Peptide conjugates of camptothecin analogs and uses thereof

Abstract

DNA-damaging agents, such as topoisomerase I inhibitors, are increasingly important as activators that enhance DNA damage response (DDR) deficiencies and as tumor suppressors. Limitations of camptothecin analogues include their side effects, such as myelosuppression and diarrhea, their relatively short half-lives, and the fact that they are substrates for efflux pump proteins. Therefore, new methods are needed to treat cancer using camptothecin analogues. This application relates to conjugates of camptothecin analogues and their salts, comprising a peptide targeting a sorting protein and a cleavable linker. In mouse models of colorectal cancer expressing the sorting protein, these conjugates have shown more potent antitumor activity than the corresponding unconjugated camptothecin analogues. This application also relates to pharmaceutical compositions comprising said conjugates or their salts, and the use of said conjugates, their salts, or pharmaceutical compositions in the treatment of cancers expressing the sorting protein.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefits of U.S. Provisional Patent Application No. 63 / 581,864, filed September 11, 2023; U.S. Provisional Patent Application No. 63 / 598,353, filed November 13, 2023; and U.S. Provisional Patent Application No. 63 / 564,151, filed March 12, 2024, which are incorporated herein by reference.

[0003] sequence list

[0004] This application contains a sequence list in a computer-readable form entitled “G11718-00480_SeqList.xml”, created on September 10, 2024, and having a size of approximately 11,401 bytes. This computer-readable form is incorporated herein by reference. Technical Field

[0005] This invention generally relates to the field of oncology, and more specifically to a method of treating cancer using topoisomerase inhibitors. Background Technology

[0006] DNA-damaging agents, as long-term mainstays of cancer chemotherapy, are becoming increasingly important as agents that enhance the activity of the deficient DNA damage response (DDR) in tumors and as tumor inhibitors. When the ability to repair damaged DNA is impaired, DNA damage can lead to selective toxicity and synthetic lethal effects (Lord CJ, Ashworth A. The DNA damage response and cancer therapy). Nature 2012;481:287-94). An important target for causing specific DNA damage is topoisomerase I (TOP1), which is inhibited by camptothecin and its analogues (Pommier, Y. DNA topoisomeraseI inhibitors: chemistry, biology, and interfacial inhibition). Chem Rev 2009;109:2894–902; Thomas, A., Pommier, Y. Targeting topoisomerase I in the era of precision medicine. Clin Cancer Res 2019;25:6581-9).

[0007] Irinotecan, a camptothecin analogue, is used to treat colon cancer and, to a lesser extent, lung cancer. Irinotecan administration is limited by its serious side effects, including bone marrow suppression and diarrhea.

[0008] 7-Ethyl-10-hydroxycamptothecin (SN-38) is the active metabolite of irinotecan, a camptothecin analogue. SN-38 has been found to be approximately 100-1000 times more cytotoxic than irinotecan, but it has not yet been used as an anticancer drug due to its poor solubility in water (< 40 μg / mL) and pharmaceutically acceptable solvents, as well as its low affinity for lipid membranes; it is also extensively metabolized in the liver. The biotransformation of irinotecan to SN-38 is slow and limited (~5%), and approximately 33-66% of irinotecan remains unhydrolyzed at the end of a 24-hour infusion (Rowinsky et al.). Cancer Research 1994;54:427-436).

[0009] Another camptothecin analogue, exatecan (Exa), is even more effective than SN-38. As a single active agent, Exa had undergone Phase III clinical trials before development was halted due to dose-limiting side effects.

[0010] A derivative of ixanotecan, delutec (Dxd), has been developed, representing a conjugate with trastuzumab (a Her2-targeting antibody).

[0011] The limitation of camptothecin analogues lies in their relatively short half-life. For example, SN-38 formed from CPT-11 has an apparent half-life of approximately 12 hours. 1 / 2 (Mathijssen, RH. et al. Clinical pharmacokinetics and metabolism of irinotecan (CPT-11). Clin Cancer Res 2001;7:2182-94), and Exa only has about 10 hours of t in the human body. 1 / 2 (Braybrooke, JP. et al. Phase I and pharmacokinetic study of the topoisomerase I inhibitor, exatecan mesylate (DX-8951f), using aweekly 30-minute intravenous infusion, in patients with advanced solidmalignancies. Ann Oncol 2003;14:913-21).

[0012] In addition, irinotecan, SN-38, and esanotecan have been reported as substrates of the efflux pump protein breast cancer resistance protein (BCRP / ABCG2), which is associated with reduced antitumor activity.

[0013] Therefore, there is a need for new methods of treating cancer using camptothecin analogues.

[0014] This manual references several documents, the contents of which are incorporated herein by reference in their entirety.

[0015] Overview

[0016] In different aspects and implementations, this disclosure provides the following items 1-52: 1. A conjugate comprising structure I or a pharmaceutically acceptable salt thereof: A — L — B (I); in A is a camptothecin analogue.

[0017] B is a peptide that binds to a sorting protein (Sortilin) ​​receptor, wherein the peptide comprises an amino acid sequence that has at least 60% identity with the amino acid sequence listed in SEQ ID NO: 1 or 2. X 1 GVRAKAGVRN(Nle)FKSESYX 2 (SEQ ID NO:1) X 1 YKSLRRKAPRWDAPLRDPALRQLLX 2 (SEQ ID NO:2); Where X 1 Cysteine ​​(C) or absent, and X 2 It is either cysteine ​​(C) or absent; L is a linker, wherein the first end of the linker is connected to one or more side chains or modified side chain residues of an amino acid of the peptide, and / or to the N- and / or C-terminus of the peptide, and the second end of the linker is connected to a camptothecin analogue.

[0018] 2. The conjugate of Item 1, wherein L is a cleavable linker, wherein a first end of the linker is attached to a side chain of one or more lysine (K) and / or cysteine ​​(C) residues of the peptide, and / or to the N- and / or C-terminus of the peptide, and a second end of the linker is attached to an oxygen or nitrogen atom of a camptothecin analogue.

[0019] 3. A conjugate of item 1 or 2 or a pharmaceutically acceptable salt thereof, wherein the conjugate or salt thereof has one of the following formulas: Ia, Ib, Ic or Id: A1 — L1 — B (Ia); A1 — L1 — B — L2 — A2 (Ib); (Ic); (Id); L1, L2, L3, and L4 can be the same or different connectors, and A1, A2, A3, and A4 can be the same or different camptothecin analogues.

[0020] 4. A conjugate of any one of items 1-3 or a pharmaceutically acceptable salt thereof, wherein the camptothecin analogue is irinotecan, SN-38, ethanotecan or delutecan.

[0021] 5. The conjugate of Item 4 or a pharmaceutically acceptable salt thereof, wherein the camptothecin analogue is SN-38.

[0022] 6. The conjugate of Item 4 or a pharmaceutically acceptable salt thereof, wherein the camptothecin analogue is ethatecan.

[0023] 7. The conjugate of Item 4 or a pharmaceutically acceptable salt thereof, wherein the camptothecin analogue is delutecan.

[0024] 8. A conjugate of any one of items 1-7 or a pharmaceutically acceptable salt thereof, wherein the peptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence listed in SEQ ID NO: 1 or 2.

[0025] 9. A conjugate of item 8 or a pharmaceutically acceptable salt thereof, wherein the peptide comprises an amino acid sequence having at least 90% identity with the amino acid sequence listed in SEQ ID NO: 1 or 2.

[0026] 10. A conjugate of item 9 or a pharmaceutically acceptable salt thereof, wherein the peptide comprises the amino acid sequence listed in SEQ ID NO: 1 or 2.

[0027] 11. The conjugate of item 9 or a pharmaceutically acceptable salt thereof, wherein the peptide comprises the amino acid sequence listed in SEQ ID NO: 1 or 2.

[0028] 12. A conjugate of any one of items 1-11 or a pharmaceutically acceptable salt thereof, wherein X 1 and / or X 2 C, and wherein the first end of the connector is connected to X. 1 and / or X 2 The side chain.

[0029] 13. A conjugate of item 12 or a pharmaceutically acceptable salt thereof, wherein X 1 It does not exist, and X 2 C, and wherein the first end of the connector is connected to X. 2 The side chain.

[0030] 14. A conjugate of any one of items 1-11 or a pharmaceutically acceptable salt thereof, wherein X 1 and X 2 It does not exist.

[0031] 15. A conjugate of any one of items 1-14 or a pharmaceutically acceptable salt thereof, wherein the linker comprises at least one amino acid.

[0032] 16. A conjugate of item 15 or a pharmaceutically acceptable salt thereof, wherein at least one amino acid comprises glycine, valine and / or citrulline.

[0033] 17. A conjugate of item 15 or 16 or a pharmaceutically acceptable salt thereof, wherein the cleavable linker is a protease-cleavable linker.

[0034] 18. A conjugate of item 17 or a pharmaceutically acceptable salt thereof, wherein the protease is a cysteine ​​protease.

[0035] 19. The conjugate of item 18 or a pharmaceutically acceptable salt thereof, wherein the cysteine ​​protease is cathepsin B.

[0036] 20. A conjugate of any one of items 1-19 or a pharmaceutically acceptable salt thereof, wherein the conjugate comprises polyethylene glycol (PEG).

[0037] 21. A conjugate of Item 20 or a pharmaceutically acceptable salt thereof, wherein the conjugate comprises 2-10 PEG units.

[0038] 22. A conjugate of any one of items 1-21 or a pharmaceutically acceptable salt thereof, wherein the linker has one of the following structures: CO(CH2) a CONH(CH2) b CO, where a and b are independently 1, 2, 3 or 4; CO(CH2) d (OCH2CH2) e CONH(CH2) f CO, where d and f are independently 1, 2, 3, or 4, and e is an integer from 2 to 8; or .

[0039] 23. A conjugate of Item 1 or a pharmaceutically acceptable salt thereof, wherein the conjugate has one of the following structures: , , , , , , , , , or .

[0040] 24. A pharmaceutical composition comprising a conjugate or salt of any one of items 1-23 and a pharmaceutically acceptable excipient.

[0041] 25. A method for treating a subject expressing a sorting protein, comprising administering to the subject an effective amount of any one of items 1-23 or the pharmaceutical composition of item 24.

[0042] 26. The method of Project 25, wherein the cancers expressing the sorting protein are hematologic cancers, ovarian cancer, endometrial cancer, cervical cancer, skin cancer, brain cancer, breast cancer, colorectal cancer, small bowel cancer, liver cancer, lung cancer, eye cancer, prostate cancer, head and neck cancer, stomach cancer, bone cancer, thyroid cancer, testicular cancer, bladder cancer, kidney cancer, or pancreatic cancer.

[0043] 27. The method of item 25 or 26, wherein the cancer expressing the sorting protein is a cancer with a poor prognosis.

[0044] 28. The method of any one of items 25-27, wherein the cancer expressing the sorting protein is an immunologically cold cancer.

[0045] 29. The method of any one of items 25-28, wherein the conjugate, its salt, or composition is administered in combination with one or more other active agents or therapies for cancer.

[0046] 30. The method of item 29, wherein one or more additional active agents or therapies for cancer include immunotherapy.

[0047] 31. The method of Project 30, wherein immunotherapy includes immune checkpoint inhibitors.

[0048] 32. The method of Project 31, wherein the immune checkpoint inhibitor is a PD1 or PD-L1 inhibitor.

[0049] 33. The method of Item 32, wherein the PD1 or PD-L1 inhibitor is an anti-PD1 or anti-PD-L1 antibody.

[0050] 34. Use of any conjugate or salt of any of items 1-23 or the pharmaceutical composition of item 24 for the treatment of a subject with cancer expressing a sorting protein.

[0051] 35. Use of any conjugate or salt of any of items 1-23 or the pharmaceutical composition of item 24 in the preparation of a medicament for treating a subject with cancer expressing a sorting protein.

[0052] 36. Uses of item 34 or 35, wherein the cancers expressing the sorting protein are hematologic cancers, ovarian cancer, endometrial cancer, cervical cancer, skin cancer, brain cancer, breast cancer, colorectal cancer, small bowel cancer, liver cancer, lung cancer, eye cancer, prostate cancer, head and neck cancer, stomach cancer, bone cancer, thyroid cancer, testicular cancer, bladder cancer, kidney cancer, or pancreatic cancer.

[0053] 37. Use of any one of items 34-36, wherein the cancer expressing the sorting protein is a cancer with a poor prognosis.

[0054] 38. Use of any one of items 34-37, wherein cancers expressing sorting proteins are immunologically cold cancers.

[0055] 39. Use of any one of items 34-38, wherein the conjugate, its salt, composition or drug is administered in combination with one or more other active agents or therapies for cancer.

[0056] 40. Use of item 39, wherein one or more additional active agents or therapies for cancer include immunotherapy.

[0057] 41. Uses of Item 40, in which immunotherapy includes immune checkpoint inhibitors.

[0058] 42. Use of Item 41, wherein the immune checkpoint inhibitor is a PD1 or PD-L1 inhibitor.

[0059] 43. Use of Item 42, wherein the PD1 or PD-L1 inhibitor is an anti-PD1 or anti-PD-L1 antibody.

[0060] 44. A conjugate or salt of any one of items 1-23 or a pharmaceutical composition of item 24, for treating a subject with cancer expressing a sorting protein.

[0061] 45. A conjugate, salt thereof, or pharmaceutical composition for use in item 44, wherein the cancer expressing the sorting protein is a hematologic cancer, ovarian cancer, endometrial cancer, cervical cancer, skin cancer, brain cancer, breast cancer, colorectal cancer, small bowel cancer, liver cancer, lung cancer, eye cancer, prostate cancer, head and neck cancer, stomach cancer, bone cancer, thyroid cancer, testicular cancer, bladder cancer, kidney cancer, or pancreatic cancer.

[0062] 46. ​​Conjugates, salts thereof, or pharmaceutical compositions used for the purposes of item 45, wherein the cancer expressing the sorting protein is a cancer with a poor prognosis.

[0063] 47. Conjugates, salts thereof, or pharmaceutical compositions used for the purposes of items 45 or 46, wherein the cancer expressing the sorting protein is an immunologically cold cancer.

[0064] 48. A conjugate, salt thereof, or pharmaceutical composition for use in any one of items 45-47, wherein the conjugate, salt thereof, or composition is to be administered in combination with one or more other active agents or therapies for cancer.

[0065] 49. Conjugates, salts thereof, or pharmaceutical compositions used for the purposes of item 48, wherein one or more additional active agents or therapies for cancer comprise immunotherapy.

[0066] 50. Conjugates, salts thereof, or pharmaceutical compositions for use in item 49, wherein the immunotherapy comprises an immune checkpoint inhibitor.

[0067] 51. A conjugate, salt thereof, or pharmaceutical composition for use in item 50, wherein the immune checkpoint inhibitor is a PD1 or PD-L1 inhibitor.

[0068] 52. A conjugate, salt thereof, or pharmaceutical composition for use in item 51, wherein the PD1 or PD-L1 inhibitor is an anti-PD1 or anti-PD-L1 antibody.

[0069] Other objects, advantages, and features of this disclosure will become more apparent when reading the following non-limiting description of specific embodiments of this disclosure given by way of example only and with reference to the accompanying drawings. Brief description of the attached diagram

[0071] In the attached diagram: Figure 1A-1D The peptide drug conjugate TH2101 was described. Figure 1A ), TH2102 Figure 1B ), TH2204 Figure 1C ) and TH2205 ( Figure 1D The structure of ).

