5Hz,1H),5.34(s ,1H),3.96(t,J=4.8Hz,2H),3.57(q,J=5.3Hz,2H),2.43(t,J=6.3Hz,2H),2.29(t,J=6.3Hz,2H),1.68(p,J=6.3Hz,2H). 13 C NMR (126MHz, CDCl3) δ171.65,171.05,152.31,124.37,119.28,108.54,104.44,96.72,59.93,45.82,28.15,25.88,19.42.

[0109] Compound 3ab, yellow solid, 54.8 mg, 88% yield, mp 173.3-174.3℃. 1 H NMR (400MHz, DMSO-d6) δ13.26(s,1H),7.88(d,J=8.3Hz,1H),7.75(d,J=8.0Hz,1H),7.31(t,J=7.6Hz,1H),7.21(t,J=7.5Hz,1H ),7.00(s,1H),4.75(s,1H),3.56(q,J=6.0Hz,4H),2.78(dt,J=19.7,6.2Hz,4H),1.93(p,J=6.3Hz,2H),1.81(p,J=6.6Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ173.84,172.58,151.50,133.13,130.90,125.35,123.32,122.17 ,122.06,111.62,98.95,95.69,58.20,40.96,32.08,29.92,26.77,20.79.HRMS(ESI)m / z calcd for C 18 H 20 N3O2[M+H] + =310.1556,found=310.1560.

[0110] Compound 3ac, yellow solid, 50.5 mg, 77% yield, mp 176.8-177.6 °C.1 H NMR (400MHz, DMSO-d6) δ13.27(s,1H),7.88(d,J=8.4Hz,1H),7.75(d,J=8.1Hz,1H),7.31(t,J=7.6Hz,1H),7.21(t,J=7.5Hz,1H),6.99(s,1H) ,4.55(s,1H),3.50(dt,J=13.9,6.6Hz,4H),2.79(dt,J=18.0,6.3Hz,4H),1.94(p,J=6.1Hz,2H),1.70(p,J=7.1Hz,2H),1.56(p,J=6.9Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ173.88,172.56,151.49,133.13,130.86,125.35,123.34,122.18,12 2.08,111.61,98.93,95.71,60.65,43.65,30.08,29.91,26.82,25.80,20.79.HRMS(ESI)m / z calcd for C 19 H 22 N3O2[M+H] + =324.1712,found=324.1714.

[0111] Compound 3ad, yellow solid, 57.6 mg, 86% yield, mp 150.5-152.3℃. 1 H NMR(400MHz,DMSO-d6)δ13.29(s,1H),7.88(s,1H),7.75(s,1H),7.31(s,1H),7.22(s,1H),7.00( s,1H),4.43(s,1H),3.53(s,4H),2.82(d,J=27.4Hz,4H),1.96(s,2H),1.68(s,2H),1.47(s,4H). 13 C NMR(101MHz,DMSO-d6)δ173.90,172.67,151.51,133.13,130.85,125.35,123.36,122.19,122.1 0,111.61,98.95,95.74,60.97,43.82,32.48,29.93,28.88,26.86,23.39,20.81.HRMS(ESI)m / z calcd for C 20 H 24 N3O2[M+H] +=338.1869,found=338.1873.

[0112] Compound 3ae, yellow solid, 57.2 mg, 82% yield, mp 137.9-139.3℃. 1 H NMR (400MHz, DMSO-d6) δ13.24(s,1H),7.88(d,J=8.3Hz,1H),7.75(d,J=8.0Hz,1H),7.31(t,J=7.5Hz,1H),7.21(t,J=7.4Hz,1H),7. 00(s,1H),3.58(s,1H),3.45-3.34(m,4H),2.84-2.72(m,4H),1.94(p,J=7.0,6.1Hz,2H),1.65(p,J=6.8Hz,2H),1.52-1.36(m,6H). 13 C NMR(101MHz,DMSO-d6)δ173.88,172.56,151.48,133.13,130.85,125.34,123.34,122.18,122.07,1 11.61,98.93,95.73,61.08,43.70,32.85,29.90,28.98,26.82,26.64,25.59,20.78.HRMS(ESI)m / z calcd for C 21 H 26 N3O2[M+H] + =352.2025,found=352.2027.

