Small molecule compound targeting folate receptor and preparation method, composition and application thereof

Abstract

The present invention provides a small molecule compound targeting folate receptors, as well as its preparation method, composition, and application. Specifically provided is a compound represented by Formula I or a pharmaceutically acceptable salt thereof. The compound or pharmaceutically acceptable salt provided by the present invention can be used as a small molecule targeted fluorescent contrast agent to assist in surgical treatment. It has a novel structure, good solubility, low steric hindrance, and strong targeting. It can significantly improve the visual field during tumor surgery and facilitate the efficient and precise removal of tumor tissue and bordering tissue, including pathological tissue. #imgabs0#

Description

[0001] This application claims priority to Chinese patent application No. 2023110325244, filed on August 16, 2023. This application incorporates the entirety of the aforementioned 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, and its preparation method, composition and application. Background Art

[0003] Folate receptor alpha (FR-α), also known as FOLR1 or folate-binding protein, is a glycoprotein anchored to the cell membrane via 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 malignancy. Similar to other GPI family proteins, FRα activates an intracellular regulatory signaling network upon binding to folate. FRɑ is primarily highly expressed in epithelial tumor tissues and is absent or expressed at very low levels in normal tissues. It has a high affinity for folate and a low affinity for reduced folate.

[0004] FRα is known to be overexpressed throughout the tumor cell surface in various cancer types, 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 is significantly elevated in certain cancer cells, making it an important target in cancer diagnosis and treatment in recent years.

[0005] Near-infrared fluorescence imaging technology. Intraoperative near-infrared (NIR) fluorescence imaging aids surgeons in making informed decisions and reduces the rate of positive surgical margins, becoming an effective solution for detecting tumor margins during surgery. Fluorescence imaging-based intraoperative navigation, based on the enrichment of fluorescein in specific tissues, can accurately display information such as blood vessels, lymph nodes, and tumor tissue during surgery. It can also specifically visualize specific molecular targets overexpressed in tumor tissue, achieving high signal-to-noise ratio tumor imaging and further reducing the rate of positive surgical margins. Near-infrared light, with a wavelength range of 650nm to 900nm, is superior to visible light for intraoperative imaging due to its tissue penetration depth of up to 10mm. Because tissue exhibits only limited autofluorescence in the NIR spectrum, the use of NIR fluorescent imaging agents can maximize the contrast between fluorescence signals in tumors and healthy tissue. Furthermore, NIR light does not interfere with the surgical field of view, as it is invisible to the human eye. Currently available imaging systems combine white light illumination of the surgical field with NIR fluorescence images, providing surgeons with both anatomical and fluorescence information.

[0006] The prior art compound OTL38 has been used 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 are highly targeted, have strong tumor detection capabilities, and are more suitable for clinical applications. Summary of the Invention

[0008] The present invention aims to provide a small molecule compound targeting the folate receptor, as well as its preparation method, composition, and application. As a small molecule targeted fluorescent contrast agent that can effectively assist in surgical treatment, this compound possesses a novel structure and strong targeting properties. It can significantly enhance the field of view of diseased tissue during tumor surgery and other treatments, helping to efficiently and effectively identify diseased and normal tissues, thereby facilitating 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 which may be 1, 2, 3 or 4. 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 a 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 objectives, an embodiment of the present invention provides a small molecule compound targeting folate receptors:

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

[0026] In the formula Refers to the covalent bond position of the R group.

[0027] In one or more embodiments of the present invention, the monovalent cation is selected from potassium ions, sodium ions, lithium ions (eg, sodium ions).

[0028] In one or more embodiments of the present invention, the small molecule compound targeting the folate receptor is

[0029] wherein M is a potassium ion, a sodium ion, or a lithium ion (eg, a sodium ion).

[0030] In one embodiment, the compound represented by formula II is

[0031]

[0032] Among them, M 1 、M 2 、M 3 and M 4 Independently K + 、Na + or Li + (e.g. Na + ).