[0072] Figures 2A-2DThe peptide drug conjugate TH2301 was described. Figure 2A ), TH2303 Figure 2B ), TH2304 Figure 2C ) and TH2207 ( Figure 2D The structure of ).

[0073] Figure 3 The structure of the peptide drug conjugate TH2302 was described.

[0074] Figure 4 The synthetic scheme for the TH2101 conjugate (TH19P01-Osu-SN-38) was described.

[0075] Figure 5 The synthetic scheme for the TH2102 conjugate (TH19P01-Osu-irinotecan) was described.

[0076] Figure 6 The synthetic scheme for the TH2205 conjugate (TH19P01-Gly-(PEG)3-SN-38) was described.

[0077] Figure 7A -B describes the synthetic scheme for the TH2302 conjugate (KBP201-Gly-(PEG)3-SN-38).

[0078] Figure 8 The synthetic scheme for the TH2301 conjugate (TH19P01-Osu-exatecan) was described.

[0079] Figure 9A -C describes the synthetic scheme for the TH2303 conjugate (TH19P01-Glyc-Osu-ethanotecan).

[0080] Figure 10 The synthetic scheme for the TH2304 conjugate (TH19P01-Osu-drutecan) was described.

[0081] Figure 11A Western blotting was performed on sorting proteins, P-gp, BCRB, and EGFR in human colorectal cancer cell lines. Cell lysates derived from different colorectal cancer cell lines were used, as well as Western blotting in TNBC MDA-MB231 cells (a positive control for sorting proteins) and in MDCK cells transfected with human MDR1 (MDCK-MDR1).

[0082] Figure 11B-11C It showed that in human colorectal cancer (CRC), Figure 11B ) and breast cancer ( Figure 11CTopoisomerase 1 (TOPO1) was detected in cell lines. Cell lysates obtained from different CRC cell lines and Western blots were performed in TNBC cell lines.

[0083] Figure 12A Examples B and B demonstrate the antiproliferative activity of PDC and the unconjugated drug against HT-29, HCT-116, and LoVo cancer cells. As shown in Example 1, cancer cells were incubated with increasing concentrations of PDC and the unconjugated drug. The half-maximal inhibitory concentrations (MCIs) are presented, evaluated by the MTT assay and calculated using GraphPad Prism software.

[0084] Figures 13A-13B The uptake of the fluorescent peptide TH19P01-Alexa488 in HT-29 cancer cells was shown to be sorting protein-dependent. As described in Example 1, the uptake of the fluorescent peptide TH19P01-Alexa488 was measured in cells transfected with either a control scrambled siRNA (siScr) or a siRNA targeting the sorting protein (siSort1). Figure 13A Sorting proteins were detected by Western blotting of cells transfected with siScr or siSort1 siRNA. Results show the detection of sorting proteins when using siSort1. Figure 13B Compared to uptake measured in cells transfected with scrambled siRNA (siScr), uptake of fluorescent TH19P01-Alexa 488 was reduced in cells transfected with a specific siRNA targeting the sorting protein (siSort1) (n = 2).

[0085] Figures 14A-14C The uptake of the TH2101 conjugate was shown to be sorting protein-dependent in HT-29 cancer cells. The uptake of the fluorescent peptide TH2101 was measured in HT-29 cancer cells transfected with either a control scrambled siRNA (siScr) or a siRNA targeting the sorting protein (siSort1). Fluorescence signal intensity was obtained by confocal microscopy as described in the examples. Figure 14A Sorting proteins were detected by Western blot analysis of cells transfected with siScr or siSort1 siRNA. Results showed that sorting protein expression was reduced by 64% when siSort1 was used. Figure 14B Images obtained by confocal microscopy showed that the uptake of fluorescent TH2101 was reduced in cells transfected with siSort1 compared with the uptake measured in cells transfected with siScr. Figure 14C Quantitative analysis of fluorescence levels showed that TH2101 uptake in cells transfected with siSort1 was almost reduced to control levels (n=5).

[0086] Figures 15A-15C This study demonstrated the in vivo efficacy of the unconjugated drug in an HT-29 xenograft model. HT-29 cells were subcutaneously implanted into the lateral flank of mice. When the tumor reached approximately 100-150 mm... 3 Animals were treated weekly with irinotecan or ethanotecan at the indicated dose. Figures 15A-15B Irinotecan was shown to be administered at doses of 12.5 mg / kg, 25 mg / kg, or 90-100 mg / kg. Figure 15A ) or 9.2 mg / kg dose of ethathecan ( Figure 15B Tumor volume in mice treated with ) Figure 15C This shows the percentage of tumor growth inhibition (TGI%) for each unconjugated drug at different doses.

[0087] Figures 16A-16C The in vivo efficacy of the peptide-irinotecan conjugate TH2102 in the HT-29 xenograft model was demonstrated. Animals were treated weekly with different formulations of irinotecan or TH2102 at escalating doses. Tumors reached approximately 100–150 mm. 3 Processing will be initiated at that time. Figure 16A The tumor volume of mice treated with different formulations of unconjugated irinotecan and peptide-irinotecan conjugate TH2102 is shown. Figure 16B This shows the percentage of tumor growth inhibition (TGI%) of irinotecan and TH2102. Figure 16C The body weight of mice in different groups was also monitored and kept within the range established for the endpoint.

[0088] Figures 17A-17C The in vivo efficacy of peptide-irinotecan-PEG-conjugate (TH2204) and peptide-SN-38-conjugate (TH2101) in an HT-29 xenograft model was demonstrated. HT-29 cells were subcutaneously implanted into the lateral ventricular region of mice. When the tumor reached approximately 100-150 mm... 3 Animals were treated weekly with escalating doses of irinotecan or TH2204 and TH2101 at the indicated dose. Figure 17A Tumor volumes in mice treated with unconjugated irinotecan, TH2204, or TH2101 are shown. Figure 17B Tumor growth inhibition percentage (TGI%) of different doses of irinotecan, TH2204 or TH2101. Figure 17C The body weight of mice in different groups was also monitored and kept within the range established for the endpoint.

[0089] Figures 18A-18EThe in vivo efficacy of peptide-SN-38 conjugates (TH2101, TH2205) and peptide-ethanotecan conjugates (TH2301 and TH2303) in an HT-29 xenograft model was demonstrated. HT-29 cells were subcutaneously implanted into the lateral ventricular region of mice. When the tumor reached approximately 100-150 mm... 3 Animals were treated weekly with ethathecan, TH2101, TH2205 or TH2301 at the indicated dose. Figure 18A The tumor volume of mice treated with ethanotecan, TH2101, TH2205, or TH2301 is shown. Figure 18B The percentage of tumor growth inhibition (TGI%) of ethanotecan, TH2101, TH2205 and TH2301 at an equivalent dose of ethanotecan (9.2 mg / kg). Figure 18C The body weights of mice treated with essanotecan, TH2101, TH2205, or TH2301 are shown. Body weights of mice in different groups were also monitored and maintained within the ranges established for the endpoint limits. Figure 18D Tumor volumes in mice treated with esanotecan (9.2 or 18.4 mg / kg) or the peptide-esanotecan-conjugate TH2303 (32.4 mg / kg, 48.6 mg / kg, or 64.8 mg / kg) are shown. Figure 18E The body weights of mice treated with ethanotecan (9.2 or 18.4 mg / kg) or the peptide-ethanotecan-conjugate TH2303 (32.4 mg / kg, 48.6 mg / kg, or 64.8 mg / kg) are shown. Body weights of mice in different groups were also monitored and maintained within the limits established for the endpoint.

[0090] Figure 19A -B demonstrates the antitumor activity of the peptide-ethanotecan conjugate (TH2303) in the HCT-116 CRC xenograft model. HCT-116 cells were subcutaneously implanted into the lateral ventricular region of mice. Animals were treated weekly with a carrier, irinotecan, etanotecan, or TH2303 at the indicated dose. Tumors reached approximately 100–150 mm. 3 Processing will be initiated at that time. Figure 19A The tumor volume was measured using calipers as described in Example 1. Figure 19B The body weight of mice in different groups was also monitored and kept within the range established for the endpoint.

[0091] Figure 20A-B demonstrates the antitumor activity of the peptide-ethanotecan conjugate (TH2303) in the MDA-MB-231 TNBC xenograft model. MDA-MB-231 cells were subcutaneously implanted into the lateral ventricular region of mice. Animals were treated weekly with the indicated dose of the medium, irinotecan, etanotecan, or TH2303. Tumors were treated when they reached approximately 100–150 mm. 3 Processing will be initiated at that time. Figure 20A The tumor volume was measured using calipers as described in Example 1. Figure 20B The body weight of mice in different groups was also monitored and kept within the range established for the endpoint.

[0092] Figure 21A -B shows the peptide-exatecan conjugate (TH2303) in an ovarian cancer SKOV3 xenograft model. Figure 21A or xenograft (PDX) models derived from colorectal cancer patients ( Figure 21B The antitumor activity of SKVO3 cells was investigated. In the ovarian xenograft tumor model, SKVO3 cells were subcutaneously implanted into the flank of mice, while in the colorectal PDX model, small tumor fragments were implanted into the flank of mice. Animals were treated weekly with the indicated dose of the medium, ezetidine, or TH2303. Tumors reached approximately 100-150 mm. 3 Processing will be initiated at that time. Invention Details

[0094] Unless otherwise indicated herein or clearly contradicted by the context, the terms “a”, “an”, and “the”, and similar designations, are to be interpreted as encompassing both the singular and the plural in the context of describing the technology (particularly in the context of the claims below).

[0095] The terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise stated.

[0096] Unless otherwise stated in this document or clearly contradicted by the context, all methods described herein may be performed in any suitable order.

[0097] Any and all embodiments or exemplary language (“e.g.”) provided herein are intended only to better illustrate implementations of the claimed technology and, unless otherwise required, do not constitute a limitation on the scope.

[0098] Nothing in this specification should be construed as indicating that any unclaimed element is essential for the implementation of the claimed technology.

[0099] The term “about” in this document has its usual meaning. The term “about” is used to indicate that a numerical value includes inherent error variations in the means or methods used to determine the value, or includes a numerical value that is close to the value, for example, within 10% of the value (or range of values).

[0100] Unless otherwise indicated herein, references to numerical ranges herein are intended only as a shorthand for referring to each individual value falling within that range, and each individual value is incorporated into this specification as if it were individually referenced herein. All subsets of values ​​within a range are also incorporated into this specification as if they were individually referenced herein.

[0101] In the case where features or aspects of this disclosure are described in the manner of a list of Markush groups or alternatives, those skilled in the art will recognize that this disclosure is therefore also described in the manner of any single member or subgroup of a list of Markush groups or alternatives.

[0102] Unless otherwise expressly defined, all technical and scientific terms used herein should be regarded as having the same meaning as commonly understood by one of ordinary skill in the art (e.g., in stem cell biology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0103] Unless otherwise stated, the recombinant proteins, cell culture, and immunotherapy techniques used in this disclosure are standard methods well known to those skilled in the art. Such technical descriptions and explanations can be found in resource literature, such as J. Perbal, *A Practical Guide to Molecular Cloning*, John Wiley and Sons (1984); J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, Cold Spring Harbour Laboratory Press (1989); TA Brown (ed.), *Essential Molecular Biology: A Practical Approach*, Volumes 1 & 2, IRL Press (1991); DM Glover and BD Hames (ed.), *DNA Cloning: A Practical Approach*, Volumes 1–4, IRL Press (1995 and 1996); and FMAusubel et al. (ed.), *Current Protocols in Molecular Biology*, Greene Pub. Associates and Wiley-Interscience (1988, including all updated versions to date); Ed Harlow and David Lane (ed.), *Antibodies: A Laboratory Manual*, Cold Spring Harbour Laboratory (1988); and JE Coligan et al. (ed.), *Current Protocols*. inImmunology, John Wiley & Sons (including all updated versions to date).

[0104] This disclosure provides conjugates comprising structure I or a pharmaceutically acceptable salt thereof: A — L — B (I); in A is a camptothecin analogue; B is a peptide that binds to a sorting protein receptor, wherein the peptide comprises an amino acid sequence that has at least 60% identity with the amino acid sequence listed in SEQ ID NO: 1 or 2. X 1 GVRAKAGVRN(Nle)FKSESYX 2(SEQ ID NO:1) X 1 YKSLRRKAPRWDAPLRDPALRQLLX 2 (SEQ ID NO:2); Where X 1 Cysteine ​​(C) or absent, and X 2 It is either cysteine ​​(C) or absent; L is a linker, wherein the first end of the linker is connected to one or more amino acid side chains or modified side chain residues (e.g., azide groups, double bonds, triple bonds, etc.) of the peptide, and / or to the N-terminus and / or C-terminus of the peptide, while the second end of the linker is connected to the hydroxyl, carboxyl, or nitrogen atom of the camptothecin analogue.

[0105] As used herein, the term "sorting protein" or "sorting protein receptor" refers to a neuronal type I membrane glycoprotein encoded by the SORT1 gene, belonging to the vacuole sorting 10 (Vps10) receptor family. The sorting protein (also known as neurotensin receptor 3; UniProtKB accession Q99523) is expressed or overexpressed in many cancers, including, for example, ovarian, breast, colon, and prostate cancer. The encoded precursor protein (residues 34-831, residues 1-33 corresponding to the signal peptide) is proteolytically processed by furin (or other homologous proteases) after amino acid 77 into a mature receptor (residues 78-831) with a molecular weight of approximately 100-110 kDa. The amino acid residues of the sorting protein discussed herein correspond to the positions in its full-length form (i.e., UniProtKB accession Q99523).

[0106] The term "amino acid" refers to the commonly known naturally occurring (genetically encoded) or synthetic amino acids and their common derivatives, as is well known to those skilled in the art. When applied to amino acids, "standard" or "protein-derived" refers to the 20 genetically encoded amino acids of their natural conformation. Similarly, when applied to amino acids, "non-standard," "non-natural," or "uncommon" refers to a wide selection of non-natural, rare, or synthetic amino acids, such as those described by Hunt, S. Chemistry and Biochemistry of the Amino Acids Those shown in Barrett, GC, ed., Chapman and Hall: New York, 1985. Some examples of non-standard amino acids include non-... Amino acids and D-amino acids. In one embodiment, the peptide compound contains only natural amino acids. In another embodiment, the peptide compound contains one or more natural or synthetic amino acids, such as D-amino acids.

[0107] As used herein, the term "sequence identity" refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced into the first amino acid or nucleic acid sequence to optimize alignment with the second amino acid or nucleotide sequence). Amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between two sequences is a function of the number of identical positions shared by these sequences throughout their entire length (i.e., %identity = number of identical overlapping positions / total number of positions x 100%). In one embodiment, the two sequences are of equal length. Mathematical algorithms can also be used to determine the percentage of identity between two sequences. BLAST protein searches can be performed using the XBLAST program parameter set, for example, 50 points, word length = 3, to obtain amino acid sequences homologous to the protein molecules of this disclosure. GappedBLAST can be used to obtain vacancy alignments for comparison. Alternatively, PSI-BLAST can be used for iterative searching, detecting distant relationships between molecules (as above). When using BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, for example, the NCBI website). Another preferred, non-limiting example of a mathematical algorithm for comparing sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. This algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, the PAM120 weighted residue table, vacancy length penalty 12, and vacancy penalty 4 can be used. The percentage of identity between two sequences can be determined using techniques similar to those described above, with or without vacancy. When calculating the percentage of identity, typically only exact matches are counted.