[0113] Compound 3af, yellow solid, 58.8 mg, 81% yield, mp 133.4-135.8 °C. 1 H NMR (400MHz, DMSO-d6) δ13.25(s,1H),7.87(d,J=8.3Hz,1H),7.75(d,J=8.1Hz,1H),7.31(t,J=7.7Hz,1H),7.21(t,J=7.5H z,1H),7.00(s,1H),4.36(s,1H),3.55-3.39(m,4H),2.79(d,J=16.2Hz,4H),1.95(s,2H),1.65(s,2H),1.46-1.29(m,8H). 13C NMR (101MHz, DMSO-d6) δ173.89,172.59,151.48,133.13,130.85,125.35,123.34,122.18,122.08,111. 61,98.93,95.73,61.14,43.73,32.94,29.91,28.98,28.90,26.84,26.80,25.89,20.79.HRMS(ESI)m / z calcd for C 22 H 28 N3O2[M+H] + =366.2182,found=366.2184.

[0114] Compound 3ag, yellow solid, 39.2mg, 64% yield, mp 169.4-170.0℃. 1 H NMR (400MHz, DMSO-d6) δ13.29(s,1H),7.87(d,J=8.3Hz,1H),7.75(d,J=8.0Hz,1H),7.34-7.28(m,1H),7.24-7.18(m,1H),6.99(s,1H),3.54(q,J =6.2Hz,2H),2.86(t,J=5.7Hz,2H),2.80-2.74(m,2H),1.96(p,J=6.1Hz, 2H), 1.66 (p, J = 7.0Hz, 2H), 1.44 (h, J = 7.4Hz, 2H), 0.96 (t, J = 7.3Hz, 3H). 13 C NMR(101MHz,DMSO-d6)δ173.90,172.69,151.50,133.13,130.85,125.34,123.35,122.19,12 2.09,111.61,98.96,95.73,43.45,30.97,29.92,26.86,20.81,19.97,14.01.HRMS(ESI)m / z calcd for C 19 H 22 N3O[M+H] + =308.1763,found=308.1767.

[0115] Compound 3ah, yellow oil, 6.6 mg, 11% yield. 1H NMR (400MHz, DMSO-d6) δ14.06(s,1H),7.93(d,J=8.4Hz,1H),7.81(d,J=8.0Hz,1H),7.37(t,J=7.6Hz,1H),7.27 (t,J=7.4Hz,1H),7.04(s,1H),3.09(t,J=6.1Hz,2H),2.86(t,J=6.0Hz,2H),2.03(t,J=6.2Hz,2H),1.59(s,9H). 13 C NMR(101MHz,DMSO-d6)δ173.80,172.87,151.38,133.03,130.63,125.39,123.32,122 .18,122.12,111.61,99.31,95.66,55.06,30.23,30.16,28.79,21.41.HRMS(ESI)m / z calcd for C 19 H 22 N3O[M+H] + =308.1763,found=308.1767.

[0116] Compound 3ai, yellow solid, 30.8 mg, 53% yield, mp 137.1-138.9 °C. 1 H NMR (400MHz, DMSO-d6) δ13.14(s,1H),7.90(d,J=8.3Hz,1H),7.78(d,J=8.1Hz,1H),7.35(t,J=7.6Hz,1H),7.24(t,J=7.4Hz,1H),7.0 3(s,1H),3.10(s,1H),3.04(t,J=5.6Hz,2H),2.85-2.76(m,2H),2.01(p,J=6.9Hz,2H),1.00(d,J=7.2Hz,2H),0.87(d,J=2.7Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ174.82,174.06,151.34,133.25,130.64,125.33,123.55,12 2.24,122.13,111.62,99.10,96.17,29.92,27.72,26.10,20.83,7.78.HRMS(ESI)m / z calcd forC 18 H 18 N3O[M+H] + =292.1450,found=292.1454.

