Folate receptor-targeted small molecule compound, preparation method, composition and application of folate receptor-targeted small molecule compound

Abstract

The invention provides a folate receptor targeting small molecule compound, a preparation method, a composition and application. Specifically provided is a compound represented by formula I or a pharmaceutically acceptable salt thereof. The compound or the pharmaceutically acceptable salt thereof provided by the invention can be used as a small-molecule targeting fluorescence contrast agent for assisting surgical treatment, has the advantages of novel structure, good solubility, small steric hindrance and strong targeting property, can obviously improve the view field of tumor surgery, and is beneficial to efficiently and accurately implementing excision of pathological tissues such as tumor tissues, boundary tissues and the like. # imgabs0 #

Description

[0001] This application claims the priority of Chinese Patent Application No. 2023110325244 filed on August 16, 2023. This application cites the entire text of the above Chinese Patent Application. Technical Field

[0002] The present invention relates to a technology for detecting and evaluating functional activity on tumors, and in particular to a small molecule compound targeting folate receptors, a preparation method, a composition and an application thereof. Background Art

[0003] Folate receptor alpha (FR-α), also known as FOLR1 or folate binding protein, is a glycoprotein anchored to the cell membrane by glycosylated phosphatidylinositol (GPI). It has a high affinity for folate and can transport folate through receptor-mediated endocytosis. FRα acts as a signaling molecule to promote tumor malignant growth. Similar to other glycosylated phosphatidylinositol family proteins, FRα activates the intracellular regulatory signaling network after binding to folate. FRɑ is mainly highly expressed in epithelial tumor tissues, not expressed or expressed at very low levels in normal tissues, has a high affinity for folate, and a low affinity for reduced folate.

[0004] FRα is known to be overexpressed on the surface of tumor cells in various cancers, including ovarian cancer, TNBC (triple negative breast cancer), endometrial cancer, mesothelioma and lung cancer. FRα is expressed at low levels in normal cells, but its expression level is significantly increased in certain cancer cells, so it has become an important target in cancer diagnosis and treatment in recent years.

[0005] Near-infrared fluorescence imaging technology, intraoperative near-infrared (NIR) fluorescence imaging helps surgeons make judgments and reduce the positive rate of surgical margins, and has become an effective solution for detecting intraoperative tumor margins. Fluorescence imaging intraoperative navigation is based on the enrichment of fluorescein in specific tissues, which can accurately display intraoperative blood vessels, lymph nodes, tumor tissues and other information. At the same time, it can specifically visualize specific molecular targets overexpressed in tumor tissues, thereby achieving the effect of high signal-to-noise ratio tumor imaging, further reducing the positive rate of surgical margins. Near-infrared light has a wavelength range of 650nm to 900nm, and because its penetration depth in tissues can reach 10mm, it is superior to visible light in intraoperative imaging. Since tissues only show limited autofluorescence in the NIR spectrum, the use of NIR fluorescent imaging agents can maximize the contrast between fluorescent signals in tumors and healthy tissues. In addition, near-infrared light does not affect the surgical field of view because near-infrared light is invisible to the human eye. Currently available imaging systems combine white light illumination of the surgical area with NIR fluorescence images to provide surgeons with anatomical information and fluorescence information at the same time.

[0006] The prior art compound OTL38 has been applied in tumor treatment. However, due to its strong structural conjugation, the binding of the molecule to the target protein is hindered.

[0007] Therefore, it is particularly important to develop compounds that have strong targeting, strong tumor detection capabilities, and are more suitable for clinical applications. Summary of the invention

[0008] The purpose of the present invention is to provide a small molecule compound targeting folate receptors, a preparation method, a composition and an application thereof. As a small molecule targeted fluorescent contrast agent that can effectively assist surgical treatment, it has a novel structure and strong targeting, and can significantly improve the field of view of diseased tissues in treatment activities such as tumor surgery, and help to identify diseased tissues and normal tissues efficiently and with high quality, thereby facilitating the conduct of surgical treatment.

[0009] The present invention solves the above technical problems through the following solutions.

[0010] The present invention provides a compound represented by formula I or a pharmaceutically acceptable salt thereof;

[0011]

[0012] in,

[0013] R is

[0014] In one scheme, R is For example

[0015] In one embodiment, the compound represented by formula I is any of the following compounds:

[0016]

[0017] In one embodiment, the pharmaceutically acceptable salt of the compound represented by Formula I contains a pharmaceutically acceptable cation, the number of cations may be 1, 2, 3 or 4, and the cation may be K + 、Na + , Li + or NH4 + , for example, the tetrasodium salt of the compound represented by formula I.

[0018] In one embodiment, the compound represented by Formula I or a pharmaceutically acceptable salt thereof is a small molecule compound targeting the folate receptor.

[0019] In one embodiment, the pharmaceutically acceptable salt of the compound represented by formula I is the compound represented by formula II;

[0020]

[0021] Among them, M 1 、M 2 、M 3 and M 4 Each is independently H or a pharmaceutically acceptable cation;

[0022] R is as described in any embodiment of the present invention.