[0033] In one embodiment, the compound represented by formula II is

[0034]

[0035]

[0036] The present invention provides a method for preparing the above-mentioned small molecule compound targeting the 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:

[0037] Prepare the first intermediate, the structural formula is

[0038] 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

[0039]

[0040] In one or more embodiments of the present invention, a method for preparing a small molecule compound targeting a folate receptor comprises the following steps:

[0041] Prepare the first intermediate, the structural formula is

[0042] 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

[0043] 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 carbonate aqueous solution has a concentration of 3-10 wt.%. Preferably, the carbonate aqueous solution is a 3.35 wt.% sodium carbonate solution.

[0044] In one or more embodiments of the present invention, the carbonate is selected from sodium carbonate and potassium carbonate.

[0045] In one or more embodiments of the present invention, the reaction temperature is 70-75°C.

[0046] In one embodiment, the reaction conditions are: 70-75° C., and the reaction time is 1-2 h.

[0047] The present invention also provides a compound represented by formula V or a pharmaceutically acceptable salt thereof;

[0048]

[0049] Wherein, R is defined as described in any embodiment of the present invention.

[0050] Preferably, the compound of formula V is any one of the following compounds:

[0051]

[0052] The present invention also provides a pharmaceutical composition comprising the aforementioned small molecule compound targeting the folate receptor. Preferably, the active ingredient of the pharmaceutical composition is selected from a small molecule compound targeting the folate receptor, or a pharmaceutically acceptable salt, conjugate, or pharmaceutically acceptable salt thereof. The composition can be administered in a suitable dosage form for intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, or intrathecal administration.

[0053] The present invention also provides a pharmaceutical composition comprising the compound of Formula I or a pharmaceutically acceptable salt thereof, the small molecule compound targeting the folate receptor, 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 the folate receptor are active ingredients.

[0054] 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 as a targeting agent targeting cancer tissue. The agent includes a detection agent and a therapeutic agent.

[0055] 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, or other cancer tissue expressing folate receptors.

[0056] The present invention provides a use of the compound represented by Formula I or a pharmaceutically acceptable salt thereof, or the small molecule compound targeting the folate receptor, in the preparation of a drug for treating or diagnosing cancerous tissue. Preferably, the drug is an imaging agent (e.g., a fluorescent contrast agent).

[0057] The cancer tissue may be a cancer tissue expressing folate receptor, and may include parenchymal tissue, interstitial tissue, or marginal tissue, such as lung cancer, breast cancer, or ovarian cancer.

[0058] Explanation of terms:

[0059] The term "pharmaceutically acceptable" means relatively non-toxic, safe, and suitable for use by patients.

[0060] The term "pharmaceutically acceptable salt" refers to a salt formed 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).

[0061] The term "pharmaceutical excipients" refers to excipients and additives used in the production of pharmaceuticals and in the preparation of prescriptions. These excipients are all substances, other than the active ingredient, contained in a pharmaceutical preparation. For details, see the Pharmacopoeia of the People's Republic of China (2020 edition) or the Handbook of Pharmaceutical Excipients (Raymond C. Rowe, 2009).

[0062] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0063] The reagents and raw materials used in the present invention are commercially available.

[0064] The positive progress of the present invention is that the composition provided by the present invention has one or more of the following advantages:

[0065] (1) Good targeting, which helps to mark diseased tissues and identify small lesions and hidden lesions;

[0066] (2) It overcomes the disadvantage of rapid clearance from cancer tissue. Generally, ICG has a half-life of 3-4 minutes. This short half-life leads to multiple perfusions during surgery, seriously affecting the quality of surgery and prolonging the operation time. The compound of the present invention can still detect fluorescence 24 hours after intravenous injection;

[0067] (3) Improved fluorescence quantum yield. The fluorescence quantum yield of ICG is generally 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 a clinical dose of 0.005 mg / kg.

[0068] (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-to-background ratio (TBR), and can more clearly distinguish between tumors and normal tissues.

[0069] (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 ability to distinguish lesion tissues as OTL38 at a lower dose in clinical practice.

[0070] (6) It has a novel structure, good solubility, and low steric hindrance, which enables 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

[0071] Figure 1 This is the near-infrared imaging image after compound 1 and OTL38 were administered at 1 nmol / mouse for 1 hour.