[0117] Compound 3aj, yellow solid, 39.3 mg, 64% yield, mp 152.6-154.1 °C. 1 H NMR (400MHz, DMSO-d6) δ13.40(d,J=5.9Hz,1H),7.87(d,J=8.3Hz,1H),7.76(d,J=8.0Hz,1H),7.32(t,J=7.6Hz,1H),7.21(t,J=7.5Hz,1H), 7.01(s,1H),4.38(h,J=7.8Hz,1H),2.76(t,J=6.3Hz,4H),2.47-2.38(m,2H),2.18-2.07(m,2H),1.93(p,J=6.3Hz,2H),1.86-1.75(m,2H). 13 C NMR(101MHz,DMSO-d6)δ174.05,171.20,151.47,133.21,130.72,125.34,123.48,122.23 ,122.12,111.61,98.80,95.99,48.18,30.72,29.88,27.00,20.80,15.40.HRMS(ESI)m / z calcd for C 19 H 20 N3O[M+H] + =306.1606,found=306.1609.

[0118] Compound 3ak, yellow solid, 52.8 mg, 82% yield, mp 167.8-168.7 °C. 1 H NMR (400MHz, DMSO-d6) δ13.52(d,J=7.9Hz,1H),7.88(d,J=8.3Hz,1H),7.75(d,J=8.0Hz,1H),7.36-7.27(m,1H),7.26-7.17(m,1H),6.99(s,1H ),4.26(h,J=7.4Hz,1H),2.88(t,J=6.2Hz,2H),2.82-2.73(m,2H),2.11 -2.00(m,2H),2.02-1.89(m,2H),1.82-1.73(m,2H),1.71-1.59(m,4H). 13C NMR(101MHz,DMSO-d6)δ173.90,171.77,151.43,133.14,130.79,125.36,123.34,122.19 ,122.09,111.62,98.87,95.71,55.25,33.58,29.95,27.13,23.90,20.89.HRMS(ESI)m / z calcd for C 20 H 22 N3O[M+H] + =320.1763,found=320.1768.

[0119] Compound 3al, yellow solid, 57.8 mg, 87% yield, mp 158.9-160.2 °C. 1 H NMR(400MHz,DMSO-d6)δ13.54(d,J=8.6Hz,1H),7.88(dd,J=8.3,1.0Hz,1H),7.7 5(d,J=8.0Hz,1H),7.31(ddd,J=8.2,6.9,1.1Hz,1H),7.21(ddd,J=8.0,6.9,1.1 Hz,1H),7.00(s,1H),3.93-3.80(m,1H),2.91(t,J=6.2Hz,2H),2.78(dd,J=7.1, 5.4Hz,2H),1.96(dt,J=12.8,7.1Hz,4H),1.77-1.67(m,2H),1.65-1.36(m,6H). 13 C NMR(101MHz,DMSO-d6)δ173.97,171.34,151.49,133.13,130.80,125.37,123.33,122.19,12 2.09,111.63,98.82,95.70,52.04,32.63,30.00,26.60,25.18,23.91,20.95.HRMS(ESI)m / z calcd for C 21 H 24 N3O[M+H] + =334.1919,found=334.1923.

[0120] The present invention performed fluorescence spectroscopy tests on the synthesized target compound, and all organic reagents used in the tests were chromatographically pure reagents (to avoid impurity peaks).

[0121] Application Example 1: Testing the fluorescence properties of the target compound of the present invention

[0122] Preparation of test solution:

[0123] (1) Preparation of the standard stock solution: Weigh a certain amount of 7-amino-4-trifluoromethylcoumarin and dissolve it in methanol (chromatographic grade), shake well, and prepare a solution with a concentration of 10. -3 The test stock solution for M will be further diluted before the specific test.

[0124] (2) Preparation of the test sample stock solution: Dissolve the synthesized target compound in an organic solvent to prepare a 10 -3 The mother liquor of M will be further diluted before testing.

[0125] (3) The test solvent is methanol (chromatographic grade).

[0126] (4) Fluorescence data testing requires the preparation of solutions at concentrations of 10 μM, 5 μM, 2.5 μM, 0.625 μM, 0.3125 μM, and 0.156 μM. This is achieved by diluting 100 μL of the stock solution to 10 mL with the required organic reagent, and then halving the solution sequentially to prepare sample test solutions of different concentration gradients. The testing process is conducted at room temperature.

