Cyanine fluorescent derivative as well as preparation method and application thereof

Abstract

The invention discloses a series of cyanine derivatives in the field of medicinal chemistry, and a preparation method and application thereof. The structural general formula of the cyanine derivatives is shown as a formula (I) or a formula (II) or a formula (III) in the text. According to the fluorescent compound, a typical cyanine intermediate is adopted, malononitrile or ethyl cyanoacetate is taken as an electron acceptor, and an active electron-donating group is introduced, so that a conjugated system for pushing and pulling electrons is formed. More additional areas are created for a structural system after cyclization of ethyl cyanoacetate, and feasibility is provided for building of a new structure. Wherein most of the compounds show good binding affinity with the A beta 42 aggregate, low cytotoxicity is verified, and the compounds can be successfully used for in-vivo and in-vitro level imaging of A beta.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry, and particularly relates to cyanine fluorescent derivatives and preparation methods and applications thereof. Background Art

[0002] Alzheimer's disease (AD), as a hidden neurodegenerative disease, has been widely seen by the public and has attracted widespread attention. The onset of the disease will lead to a decline in patients' cognitive function, behavioral disorders and other similar disorders of living ability, which will affect the family and society in many ways. The disease is more common in people over 70 years old and is more prominent in an aging society. Most countries in the world are moving towards a huge aging trend, so the global existing patient base will double on the basis of 50 million. With the gradually rising prevalence rate, its waste of resources and economic gap will deepen the social decline. At present, there is a lack of effective radical treatment methods. Currently, most of them are single-target drug treatments, and the treatment plans are relatively simple and the degree of delay in the disease is limited.

[0003] In the study of the pathological structure of AD patients' brains, it was found that β-amyloid protein (Amyloid, Aβ) is the most significant pathological marker. The predecessor of Aβ protein, β-amyloid precursor protein, lurks in the patient's brain twenty years before the onset of the disease, and then accumulates in the cerebral cortex and hippocampus after enzymatic degradation, destroying neurons to death, leading to dementia, mental confusion and emotional disorder. A major breakthrough in this main pathogenesis lies in the precise labeling and harmless removal of Aβ.

[0004] More studies have found that complex diseases are not caused by a single pathological factor, but by multiple pathogenic mechanisms. Traditional targeted drugs face difficulties in treating diseases like AD. Multi-target drugs have more advantages in treating such diseases.

[0005] Aβ monitoring is difficult. Its concentration in the body is extremely low and difficult to detect. It is not easy to obtain cerebrospinal fluid samples from patients, and its standardized application in clinical practice is limited. The limited clinical application has given rise to technological reforms. With the vigorous development of molecular imaging, innovative technologies include magnetic resonance imaging (MRI), positron imaging (Positron), electron microscopy imaging (Positron) and electron microscopy imaging (Positron). Radiation tomography (PET), single photon emission computed tomography (SPECT) and optical imaging. The creative emergence of near-infrared fluorescent probes highlights the advantages of optical imaging. Near-infrared fluorescent probes penetrate the blood-brain barrier with their unique non-invasiveness and high sensitivity, and lock unique pathological markers in the body for imaging in optical form. This emerging method of exploration simplifies the previously complex and diverse biological conformations, and provides a convenient visualization window for Aβ imaging with quick means and low cost. With the continuous deepening of research, near-infrared fluorescent probes are expected to pave the way for labeling Aβ.

[0006] Therefore, developing near-infrared fluorescent imaging agents with specific binding affinity for Aβ aggregates for imaging of Aβ levels in vivo and in vitro, achieving tracing and detection of Aβ aggregates in vivo, will greatly facilitate early diagnosis and efficacy testing of AD patients, and has important scientific significance and application value. Summary of the invention

[0007] The present invention intends to provide a series of cyanine fluorescent derivatives and preparation methods and applications thereof. The provided cyanine fluorescent derivatives can react with Aβ 42 The aggregates showed good binding affinity, low cytotoxicity, and could be successfully used for imaging Aβ levels in vitro and in vivo.