[0072] Figure 2 This is the near-infrared imaging image after compound 1 and OTL38 were administered at 1 nmol / mouse for 2 hours.

[0073] Figure 3 This is the near-infrared imaging image after compound 1 and OTL38 were administered at 1 nmol / mouse for 4 hours.

[0074] Figure 4 This is the near-infrared imaging image after compound 1 and OTL38 were administered at 1 nmol / mouse for 8 hours.

[0075] Figure 5 This is the near-infrared imaging image after compound 1 and OTL38 were administered at 1 nmol / mouse for 24 hours.

[0076] Figure 6 This is the near-infrared imaging image after compound 1 and OTL38 were administered at 10 nmol / mouse for 1 hour.

[0077] Figure 7 The near-infrared imaging images were taken after compound 1 and OTL38 were administered at 10 nmol / mouse for 2 hours.

[0078] Figure 8 The near-infrared imaging images were taken after compound 1 and OTL38 were administered at 10 nmol / mouse for 4 hours.

[0079] Figure 9 The near-infrared imaging images were taken after compound 1 and OTL38 were administered at 10 nmol / mouse for 8 hours.

[0080] Figure 10 The near-infrared imaging images were obtained after compound 1 and OTL38 were administered at 10 nmol / mouse for 24 hours.

[0081] Figure 11 These are near-infrared imaging images of mouse organs in the blank control group.

[0082] Figure 12 The figure shows the near-infrared imaging of mouse organs after administration of compound 1 at 1 nmol / mouse.

[0083] Figure 13 The figure shows the near-infrared imaging of mouse organs after administration of compound 1 at 10 nmol / mouse.

[0084] Figure 14 These are near-infrared images of mouse organs after administration of 1 nmol of OTL38.

[0085] Figure 15 These are near-infrared images of mouse organs after administration of 10 nmol / mouse of OTL38. DETAILED DESCRIPTION

[0086] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0087]

[0088]

[0089]

[0090] 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 Schematic diagram of the resin itself. eq refers to the molar ratio. "V" represents the volume / mass ratio. 7V means that when the amount of chlorine resin is The amount of reagent required for 1g is 7mL.

[0091] Example 1 Compound 1

[0092] Here M is Na + For example, Li + , K + The situation can be realized in the same way.

[0093] Synthesis route

[0094] Step 1: B01 synthesis

[0095]

[0096] Pteroic acid (600 mg) and Fmoc-ethylenediamine hydrochloride (643 mg) were weighed and added to a 250 mL single-necked bottle. DMSO (24 mL) was added to dissolve the mixture. HTAU (1.04 g) and DIEA (746 mg) were added and stirred for 2 h. Upon completion of the reaction, the reaction mixture was added to MTBE, centrifuged, and dried to obtain B01 (1.1 g, 99% yield). LCMS: [M+H] + =577.2,2.78min.

[0097] Step 2: B02 synthesis

[0098]

[0099] 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%).

[0100] Step 3: B03 Synthesis

[0101]

[0102] H-Tyr(tBu)-OtBu.HCl (490 mg) was weighed, and after being freed, it was added to a 50 mL single-necked bottle, and 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 after HPLC monitoring. 5 mL of water was added, and 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-necked bottle, DMSO (10 mL) was added to dissolve it, and the above-mentioned concentrated intermediate was added at 0-5°C, stirred at 25°C overnight, and the reaction solution was added dropwise to MTBE and centrifuged to dry to obtain B03 (809 mg, yield of about 85%).

[0103] LCMS: [M+H] + =674.4,2.92min.

[0104] Step 4: B04 synthesis

[0105]

[0106] Weigh B03 (809 mg) into a 50 mL single-necked bottle, then add 10 mL (90% TFA, 5% water, 5% triethylsilane). Stir at 25°C. HPLC monitoring of the reaction yields B04 (403 mg, approximately 60% yield). LCMS [M+H] +=562.3,1.42min.

[0107] Preparation and purification of B04

[0108]

[0109] Step 5: Synthesis of compound 1

[0110]

[0111] Weigh B04 (100 mg, 0.19 mmol) and add it to a 25 ml single-necked 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.

