Fluorescent derivatives of cyanine and methods of making and using the same

Abstract

The scheme discloses a series of corrole derivatives in the field of pharmaceutical chemistry and a preparation method and application thereof, and the structural general formula of the corrole derivatives is shown as formula (I) or formula (II) or formula (III) in the text. The fluorescent compound of the application adopts a typical corrole intermediate, takes malonitrile or ethyl cyanoacetate as an electron acceptor, and introduces an active electron-donating group to form a conjugated system of push-pull electrons. The structural system after the cyclization of ethyl cyanoacetate creates more additional regions, and provides feasibility for the construction of new structures. Most of the compounds show good binding affinity to Aβ 42 aggregates, and the low cytotoxicity is verified, and the aggregates can be successfully used for the imaging of Aβ in vivo and in vitro.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical chemistry, and particularly relates to fluorescent derivatives of the fluorin series and a preparation method and application thereof. BACKGROUND

[0002] Alzheimer's disease (AD) as a kind of insidious neurodegenerative disease has been widely present in the public view and caused widespread concern. The disease will lead to cognitive decline, behavioral disorders and other similar disorders in life ability, and will affect families 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 around 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, the resource waste and economic gap will deepen the social decline. At present, there is no effective cure, and most of the current treatments are single-target drugs, and the treatment options are relatively single and limited in slowing down the disease.

[0003] In the study of the pathological structure in the brain of AD patients, it is found that β-amyloid protein (Amyloid, Aβ) is the most significant pathological marker. The precursor of Aβ protein, β-amyloid precursor protein, is latent for 20 years before the onset of the disease, and then is accumulated in the cerebral cortex and hippocampus after enzymolysis, which destroys the neurons to the extent of dementia, mental confusion and emotional out of control, etc. A breakthrough point for the main pathogenesis is to accurately mark and remove Aβ without damage.

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

[0005] Monitoring Aβ is fraught with difficulties. Its concentration in vivo is extremely low, making it difficult to detect, and obtaining cerebrospinal fluid samples from patients is challenging, limiting its standardized clinical application. These limited clinical applications have spurred technological innovation. With the rapid development of molecular imaging, innovative techniques include magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), and optical imaging. The innovative emergence of near-infrared fluorescent probes highlights the advantages of optical imaging. Near-infrared fluorescent probes, with their unique non-invasiveness and high sensitivity, penetrate the blood-brain barrier and pinpoint unique pathological markers within the body, enabling optical imaging. This emerging approach simplifies previously complex and diverse biological conformations, providing a convenient visualization window for Aβ imaging with rapid methods and low cost. As research continues, near-infrared fluorescent probes hold promise for paving the way for Aβ labeling.

[0006] Therefore, developing near-infrared fluorescent imaging agents with specific binding affinity for Aβ aggregates for imaging at both in vivo and in vitro levels, enabling the tracing and detection of Aβ aggregates in vivo, and providing great convenience for early diagnosis and efficacy monitoring of AD patients, has significant scientific and application value. Summary of the Invention

[0007] This invention aims to provide a series of cyanine fluorescent derivatives, their preparation methods, and applications. The provided cyanine fluorescent derivatives can react with Aβ. 42 The aggregates exhibit good binding affinity and low cytotoxicity, and can be successfully used for imaging Aβ at both in vivo and in vitro levels.

[0008] In a first aspect, the cyanin-based fluorescent derivatives of this invention have the general structural formula shown in formula (I), formula (II), or formula (III):

[0009]

[0010] Where R1 is hydrogen or chlorine, and R2 is...

[0011] Secondly, the present invention also provides a method for preparing the cyanin-based fluorescent derivatives, comprising the following steps:

[0012] (1) The compound was dissolved in ethyl cyanoacetate in an organic solvent, and then reacted with piperidine as a catalyst under reflux to give the compound.