[0127] Based on the Beer-Lambert law, fluorescence spectroscopy was used to detect the fluorescence intensity of substances at different concentrations. Some compounds showed fluorescence intensity at 10... -5 The fluorescence spectrum of M in methanol solution is as follows: Figure 3 As shown, the scanning range of the fluorometer is 420-800 nm, and the scanning speed is 2000 nm / min. The normalized fluorescence emission spectra of some compounds are shown below. Figure 4 As shown, the maximum fluorescence emission wavelength of indole-fused hydrogenated boronicoline compounds is related to the electronic effects of the substituents, and the fluorescence wavelength range of these compounds is between 430-700 nm. Compared with the unsubstituent template product 3aa, most products with electron-withdrawing groups on the benzene ring (3ba, 3ca, 3da, 3fa, 3ga, 3ha, 3ka, 3la, 3ma, 3na, 3oa, 3pa, 3sa, 3ta, 3ua) show a slight blue shift. On the other hand, compounds with electron-donating groups (3ea, 3ia, 3ja) show a red shift and exhibit weaker fluorescence compared to compounds containing electron-withdrawing groups. Figure 3 The compound 3ra, substituted with a nitro group, was found to lack fluorescence, possibly because the strong electron-withdrawing ability of the nitro group disrupts the conjugated structure of the compound, leading to fluorescence quenching. When the carbon chain of the hydroxyethyl group was elongated, compounds (3ab, 3ac, 3ad, 3ae, 3af) showed a slight red shift; similarly, replacing the chain structure with a cyclic structure resulted in a slight red shift in compounds (3ai, 3aj, 3ak, 3al).

[0128] This invention uses 7-amino-4-trifluoromethylcoumarin as a reference material and its quantum yield (Ф=0.53) as a standard to calculate the fluorescence quantum yield of the target compound in methanol solution. According to literature references, the λ of 7-amino-4-trifluoromethylcoumarin... em =480nm, λ ex =376nm, the test result of this invention is λ em =484.3nm, λ ex =381.1nm( Figure 5 Fluorescence excitation and emission spectra were plotted, and concentration curves were generated to determine the fluorescence quantum yield of each target compound. The results are shown in Table 4. It can be seen that the quantum yields of the target compounds range from 15.3% to 72.9%, with the product 3pa exhibiting the highest quantum yield at 72.9%. Compound 3ja also exhibits a large Stokes shift, reaching 100 nm. These experimental results demonstrate that simple structural modifications to indole-fused hydrogenated morphine compounds can alter their fluorescence properties. These results prove that the indole-fused hydrogenated morphine compounds of this invention hold promise for applications in fluorescent materials, fluorescent imaging probes, and other fields.

[0129] Table 4 Optical properties of the compounds in methanol solution

[0130]

[0131] Application Example 2: Testing the antitumor properties of the compounds of the present invention

[0132] 1. Preparation of commonly used reagents:

[0133] (1) Complete culture medium: Use a pipette to draw 44.5 mL of cell culture medium into a 50 mL centrifuge tube, add 5 mL of fetal bovine serum and 0.5 mL of penicillin-streptomycin mixture, mix thoroughly, and store in a 4°C incubator for later use.

[0134] (2) Cell cryopreservation solution: The volume ratio of cell solution: fetal bovine serum: DMSO is 7:2:1. The volume of cell solution in each cryopreservation tube is generally 1 mL. The cryopreservation solution should be prepared and used immediately.

[0135] (3) MTT solution: Take 25mg of MTT solid into a 50mL centrifuge tube, add PBS buffer solution to dilute to 50mL, mix well, dissolve completely to make the concentration 5mg / mL, filter through a 0.22μM microporous membrane for sterilization, dispense into 2mL light-protected centrifuge tubes, and store at -20℃ in the dark. The entire preparation process should be carried out in the dark as much as possible.

[0136] (4) Drug gradient concentrations; for example, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.56 μM, 0.78 μM, 0.39 μM, containing 5‰ DMSO (dimethyl sulfoxide).

[0137] 2. Experimental Methods

[0138] 2.1 Cell Culture

[0139] (1) Cell thawing: Take the frozen cells out of liquid nitrogen or -80℃ freezer, and quickly place them in a 37℃ water bath and gently shake them to thaw. After disinfecting the cryovials with alcohol, place them in a biosafety cabinet. Use a disposable pipette to transfer the cell solution to a cell culture flask containing an appropriate amount of complete culture medium, mix by pipetting, and place it in a 37℃ incubator containing 5% CO2 until it adheres to the wall. Change the medium after culturing for 24 hours.