[0112] Post-processing: Cool down to 20 ～ 25 ° C, prepared, lyophilized to obtain 50 mg of blue solid compound 1, yield 19%, purity 96.30%), MS: [M-4Na+4H+2H] 2+ =707.3,

[0113] 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).

[0114] Preparation and purification of compound 1

[0115]

[0116]

[0117] Similarly, by using different raw materials, such as replacing the "Fmoc-ethylenediamine hydrochloride" in step 1 of Example 1 with "Fmoc-propylenediamine hydrochloride", "Fmoc-butylenediamine hydrochloride", or "Fmoc-pentanediamine hydrochloride", compounds 6, 7, and 8 can be obtained by the method of this embodiment. The specific process is described above and will not be repeated here.

[0118] Example 2

[0119] Preparation of compound 2

[0120] Here M is Na + For example, Li + , K + The situation can be realized in the same way.

[0121] Synthesis route step 1: C03 solid phase synthesis

[0122]

[0123]

[0124]

[0125] 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 reagent used is 7mL.

[0126] Step 2: Synthesis of compound 2

[0127]

[0128] Weigh CO3 (100 mg, 0.156 mmol) and add it to a 25 ml single-necked 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.

[0129] Post-processing: Cool down to 20 ～ 25 ° C, prepared, lyophilized to give 84 mg of blue solid compound 2, yield 23%, purity 97.65%, MS: [M-4Na+4H+2H] 2+ = =743.9,

[0130] 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).

[0131] Preparation and purification of compound 2

[0132]

[0133]

[0134] Similarly, by using different raw materials, such as replacing "Fmoc-PEG2-CH2CH2COOH" in step 1 in Example 2 with "Fmoc-PEG3-CH2CH2COOH", "Fmoc-PEG4-CH2CH2COOH", and "Fmoc-PEG5-CH2CH2COOH", compounds 3, 4, and 5 can be obtained by the method of this example. The specific process is described above and will not be repeated here.

[0135] Effect Test Example 1

[0136] 1. In vivo activity imaging of compound 1 (compared with OTL38, CAS:% 1628858-03-6): lung cancer model

[0137] A subcutaneous HCC827 (human lung cancer cell line ATCC_CRL-2868) lung cancer tumor-bearing mouse model was established in BALB / c nude (athymic nude mice), with an average tumor volume of approximately 300-400 mm 3The 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). 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. See the specific results for details. 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.

[0138] Table 1 In vivo imaging grouping

[0139]

[0140] At 1 hour, the tumor part of the four drug-dosing groups had some fluorescence enrichment, 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.

[0141] All four dosing groups provided a certain tumor-to-background ratio (TBR) at 1 hour and beyond (except for the OTL38 1 nmol / animal group at 1 hour). Compound 1 demonstrated superior TBR values ​​to OTL38 at every time point across both doses. The 10 nmol / animal group exhibited superior TBR values ​​to the 1 nmol / animal group at each time point across dose levels. There was no difference in TBR between the 1 nmol / animal and 10 nmol / animal OTL38 groups, indicating that increasing dose did not lead to clearer imaging boundaries. Compound 1 is expected to provide even better tumor-to-background data in subsequent dose optimization studies.

[0142] 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 OTL38.

[0143] Table 2 Comparison of tumor-to-background ratio data of OTL38 and compound 1 in vivo imaging

[0144]

[0145] 2. In vitro tissue imaging – tissue distribution studies

[0146] A subcutaneous HCC827 (human lung cancer cell line) lung cancer tumor-bearing mouse model was established in BALB / c nude (athymic nude mice), with an average tumor volume of approximately 300-400 mm 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 in vivo live imaging study above). 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. 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 their organs (tumor, heart, liver, spleen, lung, kidney, stomach, muscle, skin, ovary, 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, as shown in Table 3 below.