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

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

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

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

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

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

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

[0020] Thirdly, the application of the cyanin fluorescent derivatives described in this invention in the preparation of diagnostic and therapeutic agents for Aβ deposition and monoamine oxidase-B related diseases.

[0021] Furthermore, the relevant diseases refer to Alzheimer's disease or Parkinson's syndrome.

[0022] Fourthly, the present invention also provides a medicament for diagnosing Aβ deposition-related diseases, the medicament comprising the cyanin fluorescent derivatives described in the first aspect.

[0023] The beneficial technical effects of this invention are as follows: The cyanine fluorescent derivatives utilize typical cyanine intermediates, with malononitrile or ethyl cyanoacetate as electron acceptors, and introduce active electron-donating groups to form a push-pull conjugated system. The cyclized structure of ethyl cyanoacetate creates more addable regions, providing feasibility for constructing new structures. Most of these compounds are related to Aβ. 42 The aggregates exhibited good binding affinity and demonstrated low cytotoxicity, enabling successful imaging of Aβ levels both in vivo and in vitro. Attached Figure Description

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

[0025] Figures 10-18 These are the cytotoxic effects of nine compounds. Detailed Implementation

[0026] For parameters not specifically specified in the following embodiments, conventional techniques can be used as a reference.

[0027] Nuclear magnetic resonance (NMR) spectra were measured using a Bruker Avance III 400MHz (600MHz) NMR spectrometer with deuterated chloroform as the solvent.

[0028] I. Implementation Examples

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

[0030] The synthesis route is as follows:

[0031]

[0032] The preparation method includes the following steps:

[0033] (1) Synthetic intermediate 1

[0034] One equivalent of A3 and ten equivalents of ethyl cyanoacetate were added, with anhydrous ethanol as solvent and a small amount of piperidine as catalyst. The mixture was refluxed at 90 °C for 1 h. After cooling to room temperature, the mixture was filtered to give a blue solid. After purification by column chromatography, a green crystalline product was obtained in 65.4% yield.

[0035] (2) Synthesis intermediate 2

[0036] Replacing A3 with the same equivalent of L3, the synthesis method is the same as that of intermediate 1, with a yield of 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 1:5 ratio, with 5 equivalents of potassium carbonate (K2CO3) added as a catalyst. The reaction was carried out overnight at room temperature. After the reaction was completed, the solid base was removed by filtration to obtain a dark red solid in 32% yield. 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: A4-2, a fluorescent derivative of anthocyanins, and its preparation

[0040] The synthesis route is as follows:

[0041]

[0042] The preparation method differs from Example 1 in that: A3-B is replaced with the same equivalent of L3-B, and the synthesis method is the same as A4-1, yielding 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: Anthocyanin fluorescent derivative A4-3 and its preparation

[0044] The synthesis route is as follows:

[0045]

[0046] The preparation method differs from Example 1 in that A3-B is replaced with the same equivalent of A3-BW, and the synthesis method is the same as in A4-1, yielding 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: Anthocyanin-based fluorescent derivative Q4-1 and its preparation

[0048] The synthesis 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 at a ratio of 1:5. Five equivalents of potassium carbonate were added as a catalyst. The mixture was sealed and reacted overnight at 60°C. After the reaction was complete, the solid alkali was removed by filtration 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: Anthocyanin-based fluorescent derivative Q4-2 and its preparation

[0052] The synthesis route is as follows:

[0053]

[0054] The preparation method differs from that of Example 4 in that intermediate 1 is replaced with an equivalent amount of intermediate 2, and the synthesis method is the same as in Q4-1, yielding a bright red solid 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: Anthocyanin-based fluorescent derivative Q4-3 and its preparation

[0056] The synthesis route is as follows:

[0057]

[0058] The preparation method differs from that in Example 4 in that the same equivalent of n-propylamine is used instead of but-3-yn-1-amine. The synthesis method is the same as in Q4-1, yielding a bright red solid 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: Anthocyanin fluorescent derivative Q4-4 and its preparation

[0060] The synthesis route is as follows:

[0061]