[0008] In the first aspect, the cyanine fluorescent derivatives of the present invention have a general structural formula as shown in formula (I), formula (II), or formula (III):

[0009]

[0010] Wherein, R1 is hydrogen or chlorine, and R2 is

[0011] In a second aspect, the present invention also provides a method for preparing the cyanine fluorescent derivative, comprising the following steps:

[0012] (1) Dissolve it in an organic solvent with ethyl cyanoacetate, add piperidine as catalyst, and reflux to obtain the compound

[0013] (2) Dissolve in an organic solvent and add Potassium carbonate is used as a catalyst and reacts at room temperature to obtain a compound of formula (I) or formula (III);

[0014] (3) Dissolve in organic solvent and add or any group of R2, with potassium carbonate as a catalyst, to react to obtain the compound of formula (II).

[0015] Furthermore, the organic solvent in step (1) is anhydrous ethanol.

[0016] Furthermore, the temperature of the reflux reaction in step (1) is 90°C.

[0017] Furthermore, the organic solvent in step (2) is acetonitrile.

[0018] Furthermore, the organic solvent in step (3) is acetonitrile.

[0019] Furthermore, the reaction temperature in step (3) is 60°C.

[0020] In a third aspect, the cyanine fluorescent derivatives described in the present invention are used in the preparation of diagnostic and therapeutic agents for Aβ deposition and monoamine oxidase-B related diseases.

[0021] Furthermore, the related disease refers to Alzheimer's disease or Parkinson's syndrome.

[0022] In a fourth aspect, the present invention further provides a drug for diagnosing diseases related to Aβ deposition, wherein the drug comprises the cyanine fluorescent derivative described in the first aspect.

[0023] Beneficial technical effects of the present invention: The cyanine fluorescent derivatives adopt typical cyanine intermediates, use malononitrile or ethyl cyanoacetate as electron acceptors, and introduce active electron-donating groups to form a conjugated system that pushes and pulls electrons. The structural system after cyclization of ethyl cyanoacetate creates more attachable areas, providing feasibility for the construction of new structures. Most of the compounds are compatible with Aβ 42 The aggregates showed good binding affinity and verified low cytotoxicity and could be successfully used for imaging Aβ levels in vivo and in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figures 1 to 9 are the absorption and emission spectra of 9 compounds in PBS;

[0025] Figures 10 to 18 The cytotoxicity of the nine compounds were shown respectively. DETAILED DESCRIPTION

[0026] For parameters not specifically noted in the following embodiments, the conventional techniques can be used.

[0027] The NMR spectra were measured using an Avance III 400MHz (600MHz) NMR spectrometer from Bruker, Germany, with deuterated chloroform as solvent.

[0028] 1. Implementation

[0029] Example 1: Cyanine fluorescent derivative A4-1 and its preparation

[0030] The synthetic route is as follows:

[0031]

[0032] The preparation method comprises the following steps:

[0033] (1) Synthesis of intermediate 1

[0034] 1 equivalent of A3 and 10 equivalents of ethyl cyanoacetate were added, anhydrous ethanol was used as solvent, a small amount of piperidine was used as catalyst, and the reaction was refluxed at 90°C for 1 hour. After cooling to room temperature, a blue solid was obtained by suction filtration. After purification by column chromatography, a green crystalline product was obtained with a yield of 65.4%.

[0035] (2) Synthesis of intermediate 2

[0036] The same equivalent of L3 was used to replace A3. The synthesis method was the same as that of intermediate 1, and the yield was 69.5%.

[0037] (3) Synthesis of fluorescent derivative A4-1

[0038] A3-B and but-3-yn-1-amine were added to an appropriate amount of anhydrous acetonitrile in a ratio of 1:5, and 5 equivalents of potassium carbonate (K2CO3) were added as a catalyst. The reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the solid base was removed by suction to obtain a dark red solid with a yield of 32%. NMR data: 1 H NMR(600MHz, CDCl3)δ7.81(d,J=12.5Hz,1H),7.24(t,J=7.6Hz,2H),7.00–6.94(q,2 H),6.76(d,J=7.8Hz,1H),5.49(d,J=12.4Hz,1H),4.11(t,J=6.3Hz,2H),3.79(q,J= 7.3Hz,2H),3.76(s,1H),2.80(m,J=3.6Hz,2H),2.61–2.58(t,2H),2.36–2.33(t,2H ), 2.05 (t, J = 2.6Hz, 1H), 1.80 (m, J = 6.3Hz, 2H), 1.69 (s, 6H), 1.32 (t, J = 7.2Hz, 3H).