[0023] In one embodiment, the pharmaceutically acceptable cation is a monovalent cation, such as K + 、Na + or Li + (Preferably Na + ).

[0024] To achieve the above object, an embodiment of the present invention provides a small molecule compound targeting folate receptor:

[0025] Wherein M is a monovalent cation, and R is selected from

[0026]

[0027] In the formula Refers to the covalent bond position of the R group.

[0028] In one or more embodiments of the present invention, the monovalent cation is selected from potassium ions, sodium ions, lithium ions (eg, sodium ions).

[0029] In one or more embodiments of the present invention, the small molecule compound targeting the folate receptor is

[0030] Wherein M is potassium ion, sodium ion, lithium ion (such as sodium ion).

[0031] In one embodiment, the compound represented by formula II is

[0032]

[0033] Among them, M 1 、M 2 、M 3 and M 4 Independently for K + 、Na + or Li + (e.g. Na + ).

[0034] In one embodiment, the compound represented by formula II is

[0035]

[0036]

[0037] The present invention provides a method for preparing the above-mentioned small molecule compound targeting folate receptor, the compound represented by the above-mentioned formula I or a pharmaceutically acceptable salt thereof, characterized in that it comprises the following steps:

[0038] Prepare the first intermediate, the structural formula is

[0039] The first intermediate reacts with the first compound to obtain the target compound. The first compound is Among them, M 2 、M 3 、M 4 Each independently represents H or a pharmaceutically acceptable cation (eg, a monovalent cation), and R is selected from

[0040]

[0041] In one or more embodiments of the present invention, the method for preparing a small molecule compound targeting folate receptor is characterized by comprising the following steps:

[0042] Prepare the first intermediate, the structural formula is

[0043] The first intermediate reacts with the first compound to obtain the target compound. The first compound is Wherein M is a monovalent cation, and R is selected from

[0044] In one or more embodiments of the present invention, the reaction of the first intermediate and the first compound is carried out in a carbonate aqueous solution. Preferably, the concentration of the carbonate aqueous solution is 3-10 wt.%. Preferably, the carbonate aqueous solution is a 3.35 wt.% sodium carbonate solution.

[0045] In one or more embodiments of the present invention, the carbonate is selected from sodium carbonate and potassium carbonate.

[0046] In one or more embodiments of the present invention, the reaction conditions are: 70-75°C.

[0047] In one embodiment, the reaction conditions are: 70-75°C, reaction time 1-2h.

[0048] The present invention also provides a compound represented by formula V or a pharmaceutically acceptable salt thereof;

[0049]

[0050] Wherein, R is defined as described in any embodiment of the present invention.

[0051] Preferably, the compound of formula V is any of the following compounds:

[0052]

[0053] The present invention also provides a pharmaceutical composition, comprising the aforementioned small molecule compound targeting folate receptors. Preferably, the active ingredient of the pharmaceutical composition is selected from a small molecule compound targeting folate receptors or a pharmaceutically acceptable salt thereof, a conjugate thereof and a pharmaceutically acceptable salt thereof. The composition can be in a dosage form suitable for intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous and intrathecal administration.

[0054] The present invention also provides a pharmaceutical composition, which comprises the compound of formula I or a pharmaceutically acceptable salt thereof, the small molecule compound targeting folate receptors and a pharmaceutical excipient. Preferably, the pharmaceutically acceptable salt is a compound of formula II. Preferably, the compound of formula I or a pharmaceutically acceptable salt thereof and the small molecule compound targeting folate receptors are active ingredients.

[0055] In one or more embodiments of the present invention, the application includes the application of the aforementioned small molecule compound targeting folate receptor or the aforementioned composition in a targeting agent targeting cancer tissue. The agent includes a detection agent and a therapeutic agent.

[0056] In one or more embodiments of the present invention, the cancer tissue is a cancer tissue expressing folate receptors, which may include parenchymal tissue, interstitial tissue or marginal tissue, such as lung cancer, breast cancer, ovarian cancer and other cancer tissues expressing folate receptors.

[0057] The present invention provides a use of the compound represented by the above formula I or a pharmaceutically acceptable salt thereof, and the above small molecule compound targeting folate receptors in the preparation of a drug for treating or diagnosing cancerous tissues. Preferably, the drug is an imaging agent (eg, a fluorescent contrast agent).

[0058] The cancer tissue may be a cancer tissue expressing folate receptor, and may include parenchymal tissue or interstitial tissue or marginal tissue, such as lung cancer, breast cancer or ovarian cancer.

[0059] Terminology explanation:

[0060] The term "pharmaceutically acceptable" means relatively non-toxic, safe, and suitable for use by patients.

[0061] The term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable acid or base. When the compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. For details, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl, Camille G. Wermuth, 2011, 2nd Revised Edition).