[0108] In an embodiment, the peptide comprises or is composed of an amino acid sequence having at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence listed in SEQ ID NO: 1, wherein the peptide binds to a sorting protein.

[0109] In one embodiment, the peptide comprises 25, 24, 23, 22, 21, 20, or 19 residues or less and contains the sequence GVRAKAGVRN(Nle)FKSESY (SEQ ID NO:3). In another embodiment, the peptide compound comprises 25, 24, 23, 22, 21, 20, or 19 residues or less and contains the sequence GVRAKAGVRN(Nle)FKSESYC (SEQ ID NO:4).

[0110] In one embodiment, the peptide has an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence listed in SEQ ID NO: 2, wherein the peptide binds to a sorting protein. In one embodiment, the peptide comprises at least 15, 16, or 17 consecutive residues from SEQ ID NO: 2, wherein the peptide binds to a sorting protein.

[0111] In one embodiment, the peptide comprises 30, 29, 28, 27, or 26 residues or less and contains the sequence YKSLRRKAPRWDAPLRDPALRQLL (SEQ ID NO:5). In another embodiment, the peptide compound comprises 30, 29, 28, 27, or 26 residues or less and contains the sequence YKSLRRKAPRWDAPLRDPALRQLLC (SEQ ID NO:6).

[0112] As used herein, the term "camptothecin analogue" refers to a compound that has a similar structure (planar pentacyclic structure) to camptothecin and possesses the ability to inhibit topoisomerase 1 (Top1) activity. Examples of camptothecin analogues include belotetcan, topotecan, 9-aminocamptothecin (9-AC), irinotecan, rubitecan, lurtotecan, SN-38, ixanotecan, delutecan, and ZD06519 (FD1) (Petersen et al.). Mol Cancer Ther May 2, 2024; 23(5): 606–618), hTop1 inhibitor 12 (Khodair et al., European Journal of Medicinal Chemistry , 265, 116049 (2024)) and FLQY2 (Wang et al., Bioorganic Chemistry, 148, 107436 (2024)). Camptothecin analogues have also been described in Venditto and Simanek ( Mol Pharm April 5, 2010; 7(2): 307–349) and Chen et al. J. Med. Chem. In 2024, 67, 5, 3244–3273), the camptothecin analogue is irinotecan. In one embodiment, the camptothecin analogue is SN-38. In one embodiment, the camptothecin analogue is ethanotecan. In one embodiment, the camptothecin analogue is delutecan.

[0113] As used herein, the term "linker" refers to the chemical structure that connects the disclosed peptide compounds to camptothecin analogs. Linkers can be attached to different functional groups on the peptide compound. For example, a linker can be attached to one or more primary amines (amines (-NH2)): this group is present on the N-terminus (called α-amine) of each polypeptide chain and on the side chain of lysine (Lys, K) residues (called ε-amine). For example, a linker can be attached to one or more carboxyl groups (-COOH): this group is present on the C-terminus of each polypeptide chain and on the side chains of aspartic acid (Asp, D) and glutamic acid (Glu, E). For example, a linker can be attached to one or more thiosulfate groups (–SH): this group is present on the side chain of cysteine ​​(Cys, C). Typically, cysteine ​​residues are linked together between side chains by disulfide bonds (–S–S–) as part of the secondary or tertiary structure of proteins. They must be reduced to thiosulfate groups to allow them to be crosslinked by most types of reactive groups. For example, a linker can attach to one or more carbonyl (–CHO) groups on a peptide compound: ketone or aldehyde groups can be generated in glycoproteins, for example, by oxidizing polysaccharide post-translational modifications (glycosylation) with sodium periodate. Linkers can also attach to suitable groups / parts (e.g., azido groups, double bonds, and triple bonds) on modified side chain residues of the peptide.

[0114] In one embodiment, L is a cleavable linker, wherein a first end of the linker is connected to a side chain of one or more lysine (K) and / or cysteine ​​(C) residues of the peptide, and / or to the N- and / or C-terminus of the peptide, and a second end of the linker is connected to an oxygen or nitrogen atom of a camptothecin analogue.

[0115] By using a cleavable linker, camptothecin analogues can be cleaved from peptides on the surface of cells expressing sorting protein receptors, inside cells, or within organelles, for example by enzymes (e.g., proteases), thereby releasing the camptothecin analogue (e.g., in the vicinity of the cell or inside the cell or organelle). The linker described herein is used to attach a camptothecin analogue to a peptide that binds to cancer cells (e.g., to cancer cells expressing sorting protein receptors) and take it into the cell (e.g., via endosomes) and / or lysosomal compartments. Once taken into the cell, the linker is cleaved or degraded to release the camptothecin analogue.

[0116] Various types of cleavable linkers can be used for conjugates as described herein. For example, linkers can be acid-cleavable linkers, which utilize the natural acidity of endosomes and lysosomes (pH 4.5–6.2), opposite to the neutral pH of plasma (7.4). The most commonly used acid-cleavable linkers are hydrazones and carbonates. Cleavable linkers can also be reducible or disulfide linkers. These linkers are stable at physiological pH but are susceptible to nucleophilic attack from thiols. Glutathione (GSH), containing exposed highly reactive thiol groups, is present in the cytosol of cells, particularly in cells under oxidative stress, such as tumor cells. Other types of cleavable linkers include phosphatase-cleavable linkers (cleaved by phosphatases and pyrophosphates in lysosomes) and sulfatase-cleavable linkers (cleaved by sulfatases in lysosomes). Cleavable linkers can also be enzyme-cleavable linkers, i.e., linkers cleaved by the action of intracellular and / or extracellular enzymes expressed by the target cell. Examples of enzyme-cleavable adaptors include glycosidase-cleavable adaptors (such as β-glucuronidase or β-galactosidase cleavable adaptors) and protease-cleavable adaptors.

[0117] In one embodiment, the cleavable linker is a protease-cleavable linker. The protease-cleavable linker can be a protease such as cathepsins (e.g., cathepsin A, cathepsin B), trypsin, or other cell-expressed proteases, such as those expressed in endosomes and / or lysosomal compartments. example Camptothecin analogues are released under the action of tripeptidyl-peptidase I.

[0118] In one embodiment, the cleavable linker comprises one or more amino acid residues, such as natural and / or non-natural amino acid residues. In one embodiment, the cleavable linker comprises 2-4 amino acid residues. In one embodiment, the cleavable linker comprises one of the following dipeptides: Val-Lys, Val-Cit, Phe-Lys, Trp-Lys, Asp-Lys, Val-Arg, or Val-Ala. In other embodiments, the cleavable linker comprises one of the following tetrapeptides: Val-Phe-Gly-Sar, Val-Cit-Gly-Sar, Val-Lys-Gly-Sar, Val-Ala-Gly-Sar, Val-Phe-Gly-Pro (SEQ ID NO:7), Val-Cit-Gly-Pro, Val-Lys-Gly-Pro (SEQ ID NO:8), or Val-Ala-Gly-Pro (SEQ ID NO:9).

[0119] The linker can be prepared from a linker precursor containing reactive groups at one or both ends of a molecule. The reactive groups can be selected to allow conjugation to a camptothecin analog at one end and also promote conjugation to a peptide at the other end. It is desirable for the camptothecin analog to contain amine, hydroxyl, hydrazone, hydrazide, or thiosulfate groups to promote conjugation to the linker. For example, camptothecin analogs containing primary amine groups and / or hydroxyl groups can facilitate their conjugation to the linker. In one embodiment, the linker is conjugated to an amino group of a camptothecin analog. In another embodiment, the linker is conjugated to a hydroxyl group of a camptothecin analog.

[0120] The linker may also contain additional groups, such as spacer groups, to increase the distance between the peptide and the camptothecin analog. Suitable spacer groups may contain alkylene, alkenylene, ynylene, heteroalkylene (e.g., polyethylene glycol, PEG), carbocyclic, heterocyclic, aryl, heteroaryl, or combinations thereof. For example, the spacer group may contain a PEG group, an alkylene group, or a combination thereof. The spacer group may be substituted or unsubstituted; for example, the spacer group may contain a substituted alkylene group, a substituted heteroalkylene group, or a combination thereof. For example, the spacer group may contain a PEG group (or a PEG spacer group), an alkylene group (or an alkylene spacer group), one or more heteroatoms, and / or one or more or cyclic groups. In one embodiment, the spacer group contains 1-20 PEG groups. In another embodiment, the spacer group contains 1-10, 1-8, 1-6, 1-5, or 2-4 PEG groups. In yet another embodiment, the spacer group contains 3 PEG groups.

[0121] In one embodiment, the connector has one of the following structures: CO(CH2) a CONH(CH2) b CO, where a and b are independently 1, 2, 3 or 4; CO(CH2) d (OCH2CH2) e CONH(CH2) f CO, where d and f are independently 1, 2, 3, or 4, and e is an integer from 2 to 8; or .

[0122] It should be understood that several camptothecin analog molecules A (which may be the same or different) can be linked to a single peptide molecule B via several linkers L (which may be the same or different). In one embodiment, 1, 2, 3, or 4 camptothecin analog molecules are linked to a single peptide molecule via linkers.

[0123] Therefore, in one embodiment, the conjugate or its salt has one of the following formulas: Ia, Ib, Ic, or Id: A1 — L1 — B (Ia); A1 — L1 — B — L2 — A2 (Ib); (Ic); (Id); Wherein L1, L2, L3, and L4 are linkers (identical or different), and A1, A2, A3, and A4 are camptothecin analog molecules (identical or different). In one embodiment, A1, A2, A3, and A4 are the same camptothecin analog molecule. In another embodiment, L1, L2, L3, and L4 are the same cleavable linker.

[0124] In one implementation, X 1 and / or X 2 Let C be the connector, and the first end of one of the connectors be connected to X. 1 and / or X 2 The side chain. In another implementation, X 2 Let C be the connector, and the first end of one of the connectors be connected to X. 2 The side chain.

[0125] In one embodiment, the first end of one or more linkers is attached to the side chain of one or more lysine (K) residues of peptide B. In another embodiment, the first ends of two linkers are attached to the side chain of two lysine (K) residues of peptide B.

[0126] In one embodiment, the conjugate has one of the following structures: , , , , , , , , or .

[0127] In one embodiment, the conjugate compound disclosed herein or a pharmaceutically acceptable salt thereof is formulated into a pharmaceutical composition. In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. Such compositions can be prepared in a manner well known in the pharmaceutical industry by mixing a conjugate compound of suitable purity with one or more optional pharmaceutically acceptable carriers or excipients (see Remington: The Science and Practice of Pharmacy , Loyd V Allen, Jr, 2012, 22nd Edition, Pharmaceutical Press; Handbook of Pharmaceutical Excipients (Rowe et al., 2012, 7th ed., PharmaceuticalPress). The carrier / excipient may be suitable for administration of the conjugate compound via any conventional route of administration, such as oral, intratumoral, intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ocular, intraventricular, intracapsular, intracapsular, intrathecal, intrathecal, epidural, intracisional, intraperitoneal, intranasal, or pulmonary (e.g., aerosol) administration. In one embodiment, the carrier / excipient is suitable for administration of the conjugate compound or a salt thereof via an intravenous or subcutaneous route. In one embodiment, the carrier / excipient is suitable for administration of the conjugate via an intravenous route. In another embodiment, the carrier / excipient is suitable for administration of the conjugate compound or a salt thereof via a subcutaneous route. In yet another embodiment, the carrier / excipient is suitable for administration of the conjugate compound or a salt thereof via an oral route.

[0128] As used herein, “excipient” has its common meaning in the art and refers to any component that is not itself an active ingredient (drug). Excipients include, for example, binders, lubricants, diluents, fillers, thickeners, disintegrants, plasticizers, coatings, barrier layer articles, lubricants, stabilizers, sustained-release agents, and other components. As used herein, “pharmaceuticalally acceptable excipient” means any excipient that does not interfere with the bioavailability of the active ingredient and is non-toxic to the subject, i.e., a typical excipient and / or used in an amount that is non-toxic to the subject. Excipients are well known in the art, and this system is not limited in these respects. In some embodiments, the composition may include excipients such as one or more binders (adhesives), thickeners, surfactants, diluents, sustained-release agents, colorants, flavoring agents, fillers, disintegrants / dissolution promoters, lubricants, plasticizers, silica flow conditioners, flow aids, anti-caking agents, anti-adhesion agents, stabilizers, antistatic agents, swelling agents, and any combination thereof. As those skilled in the art will recognize, a single excipient can perform two or more functions simultaneously, for example, acting as both a binder and a thickener. As will also be recognized by those skilled in the art, these terms are not necessarily mutually exclusive. Examples of common excipients for injectable formulations include water, saline, phosphate-buffered saline, glucose, glycerol, ethanol, and combinations thereof. In many cases, isotonic agents, such as sugars, polyols such as mannitol, sorbitol, or sodium chloride, are preferably included in the composition. Other examples of pharmaceutically acceptable substances are wetting agents or excipients, such as emulsifiers, preservatives, or buffers, which can extend shelf life or efficacy.

[0129] In another aspect, this disclosure provides a method for treating a subject with cancer expressing a sorting protein, comprising administering to the subject an effective amount of the conjugate compound, salt thereof, or composition disclosed herein. This disclosure also provides the use of the conjugate compound, salt thereof, or composition disclosed herein in treating a subject with cancer expressing a sorting protein, or in the preparation of a medicament for treating a subject with cancer expressing a sorting protein. This disclosure further provides the conjugate compound, salt thereof, or composition disclosed herein for treating a subject with cancer expressing a sorting protein.