[0147] The results showed that at the 24-h time point, in the 1 nmol / animal group, the fluorescence of compound 1 and OTL38 was not sufficiently displayed in the tumor site, but due to the low uptake in other tissues, this dose group showed a relatively obvious differentiation between tumor / muscle and tumor / lung (see Table 3 for details); in the 10 nmol / animal group, both compound 1 and OTL38 could be observed to have obvious fluorescence in the tumor, and this dose group showed better differentiation between tumor / muscle and tumor / lung than the 1 nmol / animal group (see Table 3 for details), and is expected to distinguish positive resection margins in clinical use.

[0148] Significant fluorescence was observed in the kidneys, likely due to drug excretion through urine. A small amount of fluorescence was also observed in the healthy lungs, liver, stomach, and ovaries at the 10 nmol group, likely due to the limited expression of FRα in these tissues. During data processing, tumor / ovary comparisons were calculated, as ovarian cancer also overexpresses FRα. Significant differences were observed between the 10 nmol / animal group and the control group for both compounds, suggesting their potential application in ovarian cancer.

[0149] Table 3 Fluorescence ratios of subcutaneous tumor and normal tissue in dissected tissues 24 hours after injection of 1 and 10 nmol / mouse compound 1 and OTL38 into tumor-bearing mice

[0150] Grouping Tumor / muscle Tumor / lung Tumor / ovarian Blank control group 3.51 1.48 2.18 OTL38 1nmol / unit 7.04 1.60 2.78 OTL38 10nmol / each 19.21 2.80 5.43 Compound 1 1 nmol / animal 6.64 2.09 2.89 Compound 1 10 nmol / animal 27.80 5.01 5.60 .

[0151] Effect Test Example 2

[0152] In vivo live imaging studies

[0153] 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 established in BALB / c nude (athymic nude mice), with an average tumor volume of approximately 300-400 mm 3 The group dosing began at 9:00 AM (see Table 4 below for grouping details). 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. Whole-body imaging studies were then performed using a small animal in vivo imaging system, which contained a near-infrared fluorescence imaging system. Imaging was performed at 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours, respectively. The tumor and background areas 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 likely it is to provide a clearer tumor boundary, which is expected to provide a better surgical field of view during surgery. The specific results are shown in Table 5 below.

[0154] Table 4 In vivo imaging grouping

[0155]

[0156] 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 tended to be distinct at subsequent time points and persisted until the last time point of the experiment, 24 h.

[0157] All six dosing groups provided a certain tumor-to-background ratio (TBR) at 1 hour and beyond. Across dose levels, TBR values ​​at each time point were not significantly different for OTL38, compound 1, and compound 2 at 0.025 mg / kg and 0.05 mg / kg, respectively. The 0.05 mg / kg dose of compound 1 and compound 2 was superior to the lower dose groups, suggesting that: 1) increasing the dose is expected to increase the tumor-to-background ratio for compounds 1 and 2; 2) the difference was not significant at a two-fold dose interval, necessitating further dose intervals to investigate dose-related effects. Compounds 1 and 2 are expected to provide even better tumor background data in subsequent dose optimization studies. Compound 1 has a significant advantage in tumor-to-background ratio, followed by compound 2, and OTL38 has the lowest, suggesting that the structural changes in compounds 1 and 2 lead to altered pharmacokinetic properties. This conclusion is also reflected in the background fluorescence values ​​of the three compounds, as shown in Table 6. At the same dose level, background fluorescence values ​​are: OTL38 > Compound 1 > Compound 2. This background fluorescence is partially due to vascularization throughout the tissue, suggesting that the elimination rate from the blood should be: OTL38 < Compound 1 < Compound 2. Therefore, the superior tumor-to-background ratios of compounds 1 and 2 are likely due to a certain degree of tumor accumulation and lower background interference, which also widens the gap with OTL38. Good pharmacokinetic properties are not easy to achieve, and identifying changes in pharmacokinetic properties caused by structural changes is even more difficult. Compounds that eliminate too quickly from the blood generally have poor tissue accumulation due to their short distribution time. Compounds that eliminate too slowly from the blood require longer to achieve a low background, which often means longer pre-operative waiting time. Furthermore, without high tumor accumulation, a high background value means a low tumor-to-background ratio, which can provide less clear tumor boundaries. How to achieve a balance: that is, a certain blood elimination rate can not only bring about a lower background, but also allow the target tissue to have a certain accumulation that meets the time window.