[0039] Example 2: Cyanine fluorescent derivative A4-2 and its preparation

[0040] The synthetic route is as follows:

[0041]

[0042] The difference between the preparation method and Example 1 is that the same equivalent of L3-B is used to replace A3-B, and the synthesis method is the same as A4-1 to obtain a dark red solid with a yield of 33%. NMR data: 1 H NMR (400MHz, CDCl3) δ7.74(d,J=12.6Hz,1H),7.28(s,1H),6.97(s,1H),6.64(d,J=8.5Hz,1H),5.48(d,J=12.5Hz,1H),4.12(t,J=6.2Hz,2 H),3.75(q,J=7.2Hz,2H),2.80(s,2H),2.58(s,2H),2.34(s,2H),2.04(s,1H),1.79(s,2H),1.66(s,6H),1.39(s,1H),1.31–1.27(t,3H).

[0043] Example 3: Cyanine fluorescent derivative A4-3 and its preparation

[0044] The synthetic route is as follows:

[0045]

[0046] The difference between the preparation method and Example 1 is that the same equivalent of A3-BW is used to replace A3-B, and the synthesis method is the same as A4-1 to obtain a dark red solid with a yield of 35%. NMR data: 1 H NMR (400MHz, CDCl3) δ8.69(d,J=12.8Hz,1H),7.34–7.29(m,2H),7.24(s,1H),7.13(t,J=7.4Hz,1H),6.90(d,J=8.0Hz,1H),5.47(d,J= 13.1Hz,1H),4.65(t,J=5.6Hz,2H),3.89(q,J=7.3Hz,2H),3.79(t,3H),3.51(s,4H),2.08(t,1H),1.75(s,6H),1.37(t,J=6.9Hz,3H).

[0047] Example 4: Cyanine fluorescent derivative Q4-1 and its preparation

[0048] The synthetic route is as follows:

[0049]

[0050] Preparation method: Intermediate 1 and but-3-yn-1-amine were added to an appropriate amount of anhydrous acetonitrile in a ratio of 1:5, and 5 equivalents of potassium carbonate were added as a catalyst. The mixture was sealed and reacted at 60°C overnight. After the reaction, the solid base was removed by suction to obtain a bright red solid with a yield of 51%. NMR data: 1 H NMR (400MHz, CDCl3) δ7.92 (d, J = 12.5Hz, 1H), 7.25 (s, 1H), 7.23 (s, 1H), 7.16 (s, 1H), 6.97 (t,J=7.4Hz,1H),6.76(d,J=9.3Hz,1H),5.51(d,J=12.5Hz,1H),4.05(t,J=6.4Hz,2H),3. 80(q,J=7.1Hz,2H),2.73(m,J=6.4,2.6Hz,2H),2.61(m,J=6.8,2.1Hz,2H),2.43(t,J=6.3 Hz, 2H), 2.05 (t, J = 2.7Hz, 1H), 1.83 (m, J = 6.7Hz, 2H), 1.68 (s, 6H), 1.31 (t, J = 7.2Hz, 3H).

[0051] Example 5: Cyanine fluorescent derivative Q4-2 and its preparation

[0052] The synthetic route is as follows:

[0053]

[0054] The difference between the preparation method and Example 4 is that the intermediate 1 is replaced by the same equivalent of the intermediate 2, and the synthesis method is the same as Q4-1, and a bright red solid is obtained with a yield of 53%. The NMR data are as follows: 1 H NMR (600MHz, CDCl3) δ7.86(d,J=14.6Hz,1H),7.17(s,2H),6.64(d,J=7.4Hz,1H),5.50(d,J=12.4Hz,1H),4.07–4.04(t,2H),3.76(q,J=7.1Hz,2H),2. 75–2.71(t,2H),2.61(t,J=6.8Hz,2H),2.45–2.42(t,2H),2.06(s,1H),1. 83(m,J=5.4Hz,2H),1.62(s,6H),1.44–1.38(m,1H),1.30(t,J=7.1Hz,3H).