[0130] Cancers expressing sorting proteins can be of any type, including primary (or initial) cancer, recurrent cancer, or metastatic cancer, in which at least one group of tumor cells express the sorting protein. Examples of cancers include cardiac sarcoma, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma (e.g., Ewing sarcoma, Kaposi's sarcoma), lymphoma, chondroma, mesothelioma; and cancers of the gastrointestinal system, such as esophageal cancer (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach cancer (carcinoma, lymphoma, leiomyosarcoma), gastric cancer, and pancreatic cancer (ductal carcinoma). Adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, vipoma, small intestinal cancer (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestinal cancer (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); urogenital tract cancers, such as renal cancer (adenocarcinoma, Wilms' tumor [nephroblastoma], lymphoma, leukemia), bladder and / or urethral cancer (squamous cell carcinoma). Cancers include: cell carcinoma, transitional cell carcinoma, adenocarcinoma; prostate cancer (adenocarcinoma, sarcoma); testicular cancer (seminomatous carcinoma, teratoma, embryonal carcinoma, teratoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenomatous tumor, lipoma); liver cancer, such as hepatocellular carcinoma (HCC), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; pancreatic endocrine tumors (such as pheochromocytoma, insulinoma, vasoactive intestinal peptide tumor, islet cell tumor, and glucagonoma); bone cancer, such as osteoblastic sarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, malignant lymphoma (reticulocyte sarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma (osteocartilaginous exostosis). Exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumor;Cancers of the nervous system, such as central nervous system (CNS) tumors, primary CNS lymphomas, skull cancers (osteomas, hemangiomas, granulomas, xanthomas, osteitis deformans), meningeal cancers (meningiomas, meningeal sarcomas, gliomas), brain cancers (astrocytomas, medulloblastomas, gliomas, ependymomas, germ cell tumors [pineal tumors], glioblastoma multiforme, oligodendrogliomas, schwannomas, retinoblastomas, and congenital tumors), spinal neurofibromas, meningiomas, gliomas, and sarcomas; cancers of the reproductive system, such as gynecological cancers, uterine cancer (endometrial cancer), cervical cancer (cervical cancer, precancerous cervical dysplasia), ovarian cancer (ovarian cancer [serous cystadenocarcinoma, mucinous cystadenoma]). [Unclassified cancers], granulosa cell carcinoma, Sertoli-Leydig cell carcinoma, dysgerminoma, malignant teratoma), vulvar cancer (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vaginal cancer (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonic rhabdomyosarcoma), fallopian tube cancer (cancer); placental cancer, penile cancer, prostate cancer, testicular cancer; hematologic cancers, such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), myeloproliferative disorders, multiple myeloma, myelodysplastic syndromes), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; oral cancers, such as lip cancer, tongue cancer, gingival cancer, palate cancer (palate) Cancers including: oropharyngeal carcinoma, nasopharyngeal carcinoma, sinus carcinoma; skin cancers such as malignant melanoma, cutaneous melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles dysplastic nevi, lipomas, hemangiomas, dermatofibromas, and keloids; adrenal cancers: neuroblastoma; and cancers of other tissues, including connective tissue and soft tissue, retroperitoneum and peritoneum, eye cancer, intraocular melanoma and adnexa, breast cancer (e.g., ductal carcinoma of the breast), head and / or neck cancers (squamous cell carcinoma of the head and neck), anal cancer, thyroid cancer, parathyroid cancer; secondary and unexplained malignant tumors of lymph nodes, secondary malignant tumors of the respiratory and digestive systems, and secondary malignant tumors in other sites. Tumors. In one implementation, the cancers expressing the sorting protein include hematologic cancers (e.g., AML), ovarian cancer, endometrial cancer, cervical cancer, skin cancer (e.g., melanoma, squamous cell carcinoma), brain cancer (e.g., glioma, glioblastoma), breast cancer (e.g., triple-negative breast cancer), colorectal cancer, small bowel cancer, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), eye cancer (e.g., ocular melanoma), prostate cancer, head and neck cancer, stomach cancer, bone cancer, thyroid cancer, testicular cancer, bladder cancer, kidney cancer, or pancreatic cancer.

[0131] In one implementation, the cancer is defined as a cancer with a poor prognosis. As used herein, the term "cancer with a poor prognosis" refers to a specified cancer subtype that is associated with lower survival rates (e.g., 5-year or 10-year survival) relative to other subtypes of the same cancer. Cancers with poor prognosis are often associated with specific characteristics of the cancer subtype, such as the presence of certain mutations, chromosomal abnormalities, etc., which make them more resistant to treatment. Poor prognosis is also associated with cancers diagnosed at a late stage (e.g., with distant metastases). For example, in breast cancer, triple-negative breast cancer (TNBC) is considered a cancer with a poor prognosis because, relative to other breast cancer subtypes, it is associated with a lower 5-year survival rate. In ovarian cancer, invasive epithelial ovarian cancer and fallopian tube cancer are generally associated with lower 5-year relative survival rates compared to ovarian stromal tumors and germ cell tumors. The 5-year overall survival rate for pancreatic cancer is very low (approximately 3%), partly because more than half of patients are diagnosed at a late stage. A diagnosis of stage III / IV pancreatic cancer (with distant metastases) is associated with a very poor prognosis. Similarly, for prostate cancer, a stage IV diagnosis (distant metastasis) is associated with poor prognosis (5-year relative survival of less than 30% compared to at least 80-85% of stage I-III diagnoses). For lung cancer, small cell lung cancer is associated with particularly poor prognosis, especially at late diagnosis (e.g., with regional or distant metastases). Late-diagnosed non-small cell lung cancer (e.g., with distant metastases) is also associated with poor prognosis. In colorectal cancer, mucinous adenocarcinoma (characterized by abundant extracellular mucin) is associated with reduced response to chemotherapy and poor prognosis. Peritoneal involvement and BRAF mutations also constitute markers of poor prognosis in colorectal cancer. For renal cell carcinoma, clear cell RCC is associated with poorer prognosis (e.g., lower 5-year relative survival) compared to papillary RCC. In skin cancer, thicker tumors, nodular involvement, and late diagnosis (e.g., regional or distant metastases) are associated with lower survival in melanoma.

[0132] In one implementation, the cancer with poor prognosis is stage III / IV cancer. In another implementation, the cancer with poor prognosis is stage III cancer. In yet another implementation, the cancer with poor prognosis is stage IV cancer.

[0133] In one embodiment, a cancer with a poor prognosis is a cancer with a 5-year relative survival rate of less than 60%. In one embodiment, a cancer with a poor prognosis is a cancer with a 5-year relative survival rate of less than 50%. In one embodiment, a cancer with a poor prognosis is a cancer with a 5-year relative survival rate of less than 40%. In one embodiment, a cancer with a poor prognosis is a cancer with a 5-year relative survival rate of less than 30%. In one embodiment, a cancer with a poor prognosis is a cancer with a 5-year relative survival rate of less than 20%. In one embodiment, a cancer with a poor prognosis is a cancer with a 5-year relative survival rate of less than 10%. In one embodiment, a cancer with a poor prognosis is a cancer with a 5-year relative survival rate of less than 5%.

[0134] In one implementation, the cancer is classified as immune-cold (or neglected) cancer. The term "immune-cold cancer" or "cold cancer" refers to cancer that does not elicit an antitumor immune response in the patient and / or is unresponsive to cancer immunotherapy such as ICI therapy (see, for example, Bonaventura et al., "Cold Tumors: A Therapeutic Challenge for Immunotherapy." Frontiers in Immunology, Vol. 10, 168 (2019)). Immune-cold cancer is characterized by the absence of infiltration of antitumor immune cells such as TILs, TAMs, and / or NK cells in the tumor and / or the presence of high levels of immunomodulatory cells (e.g., Tregs). Numerous breast cancers, ovarian cancers, prostate cancers, pancreatic cancers, and glioblastomas are considered immune-cold cancers. Several cancer subtypes are also considered immune-cold cancers, including lung cancer subtypes such as non-small cell lung cancer (NSCLC) (Cascone et al., Tumor Immunology and Immunotherapy of Non-Small-Cell Lung Cancer, Cold Spring Harbour). Perspect Med. May 27, 2022; 12(5): a037895), renal cell carcinoma such as chromophobe renal cell carcinoma (non-clear cell renal cell carcinoma, nccRCC) (Zarrabi et al., Immune Checkpoint Inhibition in Advanced Non-Clear Cell Renal Cell Carcinoma: Leveraging Success from Clear Cell Histology into New Opportunities, Cancers 2021 Vol. 13(15): 3652), high tumor mutational burden RCC (Yakirevich et al., Tumor mutationalburden and immune signatures interplay in renal cell carcinoma. Ann Transl Med. 2020; 8(6): 269), colorectal cancer (CRC) such as consensus molecular subtype (CMS) 2 and CMS3 CRC (Roelands et al., Immunogenomic Classification of Colorectal Cancer and Therapeutic Implications, Int J Mol Sci. 2017;18(10):2229), head and neck squamous cell carcinoma (Ribbat-Idel et al., Immunologic “Cold” Squamous Cell Carcinomas of the Head and Neck Are Associated With an Unfavorable Prognosis, Frontiers in Medicine 2021;8:622330), esophageal cancer (Puhr et al., Immunotherapy for Esophageal Cancers: What Is Practice Changing in 2021?, Cancers 2021; Vol. 13(18):4632), liver cancer such as stage II hepatocellular carcinoma (Nguyen et al., Nature Communications Vol. 13, No. 1441 (2022)).It has been confirmed that homozygous deletion of 9p21.3, one of the most common genomic defects occurring in ~13% of all cancers, is associated with an immune cold phenotype, including melanoma (SKCM), bladder cancer (BLCA), pancreatic cancer (pancreatic carcinoma), gastric cancer (gastric adenocarcinoma), lung adenocarcinoma (LUAD), and squamous cell carcinoma (LUSC).

[0135] The exact dosage / follow-up of the conjugate varies depending on many factors, such as the specific cancer and cancerous disease involved; the extent or severity of the cancer; the size, age, and general health of the cancer patient; the individual patient's response; the specific compound administered; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; whether the conjugate is administered alone or in combination with other active agents; the pharmacodynamic characteristics of the conjugate and its administration mode and route; and other relevant characteristics that are readily determined by a clinician or person skilled in the art using known techniques and by observing results obtained in similar circumstances. The conjugate / composition is appropriately administered to the patient once or as part of a series of treatments. Preferably, it is desirable to determine the dose-response profile in vitro and then in a useful animal model prior to human testing. This disclosure provides dosages of the conjugate and compositions comprising the conjugate. For example, depending on the type and severity of the disease, it is approximately 1 μg / kg to 1000 mg / kg (mg / kg) body weight / day. In addition, effective doses can be 0.5 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, or 200 mg / kg, and can be increased in increments of 25 mg / kg up to 1000 mg / kg, or can be within any two of the above values. Typical daily doses range from approximately 1 μg / kg to 100 mg / kg or higher, depending on the various factors listed above. Depending on the condition, repeated administration over several days or longer is recommended, and treatment continues until the desired symptom suppression is achieved. However, other dosage regimens may be useful. Progress in this therapy can be easily monitored using routine techniques and assays.

[0136] The conjugate compounds described herein, or their salts, or compositions comprising them, can be used in combination with one or more other active agents or therapies (radiotherapy, surgery, vaccines, etc.) for the treatment of a targeted disease / condition or for the management of one or more symptoms of a targeted disease / condition (e.g., analgesics, antinausea drugs, etc.). In one embodiment, the conjugate compounds described herein are used in combination with one or more chemotherapeutic agents, immunotherapies, checkpoint inhibitors, cell-based therapies, etc. Examples of chemotherapeutic agents suitable for use in combination with the conjugate compounds described herein include, but are not limited to, vinca alkaloids, microtubule-disrupting active agents (e.g., colchicine and its derivatives), anti-angiogenic agents, therapeutic antibodies, EGFR-targeting agents, tyrosine kinase-targeting agents (e.g., tyrosine kinase inhibitors), transition metal complexes, proteasome inhibitors, antimetabolites (e.g., nucleoside analogs), alkylating agents, platinum-based active agents, anthracycline antibiotics, topoisomerase inhibitors, macrolides, retinoic acid (e.g., all-trans retinoic acid or its derivatives); gerdemycin or its derivatives (e.g., 17-AAG); and other cancer therapeutic agents recognized in the art. In some embodiments, the chemotherapeutic agents used in combination with the conjugates described herein include doxorubicin, colchicine, cyclophosphamide, actinomycin, bleomycin, daunorubicin, doxorubicin, epirubicin, mitomycin, methotrexate, mitoxantrone, fluorouracil, carboplatin, carmustine (BCNU), methyl-CCNU, cisplatin, etoposide, interferon, camptothecin and its derivatives, phenylacetic acid mustard (Phenesterine), taxanes and their derivatives (e.g., paclitaxel, paclitaxel and its derivatives, taxotere and its derivatives, etc.), topotecan, vincristine, tamoxifen, piperazine, nab-5404, nab-5800, nab-5801, irinotecan, HKP, ortataxel, gemcitabine, oxaliplatin, and Herceptin. ® Changchun Ruibin, Doxil ® Capecitabine, Alimta ® Avastin ® Velcade ® Tarceva ® Neusta ®Lapatinib, sorafenib, erlotinib, erbitux, and their derivatives are examples of such compounds. In one embodiment, the conjugate compounds described herein or compositions comprising them are used in combination with an EGFR or tyrosine kinase target, such as an EGFR inhibitor (RTK inhibitor). The conjugate compounds described herein, their salts, or compositions comprising them may also be used in combination with one or more therapeutic antibodies or antibody fragments, such as therapeutic antibodies or antibody fragments for the treatment of tumors. Examples of antibodies used to treat cancer include those targeting CD52 (e.g., alemtuzumab), VEGF / VEGFR (e.g., bevacizumab, ramucirumab), EGFR (e.g., cetuximab, nectinumumab, panitumumab), CD38 (e.g., daratumumab, isatuximab), RANKL (e.g., denosumab), GD2 (e.g., dinutuximab, naxitamab-gqgk), SLAMF7 (e.g., elotuzumab), HER2 (e.g., margetuximab-cmkb, pertuzumab), CCR4 (e.g., mogamulizumab), and CD20. (Obinutuzumab, Ofatumumab, Rituximab), BCMA (e.g., Teclistamab), CD19 (e.g., Tafasitamab), CTLA-4 (e.g., Tremelimumab), LAG-3 (e.g., Relatlimab), PD-1 (e.g., Tislelizumab, Penpulimab, Sintilimab, Toripalimab, Retifanlimab, Dostarlimab), PD-L1 (e.g., Durvalumab, Avelumab, Atezolizumab), EpCAM Antibodies such as ocportuzumab, edrecolomab, Nectin-4 (enfortumab), and CD79b (polatuzumab).

[0137] Combinations of active agents and / or compositions comprising them can be administered or co-administered (e.g., consecutively, simultaneously, at different times) in any conventional dosage form. In the context of this invention, co-administration refers to the administration of more than one therapeutic agent during a coordinated treatment process to improve clinical outcomes. Such co-administration may also be concurrent, i.e., occurring within overlapping timeframes. For example, a first active agent (e.g., the conjugate compound described herein) may be administered to a patient before, simultaneously with, before, or after the administration of a second active agent (e.g., a chemotherapeutic agent or immunotherapy). In one embodiment, the active agents are combined / formulated in a single composition and thus administered simultaneously.

[0138] In one implementation, the cancer is resistant to immunotherapy, meaning that the immunotherapy does not result in suppression of tumor growth in the patient. Cancers resistant to immunotherapy can be those that have never responded to immunotherapy (primary resistance) or those that have developed resistance to immunotherapy after a period of treatment (responsiveness) (acquired resistance).

[0139] In another implementation, the cancer develops resistance to PD-1 or PD-L1 inhibitor-based therapies (anti-PD-1 / PD-L1 therapy). In yet another implementation, cancers resistant to PD-1 or PD-L1 inhibitor-based therapies include melanoma, lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck cancer, colon cancer, liver cancer, gastric cancer, squamous cell carcinoma of the skin, or myeloma.

[0140] Immune checkpoint inhibitors have been approved or are currently being tested in phase III and IV clinical trials for several cancers, including lung cancer (e.g., non-small cell lung cancer (NSCLC) and small cell lung cancer, squamous cell lung cancer), head and neck cancer (e.g., head and neck squamous cell carcinoma, renal cell carcinoma, gastric adenocarcinoma, nasopharyngeal carcinoma, urothelial carcinoma, colorectal cancer, mesothelioma (e.g., pleural mesothelioma), breast cancer (e.g., triple-negative breast cancer, TNBC), esophageal cancer, multiple myeloma, gastric and gastroesophageal junction cancer, gastric adenocarcinoma, melanoma, Merkel-cell carcinoma (MCC), lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma, diffuse large B-cell lymphoma), liver cancer (e.g., hepatocellular carcinoma), melanoma, ovarian cancer, fallopian tube cancer, peritoneal cancer, bladder cancer, transitional cell carcinoma, prostate cancer, and biliary tract cancer (see, for example, Darvin et al., Experimental & Molecular Medicine, Vol. 50, No.: 165 (2018)). Therefore, in one implementation, cancer is one of the aforementioned cancers for which immune checkpoint inhibitors have been approved for use or are currently being tested in phase III and IV clinical trials.