[0158] The compound protected by this patent is a small molecule that targets the folate receptor. To confirm its targeting, an in vivo imaging study was conducted using the folate receptor-negative cell line A549. All six treatment groups showed no difference from the control group, confirming its receptor specificity. Tumor-to-background ratio data are shown in Table 7 below.

[0159] Table 5 Comparison of tumor-to-background ratio (TBR) data of OTL38, compound 1 and compound 2 in vivo imaging (HCC827 group)

[0160]

[0161] Table 6 Comparison of in vivo imaging background fluorescence values ​​of OTL38, compound 1, and compound 2 (unit: [p / s / cm^2 / sr] / [μW / cm^2]) (HCC827 group)

[0162]

[0163] Table 7 Comparison of tumor-to-background ratio (TBR) data of OTL38, compound 1 and compound 2 in vivo imaging (A549 group)

[0164]

[0165] In vitro tissue imaging--tissue distribution studies

[0166] A subcutaneous HCC827 (human lung cancer cell line, Jicui Yaokang) lung cancer tumor-bearing mouse model was established in BALB / c nude (athymic nude mice), with an average tumor volume of approximately 300-400 mm 3 Dosing was initiated at 1:00 AM. Mice bearing HCC827 tumor xenografts were intravenously injected with 0.025 mg / kg and 0.05 mg / kg of Compound 1, Compound 2, and OTL38 (in 5% glucose solution, the same applies below). A blank control group was injected with 5% glucose solution (grouping was the same as in the HCC827 group in the in vivo imaging study). Two hours after dosing, mice in all seven groups were euthanized en bloc. Fluorescence imaging of the tumor, heart, liver, spleen, lung, kidney, stomach, back muscle, back skin, ovary, pancreas, colon, and small intestine was performed. Fluorescence ratios of tumor to muscle, ovarian, and lung tissues were calculated and are shown in Table 8 below.

[0167] As shown in Table 8, with the exception of the two OTL38 dose groups, the ratios of tumor to muscle, healthy lung, and ovary for Compounds 1 and 2 were statistically different, indicating that positive tumor tissue can be clearly distinguished from muscle, healthy lung, and ovary, potentially enabling the identification of positive resection margins in clinical lung and ovarian cancer surgery. The reason for evaluating ovarian cancer here is that the folate receptor FRα is overexpressed in most ovarian and lung cancers. This experiment used a subcutaneous tumor-bearing model constructed from a human lung cancer cell line that overexpresses folate receptor α. The ratio of tumor to healthy ovarian tissue expressing folate receptor α suggests its potential application in ovarian cancer.

[0168] The ratio of tumor to healthy lung tissue in the two dose groups of OTL38 failed to distinguish from the blank control group, which may be due to slow elimination in the blood. The accumulation of tumors at the 2-hour time point was less than the interference of the background. It is expected that shifting the time point 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 occurred at 24 hours), the distinction between tumors and tissues at subsequent time points will be clearer. Taking both into consideration, compounds 1 and 2 are expected to provide a wider surgical time window.

[0169] Table 8 Fluorescence ratio of subcutaneous tumor to normal tissue in tumor-bearing mice 2 hours after injection of 0.025 and 0.05 mg / kg Compound 1, Compound 2, and OTL38

[0170]

[0171] Effect Test Example 3 (Ovarian Cancer)

[0172] A SKOV3 (Shanghai Cell Bank) ovarian cancer subcutaneous tumor-bearing mouse model was established in NCG mice, with an average tumor volume of approximately 300-400 mm 3 Dosing began at 1:00 p.m. Mice bearing SKOV3 tumor xenografts were intravenously injected with 0.025 mg / kg and 0.05 mg / kg of Compound 1, Compound 2, and OTL38 (in 5% glucose solution, the same applies below). A blank control group was injected with 5% glucose solution (see Table 9 below). Whole-body imaging studies were then performed using a small animal in vivo imaging system equipped with a near-infrared fluorescence imaging system. Images were taken at 1, 2, 4, 8, and 24 hours. Tumor and background areas were circled with the same diameter, and fluorescence values ​​were obtained. The tumor-to-background ratio (TBR) was calculated. A larger TBR value indicates a clearer tumor boundary, potentially providing a better surgical field during surgery. Detailed results are shown in Table 10 below.