[0062] The term "pharmaceutical excipients" refers to excipients and additives used in the production of drugs and the preparation of prescriptions. It is all substances contained in drug preparations except active ingredients. For details, please refer to the Pharmacopoeia of the People's Republic of China (2020 edition) or Handbook of Pharmaceutical Excipients (Raymond C Rowe, 2009).

[0063] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0064] The reagents and raw materials used in the present invention are commercially available.

[0065] The positive and progressive effects of the present invention are: the composition provided by the present invention has one or more of the following advantages:

[0066] (1) Good targeting, which helps to mark diseased tissues and identify tiny lesions and hidden lesions;

[0067] (2) It overcomes the disadvantage of fast clearance in cancer tissue. Generally, the half-life of ICG is 3-4 minutes. The short half-life leads to multiple perfusions during surgery, which seriously affects the quality of surgery and prolongs the operation time. The compound of the present invention can still detect fluorescence 24 hours after intravenous injection;

[0068] (3) Improved fluorescence quantum yield. The fluorescence quantum yield of ICG is usually between 0.01 and 0.1. Due to the low fluorescence quantum yield of ICG, a higher dose is required to maintain effective fluorescence intensity. The compound of the present invention is expected to provide an effective tumor-to-background ratio (TBR) at 0.005 mg / kg in clinical practice.

[0069] (4) Compared with OTL38, the compound of the present invention has more excellent pharmacokinetic properties, faster elimination rate in the blood, lower background, higher tumor background ratio (TBR), and can more clearly distinguish tumors from normal tissues.

[0070] (5) Based on the animal performance of the compound of the present invention and OTL38 at the same dose (the tumor-to-background ratio was better than that of OTL38 at each dose), it is expected that the compound of the present invention can provide the same or even better diseased tissue differentiation ability as OTL38 at a lower dose in clinical practice.

[0071] (6) The novel structure, good solubility, and small steric hindrance enable long-term enrichment of diseased tissues at low concentrations. The dynamic enrichment and elimination of diseased tissues and the elimination of background produce a significant contrast, and the diseased tissues are highlighted. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 This is the near-infrared imaging image after compound 1 and OTL38 were administered at 1 nmol / mouse for 1 hour.

[0073] Figure 2 This is the near-infrared imaging image after compound 1 and OTL38 were administered at 1 nmol / mouse for 2 hours.

[0074] Figure 3 This is the near-infrared imaging image after compound 1 and OTL38 were administered at 1 nmol / mouse for 4 hours.

[0075] Figure 4 This is the near-infrared imaging image after compound 1 and OTL38 were administered at 1 nmol / mouse for 8 hours.

[0076] Figure 5 This is the near-infrared imaging image after compound 1 and OTL38 were administered at 1 nmol / mouse for 24 hours.

[0077] Figure 6 This is the near-infrared imaging image after compound 1 and OTL38 were administered at 10 nmol / mouse for 1 hour.

[0078] Figure 7 This is the near-infrared imaging image after compound 1 and OTL38 were administered at 10 nmol / mouse for 2 hours.

[0079] Figure 8 This is the near-infrared imaging image after compound 1 and OTL38 were administered at 10 nmol / mouse for 4 hours.

[0080] Fig. 9 This is the near-infrared imaging image after compound 1 and OTL38 were administered at 10 nmol / mouse for 8 hours.

[0081] Fig.10 This is the near-infrared imaging image after compound 1 and OTL38 were administered at 10 nmol / mouse for 24 hours.

[0082] Fig.11This is the near-infrared imaging of the organs of mice in the blank control group.

[0083] Fig.12 The near-infrared imaging images of mouse organs after administration of compound 1 at 1 nmol / mouse.

[0084] Fig.13 The near-infrared imaging images of mouse organs after administration of compound 1 at 10 nmol / mouse.

[0085] Fig.14 These are near-infrared images of mouse organs after administration of 1 nmol / mouse of OTL38.

[0086] Fig.15 These are near-infrared images of mouse organs after administration of 10 nmol / mouse OTL38. DETAILED DESCRIPTION

[0087] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0088]

[0089]

[0090]

[0091] Including but not limited to the following embodiments, including but not limited to the following embodiments: Refers to the carrier that does not participate in the reaction eq refers to the molar ratio. "V" means volume / mass ratio, 7V means that when the amount of chlorine resin is The amount of reagent corresponding to 1g is 7mL.

[0092] Example 1 Compound 1

[0093] Here M is Na + For example, for Li + , K + The situation can be realized in the same way.

[0094] Synthetic route

[0095] Step 1: B01 synthesis

[0096]

[0097] Weigh pteroic acid (600 mg) and Fmoc-ethylenediamine hydrochloride (643 mg), add to a 250 mL single-mouth bottle, add DMSO (24 mL) to dissolve; add HTAU (1.04 g) and DIEA (746 mg), stir for 2 h, add the reaction solution to MTBE after the reaction is completed, centrifuge, and dry to obtain B01 (1.1 g, yield 99%). LCMS: [M+H] + =577.2,2.78min.