[0141] Currently approved immune checkpoint inhibitors include anti-CTLA-4 ipilimumab (melanoma and lung cancer), anti-PD-1 nivolumab (melanoma, lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck cancer, colon cancer, and liver cancer), pembrolizumab (melanoma, lung cancer, head and neck cancer, Hodgkin's lymphoma, renal cell carcinoma, and gastric cancer), cemiplimab (squamous cell skin cancer, melanoma, and lung cancer), anti-PD-L1 atezolizumab (NSCLC, small cell lung cancer, TNBC), avelumab (NSCLC, MCC), and durvalumab (bladder epithelial cancer, lung cancer). Therefore, in one implementation, the cancer is one of the aforementioned cancers to which the approved immune checkpoint inhibitor is applied.

[0142] As used herein, the terms "subject" or "patient" refer to mammals, such as rodents, felines, canines, and primates. Preferably, the subject or patient in this disclosure is a human.

[0143] Example

[0144] This disclosure is further illustrated in detail by means of the following non-limiting embodiments.

[0145] Example 1: Materials and Methods

[0146] Cell lines and cell cultures

[0147] Human colorectal cancer cell lines LS123 (#CCL-255), HCT-116 (#CCL-247), SK-CO-1 (#HTB-39), T-84 (#CCL-248), LoVo (#CCL-229), HT-29 (#HTB-38), and RKO (#CRL-2577) were purchased from ATCC. Human triple-negative breast cancer (TNBC)-derived MDA-MB-231 / Luc cells were obtained from Cell Biolabs Inc. (San Diego, CA, #AKR-231), and MDA-MB-468 (#HTB-132), MDA-MB-157 (#HTB-24), DU4475 (#HTB-123), and HCC70 (#CRL-2315) cells were obtained from ATCC. Canine renal epithelial MDCK-MDR1 cells were provided by Dr. Amanda Yancy (AstraZeneca Pharmaceuticals, LP, Wilmington DE, USA).

[0148] All cell lines were grown into an adhesive monolayer in a humidified atmosphere (5% CO2) at 37°C using the culture medium described in Table 1.

[0149] Table 1: Cell culture conditions for cell lines

[0150] 1 Because of the use of the L-15 medium formulation, MDA-MB-157 cells are cultured in an atmosphere of free gas exchange with the atmosphere.

[0151] For experimental applications, cells were isolated from culture flasks by treatment with trypsin (Wisent) for 5–10 minutes, followed by 10-fold dilution and neutralization with complete culture medium. Cell counts and viability were assessed using a TC20 automated cell counter (BioRad) after staining with 0.4% trypan blue (Thermo Fisher Scientific) exclusion staining.

[0152] Protein blot

[0153] Cells were homogenized in 1% SDS lysis buffer supplemented with a complete protease inhibitor mixture from Calbiochem (San Diego, CA). Cells were incubated at room temperature (RT) for 30 min, vortexed every 5 min, sonicated, and centrifuged at 15000 g for 10 min at RT. Equal amounts of protein (20 µg) were separated by SDS–polyacrylamide gel electrophoresis (PAGE). The protein was then electrotransferred onto a polyvinylidene fluoride (PVDF) membrane and blocked at room temperature for 1 h with 5% skim milk in Tris-buffered saline containing 0.1% Tween™-20 (TBST) (150 mM NaCl, 20 mM Tris–HCl, pH 7.5). The membrane was washed with TBST and incubated overnight with a primary antibody against SORT1 (1 / 1,000 dilution), P-gp (1 / 100 dilution), BCRB (1 / 500 dilution), EGFR (1 / 1,000), topoisomerase 1 (1 / 500 dilution), GAPDH (1 / 40,000 dilution), or β-actin (1 / 50,000 dilution). The membrane was then washed with TBST and incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (1 / 5000 dilution) in TBST containing 5% skim milk. The membrane was washed again with TBST and the signal was detected using chemiluminescence (Amersham Biosciences, Baie d'Urfé, QC).

[0154] animal

[0155] Female (HT-29) or male (HCT-116) athymic nude mice aged 6-8 weeks (Crl:NU(NCr)- Foxn1 nu Female CD1 naked (Crl:NU‐Foxn1nu; MDA-MB-231) mice were obtained from Charles River Laboratories, Inc. (St-Constant, QC). Animals were acclimatized to their environment for 5 days prior to the experiment. All mice were kept in a pathogen-free environment and handled in accordance with the Canadian Council on Animal Care (CCAC) guidelines for the care and use of laboratory animals.

[0156] Preparation of compounds for injection

[0157] The preparation of peptide-drug conjugates was described in Example 2 below. Docetaxel (Tecoland, Irvine, CA), irinotecan (MedChemExpress, Monmouth Junction, NJ), or ethanotecan (MedChemExpress, Monmouth Junction, NJ) were prepared on the same day as the animal administration. Docetaxel was dissolved in EtOH for injection to 50 mg / ml, diluted with polysorbate-80 to 25 mg / ml, and then diluted with sterile 5% glucose 5% aqueous solution (D5W) to the desired injection concentration (i.e., 2.5 mg / ml). Irinotecan was dissolved in DMSO to 250 mg / ml and then diluted with sterile 5% D5W to the desired injection concentration (i.e., 13 mg / ml or lower). Ethatecan was dissolved in HCl-acidified DMSO to 20 mg / ml and then diluted with HCl-acidified sterile 5% D5W to the desired injection concentration (i.e., 1.4 mg / ml). Dissolve TH2101 to 80 mg / ml in formic acid-acidified DMSO and Solutol (1:2 ratio), then dilute with sterile formic acid-acidified 5% D5W to the desired injection concentration (i.e., 5.4 mg / ml or lower). Dissolve TH2102 to 50 mg / ml in DMSO, then dilute with sterile 5% D5W to the desired injection concentration (i.e., 5.5 mg / ml). Dissolve TH2204 to 115 mg / ml in DMSO, then dilute with sterile 5% D5W to the desired injection concentration (i.e., 5.85 mg / ml). Dissolve TH2205 to 80 mg / ml in formic acid-acidified DMSO and Solutol (1:2 ratio), then dilute with sterile formic acid-acidified 5% D5W to the desired injection concentration (i.e., 5.85 mg / ml). TH2301 was dissolved to 65 mg / ml in HCl-acidified DMSO, and then diluted to the desired injection concentration (i.e., 4.7 mg / ml) with HCl-acidified sterile 5% D5W. TH2303 was dissolved to 33.3 mg / ml in HCl-acidified DMSO and Solutol (1:2 ratio), and then diluted to the desired injection concentration (i.e., 9.8 mg / ml) with HCl-acidified sterile 5% D5W. All diluted solutions were filtered before animal administration (DMSO-safe 0.2 µm injection filter, nylon membrane, PALL Corporation).

[0158] Evaluation of the in vivo efficacy of peptide-drug conjugates using the CRC HT-29 xenograft model

[0159] 7 x 10 HBSS (Sigma #H6648) were subcutaneously inoculated into 100 µl of HBSS. 6HT-29 cells were used to establish tumors. Under mild isoflurane anesthesia, all cells were injected into the right ventral region of immunocompetent male mice. For all HT-29 xenograft studies, mice were treated weekly via intravenous (IV) tail vein injection of the medium, docetaxel, irinotecan or esanotecan, TH2101, TH2102, TH2204, TH2205, and TH2301, at the doses shown in the corresponding figures. For all specified studies, tumor growth was monitored using two-dimensional measurements obtained with electronic calipers, and tumor volume was calculated using the following formula: Tumor volume (mm²) 3 = π / 6 x length x width 2 Animal body weight was measured with an accuracy of ±10 mg. Mice in the vector group reached a volume of approximately 1000 mm². 3 When the tumor is large, collect samples.

[0160] Evaluation of the in vivo efficacy of peptide drug conjugates using the CRC HCT-116 xenograft model

[0161] 3 x 10⁶ HBSS (Sigma #H6648) were subcutaneously inoculated into 100 µl of HBSS. 6 HCT-116 cells were used to establish tumors. Cells were injected into the right ventral region of immunocompetent male mice under mild isoflurane anesthesia. Mice were treated weekly via intravenous (IV) tail vein injection of a mediator, irinotecan, ethanotecan, or TH2303, at the doses shown in the corresponding figures. For all specified studies, tumor growth was monitored using two-dimensional measurements obtained with electronic calipers, and tumor volume was calculated using the following formula: Tumor volume (mm²) 3 = π / 6 x length x width 2 Animal body weight was measured with an accuracy of ±10 mg. Mice in the vector group reached a volume of approximately 1000 mm². 3 When the tumor is large, collect samples.

[0162] Evaluation of the in vivo efficacy of peptide drug conjugates using the TNBC MDA-MB-231 xenograft model

[0163] 7 x 10 HBSS (Sigma #H6648) were subcutaneously inoculated into 100 µl of HBSS. 6 MDA-MB-231 cells were used to establish tumors. Cells were injected into the right ventral region of immunocompetent male mice under mild isoflurane anesthesia. Mice were treated weekly with a mediator, irinotecan, ethanotecan, or TH2303 via intravenous (IV) tail vein injection, at the doses shown in the corresponding figures. For all specified studies, tumor growth was monitored using two-dimensional measurements obtained with electronic calipers, and tumor volume was calculated using the following formula: Tumor volume (mm²) 3 = π / 6 x length x width 2Animal body weight was measured with an accuracy of ±10 mg. Mice in the vector group reached a volume of approximately 1000 mm². 3 When the tumor is large, collect samples.

[0164] Sorting protein RNA interference

[0165] HT-29 cells were transiently transfected for 24 hours using 100 nM scrambled siRNA sequence (AllStar negative control siRNA, 1027281) or human siRNA generated targeting the sorting protein (Hs_SORT_5 FlexiTube siRNA: SI03115168; Qiagen, Valencia, CA) with Lipofectamine™ 2000 (ThermoFisher Scientific, Burlington, ON).

[0166] Fluorescent TH19P01 peptide uptake assay

[0167] HT-29 cells transfected with SiScrambled- or siSORT1- were washed twice with HBSS and incubated in HBSS with or without 200 nM Alexa488-labeled TH19P01. At 37°C... o After incubation at C for 1 hour, cells were washed with HBSS, treated with trypsin, washed again, and fluorescence was assessed in the FL1 channel using a C6 Accuri™ flow cytometer (BD Biosciences, San Jose, CA).

[0168] TH2101 uptake assay by confocal microscopy

[0169] HT-29 cells were plated on glass coverslips for 24 hours and grown to 80% confluence. Then, as described above, cells were transfected with scrambled siRNA or siSORT1 for 24 hours. Cells were washed with HBSS and then incubated in HBSS with or without 200 nM TH2101 fluorescent conjugate. After incubation at 37°C for 1 or 2 hours, cells were washed with HBSS, fixed with 4% paraformaldehyde (PFA) at RT for 15 min, washed with PBS, and fixed on slides using Prolong Gold quenching-resistant reagent. Finally, cells were digitized using confocal microscopy (Nikon A1) with excitation and emission wavelengths of 405 nm and 525 nm, respectively. Fluorescence levels were quantified using NIH ImageJ Version 1.4.21 software.

[0170] Cell proliferation assay

[0171] To evaluate the effects of free drugs (irinotecan, SN-38, and ethanotecan) and conjugates on the proliferation of HT-29, HCT-116, or LoVo cells, cells were first seeded (6000 cells / well) in complete growth medium in 96-well Perkin-Elmer plates for 24 hours. Cells were then treated with different concentrations of the drug in complete cell culture medium. After incubation for 72–96 hours, viable cell counts were estimated using the MTT (Sigma-Aldrich) assay. IC50 was calculated using GraphPad Prism software. 50 value.

[0172] Statistical analysis

[0173] Data are expressed as mean ± standard error of mean (SEM) or standard deviation (SD), as shown in the legend. Statistical analysis was performed using t-tests to compare two samples, and one-way ANOVA for three or more samples, followed by Bonferroni, Dunnett, or Tukey multiple comparisons. Values ​​p < 0.05 were considered significant, and an asterisk (...) was used. ) indicates this level of significance in the graph.

[0174] Example 2: Synthesis of peptide-drug conjugates

[0175] General analytical methods and methods for compound purification and characterization

[0176] As used herein, the term RT (min) refers to the LCMS retention time associated with a compound, in minutes. Unless otherwise indicated, the instruments and columns were used to obtain the desired compound. Instruments and columns: Waters Acquity UPLC BEH C18 1.7 mm, 2.1 x 50 mm; Agilent 1290 Infinity II LUNA C18 5 mm, 4.6 x 150 mm. AKTA pure protein purification system with 30 RPC resignation was used.

[0177] The following abbreviations can be used as follows: aq = water based cone = concentrated DCM = dichloromethane DIPEA = diisopropylethylamine DMF = dimethylformamide DMSO = dimethyl sulfoxide EtOAc = Ethyl acetate Mol = mole MeOH = Methanol RT = room temperature TEA = Triethylamine THF = Tetrahydrofuran

[0178] Synthesis of TH2102 (see...) Figure 5 )

[0179] Step 1: Synthesis of (S)-[1,4'-bipiperidine]-1'-carboxylic acid 4-(((tert-butoxycarbonyl)glycyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-9-yl ester.

[0180] Irinotecan (1.00 g, 1.70 mmol) and Boc-Gly-OH (717 mg, 4.09 mmol) were dissolved in CH2Cl2 (50.0 mL) and treated with DCC (848 mg, 4.11 mmol), followed by treatment with DMAP (292 mg, 2.39 mmol). The mixture was stirred at room temperature for 18 h, after which TLC showed the reaction was complete. The white solid was filtered through DCM-washed filter paper and washed with several portions of CH2Cl2. The filtrate was evaporated, and the residue was dissolved in a small amount of CH2Cl2 and purified with Biotage (40-g column, solvent A: CH2Cl2, solvent B: 20% MeOH / CH2Cl2, 0% B-65% B, 16 min). The pure fraction was evaporated and dried under vacuum using a rotary evaporator to give irinotecan Gly-boc (1.20 g, 95%) as a pale yellow solid. High-performance liquid chromatography / mass spectrometry (HPLC / MS) analysis showed a purity >95%. MH+ 744.4.

[0181] Step 2: Synthesis of (S)-[1,4'-bipiperidine]-1'-carboxylic acid 4,11-diethyl-4-(glycyloxy)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzazo[1,2-b]quinoline-9-yl ester

[0182] To a solid (S)-[1,4'-bipiperidine]-1'-carboxylic acid 4-(((tert-butoxycarbonyl)glycyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-9-yl ester (1.20 g, 1.61 mmol), a mixture of 45% TFA, 5% H2O, and 50% CH2Cl2 (32.1 mL, 189 mmol) was added. The compound dissolved rapidly, yielding a yellow-green fluorescent solution. After 30 minutes, volatiles were removed with rotavap, and the residue was co-evaporated several times with CH2Cl2. The residue was dissolved in 1 mL of MeOH and 60 mL of CH2Cl2. The solution was transferred to a 125-ml separatory funnel and washed with 5% sodium carbonate aqueous solution (3.42 g, 1.61 mmol) (3 x 15 ml, pH 10 on the third wash). All washes were combined, and the pH was adjusted to 9-10 with sodium carbonate solution. The mixture was extracted with CH₂Cl₂ (3 x 20 ml). These extracts were combined with the initial organic layer and washed with water (2 x 15 ml) and saturated brine (15 ml). The organic phase was separated and dried (Na₂SO₄). The solvent was evaporated and co-evaporated with CH₂Cl₂ to give a pale yellow solid, which was dried under high vacuum. The product was used in the next step. Product (MH) + 644) appears in two peaks in the LCMS analysis, but it is used in the next step accordingly.