[0140] (2) Cell culture medium change: After observing the cell adhesion under a microscope, transfer the cell culture flask to the biosafety cabinet, discard the old culture medium, add PBS to rinse the cells 3 times, and add an appropriate amount of new complete culture medium.

[0141] (3) Cell passage: Remove the cells from the incubator and observe them under a microscope. When the cells have grown to cover 80%-90% of the bottom of the culture flask, they can be passaged. Aspirate the old culture medium, rinse the cells three times with PBS, and add an appropriate amount of trypsin-EDTA digestion solution to completely cover the cell layer in the culture flask. At this time, the cells can be incubated in a 37℃ cell culture incubator for 1-2 minutes and the cell shedding is observed under a microscope. When the cell shedding reaches 80%-90%, add an appropriate amount of complete culture medium to stop the digestion. Rinse the bottom of the cell flask with a disposable pipette to completely detach the cells. Transfer the cells to a 15mL centrifuge tube, centrifuge at 1000r / min for 5 minutes, discard the supernatant, add an appropriate amount of culture medium, mix well, and culture in separate flasks. After adding an appropriate amount of complete culture medium, place the flask in a 37℃ incubator containing 5% CO2.

[0142] (4) Cell cryopreservation: After digesting cells in the logarithmic growth phase, centrifuge them, discard the supernatant, add an appropriate amount of cell cryopreservation solution, mix the cells by pipetting, dispense them into cell cryopreservation tubes, place them in a programmed cooling box, transfer them to a -80°C freezer overnight, and finally transfer them to liquid nitrogen for storage.

[0143] 2.2 MTT assay for the determination of compound IC 50

[0144] MTT, also known as thiazolyl blue, is a yellow powder commonly used to detect cell proliferation and activity. The detection principle involves mitochondrial succinate dehydrogenase reducing MTT to insoluble blue-purple crystals called formazan. In dead cells, the enzyme is inactivated and cannot form these crystals. The resulting crystals dissolve in dimethyl sulfoxide (DMSO), and their absorbance is measured at a specific wavelength using a microplate reader. The measured absorbance reflects the survival rate of cancer cells. Processing and calculating the absorbance using analytical software directly reveals the compound's inhibitory effect on cell proliferation, facilitating the analysis and summarization of the compound's efficacy.

[0145] (1) Seeding: HepG2 liver cancer, A549 lung cancer, MDA-MB-231 breast cancer, Du145 prostate cancer, and HCT-116 colon cancer cells in logarithmic growth phase were collected and digested with an appropriate amount of trypsin. Digestion was stopped when the cell exfoliation rate reached 80%-90%. Cells were collected, centrifuged at 1500 r / min for 5 min, resuspended in an appropriate amount of complete culture medium, and counted using a cell counter after thorough mixing. The cell suspension was diluted to 5 × 10⁻⁶ cells / mL with complete culture medium. 4 Cells / ml were then seeded into 96-well plates at 100 μL per well and incubated at 37°C in a 5% CO2 incubator for 24 h.

[0146] (2) Drug administration: Discard the supernatant in the 96-well plate. Add 100 μL of culture medium with compound concentrations of 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.56 μM, 0.78 μM and 0.39 μM to the drug administration group respectively. Add the same volume of the same culture medium to the blank group. Add the same volume of the same culture medium containing 5‰ DMSO to the negative control group. Each group has six replicates. Incubate at 37℃ in a 5% CO2 incubator for 48 h.

[0147] (3) Detection: Add 10 μL of 5 mg / mL MTT solution to each well, continue culturing at 37℃ for 4 h, discard the supernatant, add 100 μL of DMSO to each well, mix well on a plate shaker, and then detect the OD value with an ELISA reader at a wavelength of 490 nm.

[0148] (4) Formula for calculating tumor cell growth inhibition rate: Cell inhibition rate (%) = [1 - (OD)] (药物组) -OD (调零组) / (OD (空白组) -OD (调零组) ×100%.