[0098] Step 2: B02 Synthesis

[0099]

[0100] Weigh B01 (1.0 g) and add it into a 50 mL single-necked bottle, then add DMSO (20 mL) to dissolve it; add DBU (268 mg) and stir for 1 h. After the reaction is completed, add the reaction solution into MTBE, centrifuge and dry to obtain B02 (754 mg crude product, yield calculated as 100%).

[0101] Step 3: B03 Synthesis

[0102]

[0103] H-Tyr(tBu)-OtBu.HCl (490 mg) was weighed, and after being freed, it was added to a 50 mL single-mouth bottle. DMAP (7.2 mg) and DCM (10 mL) were added, and the temperature was lowered to 0-5°C under nitrogen protection; CDI (485 mg) was added, and the reaction was completed by HPLC monitoring, and 5 mL of water was added. The temperature was raised to 25°C and stirred for 1 h. The DCM layer was separated and concentrated; B02 (628 mg) and TEA (376 mg) were weighed and added to a 50 mL single-mouth bottle, DMSO (10 mL) was added to dissolve, and the above was added at 0-5°C to concentrate to obtain the intermediate, and stirred at 25°C overnight. The reaction solution was added dropwise to MTBE and centrifuged to dry to obtain B03 (809 mg, yield of about 85%).

[0104] LCMS: [M+H] + =674.4,2.92min.

[0105] Step 4: B04 Synthesis

[0106]

[0107] Weigh B03 (809 mg) and add it to a 50 mL single-mouth bottle, add 10 mL (90% TFA, 5% water, 5% triethylsilane), stir at 25°C, monitor the reaction by HPLC, and obtain B04 (403 mg, yield about 60%). LCMS [M+H] +=562.3,1.42min.

[0108] Preparation and purification of B04

[0109]

[0110] Step 5: Synthesis of compound 1

[0111]

[0112] Weigh B04 (100 mg, 0.19 mmol) and add it to a 25 ml single-mouth bottle. Prepare a sodium carbonate aqueous solution (sodium carbonate: 134 mg, 1.26 mmol, purified water: 4 ml) and add it to the reaction bottle. Add int1 (237 mg, 0.27 mmol) and heat to 70 ～ 75℃, maintain the temperature for one hour.

[0113] Post-processing: Cool down to 20 ～ 25°C, prepared, freeze-dried to obtain 50 mg of blue solid compound 1, yield 19%, purity 96.30%), MS: [M-4Na+4H+2H] 2+ =707.3,

[0114] 1 H NMR (400MHz, DMSO-d6 / D2O) δ8.63(s,1H),8.06(s,2H),7.78(d,J=14.0Hz,4H),7.65–7.58(m,8H),7.31(d,J=8.4 Hz,3H),7.18(d,J=8.5Hz,3H),7.00(d,J=8.5Hz,3H),6.91(t,J=6.1Hz,1H),6.61(d,J=8.7Hz,2H),6.28(s,1H),6 .19(d,J=14.2Hz,2H),6.01(d,J=7.0Hz,1H),4.46(d,J=5.9Hz,2H),4.25–4.04(m,6H),3.16–2.90(m,8H),2.78( dd,J=13.5,7.3Hz,1H),2.69(s,4H),1.90(s,3H),1.74(d,J=9.1Hz,10H),1.26(s,6H),1.23(s,2H),1.22(s,6H).

[0115] Preparation and purification of compound 1

[0116]

[0117]

[0118] Similarly, by using different raw materials, such as replacing the "Fmoc-ethylenediamine hydrochloride" in step 1 in Example 1 with "Fmoc-propylenediamine hydrochloride", "Fmoc-butylenediamine hydrochloride", or "Fmoc-pentanediamine hydrochloride", Compound 6, Compound 7, and Compound 8 can be obtained by the method of this embodiment. The specific process is described above and will not be repeated here.

[0119] Example 2

[0120] Preparation of compound 2

[0121] Here M is Na + For example, for Li + , K + The situation can be realized in the same way.

[0122] Synthesis route step 1: C03 solid phase synthesis

[0123]

[0124]

[0125]

[0126] Note: eq is the molar amount of chlororesin, V is the volume / mass ratio (mL / g), for example, 7V means that when the amount of chlororesin is 1g, the amount of the corresponding reagent is 7mL.

[0127] Step 2: Synthesis of compound 2

[0128]

[0129] Weigh CO3 (100 mg, 0.156 mmol) and add it to a 25 ml single-mouth bottle. Prepare a sodium carbonate aqueous solution (sodium carbonate: 100 mg, 0.946 mmol, purified water: 4 ml) and add it to the reaction bottle. Add int1 (152 mg, 0.156 mmol) and heat to 70 ～ 75℃, maintain the temperature for one hour.