[0183] Step 3: Synthesis of (S)-4-((2-((9-(([1,4'-bipiperidine]-1'-carbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-4-yl)oxy)-2-oxoethyl)amino)-4-oxobutyric acid

[0184] (S)-[1,4'-bipiperidine]-1'-carboxylic acid 4,11-diethyl-4-(glycyloxy)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzazo[1,2-b]quinoline-9-yl ester (800 mg, 1.24 mmol) was dissolved in anhydrous THF (62.0 mL), treated with triethylamine (520 µL, 3.73 mmol), and then with succinic anhydride (187 mg, 1.86 mmol). Note: The solid becomes a solution upon the addition of a base. The reaction mixture was stirred overnight, and acetic acid (285 µL, 4.97 mmol) (pH 5) was added. THF was removed with rotavap, and the residue was dissolved in 3 mL of DMSO. The fraction was lyophilized to purity using a 40 g C-18 column (using a water:acetonitrile gradient of 0-80%). The product was used in the next step. LCMS: MH+ 744 was determined at 2.89 min with a purity of >99%.

[0185] Step 4:

[0186] In 25 mL RBF, (S)-4-((2-((9-(([1,4'-bipiperidine]-1'-carbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-4-yl)oxy)-2-oxoethyl)amino)-4-oxobutyric acid (220 mg, 0.296 mmol) was dissolved in DMSO (3.00 mL). TBTU (95.0 mg, 0.296 mmol) was added, followed by DIPEA (129 µL, 0.739 mmol). The mixture was stirred at room temperature for 10 minutes (pH ~7). In a separate 25-mL RBF, TH19P01 (256 mg, 0.133 mmol) was dissolved in DMSO (1.00 mL). After 10 min of pre-activation, the TH19P01 solution was rapidly added to the pre-activated ester solution, and the flask was washed with two portions of DMSO (0.5 mL). Since the pH of the reaction mixture was slightly acidic (pH 5 immediately after addition), DIPEA (64.4 µL, 0.370 mmol) was added to restore the pH to 7–8, and the pH was maintained at 7–8. LCMS at t=10 min (after addition of DIPEA) showed that the reaction was pure and complete. After stirring for 20 min (total time since DIPEA addition), the reaction was stopped by adding acetic acid (102 µL, 1.77 mmol), causing the pH to drop to pH ~4. Final dilution for preparative purification: the crude solution was further diluted (~5.8 mL) with DMSO (2.1 mL). Cool to 10 °C and dilute with water (100 μl) and formic acid (20 μl), total solution volume: ~7.0 ml.

[0187] Agilent Prep LCMS purification: Purification was performed using an Agilent Prep-C18 50 x 21.2 mm, 5 µm column. Injection volume: 75 μl (~ 93 injections), yielding 120 mg TH2102. MH+3375.74 (1126.5, charge +3) > 95% purity.

[0188] Synthesis of TH2301 (see) Figure 8 ):

[0189] Step 1:

[0190] In 0 oC. To a solution of triethylamine (524 μL, 3.76 mmol) in DCM (50.0 mL), Boc anhydride (657 mg, 3.01 mmol) and ethatecan mesylate (1.00 g, 1.88 mmol) were added, and the reaction mixture was warmed to room temperature. The resulting reaction mixture was stirred at RT for 4 h. The reaction progress was monitored by LCMS. After the starting material was exhausted, the reaction mixture was directly loaded onto an 80 g normal phase column and purified using DCM:methanol as the eluent (0-10%). The pure fraction was evaporated to give N-Boc-ethatecan (950 mg, 94%). MH+ 535.96.

[0191] Step 2:

[0192] N-Boc-ethanotecan (950 mg, 1.77 mmol), succinic anhydride (599 µL, 5.32 mmol), and 4-dimethylaminopyridine (217 mg, 1.77 mmol) were dissolved in DCM (50.0 mL), and triethylamine (1.28 mL, 8.87 mmol) was added. The reaction mixture was stirred at RT for 24 hours. After 24 hours, some starting material remained, so 3 equivalents of succinic anhydride and triethylamine were added, and the reaction mixture was stirred for another 24 hours. After most of the starting material was exhausted, it was adsorbed onto an 80 g silica gel column and purified by normal-phase chromatography using 0–20% (DCM-methanol) as the eluent. The pure fraction was evaporated to obtain N-Boc-ethanotecan-Su (850 mg, 75%). MH+ 635.92

[0193] Step 3:

[0194] In a 25-mL RBF, N-Boc-ethanotecan-Su (93.3 mg, 0.142 mmol) and TBTU (33.4 mg, 0.142 mmol) were dissolved in DMSO (1.45 mL). N,N-diisopropylethylamine, DIEA, and Hunig base (62.0 µL, 0.356 mmol) were added. The mixture was stirred at room temperature for 10 min (pH ~7). In a separate 25-mL RBF, TH19P01 (123 mg, 0.0641 mmol) was dissolved in DMSO (482 µL). After 10 min of pre-activation, the TH19P01 solution was rapidly added to the pre-activated ester solution, and the flask was washed with a small amount of DMSO (241 µL). Since the reaction mixture was slightly acidic (pH 5 immediately after addition), N,N-diisopropylethylamine, DIEA, and Hunig base (31.0 µL, 0.178 mmol) were added to restore the pH to 7-8. LCMS of the reaction at t=10 min (after addition of DIPEA) showed a clean and complete reaction. After stirring for 20 min (total time since addition of DIPEA), the reaction mixture was purified by HPLC (Akta) using 20-80% acetonitrile (0.1% FA) and water (0.1% FA) to give N-Boc-ethanotecan-Su-TH19P01. N-Boc-ethanotecan-Su-TH19P01 (100.0 mg, 0.00317 mmol) was dissolved in DCM (6.00 mL) in 100 mL RBF. HCl (39.6 µL, 0.158 mmol) was immediately added, and the reaction mixture turned yellow. The reaction was monitored by LCMS. Once all the SM was exhausted, the reaction mixture was evaporated by purging nitrogen into the RBF.

[0195] The crude product was kept under high vacuum for 30 minutes, dissolved in water, and injected into an HPLC system (Akta) using 10-80% acetonitrile (0.1% FA) and water (0.1% FA). The pure fraction was freeze-dried to obtain TH2301. MH+2959.18 (986.58, charge +3) > 95% purity.

[0196] Synthesis of TH2304 (see) Figure 10 ):

[0197] Step 1: Synthesis of 2-((tert-butyldimethylsilyl)oxy)acetic acid:

[0198] Ethyl [(tert-butyldimethylsilyl)oxy]acetic acid (1.00 g, 2.99 mmol) was added to ethanol (20.0 mL) and KOH (252 mg, 4.49 mmol), and the reaction mixture was stirred at RT (at room temperature) for 12 hours. After 12 hours at 23°C, the reaction mixture was concentrated, and the residue was dissolved in water (100 mL). Concentrated HCl was carefully added to the mixture at 0°C to pH 4, and the reaction mixture was extracted with EtOAc (20 mL x 3). The organic layer was washed with H2O (30 mL) and a saturated aqueous solution of NaCl (30 mL). The organic layer was dried over sodium sulfate and concentrated under vacuum to give 2-((tert-butyldimethylsilyl)oxy)acetic acid (500 mg, 55%) as a semi-solid. 1 HNMR-DMSO-d6: 4.07 (s, 2H), 0.82 (s,9H), 0.77 (s, 6H).

[0199] Step 2: Synthesis of 2-((tert-butyldimethylsilyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)acetamide:

[0200] In 0 o C added HATU (599 mg, 1.58 mmol) and DIPEA (686 µL, 3.94 mmol) to a solution of AN-01-0125 (250 mg, 1.31 mmol) in DMF (8.33 mL), and the reaction system was kept at 0°C. oStirring at C for 10 minutes, then adding ethatecan mesylate (698 mg, 1.31 mmol) dissolved in 0.3 mL DMF, and warming the reaction mixture to room temperature. The resulting reaction mixture was stirred at RT for 4 h. The reaction progress was monitored by LCMS. After the starting material was exhausted, the reaction mixture was directly loaded onto an 80 g normal-phase column and purified using DCM:methanol as the eluent to give 2-((tert-butyldimethylsilyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)acetamide (450 mg, 56%). The MH+ of the product was determined at 2.68 min on a 4-min run.

[0201] Step 3: Synthesis of 4-(((1S,9S)-1-(2-(((tert-butyldimethylsilyl)oxy)acetamido)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-9-yl)oxy)-4-oxobutyric acid:

[0202] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)acetamide (450 mg, 0.740 mmol) was dissolved in DCM (100 mL), and succinic anhydride (222 mg, 2.22 mmol), Et3N (321 µL, 2.22 mmol), and 4-dimethylaminopyridine (90.5 mg, 0.740 mmol) were added. The reaction mixture was stirred at RT for 24 hours. The reaction was slow, and then 1 equivalent of the reagent was added. Once most of the starting material was depleted, the reaction mixture was purified by normal-phase chromatography using a 0-10% eluent (DCM:methanol). The fraction was then evaporated under vacuum to give 4-(((1S,9S)-1-(2-((tert-butyldimethylsilyl)oxy)acetamido)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-9-yl)oxy)-4-oxobutyric acid (150 mg, 29%). The MH+ of the product was determined at 2.60 min on a 4-min run (708.01), with a purity >95%.

[0203] Step 4: Synthesis of TH2304

[0204] 4-(((1S,9S)-1-(2-((tert-butyldimethylsilyl)oxy)acetamido)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-9-yl)oxy)-4-oxobutyric acid (120 mg, 0.170 mmol) was dissolved in dry DCM, and N-hydroxysuccinimide (23.4 mg, 0.203 mmol) and DCC (42.8 µL, 0.186 mmol) were added. The reaction mixture was stirred at RT for 2 hours. The reaction was monitored by LCMS. Once all the raw materials were exhausted, the reaction mixture was adsorbed onto silica gel and purified by normal-phase chromatography using DCM:methanol (0-10%) eluent. The purified fraction was concentrated to give (1S,9S)-(2,5-dioxopyrrolidone-1-yl)succinic acid 1-(2-((tert-butyldimethylsilyl)oxy)acetamido)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-9-yl ester (85.0 mg, 62%), as a colorless solid. MH+ (805.13) was determined at 2.72 min on a UPLC run of 4 min.

[0205] (1S,9S)-(2,5-dioxopyrrolidone-1-yl)succinic acid 1-(2-((tert-butyldimethylsilyl)oxy)acetamido)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-9-yl ester (28.0 mg, 0.0303 mmol), N,N-diisopropylethylamine; DIEA; Hunig base (15.8 µL, 0.0908 mmol) were dissolved in DMSO (307 µL), followed by the addition of TH19P01 (26.2 mg, 0.0136 mmol) to DMSO (102 µL). The reaction mixture was monitored by LCMS. Once all the starting materials were exhausted, the reaction mixture was purified by HPLC (Akta) using 10-80% acetonitrile (0.1% FA), and the pure fraction was lyophilized to give TH19P01-Osu-ethanotecan-OTBS (67.0 mg, 67%), which was determined at 2.28 minutes on a 4-min run to be 3303.77 (1101.81, charge +3), >94% purity, and used for the next step.

[0206] TH19P01-Osu-Exatecan: TH19P01-Osu-Exatecan-OTBS (23.0 mg, 0.00557 mmol) was dissolved in DCM (3.07 mL), and TBAF (223 µL, 0.0223 mmol) and acetic acid (1.28 µL, 0.0223 mmol) were added. The reaction was monitored for completion. Once the reaction was complete, the crude product was dried by purging with nitrogen and dissolved in 1 mL DMSO. The product was purified by HPLC (Akta) using 30–80% acetonitrile (0.1% FA) to obtain TH19P01-Osu-Exatecan (12.5 mg, 73%) as the final product. The chromatogram (3075.25, 1015.73, charge +3) was measured at 1.49 minutes of a 4-min run, indicating a purity >95%.

[0207] Synthesis of TH2101 (see...) Figure 4 ):

[0208] Step 1: ( S )-9-((tert-butyldiphenylsilyl)oxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]inzazo[1,2-b]quinoline-3,14(4H)-dione (TBDPS-SN-38)

[0209] Et3N (1.6 mL, 11.5 mmol) and tert-butyldiphenylchlorosilane (TBDPSCl, 2.65 mL, 10.2 mmol) were added to a suspension of SN-38 (1.0 g, 2.55 mmol) in 51 mL of anhydrous DCM. The reaction mixture was stirred overnight at RT and then washed with 0.2 N HCl, saturated NaHCO3, and saturated NaCl. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. The residue was dissolved in DCM and precipitated by adding hexane. The solid was filtered and dried under vacuum to give compound TBDPS-SN-38 (1.54 g, 96% yield) as a white solid.

[0210] Step 2: ( S 9-((tert-butyldiphenylsilyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzazo[1,2-b]quinoline-4-yl ester hydrochloride (TBDPS-SN-38-Gly)

[0211] DCC (1.26 g, 6.12 mmol) and DMAP (436 mg, 3.57 mmol) were added to a solution of TBDPS-SN-38 (1.61 g, 2.55 mmol) and Boc-Gly-OH (1.07 g, 6.12 mmol) in 75 mL of anhydrous DCM. The reaction mixture was stirred at RT for 20 h. The mixture was filtered. After filtration, the solvent was evaporated under vacuum to give a crude product, which was purified by a gradient of DCM solution (normal phase - Biotage) with 0-70% EtOAc to give the desired product (1.78 g, 89% yield). TBDPS-SN-38-Gly(Boc) (1.78 g, 2.25 mmol) was dissolved in 8.8 mL of dioxane, and then 8.8 mL of HCl / 4M dioxane was added to the solution. The mixture was stirred for 20 min. The reaction was monitored by LC-MS. The solvent was removed by purging with flowing air, and then diethyl ether was added. The precipitate was filtered, washed with diethyl ether, and evaporated under vacuum to give the product (1.58 mg, 97% yield) as a yellow solid.

[0212] Steps 3 and 4: ( S )-4-((2-((9-((tert-butyldiphenylsilyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-4-yl)oxy)-2-oxoethyl)amino)-4-oxobutyric acid (TBDPS-SN-38-Gly-OSu)

[0213] TBDPS-SN-38-Gly (780 mg, 1.08 mmol) was dissolved in anhydrous THF (53.8 mL), treated with triethylamine (750 µL, 5.38 mmol), and then with succinic anhydride (323 mg, 3.23 mmol). The reaction was monitored by LC-MS. After stirring overnight, the solvent was evaporated by rotavap. The residue was dissolved in 3 mL DMSO and purified by Biotage (reversed phase) using a C-18 column with a gradient of water:acetonitrile 10–80%. The pure fraction was lyophilized, and the product TBDPS-SN-38-Gly-OSu (536 mg, 63% yield) was a colorless solid. The purity of the product was assessed by HPLC-MS as >99%, with an m / z mass of 788.0 (M+H).