[0149] 2.3 Data Processing

[0150] The original data was processed using Excel to obtain the growth inhibition rates at different concentrations, and the IC50 of each compound was calculated using GraphPad Prism6. 50 value.

[0151] 3.3 Experimental Results

[0152] 3.3.1 Purity Detection of Indole-Fused Hydrogenated Caenorline Compounds

[0153] (1) High Performance Liquid Chromatography (HPLC) Conditions

[0154] Column: Gemini 5μm NX-C18; Mobile phase: methanol-water (90mL / 10mL); Detection wavelength: 254nm, 365nm; Flow rate: 1.0mL / min; Column temperature: 30℃; Injection volume: 10μL.

[0155] (2) High Performance Liquid Chromatography (HPLC) Determination Method

[0156] The synthesized target compound, an indole-fused hydrogenated oxalool, was dissolved in methanol. The dissolved sample was then filtered through a 0.22 μm filter to remove insoluble matter. The filtered solution was transferred to a liquid chromatography vial, and its purity was determined by HPLC.

[0157] The purity of the indole-fused hydrogenated morpholine compounds of this invention is shown in Table 5.

[0158] Table 5 Purity of indole-fused hydrogenated oxalool compounds

[0159]

[0160]

[0161] This invention yielded a series of indole-fused hydrogenated boronic acid derivatives. After identification by proton NMR, carbon NMR, and high-resolution mass spectrometry, the structures of the compounds were confirmed to be consistent with the expected target compounds. Furthermore, the purity of all synthesized compounds was determined by HPLC, and the purity of all compounds was above 95%.

[0162] 3.3.2 In vitro antitumor activity of the target compound

[0163] Among the synthesized indole-fused hydrogenated boronicoline compounds, only a few compounds showed good in vitro proliferation inhibitory activity against five cell lines: lung cancer A549, breast cancer MDA-MB-231, colon cancer HCT116, liver cancer HepG2, and prostate cancer DU145. The specific results are shown in Table 5.

[0164] Table 5 Summary of the in vitro antitumor activity of the target compounds

[0165]

[0166]

[0167] This invention investigated the inhibitory effects of compounds with different substituents on the indole benzene ring on the cell proliferation of five cancer cell lines. Table 5 shows that when an electron-withdrawing group -Br is introduced at the 4-position, compound 3da exhibits good antitumor activity against all five cancer cell lines: A549, MDA-MB-231, HCT116, HepG2, and DU145, with an IC50 value of [missing value]. 50 The values ​​were 20.48 μM, 17.63 μM, 7.498 μM, 11.29 μM, and 18.98 μM, respectively. When substituents of different properties are introduced at position 4 or other sites, the IC50 values ​​of most monosubstituted products are... 50 The value is greater than 50 μM, indicating that the introduction of substituents with different properties has a significant impact on the inhibitory effect on cancer cells. It can also be concluded that when the indole ring is substituted with a halogen, the compound exhibits certain biological activity. Furthermore, when an electron-withdrawing group -Br is introduced at the 7-position of the indole benzene ring, except for A549, 3pa inhibits the proliferation of the other four cell types.

[0168] Building upon research on the monosubstituted indole benzene ring, two chlorine atoms were simultaneously introduced onto the indole benzene ring, yielding compounds with dichlorinated substitutions (3sa, 3ta, 3ua). Based on this, the antitumor effects of the dihalogenated compounds were compared with those of the bromine-monosubstituted compound 3da. In general, the dichlorinated compounds 3sa and 3ua showed better inhibitory effects on cancer cell proliferation than the monosubstituted compounds 3da and 3pa. However, the IC50 of the compound 3ta, which simultaneously introduced chlorine atoms at positions 5 and 7, was significantly lower. 50 The values ​​are greater than 50 μM. This indicates that the cell viability of these five cancer cells is affected by substitutions at different positions of this type of compound. Moreover, the inhibitory effect on cancer cells is better when substituents are introduced at positions 4 and 7 than when substituents are introduced at other positions. It is speculated that the introduction of substituents at positions 4 and 7 increases the steric hindrance of the compound, thereby affecting its inhibitory effect on cancer cells.