[0130] Post-processing: Cool down to 20 ～ 25°C, prepared, freeze-dried to obtain 84 mg of blue solid compound 2, yield 23%, purity 97.65%, MS: [M-4Na+4H+2H] 2+ = =743.9,

[0131] 1H NMR (400MHz, DMSO-d6 / D2O) δ8.60(s,1H),7.77(d,J=13.8Hz,2H),7.59(dd,J=18.6,7.8Hz,6H),7.28(d,J=8.8Hz, 2H),7.18(d,J=8.4Hz,2H),6.97(d,J=8.4Hz,2H),6.59(d,J=8.7Hz,2H),6.14(d,J=14.2Hz,2H),4.45(s,2H),4.18 (s,2H),4.05(s,5H),3.54–3.38(m,6H),3.38–3.33(m,2H),3.30(d,J=5.3Hz,2H),2.95(s,1H),2.77(s,1H),2.64 (s,4H),2.56(t,J=6.7Hz,4H),2.20(d,J=5.8Hz,2H),1.86(s,2H),1.74(d,J=19.5Hz,9H),1.23(d,J=2.6Hz,12H).

[0132] Preparation and purification of compound 2

[0133]

[0134]

[0135] Similarly, by using different raw materials, such as replacing the "Fmoc-PEG2-CH2CH2COOH" in step 1 in Example 2 with "Fmoc-PEG3-CH2CH2COOH", "Fmoc-PEG4-CH2CH2COOH", "Fmoc-PEG5-CH2CH2COOH", you can obtain compound 3, compound 4 and compound 5 by the method of this example. The specific process is described above and will not be repeated here.

[0136] Effect test example 1

[0137] 1. In vivo activity imaging of compound 1 (compared with OTL38, CAS: %1628858-03-6): lung cancer model

[0138] A subcutaneous HCC827 (human lung cancer cell line ATCC_CRL-2868) lung cancer tumor-bearing mouse model was established in BALB / c nude mice (athymic nude mice), and the average tumor volume reached about 300-400mm 3The group dosing began at 1:00 p.m. The mice in the drug-treated group were divided into 4 groups, with 3 mice in each group; there were 3 mice in the blank control group. Mice bearing HCC827 tumor xenografts were intravenously injected with 1 and 10 nmol / mouse of compound 1 and OTL38 (5% glucose solution, the same below), and the blank control group was injected with 5% glucose solution (see Table 1 below). Then, whole-body imaging studies were performed using a small animal in vivo imaging system (IVIS Lumina LT serier III). The imaging equipment contains a near-infrared fluorescence imaging system. Imaging was performed at 1h, 2h, 4h, 8h, and 24h (the blank control group was imaged at 1h and 24h), and the tumor and background parts were circled with the same diameter to obtain the fluorescence value, and the tumor-to-background ratio (TBR) was calculated. The average value was calculated. The larger the value, the more it tends to provide a clearer tumor boundary, which is expected to provide a better surgical field of view during surgery. For specific results, see Figure 1-10 (The left image in the figure is the test data of compound 1, and the right image is the test data of OTL38. Radiant Efficiency is the radiation efficiency (p / sec / cm 2 / sr) / (μw / cm 2 ) and Table 2.

[0139] Table 1 Grouping of in vivo imaging

[0140]

[0141] In the four drug-treated groups, there was some fluorescence enrichment in the tumor part at 1 hour, and the tumor and non-tumor parts could be roughly distinguished from the image. The boundaries became obvious at subsequent time points and continued until the last time point of the experiment, 24 hours.

[0142] All four dosing groups can provide a certain tumor-to-background ratio at 1 hour and later (except for the 1 hour point of the OTL38 1nmol / group). In terms of compound dimension, the TBR value of compound 1 at each time point in both doses is better than that of OTL38; at different dose levels, the compound 1 10nmol / group is better than the 1nmol / group at each time point, and there is no difference in the tumor-to-background ratio (TBR) value between the OTL38 1nmol / group and the 10nmol / group, and the imaging boundary is not clearer due to the increase in dose. Compound 1 is expected to provide better tumor background data in subsequent dose optimization studies.

[0143] The results showed that compound 1 combined with a near-infrared fluorescence imaging system could distinguish tumors from background in mice in as fast as 1 hour. The TBR peaks of 1 nmol / mouse and 10 nmol / mouse were at 8 hours and 24 hours, respectively, and its performance in mice was significantly better than that of OTL38.