[0214] Step 4: Synthesis of TH2201:

[0215] TBDPS-SN-38-Gly (243 mg, 308 µmol) was dissolved in DMSO (3.1 mL). TBTU (98.9 mg, 308 µmol) was added, followed by DIPEA (134 µL, 770 µmol). The mixture was stirred at room temperature for 10 min (pH ~7). After pre-activation for 10 min, a solution of TH19P01 (267 mg, 139 µmol) in DMSO (1.04 mL) was rapidly added to the pre-activated ester solution. Since the pH of the reaction mixture was slightly acidic (pH 5 immediately after addition), DIPEA (67.1 µL, 385 µmol) was added to restore the pH to 8-9, and this was maintained. The reaction was monitored by LC-MS. After stirring for 20 min, the reaction was stopped by adding water. The precipitate was filtered and washed with water. The protected conjugate (463 mg) was obtained.

[0216] Step 5:

[0217] The obtained solid (344 mg, 99.4 µmol) was suspended in anhydrous DCM (34 mL) and TBAF (1 M THF solution) (149 µL, 149 µmol) was added. The reaction was monitored by LC-MS. After 45 min, the solid was filtered and washed with DCM and water (0.1% FA) to remove TBAF. Then, it was purified by gradient HPLC (Akta) with 10–40% acetonitrile aqueous solution to give the conjugate TH2101 (87.7 mg, 30% yield) as a colorless solid. The purity of the conjugate was assessed by HPLC-MS and was >98%, with an m / z mass of 2985 (996.5, charge +3).

[0218] Synthesis of TH2205 (see) Figure 6 ):

[0219] Steps 1-3 are similar to those described above for TH2101.

[0220] Step 4: ( S Synthesis of 1-((9-((tert-butyldiphenylsilyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-4-yl)oxy)-1,4-dioxo-7,10,13-trioxa-3-azahexadecane-16-acid (TBDPS-SN-38-Gly-PEG3)

[0221] NMM (79.8 µL, 726 µmol), TBDPS-SN-38-Gly (105 mg, 145 µmol), 6-Cl HOBt (29.5 mg, 174 µmol), and EDCI (33.4 mg, 174 µmol) in 4.5 mL of DCM were added to a solution of bis-PEG3-acid (72.6 mg, 290 µmol) in DMF (0.7 mL) and DCM (10 mL) at 0°C. The resulting reaction solution was stirred at 0°C for 4 h. The reaction progress was monitored by LC-MS. After the starting material was completely exhausted, the reaction system was concentrated. The residue was purified by reversed-phase chromatography using an aqueous solution of 10-80% acetonitrile (0.1% FA) as a gradient to give the desired product TBDPS-SN-38-Gly-PEG3 (90.8 mg, 68% yield) as a white solid. The purity of the product was assessed by HPLC-MS and was > 99%, with an m / z mass of 920.3 (M+H).

[0222] Step 5:

[0223] HATU (96.3 mg, 253 µmol) and DIEA (132 µL, 760 µmol) were added to a solution of TBDPS-SN-38-Gly-PEG3 (233 mg, 253 µmol) in DMSO (4 mL). The mixture was stirred at room temperature for 15 min. Then, a solution of TH19P01 (219 mg, 114 µmol) in DMSO (1.5 mL) was added to the reaction mixture. After stirring for 15 min, the reaction was stopped, and the mixture was purified by HPLC (Akta) using an aqueous solution of 30–80% acetonitrile (0.1% FA) as a gradient. The fraction collected by lyophilization gave the protected conjugate (162 mg, 23% yield). The obtained solid (162 mg, 43.5 µmol) was added to a suspension in anhydrous DCM (15 mL) with TBAF (1 M THF solution) (86.9 µL, 86.9 µmol). The reaction was monitored by LC-MS. After 45 min, the solvent was removed under vacuum. The solid was dissolved in DMSO and then purified by HPLC (Akta) using an aqueous solution of 30-80% acetonitrile (0.1% FA) as a gradient to give the conjugate TH2205 (57 mg, 40% yield) as a colorless solid. The purity of the conjugate was assessed by HPLC-MS and was >98%, with an m / z mass of 3249 (1084.8, charge +3).

[0224] Synthesis of TH2302 (see) Figure 7A -B):

[0225] Steps 1-4 are similar to those described above for TH2205.

[0226] Step 5:

[0227] TBDPS-SN-38-Gly-PEG3 (105 mg, 114 µmol) in DMSO (2.5 mL) was treated with TBTU (36.6 mg, 114 µmol), DIEA (59.6 µL, 342 µmol), and KBP-201 (144 mg, 48.8 µmol). The mixture was purified by HPLC (Akta) using an aqueous solution of 30-80% acetonitrile (0.1% FA) as a gradient to give the protected conjugate (87.3 mg, 18% yield). The product (87.3 mg, 18.3 µmol) was then deprotected with TBAF (36.6 µmol, 36.6 µL). Following the post-treatment and purification as described above, TH2302 (49 mg, 62% yield) was given as a white solid. The purity of the conjugate was assessed by UPLC-MS and was > 95%, with an m / z mass of 4287 (1072, charge +4).

[0228] Synthesis of TH2303 (see) Figure 9A ):

[0229] Step 1: (1 S 9 S Synthesis of 1-((tert-butoxycarbonyl)amino)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-9-yl ester (Boc-exatecan-Gly)

[0230] DCC (887 mg, 4.30 mmol) and DMAP (307 mg, 2.51 mmol) were added to a solution of Boc-ethanotecan (958 mg, 1.79 mmol) and |Z-Gly-OH (900 mg, 4.30 mmol) in 53 mL of anhydrous DCM. The reaction mixture was stirred at room temperature for 4 h. The mixture was filtered. After filtration, the solvent was evaporated under vacuum to give a crude product, which was purified by a gradient of column chromatography (normal phase - Biotage) with DCM solutions of 0-60% EtOAc to give the compound Boc-ethanotecan-Gly(Z) (1.09 g, 84% yield). This compound was dissolved in 30 mL of MeOH / DCM (1 / 1 ratio), treated with palladium on carbon (150 mg, 10 wt.%), and then bubbled with H2 for 1.5 h. The mixture obtained by filtration through diatomaceous earth was washed with MeOH and DCM. The filtrate and washings were concentrated to give Boc-ethanotecan-Gly (705 mg, 79% yield) as a white solid. The purity of the conjugate was assessed by UPLC-MS and was >95%, with an m / z mass of 593 (M+H).

[0231] Step 2: 4-((2-(((1) S 9 S Synthesis of 1-((tert-butoxycarbonyl)amino)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-9-yl)oxy)-2-oxoethyl)amino)-4-oxobutyric acid (Boc-exatecan-Gly-OSu)

[0232] Using the same protocol as for the synthesis of TBDPS-SN-38-Gly-OSu, Boc-ethanotecan-Gly (56 mg, 94.7 µmol) in THF (6 mL) was treated with triethylamine (47.9 µL, 473 µmol) and succinic anhydride (28.4 mg, 284 mmol) as described above, followed by purification to give Boc-ethanotecan-Gly-OSu (31.4 mg, 48% yield). The purity of the conjugate was assessed by UPLC-MS and was >95%, with an m / z mass of 693.06 (M+H).

[0233] Steps 3-4

[0234] In a 25-mL RBF container, TBTU (5.73 mg, 0.0244 mmol) was dissolved in DMSO (248 µL). N,N-diisopropylethylamine, DIEA, and Hunig base (10.6 µL, 0.0611 mmol) were added, followed by AN-01-123-1 (19.2 mg, 0.0244 mmol). The mixture was stirred at room temperature for 10 min (pH ~7). In a separate 25-mL RBF container, TH19P01 (21.2 mg, 0.0110 mmol) was dissolved in DMSO (82.7 µL). After pre-activation for 10 min, the TH19P01 solution was rapidly added to the pre-activated ester solution, and the flask was washed with a small amount of DMSO (41.3 µL). Since the reaction mixture was slightly acidic (pH 5 immediately after addition), N,N-diisopropylethylamine, DIEA, and Hunig base (5.32 µL, 0.0305 mmol) were added to restore the pH to 7-8. LCMS of the reaction at t=10 min (after addition of DIPEA) showed a clean and complete reaction. After stirring for 20 min (total time since addition of DIPEA), the reaction was stopped by adding water. The precipitate was filtered and washed with water to give ethanotecan with Boc protection linked to TH19P01, which was used for the next step without purification. The next step was deprotection of the Boc group using a solution of 1,4-dioxane in 4N HCl. The product was purified using HPLC (Akta) with an aqueous solution of 10-80% acetonitrile (0.1% FA) and the pure fraction was lyophilized to give TH2303 as a white solid. UPLC-MS was performed with a resolution >95%, where the m / z mass was 3073.28 (1024.41, charge +3).

[0235] Synthesis of TH2310 (see) Figures 9B-9C ):

[0236] Step 1: ( S )-9-((tert-butyldiphenylsilyl)oxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]inzazo[1,2-b]quinoline-3,14(4H)-dione (TBDPS-SN-38)

[0237] TBDPS-SN-38 was prepared as described in step 1 of the TH2101 synthesis.

[0238] Steps 2 and 3:

[0239] Step 2:

[0240] TBDPS-SN-38 (3.00 g, 4.19 mmol) was dissolved in DCM (300 mL), and DCC (2.07 g, 10.0 mmol), dimethylaminopyridine (716 mg, 5.86 mmol), and ZL-LYS(BOC)-OH (3.82 g, 10.0 mmol) were added to the solution. The reaction was monitored by LC-MS. The reaction was stopped at 70% by adding DCC and ZL-LYS(BOC)-OH (1 equivalent each time) to bring it to completion. The reaction mixture was directly loaded onto dry silica gel (30 g) and purified using an 80 g column with ethyl acetate:hexane (0-100%) as the eluting solvent. The pure fraction was evaporated to give (S)-N2-((benzyloxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysine 9-((tert-butyldiphenylsilyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-4-yl ester (3.20 g, 77 %) as the final product. MH+ 910.1 was determined at 1.87 min on a 4 min run (because ionization was not observed), >95% purity, and used accordingly for the next step.

[0241] Step 3:

[0242] (S)-9-((tert-butyldiphenylsilyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-4-yl N2-((benzyloxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysine ester (3.20 g, 3.32 mmol) was dissolved in a mixture of DCM (50.0 mL) and methanol (150 mL). The reaction mixture was bubbled with nitrogen for 10 minutes, and 10% (1.00 g) of Pd carbon was immediately added. The reaction mixture was bubbled again with nitrogen, and hydrogen was introduced by means of a balloon. The reaction mixture was stirred at RT, and the reaction was monitored by LCMS. The reaction was almost complete after 20 hours of hydrogen bubbling. The reaction mixture was filtered through Celite®, and the solvent was removed under vacuum to give (S)-N6-(tert-butoxycarbonyl)-L-lysine 9-((tert-butyldiphenylsilyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-4-yl ester (4) (2.00 g, 70 %), as a white solid. Two peaks were observed at MH+ 859. The product was used in the next step.

[0243] Steps 4 and 5: (S)-N6-(tert-butoxycarbonyl)-N2-(4-((2,5-dioxopyrrolidone-1-yl)oxy)-4-oxobutyryl)-L-lysine 9-((tert-butyldiphenylsilyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-4-yl ester (TBDPS-SN-38-Lys(Boc)-OSu)

[0244] Step 4:

[0245] (S)-N6-(tert-butoxycarbonyl)-N2-(4-((2,5-dioxopyrrolidone-1-yl)oxy)-4-oxobutyryl)-L-lysine 9-((tert-butyldiphenylsilyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-4-yl ester (4) (2 g, 2.33 mmol), succinic anhydride (699 mg, 6.98 mmol), dimethylaminopyridine (322 uL, 2.33 mmol) and triethylamine (973 uL, 6.98 mmol) were dissolved in DCM (150 mL), and the reaction mixture was stirred at RT until completion. Once the feedstock was completely depleted, the reaction mixture was adsorbed onto silica gel and purified using a 40 g column with DCM:methanol as the eluent. The purified fractions were collected and evaporated under vacuum to give 4-(((S)-6-((tert-butoxycarbonyl)amino)-1-(((S)-9-((tert-butyldiphenylsilyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-4-yl)oxy)-1-oxohexyl-2-yl)amino)-4-oxobutyric acid (5) (1.18 g, 53%), as a yellowish-white solid. MH+ was determined at 3.21 min on a 4 min run, >90% purity. The product was used for the next step.

[0246] Step 5:

[0247] 4-(((S)-6-((tert-butoxycarbonyl)amino)-1-(((S)-9-((tert-butyldiphenylsilyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-4-yl)oxy)-1-oxohex-2-yl)amino)-4-oxobutyric acid (5) (1.18 g, 1.08 mmol) and N-hydroxysuccinimide (150 mg, 1.30 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide HCl (249 mg, 1.30 mmol) were dissolved in DMF (3.00 mL), and the reaction was stirred at RT until completion. Once the reaction was complete, water was added to the reaction mixture, and the mixture was filtered through a sintered funnel. The precipitate was collected as (S)-N6-(tert-butoxycarbonyl)-N2-(4-((2,5-dioxopyrrolidone-1-yl)oxy)-4-oxobutyryl)-L-lysine 9-((tert-butyldiphenylsilyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-4-yl ester (6) (1.03 g, 90%), a yellowish-white solid. MH was determined at 3.30 minutes on a 4-minute run to be 1056.45, with a purity >95%.

[0248] Step 6:

[0249] (S)-N6-(tert-butoxycarbonyl)-N2-(4-((2,5-dioxopyrrolidone-1-yl)oxy)-4-oxobutyryl)-L-lysine 9-((tert-butyldiphenylsilyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-4-yl ester (6) (500 mg, 0.473 mmol) was dissolved in DMSO (5.74 mL), and TH19PO1 (424 mg, 0.220 mmol) was added to the mixture. Diisopropylethylamine (182 μL, 1.42 mmol) was added to the reaction mixture, and the reaction mixture was stirred at RT until all the starting material in NH-ester form was exhausted. The reaction mixture was quenched by adding water, and the precipitate was collected by filtration to obtain (2S,5S,8S,11S,14S,17S,20S,23S,26S,29S,35S,38S,41S,44S,47S)-23-(2-amino-2-oxoethyl)-17-benzyl-20-butyl-14,38-bis((S)-10-((((S)-9-((tert-butyldiphenylsilyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-4-yl)oxy)carbonyl)-2,2- Dimethyl-4,12,15-trioxo-3-oxa-5,11,16-triazaeicosano-20-yl)-8-(2-carboxyethyl)-26,44-bis(3-guanidinopropyl)-2-(4-hydroxybenzyl)-5,11-bis(hydroxymethyl)-29,47-diisopropyl-35,41-dimethyl-4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52-heptadecoxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51-heptadecoxanoic acid (7) (710 mg, crude), dried under vacuum, and used in the next step. Two peaks were observed at 2.31 and 2.77 min, corresponding to the partially deprotected product (one of the TBS groups cleaved during the reaction) and the expected product, respectively.