[0173] Table 9 In vivo imaging grouping

[0174]

[0175] Except for the OTL38 0.025 mg / kg dose group, the other five dosing groups had partial fluorescence enrichment in the tumor area at 1 hour. The tumor and non-tumor areas could be roughly distinguished from the images. The boundaries became obvious at subsequent time points and continued until the last time point of the experiment, 24 hours.

[0176] 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. 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.

[0177] 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.

[0178] Table 10 Comparison of tumor-to-background ratio (TBR) data after injection of 0.025 and 0.05 mg / kg Compound 1, Compound 2, and OTL38 into SKOV3 tumor-bearing mice

[0179]

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof; ； in, R is or .

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

3. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, wherein: The pharmaceutically acceptable salt of the compound represented by Formula I is the tetrasodium salt of the compound represented by Formula I.

4. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, wherein: The compound represented by formula I is any of the following compounds: ； n is 2 or 3.

5. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, wherein: 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 described in claim 1.

6. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 5, wherein: The pharmaceutically acceptable cation is a monovalent cation.

7. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 6, wherein: The pharmaceutically acceptable cation is K + 、Na + or Li + .

8. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 5, wherein: The compound represented by formula II is any of the following compounds: ； n is 2 or 3.

9. A small molecule compound targeting folate receptor, characterized in that: Its structure is: , where M is a monovalent cation and R is or .

10. The small molecule compound targeting folate receptor according to claim 9, characterized in that: The monovalent cation is a potassium ion, a sodium ion or a lithium ion.

11. The small molecule compound targeting folate receptor according to claim 9, characterized in that: The small molecule compound is , where M is potassium ion, sodium ion, or lithium ion.

12. A method for preparing the compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1 or the small molecule compound targeting the folate receptor according to claim 9, characterized in that: The steps include: Prepare the first intermediate, the structural formula is ; The first intermediate reacts with the first compound to obtain the target small molecule compound, and the first compound is , where M 2 、M 3 、M 4 are each independently H or a pharmaceutically acceptable cation, and R is or .

13. The preparation method according to claim 12, wherein M 2 、M 3 、M 4 Each is independently a monovalent cation.

14. The preparation method according to claim 12, wherein The preparation method meets one or more of the following conditions: (1) The reaction between the first intermediate and the first compound is carried out in a carbonate aqueous solution; (2) The reaction conditions are: 70-75°C, reaction time 1-2h.

15. The preparation method according to claim 14, wherein The carbonate is sodium carbonate, potassium carbonate or lithium carbonate.

16. An intermediate represented by formula V or a pharmaceutically acceptable salt thereof; ； V in, R is as defined in any one of claims 1-12.

17. The intermediate represented by formula V or a pharmaceutically acceptable salt thereof according to claim 16, characterized in that: It is any of the following compounds: ； n is 2 or 3.

18. A pharmaceutical composition comprising the compound of formula I according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, and the small molecule compound targeting the folate receptor according to any one of claims 9 to 11.

19. The pharmaceutical composition according to claim 18, wherein The pharmaceutically acceptable salt of the compound represented by formula I is the compound represented by formula II as claimed in any one of claims 5 to 8.

20. Use of a substance M in the preparation of a drug for treating or diagnosing cancerous tissue, or use of a substance M in a targeting agent for targeting cancerous tissue, wherein the substance M is a compound of formula I according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, a small molecule compound targeting a folate receptor according to any one of claims 9 to 11, or a pharmaceutical composition according to claim 18 or 19; The cancer tissue is a cancer tissue that overexpresses folate receptor.

21. The use according to claim 20, characterized in that The cancer tissue includes parenchymal tissue, interstitial tissue or marginal tissue.

22. The use according to claim 20, characterized in that The cancer tissue is lung cancer or ovarian cancer.

Images