[0055] Example 6: Cyanine fluorescent derivative Q4-3 and its preparation

[0056] The synthetic route is as follows:

[0057]

[0058] The difference between the preparation method and Example 4 is that the same equivalent of n-propylamine is used to replace but-3-yn-1-amine. The synthesis method is the same as Q4-1, and a bright red solid is obtained with a yield of 58%. The NMR data are as follows: 1 H NMR (400MHz, CDCl3) δ7.90 (d, J = 12.4Hz, 1H), 7.23 (t, J = 7.4Hz, 2H), 7.04 (s, 1H) ,6.97(t,J=7.4Hz,1H),6.75(d,J=8.4Hz,1H),5.50(d,J=12.5Hz,1H),3.91–3.8 6(t,2H),3.79(q,J=7.1Hz,2H),2.61(m,J=6.8,2.1Hz,2H),2.42(t,J=6.4Hz,2H ),1.87–1.78(m,4H),1.68(s,6H),1.31(t,J=7.2Hz,3H),0.99(t,J=7.4Hz,3H).

[0059] Example 7: Cyanine fluorescent derivative Q4-4 and its preparation

[0060] The synthetic route is as follows:

[0061]

[0062] The difference between the preparation method and Example 4 is that the same equivalent of n-butylamine is used to replace but-3-yn-1-amine. The synthesis method is the same as Q4-1, and a bright red solid is obtained with a yield of 55%. NMR data: 1 H NMR(400MHz, CDCl3)δ7.90(d,J=12.5Hz,1H),7.25–7.19(t,2H),7.04(s,1H),7.00– 6.94(t,1H),6.75(d,J=8.3Hz,1H),5.50(d,J=12.5Hz,1H),3.91(t,J=7.4Hz,2H),3 .79(q,J=7.2Hz,2H),2.61(m,J=6.8,2.0Hz,2H),2.42(t,J=6.3Hz,2H),1.84–1.80( m,2H),1.68(s,6H),1.46–1.37(m,4H),1.32(d,J=7.1Hz,3H),0.98(t,J=7.3Hz,3H).

[0063] Example 8: Cyanine fluorescent derivative Q4-5 and its preparation

[0064] The synthetic route is as follows:

[0065]

[0066] The difference between the preparation method and Example 4 is that the same equivalent of ethanolamine is used to replace but-3-yn-1-amine. The synthesis method is the same as Q4-1, and a bright red solid is obtained with a yield of 47%. NMR data: 1 H NMR(400MHz, CDCl3) δ7.93(d,J=12.5Hz,1H),7.24(t,J=7.3Hz,2H),7.01–6.9 6(t,1H),6.76(d,J=7.4Hz,1H),5.52(d,J=12.5Hz,1H),4.12(t,J=4.9Hz,2H) ,3.99(t,J=4.8Hz,2H),3.80(q,J=7.2Hz,2H),2.62(m,J=6.7,2.1Hz,2H),2.4 4(d,J=6.3Hz,2H),1.83(m,J=6.8Hz,4H),1.69(s,6H),1.33(t,J=7.3Hz,3H).

[0067] Example 9: Cyanine fluorescent derivative Q4-6 and its preparation

[0068] The synthetic route is as follows:

[0069]

[0070] The difference between the preparation method and Example 4 is that the same equivalent of 1-(2-aminoethyl)pyrrolidine is used to replace but-3-yn-1-amine. The synthesis method is the same as Q4-1, and a bright red solid is obtained with a yield of 48%. NMR data: 1 H NMR (400MHz, CDCl3) δ7.90(d,J=12.4Hz,1H),7.23(t,J=7.4Hz,2H),7.11(s,1H),6.97(t ,J=7.4Hz,1H),6.75(d,J=7.9Hz,1H),5.50(d,J=12.5Hz,1H),4.06(m,J=8.1Hz,3H),3.79 (q,J=7.1Hz,2H),3.67(m,J=4.5Hz,1H),2.85(t,J=6.8Hz,3H),2.61(s,J=2.4Hz,4H),2. 45–2.40(m,2H),1.68(s,6H),1.43(d,J=15.8Hz,2H),1.28(m,J=7.1Hz,4H),0.88(m,2H).

[0071] 2. The maximum absorption wavelength (λ abs ) and the maximum emission wavelength (λ em )experiment

[0072] Experimental method: 9 cyanine fluorescent derivatives were prepared into 10mM DMSO stock solution. 3μL of the above solution was taken and diluted to 3mL with PBS to obtain a 10μM solution. The maximum absorption wavelength and the maximum emission wavelength of the fluorescent derivatives were measured and recorded using an ultraviolet spectrophotometer (UV-2450, SHIMADZU) and a fluorescence spectrophotometer (F-4500, Hitachi).