[0169] Only four compounds—3da, 3pa, 3sa, and 3ua—showed good inhibitory effects on the proliferation of the five cancer cell types in this system. The remaining compounds did not show significant inhibitory effects on the cell proliferation of these cancer cells, indicating that these four compounds have a certain degree of universality against cancer cells. However, it was also found that more than four compounds inhibited HCT116 in colon cancer, suggesting that the inhibitory effects of the compounds on tumor cells have a certain degree of cell selectivity.

[0170] In a study investigating the effects of introducing substituents at different positions on the indole benzene ring on the activity of HCT116 in colon cancer, it was found that when electron-withdrawing groups -Cl or -Br were introduced at positions 4, 5, and 7, the compounds (3ca, 3da, 3ga, 3ha, 3oa, 3pa) all exhibited good inhibitory effects on cancer cells, with the obtained IC50 values... 50 The values ​​were all less than 50 μM. Compounds with -Cl or -Br introduced at the 6-position (3ma, 3na) showed less inhibitory activity against HCT116 colon cancer than those with substitutions at other positions. This indicates that introducing a halogen atom at the 6-position is not conducive to obtaining compounds with superior antitumor activity. Next, the inhibitory effect of compounds with electron-donating groups on HCT116 colon cancer was investigated. When the electron-donating group -Me was introduced at different positions on the indole benzene ring, compounds (3ea, 3ia) did not show cytotoxic activity against HCT116 colon cancer. However, when -OMe was introduced at the 5-position, 3ja showed a certain inhibitory effect, with an IC50 value of [missing value]. 50 The value was 19.68 μM, indicating that substituents of different properties have a significant impact on cytotoxicity.

[0171] This invention investigated the inhibitory effect of compounds with modified hydrocenline structures on HCT116 colon cancer. Even after elongating the carbon chain at the 5-position of hydrocenline, 3ab-3af did not exhibit significant cytotoxicity. However, after replacing the chain hydrocarbon with a cycloalkane, 3ak showed some inhibitory effect on cell proliferation, IC50. 50 The value is 33.77 μM, which is speculated to be related to steric resistance.

[0172] This invention primarily employs the MTT assay to study the in vitro antitumor activity of the obtained target indole-fused hydrogenated morpholino compounds. Cell absorbance (OD value) was measured, and based on the linear relationship between absorbance and cell viability, the measured OD values ​​can be processed, analyzed, and IC50 calculated using analytical software. 50 Value, and obtained through calculation of IC 50 In summary, the structure-activity relationships of the compounds show that, overall, the indole-fused hydrogenated oxalool compounds of the present invention exhibit better cell inhibitory activity against HCT 116 colon cancer.

[0173] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An indole-fused hydrogenated oxaloline compound, characterized in that, The structure is as follows: Where R is selected from Cl or Br, R 1 It is a C2 hydroxyalkyl group.

2. The indole-fused hydrogenated oxalool compound according to claim 1, characterized in that, Selected from the following compounds: 3da: 3sa: 3ua: 3pa: 。 3. A method for preparing an indole-fused hydrogenated oxalool compound according to any one of claims 1-2, characterized in that, The indole-fused hydrogenated oxalool compounds were synthesized in a highly polar solvent via the Stork enamine reaction and intramolecular acylation reaction using enamine ketones and amines as reaction substrates. The structural formula of the enaminoketone compound is as follows: R is selected from Cl or Br; The structural formula of the amine compound is as follows: R 1 It is a C2 hydroxyalkyl group.

4. The method for preparing indole-fused hydrogenated oxalool compounds according to claim 3, characterized in that, The molar ratio of the enamine ketone compound to the amine compound is 1:(1-10).

5. The method for preparing indole-fused hydrogenated oxalool compounds according to claim 3, characterized in that, The temperature for the Stork enamine reaction and intramolecular acylation reaction is 45-80℃.

6. The method for preparing indole-fused hydrogenated oxalool compounds according to claim 3, characterized in that, The highly polar solvent is selected from methanol, acetonitrile, and chloroform.

7. The use of an indole-fused hydrocyanoline compound as described in any one of claims 1-2 in the preparation of an antitumor drug, characterized in that, The antitumor drugs include drugs for the prevention and / or treatment of breast cancer, drugs for the prevention and / or treatment of liver cancer, and drugs for the prevention and / or treatment of prostate cancer.

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