[0144] Table 2 Comparison of tumor-to-background ratio data of OTL38 and compound 1 in vivo imaging

[0145]

[0146] 2. In vitro tissue imaging-tissue distribution research

[0147] A subcutaneous HCC827 (human lung cancer cell line) lung cancer tumor-bearing mouse model was established in BALB / c nude (athymic nude mice), and the average tumor volume reached about 300-400mm 3 The mice in the drug-treated group were divided into 4 groups, with 3 mice in each group, and 3 mice in the blank control group (the grouping was the same as the above-mentioned in vivo live imaging study). Mice bearing HCC827 tumor xenografts were intravenously injected with 1 and 10 nmol / mouse compound 1 and OTL38 (5% glucose solution, the same below), and the blank control group was injected with 5% glucose solution. Then, whole-body imaging studies were performed using a small animal live imaging system. The imaging equipment contained a near-infrared fluorescence imaging system. Imaging was performed at 1h, 2h, 4h, 8h, and 24h (the blank control group was imaged at 1h and 24h). After 24h of live fluorescence imaging, the mice were euthanized, and the organs of the mice (tumors, heart, liver, spleen, lungs, kidneys, stomach, muscles, skin, ovaries, pancreas, colon, and small intestine) were collected for measurement by the live imaging system. The results of the study are shown in Figure 11-15 At the same time, the fluorescence ratios of mouse tumors to muscle tissue, ovarian tissue, and lung tissue were calculated, see Table 3 below.

[0148] The results showed that at the 24h time point, in the 1nmol / group, the fluorescence of compound 1 and OTL38 was not fully displayed in the tumor site, but due to the low uptake in other tissues, this dose group showed a more obvious distinction between tumor / muscle and tumor / lung (see Table 3 for details); in the 10nmol / group, both compound 1 and OTL38 could be observed to have obvious fluorescence in the tumor, and this dose group showed a better distinction between tumor / muscle and tumor / lung than the 1nmol / group (see Table 3 for details), and is expected to distinguish positive resection margins in clinical use.

[0149] Obvious fluorescence was observed in the kidney, which may be related to drug excretion through urine. A small amount of fluorescence was also observed in the healthy lung, liver, stomach, and ovary in the 10 nmol group, which may be related to the fact that FRα is also slightly expressed in these tissues. During data processing, since ovarian cancer also overexpresses FRα, the tumor / ovary comparison values ​​were calculated. The 10 nmol / group of the two compounds was significantly different from the control group, indicating the application value of the two compounds in ovarian cancer.

[0150] Table 3 Fluorescence ratio of subcutaneous tumor to normal tissue in dissected tissues 24 hours after tumor-bearing mice were injected with 1 and 10 nmol / mouse compound 1 and OTL38

[0151] Grouping Tumor / Muscle Tumor / lung Tumor / ovarian Blank control group 3.51 1.48 2.18 OTL38 1nmol / each 7.04 1.60 2.78 OTL38 10nmol / each 19.21 2.80 5.43 Compound 1 1nmol / 6.64 2.09 2.89 Compound 1 10nmol / mouse 27.80 5.01 5.60 .

[0152] Effect test example 2

[0153] In vivo live imaging studies

[0154] HCC827 (human lung cancer cell line, Jicui Yaokang) and A549 (folate receptor α-negative cell line, Jicui Yaokang) lung cancer subcutaneous tumor-bearing mouse models were constructed on BALB / c nude (athymic nude mice), and the average tumor volume reached about 300-400mm 3 The group dosing began at 1h (grouping is shown in Table 4 below). Mice bearing tumor xenografts were intravenously injected with 0.025 and 0.05 mg / kg of compound 1, compound 2 and OTL38 (5% glucose solution, the same below), and the blank control group was injected with 5% glucose solution. Then, whole-body imaging studies were performed using a small animal in vivo imaging system. The imaging equipment contained a near-infrared fluorescence imaging system. Imaging was performed at 1h, 2h, 4h, 8h, and 24h, respectively. The tumor and background parts were circled with the same diameter, the fluorescence value was obtained, and the ratio of tumor to background was calculated. The larger the value, the more it tends to provide a clearer tumor boundary, which is expected to provide a better surgical field during surgery. The specific results are shown in Table 5 below.

[0155] Table 4 Grouping of in vivo imaging

[0156]

[0157] In the 6 drug-treated groups, the tumor part had some fluorescence enrichment at 1 hour, and the tumor and No The boundaries of the tumor site became more obvious at subsequent time points and lasted until the last time point of the experiment, 24 h.