[0073] The fluorescence spectra of the cyanine fluorescent derivatives in PBS of the present invention are shown in Table 1 and Figures 1 to 9 .

[0074] Table 1 Maximum absorption wavelength and emission wavelength of cyanine fluorescent derivatives in PBS

[0075] Fluorescent molecules <![CDATA[λ ex ]]> <![CDATA[λ em ]]> A4-1 568 623 A4-2 566 625 A4-3 567 617 Q4-1 535 614 Q4-2 537 614 Q4-3 521 613 Q4-4 537 610 Q4-5 521 616 Q4-6 532 618

[0076] 3. Fluorescent molecules and Aβ 1-42 Emission wavelength and fluorescence enhancement factor after aggregate binding

[0077] In this example, the fluorescent molecules and Aβ 1-42 The emission wavelengths and fluorescence enhancement spectra of monomers, oligomers, and aggregates after binding are shown in Tables 2 and Figures 1 to 9 .

[0078] Table 2. Fluorescent cyanine derivatives and Aβ 1-42 Emission wavelength and fluorescence enhancement spectrum after aggregate binding

[0079] Fluorescent molecules <![CDATA[λ em ]]> <![CDATA[λ em with Aβ]]> Fold A4-1 623 618 27 A4-2 625 620 15 A4-3 617 614 20 Q4-1 614 592 7 Q4-2 614 593 6 Q4-3 613 592 12 Q4-4 610 593 10 Q4-5 616 588 11 Q4-6 618 597 17

[0080] It can be seen from the results in Table 2 that the cyanine fluorescent derivatives of the present invention have a strong affinity with Aβ 1-42 When the aggregates bind, a reaction occurs, and the fluorescence is enhanced and accompanied by a certain amount of blue shift.

[0081] IV. Cytotoxicity test of fluorescent cyanine derivatives

[0082] Experimental method: The standard MTT method was used to test the cytotoxicity of 9 cyanine fluorescent derivatives. Five gradient concentrations of probes were added to SH-SY5Y cells with sufficient growth density, cultured at room temperature for 24 hours and their survival rate was measured. The cytotoxicity of the cyanine fluorescent derivatives is shown in Figures 10 to 18 ,from Figures 10 to 18 It can be seen that except A4-2, the other molecules have low cytotoxicity.

Claims

1. A cyanine fluorescent derivative, characterized in that The general structural formula of the cyanine fluorescent derivative is shown in formula (I), formula (II) or formula (III): Wherein, R1 is hydrogen or chlorine, and R2 is 2. The method for preparing a cyanine fluorescent derivative according to claim 1, characterized in that The following steps are involved: (1) Dissolve it in an organic solvent with ethyl cyanoacetate, add piperidine as catalyst, and reflux to obtain the compound (2) Dissolve in an organic solvent and add Potassium carbonate is used as a catalyst and reacts at room temperature to obtain a compound of formula (I) or formula (III); (3) Dissolve in organic solvent and add Or one of the R2 groups described in claim 1, potassium carbonate as a catalyst, react to obtain a compound of formula (II).

3. The preparation method according to claim 2, characterized in that: The organic solvent in step (1) is anhydrous ethanol.

4. The preparation method according to claim 2, characterized in that: The temperature of the reflux reaction in step (1) is 90°C.

5. The preparation method according to claim 2, characterized in that: The organic solvent in step (2) is acetonitrile.

6. The preparation method according to claim 2, characterized in that: The organic solvent in step (3) is acetonitrile.

7. The preparation method according to claim 2, characterized in that: The reaction temperature in step (3) is 60°C.

8. Use of the cyanine fluorescent derivative according to claim 1 in the preparation of diagnostic and therapeutic agents for Aβ deposition and monoamine oxidase-B related diseases.

9. The use according to claim 8, characterized in that: The related disease refers to Alzheimer's disease or Parkinson's syndrome.

10. A drug for diagnosing diseases related to Aβ deposition, characterized in that: It includes the cyanine fluorescent derivative described in claim 1.

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