[0062] The preparation method differs from that in Example 4 in that the same equivalent of n-butylamine is used instead of but-3-yn-1-amine. The synthesis method is the same as in Q4-1, yielding a bright red solid 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: Anthocyanin fluorescent derivative Q4-5 and its preparation

[0064] The synthesis route is as follows:

[0065]

[0066] The preparation method differs from that in Example 4 in that but-3-yn-1-amine is replaced with the same amount of ethanolamine, and the synthesis method is the same as in Q4-1, yielding a bright red solid 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: Anthocyanin fluorescent derivative Q4-6 and its preparation

[0068] The synthesis route is as follows:

[0069]

[0070] The preparation method differs from that in Example 4 in that: but-3-yn-1-amine is replaced with the same equivalent of 1-(2-aminoethyl)pyrrolidine, and the synthesis method is the same as in Q4-1, yielding a bright red solid in 48% yield. 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] II. The maximum absorption wavelength (λ) of the above nine cyanin fluorescent derivatives in PSB abs ) and maximum emission wavelength (λ) em )experiment

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

[0073] The fluorescence spectra of the anthocyanin-based fluorescent derivatives in PBS in this invention are shown in Table 1 and... Figures 1-9 .

[0074] Table 1. Maximum absorption and emission wavelengths of anthocyanin fluorescent derivatives in PBS

[0075] Fluorescent molecules ex ]]> ​ 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] III. Fluorescent molecules and Aβ 1-42 Emission wavelength and fluorescence enhancement factor after aggregate binding

[0077] In this embodiment, fluorescent molecules and Aβ 1-42 The emission wavelengths and fluorescence enhancement spectra of monomers, oligomers, and aggregates are shown in Table 2 and 2. Figures 1-9 .

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

[0079] Fluorescent molecules em ]]> ​ 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] As can be seen from the results in Table 2, the anthocyanin-based fluorescent derivatives of the present invention are similar to Aβ. 1-42 After the aggregates bind, a reaction occurs, resulting in enhanced fluorescence and a certain degree of blue shift.

[0081] IV. Cytotoxicity test of cyanide fluorescent derivatives

[0082] Experimental Methods: The cytotoxicity of nine cyanine fluorescent derivatives was tested using the standard MTT assay. Five gradient concentrations of probes were added to SH-SY5Y cells at sufficient growth density, and the cells were cultured at room temperature for 24 h, after which cell viability was measured. The cytotoxicity of the cyanine fluorescent derivatives is shown in the table below. Figures 10-18 ,from Figures 10-18 It can be seen that, except for A4-2, the other molecules have low cytotoxicity.

Claims

1. A cyanin-based fluorescent derivative, characterized in that... The general structural formulas of the cyanin fluorescent derivatives are shown in formula (I), (II), or (III): 、 、 Where R1 is hydrogen or chlorine, and R2 is... or or .

2. The method for preparing cyanine fluorescent derivatives according to claim 1, characterized in that... Includes the following steps: (1) or The compound was dissolved in ethyl cyanoacetate in an organic solvent, and then reacted with piperidine as a catalyst under reflux to give the compound. or ; (2) or or Dissolve in an organic solvent, add Potassium carbonate is used as a catalyst to react at room temperature to obtain compounds of formula (I) or formula (III); (3) or Dissolved in an organic solvent, one of but-3-yn-1-amine, n-propylamine, or n-butylamine is added, with potassium carbonate as a catalyst, and the reaction is carried out to obtain the compound in formula (II).

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

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

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

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

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

8. The application of the cyanin fluorescent derivatives according to claim 1 in the preparation of diagnostic and therapeutic agents for Aβ deposition and monoamine oxidase-B related diseases.

9. The application according to claim 8, characterized in that: The relevant diseases refer to Alzheimer's disease or Parkinson's syndrome.

10. A drug for diagnosing Aβ deposition-related diseases, characterized in that: Includes the cyanin fluorescent derivatives as described in claim 1.

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