[0158] All six dosing groups can provide a certain tumor-to-background ratio (TBR) at 1 hour and thereafter. At different dose levels, there is no significant difference in TBR values ​​at each time point for OTL38, compound 1 and compound 2 0.025mg / kg and 0.05mg / kg dose groups. The 0.05mg / kg dose groups of compound 1 and compound 2 are superior to their low dose groups, suggesting that: 1) increasing the dose is expected to increase the tumor-to-background ratio of compound 1 and compound 2; 2) the difference in the 2-fold dose interval is not significant, and the dose interval needs to be increased in the future to investigate the dose-relatedness. Compound 1 and compound 2 are expected to provide better tumor background data in subsequent dose optimization studies. In terms of compound dimensions, compound 1 has a clear advantage in tumor background ratio, followed by compound 2, and OTL38 has the lowest, suggesting that the structural changes of compound 1 and compound 2 have brought about changes in pharmacokinetic properties. This conclusion is also reflected in the background fluorescence values ​​of the three compounds. The specific data are shown in Table 6: At the same dose level, the background fluorescence value is: OTL38>Compound 1>Compound 2. Part of the background fluorescence comes from the blood vessels throughout the tissue, suggesting that the elimination rate in the blood should be: OTL38<Compound 1<Compound 2. It can be seen that the superior tumor background ratio of compound 1 and compound 2 should come from a certain tumor accumulation and lower background interference, which also widens the gap with OTL38. Good pharmacokinetic properties are not easy to obtain, and it is even more difficult to find changes in pharmacokinetic properties caused by structural changes. Compounds that are eliminated too quickly in the blood generally have unsatisfactory tissue accumulation due to their short distribution time; while compounds that are eliminated too slowly from the blood, on the one hand, take longer to reach a low background, which usually means a longer waiting time before surgery. On the other hand, if there is no higher tumor accumulation, a high background means a low tumor background ratio, and the tumor boundary provided will be more unclear. How to achieve a balance: that is, a certain blood elimination rate can bring about a lower background while allowing the target tissue to have a certain accumulation that meets the time window.

[0159] The compound protected by this patent is a small molecule compound targeting the folate receptor. To confirm its targeting, a live imaging test of the folate receptor negative cell line A549 was set up. There was no difference between the 6 drug-administered groups and the control group, confirming its receptor specificity. The tumor-to-background ratio data is shown in Table 7 below.

[0160] Table 5 Comparison of tumor-to-background ratio (TBR) data of OTL38, compound 1 and compound 2 in vivo imaging (HCC827 group)

[0161]

[0162] Table 6 Comparison of background fluorescence values ​​(unit: [p / s / cm^2 / sr] / [μW / cm^2]) of in vivo imaging OTL38, compound 1 and compound 2 (HCC827 group)

[0163]

[0164] Table 7 Comparison of tumor-to-background ratio (TBR) data of OTL38, compound 1 and compound 2 in vivo imaging (A549 group)

[0165]

[0166] In vitro tissue imaging--tissue distribution studies

[0167] A subcutaneous tumor-bearing mouse model of HCC827 (human lung cancer cell line, Jicui Yaokang) lung cancer was constructed on BALB / c nude mice (athymic nude mice), and the average tumor volume reached about 300-400mm 3 The group administration started at 14:00. The mice bearing HCC827 tumor xenografts were intravenously injected with 0.025mg / kg and 0.05mg / kg of compound 1, compound 2 and OTL38 (5% glucose solution, the same below), and the blank control group was injected with 5% glucose solution (the grouping was the same as the HCC827 group in the in vivo imaging study). 2h after administration, the mice in the 7 groups were euthanized as a whole. After euthanasia, the tumor, heart, liver, spleen, lung, kidney, stomach, back muscle, back skin, ovary, pancreas, colon and small intestine and other organs were taken for fluorescence imaging. The fluorescence ratio of mouse tumor to muscle tissue, ovarian tissue and lung tissue was calculated, as shown in Table 8 below.

[0168] From the data analysis in Table 8, it can be seen that, except for the two dose groups of OTL38, the ratios of tumor and muscle, healthy lung and ovary of compound 1 and compound 2 are statistically different, indicating that positive tumor tissue and muscle, healthy lung and ovary can be clearly distinguished, and it is expected to distinguish positive resection margins in clinical surgery of lung cancer and ovarian cancer. The reason for evaluating ovarian cancer here is that the folate receptor FRα is overexpressed in most ovarian cancers and lung cancers. This experiment is a subcutaneous tumor-bearing model constructed by human lung cancer cell line, which overexpresses folate receptor α. To a certain extent, its possible application in ovarian cancer can be speculated through the ratio of tumor expressing folate receptor to healthy ovarian tissue.

[0169] The ratio of tumor to healthy lung tissue in the two dose groups of OTL38 failed to distinguish from the blank control group, probably because of the slow elimination in the blood. The accumulation of tumor at the 2h time point was less than the interference of the background. It is expected that the time point shift may bring better results. Compared with OTL38, compounds 1 and 2 are expected to meet surgical requirements in a shorter time after clinical administration, and combined with the results of in vivo live imaging (the tumor-to-background ratio showed an upward trend, and the peak appeared at 24h), the distinction between tumors and tissues at subsequent time points will be clearer. Considering the two together, compounds 1 and 2 are expected to provide a wider surgical time window.