[0250]

[0251] Steps 7 and 8:

[0252] The (2S,5S,8S,11S,14S,17S,20S,23S,26S,29S,35S,38S,41S,44S,47S)-23-(2-amino-2-oxoethyl)-17-benzyl-20-butyl-14,38-bis((S)-10-((((S)-9-((tert-butyldiphenylsilyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-4-yl)oxy)carbonyl)-2,2-dimethyl-4,12,1 5-Trioxo-3-oxa-5,11,16-triazaeicosano-20-yl)-8-(2-carboxyethyl)-26,44-bis(3-guanidinopropyl)-2-(4-hydroxybenzyl)-5,11-bis(hydroxymethyl)-29,47-diisopropyl-35,41-dimethyl-4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52-heptadecoxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51-heptadecoxatricanoic acid (7) The crude product (710 mg, 0.187 mmol) was dissolved in DCM, and TBAF (280 µL, 0.280 mmol) was added. The reaction was monitored by LCMS to ensure complete deprotection of TBDPS. Once deprotection was complete, a dioxane solution of 4N HCl (1.87 mL, 7.46 mmol) was added, and the reaction mixture was stirred for another 1 hour. Once complete deprotection was achieved, the solvent was removed by purging with nitrogen, and the residue was dissolved in 3 mL of DMSO and purified using ACTA with water:acetonitrile as the eluent (0-80%, 0.1% formic acid).The pure fractions were pooled together to obtain (2S,5S,8S,11S,14S,17S,20S,23S,26S,29S,35S,38S,41S,44S,47S)-14,38-bis(4-(4-(((S)-6-amino-1-(((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-4-yl)oxy)-1-oxohexyl-2-yl)amino)-4-oxobutyrylamino)butyl)-23-(2 -Amino-2-oxoethyl)-17-benzyl-20-butyl-8-(2-carboxyethyl)-26,44-bis(3-guanidinopropyl)-2-(4-hydroxybenzyl)-5,11-bis(hydroxymethyl)-29,47-diisopropyl-35,41-dimethyl-4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52-heptadecano-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51-heptadecanoic acid (262 mg, 45%).

[0253] The MH+3 (1044.15) of the conjugate was determined at 1.16 min during a 4 min run, with a purity > 98%.

[0254] Example 3: Expression analysis of efflux pump, sorting protein and topoisomerase 1 in cancer cell lines

[0255] The expression of camptothecin analog P-glycoprotein (P-gp or MDR1) efflux pump and breast cancer drug resistance protein (BCRB or ABCG2), as well as sorting protein (SORT1), was evaluated in several human colorectal cancer cell lines. Epidermal growth factor receptor (EGFR) expression was also tested for comparison with the EGFR inhibitor cetuximab (Erbitux), used to treat metastatic colorectal cancer. ® Possible combinations of ) were considered. The HT-29 cell line was chosen to study the effects of the conjugates due to their influence on SORT1, BCRB, and EGFR expression. Figure 11A ).like Figure 11B As shown in -C, all colorectal and breast cancer cell lines tested, including HT-29, expressed topoisomerase 1 (TOPO1), a target of camptothecin analogues.

[0256] Example 4: Inhibitory effects of camptothecin analogues and peptide-camptothecin analogue conjugates on the growth of HT-29 cells in vitro.

[0257] Figure 12A-B demonstrates the antiproliferative activity of PDC and unconjugated drugs against HT-29, HCT-116, and LoVo cancer cells. SN-38 and esxatecan IC 50 The values ​​were lower than those obtained for irinotecan. The peptide-camptothecin analogue conjugate inhibited the growth of HT-29, HCT-116, and / or LoVo tumor cells, confirming that the conjugation of camptothecin analogues to peptides does not inhibit their antiproliferative activity.

[0258] Example 5: Internalization of peptides and peptide conjugates into sorting protein-dependent processes

[0259] Next, we evaluated whether downregulating the expression of sorting proteins in HT-29 cancer cells could affect the internalization of TH19P01 peptide (SEQ ID NO:3, present in several conjugates described herein) and TH2101 conjugate (containing TH19P01 peptide). Figures 13A-13B Results reported in 14A-14C showed that treatment with siRNA targeting the sorting protein reduced sorting protein expression in HT-29 cancer cells. Figure 13A and 14A Furthermore, reduced sorting protein expression was associated with decreased uptake of fluorescently labeled TH19P01 peptide and TH2101 conjugate. Figure 13B and 14B These results confirm that the TH19P01 peptide specifically binds to sorting proteins on the cell surface, and thus can be used in conjugates to deliver camptothecin analogs to cells expressing sorting proteins.

[0260] Example 6: Effects of camptothecin analogues and camptothecin analogue conjugates in mouse models of colorectal cancer, ovarian cancer, and breast cancer.

[0261] The antitumor effects of unconjugated camptothecin analogues and camptothecin analogue conjugates were tested in vivo in HT-29, HCT-116 and patient-derived xenograft colorectal cancer mouse models, MDA-MB-231 triple-negative breast cancer (TNBC) xenograft model and SKOV3 ovarian cancer xenograft model. Figure 15A The results reported in -C showed a weak inhibitory effect on tumor growth in mice treated with the unconjugated camptothecin analogues irinotecan and ethanotecan. For irinotecan, administration of a dose equivalent to 1 / 8 of the maximum tolerated dose (MTD) had no effect on tumor growth. Conversely, administration of this dose of irinotecan (1 / 8 MTD), but as part of a conjugate with a peptide targeting the sorting protein of SEQ ID NO:1 (TH2102), almost completely inhibited HT-29 tumor growth. Figure 16A-B). Similar effects were obtained using another irinotecan conjugate TH2204, SN-38 conjugates TH2101 and TH2205, and ethanotecan conjugates TH2301 and TH2303. Figure 17A -B and 18A-B and D). TH2303 ethatecan conjugates were also able to almost completely inhibit HCT-116 tumor growth ( Figure 19A ), and completely inhibited the growth of MDA-MB-231 tumors ( Figure 20A The administration of different conjugates was not associated with significant weight loss in any of the tested models. Figure 16C , 17C 18C, 18E, 19B and 20B). Figure 21A and 21B The results reported in the study showed that the ethatec conjugate TH2303, rather than the unconjugated ethatec, reduced the growth of ovarian tumor cells (SKOV3) and patient-derived xenograft (PDX) colorectal cancer cells, respectively.

[0262] Although the invention has been described above through specific embodiments, modifications may be made thereto without departing from the spirit and nature of the invention as defined in the appended claims. In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the word "including, but not limited to." Unless the context clearly specifies otherwise, the singular forms "a," "an," and "the" include the corresponding plural references.

Claims

1. A conjugate comprising structure I or a pharmaceutically acceptable salt thereof: A — L — B(I); in A is a camptothecin analogue. B is a peptide that binds to a sorting protein receptor, wherein the peptide comprises an amino acid sequence that has at least 60% identity with the amino acid sequence listed in SEQ ID NO:1 or 2: Where X 1 Cysteine ​​(C) or absent, and X 2 It is either cysteine ​​(C) or absent; L is a linker, wherein the first end of the linker is connected to a side chain or modified side chain residue of one or more amino acids of the peptide, and / or to the N- and / or C-terminus of the peptide, and the second end of the linker is connected to a camptothecin analogue.

2. The conjugate of claim 1, wherein L is a cleavable linker, wherein a first end of the linker is attached to a side chain of one or more lysine (K) and / or cysteine ​​(C) residues of the peptide, and / or to the N- and / or C-terminus of the peptide, and a second end of the linker is attached to an oxygen or nitrogen atom of a camptothecin analogue.

3. The conjugate of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the conjugate or salt thereof has one of the following formulas: Ia, Ib, Ic, or Id: A1 — L1 — B(Ia); A1 — L1 — B — L2 — A2(Ib); (Ic); (Id); L1, L2, L3, and L4 can be the same or different connectors, and A1, A2, A3, and A4 can be the same or different camptothecin analogues.

4. The conjugate of any one of claims 1-3 or a pharmaceutically acceptable salt thereof, wherein the camptothecin analogue is irinotecan, SN-38, ethanotecan, or delutecan.

5. The conjugate of claim 4 or a pharmaceutically acceptable salt thereof, wherein the camptothecin analogue is SN-38.

6. The conjugate of claim 4 or a pharmaceutically acceptable salt thereof, wherein the camptothecin analogue is ethatecan.

7. The conjugate of claim 4 or a pharmaceutically acceptable salt thereof, wherein the camptothecin analogue is delutecan.

8. The conjugate of any one of claims 1-7 or a pharmaceutically acceptable salt thereof, wherein the peptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence listed in SEQ ID NO: 1 or 2.

9. The conjugate of claim 8 or a pharmaceutically acceptable salt thereof, wherein the peptide comprises an amino acid sequence having at least 90% identity with the amino acid sequence listed in SEQ ID NO: 1 or 2.

10. The conjugate of claim 9 or a pharmaceutically acceptable salt thereof, wherein the peptide comprises the amino acid sequence listed in SEQ ID NO: 1 or 2.

11. The conjugate of claim 9 or a pharmaceutically acceptable salt thereof, wherein the peptide comprises the amino acid sequence listed in SEQ ID NO: 1 or 2.

12. The conjugate of any one of claims 1-11 or a pharmaceutically acceptable salt thereof, wherein X 1 and / or X 2 C, and wherein the first end of the connector is connected to X. 1 and / or X 2 The side chain.

13. The conjugate of claim 12 or a pharmaceutically acceptable salt thereof, wherein X 1 It does not exist, and X 2 C, and wherein the first end of the connector is connected to X. 2 The side chain.

14. The conjugate of any one of claims 1-11 or a pharmaceutically acceptable salt thereof, wherein X 1 and X 2 It does not exist.

15. The conjugate of any one of claims 1-14 or a pharmaceutically acceptable salt thereof, wherein the linker comprises at least one amino acid.

16. The conjugate of claim 15 or a pharmaceutically acceptable salt thereof, wherein at least one amino acid comprises glycine, valine, and / or citrulline.

17. The conjugate of claim 15 or 16 or a pharmaceutically acceptable salt thereof, wherein the cleavable linker is a protease-cleavable linker.

18. The conjugate of claim 17 or a pharmaceutically acceptable salt thereof, wherein the protease is a cysteine ​​protease.

19. The conjugate of claim 18 or a pharmaceutically acceptable salt thereof, wherein the cysteine ​​protease is cathepsin B.

20. The conjugate of any one of claims 1-19 or a pharmaceutically acceptable salt thereof, wherein the conjugate comprises polyethylene glycol (PEG).

21. The conjugate of claim 20 or a pharmaceutically acceptable salt thereof, wherein the conjugate comprises 2-10 PEG units.

22. The conjugate of any one of claims 1-21 or a pharmaceutically acceptable salt thereof, wherein the linker has one of the following structures: CO(CH2) a CONH(CH2) b CO, where a and b are independently 1, 2, 3 or 4; CO(CH2) d (OCH2CH2) e CONH(CH2) f CO, where d and f are independently 1, 2, 3, or 4, and e is an integer from 2 to 8; or 。 23. The conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the conjugate has one of the following structures: , , , , , , , , or 。 24. A pharmaceutical composition comprising the conjugate of any one of claims 1-23 or a salt thereof and a pharmaceutically acceptable excipient.

25. A method for treating a subject with cancer expressing a sorting protein, comprising administering to the subject an effective amount of the conjugate of any one of claims 1-23 or a salt thereof or the pharmaceutical composition of claim 24.

26. The method of claim 25, wherein the cancer expressing the sorting protein is a hematologic cancer, ovarian cancer, endometrial cancer, cervical cancer, skin cancer, brain cancer, breast cancer, colorectal cancer, small bowel cancer, liver cancer, lung cancer, eye cancer, prostate cancer, head and neck cancer, stomach cancer, bone cancer, thyroid cancer, testicular cancer, bladder cancer, kidney cancer, or pancreatic cancer.

27. The method of claim 25 or 26, wherein the cancer expressing the sorting protein is a cancer with a poor prognosis.

28. The method of any one of claims 25-27, wherein the cancer expressing the sorting protein is an immunologically cold cancer.

29. The method of any one of claims 25-28, wherein the conjugate, its salt, or composition is administered in combination with one or more other active agents or therapies for cancer.

30. The method of claim 29, wherein one or more additional active agents or therapies for cancer comprise immunotherapy.

31. The method of claim 30, wherein the immunotherapy comprises an immune checkpoint inhibitor.

32. The method of claim 31, wherein the immune checkpoint inhibitor is a PD1 or PD-L1 inhibitor.

33. The method of claim 32, wherein the PD1 or PD-L1 inhibitor is an anti-PD1 or anti-PD-L1 antibody.

34. Use of the conjugate of any one of claims 1-23 or a salt thereof, or the pharmaceutical composition of claim 24, for treating a subject with cancer expressing a sorting protein.

35. Use of the conjugate of any one of claims 1-23 or a salt thereof, or the pharmaceutical composition of claim 24, in the preparation of a medicament for treating a subject with cancer expressing a sorting protein.

36. The use of claim 34 or 35, wherein the cancer expressing the sorting protein is a hematologic cancer, ovarian cancer, endometrial cancer, cervical cancer, skin cancer, brain cancer, breast cancer, colorectal cancer, small bowel cancer, liver cancer, lung cancer, eye cancer, prostate cancer, head and neck cancer, stomach cancer, bone cancer, thyroid cancer, testicular cancer, bladder cancer, kidney cancer, or pancreatic cancer.

37. Use according to any one of claims 34-36, wherein the cancer expressing the sorting protein is a cancer with a poor prognosis.

38. Use according to any one of claims 34-37, wherein the cancer expressing the sorting protein is an immunologically cold cancer.

39. The use according to any one of claims 34-38, wherein the conjugate, its salt, composition, or medicine is used in combination with one or more other active agents or therapies for cancer.

40. The use of claim 39, wherein one or more additional active agents or therapies for cancer comprise immunotherapy.

41. The use of claim 40, wherein the immunotherapy comprises an immune checkpoint inhibitor.

42. The use of claim 41, wherein the immune checkpoint inhibitor is a PD1 or PD-L1 inhibitor.

43. The use of claim 42, wherein the PD1 or PD-L1 inhibitor is an anti-PD1 or anti-PD-L1 antibody.

44. The conjugate of any one of claims 1-23 or a salt thereof, or the pharmaceutical composition of claim 24, for treating a subject with cancer expressing a sorting protein.

45. A conjugate, salt thereof, or pharmaceutical composition for use in claim 44, wherein the cancer expressing the sorting protein is a hematologic cancer, ovarian cancer, endometrial cancer, cervical cancer, skin cancer, brain cancer, breast cancer, colorectal cancer, small bowel cancer, liver cancer, lung cancer, eye cancer, prostate cancer, head and neck cancer, stomach cancer, bone cancer, thyroid cancer, testicular cancer, bladder cancer, kidney cancer, or pancreatic cancer.

46. ​​A conjugate, salt thereof, or pharmaceutical composition for use in claim 45, wherein the cancer expressing the sorting protein is a cancer with a poor prognosis.

47. A conjugate, salt thereof, or pharmaceutical composition for use in claim 45 or 46, wherein the cancer expressing the sorting protein is an immunologically cold cancer.

48. A conjugate, salt thereof, or pharmaceutical composition for use in any one of claims 45-47, wherein the conjugate, salt thereof, or composition is used in combination with one or more other active agents or therapies for cancer.

49. A conjugate, salt thereof, or pharmaceutical composition for use in claim 48, wherein one or more additional active agents or therapies for cancer comprise immunotherapy.

50. A conjugate, salt thereof, or pharmaceutical composition for use in claim 49, wherein the immunotherapy comprises an immune checkpoint inhibitor.

51. A conjugate, salt thereof, or pharmaceutical composition for use in claim 50, wherein the immune checkpoint inhibitor is a PD1 or PD-L1 inhibitor.

52. A conjugate, salt thereof, or pharmaceutical composition for use in claim 51, wherein the PD1 or PD-L1 inhibitor is an anti-PD1 or anti-PD-L1 antibody.

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