[0170] Table 8 Fluorescence ratio of subcutaneous tumor to normal tissue in dissected tissue 2h after tumor-bearing mice were injected with 0.025 and 0.05 mg / kg compound 1, compound 2, and OTL38

[0171]

[0172] Effect Test Example 3 (Ovarian Cancer)

[0173] The SKOV3 (Shanghai Cell Bank) ovarian cancer subcutaneous tumor-bearing mouse model was established on NCG mice, with an average tumor volume of about 300-400 mm 3 The group dosing started at 1h. Mice bearing SKOV3 tumor xenografts were intravenously injected with 0.025mg / kg and 0.05mg / kg of compound 1, compound 2 and OTL38 (5% glucose solution, the same below), and the blank control group was injected with 5% glucose solution (see Table 9 below). Then, whole-body imaging studies were performed using a small animal in vivo imaging system, and the imaging equipment contained a near-infrared fluorescence imaging system. Imaging was performed at 1h, 2h, 4h, 8h, and 24h, respectively. The tumor and background parts were circled with the same diameter, the fluorescence value was obtained, and the tumor-to-background ratio (TBR) was calculated. The larger the value, the more it tends to provide a clearer tumor boundary, which is expected to provide a better surgical field during surgery. The specific results are shown in Table 10 below.

[0174] Table 9 Grouping of in vivo imaging

[0175]

[0176] Except for the OTL38 0.025 mg / kg dose group, the other five dosing groups had partial fluorescence enrichment in the tumor part at 1 hour. The tumor and non-tumor parts could be roughly distinguished from the image. The boundaries became obvious at subsequent time points and continued until the last time point of the experiment, 24 hours.

[0177] From the perspective of different dose levels, the two dose groups of OTL38 can provide a certain tumor-to-background ratio after 2 hours, and the other four dosing groups can provide a certain tumor-to-background ratio at 1 hour and thereafter. At different dose levels, there is little difference between high and low doses of the three compounds, and the TBR of the high-dose group is slightly higher than that of the low-dose group, suggesting that increasing the dose in the later stage may have greater application value.

[0178] Among the different compounds, compound 1 has the most obvious advantage, followed by compound 2, and OTL38 is weaker, but it can still distinguish tumors after 2 hours and provide clear surgical margins.

[0179] Table 10 Comparison of tumor-to-background ratio (TBR) data of SKOV3 tumor-bearing mice after injection of 0.025 and 0.05 mg / kg compound 1, compound 2, and OTL38

[0180]

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof; in, R is 2. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: It meets one or more of the following conditions: (1) R is For example (2) The pharmaceutically acceptable salt of the compound represented by Formula I contains a pharmaceutically acceptable cation, the number of which may be 1, 2, 3 or 4. The cation may be K + 、Na + , Li + or NH4 + , a pharmaceutically acceptable salt of the compound of formula I is, for example, a tetrasodium salt of the compound of formula I; (3) The compound represented by formula I or a pharmaceutically acceptable salt thereof is a small molecule compound targeting folate receptor.

3. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The compound represented by formula I is any of the following compounds:

4. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The pharmaceutically acceptable salt of the compound represented by formula I is the compound represented by formula II; Among them, M 1 、M 2 、M 3 and M 4 Each is independently H or a pharmaceutically acceptable cation; R as claimed in claim 1; Preferably, the pharmaceutically acceptable cation is a monovalent cation, such as K + 、Na + or Li + ; Further preferably, the compound represented by formula II is 5. A small molecule compound targeting folate receptor, characterized in that: Its structure is: Wherein M is a monovalent cation, and R is selected from Preferably, the monovalent cation is selected from potassium ions, sodium ions, and lithium ions.

6. The small molecule compound targeting folate receptor according to claim 5, characterized in that: The small molecule compound is Wherein M is potassium ion, sodium ion or lithium ion.

7. A method for preparing the compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 or the small molecule compound targeting folate receptor according to claim 5 or 6, characterized in that: The steps include: Prepare the first intermediate, the structural formula is The first intermediate reacts with the first compound to obtain a target small molecule compound, wherein the first compound is Among them, M 2 、M 3 、M 4 Each independently is H or a pharmaceutically acceptable cation, such as a monovalent cation, and R is selected from Preferably, the preparation method meets one or more of the following conditions: (1) The reaction of the first intermediate and the first compound is carried out in a carbonate aqueous solution, preferably, the carbonate is selected from sodium carbonate, potassium carbonate, and lithium carbonate; (2) The reaction conditions of the reaction are: 70-75°C, reaction time 1-2h.

8. A compound of formula V or a pharmaceutically acceptable salt thereof; in, R is defined as in any one of claims 1 to 7; Preferably, the compound of formula V is any of the following compounds:

9. A pharmaceutical composition comprising the compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, and the small molecule compound targeting folate receptor according to claim 5 or 6; Preferably, the pharmaceutically acceptable salt of the compound represented by formula I is the compound represented by formula II as claimed in claim 4.

10. Use of a substance M in the preparation of a drug for treating or diagnosing cancer tissue, or use of a substance M in a targeting agent targeting cancer tissue, wherein the substance M is a compound of formula I as described in any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, a small molecule compound targeting a folate receptor as described in claim 5 or 6, or a pharmaceutical composition as described in claim 9; Preferably, the cancer tissue is a cancer tissue expressing folate receptor, which may include parenchymal tissue, interstitial tissue or marginal tissue, such as lung cancer, breast cancer or ovarian cancer.

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